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Current Medicinal Chemistry, 2005, 12, 1361-1394 1361 Current Knowledge on the Antagonists and Inverse Agonists of Cannabinoid Receptors G.G. Muccioli and D.M. Lambert * Unité de Chimie pharmaceutique et de Radiopharmacie, Université catholique de Louvain, Avenue E. Mounier, 73, UCL-CMFA7340, B-1200 Bruxelles, Belgium Abstract: Ten years elapsed since the discovery by Sanofi of SR141716A the first selective CB 1 cannabinoid receptor antagonist. Shortly after, Sanofi also reported the synthesis of the first selective CB 2 cannabinoid receptor antagonist, SR144528. Since these two milestones in the cannabinoid field, many other compounds, more or less related to the Sanofi compounds, or based on a completely different scaffold appeared. Several of these compounds are currently involved in clinical trials for diseases such as obesity, nicotine and alcohol addictions, or allergies. Further, the cannabinoid receptors knock-out mice production strengthened the hypothesis of the existence of several other “cannabinoid” receptors for which the first antagonists begin to appear. The large amount of patents taken by many different pharmaceutical companies prove, if necessary, the great therapeutic potential expected for the cannabinoid receptors antagonists. Keywords: Cannabinoid, antagonism, inverse agonism, rimonabant, SR144528, SLV319. INTRODUCTION voltage-dependent calcium channels [15], the Mitogen Activated Protein Kinase cascade [16,17], and the phosphokinase B pathway [18]. For many years, pharmacological actions of plant derived cannabinoids were ascribed to membrane disruption effects, rather than to specific receptor-mediated interactions [1]. The development of synthetic high-affinity ligands made easier the discovery [2], and the cloning from the rat [3] and human [4] of the first cannabinoid receptor christened CB 1 , for cannabinoid type 1 receptor. This receptor, highly expressed in the CNS, especially in the allocortex, the substantia nigra, the globus pallidus, and the cerebellum [5], is also present outside the CNS, like in the testis, ileum, urinary bladder, and vas deferens. Two splice variants of this receptor, called CB 1A [6] and CB 1B [7], have also been cloned from the human. The CB 1A cannabinoid receptor exhibits all the properties of the CB 1 isoform [8], this is not the case for the 1B isoform, which essentially differs in the endocannabinoid binding [7]. However, their physiological and pharmacological significances remain, to date, unknown. Shortly after the cloning of the CB 1 receptor, a second cannabinoid receptor, the CB 2 cannabinoid receptor, was found by sequence homology analysis [9]. This receptor, sharing 44% homology with the CB 1 receptor, is mainly expressed in the immune system. Consequently to the discovery of the receptors, were characterized their endogenous ligands, the so-called “endocannabinoids”. Important representatives are arachidonylethanolamide (anandamide, AEA) [19] and 2- arachidonoylglycerol (2-AG) [20,21]. Other compounds were described as endocannabinoids – i.e. 2-arachidonyl glyceryl ether [22] (noladin ether) – but their endogenous occurrence is still under debate [23]. Nowadays, there is a growing literature dealing with the physiological role of the endocannabinoid system and some potential therapeutic applications, either for agonists or for antagonists, are already well explored [24]. This is the case for example for the anti-anorectic effect of dronabinol [25] (synthetic ∆ 9 -tetrahydrocannabinol, ∆ 9 -THC), the analgesic effect of ∆ 9 -THC containing cannabis preparations [26], or in contrast, for the anti-obesity effect of rimonabant (Acomplia ® ). However, several actions remain unclear and are currently under investigation. Since the reports, by Ledent et al. [27] and by Zimmer et al. [28], CB 1 receptor knockout mice strains became a very useful tool to further explore some of the physiological roles of this receptor. Moreover, they strengthened the hypothesis of the existence of additional non-CB 1 and non-CB 2 cannabinoid receptors in the brain and in the periphery. Wiley and Martin in 2002 reviewed the evidences for the existence of these additional “cannabinoid” receptors [29]. Despite that these putative new receptors are not yet fully characterised, the last section of this paper will describe the known antagonists of these receptors. The cloning of these two receptors was the main milestone and since, cannabinoids became a widely explored field. These cannabinoid receptors are G-protein coupled receptors (GPCR), acting mainly through G i/0 -type G- proteins [10]. Even if a G s coupling was shown to occur with the CB 1 cannabinoid receptor by Glass et al. [11] and by Calandra et al. [12], the G i/0 pathway seems to be the preferred one. The major cannabinoid signalling pathways described so far include the adenylyl cyclase inhibition [13], the inwardly rectifying potassium channels [14] and the The interest in the synthesis of new antagonists is still present as testified by the great number of new compounds reported either by the pharmaceutical companies or the academic research laboratories. Due to the great interest in the field, several reviews dealing with the cannabinoid ligands have already been published [30,31]. However, the *Address correspondence to this author at the Unité de Chimie pharmaceutique et de Radiopharmacie, Ecole de Pharmacie, Faculté de Médecine, Université catholique de Louvain, Avenue E. Mounier, 73, UCL-CMFA 7340, B-1200 Bruxelles, Belgium; Tel: +32 2 764 73 47; Fax: +32 2 764 73 63; E-mail: [email protected] 0929-8673/05 $50.00+.00 © 2005 Bentham Science Publishers Ltd.

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Page 1: Current Knowledge on the Antagonists and Inverse Agonists of

Current Medicinal Chemistry 2005 12 1361-1394 1361

Current Knowledge on the Antagonists and Inverse Agonists of CannabinoidReceptors

GG Muccioli and DM Lambert

Uniteacute de Chimie pharmaceutique et de Radiopharmacie Universiteacute catholique de Louvain Avenue E Mounier 73UCL-CMFA7340 B-1200 Bruxelles Belgium

Abstract Ten years elapsed since the discovery by Sanofi of SR141716A the first selective CB1 cannabinoidreceptor antagonist Shortly after Sanofi also reported the synthesis of the first selective CB2 cannabinoidreceptor antagonist SR144528 Since these two milestones in the cannabinoid field many other compoundsmore or less related to the Sanofi compounds or based on a completely different scaffold appeared Several ofthese compounds are currently involved in clinical trials for diseases such as obesity nicotine and alcoholaddictions or allergies Further the cannabinoid receptors knock-out mice production strengthened thehypothesis of the existence of several other ldquocannabinoidrdquo receptors for which the first antagonists begin toappear The large amount of patents taken by many different pharmaceutical companies prove if necessary thegreat therapeutic potential expected for the cannabinoid receptors antagonists

Keywords Cannabinoid antagonism inverse agonism rimonabant SR144528 SLV319

INTRODUCTION voltage-dependent calcium channels [15] the MitogenActivated Protein Kinase cascade [1617] and thephosphokinase B pathway [18]For many years pharmacological actions of plant derived

cannabinoids were ascribed to membrane disruption effectsrather than to specific receptor-mediated interactions [1] Thedevelopment of synthetic high-affinity ligands made easierthe discovery [2] and the cloning from the rat [3] and human[4] of the first cannabinoid receptor christened CB1 forcannabinoid type 1 receptor This receptor highly expressedin the CNS especially in the allocortex the substantianigra the globus pallidus and the cerebellum [5] is alsopresent outside the CNS like in the testis ileum urinarybladder and vas deferens Two splice variants of thisreceptor called CB1A [6] and CB1B [7] have also beencloned from the human The CB1A cannabinoid receptorexhibits all the properties of the CB1 isoform [8] this is notthe case for the 1B isoform which essentially differs in theendocannabinoid binding [7] However their physiologicaland pharmacological significances remain to date unknownShortly after the cloning of the CB1 receptor a secondcannabinoid receptor the CB2 cannabinoid receptor wasfound by sequence homology analysis [9] This receptorsharing 44 homology with the CB1 receptor is mainlyexpressed in the immune system

Consequently to the discovery of the receptors werecharacterized their endogenous ligands the so-calledldquoendocannabinoidsrdquo Important representatives arearachidonylethanolamide (anandamide AEA) [19] and 2-arachidonoylglycerol (2-AG) [2021] Other compounds weredescribed as endocannabinoids ndash ie 2-arachidonyl glycerylether [22] (noladin ether) ndash but their endogenous occurrenceis still under debate [23]

Nowadays there is a growing literature dealing with thephysiological role of the endocannabinoid system and somepotential therapeutic applications either for agonists or forantagonists are already well explored [24] This is the casefor example for the anti-anorectic effect of dronabinol [25](synthetic ∆9-tetrahydrocannabinol ∆9-THC) the analgesiceffect of ∆9-THC containing cannabis preparations [26] or incontrast for the anti-obesity effect of rimonabant(Acompliareg) However several actions remain unclear andare currently under investigation Since the reports byLedent et al [27] and by Zimmer et al [28] CB1 receptorknockout mice strains became a very useful tool to furtherexplore some of the physiological roles of this receptorMoreover they strengthened the hypothesis of the existenceof additional non-CB1 and non-CB2 cannabinoid receptors inthe brain and in the periphery Wiley and Martin in 2002reviewed the evidences for the existence of these additionalldquocannabinoidrdquo receptors [29] Despite that these putativenew receptors are not yet fully characterised the last sectionof this paper will describe the known antagonists of thesereceptors

The cloning of these two receptors was the mainmilestone and since cannabinoids became a widely exploredfield These cannabinoid receptors are G-protein coupledreceptors (GPCR) acting mainly through Gi0-type G-proteins [10] Even if a Gs coupling was shown to occurwith the CB1 cannabinoid receptor by Glass et al [11] andby Calandra et al [12] the Gi0 pathway seems to be thepreferred one The major cannabinoid signalling pathwaysdescribed so far include the adenylyl cyclase inhibition [13]the inwardly rectifying potassium channels [14] and the The interest in the synthesis of new antagonists is still

present as testified by the great number of new compoundsreported either by the pharmaceutical companies or theacademic research laboratories Due to the great interest inthe field several reviews dealing with the cannabinoidligands have already been published [3031] However the

Address correspondence to this author at the Uniteacute de Chimiepharmaceutique et de Radiopharmacie Ecole de Pharmacie Faculteacute deMeacutedecine Universiteacute catholique de Louvain Avenue E Mounier 73UCL-CMFA 7340 B-1200 Bruxelles Belgium Tel +32 2 764 73 47 Fax+32 2 764 73 63 E-mail lambertcmfauclacbe

0929-867305 $5000+00 copy 2005 Bentham Science Publishers Ltd

1362 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

first review only devoted to cannabinoids antagonists waspublished by Barth and Rinaldi-Carmona back in 1999 [32]

hypomotility and hypothermia upon administration in mice[33] An antagonist should reverse the effect obtained withthe agonistThe aim of this paper will be to review the cannabinoid

antagonists and inverse agonists with a particular emphasison the newest compounds Nevertheless as a matter ofcompleteness this review will also cover the earliestdevelopment in the field of cannabinoid antagonists Furtherthe great interest of the pharmaceutical companies for thefield led to the publication of a great amount of patentsTherefore this paper will cover in addition to the scientificpapers the patents covering the cannabinoid antagonists

Several ex-vivo assays are also used to characterise theagonists or antagonists properties of the cannabinoidreceptors ligands The inhibition of the electrically evokedcontractions in the guinea pig ileum and in the mouse vasdeferens are among the most widely used ex-vivo assays thelatter being more sensitive to cannabinoids [34]

However it is quite difficult in these in-vivo and ex-vivoassays to distinguish between antagonists and inverse-agonist effectsOne of the most used in-vivo assay to characterise the

pharmacological properties of the cannabinoids is thecannabinoid tetrad of effects An agonist of the CB1cannabinoid receptor must induce analgesia catalepsia

Therefore several in-vitro assays are currently used toexplore the functionality of known ligands cAMPquantification is one of the most widely used methods based

Table 1 CB1 Cannabinoid Receptor Antagonists 1 Tetrahydrocannabinol and Cannabidiol Derivatives

Some ∆8-Tetrahydrocannabinol Derivatives Possessing a Rigidified Side Chain The Affinity for the CB1 Receptor (Radioligand Cells) and the Function(Assay Used) are Given

O

OH

R

Cpd ndeg R= CB1 (Ki) Function

O-823 1

CN

077 nM([3H]-CP-55940 rat brain) c

bull inactive in Tetrad Test (up to 30 mgkg mouse) e

bull partial agonist (mouse vas deferens) c

bull antagonist (guinea pig myenteric plexus) c

bull antagonist ([35 S]-GTPγS rat cerebella) b

O-1184 2

N3

524 nM([3H]-CP-55940 hCB1-CHO

cells) a

bull partial agonist in Tetrad Test (mouse) e

bull antagonist (guinea pig myenteric plexus) d

bull partial agonist (cAMP hCB1-CHO cells) a

bull antagonist ([35 S]-GTPγS rat cerebella) b

O-584 3 426 nM([3H]-CP-55940 hCB1-CHO

cells) a

bull agonist (cAMP hCB1-CHO cells) a

bull antagonist ([35 S]-GTPγS rat cerebella) b

O-806 4

Br

12 nM([3H]-CP-55940 rat brain)

ebull partial agonist in Tetrad Test (mouse) e

bull antagonist ([35 S]-GTPγS rat cerebella) b

O-1176 5

NCS

115 nM([3H]-CP-55940 rat brain) e

bull inactive in Tetrad Test (up to 30 mgkg mouse) e

bull antagonist ([35 S]-GTPγS rat cerebella) b

O-1238 6 N3 354 nM([3H]-CP-55940 hCB1-CHO

cells) a

bull partial agonist ([35 S]-GTPγS rat cerebella) b

bull agonist (cAMP hCB1-CHO cells) a

7HN

S

O

O30 nM f bull agonist in Tetrad Test (mouse) f

O-2050 8HN

S

O

O25 nM f bull no effect per se in Tetrad Test (mouse)f

bull antagonist (mouse vas deferens) f

a[39] b[38] c[36] d[37] e[40] f[41]

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1363

on the negative coupling of cannabinoid receptors toadenylyl cyclase The binding of an agonist will produce adecrease in cAMP production which can be measured eitherdirectly (EIA) or through a gene-reporter system (fireflyluciferase) However due to the existence of a dual-couplingfor the CB1 cannabinoid receptor the [35S]-GTPγ S assayshould be preferred It is based on the property shared by allthe GPCRs to bind to a GTP molecule upon activation byan agonist Therefore the binding of an agonist will increasethe [35S]-GTPγ S a radiolabelled non-hydrolysable analogueof GTP binding [35] These assays allow to distinguishbetween full agonists (positive intrinsic activity) partialagonists neutral antagonists (no intrinsic activity) andinverse agonists (negative intrinsic activity)

antinociception nor hypothermia in mice and lacks ofagonist effects in mouse vas deferens preparation In thisassay it behaved as an antagonist devoid of inverse agonistproperties

Recently Thomas et al reported the synthesis andcharacterisation of O-2654 (9) (Fig 1) obtained bymodifying the structure of cannabidiol (or cbd) (10) a nonpsychoactive cannabinoid [42] This compound unlikecannabidiol binds to the CB1 receptor with a Ki value of114 nM against [3H]-CP-55940 on mouse brainmembranes In the mouse vas deferens model O-2654antagonises the WIN-55212-2 inhibition of current-inducedcontractions causing a rightward shift in the logconcentration response curve of the agonist (KB = 857 nM)Further in the vas deferens model O-2654 behaves as aneutral antagonist However this neutral antagonism has tobe further confirmed using other models

II CB1 CANNABINOID RECEPTOR ANTAGONISTSAND INVERSE AGONISTS

OH

OH

N3

O-2654 (9)

Compounds having antagonist or inverse agonistproperties at the CB1 cannabinoid receptor are reviewed inthis part of the paper They are classified depending on theirchemical structures

1 Tetrahydrocannabinol and Cannabidiol Derivatives

The first attempts to obtain cannabinoid ligands havingantagonist properties were conducted using the tricyclicstructure of classical cannabinoids such as ∆9-tetrahydrocannabinol (∆9-THC) as a scaffold These earlyresearches have been previously reviewed by Barth andRinaldi-Carmona [32] The most promising modulationsinvolved the side-chain of ∆8-tetrahydrocannabinol (∆8-THC) as it is the case in O-823 (1) (Table 1) Thiscompound (Ki = 077 nM) acts as a partial agonist in mousevas deferens preparations but as an antagonist in guinea-pigmyenteric plexus preparations [36] Ross and co-workers[37] showed that O-1184 (2) binds to the CB1 receptor (Ki =285 nM) and as O-823 possesses antagonist properties inthe myenteric plexus-longitudinal muscles O-823 and O-1184 were shown to act as surmountable antagonists in a[35S]-GTPγ S assay with KB values of 485 and 297 nMrespectively against CP-55940 in rat cerebellar membranes[38] Three other ∆8-tetrahydrocannabinol-3rsquo-ynylderivatives O-584 (3) O-806 (4) and O-1176 (5) alsobehaved as antagonists However in a cAMP productionassay conducted in hCB1 transfected CHO cells O-1184behaved as an agonist equipotent to CP-55940 [39] Takentogether these examples illustrate that the introduction of anacetylenic moiety into the side chain of ∆8-THC affects theactivation of the receptor more than the affinity of thesederivatives Several of these compounds were tested in-vivoin the cannabinoid tetrad test by Martin and co-workers [40]For instance O-823 which was inactive in the tetrad actedas an antagonist in the [35S]-GTPγ S assay [38]

Fig (1) CB1 cannabinoid receptor antagonists 1Tetrahydrocannabinol and cannabidiol derivatives Chemicalstructure of (-)-6-azidohex-2-yne-cannabidiol (O-2654 9)

2 Aminoalkylindole Derivatives

This family of compounds was introduced by Sterlingrsquosresearchers in the early nineties with a derivative of the anti-inflammatory drug pravadoline called WIN-55212 Albeitthis compound acts as an agonist some related compoundsshowed interesting antagonist properties in the mouse vasdeferens assay by dose-dependently antagonising ∆9-THCand levonantradol effects This is the case for instance for

N

O OMe

N

O

X

N

O

N

O

WIN-56098 (11) X=Br WIN-54461 (12)X=I AM630 (13)

Fig (2) CB1 cannabinoid receptor antagonists 2Aminoalkylindole derivatives Structures of WIN-56098 (11 )WIN-54461 (12 ) and AM630 (13 ) three aminoalkylindolederivatives acting as CB1 cannabinoid receptor antagonists

More recently Martin et al described three newcompounds possessing an alkyl sulfonamide at the end of thealkyne side chain [41] The ethyl (7) (Ki = 30 nM) andbutyl (Ki = 70 nM) derivatives were shown to behave asagonists in the cannabinoid tetrad while the methylderivative (8) (Ki = 25 nM) acted as a silent antagonistThis compound O-2050 does not induce neither

1364 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

WIN-56098 (11) and WIN-54461 (12) the latter being abromo derivative of pravadoline (Fig 2) These twocompounds however have a low affinity for the cannabinoidreceptor (IC50 gt 500 nM) [4344]

cAMP accumulation in hCB1-CHO cells with an IC50 valueof 56 nM SR141716A is able after ip or poadministration to inhibit the [3H]-CP-55940 binding tomice brain measured ex-vivo with ED50

values around 2mgkg [51] In addition to the inhibition of the classicalcannabinoid tetrad effects ndash hypothermia ring immobilityanalgesia and hypolocomotion ndash already shown by Rinaldi-Carmona et al in their first report [50] SR141716A wasshown to antagonise other in-vivo effects of cannabinoidagonists For instance the agonist-induced hypotension andbradycardia in mice are abolished by SR141716A [52] aswell as the antihyperalgesic effects of the cannabinoidagonists in a neuropathic model of pain in rat [53] This isalso the case for the behavioral effects of agonists treated rats[54] or the cannabinoid tetrad effects induced by ∆9-THC inmice [55]

In 1995 Pertwee et al [45] described the effect ofAM630 (13) another close analogue of pravadoline Thiscompound behaved as an antagonist in a [35S]-GTPγ S assayon mouse brain preparations antagonising WIN-55212-2-induced [35S]-GTPγ S binding [46] and as an inverseagonist on hCB1-CHO cells [47] (EC50 = 09 microM)However Ross et al found a weak partial agonist activityfor this compound as it decreases cAMP production byhCB1-CHO cells [48] AM630 was subsequentlycharacterised as a CB2 ligand (see the CB2 section) Thepharmacological properties of the aminoalkylindole familywere reviewed by John Huffman [49]

3 Diarylpyrazole DerivativesDespite various papers described SR141716A as an

antagonist [56-58] today this compound is considered to actas an inverse agonist based on [35S]-GTPγ S [59-62] andcAMP accumulation assays [62-64] (Table 2) An interestingreview dealing with inverse agonism at the cannabinoidreceptors and mostly with SR141716A effects was recentlypublished by Roger Pertwee [65]

The lead of this class of compounds SR141716A (14)was introduced by Sanofi back in 1994 [50] This compoundwas shown by Rinaldi-Carmona and co-workers to be ahighly selective CB1 ligand with Ki values of 56 nM for thehCB1 and over 1000 nM for the hCB2 receptors expressed inCHO cells ([3H]-CP-55940) However more recentlyevidences appeared showing that SR141716A binds toother(s) receptor(s) described as anandamide andorcannabinoid receptors Thus it is possible that some of thein-vivo effects caused by this compound are at least notsolely CB1 mediated (see the fourth section of this paper)

In 1998 Pan et al [66] demonstrated that the lysineresidue K328 (192) located in the third transmembranedomain (TMH3) of the hCB1 receptor is a key residue forthe inverse agonist action of SR141716A (Table 3) Theyshowed that SR141716A enhances calcium current in hCB1transfected neurons but not in K(192)A mutant receptortransfected neurons However SR141716A still antagonizedWIN-55212-2 inhibition of calcium currents proving that itis able to bind to the mutated receptor Later on Hurst et al[67] demonstrated using molecular modeling techniques aswell as in-vitro experiments that lysine residue K328(192)is a direct interaction site for hydrogen bonding with the C3substituent of SR141716A in CB1 receptor Binding results

In a mouse vas deferens preparation SR141716A causesa rightward shift of the CP-55940 concentration-responsecurve behaving as a competitive antagonist having a pA2value of 798 Furthermore in the cAMP accumulationmodel SR141716A produces no effect by itself butantagonizes the CP-55940 inhibition of forskolin-induced

Table 2 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

In-Vitro Functional Characterisation of SR141716A (14) The Assay Used the Cell Type and the Obtained Effect are Given

Cell type Assay Effect Function References

hCB1-CHO cells cAMP accumulation no effect by itself antagonist [50]

hCB1-CHO cells cAMP accumulation uarr [cAMP] inverse agonist [64]

hCB1-CHO cells [35 S]-GTPγS darr[35 S]-GTPγS binding inverse agonist [59]

hCB1-neurons Ca2+ currents uarr Ca2+ currents inverse agonist [66]

rCB1 (rat cerebella) [35 S]-GTPγS No effect by itself antagonist [56]

rCB1 (rat cerebella) [35 S]-GTPγS No effect by itself antagonist [57]

rCB1 (rat cerebella) [35 S]-GTPγS No effect by itself antagonist [58]

rCB1 (rat brain) [35 S]-GTPγS darr[35 S]-GTPγS binding inverse agonist [62]

rCB1 (rat cerebella) [35 S]-GTPγS darr[35 S]-GTPγS binding inverse agonist [60]

rCB1 (rat cerebella) [35 S]-GTPγS darr[35 S]-GTPγS binding inverse agonist [61]

rCB1 (rat brain) cAMP accumulation uarr [cAMP] inverse agonist [63]

mCB1 (mouse brain) cAMP accumulation uarr [cAMP] inverse agonist [62]

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1365

obtained using HEK293 cells transfected with either theK328A mutant or the wild type receptor remarkablyconfirmed the modeling results (Kd values of 396 and 23nM respectively) Interestingly a vinyl-cyclohexylSR141716A derivative VCHSR1 (15) (Table 4) lacking ofhydrogen bonding sites in C3 position is not affected bythis CB1 receptor mutation as its affinity remains unchangedwith Ki values of 31 and 35 nM for the wild-type andK328A receptors respectively Four additional compoundsCHASR1 (16) CHMSR1 (17) VPSR1 (18) and PIMSR(19) differing by their potential to form hydrogen bondswere evaluated in affinity and functional assays The resultsfurther confirm the crucial interaction between the C-3carboxamide oxygen and residue K328 to obtain an inverseagonist effect as highlighted in Table 4 [68] However it hasto be said that this lysine residue is also crucial for agonistbinding (CP-55940) andor receptor activation (WIN-55212-2) [69 70]

binding of SR141716A In contrast the substitution oftyrosine by isoleucine (Y275I) resulted in the loss of ligandrecognition Calculation studies revealed that while theY539F mutant is very similar to the wild type receptor theY539I mutant shows topology changes in the 3-4-5transmembrane region This region is considered to becrucial for agonistantagonist binding at CB1 receptor sincethe previous report by Shire et al on CB1CB2 receptorchimeras [72] In contrast the first and third extracellular(EC1 and EC3) loops of the hCB1 receptor are not essentialfor the binding of SR141716A as illustrated by Murphy etal The authors constructed several receptors mutated in theirEC1 or EC3 and none of the tested mutations affected theSR141716A binding [73] McAllister et al due the highlyaromatic nature of SR141716A further explored thehypothesis that an aromatic microdomain comprised intransmembrane helix 3-4-5-6 is the SR141716A bindingsite The modelling and mutation studies undertakensuggested to the authors that this aromatic microdomaincomprised of F336 W464 Y539 W543 and W648should represent the binding site of SR141716A Moreoverthey identified F336 and W543 as direct interaction sitesfor SR141716A [74] Residue F336 was further shown bythe same group to be a key residue for both ligand binding(WIN-55212-2 and SR141716A) and receptor activationMutation of phenylalanine 336(201) to alanine resulted in

Several other studies were undertaken to determine thecritical residues for the binding of cannabinoid compoundsUnfortunately in most of the studies SR141716A was notused However McAllister et al [71] using modeling toolsand mutagenesis explored the importance of aromaticity inposition 539(275) in the CB1 receptor The substitution oftyrosine by phenylalanine (Y275F) has no effect on the

Table 3 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Summary of the Reported CB1 Cannabinoid Receptor Single Point Mutations for which Pharmacological Data Concerning the SR141716A (14) areAvailable

Mutation Species Effect References

S114A a human no significant effect [73]

S115A a human no significant effect [73]

D250(163)N human no significant effect [263]

D250(163)E human no significant effect [263]

H181A b human no significant effect [73]

R182A b human no significant effect [73]

K183A b human no significant effect [73]

D184A b human no significant effect [73]

V324(188)A human no significant effect [73]

F325(189)A human no significant effect [73]

F325(190)A mouse no significant effect [74]

K328(192)A human loss of function [66]

K328(192)A human reduction of affinity (20 fold) [67]

F336(201)A mouse reduction of affinity (3 fold) [74]

Y539(275)F human no significant effect [71]

Y539(275)I human loss of affinity [71]

W543(280)A mouse loss of affinity [74]

W648(357)A mouse reduction of affinity (7fold) [74]

a N-terminus b First extra-cellular loop

1366 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 4 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives Five SR141716A (14) DerivativesIllustrating the Importance of the C-3 Carboxamide Oxygen in the SR141716A Inverse Agonism

The structure affinity ([3H]-SR141716A hCB1-HEK293 cells) and function (Ca++ currents) are given for each compound

NN

R

Cl

Cl

Cl

Cpd ndeg R= Affinity Function References

SR141716A 14

O

NHN

Kd=23nM Inverse agonist [67]

VCHSR1 15 Ki=313nM Neutral antagonist [67]

CHASR1 16

O

NH

Ki=17nM Inverse agonist [68]

CHMSR1 17

O

N

Ki=29nM Inverse agonist [68]

VPSR1 18

N

Ki=261nM Neutral antagonist [68]

PIMSR 19

NN

Ki=67nM Neutral antagonist [68]

an increased constitutive activity of the receptor asdemonstated by [35S]-GTPγ S binding [75]

1996 It binds with high affinity to rat brain synaptosomes(Kd=061nM) It is competitively displaced by knowncannabinoids like CP-55940 or WIN-55212-2 Usingautoradiography its rat brain distribution is similar to [3H]-CP-55940 one [77] It is now a commercially available andwidely used radioligand for competition studies

A new 3D model of the CB1 cannabinoid receptor basedon the X-ray structure of the bovine rhodopsin was recentlydeveloped by Salo and co-workers [76] It would be veryinteresting to see whether or not the results obtained withprevious models are confirmed using this new model Forinstance the lysine K328 appeared as a key residue in thismodel too

Interestingly other SR141716A radiolabeled derivativeswere synthesised as radioimaging tools among them [123I]-AM251 (20) [7879] [123I]-AM281 (21) [80-82] and [18F]-SR144385 (22) [8384] for Positon Emission Tomographyor Single Photon Emission Computed Tomography Indeedcannabinoid antagonists are a much more useful tool for

The tritiated analogue of SR141716A the [3H]-SR141716A was described by Rinaldi-Carmona et al in

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1367

human radioimaging applications than agonists as they aredevoid of cannabinoid-like effects For instance Berding etal very recently reported the use of [123I]-AM281 for SinglePhoton Emission Computed Tomography imaging of theCB1 cannabinoid receptor in six human patients [85] Inorder to further optimize the brain uptake of suchSR141716A derivatives Sanofi researchers synthesised twomethoxylated SR141716A analogues using 11C as PETtracer [86] These two compounds SR149080 (24) andSR149568 (25) possess high affinity with Ki values of 15and 38 nM respectively and selectivity for the CB1 receptoras well as an improved penetration in the brain evaluated bymeasuring the radioactivity present in various brain regionsafter tail vein injection in CD-1 mice Some of therepresentative radiolabelled SR141716A derivatives aresummarised in Table 5

Colombo et al using Wistar rats [92] Simiand et alobserved that SR141716A selectively reduces sweet foodintake in primates [93] However several authors [94-96]showed that high palatability of food is not necessary toobserve a SR141716A-induced anorectic effect at doses thatdo not cause major behavioural alterations or reduced waterintake Interestingly Gomez et al demonstrated theimplication of the peripheral CB1 receptors on themodulation of feeding and therefore the possible role ofthese receptors on the SR141716A influence on food intake[97] In addition to its effects on food consumptionSR141716A seems to be able to lower the hyperglycemiathe hyperinsulinemia as well as the insulin resistance indiet-induced obese (DIO) mice In the same DIO mice adecrease in adiposity was also observed after treatment [98]Bensaid et al using another model of obesity the obese fafarats observed that SR141716A increases mRNA expressionof Acrp30 or adiponectin a plasmatic protein exclusivelysecreted by adipose tissue through a CB1-mediatedpathway Inductions of free fatty acid oxidation body weightreduction and hyperinsulinemia decrease are some of theknown physiological actions of this protein Thus along theauthors an enhanced expression of Acrp30 followingSR141716A administration could be responsible for themetabolic effects of the compound leading to body weightreduction [99] Vickers et al using the same model (ie fafarats) showed that SR141716A significantly decreases foodconsumption and weight gain in both the obese fafa rats andthe lean Zucker rats [100] This decrease was greater in thefafa group and reversible upon SR141716A withdrawal

From a more therapeutic point of view SR141716A(rimonabant Acompliareg) is one of the promising agents totreat obesity [87] It is currently in Phase III clinical trials forthe treatment and prevention of obesity Final results areexpected for this year and FDA application filling for 2005[88] Preliminary data based on a one year treatment withrimonabant (RIO-Lipids study 1036 patients) showed a 5weight loss in 72 of the treated patients [8990] Severalreports were published before clinical trials aiming todemonstrate the anti-obesity properties of this compoundThe first report by Sanofi Recherche published in 1997described the selective inhibition of sucrose intake in ratsupon SR141716A treatment (03-3 mgkg) [91] Theanorectic and weight loss effects were firstly published by

Table 5 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structure and Affinity of SR141716A Radiolabeled Derivatives Developed as Potential Radio-Imaging Tools

NN

R3

O

NH

Cl

R4

R1

R2

Cpd ndeg R1 R2 R3 R4 Affinity CB1 References

[123I]-AM251 20 123I piperidinyl CH3 Cl Ki=25 nMa [78]

[123I]-AM281 21 123I morpholinyl CH3 Cl Ki=14 nMb [80]

[18 F]-SR144385 22 Cl piperidinyl CH2-18 F Cl IC50 =29 nMa [83]

[18 F]-SR147963 23 Cl morpholinyl CH2-18 F Cl IC50 =120 nM [84]

[11 C]-SR149080 24 O-11 CH3 piperidinyl CH3 Cl IC50 =15 nMa [86]

[11 C]-SR149568 25 O-11 CH3 morpholinyl CH3 Cl IC50 =38 nMa [86]

[18 F]-NIDA-42033 26 O-CH3 piperidinyl 18 F H Ki=18 nMc [155156]

27 O-11 CH3 piperidinyl CH3 H Ki=8nMc [155157]

arat brain homogenates [3H]-CP-55940bmouse cerebellum homogenates [3H]-SR141716crat brain homogenates [3H]-AM251

1368 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Another recent study by Higgs and co-workers furtherhighlighted the role of endocannabinoids in food taste-perception and the reduction of orosensory reward of sucrosein SR141716A treated rats [101] As it was expectedSR141716A induced effects on food consumption are absentsin CB1

-- mice [102] Very recently Cota et al obtainedresults showing that endocannabinoid system modulateshomeostasis via a dual mechanism it regulates at a centrallevel food intake while it blocks at the periphery lipogeneticprocesses [103] Moreover Cani et al showed that ghrelin(an orexigenic peptide) plasma levels are significantlyreduced 45 minutes after SR141716A administration(5mgkg ip) to fasted rats in accordance with the rapiddecrease of food intake measured by the authors It istherefore likely that SR141716 effects on body weight aredue to a conjunction of central and peripheral actions [104]

cerebrospinal fluid from patients with schizophrenia [116]Later De Marchi et al measured higher amounts ofanandamide in blood of schizophrenic patients compared tocontrols [117] On the other hand Dean et al obtainedelevated [3H]-CP-55940 binding in the dorsolateralprefrontal cortex of patients suffering from schizophrenia ascompared to controls [118] Zavitsanou using [3H]-SR141716A found elevated binding in the anterior cingulatecortex of subjects with schizophrenia [119] RecentlyMeltzer and collaborators published the results of a trialconducted to evaluate the potential of four new compoundsin treating schizophrenia and schizoaffective disorders [120]SR141716A (20 mgday) was one of the compoundsevaluated during a six weeks study The authors found noeffect of the CB1 cannabinoid receptor antagonist inimproving patientrsquos schizophrenia (72 subjects)

In addition to the anti-obesity potential the ability ofSR141716A to reduce alcohol and tobacco consumption arecurrently investigated in phase III clinical trials The effect ofSR141716A on alcohol consumption in rats was reported bythe GianLuigi Gessa team [105-108] and by Gallate et al[109] The inhibition of alcohol and nicotine induceddopamine release by SR141716A (1-3 mgkg rats) wasreported by Cohen and co-workers using a brainmicrodialysis device [110] Concerning the smokingcessation preliminary results of a clinical trial (360 subjects40 mg SR141716A) showed an increased abstinence ofsmoking [111] More recent results from the STRATUS-USstudy (787 patients) were reported 362 of patientsreceiving SR141716A (20 mgday) quit smoking against206 in the placebo group [90] Recently Le Fol andGoldberg reviewed the development of cannabinoid CB1antagonists as a new class of therapeutic agents for drugsaddictions [112]

It is known since the early nineties that theadministration of cannabinoid agonists impairs memory inrodents (for a review see Castellano et al [121]) Thusadministration of a cannabinoid antagonist was expected tosomehow improve memory [122123] However dependingon the authors SR141716A when administered alone wasreported to impair (5-10 mgkg im) [124] to have no effect(1-32 mgkg ip) [125] or to improve memory (3 mgkgip) [126] In a more recent paper Wolff and Leandershowed that SR141716A (1mgkg ip) improves memoryin rats by apparently enhancing the consolidation processesof memory [127] Further they found that at higher doses (3mgkg) this effect was lost The discrepancy in the resultsreported in the literature could be ascribed either to thedifferences in the tests used or in the doses administeredFurther experiments are needed to assess whether or not acannabinoid antagonist could be helpful in memory diseases

To conclude several patents were taken by Sanoficoncerning the therapeutic applications of their leadcompound among them being anti-obesity smokingcessation neuroinflammatory diseases and anti-diarrhoeaOne of the last patents to our knowledge concerns thetreatment of sexual dysfunctions with a cannabinoidantagonist such as SR141716A [128] However da Silva etal showed that even if this compound (2 mgkg ip)enhances the effects of apomorphine (20-80 microgkg) it has noeffect alone on penile erection [129] Meanwhile Melis et al

Since the late sixties concerns exist on the associationbetween cannabis use and schizophrenia Recent studiesshowed an association between cannabis use and an increasedrisk of developing schizophrenia [113 114] Moreover apossible role of the endocannabinoid system in schizophreniahas been suggested since its pharmacological characterisation[115] Several experimental data obtained on human subjectstend to confirm such hypothesis On the one hand Lewekeet al found elevated levels of endocannabinoids in

NN

O

NH

N

Br Cl

Cl

NN

O

NH

N

Br Cl

Cl

OH

28 29

Fig (3) CB1 cannabinoid receptor antagonists 3 Diarylpyrazole derivatives The chemical structures of 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-piperidine-carboxamide (SR147778 28 ) and 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-(4-hydroxypiperidine)-carboxamide (29 ) described by Rinaldi-Carmona et al

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1369

reported an increased rate of penile erection after directinjection of SR141716A in the paraventricular nucleus of thehypothalamus [130] This effect was dose dependent and asignificant effect was obtained with a dose of 1microgkg

AM251 in mice at a same dose range (10-30 mgkg ip) thanthe anorectic effects [143] These effects are reversed byadministration of CP-55940 and are absent in CB1

-- miceproving the implication of the CB1 receptor in theantidepressant-like effect of AM251 In 2004 Liao et alreported that AM251 was able to displace the binding of[3H]-batrachotoxin A from its binding site on sodiumchannels in mice brain synaptic preparations [144] Theyobtained an IC50 value of 112 microM and a competitivemechanism of action The authors suggested that AM251 iscapable of reducing neuronal excitability through blockade ofvoltage-sensitive sodium channels in brain

From the clinical trials involving the SR141716A themost frequently reported side-effects are nausea dizziness anddiarrhoea Depression and anxiety were not higher than inthe placebo groups Regarding one of the known side effectsof SR141716A administration the enhancement of intestinalmotility Carai et al reported recently that chronicadministration of the inverse agonists to mice induced atolerance to this prokinetic effect [131] This is not the firstreport of tolerance onset after administration of SR141716A[132] however few is known on the mechanismsresponsible for that phenomenon

Wiley and collaborators reported in 2001 a studydescribing new structure-activity relationships using thepyrazole nucleus as a central scaffold [145] Starting fromSR141716A structure they alternatively substituted one ofthe four substituents while retaining the others bysubstituents known to impart agonist activity in classicalcannabinoids One of the authors expectations was todetermine which positions are responsible respectively forthe antagonism and for the affinity of SR141716A Theaffinity of thirty compounds was assessed previous to in-vivoevaluation of their function in mice using the spontaneousactivity the tail-flick and the rectal temperature assays Theauthors showed that phenyl group in position 5 is critical foraffinity as compound O-1559 (30) lacking this phenyl has adecreased affinity (Table 6) This was already shown by Lanand co-workers [136] along with the need of a substituent inpara position on the phenyl Thomas et al previouslyshowed that this substituent could be a bromine or an iodineatom [146] However an alkyl chain is also tolerated as it isthe case in compounds O-1302 (31) O-1691 (32) or O-1704(33) All these compounds antagonise the anti-nociceptiveand hypothermic effects of ∆9-THC The authors suggestedthat the 5-substituent of pyrazoles is involved in receptorrecognition and antagonism The modulations of thesubstitution pattern of the phenyl in position 1 demonstratethat the 24-di-chloro substitution is the preferred one for theaffinity as well as for the activity Several compoundssupport this assertion Thomas et al showed that additionalhalogens result in a decreased affinity as in compounds 6-I-SR141716A (34) or 4rsquo6-di-I-SR141716A (35) (Ki values of166 and 126 nM respectively) [146] Suppression of the twochlorines (O-1300 39) or replacement of these two chlorinesby an alkyl chain like in O-1254 (40) and O-1255 (41) ledto less active compounds with Ki values ranging from 150to 430 nM However O-1254 and O-1255 behaved asantagonists in a [35S]-GTPγ S assay [61] Lan et al [136]and Wiley et al [145] also modified the substitution inposition 3 Replacement of the amido piperidinyl substituentby alkyl amides as in O-1269 (42) or O-1270 (43) etherslike in O-848 (44) and O-853 (45) ketones (O-1272 46)alcohols (O-1876 47) or alkanes (O-1877 48) resultedmostly in decreased affinity (Table 6) but also with a changeof functionality in some compounds as revealed by in-vivoassays For instance replacement of the piperidinyl by apentyl (O-1269) or by an heptyl chain (O-1270) gave agonistcompounds having Ki values of 32 and 48 nM respectivelyThus the authors suggested that the 3-substituent region isinvolved in receptor recognition and agonist activity Wileyet al on the basis of their results concluded that while the3-position seems to be involved in agonism the 1- 4- and

One close analogue of SR141716A 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-piperidinecarbox-amide (SR147778 28) was described very recently byRinaldi-Carmona et al [133 134] (Fig 3) It possesses highaffinity and selectivity for the hCB1 cannabinoid receptorwith Ki values for the hCB1 and hCB2 receptors of 35 and400 nM respectively ([3H]-CP-55940 hCB1amp2-CHO cells)Further SR147778 antagonised CP-55940 effects on mousevas deferens contractions (pA2=81) and on forskolin-stimulated adenylyl cyclase activity in U373MG cells(pA2=82) but had no effects alone In-vivo SR147778 afteroral administration reversed WIN-55212-2 inducedhypothermia and analgesia As SR141716A SR147778dose-dependently reduced ethanol and sucrose solutionintake with significant effects starting at 03 mgkg (sc) and3 mgkg (po) respectively This compound is currentlyinvestigated in Phase I clinical trials for the treatment ofobesity as well as nicotine and alcohol addictions

Another derivative the 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-(4-hydroxypiperidine)-carboxamide (29) was described in a very recent patent fromSanofi [135] (Fig 3) This antagonist possesses an IC50value for the hCB1 cannabinoid receptor of 32 nM ([3H]-CP-55940 hCB1-CHO cells)

The first structure-affinity relationships for theSR141716A derivatives were reviewed by Barth andRinaldi-Carmona in 1999 [32] Since then many papersdescribing new derivatives were published In 1999 Lan etal described around thirty 15-diarylpyrazoles derivatives[136] Among them was AM251 previously reported as aradioimaging ligand for the CB1 receptor [78] and thatappeared to be more potent (Ki = 749 nM) and selective(selectivity ratio of 306) than SR141716A (Ki = 115 nMselectivity ratio of 143) They reported a Kd value of 05 nMin the mouse vas deferens model using WIN-55212 asagonist Interestingly New et al described for the samecompound an inverse agonist effect on the hCB2 receptorusing a forskolin-induced cAMP accumulation assay (EC50= 650 nM) [137] Recently the anti-obesity effects ofAM251 were reported and are not surprisingly similar tothose of SR141716A [138 139 140] Chen et aldemonstrated that there are synergistic effects on food intakesuppression between AM251 and nalmefene an opioidantagonist [141] This synergistic effect was also presentbetween SR141716A and naloxone as shown by Kirkham[142] Shearman et al found antidepressant-like effects of

1370 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

5-positions appear to be involved in antagonism The O-derivatives reported were also described in a patent alongwith their synthetic pathways which were not given in thepaper [147]

Another investigation of the SR141716Aaminopiperidine region was conducted by Francisco et al[148] They synthesised 21 analogues possessing either analkyl amide or an alkyl hydrazide substituent of various

Table 6 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structure of some of the characterised 1- 3- 4- and 5-pyrazole derivatives Binding affinities (Ki values nM) were obtained on rat brain homogenatesusing [3H]-CP-55940 [136 145 146 148 150] or [125I]-AM-251 [155]

NN

R1

R5

R3R4

Cpd ndeg R1 R3 R4 R5 [136] [145] [146] [148] [150] [155]

SR141716 14 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Cl-Ph 115 62 62 62 13 18

O-1559 30 24-di-Cl-Ph CO-NH-piperidinyl Me 1-Methylpentyl 233

O-1302 31 24-di-Cl-Ph CO-NH-piperidinyl Me 4-((CH2)5)-Ph 21 1

O-1691 32 24-di-Cl-Ph CO-NH-piperidinyl Br 4-((CH2)5)-Ph 15

O-1704 33 24-di-Cl-Ph CO-NH-piperidinyl I 4-((CH2)5)-Ph 22

6-I-SR141716 34 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-Cl-Ph 166

4rsquo6-di-I-SR141716 35 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-I-Ph 126

4rsquo-I-SR141716 36 24-di-Cl-Ph CO-NH-piperidinyl Me 4-I-Ph 75 25 6

4rsquo-Br-SR141716 37 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Br-Ph 168 3

Cpd 25 in ref [136] 38 24-di-Cl-Ph CO-NH-morpholinyl Me 4-Br-Ph 54

O-1300 39 Ph CO-NH-piperidinyl Me 4-Cl-Ph 150

O-1254 40 4-(CH2)4-Ph CO-NH-piperidinyl Me 4-Cl-Ph 226 256

O-1255 41 4-(CH2)5-Ph CO-NH-piperidinyl Me 4-Cl-Ph 433

O-1269 42 24-di-Cl-Ph CO-NH-pentyl Me 4-Cl-Ph 32 114 3

O-1270 43 24-di-Cl-Ph CO-NH-heptyl Me 4-Cl-Ph 48 462 3

O-848 44 24-di-Cl-Ph CH2-O-(CH2)2-piperidinyl

Me 4-Cl-Ph 2450 232

O-853 45 24-di-Cl-Ph CH2-O-CH2-cyclohexyl

Me 4-Cl-Ph 388 100

O-1272 46 24-di-Cl-Ph CO-heptyl Me 4-Cl-Ph 221

O-1876 47 24-di-Cl-Ph 1rsquo-OH-heptyl Me 4-Cl-Ph 657

O-1877 48 24-di-Cl-Ph Heptyl Me 4-Cl-Ph 422

MF9725-64-17 49 24-di-Cl-Ph CO-NH-butyl Me 4-Cl-Ph 134

MF9725-179-32 50 24-di-Cl-Ph CO-NH-NH-butyl Me 4-Cl-Ph 51

MF9725-66-11 51 24-di-Cl-Ph CO-NH-cyclohexyl Me 4-Cl-Ph 25

MF9725-95-31 52 24-di-Cl-Ph CO-NH-(4-hydroxybutyl)

Me 4-Cl-Ph 154

Cpd 15 [150] 53 n-pentyl CO-NH-piperidinyl Me Ph 23

Cpd 16 [150] 54 n-pentyl CO-NH-piperidinyl Me 4-Br-Ph 63

Cpd 17 [150] 55 n-hexyl CO-NH-piperidinyl Me Ph 21

Cpd 18 [150] 56 n-heptyl CO-NH-piperidinyl Me Ph 47

NIDA-41109 57 24-di-Cl-Ph CO-NH-piperidinyl Br 4-Cl-Ph 14

NIDA-41119 58 24-di-Cl-Ph CO-NH-piperidinyl H 4-Cl-Ph 9

NIDA-41057 59 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OH-Ph 104

NIDA-41020 60 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 41

NIDA-41087 61 2-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 8

NIDA-42055 62 24-di-Cl-Ph CO-NH-piperidinyl Br 4-OMe-Ph 62

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1371

lengths in position 3 of the pyrazole moiety They observedthat until five carbons the affinity increases along with thecarbon chain length This is observed with the alkylhydrazide the alkyl amide and the hydroxyalkyl amideseries Moreover the hydrazide analogues exhibit a loweraffinity for the rCB1 than the amide analogues as illustratedby compounds MF9725-64-17 (49) and MF9725-179-32(50) having Ki values of 134 and 51 nM respectively(Table 6) From their structure-activity relationships (SAR)studies the authors concluded that the pharmacophoricrequirement of the amidopiridine region is a chain not longerthan 3 Aring and that a substituent having a positive chargedensity would probably result in increased affinity andpotency The same team described in a patent several othercompounds [149] Binding affinities (Ki) against [3H]-CP-55940 [3H]-SR141716A or [3H]-WIN55212-2 on wholerat brain or on hCB1-transfected cells were given Moreoveractivity data were obtained using the [35S]-GTPγ S assaydemonstrating that these alkyl amide and hydrazide analogsact as antagonists or inverse agonists Despite the greatnumber of derivatives claimed none of them posses asignificantly greater affinity for the CB1 receptor than theSR141716A Nevertheless they showed a slightenhancement of the selectivity for the CB1 receptor over theCB2 cannabinoid receptor

affinity of these derivatives increases with the length of thealkyl chain with an optimal length of 5-6 carbons The bestcompound of this series N-(piperidin-1-yl)-5-phenyl-1-hexyl-4-methyl-1H-pyrazole-3-carboxamide (55) possesses aKi value of 21 nM determined using [3H]-CP-55940 on ratbrain membranes which is higher than the 13 nMdetermined for SR141716A in the same conditions (Table6) In the same paper the authors starting from thehypothesis that antagonism by SR141716A is caused bybinding to the same region of the receptor as do the agonists(CP-55940 and WIN-55-2122) but preventing the agonistpromoted conformational change conducted extensiveconformational analysis as well as superimposition modelsand 3D-QSAR to propose a molecular mechanismsupporting the action of SR141716A Along with theauthors the C-5 aryl substituent of SR141716A occupyinga unique region could contribute in conferring the antagonistproperties Moreover the C-3 substituent could beresponsible for the antagonist or inverse agonist propertiesdepending on the interaction with the receptor

About this topic a very interesting review dealing withthe cannabinoid receptors pharmacophores as well as withthe activationinactivation of these receptors was recentlypublished by Reggio [151]

More recently Dyck et al described seven otherderivatives varying at the amide position [152] The onlycompound possessing a higher affinity for the CB1cannabinoid receptor than SR141716A was the 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta-

The affinity for the rat CB1 receptor of seven derivativespossessing an alkyl chain in position 1 instead of the 24-dichlorophenyl substituent was reported by Shim et al in apaper describing a molecular mechanism for the antagonistand inverse agonist activity of SR141716A [150] The

NN

O

NHN

Cl Cl

Cl

NN

Cl

Cl

NON

NN

O

NH

N

Cl

ClCl

NN

O

NH

MeO Cl

CP-272871 66

63 64

65

Fig (4) CB1 cannabinoid receptor antagonists 3 Diarylpyrazole derivatives Structures of 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63 ) 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methyl-pyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]-morpholine (64 ) N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65 ) and N-phenyl-1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-pyrazole-3-carboxamide (CP-272-871 66 )

1372 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

[c]pyrrol2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63)(Fig 4) The Ki values were 5 and 12 nM respectively([3H]-CP-55940 hCB1-HEK cells)

CP-55940 rat brain) For instance N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65) (Fig 4) showed a Ki value of15 nM However no data were provided concerning thefunctionality of such compounds

Over 200 other pyrazole derivatives acting at the CB1cannabinoid receptor were claimed in a patent from Pfizer[153] The affinity for the hCB1 receptor of 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methylpyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]ndashmorpholine (64) (Fig 4)was 79 nM ([3H]-SR141716A hCB1-HEK cells) Theothers compounds affinity was between 01 and 100 nM

In an effort to develop new SR141716A analoguespossessing lower lipophilicity to be used as PET tracersKatoch-Rouse and colleagues further explored thesubstitution pattern of the two phenyls [155] Bindingassays showed that a decreased lipophilicity lead to adecreased affinity for the CB1 receptor as determined bydisplacement of [3H]-AM251 For instance compoundNIDA-41057 (59) has a lipophilicity of 42 ( expressed asElogDoct) and a Ki value of 104 nM whereas SR141716Ahas a ElogDoct value of 54 and a Ki value of 18 nM In

Seventy five pyrazole derivatives were described in a veryrecent patent from Makriyannis et al [154] The affinity ofthe compounds for the cannabinoid receptors is given TheKi values for the best compounds are lower than 6 nM ([3H]-

Table 7 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structures and affinities for the cannabinoid receptors of some representative tricyclic pyrazole derivatives Values were obtained using [3H]-CP-55940as radioligand and mouse [160 228] rat [159 227] or human [158] cannabinoid receptors Compounds 69-78 are CB1 cannabinoid receptor ligandsCompounds 79-86 are CB2 cannabinoid receptor ligands

NN

O

NHX

R3

R1

R2

n

n˚ X R1 R2 n R3 Ki CB1 (nM) Ki CB2 (nM) Selectivity References

69 -(CH2)3- Cl 24-di-Cl 0 piperidine 126 [158]

70 -(CH2)3- H 24-di-Cl 0 azepane 100 [158]

71 -(CH2)3- H 24-di-Cl 0 piperidine 398 [158]

72 -(CH2)3- Cl 24-di-Cl 0 azepane 125 [158]

73 -(CH2)3- NO2 24-di-Cl 0 azepane 63 [158]

74 -(CH2)2-O- H 24-di-Cl 0 piperidine 501 [158]

75 -CH2-S- Br 24-di-Cl 0 piperidine lt 500 gt10 [159]

76 -CH2-SO2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

77 -(CH2)2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

78 -(CH2)3- Cl 24-di-Cl 0 piperidine 000035 21 60000 [160]

79 -CH2- Cl 24-di-Cl 0 piperidine 2050 034 00002 [228]

80 -CH2- Br 24-di-Cl 0 piperidine 1570 027 00002 [228]

81 -CH2- CH3 24-di-Cl 0 piperidine 363 0037 00001 [228]

82 -CH2- Cl 4-Cl 0 piperidine 1787 09 00005 [228]

83 -CH2- H 24-di-Cl 0 piperidine 1152 0385 00003 [228]

84 -CH2- Cl 34-di-Cl 1 133-trimethylbicyclo[221]heptyl

lt500 gt10 [227]

85 -CH2- Br 24-di-Cl 1 bicyclo[321]oct-3-yl lt500 gt10 [227]

86 -CH2- Br 4-Me 1 77-dimethylbicyclo[410]hept-3-yl

lt500 gt10 [227]

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1373

another paper Katoch-Rouse described the synthesis of[18F]-NIDA-42033 (26) a ligand useful as PET radiotracerstarting from its bromo derivative [156] Recently Kumar etal [157] proposed the synthesis of a 11C derivative ofNIDA-41087 (61) a compound previously described byKatoch-Rouse (Table 5) Post-mortem binding of thiscompound to human prefrontal cortex was also investigatedInterestingly CP-272871 (66) described by Meschler as aCB1 inverse agonist (Ki=57 nM [3H]-CP-55940 rat brainhomogenates) possesses a lipophilicity similar to theNIDA-41087 one [62] However despite the presence of amethoxy function no radiolabelled derivative has beendescribed to date One explanation could be the compoundlow selectivity (2 fold) for the CB1 cannabinoid receptor

To further explore the pharmacology of this compound afunctional assay using the mouse vas deferens model wasperformed by Ruiu and colleagues enlightening thecompetitive antagonist properties of NESS (pA2= 1246against WIN-55212-2) which had no effect by itself up to1microM Moreover NESS 0327 was unable to affect basalbinding of the [35S]-GTPγ S demonstrating its lack ofnegative intrinsic activity In-vivo studies were conductedusing the hot plate and tail flick tests NESS dose-dependently abolished the antinociceptive effect of WIN-55212-2 but had no effect by itself However the authorssuggested based on the affinity and in-vivo activity of theircompound that NESS 0327 possesses a poor centralbioavailability

Several new attempts to increase the affinity of thediarylpyrazole derivatives were recently made by rigidifyingthe SR141716A structure (Table 7) Six fused ringanalogues of SR141716A obtained by fusion of the 5-(4-chlorophenyl) substituent with the central pyrazole to forman indazole ring were published by Bass et al in 2002 [61]The compound possessing the highest affinity O-1248 (67)has a Ki value of 475 nM as determined by displacement of[3H]-CP-55940 from rat brain membranes

Another example of ring-constrained biarylpyrazole wasdescribed by Herbert Seltzman team [161] Theyinvestigated a photocyclisation reaction starting from theSR141716A leading to a pyrazolo[15-f]phenanthridinestructure (Fig 5) The new compound (68) was tested on theCB1 receptor (whole rat brain) against [3H]-CP-55940[3H]-SR141716A and [3H]-WIN-55212-2 and exhibited Kivalues of 48 35 and 50 nM respectively The CB2 affinitywas assessed using [3H]-CP-55940 and was found to be 100times lower (Ki = 3340 nM) No effect on the [35S]-GTPγ Sassay (rat brain) was observed by the authors

Stoit and colleagues from Solvay Pharmaceuticalspublished a paper describing the synthesis andpharmacological characterisation of new benzocyclohepta-pyrazole derivatives as CB1 antagonists (69-74) [158] Theaffinity of these compounds was more or less one order ofmagnitude lower than the SR141716A one with pKi valuesranging from 64 to 72 compared to 76 for the SRcompound (Table 7) Compounds were shown to beantagonists with pA2 values from 70 to 89 as theyprevent CP-55940-induced cAMP accumulation Howeverthe authors investigating the bioavailability of theircompounds either po or after ip injection found negligibleblood plasma levels

NN

Cl

Cl

O

NH

N

NN

Cl

O

NHN

Cl

Cl

O-1248 67 68However just before the publication of Stoit results

Sanofi-Synthelabo took a patent describing tricyclicpyrazolecarboxylic acid amide derivatives having antagonistproperties on the CB1 cannabinoid receptor andsubmicromolar affinities (75-77) [159] Thirty examples weregiven along with their synthetic pathway but no individualpharmacological data were shown

Fig (5) CB1 cannabinoid receptor antagonists 3Diarylpyrazole derivatives Two ring-constrained diarypyrazolederivatives O-1248 (67 ) and a pyrazolo[15-f]phenanthridinederivative (68 )

It appears from these attempts to increase SR141716Aaffinity by rigidifying its structure that only a limitedincrease in the CB1 cannabinoid receptor affinity can beexpected with the apparent exception of NESS 0327Moreover rigidifying the structure often results in a morelipophilic compound in a family of compounds being yetlipophilic

More recently Ruiu and colleagues published a paperdescribing the synthesis and complete pharmacologicalcharacterisation of a very potent CB1 receptor ligandchristened NESS 0327 (78) [160] This compound has thesame structure as compound 69 published by Stoit et al(compound 20 in the reference [158]) Nevertheless Ruiu hasobtained an affinity for the CB1 cannabinoid receptor 5000times bigger than the SR141716A one with Ki values of035 pM and 18 nM respectively using mouse forebrainhomogenates and [3H]-CP-55940 as radioligandFurthermore the selectivity ratio obtained for this compoundby Ruiu is 60000 with a Ki value for the CB2 receptor of21 nM against the [3H]-CP-55940 The discrepancybetween the data obtained independently by Stoit et al andRuiu et al could hardly be explained by the differences inthe origin of the receptor used respectively hCB1-CHO andmouse forebrain

4 Phenyl Benzofuranone Derivatives

In a 1997 patent from Eli Lilly was described thesynthesis of aryl-benzothiophene and aryl-benzofuranederivatives (87-89 Fig 6) having CB1 receptor affinity andantagonist properties [162] From ten compounds having aKi value lower than 25 microM ([3H]-CP-55940 hCB1-CHOcells) one was selected and claimed to have a Ki of 170 nMCompound 88 was shown in the patent to antagonise theeffect of anandamide on cAMP accumulation with an IC50 of

1374 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

O

O

O

O

NC

S

O

O

O

O

O

O

O

NC

LY320135 8887 89

Fig (6) CB1 cannabinoid receptor antagonists 4 Phenyl benzofuranone derivatives Chemical structures of two aryl-benzofuranederivatives (87 -88 ) and one aryl-benzothiophene (89 ) derivative from Eli Lilly

500 nM and the effect of WIN-55212-2 on calcium channelsat 1microM Moreover the intra-peritoneal injection (20 mgkg)of this compound antagonised the in-vivo effect ofanandamide in the Open Field Assay a mouse behaviourmodel Later on Felder and colleagues gave a furtherdescription of this compound called LY320135 in a paperdescribing its effect on cAMP accumulation and on ioniccurrents [163] The selectivity ratio of LY320135 was 106with Ki values of 141 and 14900 nM for hCB1 and hCB2receptors ([3H]-CP-55940) respectively LY320135antagonised anandamide inhibition of forskolin-inducedcAMP accumulation with an IC50 value of 734 nMChristopoulos et al in 2001 reported for LY320135 apKb value of 527 in the inhibition of WIN-55212-2mediated response in the rat electrically-stimulated vasdeferens while the pKb value for SR141716A was 75[164]

Other patents were taken by Aventis in which somepotential applications for their compounds were describedFor instance an association between an azetidine derivativehaving CB1 antagonists properties and sibutramine aserotonin and norepinephrine reuptake inhibitor was claimed[170] as a treatment against obesity Oral administration ofcompound 90 (3 mgkg) and sibutramine (06 mgkg) to fafaZucker rats which are genetically obese rats has resulted ina decreased food intake compared to control lean Zuckerrats In another patent the association of an azetidinederivative (1-10 mgkg po ) and a D2D3 agonist likequinpirole (01 mgkg ip) was claimed to have a therapeuticpotential in the treatment of Parkinsonrsquos disease [171] Theeffect has been evidenced using an akinesia model in the ratEvidences supporting the use of CB1 antagonists as adjuvantin the treatment of Parkinsonrsquos disease begin to appear Itseems that such compounds would be helpful in thetreatment of Parkinsonism [172] and levodopa-induceddyskinesia [173] Brotchie published an interesting paperreviewing the CB1 cannabinoid receptor signaling inParkinsonrsquos disease [174] Nevertheless further clinicalstudies should be conducted to further confirm the usefulnessof CB1 antagonists in the treatment of Parkinsonrsquos disease

5 Azetidine Derivatives

The family of azetidine compounds illustrated bycompounds 90 and 91 (Fig 7) was developed at Aventis byDaniel Achard and colleagues and was described in a seriesof patents [165-168] They claimed the IC50 values of thesecompounds for the CB1 receptor to be less than or equal to100 nM These values were obtained following the proceduredescribed by Kuster et al [169] The antagonist propertywas shown using an in-vivo model the reversal ofhypothermia induced by CP-55940 in mice and the ED50obtained were lower than 50 mgkg

6 Aryl-Imidazolidine-24-Diones Derivatives

In 1999 the Didier Lambert team published a paperdescribing the affinity of 24 new 3-alkyl-55rsquo-diphenyl-imidazolidine-24-dione derivatives for the hCB1 receptor[175] The preliminary structure-activity relationships

N

SO

O

Cl

Cl

F

F

N

SO

O

Cl

NS

90 91

Fig (7) CB1 cannabinoid receptor antagonists 5 Azetidine derivatives Two examples (90 -91 ) of the azetidine derivatives developedby Aventis

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1375

Table 8 CB1 Cannabinoid Receptor Antagonists 6 Aryl-Imidazolidine-24-Diones Derivatives

Structures affinities ([3H]-SR141716A hCB1-CHO cells) and pKb values ([35 S]-GTPγS HU210 rat brain) of three aryl-imidazolidine-24-dionederivatives (92-94)

N

NH

OO

R

Br

Br

Cpd ndeg R Ki (nM) a pKb b

DML20 92 ethylmorpholine 70 611

DML21 93 1-hydroxypropyl 103 625

DML23 94 heptyl 98 574

a [175]b [177]

showed that the substitution at the nitrogen in position 3 aswell as a bromine atom in para of the phenyl rings aremandatory for the compounds affinity Later on threecompounds termed DML20 (92) DML21 (93) and DML23(94) were characterised as neutral antagonists using the [35S]-GTPγ S assay on rat cerebellum membranes [176] (Table 8)In the same model they competitively inhibited HU-210-induced [35S]-GTPγ S binding with pKB values of 611625 and 574 respectively Moreover these compoundswere proven to be quite selective for the CB1 receptor

higher than the one for the CB2 cannabinoid receptor (IC50 =300 nM) The inverse agonism of the compound wasextrapolated by inhibiting CP-55940-induced hypothermiain mice (EC50 = 18 nM) Moreover one oral dose (10mgkg) reduces the food intake in diet-induced obese ratsInterestingly food intake of CB1

-- mice was not alteredBody weight loss was maintained all over the study during achronic administration (14 days) of 10 mgkg of Cpd A

Later on Merck published a patent [180] in which someof the 45-diaryl-pyrazoles derivatives previously describedwere claimed to be useful in the treatment or the preventionof obesity in association with compounds inhibiting the11β-hydroxysteroid dehydrogenase type 1 (1β-HSD1)enzyme The 1β-HSD1 enzyme is responsible for thesynthesis of cortisol increased levels of which according tothe inventors are associated with obesity However neitherresults on appetite suppression nor on weight loss weregiven even if some indications on the affinity of thecompounds for the cannabinoid receptors were given

Very recently the functionality of these DML compoundswas explored using the [35S]-GTPγ S assay both on rat andhuman CB1 cannabinoid receptors [177] The data obtainedconfirm that the 3-alkyl-55rsquo-diphenyl-imidazolidinedionederivatives behave as neutral antagonists on rat CB1cannabinoid receptor (rat brain) However DML 20 DML21and DML23 acted as inverse agonists on the human receptor(hCB1-CHO cells) Furthermore the authors showed thatthis different functionality is not due to the hCB1 receptorlevel of expression in the recombinant cell line as whateverthe level of expression (Bmax of 44 pmolmg or 32pmolmg) DML derivatives behaved as inverse agonists ofthe hCB1 cannabinoid receptor

Another imidazole ring pattern of substitution wasproposed by Hagmann and collaborators [181] Thecompounds described 43 examples are given possess twophenyls the first one in position 1 linked to the nitrogenand the second one in position 2 The carboxamidesubstituent is in position 4 instead of in position 2 for thediarylimidazoles described by Finke These 12-diaryl-imidazoles are claimed to be CB1 cannabinoid receptorantagonists or inverse agonists but the absence of precisepharmacological values do not allow any comparisonbetween the two substitutions patterns However a samepattern of substitution was used by other researchers Amongthem Dyck et al described in 2004 ten 1-(4-chlorophenyl)-2-(24-dichlorophenyl)-imidazole derivatives varying by theircarboxamide substituent in position 4 (101-103 Table 9)[152] For instance with a 3-azabicyclo[330]octan-3-ylsubstituent (101) they obtained a Ki value of 14 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) In a [35S]-GTPγ Sassay 101 exhibited an IC50 value of 19 nM

7 Diarylimidazoles Derivatives

Finke and colleagues at Merck developed a new class ofCB1 receptor antagonists based on an imidazole nucleusOver eighty 45-diaryl-imidazoles derivatives having CB1antagonist properties were claimed in a patent published in2003 [178] Some representative compounds of the patent areillustrated in Table 9 (95-100) The affinity of the newcompounds was determined using [3H]-CP-55940 andhCB1-CHO cells Although no detailed pharmacologicaldata were given in the patent in-vitro and in-vivo propertiesof one compound termed Cpd A (100) have been describedelsewhere [179] Its affinity for the CB1 cannabinoid receptorwas shown to be nanomolar (IC50 = 4 nM) and 75 times

1376 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 9 CB1 Cannabinoid Receptor Antagonists 7 Diaryl-Imidazole Derivatives

Structure and affinity range of some 45-diaryl-imidazole derivatives for the human cannabinoid receptors determined using [3H]-CP-55940 asradioligand and hCB1amp2-CHO cells

N

NR2

R1

R4

R3

ndeg R1 R2 R3 R4 hCB1 (IC50 or Ki) hCB2 (IC50 or Ki) References

95 CH3 CO-NH-piperidinyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

96 CH3 CO-NH-piperidinyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

97 CH3 CO-NH-cyclohexyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

98 CH3 CO-NH-phenyl 24-di-Cl-Ph 4-Cl-Ph 10 ndash 100 nM a 100 ndash1000 nM a [178]

99 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

100 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph 4 nM a 300 nM a [179]

101 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

CH3 14 nM b ND [152]

102 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

H 66 nM b ND [152]

103 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 85 nM b ND [152]

104 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 23 nM b 542 nM b [182]

105 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 30 nM b 608 nM b [182]

106 4-Br-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 60 nM b 489 nM b [182]

107 4-CF3-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 29 nM b 634 nM b [182]

108 4-Cl-Ph 24-di-Cl-Ph CO-NH-morpholinyl CH3 197 nM b 3297 nM b [182]

109 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidin-4-ol CH3 172 nM b 3959 nM b [182]

110 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CN 30 nM b 1590 nM b [182]

111 4-Cl-Ph 24-di-Cl-Ph CO-NH- piperidinyl CH2F 36 nM b 906 nM b [182]

112 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH2-CH3 14 nM b 430 nM b [182]

113 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Cl 27 nM b 823 nM b [182]

114 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Br 23 nM b 746 nM b [182]

115 4-Cl-Ph 24-di-Cl-Ph CO-NH-tetrahydroisoquinoline CH3 34 nM b 696 nM b [182]

116 4-Cl-Ph 24-di-Cl-Ph CO-NH-cycloheptyl CH3 35 nM b 349 nM b [182]

117 24-di-Cl-Ph 4-Cl-Ph CO-NH-piperidinyl CH3 403 nM b 208 nM b [182]a IC50 value b Ki value

Lange and co-workers from Solvay described twenty-eight imidazole derivatives [182] The 24-dichlorophenylsubstituent at position 2 was kept constant while thesubstitutions at the other positions were explored (Table 9)Position 5 can accommodate a large range of littlesubstituents like hydrogen (104) methyl (105) ethyl (112)chlorine (113) bromine (114) fluoromethyl (111) or cyano(110) without major changes in the affinity At position 4more bulky substituents like 1234-tetraisoquinoline (115)or cycloheptyl (116) were well tolerated whereas thepresence of a more hydrophilic moiety such as morpholine(108) or piperidin-4-ol (109) was detrimental to the affinity

Interestingly position exchange between the 24-diphenyland the 4-chlorophenyl substituents led to a 13 fold loweraffinity with Ki values of 30 and 403 nM for 105 and 117respectively ([3H]-CP-55940 hCB1-CHO cells) In thefunctional assay which was inhibition of WIN-55212-2-induced [3H]-arachidonic acid release by hCB1-CHO cellsall tested compounds behaved as antagonists Compound105 showed a pA2 value of 86 which is the same theauthors obtained for SR141716A It would be interesting toassess if these compounds are true antagonists or if they actmore as inverse agonists Compound 105 when administeredto rats was able to inhibit CP-55940-induced hypotension

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1377

with an ED50 value of 24 mgkg which is close to the 3mgkg obtained for SR141716A The compound was alsoactive in another in-vivo model the WIN-55212-2-inducedhypothermia in mice Compound 104 which has nosubstituent in position 5 of the imidazole ring was lessactive in the hypotension model and inactive in thehypothermia model Thus a methyl group is the preferredsubstituent in position 5 of the imidazole moiety Otherproperties either experimental (P-glycoprotein affinitylogPHPLC) or computational (molecular volume polarsurface area) obtained for compounds 105 were similar tothose obtained for SR141716A Finally molecularmodelling studies revealed a close structural overlap betweenthe two compounds

value of 197 nM is selective for hCB1 and is as potent asSR141716A The structural modulations of 118 led to someinteresting compounds (Table 10) Six compounds possess aKi value for the hCB1 cannabinoid receptor equal to or lowerthan 25 nM antagonist properties in the inhibition of [3H]-arachidonic acid release by WIN-55212-2-stimulated hCB1-CHO cells and Ki values for the hCB2 receptor over 1microMThe diaryl-pyrazoline derivatives contained a chiral center atposition 4 The authors resolved the racemic mixture of twocompounds (121 and 130) The levorotatory enantiomersappeared to be the eutomers For compound SLV319levorotatory enantiomer of 121 the Ki and pA2 values were78 nM and 99 respectively The Ki and pA2 values ofSLV326 levorotatory enantiomer of 130 were 359 nM and9 respectively

8 34-Diaryl-Pyrazoline Derivatives Two in-vivo models CP-55940-induced hypotension inrat and WIN-55212-2-induced hypothermia in mouse wereused to assess the potential of these compounds The resultsare of the same order of magnitude than those obtained forSR141716A For instance in the hypotension modelSLV319 ((-)121) and SLV326 ((-)130) showed ED50 values

Lange and collaborators also discovered 34-diaryl-pyrazoline derivatives after a screening of compoundsresembling to SR141716A [183184] Compound 118 thatwas initially identified during the screening possesses a Ki

Table 10 CB1 Cannabinoid Receptor Antagonists 8 34-Diaryl-Pyrazolines Derivatives

Structure affinity for the hCB1 and hCB2 cannabinoid receptors (Ki nM) expressed in CHO transfected cells ([3H]-CP-55940) and potency (pA2) ofsome 34-dirylpyrazolines derivatives Table adapted from [184]

NN

N N

SO2

R3

R2

Cl R1

R4

n˚ R1 R2 R3 R4 Ki hCB1 (nM) Ki hCB2 (nM) pA2 (CB1)

118 H H H 4-CH3 197 gt 1000 84

119 H H H 246- CH3 24 gt 1000 94

120 H H H 4-Cl 16 gt 1000 95

121 H CH3 H 4-Cl 25 gt 1000 87

122 H CH3 CH3 4-Cl 280 gt 1000 85

123 H H H 4-F 53 gt 1000 9

124 H CH3 H 4-F 338 gt 1000 85

125 H CH3 CH3 4-F gt 1000 gt 1000 lt 75

126 H CH3 H 3-Cl 14 gt 1000 86

127 4-Cl CH3 H 4-Cl 255 ND ND

128 4-F CH3 H 4-Cl 584 ND ND

129 H CH3 H H 170 ND 75

130 H CH3 H 4- CF3 221 gt 1000 93

SR141716A 25 1580 86

1378 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

N

HN

O

N

Cl Cl

Cl

N

N

HN

O

Br

Br131 132

Fig (8) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Chemical structures of two 56-diaryl-pyrazine-2-amide derivatives (131-132) developed as CB1 cannabinoid receptorantagonists by AstraZeneca

of 55 and 2 mgkg (po) respectively proving their in-vivoefficacy after oral administration The authors alsodemonstrated that SLV319 is devoid of affinity for the P-glycoprotein pump and that it possessed a goodCNSplasma ratio (17) These two compounds (SLV319and SLV326) were chosen as development candidates bySolvay and entered clinical Phase I trials in 2003 [185]

132 Fig 8) useful as CB1 cannabinoid receptor antagonists[186 187] The activity of twenty derivatives wasdetermined using a [35S]-GTPγ S assay and hCB1-CHOtransfected cells The concentration required to give halfmaximal inhibition of CP-55940-induced [35S]-GTPγ Sbinding (IC50) is lower than 200 nM for the preferredcompounds No affinity data were given for thesecompounds

9 Diaryl-Pyrazine Diphenyl-Pyridine Diphenyl-Phenyland Diaryl-Pyrimidines Derivatives

The same year in a patent from Merck Finke andcollaborators described several 56-di-phenyl-pyridinederivatives as hCB1 antagonists or inverse agonists [188]The compounds have a substituent in position 3 of thepyridine core (Fig 9) This substituent could be a cyano(133-134) or a nitro group a halogen an ester or an amide(135-136) Over 150 compounds were synthesised toillustrate the invention but no pharmacological data wasdisclosed in the patent

In 2003 appeared several patents describing new familiesof compounds that bind to the CB1 cannabinoid receptorThese compounds have in common a central six atomsaromatic ring which could be a pyrazine a pyridine aphenyl or a pyrimidine and that is substituted by at leasttwo phenyl rings

The first patents were from Berggren and collaboratorsfrom AstraZeneca who published two patents describing thesynthesis of 56-diaryl-pyrazine-2-amide derivatives (131-

A few months later another patent was taken by Sanoficlaiming the antagonist properties of 56-di-phenyl-2-pyridine carboxamide derivatives [189] These compounds

N

NH

O

Cl

Cl

N

O

Cl

O NH

N

O

Cl

ClCl

NH

CN O

N

Cl

Cl

Cl

OHN

N

O

Cl

ClCl

CNF

F

133 134

135 136 137

Fig (9) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives 56-Di-phenyl-pyridine derivatives patented by Merck (133-136) and by Sanofi (137) as CB1 cannabinoid receptorsantagonists

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1379

NH

O

N

Cl

ClCl

NH

O

N

Cl

F3CCl

N

N

O

Cl

Cl Cl F138 139 140

Fig (10) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Representative structures of the diphenyl-phenyl (138-139) and 45-diphenyl-pyrimidine (140) derivatives developed ascannabinoid antagonists at Sanofi and Merck respectively

28 examples are given structurally related to the diphenyl-phenyl derivatives have their pyridine core substituted bytwo phenyl rings and by an amide moiety (137 Fig 9) TheIC50 values were lower than 100 nM but no data were givenshowing the antagonist properties although the compoundswere assayed in the adenylate cyclase inhibition assay

10 Other Derivatives

Several amide derivatives structures recently appearedamong the CB1 cannabinoid receptor antagonists In a seriesof patents taken by Merck in 2003 Hagmann and colleaguesdescribed a large amount of compounds obtained by parallelsynthesis which are claimed to be CB1 cannabinoid receptorantagonists [192-195] They were synthesised by reacting alibrary of substituted amines with a library of carboxylicacids (141-144 Fig 11) However as no value was givenfor the affinity or activity of these amides we will notfurther discuss this class of compounds

Along this line Sanofi Synthelabo developed newdiphenyl-phenyl derivatives claimed to have CB1cannabinoid receptor antagonist properties [190] They arebased on a central phenyl ring substituted by two phenylsand by an amide moiety (138-139 Fig 10) Thirteencompounds were claimed along with their synthesis TheirIC50 values were lower than 100 nM ([3H]-CP55-940hCB1-CHO cells) but no other specific data (selectivitypA2hellip) was given

Several 15-diaryl-pyrrole-3-carboxamide derivatives weresynthesised and claimed to be CB1 cannabinoid receptorantagonists by Berggren et al [196] The affinity valueswere not disclosed in the patent (145-146 Fig 12) Otherpyrrole derivatives were synthesised by Guba et al atHofmann-La Roche [197] More specifically 2-(thiazol-4-yl)pyrrole derivatives were claimed as CB1 cannabinoidreceptor antagonist (IC50lt2microM) (147-148 Fig 12)

Finally Kopka et al from Merck described more thanone hundred 45-diaryl-pyrimidine derivatives as CB1cannabinoid receptor antagonists (140 Fig10) [191]However no precise data were provided concerning theaffinity and functionality of the compounds

R2

R1N

R3

R4

R5

O

NH

O

Cl

O F

F

R2

R1NH

R3

R4

HO R5

O

NH

O

Cl

CN

NH

O

Cl

O

O

NH

O

Cl

Cl

+

141142

143 144

Fig (11) CB1 cannabinoid receptor antagonists 10 Other derivatives Retrosynthetic scheme of a CB1 cannabinoid antagonistlibrary of amide derivatives developed at Merck The structures of four representative compounds are illustrated (141-144)

1380 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

NH

O N

Cl

Cl

Cl

N

NH

O

OMe

MeO

N

NH

O

N

S

MeO

N

NH

O

N

S

MeO

145 146

147 148

Fig (12) CB1 cannabinoid receptor antagonists 10 Other derivatives Two examples of 15-diaryl-pyrrole derivatives described byBerggren et al (145-146) While the 2-(thiazol-4yl)pyrroles derivatives 147 and 148 were described by Guba et al from Hofmann-LaRoche All these pyrroles derivatives were claimed to be CB1 cannabinoid receptor antagonists

Another new structure is represented by the 124-triazolederivatives developed by Jagerovic and collaborators In arecent paper were described the synthesis andpharmacological properties of five new 15-diphenyl-3-alkyl-

triazole derivatives [198] Among these compounds onlyone (149) behaved as a CB1 antagonist inhibiting the WIN-55212-2-induced contractions in a mouse vas deferenspreparation However despite its effects in isolated tissue

Table 11 CB1 Cannabinoid Receptor Antagonists 10 Other Derivatives

Structure and affinity of diaryl-triazole derivatives

N

NN

R3

R2

R1

Cpd R1 R2 R3 Ki CB1 (nM) References

149 24-diCl 4-Cl hexyl 855 a [198]

150 24-diCl 4-Cl2-OMe CO-NH-(3-azabicyclo[330]octan-3-yl) 270 b [152]

151 24-diCl 4-Cl CO-NH-(3-azabicyclo[330]octan-3-yl) 164 b [152]

152 4-Cl 24-diCl CO-NH-(3-azabicyclo[330]octan-3-yl) 137 b [152]

153 4-Cl 24-diCl CO-NH-benzylpyrrolidin-3-yl 29 b [152]

154 4-Cl 24-diCl CO-NH-1-(4-chlorophenyl)ethyl 66 b [152]

155 4-Cl 24-diCl CO-NH-piperidinyl 356 c [182]

156 24-diCl 4-Cl CO-NH-piperidinyl 382 c [182]

a [3H]-SR141716A rat cerebellar membranesb [3H]-CP-55940 hCB1-HEK EDNA cellsc [3H]-CP-55940 hCB1-CHO cells

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1381

N

S

O

NH

Cl

Cl

Cl

F

N

S

O

NHN

ClCl

Cl

N

S

O

NH

Br

N

S

O

NHN

Cl

Cl

Cl

156 157 158 159

Fig (13) CB1 cannabinoid receptor antagonists 10 Other derivatives Structures of 45-diaryl-thiazole derivatives patented as hCB1cannabinoid receptor antagonists by Solvay Pharmaceuticals (156 157 158) or by AstraZeneca (157 158 159)

assays (mouse vas deferens and guinea pig ileum) thecompound possessed only a reduced rCB1 affinity with Kivalues of 855 and 748 nM using [3H]-SR141716A and [3H]-WIN-55-212-2 respectively (Table 11) Further four seriesof compounds differing by the nature of the substituentaround the triazole core were also described in a patent[199] Usually the compounds claimed possess two aromaticrings possibly substituted and one linear alkyl chain Theaffinity for the cannabinoid receptors was not givenNevertheless 149 was evaluated in two isolated tissuemodels the inhibition of the electrically evoked contractionsin the guinea pig ileum and in the mouse vas deferens Inthe two models compound 149 inhibited the effect of WIN-55212-2 but had no effect by itself In the vas deferensmodel the authors obtained a pA2 value of 748 to becompared with 763 obtained for AM251

Yet another five membered ring a thiazole has beenused as central moiety of a new class of cannabinoid ligandsThe thiazole ring is substituted by two aryls in position 4and 5 and by an amide (position 2) as in compounds 156-159 (Fig 13) Forty derivatives of this type weresynthesised by Lange and colleagues from SolvayPharmaceuticals and claimed in a patent as agonists orantagonists of the CB1 cannabinoid receptor [200] Nopharmacological data were provided however their activitywas assessed by measuring the cAMP in transfected hCB1-CHO cells In a recent paper from Lange et al two of thesethiazole derivatives were further described [182] Compound13 (157) the most closely related to SR141716A has a Kivalue of 227 nM while for compound 14 (158) Ki value isover 1000 nM Thus as in the imidazole series (see Table9) the aromatic substitution pattern is of great importancefor the CB1 cannabinoid receptor affinity Compound 13however was devoid of in vivo activity

Others 124-triazole derivatives were described by Dycket al compounds 150-154 unlike the Jagerovic ones havean amide moiety in position 3 [152] The highest affinitywas obtained with a 1-benzyl-pyrrolidin-3-yl substituentThe Ki value was 29 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) to be compared to 12 nM obtained for theSR141716A

Seventeen 45-diaryl-thiazole derivatives were alsosynthesised by Berggren et al and patented by AstraZeneca(157-159 Fig 13) [201]

In a patent from Merck Toupence et al described over190 substituted furo[23]pyridines claimed to be CB1cannabinoid receptor antagonists [202] The affinity (IC50 lt1microM) was measured using recombinant CHO cells and [3H]-CP-55940 and the activity using cAMP dosage (160 Fig14)

Lange et al described a triazole derivative (155) whichcan be superimposed with SR141716A Compound 155showed a Ki value of 356 nM ([3H]-CP-55940 hCB1-CHOcells) a pA2 value of 83 in the inhibition of WIN-55212-induced release of [3H]-arachidonic acid by hCB1-CHO cellsand was active in vivo in the CP-55940-inducedhypotension in rat (ED50 = 236 mgkg) [182]

Very recently Alanine from Hoffmann-LaRoche foundthat diarylbenzo[13]dioxole derivatives act as CB1

O

O

S

N

OO

F

ClCl

O

O

F O

N

OON

NH2

O

Cl

Cl

Cl

160 161 162

Fig (14) CB1 cannabinoid receptor antagonists 10 Other derivatives One example of a furo[23]pyridine derivative (160) and twoexamples of diarylbenzo[13]dioxole derivatives (161-162) described as hCB1 cannabinoid receptor antagonists by Toupence et aland Alanine et al respectively

1382 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

cannabinoid receptor antagonists (161-162 Fig 14) [203]Approximate IC50 values for twelve compounds ([3H]-CP-55940 hCB1-HEK cells) among the three hundred andeighty compounds described were given (IC50lt2microM)

et al in 1995 [207] In their hands ∆9-THC was not able toinhibit the forskolin-stimulated adenylyl cyclase activity inan assay performed on hCB2-CHO cells Subsequently theyshowed that ∆9-THC was able to antagonise HU210-inducedinhibition of adenylyl cyclase activity thus acting as anantagonist of the hCB2 cannabinoid receptor expressed inCHO cells [208] Furthermore very recently Govaerts et al[209] reported an inverse agonist effect of ∆9-THC and ∆8-THC on hCB2-CHO cells using the [35S]-GTPγ S assayThe pEC50 values were 763 and 888 respectively whilethe Emax values were ndash27 and ndash16 as compared to basal

Griffith from Pfizer described three new series of CB1cannabinoid receptor antagonists based on a bicyclic centralaromatic ring Purine pyrazoltriazines andpyrazolo[15a]pyrimidines derivatives were synthesised andinvestigated for their ability to interact with the CB1cannabinoid receptor Albeit a [35S]-GTPγ S assay wasdescribed no data were given concerning the antagonist orinverse agonist properties of such compounds Two hundredand eighty purine derivatives were synthesised [204] Theaffinities of 163 and 164 were given in the patent (28 and12 nM [3H]-SR141716A) In a following patent thesynthesis of sixty pyrazoltriazine derivatives was described(165 Fig 15) [205] The affinity of each compound was notgiven but was said to be ranging between 01 and 590 nMAnd finally more than forty pyrazolo[15a]pyrimidines weresynthesised (166 Fig 15) [206] Their affinity for the CB1cannabinoid receptor was lower than 155 nM Despite thefact that a great number of derivatives share the samesubstitution pattern around the central bicyclic aromatic ringthe comparison of these three new series of compounds ismade difficult by the absence of precise affinity data

Some other classical cannabinoids were shown to possessan antagonist activity at the CB2 receptor This is the casefor instance for the non-selective O-1184 (2) (Ki = 74 nM)which enhances the forskolin-induced cAMP production inCB2-CHO cells (EMax = 161 EC50 = 63 nM) and alsofor O-584 (3) (EMax = 246 EC50 = 138 nM) [39]

2 Indole Derivatives

Since the disclosure in 1993 of a second cannabinoidreceptor many attempts have been made to synthesise CB2cannabinoid receptor selective ligands The first compoundreported to have a somehow greater affinity for the peripheralcannabinoid receptor was WIN-55212-2 The compoundwas introduced by DrsquoAmbra and colleagues [210] back in1992 as a new cannabinoid ligand having a nanomolaraffinity and acting as an agonist In 1996 Showalter et alused a transfected cell line expressing the CB2 cannabinoidreceptor in order to identify selective ligands for thisreceptor [211] They showed that the CB2 affinity of WIN-55212-2 was 7 times greater than the CB1 with Ki valuesof 189 and 028 nM for the CB1 and CB2 cannabinoidreceptors respectively However WIN-55212-2 is anagonist of the cannabinoid receptors Interestingly WIN-55212-3 (167 Fig 16) the so-called inactive enantiomer ofWIN-55212 was recently shown to behave as a low affinity(Ki=363 microM) inverse agonist of the hCB2 cannabinoidreceptor as it decreased the [35S]-GTPγ S binding [209]

III CB2 LIGANDS RECEPTOR ANTAGONISTS ANDINVERSE AGONISTS

The compounds acting as antagonist or inverse agonistsat the CB2 cannabinoid receptor are reviewed in this thirdpart of the paper As for the CB1 cannabinoid receptorligands they are classified depending on their chemicalstructures

1 Classical Cannabinoid Derivatives

The first report of an antagonism mediated by ∆9-THC atthe peripheral cannabinoid receptor was made by Bayewitch

N N

NN

NH COOH

Cl

Cl

N N

NN

N

Cl

Cl

NH2OH

O

N

N

N

N

N

NHNH2

O

Cl

ClN

N

N

N

Cl

Cl

N

O

163 164

165 166

Fig (15) CB1 cannabinoid receptor antagonists 10 Other derivatives Examples of purine (163-164) pyrazoltriazines (165) andpyrazolo[15-a]pyrimidines (166) derivatives described as CB1 cannabinoid receptor antagonists by Griffith

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1383

The pEC50 value was 554 and the Emax value ndash26 ascompared to basal

AM630 also acts as an inverse agonist at the hCB1cannabinoid receptor (EC50 = 900 nM) as Landsman et aldemonstrated [47] Recently Zhang and colleagues reportedthe characterisation of the microsomal metabolism of AM630[212]

N

O

N

O

O

N

O

Cl

O

N

O

(S)-WIN-55212-3 167 BML-190 168

In 1996 Gallant and colleagues identified by submittinga large number of compounds to a binding assay anotherindole analog as CB2 cannabinoid receptor ligand [213] Onthe basis of this compound christened BML-190 (168 Fig16) or called indomethacin morpholinylamide theysynthesised several derivatives that possessed a selectivityratio for the CB2 cannabinoid receptor of up to 140 Thesewere the first compounds specifically designed to be CB2cannabinoid receptor ligands The first report on thepharmacological properties of BML-190 appeared onlyrecently New and colleagues highlighted in the paper theinverse agonist properties of the compound BML-190 dose-dependently increases (103 EC50= 980plusmn70 nM) theforskolin-stimulated levels of cAMP in hCB2-HEK cellswhile WIN-55212-2 decreases (44 45plusmn42nM) thisaccumulation [137]

Fig (16) CB2 cannabinoid receptor antagonists 2 Indolederivatives Chemical structures of WIN-55212-3 (167) andBML-190 (168) two indoles derivatives acting as inverseagonists at the hCB2 cannabinoid receptor Surprisingly in 2002 Melck and collaborators used

BML-190 as a CB2 receptor agonist in a cell proliferationassay [214] BML-190 was also tested as inhibitor of thecyclooxygenase-2 enzyme by Kalgutkar et al [215] Theyobtained IC50 values higher than 33 and 66 microM for theCOX-I and COX-II enzymes respectively These valueshave to be compared with the submicromolar activity ofindomethacin the parent compound Very recently Klegerisand colleagues [216] looking for an antineurotoxic action ofcannabinoids showed that BML-190 increases TNF-αsecretion by stimulated THP-1 monocytic cells but is noteffective on the IL-1β secretion

Further on the basis of WIN-54461 (12) a CB1antagonist AM630 (13) was synthesised by replacement ofthe bromine atom by an iodine one In the initial reportPertwee et al showed the antagonist effects of AM630 butalso suggested that the CB1 cannabinoid receptor may not bethe preferential receptor of AM630 [45] Later on Ross et alreported using hCB1amp2-CHO cells and [3H]-CP-55940that AM630 binds to the CB2 cannabinoid receptor (Ki =312 nM) with a selectivity ratio over 160 [48]Furthermore using a [35S]-GTPγ S assay they showed itsinverse agonist properties (EC50 = 76 nM) However

Table 12 CB2 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Reported binding affinities (Ki) and selectivity ratio (CB1CB2) of SR144528 (169) for rat and human cannabinoid receptors determined using [3H]-CP55940 as radioligand

NN

O

NH

Cl

SR144528 169

rCB2 (spleen) rCB1 (whole brain) Selectivity Ki hCB2 (transfected CHOcells)

Ki hCB1 (transfected CHOcells)

Selectivity References

038 nM 305 nM 800 06 nM 437 nM 730 [217]

067 nM 548 nM 80 [219]

56 nM gt10microM gt1780 [48]

25 nM a gt 1microM a gt400 [224]

024 nM 276 nM 115 199 nM 503 nM 25 [232]

a COS cells

1384 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

3 Diarylpyrazole Derivatives CHO cells is constitutively active and phosphorylated onSer 352 Upon treatment by SR144528 thisphosphorylation of the receptor was inhibited Moreover theSR compound was able to induce dephosphorylation ofagonist-induced CB2 phosphorylation The question whetherSR144528 induces a CB2 receptor conformational changemaking it a better phosphatase-specific substrate or ifSR144528 binding activates specific phosphatases remainsopen However this remains a nice example of inverseagonist-induced receptor resensitisation

The same Sanofi team which synthesised the CB1antagonist SR141716A reported in 1998 the synthesis andpharmacological characterisation of a CB2 cannabinoidreceptor selective antagonist the SR144528 (169) [217218] The structure of the new compound is derived fromSR141716A one SR144528 is the first selective CB2receptor antagonist reported as the inverse agonist propertiesof BML-190 were shown only recently by New et al [137]The authors reported for 169 using [3H]-CP-55940 asradioligand Ki values of 04 nM and 06 nM for the rat andthe human CB2 receptors respectively and found a CB1-CB2 selectivity ratio around 700 Table 12 sums up thebinding affinities of SR144528 for the cannabinoid receptorsobtained by different authors Interestingly while the CB2affinity is more or less constant the affinity obtained for theCB1 receptor varies by one order of magnitude [219] Theantagonist effect of SR144528 was highlighted by evaluatingits effect on cAMP production by hCB2-CHO cells exposedto 3 nM CP-55940 (EC50=10nM) The antagonism wasalso observed on the activation of the MAP kinase pathwayby CP-55940 (IC50=39nM)

The Sanofi research team working on cannabinoidsconducted investigations in order to determine the keyresidues for SR144528 interaction with the receptor (Table13) as well as the binding mode of the compound Firstlythey identified the TM4-EL2-TM5 region as a regioncontaining crucial residues for the affinity of several ligandsamong them SR144528 [224] Among these residues themutation of two cysteine of the second extracellular loop(C174 and C179) abolishes the affinity for the CB2 receptorLater on the implication of S453(161) and S457(165) inthe binding of SR144528 was evidenced by mutationalstudies In the model proposed for the docking of SR144528into the CB2 receptor the crucial contacts are comprised intothe TM4 this is the case for S453(161) and S457(165)which are proposed to form hydrogen bonds with the ligandand W464(172) proposed to have aromatic interactions withthe 4-methylbenzyl of the SR144528 [225] In 2003 Fengand Song showed that point mutations D349(130)A andA634(241)E abolished ligand binding However mutationof the arginine R350(131) member of the highly conservedDRY motif to alanine has no influence on SR144528binding [226]

Early after the report of the synthesis and characterisationof SR144528 the same authors further explored the functionof this compound and showed its inverse agonist propertiesusing CB2-CHO cells co-transfected with the luciferasereporter gene with either the CRE or the murine krox24regulatory sequence [220] In these models SR144528 notonly inhibits CP-55940 effects but also reproduces theeffects of a pertussin toxin treatment proving its inverseagonist properties SR144528 functionality was alsoexplored by [35S]-GTPγ S binding in hCB2-CHO cells(IC50=3 nM) [221] The inverse agonist function ofSR144528 was further illustrated by Rhee and Kim usingCOS cells co-transfected with hCB2 receptor and adenylylcyclase [222]

As for the CB1 cannabinoid receptor ligands Sanofideveloped tricyclic derivatives based on the 1-benzylpyrazole-3-carboxylic scaffold More than twentycompounds were disclosed in a patent [227] in 2001 (84-86Table 7) The benzyl in position 4 characteristic of the CB2diarylpyrazoles from Sanofi is conserved as well as thebicyclic residue in position 3 The length of the second linkbetween the phenyl and the pyrazole can be made of one or

Bouaboula and co-workers investigated SR144528 effectson CB2 cannabinoid receptor phosphorylation status [223]Firstly they showed that the CB2 receptor transfected in

Table 13 CB2 Cannabinoid Receptor Antagonists 3 Di-Aryl-Pyrazole Derivatives

Some of the single point mutations reported for the hCB2 cannabinoid receptor for which pharmacological data concerning the SR144528 are available

Mutant Effect References

R350(131)A no effect [226]

S453(161)A loss of affinity [225]

V456(164)I no effect [225]

S457(165)A loss of affinity [225]

C174S a loss of affinity [224 225]

C175S a loss of affinity [225]

C177S a no effect [225]

C179S a loss of affinity [224 225]

S544(193)G no effect [225]

a Second extra-cellular loop

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1385

two methylene but according to the patent one methylenebridge is preferred The Ki values of these compounds for theCB2 receptor were said to be lower than 500 nM and theirantagonist properties have been assessed using the adenylatecyclase inhibition assay

compounds by the presence of an alkyl substituent inposition 5 instead of a phenyl and of course by position 3which remains unsubstituted However except a lecture atthe 1998 ACS meeting [230] where compound 171 wasreported to have a Ki value of 835 nM and a selectivity ratioover 100 no further report on these compounds waspublished untill now

In a recent paper Mussinu and colleagues reported thesynthesis of other tricyclic derivatives starting fromSR141716A structure [228] N-piperidin-1-yl-14-di-hydroindeno[12-c]pyrazole-3-carboxamide derivatives areCB2 receptor ligands and some of them possess high affinityand selectivity for the CB2 receptor as shown in Table 7 (79-83) These tricyclic derivatives differ from the Sanofi tricyclicderivatives as they do have a phenyl ring instead of a benzylin position 1 Another difference is the nature of thesubstituent in position 3 which is not a bicycloalkyl but acyclic amine One of the compounds synthesised byMussinu et al 81 possesses the highest affinity for the CB2receptor ever reported with a Ki value of 0037 nM for themurine CB2 receptor ([3H]-CP-55940 mice spleenhomogenate) Interestingly 79 the closest SR141716Aanalog lost almost all its affinity for the CB1 receptor andincreased by four orders of magnitude its affinity for the CB2receptor Even if the structure quite similar to the SRcompounds could suggest that these derivatives behave asantagonists no pharmacological data were given in the paperto support this hypothesis

5 2-Oxoquinoline Derivatives

Inaba and colleagues from Japan Tobacco introduced in2001 a completely new structure in the cannabinoid fieldbased on a 2-oxoquinoline scaffold Over eighty derivativeswere disclosed in a patent [231] and one compound JTE-907 (173) was subsequently characterised in-vitro as well asin-vivo [232] In Table 14 are listed some of the compounds(172-178) disclosed in the patent along with their affinity forthe cannabinoid receptors These values were obtained onhuman receptors against [3H]-WIN-55212-2 For twelvecompounds the anti-inflammatory activity per os wasassessed in a carrageenin-induced paw edema model Thehighest effect reported was obtained with JTE-907 with anED50 value lower than 01 mgkg Therefore thesecompounds were claimed to be useful as anti-inflammatoryand anti-allergic agents

To further characterize JTE-907 the authors determinedits affinity for cannabinoid receptors of different species using[3H]-CP-55940 as radioligand In their hands JTE-907behaved as a very selective hCB2 ligand with Ki values of36 and 2370 nM for the CB2 and CB1 cannabinoid receptorsrespectively even more selective than the SR144528 (Kivalues of 199 and 50 nM respectively) However it has tobe mentioned that the affinities reported in the paper forJTE-907 are quite different from those reported in the patent(hCB1 Ki values of 35nM and 009 nM respectively) Italso appears that the SR144528 affinity for the CB1 receptordetermined by Iwamura et al is higher than the affinityreported by Rinaldi-Carmona et al with values of 50 and437nM respectively The authors ascribed the difference tothe variability of SR144528 binding affinities to the assayconditions The function of JTE-907 was evaluated bymeasuring the increase in cAMP production by forskolin-stimulated hCB2-CHO cells Maximum stimulation wasattained at a concentration of 1microM of JTE-907 In the sameassay WIN-55212-2 decreased the cAMP production in adose-dependent manner

Finally it is interesting to strengthen that the five-membered ring derivatives (79-86) of the diarylpyrazolefamily are reported to possess a high CB2 receptor affinitywhile the six- and seven-membered ring derivatives (69-78)are described as CB1 receptor ligands

4 Other Pyrazole Derivatives

Other CB2 ligands based on a pyrazole nucleus wereintroduced by SmithKline Beecham in a patent describingthe synthesis of about sixty new compounds [229] Theiraffinity (Ki) for the hCB2 receptor transfected in HEK293cells was reported to be ranging between 25nM and 10microMTheir structures (170-171 Fig 17) differ from the Sanofi

NN

O

N

ON

NN

N

NN

O

N

ON

NN

N

ClCl

1

5

4 3

2

170 171

Fig (17) CB2 cannabinoid receptor antagonists 4 Otherpyrazole derivatives Structure of two compounds (170-171)among those claimed by SmithKline Beecham to be hCB2antagonists

In conclusion JTE-907 was shown to be a CB2 selectiveinverse-agonist with anti-inflammatory and anti-allergicproperties This compound is currently undergoing Phase Iclinical trials as anti-allergic drug [233]

IV NON-CB1 NON-CB2 CANNABINOIDRECEPTORS LIGANDS

In the last years the possible presence of additional non-CB1 non-CB2 cannabinoid receptors was raised frompharmacological evidences

One of the putative receptors possesses an endotheliallocalisation while other ones are localized in the CNS or atperipheral nerves For instance an additional target for ∆9-

1386 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 14 CB2 Cannabinoid Receptor Antagonists 5 2-Oxoquinoline Derivatives

Structure of six compounds among the oxoquinoline derivatives patented by Japan Tobaco Affinity (Ki nM) and selectivity (CB1CB2) for the cannabinoidreceptors are given The affinity was measured by binding against [3H]-WIN-55212-2 on either hCB2-CHO or hCB1-CHO cells Adapted from [231]

Cpd ndeg Structure hCB2 hCB1 Selectivity

1-2 172

NH

MeO O

O

OMeO

0014 3671 262215

JTE-907 173

NH

O

MeO O

O

NH

O

O

0087 3436 39495

2-2 174

OH

O

NH

MeO O

O 077 3247 4215

3-27 175

NH

O

F

NH

MeO O

O0021 336 16000

3-32 176

NH

MeO O

O

NH

F

O

0036 2398 66610

3-38 177

O

O

NH

N

O

O

NMeO

0085 935 11000

3-43 178

NMeO O

O

NH

F

O

0043 864 20100

tetrahydrocannabinol was described in capsaicin-sensitivesensory nerves by Zygmunt et al [234]

[29] and by Di Marzo et al [235] Thus in the presentreview we will focus on the description of the so faridentified ligands of the non-CB1 non-CB2 receptors withan emphasis on the compounds having antagonist properties

Pharmacological evidences for the existence of additionalcannabinoid receptors were reviewed by Wiley and Martin

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1387

(Table 15) In particular the knowledge about three not yetcloned targets has grown The growing molecularpharmacology of the three putative new cannabinoidreceptors is reviewed in the three following sections Thefourth section summarises the data on the other proposedcannabinoid targets

vasodilatator activity In addition the anandamide effect ispartially sensitive to SR141716A inhibition The authorsproposed the existence of two distinct receptors responsiblefor the vasodilatatory effect of anandamide one located in theendothelium is SR141716A-sensitive while the other onelocated on the vascular smooth muscle is SR141716A-insensitive Thus SR141716A is an antagonist of anendothelial anandamide receptor Several studies wereconducted in order to understand the molecularpharmacology of the endothelial anandamide receptor It ispertussis toxin-sensitive [237] Cannabidiol acts asantagonist and abnormal-cannabidiol (Abn-cbd 179) and itsanalogue O-1602 (180) act as agonists [238] The synthesisof O-1602 and of its analogues are disclosed in a patentdescribing the vasodilatatory activity of cannabinoid

1 Endothelial Anandamide Receptor ndash Abnormal-Cannabidiol Receptor

In 1999 Wagner et al showed that anandamide couldinduce mesenteric vasodilatation through an endotheliallylocated ldquoanandamide receptorrdquo pharmacologically distinctfrom CB1 [236] Other cannabinoids such as ∆9-THC HU-210 or WIN-55212-2 were devoid of mesenteric

Table 15 Non-CB1 Non-CB2 Cannabinoid Receptors Antagonists

An overview of the putative new non-CB1 non-CB2 anandamide-receptors ligands The antagonists are highlighted in bold

NH

N

S

HO OH

Cl

OH

OH

OH OH OH OH

OMe

OMe

RR

RR

RR

RR

HO

OH

NH

O

cannabidiol (10) Abnormal-cannabidiol 179 O-1602 180

O-1918 181 capsazepine 183capsaicin 182

Compound Endothelial AEA receptor Brain AEA receptor Cannabinoid-vanilloid brain receptor

AEA agonist a agonist de

WIN-55212-2 no effecta

agonist e agonist g h

CP-55940 no effect e agonist i

SR141716A antagonist a b j no effect ([35 S]-GTPγS binding) d e f antagonist g h

AM251 no effect k no effect h i

Cannabidiol antagonist b no effect e

O-1918 antagonist c jm

Abd-cbd agonist b l

O-1602 agonist b

Capsaicin no effectj agonist i

Capsazepine no effect b antagonist h

a [236] b [238] c [240] d [249] e [250] f [251] g [254] h [258] i [ 255] k [244] j [ 243] l [ 242] m [241]

1388 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

analogues [239] More recently Offertaler et al showed thatO-1918 (181) a cannabidiol analog acts as an antagonist ofthe endothelial anandamide receptor [240] In addition O-1918 does not bind to the CB1 and CB2 cannabinoidreceptors Furthermore O-1918 unlike cannabidiol doesnot cause vasorelaxation in the mesenteric artery preparationmodel used and thus acts as a true neutral antagonistUsing O-1918 as a pharmacological tool Begg et alproposed a coupling of this new G protein-coupledendothelial receptor through a GiGo mechanism to ionchannels in primary cultured human vascular endothelialcells [241] However in their hands SR141716A wasineffective in antagonising Abd-cbd Ho and Hiley furtherdemonstrated that Abn-cbd relaxes rat small mesentericarteries through an activation of K+ channels via aSR141716A-sensitive pathway [242] Further using thesame model they characterized the vasorelaxant activity ofvirodhamine a novel endocannabinoid apparently actingthrough the same receptor [243] The virodhamine-inducedvasorelaxation is sensitive to SR141716A and O-1918 butinsensitive to AM251 SR144528 and AM630 Moreovervanilloid receptor desensitisation by capsaicin (182) had noeffect

244] Thus position 4 on the 5-phenyl could be furtherexplored to obtain selective ligands

To come to an end despite the fact that the endothelialanandamide receptor is neither identified nor cloned at leastthree antagonists are identified SR141716A cannabidioland O-1918 It is likely that a growing number of ligandsand therapeutic applications will appear in the very nearfuture

2 Brain Anandamide Receptor

Following an in-depth evaluation of anandamide effects inCB1

-- mice Di Marzo and co-workers suggested that a non-CB1 non-CB2 G protein-coupled receptor might mediatesome of the actions of AEA in mice [249] Actually effectsof AEA but not those of ∆9-THC were not decreased inCB1 cannabinoid receptor knock-out mice For instance 30mgkg of AEA were still effective in hot-plate test whereas10 mgkg of ∆9-THC had no effect on anti-nociceptionMoreover in a [35S]-GTPγ S assay AEA stimulated with anEC50 value of 223 microM the [35S]-GTPγ S binding to CB1

--

mice brain membranes (Emax= 28) ∆9-THC showed noeffect in this assay The addition of SR141716A did notaffect the [35S]-GTPγ S binding to CB1

-- mice brainmembranes Shortly after other evidences for the existence ofa new G protein-coupled cannabinoid receptor with a distinctdistribution in the central nervous system were published[250] From a set of 24 commonly used cannabinoids (AEAWIN-55212-2 JWH-030 THChellip) only AEA and WIN-55212-2 produced an increased binding of [35S]-GTPγ S onCB1

-- mice brain membranes Interestingly in this studySR141716A alone produced a significant inhibition of [35S]-GTPγ S binding similar to the inhibition obtained usingCB1

++ mice brain membranes (IC50 values of 57 microM and47 microM respectively) However SR141716A did not affectthe anandamide-induced [35S]-GTPγ S binding in CB1

--

mice brain membranes Moreover [3H]-SR141716Aexhibited significant binding in some brain regions of CB1

--

mice which are different from those exhibiting stimulation of[35S]-GTPγ S binding by WIN-55212-2 and AEA Thusthe authors proposed that the SR141716A effects on CB1

--

mice are mediated neither by the CB1 cannabinoid receptornor by the putative new anandamide brain receptor Monoryand co-workers using CB1

-- mice described a novel nonadenylyl cyclase-coupled cannabinoid binding site in micecerebellum [251] Two agonists AEA and WIN-55212-2but not ∆9-THC or HU210 were found to bind to thisreceptor as they enhanced [35S]-GTPγ S binding NeitherSR141716A nor SR144528 were able to affect the WIN-55212-2 induced [35S]-GTPγ S binding in CB1

-- mice asfound by Breivogel et al [250] However the distributionpattern of the receptor described by Monory et al differs fromthe distribution found by Breivogel et al It has to bementioned that the knock-out strains used were different Themice used by Monory were from a CD-1 strain while theone used by Breivogel were from a C57BL61 strainInterestingly Muthane et al detected differences in nigralneurons number and sensitivity to MPTP in the two mousestrains used [252]

More recently Mo et al obtained results showing thatthe Abnormal-cannabidiol receptor is also responsible for anincreased endothelial cell migration [244] Abn-cbd enhancedhuman umbilical vein endothelial cell migration while O-1918 antagonised Abn-cbd effect The CB1 cannabinoidreceptor inverse agonist SR141716A partially inhibitedAbd-cbd action whereas AM251 and SR144528 had noeffect

In another paper from George Kunos team Batkai et alreported that SR141716A is able to inhibit the endotoxichypotension by a cardiac mechanism involving neither theCB1 nor the CB2 cannabinoid receptors [245] Theinvolvement of a non-CB1 non-CB2 site of action wasshown using CB1

-- and CB1--CB2

-- knock-out miceFurther studies are needed to identify the SR141716Asensitive myocardial site of action

In a recent patent Kunos described O-1918 and itsanalogues as vasoconstrictor agents useful to reversepathological vasodilatation of blood vessels [246] Theeffects of O-1918 on mouse blood pressure on LPS-inducedhypotension in mouse as well as the reversal of hypotensionby O-1918 injection are described Furthermore thesynthesis of O-1918 based antagonists is described startingfrom p-metha-28-dien-1-ol and the appropriate resorcinols

Interestingly the Abd-cbd receptor which was firstdetected in the vascular system also seems to be present inthe CNS [247] Indeed Walter et al found that microglialcells (BV-2) migration induced by 1 microM 2-AG is inhibitedby cannabidiol (300 nM) and O-1918 (1 microM) SR141716Awas found ineffective in this assay Thus 2-AG is an agonistof the Abd-cbd receptor Moreover Abd-cbd is able to elicita dose-dependent cell migration in the same model (EC50 =600nM) [248]

From a more structural point of view SR141716Abehaves as an antagonist while on the opposite AM251(20) a close analogue which differs from the former only bythe nature of one halogen has no effect on this receptor [242

To conclude to our knowledge no compounds havingantagonist or inverse agonist properties at this receptor havebeen identified so far

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1389

3 Cannabinoid-Vanilloid Brain Receptor a cannabinoid-vanilloid receptor at which SR141716A andcapsazepine behave as antagonists

Evidences for the presence of an additional ldquocannabinoidrdquoreceptor differing from the CB1 receptor and from the brainanandamide receptor appeared from studies focused on thehippocampus (for a review see Hajos et al [253]) Hajos etal studied the cannabinoid actions on GABAergic andglutamatergic transmission using the whole-cell patch-clamp technique [254] WIN-55212-2 induced a dose-dependent reduction in glutamatergic transmission reversedby SR141716A (1microM) Thus the authors concluded that thecannabinoid actions on the glutamatergic transmission aremediated by a non-CB1 but SR141716A-sensitive receptorIn another paper Hajos and Freund further characterised thisputative receptor [255] They showed that in addition toWIN-55212-2 CP-55940 and capsaicin (182) a TRPV1receptor agonist also act as agonists Furthermore in theirhands SR141716A as well as capsazepine (183) a TRPV1receptor antagonist were able to antagonise this effect on theglutamatergic transmission while AM251 was notSR141716A effects on CB1 knock-out mice were furtherstudied using a test of anxiety [256] Haller et al showedthat on the one hand the distribution of the CB1 receptorinduces anxiety and SR141716A has an anxiolytic effectand on the other hand SR141716A is still active in CB1

--

mice In a following paper they reported that AM-251induces anxiogenic effects in wild-type and in knock-outmice [257] Thus SR141716A in addition to CB1 bindsto a not yet identified receptor Another evidence for thisadditional receptor for SR141716A was given by Bass andco-workers [61] They conducted a structure-activityrelationship study on the stimulation of locomotor activityinduced by SR141716A and twenty analogues Nocorrelation was found between the affinity for the CB1cannabinoid receptor and the stimulation of locomotoractivity Albeit SR141716A and five of its analogues O-1803 O-1710 O-1253 O-1254 (40) O-1255 (41) behavedas inverse agonists in [35S]-GTPγ S assay none of theseanalogues stimulated the locomotor activity In conclusionthe SR141716A-induced stimulation of locomotor activity isneither due to inverse agonism nor to inhibition of anendogenous tone but to a brain receptor distinct from theCB1 receptor Di Marzo et al previously provided evidencesthat the cannabinoid-vanilloid brain receptor could mediatesome of the cannabinoid effects on locomotion [249]

4 Other Non-CB1 Non-CB2 Cannabinoid Receptors

Evidences for the existence of additional non-CB1 non-CB2 cannabinoid receptors localised in the rat isolatedmesenteric arterial bed appeared from the works of Ralevicand collaborators Several cannabinoids were evaluated intheir model of sensory neurogenic vasorelaxation evoked byelectrical field stimulation

HU210 was found to attenuate vasorelaxation in a CB1and CB2 independent way as SR141716A LY320135 andSR144528 were ineffective in inhibiting HU210 effect [259]Similar results were obtained with ∆9-THC [260]Similarly noladin ether inhibited sensory neurogenicrelaxation in a concentration-dependent manner which wasalso unaffected by SR141716A LY320135 and SR144528Moreover this effect as the ∆9-THC one was pertussistoxin sensitive suggesting the involvement of a Gio protein-coupled receptor [261]

However the inhibitory effects of CP-55940 and WIN-55212-2 were affected by the presence of SR141716A (1microM)or LY320135 (1microM) but not by the addition of SR144528(1microM) [262]

It appears that at least two receptors should be involvedin cannabinoid-mediated attenuation of sensory nerve-mediated vasorelaxation The first one is SR141716A andLY320135 sensitive while the other one is resistant toSR141716A

Some evidences for the existence of a ldquoCB2-likerdquo receptorappeared from the studies on palmitoylethanolamide (PEA)an endogenous fatty acid amide devoid of CB1 and CB2affinity [264] antinociceptive potential Calignano and co-workers showed that PEA was able to alleviate pain inseveral animal models [265 266] For instance PEA dose-dependently inhibits kaolin-evoked writhing in mice TheCB2 cannabinoid receptor inverse agonist SR144528 (02mgkg) inhibits PEA anti-nociceptive effect [266] Thus theauthors suggest that PEA acts as agonist on a SR144528-sensitive non-CB2 cannabinoid receptor (ldquoCB2-likerdquoreceptor) at which SR144528 acts as an antagonist

Further studies are needed in order to better characterisethese additional ldquocannabinoidrdquo receptors However it islikely that several therapeutic applications will soon springfor these receptors

Very recently Pistis et al published a study on theneurophysiological effects of cannabinoids on the basolateralamygdala neurons in-vivo [258] One of their findings is thatHU-210 and WIN-55212-2 did not elicit similar effects onbasal amygdala projection neurons firing rate While theformer had no effect the latter decreased the firing rateMoreover SR141716A reversed the WIN-55212-2 effectswhile AM251 had no effect The vanilloid receptorantagonist capsazepine also antagonised the WIN-55212-2effect on firing rate of these neurons Therefore the authorssuggested the presence of a novel cannabinoid-vanilloidreceptor

V CONCLUSION

Since their development cannabinoid antagonists provedto be essential tools in the understanding of cannabinoidpharmacology and biochemistry Moreover the interestshown by the pharmaceutical industry in the development ofnew cannabinoid antagonists prove if necessary the realtherapeutic potential of this class of compounds Thetreatment of eating and movement disorders memorydeficits psychosis and dependencies from various addictivedrugs are some of the most cited applications for the CB1cannabinoid receptor antagonists

From these lines it appears that in addition to theknown CB1 cannabinoid receptor it is now likely that twoother cannabinoid receptors do exist in the brain The firstone should be an anandamide brain receptor for whichSR141716A has no affinity while the second one should be

1390 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Much less wide is the knowledge on the CB2cannabinoid receptor antagonists However potentialapplications could be disorders involving the immunesystem such as inflammation or allergies

[19] Devane WA Hanus L Breuer A Pertwee RG StevensonLA Griffin G Gibson D Mandelbaum A Etinger AMechoulam R Science 1992 258 1946

[20] Mechoulam R Ben-Shabat S Hanus L Ligumsky MKaminski NE Schatz AR Gopher A Almong S MartinBR Compton DR Biochem Pharmacol 1995 50 83

Finally the growing literature on the non CB1 non CB2cannabinoid receptors should allow the development of newdrugs First examples are given by the cannabidiol andabnormal-cannabidiol derivatives described by Kunos et alfor which potential therapeutic applications on the vascularsystem are already patented

[21] Sugiura T Kondo S Sukagawa A Nakane S Shinoda AItoh K Yamashita A Waku K Biochem Biophys Res Co1995 215 89

[22] Hanus L Abu-Lafi S Fride E Breuer A Vogel Z ShalevDE Kustanovich I Mechoulam R Proc Natl Acad SciUSA 2001 98 3662

[23] Oka S Tsuchie A Tokumura A Muramatsu M Suhara YTakayama H Waku K Sugiura T J Neurochem 2003 851374It clearly appears from this paper that the antagonists of

the cannabinoid receptors are still an active research fieldfrom which should emerge new promising therapeutic toolsas well as innovative drugs in the near future

[24] Goutopoulos A and Makriyannis A Pharmacol Therapeut2002 95 103

[25] Beal JE Olson R Lefkowitz L Laubenstein L Bellman PYangco B Morales JO Murphy R Powderly W PlasseTF Mosdell KW Shepard KV J Pain Symptom Manage1997 14 7ACKNOWLEDGEMENTS [26] Notcutt W Price M Miller R Newport S Phillips CSimmons S Sansom C Anaesthesia 2004 59 440

GGM is very indebted to the ldquoFonds pour la formationagrave la Recherche dans lrsquoIndustrie et lrsquoAgriculture (FRIA)rdquo Belgium for the award of a research fellowship Theexperimental works of the authors were supported by grantsfrom the FNRS The Belgian Charcot Foundation and theUniversiteacute catholique de Louvain

[27] Ledent C Valverde O Cossu G Petitet F Aubert JFBeslot F Bohme GA Imperato A Pedrazzini T RoquesBP Vassart G Fratta W Parmentier M Science 1999 283401

[28] Zimmer A Zimmer AM Hohmann AG Herkenham MBonner TI Proc Natl Acad Sci U S A 1999 96 5780

[29] Wiley JL and Martin BR Chem Phys Lipids 2002 121 57[30] Adam J and Cowley P Expert Opin Ther Patents 2002 12

1475

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[33] Little PJ Compton DR Johnson MR Melvin LS MartinBR J Pharmacol Exp Ther 1988 247 1046

[2] Devane WA Dysarz FA Johnson MR Melvin LSHowlett AC Mol Pharmacol 1988 34 605

[34] Pertwee RG Stevenson LA Elrick DB Mechoulam RCorbett AD Brit J Pharmacol 1992 105 980

[3] Matsuda LA Lolait SJ Brownstein MJ Young ACBonner TI Nature 1990 346 561

[35] Harrison C Traynor JR Life Sci 2003 74 489-508[4] Gerard CM Molerau C Vassart G Parmentier M BiochemJ 1991 279 129 [36] Pertwee RG Fernando SR Griffin G Ryan W Razdan

RJ Compton DR Martin BR Eur J Pharmacol 1996 315195

[5] Glass M Dragunow M Faull RLM Neuroscience 1997 2299

[37] Ross RA Brockie HC Fernando SR Saha B Razdan RKPertwee RG Brit J Pharmacol 1998 125 1345

[6] Shire D Carillon C Kaghad M Calandra B Rinaldi-Carmona M LeFur G Caput D Ferrara P J Biol Chem1995 270 3726 [38] Griffin G Wray EJ Rorrer WK Crocker PJ Ryan WJ

Saha B Razdan RK Martin BR Abood ME Brit JPharmacol 1999 126 1575

[7] Ryberg E Khang Vu H Groblewski T Larsson N ElebringT Hjorth S Sjogren S Greasley PJ 2004 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 119

[39] Ross RA Gibson TM Stevenson LA Saha B Crocker PRazdan RK Pertwee RG Brit J Pharmacol 1999 128 735

[40] Martin BR Winckler JR Wiley JL Huffman JW CrockerPJ Saha B Razdan RK J Pharmacol Exp Ther 1999 2901065

[8] Rinaldi-Carmona M Calandra B Shire D Bouaboula MOustric D Barth F Casellas P Ferrara P LeFur G JPharmacol Exp Ther 1998 278 871

[41] Martin BR Stevenson LA Pertwee RG Breivogel CSWilliams A Mahadevan A Razdan RK 2002 Symposium onthe Cannabinoids Burlington Vermont International CannabinoidResearch Society page 2

[9] Munro S Thomas KL Abu-Shaar M Nature 1993 365 61[10] Howlett AC Qualy JM Khachatrian LL Mol Pharmacol

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[42] Thomas A Ross RA Saha B Mahadevan A Razdan RKPertwee RG Eur J Pharmacol 2004 487 213

[12] Calandra B Portier M Kerneacuteis A Delpech M Carillon C LeFur G Ferrara P Shire D Eur J Pharmacol 1999 374445 [43] Compton DR Gold LH Ward SJ Balster RL Martin BR

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Daum SJ Ackerman JH Gruett MD Kumar V EstepKG Olefirowicz EM Wetzel JR Alexander MD WeaverJD Haycock DA Luttinger DA Casiano FM ChippariSM Kuster JE Stevenson JI Ward SJ J Med Chem1995 38 3094

[14] Deadwyler S Hampson RE Mu J Whyte A Childers SRJ Pharmacol Exp Ther 1995 273 734

[15] Mackie K Lai Y Westenbroek R Mitchell R J Neurosci1995 15 6552

[16] Bouaboula M Poinot CC Bourrie B Canat X Calandra BRinaldi-Carmona M LeFur G Casellas P Biochem J 1995312 637 [45] Pertwee R Griffin G Fernando S Li X Hill A

Makriyannis A Life Sci 1995 56 1949[17] Bouaboula M Poinot CC Marchand J Canat X Bourrie BRinaldi-Carmona M Calandra B LeFur G Casellas P Eur JBiochem 1996 237 704

[46] Hosohata Y Quock RM Hosohata K Makriyannis AConsroe P Roeske WR Yamamura HI EurJPharmacol 1997 321 R1[18] delPulgar T Velasco G Guzman M Biochem J 2000 347

369 [47] Landsman RS Makriyannis A Deng H Consroe P RoeskeWR Yamamura HI Life Sci 1998 62 109

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[48] Ross RA Brockie HC Stevenson LA Murphy VLTempleton F Makriyannis A Pertwee RG Brit JPharmacol 1999 126 665

[81] Gatley SJ Lan R Volkow ND Pappas N King P WongCT Gifford AN Pyatt B Dewey SL Makriyannis A JNeurosci 1998 70 417

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B Congy C Martinez S Maruani J Neliat G Caput DFerrara P Soubrieacute P Breliegravere JC Le Fur G FEBS Lett1994 350 240

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[182] Lange JHM van Stuivenberg HH Coolen HKAC AdolfsT JP McCreary AC Keizer HG Walls HC Veerman WBorst AJM de Loff W Verveer PC Kruse CG J MedChem 2005 48 1823

[212] Zhang Q Ma P Wang W Cole RB Wang G J MassSpectrom 2004 39 672

[213] Gallant M Dufresne C Gareau Y Guay D Leblanc YPrasit P Rochette C Sawyer N Slipetz D M Tremblay NBioorg Med Chem Lett 1996 6 2263[183] Lange JHM Kruse CG Tipker J Hoogendorn J World

Patent Application 2002 WO02076949 [214] Melck D De Petrocellis L Orlando P Bisogno T Laezza CBifulco M Di Marzo V Endocrinology 2000 141 118[184] Lange JHM Coolen H K A C van Stuivenberg HH

Dijksman JAR Herremans AHJ Ronken E Keizer HGTipker K McCreary AC Veerman W Wals HC Stork BVerveer PC den Hartog AP de Jong NMJ Adolfs TJPHoogendoorn J Kruse CJ J Med Chem 2004 47 628

[215] Kalgutkar AS Marnett AB Crews BC Remmel RPMarnett LJ J Med Chem 2000 43 2860

[216] Klegeris A Bissonnette CJ McGeer PL Brit J Pharmacol2003 139 775

[185] httpwwwsolvaypharmaceuticalscomhtmlsciencepipelitext-html (accessed march 2004)

[217] Rinaldi-Carmona M Barth F Millan J Derocq JM CasellasP Congy C Oustric D Sarran M Bouaboula M CalandraB Portier M Shire D Breliere JC Le Fur GL JPharmacol Exp Ther 1998 284 644

[186] Wilstermann JM and Berggren AIK World PatentApplication 2003 WO03051850

[187] Berggren AIK Bostrom SJ Elebring ST Greasley PTerricabra E Wilstermann JM World Patent Application 2003WO03051851

[218] Barth F Millan J Casellas P Oustric D Sarran M RinaldiM World Patent Application 1997 WO9721682

[219] Griffin G Wray EJ Tao Q McAllister SD Rorrer WKAung MM Martin BR Abood ME Eur J Pharmacol 1999377 117

[188] Finke PE Meurer LC Debenham JS Toupence RBWalsh TE World Patent Application 2003 WO03082191

[189] Barth F Martinez S Rinaldi-Carmona M World PatentApplication 2003 WO03084930

[220] Portier M Rinaldi-Carmona M Pecceu F Combes T Poinot-Chazel C Calandra B Barth F Le Fur G Casellas P JPharmacol Exp Ther 1999 288 582[190] Barth F Martinez S Rinaldi-Carmona M World Patent

Application 2003 WO03084943 [221] Bouaboula M Desnoyer N Carayon P Combes T CasellasP Mol Pharmacol 1999 55 473[191] Kopka I E Li B Hagmann WK World Patent Application

2004 WO04029204 [222] Rhee M-H and Kim S-K J Vet Sci 2002 3 179[192] Hagmann WK Lin LS Shah SK Guthikonda RN Qi H

Chang LL Liu P Armstrong HM World Patent Application2003 WO03077847

[223] Bouaboula M Dussossoy D Casellas P J Biol Chem 1999274 20397

[224] Shire D Calandra B Bouaboula M Barth F Rinaldi-Carmona M Casellas P Ferrara P Life Sci 1999 65 627[193] Hagmann WK Lin LS Shah SK Goulet MT Jewell JP

World Patent Application 2003 WO03082190 [225] Gouldson P Calandra B Legoux P Kerneis A Rinaldi-Carmona M Barth F Le Fur G Ferrara P Shire D Eur JPharmacol 2000 401 17

[194] Castonguay LA Hagmann WK Lin LS Shah SK WorldPatent Application 2003 WO03086288

[195] Hagmann WK Lin LS Shah SK World Patent Application2003 WO03087037

[226] Feng W and Song ZH Biochem Pharmacol 2003 63 1077[227] Barth F Millan J Oustric D Rinaldi M Vernhet M World

Patent Application 2001 WO0132629[196] Berggren AIK Bostrom SJ Cheng L Elebring STGreasley P Nagard M Wilsterlmann JM Terricabras EWorld Patent Application 2004 WO04058249

[228] Mussinu JM Ruiu S Mule AC Pau A Carai MA LorigaG Murineddu G Pinna GA Bioorg Med Chem 2003 11251[197] Guba W Haap W Marty HP Narquizian R United States

Patent Application 2004 US 20040147572 [229] Xiang JN Elliott JD Atkinson ST Christensen SB WorldPatent Application 1998 WO9831227[198] Jagerovic N Hernandez Folgado L Alkorta I Goya P

Navarro M Serrano A de Fonseca FR Dannert MTDannert MT Alsasua A Suardiaz M Pascual D MartinMI J Med Chem 2004 47 2939

[230] Xiang JN Atkinson ST Wang DY Lee J Lee JCSiegfried B 216th ACS meeting Boston (MA) USA 23-27 Aug1998

[199] Jagerovic N Goya Laza MP Hernandez Folgado L AlcortaOsoro IJ Martin Fontelles MI Suardiaz Garcia MLDannert MT World Patent Application 2003 WO03082833

[231] Inaba T Kaya T Iwamura H United States Patent 2003 US6509352 B1

[232] Iwamura H Suzuki H Ueda Y Kaya T Inaba T JPharmacol Exp Ther 2001 296 420[200] Lange JHM Kruse CG Herremans AHJ VanStuivenberg

HH Dijksman JAR McCreary AC World PatentApplication 2003 WO03078413

[233] httpwwwjticojpJTI_EIR02PL20030212_Epdf (accessed02162004)

[201] Berggren AIK Bostrom SJ Elebring ST Fallefors LWilstermann JM Greasley P World Patent Application 2004WO04058255

[234] Zygmunt PM Andersson DA Houmlgestaumltt ED J Neurosci2002 22 4720

[235] Di Marzo V De Petrocellis L Fezza F Ligresti A BisognoT Prostaglandins Leukot Essent Fatty Acids 2002 66 377[202] Toupence RB Debenham JS Goulet MT Madsen-Duggan

CB Walsh TE Shah SK World Patent Application 2004WO04012671

[236] Wagner JA Varga K Jarai Z Kunos G Hypertension 199933 429

[203] Alanine A Bleicher K Guba W Haap W Kube DLuebbers T Plancher J-M Roche O Rogers-Evans MSchneider G Zuegge J World Patent Application 2004WO04013120

[237] Mukhopadhyay S Chapnick BM Howlett AC Am JPhysiol-Heart Circul Physiol 2002 282 H2046

[238] Jarai Z Wagner JA Varga K Lake KD Compton DR Martin BR Zimmer AM Bonner TI Buckley NE MezeyE Razdan RK Zimmer A Kunos G Proc Natl Acad Sci US A 1999 96 14136

[204] Griffith DA United States Patent Application 2004 US20040092520

[205] Griffith DA United States Patent Application 2004 US20040157839

[239] Kunos G Martin B Razdan R World Patent Application 2001WO0103690

[206] Griffith DA United States Patent Application 2004 US20040157838

[240] Offertaler L Mo F-M Batkai S Liu J Begg M RazdanRK Martin BR Bukoski RD Kunos G Mol Pharmacol2003 63 699[207] Bayewitch M Avidor-Reiss T Levy R Barg J Mechoulam

R Vogel Zvi FEBS letters 1995 375 143 [241] Begg M Mo F-M Offertaler L Batkai S Pacher PRazdan R K Lovinger DM Kunos G J Biol Chem 2003278 46188

[208] Bayewitch M Rhee M-H Avidor-Reiss T Breuer AMechoulam R Vogel Z J Biol Chem 1996 271 9903

[209] Govaerts SJ Hermans E Lambert DM Eur J Pharm Sci2004 23 233

[242] Ho WSV Hiley CR Brit J Pharmacol 2003 138 1320[243] Ho WSV Hiley CR J Pharm Pharmacol 2004 56 869

[210] DAmbra TE Estep KG Bell MR Eissenstat MA JosefKA Ward SJ Haycock DA Baizman ER Casiano FMBeglin NC J Med Chem 1992 35 124

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[245] Batkai S Pacher P Jarai J Wagner JA Kunos G Am JPhysiol-Heart Circul Physiol 2004 287 H595[211] Showalter VM Compton DR Martin BR Abood ME J

Pharmacol Exp Ther 1996 278 989

1394 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

[246] Kunos G World Patent Application 2003 WO03015700 [257] Haller J Varga B Ledent C Freund TF Behav Pharmacol2004 15 299[247] Franklin A and Stella N Eur J Pharmacol 2003 474 195

[248] Walter L Franklin A Witting A Wade C Xie Y KunosG Mackie K Stella N J Neurosci 2003 23 1398

[258] Pistis M Perra S Pillolla G Melis M Gessa GL MuntoniAL Neuropharmacology 2004 46 115

[249] Di Marzo V Breivogel C S Tao Q Bridgen DT RazdanRK Zimmer AM Zimmer A Martin BR J Neurochem2000 75 2434

[259] Ralevic V and Kendall DA Eur J Pharmacol 2001 418 117[260] Duncan M Kendall DA Ralevic V Brit J Pharmacol 2003

138 214P[250] Breivogel CS Griffin G Di Marzo V Martin BR Mol

Pharmacol 2001 60 155[261] Duncan M Millns P Smart D Wright JE Kendall DA

Ralevic V Brit J Pharmacol 2004 142 509[251] Monory K Tzavara ET Lexime J Ledent C Parmentier

M Borsodi A Hanoune J Biochem Bioph Res Commun2002 292 231

[262] Duncan M Kendall DA Ralevic V J Pharmacol Exp Ther2004 311 411

[263] Tao Q and Abood ME J Pharmacol Exp Ther 1998 285651[252] Muthane U Ramsay KA Jiang H Jackson-Lewis V

Donaldson D Fernando S Ferreira M Przedborki S ExpNeurol 1994 126 195

[264] Lambert DM DiPaolo FG Sonveaux P Kanyonyo MGovaerts SJ Hermans E Bueb J-L Delzenne NMTschirhart EJ Biochim Biophys Acta 1999 1440 266[253] Hajos N Freund TF Chem Phys Lipids 2002 121 73

[254] Hajos N Ledent C Freund TF Neuroscience 2001 106 1 [265] Calignano A La Rana G Giuffrida A Piomelli D Nature1998 394 277[255] Hajos N Freund TF Neuropharmacology 2002 43 503

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[266] Calignano A La Rana G Piomelli D Eur J Pharmacol 2001419 191

Page 2: Current Knowledge on the Antagonists and Inverse Agonists of

1362 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

first review only devoted to cannabinoids antagonists waspublished by Barth and Rinaldi-Carmona back in 1999 [32]

hypomotility and hypothermia upon administration in mice[33] An antagonist should reverse the effect obtained withthe agonistThe aim of this paper will be to review the cannabinoid

antagonists and inverse agonists with a particular emphasison the newest compounds Nevertheless as a matter ofcompleteness this review will also cover the earliestdevelopment in the field of cannabinoid antagonists Furtherthe great interest of the pharmaceutical companies for thefield led to the publication of a great amount of patentsTherefore this paper will cover in addition to the scientificpapers the patents covering the cannabinoid antagonists

Several ex-vivo assays are also used to characterise theagonists or antagonists properties of the cannabinoidreceptors ligands The inhibition of the electrically evokedcontractions in the guinea pig ileum and in the mouse vasdeferens are among the most widely used ex-vivo assays thelatter being more sensitive to cannabinoids [34]

However it is quite difficult in these in-vivo and ex-vivoassays to distinguish between antagonists and inverse-agonist effectsOne of the most used in-vivo assay to characterise the

pharmacological properties of the cannabinoids is thecannabinoid tetrad of effects An agonist of the CB1cannabinoid receptor must induce analgesia catalepsia

Therefore several in-vitro assays are currently used toexplore the functionality of known ligands cAMPquantification is one of the most widely used methods based

Table 1 CB1 Cannabinoid Receptor Antagonists 1 Tetrahydrocannabinol and Cannabidiol Derivatives

Some ∆8-Tetrahydrocannabinol Derivatives Possessing a Rigidified Side Chain The Affinity for the CB1 Receptor (Radioligand Cells) and the Function(Assay Used) are Given

O

OH

R

Cpd ndeg R= CB1 (Ki) Function

O-823 1

CN

077 nM([3H]-CP-55940 rat brain) c

bull inactive in Tetrad Test (up to 30 mgkg mouse) e

bull partial agonist (mouse vas deferens) c

bull antagonist (guinea pig myenteric plexus) c

bull antagonist ([35 S]-GTPγS rat cerebella) b

O-1184 2

N3

524 nM([3H]-CP-55940 hCB1-CHO

cells) a

bull partial agonist in Tetrad Test (mouse) e

bull antagonist (guinea pig myenteric plexus) d

bull partial agonist (cAMP hCB1-CHO cells) a

bull antagonist ([35 S]-GTPγS rat cerebella) b

O-584 3 426 nM([3H]-CP-55940 hCB1-CHO

cells) a

bull agonist (cAMP hCB1-CHO cells) a

bull antagonist ([35 S]-GTPγS rat cerebella) b

O-806 4

Br

12 nM([3H]-CP-55940 rat brain)

ebull partial agonist in Tetrad Test (mouse) e

bull antagonist ([35 S]-GTPγS rat cerebella) b

O-1176 5

NCS

115 nM([3H]-CP-55940 rat brain) e

bull inactive in Tetrad Test (up to 30 mgkg mouse) e

bull antagonist ([35 S]-GTPγS rat cerebella) b

O-1238 6 N3 354 nM([3H]-CP-55940 hCB1-CHO

cells) a

bull partial agonist ([35 S]-GTPγS rat cerebella) b

bull agonist (cAMP hCB1-CHO cells) a

7HN

S

O

O30 nM f bull agonist in Tetrad Test (mouse) f

O-2050 8HN

S

O

O25 nM f bull no effect per se in Tetrad Test (mouse)f

bull antagonist (mouse vas deferens) f

a[39] b[38] c[36] d[37] e[40] f[41]

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1363

on the negative coupling of cannabinoid receptors toadenylyl cyclase The binding of an agonist will produce adecrease in cAMP production which can be measured eitherdirectly (EIA) or through a gene-reporter system (fireflyluciferase) However due to the existence of a dual-couplingfor the CB1 cannabinoid receptor the [35S]-GTPγ S assayshould be preferred It is based on the property shared by allthe GPCRs to bind to a GTP molecule upon activation byan agonist Therefore the binding of an agonist will increasethe [35S]-GTPγ S a radiolabelled non-hydrolysable analogueof GTP binding [35] These assays allow to distinguishbetween full agonists (positive intrinsic activity) partialagonists neutral antagonists (no intrinsic activity) andinverse agonists (negative intrinsic activity)

antinociception nor hypothermia in mice and lacks ofagonist effects in mouse vas deferens preparation In thisassay it behaved as an antagonist devoid of inverse agonistproperties

Recently Thomas et al reported the synthesis andcharacterisation of O-2654 (9) (Fig 1) obtained bymodifying the structure of cannabidiol (or cbd) (10) a nonpsychoactive cannabinoid [42] This compound unlikecannabidiol binds to the CB1 receptor with a Ki value of114 nM against [3H]-CP-55940 on mouse brainmembranes In the mouse vas deferens model O-2654antagonises the WIN-55212-2 inhibition of current-inducedcontractions causing a rightward shift in the logconcentration response curve of the agonist (KB = 857 nM)Further in the vas deferens model O-2654 behaves as aneutral antagonist However this neutral antagonism has tobe further confirmed using other models

II CB1 CANNABINOID RECEPTOR ANTAGONISTSAND INVERSE AGONISTS

OH

OH

N3

O-2654 (9)

Compounds having antagonist or inverse agonistproperties at the CB1 cannabinoid receptor are reviewed inthis part of the paper They are classified depending on theirchemical structures

1 Tetrahydrocannabinol and Cannabidiol Derivatives

The first attempts to obtain cannabinoid ligands havingantagonist properties were conducted using the tricyclicstructure of classical cannabinoids such as ∆9-tetrahydrocannabinol (∆9-THC) as a scaffold These earlyresearches have been previously reviewed by Barth andRinaldi-Carmona [32] The most promising modulationsinvolved the side-chain of ∆8-tetrahydrocannabinol (∆8-THC) as it is the case in O-823 (1) (Table 1) Thiscompound (Ki = 077 nM) acts as a partial agonist in mousevas deferens preparations but as an antagonist in guinea-pigmyenteric plexus preparations [36] Ross and co-workers[37] showed that O-1184 (2) binds to the CB1 receptor (Ki =285 nM) and as O-823 possesses antagonist properties inthe myenteric plexus-longitudinal muscles O-823 and O-1184 were shown to act as surmountable antagonists in a[35S]-GTPγ S assay with KB values of 485 and 297 nMrespectively against CP-55940 in rat cerebellar membranes[38] Three other ∆8-tetrahydrocannabinol-3rsquo-ynylderivatives O-584 (3) O-806 (4) and O-1176 (5) alsobehaved as antagonists However in a cAMP productionassay conducted in hCB1 transfected CHO cells O-1184behaved as an agonist equipotent to CP-55940 [39] Takentogether these examples illustrate that the introduction of anacetylenic moiety into the side chain of ∆8-THC affects theactivation of the receptor more than the affinity of thesederivatives Several of these compounds were tested in-vivoin the cannabinoid tetrad test by Martin and co-workers [40]For instance O-823 which was inactive in the tetrad actedas an antagonist in the [35S]-GTPγ S assay [38]

Fig (1) CB1 cannabinoid receptor antagonists 1Tetrahydrocannabinol and cannabidiol derivatives Chemicalstructure of (-)-6-azidohex-2-yne-cannabidiol (O-2654 9)

2 Aminoalkylindole Derivatives

This family of compounds was introduced by Sterlingrsquosresearchers in the early nineties with a derivative of the anti-inflammatory drug pravadoline called WIN-55212 Albeitthis compound acts as an agonist some related compoundsshowed interesting antagonist properties in the mouse vasdeferens assay by dose-dependently antagonising ∆9-THCand levonantradol effects This is the case for instance for

N

O OMe

N

O

X

N

O

N

O

WIN-56098 (11) X=Br WIN-54461 (12)X=I AM630 (13)

Fig (2) CB1 cannabinoid receptor antagonists 2Aminoalkylindole derivatives Structures of WIN-56098 (11 )WIN-54461 (12 ) and AM630 (13 ) three aminoalkylindolederivatives acting as CB1 cannabinoid receptor antagonists

More recently Martin et al described three newcompounds possessing an alkyl sulfonamide at the end of thealkyne side chain [41] The ethyl (7) (Ki = 30 nM) andbutyl (Ki = 70 nM) derivatives were shown to behave asagonists in the cannabinoid tetrad while the methylderivative (8) (Ki = 25 nM) acted as a silent antagonistThis compound O-2050 does not induce neither

1364 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

WIN-56098 (11) and WIN-54461 (12) the latter being abromo derivative of pravadoline (Fig 2) These twocompounds however have a low affinity for the cannabinoidreceptor (IC50 gt 500 nM) [4344]

cAMP accumulation in hCB1-CHO cells with an IC50 valueof 56 nM SR141716A is able after ip or poadministration to inhibit the [3H]-CP-55940 binding tomice brain measured ex-vivo with ED50

values around 2mgkg [51] In addition to the inhibition of the classicalcannabinoid tetrad effects ndash hypothermia ring immobilityanalgesia and hypolocomotion ndash already shown by Rinaldi-Carmona et al in their first report [50] SR141716A wasshown to antagonise other in-vivo effects of cannabinoidagonists For instance the agonist-induced hypotension andbradycardia in mice are abolished by SR141716A [52] aswell as the antihyperalgesic effects of the cannabinoidagonists in a neuropathic model of pain in rat [53] This isalso the case for the behavioral effects of agonists treated rats[54] or the cannabinoid tetrad effects induced by ∆9-THC inmice [55]

In 1995 Pertwee et al [45] described the effect ofAM630 (13) another close analogue of pravadoline Thiscompound behaved as an antagonist in a [35S]-GTPγ S assayon mouse brain preparations antagonising WIN-55212-2-induced [35S]-GTPγ S binding [46] and as an inverseagonist on hCB1-CHO cells [47] (EC50 = 09 microM)However Ross et al found a weak partial agonist activityfor this compound as it decreases cAMP production byhCB1-CHO cells [48] AM630 was subsequentlycharacterised as a CB2 ligand (see the CB2 section) Thepharmacological properties of the aminoalkylindole familywere reviewed by John Huffman [49]

3 Diarylpyrazole DerivativesDespite various papers described SR141716A as an

antagonist [56-58] today this compound is considered to actas an inverse agonist based on [35S]-GTPγ S [59-62] andcAMP accumulation assays [62-64] (Table 2) An interestingreview dealing with inverse agonism at the cannabinoidreceptors and mostly with SR141716A effects was recentlypublished by Roger Pertwee [65]

The lead of this class of compounds SR141716A (14)was introduced by Sanofi back in 1994 [50] This compoundwas shown by Rinaldi-Carmona and co-workers to be ahighly selective CB1 ligand with Ki values of 56 nM for thehCB1 and over 1000 nM for the hCB2 receptors expressed inCHO cells ([3H]-CP-55940) However more recentlyevidences appeared showing that SR141716A binds toother(s) receptor(s) described as anandamide andorcannabinoid receptors Thus it is possible that some of thein-vivo effects caused by this compound are at least notsolely CB1 mediated (see the fourth section of this paper)

In 1998 Pan et al [66] demonstrated that the lysineresidue K328 (192) located in the third transmembranedomain (TMH3) of the hCB1 receptor is a key residue forthe inverse agonist action of SR141716A (Table 3) Theyshowed that SR141716A enhances calcium current in hCB1transfected neurons but not in K(192)A mutant receptortransfected neurons However SR141716A still antagonizedWIN-55212-2 inhibition of calcium currents proving that itis able to bind to the mutated receptor Later on Hurst et al[67] demonstrated using molecular modeling techniques aswell as in-vitro experiments that lysine residue K328(192)is a direct interaction site for hydrogen bonding with the C3substituent of SR141716A in CB1 receptor Binding results

In a mouse vas deferens preparation SR141716A causesa rightward shift of the CP-55940 concentration-responsecurve behaving as a competitive antagonist having a pA2value of 798 Furthermore in the cAMP accumulationmodel SR141716A produces no effect by itself butantagonizes the CP-55940 inhibition of forskolin-induced

Table 2 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

In-Vitro Functional Characterisation of SR141716A (14) The Assay Used the Cell Type and the Obtained Effect are Given

Cell type Assay Effect Function References

hCB1-CHO cells cAMP accumulation no effect by itself antagonist [50]

hCB1-CHO cells cAMP accumulation uarr [cAMP] inverse agonist [64]

hCB1-CHO cells [35 S]-GTPγS darr[35 S]-GTPγS binding inverse agonist [59]

hCB1-neurons Ca2+ currents uarr Ca2+ currents inverse agonist [66]

rCB1 (rat cerebella) [35 S]-GTPγS No effect by itself antagonist [56]

rCB1 (rat cerebella) [35 S]-GTPγS No effect by itself antagonist [57]

rCB1 (rat cerebella) [35 S]-GTPγS No effect by itself antagonist [58]

rCB1 (rat brain) [35 S]-GTPγS darr[35 S]-GTPγS binding inverse agonist [62]

rCB1 (rat cerebella) [35 S]-GTPγS darr[35 S]-GTPγS binding inverse agonist [60]

rCB1 (rat cerebella) [35 S]-GTPγS darr[35 S]-GTPγS binding inverse agonist [61]

rCB1 (rat brain) cAMP accumulation uarr [cAMP] inverse agonist [63]

mCB1 (mouse brain) cAMP accumulation uarr [cAMP] inverse agonist [62]

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1365

obtained using HEK293 cells transfected with either theK328A mutant or the wild type receptor remarkablyconfirmed the modeling results (Kd values of 396 and 23nM respectively) Interestingly a vinyl-cyclohexylSR141716A derivative VCHSR1 (15) (Table 4) lacking ofhydrogen bonding sites in C3 position is not affected bythis CB1 receptor mutation as its affinity remains unchangedwith Ki values of 31 and 35 nM for the wild-type andK328A receptors respectively Four additional compoundsCHASR1 (16) CHMSR1 (17) VPSR1 (18) and PIMSR(19) differing by their potential to form hydrogen bondswere evaluated in affinity and functional assays The resultsfurther confirm the crucial interaction between the C-3carboxamide oxygen and residue K328 to obtain an inverseagonist effect as highlighted in Table 4 [68] However it hasto be said that this lysine residue is also crucial for agonistbinding (CP-55940) andor receptor activation (WIN-55212-2) [69 70]

binding of SR141716A In contrast the substitution oftyrosine by isoleucine (Y275I) resulted in the loss of ligandrecognition Calculation studies revealed that while theY539F mutant is very similar to the wild type receptor theY539I mutant shows topology changes in the 3-4-5transmembrane region This region is considered to becrucial for agonistantagonist binding at CB1 receptor sincethe previous report by Shire et al on CB1CB2 receptorchimeras [72] In contrast the first and third extracellular(EC1 and EC3) loops of the hCB1 receptor are not essentialfor the binding of SR141716A as illustrated by Murphy etal The authors constructed several receptors mutated in theirEC1 or EC3 and none of the tested mutations affected theSR141716A binding [73] McAllister et al due the highlyaromatic nature of SR141716A further explored thehypothesis that an aromatic microdomain comprised intransmembrane helix 3-4-5-6 is the SR141716A bindingsite The modelling and mutation studies undertakensuggested to the authors that this aromatic microdomaincomprised of F336 W464 Y539 W543 and W648should represent the binding site of SR141716A Moreoverthey identified F336 and W543 as direct interaction sitesfor SR141716A [74] Residue F336 was further shown bythe same group to be a key residue for both ligand binding(WIN-55212-2 and SR141716A) and receptor activationMutation of phenylalanine 336(201) to alanine resulted in

Several other studies were undertaken to determine thecritical residues for the binding of cannabinoid compoundsUnfortunately in most of the studies SR141716A was notused However McAllister et al [71] using modeling toolsand mutagenesis explored the importance of aromaticity inposition 539(275) in the CB1 receptor The substitution oftyrosine by phenylalanine (Y275F) has no effect on the

Table 3 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Summary of the Reported CB1 Cannabinoid Receptor Single Point Mutations for which Pharmacological Data Concerning the SR141716A (14) areAvailable

Mutation Species Effect References

S114A a human no significant effect [73]

S115A a human no significant effect [73]

D250(163)N human no significant effect [263]

D250(163)E human no significant effect [263]

H181A b human no significant effect [73]

R182A b human no significant effect [73]

K183A b human no significant effect [73]

D184A b human no significant effect [73]

V324(188)A human no significant effect [73]

F325(189)A human no significant effect [73]

F325(190)A mouse no significant effect [74]

K328(192)A human loss of function [66]

K328(192)A human reduction of affinity (20 fold) [67]

F336(201)A mouse reduction of affinity (3 fold) [74]

Y539(275)F human no significant effect [71]

Y539(275)I human loss of affinity [71]

W543(280)A mouse loss of affinity [74]

W648(357)A mouse reduction of affinity (7fold) [74]

a N-terminus b First extra-cellular loop

1366 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 4 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives Five SR141716A (14) DerivativesIllustrating the Importance of the C-3 Carboxamide Oxygen in the SR141716A Inverse Agonism

The structure affinity ([3H]-SR141716A hCB1-HEK293 cells) and function (Ca++ currents) are given for each compound

NN

R

Cl

Cl

Cl

Cpd ndeg R= Affinity Function References

SR141716A 14

O

NHN

Kd=23nM Inverse agonist [67]

VCHSR1 15 Ki=313nM Neutral antagonist [67]

CHASR1 16

O

NH

Ki=17nM Inverse agonist [68]

CHMSR1 17

O

N

Ki=29nM Inverse agonist [68]

VPSR1 18

N

Ki=261nM Neutral antagonist [68]

PIMSR 19

NN

Ki=67nM Neutral antagonist [68]

an increased constitutive activity of the receptor asdemonstated by [35S]-GTPγ S binding [75]

1996 It binds with high affinity to rat brain synaptosomes(Kd=061nM) It is competitively displaced by knowncannabinoids like CP-55940 or WIN-55212-2 Usingautoradiography its rat brain distribution is similar to [3H]-CP-55940 one [77] It is now a commercially available andwidely used radioligand for competition studies

A new 3D model of the CB1 cannabinoid receptor basedon the X-ray structure of the bovine rhodopsin was recentlydeveloped by Salo and co-workers [76] It would be veryinteresting to see whether or not the results obtained withprevious models are confirmed using this new model Forinstance the lysine K328 appeared as a key residue in thismodel too

Interestingly other SR141716A radiolabeled derivativeswere synthesised as radioimaging tools among them [123I]-AM251 (20) [7879] [123I]-AM281 (21) [80-82] and [18F]-SR144385 (22) [8384] for Positon Emission Tomographyor Single Photon Emission Computed Tomography Indeedcannabinoid antagonists are a much more useful tool for

The tritiated analogue of SR141716A the [3H]-SR141716A was described by Rinaldi-Carmona et al in

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1367

human radioimaging applications than agonists as they aredevoid of cannabinoid-like effects For instance Berding etal very recently reported the use of [123I]-AM281 for SinglePhoton Emission Computed Tomography imaging of theCB1 cannabinoid receptor in six human patients [85] Inorder to further optimize the brain uptake of suchSR141716A derivatives Sanofi researchers synthesised twomethoxylated SR141716A analogues using 11C as PETtracer [86] These two compounds SR149080 (24) andSR149568 (25) possess high affinity with Ki values of 15and 38 nM respectively and selectivity for the CB1 receptoras well as an improved penetration in the brain evaluated bymeasuring the radioactivity present in various brain regionsafter tail vein injection in CD-1 mice Some of therepresentative radiolabelled SR141716A derivatives aresummarised in Table 5

Colombo et al using Wistar rats [92] Simiand et alobserved that SR141716A selectively reduces sweet foodintake in primates [93] However several authors [94-96]showed that high palatability of food is not necessary toobserve a SR141716A-induced anorectic effect at doses thatdo not cause major behavioural alterations or reduced waterintake Interestingly Gomez et al demonstrated theimplication of the peripheral CB1 receptors on themodulation of feeding and therefore the possible role ofthese receptors on the SR141716A influence on food intake[97] In addition to its effects on food consumptionSR141716A seems to be able to lower the hyperglycemiathe hyperinsulinemia as well as the insulin resistance indiet-induced obese (DIO) mice In the same DIO mice adecrease in adiposity was also observed after treatment [98]Bensaid et al using another model of obesity the obese fafarats observed that SR141716A increases mRNA expressionof Acrp30 or adiponectin a plasmatic protein exclusivelysecreted by adipose tissue through a CB1-mediatedpathway Inductions of free fatty acid oxidation body weightreduction and hyperinsulinemia decrease are some of theknown physiological actions of this protein Thus along theauthors an enhanced expression of Acrp30 followingSR141716A administration could be responsible for themetabolic effects of the compound leading to body weightreduction [99] Vickers et al using the same model (ie fafarats) showed that SR141716A significantly decreases foodconsumption and weight gain in both the obese fafa rats andthe lean Zucker rats [100] This decrease was greater in thefafa group and reversible upon SR141716A withdrawal

From a more therapeutic point of view SR141716A(rimonabant Acompliareg) is one of the promising agents totreat obesity [87] It is currently in Phase III clinical trials forthe treatment and prevention of obesity Final results areexpected for this year and FDA application filling for 2005[88] Preliminary data based on a one year treatment withrimonabant (RIO-Lipids study 1036 patients) showed a 5weight loss in 72 of the treated patients [8990] Severalreports were published before clinical trials aiming todemonstrate the anti-obesity properties of this compoundThe first report by Sanofi Recherche published in 1997described the selective inhibition of sucrose intake in ratsupon SR141716A treatment (03-3 mgkg) [91] Theanorectic and weight loss effects were firstly published by

Table 5 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structure and Affinity of SR141716A Radiolabeled Derivatives Developed as Potential Radio-Imaging Tools

NN

R3

O

NH

Cl

R4

R1

R2

Cpd ndeg R1 R2 R3 R4 Affinity CB1 References

[123I]-AM251 20 123I piperidinyl CH3 Cl Ki=25 nMa [78]

[123I]-AM281 21 123I morpholinyl CH3 Cl Ki=14 nMb [80]

[18 F]-SR144385 22 Cl piperidinyl CH2-18 F Cl IC50 =29 nMa [83]

[18 F]-SR147963 23 Cl morpholinyl CH2-18 F Cl IC50 =120 nM [84]

[11 C]-SR149080 24 O-11 CH3 piperidinyl CH3 Cl IC50 =15 nMa [86]

[11 C]-SR149568 25 O-11 CH3 morpholinyl CH3 Cl IC50 =38 nMa [86]

[18 F]-NIDA-42033 26 O-CH3 piperidinyl 18 F H Ki=18 nMc [155156]

27 O-11 CH3 piperidinyl CH3 H Ki=8nMc [155157]

arat brain homogenates [3H]-CP-55940bmouse cerebellum homogenates [3H]-SR141716crat brain homogenates [3H]-AM251

1368 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Another recent study by Higgs and co-workers furtherhighlighted the role of endocannabinoids in food taste-perception and the reduction of orosensory reward of sucrosein SR141716A treated rats [101] As it was expectedSR141716A induced effects on food consumption are absentsin CB1

-- mice [102] Very recently Cota et al obtainedresults showing that endocannabinoid system modulateshomeostasis via a dual mechanism it regulates at a centrallevel food intake while it blocks at the periphery lipogeneticprocesses [103] Moreover Cani et al showed that ghrelin(an orexigenic peptide) plasma levels are significantlyreduced 45 minutes after SR141716A administration(5mgkg ip) to fasted rats in accordance with the rapiddecrease of food intake measured by the authors It istherefore likely that SR141716 effects on body weight aredue to a conjunction of central and peripheral actions [104]

cerebrospinal fluid from patients with schizophrenia [116]Later De Marchi et al measured higher amounts ofanandamide in blood of schizophrenic patients compared tocontrols [117] On the other hand Dean et al obtainedelevated [3H]-CP-55940 binding in the dorsolateralprefrontal cortex of patients suffering from schizophrenia ascompared to controls [118] Zavitsanou using [3H]-SR141716A found elevated binding in the anterior cingulatecortex of subjects with schizophrenia [119] RecentlyMeltzer and collaborators published the results of a trialconducted to evaluate the potential of four new compoundsin treating schizophrenia and schizoaffective disorders [120]SR141716A (20 mgday) was one of the compoundsevaluated during a six weeks study The authors found noeffect of the CB1 cannabinoid receptor antagonist inimproving patientrsquos schizophrenia (72 subjects)

In addition to the anti-obesity potential the ability ofSR141716A to reduce alcohol and tobacco consumption arecurrently investigated in phase III clinical trials The effect ofSR141716A on alcohol consumption in rats was reported bythe GianLuigi Gessa team [105-108] and by Gallate et al[109] The inhibition of alcohol and nicotine induceddopamine release by SR141716A (1-3 mgkg rats) wasreported by Cohen and co-workers using a brainmicrodialysis device [110] Concerning the smokingcessation preliminary results of a clinical trial (360 subjects40 mg SR141716A) showed an increased abstinence ofsmoking [111] More recent results from the STRATUS-USstudy (787 patients) were reported 362 of patientsreceiving SR141716A (20 mgday) quit smoking against206 in the placebo group [90] Recently Le Fol andGoldberg reviewed the development of cannabinoid CB1antagonists as a new class of therapeutic agents for drugsaddictions [112]

It is known since the early nineties that theadministration of cannabinoid agonists impairs memory inrodents (for a review see Castellano et al [121]) Thusadministration of a cannabinoid antagonist was expected tosomehow improve memory [122123] However dependingon the authors SR141716A when administered alone wasreported to impair (5-10 mgkg im) [124] to have no effect(1-32 mgkg ip) [125] or to improve memory (3 mgkgip) [126] In a more recent paper Wolff and Leandershowed that SR141716A (1mgkg ip) improves memoryin rats by apparently enhancing the consolidation processesof memory [127] Further they found that at higher doses (3mgkg) this effect was lost The discrepancy in the resultsreported in the literature could be ascribed either to thedifferences in the tests used or in the doses administeredFurther experiments are needed to assess whether or not acannabinoid antagonist could be helpful in memory diseases

To conclude several patents were taken by Sanoficoncerning the therapeutic applications of their leadcompound among them being anti-obesity smokingcessation neuroinflammatory diseases and anti-diarrhoeaOne of the last patents to our knowledge concerns thetreatment of sexual dysfunctions with a cannabinoidantagonist such as SR141716A [128] However da Silva etal showed that even if this compound (2 mgkg ip)enhances the effects of apomorphine (20-80 microgkg) it has noeffect alone on penile erection [129] Meanwhile Melis et al

Since the late sixties concerns exist on the associationbetween cannabis use and schizophrenia Recent studiesshowed an association between cannabis use and an increasedrisk of developing schizophrenia [113 114] Moreover apossible role of the endocannabinoid system in schizophreniahas been suggested since its pharmacological characterisation[115] Several experimental data obtained on human subjectstend to confirm such hypothesis On the one hand Lewekeet al found elevated levels of endocannabinoids in

NN

O

NH

N

Br Cl

Cl

NN

O

NH

N

Br Cl

Cl

OH

28 29

Fig (3) CB1 cannabinoid receptor antagonists 3 Diarylpyrazole derivatives The chemical structures of 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-piperidine-carboxamide (SR147778 28 ) and 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-(4-hydroxypiperidine)-carboxamide (29 ) described by Rinaldi-Carmona et al

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1369

reported an increased rate of penile erection after directinjection of SR141716A in the paraventricular nucleus of thehypothalamus [130] This effect was dose dependent and asignificant effect was obtained with a dose of 1microgkg

AM251 in mice at a same dose range (10-30 mgkg ip) thanthe anorectic effects [143] These effects are reversed byadministration of CP-55940 and are absent in CB1

-- miceproving the implication of the CB1 receptor in theantidepressant-like effect of AM251 In 2004 Liao et alreported that AM251 was able to displace the binding of[3H]-batrachotoxin A from its binding site on sodiumchannels in mice brain synaptic preparations [144] Theyobtained an IC50 value of 112 microM and a competitivemechanism of action The authors suggested that AM251 iscapable of reducing neuronal excitability through blockade ofvoltage-sensitive sodium channels in brain

From the clinical trials involving the SR141716A themost frequently reported side-effects are nausea dizziness anddiarrhoea Depression and anxiety were not higher than inthe placebo groups Regarding one of the known side effectsof SR141716A administration the enhancement of intestinalmotility Carai et al reported recently that chronicadministration of the inverse agonists to mice induced atolerance to this prokinetic effect [131] This is not the firstreport of tolerance onset after administration of SR141716A[132] however few is known on the mechanismsresponsible for that phenomenon

Wiley and collaborators reported in 2001 a studydescribing new structure-activity relationships using thepyrazole nucleus as a central scaffold [145] Starting fromSR141716A structure they alternatively substituted one ofthe four substituents while retaining the others bysubstituents known to impart agonist activity in classicalcannabinoids One of the authors expectations was todetermine which positions are responsible respectively forthe antagonism and for the affinity of SR141716A Theaffinity of thirty compounds was assessed previous to in-vivoevaluation of their function in mice using the spontaneousactivity the tail-flick and the rectal temperature assays Theauthors showed that phenyl group in position 5 is critical foraffinity as compound O-1559 (30) lacking this phenyl has adecreased affinity (Table 6) This was already shown by Lanand co-workers [136] along with the need of a substituent inpara position on the phenyl Thomas et al previouslyshowed that this substituent could be a bromine or an iodineatom [146] However an alkyl chain is also tolerated as it isthe case in compounds O-1302 (31) O-1691 (32) or O-1704(33) All these compounds antagonise the anti-nociceptiveand hypothermic effects of ∆9-THC The authors suggestedthat the 5-substituent of pyrazoles is involved in receptorrecognition and antagonism The modulations of thesubstitution pattern of the phenyl in position 1 demonstratethat the 24-di-chloro substitution is the preferred one for theaffinity as well as for the activity Several compoundssupport this assertion Thomas et al showed that additionalhalogens result in a decreased affinity as in compounds 6-I-SR141716A (34) or 4rsquo6-di-I-SR141716A (35) (Ki values of166 and 126 nM respectively) [146] Suppression of the twochlorines (O-1300 39) or replacement of these two chlorinesby an alkyl chain like in O-1254 (40) and O-1255 (41) ledto less active compounds with Ki values ranging from 150to 430 nM However O-1254 and O-1255 behaved asantagonists in a [35S]-GTPγ S assay [61] Lan et al [136]and Wiley et al [145] also modified the substitution inposition 3 Replacement of the amido piperidinyl substituentby alkyl amides as in O-1269 (42) or O-1270 (43) etherslike in O-848 (44) and O-853 (45) ketones (O-1272 46)alcohols (O-1876 47) or alkanes (O-1877 48) resultedmostly in decreased affinity (Table 6) but also with a changeof functionality in some compounds as revealed by in-vivoassays For instance replacement of the piperidinyl by apentyl (O-1269) or by an heptyl chain (O-1270) gave agonistcompounds having Ki values of 32 and 48 nM respectivelyThus the authors suggested that the 3-substituent region isinvolved in receptor recognition and agonist activity Wileyet al on the basis of their results concluded that while the3-position seems to be involved in agonism the 1- 4- and

One close analogue of SR141716A 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-piperidinecarbox-amide (SR147778 28) was described very recently byRinaldi-Carmona et al [133 134] (Fig 3) It possesses highaffinity and selectivity for the hCB1 cannabinoid receptorwith Ki values for the hCB1 and hCB2 receptors of 35 and400 nM respectively ([3H]-CP-55940 hCB1amp2-CHO cells)Further SR147778 antagonised CP-55940 effects on mousevas deferens contractions (pA2=81) and on forskolin-stimulated adenylyl cyclase activity in U373MG cells(pA2=82) but had no effects alone In-vivo SR147778 afteroral administration reversed WIN-55212-2 inducedhypothermia and analgesia As SR141716A SR147778dose-dependently reduced ethanol and sucrose solutionintake with significant effects starting at 03 mgkg (sc) and3 mgkg (po) respectively This compound is currentlyinvestigated in Phase I clinical trials for the treatment ofobesity as well as nicotine and alcohol addictions

Another derivative the 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-(4-hydroxypiperidine)-carboxamide (29) was described in a very recent patent fromSanofi [135] (Fig 3) This antagonist possesses an IC50value for the hCB1 cannabinoid receptor of 32 nM ([3H]-CP-55940 hCB1-CHO cells)

The first structure-affinity relationships for theSR141716A derivatives were reviewed by Barth andRinaldi-Carmona in 1999 [32] Since then many papersdescribing new derivatives were published In 1999 Lan etal described around thirty 15-diarylpyrazoles derivatives[136] Among them was AM251 previously reported as aradioimaging ligand for the CB1 receptor [78] and thatappeared to be more potent (Ki = 749 nM) and selective(selectivity ratio of 306) than SR141716A (Ki = 115 nMselectivity ratio of 143) They reported a Kd value of 05 nMin the mouse vas deferens model using WIN-55212 asagonist Interestingly New et al described for the samecompound an inverse agonist effect on the hCB2 receptorusing a forskolin-induced cAMP accumulation assay (EC50= 650 nM) [137] Recently the anti-obesity effects ofAM251 were reported and are not surprisingly similar tothose of SR141716A [138 139 140] Chen et aldemonstrated that there are synergistic effects on food intakesuppression between AM251 and nalmefene an opioidantagonist [141] This synergistic effect was also presentbetween SR141716A and naloxone as shown by Kirkham[142] Shearman et al found antidepressant-like effects of

1370 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

5-positions appear to be involved in antagonism The O-derivatives reported were also described in a patent alongwith their synthetic pathways which were not given in thepaper [147]

Another investigation of the SR141716Aaminopiperidine region was conducted by Francisco et al[148] They synthesised 21 analogues possessing either analkyl amide or an alkyl hydrazide substituent of various

Table 6 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structure of some of the characterised 1- 3- 4- and 5-pyrazole derivatives Binding affinities (Ki values nM) were obtained on rat brain homogenatesusing [3H]-CP-55940 [136 145 146 148 150] or [125I]-AM-251 [155]

NN

R1

R5

R3R4

Cpd ndeg R1 R3 R4 R5 [136] [145] [146] [148] [150] [155]

SR141716 14 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Cl-Ph 115 62 62 62 13 18

O-1559 30 24-di-Cl-Ph CO-NH-piperidinyl Me 1-Methylpentyl 233

O-1302 31 24-di-Cl-Ph CO-NH-piperidinyl Me 4-((CH2)5)-Ph 21 1

O-1691 32 24-di-Cl-Ph CO-NH-piperidinyl Br 4-((CH2)5)-Ph 15

O-1704 33 24-di-Cl-Ph CO-NH-piperidinyl I 4-((CH2)5)-Ph 22

6-I-SR141716 34 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-Cl-Ph 166

4rsquo6-di-I-SR141716 35 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-I-Ph 126

4rsquo-I-SR141716 36 24-di-Cl-Ph CO-NH-piperidinyl Me 4-I-Ph 75 25 6

4rsquo-Br-SR141716 37 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Br-Ph 168 3

Cpd 25 in ref [136] 38 24-di-Cl-Ph CO-NH-morpholinyl Me 4-Br-Ph 54

O-1300 39 Ph CO-NH-piperidinyl Me 4-Cl-Ph 150

O-1254 40 4-(CH2)4-Ph CO-NH-piperidinyl Me 4-Cl-Ph 226 256

O-1255 41 4-(CH2)5-Ph CO-NH-piperidinyl Me 4-Cl-Ph 433

O-1269 42 24-di-Cl-Ph CO-NH-pentyl Me 4-Cl-Ph 32 114 3

O-1270 43 24-di-Cl-Ph CO-NH-heptyl Me 4-Cl-Ph 48 462 3

O-848 44 24-di-Cl-Ph CH2-O-(CH2)2-piperidinyl

Me 4-Cl-Ph 2450 232

O-853 45 24-di-Cl-Ph CH2-O-CH2-cyclohexyl

Me 4-Cl-Ph 388 100

O-1272 46 24-di-Cl-Ph CO-heptyl Me 4-Cl-Ph 221

O-1876 47 24-di-Cl-Ph 1rsquo-OH-heptyl Me 4-Cl-Ph 657

O-1877 48 24-di-Cl-Ph Heptyl Me 4-Cl-Ph 422

MF9725-64-17 49 24-di-Cl-Ph CO-NH-butyl Me 4-Cl-Ph 134

MF9725-179-32 50 24-di-Cl-Ph CO-NH-NH-butyl Me 4-Cl-Ph 51

MF9725-66-11 51 24-di-Cl-Ph CO-NH-cyclohexyl Me 4-Cl-Ph 25

MF9725-95-31 52 24-di-Cl-Ph CO-NH-(4-hydroxybutyl)

Me 4-Cl-Ph 154

Cpd 15 [150] 53 n-pentyl CO-NH-piperidinyl Me Ph 23

Cpd 16 [150] 54 n-pentyl CO-NH-piperidinyl Me 4-Br-Ph 63

Cpd 17 [150] 55 n-hexyl CO-NH-piperidinyl Me Ph 21

Cpd 18 [150] 56 n-heptyl CO-NH-piperidinyl Me Ph 47

NIDA-41109 57 24-di-Cl-Ph CO-NH-piperidinyl Br 4-Cl-Ph 14

NIDA-41119 58 24-di-Cl-Ph CO-NH-piperidinyl H 4-Cl-Ph 9

NIDA-41057 59 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OH-Ph 104

NIDA-41020 60 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 41

NIDA-41087 61 2-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 8

NIDA-42055 62 24-di-Cl-Ph CO-NH-piperidinyl Br 4-OMe-Ph 62

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1371

lengths in position 3 of the pyrazole moiety They observedthat until five carbons the affinity increases along with thecarbon chain length This is observed with the alkylhydrazide the alkyl amide and the hydroxyalkyl amideseries Moreover the hydrazide analogues exhibit a loweraffinity for the rCB1 than the amide analogues as illustratedby compounds MF9725-64-17 (49) and MF9725-179-32(50) having Ki values of 134 and 51 nM respectively(Table 6) From their structure-activity relationships (SAR)studies the authors concluded that the pharmacophoricrequirement of the amidopiridine region is a chain not longerthan 3 Aring and that a substituent having a positive chargedensity would probably result in increased affinity andpotency The same team described in a patent several othercompounds [149] Binding affinities (Ki) against [3H]-CP-55940 [3H]-SR141716A or [3H]-WIN55212-2 on wholerat brain or on hCB1-transfected cells were given Moreoveractivity data were obtained using the [35S]-GTPγ S assaydemonstrating that these alkyl amide and hydrazide analogsact as antagonists or inverse agonists Despite the greatnumber of derivatives claimed none of them posses asignificantly greater affinity for the CB1 receptor than theSR141716A Nevertheless they showed a slightenhancement of the selectivity for the CB1 receptor over theCB2 cannabinoid receptor

affinity of these derivatives increases with the length of thealkyl chain with an optimal length of 5-6 carbons The bestcompound of this series N-(piperidin-1-yl)-5-phenyl-1-hexyl-4-methyl-1H-pyrazole-3-carboxamide (55) possesses aKi value of 21 nM determined using [3H]-CP-55940 on ratbrain membranes which is higher than the 13 nMdetermined for SR141716A in the same conditions (Table6) In the same paper the authors starting from thehypothesis that antagonism by SR141716A is caused bybinding to the same region of the receptor as do the agonists(CP-55940 and WIN-55-2122) but preventing the agonistpromoted conformational change conducted extensiveconformational analysis as well as superimposition modelsand 3D-QSAR to propose a molecular mechanismsupporting the action of SR141716A Along with theauthors the C-5 aryl substituent of SR141716A occupyinga unique region could contribute in conferring the antagonistproperties Moreover the C-3 substituent could beresponsible for the antagonist or inverse agonist propertiesdepending on the interaction with the receptor

About this topic a very interesting review dealing withthe cannabinoid receptors pharmacophores as well as withthe activationinactivation of these receptors was recentlypublished by Reggio [151]

More recently Dyck et al described seven otherderivatives varying at the amide position [152] The onlycompound possessing a higher affinity for the CB1cannabinoid receptor than SR141716A was the 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta-

The affinity for the rat CB1 receptor of seven derivativespossessing an alkyl chain in position 1 instead of the 24-dichlorophenyl substituent was reported by Shim et al in apaper describing a molecular mechanism for the antagonistand inverse agonist activity of SR141716A [150] The

NN

O

NHN

Cl Cl

Cl

NN

Cl

Cl

NON

NN

O

NH

N

Cl

ClCl

NN

O

NH

MeO Cl

CP-272871 66

63 64

65

Fig (4) CB1 cannabinoid receptor antagonists 3 Diarylpyrazole derivatives Structures of 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63 ) 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methyl-pyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]-morpholine (64 ) N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65 ) and N-phenyl-1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-pyrazole-3-carboxamide (CP-272-871 66 )

1372 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

[c]pyrrol2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63)(Fig 4) The Ki values were 5 and 12 nM respectively([3H]-CP-55940 hCB1-HEK cells)

CP-55940 rat brain) For instance N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65) (Fig 4) showed a Ki value of15 nM However no data were provided concerning thefunctionality of such compounds

Over 200 other pyrazole derivatives acting at the CB1cannabinoid receptor were claimed in a patent from Pfizer[153] The affinity for the hCB1 receptor of 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methylpyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]ndashmorpholine (64) (Fig 4)was 79 nM ([3H]-SR141716A hCB1-HEK cells) Theothers compounds affinity was between 01 and 100 nM

In an effort to develop new SR141716A analoguespossessing lower lipophilicity to be used as PET tracersKatoch-Rouse and colleagues further explored thesubstitution pattern of the two phenyls [155] Bindingassays showed that a decreased lipophilicity lead to adecreased affinity for the CB1 receptor as determined bydisplacement of [3H]-AM251 For instance compoundNIDA-41057 (59) has a lipophilicity of 42 ( expressed asElogDoct) and a Ki value of 104 nM whereas SR141716Ahas a ElogDoct value of 54 and a Ki value of 18 nM In

Seventy five pyrazole derivatives were described in a veryrecent patent from Makriyannis et al [154] The affinity ofthe compounds for the cannabinoid receptors is given TheKi values for the best compounds are lower than 6 nM ([3H]-

Table 7 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structures and affinities for the cannabinoid receptors of some representative tricyclic pyrazole derivatives Values were obtained using [3H]-CP-55940as radioligand and mouse [160 228] rat [159 227] or human [158] cannabinoid receptors Compounds 69-78 are CB1 cannabinoid receptor ligandsCompounds 79-86 are CB2 cannabinoid receptor ligands

NN

O

NHX

R3

R1

R2

n

n˚ X R1 R2 n R3 Ki CB1 (nM) Ki CB2 (nM) Selectivity References

69 -(CH2)3- Cl 24-di-Cl 0 piperidine 126 [158]

70 -(CH2)3- H 24-di-Cl 0 azepane 100 [158]

71 -(CH2)3- H 24-di-Cl 0 piperidine 398 [158]

72 -(CH2)3- Cl 24-di-Cl 0 azepane 125 [158]

73 -(CH2)3- NO2 24-di-Cl 0 azepane 63 [158]

74 -(CH2)2-O- H 24-di-Cl 0 piperidine 501 [158]

75 -CH2-S- Br 24-di-Cl 0 piperidine lt 500 gt10 [159]

76 -CH2-SO2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

77 -(CH2)2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

78 -(CH2)3- Cl 24-di-Cl 0 piperidine 000035 21 60000 [160]

79 -CH2- Cl 24-di-Cl 0 piperidine 2050 034 00002 [228]

80 -CH2- Br 24-di-Cl 0 piperidine 1570 027 00002 [228]

81 -CH2- CH3 24-di-Cl 0 piperidine 363 0037 00001 [228]

82 -CH2- Cl 4-Cl 0 piperidine 1787 09 00005 [228]

83 -CH2- H 24-di-Cl 0 piperidine 1152 0385 00003 [228]

84 -CH2- Cl 34-di-Cl 1 133-trimethylbicyclo[221]heptyl

lt500 gt10 [227]

85 -CH2- Br 24-di-Cl 1 bicyclo[321]oct-3-yl lt500 gt10 [227]

86 -CH2- Br 4-Me 1 77-dimethylbicyclo[410]hept-3-yl

lt500 gt10 [227]

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1373

another paper Katoch-Rouse described the synthesis of[18F]-NIDA-42033 (26) a ligand useful as PET radiotracerstarting from its bromo derivative [156] Recently Kumar etal [157] proposed the synthesis of a 11C derivative ofNIDA-41087 (61) a compound previously described byKatoch-Rouse (Table 5) Post-mortem binding of thiscompound to human prefrontal cortex was also investigatedInterestingly CP-272871 (66) described by Meschler as aCB1 inverse agonist (Ki=57 nM [3H]-CP-55940 rat brainhomogenates) possesses a lipophilicity similar to theNIDA-41087 one [62] However despite the presence of amethoxy function no radiolabelled derivative has beendescribed to date One explanation could be the compoundlow selectivity (2 fold) for the CB1 cannabinoid receptor

To further explore the pharmacology of this compound afunctional assay using the mouse vas deferens model wasperformed by Ruiu and colleagues enlightening thecompetitive antagonist properties of NESS (pA2= 1246against WIN-55212-2) which had no effect by itself up to1microM Moreover NESS 0327 was unable to affect basalbinding of the [35S]-GTPγ S demonstrating its lack ofnegative intrinsic activity In-vivo studies were conductedusing the hot plate and tail flick tests NESS dose-dependently abolished the antinociceptive effect of WIN-55212-2 but had no effect by itself However the authorssuggested based on the affinity and in-vivo activity of theircompound that NESS 0327 possesses a poor centralbioavailability

Several new attempts to increase the affinity of thediarylpyrazole derivatives were recently made by rigidifyingthe SR141716A structure (Table 7) Six fused ringanalogues of SR141716A obtained by fusion of the 5-(4-chlorophenyl) substituent with the central pyrazole to forman indazole ring were published by Bass et al in 2002 [61]The compound possessing the highest affinity O-1248 (67)has a Ki value of 475 nM as determined by displacement of[3H]-CP-55940 from rat brain membranes

Another example of ring-constrained biarylpyrazole wasdescribed by Herbert Seltzman team [161] Theyinvestigated a photocyclisation reaction starting from theSR141716A leading to a pyrazolo[15-f]phenanthridinestructure (Fig 5) The new compound (68) was tested on theCB1 receptor (whole rat brain) against [3H]-CP-55940[3H]-SR141716A and [3H]-WIN-55212-2 and exhibited Kivalues of 48 35 and 50 nM respectively The CB2 affinitywas assessed using [3H]-CP-55940 and was found to be 100times lower (Ki = 3340 nM) No effect on the [35S]-GTPγ Sassay (rat brain) was observed by the authors

Stoit and colleagues from Solvay Pharmaceuticalspublished a paper describing the synthesis andpharmacological characterisation of new benzocyclohepta-pyrazole derivatives as CB1 antagonists (69-74) [158] Theaffinity of these compounds was more or less one order ofmagnitude lower than the SR141716A one with pKi valuesranging from 64 to 72 compared to 76 for the SRcompound (Table 7) Compounds were shown to beantagonists with pA2 values from 70 to 89 as theyprevent CP-55940-induced cAMP accumulation Howeverthe authors investigating the bioavailability of theircompounds either po or after ip injection found negligibleblood plasma levels

NN

Cl

Cl

O

NH

N

NN

Cl

O

NHN

Cl

Cl

O-1248 67 68However just before the publication of Stoit results

Sanofi-Synthelabo took a patent describing tricyclicpyrazolecarboxylic acid amide derivatives having antagonistproperties on the CB1 cannabinoid receptor andsubmicromolar affinities (75-77) [159] Thirty examples weregiven along with their synthetic pathway but no individualpharmacological data were shown

Fig (5) CB1 cannabinoid receptor antagonists 3Diarylpyrazole derivatives Two ring-constrained diarypyrazolederivatives O-1248 (67 ) and a pyrazolo[15-f]phenanthridinederivative (68 )

It appears from these attempts to increase SR141716Aaffinity by rigidifying its structure that only a limitedincrease in the CB1 cannabinoid receptor affinity can beexpected with the apparent exception of NESS 0327Moreover rigidifying the structure often results in a morelipophilic compound in a family of compounds being yetlipophilic

More recently Ruiu and colleagues published a paperdescribing the synthesis and complete pharmacologicalcharacterisation of a very potent CB1 receptor ligandchristened NESS 0327 (78) [160] This compound has thesame structure as compound 69 published by Stoit et al(compound 20 in the reference [158]) Nevertheless Ruiu hasobtained an affinity for the CB1 cannabinoid receptor 5000times bigger than the SR141716A one with Ki values of035 pM and 18 nM respectively using mouse forebrainhomogenates and [3H]-CP-55940 as radioligandFurthermore the selectivity ratio obtained for this compoundby Ruiu is 60000 with a Ki value for the CB2 receptor of21 nM against the [3H]-CP-55940 The discrepancybetween the data obtained independently by Stoit et al andRuiu et al could hardly be explained by the differences inthe origin of the receptor used respectively hCB1-CHO andmouse forebrain

4 Phenyl Benzofuranone Derivatives

In a 1997 patent from Eli Lilly was described thesynthesis of aryl-benzothiophene and aryl-benzofuranederivatives (87-89 Fig 6) having CB1 receptor affinity andantagonist properties [162] From ten compounds having aKi value lower than 25 microM ([3H]-CP-55940 hCB1-CHOcells) one was selected and claimed to have a Ki of 170 nMCompound 88 was shown in the patent to antagonise theeffect of anandamide on cAMP accumulation with an IC50 of

1374 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

O

O

O

O

NC

S

O

O

O

O

O

O

O

NC

LY320135 8887 89

Fig (6) CB1 cannabinoid receptor antagonists 4 Phenyl benzofuranone derivatives Chemical structures of two aryl-benzofuranederivatives (87 -88 ) and one aryl-benzothiophene (89 ) derivative from Eli Lilly

500 nM and the effect of WIN-55212-2 on calcium channelsat 1microM Moreover the intra-peritoneal injection (20 mgkg)of this compound antagonised the in-vivo effect ofanandamide in the Open Field Assay a mouse behaviourmodel Later on Felder and colleagues gave a furtherdescription of this compound called LY320135 in a paperdescribing its effect on cAMP accumulation and on ioniccurrents [163] The selectivity ratio of LY320135 was 106with Ki values of 141 and 14900 nM for hCB1 and hCB2receptors ([3H]-CP-55940) respectively LY320135antagonised anandamide inhibition of forskolin-inducedcAMP accumulation with an IC50 value of 734 nMChristopoulos et al in 2001 reported for LY320135 apKb value of 527 in the inhibition of WIN-55212-2mediated response in the rat electrically-stimulated vasdeferens while the pKb value for SR141716A was 75[164]

Other patents were taken by Aventis in which somepotential applications for their compounds were describedFor instance an association between an azetidine derivativehaving CB1 antagonists properties and sibutramine aserotonin and norepinephrine reuptake inhibitor was claimed[170] as a treatment against obesity Oral administration ofcompound 90 (3 mgkg) and sibutramine (06 mgkg) to fafaZucker rats which are genetically obese rats has resulted ina decreased food intake compared to control lean Zuckerrats In another patent the association of an azetidinederivative (1-10 mgkg po ) and a D2D3 agonist likequinpirole (01 mgkg ip) was claimed to have a therapeuticpotential in the treatment of Parkinsonrsquos disease [171] Theeffect has been evidenced using an akinesia model in the ratEvidences supporting the use of CB1 antagonists as adjuvantin the treatment of Parkinsonrsquos disease begin to appear Itseems that such compounds would be helpful in thetreatment of Parkinsonism [172] and levodopa-induceddyskinesia [173] Brotchie published an interesting paperreviewing the CB1 cannabinoid receptor signaling inParkinsonrsquos disease [174] Nevertheless further clinicalstudies should be conducted to further confirm the usefulnessof CB1 antagonists in the treatment of Parkinsonrsquos disease

5 Azetidine Derivatives

The family of azetidine compounds illustrated bycompounds 90 and 91 (Fig 7) was developed at Aventis byDaniel Achard and colleagues and was described in a seriesof patents [165-168] They claimed the IC50 values of thesecompounds for the CB1 receptor to be less than or equal to100 nM These values were obtained following the proceduredescribed by Kuster et al [169] The antagonist propertywas shown using an in-vivo model the reversal ofhypothermia induced by CP-55940 in mice and the ED50obtained were lower than 50 mgkg

6 Aryl-Imidazolidine-24-Diones Derivatives

In 1999 the Didier Lambert team published a paperdescribing the affinity of 24 new 3-alkyl-55rsquo-diphenyl-imidazolidine-24-dione derivatives for the hCB1 receptor[175] The preliminary structure-activity relationships

N

SO

O

Cl

Cl

F

F

N

SO

O

Cl

NS

90 91

Fig (7) CB1 cannabinoid receptor antagonists 5 Azetidine derivatives Two examples (90 -91 ) of the azetidine derivatives developedby Aventis

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1375

Table 8 CB1 Cannabinoid Receptor Antagonists 6 Aryl-Imidazolidine-24-Diones Derivatives

Structures affinities ([3H]-SR141716A hCB1-CHO cells) and pKb values ([35 S]-GTPγS HU210 rat brain) of three aryl-imidazolidine-24-dionederivatives (92-94)

N

NH

OO

R

Br

Br

Cpd ndeg R Ki (nM) a pKb b

DML20 92 ethylmorpholine 70 611

DML21 93 1-hydroxypropyl 103 625

DML23 94 heptyl 98 574

a [175]b [177]

showed that the substitution at the nitrogen in position 3 aswell as a bromine atom in para of the phenyl rings aremandatory for the compounds affinity Later on threecompounds termed DML20 (92) DML21 (93) and DML23(94) were characterised as neutral antagonists using the [35S]-GTPγ S assay on rat cerebellum membranes [176] (Table 8)In the same model they competitively inhibited HU-210-induced [35S]-GTPγ S binding with pKB values of 611625 and 574 respectively Moreover these compoundswere proven to be quite selective for the CB1 receptor

higher than the one for the CB2 cannabinoid receptor (IC50 =300 nM) The inverse agonism of the compound wasextrapolated by inhibiting CP-55940-induced hypothermiain mice (EC50 = 18 nM) Moreover one oral dose (10mgkg) reduces the food intake in diet-induced obese ratsInterestingly food intake of CB1

-- mice was not alteredBody weight loss was maintained all over the study during achronic administration (14 days) of 10 mgkg of Cpd A

Later on Merck published a patent [180] in which someof the 45-diaryl-pyrazoles derivatives previously describedwere claimed to be useful in the treatment or the preventionof obesity in association with compounds inhibiting the11β-hydroxysteroid dehydrogenase type 1 (1β-HSD1)enzyme The 1β-HSD1 enzyme is responsible for thesynthesis of cortisol increased levels of which according tothe inventors are associated with obesity However neitherresults on appetite suppression nor on weight loss weregiven even if some indications on the affinity of thecompounds for the cannabinoid receptors were given

Very recently the functionality of these DML compoundswas explored using the [35S]-GTPγ S assay both on rat andhuman CB1 cannabinoid receptors [177] The data obtainedconfirm that the 3-alkyl-55rsquo-diphenyl-imidazolidinedionederivatives behave as neutral antagonists on rat CB1cannabinoid receptor (rat brain) However DML 20 DML21and DML23 acted as inverse agonists on the human receptor(hCB1-CHO cells) Furthermore the authors showed thatthis different functionality is not due to the hCB1 receptorlevel of expression in the recombinant cell line as whateverthe level of expression (Bmax of 44 pmolmg or 32pmolmg) DML derivatives behaved as inverse agonists ofthe hCB1 cannabinoid receptor

Another imidazole ring pattern of substitution wasproposed by Hagmann and collaborators [181] Thecompounds described 43 examples are given possess twophenyls the first one in position 1 linked to the nitrogenand the second one in position 2 The carboxamidesubstituent is in position 4 instead of in position 2 for thediarylimidazoles described by Finke These 12-diaryl-imidazoles are claimed to be CB1 cannabinoid receptorantagonists or inverse agonists but the absence of precisepharmacological values do not allow any comparisonbetween the two substitutions patterns However a samepattern of substitution was used by other researchers Amongthem Dyck et al described in 2004 ten 1-(4-chlorophenyl)-2-(24-dichlorophenyl)-imidazole derivatives varying by theircarboxamide substituent in position 4 (101-103 Table 9)[152] For instance with a 3-azabicyclo[330]octan-3-ylsubstituent (101) they obtained a Ki value of 14 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) In a [35S]-GTPγ Sassay 101 exhibited an IC50 value of 19 nM

7 Diarylimidazoles Derivatives

Finke and colleagues at Merck developed a new class ofCB1 receptor antagonists based on an imidazole nucleusOver eighty 45-diaryl-imidazoles derivatives having CB1antagonist properties were claimed in a patent published in2003 [178] Some representative compounds of the patent areillustrated in Table 9 (95-100) The affinity of the newcompounds was determined using [3H]-CP-55940 andhCB1-CHO cells Although no detailed pharmacologicaldata were given in the patent in-vitro and in-vivo propertiesof one compound termed Cpd A (100) have been describedelsewhere [179] Its affinity for the CB1 cannabinoid receptorwas shown to be nanomolar (IC50 = 4 nM) and 75 times

1376 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 9 CB1 Cannabinoid Receptor Antagonists 7 Diaryl-Imidazole Derivatives

Structure and affinity range of some 45-diaryl-imidazole derivatives for the human cannabinoid receptors determined using [3H]-CP-55940 asradioligand and hCB1amp2-CHO cells

N

NR2

R1

R4

R3

ndeg R1 R2 R3 R4 hCB1 (IC50 or Ki) hCB2 (IC50 or Ki) References

95 CH3 CO-NH-piperidinyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

96 CH3 CO-NH-piperidinyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

97 CH3 CO-NH-cyclohexyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

98 CH3 CO-NH-phenyl 24-di-Cl-Ph 4-Cl-Ph 10 ndash 100 nM a 100 ndash1000 nM a [178]

99 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

100 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph 4 nM a 300 nM a [179]

101 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

CH3 14 nM b ND [152]

102 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

H 66 nM b ND [152]

103 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 85 nM b ND [152]

104 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 23 nM b 542 nM b [182]

105 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 30 nM b 608 nM b [182]

106 4-Br-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 60 nM b 489 nM b [182]

107 4-CF3-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 29 nM b 634 nM b [182]

108 4-Cl-Ph 24-di-Cl-Ph CO-NH-morpholinyl CH3 197 nM b 3297 nM b [182]

109 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidin-4-ol CH3 172 nM b 3959 nM b [182]

110 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CN 30 nM b 1590 nM b [182]

111 4-Cl-Ph 24-di-Cl-Ph CO-NH- piperidinyl CH2F 36 nM b 906 nM b [182]

112 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH2-CH3 14 nM b 430 nM b [182]

113 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Cl 27 nM b 823 nM b [182]

114 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Br 23 nM b 746 nM b [182]

115 4-Cl-Ph 24-di-Cl-Ph CO-NH-tetrahydroisoquinoline CH3 34 nM b 696 nM b [182]

116 4-Cl-Ph 24-di-Cl-Ph CO-NH-cycloheptyl CH3 35 nM b 349 nM b [182]

117 24-di-Cl-Ph 4-Cl-Ph CO-NH-piperidinyl CH3 403 nM b 208 nM b [182]a IC50 value b Ki value

Lange and co-workers from Solvay described twenty-eight imidazole derivatives [182] The 24-dichlorophenylsubstituent at position 2 was kept constant while thesubstitutions at the other positions were explored (Table 9)Position 5 can accommodate a large range of littlesubstituents like hydrogen (104) methyl (105) ethyl (112)chlorine (113) bromine (114) fluoromethyl (111) or cyano(110) without major changes in the affinity At position 4more bulky substituents like 1234-tetraisoquinoline (115)or cycloheptyl (116) were well tolerated whereas thepresence of a more hydrophilic moiety such as morpholine(108) or piperidin-4-ol (109) was detrimental to the affinity

Interestingly position exchange between the 24-diphenyland the 4-chlorophenyl substituents led to a 13 fold loweraffinity with Ki values of 30 and 403 nM for 105 and 117respectively ([3H]-CP-55940 hCB1-CHO cells) In thefunctional assay which was inhibition of WIN-55212-2-induced [3H]-arachidonic acid release by hCB1-CHO cellsall tested compounds behaved as antagonists Compound105 showed a pA2 value of 86 which is the same theauthors obtained for SR141716A It would be interesting toassess if these compounds are true antagonists or if they actmore as inverse agonists Compound 105 when administeredto rats was able to inhibit CP-55940-induced hypotension

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1377

with an ED50 value of 24 mgkg which is close to the 3mgkg obtained for SR141716A The compound was alsoactive in another in-vivo model the WIN-55212-2-inducedhypothermia in mice Compound 104 which has nosubstituent in position 5 of the imidazole ring was lessactive in the hypotension model and inactive in thehypothermia model Thus a methyl group is the preferredsubstituent in position 5 of the imidazole moiety Otherproperties either experimental (P-glycoprotein affinitylogPHPLC) or computational (molecular volume polarsurface area) obtained for compounds 105 were similar tothose obtained for SR141716A Finally molecularmodelling studies revealed a close structural overlap betweenthe two compounds

value of 197 nM is selective for hCB1 and is as potent asSR141716A The structural modulations of 118 led to someinteresting compounds (Table 10) Six compounds possess aKi value for the hCB1 cannabinoid receptor equal to or lowerthan 25 nM antagonist properties in the inhibition of [3H]-arachidonic acid release by WIN-55212-2-stimulated hCB1-CHO cells and Ki values for the hCB2 receptor over 1microMThe diaryl-pyrazoline derivatives contained a chiral center atposition 4 The authors resolved the racemic mixture of twocompounds (121 and 130) The levorotatory enantiomersappeared to be the eutomers For compound SLV319levorotatory enantiomer of 121 the Ki and pA2 values were78 nM and 99 respectively The Ki and pA2 values ofSLV326 levorotatory enantiomer of 130 were 359 nM and9 respectively

8 34-Diaryl-Pyrazoline Derivatives Two in-vivo models CP-55940-induced hypotension inrat and WIN-55212-2-induced hypothermia in mouse wereused to assess the potential of these compounds The resultsare of the same order of magnitude than those obtained forSR141716A For instance in the hypotension modelSLV319 ((-)121) and SLV326 ((-)130) showed ED50 values

Lange and collaborators also discovered 34-diaryl-pyrazoline derivatives after a screening of compoundsresembling to SR141716A [183184] Compound 118 thatwas initially identified during the screening possesses a Ki

Table 10 CB1 Cannabinoid Receptor Antagonists 8 34-Diaryl-Pyrazolines Derivatives

Structure affinity for the hCB1 and hCB2 cannabinoid receptors (Ki nM) expressed in CHO transfected cells ([3H]-CP-55940) and potency (pA2) ofsome 34-dirylpyrazolines derivatives Table adapted from [184]

NN

N N

SO2

R3

R2

Cl R1

R4

n˚ R1 R2 R3 R4 Ki hCB1 (nM) Ki hCB2 (nM) pA2 (CB1)

118 H H H 4-CH3 197 gt 1000 84

119 H H H 246- CH3 24 gt 1000 94

120 H H H 4-Cl 16 gt 1000 95

121 H CH3 H 4-Cl 25 gt 1000 87

122 H CH3 CH3 4-Cl 280 gt 1000 85

123 H H H 4-F 53 gt 1000 9

124 H CH3 H 4-F 338 gt 1000 85

125 H CH3 CH3 4-F gt 1000 gt 1000 lt 75

126 H CH3 H 3-Cl 14 gt 1000 86

127 4-Cl CH3 H 4-Cl 255 ND ND

128 4-F CH3 H 4-Cl 584 ND ND

129 H CH3 H H 170 ND 75

130 H CH3 H 4- CF3 221 gt 1000 93

SR141716A 25 1580 86

1378 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

N

HN

O

N

Cl Cl

Cl

N

N

HN

O

Br

Br131 132

Fig (8) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Chemical structures of two 56-diaryl-pyrazine-2-amide derivatives (131-132) developed as CB1 cannabinoid receptorantagonists by AstraZeneca

of 55 and 2 mgkg (po) respectively proving their in-vivoefficacy after oral administration The authors alsodemonstrated that SLV319 is devoid of affinity for the P-glycoprotein pump and that it possessed a goodCNSplasma ratio (17) These two compounds (SLV319and SLV326) were chosen as development candidates bySolvay and entered clinical Phase I trials in 2003 [185]

132 Fig 8) useful as CB1 cannabinoid receptor antagonists[186 187] The activity of twenty derivatives wasdetermined using a [35S]-GTPγ S assay and hCB1-CHOtransfected cells The concentration required to give halfmaximal inhibition of CP-55940-induced [35S]-GTPγ Sbinding (IC50) is lower than 200 nM for the preferredcompounds No affinity data were given for thesecompounds

9 Diaryl-Pyrazine Diphenyl-Pyridine Diphenyl-Phenyland Diaryl-Pyrimidines Derivatives

The same year in a patent from Merck Finke andcollaborators described several 56-di-phenyl-pyridinederivatives as hCB1 antagonists or inverse agonists [188]The compounds have a substituent in position 3 of thepyridine core (Fig 9) This substituent could be a cyano(133-134) or a nitro group a halogen an ester or an amide(135-136) Over 150 compounds were synthesised toillustrate the invention but no pharmacological data wasdisclosed in the patent

In 2003 appeared several patents describing new familiesof compounds that bind to the CB1 cannabinoid receptorThese compounds have in common a central six atomsaromatic ring which could be a pyrazine a pyridine aphenyl or a pyrimidine and that is substituted by at leasttwo phenyl rings

The first patents were from Berggren and collaboratorsfrom AstraZeneca who published two patents describing thesynthesis of 56-diaryl-pyrazine-2-amide derivatives (131-

A few months later another patent was taken by Sanoficlaiming the antagonist properties of 56-di-phenyl-2-pyridine carboxamide derivatives [189] These compounds

N

NH

O

Cl

Cl

N

O

Cl

O NH

N

O

Cl

ClCl

NH

CN O

N

Cl

Cl

Cl

OHN

N

O

Cl

ClCl

CNF

F

133 134

135 136 137

Fig (9) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives 56-Di-phenyl-pyridine derivatives patented by Merck (133-136) and by Sanofi (137) as CB1 cannabinoid receptorsantagonists

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1379

NH

O

N

Cl

ClCl

NH

O

N

Cl

F3CCl

N

N

O

Cl

Cl Cl F138 139 140

Fig (10) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Representative structures of the diphenyl-phenyl (138-139) and 45-diphenyl-pyrimidine (140) derivatives developed ascannabinoid antagonists at Sanofi and Merck respectively

28 examples are given structurally related to the diphenyl-phenyl derivatives have their pyridine core substituted bytwo phenyl rings and by an amide moiety (137 Fig 9) TheIC50 values were lower than 100 nM but no data were givenshowing the antagonist properties although the compoundswere assayed in the adenylate cyclase inhibition assay

10 Other Derivatives

Several amide derivatives structures recently appearedamong the CB1 cannabinoid receptor antagonists In a seriesof patents taken by Merck in 2003 Hagmann and colleaguesdescribed a large amount of compounds obtained by parallelsynthesis which are claimed to be CB1 cannabinoid receptorantagonists [192-195] They were synthesised by reacting alibrary of substituted amines with a library of carboxylicacids (141-144 Fig 11) However as no value was givenfor the affinity or activity of these amides we will notfurther discuss this class of compounds

Along this line Sanofi Synthelabo developed newdiphenyl-phenyl derivatives claimed to have CB1cannabinoid receptor antagonist properties [190] They arebased on a central phenyl ring substituted by two phenylsand by an amide moiety (138-139 Fig 10) Thirteencompounds were claimed along with their synthesis TheirIC50 values were lower than 100 nM ([3H]-CP55-940hCB1-CHO cells) but no other specific data (selectivitypA2hellip) was given

Several 15-diaryl-pyrrole-3-carboxamide derivatives weresynthesised and claimed to be CB1 cannabinoid receptorantagonists by Berggren et al [196] The affinity valueswere not disclosed in the patent (145-146 Fig 12) Otherpyrrole derivatives were synthesised by Guba et al atHofmann-La Roche [197] More specifically 2-(thiazol-4-yl)pyrrole derivatives were claimed as CB1 cannabinoidreceptor antagonist (IC50lt2microM) (147-148 Fig 12)

Finally Kopka et al from Merck described more thanone hundred 45-diaryl-pyrimidine derivatives as CB1cannabinoid receptor antagonists (140 Fig10) [191]However no precise data were provided concerning theaffinity and functionality of the compounds

R2

R1N

R3

R4

R5

O

NH

O

Cl

O F

F

R2

R1NH

R3

R4

HO R5

O

NH

O

Cl

CN

NH

O

Cl

O

O

NH

O

Cl

Cl

+

141142

143 144

Fig (11) CB1 cannabinoid receptor antagonists 10 Other derivatives Retrosynthetic scheme of a CB1 cannabinoid antagonistlibrary of amide derivatives developed at Merck The structures of four representative compounds are illustrated (141-144)

1380 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

NH

O N

Cl

Cl

Cl

N

NH

O

OMe

MeO

N

NH

O

N

S

MeO

N

NH

O

N

S

MeO

145 146

147 148

Fig (12) CB1 cannabinoid receptor antagonists 10 Other derivatives Two examples of 15-diaryl-pyrrole derivatives described byBerggren et al (145-146) While the 2-(thiazol-4yl)pyrroles derivatives 147 and 148 were described by Guba et al from Hofmann-LaRoche All these pyrroles derivatives were claimed to be CB1 cannabinoid receptor antagonists

Another new structure is represented by the 124-triazolederivatives developed by Jagerovic and collaborators In arecent paper were described the synthesis andpharmacological properties of five new 15-diphenyl-3-alkyl-

triazole derivatives [198] Among these compounds onlyone (149) behaved as a CB1 antagonist inhibiting the WIN-55212-2-induced contractions in a mouse vas deferenspreparation However despite its effects in isolated tissue

Table 11 CB1 Cannabinoid Receptor Antagonists 10 Other Derivatives

Structure and affinity of diaryl-triazole derivatives

N

NN

R3

R2

R1

Cpd R1 R2 R3 Ki CB1 (nM) References

149 24-diCl 4-Cl hexyl 855 a [198]

150 24-diCl 4-Cl2-OMe CO-NH-(3-azabicyclo[330]octan-3-yl) 270 b [152]

151 24-diCl 4-Cl CO-NH-(3-azabicyclo[330]octan-3-yl) 164 b [152]

152 4-Cl 24-diCl CO-NH-(3-azabicyclo[330]octan-3-yl) 137 b [152]

153 4-Cl 24-diCl CO-NH-benzylpyrrolidin-3-yl 29 b [152]

154 4-Cl 24-diCl CO-NH-1-(4-chlorophenyl)ethyl 66 b [152]

155 4-Cl 24-diCl CO-NH-piperidinyl 356 c [182]

156 24-diCl 4-Cl CO-NH-piperidinyl 382 c [182]

a [3H]-SR141716A rat cerebellar membranesb [3H]-CP-55940 hCB1-HEK EDNA cellsc [3H]-CP-55940 hCB1-CHO cells

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1381

N

S

O

NH

Cl

Cl

Cl

F

N

S

O

NHN

ClCl

Cl

N

S

O

NH

Br

N

S

O

NHN

Cl

Cl

Cl

156 157 158 159

Fig (13) CB1 cannabinoid receptor antagonists 10 Other derivatives Structures of 45-diaryl-thiazole derivatives patented as hCB1cannabinoid receptor antagonists by Solvay Pharmaceuticals (156 157 158) or by AstraZeneca (157 158 159)

assays (mouse vas deferens and guinea pig ileum) thecompound possessed only a reduced rCB1 affinity with Kivalues of 855 and 748 nM using [3H]-SR141716A and [3H]-WIN-55-212-2 respectively (Table 11) Further four seriesof compounds differing by the nature of the substituentaround the triazole core were also described in a patent[199] Usually the compounds claimed possess two aromaticrings possibly substituted and one linear alkyl chain Theaffinity for the cannabinoid receptors was not givenNevertheless 149 was evaluated in two isolated tissuemodels the inhibition of the electrically evoked contractionsin the guinea pig ileum and in the mouse vas deferens Inthe two models compound 149 inhibited the effect of WIN-55212-2 but had no effect by itself In the vas deferensmodel the authors obtained a pA2 value of 748 to becompared with 763 obtained for AM251

Yet another five membered ring a thiazole has beenused as central moiety of a new class of cannabinoid ligandsThe thiazole ring is substituted by two aryls in position 4and 5 and by an amide (position 2) as in compounds 156-159 (Fig 13) Forty derivatives of this type weresynthesised by Lange and colleagues from SolvayPharmaceuticals and claimed in a patent as agonists orantagonists of the CB1 cannabinoid receptor [200] Nopharmacological data were provided however their activitywas assessed by measuring the cAMP in transfected hCB1-CHO cells In a recent paper from Lange et al two of thesethiazole derivatives were further described [182] Compound13 (157) the most closely related to SR141716A has a Kivalue of 227 nM while for compound 14 (158) Ki value isover 1000 nM Thus as in the imidazole series (see Table9) the aromatic substitution pattern is of great importancefor the CB1 cannabinoid receptor affinity Compound 13however was devoid of in vivo activity

Others 124-triazole derivatives were described by Dycket al compounds 150-154 unlike the Jagerovic ones havean amide moiety in position 3 [152] The highest affinitywas obtained with a 1-benzyl-pyrrolidin-3-yl substituentThe Ki value was 29 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) to be compared to 12 nM obtained for theSR141716A

Seventeen 45-diaryl-thiazole derivatives were alsosynthesised by Berggren et al and patented by AstraZeneca(157-159 Fig 13) [201]

In a patent from Merck Toupence et al described over190 substituted furo[23]pyridines claimed to be CB1cannabinoid receptor antagonists [202] The affinity (IC50 lt1microM) was measured using recombinant CHO cells and [3H]-CP-55940 and the activity using cAMP dosage (160 Fig14)

Lange et al described a triazole derivative (155) whichcan be superimposed with SR141716A Compound 155showed a Ki value of 356 nM ([3H]-CP-55940 hCB1-CHOcells) a pA2 value of 83 in the inhibition of WIN-55212-induced release of [3H]-arachidonic acid by hCB1-CHO cellsand was active in vivo in the CP-55940-inducedhypotension in rat (ED50 = 236 mgkg) [182]

Very recently Alanine from Hoffmann-LaRoche foundthat diarylbenzo[13]dioxole derivatives act as CB1

O

O

S

N

OO

F

ClCl

O

O

F O

N

OON

NH2

O

Cl

Cl

Cl

160 161 162

Fig (14) CB1 cannabinoid receptor antagonists 10 Other derivatives One example of a furo[23]pyridine derivative (160) and twoexamples of diarylbenzo[13]dioxole derivatives (161-162) described as hCB1 cannabinoid receptor antagonists by Toupence et aland Alanine et al respectively

1382 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

cannabinoid receptor antagonists (161-162 Fig 14) [203]Approximate IC50 values for twelve compounds ([3H]-CP-55940 hCB1-HEK cells) among the three hundred andeighty compounds described were given (IC50lt2microM)

et al in 1995 [207] In their hands ∆9-THC was not able toinhibit the forskolin-stimulated adenylyl cyclase activity inan assay performed on hCB2-CHO cells Subsequently theyshowed that ∆9-THC was able to antagonise HU210-inducedinhibition of adenylyl cyclase activity thus acting as anantagonist of the hCB2 cannabinoid receptor expressed inCHO cells [208] Furthermore very recently Govaerts et al[209] reported an inverse agonist effect of ∆9-THC and ∆8-THC on hCB2-CHO cells using the [35S]-GTPγ S assayThe pEC50 values were 763 and 888 respectively whilethe Emax values were ndash27 and ndash16 as compared to basal

Griffith from Pfizer described three new series of CB1cannabinoid receptor antagonists based on a bicyclic centralaromatic ring Purine pyrazoltriazines andpyrazolo[15a]pyrimidines derivatives were synthesised andinvestigated for their ability to interact with the CB1cannabinoid receptor Albeit a [35S]-GTPγ S assay wasdescribed no data were given concerning the antagonist orinverse agonist properties of such compounds Two hundredand eighty purine derivatives were synthesised [204] Theaffinities of 163 and 164 were given in the patent (28 and12 nM [3H]-SR141716A) In a following patent thesynthesis of sixty pyrazoltriazine derivatives was described(165 Fig 15) [205] The affinity of each compound was notgiven but was said to be ranging between 01 and 590 nMAnd finally more than forty pyrazolo[15a]pyrimidines weresynthesised (166 Fig 15) [206] Their affinity for the CB1cannabinoid receptor was lower than 155 nM Despite thefact that a great number of derivatives share the samesubstitution pattern around the central bicyclic aromatic ringthe comparison of these three new series of compounds ismade difficult by the absence of precise affinity data

Some other classical cannabinoids were shown to possessan antagonist activity at the CB2 receptor This is the casefor instance for the non-selective O-1184 (2) (Ki = 74 nM)which enhances the forskolin-induced cAMP production inCB2-CHO cells (EMax = 161 EC50 = 63 nM) and alsofor O-584 (3) (EMax = 246 EC50 = 138 nM) [39]

2 Indole Derivatives

Since the disclosure in 1993 of a second cannabinoidreceptor many attempts have been made to synthesise CB2cannabinoid receptor selective ligands The first compoundreported to have a somehow greater affinity for the peripheralcannabinoid receptor was WIN-55212-2 The compoundwas introduced by DrsquoAmbra and colleagues [210] back in1992 as a new cannabinoid ligand having a nanomolaraffinity and acting as an agonist In 1996 Showalter et alused a transfected cell line expressing the CB2 cannabinoidreceptor in order to identify selective ligands for thisreceptor [211] They showed that the CB2 affinity of WIN-55212-2 was 7 times greater than the CB1 with Ki valuesof 189 and 028 nM for the CB1 and CB2 cannabinoidreceptors respectively However WIN-55212-2 is anagonist of the cannabinoid receptors Interestingly WIN-55212-3 (167 Fig 16) the so-called inactive enantiomer ofWIN-55212 was recently shown to behave as a low affinity(Ki=363 microM) inverse agonist of the hCB2 cannabinoidreceptor as it decreased the [35S]-GTPγ S binding [209]

III CB2 LIGANDS RECEPTOR ANTAGONISTS ANDINVERSE AGONISTS

The compounds acting as antagonist or inverse agonistsat the CB2 cannabinoid receptor are reviewed in this thirdpart of the paper As for the CB1 cannabinoid receptorligands they are classified depending on their chemicalstructures

1 Classical Cannabinoid Derivatives

The first report of an antagonism mediated by ∆9-THC atthe peripheral cannabinoid receptor was made by Bayewitch

N N

NN

NH COOH

Cl

Cl

N N

NN

N

Cl

Cl

NH2OH

O

N

N

N

N

N

NHNH2

O

Cl

ClN

N

N

N

Cl

Cl

N

O

163 164

165 166

Fig (15) CB1 cannabinoid receptor antagonists 10 Other derivatives Examples of purine (163-164) pyrazoltriazines (165) andpyrazolo[15-a]pyrimidines (166) derivatives described as CB1 cannabinoid receptor antagonists by Griffith

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1383

The pEC50 value was 554 and the Emax value ndash26 ascompared to basal

AM630 also acts as an inverse agonist at the hCB1cannabinoid receptor (EC50 = 900 nM) as Landsman et aldemonstrated [47] Recently Zhang and colleagues reportedthe characterisation of the microsomal metabolism of AM630[212]

N

O

N

O

O

N

O

Cl

O

N

O

(S)-WIN-55212-3 167 BML-190 168

In 1996 Gallant and colleagues identified by submittinga large number of compounds to a binding assay anotherindole analog as CB2 cannabinoid receptor ligand [213] Onthe basis of this compound christened BML-190 (168 Fig16) or called indomethacin morpholinylamide theysynthesised several derivatives that possessed a selectivityratio for the CB2 cannabinoid receptor of up to 140 Thesewere the first compounds specifically designed to be CB2cannabinoid receptor ligands The first report on thepharmacological properties of BML-190 appeared onlyrecently New and colleagues highlighted in the paper theinverse agonist properties of the compound BML-190 dose-dependently increases (103 EC50= 980plusmn70 nM) theforskolin-stimulated levels of cAMP in hCB2-HEK cellswhile WIN-55212-2 decreases (44 45plusmn42nM) thisaccumulation [137]

Fig (16) CB2 cannabinoid receptor antagonists 2 Indolederivatives Chemical structures of WIN-55212-3 (167) andBML-190 (168) two indoles derivatives acting as inverseagonists at the hCB2 cannabinoid receptor Surprisingly in 2002 Melck and collaborators used

BML-190 as a CB2 receptor agonist in a cell proliferationassay [214] BML-190 was also tested as inhibitor of thecyclooxygenase-2 enzyme by Kalgutkar et al [215] Theyobtained IC50 values higher than 33 and 66 microM for theCOX-I and COX-II enzymes respectively These valueshave to be compared with the submicromolar activity ofindomethacin the parent compound Very recently Klegerisand colleagues [216] looking for an antineurotoxic action ofcannabinoids showed that BML-190 increases TNF-αsecretion by stimulated THP-1 monocytic cells but is noteffective on the IL-1β secretion

Further on the basis of WIN-54461 (12) a CB1antagonist AM630 (13) was synthesised by replacement ofthe bromine atom by an iodine one In the initial reportPertwee et al showed the antagonist effects of AM630 butalso suggested that the CB1 cannabinoid receptor may not bethe preferential receptor of AM630 [45] Later on Ross et alreported using hCB1amp2-CHO cells and [3H]-CP-55940that AM630 binds to the CB2 cannabinoid receptor (Ki =312 nM) with a selectivity ratio over 160 [48]Furthermore using a [35S]-GTPγ S assay they showed itsinverse agonist properties (EC50 = 76 nM) However

Table 12 CB2 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Reported binding affinities (Ki) and selectivity ratio (CB1CB2) of SR144528 (169) for rat and human cannabinoid receptors determined using [3H]-CP55940 as radioligand

NN

O

NH

Cl

SR144528 169

rCB2 (spleen) rCB1 (whole brain) Selectivity Ki hCB2 (transfected CHOcells)

Ki hCB1 (transfected CHOcells)

Selectivity References

038 nM 305 nM 800 06 nM 437 nM 730 [217]

067 nM 548 nM 80 [219]

56 nM gt10microM gt1780 [48]

25 nM a gt 1microM a gt400 [224]

024 nM 276 nM 115 199 nM 503 nM 25 [232]

a COS cells

1384 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

3 Diarylpyrazole Derivatives CHO cells is constitutively active and phosphorylated onSer 352 Upon treatment by SR144528 thisphosphorylation of the receptor was inhibited Moreover theSR compound was able to induce dephosphorylation ofagonist-induced CB2 phosphorylation The question whetherSR144528 induces a CB2 receptor conformational changemaking it a better phosphatase-specific substrate or ifSR144528 binding activates specific phosphatases remainsopen However this remains a nice example of inverseagonist-induced receptor resensitisation

The same Sanofi team which synthesised the CB1antagonist SR141716A reported in 1998 the synthesis andpharmacological characterisation of a CB2 cannabinoidreceptor selective antagonist the SR144528 (169) [217218] The structure of the new compound is derived fromSR141716A one SR144528 is the first selective CB2receptor antagonist reported as the inverse agonist propertiesof BML-190 were shown only recently by New et al [137]The authors reported for 169 using [3H]-CP-55940 asradioligand Ki values of 04 nM and 06 nM for the rat andthe human CB2 receptors respectively and found a CB1-CB2 selectivity ratio around 700 Table 12 sums up thebinding affinities of SR144528 for the cannabinoid receptorsobtained by different authors Interestingly while the CB2affinity is more or less constant the affinity obtained for theCB1 receptor varies by one order of magnitude [219] Theantagonist effect of SR144528 was highlighted by evaluatingits effect on cAMP production by hCB2-CHO cells exposedto 3 nM CP-55940 (EC50=10nM) The antagonism wasalso observed on the activation of the MAP kinase pathwayby CP-55940 (IC50=39nM)

The Sanofi research team working on cannabinoidsconducted investigations in order to determine the keyresidues for SR144528 interaction with the receptor (Table13) as well as the binding mode of the compound Firstlythey identified the TM4-EL2-TM5 region as a regioncontaining crucial residues for the affinity of several ligandsamong them SR144528 [224] Among these residues themutation of two cysteine of the second extracellular loop(C174 and C179) abolishes the affinity for the CB2 receptorLater on the implication of S453(161) and S457(165) inthe binding of SR144528 was evidenced by mutationalstudies In the model proposed for the docking of SR144528into the CB2 receptor the crucial contacts are comprised intothe TM4 this is the case for S453(161) and S457(165)which are proposed to form hydrogen bonds with the ligandand W464(172) proposed to have aromatic interactions withthe 4-methylbenzyl of the SR144528 [225] In 2003 Fengand Song showed that point mutations D349(130)A andA634(241)E abolished ligand binding However mutationof the arginine R350(131) member of the highly conservedDRY motif to alanine has no influence on SR144528binding [226]

Early after the report of the synthesis and characterisationof SR144528 the same authors further explored the functionof this compound and showed its inverse agonist propertiesusing CB2-CHO cells co-transfected with the luciferasereporter gene with either the CRE or the murine krox24regulatory sequence [220] In these models SR144528 notonly inhibits CP-55940 effects but also reproduces theeffects of a pertussin toxin treatment proving its inverseagonist properties SR144528 functionality was alsoexplored by [35S]-GTPγ S binding in hCB2-CHO cells(IC50=3 nM) [221] The inverse agonist function ofSR144528 was further illustrated by Rhee and Kim usingCOS cells co-transfected with hCB2 receptor and adenylylcyclase [222]

As for the CB1 cannabinoid receptor ligands Sanofideveloped tricyclic derivatives based on the 1-benzylpyrazole-3-carboxylic scaffold More than twentycompounds were disclosed in a patent [227] in 2001 (84-86Table 7) The benzyl in position 4 characteristic of the CB2diarylpyrazoles from Sanofi is conserved as well as thebicyclic residue in position 3 The length of the second linkbetween the phenyl and the pyrazole can be made of one or

Bouaboula and co-workers investigated SR144528 effectson CB2 cannabinoid receptor phosphorylation status [223]Firstly they showed that the CB2 receptor transfected in

Table 13 CB2 Cannabinoid Receptor Antagonists 3 Di-Aryl-Pyrazole Derivatives

Some of the single point mutations reported for the hCB2 cannabinoid receptor for which pharmacological data concerning the SR144528 are available

Mutant Effect References

R350(131)A no effect [226]

S453(161)A loss of affinity [225]

V456(164)I no effect [225]

S457(165)A loss of affinity [225]

C174S a loss of affinity [224 225]

C175S a loss of affinity [225]

C177S a no effect [225]

C179S a loss of affinity [224 225]

S544(193)G no effect [225]

a Second extra-cellular loop

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1385

two methylene but according to the patent one methylenebridge is preferred The Ki values of these compounds for theCB2 receptor were said to be lower than 500 nM and theirantagonist properties have been assessed using the adenylatecyclase inhibition assay

compounds by the presence of an alkyl substituent inposition 5 instead of a phenyl and of course by position 3which remains unsubstituted However except a lecture atthe 1998 ACS meeting [230] where compound 171 wasreported to have a Ki value of 835 nM and a selectivity ratioover 100 no further report on these compounds waspublished untill now

In a recent paper Mussinu and colleagues reported thesynthesis of other tricyclic derivatives starting fromSR141716A structure [228] N-piperidin-1-yl-14-di-hydroindeno[12-c]pyrazole-3-carboxamide derivatives areCB2 receptor ligands and some of them possess high affinityand selectivity for the CB2 receptor as shown in Table 7 (79-83) These tricyclic derivatives differ from the Sanofi tricyclicderivatives as they do have a phenyl ring instead of a benzylin position 1 Another difference is the nature of thesubstituent in position 3 which is not a bicycloalkyl but acyclic amine One of the compounds synthesised byMussinu et al 81 possesses the highest affinity for the CB2receptor ever reported with a Ki value of 0037 nM for themurine CB2 receptor ([3H]-CP-55940 mice spleenhomogenate) Interestingly 79 the closest SR141716Aanalog lost almost all its affinity for the CB1 receptor andincreased by four orders of magnitude its affinity for the CB2receptor Even if the structure quite similar to the SRcompounds could suggest that these derivatives behave asantagonists no pharmacological data were given in the paperto support this hypothesis

5 2-Oxoquinoline Derivatives

Inaba and colleagues from Japan Tobacco introduced in2001 a completely new structure in the cannabinoid fieldbased on a 2-oxoquinoline scaffold Over eighty derivativeswere disclosed in a patent [231] and one compound JTE-907 (173) was subsequently characterised in-vitro as well asin-vivo [232] In Table 14 are listed some of the compounds(172-178) disclosed in the patent along with their affinity forthe cannabinoid receptors These values were obtained onhuman receptors against [3H]-WIN-55212-2 For twelvecompounds the anti-inflammatory activity per os wasassessed in a carrageenin-induced paw edema model Thehighest effect reported was obtained with JTE-907 with anED50 value lower than 01 mgkg Therefore thesecompounds were claimed to be useful as anti-inflammatoryand anti-allergic agents

To further characterize JTE-907 the authors determinedits affinity for cannabinoid receptors of different species using[3H]-CP-55940 as radioligand In their hands JTE-907behaved as a very selective hCB2 ligand with Ki values of36 and 2370 nM for the CB2 and CB1 cannabinoid receptorsrespectively even more selective than the SR144528 (Kivalues of 199 and 50 nM respectively) However it has tobe mentioned that the affinities reported in the paper forJTE-907 are quite different from those reported in the patent(hCB1 Ki values of 35nM and 009 nM respectively) Italso appears that the SR144528 affinity for the CB1 receptordetermined by Iwamura et al is higher than the affinityreported by Rinaldi-Carmona et al with values of 50 and437nM respectively The authors ascribed the difference tothe variability of SR144528 binding affinities to the assayconditions The function of JTE-907 was evaluated bymeasuring the increase in cAMP production by forskolin-stimulated hCB2-CHO cells Maximum stimulation wasattained at a concentration of 1microM of JTE-907 In the sameassay WIN-55212-2 decreased the cAMP production in adose-dependent manner

Finally it is interesting to strengthen that the five-membered ring derivatives (79-86) of the diarylpyrazolefamily are reported to possess a high CB2 receptor affinitywhile the six- and seven-membered ring derivatives (69-78)are described as CB1 receptor ligands

4 Other Pyrazole Derivatives

Other CB2 ligands based on a pyrazole nucleus wereintroduced by SmithKline Beecham in a patent describingthe synthesis of about sixty new compounds [229] Theiraffinity (Ki) for the hCB2 receptor transfected in HEK293cells was reported to be ranging between 25nM and 10microMTheir structures (170-171 Fig 17) differ from the Sanofi

NN

O

N

ON

NN

N

NN

O

N

ON

NN

N

ClCl

1

5

4 3

2

170 171

Fig (17) CB2 cannabinoid receptor antagonists 4 Otherpyrazole derivatives Structure of two compounds (170-171)among those claimed by SmithKline Beecham to be hCB2antagonists

In conclusion JTE-907 was shown to be a CB2 selectiveinverse-agonist with anti-inflammatory and anti-allergicproperties This compound is currently undergoing Phase Iclinical trials as anti-allergic drug [233]

IV NON-CB1 NON-CB2 CANNABINOIDRECEPTORS LIGANDS

In the last years the possible presence of additional non-CB1 non-CB2 cannabinoid receptors was raised frompharmacological evidences

One of the putative receptors possesses an endotheliallocalisation while other ones are localized in the CNS or atperipheral nerves For instance an additional target for ∆9-

1386 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 14 CB2 Cannabinoid Receptor Antagonists 5 2-Oxoquinoline Derivatives

Structure of six compounds among the oxoquinoline derivatives patented by Japan Tobaco Affinity (Ki nM) and selectivity (CB1CB2) for the cannabinoidreceptors are given The affinity was measured by binding against [3H]-WIN-55212-2 on either hCB2-CHO or hCB1-CHO cells Adapted from [231]

Cpd ndeg Structure hCB2 hCB1 Selectivity

1-2 172

NH

MeO O

O

OMeO

0014 3671 262215

JTE-907 173

NH

O

MeO O

O

NH

O

O

0087 3436 39495

2-2 174

OH

O

NH

MeO O

O 077 3247 4215

3-27 175

NH

O

F

NH

MeO O

O0021 336 16000

3-32 176

NH

MeO O

O

NH

F

O

0036 2398 66610

3-38 177

O

O

NH

N

O

O

NMeO

0085 935 11000

3-43 178

NMeO O

O

NH

F

O

0043 864 20100

tetrahydrocannabinol was described in capsaicin-sensitivesensory nerves by Zygmunt et al [234]

[29] and by Di Marzo et al [235] Thus in the presentreview we will focus on the description of the so faridentified ligands of the non-CB1 non-CB2 receptors withan emphasis on the compounds having antagonist properties

Pharmacological evidences for the existence of additionalcannabinoid receptors were reviewed by Wiley and Martin

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1387

(Table 15) In particular the knowledge about three not yetcloned targets has grown The growing molecularpharmacology of the three putative new cannabinoidreceptors is reviewed in the three following sections Thefourth section summarises the data on the other proposedcannabinoid targets

vasodilatator activity In addition the anandamide effect ispartially sensitive to SR141716A inhibition The authorsproposed the existence of two distinct receptors responsiblefor the vasodilatatory effect of anandamide one located in theendothelium is SR141716A-sensitive while the other onelocated on the vascular smooth muscle is SR141716A-insensitive Thus SR141716A is an antagonist of anendothelial anandamide receptor Several studies wereconducted in order to understand the molecularpharmacology of the endothelial anandamide receptor It ispertussis toxin-sensitive [237] Cannabidiol acts asantagonist and abnormal-cannabidiol (Abn-cbd 179) and itsanalogue O-1602 (180) act as agonists [238] The synthesisof O-1602 and of its analogues are disclosed in a patentdescribing the vasodilatatory activity of cannabinoid

1 Endothelial Anandamide Receptor ndash Abnormal-Cannabidiol Receptor

In 1999 Wagner et al showed that anandamide couldinduce mesenteric vasodilatation through an endotheliallylocated ldquoanandamide receptorrdquo pharmacologically distinctfrom CB1 [236] Other cannabinoids such as ∆9-THC HU-210 or WIN-55212-2 were devoid of mesenteric

Table 15 Non-CB1 Non-CB2 Cannabinoid Receptors Antagonists

An overview of the putative new non-CB1 non-CB2 anandamide-receptors ligands The antagonists are highlighted in bold

NH

N

S

HO OH

Cl

OH

OH

OH OH OH OH

OMe

OMe

RR

RR

RR

RR

HO

OH

NH

O

cannabidiol (10) Abnormal-cannabidiol 179 O-1602 180

O-1918 181 capsazepine 183capsaicin 182

Compound Endothelial AEA receptor Brain AEA receptor Cannabinoid-vanilloid brain receptor

AEA agonist a agonist de

WIN-55212-2 no effecta

agonist e agonist g h

CP-55940 no effect e agonist i

SR141716A antagonist a b j no effect ([35 S]-GTPγS binding) d e f antagonist g h

AM251 no effect k no effect h i

Cannabidiol antagonist b no effect e

O-1918 antagonist c jm

Abd-cbd agonist b l

O-1602 agonist b

Capsaicin no effectj agonist i

Capsazepine no effect b antagonist h

a [236] b [238] c [240] d [249] e [250] f [251] g [254] h [258] i [ 255] k [244] j [ 243] l [ 242] m [241]

1388 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

analogues [239] More recently Offertaler et al showed thatO-1918 (181) a cannabidiol analog acts as an antagonist ofthe endothelial anandamide receptor [240] In addition O-1918 does not bind to the CB1 and CB2 cannabinoidreceptors Furthermore O-1918 unlike cannabidiol doesnot cause vasorelaxation in the mesenteric artery preparationmodel used and thus acts as a true neutral antagonistUsing O-1918 as a pharmacological tool Begg et alproposed a coupling of this new G protein-coupledendothelial receptor through a GiGo mechanism to ionchannels in primary cultured human vascular endothelialcells [241] However in their hands SR141716A wasineffective in antagonising Abd-cbd Ho and Hiley furtherdemonstrated that Abn-cbd relaxes rat small mesentericarteries through an activation of K+ channels via aSR141716A-sensitive pathway [242] Further using thesame model they characterized the vasorelaxant activity ofvirodhamine a novel endocannabinoid apparently actingthrough the same receptor [243] The virodhamine-inducedvasorelaxation is sensitive to SR141716A and O-1918 butinsensitive to AM251 SR144528 and AM630 Moreovervanilloid receptor desensitisation by capsaicin (182) had noeffect

244] Thus position 4 on the 5-phenyl could be furtherexplored to obtain selective ligands

To come to an end despite the fact that the endothelialanandamide receptor is neither identified nor cloned at leastthree antagonists are identified SR141716A cannabidioland O-1918 It is likely that a growing number of ligandsand therapeutic applications will appear in the very nearfuture

2 Brain Anandamide Receptor

Following an in-depth evaluation of anandamide effects inCB1

-- mice Di Marzo and co-workers suggested that a non-CB1 non-CB2 G protein-coupled receptor might mediatesome of the actions of AEA in mice [249] Actually effectsof AEA but not those of ∆9-THC were not decreased inCB1 cannabinoid receptor knock-out mice For instance 30mgkg of AEA were still effective in hot-plate test whereas10 mgkg of ∆9-THC had no effect on anti-nociceptionMoreover in a [35S]-GTPγ S assay AEA stimulated with anEC50 value of 223 microM the [35S]-GTPγ S binding to CB1

--

mice brain membranes (Emax= 28) ∆9-THC showed noeffect in this assay The addition of SR141716A did notaffect the [35S]-GTPγ S binding to CB1

-- mice brainmembranes Shortly after other evidences for the existence ofa new G protein-coupled cannabinoid receptor with a distinctdistribution in the central nervous system were published[250] From a set of 24 commonly used cannabinoids (AEAWIN-55212-2 JWH-030 THChellip) only AEA and WIN-55212-2 produced an increased binding of [35S]-GTPγ S onCB1

-- mice brain membranes Interestingly in this studySR141716A alone produced a significant inhibition of [35S]-GTPγ S binding similar to the inhibition obtained usingCB1

++ mice brain membranes (IC50 values of 57 microM and47 microM respectively) However SR141716A did not affectthe anandamide-induced [35S]-GTPγ S binding in CB1

--

mice brain membranes Moreover [3H]-SR141716Aexhibited significant binding in some brain regions of CB1

--

mice which are different from those exhibiting stimulation of[35S]-GTPγ S binding by WIN-55212-2 and AEA Thusthe authors proposed that the SR141716A effects on CB1

--

mice are mediated neither by the CB1 cannabinoid receptornor by the putative new anandamide brain receptor Monoryand co-workers using CB1

-- mice described a novel nonadenylyl cyclase-coupled cannabinoid binding site in micecerebellum [251] Two agonists AEA and WIN-55212-2but not ∆9-THC or HU210 were found to bind to thisreceptor as they enhanced [35S]-GTPγ S binding NeitherSR141716A nor SR144528 were able to affect the WIN-55212-2 induced [35S]-GTPγ S binding in CB1

-- mice asfound by Breivogel et al [250] However the distributionpattern of the receptor described by Monory et al differs fromthe distribution found by Breivogel et al It has to bementioned that the knock-out strains used were different Themice used by Monory were from a CD-1 strain while theone used by Breivogel were from a C57BL61 strainInterestingly Muthane et al detected differences in nigralneurons number and sensitivity to MPTP in the two mousestrains used [252]

More recently Mo et al obtained results showing thatthe Abnormal-cannabidiol receptor is also responsible for anincreased endothelial cell migration [244] Abn-cbd enhancedhuman umbilical vein endothelial cell migration while O-1918 antagonised Abn-cbd effect The CB1 cannabinoidreceptor inverse agonist SR141716A partially inhibitedAbd-cbd action whereas AM251 and SR144528 had noeffect

In another paper from George Kunos team Batkai et alreported that SR141716A is able to inhibit the endotoxichypotension by a cardiac mechanism involving neither theCB1 nor the CB2 cannabinoid receptors [245] Theinvolvement of a non-CB1 non-CB2 site of action wasshown using CB1

-- and CB1--CB2

-- knock-out miceFurther studies are needed to identify the SR141716Asensitive myocardial site of action

In a recent patent Kunos described O-1918 and itsanalogues as vasoconstrictor agents useful to reversepathological vasodilatation of blood vessels [246] Theeffects of O-1918 on mouse blood pressure on LPS-inducedhypotension in mouse as well as the reversal of hypotensionby O-1918 injection are described Furthermore thesynthesis of O-1918 based antagonists is described startingfrom p-metha-28-dien-1-ol and the appropriate resorcinols

Interestingly the Abd-cbd receptor which was firstdetected in the vascular system also seems to be present inthe CNS [247] Indeed Walter et al found that microglialcells (BV-2) migration induced by 1 microM 2-AG is inhibitedby cannabidiol (300 nM) and O-1918 (1 microM) SR141716Awas found ineffective in this assay Thus 2-AG is an agonistof the Abd-cbd receptor Moreover Abd-cbd is able to elicita dose-dependent cell migration in the same model (EC50 =600nM) [248]

From a more structural point of view SR141716Abehaves as an antagonist while on the opposite AM251(20) a close analogue which differs from the former only bythe nature of one halogen has no effect on this receptor [242

To conclude to our knowledge no compounds havingantagonist or inverse agonist properties at this receptor havebeen identified so far

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1389

3 Cannabinoid-Vanilloid Brain Receptor a cannabinoid-vanilloid receptor at which SR141716A andcapsazepine behave as antagonists

Evidences for the presence of an additional ldquocannabinoidrdquoreceptor differing from the CB1 receptor and from the brainanandamide receptor appeared from studies focused on thehippocampus (for a review see Hajos et al [253]) Hajos etal studied the cannabinoid actions on GABAergic andglutamatergic transmission using the whole-cell patch-clamp technique [254] WIN-55212-2 induced a dose-dependent reduction in glutamatergic transmission reversedby SR141716A (1microM) Thus the authors concluded that thecannabinoid actions on the glutamatergic transmission aremediated by a non-CB1 but SR141716A-sensitive receptorIn another paper Hajos and Freund further characterised thisputative receptor [255] They showed that in addition toWIN-55212-2 CP-55940 and capsaicin (182) a TRPV1receptor agonist also act as agonists Furthermore in theirhands SR141716A as well as capsazepine (183) a TRPV1receptor antagonist were able to antagonise this effect on theglutamatergic transmission while AM251 was notSR141716A effects on CB1 knock-out mice were furtherstudied using a test of anxiety [256] Haller et al showedthat on the one hand the distribution of the CB1 receptorinduces anxiety and SR141716A has an anxiolytic effectand on the other hand SR141716A is still active in CB1

--

mice In a following paper they reported that AM-251induces anxiogenic effects in wild-type and in knock-outmice [257] Thus SR141716A in addition to CB1 bindsto a not yet identified receptor Another evidence for thisadditional receptor for SR141716A was given by Bass andco-workers [61] They conducted a structure-activityrelationship study on the stimulation of locomotor activityinduced by SR141716A and twenty analogues Nocorrelation was found between the affinity for the CB1cannabinoid receptor and the stimulation of locomotoractivity Albeit SR141716A and five of its analogues O-1803 O-1710 O-1253 O-1254 (40) O-1255 (41) behavedas inverse agonists in [35S]-GTPγ S assay none of theseanalogues stimulated the locomotor activity In conclusionthe SR141716A-induced stimulation of locomotor activity isneither due to inverse agonism nor to inhibition of anendogenous tone but to a brain receptor distinct from theCB1 receptor Di Marzo et al previously provided evidencesthat the cannabinoid-vanilloid brain receptor could mediatesome of the cannabinoid effects on locomotion [249]

4 Other Non-CB1 Non-CB2 Cannabinoid Receptors

Evidences for the existence of additional non-CB1 non-CB2 cannabinoid receptors localised in the rat isolatedmesenteric arterial bed appeared from the works of Ralevicand collaborators Several cannabinoids were evaluated intheir model of sensory neurogenic vasorelaxation evoked byelectrical field stimulation

HU210 was found to attenuate vasorelaxation in a CB1and CB2 independent way as SR141716A LY320135 andSR144528 were ineffective in inhibiting HU210 effect [259]Similar results were obtained with ∆9-THC [260]Similarly noladin ether inhibited sensory neurogenicrelaxation in a concentration-dependent manner which wasalso unaffected by SR141716A LY320135 and SR144528Moreover this effect as the ∆9-THC one was pertussistoxin sensitive suggesting the involvement of a Gio protein-coupled receptor [261]

However the inhibitory effects of CP-55940 and WIN-55212-2 were affected by the presence of SR141716A (1microM)or LY320135 (1microM) but not by the addition of SR144528(1microM) [262]

It appears that at least two receptors should be involvedin cannabinoid-mediated attenuation of sensory nerve-mediated vasorelaxation The first one is SR141716A andLY320135 sensitive while the other one is resistant toSR141716A

Some evidences for the existence of a ldquoCB2-likerdquo receptorappeared from the studies on palmitoylethanolamide (PEA)an endogenous fatty acid amide devoid of CB1 and CB2affinity [264] antinociceptive potential Calignano and co-workers showed that PEA was able to alleviate pain inseveral animal models [265 266] For instance PEA dose-dependently inhibits kaolin-evoked writhing in mice TheCB2 cannabinoid receptor inverse agonist SR144528 (02mgkg) inhibits PEA anti-nociceptive effect [266] Thus theauthors suggest that PEA acts as agonist on a SR144528-sensitive non-CB2 cannabinoid receptor (ldquoCB2-likerdquoreceptor) at which SR144528 acts as an antagonist

Further studies are needed in order to better characterisethese additional ldquocannabinoidrdquo receptors However it islikely that several therapeutic applications will soon springfor these receptors

Very recently Pistis et al published a study on theneurophysiological effects of cannabinoids on the basolateralamygdala neurons in-vivo [258] One of their findings is thatHU-210 and WIN-55212-2 did not elicit similar effects onbasal amygdala projection neurons firing rate While theformer had no effect the latter decreased the firing rateMoreover SR141716A reversed the WIN-55212-2 effectswhile AM251 had no effect The vanilloid receptorantagonist capsazepine also antagonised the WIN-55212-2effect on firing rate of these neurons Therefore the authorssuggested the presence of a novel cannabinoid-vanilloidreceptor

V CONCLUSION

Since their development cannabinoid antagonists provedto be essential tools in the understanding of cannabinoidpharmacology and biochemistry Moreover the interestshown by the pharmaceutical industry in the development ofnew cannabinoid antagonists prove if necessary the realtherapeutic potential of this class of compounds Thetreatment of eating and movement disorders memorydeficits psychosis and dependencies from various addictivedrugs are some of the most cited applications for the CB1cannabinoid receptor antagonists

From these lines it appears that in addition to theknown CB1 cannabinoid receptor it is now likely that twoother cannabinoid receptors do exist in the brain The firstone should be an anandamide brain receptor for whichSR141716A has no affinity while the second one should be

1390 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Much less wide is the knowledge on the CB2cannabinoid receptor antagonists However potentialapplications could be disorders involving the immunesystem such as inflammation or allergies

[19] Devane WA Hanus L Breuer A Pertwee RG StevensonLA Griffin G Gibson D Mandelbaum A Etinger AMechoulam R Science 1992 258 1946

[20] Mechoulam R Ben-Shabat S Hanus L Ligumsky MKaminski NE Schatz AR Gopher A Almong S MartinBR Compton DR Biochem Pharmacol 1995 50 83

Finally the growing literature on the non CB1 non CB2cannabinoid receptors should allow the development of newdrugs First examples are given by the cannabidiol andabnormal-cannabidiol derivatives described by Kunos et alfor which potential therapeutic applications on the vascularsystem are already patented

[21] Sugiura T Kondo S Sukagawa A Nakane S Shinoda AItoh K Yamashita A Waku K Biochem Biophys Res Co1995 215 89

[22] Hanus L Abu-Lafi S Fride E Breuer A Vogel Z ShalevDE Kustanovich I Mechoulam R Proc Natl Acad SciUSA 2001 98 3662

[23] Oka S Tsuchie A Tokumura A Muramatsu M Suhara YTakayama H Waku K Sugiura T J Neurochem 2003 851374It clearly appears from this paper that the antagonists of

the cannabinoid receptors are still an active research fieldfrom which should emerge new promising therapeutic toolsas well as innovative drugs in the near future

[24] Goutopoulos A and Makriyannis A Pharmacol Therapeut2002 95 103

[25] Beal JE Olson R Lefkowitz L Laubenstein L Bellman PYangco B Morales JO Murphy R Powderly W PlasseTF Mosdell KW Shepard KV J Pain Symptom Manage1997 14 7ACKNOWLEDGEMENTS [26] Notcutt W Price M Miller R Newport S Phillips CSimmons S Sansom C Anaesthesia 2004 59 440

GGM is very indebted to the ldquoFonds pour la formationagrave la Recherche dans lrsquoIndustrie et lrsquoAgriculture (FRIA)rdquo Belgium for the award of a research fellowship Theexperimental works of the authors were supported by grantsfrom the FNRS The Belgian Charcot Foundation and theUniversiteacute catholique de Louvain

[27] Ledent C Valverde O Cossu G Petitet F Aubert JFBeslot F Bohme GA Imperato A Pedrazzini T RoquesBP Vassart G Fratta W Parmentier M Science 1999 283401

[28] Zimmer A Zimmer AM Hohmann AG Herkenham MBonner TI Proc Natl Acad Sci U S A 1999 96 5780

[29] Wiley JL and Martin BR Chem Phys Lipids 2002 121 57[30] Adam J and Cowley P Expert Opin Ther Patents 2002 12

1475

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[33] Little PJ Compton DR Johnson MR Melvin LS MartinBR J Pharmacol Exp Ther 1988 247 1046

[2] Devane WA Dysarz FA Johnson MR Melvin LSHowlett AC Mol Pharmacol 1988 34 605

[34] Pertwee RG Stevenson LA Elrick DB Mechoulam RCorbett AD Brit J Pharmacol 1992 105 980

[3] Matsuda LA Lolait SJ Brownstein MJ Young ACBonner TI Nature 1990 346 561

[35] Harrison C Traynor JR Life Sci 2003 74 489-508[4] Gerard CM Molerau C Vassart G Parmentier M BiochemJ 1991 279 129 [36] Pertwee RG Fernando SR Griffin G Ryan W Razdan

RJ Compton DR Martin BR Eur J Pharmacol 1996 315195

[5] Glass M Dragunow M Faull RLM Neuroscience 1997 2299

[37] Ross RA Brockie HC Fernando SR Saha B Razdan RKPertwee RG Brit J Pharmacol 1998 125 1345

[6] Shire D Carillon C Kaghad M Calandra B Rinaldi-Carmona M LeFur G Caput D Ferrara P J Biol Chem1995 270 3726 [38] Griffin G Wray EJ Rorrer WK Crocker PJ Ryan WJ

Saha B Razdan RK Martin BR Abood ME Brit JPharmacol 1999 126 1575

[7] Ryberg E Khang Vu H Groblewski T Larsson N ElebringT Hjorth S Sjogren S Greasley PJ 2004 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 119

[39] Ross RA Gibson TM Stevenson LA Saha B Crocker PRazdan RK Pertwee RG Brit J Pharmacol 1999 128 735

[40] Martin BR Winckler JR Wiley JL Huffman JW CrockerPJ Saha B Razdan RK J Pharmacol Exp Ther 1999 2901065

[8] Rinaldi-Carmona M Calandra B Shire D Bouaboula MOustric D Barth F Casellas P Ferrara P LeFur G JPharmacol Exp Ther 1998 278 871

[41] Martin BR Stevenson LA Pertwee RG Breivogel CSWilliams A Mahadevan A Razdan RK 2002 Symposium onthe Cannabinoids Burlington Vermont International CannabinoidResearch Society page 2

[9] Munro S Thomas KL Abu-Shaar M Nature 1993 365 61[10] Howlett AC Qualy JM Khachatrian LL Mol Pharmacol

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[42] Thomas A Ross RA Saha B Mahadevan A Razdan RKPertwee RG Eur J Pharmacol 2004 487 213

[12] Calandra B Portier M Kerneacuteis A Delpech M Carillon C LeFur G Ferrara P Shire D Eur J Pharmacol 1999 374445 [43] Compton DR Gold LH Ward SJ Balster RL Martin BR

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Daum SJ Ackerman JH Gruett MD Kumar V EstepKG Olefirowicz EM Wetzel JR Alexander MD WeaverJD Haycock DA Luttinger DA Casiano FM ChippariSM Kuster JE Stevenson JI Ward SJ J Med Chem1995 38 3094

[14] Deadwyler S Hampson RE Mu J Whyte A Childers SRJ Pharmacol Exp Ther 1995 273 734

[15] Mackie K Lai Y Westenbroek R Mitchell R J Neurosci1995 15 6552

[16] Bouaboula M Poinot CC Bourrie B Canat X Calandra BRinaldi-Carmona M LeFur G Casellas P Biochem J 1995312 637 [45] Pertwee R Griffin G Fernando S Li X Hill A

Makriyannis A Life Sci 1995 56 1949[17] Bouaboula M Poinot CC Marchand J Canat X Bourrie BRinaldi-Carmona M Calandra B LeFur G Casellas P Eur JBiochem 1996 237 704

[46] Hosohata Y Quock RM Hosohata K Makriyannis AConsroe P Roeske WR Yamamura HI EurJPharmacol 1997 321 R1[18] delPulgar T Velasco G Guzman M Biochem J 2000 347

369 [47] Landsman RS Makriyannis A Deng H Consroe P RoeskeWR Yamamura HI Life Sci 1998 62 109

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[48] Ross RA Brockie HC Stevenson LA Murphy VLTempleton F Makriyannis A Pertwee RG Brit JPharmacol 1999 126 665

[81] Gatley SJ Lan R Volkow ND Pappas N King P WongCT Gifford AN Pyatt B Dewey SL Makriyannis A JNeurosci 1998 70 417

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B Congy C Martinez S Maruani J Neliat G Caput DFerrara P Soubrieacute P Breliegravere JC Le Fur G FEBS Lett1994 350 240

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[182] Lange JHM van Stuivenberg HH Coolen HKAC AdolfsT JP McCreary AC Keizer HG Walls HC Veerman WBorst AJM de Loff W Verveer PC Kruse CG J MedChem 2005 48 1823

[212] Zhang Q Ma P Wang W Cole RB Wang G J MassSpectrom 2004 39 672

[213] Gallant M Dufresne C Gareau Y Guay D Leblanc YPrasit P Rochette C Sawyer N Slipetz D M Tremblay NBioorg Med Chem Lett 1996 6 2263[183] Lange JHM Kruse CG Tipker J Hoogendorn J World

Patent Application 2002 WO02076949 [214] Melck D De Petrocellis L Orlando P Bisogno T Laezza CBifulco M Di Marzo V Endocrinology 2000 141 118[184] Lange JHM Coolen H K A C van Stuivenberg HH

Dijksman JAR Herremans AHJ Ronken E Keizer HGTipker K McCreary AC Veerman W Wals HC Stork BVerveer PC den Hartog AP de Jong NMJ Adolfs TJPHoogendoorn J Kruse CJ J Med Chem 2004 47 628

[215] Kalgutkar AS Marnett AB Crews BC Remmel RPMarnett LJ J Med Chem 2000 43 2860

[216] Klegeris A Bissonnette CJ McGeer PL Brit J Pharmacol2003 139 775

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[217] Rinaldi-Carmona M Barth F Millan J Derocq JM CasellasP Congy C Oustric D Sarran M Bouaboula M CalandraB Portier M Shire D Breliere JC Le Fur GL JPharmacol Exp Ther 1998 284 644

[186] Wilstermann JM and Berggren AIK World PatentApplication 2003 WO03051850

[187] Berggren AIK Bostrom SJ Elebring ST Greasley PTerricabra E Wilstermann JM World Patent Application 2003WO03051851

[218] Barth F Millan J Casellas P Oustric D Sarran M RinaldiM World Patent Application 1997 WO9721682

[219] Griffin G Wray EJ Tao Q McAllister SD Rorrer WKAung MM Martin BR Abood ME Eur J Pharmacol 1999377 117

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[189] Barth F Martinez S Rinaldi-Carmona M World PatentApplication 2003 WO03084930

[220] Portier M Rinaldi-Carmona M Pecceu F Combes T Poinot-Chazel C Calandra B Barth F Le Fur G Casellas P JPharmacol Exp Ther 1999 288 582[190] Barth F Martinez S Rinaldi-Carmona M World Patent

Application 2003 WO03084943 [221] Bouaboula M Desnoyer N Carayon P Combes T CasellasP Mol Pharmacol 1999 55 473[191] Kopka I E Li B Hagmann WK World Patent Application

2004 WO04029204 [222] Rhee M-H and Kim S-K J Vet Sci 2002 3 179[192] Hagmann WK Lin LS Shah SK Guthikonda RN Qi H

Chang LL Liu P Armstrong HM World Patent Application2003 WO03077847

[223] Bouaboula M Dussossoy D Casellas P J Biol Chem 1999274 20397

[224] Shire D Calandra B Bouaboula M Barth F Rinaldi-Carmona M Casellas P Ferrara P Life Sci 1999 65 627[193] Hagmann WK Lin LS Shah SK Goulet MT Jewell JP

World Patent Application 2003 WO03082190 [225] Gouldson P Calandra B Legoux P Kerneis A Rinaldi-Carmona M Barth F Le Fur G Ferrara P Shire D Eur JPharmacol 2000 401 17

[194] Castonguay LA Hagmann WK Lin LS Shah SK WorldPatent Application 2003 WO03086288

[195] Hagmann WK Lin LS Shah SK World Patent Application2003 WO03087037

[226] Feng W and Song ZH Biochem Pharmacol 2003 63 1077[227] Barth F Millan J Oustric D Rinaldi M Vernhet M World

Patent Application 2001 WO0132629[196] Berggren AIK Bostrom SJ Cheng L Elebring STGreasley P Nagard M Wilsterlmann JM Terricabras EWorld Patent Application 2004 WO04058249

[228] Mussinu JM Ruiu S Mule AC Pau A Carai MA LorigaG Murineddu G Pinna GA Bioorg Med Chem 2003 11251[197] Guba W Haap W Marty HP Narquizian R United States

Patent Application 2004 US 20040147572 [229] Xiang JN Elliott JD Atkinson ST Christensen SB WorldPatent Application 1998 WO9831227[198] Jagerovic N Hernandez Folgado L Alkorta I Goya P

Navarro M Serrano A de Fonseca FR Dannert MTDannert MT Alsasua A Suardiaz M Pascual D MartinMI J Med Chem 2004 47 2939

[230] Xiang JN Atkinson ST Wang DY Lee J Lee JCSiegfried B 216th ACS meeting Boston (MA) USA 23-27 Aug1998

[199] Jagerovic N Goya Laza MP Hernandez Folgado L AlcortaOsoro IJ Martin Fontelles MI Suardiaz Garcia MLDannert MT World Patent Application 2003 WO03082833

[231] Inaba T Kaya T Iwamura H United States Patent 2003 US6509352 B1

[232] Iwamura H Suzuki H Ueda Y Kaya T Inaba T JPharmacol Exp Ther 2001 296 420[200] Lange JHM Kruse CG Herremans AHJ VanStuivenberg

HH Dijksman JAR McCreary AC World PatentApplication 2003 WO03078413

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[201] Berggren AIK Bostrom SJ Elebring ST Fallefors LWilstermann JM Greasley P World Patent Application 2004WO04058255

[234] Zygmunt PM Andersson DA Houmlgestaumltt ED J Neurosci2002 22 4720

[235] Di Marzo V De Petrocellis L Fezza F Ligresti A BisognoT Prostaglandins Leukot Essent Fatty Acids 2002 66 377[202] Toupence RB Debenham JS Goulet MT Madsen-Duggan

CB Walsh TE Shah SK World Patent Application 2004WO04012671

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[238] Jarai Z Wagner JA Varga K Lake KD Compton DR Martin BR Zimmer AM Bonner TI Buckley NE MezeyE Razdan RK Zimmer A Kunos G Proc Natl Acad Sci US A 1999 96 14136

[204] Griffith DA United States Patent Application 2004 US20040092520

[205] Griffith DA United States Patent Application 2004 US20040157839

[239] Kunos G Martin B Razdan R World Patent Application 2001WO0103690

[206] Griffith DA United States Patent Application 2004 US20040157838

[240] Offertaler L Mo F-M Batkai S Liu J Begg M RazdanRK Martin BR Bukoski RD Kunos G Mol Pharmacol2003 63 699[207] Bayewitch M Avidor-Reiss T Levy R Barg J Mechoulam

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[245] Batkai S Pacher P Jarai J Wagner JA Kunos G Am JPhysiol-Heart Circul Physiol 2004 287 H595[211] Showalter VM Compton DR Martin BR Abood ME J

Pharmacol Exp Ther 1996 278 989

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[246] Kunos G World Patent Application 2003 WO03015700 [257] Haller J Varga B Ledent C Freund TF Behav Pharmacol2004 15 299[247] Franklin A and Stella N Eur J Pharmacol 2003 474 195

[248] Walter L Franklin A Witting A Wade C Xie Y KunosG Mackie K Stella N J Neurosci 2003 23 1398

[258] Pistis M Perra S Pillolla G Melis M Gessa GL MuntoniAL Neuropharmacology 2004 46 115

[249] Di Marzo V Breivogel C S Tao Q Bridgen DT RazdanRK Zimmer AM Zimmer A Martin BR J Neurochem2000 75 2434

[259] Ralevic V and Kendall DA Eur J Pharmacol 2001 418 117[260] Duncan M Kendall DA Ralevic V Brit J Pharmacol 2003

138 214P[250] Breivogel CS Griffin G Di Marzo V Martin BR Mol

Pharmacol 2001 60 155[261] Duncan M Millns P Smart D Wright JE Kendall DA

Ralevic V Brit J Pharmacol 2004 142 509[251] Monory K Tzavara ET Lexime J Ledent C Parmentier

M Borsodi A Hanoune J Biochem Bioph Res Commun2002 292 231

[262] Duncan M Kendall DA Ralevic V J Pharmacol Exp Ther2004 311 411

[263] Tao Q and Abood ME J Pharmacol Exp Ther 1998 285651[252] Muthane U Ramsay KA Jiang H Jackson-Lewis V

Donaldson D Fernando S Ferreira M Przedborki S ExpNeurol 1994 126 195

[264] Lambert DM DiPaolo FG Sonveaux P Kanyonyo MGovaerts SJ Hermans E Bueb J-L Delzenne NMTschirhart EJ Biochim Biophys Acta 1999 1440 266[253] Hajos N Freund TF Chem Phys Lipids 2002 121 73

[254] Hajos N Ledent C Freund TF Neuroscience 2001 106 1 [265] Calignano A La Rana G Giuffrida A Piomelli D Nature1998 394 277[255] Hajos N Freund TF Neuropharmacology 2002 43 503

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[266] Calignano A La Rana G Piomelli D Eur J Pharmacol 2001419 191

Page 3: Current Knowledge on the Antagonists and Inverse Agonists of

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1363

on the negative coupling of cannabinoid receptors toadenylyl cyclase The binding of an agonist will produce adecrease in cAMP production which can be measured eitherdirectly (EIA) or through a gene-reporter system (fireflyluciferase) However due to the existence of a dual-couplingfor the CB1 cannabinoid receptor the [35S]-GTPγ S assayshould be preferred It is based on the property shared by allthe GPCRs to bind to a GTP molecule upon activation byan agonist Therefore the binding of an agonist will increasethe [35S]-GTPγ S a radiolabelled non-hydrolysable analogueof GTP binding [35] These assays allow to distinguishbetween full agonists (positive intrinsic activity) partialagonists neutral antagonists (no intrinsic activity) andinverse agonists (negative intrinsic activity)

antinociception nor hypothermia in mice and lacks ofagonist effects in mouse vas deferens preparation In thisassay it behaved as an antagonist devoid of inverse agonistproperties

Recently Thomas et al reported the synthesis andcharacterisation of O-2654 (9) (Fig 1) obtained bymodifying the structure of cannabidiol (or cbd) (10) a nonpsychoactive cannabinoid [42] This compound unlikecannabidiol binds to the CB1 receptor with a Ki value of114 nM against [3H]-CP-55940 on mouse brainmembranes In the mouse vas deferens model O-2654antagonises the WIN-55212-2 inhibition of current-inducedcontractions causing a rightward shift in the logconcentration response curve of the agonist (KB = 857 nM)Further in the vas deferens model O-2654 behaves as aneutral antagonist However this neutral antagonism has tobe further confirmed using other models

II CB1 CANNABINOID RECEPTOR ANTAGONISTSAND INVERSE AGONISTS

OH

OH

N3

O-2654 (9)

Compounds having antagonist or inverse agonistproperties at the CB1 cannabinoid receptor are reviewed inthis part of the paper They are classified depending on theirchemical structures

1 Tetrahydrocannabinol and Cannabidiol Derivatives

The first attempts to obtain cannabinoid ligands havingantagonist properties were conducted using the tricyclicstructure of classical cannabinoids such as ∆9-tetrahydrocannabinol (∆9-THC) as a scaffold These earlyresearches have been previously reviewed by Barth andRinaldi-Carmona [32] The most promising modulationsinvolved the side-chain of ∆8-tetrahydrocannabinol (∆8-THC) as it is the case in O-823 (1) (Table 1) Thiscompound (Ki = 077 nM) acts as a partial agonist in mousevas deferens preparations but as an antagonist in guinea-pigmyenteric plexus preparations [36] Ross and co-workers[37] showed that O-1184 (2) binds to the CB1 receptor (Ki =285 nM) and as O-823 possesses antagonist properties inthe myenteric plexus-longitudinal muscles O-823 and O-1184 were shown to act as surmountable antagonists in a[35S]-GTPγ S assay with KB values of 485 and 297 nMrespectively against CP-55940 in rat cerebellar membranes[38] Three other ∆8-tetrahydrocannabinol-3rsquo-ynylderivatives O-584 (3) O-806 (4) and O-1176 (5) alsobehaved as antagonists However in a cAMP productionassay conducted in hCB1 transfected CHO cells O-1184behaved as an agonist equipotent to CP-55940 [39] Takentogether these examples illustrate that the introduction of anacetylenic moiety into the side chain of ∆8-THC affects theactivation of the receptor more than the affinity of thesederivatives Several of these compounds were tested in-vivoin the cannabinoid tetrad test by Martin and co-workers [40]For instance O-823 which was inactive in the tetrad actedas an antagonist in the [35S]-GTPγ S assay [38]

Fig (1) CB1 cannabinoid receptor antagonists 1Tetrahydrocannabinol and cannabidiol derivatives Chemicalstructure of (-)-6-azidohex-2-yne-cannabidiol (O-2654 9)

2 Aminoalkylindole Derivatives

This family of compounds was introduced by Sterlingrsquosresearchers in the early nineties with a derivative of the anti-inflammatory drug pravadoline called WIN-55212 Albeitthis compound acts as an agonist some related compoundsshowed interesting antagonist properties in the mouse vasdeferens assay by dose-dependently antagonising ∆9-THCand levonantradol effects This is the case for instance for

N

O OMe

N

O

X

N

O

N

O

WIN-56098 (11) X=Br WIN-54461 (12)X=I AM630 (13)

Fig (2) CB1 cannabinoid receptor antagonists 2Aminoalkylindole derivatives Structures of WIN-56098 (11 )WIN-54461 (12 ) and AM630 (13 ) three aminoalkylindolederivatives acting as CB1 cannabinoid receptor antagonists

More recently Martin et al described three newcompounds possessing an alkyl sulfonamide at the end of thealkyne side chain [41] The ethyl (7) (Ki = 30 nM) andbutyl (Ki = 70 nM) derivatives were shown to behave asagonists in the cannabinoid tetrad while the methylderivative (8) (Ki = 25 nM) acted as a silent antagonistThis compound O-2050 does not induce neither

1364 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

WIN-56098 (11) and WIN-54461 (12) the latter being abromo derivative of pravadoline (Fig 2) These twocompounds however have a low affinity for the cannabinoidreceptor (IC50 gt 500 nM) [4344]

cAMP accumulation in hCB1-CHO cells with an IC50 valueof 56 nM SR141716A is able after ip or poadministration to inhibit the [3H]-CP-55940 binding tomice brain measured ex-vivo with ED50

values around 2mgkg [51] In addition to the inhibition of the classicalcannabinoid tetrad effects ndash hypothermia ring immobilityanalgesia and hypolocomotion ndash already shown by Rinaldi-Carmona et al in their first report [50] SR141716A wasshown to antagonise other in-vivo effects of cannabinoidagonists For instance the agonist-induced hypotension andbradycardia in mice are abolished by SR141716A [52] aswell as the antihyperalgesic effects of the cannabinoidagonists in a neuropathic model of pain in rat [53] This isalso the case for the behavioral effects of agonists treated rats[54] or the cannabinoid tetrad effects induced by ∆9-THC inmice [55]

In 1995 Pertwee et al [45] described the effect ofAM630 (13) another close analogue of pravadoline Thiscompound behaved as an antagonist in a [35S]-GTPγ S assayon mouse brain preparations antagonising WIN-55212-2-induced [35S]-GTPγ S binding [46] and as an inverseagonist on hCB1-CHO cells [47] (EC50 = 09 microM)However Ross et al found a weak partial agonist activityfor this compound as it decreases cAMP production byhCB1-CHO cells [48] AM630 was subsequentlycharacterised as a CB2 ligand (see the CB2 section) Thepharmacological properties of the aminoalkylindole familywere reviewed by John Huffman [49]

3 Diarylpyrazole DerivativesDespite various papers described SR141716A as an

antagonist [56-58] today this compound is considered to actas an inverse agonist based on [35S]-GTPγ S [59-62] andcAMP accumulation assays [62-64] (Table 2) An interestingreview dealing with inverse agonism at the cannabinoidreceptors and mostly with SR141716A effects was recentlypublished by Roger Pertwee [65]

The lead of this class of compounds SR141716A (14)was introduced by Sanofi back in 1994 [50] This compoundwas shown by Rinaldi-Carmona and co-workers to be ahighly selective CB1 ligand with Ki values of 56 nM for thehCB1 and over 1000 nM for the hCB2 receptors expressed inCHO cells ([3H]-CP-55940) However more recentlyevidences appeared showing that SR141716A binds toother(s) receptor(s) described as anandamide andorcannabinoid receptors Thus it is possible that some of thein-vivo effects caused by this compound are at least notsolely CB1 mediated (see the fourth section of this paper)

In 1998 Pan et al [66] demonstrated that the lysineresidue K328 (192) located in the third transmembranedomain (TMH3) of the hCB1 receptor is a key residue forthe inverse agonist action of SR141716A (Table 3) Theyshowed that SR141716A enhances calcium current in hCB1transfected neurons but not in K(192)A mutant receptortransfected neurons However SR141716A still antagonizedWIN-55212-2 inhibition of calcium currents proving that itis able to bind to the mutated receptor Later on Hurst et al[67] demonstrated using molecular modeling techniques aswell as in-vitro experiments that lysine residue K328(192)is a direct interaction site for hydrogen bonding with the C3substituent of SR141716A in CB1 receptor Binding results

In a mouse vas deferens preparation SR141716A causesa rightward shift of the CP-55940 concentration-responsecurve behaving as a competitive antagonist having a pA2value of 798 Furthermore in the cAMP accumulationmodel SR141716A produces no effect by itself butantagonizes the CP-55940 inhibition of forskolin-induced

Table 2 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

In-Vitro Functional Characterisation of SR141716A (14) The Assay Used the Cell Type and the Obtained Effect are Given

Cell type Assay Effect Function References

hCB1-CHO cells cAMP accumulation no effect by itself antagonist [50]

hCB1-CHO cells cAMP accumulation uarr [cAMP] inverse agonist [64]

hCB1-CHO cells [35 S]-GTPγS darr[35 S]-GTPγS binding inverse agonist [59]

hCB1-neurons Ca2+ currents uarr Ca2+ currents inverse agonist [66]

rCB1 (rat cerebella) [35 S]-GTPγS No effect by itself antagonist [56]

rCB1 (rat cerebella) [35 S]-GTPγS No effect by itself antagonist [57]

rCB1 (rat cerebella) [35 S]-GTPγS No effect by itself antagonist [58]

rCB1 (rat brain) [35 S]-GTPγS darr[35 S]-GTPγS binding inverse agonist [62]

rCB1 (rat cerebella) [35 S]-GTPγS darr[35 S]-GTPγS binding inverse agonist [60]

rCB1 (rat cerebella) [35 S]-GTPγS darr[35 S]-GTPγS binding inverse agonist [61]

rCB1 (rat brain) cAMP accumulation uarr [cAMP] inverse agonist [63]

mCB1 (mouse brain) cAMP accumulation uarr [cAMP] inverse agonist [62]

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1365

obtained using HEK293 cells transfected with either theK328A mutant or the wild type receptor remarkablyconfirmed the modeling results (Kd values of 396 and 23nM respectively) Interestingly a vinyl-cyclohexylSR141716A derivative VCHSR1 (15) (Table 4) lacking ofhydrogen bonding sites in C3 position is not affected bythis CB1 receptor mutation as its affinity remains unchangedwith Ki values of 31 and 35 nM for the wild-type andK328A receptors respectively Four additional compoundsCHASR1 (16) CHMSR1 (17) VPSR1 (18) and PIMSR(19) differing by their potential to form hydrogen bondswere evaluated in affinity and functional assays The resultsfurther confirm the crucial interaction between the C-3carboxamide oxygen and residue K328 to obtain an inverseagonist effect as highlighted in Table 4 [68] However it hasto be said that this lysine residue is also crucial for agonistbinding (CP-55940) andor receptor activation (WIN-55212-2) [69 70]

binding of SR141716A In contrast the substitution oftyrosine by isoleucine (Y275I) resulted in the loss of ligandrecognition Calculation studies revealed that while theY539F mutant is very similar to the wild type receptor theY539I mutant shows topology changes in the 3-4-5transmembrane region This region is considered to becrucial for agonistantagonist binding at CB1 receptor sincethe previous report by Shire et al on CB1CB2 receptorchimeras [72] In contrast the first and third extracellular(EC1 and EC3) loops of the hCB1 receptor are not essentialfor the binding of SR141716A as illustrated by Murphy etal The authors constructed several receptors mutated in theirEC1 or EC3 and none of the tested mutations affected theSR141716A binding [73] McAllister et al due the highlyaromatic nature of SR141716A further explored thehypothesis that an aromatic microdomain comprised intransmembrane helix 3-4-5-6 is the SR141716A bindingsite The modelling and mutation studies undertakensuggested to the authors that this aromatic microdomaincomprised of F336 W464 Y539 W543 and W648should represent the binding site of SR141716A Moreoverthey identified F336 and W543 as direct interaction sitesfor SR141716A [74] Residue F336 was further shown bythe same group to be a key residue for both ligand binding(WIN-55212-2 and SR141716A) and receptor activationMutation of phenylalanine 336(201) to alanine resulted in

Several other studies were undertaken to determine thecritical residues for the binding of cannabinoid compoundsUnfortunately in most of the studies SR141716A was notused However McAllister et al [71] using modeling toolsand mutagenesis explored the importance of aromaticity inposition 539(275) in the CB1 receptor The substitution oftyrosine by phenylalanine (Y275F) has no effect on the

Table 3 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Summary of the Reported CB1 Cannabinoid Receptor Single Point Mutations for which Pharmacological Data Concerning the SR141716A (14) areAvailable

Mutation Species Effect References

S114A a human no significant effect [73]

S115A a human no significant effect [73]

D250(163)N human no significant effect [263]

D250(163)E human no significant effect [263]

H181A b human no significant effect [73]

R182A b human no significant effect [73]

K183A b human no significant effect [73]

D184A b human no significant effect [73]

V324(188)A human no significant effect [73]

F325(189)A human no significant effect [73]

F325(190)A mouse no significant effect [74]

K328(192)A human loss of function [66]

K328(192)A human reduction of affinity (20 fold) [67]

F336(201)A mouse reduction of affinity (3 fold) [74]

Y539(275)F human no significant effect [71]

Y539(275)I human loss of affinity [71]

W543(280)A mouse loss of affinity [74]

W648(357)A mouse reduction of affinity (7fold) [74]

a N-terminus b First extra-cellular loop

1366 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 4 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives Five SR141716A (14) DerivativesIllustrating the Importance of the C-3 Carboxamide Oxygen in the SR141716A Inverse Agonism

The structure affinity ([3H]-SR141716A hCB1-HEK293 cells) and function (Ca++ currents) are given for each compound

NN

R

Cl

Cl

Cl

Cpd ndeg R= Affinity Function References

SR141716A 14

O

NHN

Kd=23nM Inverse agonist [67]

VCHSR1 15 Ki=313nM Neutral antagonist [67]

CHASR1 16

O

NH

Ki=17nM Inverse agonist [68]

CHMSR1 17

O

N

Ki=29nM Inverse agonist [68]

VPSR1 18

N

Ki=261nM Neutral antagonist [68]

PIMSR 19

NN

Ki=67nM Neutral antagonist [68]

an increased constitutive activity of the receptor asdemonstated by [35S]-GTPγ S binding [75]

1996 It binds with high affinity to rat brain synaptosomes(Kd=061nM) It is competitively displaced by knowncannabinoids like CP-55940 or WIN-55212-2 Usingautoradiography its rat brain distribution is similar to [3H]-CP-55940 one [77] It is now a commercially available andwidely used radioligand for competition studies

A new 3D model of the CB1 cannabinoid receptor basedon the X-ray structure of the bovine rhodopsin was recentlydeveloped by Salo and co-workers [76] It would be veryinteresting to see whether or not the results obtained withprevious models are confirmed using this new model Forinstance the lysine K328 appeared as a key residue in thismodel too

Interestingly other SR141716A radiolabeled derivativeswere synthesised as radioimaging tools among them [123I]-AM251 (20) [7879] [123I]-AM281 (21) [80-82] and [18F]-SR144385 (22) [8384] for Positon Emission Tomographyor Single Photon Emission Computed Tomography Indeedcannabinoid antagonists are a much more useful tool for

The tritiated analogue of SR141716A the [3H]-SR141716A was described by Rinaldi-Carmona et al in

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1367

human radioimaging applications than agonists as they aredevoid of cannabinoid-like effects For instance Berding etal very recently reported the use of [123I]-AM281 for SinglePhoton Emission Computed Tomography imaging of theCB1 cannabinoid receptor in six human patients [85] Inorder to further optimize the brain uptake of suchSR141716A derivatives Sanofi researchers synthesised twomethoxylated SR141716A analogues using 11C as PETtracer [86] These two compounds SR149080 (24) andSR149568 (25) possess high affinity with Ki values of 15and 38 nM respectively and selectivity for the CB1 receptoras well as an improved penetration in the brain evaluated bymeasuring the radioactivity present in various brain regionsafter tail vein injection in CD-1 mice Some of therepresentative radiolabelled SR141716A derivatives aresummarised in Table 5

Colombo et al using Wistar rats [92] Simiand et alobserved that SR141716A selectively reduces sweet foodintake in primates [93] However several authors [94-96]showed that high palatability of food is not necessary toobserve a SR141716A-induced anorectic effect at doses thatdo not cause major behavioural alterations or reduced waterintake Interestingly Gomez et al demonstrated theimplication of the peripheral CB1 receptors on themodulation of feeding and therefore the possible role ofthese receptors on the SR141716A influence on food intake[97] In addition to its effects on food consumptionSR141716A seems to be able to lower the hyperglycemiathe hyperinsulinemia as well as the insulin resistance indiet-induced obese (DIO) mice In the same DIO mice adecrease in adiposity was also observed after treatment [98]Bensaid et al using another model of obesity the obese fafarats observed that SR141716A increases mRNA expressionof Acrp30 or adiponectin a plasmatic protein exclusivelysecreted by adipose tissue through a CB1-mediatedpathway Inductions of free fatty acid oxidation body weightreduction and hyperinsulinemia decrease are some of theknown physiological actions of this protein Thus along theauthors an enhanced expression of Acrp30 followingSR141716A administration could be responsible for themetabolic effects of the compound leading to body weightreduction [99] Vickers et al using the same model (ie fafarats) showed that SR141716A significantly decreases foodconsumption and weight gain in both the obese fafa rats andthe lean Zucker rats [100] This decrease was greater in thefafa group and reversible upon SR141716A withdrawal

From a more therapeutic point of view SR141716A(rimonabant Acompliareg) is one of the promising agents totreat obesity [87] It is currently in Phase III clinical trials forthe treatment and prevention of obesity Final results areexpected for this year and FDA application filling for 2005[88] Preliminary data based on a one year treatment withrimonabant (RIO-Lipids study 1036 patients) showed a 5weight loss in 72 of the treated patients [8990] Severalreports were published before clinical trials aiming todemonstrate the anti-obesity properties of this compoundThe first report by Sanofi Recherche published in 1997described the selective inhibition of sucrose intake in ratsupon SR141716A treatment (03-3 mgkg) [91] Theanorectic and weight loss effects were firstly published by

Table 5 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structure and Affinity of SR141716A Radiolabeled Derivatives Developed as Potential Radio-Imaging Tools

NN

R3

O

NH

Cl

R4

R1

R2

Cpd ndeg R1 R2 R3 R4 Affinity CB1 References

[123I]-AM251 20 123I piperidinyl CH3 Cl Ki=25 nMa [78]

[123I]-AM281 21 123I morpholinyl CH3 Cl Ki=14 nMb [80]

[18 F]-SR144385 22 Cl piperidinyl CH2-18 F Cl IC50 =29 nMa [83]

[18 F]-SR147963 23 Cl morpholinyl CH2-18 F Cl IC50 =120 nM [84]

[11 C]-SR149080 24 O-11 CH3 piperidinyl CH3 Cl IC50 =15 nMa [86]

[11 C]-SR149568 25 O-11 CH3 morpholinyl CH3 Cl IC50 =38 nMa [86]

[18 F]-NIDA-42033 26 O-CH3 piperidinyl 18 F H Ki=18 nMc [155156]

27 O-11 CH3 piperidinyl CH3 H Ki=8nMc [155157]

arat brain homogenates [3H]-CP-55940bmouse cerebellum homogenates [3H]-SR141716crat brain homogenates [3H]-AM251

1368 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Another recent study by Higgs and co-workers furtherhighlighted the role of endocannabinoids in food taste-perception and the reduction of orosensory reward of sucrosein SR141716A treated rats [101] As it was expectedSR141716A induced effects on food consumption are absentsin CB1

-- mice [102] Very recently Cota et al obtainedresults showing that endocannabinoid system modulateshomeostasis via a dual mechanism it regulates at a centrallevel food intake while it blocks at the periphery lipogeneticprocesses [103] Moreover Cani et al showed that ghrelin(an orexigenic peptide) plasma levels are significantlyreduced 45 minutes after SR141716A administration(5mgkg ip) to fasted rats in accordance with the rapiddecrease of food intake measured by the authors It istherefore likely that SR141716 effects on body weight aredue to a conjunction of central and peripheral actions [104]

cerebrospinal fluid from patients with schizophrenia [116]Later De Marchi et al measured higher amounts ofanandamide in blood of schizophrenic patients compared tocontrols [117] On the other hand Dean et al obtainedelevated [3H]-CP-55940 binding in the dorsolateralprefrontal cortex of patients suffering from schizophrenia ascompared to controls [118] Zavitsanou using [3H]-SR141716A found elevated binding in the anterior cingulatecortex of subjects with schizophrenia [119] RecentlyMeltzer and collaborators published the results of a trialconducted to evaluate the potential of four new compoundsin treating schizophrenia and schizoaffective disorders [120]SR141716A (20 mgday) was one of the compoundsevaluated during a six weeks study The authors found noeffect of the CB1 cannabinoid receptor antagonist inimproving patientrsquos schizophrenia (72 subjects)

In addition to the anti-obesity potential the ability ofSR141716A to reduce alcohol and tobacco consumption arecurrently investigated in phase III clinical trials The effect ofSR141716A on alcohol consumption in rats was reported bythe GianLuigi Gessa team [105-108] and by Gallate et al[109] The inhibition of alcohol and nicotine induceddopamine release by SR141716A (1-3 mgkg rats) wasreported by Cohen and co-workers using a brainmicrodialysis device [110] Concerning the smokingcessation preliminary results of a clinical trial (360 subjects40 mg SR141716A) showed an increased abstinence ofsmoking [111] More recent results from the STRATUS-USstudy (787 patients) were reported 362 of patientsreceiving SR141716A (20 mgday) quit smoking against206 in the placebo group [90] Recently Le Fol andGoldberg reviewed the development of cannabinoid CB1antagonists as a new class of therapeutic agents for drugsaddictions [112]

It is known since the early nineties that theadministration of cannabinoid agonists impairs memory inrodents (for a review see Castellano et al [121]) Thusadministration of a cannabinoid antagonist was expected tosomehow improve memory [122123] However dependingon the authors SR141716A when administered alone wasreported to impair (5-10 mgkg im) [124] to have no effect(1-32 mgkg ip) [125] or to improve memory (3 mgkgip) [126] In a more recent paper Wolff and Leandershowed that SR141716A (1mgkg ip) improves memoryin rats by apparently enhancing the consolidation processesof memory [127] Further they found that at higher doses (3mgkg) this effect was lost The discrepancy in the resultsreported in the literature could be ascribed either to thedifferences in the tests used or in the doses administeredFurther experiments are needed to assess whether or not acannabinoid antagonist could be helpful in memory diseases

To conclude several patents were taken by Sanoficoncerning the therapeutic applications of their leadcompound among them being anti-obesity smokingcessation neuroinflammatory diseases and anti-diarrhoeaOne of the last patents to our knowledge concerns thetreatment of sexual dysfunctions with a cannabinoidantagonist such as SR141716A [128] However da Silva etal showed that even if this compound (2 mgkg ip)enhances the effects of apomorphine (20-80 microgkg) it has noeffect alone on penile erection [129] Meanwhile Melis et al

Since the late sixties concerns exist on the associationbetween cannabis use and schizophrenia Recent studiesshowed an association between cannabis use and an increasedrisk of developing schizophrenia [113 114] Moreover apossible role of the endocannabinoid system in schizophreniahas been suggested since its pharmacological characterisation[115] Several experimental data obtained on human subjectstend to confirm such hypothesis On the one hand Lewekeet al found elevated levels of endocannabinoids in

NN

O

NH

N

Br Cl

Cl

NN

O

NH

N

Br Cl

Cl

OH

28 29

Fig (3) CB1 cannabinoid receptor antagonists 3 Diarylpyrazole derivatives The chemical structures of 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-piperidine-carboxamide (SR147778 28 ) and 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-(4-hydroxypiperidine)-carboxamide (29 ) described by Rinaldi-Carmona et al

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1369

reported an increased rate of penile erection after directinjection of SR141716A in the paraventricular nucleus of thehypothalamus [130] This effect was dose dependent and asignificant effect was obtained with a dose of 1microgkg

AM251 in mice at a same dose range (10-30 mgkg ip) thanthe anorectic effects [143] These effects are reversed byadministration of CP-55940 and are absent in CB1

-- miceproving the implication of the CB1 receptor in theantidepressant-like effect of AM251 In 2004 Liao et alreported that AM251 was able to displace the binding of[3H]-batrachotoxin A from its binding site on sodiumchannels in mice brain synaptic preparations [144] Theyobtained an IC50 value of 112 microM and a competitivemechanism of action The authors suggested that AM251 iscapable of reducing neuronal excitability through blockade ofvoltage-sensitive sodium channels in brain

From the clinical trials involving the SR141716A themost frequently reported side-effects are nausea dizziness anddiarrhoea Depression and anxiety were not higher than inthe placebo groups Regarding one of the known side effectsof SR141716A administration the enhancement of intestinalmotility Carai et al reported recently that chronicadministration of the inverse agonists to mice induced atolerance to this prokinetic effect [131] This is not the firstreport of tolerance onset after administration of SR141716A[132] however few is known on the mechanismsresponsible for that phenomenon

Wiley and collaborators reported in 2001 a studydescribing new structure-activity relationships using thepyrazole nucleus as a central scaffold [145] Starting fromSR141716A structure they alternatively substituted one ofthe four substituents while retaining the others bysubstituents known to impart agonist activity in classicalcannabinoids One of the authors expectations was todetermine which positions are responsible respectively forthe antagonism and for the affinity of SR141716A Theaffinity of thirty compounds was assessed previous to in-vivoevaluation of their function in mice using the spontaneousactivity the tail-flick and the rectal temperature assays Theauthors showed that phenyl group in position 5 is critical foraffinity as compound O-1559 (30) lacking this phenyl has adecreased affinity (Table 6) This was already shown by Lanand co-workers [136] along with the need of a substituent inpara position on the phenyl Thomas et al previouslyshowed that this substituent could be a bromine or an iodineatom [146] However an alkyl chain is also tolerated as it isthe case in compounds O-1302 (31) O-1691 (32) or O-1704(33) All these compounds antagonise the anti-nociceptiveand hypothermic effects of ∆9-THC The authors suggestedthat the 5-substituent of pyrazoles is involved in receptorrecognition and antagonism The modulations of thesubstitution pattern of the phenyl in position 1 demonstratethat the 24-di-chloro substitution is the preferred one for theaffinity as well as for the activity Several compoundssupport this assertion Thomas et al showed that additionalhalogens result in a decreased affinity as in compounds 6-I-SR141716A (34) or 4rsquo6-di-I-SR141716A (35) (Ki values of166 and 126 nM respectively) [146] Suppression of the twochlorines (O-1300 39) or replacement of these two chlorinesby an alkyl chain like in O-1254 (40) and O-1255 (41) ledto less active compounds with Ki values ranging from 150to 430 nM However O-1254 and O-1255 behaved asantagonists in a [35S]-GTPγ S assay [61] Lan et al [136]and Wiley et al [145] also modified the substitution inposition 3 Replacement of the amido piperidinyl substituentby alkyl amides as in O-1269 (42) or O-1270 (43) etherslike in O-848 (44) and O-853 (45) ketones (O-1272 46)alcohols (O-1876 47) or alkanes (O-1877 48) resultedmostly in decreased affinity (Table 6) but also with a changeof functionality in some compounds as revealed by in-vivoassays For instance replacement of the piperidinyl by apentyl (O-1269) or by an heptyl chain (O-1270) gave agonistcompounds having Ki values of 32 and 48 nM respectivelyThus the authors suggested that the 3-substituent region isinvolved in receptor recognition and agonist activity Wileyet al on the basis of their results concluded that while the3-position seems to be involved in agonism the 1- 4- and

One close analogue of SR141716A 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-piperidinecarbox-amide (SR147778 28) was described very recently byRinaldi-Carmona et al [133 134] (Fig 3) It possesses highaffinity and selectivity for the hCB1 cannabinoid receptorwith Ki values for the hCB1 and hCB2 receptors of 35 and400 nM respectively ([3H]-CP-55940 hCB1amp2-CHO cells)Further SR147778 antagonised CP-55940 effects on mousevas deferens contractions (pA2=81) and on forskolin-stimulated adenylyl cyclase activity in U373MG cells(pA2=82) but had no effects alone In-vivo SR147778 afteroral administration reversed WIN-55212-2 inducedhypothermia and analgesia As SR141716A SR147778dose-dependently reduced ethanol and sucrose solutionintake with significant effects starting at 03 mgkg (sc) and3 mgkg (po) respectively This compound is currentlyinvestigated in Phase I clinical trials for the treatment ofobesity as well as nicotine and alcohol addictions

Another derivative the 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-(4-hydroxypiperidine)-carboxamide (29) was described in a very recent patent fromSanofi [135] (Fig 3) This antagonist possesses an IC50value for the hCB1 cannabinoid receptor of 32 nM ([3H]-CP-55940 hCB1-CHO cells)

The first structure-affinity relationships for theSR141716A derivatives were reviewed by Barth andRinaldi-Carmona in 1999 [32] Since then many papersdescribing new derivatives were published In 1999 Lan etal described around thirty 15-diarylpyrazoles derivatives[136] Among them was AM251 previously reported as aradioimaging ligand for the CB1 receptor [78] and thatappeared to be more potent (Ki = 749 nM) and selective(selectivity ratio of 306) than SR141716A (Ki = 115 nMselectivity ratio of 143) They reported a Kd value of 05 nMin the mouse vas deferens model using WIN-55212 asagonist Interestingly New et al described for the samecompound an inverse agonist effect on the hCB2 receptorusing a forskolin-induced cAMP accumulation assay (EC50= 650 nM) [137] Recently the anti-obesity effects ofAM251 were reported and are not surprisingly similar tothose of SR141716A [138 139 140] Chen et aldemonstrated that there are synergistic effects on food intakesuppression between AM251 and nalmefene an opioidantagonist [141] This synergistic effect was also presentbetween SR141716A and naloxone as shown by Kirkham[142] Shearman et al found antidepressant-like effects of

1370 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

5-positions appear to be involved in antagonism The O-derivatives reported were also described in a patent alongwith their synthetic pathways which were not given in thepaper [147]

Another investigation of the SR141716Aaminopiperidine region was conducted by Francisco et al[148] They synthesised 21 analogues possessing either analkyl amide or an alkyl hydrazide substituent of various

Table 6 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structure of some of the characterised 1- 3- 4- and 5-pyrazole derivatives Binding affinities (Ki values nM) were obtained on rat brain homogenatesusing [3H]-CP-55940 [136 145 146 148 150] or [125I]-AM-251 [155]

NN

R1

R5

R3R4

Cpd ndeg R1 R3 R4 R5 [136] [145] [146] [148] [150] [155]

SR141716 14 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Cl-Ph 115 62 62 62 13 18

O-1559 30 24-di-Cl-Ph CO-NH-piperidinyl Me 1-Methylpentyl 233

O-1302 31 24-di-Cl-Ph CO-NH-piperidinyl Me 4-((CH2)5)-Ph 21 1

O-1691 32 24-di-Cl-Ph CO-NH-piperidinyl Br 4-((CH2)5)-Ph 15

O-1704 33 24-di-Cl-Ph CO-NH-piperidinyl I 4-((CH2)5)-Ph 22

6-I-SR141716 34 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-Cl-Ph 166

4rsquo6-di-I-SR141716 35 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-I-Ph 126

4rsquo-I-SR141716 36 24-di-Cl-Ph CO-NH-piperidinyl Me 4-I-Ph 75 25 6

4rsquo-Br-SR141716 37 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Br-Ph 168 3

Cpd 25 in ref [136] 38 24-di-Cl-Ph CO-NH-morpholinyl Me 4-Br-Ph 54

O-1300 39 Ph CO-NH-piperidinyl Me 4-Cl-Ph 150

O-1254 40 4-(CH2)4-Ph CO-NH-piperidinyl Me 4-Cl-Ph 226 256

O-1255 41 4-(CH2)5-Ph CO-NH-piperidinyl Me 4-Cl-Ph 433

O-1269 42 24-di-Cl-Ph CO-NH-pentyl Me 4-Cl-Ph 32 114 3

O-1270 43 24-di-Cl-Ph CO-NH-heptyl Me 4-Cl-Ph 48 462 3

O-848 44 24-di-Cl-Ph CH2-O-(CH2)2-piperidinyl

Me 4-Cl-Ph 2450 232

O-853 45 24-di-Cl-Ph CH2-O-CH2-cyclohexyl

Me 4-Cl-Ph 388 100

O-1272 46 24-di-Cl-Ph CO-heptyl Me 4-Cl-Ph 221

O-1876 47 24-di-Cl-Ph 1rsquo-OH-heptyl Me 4-Cl-Ph 657

O-1877 48 24-di-Cl-Ph Heptyl Me 4-Cl-Ph 422

MF9725-64-17 49 24-di-Cl-Ph CO-NH-butyl Me 4-Cl-Ph 134

MF9725-179-32 50 24-di-Cl-Ph CO-NH-NH-butyl Me 4-Cl-Ph 51

MF9725-66-11 51 24-di-Cl-Ph CO-NH-cyclohexyl Me 4-Cl-Ph 25

MF9725-95-31 52 24-di-Cl-Ph CO-NH-(4-hydroxybutyl)

Me 4-Cl-Ph 154

Cpd 15 [150] 53 n-pentyl CO-NH-piperidinyl Me Ph 23

Cpd 16 [150] 54 n-pentyl CO-NH-piperidinyl Me 4-Br-Ph 63

Cpd 17 [150] 55 n-hexyl CO-NH-piperidinyl Me Ph 21

Cpd 18 [150] 56 n-heptyl CO-NH-piperidinyl Me Ph 47

NIDA-41109 57 24-di-Cl-Ph CO-NH-piperidinyl Br 4-Cl-Ph 14

NIDA-41119 58 24-di-Cl-Ph CO-NH-piperidinyl H 4-Cl-Ph 9

NIDA-41057 59 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OH-Ph 104

NIDA-41020 60 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 41

NIDA-41087 61 2-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 8

NIDA-42055 62 24-di-Cl-Ph CO-NH-piperidinyl Br 4-OMe-Ph 62

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1371

lengths in position 3 of the pyrazole moiety They observedthat until five carbons the affinity increases along with thecarbon chain length This is observed with the alkylhydrazide the alkyl amide and the hydroxyalkyl amideseries Moreover the hydrazide analogues exhibit a loweraffinity for the rCB1 than the amide analogues as illustratedby compounds MF9725-64-17 (49) and MF9725-179-32(50) having Ki values of 134 and 51 nM respectively(Table 6) From their structure-activity relationships (SAR)studies the authors concluded that the pharmacophoricrequirement of the amidopiridine region is a chain not longerthan 3 Aring and that a substituent having a positive chargedensity would probably result in increased affinity andpotency The same team described in a patent several othercompounds [149] Binding affinities (Ki) against [3H]-CP-55940 [3H]-SR141716A or [3H]-WIN55212-2 on wholerat brain or on hCB1-transfected cells were given Moreoveractivity data were obtained using the [35S]-GTPγ S assaydemonstrating that these alkyl amide and hydrazide analogsact as antagonists or inverse agonists Despite the greatnumber of derivatives claimed none of them posses asignificantly greater affinity for the CB1 receptor than theSR141716A Nevertheless they showed a slightenhancement of the selectivity for the CB1 receptor over theCB2 cannabinoid receptor

affinity of these derivatives increases with the length of thealkyl chain with an optimal length of 5-6 carbons The bestcompound of this series N-(piperidin-1-yl)-5-phenyl-1-hexyl-4-methyl-1H-pyrazole-3-carboxamide (55) possesses aKi value of 21 nM determined using [3H]-CP-55940 on ratbrain membranes which is higher than the 13 nMdetermined for SR141716A in the same conditions (Table6) In the same paper the authors starting from thehypothesis that antagonism by SR141716A is caused bybinding to the same region of the receptor as do the agonists(CP-55940 and WIN-55-2122) but preventing the agonistpromoted conformational change conducted extensiveconformational analysis as well as superimposition modelsand 3D-QSAR to propose a molecular mechanismsupporting the action of SR141716A Along with theauthors the C-5 aryl substituent of SR141716A occupyinga unique region could contribute in conferring the antagonistproperties Moreover the C-3 substituent could beresponsible for the antagonist or inverse agonist propertiesdepending on the interaction with the receptor

About this topic a very interesting review dealing withthe cannabinoid receptors pharmacophores as well as withthe activationinactivation of these receptors was recentlypublished by Reggio [151]

More recently Dyck et al described seven otherderivatives varying at the amide position [152] The onlycompound possessing a higher affinity for the CB1cannabinoid receptor than SR141716A was the 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta-

The affinity for the rat CB1 receptor of seven derivativespossessing an alkyl chain in position 1 instead of the 24-dichlorophenyl substituent was reported by Shim et al in apaper describing a molecular mechanism for the antagonistand inverse agonist activity of SR141716A [150] The

NN

O

NHN

Cl Cl

Cl

NN

Cl

Cl

NON

NN

O

NH

N

Cl

ClCl

NN

O

NH

MeO Cl

CP-272871 66

63 64

65

Fig (4) CB1 cannabinoid receptor antagonists 3 Diarylpyrazole derivatives Structures of 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63 ) 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methyl-pyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]-morpholine (64 ) N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65 ) and N-phenyl-1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-pyrazole-3-carboxamide (CP-272-871 66 )

1372 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

[c]pyrrol2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63)(Fig 4) The Ki values were 5 and 12 nM respectively([3H]-CP-55940 hCB1-HEK cells)

CP-55940 rat brain) For instance N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65) (Fig 4) showed a Ki value of15 nM However no data were provided concerning thefunctionality of such compounds

Over 200 other pyrazole derivatives acting at the CB1cannabinoid receptor were claimed in a patent from Pfizer[153] The affinity for the hCB1 receptor of 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methylpyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]ndashmorpholine (64) (Fig 4)was 79 nM ([3H]-SR141716A hCB1-HEK cells) Theothers compounds affinity was between 01 and 100 nM

In an effort to develop new SR141716A analoguespossessing lower lipophilicity to be used as PET tracersKatoch-Rouse and colleagues further explored thesubstitution pattern of the two phenyls [155] Bindingassays showed that a decreased lipophilicity lead to adecreased affinity for the CB1 receptor as determined bydisplacement of [3H]-AM251 For instance compoundNIDA-41057 (59) has a lipophilicity of 42 ( expressed asElogDoct) and a Ki value of 104 nM whereas SR141716Ahas a ElogDoct value of 54 and a Ki value of 18 nM In

Seventy five pyrazole derivatives were described in a veryrecent patent from Makriyannis et al [154] The affinity ofthe compounds for the cannabinoid receptors is given TheKi values for the best compounds are lower than 6 nM ([3H]-

Table 7 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structures and affinities for the cannabinoid receptors of some representative tricyclic pyrazole derivatives Values were obtained using [3H]-CP-55940as radioligand and mouse [160 228] rat [159 227] or human [158] cannabinoid receptors Compounds 69-78 are CB1 cannabinoid receptor ligandsCompounds 79-86 are CB2 cannabinoid receptor ligands

NN

O

NHX

R3

R1

R2

n

n˚ X R1 R2 n R3 Ki CB1 (nM) Ki CB2 (nM) Selectivity References

69 -(CH2)3- Cl 24-di-Cl 0 piperidine 126 [158]

70 -(CH2)3- H 24-di-Cl 0 azepane 100 [158]

71 -(CH2)3- H 24-di-Cl 0 piperidine 398 [158]

72 -(CH2)3- Cl 24-di-Cl 0 azepane 125 [158]

73 -(CH2)3- NO2 24-di-Cl 0 azepane 63 [158]

74 -(CH2)2-O- H 24-di-Cl 0 piperidine 501 [158]

75 -CH2-S- Br 24-di-Cl 0 piperidine lt 500 gt10 [159]

76 -CH2-SO2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

77 -(CH2)2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

78 -(CH2)3- Cl 24-di-Cl 0 piperidine 000035 21 60000 [160]

79 -CH2- Cl 24-di-Cl 0 piperidine 2050 034 00002 [228]

80 -CH2- Br 24-di-Cl 0 piperidine 1570 027 00002 [228]

81 -CH2- CH3 24-di-Cl 0 piperidine 363 0037 00001 [228]

82 -CH2- Cl 4-Cl 0 piperidine 1787 09 00005 [228]

83 -CH2- H 24-di-Cl 0 piperidine 1152 0385 00003 [228]

84 -CH2- Cl 34-di-Cl 1 133-trimethylbicyclo[221]heptyl

lt500 gt10 [227]

85 -CH2- Br 24-di-Cl 1 bicyclo[321]oct-3-yl lt500 gt10 [227]

86 -CH2- Br 4-Me 1 77-dimethylbicyclo[410]hept-3-yl

lt500 gt10 [227]

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1373

another paper Katoch-Rouse described the synthesis of[18F]-NIDA-42033 (26) a ligand useful as PET radiotracerstarting from its bromo derivative [156] Recently Kumar etal [157] proposed the synthesis of a 11C derivative ofNIDA-41087 (61) a compound previously described byKatoch-Rouse (Table 5) Post-mortem binding of thiscompound to human prefrontal cortex was also investigatedInterestingly CP-272871 (66) described by Meschler as aCB1 inverse agonist (Ki=57 nM [3H]-CP-55940 rat brainhomogenates) possesses a lipophilicity similar to theNIDA-41087 one [62] However despite the presence of amethoxy function no radiolabelled derivative has beendescribed to date One explanation could be the compoundlow selectivity (2 fold) for the CB1 cannabinoid receptor

To further explore the pharmacology of this compound afunctional assay using the mouse vas deferens model wasperformed by Ruiu and colleagues enlightening thecompetitive antagonist properties of NESS (pA2= 1246against WIN-55212-2) which had no effect by itself up to1microM Moreover NESS 0327 was unable to affect basalbinding of the [35S]-GTPγ S demonstrating its lack ofnegative intrinsic activity In-vivo studies were conductedusing the hot plate and tail flick tests NESS dose-dependently abolished the antinociceptive effect of WIN-55212-2 but had no effect by itself However the authorssuggested based on the affinity and in-vivo activity of theircompound that NESS 0327 possesses a poor centralbioavailability

Several new attempts to increase the affinity of thediarylpyrazole derivatives were recently made by rigidifyingthe SR141716A structure (Table 7) Six fused ringanalogues of SR141716A obtained by fusion of the 5-(4-chlorophenyl) substituent with the central pyrazole to forman indazole ring were published by Bass et al in 2002 [61]The compound possessing the highest affinity O-1248 (67)has a Ki value of 475 nM as determined by displacement of[3H]-CP-55940 from rat brain membranes

Another example of ring-constrained biarylpyrazole wasdescribed by Herbert Seltzman team [161] Theyinvestigated a photocyclisation reaction starting from theSR141716A leading to a pyrazolo[15-f]phenanthridinestructure (Fig 5) The new compound (68) was tested on theCB1 receptor (whole rat brain) against [3H]-CP-55940[3H]-SR141716A and [3H]-WIN-55212-2 and exhibited Kivalues of 48 35 and 50 nM respectively The CB2 affinitywas assessed using [3H]-CP-55940 and was found to be 100times lower (Ki = 3340 nM) No effect on the [35S]-GTPγ Sassay (rat brain) was observed by the authors

Stoit and colleagues from Solvay Pharmaceuticalspublished a paper describing the synthesis andpharmacological characterisation of new benzocyclohepta-pyrazole derivatives as CB1 antagonists (69-74) [158] Theaffinity of these compounds was more or less one order ofmagnitude lower than the SR141716A one with pKi valuesranging from 64 to 72 compared to 76 for the SRcompound (Table 7) Compounds were shown to beantagonists with pA2 values from 70 to 89 as theyprevent CP-55940-induced cAMP accumulation Howeverthe authors investigating the bioavailability of theircompounds either po or after ip injection found negligibleblood plasma levels

NN

Cl

Cl

O

NH

N

NN

Cl

O

NHN

Cl

Cl

O-1248 67 68However just before the publication of Stoit results

Sanofi-Synthelabo took a patent describing tricyclicpyrazolecarboxylic acid amide derivatives having antagonistproperties on the CB1 cannabinoid receptor andsubmicromolar affinities (75-77) [159] Thirty examples weregiven along with their synthetic pathway but no individualpharmacological data were shown

Fig (5) CB1 cannabinoid receptor antagonists 3Diarylpyrazole derivatives Two ring-constrained diarypyrazolederivatives O-1248 (67 ) and a pyrazolo[15-f]phenanthridinederivative (68 )

It appears from these attempts to increase SR141716Aaffinity by rigidifying its structure that only a limitedincrease in the CB1 cannabinoid receptor affinity can beexpected with the apparent exception of NESS 0327Moreover rigidifying the structure often results in a morelipophilic compound in a family of compounds being yetlipophilic

More recently Ruiu and colleagues published a paperdescribing the synthesis and complete pharmacologicalcharacterisation of a very potent CB1 receptor ligandchristened NESS 0327 (78) [160] This compound has thesame structure as compound 69 published by Stoit et al(compound 20 in the reference [158]) Nevertheless Ruiu hasobtained an affinity for the CB1 cannabinoid receptor 5000times bigger than the SR141716A one with Ki values of035 pM and 18 nM respectively using mouse forebrainhomogenates and [3H]-CP-55940 as radioligandFurthermore the selectivity ratio obtained for this compoundby Ruiu is 60000 with a Ki value for the CB2 receptor of21 nM against the [3H]-CP-55940 The discrepancybetween the data obtained independently by Stoit et al andRuiu et al could hardly be explained by the differences inthe origin of the receptor used respectively hCB1-CHO andmouse forebrain

4 Phenyl Benzofuranone Derivatives

In a 1997 patent from Eli Lilly was described thesynthesis of aryl-benzothiophene and aryl-benzofuranederivatives (87-89 Fig 6) having CB1 receptor affinity andantagonist properties [162] From ten compounds having aKi value lower than 25 microM ([3H]-CP-55940 hCB1-CHOcells) one was selected and claimed to have a Ki of 170 nMCompound 88 was shown in the patent to antagonise theeffect of anandamide on cAMP accumulation with an IC50 of

1374 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

O

O

O

O

NC

S

O

O

O

O

O

O

O

NC

LY320135 8887 89

Fig (6) CB1 cannabinoid receptor antagonists 4 Phenyl benzofuranone derivatives Chemical structures of two aryl-benzofuranederivatives (87 -88 ) and one aryl-benzothiophene (89 ) derivative from Eli Lilly

500 nM and the effect of WIN-55212-2 on calcium channelsat 1microM Moreover the intra-peritoneal injection (20 mgkg)of this compound antagonised the in-vivo effect ofanandamide in the Open Field Assay a mouse behaviourmodel Later on Felder and colleagues gave a furtherdescription of this compound called LY320135 in a paperdescribing its effect on cAMP accumulation and on ioniccurrents [163] The selectivity ratio of LY320135 was 106with Ki values of 141 and 14900 nM for hCB1 and hCB2receptors ([3H]-CP-55940) respectively LY320135antagonised anandamide inhibition of forskolin-inducedcAMP accumulation with an IC50 value of 734 nMChristopoulos et al in 2001 reported for LY320135 apKb value of 527 in the inhibition of WIN-55212-2mediated response in the rat electrically-stimulated vasdeferens while the pKb value for SR141716A was 75[164]

Other patents were taken by Aventis in which somepotential applications for their compounds were describedFor instance an association between an azetidine derivativehaving CB1 antagonists properties and sibutramine aserotonin and norepinephrine reuptake inhibitor was claimed[170] as a treatment against obesity Oral administration ofcompound 90 (3 mgkg) and sibutramine (06 mgkg) to fafaZucker rats which are genetically obese rats has resulted ina decreased food intake compared to control lean Zuckerrats In another patent the association of an azetidinederivative (1-10 mgkg po ) and a D2D3 agonist likequinpirole (01 mgkg ip) was claimed to have a therapeuticpotential in the treatment of Parkinsonrsquos disease [171] Theeffect has been evidenced using an akinesia model in the ratEvidences supporting the use of CB1 antagonists as adjuvantin the treatment of Parkinsonrsquos disease begin to appear Itseems that such compounds would be helpful in thetreatment of Parkinsonism [172] and levodopa-induceddyskinesia [173] Brotchie published an interesting paperreviewing the CB1 cannabinoid receptor signaling inParkinsonrsquos disease [174] Nevertheless further clinicalstudies should be conducted to further confirm the usefulnessof CB1 antagonists in the treatment of Parkinsonrsquos disease

5 Azetidine Derivatives

The family of azetidine compounds illustrated bycompounds 90 and 91 (Fig 7) was developed at Aventis byDaniel Achard and colleagues and was described in a seriesof patents [165-168] They claimed the IC50 values of thesecompounds for the CB1 receptor to be less than or equal to100 nM These values were obtained following the proceduredescribed by Kuster et al [169] The antagonist propertywas shown using an in-vivo model the reversal ofhypothermia induced by CP-55940 in mice and the ED50obtained were lower than 50 mgkg

6 Aryl-Imidazolidine-24-Diones Derivatives

In 1999 the Didier Lambert team published a paperdescribing the affinity of 24 new 3-alkyl-55rsquo-diphenyl-imidazolidine-24-dione derivatives for the hCB1 receptor[175] The preliminary structure-activity relationships

N

SO

O

Cl

Cl

F

F

N

SO

O

Cl

NS

90 91

Fig (7) CB1 cannabinoid receptor antagonists 5 Azetidine derivatives Two examples (90 -91 ) of the azetidine derivatives developedby Aventis

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1375

Table 8 CB1 Cannabinoid Receptor Antagonists 6 Aryl-Imidazolidine-24-Diones Derivatives

Structures affinities ([3H]-SR141716A hCB1-CHO cells) and pKb values ([35 S]-GTPγS HU210 rat brain) of three aryl-imidazolidine-24-dionederivatives (92-94)

N

NH

OO

R

Br

Br

Cpd ndeg R Ki (nM) a pKb b

DML20 92 ethylmorpholine 70 611

DML21 93 1-hydroxypropyl 103 625

DML23 94 heptyl 98 574

a [175]b [177]

showed that the substitution at the nitrogen in position 3 aswell as a bromine atom in para of the phenyl rings aremandatory for the compounds affinity Later on threecompounds termed DML20 (92) DML21 (93) and DML23(94) were characterised as neutral antagonists using the [35S]-GTPγ S assay on rat cerebellum membranes [176] (Table 8)In the same model they competitively inhibited HU-210-induced [35S]-GTPγ S binding with pKB values of 611625 and 574 respectively Moreover these compoundswere proven to be quite selective for the CB1 receptor

higher than the one for the CB2 cannabinoid receptor (IC50 =300 nM) The inverse agonism of the compound wasextrapolated by inhibiting CP-55940-induced hypothermiain mice (EC50 = 18 nM) Moreover one oral dose (10mgkg) reduces the food intake in diet-induced obese ratsInterestingly food intake of CB1

-- mice was not alteredBody weight loss was maintained all over the study during achronic administration (14 days) of 10 mgkg of Cpd A

Later on Merck published a patent [180] in which someof the 45-diaryl-pyrazoles derivatives previously describedwere claimed to be useful in the treatment or the preventionof obesity in association with compounds inhibiting the11β-hydroxysteroid dehydrogenase type 1 (1β-HSD1)enzyme The 1β-HSD1 enzyme is responsible for thesynthesis of cortisol increased levels of which according tothe inventors are associated with obesity However neitherresults on appetite suppression nor on weight loss weregiven even if some indications on the affinity of thecompounds for the cannabinoid receptors were given

Very recently the functionality of these DML compoundswas explored using the [35S]-GTPγ S assay both on rat andhuman CB1 cannabinoid receptors [177] The data obtainedconfirm that the 3-alkyl-55rsquo-diphenyl-imidazolidinedionederivatives behave as neutral antagonists on rat CB1cannabinoid receptor (rat brain) However DML 20 DML21and DML23 acted as inverse agonists on the human receptor(hCB1-CHO cells) Furthermore the authors showed thatthis different functionality is not due to the hCB1 receptorlevel of expression in the recombinant cell line as whateverthe level of expression (Bmax of 44 pmolmg or 32pmolmg) DML derivatives behaved as inverse agonists ofthe hCB1 cannabinoid receptor

Another imidazole ring pattern of substitution wasproposed by Hagmann and collaborators [181] Thecompounds described 43 examples are given possess twophenyls the first one in position 1 linked to the nitrogenand the second one in position 2 The carboxamidesubstituent is in position 4 instead of in position 2 for thediarylimidazoles described by Finke These 12-diaryl-imidazoles are claimed to be CB1 cannabinoid receptorantagonists or inverse agonists but the absence of precisepharmacological values do not allow any comparisonbetween the two substitutions patterns However a samepattern of substitution was used by other researchers Amongthem Dyck et al described in 2004 ten 1-(4-chlorophenyl)-2-(24-dichlorophenyl)-imidazole derivatives varying by theircarboxamide substituent in position 4 (101-103 Table 9)[152] For instance with a 3-azabicyclo[330]octan-3-ylsubstituent (101) they obtained a Ki value of 14 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) In a [35S]-GTPγ Sassay 101 exhibited an IC50 value of 19 nM

7 Diarylimidazoles Derivatives

Finke and colleagues at Merck developed a new class ofCB1 receptor antagonists based on an imidazole nucleusOver eighty 45-diaryl-imidazoles derivatives having CB1antagonist properties were claimed in a patent published in2003 [178] Some representative compounds of the patent areillustrated in Table 9 (95-100) The affinity of the newcompounds was determined using [3H]-CP-55940 andhCB1-CHO cells Although no detailed pharmacologicaldata were given in the patent in-vitro and in-vivo propertiesof one compound termed Cpd A (100) have been describedelsewhere [179] Its affinity for the CB1 cannabinoid receptorwas shown to be nanomolar (IC50 = 4 nM) and 75 times

1376 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 9 CB1 Cannabinoid Receptor Antagonists 7 Diaryl-Imidazole Derivatives

Structure and affinity range of some 45-diaryl-imidazole derivatives for the human cannabinoid receptors determined using [3H]-CP-55940 asradioligand and hCB1amp2-CHO cells

N

NR2

R1

R4

R3

ndeg R1 R2 R3 R4 hCB1 (IC50 or Ki) hCB2 (IC50 or Ki) References

95 CH3 CO-NH-piperidinyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

96 CH3 CO-NH-piperidinyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

97 CH3 CO-NH-cyclohexyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

98 CH3 CO-NH-phenyl 24-di-Cl-Ph 4-Cl-Ph 10 ndash 100 nM a 100 ndash1000 nM a [178]

99 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

100 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph 4 nM a 300 nM a [179]

101 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

CH3 14 nM b ND [152]

102 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

H 66 nM b ND [152]

103 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 85 nM b ND [152]

104 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 23 nM b 542 nM b [182]

105 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 30 nM b 608 nM b [182]

106 4-Br-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 60 nM b 489 nM b [182]

107 4-CF3-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 29 nM b 634 nM b [182]

108 4-Cl-Ph 24-di-Cl-Ph CO-NH-morpholinyl CH3 197 nM b 3297 nM b [182]

109 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidin-4-ol CH3 172 nM b 3959 nM b [182]

110 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CN 30 nM b 1590 nM b [182]

111 4-Cl-Ph 24-di-Cl-Ph CO-NH- piperidinyl CH2F 36 nM b 906 nM b [182]

112 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH2-CH3 14 nM b 430 nM b [182]

113 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Cl 27 nM b 823 nM b [182]

114 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Br 23 nM b 746 nM b [182]

115 4-Cl-Ph 24-di-Cl-Ph CO-NH-tetrahydroisoquinoline CH3 34 nM b 696 nM b [182]

116 4-Cl-Ph 24-di-Cl-Ph CO-NH-cycloheptyl CH3 35 nM b 349 nM b [182]

117 24-di-Cl-Ph 4-Cl-Ph CO-NH-piperidinyl CH3 403 nM b 208 nM b [182]a IC50 value b Ki value

Lange and co-workers from Solvay described twenty-eight imidazole derivatives [182] The 24-dichlorophenylsubstituent at position 2 was kept constant while thesubstitutions at the other positions were explored (Table 9)Position 5 can accommodate a large range of littlesubstituents like hydrogen (104) methyl (105) ethyl (112)chlorine (113) bromine (114) fluoromethyl (111) or cyano(110) without major changes in the affinity At position 4more bulky substituents like 1234-tetraisoquinoline (115)or cycloheptyl (116) were well tolerated whereas thepresence of a more hydrophilic moiety such as morpholine(108) or piperidin-4-ol (109) was detrimental to the affinity

Interestingly position exchange between the 24-diphenyland the 4-chlorophenyl substituents led to a 13 fold loweraffinity with Ki values of 30 and 403 nM for 105 and 117respectively ([3H]-CP-55940 hCB1-CHO cells) In thefunctional assay which was inhibition of WIN-55212-2-induced [3H]-arachidonic acid release by hCB1-CHO cellsall tested compounds behaved as antagonists Compound105 showed a pA2 value of 86 which is the same theauthors obtained for SR141716A It would be interesting toassess if these compounds are true antagonists or if they actmore as inverse agonists Compound 105 when administeredto rats was able to inhibit CP-55940-induced hypotension

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1377

with an ED50 value of 24 mgkg which is close to the 3mgkg obtained for SR141716A The compound was alsoactive in another in-vivo model the WIN-55212-2-inducedhypothermia in mice Compound 104 which has nosubstituent in position 5 of the imidazole ring was lessactive in the hypotension model and inactive in thehypothermia model Thus a methyl group is the preferredsubstituent in position 5 of the imidazole moiety Otherproperties either experimental (P-glycoprotein affinitylogPHPLC) or computational (molecular volume polarsurface area) obtained for compounds 105 were similar tothose obtained for SR141716A Finally molecularmodelling studies revealed a close structural overlap betweenthe two compounds

value of 197 nM is selective for hCB1 and is as potent asSR141716A The structural modulations of 118 led to someinteresting compounds (Table 10) Six compounds possess aKi value for the hCB1 cannabinoid receptor equal to or lowerthan 25 nM antagonist properties in the inhibition of [3H]-arachidonic acid release by WIN-55212-2-stimulated hCB1-CHO cells and Ki values for the hCB2 receptor over 1microMThe diaryl-pyrazoline derivatives contained a chiral center atposition 4 The authors resolved the racemic mixture of twocompounds (121 and 130) The levorotatory enantiomersappeared to be the eutomers For compound SLV319levorotatory enantiomer of 121 the Ki and pA2 values were78 nM and 99 respectively The Ki and pA2 values ofSLV326 levorotatory enantiomer of 130 were 359 nM and9 respectively

8 34-Diaryl-Pyrazoline Derivatives Two in-vivo models CP-55940-induced hypotension inrat and WIN-55212-2-induced hypothermia in mouse wereused to assess the potential of these compounds The resultsare of the same order of magnitude than those obtained forSR141716A For instance in the hypotension modelSLV319 ((-)121) and SLV326 ((-)130) showed ED50 values

Lange and collaborators also discovered 34-diaryl-pyrazoline derivatives after a screening of compoundsresembling to SR141716A [183184] Compound 118 thatwas initially identified during the screening possesses a Ki

Table 10 CB1 Cannabinoid Receptor Antagonists 8 34-Diaryl-Pyrazolines Derivatives

Structure affinity for the hCB1 and hCB2 cannabinoid receptors (Ki nM) expressed in CHO transfected cells ([3H]-CP-55940) and potency (pA2) ofsome 34-dirylpyrazolines derivatives Table adapted from [184]

NN

N N

SO2

R3

R2

Cl R1

R4

n˚ R1 R2 R3 R4 Ki hCB1 (nM) Ki hCB2 (nM) pA2 (CB1)

118 H H H 4-CH3 197 gt 1000 84

119 H H H 246- CH3 24 gt 1000 94

120 H H H 4-Cl 16 gt 1000 95

121 H CH3 H 4-Cl 25 gt 1000 87

122 H CH3 CH3 4-Cl 280 gt 1000 85

123 H H H 4-F 53 gt 1000 9

124 H CH3 H 4-F 338 gt 1000 85

125 H CH3 CH3 4-F gt 1000 gt 1000 lt 75

126 H CH3 H 3-Cl 14 gt 1000 86

127 4-Cl CH3 H 4-Cl 255 ND ND

128 4-F CH3 H 4-Cl 584 ND ND

129 H CH3 H H 170 ND 75

130 H CH3 H 4- CF3 221 gt 1000 93

SR141716A 25 1580 86

1378 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

N

HN

O

N

Cl Cl

Cl

N

N

HN

O

Br

Br131 132

Fig (8) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Chemical structures of two 56-diaryl-pyrazine-2-amide derivatives (131-132) developed as CB1 cannabinoid receptorantagonists by AstraZeneca

of 55 and 2 mgkg (po) respectively proving their in-vivoefficacy after oral administration The authors alsodemonstrated that SLV319 is devoid of affinity for the P-glycoprotein pump and that it possessed a goodCNSplasma ratio (17) These two compounds (SLV319and SLV326) were chosen as development candidates bySolvay and entered clinical Phase I trials in 2003 [185]

132 Fig 8) useful as CB1 cannabinoid receptor antagonists[186 187] The activity of twenty derivatives wasdetermined using a [35S]-GTPγ S assay and hCB1-CHOtransfected cells The concentration required to give halfmaximal inhibition of CP-55940-induced [35S]-GTPγ Sbinding (IC50) is lower than 200 nM for the preferredcompounds No affinity data were given for thesecompounds

9 Diaryl-Pyrazine Diphenyl-Pyridine Diphenyl-Phenyland Diaryl-Pyrimidines Derivatives

The same year in a patent from Merck Finke andcollaborators described several 56-di-phenyl-pyridinederivatives as hCB1 antagonists or inverse agonists [188]The compounds have a substituent in position 3 of thepyridine core (Fig 9) This substituent could be a cyano(133-134) or a nitro group a halogen an ester or an amide(135-136) Over 150 compounds were synthesised toillustrate the invention but no pharmacological data wasdisclosed in the patent

In 2003 appeared several patents describing new familiesof compounds that bind to the CB1 cannabinoid receptorThese compounds have in common a central six atomsaromatic ring which could be a pyrazine a pyridine aphenyl or a pyrimidine and that is substituted by at leasttwo phenyl rings

The first patents were from Berggren and collaboratorsfrom AstraZeneca who published two patents describing thesynthesis of 56-diaryl-pyrazine-2-amide derivatives (131-

A few months later another patent was taken by Sanoficlaiming the antagonist properties of 56-di-phenyl-2-pyridine carboxamide derivatives [189] These compounds

N

NH

O

Cl

Cl

N

O

Cl

O NH

N

O

Cl

ClCl

NH

CN O

N

Cl

Cl

Cl

OHN

N

O

Cl

ClCl

CNF

F

133 134

135 136 137

Fig (9) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives 56-Di-phenyl-pyridine derivatives patented by Merck (133-136) and by Sanofi (137) as CB1 cannabinoid receptorsantagonists

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1379

NH

O

N

Cl

ClCl

NH

O

N

Cl

F3CCl

N

N

O

Cl

Cl Cl F138 139 140

Fig (10) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Representative structures of the diphenyl-phenyl (138-139) and 45-diphenyl-pyrimidine (140) derivatives developed ascannabinoid antagonists at Sanofi and Merck respectively

28 examples are given structurally related to the diphenyl-phenyl derivatives have their pyridine core substituted bytwo phenyl rings and by an amide moiety (137 Fig 9) TheIC50 values were lower than 100 nM but no data were givenshowing the antagonist properties although the compoundswere assayed in the adenylate cyclase inhibition assay

10 Other Derivatives

Several amide derivatives structures recently appearedamong the CB1 cannabinoid receptor antagonists In a seriesof patents taken by Merck in 2003 Hagmann and colleaguesdescribed a large amount of compounds obtained by parallelsynthesis which are claimed to be CB1 cannabinoid receptorantagonists [192-195] They were synthesised by reacting alibrary of substituted amines with a library of carboxylicacids (141-144 Fig 11) However as no value was givenfor the affinity or activity of these amides we will notfurther discuss this class of compounds

Along this line Sanofi Synthelabo developed newdiphenyl-phenyl derivatives claimed to have CB1cannabinoid receptor antagonist properties [190] They arebased on a central phenyl ring substituted by two phenylsand by an amide moiety (138-139 Fig 10) Thirteencompounds were claimed along with their synthesis TheirIC50 values were lower than 100 nM ([3H]-CP55-940hCB1-CHO cells) but no other specific data (selectivitypA2hellip) was given

Several 15-diaryl-pyrrole-3-carboxamide derivatives weresynthesised and claimed to be CB1 cannabinoid receptorantagonists by Berggren et al [196] The affinity valueswere not disclosed in the patent (145-146 Fig 12) Otherpyrrole derivatives were synthesised by Guba et al atHofmann-La Roche [197] More specifically 2-(thiazol-4-yl)pyrrole derivatives were claimed as CB1 cannabinoidreceptor antagonist (IC50lt2microM) (147-148 Fig 12)

Finally Kopka et al from Merck described more thanone hundred 45-diaryl-pyrimidine derivatives as CB1cannabinoid receptor antagonists (140 Fig10) [191]However no precise data were provided concerning theaffinity and functionality of the compounds

R2

R1N

R3

R4

R5

O

NH

O

Cl

O F

F

R2

R1NH

R3

R4

HO R5

O

NH

O

Cl

CN

NH

O

Cl

O

O

NH

O

Cl

Cl

+

141142

143 144

Fig (11) CB1 cannabinoid receptor antagonists 10 Other derivatives Retrosynthetic scheme of a CB1 cannabinoid antagonistlibrary of amide derivatives developed at Merck The structures of four representative compounds are illustrated (141-144)

1380 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

NH

O N

Cl

Cl

Cl

N

NH

O

OMe

MeO

N

NH

O

N

S

MeO

N

NH

O

N

S

MeO

145 146

147 148

Fig (12) CB1 cannabinoid receptor antagonists 10 Other derivatives Two examples of 15-diaryl-pyrrole derivatives described byBerggren et al (145-146) While the 2-(thiazol-4yl)pyrroles derivatives 147 and 148 were described by Guba et al from Hofmann-LaRoche All these pyrroles derivatives were claimed to be CB1 cannabinoid receptor antagonists

Another new structure is represented by the 124-triazolederivatives developed by Jagerovic and collaborators In arecent paper were described the synthesis andpharmacological properties of five new 15-diphenyl-3-alkyl-

triazole derivatives [198] Among these compounds onlyone (149) behaved as a CB1 antagonist inhibiting the WIN-55212-2-induced contractions in a mouse vas deferenspreparation However despite its effects in isolated tissue

Table 11 CB1 Cannabinoid Receptor Antagonists 10 Other Derivatives

Structure and affinity of diaryl-triazole derivatives

N

NN

R3

R2

R1

Cpd R1 R2 R3 Ki CB1 (nM) References

149 24-diCl 4-Cl hexyl 855 a [198]

150 24-diCl 4-Cl2-OMe CO-NH-(3-azabicyclo[330]octan-3-yl) 270 b [152]

151 24-diCl 4-Cl CO-NH-(3-azabicyclo[330]octan-3-yl) 164 b [152]

152 4-Cl 24-diCl CO-NH-(3-azabicyclo[330]octan-3-yl) 137 b [152]

153 4-Cl 24-diCl CO-NH-benzylpyrrolidin-3-yl 29 b [152]

154 4-Cl 24-diCl CO-NH-1-(4-chlorophenyl)ethyl 66 b [152]

155 4-Cl 24-diCl CO-NH-piperidinyl 356 c [182]

156 24-diCl 4-Cl CO-NH-piperidinyl 382 c [182]

a [3H]-SR141716A rat cerebellar membranesb [3H]-CP-55940 hCB1-HEK EDNA cellsc [3H]-CP-55940 hCB1-CHO cells

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1381

N

S

O

NH

Cl

Cl

Cl

F

N

S

O

NHN

ClCl

Cl

N

S

O

NH

Br

N

S

O

NHN

Cl

Cl

Cl

156 157 158 159

Fig (13) CB1 cannabinoid receptor antagonists 10 Other derivatives Structures of 45-diaryl-thiazole derivatives patented as hCB1cannabinoid receptor antagonists by Solvay Pharmaceuticals (156 157 158) or by AstraZeneca (157 158 159)

assays (mouse vas deferens and guinea pig ileum) thecompound possessed only a reduced rCB1 affinity with Kivalues of 855 and 748 nM using [3H]-SR141716A and [3H]-WIN-55-212-2 respectively (Table 11) Further four seriesof compounds differing by the nature of the substituentaround the triazole core were also described in a patent[199] Usually the compounds claimed possess two aromaticrings possibly substituted and one linear alkyl chain Theaffinity for the cannabinoid receptors was not givenNevertheless 149 was evaluated in two isolated tissuemodels the inhibition of the electrically evoked contractionsin the guinea pig ileum and in the mouse vas deferens Inthe two models compound 149 inhibited the effect of WIN-55212-2 but had no effect by itself In the vas deferensmodel the authors obtained a pA2 value of 748 to becompared with 763 obtained for AM251

Yet another five membered ring a thiazole has beenused as central moiety of a new class of cannabinoid ligandsThe thiazole ring is substituted by two aryls in position 4and 5 and by an amide (position 2) as in compounds 156-159 (Fig 13) Forty derivatives of this type weresynthesised by Lange and colleagues from SolvayPharmaceuticals and claimed in a patent as agonists orantagonists of the CB1 cannabinoid receptor [200] Nopharmacological data were provided however their activitywas assessed by measuring the cAMP in transfected hCB1-CHO cells In a recent paper from Lange et al two of thesethiazole derivatives were further described [182] Compound13 (157) the most closely related to SR141716A has a Kivalue of 227 nM while for compound 14 (158) Ki value isover 1000 nM Thus as in the imidazole series (see Table9) the aromatic substitution pattern is of great importancefor the CB1 cannabinoid receptor affinity Compound 13however was devoid of in vivo activity

Others 124-triazole derivatives were described by Dycket al compounds 150-154 unlike the Jagerovic ones havean amide moiety in position 3 [152] The highest affinitywas obtained with a 1-benzyl-pyrrolidin-3-yl substituentThe Ki value was 29 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) to be compared to 12 nM obtained for theSR141716A

Seventeen 45-diaryl-thiazole derivatives were alsosynthesised by Berggren et al and patented by AstraZeneca(157-159 Fig 13) [201]

In a patent from Merck Toupence et al described over190 substituted furo[23]pyridines claimed to be CB1cannabinoid receptor antagonists [202] The affinity (IC50 lt1microM) was measured using recombinant CHO cells and [3H]-CP-55940 and the activity using cAMP dosage (160 Fig14)

Lange et al described a triazole derivative (155) whichcan be superimposed with SR141716A Compound 155showed a Ki value of 356 nM ([3H]-CP-55940 hCB1-CHOcells) a pA2 value of 83 in the inhibition of WIN-55212-induced release of [3H]-arachidonic acid by hCB1-CHO cellsand was active in vivo in the CP-55940-inducedhypotension in rat (ED50 = 236 mgkg) [182]

Very recently Alanine from Hoffmann-LaRoche foundthat diarylbenzo[13]dioxole derivatives act as CB1

O

O

S

N

OO

F

ClCl

O

O

F O

N

OON

NH2

O

Cl

Cl

Cl

160 161 162

Fig (14) CB1 cannabinoid receptor antagonists 10 Other derivatives One example of a furo[23]pyridine derivative (160) and twoexamples of diarylbenzo[13]dioxole derivatives (161-162) described as hCB1 cannabinoid receptor antagonists by Toupence et aland Alanine et al respectively

1382 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

cannabinoid receptor antagonists (161-162 Fig 14) [203]Approximate IC50 values for twelve compounds ([3H]-CP-55940 hCB1-HEK cells) among the three hundred andeighty compounds described were given (IC50lt2microM)

et al in 1995 [207] In their hands ∆9-THC was not able toinhibit the forskolin-stimulated adenylyl cyclase activity inan assay performed on hCB2-CHO cells Subsequently theyshowed that ∆9-THC was able to antagonise HU210-inducedinhibition of adenylyl cyclase activity thus acting as anantagonist of the hCB2 cannabinoid receptor expressed inCHO cells [208] Furthermore very recently Govaerts et al[209] reported an inverse agonist effect of ∆9-THC and ∆8-THC on hCB2-CHO cells using the [35S]-GTPγ S assayThe pEC50 values were 763 and 888 respectively whilethe Emax values were ndash27 and ndash16 as compared to basal

Griffith from Pfizer described three new series of CB1cannabinoid receptor antagonists based on a bicyclic centralaromatic ring Purine pyrazoltriazines andpyrazolo[15a]pyrimidines derivatives were synthesised andinvestigated for their ability to interact with the CB1cannabinoid receptor Albeit a [35S]-GTPγ S assay wasdescribed no data were given concerning the antagonist orinverse agonist properties of such compounds Two hundredand eighty purine derivatives were synthesised [204] Theaffinities of 163 and 164 were given in the patent (28 and12 nM [3H]-SR141716A) In a following patent thesynthesis of sixty pyrazoltriazine derivatives was described(165 Fig 15) [205] The affinity of each compound was notgiven but was said to be ranging between 01 and 590 nMAnd finally more than forty pyrazolo[15a]pyrimidines weresynthesised (166 Fig 15) [206] Their affinity for the CB1cannabinoid receptor was lower than 155 nM Despite thefact that a great number of derivatives share the samesubstitution pattern around the central bicyclic aromatic ringthe comparison of these three new series of compounds ismade difficult by the absence of precise affinity data

Some other classical cannabinoids were shown to possessan antagonist activity at the CB2 receptor This is the casefor instance for the non-selective O-1184 (2) (Ki = 74 nM)which enhances the forskolin-induced cAMP production inCB2-CHO cells (EMax = 161 EC50 = 63 nM) and alsofor O-584 (3) (EMax = 246 EC50 = 138 nM) [39]

2 Indole Derivatives

Since the disclosure in 1993 of a second cannabinoidreceptor many attempts have been made to synthesise CB2cannabinoid receptor selective ligands The first compoundreported to have a somehow greater affinity for the peripheralcannabinoid receptor was WIN-55212-2 The compoundwas introduced by DrsquoAmbra and colleagues [210] back in1992 as a new cannabinoid ligand having a nanomolaraffinity and acting as an agonist In 1996 Showalter et alused a transfected cell line expressing the CB2 cannabinoidreceptor in order to identify selective ligands for thisreceptor [211] They showed that the CB2 affinity of WIN-55212-2 was 7 times greater than the CB1 with Ki valuesof 189 and 028 nM for the CB1 and CB2 cannabinoidreceptors respectively However WIN-55212-2 is anagonist of the cannabinoid receptors Interestingly WIN-55212-3 (167 Fig 16) the so-called inactive enantiomer ofWIN-55212 was recently shown to behave as a low affinity(Ki=363 microM) inverse agonist of the hCB2 cannabinoidreceptor as it decreased the [35S]-GTPγ S binding [209]

III CB2 LIGANDS RECEPTOR ANTAGONISTS ANDINVERSE AGONISTS

The compounds acting as antagonist or inverse agonistsat the CB2 cannabinoid receptor are reviewed in this thirdpart of the paper As for the CB1 cannabinoid receptorligands they are classified depending on their chemicalstructures

1 Classical Cannabinoid Derivatives

The first report of an antagonism mediated by ∆9-THC atthe peripheral cannabinoid receptor was made by Bayewitch

N N

NN

NH COOH

Cl

Cl

N N

NN

N

Cl

Cl

NH2OH

O

N

N

N

N

N

NHNH2

O

Cl

ClN

N

N

N

Cl

Cl

N

O

163 164

165 166

Fig (15) CB1 cannabinoid receptor antagonists 10 Other derivatives Examples of purine (163-164) pyrazoltriazines (165) andpyrazolo[15-a]pyrimidines (166) derivatives described as CB1 cannabinoid receptor antagonists by Griffith

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1383

The pEC50 value was 554 and the Emax value ndash26 ascompared to basal

AM630 also acts as an inverse agonist at the hCB1cannabinoid receptor (EC50 = 900 nM) as Landsman et aldemonstrated [47] Recently Zhang and colleagues reportedthe characterisation of the microsomal metabolism of AM630[212]

N

O

N

O

O

N

O

Cl

O

N

O

(S)-WIN-55212-3 167 BML-190 168

In 1996 Gallant and colleagues identified by submittinga large number of compounds to a binding assay anotherindole analog as CB2 cannabinoid receptor ligand [213] Onthe basis of this compound christened BML-190 (168 Fig16) or called indomethacin morpholinylamide theysynthesised several derivatives that possessed a selectivityratio for the CB2 cannabinoid receptor of up to 140 Thesewere the first compounds specifically designed to be CB2cannabinoid receptor ligands The first report on thepharmacological properties of BML-190 appeared onlyrecently New and colleagues highlighted in the paper theinverse agonist properties of the compound BML-190 dose-dependently increases (103 EC50= 980plusmn70 nM) theforskolin-stimulated levels of cAMP in hCB2-HEK cellswhile WIN-55212-2 decreases (44 45plusmn42nM) thisaccumulation [137]

Fig (16) CB2 cannabinoid receptor antagonists 2 Indolederivatives Chemical structures of WIN-55212-3 (167) andBML-190 (168) two indoles derivatives acting as inverseagonists at the hCB2 cannabinoid receptor Surprisingly in 2002 Melck and collaborators used

BML-190 as a CB2 receptor agonist in a cell proliferationassay [214] BML-190 was also tested as inhibitor of thecyclooxygenase-2 enzyme by Kalgutkar et al [215] Theyobtained IC50 values higher than 33 and 66 microM for theCOX-I and COX-II enzymes respectively These valueshave to be compared with the submicromolar activity ofindomethacin the parent compound Very recently Klegerisand colleagues [216] looking for an antineurotoxic action ofcannabinoids showed that BML-190 increases TNF-αsecretion by stimulated THP-1 monocytic cells but is noteffective on the IL-1β secretion

Further on the basis of WIN-54461 (12) a CB1antagonist AM630 (13) was synthesised by replacement ofthe bromine atom by an iodine one In the initial reportPertwee et al showed the antagonist effects of AM630 butalso suggested that the CB1 cannabinoid receptor may not bethe preferential receptor of AM630 [45] Later on Ross et alreported using hCB1amp2-CHO cells and [3H]-CP-55940that AM630 binds to the CB2 cannabinoid receptor (Ki =312 nM) with a selectivity ratio over 160 [48]Furthermore using a [35S]-GTPγ S assay they showed itsinverse agonist properties (EC50 = 76 nM) However

Table 12 CB2 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Reported binding affinities (Ki) and selectivity ratio (CB1CB2) of SR144528 (169) for rat and human cannabinoid receptors determined using [3H]-CP55940 as radioligand

NN

O

NH

Cl

SR144528 169

rCB2 (spleen) rCB1 (whole brain) Selectivity Ki hCB2 (transfected CHOcells)

Ki hCB1 (transfected CHOcells)

Selectivity References

038 nM 305 nM 800 06 nM 437 nM 730 [217]

067 nM 548 nM 80 [219]

56 nM gt10microM gt1780 [48]

25 nM a gt 1microM a gt400 [224]

024 nM 276 nM 115 199 nM 503 nM 25 [232]

a COS cells

1384 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

3 Diarylpyrazole Derivatives CHO cells is constitutively active and phosphorylated onSer 352 Upon treatment by SR144528 thisphosphorylation of the receptor was inhibited Moreover theSR compound was able to induce dephosphorylation ofagonist-induced CB2 phosphorylation The question whetherSR144528 induces a CB2 receptor conformational changemaking it a better phosphatase-specific substrate or ifSR144528 binding activates specific phosphatases remainsopen However this remains a nice example of inverseagonist-induced receptor resensitisation

The same Sanofi team which synthesised the CB1antagonist SR141716A reported in 1998 the synthesis andpharmacological characterisation of a CB2 cannabinoidreceptor selective antagonist the SR144528 (169) [217218] The structure of the new compound is derived fromSR141716A one SR144528 is the first selective CB2receptor antagonist reported as the inverse agonist propertiesof BML-190 were shown only recently by New et al [137]The authors reported for 169 using [3H]-CP-55940 asradioligand Ki values of 04 nM and 06 nM for the rat andthe human CB2 receptors respectively and found a CB1-CB2 selectivity ratio around 700 Table 12 sums up thebinding affinities of SR144528 for the cannabinoid receptorsobtained by different authors Interestingly while the CB2affinity is more or less constant the affinity obtained for theCB1 receptor varies by one order of magnitude [219] Theantagonist effect of SR144528 was highlighted by evaluatingits effect on cAMP production by hCB2-CHO cells exposedto 3 nM CP-55940 (EC50=10nM) The antagonism wasalso observed on the activation of the MAP kinase pathwayby CP-55940 (IC50=39nM)

The Sanofi research team working on cannabinoidsconducted investigations in order to determine the keyresidues for SR144528 interaction with the receptor (Table13) as well as the binding mode of the compound Firstlythey identified the TM4-EL2-TM5 region as a regioncontaining crucial residues for the affinity of several ligandsamong them SR144528 [224] Among these residues themutation of two cysteine of the second extracellular loop(C174 and C179) abolishes the affinity for the CB2 receptorLater on the implication of S453(161) and S457(165) inthe binding of SR144528 was evidenced by mutationalstudies In the model proposed for the docking of SR144528into the CB2 receptor the crucial contacts are comprised intothe TM4 this is the case for S453(161) and S457(165)which are proposed to form hydrogen bonds with the ligandand W464(172) proposed to have aromatic interactions withthe 4-methylbenzyl of the SR144528 [225] In 2003 Fengand Song showed that point mutations D349(130)A andA634(241)E abolished ligand binding However mutationof the arginine R350(131) member of the highly conservedDRY motif to alanine has no influence on SR144528binding [226]

Early after the report of the synthesis and characterisationof SR144528 the same authors further explored the functionof this compound and showed its inverse agonist propertiesusing CB2-CHO cells co-transfected with the luciferasereporter gene with either the CRE or the murine krox24regulatory sequence [220] In these models SR144528 notonly inhibits CP-55940 effects but also reproduces theeffects of a pertussin toxin treatment proving its inverseagonist properties SR144528 functionality was alsoexplored by [35S]-GTPγ S binding in hCB2-CHO cells(IC50=3 nM) [221] The inverse agonist function ofSR144528 was further illustrated by Rhee and Kim usingCOS cells co-transfected with hCB2 receptor and adenylylcyclase [222]

As for the CB1 cannabinoid receptor ligands Sanofideveloped tricyclic derivatives based on the 1-benzylpyrazole-3-carboxylic scaffold More than twentycompounds were disclosed in a patent [227] in 2001 (84-86Table 7) The benzyl in position 4 characteristic of the CB2diarylpyrazoles from Sanofi is conserved as well as thebicyclic residue in position 3 The length of the second linkbetween the phenyl and the pyrazole can be made of one or

Bouaboula and co-workers investigated SR144528 effectson CB2 cannabinoid receptor phosphorylation status [223]Firstly they showed that the CB2 receptor transfected in

Table 13 CB2 Cannabinoid Receptor Antagonists 3 Di-Aryl-Pyrazole Derivatives

Some of the single point mutations reported for the hCB2 cannabinoid receptor for which pharmacological data concerning the SR144528 are available

Mutant Effect References

R350(131)A no effect [226]

S453(161)A loss of affinity [225]

V456(164)I no effect [225]

S457(165)A loss of affinity [225]

C174S a loss of affinity [224 225]

C175S a loss of affinity [225]

C177S a no effect [225]

C179S a loss of affinity [224 225]

S544(193)G no effect [225]

a Second extra-cellular loop

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1385

two methylene but according to the patent one methylenebridge is preferred The Ki values of these compounds for theCB2 receptor were said to be lower than 500 nM and theirantagonist properties have been assessed using the adenylatecyclase inhibition assay

compounds by the presence of an alkyl substituent inposition 5 instead of a phenyl and of course by position 3which remains unsubstituted However except a lecture atthe 1998 ACS meeting [230] where compound 171 wasreported to have a Ki value of 835 nM and a selectivity ratioover 100 no further report on these compounds waspublished untill now

In a recent paper Mussinu and colleagues reported thesynthesis of other tricyclic derivatives starting fromSR141716A structure [228] N-piperidin-1-yl-14-di-hydroindeno[12-c]pyrazole-3-carboxamide derivatives areCB2 receptor ligands and some of them possess high affinityand selectivity for the CB2 receptor as shown in Table 7 (79-83) These tricyclic derivatives differ from the Sanofi tricyclicderivatives as they do have a phenyl ring instead of a benzylin position 1 Another difference is the nature of thesubstituent in position 3 which is not a bicycloalkyl but acyclic amine One of the compounds synthesised byMussinu et al 81 possesses the highest affinity for the CB2receptor ever reported with a Ki value of 0037 nM for themurine CB2 receptor ([3H]-CP-55940 mice spleenhomogenate) Interestingly 79 the closest SR141716Aanalog lost almost all its affinity for the CB1 receptor andincreased by four orders of magnitude its affinity for the CB2receptor Even if the structure quite similar to the SRcompounds could suggest that these derivatives behave asantagonists no pharmacological data were given in the paperto support this hypothesis

5 2-Oxoquinoline Derivatives

Inaba and colleagues from Japan Tobacco introduced in2001 a completely new structure in the cannabinoid fieldbased on a 2-oxoquinoline scaffold Over eighty derivativeswere disclosed in a patent [231] and one compound JTE-907 (173) was subsequently characterised in-vitro as well asin-vivo [232] In Table 14 are listed some of the compounds(172-178) disclosed in the patent along with their affinity forthe cannabinoid receptors These values were obtained onhuman receptors against [3H]-WIN-55212-2 For twelvecompounds the anti-inflammatory activity per os wasassessed in a carrageenin-induced paw edema model Thehighest effect reported was obtained with JTE-907 with anED50 value lower than 01 mgkg Therefore thesecompounds were claimed to be useful as anti-inflammatoryand anti-allergic agents

To further characterize JTE-907 the authors determinedits affinity for cannabinoid receptors of different species using[3H]-CP-55940 as radioligand In their hands JTE-907behaved as a very selective hCB2 ligand with Ki values of36 and 2370 nM for the CB2 and CB1 cannabinoid receptorsrespectively even more selective than the SR144528 (Kivalues of 199 and 50 nM respectively) However it has tobe mentioned that the affinities reported in the paper forJTE-907 are quite different from those reported in the patent(hCB1 Ki values of 35nM and 009 nM respectively) Italso appears that the SR144528 affinity for the CB1 receptordetermined by Iwamura et al is higher than the affinityreported by Rinaldi-Carmona et al with values of 50 and437nM respectively The authors ascribed the difference tothe variability of SR144528 binding affinities to the assayconditions The function of JTE-907 was evaluated bymeasuring the increase in cAMP production by forskolin-stimulated hCB2-CHO cells Maximum stimulation wasattained at a concentration of 1microM of JTE-907 In the sameassay WIN-55212-2 decreased the cAMP production in adose-dependent manner

Finally it is interesting to strengthen that the five-membered ring derivatives (79-86) of the diarylpyrazolefamily are reported to possess a high CB2 receptor affinitywhile the six- and seven-membered ring derivatives (69-78)are described as CB1 receptor ligands

4 Other Pyrazole Derivatives

Other CB2 ligands based on a pyrazole nucleus wereintroduced by SmithKline Beecham in a patent describingthe synthesis of about sixty new compounds [229] Theiraffinity (Ki) for the hCB2 receptor transfected in HEK293cells was reported to be ranging between 25nM and 10microMTheir structures (170-171 Fig 17) differ from the Sanofi

NN

O

N

ON

NN

N

NN

O

N

ON

NN

N

ClCl

1

5

4 3

2

170 171

Fig (17) CB2 cannabinoid receptor antagonists 4 Otherpyrazole derivatives Structure of two compounds (170-171)among those claimed by SmithKline Beecham to be hCB2antagonists

In conclusion JTE-907 was shown to be a CB2 selectiveinverse-agonist with anti-inflammatory and anti-allergicproperties This compound is currently undergoing Phase Iclinical trials as anti-allergic drug [233]

IV NON-CB1 NON-CB2 CANNABINOIDRECEPTORS LIGANDS

In the last years the possible presence of additional non-CB1 non-CB2 cannabinoid receptors was raised frompharmacological evidences

One of the putative receptors possesses an endotheliallocalisation while other ones are localized in the CNS or atperipheral nerves For instance an additional target for ∆9-

1386 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 14 CB2 Cannabinoid Receptor Antagonists 5 2-Oxoquinoline Derivatives

Structure of six compounds among the oxoquinoline derivatives patented by Japan Tobaco Affinity (Ki nM) and selectivity (CB1CB2) for the cannabinoidreceptors are given The affinity was measured by binding against [3H]-WIN-55212-2 on either hCB2-CHO or hCB1-CHO cells Adapted from [231]

Cpd ndeg Structure hCB2 hCB1 Selectivity

1-2 172

NH

MeO O

O

OMeO

0014 3671 262215

JTE-907 173

NH

O

MeO O

O

NH

O

O

0087 3436 39495

2-2 174

OH

O

NH

MeO O

O 077 3247 4215

3-27 175

NH

O

F

NH

MeO O

O0021 336 16000

3-32 176

NH

MeO O

O

NH

F

O

0036 2398 66610

3-38 177

O

O

NH

N

O

O

NMeO

0085 935 11000

3-43 178

NMeO O

O

NH

F

O

0043 864 20100

tetrahydrocannabinol was described in capsaicin-sensitivesensory nerves by Zygmunt et al [234]

[29] and by Di Marzo et al [235] Thus in the presentreview we will focus on the description of the so faridentified ligands of the non-CB1 non-CB2 receptors withan emphasis on the compounds having antagonist properties

Pharmacological evidences for the existence of additionalcannabinoid receptors were reviewed by Wiley and Martin

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1387

(Table 15) In particular the knowledge about three not yetcloned targets has grown The growing molecularpharmacology of the three putative new cannabinoidreceptors is reviewed in the three following sections Thefourth section summarises the data on the other proposedcannabinoid targets

vasodilatator activity In addition the anandamide effect ispartially sensitive to SR141716A inhibition The authorsproposed the existence of two distinct receptors responsiblefor the vasodilatatory effect of anandamide one located in theendothelium is SR141716A-sensitive while the other onelocated on the vascular smooth muscle is SR141716A-insensitive Thus SR141716A is an antagonist of anendothelial anandamide receptor Several studies wereconducted in order to understand the molecularpharmacology of the endothelial anandamide receptor It ispertussis toxin-sensitive [237] Cannabidiol acts asantagonist and abnormal-cannabidiol (Abn-cbd 179) and itsanalogue O-1602 (180) act as agonists [238] The synthesisof O-1602 and of its analogues are disclosed in a patentdescribing the vasodilatatory activity of cannabinoid

1 Endothelial Anandamide Receptor ndash Abnormal-Cannabidiol Receptor

In 1999 Wagner et al showed that anandamide couldinduce mesenteric vasodilatation through an endotheliallylocated ldquoanandamide receptorrdquo pharmacologically distinctfrom CB1 [236] Other cannabinoids such as ∆9-THC HU-210 or WIN-55212-2 were devoid of mesenteric

Table 15 Non-CB1 Non-CB2 Cannabinoid Receptors Antagonists

An overview of the putative new non-CB1 non-CB2 anandamide-receptors ligands The antagonists are highlighted in bold

NH

N

S

HO OH

Cl

OH

OH

OH OH OH OH

OMe

OMe

RR

RR

RR

RR

HO

OH

NH

O

cannabidiol (10) Abnormal-cannabidiol 179 O-1602 180

O-1918 181 capsazepine 183capsaicin 182

Compound Endothelial AEA receptor Brain AEA receptor Cannabinoid-vanilloid brain receptor

AEA agonist a agonist de

WIN-55212-2 no effecta

agonist e agonist g h

CP-55940 no effect e agonist i

SR141716A antagonist a b j no effect ([35 S]-GTPγS binding) d e f antagonist g h

AM251 no effect k no effect h i

Cannabidiol antagonist b no effect e

O-1918 antagonist c jm

Abd-cbd agonist b l

O-1602 agonist b

Capsaicin no effectj agonist i

Capsazepine no effect b antagonist h

a [236] b [238] c [240] d [249] e [250] f [251] g [254] h [258] i [ 255] k [244] j [ 243] l [ 242] m [241]

1388 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

analogues [239] More recently Offertaler et al showed thatO-1918 (181) a cannabidiol analog acts as an antagonist ofthe endothelial anandamide receptor [240] In addition O-1918 does not bind to the CB1 and CB2 cannabinoidreceptors Furthermore O-1918 unlike cannabidiol doesnot cause vasorelaxation in the mesenteric artery preparationmodel used and thus acts as a true neutral antagonistUsing O-1918 as a pharmacological tool Begg et alproposed a coupling of this new G protein-coupledendothelial receptor through a GiGo mechanism to ionchannels in primary cultured human vascular endothelialcells [241] However in their hands SR141716A wasineffective in antagonising Abd-cbd Ho and Hiley furtherdemonstrated that Abn-cbd relaxes rat small mesentericarteries through an activation of K+ channels via aSR141716A-sensitive pathway [242] Further using thesame model they characterized the vasorelaxant activity ofvirodhamine a novel endocannabinoid apparently actingthrough the same receptor [243] The virodhamine-inducedvasorelaxation is sensitive to SR141716A and O-1918 butinsensitive to AM251 SR144528 and AM630 Moreovervanilloid receptor desensitisation by capsaicin (182) had noeffect

244] Thus position 4 on the 5-phenyl could be furtherexplored to obtain selective ligands

To come to an end despite the fact that the endothelialanandamide receptor is neither identified nor cloned at leastthree antagonists are identified SR141716A cannabidioland O-1918 It is likely that a growing number of ligandsand therapeutic applications will appear in the very nearfuture

2 Brain Anandamide Receptor

Following an in-depth evaluation of anandamide effects inCB1

-- mice Di Marzo and co-workers suggested that a non-CB1 non-CB2 G protein-coupled receptor might mediatesome of the actions of AEA in mice [249] Actually effectsof AEA but not those of ∆9-THC were not decreased inCB1 cannabinoid receptor knock-out mice For instance 30mgkg of AEA were still effective in hot-plate test whereas10 mgkg of ∆9-THC had no effect on anti-nociceptionMoreover in a [35S]-GTPγ S assay AEA stimulated with anEC50 value of 223 microM the [35S]-GTPγ S binding to CB1

--

mice brain membranes (Emax= 28) ∆9-THC showed noeffect in this assay The addition of SR141716A did notaffect the [35S]-GTPγ S binding to CB1

-- mice brainmembranes Shortly after other evidences for the existence ofa new G protein-coupled cannabinoid receptor with a distinctdistribution in the central nervous system were published[250] From a set of 24 commonly used cannabinoids (AEAWIN-55212-2 JWH-030 THChellip) only AEA and WIN-55212-2 produced an increased binding of [35S]-GTPγ S onCB1

-- mice brain membranes Interestingly in this studySR141716A alone produced a significant inhibition of [35S]-GTPγ S binding similar to the inhibition obtained usingCB1

++ mice brain membranes (IC50 values of 57 microM and47 microM respectively) However SR141716A did not affectthe anandamide-induced [35S]-GTPγ S binding in CB1

--

mice brain membranes Moreover [3H]-SR141716Aexhibited significant binding in some brain regions of CB1

--

mice which are different from those exhibiting stimulation of[35S]-GTPγ S binding by WIN-55212-2 and AEA Thusthe authors proposed that the SR141716A effects on CB1

--

mice are mediated neither by the CB1 cannabinoid receptornor by the putative new anandamide brain receptor Monoryand co-workers using CB1

-- mice described a novel nonadenylyl cyclase-coupled cannabinoid binding site in micecerebellum [251] Two agonists AEA and WIN-55212-2but not ∆9-THC or HU210 were found to bind to thisreceptor as they enhanced [35S]-GTPγ S binding NeitherSR141716A nor SR144528 were able to affect the WIN-55212-2 induced [35S]-GTPγ S binding in CB1

-- mice asfound by Breivogel et al [250] However the distributionpattern of the receptor described by Monory et al differs fromthe distribution found by Breivogel et al It has to bementioned that the knock-out strains used were different Themice used by Monory were from a CD-1 strain while theone used by Breivogel were from a C57BL61 strainInterestingly Muthane et al detected differences in nigralneurons number and sensitivity to MPTP in the two mousestrains used [252]

More recently Mo et al obtained results showing thatthe Abnormal-cannabidiol receptor is also responsible for anincreased endothelial cell migration [244] Abn-cbd enhancedhuman umbilical vein endothelial cell migration while O-1918 antagonised Abn-cbd effect The CB1 cannabinoidreceptor inverse agonist SR141716A partially inhibitedAbd-cbd action whereas AM251 and SR144528 had noeffect

In another paper from George Kunos team Batkai et alreported that SR141716A is able to inhibit the endotoxichypotension by a cardiac mechanism involving neither theCB1 nor the CB2 cannabinoid receptors [245] Theinvolvement of a non-CB1 non-CB2 site of action wasshown using CB1

-- and CB1--CB2

-- knock-out miceFurther studies are needed to identify the SR141716Asensitive myocardial site of action

In a recent patent Kunos described O-1918 and itsanalogues as vasoconstrictor agents useful to reversepathological vasodilatation of blood vessels [246] Theeffects of O-1918 on mouse blood pressure on LPS-inducedhypotension in mouse as well as the reversal of hypotensionby O-1918 injection are described Furthermore thesynthesis of O-1918 based antagonists is described startingfrom p-metha-28-dien-1-ol and the appropriate resorcinols

Interestingly the Abd-cbd receptor which was firstdetected in the vascular system also seems to be present inthe CNS [247] Indeed Walter et al found that microglialcells (BV-2) migration induced by 1 microM 2-AG is inhibitedby cannabidiol (300 nM) and O-1918 (1 microM) SR141716Awas found ineffective in this assay Thus 2-AG is an agonistof the Abd-cbd receptor Moreover Abd-cbd is able to elicita dose-dependent cell migration in the same model (EC50 =600nM) [248]

From a more structural point of view SR141716Abehaves as an antagonist while on the opposite AM251(20) a close analogue which differs from the former only bythe nature of one halogen has no effect on this receptor [242

To conclude to our knowledge no compounds havingantagonist or inverse agonist properties at this receptor havebeen identified so far

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1389

3 Cannabinoid-Vanilloid Brain Receptor a cannabinoid-vanilloid receptor at which SR141716A andcapsazepine behave as antagonists

Evidences for the presence of an additional ldquocannabinoidrdquoreceptor differing from the CB1 receptor and from the brainanandamide receptor appeared from studies focused on thehippocampus (for a review see Hajos et al [253]) Hajos etal studied the cannabinoid actions on GABAergic andglutamatergic transmission using the whole-cell patch-clamp technique [254] WIN-55212-2 induced a dose-dependent reduction in glutamatergic transmission reversedby SR141716A (1microM) Thus the authors concluded that thecannabinoid actions on the glutamatergic transmission aremediated by a non-CB1 but SR141716A-sensitive receptorIn another paper Hajos and Freund further characterised thisputative receptor [255] They showed that in addition toWIN-55212-2 CP-55940 and capsaicin (182) a TRPV1receptor agonist also act as agonists Furthermore in theirhands SR141716A as well as capsazepine (183) a TRPV1receptor antagonist were able to antagonise this effect on theglutamatergic transmission while AM251 was notSR141716A effects on CB1 knock-out mice were furtherstudied using a test of anxiety [256] Haller et al showedthat on the one hand the distribution of the CB1 receptorinduces anxiety and SR141716A has an anxiolytic effectand on the other hand SR141716A is still active in CB1

--

mice In a following paper they reported that AM-251induces anxiogenic effects in wild-type and in knock-outmice [257] Thus SR141716A in addition to CB1 bindsto a not yet identified receptor Another evidence for thisadditional receptor for SR141716A was given by Bass andco-workers [61] They conducted a structure-activityrelationship study on the stimulation of locomotor activityinduced by SR141716A and twenty analogues Nocorrelation was found between the affinity for the CB1cannabinoid receptor and the stimulation of locomotoractivity Albeit SR141716A and five of its analogues O-1803 O-1710 O-1253 O-1254 (40) O-1255 (41) behavedas inverse agonists in [35S]-GTPγ S assay none of theseanalogues stimulated the locomotor activity In conclusionthe SR141716A-induced stimulation of locomotor activity isneither due to inverse agonism nor to inhibition of anendogenous tone but to a brain receptor distinct from theCB1 receptor Di Marzo et al previously provided evidencesthat the cannabinoid-vanilloid brain receptor could mediatesome of the cannabinoid effects on locomotion [249]

4 Other Non-CB1 Non-CB2 Cannabinoid Receptors

Evidences for the existence of additional non-CB1 non-CB2 cannabinoid receptors localised in the rat isolatedmesenteric arterial bed appeared from the works of Ralevicand collaborators Several cannabinoids were evaluated intheir model of sensory neurogenic vasorelaxation evoked byelectrical field stimulation

HU210 was found to attenuate vasorelaxation in a CB1and CB2 independent way as SR141716A LY320135 andSR144528 were ineffective in inhibiting HU210 effect [259]Similar results were obtained with ∆9-THC [260]Similarly noladin ether inhibited sensory neurogenicrelaxation in a concentration-dependent manner which wasalso unaffected by SR141716A LY320135 and SR144528Moreover this effect as the ∆9-THC one was pertussistoxin sensitive suggesting the involvement of a Gio protein-coupled receptor [261]

However the inhibitory effects of CP-55940 and WIN-55212-2 were affected by the presence of SR141716A (1microM)or LY320135 (1microM) but not by the addition of SR144528(1microM) [262]

It appears that at least two receptors should be involvedin cannabinoid-mediated attenuation of sensory nerve-mediated vasorelaxation The first one is SR141716A andLY320135 sensitive while the other one is resistant toSR141716A

Some evidences for the existence of a ldquoCB2-likerdquo receptorappeared from the studies on palmitoylethanolamide (PEA)an endogenous fatty acid amide devoid of CB1 and CB2affinity [264] antinociceptive potential Calignano and co-workers showed that PEA was able to alleviate pain inseveral animal models [265 266] For instance PEA dose-dependently inhibits kaolin-evoked writhing in mice TheCB2 cannabinoid receptor inverse agonist SR144528 (02mgkg) inhibits PEA anti-nociceptive effect [266] Thus theauthors suggest that PEA acts as agonist on a SR144528-sensitive non-CB2 cannabinoid receptor (ldquoCB2-likerdquoreceptor) at which SR144528 acts as an antagonist

Further studies are needed in order to better characterisethese additional ldquocannabinoidrdquo receptors However it islikely that several therapeutic applications will soon springfor these receptors

Very recently Pistis et al published a study on theneurophysiological effects of cannabinoids on the basolateralamygdala neurons in-vivo [258] One of their findings is thatHU-210 and WIN-55212-2 did not elicit similar effects onbasal amygdala projection neurons firing rate While theformer had no effect the latter decreased the firing rateMoreover SR141716A reversed the WIN-55212-2 effectswhile AM251 had no effect The vanilloid receptorantagonist capsazepine also antagonised the WIN-55212-2effect on firing rate of these neurons Therefore the authorssuggested the presence of a novel cannabinoid-vanilloidreceptor

V CONCLUSION

Since their development cannabinoid antagonists provedto be essential tools in the understanding of cannabinoidpharmacology and biochemistry Moreover the interestshown by the pharmaceutical industry in the development ofnew cannabinoid antagonists prove if necessary the realtherapeutic potential of this class of compounds Thetreatment of eating and movement disorders memorydeficits psychosis and dependencies from various addictivedrugs are some of the most cited applications for the CB1cannabinoid receptor antagonists

From these lines it appears that in addition to theknown CB1 cannabinoid receptor it is now likely that twoother cannabinoid receptors do exist in the brain The firstone should be an anandamide brain receptor for whichSR141716A has no affinity while the second one should be

1390 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Much less wide is the knowledge on the CB2cannabinoid receptor antagonists However potentialapplications could be disorders involving the immunesystem such as inflammation or allergies

[19] Devane WA Hanus L Breuer A Pertwee RG StevensonLA Griffin G Gibson D Mandelbaum A Etinger AMechoulam R Science 1992 258 1946

[20] Mechoulam R Ben-Shabat S Hanus L Ligumsky MKaminski NE Schatz AR Gopher A Almong S MartinBR Compton DR Biochem Pharmacol 1995 50 83

Finally the growing literature on the non CB1 non CB2cannabinoid receptors should allow the development of newdrugs First examples are given by the cannabidiol andabnormal-cannabidiol derivatives described by Kunos et alfor which potential therapeutic applications on the vascularsystem are already patented

[21] Sugiura T Kondo S Sukagawa A Nakane S Shinoda AItoh K Yamashita A Waku K Biochem Biophys Res Co1995 215 89

[22] Hanus L Abu-Lafi S Fride E Breuer A Vogel Z ShalevDE Kustanovich I Mechoulam R Proc Natl Acad SciUSA 2001 98 3662

[23] Oka S Tsuchie A Tokumura A Muramatsu M Suhara YTakayama H Waku K Sugiura T J Neurochem 2003 851374It clearly appears from this paper that the antagonists of

the cannabinoid receptors are still an active research fieldfrom which should emerge new promising therapeutic toolsas well as innovative drugs in the near future

[24] Goutopoulos A and Makriyannis A Pharmacol Therapeut2002 95 103

[25] Beal JE Olson R Lefkowitz L Laubenstein L Bellman PYangco B Morales JO Murphy R Powderly W PlasseTF Mosdell KW Shepard KV J Pain Symptom Manage1997 14 7ACKNOWLEDGEMENTS [26] Notcutt W Price M Miller R Newport S Phillips CSimmons S Sansom C Anaesthesia 2004 59 440

GGM is very indebted to the ldquoFonds pour la formationagrave la Recherche dans lrsquoIndustrie et lrsquoAgriculture (FRIA)rdquo Belgium for the award of a research fellowship Theexperimental works of the authors were supported by grantsfrom the FNRS The Belgian Charcot Foundation and theUniversiteacute catholique de Louvain

[27] Ledent C Valverde O Cossu G Petitet F Aubert JFBeslot F Bohme GA Imperato A Pedrazzini T RoquesBP Vassart G Fratta W Parmentier M Science 1999 283401

[28] Zimmer A Zimmer AM Hohmann AG Herkenham MBonner TI Proc Natl Acad Sci U S A 1999 96 5780

[29] Wiley JL and Martin BR Chem Phys Lipids 2002 121 57[30] Adam J and Cowley P Expert Opin Ther Patents 2002 12

1475

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[32] Barth F and Rinaldi-Carmona M Curr Med Chem 1999 6745

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[33] Little PJ Compton DR Johnson MR Melvin LS MartinBR J Pharmacol Exp Ther 1988 247 1046

[2] Devane WA Dysarz FA Johnson MR Melvin LSHowlett AC Mol Pharmacol 1988 34 605

[34] Pertwee RG Stevenson LA Elrick DB Mechoulam RCorbett AD Brit J Pharmacol 1992 105 980

[3] Matsuda LA Lolait SJ Brownstein MJ Young ACBonner TI Nature 1990 346 561

[35] Harrison C Traynor JR Life Sci 2003 74 489-508[4] Gerard CM Molerau C Vassart G Parmentier M BiochemJ 1991 279 129 [36] Pertwee RG Fernando SR Griffin G Ryan W Razdan

RJ Compton DR Martin BR Eur J Pharmacol 1996 315195

[5] Glass M Dragunow M Faull RLM Neuroscience 1997 2299

[37] Ross RA Brockie HC Fernando SR Saha B Razdan RKPertwee RG Brit J Pharmacol 1998 125 1345

[6] Shire D Carillon C Kaghad M Calandra B Rinaldi-Carmona M LeFur G Caput D Ferrara P J Biol Chem1995 270 3726 [38] Griffin G Wray EJ Rorrer WK Crocker PJ Ryan WJ

Saha B Razdan RK Martin BR Abood ME Brit JPharmacol 1999 126 1575

[7] Ryberg E Khang Vu H Groblewski T Larsson N ElebringT Hjorth S Sjogren S Greasley PJ 2004 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 119

[39] Ross RA Gibson TM Stevenson LA Saha B Crocker PRazdan RK Pertwee RG Brit J Pharmacol 1999 128 735

[40] Martin BR Winckler JR Wiley JL Huffman JW CrockerPJ Saha B Razdan RK J Pharmacol Exp Ther 1999 2901065

[8] Rinaldi-Carmona M Calandra B Shire D Bouaboula MOustric D Barth F Casellas P Ferrara P LeFur G JPharmacol Exp Ther 1998 278 871

[41] Martin BR Stevenson LA Pertwee RG Breivogel CSWilliams A Mahadevan A Razdan RK 2002 Symposium onthe Cannabinoids Burlington Vermont International CannabinoidResearch Society page 2

[9] Munro S Thomas KL Abu-Shaar M Nature 1993 365 61[10] Howlett AC Qualy JM Khachatrian LL Mol Pharmacol

1986 29 307[11] Glass M Felder CC J Neurosci 1997 17 5327

[42] Thomas A Ross RA Saha B Mahadevan A Razdan RKPertwee RG Eur J Pharmacol 2004 487 213

[12] Calandra B Portier M Kerneacuteis A Delpech M Carillon C LeFur G Ferrara P Shire D Eur J Pharmacol 1999 374445 [43] Compton DR Gold LH Ward SJ Balster RL Martin BR

J Pharmacol Exp Ther 1992 263 1118[13] Howlett AC Life Sci 1984 35 1803[44] Eissenstat MA Bell MR DAmbra TE Alexander EJ

Daum SJ Ackerman JH Gruett MD Kumar V EstepKG Olefirowicz EM Wetzel JR Alexander MD WeaverJD Haycock DA Luttinger DA Casiano FM ChippariSM Kuster JE Stevenson JI Ward SJ J Med Chem1995 38 3094

[14] Deadwyler S Hampson RE Mu J Whyte A Childers SRJ Pharmacol Exp Ther 1995 273 734

[15] Mackie K Lai Y Westenbroek R Mitchell R J Neurosci1995 15 6552

[16] Bouaboula M Poinot CC Bourrie B Canat X Calandra BRinaldi-Carmona M LeFur G Casellas P Biochem J 1995312 637 [45] Pertwee R Griffin G Fernando S Li X Hill A

Makriyannis A Life Sci 1995 56 1949[17] Bouaboula M Poinot CC Marchand J Canat X Bourrie BRinaldi-Carmona M Calandra B LeFur G Casellas P Eur JBiochem 1996 237 704

[46] Hosohata Y Quock RM Hosohata K Makriyannis AConsroe P Roeske WR Yamamura HI EurJPharmacol 1997 321 R1[18] delPulgar T Velasco G Guzman M Biochem J 2000 347

369 [47] Landsman RS Makriyannis A Deng H Consroe P RoeskeWR Yamamura HI Life Sci 1998 62 109

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[48] Ross RA Brockie HC Stevenson LA Murphy VLTempleton F Makriyannis A Pertwee RG Brit JPharmacol 1999 126 665

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[49] Huffmann JW Curr Med Chem 1999 6 705 [82] Lan R Lu Q Fan P Gatley J Volkow ND Fernando SRPertwee R Makriyannis A AAPS Pharmsci 1999 1 article 4[50] Rinaldi-Carmona M Barth F Heaulme M Shire D Calandra

B Congy C Martinez S Maruani J Neliat G Caput DFerrara P Soubrieacute P Breliegravere JC Le Fur G FEBS Lett1994 350 240

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[232] Iwamura H Suzuki H Ueda Y Kaya T Inaba T JPharmacol Exp Ther 2001 296 420[200] Lange JHM Kruse CG Herremans AHJ VanStuivenberg

HH Dijksman JAR McCreary AC World PatentApplication 2003 WO03078413

[233] httpwwwjticojpJTI_EIR02PL20030212_Epdf (accessed02162004)

[201] Berggren AIK Bostrom SJ Elebring ST Fallefors LWilstermann JM Greasley P World Patent Application 2004WO04058255

[234] Zygmunt PM Andersson DA Houmlgestaumltt ED J Neurosci2002 22 4720

[235] Di Marzo V De Petrocellis L Fezza F Ligresti A BisognoT Prostaglandins Leukot Essent Fatty Acids 2002 66 377[202] Toupence RB Debenham JS Goulet MT Madsen-Duggan

CB Walsh TE Shah SK World Patent Application 2004WO04012671

[236] Wagner JA Varga K Jarai Z Kunos G Hypertension 199933 429

[203] Alanine A Bleicher K Guba W Haap W Kube DLuebbers T Plancher J-M Roche O Rogers-Evans MSchneider G Zuegge J World Patent Application 2004WO04013120

[237] Mukhopadhyay S Chapnick BM Howlett AC Am JPhysiol-Heart Circul Physiol 2002 282 H2046

[238] Jarai Z Wagner JA Varga K Lake KD Compton DR Martin BR Zimmer AM Bonner TI Buckley NE MezeyE Razdan RK Zimmer A Kunos G Proc Natl Acad Sci US A 1999 96 14136

[204] Griffith DA United States Patent Application 2004 US20040092520

[205] Griffith DA United States Patent Application 2004 US20040157839

[239] Kunos G Martin B Razdan R World Patent Application 2001WO0103690

[206] Griffith DA United States Patent Application 2004 US20040157838

[240] Offertaler L Mo F-M Batkai S Liu J Begg M RazdanRK Martin BR Bukoski RD Kunos G Mol Pharmacol2003 63 699[207] Bayewitch M Avidor-Reiss T Levy R Barg J Mechoulam

R Vogel Zvi FEBS letters 1995 375 143 [241] Begg M Mo F-M Offertaler L Batkai S Pacher PRazdan R K Lovinger DM Kunos G J Biol Chem 2003278 46188

[208] Bayewitch M Rhee M-H Avidor-Reiss T Breuer AMechoulam R Vogel Z J Biol Chem 1996 271 9903

[209] Govaerts SJ Hermans E Lambert DM Eur J Pharm Sci2004 23 233

[242] Ho WSV Hiley CR Brit J Pharmacol 2003 138 1320[243] Ho WSV Hiley CR J Pharm Pharmacol 2004 56 869

[210] DAmbra TE Estep KG Bell MR Eissenstat MA JosefKA Ward SJ Haycock DA Baizman ER Casiano FMBeglin NC J Med Chem 1992 35 124

[244] Mo FM Offertaler L Kunos G Eur J Pharmacol 2004489 21

[245] Batkai S Pacher P Jarai J Wagner JA Kunos G Am JPhysiol-Heart Circul Physiol 2004 287 H595[211] Showalter VM Compton DR Martin BR Abood ME J

Pharmacol Exp Ther 1996 278 989

1394 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

[246] Kunos G World Patent Application 2003 WO03015700 [257] Haller J Varga B Ledent C Freund TF Behav Pharmacol2004 15 299[247] Franklin A and Stella N Eur J Pharmacol 2003 474 195

[248] Walter L Franklin A Witting A Wade C Xie Y KunosG Mackie K Stella N J Neurosci 2003 23 1398

[258] Pistis M Perra S Pillolla G Melis M Gessa GL MuntoniAL Neuropharmacology 2004 46 115

[249] Di Marzo V Breivogel C S Tao Q Bridgen DT RazdanRK Zimmer AM Zimmer A Martin BR J Neurochem2000 75 2434

[259] Ralevic V and Kendall DA Eur J Pharmacol 2001 418 117[260] Duncan M Kendall DA Ralevic V Brit J Pharmacol 2003

138 214P[250] Breivogel CS Griffin G Di Marzo V Martin BR Mol

Pharmacol 2001 60 155[261] Duncan M Millns P Smart D Wright JE Kendall DA

Ralevic V Brit J Pharmacol 2004 142 509[251] Monory K Tzavara ET Lexime J Ledent C Parmentier

M Borsodi A Hanoune J Biochem Bioph Res Commun2002 292 231

[262] Duncan M Kendall DA Ralevic V J Pharmacol Exp Ther2004 311 411

[263] Tao Q and Abood ME J Pharmacol Exp Ther 1998 285651[252] Muthane U Ramsay KA Jiang H Jackson-Lewis V

Donaldson D Fernando S Ferreira M Przedborki S ExpNeurol 1994 126 195

[264] Lambert DM DiPaolo FG Sonveaux P Kanyonyo MGovaerts SJ Hermans E Bueb J-L Delzenne NMTschirhart EJ Biochim Biophys Acta 1999 1440 266[253] Hajos N Freund TF Chem Phys Lipids 2002 121 73

[254] Hajos N Ledent C Freund TF Neuroscience 2001 106 1 [265] Calignano A La Rana G Giuffrida A Piomelli D Nature1998 394 277[255] Hajos N Freund TF Neuropharmacology 2002 43 503

[256] Haller J Bakos N Szirmay M Ledent C Freund TF Eur JNeurosci 2002 16 1395

[266] Calignano A La Rana G Piomelli D Eur J Pharmacol 2001419 191

Page 4: Current Knowledge on the Antagonists and Inverse Agonists of

1364 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

WIN-56098 (11) and WIN-54461 (12) the latter being abromo derivative of pravadoline (Fig 2) These twocompounds however have a low affinity for the cannabinoidreceptor (IC50 gt 500 nM) [4344]

cAMP accumulation in hCB1-CHO cells with an IC50 valueof 56 nM SR141716A is able after ip or poadministration to inhibit the [3H]-CP-55940 binding tomice brain measured ex-vivo with ED50

values around 2mgkg [51] In addition to the inhibition of the classicalcannabinoid tetrad effects ndash hypothermia ring immobilityanalgesia and hypolocomotion ndash already shown by Rinaldi-Carmona et al in their first report [50] SR141716A wasshown to antagonise other in-vivo effects of cannabinoidagonists For instance the agonist-induced hypotension andbradycardia in mice are abolished by SR141716A [52] aswell as the antihyperalgesic effects of the cannabinoidagonists in a neuropathic model of pain in rat [53] This isalso the case for the behavioral effects of agonists treated rats[54] or the cannabinoid tetrad effects induced by ∆9-THC inmice [55]

In 1995 Pertwee et al [45] described the effect ofAM630 (13) another close analogue of pravadoline Thiscompound behaved as an antagonist in a [35S]-GTPγ S assayon mouse brain preparations antagonising WIN-55212-2-induced [35S]-GTPγ S binding [46] and as an inverseagonist on hCB1-CHO cells [47] (EC50 = 09 microM)However Ross et al found a weak partial agonist activityfor this compound as it decreases cAMP production byhCB1-CHO cells [48] AM630 was subsequentlycharacterised as a CB2 ligand (see the CB2 section) Thepharmacological properties of the aminoalkylindole familywere reviewed by John Huffman [49]

3 Diarylpyrazole DerivativesDespite various papers described SR141716A as an

antagonist [56-58] today this compound is considered to actas an inverse agonist based on [35S]-GTPγ S [59-62] andcAMP accumulation assays [62-64] (Table 2) An interestingreview dealing with inverse agonism at the cannabinoidreceptors and mostly with SR141716A effects was recentlypublished by Roger Pertwee [65]

The lead of this class of compounds SR141716A (14)was introduced by Sanofi back in 1994 [50] This compoundwas shown by Rinaldi-Carmona and co-workers to be ahighly selective CB1 ligand with Ki values of 56 nM for thehCB1 and over 1000 nM for the hCB2 receptors expressed inCHO cells ([3H]-CP-55940) However more recentlyevidences appeared showing that SR141716A binds toother(s) receptor(s) described as anandamide andorcannabinoid receptors Thus it is possible that some of thein-vivo effects caused by this compound are at least notsolely CB1 mediated (see the fourth section of this paper)

In 1998 Pan et al [66] demonstrated that the lysineresidue K328 (192) located in the third transmembranedomain (TMH3) of the hCB1 receptor is a key residue forthe inverse agonist action of SR141716A (Table 3) Theyshowed that SR141716A enhances calcium current in hCB1transfected neurons but not in K(192)A mutant receptortransfected neurons However SR141716A still antagonizedWIN-55212-2 inhibition of calcium currents proving that itis able to bind to the mutated receptor Later on Hurst et al[67] demonstrated using molecular modeling techniques aswell as in-vitro experiments that lysine residue K328(192)is a direct interaction site for hydrogen bonding with the C3substituent of SR141716A in CB1 receptor Binding results

In a mouse vas deferens preparation SR141716A causesa rightward shift of the CP-55940 concentration-responsecurve behaving as a competitive antagonist having a pA2value of 798 Furthermore in the cAMP accumulationmodel SR141716A produces no effect by itself butantagonizes the CP-55940 inhibition of forskolin-induced

Table 2 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

In-Vitro Functional Characterisation of SR141716A (14) The Assay Used the Cell Type and the Obtained Effect are Given

Cell type Assay Effect Function References

hCB1-CHO cells cAMP accumulation no effect by itself antagonist [50]

hCB1-CHO cells cAMP accumulation uarr [cAMP] inverse agonist [64]

hCB1-CHO cells [35 S]-GTPγS darr[35 S]-GTPγS binding inverse agonist [59]

hCB1-neurons Ca2+ currents uarr Ca2+ currents inverse agonist [66]

rCB1 (rat cerebella) [35 S]-GTPγS No effect by itself antagonist [56]

rCB1 (rat cerebella) [35 S]-GTPγS No effect by itself antagonist [57]

rCB1 (rat cerebella) [35 S]-GTPγS No effect by itself antagonist [58]

rCB1 (rat brain) [35 S]-GTPγS darr[35 S]-GTPγS binding inverse agonist [62]

rCB1 (rat cerebella) [35 S]-GTPγS darr[35 S]-GTPγS binding inverse agonist [60]

rCB1 (rat cerebella) [35 S]-GTPγS darr[35 S]-GTPγS binding inverse agonist [61]

rCB1 (rat brain) cAMP accumulation uarr [cAMP] inverse agonist [63]

mCB1 (mouse brain) cAMP accumulation uarr [cAMP] inverse agonist [62]

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1365

obtained using HEK293 cells transfected with either theK328A mutant or the wild type receptor remarkablyconfirmed the modeling results (Kd values of 396 and 23nM respectively) Interestingly a vinyl-cyclohexylSR141716A derivative VCHSR1 (15) (Table 4) lacking ofhydrogen bonding sites in C3 position is not affected bythis CB1 receptor mutation as its affinity remains unchangedwith Ki values of 31 and 35 nM for the wild-type andK328A receptors respectively Four additional compoundsCHASR1 (16) CHMSR1 (17) VPSR1 (18) and PIMSR(19) differing by their potential to form hydrogen bondswere evaluated in affinity and functional assays The resultsfurther confirm the crucial interaction between the C-3carboxamide oxygen and residue K328 to obtain an inverseagonist effect as highlighted in Table 4 [68] However it hasto be said that this lysine residue is also crucial for agonistbinding (CP-55940) andor receptor activation (WIN-55212-2) [69 70]

binding of SR141716A In contrast the substitution oftyrosine by isoleucine (Y275I) resulted in the loss of ligandrecognition Calculation studies revealed that while theY539F mutant is very similar to the wild type receptor theY539I mutant shows topology changes in the 3-4-5transmembrane region This region is considered to becrucial for agonistantagonist binding at CB1 receptor sincethe previous report by Shire et al on CB1CB2 receptorchimeras [72] In contrast the first and third extracellular(EC1 and EC3) loops of the hCB1 receptor are not essentialfor the binding of SR141716A as illustrated by Murphy etal The authors constructed several receptors mutated in theirEC1 or EC3 and none of the tested mutations affected theSR141716A binding [73] McAllister et al due the highlyaromatic nature of SR141716A further explored thehypothesis that an aromatic microdomain comprised intransmembrane helix 3-4-5-6 is the SR141716A bindingsite The modelling and mutation studies undertakensuggested to the authors that this aromatic microdomaincomprised of F336 W464 Y539 W543 and W648should represent the binding site of SR141716A Moreoverthey identified F336 and W543 as direct interaction sitesfor SR141716A [74] Residue F336 was further shown bythe same group to be a key residue for both ligand binding(WIN-55212-2 and SR141716A) and receptor activationMutation of phenylalanine 336(201) to alanine resulted in

Several other studies were undertaken to determine thecritical residues for the binding of cannabinoid compoundsUnfortunately in most of the studies SR141716A was notused However McAllister et al [71] using modeling toolsand mutagenesis explored the importance of aromaticity inposition 539(275) in the CB1 receptor The substitution oftyrosine by phenylalanine (Y275F) has no effect on the

Table 3 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Summary of the Reported CB1 Cannabinoid Receptor Single Point Mutations for which Pharmacological Data Concerning the SR141716A (14) areAvailable

Mutation Species Effect References

S114A a human no significant effect [73]

S115A a human no significant effect [73]

D250(163)N human no significant effect [263]

D250(163)E human no significant effect [263]

H181A b human no significant effect [73]

R182A b human no significant effect [73]

K183A b human no significant effect [73]

D184A b human no significant effect [73]

V324(188)A human no significant effect [73]

F325(189)A human no significant effect [73]

F325(190)A mouse no significant effect [74]

K328(192)A human loss of function [66]

K328(192)A human reduction of affinity (20 fold) [67]

F336(201)A mouse reduction of affinity (3 fold) [74]

Y539(275)F human no significant effect [71]

Y539(275)I human loss of affinity [71]

W543(280)A mouse loss of affinity [74]

W648(357)A mouse reduction of affinity (7fold) [74]

a N-terminus b First extra-cellular loop

1366 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 4 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives Five SR141716A (14) DerivativesIllustrating the Importance of the C-3 Carboxamide Oxygen in the SR141716A Inverse Agonism

The structure affinity ([3H]-SR141716A hCB1-HEK293 cells) and function (Ca++ currents) are given for each compound

NN

R

Cl

Cl

Cl

Cpd ndeg R= Affinity Function References

SR141716A 14

O

NHN

Kd=23nM Inverse agonist [67]

VCHSR1 15 Ki=313nM Neutral antagonist [67]

CHASR1 16

O

NH

Ki=17nM Inverse agonist [68]

CHMSR1 17

O

N

Ki=29nM Inverse agonist [68]

VPSR1 18

N

Ki=261nM Neutral antagonist [68]

PIMSR 19

NN

Ki=67nM Neutral antagonist [68]

an increased constitutive activity of the receptor asdemonstated by [35S]-GTPγ S binding [75]

1996 It binds with high affinity to rat brain synaptosomes(Kd=061nM) It is competitively displaced by knowncannabinoids like CP-55940 or WIN-55212-2 Usingautoradiography its rat brain distribution is similar to [3H]-CP-55940 one [77] It is now a commercially available andwidely used radioligand for competition studies

A new 3D model of the CB1 cannabinoid receptor basedon the X-ray structure of the bovine rhodopsin was recentlydeveloped by Salo and co-workers [76] It would be veryinteresting to see whether or not the results obtained withprevious models are confirmed using this new model Forinstance the lysine K328 appeared as a key residue in thismodel too

Interestingly other SR141716A radiolabeled derivativeswere synthesised as radioimaging tools among them [123I]-AM251 (20) [7879] [123I]-AM281 (21) [80-82] and [18F]-SR144385 (22) [8384] for Positon Emission Tomographyor Single Photon Emission Computed Tomography Indeedcannabinoid antagonists are a much more useful tool for

The tritiated analogue of SR141716A the [3H]-SR141716A was described by Rinaldi-Carmona et al in

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1367

human radioimaging applications than agonists as they aredevoid of cannabinoid-like effects For instance Berding etal very recently reported the use of [123I]-AM281 for SinglePhoton Emission Computed Tomography imaging of theCB1 cannabinoid receptor in six human patients [85] Inorder to further optimize the brain uptake of suchSR141716A derivatives Sanofi researchers synthesised twomethoxylated SR141716A analogues using 11C as PETtracer [86] These two compounds SR149080 (24) andSR149568 (25) possess high affinity with Ki values of 15and 38 nM respectively and selectivity for the CB1 receptoras well as an improved penetration in the brain evaluated bymeasuring the radioactivity present in various brain regionsafter tail vein injection in CD-1 mice Some of therepresentative radiolabelled SR141716A derivatives aresummarised in Table 5

Colombo et al using Wistar rats [92] Simiand et alobserved that SR141716A selectively reduces sweet foodintake in primates [93] However several authors [94-96]showed that high palatability of food is not necessary toobserve a SR141716A-induced anorectic effect at doses thatdo not cause major behavioural alterations or reduced waterintake Interestingly Gomez et al demonstrated theimplication of the peripheral CB1 receptors on themodulation of feeding and therefore the possible role ofthese receptors on the SR141716A influence on food intake[97] In addition to its effects on food consumptionSR141716A seems to be able to lower the hyperglycemiathe hyperinsulinemia as well as the insulin resistance indiet-induced obese (DIO) mice In the same DIO mice adecrease in adiposity was also observed after treatment [98]Bensaid et al using another model of obesity the obese fafarats observed that SR141716A increases mRNA expressionof Acrp30 or adiponectin a plasmatic protein exclusivelysecreted by adipose tissue through a CB1-mediatedpathway Inductions of free fatty acid oxidation body weightreduction and hyperinsulinemia decrease are some of theknown physiological actions of this protein Thus along theauthors an enhanced expression of Acrp30 followingSR141716A administration could be responsible for themetabolic effects of the compound leading to body weightreduction [99] Vickers et al using the same model (ie fafarats) showed that SR141716A significantly decreases foodconsumption and weight gain in both the obese fafa rats andthe lean Zucker rats [100] This decrease was greater in thefafa group and reversible upon SR141716A withdrawal

From a more therapeutic point of view SR141716A(rimonabant Acompliareg) is one of the promising agents totreat obesity [87] It is currently in Phase III clinical trials forthe treatment and prevention of obesity Final results areexpected for this year and FDA application filling for 2005[88] Preliminary data based on a one year treatment withrimonabant (RIO-Lipids study 1036 patients) showed a 5weight loss in 72 of the treated patients [8990] Severalreports were published before clinical trials aiming todemonstrate the anti-obesity properties of this compoundThe first report by Sanofi Recherche published in 1997described the selective inhibition of sucrose intake in ratsupon SR141716A treatment (03-3 mgkg) [91] Theanorectic and weight loss effects were firstly published by

Table 5 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structure and Affinity of SR141716A Radiolabeled Derivatives Developed as Potential Radio-Imaging Tools

NN

R3

O

NH

Cl

R4

R1

R2

Cpd ndeg R1 R2 R3 R4 Affinity CB1 References

[123I]-AM251 20 123I piperidinyl CH3 Cl Ki=25 nMa [78]

[123I]-AM281 21 123I morpholinyl CH3 Cl Ki=14 nMb [80]

[18 F]-SR144385 22 Cl piperidinyl CH2-18 F Cl IC50 =29 nMa [83]

[18 F]-SR147963 23 Cl morpholinyl CH2-18 F Cl IC50 =120 nM [84]

[11 C]-SR149080 24 O-11 CH3 piperidinyl CH3 Cl IC50 =15 nMa [86]

[11 C]-SR149568 25 O-11 CH3 morpholinyl CH3 Cl IC50 =38 nMa [86]

[18 F]-NIDA-42033 26 O-CH3 piperidinyl 18 F H Ki=18 nMc [155156]

27 O-11 CH3 piperidinyl CH3 H Ki=8nMc [155157]

arat brain homogenates [3H]-CP-55940bmouse cerebellum homogenates [3H]-SR141716crat brain homogenates [3H]-AM251

1368 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Another recent study by Higgs and co-workers furtherhighlighted the role of endocannabinoids in food taste-perception and the reduction of orosensory reward of sucrosein SR141716A treated rats [101] As it was expectedSR141716A induced effects on food consumption are absentsin CB1

-- mice [102] Very recently Cota et al obtainedresults showing that endocannabinoid system modulateshomeostasis via a dual mechanism it regulates at a centrallevel food intake while it blocks at the periphery lipogeneticprocesses [103] Moreover Cani et al showed that ghrelin(an orexigenic peptide) plasma levels are significantlyreduced 45 minutes after SR141716A administration(5mgkg ip) to fasted rats in accordance with the rapiddecrease of food intake measured by the authors It istherefore likely that SR141716 effects on body weight aredue to a conjunction of central and peripheral actions [104]

cerebrospinal fluid from patients with schizophrenia [116]Later De Marchi et al measured higher amounts ofanandamide in blood of schizophrenic patients compared tocontrols [117] On the other hand Dean et al obtainedelevated [3H]-CP-55940 binding in the dorsolateralprefrontal cortex of patients suffering from schizophrenia ascompared to controls [118] Zavitsanou using [3H]-SR141716A found elevated binding in the anterior cingulatecortex of subjects with schizophrenia [119] RecentlyMeltzer and collaborators published the results of a trialconducted to evaluate the potential of four new compoundsin treating schizophrenia and schizoaffective disorders [120]SR141716A (20 mgday) was one of the compoundsevaluated during a six weeks study The authors found noeffect of the CB1 cannabinoid receptor antagonist inimproving patientrsquos schizophrenia (72 subjects)

In addition to the anti-obesity potential the ability ofSR141716A to reduce alcohol and tobacco consumption arecurrently investigated in phase III clinical trials The effect ofSR141716A on alcohol consumption in rats was reported bythe GianLuigi Gessa team [105-108] and by Gallate et al[109] The inhibition of alcohol and nicotine induceddopamine release by SR141716A (1-3 mgkg rats) wasreported by Cohen and co-workers using a brainmicrodialysis device [110] Concerning the smokingcessation preliminary results of a clinical trial (360 subjects40 mg SR141716A) showed an increased abstinence ofsmoking [111] More recent results from the STRATUS-USstudy (787 patients) were reported 362 of patientsreceiving SR141716A (20 mgday) quit smoking against206 in the placebo group [90] Recently Le Fol andGoldberg reviewed the development of cannabinoid CB1antagonists as a new class of therapeutic agents for drugsaddictions [112]

It is known since the early nineties that theadministration of cannabinoid agonists impairs memory inrodents (for a review see Castellano et al [121]) Thusadministration of a cannabinoid antagonist was expected tosomehow improve memory [122123] However dependingon the authors SR141716A when administered alone wasreported to impair (5-10 mgkg im) [124] to have no effect(1-32 mgkg ip) [125] or to improve memory (3 mgkgip) [126] In a more recent paper Wolff and Leandershowed that SR141716A (1mgkg ip) improves memoryin rats by apparently enhancing the consolidation processesof memory [127] Further they found that at higher doses (3mgkg) this effect was lost The discrepancy in the resultsreported in the literature could be ascribed either to thedifferences in the tests used or in the doses administeredFurther experiments are needed to assess whether or not acannabinoid antagonist could be helpful in memory diseases

To conclude several patents were taken by Sanoficoncerning the therapeutic applications of their leadcompound among them being anti-obesity smokingcessation neuroinflammatory diseases and anti-diarrhoeaOne of the last patents to our knowledge concerns thetreatment of sexual dysfunctions with a cannabinoidantagonist such as SR141716A [128] However da Silva etal showed that even if this compound (2 mgkg ip)enhances the effects of apomorphine (20-80 microgkg) it has noeffect alone on penile erection [129] Meanwhile Melis et al

Since the late sixties concerns exist on the associationbetween cannabis use and schizophrenia Recent studiesshowed an association between cannabis use and an increasedrisk of developing schizophrenia [113 114] Moreover apossible role of the endocannabinoid system in schizophreniahas been suggested since its pharmacological characterisation[115] Several experimental data obtained on human subjectstend to confirm such hypothesis On the one hand Lewekeet al found elevated levels of endocannabinoids in

NN

O

NH

N

Br Cl

Cl

NN

O

NH

N

Br Cl

Cl

OH

28 29

Fig (3) CB1 cannabinoid receptor antagonists 3 Diarylpyrazole derivatives The chemical structures of 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-piperidine-carboxamide (SR147778 28 ) and 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-(4-hydroxypiperidine)-carboxamide (29 ) described by Rinaldi-Carmona et al

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1369

reported an increased rate of penile erection after directinjection of SR141716A in the paraventricular nucleus of thehypothalamus [130] This effect was dose dependent and asignificant effect was obtained with a dose of 1microgkg

AM251 in mice at a same dose range (10-30 mgkg ip) thanthe anorectic effects [143] These effects are reversed byadministration of CP-55940 and are absent in CB1

-- miceproving the implication of the CB1 receptor in theantidepressant-like effect of AM251 In 2004 Liao et alreported that AM251 was able to displace the binding of[3H]-batrachotoxin A from its binding site on sodiumchannels in mice brain synaptic preparations [144] Theyobtained an IC50 value of 112 microM and a competitivemechanism of action The authors suggested that AM251 iscapable of reducing neuronal excitability through blockade ofvoltage-sensitive sodium channels in brain

From the clinical trials involving the SR141716A themost frequently reported side-effects are nausea dizziness anddiarrhoea Depression and anxiety were not higher than inthe placebo groups Regarding one of the known side effectsof SR141716A administration the enhancement of intestinalmotility Carai et al reported recently that chronicadministration of the inverse agonists to mice induced atolerance to this prokinetic effect [131] This is not the firstreport of tolerance onset after administration of SR141716A[132] however few is known on the mechanismsresponsible for that phenomenon

Wiley and collaborators reported in 2001 a studydescribing new structure-activity relationships using thepyrazole nucleus as a central scaffold [145] Starting fromSR141716A structure they alternatively substituted one ofthe four substituents while retaining the others bysubstituents known to impart agonist activity in classicalcannabinoids One of the authors expectations was todetermine which positions are responsible respectively forthe antagonism and for the affinity of SR141716A Theaffinity of thirty compounds was assessed previous to in-vivoevaluation of their function in mice using the spontaneousactivity the tail-flick and the rectal temperature assays Theauthors showed that phenyl group in position 5 is critical foraffinity as compound O-1559 (30) lacking this phenyl has adecreased affinity (Table 6) This was already shown by Lanand co-workers [136] along with the need of a substituent inpara position on the phenyl Thomas et al previouslyshowed that this substituent could be a bromine or an iodineatom [146] However an alkyl chain is also tolerated as it isthe case in compounds O-1302 (31) O-1691 (32) or O-1704(33) All these compounds antagonise the anti-nociceptiveand hypothermic effects of ∆9-THC The authors suggestedthat the 5-substituent of pyrazoles is involved in receptorrecognition and antagonism The modulations of thesubstitution pattern of the phenyl in position 1 demonstratethat the 24-di-chloro substitution is the preferred one for theaffinity as well as for the activity Several compoundssupport this assertion Thomas et al showed that additionalhalogens result in a decreased affinity as in compounds 6-I-SR141716A (34) or 4rsquo6-di-I-SR141716A (35) (Ki values of166 and 126 nM respectively) [146] Suppression of the twochlorines (O-1300 39) or replacement of these two chlorinesby an alkyl chain like in O-1254 (40) and O-1255 (41) ledto less active compounds with Ki values ranging from 150to 430 nM However O-1254 and O-1255 behaved asantagonists in a [35S]-GTPγ S assay [61] Lan et al [136]and Wiley et al [145] also modified the substitution inposition 3 Replacement of the amido piperidinyl substituentby alkyl amides as in O-1269 (42) or O-1270 (43) etherslike in O-848 (44) and O-853 (45) ketones (O-1272 46)alcohols (O-1876 47) or alkanes (O-1877 48) resultedmostly in decreased affinity (Table 6) but also with a changeof functionality in some compounds as revealed by in-vivoassays For instance replacement of the piperidinyl by apentyl (O-1269) or by an heptyl chain (O-1270) gave agonistcompounds having Ki values of 32 and 48 nM respectivelyThus the authors suggested that the 3-substituent region isinvolved in receptor recognition and agonist activity Wileyet al on the basis of their results concluded that while the3-position seems to be involved in agonism the 1- 4- and

One close analogue of SR141716A 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-piperidinecarbox-amide (SR147778 28) was described very recently byRinaldi-Carmona et al [133 134] (Fig 3) It possesses highaffinity and selectivity for the hCB1 cannabinoid receptorwith Ki values for the hCB1 and hCB2 receptors of 35 and400 nM respectively ([3H]-CP-55940 hCB1amp2-CHO cells)Further SR147778 antagonised CP-55940 effects on mousevas deferens contractions (pA2=81) and on forskolin-stimulated adenylyl cyclase activity in U373MG cells(pA2=82) but had no effects alone In-vivo SR147778 afteroral administration reversed WIN-55212-2 inducedhypothermia and analgesia As SR141716A SR147778dose-dependently reduced ethanol and sucrose solutionintake with significant effects starting at 03 mgkg (sc) and3 mgkg (po) respectively This compound is currentlyinvestigated in Phase I clinical trials for the treatment ofobesity as well as nicotine and alcohol addictions

Another derivative the 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-(4-hydroxypiperidine)-carboxamide (29) was described in a very recent patent fromSanofi [135] (Fig 3) This antagonist possesses an IC50value for the hCB1 cannabinoid receptor of 32 nM ([3H]-CP-55940 hCB1-CHO cells)

The first structure-affinity relationships for theSR141716A derivatives were reviewed by Barth andRinaldi-Carmona in 1999 [32] Since then many papersdescribing new derivatives were published In 1999 Lan etal described around thirty 15-diarylpyrazoles derivatives[136] Among them was AM251 previously reported as aradioimaging ligand for the CB1 receptor [78] and thatappeared to be more potent (Ki = 749 nM) and selective(selectivity ratio of 306) than SR141716A (Ki = 115 nMselectivity ratio of 143) They reported a Kd value of 05 nMin the mouse vas deferens model using WIN-55212 asagonist Interestingly New et al described for the samecompound an inverse agonist effect on the hCB2 receptorusing a forskolin-induced cAMP accumulation assay (EC50= 650 nM) [137] Recently the anti-obesity effects ofAM251 were reported and are not surprisingly similar tothose of SR141716A [138 139 140] Chen et aldemonstrated that there are synergistic effects on food intakesuppression between AM251 and nalmefene an opioidantagonist [141] This synergistic effect was also presentbetween SR141716A and naloxone as shown by Kirkham[142] Shearman et al found antidepressant-like effects of

1370 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

5-positions appear to be involved in antagonism The O-derivatives reported were also described in a patent alongwith their synthetic pathways which were not given in thepaper [147]

Another investigation of the SR141716Aaminopiperidine region was conducted by Francisco et al[148] They synthesised 21 analogues possessing either analkyl amide or an alkyl hydrazide substituent of various

Table 6 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structure of some of the characterised 1- 3- 4- and 5-pyrazole derivatives Binding affinities (Ki values nM) were obtained on rat brain homogenatesusing [3H]-CP-55940 [136 145 146 148 150] or [125I]-AM-251 [155]

NN

R1

R5

R3R4

Cpd ndeg R1 R3 R4 R5 [136] [145] [146] [148] [150] [155]

SR141716 14 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Cl-Ph 115 62 62 62 13 18

O-1559 30 24-di-Cl-Ph CO-NH-piperidinyl Me 1-Methylpentyl 233

O-1302 31 24-di-Cl-Ph CO-NH-piperidinyl Me 4-((CH2)5)-Ph 21 1

O-1691 32 24-di-Cl-Ph CO-NH-piperidinyl Br 4-((CH2)5)-Ph 15

O-1704 33 24-di-Cl-Ph CO-NH-piperidinyl I 4-((CH2)5)-Ph 22

6-I-SR141716 34 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-Cl-Ph 166

4rsquo6-di-I-SR141716 35 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-I-Ph 126

4rsquo-I-SR141716 36 24-di-Cl-Ph CO-NH-piperidinyl Me 4-I-Ph 75 25 6

4rsquo-Br-SR141716 37 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Br-Ph 168 3

Cpd 25 in ref [136] 38 24-di-Cl-Ph CO-NH-morpholinyl Me 4-Br-Ph 54

O-1300 39 Ph CO-NH-piperidinyl Me 4-Cl-Ph 150

O-1254 40 4-(CH2)4-Ph CO-NH-piperidinyl Me 4-Cl-Ph 226 256

O-1255 41 4-(CH2)5-Ph CO-NH-piperidinyl Me 4-Cl-Ph 433

O-1269 42 24-di-Cl-Ph CO-NH-pentyl Me 4-Cl-Ph 32 114 3

O-1270 43 24-di-Cl-Ph CO-NH-heptyl Me 4-Cl-Ph 48 462 3

O-848 44 24-di-Cl-Ph CH2-O-(CH2)2-piperidinyl

Me 4-Cl-Ph 2450 232

O-853 45 24-di-Cl-Ph CH2-O-CH2-cyclohexyl

Me 4-Cl-Ph 388 100

O-1272 46 24-di-Cl-Ph CO-heptyl Me 4-Cl-Ph 221

O-1876 47 24-di-Cl-Ph 1rsquo-OH-heptyl Me 4-Cl-Ph 657

O-1877 48 24-di-Cl-Ph Heptyl Me 4-Cl-Ph 422

MF9725-64-17 49 24-di-Cl-Ph CO-NH-butyl Me 4-Cl-Ph 134

MF9725-179-32 50 24-di-Cl-Ph CO-NH-NH-butyl Me 4-Cl-Ph 51

MF9725-66-11 51 24-di-Cl-Ph CO-NH-cyclohexyl Me 4-Cl-Ph 25

MF9725-95-31 52 24-di-Cl-Ph CO-NH-(4-hydroxybutyl)

Me 4-Cl-Ph 154

Cpd 15 [150] 53 n-pentyl CO-NH-piperidinyl Me Ph 23

Cpd 16 [150] 54 n-pentyl CO-NH-piperidinyl Me 4-Br-Ph 63

Cpd 17 [150] 55 n-hexyl CO-NH-piperidinyl Me Ph 21

Cpd 18 [150] 56 n-heptyl CO-NH-piperidinyl Me Ph 47

NIDA-41109 57 24-di-Cl-Ph CO-NH-piperidinyl Br 4-Cl-Ph 14

NIDA-41119 58 24-di-Cl-Ph CO-NH-piperidinyl H 4-Cl-Ph 9

NIDA-41057 59 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OH-Ph 104

NIDA-41020 60 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 41

NIDA-41087 61 2-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 8

NIDA-42055 62 24-di-Cl-Ph CO-NH-piperidinyl Br 4-OMe-Ph 62

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1371

lengths in position 3 of the pyrazole moiety They observedthat until five carbons the affinity increases along with thecarbon chain length This is observed with the alkylhydrazide the alkyl amide and the hydroxyalkyl amideseries Moreover the hydrazide analogues exhibit a loweraffinity for the rCB1 than the amide analogues as illustratedby compounds MF9725-64-17 (49) and MF9725-179-32(50) having Ki values of 134 and 51 nM respectively(Table 6) From their structure-activity relationships (SAR)studies the authors concluded that the pharmacophoricrequirement of the amidopiridine region is a chain not longerthan 3 Aring and that a substituent having a positive chargedensity would probably result in increased affinity andpotency The same team described in a patent several othercompounds [149] Binding affinities (Ki) against [3H]-CP-55940 [3H]-SR141716A or [3H]-WIN55212-2 on wholerat brain or on hCB1-transfected cells were given Moreoveractivity data were obtained using the [35S]-GTPγ S assaydemonstrating that these alkyl amide and hydrazide analogsact as antagonists or inverse agonists Despite the greatnumber of derivatives claimed none of them posses asignificantly greater affinity for the CB1 receptor than theSR141716A Nevertheless they showed a slightenhancement of the selectivity for the CB1 receptor over theCB2 cannabinoid receptor

affinity of these derivatives increases with the length of thealkyl chain with an optimal length of 5-6 carbons The bestcompound of this series N-(piperidin-1-yl)-5-phenyl-1-hexyl-4-methyl-1H-pyrazole-3-carboxamide (55) possesses aKi value of 21 nM determined using [3H]-CP-55940 on ratbrain membranes which is higher than the 13 nMdetermined for SR141716A in the same conditions (Table6) In the same paper the authors starting from thehypothesis that antagonism by SR141716A is caused bybinding to the same region of the receptor as do the agonists(CP-55940 and WIN-55-2122) but preventing the agonistpromoted conformational change conducted extensiveconformational analysis as well as superimposition modelsand 3D-QSAR to propose a molecular mechanismsupporting the action of SR141716A Along with theauthors the C-5 aryl substituent of SR141716A occupyinga unique region could contribute in conferring the antagonistproperties Moreover the C-3 substituent could beresponsible for the antagonist or inverse agonist propertiesdepending on the interaction with the receptor

About this topic a very interesting review dealing withthe cannabinoid receptors pharmacophores as well as withthe activationinactivation of these receptors was recentlypublished by Reggio [151]

More recently Dyck et al described seven otherderivatives varying at the amide position [152] The onlycompound possessing a higher affinity for the CB1cannabinoid receptor than SR141716A was the 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta-

The affinity for the rat CB1 receptor of seven derivativespossessing an alkyl chain in position 1 instead of the 24-dichlorophenyl substituent was reported by Shim et al in apaper describing a molecular mechanism for the antagonistand inverse agonist activity of SR141716A [150] The

NN

O

NHN

Cl Cl

Cl

NN

Cl

Cl

NON

NN

O

NH

N

Cl

ClCl

NN

O

NH

MeO Cl

CP-272871 66

63 64

65

Fig (4) CB1 cannabinoid receptor antagonists 3 Diarylpyrazole derivatives Structures of 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63 ) 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methyl-pyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]-morpholine (64 ) N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65 ) and N-phenyl-1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-pyrazole-3-carboxamide (CP-272-871 66 )

1372 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

[c]pyrrol2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63)(Fig 4) The Ki values were 5 and 12 nM respectively([3H]-CP-55940 hCB1-HEK cells)

CP-55940 rat brain) For instance N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65) (Fig 4) showed a Ki value of15 nM However no data were provided concerning thefunctionality of such compounds

Over 200 other pyrazole derivatives acting at the CB1cannabinoid receptor were claimed in a patent from Pfizer[153] The affinity for the hCB1 receptor of 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methylpyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]ndashmorpholine (64) (Fig 4)was 79 nM ([3H]-SR141716A hCB1-HEK cells) Theothers compounds affinity was between 01 and 100 nM

In an effort to develop new SR141716A analoguespossessing lower lipophilicity to be used as PET tracersKatoch-Rouse and colleagues further explored thesubstitution pattern of the two phenyls [155] Bindingassays showed that a decreased lipophilicity lead to adecreased affinity for the CB1 receptor as determined bydisplacement of [3H]-AM251 For instance compoundNIDA-41057 (59) has a lipophilicity of 42 ( expressed asElogDoct) and a Ki value of 104 nM whereas SR141716Ahas a ElogDoct value of 54 and a Ki value of 18 nM In

Seventy five pyrazole derivatives were described in a veryrecent patent from Makriyannis et al [154] The affinity ofthe compounds for the cannabinoid receptors is given TheKi values for the best compounds are lower than 6 nM ([3H]-

Table 7 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structures and affinities for the cannabinoid receptors of some representative tricyclic pyrazole derivatives Values were obtained using [3H]-CP-55940as radioligand and mouse [160 228] rat [159 227] or human [158] cannabinoid receptors Compounds 69-78 are CB1 cannabinoid receptor ligandsCompounds 79-86 are CB2 cannabinoid receptor ligands

NN

O

NHX

R3

R1

R2

n

n˚ X R1 R2 n R3 Ki CB1 (nM) Ki CB2 (nM) Selectivity References

69 -(CH2)3- Cl 24-di-Cl 0 piperidine 126 [158]

70 -(CH2)3- H 24-di-Cl 0 azepane 100 [158]

71 -(CH2)3- H 24-di-Cl 0 piperidine 398 [158]

72 -(CH2)3- Cl 24-di-Cl 0 azepane 125 [158]

73 -(CH2)3- NO2 24-di-Cl 0 azepane 63 [158]

74 -(CH2)2-O- H 24-di-Cl 0 piperidine 501 [158]

75 -CH2-S- Br 24-di-Cl 0 piperidine lt 500 gt10 [159]

76 -CH2-SO2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

77 -(CH2)2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

78 -(CH2)3- Cl 24-di-Cl 0 piperidine 000035 21 60000 [160]

79 -CH2- Cl 24-di-Cl 0 piperidine 2050 034 00002 [228]

80 -CH2- Br 24-di-Cl 0 piperidine 1570 027 00002 [228]

81 -CH2- CH3 24-di-Cl 0 piperidine 363 0037 00001 [228]

82 -CH2- Cl 4-Cl 0 piperidine 1787 09 00005 [228]

83 -CH2- H 24-di-Cl 0 piperidine 1152 0385 00003 [228]

84 -CH2- Cl 34-di-Cl 1 133-trimethylbicyclo[221]heptyl

lt500 gt10 [227]

85 -CH2- Br 24-di-Cl 1 bicyclo[321]oct-3-yl lt500 gt10 [227]

86 -CH2- Br 4-Me 1 77-dimethylbicyclo[410]hept-3-yl

lt500 gt10 [227]

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1373

another paper Katoch-Rouse described the synthesis of[18F]-NIDA-42033 (26) a ligand useful as PET radiotracerstarting from its bromo derivative [156] Recently Kumar etal [157] proposed the synthesis of a 11C derivative ofNIDA-41087 (61) a compound previously described byKatoch-Rouse (Table 5) Post-mortem binding of thiscompound to human prefrontal cortex was also investigatedInterestingly CP-272871 (66) described by Meschler as aCB1 inverse agonist (Ki=57 nM [3H]-CP-55940 rat brainhomogenates) possesses a lipophilicity similar to theNIDA-41087 one [62] However despite the presence of amethoxy function no radiolabelled derivative has beendescribed to date One explanation could be the compoundlow selectivity (2 fold) for the CB1 cannabinoid receptor

To further explore the pharmacology of this compound afunctional assay using the mouse vas deferens model wasperformed by Ruiu and colleagues enlightening thecompetitive antagonist properties of NESS (pA2= 1246against WIN-55212-2) which had no effect by itself up to1microM Moreover NESS 0327 was unable to affect basalbinding of the [35S]-GTPγ S demonstrating its lack ofnegative intrinsic activity In-vivo studies were conductedusing the hot plate and tail flick tests NESS dose-dependently abolished the antinociceptive effect of WIN-55212-2 but had no effect by itself However the authorssuggested based on the affinity and in-vivo activity of theircompound that NESS 0327 possesses a poor centralbioavailability

Several new attempts to increase the affinity of thediarylpyrazole derivatives were recently made by rigidifyingthe SR141716A structure (Table 7) Six fused ringanalogues of SR141716A obtained by fusion of the 5-(4-chlorophenyl) substituent with the central pyrazole to forman indazole ring were published by Bass et al in 2002 [61]The compound possessing the highest affinity O-1248 (67)has a Ki value of 475 nM as determined by displacement of[3H]-CP-55940 from rat brain membranes

Another example of ring-constrained biarylpyrazole wasdescribed by Herbert Seltzman team [161] Theyinvestigated a photocyclisation reaction starting from theSR141716A leading to a pyrazolo[15-f]phenanthridinestructure (Fig 5) The new compound (68) was tested on theCB1 receptor (whole rat brain) against [3H]-CP-55940[3H]-SR141716A and [3H]-WIN-55212-2 and exhibited Kivalues of 48 35 and 50 nM respectively The CB2 affinitywas assessed using [3H]-CP-55940 and was found to be 100times lower (Ki = 3340 nM) No effect on the [35S]-GTPγ Sassay (rat brain) was observed by the authors

Stoit and colleagues from Solvay Pharmaceuticalspublished a paper describing the synthesis andpharmacological characterisation of new benzocyclohepta-pyrazole derivatives as CB1 antagonists (69-74) [158] Theaffinity of these compounds was more or less one order ofmagnitude lower than the SR141716A one with pKi valuesranging from 64 to 72 compared to 76 for the SRcompound (Table 7) Compounds were shown to beantagonists with pA2 values from 70 to 89 as theyprevent CP-55940-induced cAMP accumulation Howeverthe authors investigating the bioavailability of theircompounds either po or after ip injection found negligibleblood plasma levels

NN

Cl

Cl

O

NH

N

NN

Cl

O

NHN

Cl

Cl

O-1248 67 68However just before the publication of Stoit results

Sanofi-Synthelabo took a patent describing tricyclicpyrazolecarboxylic acid amide derivatives having antagonistproperties on the CB1 cannabinoid receptor andsubmicromolar affinities (75-77) [159] Thirty examples weregiven along with their synthetic pathway but no individualpharmacological data were shown

Fig (5) CB1 cannabinoid receptor antagonists 3Diarylpyrazole derivatives Two ring-constrained diarypyrazolederivatives O-1248 (67 ) and a pyrazolo[15-f]phenanthridinederivative (68 )

It appears from these attempts to increase SR141716Aaffinity by rigidifying its structure that only a limitedincrease in the CB1 cannabinoid receptor affinity can beexpected with the apparent exception of NESS 0327Moreover rigidifying the structure often results in a morelipophilic compound in a family of compounds being yetlipophilic

More recently Ruiu and colleagues published a paperdescribing the synthesis and complete pharmacologicalcharacterisation of a very potent CB1 receptor ligandchristened NESS 0327 (78) [160] This compound has thesame structure as compound 69 published by Stoit et al(compound 20 in the reference [158]) Nevertheless Ruiu hasobtained an affinity for the CB1 cannabinoid receptor 5000times bigger than the SR141716A one with Ki values of035 pM and 18 nM respectively using mouse forebrainhomogenates and [3H]-CP-55940 as radioligandFurthermore the selectivity ratio obtained for this compoundby Ruiu is 60000 with a Ki value for the CB2 receptor of21 nM against the [3H]-CP-55940 The discrepancybetween the data obtained independently by Stoit et al andRuiu et al could hardly be explained by the differences inthe origin of the receptor used respectively hCB1-CHO andmouse forebrain

4 Phenyl Benzofuranone Derivatives

In a 1997 patent from Eli Lilly was described thesynthesis of aryl-benzothiophene and aryl-benzofuranederivatives (87-89 Fig 6) having CB1 receptor affinity andantagonist properties [162] From ten compounds having aKi value lower than 25 microM ([3H]-CP-55940 hCB1-CHOcells) one was selected and claimed to have a Ki of 170 nMCompound 88 was shown in the patent to antagonise theeffect of anandamide on cAMP accumulation with an IC50 of

1374 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

O

O

O

O

NC

S

O

O

O

O

O

O

O

NC

LY320135 8887 89

Fig (6) CB1 cannabinoid receptor antagonists 4 Phenyl benzofuranone derivatives Chemical structures of two aryl-benzofuranederivatives (87 -88 ) and one aryl-benzothiophene (89 ) derivative from Eli Lilly

500 nM and the effect of WIN-55212-2 on calcium channelsat 1microM Moreover the intra-peritoneal injection (20 mgkg)of this compound antagonised the in-vivo effect ofanandamide in the Open Field Assay a mouse behaviourmodel Later on Felder and colleagues gave a furtherdescription of this compound called LY320135 in a paperdescribing its effect on cAMP accumulation and on ioniccurrents [163] The selectivity ratio of LY320135 was 106with Ki values of 141 and 14900 nM for hCB1 and hCB2receptors ([3H]-CP-55940) respectively LY320135antagonised anandamide inhibition of forskolin-inducedcAMP accumulation with an IC50 value of 734 nMChristopoulos et al in 2001 reported for LY320135 apKb value of 527 in the inhibition of WIN-55212-2mediated response in the rat electrically-stimulated vasdeferens while the pKb value for SR141716A was 75[164]

Other patents were taken by Aventis in which somepotential applications for their compounds were describedFor instance an association between an azetidine derivativehaving CB1 antagonists properties and sibutramine aserotonin and norepinephrine reuptake inhibitor was claimed[170] as a treatment against obesity Oral administration ofcompound 90 (3 mgkg) and sibutramine (06 mgkg) to fafaZucker rats which are genetically obese rats has resulted ina decreased food intake compared to control lean Zuckerrats In another patent the association of an azetidinederivative (1-10 mgkg po ) and a D2D3 agonist likequinpirole (01 mgkg ip) was claimed to have a therapeuticpotential in the treatment of Parkinsonrsquos disease [171] Theeffect has been evidenced using an akinesia model in the ratEvidences supporting the use of CB1 antagonists as adjuvantin the treatment of Parkinsonrsquos disease begin to appear Itseems that such compounds would be helpful in thetreatment of Parkinsonism [172] and levodopa-induceddyskinesia [173] Brotchie published an interesting paperreviewing the CB1 cannabinoid receptor signaling inParkinsonrsquos disease [174] Nevertheless further clinicalstudies should be conducted to further confirm the usefulnessof CB1 antagonists in the treatment of Parkinsonrsquos disease

5 Azetidine Derivatives

The family of azetidine compounds illustrated bycompounds 90 and 91 (Fig 7) was developed at Aventis byDaniel Achard and colleagues and was described in a seriesof patents [165-168] They claimed the IC50 values of thesecompounds for the CB1 receptor to be less than or equal to100 nM These values were obtained following the proceduredescribed by Kuster et al [169] The antagonist propertywas shown using an in-vivo model the reversal ofhypothermia induced by CP-55940 in mice and the ED50obtained were lower than 50 mgkg

6 Aryl-Imidazolidine-24-Diones Derivatives

In 1999 the Didier Lambert team published a paperdescribing the affinity of 24 new 3-alkyl-55rsquo-diphenyl-imidazolidine-24-dione derivatives for the hCB1 receptor[175] The preliminary structure-activity relationships

N

SO

O

Cl

Cl

F

F

N

SO

O

Cl

NS

90 91

Fig (7) CB1 cannabinoid receptor antagonists 5 Azetidine derivatives Two examples (90 -91 ) of the azetidine derivatives developedby Aventis

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1375

Table 8 CB1 Cannabinoid Receptor Antagonists 6 Aryl-Imidazolidine-24-Diones Derivatives

Structures affinities ([3H]-SR141716A hCB1-CHO cells) and pKb values ([35 S]-GTPγS HU210 rat brain) of three aryl-imidazolidine-24-dionederivatives (92-94)

N

NH

OO

R

Br

Br

Cpd ndeg R Ki (nM) a pKb b

DML20 92 ethylmorpholine 70 611

DML21 93 1-hydroxypropyl 103 625

DML23 94 heptyl 98 574

a [175]b [177]

showed that the substitution at the nitrogen in position 3 aswell as a bromine atom in para of the phenyl rings aremandatory for the compounds affinity Later on threecompounds termed DML20 (92) DML21 (93) and DML23(94) were characterised as neutral antagonists using the [35S]-GTPγ S assay on rat cerebellum membranes [176] (Table 8)In the same model they competitively inhibited HU-210-induced [35S]-GTPγ S binding with pKB values of 611625 and 574 respectively Moreover these compoundswere proven to be quite selective for the CB1 receptor

higher than the one for the CB2 cannabinoid receptor (IC50 =300 nM) The inverse agonism of the compound wasextrapolated by inhibiting CP-55940-induced hypothermiain mice (EC50 = 18 nM) Moreover one oral dose (10mgkg) reduces the food intake in diet-induced obese ratsInterestingly food intake of CB1

-- mice was not alteredBody weight loss was maintained all over the study during achronic administration (14 days) of 10 mgkg of Cpd A

Later on Merck published a patent [180] in which someof the 45-diaryl-pyrazoles derivatives previously describedwere claimed to be useful in the treatment or the preventionof obesity in association with compounds inhibiting the11β-hydroxysteroid dehydrogenase type 1 (1β-HSD1)enzyme The 1β-HSD1 enzyme is responsible for thesynthesis of cortisol increased levels of which according tothe inventors are associated with obesity However neitherresults on appetite suppression nor on weight loss weregiven even if some indications on the affinity of thecompounds for the cannabinoid receptors were given

Very recently the functionality of these DML compoundswas explored using the [35S]-GTPγ S assay both on rat andhuman CB1 cannabinoid receptors [177] The data obtainedconfirm that the 3-alkyl-55rsquo-diphenyl-imidazolidinedionederivatives behave as neutral antagonists on rat CB1cannabinoid receptor (rat brain) However DML 20 DML21and DML23 acted as inverse agonists on the human receptor(hCB1-CHO cells) Furthermore the authors showed thatthis different functionality is not due to the hCB1 receptorlevel of expression in the recombinant cell line as whateverthe level of expression (Bmax of 44 pmolmg or 32pmolmg) DML derivatives behaved as inverse agonists ofthe hCB1 cannabinoid receptor

Another imidazole ring pattern of substitution wasproposed by Hagmann and collaborators [181] Thecompounds described 43 examples are given possess twophenyls the first one in position 1 linked to the nitrogenand the second one in position 2 The carboxamidesubstituent is in position 4 instead of in position 2 for thediarylimidazoles described by Finke These 12-diaryl-imidazoles are claimed to be CB1 cannabinoid receptorantagonists or inverse agonists but the absence of precisepharmacological values do not allow any comparisonbetween the two substitutions patterns However a samepattern of substitution was used by other researchers Amongthem Dyck et al described in 2004 ten 1-(4-chlorophenyl)-2-(24-dichlorophenyl)-imidazole derivatives varying by theircarboxamide substituent in position 4 (101-103 Table 9)[152] For instance with a 3-azabicyclo[330]octan-3-ylsubstituent (101) they obtained a Ki value of 14 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) In a [35S]-GTPγ Sassay 101 exhibited an IC50 value of 19 nM

7 Diarylimidazoles Derivatives

Finke and colleagues at Merck developed a new class ofCB1 receptor antagonists based on an imidazole nucleusOver eighty 45-diaryl-imidazoles derivatives having CB1antagonist properties were claimed in a patent published in2003 [178] Some representative compounds of the patent areillustrated in Table 9 (95-100) The affinity of the newcompounds was determined using [3H]-CP-55940 andhCB1-CHO cells Although no detailed pharmacologicaldata were given in the patent in-vitro and in-vivo propertiesof one compound termed Cpd A (100) have been describedelsewhere [179] Its affinity for the CB1 cannabinoid receptorwas shown to be nanomolar (IC50 = 4 nM) and 75 times

1376 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 9 CB1 Cannabinoid Receptor Antagonists 7 Diaryl-Imidazole Derivatives

Structure and affinity range of some 45-diaryl-imidazole derivatives for the human cannabinoid receptors determined using [3H]-CP-55940 asradioligand and hCB1amp2-CHO cells

N

NR2

R1

R4

R3

ndeg R1 R2 R3 R4 hCB1 (IC50 or Ki) hCB2 (IC50 or Ki) References

95 CH3 CO-NH-piperidinyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

96 CH3 CO-NH-piperidinyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

97 CH3 CO-NH-cyclohexyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

98 CH3 CO-NH-phenyl 24-di-Cl-Ph 4-Cl-Ph 10 ndash 100 nM a 100 ndash1000 nM a [178]

99 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

100 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph 4 nM a 300 nM a [179]

101 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

CH3 14 nM b ND [152]

102 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

H 66 nM b ND [152]

103 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 85 nM b ND [152]

104 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 23 nM b 542 nM b [182]

105 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 30 nM b 608 nM b [182]

106 4-Br-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 60 nM b 489 nM b [182]

107 4-CF3-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 29 nM b 634 nM b [182]

108 4-Cl-Ph 24-di-Cl-Ph CO-NH-morpholinyl CH3 197 nM b 3297 nM b [182]

109 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidin-4-ol CH3 172 nM b 3959 nM b [182]

110 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CN 30 nM b 1590 nM b [182]

111 4-Cl-Ph 24-di-Cl-Ph CO-NH- piperidinyl CH2F 36 nM b 906 nM b [182]

112 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH2-CH3 14 nM b 430 nM b [182]

113 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Cl 27 nM b 823 nM b [182]

114 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Br 23 nM b 746 nM b [182]

115 4-Cl-Ph 24-di-Cl-Ph CO-NH-tetrahydroisoquinoline CH3 34 nM b 696 nM b [182]

116 4-Cl-Ph 24-di-Cl-Ph CO-NH-cycloheptyl CH3 35 nM b 349 nM b [182]

117 24-di-Cl-Ph 4-Cl-Ph CO-NH-piperidinyl CH3 403 nM b 208 nM b [182]a IC50 value b Ki value

Lange and co-workers from Solvay described twenty-eight imidazole derivatives [182] The 24-dichlorophenylsubstituent at position 2 was kept constant while thesubstitutions at the other positions were explored (Table 9)Position 5 can accommodate a large range of littlesubstituents like hydrogen (104) methyl (105) ethyl (112)chlorine (113) bromine (114) fluoromethyl (111) or cyano(110) without major changes in the affinity At position 4more bulky substituents like 1234-tetraisoquinoline (115)or cycloheptyl (116) were well tolerated whereas thepresence of a more hydrophilic moiety such as morpholine(108) or piperidin-4-ol (109) was detrimental to the affinity

Interestingly position exchange between the 24-diphenyland the 4-chlorophenyl substituents led to a 13 fold loweraffinity with Ki values of 30 and 403 nM for 105 and 117respectively ([3H]-CP-55940 hCB1-CHO cells) In thefunctional assay which was inhibition of WIN-55212-2-induced [3H]-arachidonic acid release by hCB1-CHO cellsall tested compounds behaved as antagonists Compound105 showed a pA2 value of 86 which is the same theauthors obtained for SR141716A It would be interesting toassess if these compounds are true antagonists or if they actmore as inverse agonists Compound 105 when administeredto rats was able to inhibit CP-55940-induced hypotension

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1377

with an ED50 value of 24 mgkg which is close to the 3mgkg obtained for SR141716A The compound was alsoactive in another in-vivo model the WIN-55212-2-inducedhypothermia in mice Compound 104 which has nosubstituent in position 5 of the imidazole ring was lessactive in the hypotension model and inactive in thehypothermia model Thus a methyl group is the preferredsubstituent in position 5 of the imidazole moiety Otherproperties either experimental (P-glycoprotein affinitylogPHPLC) or computational (molecular volume polarsurface area) obtained for compounds 105 were similar tothose obtained for SR141716A Finally molecularmodelling studies revealed a close structural overlap betweenthe two compounds

value of 197 nM is selective for hCB1 and is as potent asSR141716A The structural modulations of 118 led to someinteresting compounds (Table 10) Six compounds possess aKi value for the hCB1 cannabinoid receptor equal to or lowerthan 25 nM antagonist properties in the inhibition of [3H]-arachidonic acid release by WIN-55212-2-stimulated hCB1-CHO cells and Ki values for the hCB2 receptor over 1microMThe diaryl-pyrazoline derivatives contained a chiral center atposition 4 The authors resolved the racemic mixture of twocompounds (121 and 130) The levorotatory enantiomersappeared to be the eutomers For compound SLV319levorotatory enantiomer of 121 the Ki and pA2 values were78 nM and 99 respectively The Ki and pA2 values ofSLV326 levorotatory enantiomer of 130 were 359 nM and9 respectively

8 34-Diaryl-Pyrazoline Derivatives Two in-vivo models CP-55940-induced hypotension inrat and WIN-55212-2-induced hypothermia in mouse wereused to assess the potential of these compounds The resultsare of the same order of magnitude than those obtained forSR141716A For instance in the hypotension modelSLV319 ((-)121) and SLV326 ((-)130) showed ED50 values

Lange and collaborators also discovered 34-diaryl-pyrazoline derivatives after a screening of compoundsresembling to SR141716A [183184] Compound 118 thatwas initially identified during the screening possesses a Ki

Table 10 CB1 Cannabinoid Receptor Antagonists 8 34-Diaryl-Pyrazolines Derivatives

Structure affinity for the hCB1 and hCB2 cannabinoid receptors (Ki nM) expressed in CHO transfected cells ([3H]-CP-55940) and potency (pA2) ofsome 34-dirylpyrazolines derivatives Table adapted from [184]

NN

N N

SO2

R3

R2

Cl R1

R4

n˚ R1 R2 R3 R4 Ki hCB1 (nM) Ki hCB2 (nM) pA2 (CB1)

118 H H H 4-CH3 197 gt 1000 84

119 H H H 246- CH3 24 gt 1000 94

120 H H H 4-Cl 16 gt 1000 95

121 H CH3 H 4-Cl 25 gt 1000 87

122 H CH3 CH3 4-Cl 280 gt 1000 85

123 H H H 4-F 53 gt 1000 9

124 H CH3 H 4-F 338 gt 1000 85

125 H CH3 CH3 4-F gt 1000 gt 1000 lt 75

126 H CH3 H 3-Cl 14 gt 1000 86

127 4-Cl CH3 H 4-Cl 255 ND ND

128 4-F CH3 H 4-Cl 584 ND ND

129 H CH3 H H 170 ND 75

130 H CH3 H 4- CF3 221 gt 1000 93

SR141716A 25 1580 86

1378 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

N

HN

O

N

Cl Cl

Cl

N

N

HN

O

Br

Br131 132

Fig (8) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Chemical structures of two 56-diaryl-pyrazine-2-amide derivatives (131-132) developed as CB1 cannabinoid receptorantagonists by AstraZeneca

of 55 and 2 mgkg (po) respectively proving their in-vivoefficacy after oral administration The authors alsodemonstrated that SLV319 is devoid of affinity for the P-glycoprotein pump and that it possessed a goodCNSplasma ratio (17) These two compounds (SLV319and SLV326) were chosen as development candidates bySolvay and entered clinical Phase I trials in 2003 [185]

132 Fig 8) useful as CB1 cannabinoid receptor antagonists[186 187] The activity of twenty derivatives wasdetermined using a [35S]-GTPγ S assay and hCB1-CHOtransfected cells The concentration required to give halfmaximal inhibition of CP-55940-induced [35S]-GTPγ Sbinding (IC50) is lower than 200 nM for the preferredcompounds No affinity data were given for thesecompounds

9 Diaryl-Pyrazine Diphenyl-Pyridine Diphenyl-Phenyland Diaryl-Pyrimidines Derivatives

The same year in a patent from Merck Finke andcollaborators described several 56-di-phenyl-pyridinederivatives as hCB1 antagonists or inverse agonists [188]The compounds have a substituent in position 3 of thepyridine core (Fig 9) This substituent could be a cyano(133-134) or a nitro group a halogen an ester or an amide(135-136) Over 150 compounds were synthesised toillustrate the invention but no pharmacological data wasdisclosed in the patent

In 2003 appeared several patents describing new familiesof compounds that bind to the CB1 cannabinoid receptorThese compounds have in common a central six atomsaromatic ring which could be a pyrazine a pyridine aphenyl or a pyrimidine and that is substituted by at leasttwo phenyl rings

The first patents were from Berggren and collaboratorsfrom AstraZeneca who published two patents describing thesynthesis of 56-diaryl-pyrazine-2-amide derivatives (131-

A few months later another patent was taken by Sanoficlaiming the antagonist properties of 56-di-phenyl-2-pyridine carboxamide derivatives [189] These compounds

N

NH

O

Cl

Cl

N

O

Cl

O NH

N

O

Cl

ClCl

NH

CN O

N

Cl

Cl

Cl

OHN

N

O

Cl

ClCl

CNF

F

133 134

135 136 137

Fig (9) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives 56-Di-phenyl-pyridine derivatives patented by Merck (133-136) and by Sanofi (137) as CB1 cannabinoid receptorsantagonists

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1379

NH

O

N

Cl

ClCl

NH

O

N

Cl

F3CCl

N

N

O

Cl

Cl Cl F138 139 140

Fig (10) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Representative structures of the diphenyl-phenyl (138-139) and 45-diphenyl-pyrimidine (140) derivatives developed ascannabinoid antagonists at Sanofi and Merck respectively

28 examples are given structurally related to the diphenyl-phenyl derivatives have their pyridine core substituted bytwo phenyl rings and by an amide moiety (137 Fig 9) TheIC50 values were lower than 100 nM but no data were givenshowing the antagonist properties although the compoundswere assayed in the adenylate cyclase inhibition assay

10 Other Derivatives

Several amide derivatives structures recently appearedamong the CB1 cannabinoid receptor antagonists In a seriesof patents taken by Merck in 2003 Hagmann and colleaguesdescribed a large amount of compounds obtained by parallelsynthesis which are claimed to be CB1 cannabinoid receptorantagonists [192-195] They were synthesised by reacting alibrary of substituted amines with a library of carboxylicacids (141-144 Fig 11) However as no value was givenfor the affinity or activity of these amides we will notfurther discuss this class of compounds

Along this line Sanofi Synthelabo developed newdiphenyl-phenyl derivatives claimed to have CB1cannabinoid receptor antagonist properties [190] They arebased on a central phenyl ring substituted by two phenylsand by an amide moiety (138-139 Fig 10) Thirteencompounds were claimed along with their synthesis TheirIC50 values were lower than 100 nM ([3H]-CP55-940hCB1-CHO cells) but no other specific data (selectivitypA2hellip) was given

Several 15-diaryl-pyrrole-3-carboxamide derivatives weresynthesised and claimed to be CB1 cannabinoid receptorantagonists by Berggren et al [196] The affinity valueswere not disclosed in the patent (145-146 Fig 12) Otherpyrrole derivatives were synthesised by Guba et al atHofmann-La Roche [197] More specifically 2-(thiazol-4-yl)pyrrole derivatives were claimed as CB1 cannabinoidreceptor antagonist (IC50lt2microM) (147-148 Fig 12)

Finally Kopka et al from Merck described more thanone hundred 45-diaryl-pyrimidine derivatives as CB1cannabinoid receptor antagonists (140 Fig10) [191]However no precise data were provided concerning theaffinity and functionality of the compounds

R2

R1N

R3

R4

R5

O

NH

O

Cl

O F

F

R2

R1NH

R3

R4

HO R5

O

NH

O

Cl

CN

NH

O

Cl

O

O

NH

O

Cl

Cl

+

141142

143 144

Fig (11) CB1 cannabinoid receptor antagonists 10 Other derivatives Retrosynthetic scheme of a CB1 cannabinoid antagonistlibrary of amide derivatives developed at Merck The structures of four representative compounds are illustrated (141-144)

1380 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

NH

O N

Cl

Cl

Cl

N

NH

O

OMe

MeO

N

NH

O

N

S

MeO

N

NH

O

N

S

MeO

145 146

147 148

Fig (12) CB1 cannabinoid receptor antagonists 10 Other derivatives Two examples of 15-diaryl-pyrrole derivatives described byBerggren et al (145-146) While the 2-(thiazol-4yl)pyrroles derivatives 147 and 148 were described by Guba et al from Hofmann-LaRoche All these pyrroles derivatives were claimed to be CB1 cannabinoid receptor antagonists

Another new structure is represented by the 124-triazolederivatives developed by Jagerovic and collaborators In arecent paper were described the synthesis andpharmacological properties of five new 15-diphenyl-3-alkyl-

triazole derivatives [198] Among these compounds onlyone (149) behaved as a CB1 antagonist inhibiting the WIN-55212-2-induced contractions in a mouse vas deferenspreparation However despite its effects in isolated tissue

Table 11 CB1 Cannabinoid Receptor Antagonists 10 Other Derivatives

Structure and affinity of diaryl-triazole derivatives

N

NN

R3

R2

R1

Cpd R1 R2 R3 Ki CB1 (nM) References

149 24-diCl 4-Cl hexyl 855 a [198]

150 24-diCl 4-Cl2-OMe CO-NH-(3-azabicyclo[330]octan-3-yl) 270 b [152]

151 24-diCl 4-Cl CO-NH-(3-azabicyclo[330]octan-3-yl) 164 b [152]

152 4-Cl 24-diCl CO-NH-(3-azabicyclo[330]octan-3-yl) 137 b [152]

153 4-Cl 24-diCl CO-NH-benzylpyrrolidin-3-yl 29 b [152]

154 4-Cl 24-diCl CO-NH-1-(4-chlorophenyl)ethyl 66 b [152]

155 4-Cl 24-diCl CO-NH-piperidinyl 356 c [182]

156 24-diCl 4-Cl CO-NH-piperidinyl 382 c [182]

a [3H]-SR141716A rat cerebellar membranesb [3H]-CP-55940 hCB1-HEK EDNA cellsc [3H]-CP-55940 hCB1-CHO cells

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1381

N

S

O

NH

Cl

Cl

Cl

F

N

S

O

NHN

ClCl

Cl

N

S

O

NH

Br

N

S

O

NHN

Cl

Cl

Cl

156 157 158 159

Fig (13) CB1 cannabinoid receptor antagonists 10 Other derivatives Structures of 45-diaryl-thiazole derivatives patented as hCB1cannabinoid receptor antagonists by Solvay Pharmaceuticals (156 157 158) or by AstraZeneca (157 158 159)

assays (mouse vas deferens and guinea pig ileum) thecompound possessed only a reduced rCB1 affinity with Kivalues of 855 and 748 nM using [3H]-SR141716A and [3H]-WIN-55-212-2 respectively (Table 11) Further four seriesof compounds differing by the nature of the substituentaround the triazole core were also described in a patent[199] Usually the compounds claimed possess two aromaticrings possibly substituted and one linear alkyl chain Theaffinity for the cannabinoid receptors was not givenNevertheless 149 was evaluated in two isolated tissuemodels the inhibition of the electrically evoked contractionsin the guinea pig ileum and in the mouse vas deferens Inthe two models compound 149 inhibited the effect of WIN-55212-2 but had no effect by itself In the vas deferensmodel the authors obtained a pA2 value of 748 to becompared with 763 obtained for AM251

Yet another five membered ring a thiazole has beenused as central moiety of a new class of cannabinoid ligandsThe thiazole ring is substituted by two aryls in position 4and 5 and by an amide (position 2) as in compounds 156-159 (Fig 13) Forty derivatives of this type weresynthesised by Lange and colleagues from SolvayPharmaceuticals and claimed in a patent as agonists orantagonists of the CB1 cannabinoid receptor [200] Nopharmacological data were provided however their activitywas assessed by measuring the cAMP in transfected hCB1-CHO cells In a recent paper from Lange et al two of thesethiazole derivatives were further described [182] Compound13 (157) the most closely related to SR141716A has a Kivalue of 227 nM while for compound 14 (158) Ki value isover 1000 nM Thus as in the imidazole series (see Table9) the aromatic substitution pattern is of great importancefor the CB1 cannabinoid receptor affinity Compound 13however was devoid of in vivo activity

Others 124-triazole derivatives were described by Dycket al compounds 150-154 unlike the Jagerovic ones havean amide moiety in position 3 [152] The highest affinitywas obtained with a 1-benzyl-pyrrolidin-3-yl substituentThe Ki value was 29 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) to be compared to 12 nM obtained for theSR141716A

Seventeen 45-diaryl-thiazole derivatives were alsosynthesised by Berggren et al and patented by AstraZeneca(157-159 Fig 13) [201]

In a patent from Merck Toupence et al described over190 substituted furo[23]pyridines claimed to be CB1cannabinoid receptor antagonists [202] The affinity (IC50 lt1microM) was measured using recombinant CHO cells and [3H]-CP-55940 and the activity using cAMP dosage (160 Fig14)

Lange et al described a triazole derivative (155) whichcan be superimposed with SR141716A Compound 155showed a Ki value of 356 nM ([3H]-CP-55940 hCB1-CHOcells) a pA2 value of 83 in the inhibition of WIN-55212-induced release of [3H]-arachidonic acid by hCB1-CHO cellsand was active in vivo in the CP-55940-inducedhypotension in rat (ED50 = 236 mgkg) [182]

Very recently Alanine from Hoffmann-LaRoche foundthat diarylbenzo[13]dioxole derivatives act as CB1

O

O

S

N

OO

F

ClCl

O

O

F O

N

OON

NH2

O

Cl

Cl

Cl

160 161 162

Fig (14) CB1 cannabinoid receptor antagonists 10 Other derivatives One example of a furo[23]pyridine derivative (160) and twoexamples of diarylbenzo[13]dioxole derivatives (161-162) described as hCB1 cannabinoid receptor antagonists by Toupence et aland Alanine et al respectively

1382 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

cannabinoid receptor antagonists (161-162 Fig 14) [203]Approximate IC50 values for twelve compounds ([3H]-CP-55940 hCB1-HEK cells) among the three hundred andeighty compounds described were given (IC50lt2microM)

et al in 1995 [207] In their hands ∆9-THC was not able toinhibit the forskolin-stimulated adenylyl cyclase activity inan assay performed on hCB2-CHO cells Subsequently theyshowed that ∆9-THC was able to antagonise HU210-inducedinhibition of adenylyl cyclase activity thus acting as anantagonist of the hCB2 cannabinoid receptor expressed inCHO cells [208] Furthermore very recently Govaerts et al[209] reported an inverse agonist effect of ∆9-THC and ∆8-THC on hCB2-CHO cells using the [35S]-GTPγ S assayThe pEC50 values were 763 and 888 respectively whilethe Emax values were ndash27 and ndash16 as compared to basal

Griffith from Pfizer described three new series of CB1cannabinoid receptor antagonists based on a bicyclic centralaromatic ring Purine pyrazoltriazines andpyrazolo[15a]pyrimidines derivatives were synthesised andinvestigated for their ability to interact with the CB1cannabinoid receptor Albeit a [35S]-GTPγ S assay wasdescribed no data were given concerning the antagonist orinverse agonist properties of such compounds Two hundredand eighty purine derivatives were synthesised [204] Theaffinities of 163 and 164 were given in the patent (28 and12 nM [3H]-SR141716A) In a following patent thesynthesis of sixty pyrazoltriazine derivatives was described(165 Fig 15) [205] The affinity of each compound was notgiven but was said to be ranging between 01 and 590 nMAnd finally more than forty pyrazolo[15a]pyrimidines weresynthesised (166 Fig 15) [206] Their affinity for the CB1cannabinoid receptor was lower than 155 nM Despite thefact that a great number of derivatives share the samesubstitution pattern around the central bicyclic aromatic ringthe comparison of these three new series of compounds ismade difficult by the absence of precise affinity data

Some other classical cannabinoids were shown to possessan antagonist activity at the CB2 receptor This is the casefor instance for the non-selective O-1184 (2) (Ki = 74 nM)which enhances the forskolin-induced cAMP production inCB2-CHO cells (EMax = 161 EC50 = 63 nM) and alsofor O-584 (3) (EMax = 246 EC50 = 138 nM) [39]

2 Indole Derivatives

Since the disclosure in 1993 of a second cannabinoidreceptor many attempts have been made to synthesise CB2cannabinoid receptor selective ligands The first compoundreported to have a somehow greater affinity for the peripheralcannabinoid receptor was WIN-55212-2 The compoundwas introduced by DrsquoAmbra and colleagues [210] back in1992 as a new cannabinoid ligand having a nanomolaraffinity and acting as an agonist In 1996 Showalter et alused a transfected cell line expressing the CB2 cannabinoidreceptor in order to identify selective ligands for thisreceptor [211] They showed that the CB2 affinity of WIN-55212-2 was 7 times greater than the CB1 with Ki valuesof 189 and 028 nM for the CB1 and CB2 cannabinoidreceptors respectively However WIN-55212-2 is anagonist of the cannabinoid receptors Interestingly WIN-55212-3 (167 Fig 16) the so-called inactive enantiomer ofWIN-55212 was recently shown to behave as a low affinity(Ki=363 microM) inverse agonist of the hCB2 cannabinoidreceptor as it decreased the [35S]-GTPγ S binding [209]

III CB2 LIGANDS RECEPTOR ANTAGONISTS ANDINVERSE AGONISTS

The compounds acting as antagonist or inverse agonistsat the CB2 cannabinoid receptor are reviewed in this thirdpart of the paper As for the CB1 cannabinoid receptorligands they are classified depending on their chemicalstructures

1 Classical Cannabinoid Derivatives

The first report of an antagonism mediated by ∆9-THC atthe peripheral cannabinoid receptor was made by Bayewitch

N N

NN

NH COOH

Cl

Cl

N N

NN

N

Cl

Cl

NH2OH

O

N

N

N

N

N

NHNH2

O

Cl

ClN

N

N

N

Cl

Cl

N

O

163 164

165 166

Fig (15) CB1 cannabinoid receptor antagonists 10 Other derivatives Examples of purine (163-164) pyrazoltriazines (165) andpyrazolo[15-a]pyrimidines (166) derivatives described as CB1 cannabinoid receptor antagonists by Griffith

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1383

The pEC50 value was 554 and the Emax value ndash26 ascompared to basal

AM630 also acts as an inverse agonist at the hCB1cannabinoid receptor (EC50 = 900 nM) as Landsman et aldemonstrated [47] Recently Zhang and colleagues reportedthe characterisation of the microsomal metabolism of AM630[212]

N

O

N

O

O

N

O

Cl

O

N

O

(S)-WIN-55212-3 167 BML-190 168

In 1996 Gallant and colleagues identified by submittinga large number of compounds to a binding assay anotherindole analog as CB2 cannabinoid receptor ligand [213] Onthe basis of this compound christened BML-190 (168 Fig16) or called indomethacin morpholinylamide theysynthesised several derivatives that possessed a selectivityratio for the CB2 cannabinoid receptor of up to 140 Thesewere the first compounds specifically designed to be CB2cannabinoid receptor ligands The first report on thepharmacological properties of BML-190 appeared onlyrecently New and colleagues highlighted in the paper theinverse agonist properties of the compound BML-190 dose-dependently increases (103 EC50= 980plusmn70 nM) theforskolin-stimulated levels of cAMP in hCB2-HEK cellswhile WIN-55212-2 decreases (44 45plusmn42nM) thisaccumulation [137]

Fig (16) CB2 cannabinoid receptor antagonists 2 Indolederivatives Chemical structures of WIN-55212-3 (167) andBML-190 (168) two indoles derivatives acting as inverseagonists at the hCB2 cannabinoid receptor Surprisingly in 2002 Melck and collaborators used

BML-190 as a CB2 receptor agonist in a cell proliferationassay [214] BML-190 was also tested as inhibitor of thecyclooxygenase-2 enzyme by Kalgutkar et al [215] Theyobtained IC50 values higher than 33 and 66 microM for theCOX-I and COX-II enzymes respectively These valueshave to be compared with the submicromolar activity ofindomethacin the parent compound Very recently Klegerisand colleagues [216] looking for an antineurotoxic action ofcannabinoids showed that BML-190 increases TNF-αsecretion by stimulated THP-1 monocytic cells but is noteffective on the IL-1β secretion

Further on the basis of WIN-54461 (12) a CB1antagonist AM630 (13) was synthesised by replacement ofthe bromine atom by an iodine one In the initial reportPertwee et al showed the antagonist effects of AM630 butalso suggested that the CB1 cannabinoid receptor may not bethe preferential receptor of AM630 [45] Later on Ross et alreported using hCB1amp2-CHO cells and [3H]-CP-55940that AM630 binds to the CB2 cannabinoid receptor (Ki =312 nM) with a selectivity ratio over 160 [48]Furthermore using a [35S]-GTPγ S assay they showed itsinverse agonist properties (EC50 = 76 nM) However

Table 12 CB2 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Reported binding affinities (Ki) and selectivity ratio (CB1CB2) of SR144528 (169) for rat and human cannabinoid receptors determined using [3H]-CP55940 as radioligand

NN

O

NH

Cl

SR144528 169

rCB2 (spleen) rCB1 (whole brain) Selectivity Ki hCB2 (transfected CHOcells)

Ki hCB1 (transfected CHOcells)

Selectivity References

038 nM 305 nM 800 06 nM 437 nM 730 [217]

067 nM 548 nM 80 [219]

56 nM gt10microM gt1780 [48]

25 nM a gt 1microM a gt400 [224]

024 nM 276 nM 115 199 nM 503 nM 25 [232]

a COS cells

1384 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

3 Diarylpyrazole Derivatives CHO cells is constitutively active and phosphorylated onSer 352 Upon treatment by SR144528 thisphosphorylation of the receptor was inhibited Moreover theSR compound was able to induce dephosphorylation ofagonist-induced CB2 phosphorylation The question whetherSR144528 induces a CB2 receptor conformational changemaking it a better phosphatase-specific substrate or ifSR144528 binding activates specific phosphatases remainsopen However this remains a nice example of inverseagonist-induced receptor resensitisation

The same Sanofi team which synthesised the CB1antagonist SR141716A reported in 1998 the synthesis andpharmacological characterisation of a CB2 cannabinoidreceptor selective antagonist the SR144528 (169) [217218] The structure of the new compound is derived fromSR141716A one SR144528 is the first selective CB2receptor antagonist reported as the inverse agonist propertiesof BML-190 were shown only recently by New et al [137]The authors reported for 169 using [3H]-CP-55940 asradioligand Ki values of 04 nM and 06 nM for the rat andthe human CB2 receptors respectively and found a CB1-CB2 selectivity ratio around 700 Table 12 sums up thebinding affinities of SR144528 for the cannabinoid receptorsobtained by different authors Interestingly while the CB2affinity is more or less constant the affinity obtained for theCB1 receptor varies by one order of magnitude [219] Theantagonist effect of SR144528 was highlighted by evaluatingits effect on cAMP production by hCB2-CHO cells exposedto 3 nM CP-55940 (EC50=10nM) The antagonism wasalso observed on the activation of the MAP kinase pathwayby CP-55940 (IC50=39nM)

The Sanofi research team working on cannabinoidsconducted investigations in order to determine the keyresidues for SR144528 interaction with the receptor (Table13) as well as the binding mode of the compound Firstlythey identified the TM4-EL2-TM5 region as a regioncontaining crucial residues for the affinity of several ligandsamong them SR144528 [224] Among these residues themutation of two cysteine of the second extracellular loop(C174 and C179) abolishes the affinity for the CB2 receptorLater on the implication of S453(161) and S457(165) inthe binding of SR144528 was evidenced by mutationalstudies In the model proposed for the docking of SR144528into the CB2 receptor the crucial contacts are comprised intothe TM4 this is the case for S453(161) and S457(165)which are proposed to form hydrogen bonds with the ligandand W464(172) proposed to have aromatic interactions withthe 4-methylbenzyl of the SR144528 [225] In 2003 Fengand Song showed that point mutations D349(130)A andA634(241)E abolished ligand binding However mutationof the arginine R350(131) member of the highly conservedDRY motif to alanine has no influence on SR144528binding [226]

Early after the report of the synthesis and characterisationof SR144528 the same authors further explored the functionof this compound and showed its inverse agonist propertiesusing CB2-CHO cells co-transfected with the luciferasereporter gene with either the CRE or the murine krox24regulatory sequence [220] In these models SR144528 notonly inhibits CP-55940 effects but also reproduces theeffects of a pertussin toxin treatment proving its inverseagonist properties SR144528 functionality was alsoexplored by [35S]-GTPγ S binding in hCB2-CHO cells(IC50=3 nM) [221] The inverse agonist function ofSR144528 was further illustrated by Rhee and Kim usingCOS cells co-transfected with hCB2 receptor and adenylylcyclase [222]

As for the CB1 cannabinoid receptor ligands Sanofideveloped tricyclic derivatives based on the 1-benzylpyrazole-3-carboxylic scaffold More than twentycompounds were disclosed in a patent [227] in 2001 (84-86Table 7) The benzyl in position 4 characteristic of the CB2diarylpyrazoles from Sanofi is conserved as well as thebicyclic residue in position 3 The length of the second linkbetween the phenyl and the pyrazole can be made of one or

Bouaboula and co-workers investigated SR144528 effectson CB2 cannabinoid receptor phosphorylation status [223]Firstly they showed that the CB2 receptor transfected in

Table 13 CB2 Cannabinoid Receptor Antagonists 3 Di-Aryl-Pyrazole Derivatives

Some of the single point mutations reported for the hCB2 cannabinoid receptor for which pharmacological data concerning the SR144528 are available

Mutant Effect References

R350(131)A no effect [226]

S453(161)A loss of affinity [225]

V456(164)I no effect [225]

S457(165)A loss of affinity [225]

C174S a loss of affinity [224 225]

C175S a loss of affinity [225]

C177S a no effect [225]

C179S a loss of affinity [224 225]

S544(193)G no effect [225]

a Second extra-cellular loop

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1385

two methylene but according to the patent one methylenebridge is preferred The Ki values of these compounds for theCB2 receptor were said to be lower than 500 nM and theirantagonist properties have been assessed using the adenylatecyclase inhibition assay

compounds by the presence of an alkyl substituent inposition 5 instead of a phenyl and of course by position 3which remains unsubstituted However except a lecture atthe 1998 ACS meeting [230] where compound 171 wasreported to have a Ki value of 835 nM and a selectivity ratioover 100 no further report on these compounds waspublished untill now

In a recent paper Mussinu and colleagues reported thesynthesis of other tricyclic derivatives starting fromSR141716A structure [228] N-piperidin-1-yl-14-di-hydroindeno[12-c]pyrazole-3-carboxamide derivatives areCB2 receptor ligands and some of them possess high affinityand selectivity for the CB2 receptor as shown in Table 7 (79-83) These tricyclic derivatives differ from the Sanofi tricyclicderivatives as they do have a phenyl ring instead of a benzylin position 1 Another difference is the nature of thesubstituent in position 3 which is not a bicycloalkyl but acyclic amine One of the compounds synthesised byMussinu et al 81 possesses the highest affinity for the CB2receptor ever reported with a Ki value of 0037 nM for themurine CB2 receptor ([3H]-CP-55940 mice spleenhomogenate) Interestingly 79 the closest SR141716Aanalog lost almost all its affinity for the CB1 receptor andincreased by four orders of magnitude its affinity for the CB2receptor Even if the structure quite similar to the SRcompounds could suggest that these derivatives behave asantagonists no pharmacological data were given in the paperto support this hypothesis

5 2-Oxoquinoline Derivatives

Inaba and colleagues from Japan Tobacco introduced in2001 a completely new structure in the cannabinoid fieldbased on a 2-oxoquinoline scaffold Over eighty derivativeswere disclosed in a patent [231] and one compound JTE-907 (173) was subsequently characterised in-vitro as well asin-vivo [232] In Table 14 are listed some of the compounds(172-178) disclosed in the patent along with their affinity forthe cannabinoid receptors These values were obtained onhuman receptors against [3H]-WIN-55212-2 For twelvecompounds the anti-inflammatory activity per os wasassessed in a carrageenin-induced paw edema model Thehighest effect reported was obtained with JTE-907 with anED50 value lower than 01 mgkg Therefore thesecompounds were claimed to be useful as anti-inflammatoryand anti-allergic agents

To further characterize JTE-907 the authors determinedits affinity for cannabinoid receptors of different species using[3H]-CP-55940 as radioligand In their hands JTE-907behaved as a very selective hCB2 ligand with Ki values of36 and 2370 nM for the CB2 and CB1 cannabinoid receptorsrespectively even more selective than the SR144528 (Kivalues of 199 and 50 nM respectively) However it has tobe mentioned that the affinities reported in the paper forJTE-907 are quite different from those reported in the patent(hCB1 Ki values of 35nM and 009 nM respectively) Italso appears that the SR144528 affinity for the CB1 receptordetermined by Iwamura et al is higher than the affinityreported by Rinaldi-Carmona et al with values of 50 and437nM respectively The authors ascribed the difference tothe variability of SR144528 binding affinities to the assayconditions The function of JTE-907 was evaluated bymeasuring the increase in cAMP production by forskolin-stimulated hCB2-CHO cells Maximum stimulation wasattained at a concentration of 1microM of JTE-907 In the sameassay WIN-55212-2 decreased the cAMP production in adose-dependent manner

Finally it is interesting to strengthen that the five-membered ring derivatives (79-86) of the diarylpyrazolefamily are reported to possess a high CB2 receptor affinitywhile the six- and seven-membered ring derivatives (69-78)are described as CB1 receptor ligands

4 Other Pyrazole Derivatives

Other CB2 ligands based on a pyrazole nucleus wereintroduced by SmithKline Beecham in a patent describingthe synthesis of about sixty new compounds [229] Theiraffinity (Ki) for the hCB2 receptor transfected in HEK293cells was reported to be ranging between 25nM and 10microMTheir structures (170-171 Fig 17) differ from the Sanofi

NN

O

N

ON

NN

N

NN

O

N

ON

NN

N

ClCl

1

5

4 3

2

170 171

Fig (17) CB2 cannabinoid receptor antagonists 4 Otherpyrazole derivatives Structure of two compounds (170-171)among those claimed by SmithKline Beecham to be hCB2antagonists

In conclusion JTE-907 was shown to be a CB2 selectiveinverse-agonist with anti-inflammatory and anti-allergicproperties This compound is currently undergoing Phase Iclinical trials as anti-allergic drug [233]

IV NON-CB1 NON-CB2 CANNABINOIDRECEPTORS LIGANDS

In the last years the possible presence of additional non-CB1 non-CB2 cannabinoid receptors was raised frompharmacological evidences

One of the putative receptors possesses an endotheliallocalisation while other ones are localized in the CNS or atperipheral nerves For instance an additional target for ∆9-

1386 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 14 CB2 Cannabinoid Receptor Antagonists 5 2-Oxoquinoline Derivatives

Structure of six compounds among the oxoquinoline derivatives patented by Japan Tobaco Affinity (Ki nM) and selectivity (CB1CB2) for the cannabinoidreceptors are given The affinity was measured by binding against [3H]-WIN-55212-2 on either hCB2-CHO or hCB1-CHO cells Adapted from [231]

Cpd ndeg Structure hCB2 hCB1 Selectivity

1-2 172

NH

MeO O

O

OMeO

0014 3671 262215

JTE-907 173

NH

O

MeO O

O

NH

O

O

0087 3436 39495

2-2 174

OH

O

NH

MeO O

O 077 3247 4215

3-27 175

NH

O

F

NH

MeO O

O0021 336 16000

3-32 176

NH

MeO O

O

NH

F

O

0036 2398 66610

3-38 177

O

O

NH

N

O

O

NMeO

0085 935 11000

3-43 178

NMeO O

O

NH

F

O

0043 864 20100

tetrahydrocannabinol was described in capsaicin-sensitivesensory nerves by Zygmunt et al [234]

[29] and by Di Marzo et al [235] Thus in the presentreview we will focus on the description of the so faridentified ligands of the non-CB1 non-CB2 receptors withan emphasis on the compounds having antagonist properties

Pharmacological evidences for the existence of additionalcannabinoid receptors were reviewed by Wiley and Martin

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1387

(Table 15) In particular the knowledge about three not yetcloned targets has grown The growing molecularpharmacology of the three putative new cannabinoidreceptors is reviewed in the three following sections Thefourth section summarises the data on the other proposedcannabinoid targets

vasodilatator activity In addition the anandamide effect ispartially sensitive to SR141716A inhibition The authorsproposed the existence of two distinct receptors responsiblefor the vasodilatatory effect of anandamide one located in theendothelium is SR141716A-sensitive while the other onelocated on the vascular smooth muscle is SR141716A-insensitive Thus SR141716A is an antagonist of anendothelial anandamide receptor Several studies wereconducted in order to understand the molecularpharmacology of the endothelial anandamide receptor It ispertussis toxin-sensitive [237] Cannabidiol acts asantagonist and abnormal-cannabidiol (Abn-cbd 179) and itsanalogue O-1602 (180) act as agonists [238] The synthesisof O-1602 and of its analogues are disclosed in a patentdescribing the vasodilatatory activity of cannabinoid

1 Endothelial Anandamide Receptor ndash Abnormal-Cannabidiol Receptor

In 1999 Wagner et al showed that anandamide couldinduce mesenteric vasodilatation through an endotheliallylocated ldquoanandamide receptorrdquo pharmacologically distinctfrom CB1 [236] Other cannabinoids such as ∆9-THC HU-210 or WIN-55212-2 were devoid of mesenteric

Table 15 Non-CB1 Non-CB2 Cannabinoid Receptors Antagonists

An overview of the putative new non-CB1 non-CB2 anandamide-receptors ligands The antagonists are highlighted in bold

NH

N

S

HO OH

Cl

OH

OH

OH OH OH OH

OMe

OMe

RR

RR

RR

RR

HO

OH

NH

O

cannabidiol (10) Abnormal-cannabidiol 179 O-1602 180

O-1918 181 capsazepine 183capsaicin 182

Compound Endothelial AEA receptor Brain AEA receptor Cannabinoid-vanilloid brain receptor

AEA agonist a agonist de

WIN-55212-2 no effecta

agonist e agonist g h

CP-55940 no effect e agonist i

SR141716A antagonist a b j no effect ([35 S]-GTPγS binding) d e f antagonist g h

AM251 no effect k no effect h i

Cannabidiol antagonist b no effect e

O-1918 antagonist c jm

Abd-cbd agonist b l

O-1602 agonist b

Capsaicin no effectj agonist i

Capsazepine no effect b antagonist h

a [236] b [238] c [240] d [249] e [250] f [251] g [254] h [258] i [ 255] k [244] j [ 243] l [ 242] m [241]

1388 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

analogues [239] More recently Offertaler et al showed thatO-1918 (181) a cannabidiol analog acts as an antagonist ofthe endothelial anandamide receptor [240] In addition O-1918 does not bind to the CB1 and CB2 cannabinoidreceptors Furthermore O-1918 unlike cannabidiol doesnot cause vasorelaxation in the mesenteric artery preparationmodel used and thus acts as a true neutral antagonistUsing O-1918 as a pharmacological tool Begg et alproposed a coupling of this new G protein-coupledendothelial receptor through a GiGo mechanism to ionchannels in primary cultured human vascular endothelialcells [241] However in their hands SR141716A wasineffective in antagonising Abd-cbd Ho and Hiley furtherdemonstrated that Abn-cbd relaxes rat small mesentericarteries through an activation of K+ channels via aSR141716A-sensitive pathway [242] Further using thesame model they characterized the vasorelaxant activity ofvirodhamine a novel endocannabinoid apparently actingthrough the same receptor [243] The virodhamine-inducedvasorelaxation is sensitive to SR141716A and O-1918 butinsensitive to AM251 SR144528 and AM630 Moreovervanilloid receptor desensitisation by capsaicin (182) had noeffect

244] Thus position 4 on the 5-phenyl could be furtherexplored to obtain selective ligands

To come to an end despite the fact that the endothelialanandamide receptor is neither identified nor cloned at leastthree antagonists are identified SR141716A cannabidioland O-1918 It is likely that a growing number of ligandsand therapeutic applications will appear in the very nearfuture

2 Brain Anandamide Receptor

Following an in-depth evaluation of anandamide effects inCB1

-- mice Di Marzo and co-workers suggested that a non-CB1 non-CB2 G protein-coupled receptor might mediatesome of the actions of AEA in mice [249] Actually effectsof AEA but not those of ∆9-THC were not decreased inCB1 cannabinoid receptor knock-out mice For instance 30mgkg of AEA were still effective in hot-plate test whereas10 mgkg of ∆9-THC had no effect on anti-nociceptionMoreover in a [35S]-GTPγ S assay AEA stimulated with anEC50 value of 223 microM the [35S]-GTPγ S binding to CB1

--

mice brain membranes (Emax= 28) ∆9-THC showed noeffect in this assay The addition of SR141716A did notaffect the [35S]-GTPγ S binding to CB1

-- mice brainmembranes Shortly after other evidences for the existence ofa new G protein-coupled cannabinoid receptor with a distinctdistribution in the central nervous system were published[250] From a set of 24 commonly used cannabinoids (AEAWIN-55212-2 JWH-030 THChellip) only AEA and WIN-55212-2 produced an increased binding of [35S]-GTPγ S onCB1

-- mice brain membranes Interestingly in this studySR141716A alone produced a significant inhibition of [35S]-GTPγ S binding similar to the inhibition obtained usingCB1

++ mice brain membranes (IC50 values of 57 microM and47 microM respectively) However SR141716A did not affectthe anandamide-induced [35S]-GTPγ S binding in CB1

--

mice brain membranes Moreover [3H]-SR141716Aexhibited significant binding in some brain regions of CB1

--

mice which are different from those exhibiting stimulation of[35S]-GTPγ S binding by WIN-55212-2 and AEA Thusthe authors proposed that the SR141716A effects on CB1

--

mice are mediated neither by the CB1 cannabinoid receptornor by the putative new anandamide brain receptor Monoryand co-workers using CB1

-- mice described a novel nonadenylyl cyclase-coupled cannabinoid binding site in micecerebellum [251] Two agonists AEA and WIN-55212-2but not ∆9-THC or HU210 were found to bind to thisreceptor as they enhanced [35S]-GTPγ S binding NeitherSR141716A nor SR144528 were able to affect the WIN-55212-2 induced [35S]-GTPγ S binding in CB1

-- mice asfound by Breivogel et al [250] However the distributionpattern of the receptor described by Monory et al differs fromthe distribution found by Breivogel et al It has to bementioned that the knock-out strains used were different Themice used by Monory were from a CD-1 strain while theone used by Breivogel were from a C57BL61 strainInterestingly Muthane et al detected differences in nigralneurons number and sensitivity to MPTP in the two mousestrains used [252]

More recently Mo et al obtained results showing thatthe Abnormal-cannabidiol receptor is also responsible for anincreased endothelial cell migration [244] Abn-cbd enhancedhuman umbilical vein endothelial cell migration while O-1918 antagonised Abn-cbd effect The CB1 cannabinoidreceptor inverse agonist SR141716A partially inhibitedAbd-cbd action whereas AM251 and SR144528 had noeffect

In another paper from George Kunos team Batkai et alreported that SR141716A is able to inhibit the endotoxichypotension by a cardiac mechanism involving neither theCB1 nor the CB2 cannabinoid receptors [245] Theinvolvement of a non-CB1 non-CB2 site of action wasshown using CB1

-- and CB1--CB2

-- knock-out miceFurther studies are needed to identify the SR141716Asensitive myocardial site of action

In a recent patent Kunos described O-1918 and itsanalogues as vasoconstrictor agents useful to reversepathological vasodilatation of blood vessels [246] Theeffects of O-1918 on mouse blood pressure on LPS-inducedhypotension in mouse as well as the reversal of hypotensionby O-1918 injection are described Furthermore thesynthesis of O-1918 based antagonists is described startingfrom p-metha-28-dien-1-ol and the appropriate resorcinols

Interestingly the Abd-cbd receptor which was firstdetected in the vascular system also seems to be present inthe CNS [247] Indeed Walter et al found that microglialcells (BV-2) migration induced by 1 microM 2-AG is inhibitedby cannabidiol (300 nM) and O-1918 (1 microM) SR141716Awas found ineffective in this assay Thus 2-AG is an agonistof the Abd-cbd receptor Moreover Abd-cbd is able to elicita dose-dependent cell migration in the same model (EC50 =600nM) [248]

From a more structural point of view SR141716Abehaves as an antagonist while on the opposite AM251(20) a close analogue which differs from the former only bythe nature of one halogen has no effect on this receptor [242

To conclude to our knowledge no compounds havingantagonist or inverse agonist properties at this receptor havebeen identified so far

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1389

3 Cannabinoid-Vanilloid Brain Receptor a cannabinoid-vanilloid receptor at which SR141716A andcapsazepine behave as antagonists

Evidences for the presence of an additional ldquocannabinoidrdquoreceptor differing from the CB1 receptor and from the brainanandamide receptor appeared from studies focused on thehippocampus (for a review see Hajos et al [253]) Hajos etal studied the cannabinoid actions on GABAergic andglutamatergic transmission using the whole-cell patch-clamp technique [254] WIN-55212-2 induced a dose-dependent reduction in glutamatergic transmission reversedby SR141716A (1microM) Thus the authors concluded that thecannabinoid actions on the glutamatergic transmission aremediated by a non-CB1 but SR141716A-sensitive receptorIn another paper Hajos and Freund further characterised thisputative receptor [255] They showed that in addition toWIN-55212-2 CP-55940 and capsaicin (182) a TRPV1receptor agonist also act as agonists Furthermore in theirhands SR141716A as well as capsazepine (183) a TRPV1receptor antagonist were able to antagonise this effect on theglutamatergic transmission while AM251 was notSR141716A effects on CB1 knock-out mice were furtherstudied using a test of anxiety [256] Haller et al showedthat on the one hand the distribution of the CB1 receptorinduces anxiety and SR141716A has an anxiolytic effectand on the other hand SR141716A is still active in CB1

--

mice In a following paper they reported that AM-251induces anxiogenic effects in wild-type and in knock-outmice [257] Thus SR141716A in addition to CB1 bindsto a not yet identified receptor Another evidence for thisadditional receptor for SR141716A was given by Bass andco-workers [61] They conducted a structure-activityrelationship study on the stimulation of locomotor activityinduced by SR141716A and twenty analogues Nocorrelation was found between the affinity for the CB1cannabinoid receptor and the stimulation of locomotoractivity Albeit SR141716A and five of its analogues O-1803 O-1710 O-1253 O-1254 (40) O-1255 (41) behavedas inverse agonists in [35S]-GTPγ S assay none of theseanalogues stimulated the locomotor activity In conclusionthe SR141716A-induced stimulation of locomotor activity isneither due to inverse agonism nor to inhibition of anendogenous tone but to a brain receptor distinct from theCB1 receptor Di Marzo et al previously provided evidencesthat the cannabinoid-vanilloid brain receptor could mediatesome of the cannabinoid effects on locomotion [249]

4 Other Non-CB1 Non-CB2 Cannabinoid Receptors

Evidences for the existence of additional non-CB1 non-CB2 cannabinoid receptors localised in the rat isolatedmesenteric arterial bed appeared from the works of Ralevicand collaborators Several cannabinoids were evaluated intheir model of sensory neurogenic vasorelaxation evoked byelectrical field stimulation

HU210 was found to attenuate vasorelaxation in a CB1and CB2 independent way as SR141716A LY320135 andSR144528 were ineffective in inhibiting HU210 effect [259]Similar results were obtained with ∆9-THC [260]Similarly noladin ether inhibited sensory neurogenicrelaxation in a concentration-dependent manner which wasalso unaffected by SR141716A LY320135 and SR144528Moreover this effect as the ∆9-THC one was pertussistoxin sensitive suggesting the involvement of a Gio protein-coupled receptor [261]

However the inhibitory effects of CP-55940 and WIN-55212-2 were affected by the presence of SR141716A (1microM)or LY320135 (1microM) but not by the addition of SR144528(1microM) [262]

It appears that at least two receptors should be involvedin cannabinoid-mediated attenuation of sensory nerve-mediated vasorelaxation The first one is SR141716A andLY320135 sensitive while the other one is resistant toSR141716A

Some evidences for the existence of a ldquoCB2-likerdquo receptorappeared from the studies on palmitoylethanolamide (PEA)an endogenous fatty acid amide devoid of CB1 and CB2affinity [264] antinociceptive potential Calignano and co-workers showed that PEA was able to alleviate pain inseveral animal models [265 266] For instance PEA dose-dependently inhibits kaolin-evoked writhing in mice TheCB2 cannabinoid receptor inverse agonist SR144528 (02mgkg) inhibits PEA anti-nociceptive effect [266] Thus theauthors suggest that PEA acts as agonist on a SR144528-sensitive non-CB2 cannabinoid receptor (ldquoCB2-likerdquoreceptor) at which SR144528 acts as an antagonist

Further studies are needed in order to better characterisethese additional ldquocannabinoidrdquo receptors However it islikely that several therapeutic applications will soon springfor these receptors

Very recently Pistis et al published a study on theneurophysiological effects of cannabinoids on the basolateralamygdala neurons in-vivo [258] One of their findings is thatHU-210 and WIN-55212-2 did not elicit similar effects onbasal amygdala projection neurons firing rate While theformer had no effect the latter decreased the firing rateMoreover SR141716A reversed the WIN-55212-2 effectswhile AM251 had no effect The vanilloid receptorantagonist capsazepine also antagonised the WIN-55212-2effect on firing rate of these neurons Therefore the authorssuggested the presence of a novel cannabinoid-vanilloidreceptor

V CONCLUSION

Since their development cannabinoid antagonists provedto be essential tools in the understanding of cannabinoidpharmacology and biochemistry Moreover the interestshown by the pharmaceutical industry in the development ofnew cannabinoid antagonists prove if necessary the realtherapeutic potential of this class of compounds Thetreatment of eating and movement disorders memorydeficits psychosis and dependencies from various addictivedrugs are some of the most cited applications for the CB1cannabinoid receptor antagonists

From these lines it appears that in addition to theknown CB1 cannabinoid receptor it is now likely that twoother cannabinoid receptors do exist in the brain The firstone should be an anandamide brain receptor for whichSR141716A has no affinity while the second one should be

1390 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Much less wide is the knowledge on the CB2cannabinoid receptor antagonists However potentialapplications could be disorders involving the immunesystem such as inflammation or allergies

[19] Devane WA Hanus L Breuer A Pertwee RG StevensonLA Griffin G Gibson D Mandelbaum A Etinger AMechoulam R Science 1992 258 1946

[20] Mechoulam R Ben-Shabat S Hanus L Ligumsky MKaminski NE Schatz AR Gopher A Almong S MartinBR Compton DR Biochem Pharmacol 1995 50 83

Finally the growing literature on the non CB1 non CB2cannabinoid receptors should allow the development of newdrugs First examples are given by the cannabidiol andabnormal-cannabidiol derivatives described by Kunos et alfor which potential therapeutic applications on the vascularsystem are already patented

[21] Sugiura T Kondo S Sukagawa A Nakane S Shinoda AItoh K Yamashita A Waku K Biochem Biophys Res Co1995 215 89

[22] Hanus L Abu-Lafi S Fride E Breuer A Vogel Z ShalevDE Kustanovich I Mechoulam R Proc Natl Acad SciUSA 2001 98 3662

[23] Oka S Tsuchie A Tokumura A Muramatsu M Suhara YTakayama H Waku K Sugiura T J Neurochem 2003 851374It clearly appears from this paper that the antagonists of

the cannabinoid receptors are still an active research fieldfrom which should emerge new promising therapeutic toolsas well as innovative drugs in the near future

[24] Goutopoulos A and Makriyannis A Pharmacol Therapeut2002 95 103

[25] Beal JE Olson R Lefkowitz L Laubenstein L Bellman PYangco B Morales JO Murphy R Powderly W PlasseTF Mosdell KW Shepard KV J Pain Symptom Manage1997 14 7ACKNOWLEDGEMENTS [26] Notcutt W Price M Miller R Newport S Phillips CSimmons S Sansom C Anaesthesia 2004 59 440

GGM is very indebted to the ldquoFonds pour la formationagrave la Recherche dans lrsquoIndustrie et lrsquoAgriculture (FRIA)rdquo Belgium for the award of a research fellowship Theexperimental works of the authors were supported by grantsfrom the FNRS The Belgian Charcot Foundation and theUniversiteacute catholique de Louvain

[27] Ledent C Valverde O Cossu G Petitet F Aubert JFBeslot F Bohme GA Imperato A Pedrazzini T RoquesBP Vassart G Fratta W Parmentier M Science 1999 283401

[28] Zimmer A Zimmer AM Hohmann AG Herkenham MBonner TI Proc Natl Acad Sci U S A 1999 96 5780

[29] Wiley JL and Martin BR Chem Phys Lipids 2002 121 57[30] Adam J and Cowley P Expert Opin Ther Patents 2002 12

1475

REFERENCES [31] Razdan RK and Mahadevan A Chem Phys Lipids 2002 12121

[32] Barth F and Rinaldi-Carmona M Curr Med Chem 1999 6745

[1] Hillard CJ Harris RE Bloom AS J Pharmacol Exp Ther1985 232 579

[33] Little PJ Compton DR Johnson MR Melvin LS MartinBR J Pharmacol Exp Ther 1988 247 1046

[2] Devane WA Dysarz FA Johnson MR Melvin LSHowlett AC Mol Pharmacol 1988 34 605

[34] Pertwee RG Stevenson LA Elrick DB Mechoulam RCorbett AD Brit J Pharmacol 1992 105 980

[3] Matsuda LA Lolait SJ Brownstein MJ Young ACBonner TI Nature 1990 346 561

[35] Harrison C Traynor JR Life Sci 2003 74 489-508[4] Gerard CM Molerau C Vassart G Parmentier M BiochemJ 1991 279 129 [36] Pertwee RG Fernando SR Griffin G Ryan W Razdan

RJ Compton DR Martin BR Eur J Pharmacol 1996 315195

[5] Glass M Dragunow M Faull RLM Neuroscience 1997 2299

[37] Ross RA Brockie HC Fernando SR Saha B Razdan RKPertwee RG Brit J Pharmacol 1998 125 1345

[6] Shire D Carillon C Kaghad M Calandra B Rinaldi-Carmona M LeFur G Caput D Ferrara P J Biol Chem1995 270 3726 [38] Griffin G Wray EJ Rorrer WK Crocker PJ Ryan WJ

Saha B Razdan RK Martin BR Abood ME Brit JPharmacol 1999 126 1575

[7] Ryberg E Khang Vu H Groblewski T Larsson N ElebringT Hjorth S Sjogren S Greasley PJ 2004 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 119

[39] Ross RA Gibson TM Stevenson LA Saha B Crocker PRazdan RK Pertwee RG Brit J Pharmacol 1999 128 735

[40] Martin BR Winckler JR Wiley JL Huffman JW CrockerPJ Saha B Razdan RK J Pharmacol Exp Ther 1999 2901065

[8] Rinaldi-Carmona M Calandra B Shire D Bouaboula MOustric D Barth F Casellas P Ferrara P LeFur G JPharmacol Exp Ther 1998 278 871

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Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1365

obtained using HEK293 cells transfected with either theK328A mutant or the wild type receptor remarkablyconfirmed the modeling results (Kd values of 396 and 23nM respectively) Interestingly a vinyl-cyclohexylSR141716A derivative VCHSR1 (15) (Table 4) lacking ofhydrogen bonding sites in C3 position is not affected bythis CB1 receptor mutation as its affinity remains unchangedwith Ki values of 31 and 35 nM for the wild-type andK328A receptors respectively Four additional compoundsCHASR1 (16) CHMSR1 (17) VPSR1 (18) and PIMSR(19) differing by their potential to form hydrogen bondswere evaluated in affinity and functional assays The resultsfurther confirm the crucial interaction between the C-3carboxamide oxygen and residue K328 to obtain an inverseagonist effect as highlighted in Table 4 [68] However it hasto be said that this lysine residue is also crucial for agonistbinding (CP-55940) andor receptor activation (WIN-55212-2) [69 70]

binding of SR141716A In contrast the substitution oftyrosine by isoleucine (Y275I) resulted in the loss of ligandrecognition Calculation studies revealed that while theY539F mutant is very similar to the wild type receptor theY539I mutant shows topology changes in the 3-4-5transmembrane region This region is considered to becrucial for agonistantagonist binding at CB1 receptor sincethe previous report by Shire et al on CB1CB2 receptorchimeras [72] In contrast the first and third extracellular(EC1 and EC3) loops of the hCB1 receptor are not essentialfor the binding of SR141716A as illustrated by Murphy etal The authors constructed several receptors mutated in theirEC1 or EC3 and none of the tested mutations affected theSR141716A binding [73] McAllister et al due the highlyaromatic nature of SR141716A further explored thehypothesis that an aromatic microdomain comprised intransmembrane helix 3-4-5-6 is the SR141716A bindingsite The modelling and mutation studies undertakensuggested to the authors that this aromatic microdomaincomprised of F336 W464 Y539 W543 and W648should represent the binding site of SR141716A Moreoverthey identified F336 and W543 as direct interaction sitesfor SR141716A [74] Residue F336 was further shown bythe same group to be a key residue for both ligand binding(WIN-55212-2 and SR141716A) and receptor activationMutation of phenylalanine 336(201) to alanine resulted in

Several other studies were undertaken to determine thecritical residues for the binding of cannabinoid compoundsUnfortunately in most of the studies SR141716A was notused However McAllister et al [71] using modeling toolsand mutagenesis explored the importance of aromaticity inposition 539(275) in the CB1 receptor The substitution oftyrosine by phenylalanine (Y275F) has no effect on the

Table 3 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Summary of the Reported CB1 Cannabinoid Receptor Single Point Mutations for which Pharmacological Data Concerning the SR141716A (14) areAvailable

Mutation Species Effect References

S114A a human no significant effect [73]

S115A a human no significant effect [73]

D250(163)N human no significant effect [263]

D250(163)E human no significant effect [263]

H181A b human no significant effect [73]

R182A b human no significant effect [73]

K183A b human no significant effect [73]

D184A b human no significant effect [73]

V324(188)A human no significant effect [73]

F325(189)A human no significant effect [73]

F325(190)A mouse no significant effect [74]

K328(192)A human loss of function [66]

K328(192)A human reduction of affinity (20 fold) [67]

F336(201)A mouse reduction of affinity (3 fold) [74]

Y539(275)F human no significant effect [71]

Y539(275)I human loss of affinity [71]

W543(280)A mouse loss of affinity [74]

W648(357)A mouse reduction of affinity (7fold) [74]

a N-terminus b First extra-cellular loop

1366 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 4 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives Five SR141716A (14) DerivativesIllustrating the Importance of the C-3 Carboxamide Oxygen in the SR141716A Inverse Agonism

The structure affinity ([3H]-SR141716A hCB1-HEK293 cells) and function (Ca++ currents) are given for each compound

NN

R

Cl

Cl

Cl

Cpd ndeg R= Affinity Function References

SR141716A 14

O

NHN

Kd=23nM Inverse agonist [67]

VCHSR1 15 Ki=313nM Neutral antagonist [67]

CHASR1 16

O

NH

Ki=17nM Inverse agonist [68]

CHMSR1 17

O

N

Ki=29nM Inverse agonist [68]

VPSR1 18

N

Ki=261nM Neutral antagonist [68]

PIMSR 19

NN

Ki=67nM Neutral antagonist [68]

an increased constitutive activity of the receptor asdemonstated by [35S]-GTPγ S binding [75]

1996 It binds with high affinity to rat brain synaptosomes(Kd=061nM) It is competitively displaced by knowncannabinoids like CP-55940 or WIN-55212-2 Usingautoradiography its rat brain distribution is similar to [3H]-CP-55940 one [77] It is now a commercially available andwidely used radioligand for competition studies

A new 3D model of the CB1 cannabinoid receptor basedon the X-ray structure of the bovine rhodopsin was recentlydeveloped by Salo and co-workers [76] It would be veryinteresting to see whether or not the results obtained withprevious models are confirmed using this new model Forinstance the lysine K328 appeared as a key residue in thismodel too

Interestingly other SR141716A radiolabeled derivativeswere synthesised as radioimaging tools among them [123I]-AM251 (20) [7879] [123I]-AM281 (21) [80-82] and [18F]-SR144385 (22) [8384] for Positon Emission Tomographyor Single Photon Emission Computed Tomography Indeedcannabinoid antagonists are a much more useful tool for

The tritiated analogue of SR141716A the [3H]-SR141716A was described by Rinaldi-Carmona et al in

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1367

human radioimaging applications than agonists as they aredevoid of cannabinoid-like effects For instance Berding etal very recently reported the use of [123I]-AM281 for SinglePhoton Emission Computed Tomography imaging of theCB1 cannabinoid receptor in six human patients [85] Inorder to further optimize the brain uptake of suchSR141716A derivatives Sanofi researchers synthesised twomethoxylated SR141716A analogues using 11C as PETtracer [86] These two compounds SR149080 (24) andSR149568 (25) possess high affinity with Ki values of 15and 38 nM respectively and selectivity for the CB1 receptoras well as an improved penetration in the brain evaluated bymeasuring the radioactivity present in various brain regionsafter tail vein injection in CD-1 mice Some of therepresentative radiolabelled SR141716A derivatives aresummarised in Table 5

Colombo et al using Wistar rats [92] Simiand et alobserved that SR141716A selectively reduces sweet foodintake in primates [93] However several authors [94-96]showed that high palatability of food is not necessary toobserve a SR141716A-induced anorectic effect at doses thatdo not cause major behavioural alterations or reduced waterintake Interestingly Gomez et al demonstrated theimplication of the peripheral CB1 receptors on themodulation of feeding and therefore the possible role ofthese receptors on the SR141716A influence on food intake[97] In addition to its effects on food consumptionSR141716A seems to be able to lower the hyperglycemiathe hyperinsulinemia as well as the insulin resistance indiet-induced obese (DIO) mice In the same DIO mice adecrease in adiposity was also observed after treatment [98]Bensaid et al using another model of obesity the obese fafarats observed that SR141716A increases mRNA expressionof Acrp30 or adiponectin a plasmatic protein exclusivelysecreted by adipose tissue through a CB1-mediatedpathway Inductions of free fatty acid oxidation body weightreduction and hyperinsulinemia decrease are some of theknown physiological actions of this protein Thus along theauthors an enhanced expression of Acrp30 followingSR141716A administration could be responsible for themetabolic effects of the compound leading to body weightreduction [99] Vickers et al using the same model (ie fafarats) showed that SR141716A significantly decreases foodconsumption and weight gain in both the obese fafa rats andthe lean Zucker rats [100] This decrease was greater in thefafa group and reversible upon SR141716A withdrawal

From a more therapeutic point of view SR141716A(rimonabant Acompliareg) is one of the promising agents totreat obesity [87] It is currently in Phase III clinical trials forthe treatment and prevention of obesity Final results areexpected for this year and FDA application filling for 2005[88] Preliminary data based on a one year treatment withrimonabant (RIO-Lipids study 1036 patients) showed a 5weight loss in 72 of the treated patients [8990] Severalreports were published before clinical trials aiming todemonstrate the anti-obesity properties of this compoundThe first report by Sanofi Recherche published in 1997described the selective inhibition of sucrose intake in ratsupon SR141716A treatment (03-3 mgkg) [91] Theanorectic and weight loss effects were firstly published by

Table 5 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structure and Affinity of SR141716A Radiolabeled Derivatives Developed as Potential Radio-Imaging Tools

NN

R3

O

NH

Cl

R4

R1

R2

Cpd ndeg R1 R2 R3 R4 Affinity CB1 References

[123I]-AM251 20 123I piperidinyl CH3 Cl Ki=25 nMa [78]

[123I]-AM281 21 123I morpholinyl CH3 Cl Ki=14 nMb [80]

[18 F]-SR144385 22 Cl piperidinyl CH2-18 F Cl IC50 =29 nMa [83]

[18 F]-SR147963 23 Cl morpholinyl CH2-18 F Cl IC50 =120 nM [84]

[11 C]-SR149080 24 O-11 CH3 piperidinyl CH3 Cl IC50 =15 nMa [86]

[11 C]-SR149568 25 O-11 CH3 morpholinyl CH3 Cl IC50 =38 nMa [86]

[18 F]-NIDA-42033 26 O-CH3 piperidinyl 18 F H Ki=18 nMc [155156]

27 O-11 CH3 piperidinyl CH3 H Ki=8nMc [155157]

arat brain homogenates [3H]-CP-55940bmouse cerebellum homogenates [3H]-SR141716crat brain homogenates [3H]-AM251

1368 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Another recent study by Higgs and co-workers furtherhighlighted the role of endocannabinoids in food taste-perception and the reduction of orosensory reward of sucrosein SR141716A treated rats [101] As it was expectedSR141716A induced effects on food consumption are absentsin CB1

-- mice [102] Very recently Cota et al obtainedresults showing that endocannabinoid system modulateshomeostasis via a dual mechanism it regulates at a centrallevel food intake while it blocks at the periphery lipogeneticprocesses [103] Moreover Cani et al showed that ghrelin(an orexigenic peptide) plasma levels are significantlyreduced 45 minutes after SR141716A administration(5mgkg ip) to fasted rats in accordance with the rapiddecrease of food intake measured by the authors It istherefore likely that SR141716 effects on body weight aredue to a conjunction of central and peripheral actions [104]

cerebrospinal fluid from patients with schizophrenia [116]Later De Marchi et al measured higher amounts ofanandamide in blood of schizophrenic patients compared tocontrols [117] On the other hand Dean et al obtainedelevated [3H]-CP-55940 binding in the dorsolateralprefrontal cortex of patients suffering from schizophrenia ascompared to controls [118] Zavitsanou using [3H]-SR141716A found elevated binding in the anterior cingulatecortex of subjects with schizophrenia [119] RecentlyMeltzer and collaborators published the results of a trialconducted to evaluate the potential of four new compoundsin treating schizophrenia and schizoaffective disorders [120]SR141716A (20 mgday) was one of the compoundsevaluated during a six weeks study The authors found noeffect of the CB1 cannabinoid receptor antagonist inimproving patientrsquos schizophrenia (72 subjects)

In addition to the anti-obesity potential the ability ofSR141716A to reduce alcohol and tobacco consumption arecurrently investigated in phase III clinical trials The effect ofSR141716A on alcohol consumption in rats was reported bythe GianLuigi Gessa team [105-108] and by Gallate et al[109] The inhibition of alcohol and nicotine induceddopamine release by SR141716A (1-3 mgkg rats) wasreported by Cohen and co-workers using a brainmicrodialysis device [110] Concerning the smokingcessation preliminary results of a clinical trial (360 subjects40 mg SR141716A) showed an increased abstinence ofsmoking [111] More recent results from the STRATUS-USstudy (787 patients) were reported 362 of patientsreceiving SR141716A (20 mgday) quit smoking against206 in the placebo group [90] Recently Le Fol andGoldberg reviewed the development of cannabinoid CB1antagonists as a new class of therapeutic agents for drugsaddictions [112]

It is known since the early nineties that theadministration of cannabinoid agonists impairs memory inrodents (for a review see Castellano et al [121]) Thusadministration of a cannabinoid antagonist was expected tosomehow improve memory [122123] However dependingon the authors SR141716A when administered alone wasreported to impair (5-10 mgkg im) [124] to have no effect(1-32 mgkg ip) [125] or to improve memory (3 mgkgip) [126] In a more recent paper Wolff and Leandershowed that SR141716A (1mgkg ip) improves memoryin rats by apparently enhancing the consolidation processesof memory [127] Further they found that at higher doses (3mgkg) this effect was lost The discrepancy in the resultsreported in the literature could be ascribed either to thedifferences in the tests used or in the doses administeredFurther experiments are needed to assess whether or not acannabinoid antagonist could be helpful in memory diseases

To conclude several patents were taken by Sanoficoncerning the therapeutic applications of their leadcompound among them being anti-obesity smokingcessation neuroinflammatory diseases and anti-diarrhoeaOne of the last patents to our knowledge concerns thetreatment of sexual dysfunctions with a cannabinoidantagonist such as SR141716A [128] However da Silva etal showed that even if this compound (2 mgkg ip)enhances the effects of apomorphine (20-80 microgkg) it has noeffect alone on penile erection [129] Meanwhile Melis et al

Since the late sixties concerns exist on the associationbetween cannabis use and schizophrenia Recent studiesshowed an association between cannabis use and an increasedrisk of developing schizophrenia [113 114] Moreover apossible role of the endocannabinoid system in schizophreniahas been suggested since its pharmacological characterisation[115] Several experimental data obtained on human subjectstend to confirm such hypothesis On the one hand Lewekeet al found elevated levels of endocannabinoids in

NN

O

NH

N

Br Cl

Cl

NN

O

NH

N

Br Cl

Cl

OH

28 29

Fig (3) CB1 cannabinoid receptor antagonists 3 Diarylpyrazole derivatives The chemical structures of 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-piperidine-carboxamide (SR147778 28 ) and 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-(4-hydroxypiperidine)-carboxamide (29 ) described by Rinaldi-Carmona et al

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1369

reported an increased rate of penile erection after directinjection of SR141716A in the paraventricular nucleus of thehypothalamus [130] This effect was dose dependent and asignificant effect was obtained with a dose of 1microgkg

AM251 in mice at a same dose range (10-30 mgkg ip) thanthe anorectic effects [143] These effects are reversed byadministration of CP-55940 and are absent in CB1

-- miceproving the implication of the CB1 receptor in theantidepressant-like effect of AM251 In 2004 Liao et alreported that AM251 was able to displace the binding of[3H]-batrachotoxin A from its binding site on sodiumchannels in mice brain synaptic preparations [144] Theyobtained an IC50 value of 112 microM and a competitivemechanism of action The authors suggested that AM251 iscapable of reducing neuronal excitability through blockade ofvoltage-sensitive sodium channels in brain

From the clinical trials involving the SR141716A themost frequently reported side-effects are nausea dizziness anddiarrhoea Depression and anxiety were not higher than inthe placebo groups Regarding one of the known side effectsof SR141716A administration the enhancement of intestinalmotility Carai et al reported recently that chronicadministration of the inverse agonists to mice induced atolerance to this prokinetic effect [131] This is not the firstreport of tolerance onset after administration of SR141716A[132] however few is known on the mechanismsresponsible for that phenomenon

Wiley and collaborators reported in 2001 a studydescribing new structure-activity relationships using thepyrazole nucleus as a central scaffold [145] Starting fromSR141716A structure they alternatively substituted one ofthe four substituents while retaining the others bysubstituents known to impart agonist activity in classicalcannabinoids One of the authors expectations was todetermine which positions are responsible respectively forthe antagonism and for the affinity of SR141716A Theaffinity of thirty compounds was assessed previous to in-vivoevaluation of their function in mice using the spontaneousactivity the tail-flick and the rectal temperature assays Theauthors showed that phenyl group in position 5 is critical foraffinity as compound O-1559 (30) lacking this phenyl has adecreased affinity (Table 6) This was already shown by Lanand co-workers [136] along with the need of a substituent inpara position on the phenyl Thomas et al previouslyshowed that this substituent could be a bromine or an iodineatom [146] However an alkyl chain is also tolerated as it isthe case in compounds O-1302 (31) O-1691 (32) or O-1704(33) All these compounds antagonise the anti-nociceptiveand hypothermic effects of ∆9-THC The authors suggestedthat the 5-substituent of pyrazoles is involved in receptorrecognition and antagonism The modulations of thesubstitution pattern of the phenyl in position 1 demonstratethat the 24-di-chloro substitution is the preferred one for theaffinity as well as for the activity Several compoundssupport this assertion Thomas et al showed that additionalhalogens result in a decreased affinity as in compounds 6-I-SR141716A (34) or 4rsquo6-di-I-SR141716A (35) (Ki values of166 and 126 nM respectively) [146] Suppression of the twochlorines (O-1300 39) or replacement of these two chlorinesby an alkyl chain like in O-1254 (40) and O-1255 (41) ledto less active compounds with Ki values ranging from 150to 430 nM However O-1254 and O-1255 behaved asantagonists in a [35S]-GTPγ S assay [61] Lan et al [136]and Wiley et al [145] also modified the substitution inposition 3 Replacement of the amido piperidinyl substituentby alkyl amides as in O-1269 (42) or O-1270 (43) etherslike in O-848 (44) and O-853 (45) ketones (O-1272 46)alcohols (O-1876 47) or alkanes (O-1877 48) resultedmostly in decreased affinity (Table 6) but also with a changeof functionality in some compounds as revealed by in-vivoassays For instance replacement of the piperidinyl by apentyl (O-1269) or by an heptyl chain (O-1270) gave agonistcompounds having Ki values of 32 and 48 nM respectivelyThus the authors suggested that the 3-substituent region isinvolved in receptor recognition and agonist activity Wileyet al on the basis of their results concluded that while the3-position seems to be involved in agonism the 1- 4- and

One close analogue of SR141716A 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-piperidinecarbox-amide (SR147778 28) was described very recently byRinaldi-Carmona et al [133 134] (Fig 3) It possesses highaffinity and selectivity for the hCB1 cannabinoid receptorwith Ki values for the hCB1 and hCB2 receptors of 35 and400 nM respectively ([3H]-CP-55940 hCB1amp2-CHO cells)Further SR147778 antagonised CP-55940 effects on mousevas deferens contractions (pA2=81) and on forskolin-stimulated adenylyl cyclase activity in U373MG cells(pA2=82) but had no effects alone In-vivo SR147778 afteroral administration reversed WIN-55212-2 inducedhypothermia and analgesia As SR141716A SR147778dose-dependently reduced ethanol and sucrose solutionintake with significant effects starting at 03 mgkg (sc) and3 mgkg (po) respectively This compound is currentlyinvestigated in Phase I clinical trials for the treatment ofobesity as well as nicotine and alcohol addictions

Another derivative the 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-(4-hydroxypiperidine)-carboxamide (29) was described in a very recent patent fromSanofi [135] (Fig 3) This antagonist possesses an IC50value for the hCB1 cannabinoid receptor of 32 nM ([3H]-CP-55940 hCB1-CHO cells)

The first structure-affinity relationships for theSR141716A derivatives were reviewed by Barth andRinaldi-Carmona in 1999 [32] Since then many papersdescribing new derivatives were published In 1999 Lan etal described around thirty 15-diarylpyrazoles derivatives[136] Among them was AM251 previously reported as aradioimaging ligand for the CB1 receptor [78] and thatappeared to be more potent (Ki = 749 nM) and selective(selectivity ratio of 306) than SR141716A (Ki = 115 nMselectivity ratio of 143) They reported a Kd value of 05 nMin the mouse vas deferens model using WIN-55212 asagonist Interestingly New et al described for the samecompound an inverse agonist effect on the hCB2 receptorusing a forskolin-induced cAMP accumulation assay (EC50= 650 nM) [137] Recently the anti-obesity effects ofAM251 were reported and are not surprisingly similar tothose of SR141716A [138 139 140] Chen et aldemonstrated that there are synergistic effects on food intakesuppression between AM251 and nalmefene an opioidantagonist [141] This synergistic effect was also presentbetween SR141716A and naloxone as shown by Kirkham[142] Shearman et al found antidepressant-like effects of

1370 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

5-positions appear to be involved in antagonism The O-derivatives reported were also described in a patent alongwith their synthetic pathways which were not given in thepaper [147]

Another investigation of the SR141716Aaminopiperidine region was conducted by Francisco et al[148] They synthesised 21 analogues possessing either analkyl amide or an alkyl hydrazide substituent of various

Table 6 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structure of some of the characterised 1- 3- 4- and 5-pyrazole derivatives Binding affinities (Ki values nM) were obtained on rat brain homogenatesusing [3H]-CP-55940 [136 145 146 148 150] or [125I]-AM-251 [155]

NN

R1

R5

R3R4

Cpd ndeg R1 R3 R4 R5 [136] [145] [146] [148] [150] [155]

SR141716 14 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Cl-Ph 115 62 62 62 13 18

O-1559 30 24-di-Cl-Ph CO-NH-piperidinyl Me 1-Methylpentyl 233

O-1302 31 24-di-Cl-Ph CO-NH-piperidinyl Me 4-((CH2)5)-Ph 21 1

O-1691 32 24-di-Cl-Ph CO-NH-piperidinyl Br 4-((CH2)5)-Ph 15

O-1704 33 24-di-Cl-Ph CO-NH-piperidinyl I 4-((CH2)5)-Ph 22

6-I-SR141716 34 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-Cl-Ph 166

4rsquo6-di-I-SR141716 35 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-I-Ph 126

4rsquo-I-SR141716 36 24-di-Cl-Ph CO-NH-piperidinyl Me 4-I-Ph 75 25 6

4rsquo-Br-SR141716 37 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Br-Ph 168 3

Cpd 25 in ref [136] 38 24-di-Cl-Ph CO-NH-morpholinyl Me 4-Br-Ph 54

O-1300 39 Ph CO-NH-piperidinyl Me 4-Cl-Ph 150

O-1254 40 4-(CH2)4-Ph CO-NH-piperidinyl Me 4-Cl-Ph 226 256

O-1255 41 4-(CH2)5-Ph CO-NH-piperidinyl Me 4-Cl-Ph 433

O-1269 42 24-di-Cl-Ph CO-NH-pentyl Me 4-Cl-Ph 32 114 3

O-1270 43 24-di-Cl-Ph CO-NH-heptyl Me 4-Cl-Ph 48 462 3

O-848 44 24-di-Cl-Ph CH2-O-(CH2)2-piperidinyl

Me 4-Cl-Ph 2450 232

O-853 45 24-di-Cl-Ph CH2-O-CH2-cyclohexyl

Me 4-Cl-Ph 388 100

O-1272 46 24-di-Cl-Ph CO-heptyl Me 4-Cl-Ph 221

O-1876 47 24-di-Cl-Ph 1rsquo-OH-heptyl Me 4-Cl-Ph 657

O-1877 48 24-di-Cl-Ph Heptyl Me 4-Cl-Ph 422

MF9725-64-17 49 24-di-Cl-Ph CO-NH-butyl Me 4-Cl-Ph 134

MF9725-179-32 50 24-di-Cl-Ph CO-NH-NH-butyl Me 4-Cl-Ph 51

MF9725-66-11 51 24-di-Cl-Ph CO-NH-cyclohexyl Me 4-Cl-Ph 25

MF9725-95-31 52 24-di-Cl-Ph CO-NH-(4-hydroxybutyl)

Me 4-Cl-Ph 154

Cpd 15 [150] 53 n-pentyl CO-NH-piperidinyl Me Ph 23

Cpd 16 [150] 54 n-pentyl CO-NH-piperidinyl Me 4-Br-Ph 63

Cpd 17 [150] 55 n-hexyl CO-NH-piperidinyl Me Ph 21

Cpd 18 [150] 56 n-heptyl CO-NH-piperidinyl Me Ph 47

NIDA-41109 57 24-di-Cl-Ph CO-NH-piperidinyl Br 4-Cl-Ph 14

NIDA-41119 58 24-di-Cl-Ph CO-NH-piperidinyl H 4-Cl-Ph 9

NIDA-41057 59 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OH-Ph 104

NIDA-41020 60 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 41

NIDA-41087 61 2-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 8

NIDA-42055 62 24-di-Cl-Ph CO-NH-piperidinyl Br 4-OMe-Ph 62

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1371

lengths in position 3 of the pyrazole moiety They observedthat until five carbons the affinity increases along with thecarbon chain length This is observed with the alkylhydrazide the alkyl amide and the hydroxyalkyl amideseries Moreover the hydrazide analogues exhibit a loweraffinity for the rCB1 than the amide analogues as illustratedby compounds MF9725-64-17 (49) and MF9725-179-32(50) having Ki values of 134 and 51 nM respectively(Table 6) From their structure-activity relationships (SAR)studies the authors concluded that the pharmacophoricrequirement of the amidopiridine region is a chain not longerthan 3 Aring and that a substituent having a positive chargedensity would probably result in increased affinity andpotency The same team described in a patent several othercompounds [149] Binding affinities (Ki) against [3H]-CP-55940 [3H]-SR141716A or [3H]-WIN55212-2 on wholerat brain or on hCB1-transfected cells were given Moreoveractivity data were obtained using the [35S]-GTPγ S assaydemonstrating that these alkyl amide and hydrazide analogsact as antagonists or inverse agonists Despite the greatnumber of derivatives claimed none of them posses asignificantly greater affinity for the CB1 receptor than theSR141716A Nevertheless they showed a slightenhancement of the selectivity for the CB1 receptor over theCB2 cannabinoid receptor

affinity of these derivatives increases with the length of thealkyl chain with an optimal length of 5-6 carbons The bestcompound of this series N-(piperidin-1-yl)-5-phenyl-1-hexyl-4-methyl-1H-pyrazole-3-carboxamide (55) possesses aKi value of 21 nM determined using [3H]-CP-55940 on ratbrain membranes which is higher than the 13 nMdetermined for SR141716A in the same conditions (Table6) In the same paper the authors starting from thehypothesis that antagonism by SR141716A is caused bybinding to the same region of the receptor as do the agonists(CP-55940 and WIN-55-2122) but preventing the agonistpromoted conformational change conducted extensiveconformational analysis as well as superimposition modelsand 3D-QSAR to propose a molecular mechanismsupporting the action of SR141716A Along with theauthors the C-5 aryl substituent of SR141716A occupyinga unique region could contribute in conferring the antagonistproperties Moreover the C-3 substituent could beresponsible for the antagonist or inverse agonist propertiesdepending on the interaction with the receptor

About this topic a very interesting review dealing withthe cannabinoid receptors pharmacophores as well as withthe activationinactivation of these receptors was recentlypublished by Reggio [151]

More recently Dyck et al described seven otherderivatives varying at the amide position [152] The onlycompound possessing a higher affinity for the CB1cannabinoid receptor than SR141716A was the 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta-

The affinity for the rat CB1 receptor of seven derivativespossessing an alkyl chain in position 1 instead of the 24-dichlorophenyl substituent was reported by Shim et al in apaper describing a molecular mechanism for the antagonistand inverse agonist activity of SR141716A [150] The

NN

O

NHN

Cl Cl

Cl

NN

Cl

Cl

NON

NN

O

NH

N

Cl

ClCl

NN

O

NH

MeO Cl

CP-272871 66

63 64

65

Fig (4) CB1 cannabinoid receptor antagonists 3 Diarylpyrazole derivatives Structures of 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63 ) 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methyl-pyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]-morpholine (64 ) N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65 ) and N-phenyl-1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-pyrazole-3-carboxamide (CP-272-871 66 )

1372 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

[c]pyrrol2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63)(Fig 4) The Ki values were 5 and 12 nM respectively([3H]-CP-55940 hCB1-HEK cells)

CP-55940 rat brain) For instance N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65) (Fig 4) showed a Ki value of15 nM However no data were provided concerning thefunctionality of such compounds

Over 200 other pyrazole derivatives acting at the CB1cannabinoid receptor were claimed in a patent from Pfizer[153] The affinity for the hCB1 receptor of 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methylpyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]ndashmorpholine (64) (Fig 4)was 79 nM ([3H]-SR141716A hCB1-HEK cells) Theothers compounds affinity was between 01 and 100 nM

In an effort to develop new SR141716A analoguespossessing lower lipophilicity to be used as PET tracersKatoch-Rouse and colleagues further explored thesubstitution pattern of the two phenyls [155] Bindingassays showed that a decreased lipophilicity lead to adecreased affinity for the CB1 receptor as determined bydisplacement of [3H]-AM251 For instance compoundNIDA-41057 (59) has a lipophilicity of 42 ( expressed asElogDoct) and a Ki value of 104 nM whereas SR141716Ahas a ElogDoct value of 54 and a Ki value of 18 nM In

Seventy five pyrazole derivatives were described in a veryrecent patent from Makriyannis et al [154] The affinity ofthe compounds for the cannabinoid receptors is given TheKi values for the best compounds are lower than 6 nM ([3H]-

Table 7 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structures and affinities for the cannabinoid receptors of some representative tricyclic pyrazole derivatives Values were obtained using [3H]-CP-55940as radioligand and mouse [160 228] rat [159 227] or human [158] cannabinoid receptors Compounds 69-78 are CB1 cannabinoid receptor ligandsCompounds 79-86 are CB2 cannabinoid receptor ligands

NN

O

NHX

R3

R1

R2

n

n˚ X R1 R2 n R3 Ki CB1 (nM) Ki CB2 (nM) Selectivity References

69 -(CH2)3- Cl 24-di-Cl 0 piperidine 126 [158]

70 -(CH2)3- H 24-di-Cl 0 azepane 100 [158]

71 -(CH2)3- H 24-di-Cl 0 piperidine 398 [158]

72 -(CH2)3- Cl 24-di-Cl 0 azepane 125 [158]

73 -(CH2)3- NO2 24-di-Cl 0 azepane 63 [158]

74 -(CH2)2-O- H 24-di-Cl 0 piperidine 501 [158]

75 -CH2-S- Br 24-di-Cl 0 piperidine lt 500 gt10 [159]

76 -CH2-SO2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

77 -(CH2)2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

78 -(CH2)3- Cl 24-di-Cl 0 piperidine 000035 21 60000 [160]

79 -CH2- Cl 24-di-Cl 0 piperidine 2050 034 00002 [228]

80 -CH2- Br 24-di-Cl 0 piperidine 1570 027 00002 [228]

81 -CH2- CH3 24-di-Cl 0 piperidine 363 0037 00001 [228]

82 -CH2- Cl 4-Cl 0 piperidine 1787 09 00005 [228]

83 -CH2- H 24-di-Cl 0 piperidine 1152 0385 00003 [228]

84 -CH2- Cl 34-di-Cl 1 133-trimethylbicyclo[221]heptyl

lt500 gt10 [227]

85 -CH2- Br 24-di-Cl 1 bicyclo[321]oct-3-yl lt500 gt10 [227]

86 -CH2- Br 4-Me 1 77-dimethylbicyclo[410]hept-3-yl

lt500 gt10 [227]

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1373

another paper Katoch-Rouse described the synthesis of[18F]-NIDA-42033 (26) a ligand useful as PET radiotracerstarting from its bromo derivative [156] Recently Kumar etal [157] proposed the synthesis of a 11C derivative ofNIDA-41087 (61) a compound previously described byKatoch-Rouse (Table 5) Post-mortem binding of thiscompound to human prefrontal cortex was also investigatedInterestingly CP-272871 (66) described by Meschler as aCB1 inverse agonist (Ki=57 nM [3H]-CP-55940 rat brainhomogenates) possesses a lipophilicity similar to theNIDA-41087 one [62] However despite the presence of amethoxy function no radiolabelled derivative has beendescribed to date One explanation could be the compoundlow selectivity (2 fold) for the CB1 cannabinoid receptor

To further explore the pharmacology of this compound afunctional assay using the mouse vas deferens model wasperformed by Ruiu and colleagues enlightening thecompetitive antagonist properties of NESS (pA2= 1246against WIN-55212-2) which had no effect by itself up to1microM Moreover NESS 0327 was unable to affect basalbinding of the [35S]-GTPγ S demonstrating its lack ofnegative intrinsic activity In-vivo studies were conductedusing the hot plate and tail flick tests NESS dose-dependently abolished the antinociceptive effect of WIN-55212-2 but had no effect by itself However the authorssuggested based on the affinity and in-vivo activity of theircompound that NESS 0327 possesses a poor centralbioavailability

Several new attempts to increase the affinity of thediarylpyrazole derivatives were recently made by rigidifyingthe SR141716A structure (Table 7) Six fused ringanalogues of SR141716A obtained by fusion of the 5-(4-chlorophenyl) substituent with the central pyrazole to forman indazole ring were published by Bass et al in 2002 [61]The compound possessing the highest affinity O-1248 (67)has a Ki value of 475 nM as determined by displacement of[3H]-CP-55940 from rat brain membranes

Another example of ring-constrained biarylpyrazole wasdescribed by Herbert Seltzman team [161] Theyinvestigated a photocyclisation reaction starting from theSR141716A leading to a pyrazolo[15-f]phenanthridinestructure (Fig 5) The new compound (68) was tested on theCB1 receptor (whole rat brain) against [3H]-CP-55940[3H]-SR141716A and [3H]-WIN-55212-2 and exhibited Kivalues of 48 35 and 50 nM respectively The CB2 affinitywas assessed using [3H]-CP-55940 and was found to be 100times lower (Ki = 3340 nM) No effect on the [35S]-GTPγ Sassay (rat brain) was observed by the authors

Stoit and colleagues from Solvay Pharmaceuticalspublished a paper describing the synthesis andpharmacological characterisation of new benzocyclohepta-pyrazole derivatives as CB1 antagonists (69-74) [158] Theaffinity of these compounds was more or less one order ofmagnitude lower than the SR141716A one with pKi valuesranging from 64 to 72 compared to 76 for the SRcompound (Table 7) Compounds were shown to beantagonists with pA2 values from 70 to 89 as theyprevent CP-55940-induced cAMP accumulation Howeverthe authors investigating the bioavailability of theircompounds either po or after ip injection found negligibleblood plasma levels

NN

Cl

Cl

O

NH

N

NN

Cl

O

NHN

Cl

Cl

O-1248 67 68However just before the publication of Stoit results

Sanofi-Synthelabo took a patent describing tricyclicpyrazolecarboxylic acid amide derivatives having antagonistproperties on the CB1 cannabinoid receptor andsubmicromolar affinities (75-77) [159] Thirty examples weregiven along with their synthetic pathway but no individualpharmacological data were shown

Fig (5) CB1 cannabinoid receptor antagonists 3Diarylpyrazole derivatives Two ring-constrained diarypyrazolederivatives O-1248 (67 ) and a pyrazolo[15-f]phenanthridinederivative (68 )

It appears from these attempts to increase SR141716Aaffinity by rigidifying its structure that only a limitedincrease in the CB1 cannabinoid receptor affinity can beexpected with the apparent exception of NESS 0327Moreover rigidifying the structure often results in a morelipophilic compound in a family of compounds being yetlipophilic

More recently Ruiu and colleagues published a paperdescribing the synthesis and complete pharmacologicalcharacterisation of a very potent CB1 receptor ligandchristened NESS 0327 (78) [160] This compound has thesame structure as compound 69 published by Stoit et al(compound 20 in the reference [158]) Nevertheless Ruiu hasobtained an affinity for the CB1 cannabinoid receptor 5000times bigger than the SR141716A one with Ki values of035 pM and 18 nM respectively using mouse forebrainhomogenates and [3H]-CP-55940 as radioligandFurthermore the selectivity ratio obtained for this compoundby Ruiu is 60000 with a Ki value for the CB2 receptor of21 nM against the [3H]-CP-55940 The discrepancybetween the data obtained independently by Stoit et al andRuiu et al could hardly be explained by the differences inthe origin of the receptor used respectively hCB1-CHO andmouse forebrain

4 Phenyl Benzofuranone Derivatives

In a 1997 patent from Eli Lilly was described thesynthesis of aryl-benzothiophene and aryl-benzofuranederivatives (87-89 Fig 6) having CB1 receptor affinity andantagonist properties [162] From ten compounds having aKi value lower than 25 microM ([3H]-CP-55940 hCB1-CHOcells) one was selected and claimed to have a Ki of 170 nMCompound 88 was shown in the patent to antagonise theeffect of anandamide on cAMP accumulation with an IC50 of

1374 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

O

O

O

O

NC

S

O

O

O

O

O

O

O

NC

LY320135 8887 89

Fig (6) CB1 cannabinoid receptor antagonists 4 Phenyl benzofuranone derivatives Chemical structures of two aryl-benzofuranederivatives (87 -88 ) and one aryl-benzothiophene (89 ) derivative from Eli Lilly

500 nM and the effect of WIN-55212-2 on calcium channelsat 1microM Moreover the intra-peritoneal injection (20 mgkg)of this compound antagonised the in-vivo effect ofanandamide in the Open Field Assay a mouse behaviourmodel Later on Felder and colleagues gave a furtherdescription of this compound called LY320135 in a paperdescribing its effect on cAMP accumulation and on ioniccurrents [163] The selectivity ratio of LY320135 was 106with Ki values of 141 and 14900 nM for hCB1 and hCB2receptors ([3H]-CP-55940) respectively LY320135antagonised anandamide inhibition of forskolin-inducedcAMP accumulation with an IC50 value of 734 nMChristopoulos et al in 2001 reported for LY320135 apKb value of 527 in the inhibition of WIN-55212-2mediated response in the rat electrically-stimulated vasdeferens while the pKb value for SR141716A was 75[164]

Other patents were taken by Aventis in which somepotential applications for their compounds were describedFor instance an association between an azetidine derivativehaving CB1 antagonists properties and sibutramine aserotonin and norepinephrine reuptake inhibitor was claimed[170] as a treatment against obesity Oral administration ofcompound 90 (3 mgkg) and sibutramine (06 mgkg) to fafaZucker rats which are genetically obese rats has resulted ina decreased food intake compared to control lean Zuckerrats In another patent the association of an azetidinederivative (1-10 mgkg po ) and a D2D3 agonist likequinpirole (01 mgkg ip) was claimed to have a therapeuticpotential in the treatment of Parkinsonrsquos disease [171] Theeffect has been evidenced using an akinesia model in the ratEvidences supporting the use of CB1 antagonists as adjuvantin the treatment of Parkinsonrsquos disease begin to appear Itseems that such compounds would be helpful in thetreatment of Parkinsonism [172] and levodopa-induceddyskinesia [173] Brotchie published an interesting paperreviewing the CB1 cannabinoid receptor signaling inParkinsonrsquos disease [174] Nevertheless further clinicalstudies should be conducted to further confirm the usefulnessof CB1 antagonists in the treatment of Parkinsonrsquos disease

5 Azetidine Derivatives

The family of azetidine compounds illustrated bycompounds 90 and 91 (Fig 7) was developed at Aventis byDaniel Achard and colleagues and was described in a seriesof patents [165-168] They claimed the IC50 values of thesecompounds for the CB1 receptor to be less than or equal to100 nM These values were obtained following the proceduredescribed by Kuster et al [169] The antagonist propertywas shown using an in-vivo model the reversal ofhypothermia induced by CP-55940 in mice and the ED50obtained were lower than 50 mgkg

6 Aryl-Imidazolidine-24-Diones Derivatives

In 1999 the Didier Lambert team published a paperdescribing the affinity of 24 new 3-alkyl-55rsquo-diphenyl-imidazolidine-24-dione derivatives for the hCB1 receptor[175] The preliminary structure-activity relationships

N

SO

O

Cl

Cl

F

F

N

SO

O

Cl

NS

90 91

Fig (7) CB1 cannabinoid receptor antagonists 5 Azetidine derivatives Two examples (90 -91 ) of the azetidine derivatives developedby Aventis

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1375

Table 8 CB1 Cannabinoid Receptor Antagonists 6 Aryl-Imidazolidine-24-Diones Derivatives

Structures affinities ([3H]-SR141716A hCB1-CHO cells) and pKb values ([35 S]-GTPγS HU210 rat brain) of three aryl-imidazolidine-24-dionederivatives (92-94)

N

NH

OO

R

Br

Br

Cpd ndeg R Ki (nM) a pKb b

DML20 92 ethylmorpholine 70 611

DML21 93 1-hydroxypropyl 103 625

DML23 94 heptyl 98 574

a [175]b [177]

showed that the substitution at the nitrogen in position 3 aswell as a bromine atom in para of the phenyl rings aremandatory for the compounds affinity Later on threecompounds termed DML20 (92) DML21 (93) and DML23(94) were characterised as neutral antagonists using the [35S]-GTPγ S assay on rat cerebellum membranes [176] (Table 8)In the same model they competitively inhibited HU-210-induced [35S]-GTPγ S binding with pKB values of 611625 and 574 respectively Moreover these compoundswere proven to be quite selective for the CB1 receptor

higher than the one for the CB2 cannabinoid receptor (IC50 =300 nM) The inverse agonism of the compound wasextrapolated by inhibiting CP-55940-induced hypothermiain mice (EC50 = 18 nM) Moreover one oral dose (10mgkg) reduces the food intake in diet-induced obese ratsInterestingly food intake of CB1

-- mice was not alteredBody weight loss was maintained all over the study during achronic administration (14 days) of 10 mgkg of Cpd A

Later on Merck published a patent [180] in which someof the 45-diaryl-pyrazoles derivatives previously describedwere claimed to be useful in the treatment or the preventionof obesity in association with compounds inhibiting the11β-hydroxysteroid dehydrogenase type 1 (1β-HSD1)enzyme The 1β-HSD1 enzyme is responsible for thesynthesis of cortisol increased levels of which according tothe inventors are associated with obesity However neitherresults on appetite suppression nor on weight loss weregiven even if some indications on the affinity of thecompounds for the cannabinoid receptors were given

Very recently the functionality of these DML compoundswas explored using the [35S]-GTPγ S assay both on rat andhuman CB1 cannabinoid receptors [177] The data obtainedconfirm that the 3-alkyl-55rsquo-diphenyl-imidazolidinedionederivatives behave as neutral antagonists on rat CB1cannabinoid receptor (rat brain) However DML 20 DML21and DML23 acted as inverse agonists on the human receptor(hCB1-CHO cells) Furthermore the authors showed thatthis different functionality is not due to the hCB1 receptorlevel of expression in the recombinant cell line as whateverthe level of expression (Bmax of 44 pmolmg or 32pmolmg) DML derivatives behaved as inverse agonists ofthe hCB1 cannabinoid receptor

Another imidazole ring pattern of substitution wasproposed by Hagmann and collaborators [181] Thecompounds described 43 examples are given possess twophenyls the first one in position 1 linked to the nitrogenand the second one in position 2 The carboxamidesubstituent is in position 4 instead of in position 2 for thediarylimidazoles described by Finke These 12-diaryl-imidazoles are claimed to be CB1 cannabinoid receptorantagonists or inverse agonists but the absence of precisepharmacological values do not allow any comparisonbetween the two substitutions patterns However a samepattern of substitution was used by other researchers Amongthem Dyck et al described in 2004 ten 1-(4-chlorophenyl)-2-(24-dichlorophenyl)-imidazole derivatives varying by theircarboxamide substituent in position 4 (101-103 Table 9)[152] For instance with a 3-azabicyclo[330]octan-3-ylsubstituent (101) they obtained a Ki value of 14 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) In a [35S]-GTPγ Sassay 101 exhibited an IC50 value of 19 nM

7 Diarylimidazoles Derivatives

Finke and colleagues at Merck developed a new class ofCB1 receptor antagonists based on an imidazole nucleusOver eighty 45-diaryl-imidazoles derivatives having CB1antagonist properties were claimed in a patent published in2003 [178] Some representative compounds of the patent areillustrated in Table 9 (95-100) The affinity of the newcompounds was determined using [3H]-CP-55940 andhCB1-CHO cells Although no detailed pharmacologicaldata were given in the patent in-vitro and in-vivo propertiesof one compound termed Cpd A (100) have been describedelsewhere [179] Its affinity for the CB1 cannabinoid receptorwas shown to be nanomolar (IC50 = 4 nM) and 75 times

1376 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 9 CB1 Cannabinoid Receptor Antagonists 7 Diaryl-Imidazole Derivatives

Structure and affinity range of some 45-diaryl-imidazole derivatives for the human cannabinoid receptors determined using [3H]-CP-55940 asradioligand and hCB1amp2-CHO cells

N

NR2

R1

R4

R3

ndeg R1 R2 R3 R4 hCB1 (IC50 or Ki) hCB2 (IC50 or Ki) References

95 CH3 CO-NH-piperidinyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

96 CH3 CO-NH-piperidinyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

97 CH3 CO-NH-cyclohexyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

98 CH3 CO-NH-phenyl 24-di-Cl-Ph 4-Cl-Ph 10 ndash 100 nM a 100 ndash1000 nM a [178]

99 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

100 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph 4 nM a 300 nM a [179]

101 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

CH3 14 nM b ND [152]

102 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

H 66 nM b ND [152]

103 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 85 nM b ND [152]

104 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 23 nM b 542 nM b [182]

105 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 30 nM b 608 nM b [182]

106 4-Br-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 60 nM b 489 nM b [182]

107 4-CF3-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 29 nM b 634 nM b [182]

108 4-Cl-Ph 24-di-Cl-Ph CO-NH-morpholinyl CH3 197 nM b 3297 nM b [182]

109 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidin-4-ol CH3 172 nM b 3959 nM b [182]

110 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CN 30 nM b 1590 nM b [182]

111 4-Cl-Ph 24-di-Cl-Ph CO-NH- piperidinyl CH2F 36 nM b 906 nM b [182]

112 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH2-CH3 14 nM b 430 nM b [182]

113 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Cl 27 nM b 823 nM b [182]

114 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Br 23 nM b 746 nM b [182]

115 4-Cl-Ph 24-di-Cl-Ph CO-NH-tetrahydroisoquinoline CH3 34 nM b 696 nM b [182]

116 4-Cl-Ph 24-di-Cl-Ph CO-NH-cycloheptyl CH3 35 nM b 349 nM b [182]

117 24-di-Cl-Ph 4-Cl-Ph CO-NH-piperidinyl CH3 403 nM b 208 nM b [182]a IC50 value b Ki value

Lange and co-workers from Solvay described twenty-eight imidazole derivatives [182] The 24-dichlorophenylsubstituent at position 2 was kept constant while thesubstitutions at the other positions were explored (Table 9)Position 5 can accommodate a large range of littlesubstituents like hydrogen (104) methyl (105) ethyl (112)chlorine (113) bromine (114) fluoromethyl (111) or cyano(110) without major changes in the affinity At position 4more bulky substituents like 1234-tetraisoquinoline (115)or cycloheptyl (116) were well tolerated whereas thepresence of a more hydrophilic moiety such as morpholine(108) or piperidin-4-ol (109) was detrimental to the affinity

Interestingly position exchange between the 24-diphenyland the 4-chlorophenyl substituents led to a 13 fold loweraffinity with Ki values of 30 and 403 nM for 105 and 117respectively ([3H]-CP-55940 hCB1-CHO cells) In thefunctional assay which was inhibition of WIN-55212-2-induced [3H]-arachidonic acid release by hCB1-CHO cellsall tested compounds behaved as antagonists Compound105 showed a pA2 value of 86 which is the same theauthors obtained for SR141716A It would be interesting toassess if these compounds are true antagonists or if they actmore as inverse agonists Compound 105 when administeredto rats was able to inhibit CP-55940-induced hypotension

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1377

with an ED50 value of 24 mgkg which is close to the 3mgkg obtained for SR141716A The compound was alsoactive in another in-vivo model the WIN-55212-2-inducedhypothermia in mice Compound 104 which has nosubstituent in position 5 of the imidazole ring was lessactive in the hypotension model and inactive in thehypothermia model Thus a methyl group is the preferredsubstituent in position 5 of the imidazole moiety Otherproperties either experimental (P-glycoprotein affinitylogPHPLC) or computational (molecular volume polarsurface area) obtained for compounds 105 were similar tothose obtained for SR141716A Finally molecularmodelling studies revealed a close structural overlap betweenthe two compounds

value of 197 nM is selective for hCB1 and is as potent asSR141716A The structural modulations of 118 led to someinteresting compounds (Table 10) Six compounds possess aKi value for the hCB1 cannabinoid receptor equal to or lowerthan 25 nM antagonist properties in the inhibition of [3H]-arachidonic acid release by WIN-55212-2-stimulated hCB1-CHO cells and Ki values for the hCB2 receptor over 1microMThe diaryl-pyrazoline derivatives contained a chiral center atposition 4 The authors resolved the racemic mixture of twocompounds (121 and 130) The levorotatory enantiomersappeared to be the eutomers For compound SLV319levorotatory enantiomer of 121 the Ki and pA2 values were78 nM and 99 respectively The Ki and pA2 values ofSLV326 levorotatory enantiomer of 130 were 359 nM and9 respectively

8 34-Diaryl-Pyrazoline Derivatives Two in-vivo models CP-55940-induced hypotension inrat and WIN-55212-2-induced hypothermia in mouse wereused to assess the potential of these compounds The resultsare of the same order of magnitude than those obtained forSR141716A For instance in the hypotension modelSLV319 ((-)121) and SLV326 ((-)130) showed ED50 values

Lange and collaborators also discovered 34-diaryl-pyrazoline derivatives after a screening of compoundsresembling to SR141716A [183184] Compound 118 thatwas initially identified during the screening possesses a Ki

Table 10 CB1 Cannabinoid Receptor Antagonists 8 34-Diaryl-Pyrazolines Derivatives

Structure affinity for the hCB1 and hCB2 cannabinoid receptors (Ki nM) expressed in CHO transfected cells ([3H]-CP-55940) and potency (pA2) ofsome 34-dirylpyrazolines derivatives Table adapted from [184]

NN

N N

SO2

R3

R2

Cl R1

R4

n˚ R1 R2 R3 R4 Ki hCB1 (nM) Ki hCB2 (nM) pA2 (CB1)

118 H H H 4-CH3 197 gt 1000 84

119 H H H 246- CH3 24 gt 1000 94

120 H H H 4-Cl 16 gt 1000 95

121 H CH3 H 4-Cl 25 gt 1000 87

122 H CH3 CH3 4-Cl 280 gt 1000 85

123 H H H 4-F 53 gt 1000 9

124 H CH3 H 4-F 338 gt 1000 85

125 H CH3 CH3 4-F gt 1000 gt 1000 lt 75

126 H CH3 H 3-Cl 14 gt 1000 86

127 4-Cl CH3 H 4-Cl 255 ND ND

128 4-F CH3 H 4-Cl 584 ND ND

129 H CH3 H H 170 ND 75

130 H CH3 H 4- CF3 221 gt 1000 93

SR141716A 25 1580 86

1378 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

N

HN

O

N

Cl Cl

Cl

N

N

HN

O

Br

Br131 132

Fig (8) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Chemical structures of two 56-diaryl-pyrazine-2-amide derivatives (131-132) developed as CB1 cannabinoid receptorantagonists by AstraZeneca

of 55 and 2 mgkg (po) respectively proving their in-vivoefficacy after oral administration The authors alsodemonstrated that SLV319 is devoid of affinity for the P-glycoprotein pump and that it possessed a goodCNSplasma ratio (17) These two compounds (SLV319and SLV326) were chosen as development candidates bySolvay and entered clinical Phase I trials in 2003 [185]

132 Fig 8) useful as CB1 cannabinoid receptor antagonists[186 187] The activity of twenty derivatives wasdetermined using a [35S]-GTPγ S assay and hCB1-CHOtransfected cells The concentration required to give halfmaximal inhibition of CP-55940-induced [35S]-GTPγ Sbinding (IC50) is lower than 200 nM for the preferredcompounds No affinity data were given for thesecompounds

9 Diaryl-Pyrazine Diphenyl-Pyridine Diphenyl-Phenyland Diaryl-Pyrimidines Derivatives

The same year in a patent from Merck Finke andcollaborators described several 56-di-phenyl-pyridinederivatives as hCB1 antagonists or inverse agonists [188]The compounds have a substituent in position 3 of thepyridine core (Fig 9) This substituent could be a cyano(133-134) or a nitro group a halogen an ester or an amide(135-136) Over 150 compounds were synthesised toillustrate the invention but no pharmacological data wasdisclosed in the patent

In 2003 appeared several patents describing new familiesof compounds that bind to the CB1 cannabinoid receptorThese compounds have in common a central six atomsaromatic ring which could be a pyrazine a pyridine aphenyl or a pyrimidine and that is substituted by at leasttwo phenyl rings

The first patents were from Berggren and collaboratorsfrom AstraZeneca who published two patents describing thesynthesis of 56-diaryl-pyrazine-2-amide derivatives (131-

A few months later another patent was taken by Sanoficlaiming the antagonist properties of 56-di-phenyl-2-pyridine carboxamide derivatives [189] These compounds

N

NH

O

Cl

Cl

N

O

Cl

O NH

N

O

Cl

ClCl

NH

CN O

N

Cl

Cl

Cl

OHN

N

O

Cl

ClCl

CNF

F

133 134

135 136 137

Fig (9) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives 56-Di-phenyl-pyridine derivatives patented by Merck (133-136) and by Sanofi (137) as CB1 cannabinoid receptorsantagonists

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1379

NH

O

N

Cl

ClCl

NH

O

N

Cl

F3CCl

N

N

O

Cl

Cl Cl F138 139 140

Fig (10) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Representative structures of the diphenyl-phenyl (138-139) and 45-diphenyl-pyrimidine (140) derivatives developed ascannabinoid antagonists at Sanofi and Merck respectively

28 examples are given structurally related to the diphenyl-phenyl derivatives have their pyridine core substituted bytwo phenyl rings and by an amide moiety (137 Fig 9) TheIC50 values were lower than 100 nM but no data were givenshowing the antagonist properties although the compoundswere assayed in the adenylate cyclase inhibition assay

10 Other Derivatives

Several amide derivatives structures recently appearedamong the CB1 cannabinoid receptor antagonists In a seriesof patents taken by Merck in 2003 Hagmann and colleaguesdescribed a large amount of compounds obtained by parallelsynthesis which are claimed to be CB1 cannabinoid receptorantagonists [192-195] They were synthesised by reacting alibrary of substituted amines with a library of carboxylicacids (141-144 Fig 11) However as no value was givenfor the affinity or activity of these amides we will notfurther discuss this class of compounds

Along this line Sanofi Synthelabo developed newdiphenyl-phenyl derivatives claimed to have CB1cannabinoid receptor antagonist properties [190] They arebased on a central phenyl ring substituted by two phenylsand by an amide moiety (138-139 Fig 10) Thirteencompounds were claimed along with their synthesis TheirIC50 values were lower than 100 nM ([3H]-CP55-940hCB1-CHO cells) but no other specific data (selectivitypA2hellip) was given

Several 15-diaryl-pyrrole-3-carboxamide derivatives weresynthesised and claimed to be CB1 cannabinoid receptorantagonists by Berggren et al [196] The affinity valueswere not disclosed in the patent (145-146 Fig 12) Otherpyrrole derivatives were synthesised by Guba et al atHofmann-La Roche [197] More specifically 2-(thiazol-4-yl)pyrrole derivatives were claimed as CB1 cannabinoidreceptor antagonist (IC50lt2microM) (147-148 Fig 12)

Finally Kopka et al from Merck described more thanone hundred 45-diaryl-pyrimidine derivatives as CB1cannabinoid receptor antagonists (140 Fig10) [191]However no precise data were provided concerning theaffinity and functionality of the compounds

R2

R1N

R3

R4

R5

O

NH

O

Cl

O F

F

R2

R1NH

R3

R4

HO R5

O

NH

O

Cl

CN

NH

O

Cl

O

O

NH

O

Cl

Cl

+

141142

143 144

Fig (11) CB1 cannabinoid receptor antagonists 10 Other derivatives Retrosynthetic scheme of a CB1 cannabinoid antagonistlibrary of amide derivatives developed at Merck The structures of four representative compounds are illustrated (141-144)

1380 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

NH

O N

Cl

Cl

Cl

N

NH

O

OMe

MeO

N

NH

O

N

S

MeO

N

NH

O

N

S

MeO

145 146

147 148

Fig (12) CB1 cannabinoid receptor antagonists 10 Other derivatives Two examples of 15-diaryl-pyrrole derivatives described byBerggren et al (145-146) While the 2-(thiazol-4yl)pyrroles derivatives 147 and 148 were described by Guba et al from Hofmann-LaRoche All these pyrroles derivatives were claimed to be CB1 cannabinoid receptor antagonists

Another new structure is represented by the 124-triazolederivatives developed by Jagerovic and collaborators In arecent paper were described the synthesis andpharmacological properties of five new 15-diphenyl-3-alkyl-

triazole derivatives [198] Among these compounds onlyone (149) behaved as a CB1 antagonist inhibiting the WIN-55212-2-induced contractions in a mouse vas deferenspreparation However despite its effects in isolated tissue

Table 11 CB1 Cannabinoid Receptor Antagonists 10 Other Derivatives

Structure and affinity of diaryl-triazole derivatives

N

NN

R3

R2

R1

Cpd R1 R2 R3 Ki CB1 (nM) References

149 24-diCl 4-Cl hexyl 855 a [198]

150 24-diCl 4-Cl2-OMe CO-NH-(3-azabicyclo[330]octan-3-yl) 270 b [152]

151 24-diCl 4-Cl CO-NH-(3-azabicyclo[330]octan-3-yl) 164 b [152]

152 4-Cl 24-diCl CO-NH-(3-azabicyclo[330]octan-3-yl) 137 b [152]

153 4-Cl 24-diCl CO-NH-benzylpyrrolidin-3-yl 29 b [152]

154 4-Cl 24-diCl CO-NH-1-(4-chlorophenyl)ethyl 66 b [152]

155 4-Cl 24-diCl CO-NH-piperidinyl 356 c [182]

156 24-diCl 4-Cl CO-NH-piperidinyl 382 c [182]

a [3H]-SR141716A rat cerebellar membranesb [3H]-CP-55940 hCB1-HEK EDNA cellsc [3H]-CP-55940 hCB1-CHO cells

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1381

N

S

O

NH

Cl

Cl

Cl

F

N

S

O

NHN

ClCl

Cl

N

S

O

NH

Br

N

S

O

NHN

Cl

Cl

Cl

156 157 158 159

Fig (13) CB1 cannabinoid receptor antagonists 10 Other derivatives Structures of 45-diaryl-thiazole derivatives patented as hCB1cannabinoid receptor antagonists by Solvay Pharmaceuticals (156 157 158) or by AstraZeneca (157 158 159)

assays (mouse vas deferens and guinea pig ileum) thecompound possessed only a reduced rCB1 affinity with Kivalues of 855 and 748 nM using [3H]-SR141716A and [3H]-WIN-55-212-2 respectively (Table 11) Further four seriesof compounds differing by the nature of the substituentaround the triazole core were also described in a patent[199] Usually the compounds claimed possess two aromaticrings possibly substituted and one linear alkyl chain Theaffinity for the cannabinoid receptors was not givenNevertheless 149 was evaluated in two isolated tissuemodels the inhibition of the electrically evoked contractionsin the guinea pig ileum and in the mouse vas deferens Inthe two models compound 149 inhibited the effect of WIN-55212-2 but had no effect by itself In the vas deferensmodel the authors obtained a pA2 value of 748 to becompared with 763 obtained for AM251

Yet another five membered ring a thiazole has beenused as central moiety of a new class of cannabinoid ligandsThe thiazole ring is substituted by two aryls in position 4and 5 and by an amide (position 2) as in compounds 156-159 (Fig 13) Forty derivatives of this type weresynthesised by Lange and colleagues from SolvayPharmaceuticals and claimed in a patent as agonists orantagonists of the CB1 cannabinoid receptor [200] Nopharmacological data were provided however their activitywas assessed by measuring the cAMP in transfected hCB1-CHO cells In a recent paper from Lange et al two of thesethiazole derivatives were further described [182] Compound13 (157) the most closely related to SR141716A has a Kivalue of 227 nM while for compound 14 (158) Ki value isover 1000 nM Thus as in the imidazole series (see Table9) the aromatic substitution pattern is of great importancefor the CB1 cannabinoid receptor affinity Compound 13however was devoid of in vivo activity

Others 124-triazole derivatives were described by Dycket al compounds 150-154 unlike the Jagerovic ones havean amide moiety in position 3 [152] The highest affinitywas obtained with a 1-benzyl-pyrrolidin-3-yl substituentThe Ki value was 29 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) to be compared to 12 nM obtained for theSR141716A

Seventeen 45-diaryl-thiazole derivatives were alsosynthesised by Berggren et al and patented by AstraZeneca(157-159 Fig 13) [201]

In a patent from Merck Toupence et al described over190 substituted furo[23]pyridines claimed to be CB1cannabinoid receptor antagonists [202] The affinity (IC50 lt1microM) was measured using recombinant CHO cells and [3H]-CP-55940 and the activity using cAMP dosage (160 Fig14)

Lange et al described a triazole derivative (155) whichcan be superimposed with SR141716A Compound 155showed a Ki value of 356 nM ([3H]-CP-55940 hCB1-CHOcells) a pA2 value of 83 in the inhibition of WIN-55212-induced release of [3H]-arachidonic acid by hCB1-CHO cellsand was active in vivo in the CP-55940-inducedhypotension in rat (ED50 = 236 mgkg) [182]

Very recently Alanine from Hoffmann-LaRoche foundthat diarylbenzo[13]dioxole derivatives act as CB1

O

O

S

N

OO

F

ClCl

O

O

F O

N

OON

NH2

O

Cl

Cl

Cl

160 161 162

Fig (14) CB1 cannabinoid receptor antagonists 10 Other derivatives One example of a furo[23]pyridine derivative (160) and twoexamples of diarylbenzo[13]dioxole derivatives (161-162) described as hCB1 cannabinoid receptor antagonists by Toupence et aland Alanine et al respectively

1382 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

cannabinoid receptor antagonists (161-162 Fig 14) [203]Approximate IC50 values for twelve compounds ([3H]-CP-55940 hCB1-HEK cells) among the three hundred andeighty compounds described were given (IC50lt2microM)

et al in 1995 [207] In their hands ∆9-THC was not able toinhibit the forskolin-stimulated adenylyl cyclase activity inan assay performed on hCB2-CHO cells Subsequently theyshowed that ∆9-THC was able to antagonise HU210-inducedinhibition of adenylyl cyclase activity thus acting as anantagonist of the hCB2 cannabinoid receptor expressed inCHO cells [208] Furthermore very recently Govaerts et al[209] reported an inverse agonist effect of ∆9-THC and ∆8-THC on hCB2-CHO cells using the [35S]-GTPγ S assayThe pEC50 values were 763 and 888 respectively whilethe Emax values were ndash27 and ndash16 as compared to basal

Griffith from Pfizer described three new series of CB1cannabinoid receptor antagonists based on a bicyclic centralaromatic ring Purine pyrazoltriazines andpyrazolo[15a]pyrimidines derivatives were synthesised andinvestigated for their ability to interact with the CB1cannabinoid receptor Albeit a [35S]-GTPγ S assay wasdescribed no data were given concerning the antagonist orinverse agonist properties of such compounds Two hundredand eighty purine derivatives were synthesised [204] Theaffinities of 163 and 164 were given in the patent (28 and12 nM [3H]-SR141716A) In a following patent thesynthesis of sixty pyrazoltriazine derivatives was described(165 Fig 15) [205] The affinity of each compound was notgiven but was said to be ranging between 01 and 590 nMAnd finally more than forty pyrazolo[15a]pyrimidines weresynthesised (166 Fig 15) [206] Their affinity for the CB1cannabinoid receptor was lower than 155 nM Despite thefact that a great number of derivatives share the samesubstitution pattern around the central bicyclic aromatic ringthe comparison of these three new series of compounds ismade difficult by the absence of precise affinity data

Some other classical cannabinoids were shown to possessan antagonist activity at the CB2 receptor This is the casefor instance for the non-selective O-1184 (2) (Ki = 74 nM)which enhances the forskolin-induced cAMP production inCB2-CHO cells (EMax = 161 EC50 = 63 nM) and alsofor O-584 (3) (EMax = 246 EC50 = 138 nM) [39]

2 Indole Derivatives

Since the disclosure in 1993 of a second cannabinoidreceptor many attempts have been made to synthesise CB2cannabinoid receptor selective ligands The first compoundreported to have a somehow greater affinity for the peripheralcannabinoid receptor was WIN-55212-2 The compoundwas introduced by DrsquoAmbra and colleagues [210] back in1992 as a new cannabinoid ligand having a nanomolaraffinity and acting as an agonist In 1996 Showalter et alused a transfected cell line expressing the CB2 cannabinoidreceptor in order to identify selective ligands for thisreceptor [211] They showed that the CB2 affinity of WIN-55212-2 was 7 times greater than the CB1 with Ki valuesof 189 and 028 nM for the CB1 and CB2 cannabinoidreceptors respectively However WIN-55212-2 is anagonist of the cannabinoid receptors Interestingly WIN-55212-3 (167 Fig 16) the so-called inactive enantiomer ofWIN-55212 was recently shown to behave as a low affinity(Ki=363 microM) inverse agonist of the hCB2 cannabinoidreceptor as it decreased the [35S]-GTPγ S binding [209]

III CB2 LIGANDS RECEPTOR ANTAGONISTS ANDINVERSE AGONISTS

The compounds acting as antagonist or inverse agonistsat the CB2 cannabinoid receptor are reviewed in this thirdpart of the paper As for the CB1 cannabinoid receptorligands they are classified depending on their chemicalstructures

1 Classical Cannabinoid Derivatives

The first report of an antagonism mediated by ∆9-THC atthe peripheral cannabinoid receptor was made by Bayewitch

N N

NN

NH COOH

Cl

Cl

N N

NN

N

Cl

Cl

NH2OH

O

N

N

N

N

N

NHNH2

O

Cl

ClN

N

N

N

Cl

Cl

N

O

163 164

165 166

Fig (15) CB1 cannabinoid receptor antagonists 10 Other derivatives Examples of purine (163-164) pyrazoltriazines (165) andpyrazolo[15-a]pyrimidines (166) derivatives described as CB1 cannabinoid receptor antagonists by Griffith

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1383

The pEC50 value was 554 and the Emax value ndash26 ascompared to basal

AM630 also acts as an inverse agonist at the hCB1cannabinoid receptor (EC50 = 900 nM) as Landsman et aldemonstrated [47] Recently Zhang and colleagues reportedthe characterisation of the microsomal metabolism of AM630[212]

N

O

N

O

O

N

O

Cl

O

N

O

(S)-WIN-55212-3 167 BML-190 168

In 1996 Gallant and colleagues identified by submittinga large number of compounds to a binding assay anotherindole analog as CB2 cannabinoid receptor ligand [213] Onthe basis of this compound christened BML-190 (168 Fig16) or called indomethacin morpholinylamide theysynthesised several derivatives that possessed a selectivityratio for the CB2 cannabinoid receptor of up to 140 Thesewere the first compounds specifically designed to be CB2cannabinoid receptor ligands The first report on thepharmacological properties of BML-190 appeared onlyrecently New and colleagues highlighted in the paper theinverse agonist properties of the compound BML-190 dose-dependently increases (103 EC50= 980plusmn70 nM) theforskolin-stimulated levels of cAMP in hCB2-HEK cellswhile WIN-55212-2 decreases (44 45plusmn42nM) thisaccumulation [137]

Fig (16) CB2 cannabinoid receptor antagonists 2 Indolederivatives Chemical structures of WIN-55212-3 (167) andBML-190 (168) two indoles derivatives acting as inverseagonists at the hCB2 cannabinoid receptor Surprisingly in 2002 Melck and collaborators used

BML-190 as a CB2 receptor agonist in a cell proliferationassay [214] BML-190 was also tested as inhibitor of thecyclooxygenase-2 enzyme by Kalgutkar et al [215] Theyobtained IC50 values higher than 33 and 66 microM for theCOX-I and COX-II enzymes respectively These valueshave to be compared with the submicromolar activity ofindomethacin the parent compound Very recently Klegerisand colleagues [216] looking for an antineurotoxic action ofcannabinoids showed that BML-190 increases TNF-αsecretion by stimulated THP-1 monocytic cells but is noteffective on the IL-1β secretion

Further on the basis of WIN-54461 (12) a CB1antagonist AM630 (13) was synthesised by replacement ofthe bromine atom by an iodine one In the initial reportPertwee et al showed the antagonist effects of AM630 butalso suggested that the CB1 cannabinoid receptor may not bethe preferential receptor of AM630 [45] Later on Ross et alreported using hCB1amp2-CHO cells and [3H]-CP-55940that AM630 binds to the CB2 cannabinoid receptor (Ki =312 nM) with a selectivity ratio over 160 [48]Furthermore using a [35S]-GTPγ S assay they showed itsinverse agonist properties (EC50 = 76 nM) However

Table 12 CB2 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Reported binding affinities (Ki) and selectivity ratio (CB1CB2) of SR144528 (169) for rat and human cannabinoid receptors determined using [3H]-CP55940 as radioligand

NN

O

NH

Cl

SR144528 169

rCB2 (spleen) rCB1 (whole brain) Selectivity Ki hCB2 (transfected CHOcells)

Ki hCB1 (transfected CHOcells)

Selectivity References

038 nM 305 nM 800 06 nM 437 nM 730 [217]

067 nM 548 nM 80 [219]

56 nM gt10microM gt1780 [48]

25 nM a gt 1microM a gt400 [224]

024 nM 276 nM 115 199 nM 503 nM 25 [232]

a COS cells

1384 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

3 Diarylpyrazole Derivatives CHO cells is constitutively active and phosphorylated onSer 352 Upon treatment by SR144528 thisphosphorylation of the receptor was inhibited Moreover theSR compound was able to induce dephosphorylation ofagonist-induced CB2 phosphorylation The question whetherSR144528 induces a CB2 receptor conformational changemaking it a better phosphatase-specific substrate or ifSR144528 binding activates specific phosphatases remainsopen However this remains a nice example of inverseagonist-induced receptor resensitisation

The same Sanofi team which synthesised the CB1antagonist SR141716A reported in 1998 the synthesis andpharmacological characterisation of a CB2 cannabinoidreceptor selective antagonist the SR144528 (169) [217218] The structure of the new compound is derived fromSR141716A one SR144528 is the first selective CB2receptor antagonist reported as the inverse agonist propertiesof BML-190 were shown only recently by New et al [137]The authors reported for 169 using [3H]-CP-55940 asradioligand Ki values of 04 nM and 06 nM for the rat andthe human CB2 receptors respectively and found a CB1-CB2 selectivity ratio around 700 Table 12 sums up thebinding affinities of SR144528 for the cannabinoid receptorsobtained by different authors Interestingly while the CB2affinity is more or less constant the affinity obtained for theCB1 receptor varies by one order of magnitude [219] Theantagonist effect of SR144528 was highlighted by evaluatingits effect on cAMP production by hCB2-CHO cells exposedto 3 nM CP-55940 (EC50=10nM) The antagonism wasalso observed on the activation of the MAP kinase pathwayby CP-55940 (IC50=39nM)

The Sanofi research team working on cannabinoidsconducted investigations in order to determine the keyresidues for SR144528 interaction with the receptor (Table13) as well as the binding mode of the compound Firstlythey identified the TM4-EL2-TM5 region as a regioncontaining crucial residues for the affinity of several ligandsamong them SR144528 [224] Among these residues themutation of two cysteine of the second extracellular loop(C174 and C179) abolishes the affinity for the CB2 receptorLater on the implication of S453(161) and S457(165) inthe binding of SR144528 was evidenced by mutationalstudies In the model proposed for the docking of SR144528into the CB2 receptor the crucial contacts are comprised intothe TM4 this is the case for S453(161) and S457(165)which are proposed to form hydrogen bonds with the ligandand W464(172) proposed to have aromatic interactions withthe 4-methylbenzyl of the SR144528 [225] In 2003 Fengand Song showed that point mutations D349(130)A andA634(241)E abolished ligand binding However mutationof the arginine R350(131) member of the highly conservedDRY motif to alanine has no influence on SR144528binding [226]

Early after the report of the synthesis and characterisationof SR144528 the same authors further explored the functionof this compound and showed its inverse agonist propertiesusing CB2-CHO cells co-transfected with the luciferasereporter gene with either the CRE or the murine krox24regulatory sequence [220] In these models SR144528 notonly inhibits CP-55940 effects but also reproduces theeffects of a pertussin toxin treatment proving its inverseagonist properties SR144528 functionality was alsoexplored by [35S]-GTPγ S binding in hCB2-CHO cells(IC50=3 nM) [221] The inverse agonist function ofSR144528 was further illustrated by Rhee and Kim usingCOS cells co-transfected with hCB2 receptor and adenylylcyclase [222]

As for the CB1 cannabinoid receptor ligands Sanofideveloped tricyclic derivatives based on the 1-benzylpyrazole-3-carboxylic scaffold More than twentycompounds were disclosed in a patent [227] in 2001 (84-86Table 7) The benzyl in position 4 characteristic of the CB2diarylpyrazoles from Sanofi is conserved as well as thebicyclic residue in position 3 The length of the second linkbetween the phenyl and the pyrazole can be made of one or

Bouaboula and co-workers investigated SR144528 effectson CB2 cannabinoid receptor phosphorylation status [223]Firstly they showed that the CB2 receptor transfected in

Table 13 CB2 Cannabinoid Receptor Antagonists 3 Di-Aryl-Pyrazole Derivatives

Some of the single point mutations reported for the hCB2 cannabinoid receptor for which pharmacological data concerning the SR144528 are available

Mutant Effect References

R350(131)A no effect [226]

S453(161)A loss of affinity [225]

V456(164)I no effect [225]

S457(165)A loss of affinity [225]

C174S a loss of affinity [224 225]

C175S a loss of affinity [225]

C177S a no effect [225]

C179S a loss of affinity [224 225]

S544(193)G no effect [225]

a Second extra-cellular loop

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1385

two methylene but according to the patent one methylenebridge is preferred The Ki values of these compounds for theCB2 receptor were said to be lower than 500 nM and theirantagonist properties have been assessed using the adenylatecyclase inhibition assay

compounds by the presence of an alkyl substituent inposition 5 instead of a phenyl and of course by position 3which remains unsubstituted However except a lecture atthe 1998 ACS meeting [230] where compound 171 wasreported to have a Ki value of 835 nM and a selectivity ratioover 100 no further report on these compounds waspublished untill now

In a recent paper Mussinu and colleagues reported thesynthesis of other tricyclic derivatives starting fromSR141716A structure [228] N-piperidin-1-yl-14-di-hydroindeno[12-c]pyrazole-3-carboxamide derivatives areCB2 receptor ligands and some of them possess high affinityand selectivity for the CB2 receptor as shown in Table 7 (79-83) These tricyclic derivatives differ from the Sanofi tricyclicderivatives as they do have a phenyl ring instead of a benzylin position 1 Another difference is the nature of thesubstituent in position 3 which is not a bicycloalkyl but acyclic amine One of the compounds synthesised byMussinu et al 81 possesses the highest affinity for the CB2receptor ever reported with a Ki value of 0037 nM for themurine CB2 receptor ([3H]-CP-55940 mice spleenhomogenate) Interestingly 79 the closest SR141716Aanalog lost almost all its affinity for the CB1 receptor andincreased by four orders of magnitude its affinity for the CB2receptor Even if the structure quite similar to the SRcompounds could suggest that these derivatives behave asantagonists no pharmacological data were given in the paperto support this hypothesis

5 2-Oxoquinoline Derivatives

Inaba and colleagues from Japan Tobacco introduced in2001 a completely new structure in the cannabinoid fieldbased on a 2-oxoquinoline scaffold Over eighty derivativeswere disclosed in a patent [231] and one compound JTE-907 (173) was subsequently characterised in-vitro as well asin-vivo [232] In Table 14 are listed some of the compounds(172-178) disclosed in the patent along with their affinity forthe cannabinoid receptors These values were obtained onhuman receptors against [3H]-WIN-55212-2 For twelvecompounds the anti-inflammatory activity per os wasassessed in a carrageenin-induced paw edema model Thehighest effect reported was obtained with JTE-907 with anED50 value lower than 01 mgkg Therefore thesecompounds were claimed to be useful as anti-inflammatoryand anti-allergic agents

To further characterize JTE-907 the authors determinedits affinity for cannabinoid receptors of different species using[3H]-CP-55940 as radioligand In their hands JTE-907behaved as a very selective hCB2 ligand with Ki values of36 and 2370 nM for the CB2 and CB1 cannabinoid receptorsrespectively even more selective than the SR144528 (Kivalues of 199 and 50 nM respectively) However it has tobe mentioned that the affinities reported in the paper forJTE-907 are quite different from those reported in the patent(hCB1 Ki values of 35nM and 009 nM respectively) Italso appears that the SR144528 affinity for the CB1 receptordetermined by Iwamura et al is higher than the affinityreported by Rinaldi-Carmona et al with values of 50 and437nM respectively The authors ascribed the difference tothe variability of SR144528 binding affinities to the assayconditions The function of JTE-907 was evaluated bymeasuring the increase in cAMP production by forskolin-stimulated hCB2-CHO cells Maximum stimulation wasattained at a concentration of 1microM of JTE-907 In the sameassay WIN-55212-2 decreased the cAMP production in adose-dependent manner

Finally it is interesting to strengthen that the five-membered ring derivatives (79-86) of the diarylpyrazolefamily are reported to possess a high CB2 receptor affinitywhile the six- and seven-membered ring derivatives (69-78)are described as CB1 receptor ligands

4 Other Pyrazole Derivatives

Other CB2 ligands based on a pyrazole nucleus wereintroduced by SmithKline Beecham in a patent describingthe synthesis of about sixty new compounds [229] Theiraffinity (Ki) for the hCB2 receptor transfected in HEK293cells was reported to be ranging between 25nM and 10microMTheir structures (170-171 Fig 17) differ from the Sanofi

NN

O

N

ON

NN

N

NN

O

N

ON

NN

N

ClCl

1

5

4 3

2

170 171

Fig (17) CB2 cannabinoid receptor antagonists 4 Otherpyrazole derivatives Structure of two compounds (170-171)among those claimed by SmithKline Beecham to be hCB2antagonists

In conclusion JTE-907 was shown to be a CB2 selectiveinverse-agonist with anti-inflammatory and anti-allergicproperties This compound is currently undergoing Phase Iclinical trials as anti-allergic drug [233]

IV NON-CB1 NON-CB2 CANNABINOIDRECEPTORS LIGANDS

In the last years the possible presence of additional non-CB1 non-CB2 cannabinoid receptors was raised frompharmacological evidences

One of the putative receptors possesses an endotheliallocalisation while other ones are localized in the CNS or atperipheral nerves For instance an additional target for ∆9-

1386 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 14 CB2 Cannabinoid Receptor Antagonists 5 2-Oxoquinoline Derivatives

Structure of six compounds among the oxoquinoline derivatives patented by Japan Tobaco Affinity (Ki nM) and selectivity (CB1CB2) for the cannabinoidreceptors are given The affinity was measured by binding against [3H]-WIN-55212-2 on either hCB2-CHO or hCB1-CHO cells Adapted from [231]

Cpd ndeg Structure hCB2 hCB1 Selectivity

1-2 172

NH

MeO O

O

OMeO

0014 3671 262215

JTE-907 173

NH

O

MeO O

O

NH

O

O

0087 3436 39495

2-2 174

OH

O

NH

MeO O

O 077 3247 4215

3-27 175

NH

O

F

NH

MeO O

O0021 336 16000

3-32 176

NH

MeO O

O

NH

F

O

0036 2398 66610

3-38 177

O

O

NH

N

O

O

NMeO

0085 935 11000

3-43 178

NMeO O

O

NH

F

O

0043 864 20100

tetrahydrocannabinol was described in capsaicin-sensitivesensory nerves by Zygmunt et al [234]

[29] and by Di Marzo et al [235] Thus in the presentreview we will focus on the description of the so faridentified ligands of the non-CB1 non-CB2 receptors withan emphasis on the compounds having antagonist properties

Pharmacological evidences for the existence of additionalcannabinoid receptors were reviewed by Wiley and Martin

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1387

(Table 15) In particular the knowledge about three not yetcloned targets has grown The growing molecularpharmacology of the three putative new cannabinoidreceptors is reviewed in the three following sections Thefourth section summarises the data on the other proposedcannabinoid targets

vasodilatator activity In addition the anandamide effect ispartially sensitive to SR141716A inhibition The authorsproposed the existence of two distinct receptors responsiblefor the vasodilatatory effect of anandamide one located in theendothelium is SR141716A-sensitive while the other onelocated on the vascular smooth muscle is SR141716A-insensitive Thus SR141716A is an antagonist of anendothelial anandamide receptor Several studies wereconducted in order to understand the molecularpharmacology of the endothelial anandamide receptor It ispertussis toxin-sensitive [237] Cannabidiol acts asantagonist and abnormal-cannabidiol (Abn-cbd 179) and itsanalogue O-1602 (180) act as agonists [238] The synthesisof O-1602 and of its analogues are disclosed in a patentdescribing the vasodilatatory activity of cannabinoid

1 Endothelial Anandamide Receptor ndash Abnormal-Cannabidiol Receptor

In 1999 Wagner et al showed that anandamide couldinduce mesenteric vasodilatation through an endotheliallylocated ldquoanandamide receptorrdquo pharmacologically distinctfrom CB1 [236] Other cannabinoids such as ∆9-THC HU-210 or WIN-55212-2 were devoid of mesenteric

Table 15 Non-CB1 Non-CB2 Cannabinoid Receptors Antagonists

An overview of the putative new non-CB1 non-CB2 anandamide-receptors ligands The antagonists are highlighted in bold

NH

N

S

HO OH

Cl

OH

OH

OH OH OH OH

OMe

OMe

RR

RR

RR

RR

HO

OH

NH

O

cannabidiol (10) Abnormal-cannabidiol 179 O-1602 180

O-1918 181 capsazepine 183capsaicin 182

Compound Endothelial AEA receptor Brain AEA receptor Cannabinoid-vanilloid brain receptor

AEA agonist a agonist de

WIN-55212-2 no effecta

agonist e agonist g h

CP-55940 no effect e agonist i

SR141716A antagonist a b j no effect ([35 S]-GTPγS binding) d e f antagonist g h

AM251 no effect k no effect h i

Cannabidiol antagonist b no effect e

O-1918 antagonist c jm

Abd-cbd agonist b l

O-1602 agonist b

Capsaicin no effectj agonist i

Capsazepine no effect b antagonist h

a [236] b [238] c [240] d [249] e [250] f [251] g [254] h [258] i [ 255] k [244] j [ 243] l [ 242] m [241]

1388 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

analogues [239] More recently Offertaler et al showed thatO-1918 (181) a cannabidiol analog acts as an antagonist ofthe endothelial anandamide receptor [240] In addition O-1918 does not bind to the CB1 and CB2 cannabinoidreceptors Furthermore O-1918 unlike cannabidiol doesnot cause vasorelaxation in the mesenteric artery preparationmodel used and thus acts as a true neutral antagonistUsing O-1918 as a pharmacological tool Begg et alproposed a coupling of this new G protein-coupledendothelial receptor through a GiGo mechanism to ionchannels in primary cultured human vascular endothelialcells [241] However in their hands SR141716A wasineffective in antagonising Abd-cbd Ho and Hiley furtherdemonstrated that Abn-cbd relaxes rat small mesentericarteries through an activation of K+ channels via aSR141716A-sensitive pathway [242] Further using thesame model they characterized the vasorelaxant activity ofvirodhamine a novel endocannabinoid apparently actingthrough the same receptor [243] The virodhamine-inducedvasorelaxation is sensitive to SR141716A and O-1918 butinsensitive to AM251 SR144528 and AM630 Moreovervanilloid receptor desensitisation by capsaicin (182) had noeffect

244] Thus position 4 on the 5-phenyl could be furtherexplored to obtain selective ligands

To come to an end despite the fact that the endothelialanandamide receptor is neither identified nor cloned at leastthree antagonists are identified SR141716A cannabidioland O-1918 It is likely that a growing number of ligandsand therapeutic applications will appear in the very nearfuture

2 Brain Anandamide Receptor

Following an in-depth evaluation of anandamide effects inCB1

-- mice Di Marzo and co-workers suggested that a non-CB1 non-CB2 G protein-coupled receptor might mediatesome of the actions of AEA in mice [249] Actually effectsof AEA but not those of ∆9-THC were not decreased inCB1 cannabinoid receptor knock-out mice For instance 30mgkg of AEA were still effective in hot-plate test whereas10 mgkg of ∆9-THC had no effect on anti-nociceptionMoreover in a [35S]-GTPγ S assay AEA stimulated with anEC50 value of 223 microM the [35S]-GTPγ S binding to CB1

--

mice brain membranes (Emax= 28) ∆9-THC showed noeffect in this assay The addition of SR141716A did notaffect the [35S]-GTPγ S binding to CB1

-- mice brainmembranes Shortly after other evidences for the existence ofa new G protein-coupled cannabinoid receptor with a distinctdistribution in the central nervous system were published[250] From a set of 24 commonly used cannabinoids (AEAWIN-55212-2 JWH-030 THChellip) only AEA and WIN-55212-2 produced an increased binding of [35S]-GTPγ S onCB1

-- mice brain membranes Interestingly in this studySR141716A alone produced a significant inhibition of [35S]-GTPγ S binding similar to the inhibition obtained usingCB1

++ mice brain membranes (IC50 values of 57 microM and47 microM respectively) However SR141716A did not affectthe anandamide-induced [35S]-GTPγ S binding in CB1

--

mice brain membranes Moreover [3H]-SR141716Aexhibited significant binding in some brain regions of CB1

--

mice which are different from those exhibiting stimulation of[35S]-GTPγ S binding by WIN-55212-2 and AEA Thusthe authors proposed that the SR141716A effects on CB1

--

mice are mediated neither by the CB1 cannabinoid receptornor by the putative new anandamide brain receptor Monoryand co-workers using CB1

-- mice described a novel nonadenylyl cyclase-coupled cannabinoid binding site in micecerebellum [251] Two agonists AEA and WIN-55212-2but not ∆9-THC or HU210 were found to bind to thisreceptor as they enhanced [35S]-GTPγ S binding NeitherSR141716A nor SR144528 were able to affect the WIN-55212-2 induced [35S]-GTPγ S binding in CB1

-- mice asfound by Breivogel et al [250] However the distributionpattern of the receptor described by Monory et al differs fromthe distribution found by Breivogel et al It has to bementioned that the knock-out strains used were different Themice used by Monory were from a CD-1 strain while theone used by Breivogel were from a C57BL61 strainInterestingly Muthane et al detected differences in nigralneurons number and sensitivity to MPTP in the two mousestrains used [252]

More recently Mo et al obtained results showing thatthe Abnormal-cannabidiol receptor is also responsible for anincreased endothelial cell migration [244] Abn-cbd enhancedhuman umbilical vein endothelial cell migration while O-1918 antagonised Abn-cbd effect The CB1 cannabinoidreceptor inverse agonist SR141716A partially inhibitedAbd-cbd action whereas AM251 and SR144528 had noeffect

In another paper from George Kunos team Batkai et alreported that SR141716A is able to inhibit the endotoxichypotension by a cardiac mechanism involving neither theCB1 nor the CB2 cannabinoid receptors [245] Theinvolvement of a non-CB1 non-CB2 site of action wasshown using CB1

-- and CB1--CB2

-- knock-out miceFurther studies are needed to identify the SR141716Asensitive myocardial site of action

In a recent patent Kunos described O-1918 and itsanalogues as vasoconstrictor agents useful to reversepathological vasodilatation of blood vessels [246] Theeffects of O-1918 on mouse blood pressure on LPS-inducedhypotension in mouse as well as the reversal of hypotensionby O-1918 injection are described Furthermore thesynthesis of O-1918 based antagonists is described startingfrom p-metha-28-dien-1-ol and the appropriate resorcinols

Interestingly the Abd-cbd receptor which was firstdetected in the vascular system also seems to be present inthe CNS [247] Indeed Walter et al found that microglialcells (BV-2) migration induced by 1 microM 2-AG is inhibitedby cannabidiol (300 nM) and O-1918 (1 microM) SR141716Awas found ineffective in this assay Thus 2-AG is an agonistof the Abd-cbd receptor Moreover Abd-cbd is able to elicita dose-dependent cell migration in the same model (EC50 =600nM) [248]

From a more structural point of view SR141716Abehaves as an antagonist while on the opposite AM251(20) a close analogue which differs from the former only bythe nature of one halogen has no effect on this receptor [242

To conclude to our knowledge no compounds havingantagonist or inverse agonist properties at this receptor havebeen identified so far

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1389

3 Cannabinoid-Vanilloid Brain Receptor a cannabinoid-vanilloid receptor at which SR141716A andcapsazepine behave as antagonists

Evidences for the presence of an additional ldquocannabinoidrdquoreceptor differing from the CB1 receptor and from the brainanandamide receptor appeared from studies focused on thehippocampus (for a review see Hajos et al [253]) Hajos etal studied the cannabinoid actions on GABAergic andglutamatergic transmission using the whole-cell patch-clamp technique [254] WIN-55212-2 induced a dose-dependent reduction in glutamatergic transmission reversedby SR141716A (1microM) Thus the authors concluded that thecannabinoid actions on the glutamatergic transmission aremediated by a non-CB1 but SR141716A-sensitive receptorIn another paper Hajos and Freund further characterised thisputative receptor [255] They showed that in addition toWIN-55212-2 CP-55940 and capsaicin (182) a TRPV1receptor agonist also act as agonists Furthermore in theirhands SR141716A as well as capsazepine (183) a TRPV1receptor antagonist were able to antagonise this effect on theglutamatergic transmission while AM251 was notSR141716A effects on CB1 knock-out mice were furtherstudied using a test of anxiety [256] Haller et al showedthat on the one hand the distribution of the CB1 receptorinduces anxiety and SR141716A has an anxiolytic effectand on the other hand SR141716A is still active in CB1

--

mice In a following paper they reported that AM-251induces anxiogenic effects in wild-type and in knock-outmice [257] Thus SR141716A in addition to CB1 bindsto a not yet identified receptor Another evidence for thisadditional receptor for SR141716A was given by Bass andco-workers [61] They conducted a structure-activityrelationship study on the stimulation of locomotor activityinduced by SR141716A and twenty analogues Nocorrelation was found between the affinity for the CB1cannabinoid receptor and the stimulation of locomotoractivity Albeit SR141716A and five of its analogues O-1803 O-1710 O-1253 O-1254 (40) O-1255 (41) behavedas inverse agonists in [35S]-GTPγ S assay none of theseanalogues stimulated the locomotor activity In conclusionthe SR141716A-induced stimulation of locomotor activity isneither due to inverse agonism nor to inhibition of anendogenous tone but to a brain receptor distinct from theCB1 receptor Di Marzo et al previously provided evidencesthat the cannabinoid-vanilloid brain receptor could mediatesome of the cannabinoid effects on locomotion [249]

4 Other Non-CB1 Non-CB2 Cannabinoid Receptors

Evidences for the existence of additional non-CB1 non-CB2 cannabinoid receptors localised in the rat isolatedmesenteric arterial bed appeared from the works of Ralevicand collaborators Several cannabinoids were evaluated intheir model of sensory neurogenic vasorelaxation evoked byelectrical field stimulation

HU210 was found to attenuate vasorelaxation in a CB1and CB2 independent way as SR141716A LY320135 andSR144528 were ineffective in inhibiting HU210 effect [259]Similar results were obtained with ∆9-THC [260]Similarly noladin ether inhibited sensory neurogenicrelaxation in a concentration-dependent manner which wasalso unaffected by SR141716A LY320135 and SR144528Moreover this effect as the ∆9-THC one was pertussistoxin sensitive suggesting the involvement of a Gio protein-coupled receptor [261]

However the inhibitory effects of CP-55940 and WIN-55212-2 were affected by the presence of SR141716A (1microM)or LY320135 (1microM) but not by the addition of SR144528(1microM) [262]

It appears that at least two receptors should be involvedin cannabinoid-mediated attenuation of sensory nerve-mediated vasorelaxation The first one is SR141716A andLY320135 sensitive while the other one is resistant toSR141716A

Some evidences for the existence of a ldquoCB2-likerdquo receptorappeared from the studies on palmitoylethanolamide (PEA)an endogenous fatty acid amide devoid of CB1 and CB2affinity [264] antinociceptive potential Calignano and co-workers showed that PEA was able to alleviate pain inseveral animal models [265 266] For instance PEA dose-dependently inhibits kaolin-evoked writhing in mice TheCB2 cannabinoid receptor inverse agonist SR144528 (02mgkg) inhibits PEA anti-nociceptive effect [266] Thus theauthors suggest that PEA acts as agonist on a SR144528-sensitive non-CB2 cannabinoid receptor (ldquoCB2-likerdquoreceptor) at which SR144528 acts as an antagonist

Further studies are needed in order to better characterisethese additional ldquocannabinoidrdquo receptors However it islikely that several therapeutic applications will soon springfor these receptors

Very recently Pistis et al published a study on theneurophysiological effects of cannabinoids on the basolateralamygdala neurons in-vivo [258] One of their findings is thatHU-210 and WIN-55212-2 did not elicit similar effects onbasal amygdala projection neurons firing rate While theformer had no effect the latter decreased the firing rateMoreover SR141716A reversed the WIN-55212-2 effectswhile AM251 had no effect The vanilloid receptorantagonist capsazepine also antagonised the WIN-55212-2effect on firing rate of these neurons Therefore the authorssuggested the presence of a novel cannabinoid-vanilloidreceptor

V CONCLUSION

Since their development cannabinoid antagonists provedto be essential tools in the understanding of cannabinoidpharmacology and biochemistry Moreover the interestshown by the pharmaceutical industry in the development ofnew cannabinoid antagonists prove if necessary the realtherapeutic potential of this class of compounds Thetreatment of eating and movement disorders memorydeficits psychosis and dependencies from various addictivedrugs are some of the most cited applications for the CB1cannabinoid receptor antagonists

From these lines it appears that in addition to theknown CB1 cannabinoid receptor it is now likely that twoother cannabinoid receptors do exist in the brain The firstone should be an anandamide brain receptor for whichSR141716A has no affinity while the second one should be

1390 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Much less wide is the knowledge on the CB2cannabinoid receptor antagonists However potentialapplications could be disorders involving the immunesystem such as inflammation or allergies

[19] Devane WA Hanus L Breuer A Pertwee RG StevensonLA Griffin G Gibson D Mandelbaum A Etinger AMechoulam R Science 1992 258 1946

[20] Mechoulam R Ben-Shabat S Hanus L Ligumsky MKaminski NE Schatz AR Gopher A Almong S MartinBR Compton DR Biochem Pharmacol 1995 50 83

Finally the growing literature on the non CB1 non CB2cannabinoid receptors should allow the development of newdrugs First examples are given by the cannabidiol andabnormal-cannabidiol derivatives described by Kunos et alfor which potential therapeutic applications on the vascularsystem are already patented

[21] Sugiura T Kondo S Sukagawa A Nakane S Shinoda AItoh K Yamashita A Waku K Biochem Biophys Res Co1995 215 89

[22] Hanus L Abu-Lafi S Fride E Breuer A Vogel Z ShalevDE Kustanovich I Mechoulam R Proc Natl Acad SciUSA 2001 98 3662

[23] Oka S Tsuchie A Tokumura A Muramatsu M Suhara YTakayama H Waku K Sugiura T J Neurochem 2003 851374It clearly appears from this paper that the antagonists of

the cannabinoid receptors are still an active research fieldfrom which should emerge new promising therapeutic toolsas well as innovative drugs in the near future

[24] Goutopoulos A and Makriyannis A Pharmacol Therapeut2002 95 103

[25] Beal JE Olson R Lefkowitz L Laubenstein L Bellman PYangco B Morales JO Murphy R Powderly W PlasseTF Mosdell KW Shepard KV J Pain Symptom Manage1997 14 7ACKNOWLEDGEMENTS [26] Notcutt W Price M Miller R Newport S Phillips CSimmons S Sansom C Anaesthesia 2004 59 440

GGM is very indebted to the ldquoFonds pour la formationagrave la Recherche dans lrsquoIndustrie et lrsquoAgriculture (FRIA)rdquo Belgium for the award of a research fellowship Theexperimental works of the authors were supported by grantsfrom the FNRS The Belgian Charcot Foundation and theUniversiteacute catholique de Louvain

[27] Ledent C Valverde O Cossu G Petitet F Aubert JFBeslot F Bohme GA Imperato A Pedrazzini T RoquesBP Vassart G Fratta W Parmentier M Science 1999 283401

[28] Zimmer A Zimmer AM Hohmann AG Herkenham MBonner TI Proc Natl Acad Sci U S A 1999 96 5780

[29] Wiley JL and Martin BR Chem Phys Lipids 2002 121 57[30] Adam J and Cowley P Expert Opin Ther Patents 2002 12

1475

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[32] Barth F and Rinaldi-Carmona M Curr Med Chem 1999 6745

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[33] Little PJ Compton DR Johnson MR Melvin LS MartinBR J Pharmacol Exp Ther 1988 247 1046

[2] Devane WA Dysarz FA Johnson MR Melvin LSHowlett AC Mol Pharmacol 1988 34 605

[34] Pertwee RG Stevenson LA Elrick DB Mechoulam RCorbett AD Brit J Pharmacol 1992 105 980

[3] Matsuda LA Lolait SJ Brownstein MJ Young ACBonner TI Nature 1990 346 561

[35] Harrison C Traynor JR Life Sci 2003 74 489-508[4] Gerard CM Molerau C Vassart G Parmentier M BiochemJ 1991 279 129 [36] Pertwee RG Fernando SR Griffin G Ryan W Razdan

RJ Compton DR Martin BR Eur J Pharmacol 1996 315195

[5] Glass M Dragunow M Faull RLM Neuroscience 1997 2299

[37] Ross RA Brockie HC Fernando SR Saha B Razdan RKPertwee RG Brit J Pharmacol 1998 125 1345

[6] Shire D Carillon C Kaghad M Calandra B Rinaldi-Carmona M LeFur G Caput D Ferrara P J Biol Chem1995 270 3726 [38] Griffin G Wray EJ Rorrer WK Crocker PJ Ryan WJ

Saha B Razdan RK Martin BR Abood ME Brit JPharmacol 1999 126 1575

[7] Ryberg E Khang Vu H Groblewski T Larsson N ElebringT Hjorth S Sjogren S Greasley PJ 2004 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 119

[39] Ross RA Gibson TM Stevenson LA Saha B Crocker PRazdan RK Pertwee RG Brit J Pharmacol 1999 128 735

[40] Martin BR Winckler JR Wiley JL Huffman JW CrockerPJ Saha B Razdan RK J Pharmacol Exp Ther 1999 2901065

[8] Rinaldi-Carmona M Calandra B Shire D Bouaboula MOustric D Barth F Casellas P Ferrara P LeFur G JPharmacol Exp Ther 1998 278 871

[41] Martin BR Stevenson LA Pertwee RG Breivogel CSWilliams A Mahadevan A Razdan RK 2002 Symposium onthe Cannabinoids Burlington Vermont International CannabinoidResearch Society page 2

[9] Munro S Thomas KL Abu-Shaar M Nature 1993 365 61[10] Howlett AC Qualy JM Khachatrian LL Mol Pharmacol

1986 29 307[11] Glass M Felder CC J Neurosci 1997 17 5327

[42] Thomas A Ross RA Saha B Mahadevan A Razdan RKPertwee RG Eur J Pharmacol 2004 487 213

[12] Calandra B Portier M Kerneacuteis A Delpech M Carillon C LeFur G Ferrara P Shire D Eur J Pharmacol 1999 374445 [43] Compton DR Gold LH Ward SJ Balster RL Martin BR

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Daum SJ Ackerman JH Gruett MD Kumar V EstepKG Olefirowicz EM Wetzel JR Alexander MD WeaverJD Haycock DA Luttinger DA Casiano FM ChippariSM Kuster JE Stevenson JI Ward SJ J Med Chem1995 38 3094

[14] Deadwyler S Hampson RE Mu J Whyte A Childers SRJ Pharmacol Exp Ther 1995 273 734

[15] Mackie K Lai Y Westenbroek R Mitchell R J Neurosci1995 15 6552

[16] Bouaboula M Poinot CC Bourrie B Canat X Calandra BRinaldi-Carmona M LeFur G Casellas P Biochem J 1995312 637 [45] Pertwee R Griffin G Fernando S Li X Hill A

Makriyannis A Life Sci 1995 56 1949[17] Bouaboula M Poinot CC Marchand J Canat X Bourrie BRinaldi-Carmona M Calandra B LeFur G Casellas P Eur JBiochem 1996 237 704

[46] Hosohata Y Quock RM Hosohata K Makriyannis AConsroe P Roeske WR Yamamura HI EurJPharmacol 1997 321 R1[18] delPulgar T Velasco G Guzman M Biochem J 2000 347

369 [47] Landsman RS Makriyannis A Deng H Consroe P RoeskeWR Yamamura HI Life Sci 1998 62 109

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[48] Ross RA Brockie HC Stevenson LA Murphy VLTempleton F Makriyannis A Pertwee RG Brit JPharmacol 1999 126 665

[81] Gatley SJ Lan R Volkow ND Pappas N King P WongCT Gifford AN Pyatt B Dewey SL Makriyannis A JNeurosci 1998 70 417

[49] Huffmann JW Curr Med Chem 1999 6 705 [82] Lan R Lu Q Fan P Gatley J Volkow ND Fernando SRPertwee R Makriyannis A AAPS Pharmsci 1999 1 article 4[50] Rinaldi-Carmona M Barth F Heaulme M Shire D Calandra

B Congy C Martinez S Maruani J Neliat G Caput DFerrara P Soubrieacute P Breliegravere JC Le Fur G FEBS Lett1994 350 240

[83] Mathews WB Ravert HT Musachio JL Franck RARinaldi-Carmona M Barth F Dannals RF J Labelled CompdRad 1999 42 589

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GA Psychopharmacology 2003 167 103[68] Reggio P Umijiego U Hurst D Seltzman H Hyatt SRoche M McAllister S Fleisher D 2004 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 2

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[102] Ravinet Trillou C Delgorge C Menet M Arnone M SoubrieP Int J Obesity 2004 28 640[69] Song ZH and Bonner TI Mol Pharmacol 1996 49 891

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[103] Cota D Marsicano G Tschoumlp M Gruumlbler Y FlachskammC Schubert M Auer D Yassouridis A Thoumlne-Reineke COrtmann S Tomassoni F Cervino C Nisoli E LinthorstACE Pasquali R Lutz B Stalla GK Pagotto U J ClinInvest 2003 112 423

[71] McAllister SD Tao Q Barnett-Norris J Buehner K HurstDP Guarnieri F Reggio PH Nowell Harmon KW CabralGA Abood ME Biochem Pharmacol 2002 63 2121

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[104] Cani PD Lasa Montoya M Neyrinck AM Delzenne NMLambert DM Br J Nutr 2004 92 757

[73] Murphy JW and Kendall DA Biochem Pharmacol 2003 651623

[105] Colombo G Agabio R Fa M Guano L Lobina C LocheA Reali R Gessa GL Alcohol Alcohol 1998 33 126

[74] McAllister SD Rizvi G Anavi-Goffer S Hurst DP Barnett-Norris J Lynch DL Reggio PH Abood ME J Med Chem2003 46 5139

[106] Serra S Carai MA Brunetti G Gomez R Melis S VaccaG Colombo G Gessa GL Eur J Pharmacol 2001 430 369

[107] Serra S Brunetti G Pani M Vacca G Carai MAM GessaGL Colombo G Eur J Pharmacol 2002 443 95[75] McAllister SD Hurst DP Barnett-Norris J Lynch D

Reggio PH Abood ME J Biol Chem 2004 279 48024 [108] Colombo G Vacca G Serra S Carai MAM Gessa GLEur J Pharmacol 2004 498 119[76] Salo OM Lahtela-Kakkonen M Gynther J Jarvinen T

Poso A J Med Chem 2004 47 3048 [109] Gallate JE and McGregor IS Psychopharmacology 1999 142302[77] Rinaldi-Carmona M Pialot F Congy C Redon E Barth F

Bachy A Breliere JC Soubrie P Le Fur G Life Sci 199658 1239

[110] Cohen C Perrault G Voltz C Steinberg R Soubrie P BehavPharmacol 2002 13 451

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[111] Le Fur G Cohen C Steinberg R Soubrie P 2003 Symposiumon the Cannabinoids Burlington Vermont InternationalCannabinoid Research Society page 55[79] Gatley SJ Lan R Volkow ND Pappas N King P Wong

CT Gifford AN Pyatt B Dewey SL Makriyannis A JNeurochem 1998 70 417

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Behav 1997 56 803 [153] Dow RL Hammond M World Patent Application 2004WO04052864[116] Leweke FM Giuffrida A Wurster U Emrich HM Piomelli

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F Di Marzo V Lipids Health Dis 2003 2 5 [155] Katoch-Rouse R Pavlova OA Caulder T Hoffman AFMukhin AG Horti AG J Med Chem 2003 46 642[118] Dean B Sundram S Bradbury R Scarr E Copolov D

Neuroscience 2001 103 9 [156] Katoch-Rouse R and Horti AG J Labelelled Compd Rad2003 46 93[119] Zavitsanou K Garrick T Huang XF Prog Neuro-

Psychopharmacol Biol Psychiatry 2004 28 355 [157] Kumar JSD Prabhakaran J Arango V Parsey RV Underwood MD Simpson NR Kassir SA Majo VJ VanHeertum RL Mann JJ Bioorg Med Chem Lett 2004 142393

[120] Meltzer HY Arvanitis L Bauer D Rein W Am JPsychiatry 2004 161 975

[121] Castellano C Rossi-Arnaud C Cestari V Constanzi M CurrDrug Targets 2003 2 61 [158] Stoit AR Lange JHM den Hartog AP Ronken E Tipker

K van Stuivenberg HH Dijksman JAR Wals HC KruseCG Chem Pharm Bull 2002 50 1109

[122] Terranova JP Storme JJ Lafon N Perio A Rinaldi-Carmona M Le Fur G Soubrie P Psychopharmacology 1996126 165 [159] Barth F Congy C Martinez S Rinaldi M World Patent

Application 2001 WO0132663[123] Mallet PE Beninger RJ Psychopharmacology 1998 140 11[124] Nakamura-Palacios EM Winsaure PJ Moerschbaecher JM

Behav Pharmacol 2000 11 377[160] Ruiu S Pinna GA Marchese G Mussinu J-M Saba P

Tambaro S Casti P Vargiu R Pani L J Pharmacol ExpTher 2003 306 363[125] Brodkin J Moerschbaecher JM J Pharmacol Exp Ther

1997 282 1526 [161] Francisco MEY Burgess JP George C Bailey GSGilliam AF Seltzman HH Thomas BF Magn Reson Chem2003 41 265

[126] Lichtman AH Eur J Pharmacol 2000 404 175[127] Wolff MC Leander JD Eur J Pharmacol 2003 477 213[128] Arnone M World Patent Application 2003 WO03082256 [162] Cullinan GJ Fahey KJ Koppel GA United States Patent

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CB Walsh TE Shah SK World Patent Application 2004WO04012671

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[246] Kunos G World Patent Application 2003 WO03015700 [257] Haller J Varga B Ledent C Freund TF Behav Pharmacol2004 15 299[247] Franklin A and Stella N Eur J Pharmacol 2003 474 195

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[266] Calignano A La Rana G Piomelli D Eur J Pharmacol 2001419 191

Page 6: Current Knowledge on the Antagonists and Inverse Agonists of

1366 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 4 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives Five SR141716A (14) DerivativesIllustrating the Importance of the C-3 Carboxamide Oxygen in the SR141716A Inverse Agonism

The structure affinity ([3H]-SR141716A hCB1-HEK293 cells) and function (Ca++ currents) are given for each compound

NN

R

Cl

Cl

Cl

Cpd ndeg R= Affinity Function References

SR141716A 14

O

NHN

Kd=23nM Inverse agonist [67]

VCHSR1 15 Ki=313nM Neutral antagonist [67]

CHASR1 16

O

NH

Ki=17nM Inverse agonist [68]

CHMSR1 17

O

N

Ki=29nM Inverse agonist [68]

VPSR1 18

N

Ki=261nM Neutral antagonist [68]

PIMSR 19

NN

Ki=67nM Neutral antagonist [68]

an increased constitutive activity of the receptor asdemonstated by [35S]-GTPγ S binding [75]

1996 It binds with high affinity to rat brain synaptosomes(Kd=061nM) It is competitively displaced by knowncannabinoids like CP-55940 or WIN-55212-2 Usingautoradiography its rat brain distribution is similar to [3H]-CP-55940 one [77] It is now a commercially available andwidely used radioligand for competition studies

A new 3D model of the CB1 cannabinoid receptor basedon the X-ray structure of the bovine rhodopsin was recentlydeveloped by Salo and co-workers [76] It would be veryinteresting to see whether or not the results obtained withprevious models are confirmed using this new model Forinstance the lysine K328 appeared as a key residue in thismodel too

Interestingly other SR141716A radiolabeled derivativeswere synthesised as radioimaging tools among them [123I]-AM251 (20) [7879] [123I]-AM281 (21) [80-82] and [18F]-SR144385 (22) [8384] for Positon Emission Tomographyor Single Photon Emission Computed Tomography Indeedcannabinoid antagonists are a much more useful tool for

The tritiated analogue of SR141716A the [3H]-SR141716A was described by Rinaldi-Carmona et al in

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1367

human radioimaging applications than agonists as they aredevoid of cannabinoid-like effects For instance Berding etal very recently reported the use of [123I]-AM281 for SinglePhoton Emission Computed Tomography imaging of theCB1 cannabinoid receptor in six human patients [85] Inorder to further optimize the brain uptake of suchSR141716A derivatives Sanofi researchers synthesised twomethoxylated SR141716A analogues using 11C as PETtracer [86] These two compounds SR149080 (24) andSR149568 (25) possess high affinity with Ki values of 15and 38 nM respectively and selectivity for the CB1 receptoras well as an improved penetration in the brain evaluated bymeasuring the radioactivity present in various brain regionsafter tail vein injection in CD-1 mice Some of therepresentative radiolabelled SR141716A derivatives aresummarised in Table 5

Colombo et al using Wistar rats [92] Simiand et alobserved that SR141716A selectively reduces sweet foodintake in primates [93] However several authors [94-96]showed that high palatability of food is not necessary toobserve a SR141716A-induced anorectic effect at doses thatdo not cause major behavioural alterations or reduced waterintake Interestingly Gomez et al demonstrated theimplication of the peripheral CB1 receptors on themodulation of feeding and therefore the possible role ofthese receptors on the SR141716A influence on food intake[97] In addition to its effects on food consumptionSR141716A seems to be able to lower the hyperglycemiathe hyperinsulinemia as well as the insulin resistance indiet-induced obese (DIO) mice In the same DIO mice adecrease in adiposity was also observed after treatment [98]Bensaid et al using another model of obesity the obese fafarats observed that SR141716A increases mRNA expressionof Acrp30 or adiponectin a plasmatic protein exclusivelysecreted by adipose tissue through a CB1-mediatedpathway Inductions of free fatty acid oxidation body weightreduction and hyperinsulinemia decrease are some of theknown physiological actions of this protein Thus along theauthors an enhanced expression of Acrp30 followingSR141716A administration could be responsible for themetabolic effects of the compound leading to body weightreduction [99] Vickers et al using the same model (ie fafarats) showed that SR141716A significantly decreases foodconsumption and weight gain in both the obese fafa rats andthe lean Zucker rats [100] This decrease was greater in thefafa group and reversible upon SR141716A withdrawal

From a more therapeutic point of view SR141716A(rimonabant Acompliareg) is one of the promising agents totreat obesity [87] It is currently in Phase III clinical trials forthe treatment and prevention of obesity Final results areexpected for this year and FDA application filling for 2005[88] Preliminary data based on a one year treatment withrimonabant (RIO-Lipids study 1036 patients) showed a 5weight loss in 72 of the treated patients [8990] Severalreports were published before clinical trials aiming todemonstrate the anti-obesity properties of this compoundThe first report by Sanofi Recherche published in 1997described the selective inhibition of sucrose intake in ratsupon SR141716A treatment (03-3 mgkg) [91] Theanorectic and weight loss effects were firstly published by

Table 5 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structure and Affinity of SR141716A Radiolabeled Derivatives Developed as Potential Radio-Imaging Tools

NN

R3

O

NH

Cl

R4

R1

R2

Cpd ndeg R1 R2 R3 R4 Affinity CB1 References

[123I]-AM251 20 123I piperidinyl CH3 Cl Ki=25 nMa [78]

[123I]-AM281 21 123I morpholinyl CH3 Cl Ki=14 nMb [80]

[18 F]-SR144385 22 Cl piperidinyl CH2-18 F Cl IC50 =29 nMa [83]

[18 F]-SR147963 23 Cl morpholinyl CH2-18 F Cl IC50 =120 nM [84]

[11 C]-SR149080 24 O-11 CH3 piperidinyl CH3 Cl IC50 =15 nMa [86]

[11 C]-SR149568 25 O-11 CH3 morpholinyl CH3 Cl IC50 =38 nMa [86]

[18 F]-NIDA-42033 26 O-CH3 piperidinyl 18 F H Ki=18 nMc [155156]

27 O-11 CH3 piperidinyl CH3 H Ki=8nMc [155157]

arat brain homogenates [3H]-CP-55940bmouse cerebellum homogenates [3H]-SR141716crat brain homogenates [3H]-AM251

1368 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Another recent study by Higgs and co-workers furtherhighlighted the role of endocannabinoids in food taste-perception and the reduction of orosensory reward of sucrosein SR141716A treated rats [101] As it was expectedSR141716A induced effects on food consumption are absentsin CB1

-- mice [102] Very recently Cota et al obtainedresults showing that endocannabinoid system modulateshomeostasis via a dual mechanism it regulates at a centrallevel food intake while it blocks at the periphery lipogeneticprocesses [103] Moreover Cani et al showed that ghrelin(an orexigenic peptide) plasma levels are significantlyreduced 45 minutes after SR141716A administration(5mgkg ip) to fasted rats in accordance with the rapiddecrease of food intake measured by the authors It istherefore likely that SR141716 effects on body weight aredue to a conjunction of central and peripheral actions [104]

cerebrospinal fluid from patients with schizophrenia [116]Later De Marchi et al measured higher amounts ofanandamide in blood of schizophrenic patients compared tocontrols [117] On the other hand Dean et al obtainedelevated [3H]-CP-55940 binding in the dorsolateralprefrontal cortex of patients suffering from schizophrenia ascompared to controls [118] Zavitsanou using [3H]-SR141716A found elevated binding in the anterior cingulatecortex of subjects with schizophrenia [119] RecentlyMeltzer and collaborators published the results of a trialconducted to evaluate the potential of four new compoundsin treating schizophrenia and schizoaffective disorders [120]SR141716A (20 mgday) was one of the compoundsevaluated during a six weeks study The authors found noeffect of the CB1 cannabinoid receptor antagonist inimproving patientrsquos schizophrenia (72 subjects)

In addition to the anti-obesity potential the ability ofSR141716A to reduce alcohol and tobacco consumption arecurrently investigated in phase III clinical trials The effect ofSR141716A on alcohol consumption in rats was reported bythe GianLuigi Gessa team [105-108] and by Gallate et al[109] The inhibition of alcohol and nicotine induceddopamine release by SR141716A (1-3 mgkg rats) wasreported by Cohen and co-workers using a brainmicrodialysis device [110] Concerning the smokingcessation preliminary results of a clinical trial (360 subjects40 mg SR141716A) showed an increased abstinence ofsmoking [111] More recent results from the STRATUS-USstudy (787 patients) were reported 362 of patientsreceiving SR141716A (20 mgday) quit smoking against206 in the placebo group [90] Recently Le Fol andGoldberg reviewed the development of cannabinoid CB1antagonists as a new class of therapeutic agents for drugsaddictions [112]

It is known since the early nineties that theadministration of cannabinoid agonists impairs memory inrodents (for a review see Castellano et al [121]) Thusadministration of a cannabinoid antagonist was expected tosomehow improve memory [122123] However dependingon the authors SR141716A when administered alone wasreported to impair (5-10 mgkg im) [124] to have no effect(1-32 mgkg ip) [125] or to improve memory (3 mgkgip) [126] In a more recent paper Wolff and Leandershowed that SR141716A (1mgkg ip) improves memoryin rats by apparently enhancing the consolidation processesof memory [127] Further they found that at higher doses (3mgkg) this effect was lost The discrepancy in the resultsreported in the literature could be ascribed either to thedifferences in the tests used or in the doses administeredFurther experiments are needed to assess whether or not acannabinoid antagonist could be helpful in memory diseases

To conclude several patents were taken by Sanoficoncerning the therapeutic applications of their leadcompound among them being anti-obesity smokingcessation neuroinflammatory diseases and anti-diarrhoeaOne of the last patents to our knowledge concerns thetreatment of sexual dysfunctions with a cannabinoidantagonist such as SR141716A [128] However da Silva etal showed that even if this compound (2 mgkg ip)enhances the effects of apomorphine (20-80 microgkg) it has noeffect alone on penile erection [129] Meanwhile Melis et al

Since the late sixties concerns exist on the associationbetween cannabis use and schizophrenia Recent studiesshowed an association between cannabis use and an increasedrisk of developing schizophrenia [113 114] Moreover apossible role of the endocannabinoid system in schizophreniahas been suggested since its pharmacological characterisation[115] Several experimental data obtained on human subjectstend to confirm such hypothesis On the one hand Lewekeet al found elevated levels of endocannabinoids in

NN

O

NH

N

Br Cl

Cl

NN

O

NH

N

Br Cl

Cl

OH

28 29

Fig (3) CB1 cannabinoid receptor antagonists 3 Diarylpyrazole derivatives The chemical structures of 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-piperidine-carboxamide (SR147778 28 ) and 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-(4-hydroxypiperidine)-carboxamide (29 ) described by Rinaldi-Carmona et al

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1369

reported an increased rate of penile erection after directinjection of SR141716A in the paraventricular nucleus of thehypothalamus [130] This effect was dose dependent and asignificant effect was obtained with a dose of 1microgkg

AM251 in mice at a same dose range (10-30 mgkg ip) thanthe anorectic effects [143] These effects are reversed byadministration of CP-55940 and are absent in CB1

-- miceproving the implication of the CB1 receptor in theantidepressant-like effect of AM251 In 2004 Liao et alreported that AM251 was able to displace the binding of[3H]-batrachotoxin A from its binding site on sodiumchannels in mice brain synaptic preparations [144] Theyobtained an IC50 value of 112 microM and a competitivemechanism of action The authors suggested that AM251 iscapable of reducing neuronal excitability through blockade ofvoltage-sensitive sodium channels in brain

From the clinical trials involving the SR141716A themost frequently reported side-effects are nausea dizziness anddiarrhoea Depression and anxiety were not higher than inthe placebo groups Regarding one of the known side effectsof SR141716A administration the enhancement of intestinalmotility Carai et al reported recently that chronicadministration of the inverse agonists to mice induced atolerance to this prokinetic effect [131] This is not the firstreport of tolerance onset after administration of SR141716A[132] however few is known on the mechanismsresponsible for that phenomenon

Wiley and collaborators reported in 2001 a studydescribing new structure-activity relationships using thepyrazole nucleus as a central scaffold [145] Starting fromSR141716A structure they alternatively substituted one ofthe four substituents while retaining the others bysubstituents known to impart agonist activity in classicalcannabinoids One of the authors expectations was todetermine which positions are responsible respectively forthe antagonism and for the affinity of SR141716A Theaffinity of thirty compounds was assessed previous to in-vivoevaluation of their function in mice using the spontaneousactivity the tail-flick and the rectal temperature assays Theauthors showed that phenyl group in position 5 is critical foraffinity as compound O-1559 (30) lacking this phenyl has adecreased affinity (Table 6) This was already shown by Lanand co-workers [136] along with the need of a substituent inpara position on the phenyl Thomas et al previouslyshowed that this substituent could be a bromine or an iodineatom [146] However an alkyl chain is also tolerated as it isthe case in compounds O-1302 (31) O-1691 (32) or O-1704(33) All these compounds antagonise the anti-nociceptiveand hypothermic effects of ∆9-THC The authors suggestedthat the 5-substituent of pyrazoles is involved in receptorrecognition and antagonism The modulations of thesubstitution pattern of the phenyl in position 1 demonstratethat the 24-di-chloro substitution is the preferred one for theaffinity as well as for the activity Several compoundssupport this assertion Thomas et al showed that additionalhalogens result in a decreased affinity as in compounds 6-I-SR141716A (34) or 4rsquo6-di-I-SR141716A (35) (Ki values of166 and 126 nM respectively) [146] Suppression of the twochlorines (O-1300 39) or replacement of these two chlorinesby an alkyl chain like in O-1254 (40) and O-1255 (41) ledto less active compounds with Ki values ranging from 150to 430 nM However O-1254 and O-1255 behaved asantagonists in a [35S]-GTPγ S assay [61] Lan et al [136]and Wiley et al [145] also modified the substitution inposition 3 Replacement of the amido piperidinyl substituentby alkyl amides as in O-1269 (42) or O-1270 (43) etherslike in O-848 (44) and O-853 (45) ketones (O-1272 46)alcohols (O-1876 47) or alkanes (O-1877 48) resultedmostly in decreased affinity (Table 6) but also with a changeof functionality in some compounds as revealed by in-vivoassays For instance replacement of the piperidinyl by apentyl (O-1269) or by an heptyl chain (O-1270) gave agonistcompounds having Ki values of 32 and 48 nM respectivelyThus the authors suggested that the 3-substituent region isinvolved in receptor recognition and agonist activity Wileyet al on the basis of their results concluded that while the3-position seems to be involved in agonism the 1- 4- and

One close analogue of SR141716A 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-piperidinecarbox-amide (SR147778 28) was described very recently byRinaldi-Carmona et al [133 134] (Fig 3) It possesses highaffinity and selectivity for the hCB1 cannabinoid receptorwith Ki values for the hCB1 and hCB2 receptors of 35 and400 nM respectively ([3H]-CP-55940 hCB1amp2-CHO cells)Further SR147778 antagonised CP-55940 effects on mousevas deferens contractions (pA2=81) and on forskolin-stimulated adenylyl cyclase activity in U373MG cells(pA2=82) but had no effects alone In-vivo SR147778 afteroral administration reversed WIN-55212-2 inducedhypothermia and analgesia As SR141716A SR147778dose-dependently reduced ethanol and sucrose solutionintake with significant effects starting at 03 mgkg (sc) and3 mgkg (po) respectively This compound is currentlyinvestigated in Phase I clinical trials for the treatment ofobesity as well as nicotine and alcohol addictions

Another derivative the 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-(4-hydroxypiperidine)-carboxamide (29) was described in a very recent patent fromSanofi [135] (Fig 3) This antagonist possesses an IC50value for the hCB1 cannabinoid receptor of 32 nM ([3H]-CP-55940 hCB1-CHO cells)

The first structure-affinity relationships for theSR141716A derivatives were reviewed by Barth andRinaldi-Carmona in 1999 [32] Since then many papersdescribing new derivatives were published In 1999 Lan etal described around thirty 15-diarylpyrazoles derivatives[136] Among them was AM251 previously reported as aradioimaging ligand for the CB1 receptor [78] and thatappeared to be more potent (Ki = 749 nM) and selective(selectivity ratio of 306) than SR141716A (Ki = 115 nMselectivity ratio of 143) They reported a Kd value of 05 nMin the mouse vas deferens model using WIN-55212 asagonist Interestingly New et al described for the samecompound an inverse agonist effect on the hCB2 receptorusing a forskolin-induced cAMP accumulation assay (EC50= 650 nM) [137] Recently the anti-obesity effects ofAM251 were reported and are not surprisingly similar tothose of SR141716A [138 139 140] Chen et aldemonstrated that there are synergistic effects on food intakesuppression between AM251 and nalmefene an opioidantagonist [141] This synergistic effect was also presentbetween SR141716A and naloxone as shown by Kirkham[142] Shearman et al found antidepressant-like effects of

1370 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

5-positions appear to be involved in antagonism The O-derivatives reported were also described in a patent alongwith their synthetic pathways which were not given in thepaper [147]

Another investigation of the SR141716Aaminopiperidine region was conducted by Francisco et al[148] They synthesised 21 analogues possessing either analkyl amide or an alkyl hydrazide substituent of various

Table 6 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structure of some of the characterised 1- 3- 4- and 5-pyrazole derivatives Binding affinities (Ki values nM) were obtained on rat brain homogenatesusing [3H]-CP-55940 [136 145 146 148 150] or [125I]-AM-251 [155]

NN

R1

R5

R3R4

Cpd ndeg R1 R3 R4 R5 [136] [145] [146] [148] [150] [155]

SR141716 14 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Cl-Ph 115 62 62 62 13 18

O-1559 30 24-di-Cl-Ph CO-NH-piperidinyl Me 1-Methylpentyl 233

O-1302 31 24-di-Cl-Ph CO-NH-piperidinyl Me 4-((CH2)5)-Ph 21 1

O-1691 32 24-di-Cl-Ph CO-NH-piperidinyl Br 4-((CH2)5)-Ph 15

O-1704 33 24-di-Cl-Ph CO-NH-piperidinyl I 4-((CH2)5)-Ph 22

6-I-SR141716 34 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-Cl-Ph 166

4rsquo6-di-I-SR141716 35 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-I-Ph 126

4rsquo-I-SR141716 36 24-di-Cl-Ph CO-NH-piperidinyl Me 4-I-Ph 75 25 6

4rsquo-Br-SR141716 37 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Br-Ph 168 3

Cpd 25 in ref [136] 38 24-di-Cl-Ph CO-NH-morpholinyl Me 4-Br-Ph 54

O-1300 39 Ph CO-NH-piperidinyl Me 4-Cl-Ph 150

O-1254 40 4-(CH2)4-Ph CO-NH-piperidinyl Me 4-Cl-Ph 226 256

O-1255 41 4-(CH2)5-Ph CO-NH-piperidinyl Me 4-Cl-Ph 433

O-1269 42 24-di-Cl-Ph CO-NH-pentyl Me 4-Cl-Ph 32 114 3

O-1270 43 24-di-Cl-Ph CO-NH-heptyl Me 4-Cl-Ph 48 462 3

O-848 44 24-di-Cl-Ph CH2-O-(CH2)2-piperidinyl

Me 4-Cl-Ph 2450 232

O-853 45 24-di-Cl-Ph CH2-O-CH2-cyclohexyl

Me 4-Cl-Ph 388 100

O-1272 46 24-di-Cl-Ph CO-heptyl Me 4-Cl-Ph 221

O-1876 47 24-di-Cl-Ph 1rsquo-OH-heptyl Me 4-Cl-Ph 657

O-1877 48 24-di-Cl-Ph Heptyl Me 4-Cl-Ph 422

MF9725-64-17 49 24-di-Cl-Ph CO-NH-butyl Me 4-Cl-Ph 134

MF9725-179-32 50 24-di-Cl-Ph CO-NH-NH-butyl Me 4-Cl-Ph 51

MF9725-66-11 51 24-di-Cl-Ph CO-NH-cyclohexyl Me 4-Cl-Ph 25

MF9725-95-31 52 24-di-Cl-Ph CO-NH-(4-hydroxybutyl)

Me 4-Cl-Ph 154

Cpd 15 [150] 53 n-pentyl CO-NH-piperidinyl Me Ph 23

Cpd 16 [150] 54 n-pentyl CO-NH-piperidinyl Me 4-Br-Ph 63

Cpd 17 [150] 55 n-hexyl CO-NH-piperidinyl Me Ph 21

Cpd 18 [150] 56 n-heptyl CO-NH-piperidinyl Me Ph 47

NIDA-41109 57 24-di-Cl-Ph CO-NH-piperidinyl Br 4-Cl-Ph 14

NIDA-41119 58 24-di-Cl-Ph CO-NH-piperidinyl H 4-Cl-Ph 9

NIDA-41057 59 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OH-Ph 104

NIDA-41020 60 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 41

NIDA-41087 61 2-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 8

NIDA-42055 62 24-di-Cl-Ph CO-NH-piperidinyl Br 4-OMe-Ph 62

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1371

lengths in position 3 of the pyrazole moiety They observedthat until five carbons the affinity increases along with thecarbon chain length This is observed with the alkylhydrazide the alkyl amide and the hydroxyalkyl amideseries Moreover the hydrazide analogues exhibit a loweraffinity for the rCB1 than the amide analogues as illustratedby compounds MF9725-64-17 (49) and MF9725-179-32(50) having Ki values of 134 and 51 nM respectively(Table 6) From their structure-activity relationships (SAR)studies the authors concluded that the pharmacophoricrequirement of the amidopiridine region is a chain not longerthan 3 Aring and that a substituent having a positive chargedensity would probably result in increased affinity andpotency The same team described in a patent several othercompounds [149] Binding affinities (Ki) against [3H]-CP-55940 [3H]-SR141716A or [3H]-WIN55212-2 on wholerat brain or on hCB1-transfected cells were given Moreoveractivity data were obtained using the [35S]-GTPγ S assaydemonstrating that these alkyl amide and hydrazide analogsact as antagonists or inverse agonists Despite the greatnumber of derivatives claimed none of them posses asignificantly greater affinity for the CB1 receptor than theSR141716A Nevertheless they showed a slightenhancement of the selectivity for the CB1 receptor over theCB2 cannabinoid receptor

affinity of these derivatives increases with the length of thealkyl chain with an optimal length of 5-6 carbons The bestcompound of this series N-(piperidin-1-yl)-5-phenyl-1-hexyl-4-methyl-1H-pyrazole-3-carboxamide (55) possesses aKi value of 21 nM determined using [3H]-CP-55940 on ratbrain membranes which is higher than the 13 nMdetermined for SR141716A in the same conditions (Table6) In the same paper the authors starting from thehypothesis that antagonism by SR141716A is caused bybinding to the same region of the receptor as do the agonists(CP-55940 and WIN-55-2122) but preventing the agonistpromoted conformational change conducted extensiveconformational analysis as well as superimposition modelsand 3D-QSAR to propose a molecular mechanismsupporting the action of SR141716A Along with theauthors the C-5 aryl substituent of SR141716A occupyinga unique region could contribute in conferring the antagonistproperties Moreover the C-3 substituent could beresponsible for the antagonist or inverse agonist propertiesdepending on the interaction with the receptor

About this topic a very interesting review dealing withthe cannabinoid receptors pharmacophores as well as withthe activationinactivation of these receptors was recentlypublished by Reggio [151]

More recently Dyck et al described seven otherderivatives varying at the amide position [152] The onlycompound possessing a higher affinity for the CB1cannabinoid receptor than SR141716A was the 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta-

The affinity for the rat CB1 receptor of seven derivativespossessing an alkyl chain in position 1 instead of the 24-dichlorophenyl substituent was reported by Shim et al in apaper describing a molecular mechanism for the antagonistand inverse agonist activity of SR141716A [150] The

NN

O

NHN

Cl Cl

Cl

NN

Cl

Cl

NON

NN

O

NH

N

Cl

ClCl

NN

O

NH

MeO Cl

CP-272871 66

63 64

65

Fig (4) CB1 cannabinoid receptor antagonists 3 Diarylpyrazole derivatives Structures of 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63 ) 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methyl-pyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]-morpholine (64 ) N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65 ) and N-phenyl-1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-pyrazole-3-carboxamide (CP-272-871 66 )

1372 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

[c]pyrrol2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63)(Fig 4) The Ki values were 5 and 12 nM respectively([3H]-CP-55940 hCB1-HEK cells)

CP-55940 rat brain) For instance N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65) (Fig 4) showed a Ki value of15 nM However no data were provided concerning thefunctionality of such compounds

Over 200 other pyrazole derivatives acting at the CB1cannabinoid receptor were claimed in a patent from Pfizer[153] The affinity for the hCB1 receptor of 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methylpyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]ndashmorpholine (64) (Fig 4)was 79 nM ([3H]-SR141716A hCB1-HEK cells) Theothers compounds affinity was between 01 and 100 nM

In an effort to develop new SR141716A analoguespossessing lower lipophilicity to be used as PET tracersKatoch-Rouse and colleagues further explored thesubstitution pattern of the two phenyls [155] Bindingassays showed that a decreased lipophilicity lead to adecreased affinity for the CB1 receptor as determined bydisplacement of [3H]-AM251 For instance compoundNIDA-41057 (59) has a lipophilicity of 42 ( expressed asElogDoct) and a Ki value of 104 nM whereas SR141716Ahas a ElogDoct value of 54 and a Ki value of 18 nM In

Seventy five pyrazole derivatives were described in a veryrecent patent from Makriyannis et al [154] The affinity ofthe compounds for the cannabinoid receptors is given TheKi values for the best compounds are lower than 6 nM ([3H]-

Table 7 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structures and affinities for the cannabinoid receptors of some representative tricyclic pyrazole derivatives Values were obtained using [3H]-CP-55940as radioligand and mouse [160 228] rat [159 227] or human [158] cannabinoid receptors Compounds 69-78 are CB1 cannabinoid receptor ligandsCompounds 79-86 are CB2 cannabinoid receptor ligands

NN

O

NHX

R3

R1

R2

n

n˚ X R1 R2 n R3 Ki CB1 (nM) Ki CB2 (nM) Selectivity References

69 -(CH2)3- Cl 24-di-Cl 0 piperidine 126 [158]

70 -(CH2)3- H 24-di-Cl 0 azepane 100 [158]

71 -(CH2)3- H 24-di-Cl 0 piperidine 398 [158]

72 -(CH2)3- Cl 24-di-Cl 0 azepane 125 [158]

73 -(CH2)3- NO2 24-di-Cl 0 azepane 63 [158]

74 -(CH2)2-O- H 24-di-Cl 0 piperidine 501 [158]

75 -CH2-S- Br 24-di-Cl 0 piperidine lt 500 gt10 [159]

76 -CH2-SO2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

77 -(CH2)2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

78 -(CH2)3- Cl 24-di-Cl 0 piperidine 000035 21 60000 [160]

79 -CH2- Cl 24-di-Cl 0 piperidine 2050 034 00002 [228]

80 -CH2- Br 24-di-Cl 0 piperidine 1570 027 00002 [228]

81 -CH2- CH3 24-di-Cl 0 piperidine 363 0037 00001 [228]

82 -CH2- Cl 4-Cl 0 piperidine 1787 09 00005 [228]

83 -CH2- H 24-di-Cl 0 piperidine 1152 0385 00003 [228]

84 -CH2- Cl 34-di-Cl 1 133-trimethylbicyclo[221]heptyl

lt500 gt10 [227]

85 -CH2- Br 24-di-Cl 1 bicyclo[321]oct-3-yl lt500 gt10 [227]

86 -CH2- Br 4-Me 1 77-dimethylbicyclo[410]hept-3-yl

lt500 gt10 [227]

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1373

another paper Katoch-Rouse described the synthesis of[18F]-NIDA-42033 (26) a ligand useful as PET radiotracerstarting from its bromo derivative [156] Recently Kumar etal [157] proposed the synthesis of a 11C derivative ofNIDA-41087 (61) a compound previously described byKatoch-Rouse (Table 5) Post-mortem binding of thiscompound to human prefrontal cortex was also investigatedInterestingly CP-272871 (66) described by Meschler as aCB1 inverse agonist (Ki=57 nM [3H]-CP-55940 rat brainhomogenates) possesses a lipophilicity similar to theNIDA-41087 one [62] However despite the presence of amethoxy function no radiolabelled derivative has beendescribed to date One explanation could be the compoundlow selectivity (2 fold) for the CB1 cannabinoid receptor

To further explore the pharmacology of this compound afunctional assay using the mouse vas deferens model wasperformed by Ruiu and colleagues enlightening thecompetitive antagonist properties of NESS (pA2= 1246against WIN-55212-2) which had no effect by itself up to1microM Moreover NESS 0327 was unable to affect basalbinding of the [35S]-GTPγ S demonstrating its lack ofnegative intrinsic activity In-vivo studies were conductedusing the hot plate and tail flick tests NESS dose-dependently abolished the antinociceptive effect of WIN-55212-2 but had no effect by itself However the authorssuggested based on the affinity and in-vivo activity of theircompound that NESS 0327 possesses a poor centralbioavailability

Several new attempts to increase the affinity of thediarylpyrazole derivatives were recently made by rigidifyingthe SR141716A structure (Table 7) Six fused ringanalogues of SR141716A obtained by fusion of the 5-(4-chlorophenyl) substituent with the central pyrazole to forman indazole ring were published by Bass et al in 2002 [61]The compound possessing the highest affinity O-1248 (67)has a Ki value of 475 nM as determined by displacement of[3H]-CP-55940 from rat brain membranes

Another example of ring-constrained biarylpyrazole wasdescribed by Herbert Seltzman team [161] Theyinvestigated a photocyclisation reaction starting from theSR141716A leading to a pyrazolo[15-f]phenanthridinestructure (Fig 5) The new compound (68) was tested on theCB1 receptor (whole rat brain) against [3H]-CP-55940[3H]-SR141716A and [3H]-WIN-55212-2 and exhibited Kivalues of 48 35 and 50 nM respectively The CB2 affinitywas assessed using [3H]-CP-55940 and was found to be 100times lower (Ki = 3340 nM) No effect on the [35S]-GTPγ Sassay (rat brain) was observed by the authors

Stoit and colleagues from Solvay Pharmaceuticalspublished a paper describing the synthesis andpharmacological characterisation of new benzocyclohepta-pyrazole derivatives as CB1 antagonists (69-74) [158] Theaffinity of these compounds was more or less one order ofmagnitude lower than the SR141716A one with pKi valuesranging from 64 to 72 compared to 76 for the SRcompound (Table 7) Compounds were shown to beantagonists with pA2 values from 70 to 89 as theyprevent CP-55940-induced cAMP accumulation Howeverthe authors investigating the bioavailability of theircompounds either po or after ip injection found negligibleblood plasma levels

NN

Cl

Cl

O

NH

N

NN

Cl

O

NHN

Cl

Cl

O-1248 67 68However just before the publication of Stoit results

Sanofi-Synthelabo took a patent describing tricyclicpyrazolecarboxylic acid amide derivatives having antagonistproperties on the CB1 cannabinoid receptor andsubmicromolar affinities (75-77) [159] Thirty examples weregiven along with their synthetic pathway but no individualpharmacological data were shown

Fig (5) CB1 cannabinoid receptor antagonists 3Diarylpyrazole derivatives Two ring-constrained diarypyrazolederivatives O-1248 (67 ) and a pyrazolo[15-f]phenanthridinederivative (68 )

It appears from these attempts to increase SR141716Aaffinity by rigidifying its structure that only a limitedincrease in the CB1 cannabinoid receptor affinity can beexpected with the apparent exception of NESS 0327Moreover rigidifying the structure often results in a morelipophilic compound in a family of compounds being yetlipophilic

More recently Ruiu and colleagues published a paperdescribing the synthesis and complete pharmacologicalcharacterisation of a very potent CB1 receptor ligandchristened NESS 0327 (78) [160] This compound has thesame structure as compound 69 published by Stoit et al(compound 20 in the reference [158]) Nevertheless Ruiu hasobtained an affinity for the CB1 cannabinoid receptor 5000times bigger than the SR141716A one with Ki values of035 pM and 18 nM respectively using mouse forebrainhomogenates and [3H]-CP-55940 as radioligandFurthermore the selectivity ratio obtained for this compoundby Ruiu is 60000 with a Ki value for the CB2 receptor of21 nM against the [3H]-CP-55940 The discrepancybetween the data obtained independently by Stoit et al andRuiu et al could hardly be explained by the differences inthe origin of the receptor used respectively hCB1-CHO andmouse forebrain

4 Phenyl Benzofuranone Derivatives

In a 1997 patent from Eli Lilly was described thesynthesis of aryl-benzothiophene and aryl-benzofuranederivatives (87-89 Fig 6) having CB1 receptor affinity andantagonist properties [162] From ten compounds having aKi value lower than 25 microM ([3H]-CP-55940 hCB1-CHOcells) one was selected and claimed to have a Ki of 170 nMCompound 88 was shown in the patent to antagonise theeffect of anandamide on cAMP accumulation with an IC50 of

1374 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

O

O

O

O

NC

S

O

O

O

O

O

O

O

NC

LY320135 8887 89

Fig (6) CB1 cannabinoid receptor antagonists 4 Phenyl benzofuranone derivatives Chemical structures of two aryl-benzofuranederivatives (87 -88 ) and one aryl-benzothiophene (89 ) derivative from Eli Lilly

500 nM and the effect of WIN-55212-2 on calcium channelsat 1microM Moreover the intra-peritoneal injection (20 mgkg)of this compound antagonised the in-vivo effect ofanandamide in the Open Field Assay a mouse behaviourmodel Later on Felder and colleagues gave a furtherdescription of this compound called LY320135 in a paperdescribing its effect on cAMP accumulation and on ioniccurrents [163] The selectivity ratio of LY320135 was 106with Ki values of 141 and 14900 nM for hCB1 and hCB2receptors ([3H]-CP-55940) respectively LY320135antagonised anandamide inhibition of forskolin-inducedcAMP accumulation with an IC50 value of 734 nMChristopoulos et al in 2001 reported for LY320135 apKb value of 527 in the inhibition of WIN-55212-2mediated response in the rat electrically-stimulated vasdeferens while the pKb value for SR141716A was 75[164]

Other patents were taken by Aventis in which somepotential applications for their compounds were describedFor instance an association between an azetidine derivativehaving CB1 antagonists properties and sibutramine aserotonin and norepinephrine reuptake inhibitor was claimed[170] as a treatment against obesity Oral administration ofcompound 90 (3 mgkg) and sibutramine (06 mgkg) to fafaZucker rats which are genetically obese rats has resulted ina decreased food intake compared to control lean Zuckerrats In another patent the association of an azetidinederivative (1-10 mgkg po ) and a D2D3 agonist likequinpirole (01 mgkg ip) was claimed to have a therapeuticpotential in the treatment of Parkinsonrsquos disease [171] Theeffect has been evidenced using an akinesia model in the ratEvidences supporting the use of CB1 antagonists as adjuvantin the treatment of Parkinsonrsquos disease begin to appear Itseems that such compounds would be helpful in thetreatment of Parkinsonism [172] and levodopa-induceddyskinesia [173] Brotchie published an interesting paperreviewing the CB1 cannabinoid receptor signaling inParkinsonrsquos disease [174] Nevertheless further clinicalstudies should be conducted to further confirm the usefulnessof CB1 antagonists in the treatment of Parkinsonrsquos disease

5 Azetidine Derivatives

The family of azetidine compounds illustrated bycompounds 90 and 91 (Fig 7) was developed at Aventis byDaniel Achard and colleagues and was described in a seriesof patents [165-168] They claimed the IC50 values of thesecompounds for the CB1 receptor to be less than or equal to100 nM These values were obtained following the proceduredescribed by Kuster et al [169] The antagonist propertywas shown using an in-vivo model the reversal ofhypothermia induced by CP-55940 in mice and the ED50obtained were lower than 50 mgkg

6 Aryl-Imidazolidine-24-Diones Derivatives

In 1999 the Didier Lambert team published a paperdescribing the affinity of 24 new 3-alkyl-55rsquo-diphenyl-imidazolidine-24-dione derivatives for the hCB1 receptor[175] The preliminary structure-activity relationships

N

SO

O

Cl

Cl

F

F

N

SO

O

Cl

NS

90 91

Fig (7) CB1 cannabinoid receptor antagonists 5 Azetidine derivatives Two examples (90 -91 ) of the azetidine derivatives developedby Aventis

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1375

Table 8 CB1 Cannabinoid Receptor Antagonists 6 Aryl-Imidazolidine-24-Diones Derivatives

Structures affinities ([3H]-SR141716A hCB1-CHO cells) and pKb values ([35 S]-GTPγS HU210 rat brain) of three aryl-imidazolidine-24-dionederivatives (92-94)

N

NH

OO

R

Br

Br

Cpd ndeg R Ki (nM) a pKb b

DML20 92 ethylmorpholine 70 611

DML21 93 1-hydroxypropyl 103 625

DML23 94 heptyl 98 574

a [175]b [177]

showed that the substitution at the nitrogen in position 3 aswell as a bromine atom in para of the phenyl rings aremandatory for the compounds affinity Later on threecompounds termed DML20 (92) DML21 (93) and DML23(94) were characterised as neutral antagonists using the [35S]-GTPγ S assay on rat cerebellum membranes [176] (Table 8)In the same model they competitively inhibited HU-210-induced [35S]-GTPγ S binding with pKB values of 611625 and 574 respectively Moreover these compoundswere proven to be quite selective for the CB1 receptor

higher than the one for the CB2 cannabinoid receptor (IC50 =300 nM) The inverse agonism of the compound wasextrapolated by inhibiting CP-55940-induced hypothermiain mice (EC50 = 18 nM) Moreover one oral dose (10mgkg) reduces the food intake in diet-induced obese ratsInterestingly food intake of CB1

-- mice was not alteredBody weight loss was maintained all over the study during achronic administration (14 days) of 10 mgkg of Cpd A

Later on Merck published a patent [180] in which someof the 45-diaryl-pyrazoles derivatives previously describedwere claimed to be useful in the treatment or the preventionof obesity in association with compounds inhibiting the11β-hydroxysteroid dehydrogenase type 1 (1β-HSD1)enzyme The 1β-HSD1 enzyme is responsible for thesynthesis of cortisol increased levels of which according tothe inventors are associated with obesity However neitherresults on appetite suppression nor on weight loss weregiven even if some indications on the affinity of thecompounds for the cannabinoid receptors were given

Very recently the functionality of these DML compoundswas explored using the [35S]-GTPγ S assay both on rat andhuman CB1 cannabinoid receptors [177] The data obtainedconfirm that the 3-alkyl-55rsquo-diphenyl-imidazolidinedionederivatives behave as neutral antagonists on rat CB1cannabinoid receptor (rat brain) However DML 20 DML21and DML23 acted as inverse agonists on the human receptor(hCB1-CHO cells) Furthermore the authors showed thatthis different functionality is not due to the hCB1 receptorlevel of expression in the recombinant cell line as whateverthe level of expression (Bmax of 44 pmolmg or 32pmolmg) DML derivatives behaved as inverse agonists ofthe hCB1 cannabinoid receptor

Another imidazole ring pattern of substitution wasproposed by Hagmann and collaborators [181] Thecompounds described 43 examples are given possess twophenyls the first one in position 1 linked to the nitrogenand the second one in position 2 The carboxamidesubstituent is in position 4 instead of in position 2 for thediarylimidazoles described by Finke These 12-diaryl-imidazoles are claimed to be CB1 cannabinoid receptorantagonists or inverse agonists but the absence of precisepharmacological values do not allow any comparisonbetween the two substitutions patterns However a samepattern of substitution was used by other researchers Amongthem Dyck et al described in 2004 ten 1-(4-chlorophenyl)-2-(24-dichlorophenyl)-imidazole derivatives varying by theircarboxamide substituent in position 4 (101-103 Table 9)[152] For instance with a 3-azabicyclo[330]octan-3-ylsubstituent (101) they obtained a Ki value of 14 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) In a [35S]-GTPγ Sassay 101 exhibited an IC50 value of 19 nM

7 Diarylimidazoles Derivatives

Finke and colleagues at Merck developed a new class ofCB1 receptor antagonists based on an imidazole nucleusOver eighty 45-diaryl-imidazoles derivatives having CB1antagonist properties were claimed in a patent published in2003 [178] Some representative compounds of the patent areillustrated in Table 9 (95-100) The affinity of the newcompounds was determined using [3H]-CP-55940 andhCB1-CHO cells Although no detailed pharmacologicaldata were given in the patent in-vitro and in-vivo propertiesof one compound termed Cpd A (100) have been describedelsewhere [179] Its affinity for the CB1 cannabinoid receptorwas shown to be nanomolar (IC50 = 4 nM) and 75 times

1376 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 9 CB1 Cannabinoid Receptor Antagonists 7 Diaryl-Imidazole Derivatives

Structure and affinity range of some 45-diaryl-imidazole derivatives for the human cannabinoid receptors determined using [3H]-CP-55940 asradioligand and hCB1amp2-CHO cells

N

NR2

R1

R4

R3

ndeg R1 R2 R3 R4 hCB1 (IC50 or Ki) hCB2 (IC50 or Ki) References

95 CH3 CO-NH-piperidinyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

96 CH3 CO-NH-piperidinyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

97 CH3 CO-NH-cyclohexyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

98 CH3 CO-NH-phenyl 24-di-Cl-Ph 4-Cl-Ph 10 ndash 100 nM a 100 ndash1000 nM a [178]

99 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

100 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph 4 nM a 300 nM a [179]

101 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

CH3 14 nM b ND [152]

102 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

H 66 nM b ND [152]

103 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 85 nM b ND [152]

104 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 23 nM b 542 nM b [182]

105 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 30 nM b 608 nM b [182]

106 4-Br-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 60 nM b 489 nM b [182]

107 4-CF3-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 29 nM b 634 nM b [182]

108 4-Cl-Ph 24-di-Cl-Ph CO-NH-morpholinyl CH3 197 nM b 3297 nM b [182]

109 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidin-4-ol CH3 172 nM b 3959 nM b [182]

110 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CN 30 nM b 1590 nM b [182]

111 4-Cl-Ph 24-di-Cl-Ph CO-NH- piperidinyl CH2F 36 nM b 906 nM b [182]

112 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH2-CH3 14 nM b 430 nM b [182]

113 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Cl 27 nM b 823 nM b [182]

114 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Br 23 nM b 746 nM b [182]

115 4-Cl-Ph 24-di-Cl-Ph CO-NH-tetrahydroisoquinoline CH3 34 nM b 696 nM b [182]

116 4-Cl-Ph 24-di-Cl-Ph CO-NH-cycloheptyl CH3 35 nM b 349 nM b [182]

117 24-di-Cl-Ph 4-Cl-Ph CO-NH-piperidinyl CH3 403 nM b 208 nM b [182]a IC50 value b Ki value

Lange and co-workers from Solvay described twenty-eight imidazole derivatives [182] The 24-dichlorophenylsubstituent at position 2 was kept constant while thesubstitutions at the other positions were explored (Table 9)Position 5 can accommodate a large range of littlesubstituents like hydrogen (104) methyl (105) ethyl (112)chlorine (113) bromine (114) fluoromethyl (111) or cyano(110) without major changes in the affinity At position 4more bulky substituents like 1234-tetraisoquinoline (115)or cycloheptyl (116) were well tolerated whereas thepresence of a more hydrophilic moiety such as morpholine(108) or piperidin-4-ol (109) was detrimental to the affinity

Interestingly position exchange between the 24-diphenyland the 4-chlorophenyl substituents led to a 13 fold loweraffinity with Ki values of 30 and 403 nM for 105 and 117respectively ([3H]-CP-55940 hCB1-CHO cells) In thefunctional assay which was inhibition of WIN-55212-2-induced [3H]-arachidonic acid release by hCB1-CHO cellsall tested compounds behaved as antagonists Compound105 showed a pA2 value of 86 which is the same theauthors obtained for SR141716A It would be interesting toassess if these compounds are true antagonists or if they actmore as inverse agonists Compound 105 when administeredto rats was able to inhibit CP-55940-induced hypotension

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1377

with an ED50 value of 24 mgkg which is close to the 3mgkg obtained for SR141716A The compound was alsoactive in another in-vivo model the WIN-55212-2-inducedhypothermia in mice Compound 104 which has nosubstituent in position 5 of the imidazole ring was lessactive in the hypotension model and inactive in thehypothermia model Thus a methyl group is the preferredsubstituent in position 5 of the imidazole moiety Otherproperties either experimental (P-glycoprotein affinitylogPHPLC) or computational (molecular volume polarsurface area) obtained for compounds 105 were similar tothose obtained for SR141716A Finally molecularmodelling studies revealed a close structural overlap betweenthe two compounds

value of 197 nM is selective for hCB1 and is as potent asSR141716A The structural modulations of 118 led to someinteresting compounds (Table 10) Six compounds possess aKi value for the hCB1 cannabinoid receptor equal to or lowerthan 25 nM antagonist properties in the inhibition of [3H]-arachidonic acid release by WIN-55212-2-stimulated hCB1-CHO cells and Ki values for the hCB2 receptor over 1microMThe diaryl-pyrazoline derivatives contained a chiral center atposition 4 The authors resolved the racemic mixture of twocompounds (121 and 130) The levorotatory enantiomersappeared to be the eutomers For compound SLV319levorotatory enantiomer of 121 the Ki and pA2 values were78 nM and 99 respectively The Ki and pA2 values ofSLV326 levorotatory enantiomer of 130 were 359 nM and9 respectively

8 34-Diaryl-Pyrazoline Derivatives Two in-vivo models CP-55940-induced hypotension inrat and WIN-55212-2-induced hypothermia in mouse wereused to assess the potential of these compounds The resultsare of the same order of magnitude than those obtained forSR141716A For instance in the hypotension modelSLV319 ((-)121) and SLV326 ((-)130) showed ED50 values

Lange and collaborators also discovered 34-diaryl-pyrazoline derivatives after a screening of compoundsresembling to SR141716A [183184] Compound 118 thatwas initially identified during the screening possesses a Ki

Table 10 CB1 Cannabinoid Receptor Antagonists 8 34-Diaryl-Pyrazolines Derivatives

Structure affinity for the hCB1 and hCB2 cannabinoid receptors (Ki nM) expressed in CHO transfected cells ([3H]-CP-55940) and potency (pA2) ofsome 34-dirylpyrazolines derivatives Table adapted from [184]

NN

N N

SO2

R3

R2

Cl R1

R4

n˚ R1 R2 R3 R4 Ki hCB1 (nM) Ki hCB2 (nM) pA2 (CB1)

118 H H H 4-CH3 197 gt 1000 84

119 H H H 246- CH3 24 gt 1000 94

120 H H H 4-Cl 16 gt 1000 95

121 H CH3 H 4-Cl 25 gt 1000 87

122 H CH3 CH3 4-Cl 280 gt 1000 85

123 H H H 4-F 53 gt 1000 9

124 H CH3 H 4-F 338 gt 1000 85

125 H CH3 CH3 4-F gt 1000 gt 1000 lt 75

126 H CH3 H 3-Cl 14 gt 1000 86

127 4-Cl CH3 H 4-Cl 255 ND ND

128 4-F CH3 H 4-Cl 584 ND ND

129 H CH3 H H 170 ND 75

130 H CH3 H 4- CF3 221 gt 1000 93

SR141716A 25 1580 86

1378 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

N

HN

O

N

Cl Cl

Cl

N

N

HN

O

Br

Br131 132

Fig (8) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Chemical structures of two 56-diaryl-pyrazine-2-amide derivatives (131-132) developed as CB1 cannabinoid receptorantagonists by AstraZeneca

of 55 and 2 mgkg (po) respectively proving their in-vivoefficacy after oral administration The authors alsodemonstrated that SLV319 is devoid of affinity for the P-glycoprotein pump and that it possessed a goodCNSplasma ratio (17) These two compounds (SLV319and SLV326) were chosen as development candidates bySolvay and entered clinical Phase I trials in 2003 [185]

132 Fig 8) useful as CB1 cannabinoid receptor antagonists[186 187] The activity of twenty derivatives wasdetermined using a [35S]-GTPγ S assay and hCB1-CHOtransfected cells The concentration required to give halfmaximal inhibition of CP-55940-induced [35S]-GTPγ Sbinding (IC50) is lower than 200 nM for the preferredcompounds No affinity data were given for thesecompounds

9 Diaryl-Pyrazine Diphenyl-Pyridine Diphenyl-Phenyland Diaryl-Pyrimidines Derivatives

The same year in a patent from Merck Finke andcollaborators described several 56-di-phenyl-pyridinederivatives as hCB1 antagonists or inverse agonists [188]The compounds have a substituent in position 3 of thepyridine core (Fig 9) This substituent could be a cyano(133-134) or a nitro group a halogen an ester or an amide(135-136) Over 150 compounds were synthesised toillustrate the invention but no pharmacological data wasdisclosed in the patent

In 2003 appeared several patents describing new familiesof compounds that bind to the CB1 cannabinoid receptorThese compounds have in common a central six atomsaromatic ring which could be a pyrazine a pyridine aphenyl or a pyrimidine and that is substituted by at leasttwo phenyl rings

The first patents were from Berggren and collaboratorsfrom AstraZeneca who published two patents describing thesynthesis of 56-diaryl-pyrazine-2-amide derivatives (131-

A few months later another patent was taken by Sanoficlaiming the antagonist properties of 56-di-phenyl-2-pyridine carboxamide derivatives [189] These compounds

N

NH

O

Cl

Cl

N

O

Cl

O NH

N

O

Cl

ClCl

NH

CN O

N

Cl

Cl

Cl

OHN

N

O

Cl

ClCl

CNF

F

133 134

135 136 137

Fig (9) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives 56-Di-phenyl-pyridine derivatives patented by Merck (133-136) and by Sanofi (137) as CB1 cannabinoid receptorsantagonists

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1379

NH

O

N

Cl

ClCl

NH

O

N

Cl

F3CCl

N

N

O

Cl

Cl Cl F138 139 140

Fig (10) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Representative structures of the diphenyl-phenyl (138-139) and 45-diphenyl-pyrimidine (140) derivatives developed ascannabinoid antagonists at Sanofi and Merck respectively

28 examples are given structurally related to the diphenyl-phenyl derivatives have their pyridine core substituted bytwo phenyl rings and by an amide moiety (137 Fig 9) TheIC50 values were lower than 100 nM but no data were givenshowing the antagonist properties although the compoundswere assayed in the adenylate cyclase inhibition assay

10 Other Derivatives

Several amide derivatives structures recently appearedamong the CB1 cannabinoid receptor antagonists In a seriesof patents taken by Merck in 2003 Hagmann and colleaguesdescribed a large amount of compounds obtained by parallelsynthesis which are claimed to be CB1 cannabinoid receptorantagonists [192-195] They were synthesised by reacting alibrary of substituted amines with a library of carboxylicacids (141-144 Fig 11) However as no value was givenfor the affinity or activity of these amides we will notfurther discuss this class of compounds

Along this line Sanofi Synthelabo developed newdiphenyl-phenyl derivatives claimed to have CB1cannabinoid receptor antagonist properties [190] They arebased on a central phenyl ring substituted by two phenylsand by an amide moiety (138-139 Fig 10) Thirteencompounds were claimed along with their synthesis TheirIC50 values were lower than 100 nM ([3H]-CP55-940hCB1-CHO cells) but no other specific data (selectivitypA2hellip) was given

Several 15-diaryl-pyrrole-3-carboxamide derivatives weresynthesised and claimed to be CB1 cannabinoid receptorantagonists by Berggren et al [196] The affinity valueswere not disclosed in the patent (145-146 Fig 12) Otherpyrrole derivatives were synthesised by Guba et al atHofmann-La Roche [197] More specifically 2-(thiazol-4-yl)pyrrole derivatives were claimed as CB1 cannabinoidreceptor antagonist (IC50lt2microM) (147-148 Fig 12)

Finally Kopka et al from Merck described more thanone hundred 45-diaryl-pyrimidine derivatives as CB1cannabinoid receptor antagonists (140 Fig10) [191]However no precise data were provided concerning theaffinity and functionality of the compounds

R2

R1N

R3

R4

R5

O

NH

O

Cl

O F

F

R2

R1NH

R3

R4

HO R5

O

NH

O

Cl

CN

NH

O

Cl

O

O

NH

O

Cl

Cl

+

141142

143 144

Fig (11) CB1 cannabinoid receptor antagonists 10 Other derivatives Retrosynthetic scheme of a CB1 cannabinoid antagonistlibrary of amide derivatives developed at Merck The structures of four representative compounds are illustrated (141-144)

1380 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

NH

O N

Cl

Cl

Cl

N

NH

O

OMe

MeO

N

NH

O

N

S

MeO

N

NH

O

N

S

MeO

145 146

147 148

Fig (12) CB1 cannabinoid receptor antagonists 10 Other derivatives Two examples of 15-diaryl-pyrrole derivatives described byBerggren et al (145-146) While the 2-(thiazol-4yl)pyrroles derivatives 147 and 148 were described by Guba et al from Hofmann-LaRoche All these pyrroles derivatives were claimed to be CB1 cannabinoid receptor antagonists

Another new structure is represented by the 124-triazolederivatives developed by Jagerovic and collaborators In arecent paper were described the synthesis andpharmacological properties of five new 15-diphenyl-3-alkyl-

triazole derivatives [198] Among these compounds onlyone (149) behaved as a CB1 antagonist inhibiting the WIN-55212-2-induced contractions in a mouse vas deferenspreparation However despite its effects in isolated tissue

Table 11 CB1 Cannabinoid Receptor Antagonists 10 Other Derivatives

Structure and affinity of diaryl-triazole derivatives

N

NN

R3

R2

R1

Cpd R1 R2 R3 Ki CB1 (nM) References

149 24-diCl 4-Cl hexyl 855 a [198]

150 24-diCl 4-Cl2-OMe CO-NH-(3-azabicyclo[330]octan-3-yl) 270 b [152]

151 24-diCl 4-Cl CO-NH-(3-azabicyclo[330]octan-3-yl) 164 b [152]

152 4-Cl 24-diCl CO-NH-(3-azabicyclo[330]octan-3-yl) 137 b [152]

153 4-Cl 24-diCl CO-NH-benzylpyrrolidin-3-yl 29 b [152]

154 4-Cl 24-diCl CO-NH-1-(4-chlorophenyl)ethyl 66 b [152]

155 4-Cl 24-diCl CO-NH-piperidinyl 356 c [182]

156 24-diCl 4-Cl CO-NH-piperidinyl 382 c [182]

a [3H]-SR141716A rat cerebellar membranesb [3H]-CP-55940 hCB1-HEK EDNA cellsc [3H]-CP-55940 hCB1-CHO cells

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1381

N

S

O

NH

Cl

Cl

Cl

F

N

S

O

NHN

ClCl

Cl

N

S

O

NH

Br

N

S

O

NHN

Cl

Cl

Cl

156 157 158 159

Fig (13) CB1 cannabinoid receptor antagonists 10 Other derivatives Structures of 45-diaryl-thiazole derivatives patented as hCB1cannabinoid receptor antagonists by Solvay Pharmaceuticals (156 157 158) or by AstraZeneca (157 158 159)

assays (mouse vas deferens and guinea pig ileum) thecompound possessed only a reduced rCB1 affinity with Kivalues of 855 and 748 nM using [3H]-SR141716A and [3H]-WIN-55-212-2 respectively (Table 11) Further four seriesof compounds differing by the nature of the substituentaround the triazole core were also described in a patent[199] Usually the compounds claimed possess two aromaticrings possibly substituted and one linear alkyl chain Theaffinity for the cannabinoid receptors was not givenNevertheless 149 was evaluated in two isolated tissuemodels the inhibition of the electrically evoked contractionsin the guinea pig ileum and in the mouse vas deferens Inthe two models compound 149 inhibited the effect of WIN-55212-2 but had no effect by itself In the vas deferensmodel the authors obtained a pA2 value of 748 to becompared with 763 obtained for AM251

Yet another five membered ring a thiazole has beenused as central moiety of a new class of cannabinoid ligandsThe thiazole ring is substituted by two aryls in position 4and 5 and by an amide (position 2) as in compounds 156-159 (Fig 13) Forty derivatives of this type weresynthesised by Lange and colleagues from SolvayPharmaceuticals and claimed in a patent as agonists orantagonists of the CB1 cannabinoid receptor [200] Nopharmacological data were provided however their activitywas assessed by measuring the cAMP in transfected hCB1-CHO cells In a recent paper from Lange et al two of thesethiazole derivatives were further described [182] Compound13 (157) the most closely related to SR141716A has a Kivalue of 227 nM while for compound 14 (158) Ki value isover 1000 nM Thus as in the imidazole series (see Table9) the aromatic substitution pattern is of great importancefor the CB1 cannabinoid receptor affinity Compound 13however was devoid of in vivo activity

Others 124-triazole derivatives were described by Dycket al compounds 150-154 unlike the Jagerovic ones havean amide moiety in position 3 [152] The highest affinitywas obtained with a 1-benzyl-pyrrolidin-3-yl substituentThe Ki value was 29 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) to be compared to 12 nM obtained for theSR141716A

Seventeen 45-diaryl-thiazole derivatives were alsosynthesised by Berggren et al and patented by AstraZeneca(157-159 Fig 13) [201]

In a patent from Merck Toupence et al described over190 substituted furo[23]pyridines claimed to be CB1cannabinoid receptor antagonists [202] The affinity (IC50 lt1microM) was measured using recombinant CHO cells and [3H]-CP-55940 and the activity using cAMP dosage (160 Fig14)

Lange et al described a triazole derivative (155) whichcan be superimposed with SR141716A Compound 155showed a Ki value of 356 nM ([3H]-CP-55940 hCB1-CHOcells) a pA2 value of 83 in the inhibition of WIN-55212-induced release of [3H]-arachidonic acid by hCB1-CHO cellsand was active in vivo in the CP-55940-inducedhypotension in rat (ED50 = 236 mgkg) [182]

Very recently Alanine from Hoffmann-LaRoche foundthat diarylbenzo[13]dioxole derivatives act as CB1

O

O

S

N

OO

F

ClCl

O

O

F O

N

OON

NH2

O

Cl

Cl

Cl

160 161 162

Fig (14) CB1 cannabinoid receptor antagonists 10 Other derivatives One example of a furo[23]pyridine derivative (160) and twoexamples of diarylbenzo[13]dioxole derivatives (161-162) described as hCB1 cannabinoid receptor antagonists by Toupence et aland Alanine et al respectively

1382 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

cannabinoid receptor antagonists (161-162 Fig 14) [203]Approximate IC50 values for twelve compounds ([3H]-CP-55940 hCB1-HEK cells) among the three hundred andeighty compounds described were given (IC50lt2microM)

et al in 1995 [207] In their hands ∆9-THC was not able toinhibit the forskolin-stimulated adenylyl cyclase activity inan assay performed on hCB2-CHO cells Subsequently theyshowed that ∆9-THC was able to antagonise HU210-inducedinhibition of adenylyl cyclase activity thus acting as anantagonist of the hCB2 cannabinoid receptor expressed inCHO cells [208] Furthermore very recently Govaerts et al[209] reported an inverse agonist effect of ∆9-THC and ∆8-THC on hCB2-CHO cells using the [35S]-GTPγ S assayThe pEC50 values were 763 and 888 respectively whilethe Emax values were ndash27 and ndash16 as compared to basal

Griffith from Pfizer described three new series of CB1cannabinoid receptor antagonists based on a bicyclic centralaromatic ring Purine pyrazoltriazines andpyrazolo[15a]pyrimidines derivatives were synthesised andinvestigated for their ability to interact with the CB1cannabinoid receptor Albeit a [35S]-GTPγ S assay wasdescribed no data were given concerning the antagonist orinverse agonist properties of such compounds Two hundredand eighty purine derivatives were synthesised [204] Theaffinities of 163 and 164 were given in the patent (28 and12 nM [3H]-SR141716A) In a following patent thesynthesis of sixty pyrazoltriazine derivatives was described(165 Fig 15) [205] The affinity of each compound was notgiven but was said to be ranging between 01 and 590 nMAnd finally more than forty pyrazolo[15a]pyrimidines weresynthesised (166 Fig 15) [206] Their affinity for the CB1cannabinoid receptor was lower than 155 nM Despite thefact that a great number of derivatives share the samesubstitution pattern around the central bicyclic aromatic ringthe comparison of these three new series of compounds ismade difficult by the absence of precise affinity data

Some other classical cannabinoids were shown to possessan antagonist activity at the CB2 receptor This is the casefor instance for the non-selective O-1184 (2) (Ki = 74 nM)which enhances the forskolin-induced cAMP production inCB2-CHO cells (EMax = 161 EC50 = 63 nM) and alsofor O-584 (3) (EMax = 246 EC50 = 138 nM) [39]

2 Indole Derivatives

Since the disclosure in 1993 of a second cannabinoidreceptor many attempts have been made to synthesise CB2cannabinoid receptor selective ligands The first compoundreported to have a somehow greater affinity for the peripheralcannabinoid receptor was WIN-55212-2 The compoundwas introduced by DrsquoAmbra and colleagues [210] back in1992 as a new cannabinoid ligand having a nanomolaraffinity and acting as an agonist In 1996 Showalter et alused a transfected cell line expressing the CB2 cannabinoidreceptor in order to identify selective ligands for thisreceptor [211] They showed that the CB2 affinity of WIN-55212-2 was 7 times greater than the CB1 with Ki valuesof 189 and 028 nM for the CB1 and CB2 cannabinoidreceptors respectively However WIN-55212-2 is anagonist of the cannabinoid receptors Interestingly WIN-55212-3 (167 Fig 16) the so-called inactive enantiomer ofWIN-55212 was recently shown to behave as a low affinity(Ki=363 microM) inverse agonist of the hCB2 cannabinoidreceptor as it decreased the [35S]-GTPγ S binding [209]

III CB2 LIGANDS RECEPTOR ANTAGONISTS ANDINVERSE AGONISTS

The compounds acting as antagonist or inverse agonistsat the CB2 cannabinoid receptor are reviewed in this thirdpart of the paper As for the CB1 cannabinoid receptorligands they are classified depending on their chemicalstructures

1 Classical Cannabinoid Derivatives

The first report of an antagonism mediated by ∆9-THC atthe peripheral cannabinoid receptor was made by Bayewitch

N N

NN

NH COOH

Cl

Cl

N N

NN

N

Cl

Cl

NH2OH

O

N

N

N

N

N

NHNH2

O

Cl

ClN

N

N

N

Cl

Cl

N

O

163 164

165 166

Fig (15) CB1 cannabinoid receptor antagonists 10 Other derivatives Examples of purine (163-164) pyrazoltriazines (165) andpyrazolo[15-a]pyrimidines (166) derivatives described as CB1 cannabinoid receptor antagonists by Griffith

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1383

The pEC50 value was 554 and the Emax value ndash26 ascompared to basal

AM630 also acts as an inverse agonist at the hCB1cannabinoid receptor (EC50 = 900 nM) as Landsman et aldemonstrated [47] Recently Zhang and colleagues reportedthe characterisation of the microsomal metabolism of AM630[212]

N

O

N

O

O

N

O

Cl

O

N

O

(S)-WIN-55212-3 167 BML-190 168

In 1996 Gallant and colleagues identified by submittinga large number of compounds to a binding assay anotherindole analog as CB2 cannabinoid receptor ligand [213] Onthe basis of this compound christened BML-190 (168 Fig16) or called indomethacin morpholinylamide theysynthesised several derivatives that possessed a selectivityratio for the CB2 cannabinoid receptor of up to 140 Thesewere the first compounds specifically designed to be CB2cannabinoid receptor ligands The first report on thepharmacological properties of BML-190 appeared onlyrecently New and colleagues highlighted in the paper theinverse agonist properties of the compound BML-190 dose-dependently increases (103 EC50= 980plusmn70 nM) theforskolin-stimulated levels of cAMP in hCB2-HEK cellswhile WIN-55212-2 decreases (44 45plusmn42nM) thisaccumulation [137]

Fig (16) CB2 cannabinoid receptor antagonists 2 Indolederivatives Chemical structures of WIN-55212-3 (167) andBML-190 (168) two indoles derivatives acting as inverseagonists at the hCB2 cannabinoid receptor Surprisingly in 2002 Melck and collaborators used

BML-190 as a CB2 receptor agonist in a cell proliferationassay [214] BML-190 was also tested as inhibitor of thecyclooxygenase-2 enzyme by Kalgutkar et al [215] Theyobtained IC50 values higher than 33 and 66 microM for theCOX-I and COX-II enzymes respectively These valueshave to be compared with the submicromolar activity ofindomethacin the parent compound Very recently Klegerisand colleagues [216] looking for an antineurotoxic action ofcannabinoids showed that BML-190 increases TNF-αsecretion by stimulated THP-1 monocytic cells but is noteffective on the IL-1β secretion

Further on the basis of WIN-54461 (12) a CB1antagonist AM630 (13) was synthesised by replacement ofthe bromine atom by an iodine one In the initial reportPertwee et al showed the antagonist effects of AM630 butalso suggested that the CB1 cannabinoid receptor may not bethe preferential receptor of AM630 [45] Later on Ross et alreported using hCB1amp2-CHO cells and [3H]-CP-55940that AM630 binds to the CB2 cannabinoid receptor (Ki =312 nM) with a selectivity ratio over 160 [48]Furthermore using a [35S]-GTPγ S assay they showed itsinverse agonist properties (EC50 = 76 nM) However

Table 12 CB2 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Reported binding affinities (Ki) and selectivity ratio (CB1CB2) of SR144528 (169) for rat and human cannabinoid receptors determined using [3H]-CP55940 as radioligand

NN

O

NH

Cl

SR144528 169

rCB2 (spleen) rCB1 (whole brain) Selectivity Ki hCB2 (transfected CHOcells)

Ki hCB1 (transfected CHOcells)

Selectivity References

038 nM 305 nM 800 06 nM 437 nM 730 [217]

067 nM 548 nM 80 [219]

56 nM gt10microM gt1780 [48]

25 nM a gt 1microM a gt400 [224]

024 nM 276 nM 115 199 nM 503 nM 25 [232]

a COS cells

1384 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

3 Diarylpyrazole Derivatives CHO cells is constitutively active and phosphorylated onSer 352 Upon treatment by SR144528 thisphosphorylation of the receptor was inhibited Moreover theSR compound was able to induce dephosphorylation ofagonist-induced CB2 phosphorylation The question whetherSR144528 induces a CB2 receptor conformational changemaking it a better phosphatase-specific substrate or ifSR144528 binding activates specific phosphatases remainsopen However this remains a nice example of inverseagonist-induced receptor resensitisation

The same Sanofi team which synthesised the CB1antagonist SR141716A reported in 1998 the synthesis andpharmacological characterisation of a CB2 cannabinoidreceptor selective antagonist the SR144528 (169) [217218] The structure of the new compound is derived fromSR141716A one SR144528 is the first selective CB2receptor antagonist reported as the inverse agonist propertiesof BML-190 were shown only recently by New et al [137]The authors reported for 169 using [3H]-CP-55940 asradioligand Ki values of 04 nM and 06 nM for the rat andthe human CB2 receptors respectively and found a CB1-CB2 selectivity ratio around 700 Table 12 sums up thebinding affinities of SR144528 for the cannabinoid receptorsobtained by different authors Interestingly while the CB2affinity is more or less constant the affinity obtained for theCB1 receptor varies by one order of magnitude [219] Theantagonist effect of SR144528 was highlighted by evaluatingits effect on cAMP production by hCB2-CHO cells exposedto 3 nM CP-55940 (EC50=10nM) The antagonism wasalso observed on the activation of the MAP kinase pathwayby CP-55940 (IC50=39nM)

The Sanofi research team working on cannabinoidsconducted investigations in order to determine the keyresidues for SR144528 interaction with the receptor (Table13) as well as the binding mode of the compound Firstlythey identified the TM4-EL2-TM5 region as a regioncontaining crucial residues for the affinity of several ligandsamong them SR144528 [224] Among these residues themutation of two cysteine of the second extracellular loop(C174 and C179) abolishes the affinity for the CB2 receptorLater on the implication of S453(161) and S457(165) inthe binding of SR144528 was evidenced by mutationalstudies In the model proposed for the docking of SR144528into the CB2 receptor the crucial contacts are comprised intothe TM4 this is the case for S453(161) and S457(165)which are proposed to form hydrogen bonds with the ligandand W464(172) proposed to have aromatic interactions withthe 4-methylbenzyl of the SR144528 [225] In 2003 Fengand Song showed that point mutations D349(130)A andA634(241)E abolished ligand binding However mutationof the arginine R350(131) member of the highly conservedDRY motif to alanine has no influence on SR144528binding [226]

Early after the report of the synthesis and characterisationof SR144528 the same authors further explored the functionof this compound and showed its inverse agonist propertiesusing CB2-CHO cells co-transfected with the luciferasereporter gene with either the CRE or the murine krox24regulatory sequence [220] In these models SR144528 notonly inhibits CP-55940 effects but also reproduces theeffects of a pertussin toxin treatment proving its inverseagonist properties SR144528 functionality was alsoexplored by [35S]-GTPγ S binding in hCB2-CHO cells(IC50=3 nM) [221] The inverse agonist function ofSR144528 was further illustrated by Rhee and Kim usingCOS cells co-transfected with hCB2 receptor and adenylylcyclase [222]

As for the CB1 cannabinoid receptor ligands Sanofideveloped tricyclic derivatives based on the 1-benzylpyrazole-3-carboxylic scaffold More than twentycompounds were disclosed in a patent [227] in 2001 (84-86Table 7) The benzyl in position 4 characteristic of the CB2diarylpyrazoles from Sanofi is conserved as well as thebicyclic residue in position 3 The length of the second linkbetween the phenyl and the pyrazole can be made of one or

Bouaboula and co-workers investigated SR144528 effectson CB2 cannabinoid receptor phosphorylation status [223]Firstly they showed that the CB2 receptor transfected in

Table 13 CB2 Cannabinoid Receptor Antagonists 3 Di-Aryl-Pyrazole Derivatives

Some of the single point mutations reported for the hCB2 cannabinoid receptor for which pharmacological data concerning the SR144528 are available

Mutant Effect References

R350(131)A no effect [226]

S453(161)A loss of affinity [225]

V456(164)I no effect [225]

S457(165)A loss of affinity [225]

C174S a loss of affinity [224 225]

C175S a loss of affinity [225]

C177S a no effect [225]

C179S a loss of affinity [224 225]

S544(193)G no effect [225]

a Second extra-cellular loop

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1385

two methylene but according to the patent one methylenebridge is preferred The Ki values of these compounds for theCB2 receptor were said to be lower than 500 nM and theirantagonist properties have been assessed using the adenylatecyclase inhibition assay

compounds by the presence of an alkyl substituent inposition 5 instead of a phenyl and of course by position 3which remains unsubstituted However except a lecture atthe 1998 ACS meeting [230] where compound 171 wasreported to have a Ki value of 835 nM and a selectivity ratioover 100 no further report on these compounds waspublished untill now

In a recent paper Mussinu and colleagues reported thesynthesis of other tricyclic derivatives starting fromSR141716A structure [228] N-piperidin-1-yl-14-di-hydroindeno[12-c]pyrazole-3-carboxamide derivatives areCB2 receptor ligands and some of them possess high affinityand selectivity for the CB2 receptor as shown in Table 7 (79-83) These tricyclic derivatives differ from the Sanofi tricyclicderivatives as they do have a phenyl ring instead of a benzylin position 1 Another difference is the nature of thesubstituent in position 3 which is not a bicycloalkyl but acyclic amine One of the compounds synthesised byMussinu et al 81 possesses the highest affinity for the CB2receptor ever reported with a Ki value of 0037 nM for themurine CB2 receptor ([3H]-CP-55940 mice spleenhomogenate) Interestingly 79 the closest SR141716Aanalog lost almost all its affinity for the CB1 receptor andincreased by four orders of magnitude its affinity for the CB2receptor Even if the structure quite similar to the SRcompounds could suggest that these derivatives behave asantagonists no pharmacological data were given in the paperto support this hypothesis

5 2-Oxoquinoline Derivatives

Inaba and colleagues from Japan Tobacco introduced in2001 a completely new structure in the cannabinoid fieldbased on a 2-oxoquinoline scaffold Over eighty derivativeswere disclosed in a patent [231] and one compound JTE-907 (173) was subsequently characterised in-vitro as well asin-vivo [232] In Table 14 are listed some of the compounds(172-178) disclosed in the patent along with their affinity forthe cannabinoid receptors These values were obtained onhuman receptors against [3H]-WIN-55212-2 For twelvecompounds the anti-inflammatory activity per os wasassessed in a carrageenin-induced paw edema model Thehighest effect reported was obtained with JTE-907 with anED50 value lower than 01 mgkg Therefore thesecompounds were claimed to be useful as anti-inflammatoryand anti-allergic agents

To further characterize JTE-907 the authors determinedits affinity for cannabinoid receptors of different species using[3H]-CP-55940 as radioligand In their hands JTE-907behaved as a very selective hCB2 ligand with Ki values of36 and 2370 nM for the CB2 and CB1 cannabinoid receptorsrespectively even more selective than the SR144528 (Kivalues of 199 and 50 nM respectively) However it has tobe mentioned that the affinities reported in the paper forJTE-907 are quite different from those reported in the patent(hCB1 Ki values of 35nM and 009 nM respectively) Italso appears that the SR144528 affinity for the CB1 receptordetermined by Iwamura et al is higher than the affinityreported by Rinaldi-Carmona et al with values of 50 and437nM respectively The authors ascribed the difference tothe variability of SR144528 binding affinities to the assayconditions The function of JTE-907 was evaluated bymeasuring the increase in cAMP production by forskolin-stimulated hCB2-CHO cells Maximum stimulation wasattained at a concentration of 1microM of JTE-907 In the sameassay WIN-55212-2 decreased the cAMP production in adose-dependent manner

Finally it is interesting to strengthen that the five-membered ring derivatives (79-86) of the diarylpyrazolefamily are reported to possess a high CB2 receptor affinitywhile the six- and seven-membered ring derivatives (69-78)are described as CB1 receptor ligands

4 Other Pyrazole Derivatives

Other CB2 ligands based on a pyrazole nucleus wereintroduced by SmithKline Beecham in a patent describingthe synthesis of about sixty new compounds [229] Theiraffinity (Ki) for the hCB2 receptor transfected in HEK293cells was reported to be ranging between 25nM and 10microMTheir structures (170-171 Fig 17) differ from the Sanofi

NN

O

N

ON

NN

N

NN

O

N

ON

NN

N

ClCl

1

5

4 3

2

170 171

Fig (17) CB2 cannabinoid receptor antagonists 4 Otherpyrazole derivatives Structure of two compounds (170-171)among those claimed by SmithKline Beecham to be hCB2antagonists

In conclusion JTE-907 was shown to be a CB2 selectiveinverse-agonist with anti-inflammatory and anti-allergicproperties This compound is currently undergoing Phase Iclinical trials as anti-allergic drug [233]

IV NON-CB1 NON-CB2 CANNABINOIDRECEPTORS LIGANDS

In the last years the possible presence of additional non-CB1 non-CB2 cannabinoid receptors was raised frompharmacological evidences

One of the putative receptors possesses an endotheliallocalisation while other ones are localized in the CNS or atperipheral nerves For instance an additional target for ∆9-

1386 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 14 CB2 Cannabinoid Receptor Antagonists 5 2-Oxoquinoline Derivatives

Structure of six compounds among the oxoquinoline derivatives patented by Japan Tobaco Affinity (Ki nM) and selectivity (CB1CB2) for the cannabinoidreceptors are given The affinity was measured by binding against [3H]-WIN-55212-2 on either hCB2-CHO or hCB1-CHO cells Adapted from [231]

Cpd ndeg Structure hCB2 hCB1 Selectivity

1-2 172

NH

MeO O

O

OMeO

0014 3671 262215

JTE-907 173

NH

O

MeO O

O

NH

O

O

0087 3436 39495

2-2 174

OH

O

NH

MeO O

O 077 3247 4215

3-27 175

NH

O

F

NH

MeO O

O0021 336 16000

3-32 176

NH

MeO O

O

NH

F

O

0036 2398 66610

3-38 177

O

O

NH

N

O

O

NMeO

0085 935 11000

3-43 178

NMeO O

O

NH

F

O

0043 864 20100

tetrahydrocannabinol was described in capsaicin-sensitivesensory nerves by Zygmunt et al [234]

[29] and by Di Marzo et al [235] Thus in the presentreview we will focus on the description of the so faridentified ligands of the non-CB1 non-CB2 receptors withan emphasis on the compounds having antagonist properties

Pharmacological evidences for the existence of additionalcannabinoid receptors were reviewed by Wiley and Martin

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1387

(Table 15) In particular the knowledge about three not yetcloned targets has grown The growing molecularpharmacology of the three putative new cannabinoidreceptors is reviewed in the three following sections Thefourth section summarises the data on the other proposedcannabinoid targets

vasodilatator activity In addition the anandamide effect ispartially sensitive to SR141716A inhibition The authorsproposed the existence of two distinct receptors responsiblefor the vasodilatatory effect of anandamide one located in theendothelium is SR141716A-sensitive while the other onelocated on the vascular smooth muscle is SR141716A-insensitive Thus SR141716A is an antagonist of anendothelial anandamide receptor Several studies wereconducted in order to understand the molecularpharmacology of the endothelial anandamide receptor It ispertussis toxin-sensitive [237] Cannabidiol acts asantagonist and abnormal-cannabidiol (Abn-cbd 179) and itsanalogue O-1602 (180) act as agonists [238] The synthesisof O-1602 and of its analogues are disclosed in a patentdescribing the vasodilatatory activity of cannabinoid

1 Endothelial Anandamide Receptor ndash Abnormal-Cannabidiol Receptor

In 1999 Wagner et al showed that anandamide couldinduce mesenteric vasodilatation through an endotheliallylocated ldquoanandamide receptorrdquo pharmacologically distinctfrom CB1 [236] Other cannabinoids such as ∆9-THC HU-210 or WIN-55212-2 were devoid of mesenteric

Table 15 Non-CB1 Non-CB2 Cannabinoid Receptors Antagonists

An overview of the putative new non-CB1 non-CB2 anandamide-receptors ligands The antagonists are highlighted in bold

NH

N

S

HO OH

Cl

OH

OH

OH OH OH OH

OMe

OMe

RR

RR

RR

RR

HO

OH

NH

O

cannabidiol (10) Abnormal-cannabidiol 179 O-1602 180

O-1918 181 capsazepine 183capsaicin 182

Compound Endothelial AEA receptor Brain AEA receptor Cannabinoid-vanilloid brain receptor

AEA agonist a agonist de

WIN-55212-2 no effecta

agonist e agonist g h

CP-55940 no effect e agonist i

SR141716A antagonist a b j no effect ([35 S]-GTPγS binding) d e f antagonist g h

AM251 no effect k no effect h i

Cannabidiol antagonist b no effect e

O-1918 antagonist c jm

Abd-cbd agonist b l

O-1602 agonist b

Capsaicin no effectj agonist i

Capsazepine no effect b antagonist h

a [236] b [238] c [240] d [249] e [250] f [251] g [254] h [258] i [ 255] k [244] j [ 243] l [ 242] m [241]

1388 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

analogues [239] More recently Offertaler et al showed thatO-1918 (181) a cannabidiol analog acts as an antagonist ofthe endothelial anandamide receptor [240] In addition O-1918 does not bind to the CB1 and CB2 cannabinoidreceptors Furthermore O-1918 unlike cannabidiol doesnot cause vasorelaxation in the mesenteric artery preparationmodel used and thus acts as a true neutral antagonistUsing O-1918 as a pharmacological tool Begg et alproposed a coupling of this new G protein-coupledendothelial receptor through a GiGo mechanism to ionchannels in primary cultured human vascular endothelialcells [241] However in their hands SR141716A wasineffective in antagonising Abd-cbd Ho and Hiley furtherdemonstrated that Abn-cbd relaxes rat small mesentericarteries through an activation of K+ channels via aSR141716A-sensitive pathway [242] Further using thesame model they characterized the vasorelaxant activity ofvirodhamine a novel endocannabinoid apparently actingthrough the same receptor [243] The virodhamine-inducedvasorelaxation is sensitive to SR141716A and O-1918 butinsensitive to AM251 SR144528 and AM630 Moreovervanilloid receptor desensitisation by capsaicin (182) had noeffect

244] Thus position 4 on the 5-phenyl could be furtherexplored to obtain selective ligands

To come to an end despite the fact that the endothelialanandamide receptor is neither identified nor cloned at leastthree antagonists are identified SR141716A cannabidioland O-1918 It is likely that a growing number of ligandsand therapeutic applications will appear in the very nearfuture

2 Brain Anandamide Receptor

Following an in-depth evaluation of anandamide effects inCB1

-- mice Di Marzo and co-workers suggested that a non-CB1 non-CB2 G protein-coupled receptor might mediatesome of the actions of AEA in mice [249] Actually effectsof AEA but not those of ∆9-THC were not decreased inCB1 cannabinoid receptor knock-out mice For instance 30mgkg of AEA were still effective in hot-plate test whereas10 mgkg of ∆9-THC had no effect on anti-nociceptionMoreover in a [35S]-GTPγ S assay AEA stimulated with anEC50 value of 223 microM the [35S]-GTPγ S binding to CB1

--

mice brain membranes (Emax= 28) ∆9-THC showed noeffect in this assay The addition of SR141716A did notaffect the [35S]-GTPγ S binding to CB1

-- mice brainmembranes Shortly after other evidences for the existence ofa new G protein-coupled cannabinoid receptor with a distinctdistribution in the central nervous system were published[250] From a set of 24 commonly used cannabinoids (AEAWIN-55212-2 JWH-030 THChellip) only AEA and WIN-55212-2 produced an increased binding of [35S]-GTPγ S onCB1

-- mice brain membranes Interestingly in this studySR141716A alone produced a significant inhibition of [35S]-GTPγ S binding similar to the inhibition obtained usingCB1

++ mice brain membranes (IC50 values of 57 microM and47 microM respectively) However SR141716A did not affectthe anandamide-induced [35S]-GTPγ S binding in CB1

--

mice brain membranes Moreover [3H]-SR141716Aexhibited significant binding in some brain regions of CB1

--

mice which are different from those exhibiting stimulation of[35S]-GTPγ S binding by WIN-55212-2 and AEA Thusthe authors proposed that the SR141716A effects on CB1

--

mice are mediated neither by the CB1 cannabinoid receptornor by the putative new anandamide brain receptor Monoryand co-workers using CB1

-- mice described a novel nonadenylyl cyclase-coupled cannabinoid binding site in micecerebellum [251] Two agonists AEA and WIN-55212-2but not ∆9-THC or HU210 were found to bind to thisreceptor as they enhanced [35S]-GTPγ S binding NeitherSR141716A nor SR144528 were able to affect the WIN-55212-2 induced [35S]-GTPγ S binding in CB1

-- mice asfound by Breivogel et al [250] However the distributionpattern of the receptor described by Monory et al differs fromthe distribution found by Breivogel et al It has to bementioned that the knock-out strains used were different Themice used by Monory were from a CD-1 strain while theone used by Breivogel were from a C57BL61 strainInterestingly Muthane et al detected differences in nigralneurons number and sensitivity to MPTP in the two mousestrains used [252]

More recently Mo et al obtained results showing thatthe Abnormal-cannabidiol receptor is also responsible for anincreased endothelial cell migration [244] Abn-cbd enhancedhuman umbilical vein endothelial cell migration while O-1918 antagonised Abn-cbd effect The CB1 cannabinoidreceptor inverse agonist SR141716A partially inhibitedAbd-cbd action whereas AM251 and SR144528 had noeffect

In another paper from George Kunos team Batkai et alreported that SR141716A is able to inhibit the endotoxichypotension by a cardiac mechanism involving neither theCB1 nor the CB2 cannabinoid receptors [245] Theinvolvement of a non-CB1 non-CB2 site of action wasshown using CB1

-- and CB1--CB2

-- knock-out miceFurther studies are needed to identify the SR141716Asensitive myocardial site of action

In a recent patent Kunos described O-1918 and itsanalogues as vasoconstrictor agents useful to reversepathological vasodilatation of blood vessels [246] Theeffects of O-1918 on mouse blood pressure on LPS-inducedhypotension in mouse as well as the reversal of hypotensionby O-1918 injection are described Furthermore thesynthesis of O-1918 based antagonists is described startingfrom p-metha-28-dien-1-ol and the appropriate resorcinols

Interestingly the Abd-cbd receptor which was firstdetected in the vascular system also seems to be present inthe CNS [247] Indeed Walter et al found that microglialcells (BV-2) migration induced by 1 microM 2-AG is inhibitedby cannabidiol (300 nM) and O-1918 (1 microM) SR141716Awas found ineffective in this assay Thus 2-AG is an agonistof the Abd-cbd receptor Moreover Abd-cbd is able to elicita dose-dependent cell migration in the same model (EC50 =600nM) [248]

From a more structural point of view SR141716Abehaves as an antagonist while on the opposite AM251(20) a close analogue which differs from the former only bythe nature of one halogen has no effect on this receptor [242

To conclude to our knowledge no compounds havingantagonist or inverse agonist properties at this receptor havebeen identified so far

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1389

3 Cannabinoid-Vanilloid Brain Receptor a cannabinoid-vanilloid receptor at which SR141716A andcapsazepine behave as antagonists

Evidences for the presence of an additional ldquocannabinoidrdquoreceptor differing from the CB1 receptor and from the brainanandamide receptor appeared from studies focused on thehippocampus (for a review see Hajos et al [253]) Hajos etal studied the cannabinoid actions on GABAergic andglutamatergic transmission using the whole-cell patch-clamp technique [254] WIN-55212-2 induced a dose-dependent reduction in glutamatergic transmission reversedby SR141716A (1microM) Thus the authors concluded that thecannabinoid actions on the glutamatergic transmission aremediated by a non-CB1 but SR141716A-sensitive receptorIn another paper Hajos and Freund further characterised thisputative receptor [255] They showed that in addition toWIN-55212-2 CP-55940 and capsaicin (182) a TRPV1receptor agonist also act as agonists Furthermore in theirhands SR141716A as well as capsazepine (183) a TRPV1receptor antagonist were able to antagonise this effect on theglutamatergic transmission while AM251 was notSR141716A effects on CB1 knock-out mice were furtherstudied using a test of anxiety [256] Haller et al showedthat on the one hand the distribution of the CB1 receptorinduces anxiety and SR141716A has an anxiolytic effectand on the other hand SR141716A is still active in CB1

--

mice In a following paper they reported that AM-251induces anxiogenic effects in wild-type and in knock-outmice [257] Thus SR141716A in addition to CB1 bindsto a not yet identified receptor Another evidence for thisadditional receptor for SR141716A was given by Bass andco-workers [61] They conducted a structure-activityrelationship study on the stimulation of locomotor activityinduced by SR141716A and twenty analogues Nocorrelation was found between the affinity for the CB1cannabinoid receptor and the stimulation of locomotoractivity Albeit SR141716A and five of its analogues O-1803 O-1710 O-1253 O-1254 (40) O-1255 (41) behavedas inverse agonists in [35S]-GTPγ S assay none of theseanalogues stimulated the locomotor activity In conclusionthe SR141716A-induced stimulation of locomotor activity isneither due to inverse agonism nor to inhibition of anendogenous tone but to a brain receptor distinct from theCB1 receptor Di Marzo et al previously provided evidencesthat the cannabinoid-vanilloid brain receptor could mediatesome of the cannabinoid effects on locomotion [249]

4 Other Non-CB1 Non-CB2 Cannabinoid Receptors

Evidences for the existence of additional non-CB1 non-CB2 cannabinoid receptors localised in the rat isolatedmesenteric arterial bed appeared from the works of Ralevicand collaborators Several cannabinoids were evaluated intheir model of sensory neurogenic vasorelaxation evoked byelectrical field stimulation

HU210 was found to attenuate vasorelaxation in a CB1and CB2 independent way as SR141716A LY320135 andSR144528 were ineffective in inhibiting HU210 effect [259]Similar results were obtained with ∆9-THC [260]Similarly noladin ether inhibited sensory neurogenicrelaxation in a concentration-dependent manner which wasalso unaffected by SR141716A LY320135 and SR144528Moreover this effect as the ∆9-THC one was pertussistoxin sensitive suggesting the involvement of a Gio protein-coupled receptor [261]

However the inhibitory effects of CP-55940 and WIN-55212-2 were affected by the presence of SR141716A (1microM)or LY320135 (1microM) but not by the addition of SR144528(1microM) [262]

It appears that at least two receptors should be involvedin cannabinoid-mediated attenuation of sensory nerve-mediated vasorelaxation The first one is SR141716A andLY320135 sensitive while the other one is resistant toSR141716A

Some evidences for the existence of a ldquoCB2-likerdquo receptorappeared from the studies on palmitoylethanolamide (PEA)an endogenous fatty acid amide devoid of CB1 and CB2affinity [264] antinociceptive potential Calignano and co-workers showed that PEA was able to alleviate pain inseveral animal models [265 266] For instance PEA dose-dependently inhibits kaolin-evoked writhing in mice TheCB2 cannabinoid receptor inverse agonist SR144528 (02mgkg) inhibits PEA anti-nociceptive effect [266] Thus theauthors suggest that PEA acts as agonist on a SR144528-sensitive non-CB2 cannabinoid receptor (ldquoCB2-likerdquoreceptor) at which SR144528 acts as an antagonist

Further studies are needed in order to better characterisethese additional ldquocannabinoidrdquo receptors However it islikely that several therapeutic applications will soon springfor these receptors

Very recently Pistis et al published a study on theneurophysiological effects of cannabinoids on the basolateralamygdala neurons in-vivo [258] One of their findings is thatHU-210 and WIN-55212-2 did not elicit similar effects onbasal amygdala projection neurons firing rate While theformer had no effect the latter decreased the firing rateMoreover SR141716A reversed the WIN-55212-2 effectswhile AM251 had no effect The vanilloid receptorantagonist capsazepine also antagonised the WIN-55212-2effect on firing rate of these neurons Therefore the authorssuggested the presence of a novel cannabinoid-vanilloidreceptor

V CONCLUSION

Since their development cannabinoid antagonists provedto be essential tools in the understanding of cannabinoidpharmacology and biochemistry Moreover the interestshown by the pharmaceutical industry in the development ofnew cannabinoid antagonists prove if necessary the realtherapeutic potential of this class of compounds Thetreatment of eating and movement disorders memorydeficits psychosis and dependencies from various addictivedrugs are some of the most cited applications for the CB1cannabinoid receptor antagonists

From these lines it appears that in addition to theknown CB1 cannabinoid receptor it is now likely that twoother cannabinoid receptors do exist in the brain The firstone should be an anandamide brain receptor for whichSR141716A has no affinity while the second one should be

1390 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Much less wide is the knowledge on the CB2cannabinoid receptor antagonists However potentialapplications could be disorders involving the immunesystem such as inflammation or allergies

[19] Devane WA Hanus L Breuer A Pertwee RG StevensonLA Griffin G Gibson D Mandelbaum A Etinger AMechoulam R Science 1992 258 1946

[20] Mechoulam R Ben-Shabat S Hanus L Ligumsky MKaminski NE Schatz AR Gopher A Almong S MartinBR Compton DR Biochem Pharmacol 1995 50 83

Finally the growing literature on the non CB1 non CB2cannabinoid receptors should allow the development of newdrugs First examples are given by the cannabidiol andabnormal-cannabidiol derivatives described by Kunos et alfor which potential therapeutic applications on the vascularsystem are already patented

[21] Sugiura T Kondo S Sukagawa A Nakane S Shinoda AItoh K Yamashita A Waku K Biochem Biophys Res Co1995 215 89

[22] Hanus L Abu-Lafi S Fride E Breuer A Vogel Z ShalevDE Kustanovich I Mechoulam R Proc Natl Acad SciUSA 2001 98 3662

[23] Oka S Tsuchie A Tokumura A Muramatsu M Suhara YTakayama H Waku K Sugiura T J Neurochem 2003 851374It clearly appears from this paper that the antagonists of

the cannabinoid receptors are still an active research fieldfrom which should emerge new promising therapeutic toolsas well as innovative drugs in the near future

[24] Goutopoulos A and Makriyannis A Pharmacol Therapeut2002 95 103

[25] Beal JE Olson R Lefkowitz L Laubenstein L Bellman PYangco B Morales JO Murphy R Powderly W PlasseTF Mosdell KW Shepard KV J Pain Symptom Manage1997 14 7ACKNOWLEDGEMENTS [26] Notcutt W Price M Miller R Newport S Phillips CSimmons S Sansom C Anaesthesia 2004 59 440

GGM is very indebted to the ldquoFonds pour la formationagrave la Recherche dans lrsquoIndustrie et lrsquoAgriculture (FRIA)rdquo Belgium for the award of a research fellowship Theexperimental works of the authors were supported by grantsfrom the FNRS The Belgian Charcot Foundation and theUniversiteacute catholique de Louvain

[27] Ledent C Valverde O Cossu G Petitet F Aubert JFBeslot F Bohme GA Imperato A Pedrazzini T RoquesBP Vassart G Fratta W Parmentier M Science 1999 283401

[28] Zimmer A Zimmer AM Hohmann AG Herkenham MBonner TI Proc Natl Acad Sci U S A 1999 96 5780

[29] Wiley JL and Martin BR Chem Phys Lipids 2002 121 57[30] Adam J and Cowley P Expert Opin Ther Patents 2002 12

1475

REFERENCES [31] Razdan RK and Mahadevan A Chem Phys Lipids 2002 12121

[32] Barth F and Rinaldi-Carmona M Curr Med Chem 1999 6745

[1] Hillard CJ Harris RE Bloom AS J Pharmacol Exp Ther1985 232 579

[33] Little PJ Compton DR Johnson MR Melvin LS MartinBR J Pharmacol Exp Ther 1988 247 1046

[2] Devane WA Dysarz FA Johnson MR Melvin LSHowlett AC Mol Pharmacol 1988 34 605

[34] Pertwee RG Stevenson LA Elrick DB Mechoulam RCorbett AD Brit J Pharmacol 1992 105 980

[3] Matsuda LA Lolait SJ Brownstein MJ Young ACBonner TI Nature 1990 346 561

[35] Harrison C Traynor JR Life Sci 2003 74 489-508[4] Gerard CM Molerau C Vassart G Parmentier M BiochemJ 1991 279 129 [36] Pertwee RG Fernando SR Griffin G Ryan W Razdan

RJ Compton DR Martin BR Eur J Pharmacol 1996 315195

[5] Glass M Dragunow M Faull RLM Neuroscience 1997 2299

[37] Ross RA Brockie HC Fernando SR Saha B Razdan RKPertwee RG Brit J Pharmacol 1998 125 1345

[6] Shire D Carillon C Kaghad M Calandra B Rinaldi-Carmona M LeFur G Caput D Ferrara P J Biol Chem1995 270 3726 [38] Griffin G Wray EJ Rorrer WK Crocker PJ Ryan WJ

Saha B Razdan RK Martin BR Abood ME Brit JPharmacol 1999 126 1575

[7] Ryberg E Khang Vu H Groblewski T Larsson N ElebringT Hjorth S Sjogren S Greasley PJ 2004 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 119

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Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1367

human radioimaging applications than agonists as they aredevoid of cannabinoid-like effects For instance Berding etal very recently reported the use of [123I]-AM281 for SinglePhoton Emission Computed Tomography imaging of theCB1 cannabinoid receptor in six human patients [85] Inorder to further optimize the brain uptake of suchSR141716A derivatives Sanofi researchers synthesised twomethoxylated SR141716A analogues using 11C as PETtracer [86] These two compounds SR149080 (24) andSR149568 (25) possess high affinity with Ki values of 15and 38 nM respectively and selectivity for the CB1 receptoras well as an improved penetration in the brain evaluated bymeasuring the radioactivity present in various brain regionsafter tail vein injection in CD-1 mice Some of therepresentative radiolabelled SR141716A derivatives aresummarised in Table 5

Colombo et al using Wistar rats [92] Simiand et alobserved that SR141716A selectively reduces sweet foodintake in primates [93] However several authors [94-96]showed that high palatability of food is not necessary toobserve a SR141716A-induced anorectic effect at doses thatdo not cause major behavioural alterations or reduced waterintake Interestingly Gomez et al demonstrated theimplication of the peripheral CB1 receptors on themodulation of feeding and therefore the possible role ofthese receptors on the SR141716A influence on food intake[97] In addition to its effects on food consumptionSR141716A seems to be able to lower the hyperglycemiathe hyperinsulinemia as well as the insulin resistance indiet-induced obese (DIO) mice In the same DIO mice adecrease in adiposity was also observed after treatment [98]Bensaid et al using another model of obesity the obese fafarats observed that SR141716A increases mRNA expressionof Acrp30 or adiponectin a plasmatic protein exclusivelysecreted by adipose tissue through a CB1-mediatedpathway Inductions of free fatty acid oxidation body weightreduction and hyperinsulinemia decrease are some of theknown physiological actions of this protein Thus along theauthors an enhanced expression of Acrp30 followingSR141716A administration could be responsible for themetabolic effects of the compound leading to body weightreduction [99] Vickers et al using the same model (ie fafarats) showed that SR141716A significantly decreases foodconsumption and weight gain in both the obese fafa rats andthe lean Zucker rats [100] This decrease was greater in thefafa group and reversible upon SR141716A withdrawal

From a more therapeutic point of view SR141716A(rimonabant Acompliareg) is one of the promising agents totreat obesity [87] It is currently in Phase III clinical trials forthe treatment and prevention of obesity Final results areexpected for this year and FDA application filling for 2005[88] Preliminary data based on a one year treatment withrimonabant (RIO-Lipids study 1036 patients) showed a 5weight loss in 72 of the treated patients [8990] Severalreports were published before clinical trials aiming todemonstrate the anti-obesity properties of this compoundThe first report by Sanofi Recherche published in 1997described the selective inhibition of sucrose intake in ratsupon SR141716A treatment (03-3 mgkg) [91] Theanorectic and weight loss effects were firstly published by

Table 5 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structure and Affinity of SR141716A Radiolabeled Derivatives Developed as Potential Radio-Imaging Tools

NN

R3

O

NH

Cl

R4

R1

R2

Cpd ndeg R1 R2 R3 R4 Affinity CB1 References

[123I]-AM251 20 123I piperidinyl CH3 Cl Ki=25 nMa [78]

[123I]-AM281 21 123I morpholinyl CH3 Cl Ki=14 nMb [80]

[18 F]-SR144385 22 Cl piperidinyl CH2-18 F Cl IC50 =29 nMa [83]

[18 F]-SR147963 23 Cl morpholinyl CH2-18 F Cl IC50 =120 nM [84]

[11 C]-SR149080 24 O-11 CH3 piperidinyl CH3 Cl IC50 =15 nMa [86]

[11 C]-SR149568 25 O-11 CH3 morpholinyl CH3 Cl IC50 =38 nMa [86]

[18 F]-NIDA-42033 26 O-CH3 piperidinyl 18 F H Ki=18 nMc [155156]

27 O-11 CH3 piperidinyl CH3 H Ki=8nMc [155157]

arat brain homogenates [3H]-CP-55940bmouse cerebellum homogenates [3H]-SR141716crat brain homogenates [3H]-AM251

1368 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Another recent study by Higgs and co-workers furtherhighlighted the role of endocannabinoids in food taste-perception and the reduction of orosensory reward of sucrosein SR141716A treated rats [101] As it was expectedSR141716A induced effects on food consumption are absentsin CB1

-- mice [102] Very recently Cota et al obtainedresults showing that endocannabinoid system modulateshomeostasis via a dual mechanism it regulates at a centrallevel food intake while it blocks at the periphery lipogeneticprocesses [103] Moreover Cani et al showed that ghrelin(an orexigenic peptide) plasma levels are significantlyreduced 45 minutes after SR141716A administration(5mgkg ip) to fasted rats in accordance with the rapiddecrease of food intake measured by the authors It istherefore likely that SR141716 effects on body weight aredue to a conjunction of central and peripheral actions [104]

cerebrospinal fluid from patients with schizophrenia [116]Later De Marchi et al measured higher amounts ofanandamide in blood of schizophrenic patients compared tocontrols [117] On the other hand Dean et al obtainedelevated [3H]-CP-55940 binding in the dorsolateralprefrontal cortex of patients suffering from schizophrenia ascompared to controls [118] Zavitsanou using [3H]-SR141716A found elevated binding in the anterior cingulatecortex of subjects with schizophrenia [119] RecentlyMeltzer and collaborators published the results of a trialconducted to evaluate the potential of four new compoundsin treating schizophrenia and schizoaffective disorders [120]SR141716A (20 mgday) was one of the compoundsevaluated during a six weeks study The authors found noeffect of the CB1 cannabinoid receptor antagonist inimproving patientrsquos schizophrenia (72 subjects)

In addition to the anti-obesity potential the ability ofSR141716A to reduce alcohol and tobacco consumption arecurrently investigated in phase III clinical trials The effect ofSR141716A on alcohol consumption in rats was reported bythe GianLuigi Gessa team [105-108] and by Gallate et al[109] The inhibition of alcohol and nicotine induceddopamine release by SR141716A (1-3 mgkg rats) wasreported by Cohen and co-workers using a brainmicrodialysis device [110] Concerning the smokingcessation preliminary results of a clinical trial (360 subjects40 mg SR141716A) showed an increased abstinence ofsmoking [111] More recent results from the STRATUS-USstudy (787 patients) were reported 362 of patientsreceiving SR141716A (20 mgday) quit smoking against206 in the placebo group [90] Recently Le Fol andGoldberg reviewed the development of cannabinoid CB1antagonists as a new class of therapeutic agents for drugsaddictions [112]

It is known since the early nineties that theadministration of cannabinoid agonists impairs memory inrodents (for a review see Castellano et al [121]) Thusadministration of a cannabinoid antagonist was expected tosomehow improve memory [122123] However dependingon the authors SR141716A when administered alone wasreported to impair (5-10 mgkg im) [124] to have no effect(1-32 mgkg ip) [125] or to improve memory (3 mgkgip) [126] In a more recent paper Wolff and Leandershowed that SR141716A (1mgkg ip) improves memoryin rats by apparently enhancing the consolidation processesof memory [127] Further they found that at higher doses (3mgkg) this effect was lost The discrepancy in the resultsreported in the literature could be ascribed either to thedifferences in the tests used or in the doses administeredFurther experiments are needed to assess whether or not acannabinoid antagonist could be helpful in memory diseases

To conclude several patents were taken by Sanoficoncerning the therapeutic applications of their leadcompound among them being anti-obesity smokingcessation neuroinflammatory diseases and anti-diarrhoeaOne of the last patents to our knowledge concerns thetreatment of sexual dysfunctions with a cannabinoidantagonist such as SR141716A [128] However da Silva etal showed that even if this compound (2 mgkg ip)enhances the effects of apomorphine (20-80 microgkg) it has noeffect alone on penile erection [129] Meanwhile Melis et al

Since the late sixties concerns exist on the associationbetween cannabis use and schizophrenia Recent studiesshowed an association between cannabis use and an increasedrisk of developing schizophrenia [113 114] Moreover apossible role of the endocannabinoid system in schizophreniahas been suggested since its pharmacological characterisation[115] Several experimental data obtained on human subjectstend to confirm such hypothesis On the one hand Lewekeet al found elevated levels of endocannabinoids in

NN

O

NH

N

Br Cl

Cl

NN

O

NH

N

Br Cl

Cl

OH

28 29

Fig (3) CB1 cannabinoid receptor antagonists 3 Diarylpyrazole derivatives The chemical structures of 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-piperidine-carboxamide (SR147778 28 ) and 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-(4-hydroxypiperidine)-carboxamide (29 ) described by Rinaldi-Carmona et al

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1369

reported an increased rate of penile erection after directinjection of SR141716A in the paraventricular nucleus of thehypothalamus [130] This effect was dose dependent and asignificant effect was obtained with a dose of 1microgkg

AM251 in mice at a same dose range (10-30 mgkg ip) thanthe anorectic effects [143] These effects are reversed byadministration of CP-55940 and are absent in CB1

-- miceproving the implication of the CB1 receptor in theantidepressant-like effect of AM251 In 2004 Liao et alreported that AM251 was able to displace the binding of[3H]-batrachotoxin A from its binding site on sodiumchannels in mice brain synaptic preparations [144] Theyobtained an IC50 value of 112 microM and a competitivemechanism of action The authors suggested that AM251 iscapable of reducing neuronal excitability through blockade ofvoltage-sensitive sodium channels in brain

From the clinical trials involving the SR141716A themost frequently reported side-effects are nausea dizziness anddiarrhoea Depression and anxiety were not higher than inthe placebo groups Regarding one of the known side effectsof SR141716A administration the enhancement of intestinalmotility Carai et al reported recently that chronicadministration of the inverse agonists to mice induced atolerance to this prokinetic effect [131] This is not the firstreport of tolerance onset after administration of SR141716A[132] however few is known on the mechanismsresponsible for that phenomenon

Wiley and collaborators reported in 2001 a studydescribing new structure-activity relationships using thepyrazole nucleus as a central scaffold [145] Starting fromSR141716A structure they alternatively substituted one ofthe four substituents while retaining the others bysubstituents known to impart agonist activity in classicalcannabinoids One of the authors expectations was todetermine which positions are responsible respectively forthe antagonism and for the affinity of SR141716A Theaffinity of thirty compounds was assessed previous to in-vivoevaluation of their function in mice using the spontaneousactivity the tail-flick and the rectal temperature assays Theauthors showed that phenyl group in position 5 is critical foraffinity as compound O-1559 (30) lacking this phenyl has adecreased affinity (Table 6) This was already shown by Lanand co-workers [136] along with the need of a substituent inpara position on the phenyl Thomas et al previouslyshowed that this substituent could be a bromine or an iodineatom [146] However an alkyl chain is also tolerated as it isthe case in compounds O-1302 (31) O-1691 (32) or O-1704(33) All these compounds antagonise the anti-nociceptiveand hypothermic effects of ∆9-THC The authors suggestedthat the 5-substituent of pyrazoles is involved in receptorrecognition and antagonism The modulations of thesubstitution pattern of the phenyl in position 1 demonstratethat the 24-di-chloro substitution is the preferred one for theaffinity as well as for the activity Several compoundssupport this assertion Thomas et al showed that additionalhalogens result in a decreased affinity as in compounds 6-I-SR141716A (34) or 4rsquo6-di-I-SR141716A (35) (Ki values of166 and 126 nM respectively) [146] Suppression of the twochlorines (O-1300 39) or replacement of these two chlorinesby an alkyl chain like in O-1254 (40) and O-1255 (41) ledto less active compounds with Ki values ranging from 150to 430 nM However O-1254 and O-1255 behaved asantagonists in a [35S]-GTPγ S assay [61] Lan et al [136]and Wiley et al [145] also modified the substitution inposition 3 Replacement of the amido piperidinyl substituentby alkyl amides as in O-1269 (42) or O-1270 (43) etherslike in O-848 (44) and O-853 (45) ketones (O-1272 46)alcohols (O-1876 47) or alkanes (O-1877 48) resultedmostly in decreased affinity (Table 6) but also with a changeof functionality in some compounds as revealed by in-vivoassays For instance replacement of the piperidinyl by apentyl (O-1269) or by an heptyl chain (O-1270) gave agonistcompounds having Ki values of 32 and 48 nM respectivelyThus the authors suggested that the 3-substituent region isinvolved in receptor recognition and agonist activity Wileyet al on the basis of their results concluded that while the3-position seems to be involved in agonism the 1- 4- and

One close analogue of SR141716A 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-piperidinecarbox-amide (SR147778 28) was described very recently byRinaldi-Carmona et al [133 134] (Fig 3) It possesses highaffinity and selectivity for the hCB1 cannabinoid receptorwith Ki values for the hCB1 and hCB2 receptors of 35 and400 nM respectively ([3H]-CP-55940 hCB1amp2-CHO cells)Further SR147778 antagonised CP-55940 effects on mousevas deferens contractions (pA2=81) and on forskolin-stimulated adenylyl cyclase activity in U373MG cells(pA2=82) but had no effects alone In-vivo SR147778 afteroral administration reversed WIN-55212-2 inducedhypothermia and analgesia As SR141716A SR147778dose-dependently reduced ethanol and sucrose solutionintake with significant effects starting at 03 mgkg (sc) and3 mgkg (po) respectively This compound is currentlyinvestigated in Phase I clinical trials for the treatment ofobesity as well as nicotine and alcohol addictions

Another derivative the 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-(4-hydroxypiperidine)-carboxamide (29) was described in a very recent patent fromSanofi [135] (Fig 3) This antagonist possesses an IC50value for the hCB1 cannabinoid receptor of 32 nM ([3H]-CP-55940 hCB1-CHO cells)

The first structure-affinity relationships for theSR141716A derivatives were reviewed by Barth andRinaldi-Carmona in 1999 [32] Since then many papersdescribing new derivatives were published In 1999 Lan etal described around thirty 15-diarylpyrazoles derivatives[136] Among them was AM251 previously reported as aradioimaging ligand for the CB1 receptor [78] and thatappeared to be more potent (Ki = 749 nM) and selective(selectivity ratio of 306) than SR141716A (Ki = 115 nMselectivity ratio of 143) They reported a Kd value of 05 nMin the mouse vas deferens model using WIN-55212 asagonist Interestingly New et al described for the samecompound an inverse agonist effect on the hCB2 receptorusing a forskolin-induced cAMP accumulation assay (EC50= 650 nM) [137] Recently the anti-obesity effects ofAM251 were reported and are not surprisingly similar tothose of SR141716A [138 139 140] Chen et aldemonstrated that there are synergistic effects on food intakesuppression between AM251 and nalmefene an opioidantagonist [141] This synergistic effect was also presentbetween SR141716A and naloxone as shown by Kirkham[142] Shearman et al found antidepressant-like effects of

1370 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

5-positions appear to be involved in antagonism The O-derivatives reported were also described in a patent alongwith their synthetic pathways which were not given in thepaper [147]

Another investigation of the SR141716Aaminopiperidine region was conducted by Francisco et al[148] They synthesised 21 analogues possessing either analkyl amide or an alkyl hydrazide substituent of various

Table 6 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structure of some of the characterised 1- 3- 4- and 5-pyrazole derivatives Binding affinities (Ki values nM) were obtained on rat brain homogenatesusing [3H]-CP-55940 [136 145 146 148 150] or [125I]-AM-251 [155]

NN

R1

R5

R3R4

Cpd ndeg R1 R3 R4 R5 [136] [145] [146] [148] [150] [155]

SR141716 14 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Cl-Ph 115 62 62 62 13 18

O-1559 30 24-di-Cl-Ph CO-NH-piperidinyl Me 1-Methylpentyl 233

O-1302 31 24-di-Cl-Ph CO-NH-piperidinyl Me 4-((CH2)5)-Ph 21 1

O-1691 32 24-di-Cl-Ph CO-NH-piperidinyl Br 4-((CH2)5)-Ph 15

O-1704 33 24-di-Cl-Ph CO-NH-piperidinyl I 4-((CH2)5)-Ph 22

6-I-SR141716 34 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-Cl-Ph 166

4rsquo6-di-I-SR141716 35 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-I-Ph 126

4rsquo-I-SR141716 36 24-di-Cl-Ph CO-NH-piperidinyl Me 4-I-Ph 75 25 6

4rsquo-Br-SR141716 37 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Br-Ph 168 3

Cpd 25 in ref [136] 38 24-di-Cl-Ph CO-NH-morpholinyl Me 4-Br-Ph 54

O-1300 39 Ph CO-NH-piperidinyl Me 4-Cl-Ph 150

O-1254 40 4-(CH2)4-Ph CO-NH-piperidinyl Me 4-Cl-Ph 226 256

O-1255 41 4-(CH2)5-Ph CO-NH-piperidinyl Me 4-Cl-Ph 433

O-1269 42 24-di-Cl-Ph CO-NH-pentyl Me 4-Cl-Ph 32 114 3

O-1270 43 24-di-Cl-Ph CO-NH-heptyl Me 4-Cl-Ph 48 462 3

O-848 44 24-di-Cl-Ph CH2-O-(CH2)2-piperidinyl

Me 4-Cl-Ph 2450 232

O-853 45 24-di-Cl-Ph CH2-O-CH2-cyclohexyl

Me 4-Cl-Ph 388 100

O-1272 46 24-di-Cl-Ph CO-heptyl Me 4-Cl-Ph 221

O-1876 47 24-di-Cl-Ph 1rsquo-OH-heptyl Me 4-Cl-Ph 657

O-1877 48 24-di-Cl-Ph Heptyl Me 4-Cl-Ph 422

MF9725-64-17 49 24-di-Cl-Ph CO-NH-butyl Me 4-Cl-Ph 134

MF9725-179-32 50 24-di-Cl-Ph CO-NH-NH-butyl Me 4-Cl-Ph 51

MF9725-66-11 51 24-di-Cl-Ph CO-NH-cyclohexyl Me 4-Cl-Ph 25

MF9725-95-31 52 24-di-Cl-Ph CO-NH-(4-hydroxybutyl)

Me 4-Cl-Ph 154

Cpd 15 [150] 53 n-pentyl CO-NH-piperidinyl Me Ph 23

Cpd 16 [150] 54 n-pentyl CO-NH-piperidinyl Me 4-Br-Ph 63

Cpd 17 [150] 55 n-hexyl CO-NH-piperidinyl Me Ph 21

Cpd 18 [150] 56 n-heptyl CO-NH-piperidinyl Me Ph 47

NIDA-41109 57 24-di-Cl-Ph CO-NH-piperidinyl Br 4-Cl-Ph 14

NIDA-41119 58 24-di-Cl-Ph CO-NH-piperidinyl H 4-Cl-Ph 9

NIDA-41057 59 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OH-Ph 104

NIDA-41020 60 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 41

NIDA-41087 61 2-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 8

NIDA-42055 62 24-di-Cl-Ph CO-NH-piperidinyl Br 4-OMe-Ph 62

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1371

lengths in position 3 of the pyrazole moiety They observedthat until five carbons the affinity increases along with thecarbon chain length This is observed with the alkylhydrazide the alkyl amide and the hydroxyalkyl amideseries Moreover the hydrazide analogues exhibit a loweraffinity for the rCB1 than the amide analogues as illustratedby compounds MF9725-64-17 (49) and MF9725-179-32(50) having Ki values of 134 and 51 nM respectively(Table 6) From their structure-activity relationships (SAR)studies the authors concluded that the pharmacophoricrequirement of the amidopiridine region is a chain not longerthan 3 Aring and that a substituent having a positive chargedensity would probably result in increased affinity andpotency The same team described in a patent several othercompounds [149] Binding affinities (Ki) against [3H]-CP-55940 [3H]-SR141716A or [3H]-WIN55212-2 on wholerat brain or on hCB1-transfected cells were given Moreoveractivity data were obtained using the [35S]-GTPγ S assaydemonstrating that these alkyl amide and hydrazide analogsact as antagonists or inverse agonists Despite the greatnumber of derivatives claimed none of them posses asignificantly greater affinity for the CB1 receptor than theSR141716A Nevertheless they showed a slightenhancement of the selectivity for the CB1 receptor over theCB2 cannabinoid receptor

affinity of these derivatives increases with the length of thealkyl chain with an optimal length of 5-6 carbons The bestcompound of this series N-(piperidin-1-yl)-5-phenyl-1-hexyl-4-methyl-1H-pyrazole-3-carboxamide (55) possesses aKi value of 21 nM determined using [3H]-CP-55940 on ratbrain membranes which is higher than the 13 nMdetermined for SR141716A in the same conditions (Table6) In the same paper the authors starting from thehypothesis that antagonism by SR141716A is caused bybinding to the same region of the receptor as do the agonists(CP-55940 and WIN-55-2122) but preventing the agonistpromoted conformational change conducted extensiveconformational analysis as well as superimposition modelsand 3D-QSAR to propose a molecular mechanismsupporting the action of SR141716A Along with theauthors the C-5 aryl substituent of SR141716A occupyinga unique region could contribute in conferring the antagonistproperties Moreover the C-3 substituent could beresponsible for the antagonist or inverse agonist propertiesdepending on the interaction with the receptor

About this topic a very interesting review dealing withthe cannabinoid receptors pharmacophores as well as withthe activationinactivation of these receptors was recentlypublished by Reggio [151]

More recently Dyck et al described seven otherderivatives varying at the amide position [152] The onlycompound possessing a higher affinity for the CB1cannabinoid receptor than SR141716A was the 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta-

The affinity for the rat CB1 receptor of seven derivativespossessing an alkyl chain in position 1 instead of the 24-dichlorophenyl substituent was reported by Shim et al in apaper describing a molecular mechanism for the antagonistand inverse agonist activity of SR141716A [150] The

NN

O

NHN

Cl Cl

Cl

NN

Cl

Cl

NON

NN

O

NH

N

Cl

ClCl

NN

O

NH

MeO Cl

CP-272871 66

63 64

65

Fig (4) CB1 cannabinoid receptor antagonists 3 Diarylpyrazole derivatives Structures of 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63 ) 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methyl-pyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]-morpholine (64 ) N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65 ) and N-phenyl-1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-pyrazole-3-carboxamide (CP-272-871 66 )

1372 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

[c]pyrrol2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63)(Fig 4) The Ki values were 5 and 12 nM respectively([3H]-CP-55940 hCB1-HEK cells)

CP-55940 rat brain) For instance N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65) (Fig 4) showed a Ki value of15 nM However no data were provided concerning thefunctionality of such compounds

Over 200 other pyrazole derivatives acting at the CB1cannabinoid receptor were claimed in a patent from Pfizer[153] The affinity for the hCB1 receptor of 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methylpyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]ndashmorpholine (64) (Fig 4)was 79 nM ([3H]-SR141716A hCB1-HEK cells) Theothers compounds affinity was between 01 and 100 nM

In an effort to develop new SR141716A analoguespossessing lower lipophilicity to be used as PET tracersKatoch-Rouse and colleagues further explored thesubstitution pattern of the two phenyls [155] Bindingassays showed that a decreased lipophilicity lead to adecreased affinity for the CB1 receptor as determined bydisplacement of [3H]-AM251 For instance compoundNIDA-41057 (59) has a lipophilicity of 42 ( expressed asElogDoct) and a Ki value of 104 nM whereas SR141716Ahas a ElogDoct value of 54 and a Ki value of 18 nM In

Seventy five pyrazole derivatives were described in a veryrecent patent from Makriyannis et al [154] The affinity ofthe compounds for the cannabinoid receptors is given TheKi values for the best compounds are lower than 6 nM ([3H]-

Table 7 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structures and affinities for the cannabinoid receptors of some representative tricyclic pyrazole derivatives Values were obtained using [3H]-CP-55940as radioligand and mouse [160 228] rat [159 227] or human [158] cannabinoid receptors Compounds 69-78 are CB1 cannabinoid receptor ligandsCompounds 79-86 are CB2 cannabinoid receptor ligands

NN

O

NHX

R3

R1

R2

n

n˚ X R1 R2 n R3 Ki CB1 (nM) Ki CB2 (nM) Selectivity References

69 -(CH2)3- Cl 24-di-Cl 0 piperidine 126 [158]

70 -(CH2)3- H 24-di-Cl 0 azepane 100 [158]

71 -(CH2)3- H 24-di-Cl 0 piperidine 398 [158]

72 -(CH2)3- Cl 24-di-Cl 0 azepane 125 [158]

73 -(CH2)3- NO2 24-di-Cl 0 azepane 63 [158]

74 -(CH2)2-O- H 24-di-Cl 0 piperidine 501 [158]

75 -CH2-S- Br 24-di-Cl 0 piperidine lt 500 gt10 [159]

76 -CH2-SO2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

77 -(CH2)2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

78 -(CH2)3- Cl 24-di-Cl 0 piperidine 000035 21 60000 [160]

79 -CH2- Cl 24-di-Cl 0 piperidine 2050 034 00002 [228]

80 -CH2- Br 24-di-Cl 0 piperidine 1570 027 00002 [228]

81 -CH2- CH3 24-di-Cl 0 piperidine 363 0037 00001 [228]

82 -CH2- Cl 4-Cl 0 piperidine 1787 09 00005 [228]

83 -CH2- H 24-di-Cl 0 piperidine 1152 0385 00003 [228]

84 -CH2- Cl 34-di-Cl 1 133-trimethylbicyclo[221]heptyl

lt500 gt10 [227]

85 -CH2- Br 24-di-Cl 1 bicyclo[321]oct-3-yl lt500 gt10 [227]

86 -CH2- Br 4-Me 1 77-dimethylbicyclo[410]hept-3-yl

lt500 gt10 [227]

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1373

another paper Katoch-Rouse described the synthesis of[18F]-NIDA-42033 (26) a ligand useful as PET radiotracerstarting from its bromo derivative [156] Recently Kumar etal [157] proposed the synthesis of a 11C derivative ofNIDA-41087 (61) a compound previously described byKatoch-Rouse (Table 5) Post-mortem binding of thiscompound to human prefrontal cortex was also investigatedInterestingly CP-272871 (66) described by Meschler as aCB1 inverse agonist (Ki=57 nM [3H]-CP-55940 rat brainhomogenates) possesses a lipophilicity similar to theNIDA-41087 one [62] However despite the presence of amethoxy function no radiolabelled derivative has beendescribed to date One explanation could be the compoundlow selectivity (2 fold) for the CB1 cannabinoid receptor

To further explore the pharmacology of this compound afunctional assay using the mouse vas deferens model wasperformed by Ruiu and colleagues enlightening thecompetitive antagonist properties of NESS (pA2= 1246against WIN-55212-2) which had no effect by itself up to1microM Moreover NESS 0327 was unable to affect basalbinding of the [35S]-GTPγ S demonstrating its lack ofnegative intrinsic activity In-vivo studies were conductedusing the hot plate and tail flick tests NESS dose-dependently abolished the antinociceptive effect of WIN-55212-2 but had no effect by itself However the authorssuggested based on the affinity and in-vivo activity of theircompound that NESS 0327 possesses a poor centralbioavailability

Several new attempts to increase the affinity of thediarylpyrazole derivatives were recently made by rigidifyingthe SR141716A structure (Table 7) Six fused ringanalogues of SR141716A obtained by fusion of the 5-(4-chlorophenyl) substituent with the central pyrazole to forman indazole ring were published by Bass et al in 2002 [61]The compound possessing the highest affinity O-1248 (67)has a Ki value of 475 nM as determined by displacement of[3H]-CP-55940 from rat brain membranes

Another example of ring-constrained biarylpyrazole wasdescribed by Herbert Seltzman team [161] Theyinvestigated a photocyclisation reaction starting from theSR141716A leading to a pyrazolo[15-f]phenanthridinestructure (Fig 5) The new compound (68) was tested on theCB1 receptor (whole rat brain) against [3H]-CP-55940[3H]-SR141716A and [3H]-WIN-55212-2 and exhibited Kivalues of 48 35 and 50 nM respectively The CB2 affinitywas assessed using [3H]-CP-55940 and was found to be 100times lower (Ki = 3340 nM) No effect on the [35S]-GTPγ Sassay (rat brain) was observed by the authors

Stoit and colleagues from Solvay Pharmaceuticalspublished a paper describing the synthesis andpharmacological characterisation of new benzocyclohepta-pyrazole derivatives as CB1 antagonists (69-74) [158] Theaffinity of these compounds was more or less one order ofmagnitude lower than the SR141716A one with pKi valuesranging from 64 to 72 compared to 76 for the SRcompound (Table 7) Compounds were shown to beantagonists with pA2 values from 70 to 89 as theyprevent CP-55940-induced cAMP accumulation Howeverthe authors investigating the bioavailability of theircompounds either po or after ip injection found negligibleblood plasma levels

NN

Cl

Cl

O

NH

N

NN

Cl

O

NHN

Cl

Cl

O-1248 67 68However just before the publication of Stoit results

Sanofi-Synthelabo took a patent describing tricyclicpyrazolecarboxylic acid amide derivatives having antagonistproperties on the CB1 cannabinoid receptor andsubmicromolar affinities (75-77) [159] Thirty examples weregiven along with their synthetic pathway but no individualpharmacological data were shown

Fig (5) CB1 cannabinoid receptor antagonists 3Diarylpyrazole derivatives Two ring-constrained diarypyrazolederivatives O-1248 (67 ) and a pyrazolo[15-f]phenanthridinederivative (68 )

It appears from these attempts to increase SR141716Aaffinity by rigidifying its structure that only a limitedincrease in the CB1 cannabinoid receptor affinity can beexpected with the apparent exception of NESS 0327Moreover rigidifying the structure often results in a morelipophilic compound in a family of compounds being yetlipophilic

More recently Ruiu and colleagues published a paperdescribing the synthesis and complete pharmacologicalcharacterisation of a very potent CB1 receptor ligandchristened NESS 0327 (78) [160] This compound has thesame structure as compound 69 published by Stoit et al(compound 20 in the reference [158]) Nevertheless Ruiu hasobtained an affinity for the CB1 cannabinoid receptor 5000times bigger than the SR141716A one with Ki values of035 pM and 18 nM respectively using mouse forebrainhomogenates and [3H]-CP-55940 as radioligandFurthermore the selectivity ratio obtained for this compoundby Ruiu is 60000 with a Ki value for the CB2 receptor of21 nM against the [3H]-CP-55940 The discrepancybetween the data obtained independently by Stoit et al andRuiu et al could hardly be explained by the differences inthe origin of the receptor used respectively hCB1-CHO andmouse forebrain

4 Phenyl Benzofuranone Derivatives

In a 1997 patent from Eli Lilly was described thesynthesis of aryl-benzothiophene and aryl-benzofuranederivatives (87-89 Fig 6) having CB1 receptor affinity andantagonist properties [162] From ten compounds having aKi value lower than 25 microM ([3H]-CP-55940 hCB1-CHOcells) one was selected and claimed to have a Ki of 170 nMCompound 88 was shown in the patent to antagonise theeffect of anandamide on cAMP accumulation with an IC50 of

1374 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

O

O

O

O

NC

S

O

O

O

O

O

O

O

NC

LY320135 8887 89

Fig (6) CB1 cannabinoid receptor antagonists 4 Phenyl benzofuranone derivatives Chemical structures of two aryl-benzofuranederivatives (87 -88 ) and one aryl-benzothiophene (89 ) derivative from Eli Lilly

500 nM and the effect of WIN-55212-2 on calcium channelsat 1microM Moreover the intra-peritoneal injection (20 mgkg)of this compound antagonised the in-vivo effect ofanandamide in the Open Field Assay a mouse behaviourmodel Later on Felder and colleagues gave a furtherdescription of this compound called LY320135 in a paperdescribing its effect on cAMP accumulation and on ioniccurrents [163] The selectivity ratio of LY320135 was 106with Ki values of 141 and 14900 nM for hCB1 and hCB2receptors ([3H]-CP-55940) respectively LY320135antagonised anandamide inhibition of forskolin-inducedcAMP accumulation with an IC50 value of 734 nMChristopoulos et al in 2001 reported for LY320135 apKb value of 527 in the inhibition of WIN-55212-2mediated response in the rat electrically-stimulated vasdeferens while the pKb value for SR141716A was 75[164]

Other patents were taken by Aventis in which somepotential applications for their compounds were describedFor instance an association between an azetidine derivativehaving CB1 antagonists properties and sibutramine aserotonin and norepinephrine reuptake inhibitor was claimed[170] as a treatment against obesity Oral administration ofcompound 90 (3 mgkg) and sibutramine (06 mgkg) to fafaZucker rats which are genetically obese rats has resulted ina decreased food intake compared to control lean Zuckerrats In another patent the association of an azetidinederivative (1-10 mgkg po ) and a D2D3 agonist likequinpirole (01 mgkg ip) was claimed to have a therapeuticpotential in the treatment of Parkinsonrsquos disease [171] Theeffect has been evidenced using an akinesia model in the ratEvidences supporting the use of CB1 antagonists as adjuvantin the treatment of Parkinsonrsquos disease begin to appear Itseems that such compounds would be helpful in thetreatment of Parkinsonism [172] and levodopa-induceddyskinesia [173] Brotchie published an interesting paperreviewing the CB1 cannabinoid receptor signaling inParkinsonrsquos disease [174] Nevertheless further clinicalstudies should be conducted to further confirm the usefulnessof CB1 antagonists in the treatment of Parkinsonrsquos disease

5 Azetidine Derivatives

The family of azetidine compounds illustrated bycompounds 90 and 91 (Fig 7) was developed at Aventis byDaniel Achard and colleagues and was described in a seriesof patents [165-168] They claimed the IC50 values of thesecompounds for the CB1 receptor to be less than or equal to100 nM These values were obtained following the proceduredescribed by Kuster et al [169] The antagonist propertywas shown using an in-vivo model the reversal ofhypothermia induced by CP-55940 in mice and the ED50obtained were lower than 50 mgkg

6 Aryl-Imidazolidine-24-Diones Derivatives

In 1999 the Didier Lambert team published a paperdescribing the affinity of 24 new 3-alkyl-55rsquo-diphenyl-imidazolidine-24-dione derivatives for the hCB1 receptor[175] The preliminary structure-activity relationships

N

SO

O

Cl

Cl

F

F

N

SO

O

Cl

NS

90 91

Fig (7) CB1 cannabinoid receptor antagonists 5 Azetidine derivatives Two examples (90 -91 ) of the azetidine derivatives developedby Aventis

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1375

Table 8 CB1 Cannabinoid Receptor Antagonists 6 Aryl-Imidazolidine-24-Diones Derivatives

Structures affinities ([3H]-SR141716A hCB1-CHO cells) and pKb values ([35 S]-GTPγS HU210 rat brain) of three aryl-imidazolidine-24-dionederivatives (92-94)

N

NH

OO

R

Br

Br

Cpd ndeg R Ki (nM) a pKb b

DML20 92 ethylmorpholine 70 611

DML21 93 1-hydroxypropyl 103 625

DML23 94 heptyl 98 574

a [175]b [177]

showed that the substitution at the nitrogen in position 3 aswell as a bromine atom in para of the phenyl rings aremandatory for the compounds affinity Later on threecompounds termed DML20 (92) DML21 (93) and DML23(94) were characterised as neutral antagonists using the [35S]-GTPγ S assay on rat cerebellum membranes [176] (Table 8)In the same model they competitively inhibited HU-210-induced [35S]-GTPγ S binding with pKB values of 611625 and 574 respectively Moreover these compoundswere proven to be quite selective for the CB1 receptor

higher than the one for the CB2 cannabinoid receptor (IC50 =300 nM) The inverse agonism of the compound wasextrapolated by inhibiting CP-55940-induced hypothermiain mice (EC50 = 18 nM) Moreover one oral dose (10mgkg) reduces the food intake in diet-induced obese ratsInterestingly food intake of CB1

-- mice was not alteredBody weight loss was maintained all over the study during achronic administration (14 days) of 10 mgkg of Cpd A

Later on Merck published a patent [180] in which someof the 45-diaryl-pyrazoles derivatives previously describedwere claimed to be useful in the treatment or the preventionof obesity in association with compounds inhibiting the11β-hydroxysteroid dehydrogenase type 1 (1β-HSD1)enzyme The 1β-HSD1 enzyme is responsible for thesynthesis of cortisol increased levels of which according tothe inventors are associated with obesity However neitherresults on appetite suppression nor on weight loss weregiven even if some indications on the affinity of thecompounds for the cannabinoid receptors were given

Very recently the functionality of these DML compoundswas explored using the [35S]-GTPγ S assay both on rat andhuman CB1 cannabinoid receptors [177] The data obtainedconfirm that the 3-alkyl-55rsquo-diphenyl-imidazolidinedionederivatives behave as neutral antagonists on rat CB1cannabinoid receptor (rat brain) However DML 20 DML21and DML23 acted as inverse agonists on the human receptor(hCB1-CHO cells) Furthermore the authors showed thatthis different functionality is not due to the hCB1 receptorlevel of expression in the recombinant cell line as whateverthe level of expression (Bmax of 44 pmolmg or 32pmolmg) DML derivatives behaved as inverse agonists ofthe hCB1 cannabinoid receptor

Another imidazole ring pattern of substitution wasproposed by Hagmann and collaborators [181] Thecompounds described 43 examples are given possess twophenyls the first one in position 1 linked to the nitrogenand the second one in position 2 The carboxamidesubstituent is in position 4 instead of in position 2 for thediarylimidazoles described by Finke These 12-diaryl-imidazoles are claimed to be CB1 cannabinoid receptorantagonists or inverse agonists but the absence of precisepharmacological values do not allow any comparisonbetween the two substitutions patterns However a samepattern of substitution was used by other researchers Amongthem Dyck et al described in 2004 ten 1-(4-chlorophenyl)-2-(24-dichlorophenyl)-imidazole derivatives varying by theircarboxamide substituent in position 4 (101-103 Table 9)[152] For instance with a 3-azabicyclo[330]octan-3-ylsubstituent (101) they obtained a Ki value of 14 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) In a [35S]-GTPγ Sassay 101 exhibited an IC50 value of 19 nM

7 Diarylimidazoles Derivatives

Finke and colleagues at Merck developed a new class ofCB1 receptor antagonists based on an imidazole nucleusOver eighty 45-diaryl-imidazoles derivatives having CB1antagonist properties were claimed in a patent published in2003 [178] Some representative compounds of the patent areillustrated in Table 9 (95-100) The affinity of the newcompounds was determined using [3H]-CP-55940 andhCB1-CHO cells Although no detailed pharmacologicaldata were given in the patent in-vitro and in-vivo propertiesof one compound termed Cpd A (100) have been describedelsewhere [179] Its affinity for the CB1 cannabinoid receptorwas shown to be nanomolar (IC50 = 4 nM) and 75 times

1376 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 9 CB1 Cannabinoid Receptor Antagonists 7 Diaryl-Imidazole Derivatives

Structure and affinity range of some 45-diaryl-imidazole derivatives for the human cannabinoid receptors determined using [3H]-CP-55940 asradioligand and hCB1amp2-CHO cells

N

NR2

R1

R4

R3

ndeg R1 R2 R3 R4 hCB1 (IC50 or Ki) hCB2 (IC50 or Ki) References

95 CH3 CO-NH-piperidinyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

96 CH3 CO-NH-piperidinyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

97 CH3 CO-NH-cyclohexyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

98 CH3 CO-NH-phenyl 24-di-Cl-Ph 4-Cl-Ph 10 ndash 100 nM a 100 ndash1000 nM a [178]

99 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

100 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph 4 nM a 300 nM a [179]

101 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

CH3 14 nM b ND [152]

102 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

H 66 nM b ND [152]

103 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 85 nM b ND [152]

104 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 23 nM b 542 nM b [182]

105 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 30 nM b 608 nM b [182]

106 4-Br-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 60 nM b 489 nM b [182]

107 4-CF3-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 29 nM b 634 nM b [182]

108 4-Cl-Ph 24-di-Cl-Ph CO-NH-morpholinyl CH3 197 nM b 3297 nM b [182]

109 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidin-4-ol CH3 172 nM b 3959 nM b [182]

110 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CN 30 nM b 1590 nM b [182]

111 4-Cl-Ph 24-di-Cl-Ph CO-NH- piperidinyl CH2F 36 nM b 906 nM b [182]

112 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH2-CH3 14 nM b 430 nM b [182]

113 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Cl 27 nM b 823 nM b [182]

114 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Br 23 nM b 746 nM b [182]

115 4-Cl-Ph 24-di-Cl-Ph CO-NH-tetrahydroisoquinoline CH3 34 nM b 696 nM b [182]

116 4-Cl-Ph 24-di-Cl-Ph CO-NH-cycloheptyl CH3 35 nM b 349 nM b [182]

117 24-di-Cl-Ph 4-Cl-Ph CO-NH-piperidinyl CH3 403 nM b 208 nM b [182]a IC50 value b Ki value

Lange and co-workers from Solvay described twenty-eight imidazole derivatives [182] The 24-dichlorophenylsubstituent at position 2 was kept constant while thesubstitutions at the other positions were explored (Table 9)Position 5 can accommodate a large range of littlesubstituents like hydrogen (104) methyl (105) ethyl (112)chlorine (113) bromine (114) fluoromethyl (111) or cyano(110) without major changes in the affinity At position 4more bulky substituents like 1234-tetraisoquinoline (115)or cycloheptyl (116) were well tolerated whereas thepresence of a more hydrophilic moiety such as morpholine(108) or piperidin-4-ol (109) was detrimental to the affinity

Interestingly position exchange between the 24-diphenyland the 4-chlorophenyl substituents led to a 13 fold loweraffinity with Ki values of 30 and 403 nM for 105 and 117respectively ([3H]-CP-55940 hCB1-CHO cells) In thefunctional assay which was inhibition of WIN-55212-2-induced [3H]-arachidonic acid release by hCB1-CHO cellsall tested compounds behaved as antagonists Compound105 showed a pA2 value of 86 which is the same theauthors obtained for SR141716A It would be interesting toassess if these compounds are true antagonists or if they actmore as inverse agonists Compound 105 when administeredto rats was able to inhibit CP-55940-induced hypotension

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1377

with an ED50 value of 24 mgkg which is close to the 3mgkg obtained for SR141716A The compound was alsoactive in another in-vivo model the WIN-55212-2-inducedhypothermia in mice Compound 104 which has nosubstituent in position 5 of the imidazole ring was lessactive in the hypotension model and inactive in thehypothermia model Thus a methyl group is the preferredsubstituent in position 5 of the imidazole moiety Otherproperties either experimental (P-glycoprotein affinitylogPHPLC) or computational (molecular volume polarsurface area) obtained for compounds 105 were similar tothose obtained for SR141716A Finally molecularmodelling studies revealed a close structural overlap betweenthe two compounds

value of 197 nM is selective for hCB1 and is as potent asSR141716A The structural modulations of 118 led to someinteresting compounds (Table 10) Six compounds possess aKi value for the hCB1 cannabinoid receptor equal to or lowerthan 25 nM antagonist properties in the inhibition of [3H]-arachidonic acid release by WIN-55212-2-stimulated hCB1-CHO cells and Ki values for the hCB2 receptor over 1microMThe diaryl-pyrazoline derivatives contained a chiral center atposition 4 The authors resolved the racemic mixture of twocompounds (121 and 130) The levorotatory enantiomersappeared to be the eutomers For compound SLV319levorotatory enantiomer of 121 the Ki and pA2 values were78 nM and 99 respectively The Ki and pA2 values ofSLV326 levorotatory enantiomer of 130 were 359 nM and9 respectively

8 34-Diaryl-Pyrazoline Derivatives Two in-vivo models CP-55940-induced hypotension inrat and WIN-55212-2-induced hypothermia in mouse wereused to assess the potential of these compounds The resultsare of the same order of magnitude than those obtained forSR141716A For instance in the hypotension modelSLV319 ((-)121) and SLV326 ((-)130) showed ED50 values

Lange and collaborators also discovered 34-diaryl-pyrazoline derivatives after a screening of compoundsresembling to SR141716A [183184] Compound 118 thatwas initially identified during the screening possesses a Ki

Table 10 CB1 Cannabinoid Receptor Antagonists 8 34-Diaryl-Pyrazolines Derivatives

Structure affinity for the hCB1 and hCB2 cannabinoid receptors (Ki nM) expressed in CHO transfected cells ([3H]-CP-55940) and potency (pA2) ofsome 34-dirylpyrazolines derivatives Table adapted from [184]

NN

N N

SO2

R3

R2

Cl R1

R4

n˚ R1 R2 R3 R4 Ki hCB1 (nM) Ki hCB2 (nM) pA2 (CB1)

118 H H H 4-CH3 197 gt 1000 84

119 H H H 246- CH3 24 gt 1000 94

120 H H H 4-Cl 16 gt 1000 95

121 H CH3 H 4-Cl 25 gt 1000 87

122 H CH3 CH3 4-Cl 280 gt 1000 85

123 H H H 4-F 53 gt 1000 9

124 H CH3 H 4-F 338 gt 1000 85

125 H CH3 CH3 4-F gt 1000 gt 1000 lt 75

126 H CH3 H 3-Cl 14 gt 1000 86

127 4-Cl CH3 H 4-Cl 255 ND ND

128 4-F CH3 H 4-Cl 584 ND ND

129 H CH3 H H 170 ND 75

130 H CH3 H 4- CF3 221 gt 1000 93

SR141716A 25 1580 86

1378 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

N

HN

O

N

Cl Cl

Cl

N

N

HN

O

Br

Br131 132

Fig (8) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Chemical structures of two 56-diaryl-pyrazine-2-amide derivatives (131-132) developed as CB1 cannabinoid receptorantagonists by AstraZeneca

of 55 and 2 mgkg (po) respectively proving their in-vivoefficacy after oral administration The authors alsodemonstrated that SLV319 is devoid of affinity for the P-glycoprotein pump and that it possessed a goodCNSplasma ratio (17) These two compounds (SLV319and SLV326) were chosen as development candidates bySolvay and entered clinical Phase I trials in 2003 [185]

132 Fig 8) useful as CB1 cannabinoid receptor antagonists[186 187] The activity of twenty derivatives wasdetermined using a [35S]-GTPγ S assay and hCB1-CHOtransfected cells The concentration required to give halfmaximal inhibition of CP-55940-induced [35S]-GTPγ Sbinding (IC50) is lower than 200 nM for the preferredcompounds No affinity data were given for thesecompounds

9 Diaryl-Pyrazine Diphenyl-Pyridine Diphenyl-Phenyland Diaryl-Pyrimidines Derivatives

The same year in a patent from Merck Finke andcollaborators described several 56-di-phenyl-pyridinederivatives as hCB1 antagonists or inverse agonists [188]The compounds have a substituent in position 3 of thepyridine core (Fig 9) This substituent could be a cyano(133-134) or a nitro group a halogen an ester or an amide(135-136) Over 150 compounds were synthesised toillustrate the invention but no pharmacological data wasdisclosed in the patent

In 2003 appeared several patents describing new familiesof compounds that bind to the CB1 cannabinoid receptorThese compounds have in common a central six atomsaromatic ring which could be a pyrazine a pyridine aphenyl or a pyrimidine and that is substituted by at leasttwo phenyl rings

The first patents were from Berggren and collaboratorsfrom AstraZeneca who published two patents describing thesynthesis of 56-diaryl-pyrazine-2-amide derivatives (131-

A few months later another patent was taken by Sanoficlaiming the antagonist properties of 56-di-phenyl-2-pyridine carboxamide derivatives [189] These compounds

N

NH

O

Cl

Cl

N

O

Cl

O NH

N

O

Cl

ClCl

NH

CN O

N

Cl

Cl

Cl

OHN

N

O

Cl

ClCl

CNF

F

133 134

135 136 137

Fig (9) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives 56-Di-phenyl-pyridine derivatives patented by Merck (133-136) and by Sanofi (137) as CB1 cannabinoid receptorsantagonists

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1379

NH

O

N

Cl

ClCl

NH

O

N

Cl

F3CCl

N

N

O

Cl

Cl Cl F138 139 140

Fig (10) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Representative structures of the diphenyl-phenyl (138-139) and 45-diphenyl-pyrimidine (140) derivatives developed ascannabinoid antagonists at Sanofi and Merck respectively

28 examples are given structurally related to the diphenyl-phenyl derivatives have their pyridine core substituted bytwo phenyl rings and by an amide moiety (137 Fig 9) TheIC50 values were lower than 100 nM but no data were givenshowing the antagonist properties although the compoundswere assayed in the adenylate cyclase inhibition assay

10 Other Derivatives

Several amide derivatives structures recently appearedamong the CB1 cannabinoid receptor antagonists In a seriesof patents taken by Merck in 2003 Hagmann and colleaguesdescribed a large amount of compounds obtained by parallelsynthesis which are claimed to be CB1 cannabinoid receptorantagonists [192-195] They were synthesised by reacting alibrary of substituted amines with a library of carboxylicacids (141-144 Fig 11) However as no value was givenfor the affinity or activity of these amides we will notfurther discuss this class of compounds

Along this line Sanofi Synthelabo developed newdiphenyl-phenyl derivatives claimed to have CB1cannabinoid receptor antagonist properties [190] They arebased on a central phenyl ring substituted by two phenylsand by an amide moiety (138-139 Fig 10) Thirteencompounds were claimed along with their synthesis TheirIC50 values were lower than 100 nM ([3H]-CP55-940hCB1-CHO cells) but no other specific data (selectivitypA2hellip) was given

Several 15-diaryl-pyrrole-3-carboxamide derivatives weresynthesised and claimed to be CB1 cannabinoid receptorantagonists by Berggren et al [196] The affinity valueswere not disclosed in the patent (145-146 Fig 12) Otherpyrrole derivatives were synthesised by Guba et al atHofmann-La Roche [197] More specifically 2-(thiazol-4-yl)pyrrole derivatives were claimed as CB1 cannabinoidreceptor antagonist (IC50lt2microM) (147-148 Fig 12)

Finally Kopka et al from Merck described more thanone hundred 45-diaryl-pyrimidine derivatives as CB1cannabinoid receptor antagonists (140 Fig10) [191]However no precise data were provided concerning theaffinity and functionality of the compounds

R2

R1N

R3

R4

R5

O

NH

O

Cl

O F

F

R2

R1NH

R3

R4

HO R5

O

NH

O

Cl

CN

NH

O

Cl

O

O

NH

O

Cl

Cl

+

141142

143 144

Fig (11) CB1 cannabinoid receptor antagonists 10 Other derivatives Retrosynthetic scheme of a CB1 cannabinoid antagonistlibrary of amide derivatives developed at Merck The structures of four representative compounds are illustrated (141-144)

1380 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

NH

O N

Cl

Cl

Cl

N

NH

O

OMe

MeO

N

NH

O

N

S

MeO

N

NH

O

N

S

MeO

145 146

147 148

Fig (12) CB1 cannabinoid receptor antagonists 10 Other derivatives Two examples of 15-diaryl-pyrrole derivatives described byBerggren et al (145-146) While the 2-(thiazol-4yl)pyrroles derivatives 147 and 148 were described by Guba et al from Hofmann-LaRoche All these pyrroles derivatives were claimed to be CB1 cannabinoid receptor antagonists

Another new structure is represented by the 124-triazolederivatives developed by Jagerovic and collaborators In arecent paper were described the synthesis andpharmacological properties of five new 15-diphenyl-3-alkyl-

triazole derivatives [198] Among these compounds onlyone (149) behaved as a CB1 antagonist inhibiting the WIN-55212-2-induced contractions in a mouse vas deferenspreparation However despite its effects in isolated tissue

Table 11 CB1 Cannabinoid Receptor Antagonists 10 Other Derivatives

Structure and affinity of diaryl-triazole derivatives

N

NN

R3

R2

R1

Cpd R1 R2 R3 Ki CB1 (nM) References

149 24-diCl 4-Cl hexyl 855 a [198]

150 24-diCl 4-Cl2-OMe CO-NH-(3-azabicyclo[330]octan-3-yl) 270 b [152]

151 24-diCl 4-Cl CO-NH-(3-azabicyclo[330]octan-3-yl) 164 b [152]

152 4-Cl 24-diCl CO-NH-(3-azabicyclo[330]octan-3-yl) 137 b [152]

153 4-Cl 24-diCl CO-NH-benzylpyrrolidin-3-yl 29 b [152]

154 4-Cl 24-diCl CO-NH-1-(4-chlorophenyl)ethyl 66 b [152]

155 4-Cl 24-diCl CO-NH-piperidinyl 356 c [182]

156 24-diCl 4-Cl CO-NH-piperidinyl 382 c [182]

a [3H]-SR141716A rat cerebellar membranesb [3H]-CP-55940 hCB1-HEK EDNA cellsc [3H]-CP-55940 hCB1-CHO cells

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1381

N

S

O

NH

Cl

Cl

Cl

F

N

S

O

NHN

ClCl

Cl

N

S

O

NH

Br

N

S

O

NHN

Cl

Cl

Cl

156 157 158 159

Fig (13) CB1 cannabinoid receptor antagonists 10 Other derivatives Structures of 45-diaryl-thiazole derivatives patented as hCB1cannabinoid receptor antagonists by Solvay Pharmaceuticals (156 157 158) or by AstraZeneca (157 158 159)

assays (mouse vas deferens and guinea pig ileum) thecompound possessed only a reduced rCB1 affinity with Kivalues of 855 and 748 nM using [3H]-SR141716A and [3H]-WIN-55-212-2 respectively (Table 11) Further four seriesof compounds differing by the nature of the substituentaround the triazole core were also described in a patent[199] Usually the compounds claimed possess two aromaticrings possibly substituted and one linear alkyl chain Theaffinity for the cannabinoid receptors was not givenNevertheless 149 was evaluated in two isolated tissuemodels the inhibition of the electrically evoked contractionsin the guinea pig ileum and in the mouse vas deferens Inthe two models compound 149 inhibited the effect of WIN-55212-2 but had no effect by itself In the vas deferensmodel the authors obtained a pA2 value of 748 to becompared with 763 obtained for AM251

Yet another five membered ring a thiazole has beenused as central moiety of a new class of cannabinoid ligandsThe thiazole ring is substituted by two aryls in position 4and 5 and by an amide (position 2) as in compounds 156-159 (Fig 13) Forty derivatives of this type weresynthesised by Lange and colleagues from SolvayPharmaceuticals and claimed in a patent as agonists orantagonists of the CB1 cannabinoid receptor [200] Nopharmacological data were provided however their activitywas assessed by measuring the cAMP in transfected hCB1-CHO cells In a recent paper from Lange et al two of thesethiazole derivatives were further described [182] Compound13 (157) the most closely related to SR141716A has a Kivalue of 227 nM while for compound 14 (158) Ki value isover 1000 nM Thus as in the imidazole series (see Table9) the aromatic substitution pattern is of great importancefor the CB1 cannabinoid receptor affinity Compound 13however was devoid of in vivo activity

Others 124-triazole derivatives were described by Dycket al compounds 150-154 unlike the Jagerovic ones havean amide moiety in position 3 [152] The highest affinitywas obtained with a 1-benzyl-pyrrolidin-3-yl substituentThe Ki value was 29 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) to be compared to 12 nM obtained for theSR141716A

Seventeen 45-diaryl-thiazole derivatives were alsosynthesised by Berggren et al and patented by AstraZeneca(157-159 Fig 13) [201]

In a patent from Merck Toupence et al described over190 substituted furo[23]pyridines claimed to be CB1cannabinoid receptor antagonists [202] The affinity (IC50 lt1microM) was measured using recombinant CHO cells and [3H]-CP-55940 and the activity using cAMP dosage (160 Fig14)

Lange et al described a triazole derivative (155) whichcan be superimposed with SR141716A Compound 155showed a Ki value of 356 nM ([3H]-CP-55940 hCB1-CHOcells) a pA2 value of 83 in the inhibition of WIN-55212-induced release of [3H]-arachidonic acid by hCB1-CHO cellsand was active in vivo in the CP-55940-inducedhypotension in rat (ED50 = 236 mgkg) [182]

Very recently Alanine from Hoffmann-LaRoche foundthat diarylbenzo[13]dioxole derivatives act as CB1

O

O

S

N

OO

F

ClCl

O

O

F O

N

OON

NH2

O

Cl

Cl

Cl

160 161 162

Fig (14) CB1 cannabinoid receptor antagonists 10 Other derivatives One example of a furo[23]pyridine derivative (160) and twoexamples of diarylbenzo[13]dioxole derivatives (161-162) described as hCB1 cannabinoid receptor antagonists by Toupence et aland Alanine et al respectively

1382 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

cannabinoid receptor antagonists (161-162 Fig 14) [203]Approximate IC50 values for twelve compounds ([3H]-CP-55940 hCB1-HEK cells) among the three hundred andeighty compounds described were given (IC50lt2microM)

et al in 1995 [207] In their hands ∆9-THC was not able toinhibit the forskolin-stimulated adenylyl cyclase activity inan assay performed on hCB2-CHO cells Subsequently theyshowed that ∆9-THC was able to antagonise HU210-inducedinhibition of adenylyl cyclase activity thus acting as anantagonist of the hCB2 cannabinoid receptor expressed inCHO cells [208] Furthermore very recently Govaerts et al[209] reported an inverse agonist effect of ∆9-THC and ∆8-THC on hCB2-CHO cells using the [35S]-GTPγ S assayThe pEC50 values were 763 and 888 respectively whilethe Emax values were ndash27 and ndash16 as compared to basal

Griffith from Pfizer described three new series of CB1cannabinoid receptor antagonists based on a bicyclic centralaromatic ring Purine pyrazoltriazines andpyrazolo[15a]pyrimidines derivatives were synthesised andinvestigated for their ability to interact with the CB1cannabinoid receptor Albeit a [35S]-GTPγ S assay wasdescribed no data were given concerning the antagonist orinverse agonist properties of such compounds Two hundredand eighty purine derivatives were synthesised [204] Theaffinities of 163 and 164 were given in the patent (28 and12 nM [3H]-SR141716A) In a following patent thesynthesis of sixty pyrazoltriazine derivatives was described(165 Fig 15) [205] The affinity of each compound was notgiven but was said to be ranging between 01 and 590 nMAnd finally more than forty pyrazolo[15a]pyrimidines weresynthesised (166 Fig 15) [206] Their affinity for the CB1cannabinoid receptor was lower than 155 nM Despite thefact that a great number of derivatives share the samesubstitution pattern around the central bicyclic aromatic ringthe comparison of these three new series of compounds ismade difficult by the absence of precise affinity data

Some other classical cannabinoids were shown to possessan antagonist activity at the CB2 receptor This is the casefor instance for the non-selective O-1184 (2) (Ki = 74 nM)which enhances the forskolin-induced cAMP production inCB2-CHO cells (EMax = 161 EC50 = 63 nM) and alsofor O-584 (3) (EMax = 246 EC50 = 138 nM) [39]

2 Indole Derivatives

Since the disclosure in 1993 of a second cannabinoidreceptor many attempts have been made to synthesise CB2cannabinoid receptor selective ligands The first compoundreported to have a somehow greater affinity for the peripheralcannabinoid receptor was WIN-55212-2 The compoundwas introduced by DrsquoAmbra and colleagues [210] back in1992 as a new cannabinoid ligand having a nanomolaraffinity and acting as an agonist In 1996 Showalter et alused a transfected cell line expressing the CB2 cannabinoidreceptor in order to identify selective ligands for thisreceptor [211] They showed that the CB2 affinity of WIN-55212-2 was 7 times greater than the CB1 with Ki valuesof 189 and 028 nM for the CB1 and CB2 cannabinoidreceptors respectively However WIN-55212-2 is anagonist of the cannabinoid receptors Interestingly WIN-55212-3 (167 Fig 16) the so-called inactive enantiomer ofWIN-55212 was recently shown to behave as a low affinity(Ki=363 microM) inverse agonist of the hCB2 cannabinoidreceptor as it decreased the [35S]-GTPγ S binding [209]

III CB2 LIGANDS RECEPTOR ANTAGONISTS ANDINVERSE AGONISTS

The compounds acting as antagonist or inverse agonistsat the CB2 cannabinoid receptor are reviewed in this thirdpart of the paper As for the CB1 cannabinoid receptorligands they are classified depending on their chemicalstructures

1 Classical Cannabinoid Derivatives

The first report of an antagonism mediated by ∆9-THC atthe peripheral cannabinoid receptor was made by Bayewitch

N N

NN

NH COOH

Cl

Cl

N N

NN

N

Cl

Cl

NH2OH

O

N

N

N

N

N

NHNH2

O

Cl

ClN

N

N

N

Cl

Cl

N

O

163 164

165 166

Fig (15) CB1 cannabinoid receptor antagonists 10 Other derivatives Examples of purine (163-164) pyrazoltriazines (165) andpyrazolo[15-a]pyrimidines (166) derivatives described as CB1 cannabinoid receptor antagonists by Griffith

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1383

The pEC50 value was 554 and the Emax value ndash26 ascompared to basal

AM630 also acts as an inverse agonist at the hCB1cannabinoid receptor (EC50 = 900 nM) as Landsman et aldemonstrated [47] Recently Zhang and colleagues reportedthe characterisation of the microsomal metabolism of AM630[212]

N

O

N

O

O

N

O

Cl

O

N

O

(S)-WIN-55212-3 167 BML-190 168

In 1996 Gallant and colleagues identified by submittinga large number of compounds to a binding assay anotherindole analog as CB2 cannabinoid receptor ligand [213] Onthe basis of this compound christened BML-190 (168 Fig16) or called indomethacin morpholinylamide theysynthesised several derivatives that possessed a selectivityratio for the CB2 cannabinoid receptor of up to 140 Thesewere the first compounds specifically designed to be CB2cannabinoid receptor ligands The first report on thepharmacological properties of BML-190 appeared onlyrecently New and colleagues highlighted in the paper theinverse agonist properties of the compound BML-190 dose-dependently increases (103 EC50= 980plusmn70 nM) theforskolin-stimulated levels of cAMP in hCB2-HEK cellswhile WIN-55212-2 decreases (44 45plusmn42nM) thisaccumulation [137]

Fig (16) CB2 cannabinoid receptor antagonists 2 Indolederivatives Chemical structures of WIN-55212-3 (167) andBML-190 (168) two indoles derivatives acting as inverseagonists at the hCB2 cannabinoid receptor Surprisingly in 2002 Melck and collaborators used

BML-190 as a CB2 receptor agonist in a cell proliferationassay [214] BML-190 was also tested as inhibitor of thecyclooxygenase-2 enzyme by Kalgutkar et al [215] Theyobtained IC50 values higher than 33 and 66 microM for theCOX-I and COX-II enzymes respectively These valueshave to be compared with the submicromolar activity ofindomethacin the parent compound Very recently Klegerisand colleagues [216] looking for an antineurotoxic action ofcannabinoids showed that BML-190 increases TNF-αsecretion by stimulated THP-1 monocytic cells but is noteffective on the IL-1β secretion

Further on the basis of WIN-54461 (12) a CB1antagonist AM630 (13) was synthesised by replacement ofthe bromine atom by an iodine one In the initial reportPertwee et al showed the antagonist effects of AM630 butalso suggested that the CB1 cannabinoid receptor may not bethe preferential receptor of AM630 [45] Later on Ross et alreported using hCB1amp2-CHO cells and [3H]-CP-55940that AM630 binds to the CB2 cannabinoid receptor (Ki =312 nM) with a selectivity ratio over 160 [48]Furthermore using a [35S]-GTPγ S assay they showed itsinverse agonist properties (EC50 = 76 nM) However

Table 12 CB2 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Reported binding affinities (Ki) and selectivity ratio (CB1CB2) of SR144528 (169) for rat and human cannabinoid receptors determined using [3H]-CP55940 as radioligand

NN

O

NH

Cl

SR144528 169

rCB2 (spleen) rCB1 (whole brain) Selectivity Ki hCB2 (transfected CHOcells)

Ki hCB1 (transfected CHOcells)

Selectivity References

038 nM 305 nM 800 06 nM 437 nM 730 [217]

067 nM 548 nM 80 [219]

56 nM gt10microM gt1780 [48]

25 nM a gt 1microM a gt400 [224]

024 nM 276 nM 115 199 nM 503 nM 25 [232]

a COS cells

1384 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

3 Diarylpyrazole Derivatives CHO cells is constitutively active and phosphorylated onSer 352 Upon treatment by SR144528 thisphosphorylation of the receptor was inhibited Moreover theSR compound was able to induce dephosphorylation ofagonist-induced CB2 phosphorylation The question whetherSR144528 induces a CB2 receptor conformational changemaking it a better phosphatase-specific substrate or ifSR144528 binding activates specific phosphatases remainsopen However this remains a nice example of inverseagonist-induced receptor resensitisation

The same Sanofi team which synthesised the CB1antagonist SR141716A reported in 1998 the synthesis andpharmacological characterisation of a CB2 cannabinoidreceptor selective antagonist the SR144528 (169) [217218] The structure of the new compound is derived fromSR141716A one SR144528 is the first selective CB2receptor antagonist reported as the inverse agonist propertiesof BML-190 were shown only recently by New et al [137]The authors reported for 169 using [3H]-CP-55940 asradioligand Ki values of 04 nM and 06 nM for the rat andthe human CB2 receptors respectively and found a CB1-CB2 selectivity ratio around 700 Table 12 sums up thebinding affinities of SR144528 for the cannabinoid receptorsobtained by different authors Interestingly while the CB2affinity is more or less constant the affinity obtained for theCB1 receptor varies by one order of magnitude [219] Theantagonist effect of SR144528 was highlighted by evaluatingits effect on cAMP production by hCB2-CHO cells exposedto 3 nM CP-55940 (EC50=10nM) The antagonism wasalso observed on the activation of the MAP kinase pathwayby CP-55940 (IC50=39nM)

The Sanofi research team working on cannabinoidsconducted investigations in order to determine the keyresidues for SR144528 interaction with the receptor (Table13) as well as the binding mode of the compound Firstlythey identified the TM4-EL2-TM5 region as a regioncontaining crucial residues for the affinity of several ligandsamong them SR144528 [224] Among these residues themutation of two cysteine of the second extracellular loop(C174 and C179) abolishes the affinity for the CB2 receptorLater on the implication of S453(161) and S457(165) inthe binding of SR144528 was evidenced by mutationalstudies In the model proposed for the docking of SR144528into the CB2 receptor the crucial contacts are comprised intothe TM4 this is the case for S453(161) and S457(165)which are proposed to form hydrogen bonds with the ligandand W464(172) proposed to have aromatic interactions withthe 4-methylbenzyl of the SR144528 [225] In 2003 Fengand Song showed that point mutations D349(130)A andA634(241)E abolished ligand binding However mutationof the arginine R350(131) member of the highly conservedDRY motif to alanine has no influence on SR144528binding [226]

Early after the report of the synthesis and characterisationof SR144528 the same authors further explored the functionof this compound and showed its inverse agonist propertiesusing CB2-CHO cells co-transfected with the luciferasereporter gene with either the CRE or the murine krox24regulatory sequence [220] In these models SR144528 notonly inhibits CP-55940 effects but also reproduces theeffects of a pertussin toxin treatment proving its inverseagonist properties SR144528 functionality was alsoexplored by [35S]-GTPγ S binding in hCB2-CHO cells(IC50=3 nM) [221] The inverse agonist function ofSR144528 was further illustrated by Rhee and Kim usingCOS cells co-transfected with hCB2 receptor and adenylylcyclase [222]

As for the CB1 cannabinoid receptor ligands Sanofideveloped tricyclic derivatives based on the 1-benzylpyrazole-3-carboxylic scaffold More than twentycompounds were disclosed in a patent [227] in 2001 (84-86Table 7) The benzyl in position 4 characteristic of the CB2diarylpyrazoles from Sanofi is conserved as well as thebicyclic residue in position 3 The length of the second linkbetween the phenyl and the pyrazole can be made of one or

Bouaboula and co-workers investigated SR144528 effectson CB2 cannabinoid receptor phosphorylation status [223]Firstly they showed that the CB2 receptor transfected in

Table 13 CB2 Cannabinoid Receptor Antagonists 3 Di-Aryl-Pyrazole Derivatives

Some of the single point mutations reported for the hCB2 cannabinoid receptor for which pharmacological data concerning the SR144528 are available

Mutant Effect References

R350(131)A no effect [226]

S453(161)A loss of affinity [225]

V456(164)I no effect [225]

S457(165)A loss of affinity [225]

C174S a loss of affinity [224 225]

C175S a loss of affinity [225]

C177S a no effect [225]

C179S a loss of affinity [224 225]

S544(193)G no effect [225]

a Second extra-cellular loop

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1385

two methylene but according to the patent one methylenebridge is preferred The Ki values of these compounds for theCB2 receptor were said to be lower than 500 nM and theirantagonist properties have been assessed using the adenylatecyclase inhibition assay

compounds by the presence of an alkyl substituent inposition 5 instead of a phenyl and of course by position 3which remains unsubstituted However except a lecture atthe 1998 ACS meeting [230] where compound 171 wasreported to have a Ki value of 835 nM and a selectivity ratioover 100 no further report on these compounds waspublished untill now

In a recent paper Mussinu and colleagues reported thesynthesis of other tricyclic derivatives starting fromSR141716A structure [228] N-piperidin-1-yl-14-di-hydroindeno[12-c]pyrazole-3-carboxamide derivatives areCB2 receptor ligands and some of them possess high affinityand selectivity for the CB2 receptor as shown in Table 7 (79-83) These tricyclic derivatives differ from the Sanofi tricyclicderivatives as they do have a phenyl ring instead of a benzylin position 1 Another difference is the nature of thesubstituent in position 3 which is not a bicycloalkyl but acyclic amine One of the compounds synthesised byMussinu et al 81 possesses the highest affinity for the CB2receptor ever reported with a Ki value of 0037 nM for themurine CB2 receptor ([3H]-CP-55940 mice spleenhomogenate) Interestingly 79 the closest SR141716Aanalog lost almost all its affinity for the CB1 receptor andincreased by four orders of magnitude its affinity for the CB2receptor Even if the structure quite similar to the SRcompounds could suggest that these derivatives behave asantagonists no pharmacological data were given in the paperto support this hypothesis

5 2-Oxoquinoline Derivatives

Inaba and colleagues from Japan Tobacco introduced in2001 a completely new structure in the cannabinoid fieldbased on a 2-oxoquinoline scaffold Over eighty derivativeswere disclosed in a patent [231] and one compound JTE-907 (173) was subsequently characterised in-vitro as well asin-vivo [232] In Table 14 are listed some of the compounds(172-178) disclosed in the patent along with their affinity forthe cannabinoid receptors These values were obtained onhuman receptors against [3H]-WIN-55212-2 For twelvecompounds the anti-inflammatory activity per os wasassessed in a carrageenin-induced paw edema model Thehighest effect reported was obtained with JTE-907 with anED50 value lower than 01 mgkg Therefore thesecompounds were claimed to be useful as anti-inflammatoryand anti-allergic agents

To further characterize JTE-907 the authors determinedits affinity for cannabinoid receptors of different species using[3H]-CP-55940 as radioligand In their hands JTE-907behaved as a very selective hCB2 ligand with Ki values of36 and 2370 nM for the CB2 and CB1 cannabinoid receptorsrespectively even more selective than the SR144528 (Kivalues of 199 and 50 nM respectively) However it has tobe mentioned that the affinities reported in the paper forJTE-907 are quite different from those reported in the patent(hCB1 Ki values of 35nM and 009 nM respectively) Italso appears that the SR144528 affinity for the CB1 receptordetermined by Iwamura et al is higher than the affinityreported by Rinaldi-Carmona et al with values of 50 and437nM respectively The authors ascribed the difference tothe variability of SR144528 binding affinities to the assayconditions The function of JTE-907 was evaluated bymeasuring the increase in cAMP production by forskolin-stimulated hCB2-CHO cells Maximum stimulation wasattained at a concentration of 1microM of JTE-907 In the sameassay WIN-55212-2 decreased the cAMP production in adose-dependent manner

Finally it is interesting to strengthen that the five-membered ring derivatives (79-86) of the diarylpyrazolefamily are reported to possess a high CB2 receptor affinitywhile the six- and seven-membered ring derivatives (69-78)are described as CB1 receptor ligands

4 Other Pyrazole Derivatives

Other CB2 ligands based on a pyrazole nucleus wereintroduced by SmithKline Beecham in a patent describingthe synthesis of about sixty new compounds [229] Theiraffinity (Ki) for the hCB2 receptor transfected in HEK293cells was reported to be ranging between 25nM and 10microMTheir structures (170-171 Fig 17) differ from the Sanofi

NN

O

N

ON

NN

N

NN

O

N

ON

NN

N

ClCl

1

5

4 3

2

170 171

Fig (17) CB2 cannabinoid receptor antagonists 4 Otherpyrazole derivatives Structure of two compounds (170-171)among those claimed by SmithKline Beecham to be hCB2antagonists

In conclusion JTE-907 was shown to be a CB2 selectiveinverse-agonist with anti-inflammatory and anti-allergicproperties This compound is currently undergoing Phase Iclinical trials as anti-allergic drug [233]

IV NON-CB1 NON-CB2 CANNABINOIDRECEPTORS LIGANDS

In the last years the possible presence of additional non-CB1 non-CB2 cannabinoid receptors was raised frompharmacological evidences

One of the putative receptors possesses an endotheliallocalisation while other ones are localized in the CNS or atperipheral nerves For instance an additional target for ∆9-

1386 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 14 CB2 Cannabinoid Receptor Antagonists 5 2-Oxoquinoline Derivatives

Structure of six compounds among the oxoquinoline derivatives patented by Japan Tobaco Affinity (Ki nM) and selectivity (CB1CB2) for the cannabinoidreceptors are given The affinity was measured by binding against [3H]-WIN-55212-2 on either hCB2-CHO or hCB1-CHO cells Adapted from [231]

Cpd ndeg Structure hCB2 hCB1 Selectivity

1-2 172

NH

MeO O

O

OMeO

0014 3671 262215

JTE-907 173

NH

O

MeO O

O

NH

O

O

0087 3436 39495

2-2 174

OH

O

NH

MeO O

O 077 3247 4215

3-27 175

NH

O

F

NH

MeO O

O0021 336 16000

3-32 176

NH

MeO O

O

NH

F

O

0036 2398 66610

3-38 177

O

O

NH

N

O

O

NMeO

0085 935 11000

3-43 178

NMeO O

O

NH

F

O

0043 864 20100

tetrahydrocannabinol was described in capsaicin-sensitivesensory nerves by Zygmunt et al [234]

[29] and by Di Marzo et al [235] Thus in the presentreview we will focus on the description of the so faridentified ligands of the non-CB1 non-CB2 receptors withan emphasis on the compounds having antagonist properties

Pharmacological evidences for the existence of additionalcannabinoid receptors were reviewed by Wiley and Martin

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1387

(Table 15) In particular the knowledge about three not yetcloned targets has grown The growing molecularpharmacology of the three putative new cannabinoidreceptors is reviewed in the three following sections Thefourth section summarises the data on the other proposedcannabinoid targets

vasodilatator activity In addition the anandamide effect ispartially sensitive to SR141716A inhibition The authorsproposed the existence of two distinct receptors responsiblefor the vasodilatatory effect of anandamide one located in theendothelium is SR141716A-sensitive while the other onelocated on the vascular smooth muscle is SR141716A-insensitive Thus SR141716A is an antagonist of anendothelial anandamide receptor Several studies wereconducted in order to understand the molecularpharmacology of the endothelial anandamide receptor It ispertussis toxin-sensitive [237] Cannabidiol acts asantagonist and abnormal-cannabidiol (Abn-cbd 179) and itsanalogue O-1602 (180) act as agonists [238] The synthesisof O-1602 and of its analogues are disclosed in a patentdescribing the vasodilatatory activity of cannabinoid

1 Endothelial Anandamide Receptor ndash Abnormal-Cannabidiol Receptor

In 1999 Wagner et al showed that anandamide couldinduce mesenteric vasodilatation through an endotheliallylocated ldquoanandamide receptorrdquo pharmacologically distinctfrom CB1 [236] Other cannabinoids such as ∆9-THC HU-210 or WIN-55212-2 were devoid of mesenteric

Table 15 Non-CB1 Non-CB2 Cannabinoid Receptors Antagonists

An overview of the putative new non-CB1 non-CB2 anandamide-receptors ligands The antagonists are highlighted in bold

NH

N

S

HO OH

Cl

OH

OH

OH OH OH OH

OMe

OMe

RR

RR

RR

RR

HO

OH

NH

O

cannabidiol (10) Abnormal-cannabidiol 179 O-1602 180

O-1918 181 capsazepine 183capsaicin 182

Compound Endothelial AEA receptor Brain AEA receptor Cannabinoid-vanilloid brain receptor

AEA agonist a agonist de

WIN-55212-2 no effecta

agonist e agonist g h

CP-55940 no effect e agonist i

SR141716A antagonist a b j no effect ([35 S]-GTPγS binding) d e f antagonist g h

AM251 no effect k no effect h i

Cannabidiol antagonist b no effect e

O-1918 antagonist c jm

Abd-cbd agonist b l

O-1602 agonist b

Capsaicin no effectj agonist i

Capsazepine no effect b antagonist h

a [236] b [238] c [240] d [249] e [250] f [251] g [254] h [258] i [ 255] k [244] j [ 243] l [ 242] m [241]

1388 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

analogues [239] More recently Offertaler et al showed thatO-1918 (181) a cannabidiol analog acts as an antagonist ofthe endothelial anandamide receptor [240] In addition O-1918 does not bind to the CB1 and CB2 cannabinoidreceptors Furthermore O-1918 unlike cannabidiol doesnot cause vasorelaxation in the mesenteric artery preparationmodel used and thus acts as a true neutral antagonistUsing O-1918 as a pharmacological tool Begg et alproposed a coupling of this new G protein-coupledendothelial receptor through a GiGo mechanism to ionchannels in primary cultured human vascular endothelialcells [241] However in their hands SR141716A wasineffective in antagonising Abd-cbd Ho and Hiley furtherdemonstrated that Abn-cbd relaxes rat small mesentericarteries through an activation of K+ channels via aSR141716A-sensitive pathway [242] Further using thesame model they characterized the vasorelaxant activity ofvirodhamine a novel endocannabinoid apparently actingthrough the same receptor [243] The virodhamine-inducedvasorelaxation is sensitive to SR141716A and O-1918 butinsensitive to AM251 SR144528 and AM630 Moreovervanilloid receptor desensitisation by capsaicin (182) had noeffect

244] Thus position 4 on the 5-phenyl could be furtherexplored to obtain selective ligands

To come to an end despite the fact that the endothelialanandamide receptor is neither identified nor cloned at leastthree antagonists are identified SR141716A cannabidioland O-1918 It is likely that a growing number of ligandsand therapeutic applications will appear in the very nearfuture

2 Brain Anandamide Receptor

Following an in-depth evaluation of anandamide effects inCB1

-- mice Di Marzo and co-workers suggested that a non-CB1 non-CB2 G protein-coupled receptor might mediatesome of the actions of AEA in mice [249] Actually effectsof AEA but not those of ∆9-THC were not decreased inCB1 cannabinoid receptor knock-out mice For instance 30mgkg of AEA were still effective in hot-plate test whereas10 mgkg of ∆9-THC had no effect on anti-nociceptionMoreover in a [35S]-GTPγ S assay AEA stimulated with anEC50 value of 223 microM the [35S]-GTPγ S binding to CB1

--

mice brain membranes (Emax= 28) ∆9-THC showed noeffect in this assay The addition of SR141716A did notaffect the [35S]-GTPγ S binding to CB1

-- mice brainmembranes Shortly after other evidences for the existence ofa new G protein-coupled cannabinoid receptor with a distinctdistribution in the central nervous system were published[250] From a set of 24 commonly used cannabinoids (AEAWIN-55212-2 JWH-030 THChellip) only AEA and WIN-55212-2 produced an increased binding of [35S]-GTPγ S onCB1

-- mice brain membranes Interestingly in this studySR141716A alone produced a significant inhibition of [35S]-GTPγ S binding similar to the inhibition obtained usingCB1

++ mice brain membranes (IC50 values of 57 microM and47 microM respectively) However SR141716A did not affectthe anandamide-induced [35S]-GTPγ S binding in CB1

--

mice brain membranes Moreover [3H]-SR141716Aexhibited significant binding in some brain regions of CB1

--

mice which are different from those exhibiting stimulation of[35S]-GTPγ S binding by WIN-55212-2 and AEA Thusthe authors proposed that the SR141716A effects on CB1

--

mice are mediated neither by the CB1 cannabinoid receptornor by the putative new anandamide brain receptor Monoryand co-workers using CB1

-- mice described a novel nonadenylyl cyclase-coupled cannabinoid binding site in micecerebellum [251] Two agonists AEA and WIN-55212-2but not ∆9-THC or HU210 were found to bind to thisreceptor as they enhanced [35S]-GTPγ S binding NeitherSR141716A nor SR144528 were able to affect the WIN-55212-2 induced [35S]-GTPγ S binding in CB1

-- mice asfound by Breivogel et al [250] However the distributionpattern of the receptor described by Monory et al differs fromthe distribution found by Breivogel et al It has to bementioned that the knock-out strains used were different Themice used by Monory were from a CD-1 strain while theone used by Breivogel were from a C57BL61 strainInterestingly Muthane et al detected differences in nigralneurons number and sensitivity to MPTP in the two mousestrains used [252]

More recently Mo et al obtained results showing thatthe Abnormal-cannabidiol receptor is also responsible for anincreased endothelial cell migration [244] Abn-cbd enhancedhuman umbilical vein endothelial cell migration while O-1918 antagonised Abn-cbd effect The CB1 cannabinoidreceptor inverse agonist SR141716A partially inhibitedAbd-cbd action whereas AM251 and SR144528 had noeffect

In another paper from George Kunos team Batkai et alreported that SR141716A is able to inhibit the endotoxichypotension by a cardiac mechanism involving neither theCB1 nor the CB2 cannabinoid receptors [245] Theinvolvement of a non-CB1 non-CB2 site of action wasshown using CB1

-- and CB1--CB2

-- knock-out miceFurther studies are needed to identify the SR141716Asensitive myocardial site of action

In a recent patent Kunos described O-1918 and itsanalogues as vasoconstrictor agents useful to reversepathological vasodilatation of blood vessels [246] Theeffects of O-1918 on mouse blood pressure on LPS-inducedhypotension in mouse as well as the reversal of hypotensionby O-1918 injection are described Furthermore thesynthesis of O-1918 based antagonists is described startingfrom p-metha-28-dien-1-ol and the appropriate resorcinols

Interestingly the Abd-cbd receptor which was firstdetected in the vascular system also seems to be present inthe CNS [247] Indeed Walter et al found that microglialcells (BV-2) migration induced by 1 microM 2-AG is inhibitedby cannabidiol (300 nM) and O-1918 (1 microM) SR141716Awas found ineffective in this assay Thus 2-AG is an agonistof the Abd-cbd receptor Moreover Abd-cbd is able to elicita dose-dependent cell migration in the same model (EC50 =600nM) [248]

From a more structural point of view SR141716Abehaves as an antagonist while on the opposite AM251(20) a close analogue which differs from the former only bythe nature of one halogen has no effect on this receptor [242

To conclude to our knowledge no compounds havingantagonist or inverse agonist properties at this receptor havebeen identified so far

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1389

3 Cannabinoid-Vanilloid Brain Receptor a cannabinoid-vanilloid receptor at which SR141716A andcapsazepine behave as antagonists

Evidences for the presence of an additional ldquocannabinoidrdquoreceptor differing from the CB1 receptor and from the brainanandamide receptor appeared from studies focused on thehippocampus (for a review see Hajos et al [253]) Hajos etal studied the cannabinoid actions on GABAergic andglutamatergic transmission using the whole-cell patch-clamp technique [254] WIN-55212-2 induced a dose-dependent reduction in glutamatergic transmission reversedby SR141716A (1microM) Thus the authors concluded that thecannabinoid actions on the glutamatergic transmission aremediated by a non-CB1 but SR141716A-sensitive receptorIn another paper Hajos and Freund further characterised thisputative receptor [255] They showed that in addition toWIN-55212-2 CP-55940 and capsaicin (182) a TRPV1receptor agonist also act as agonists Furthermore in theirhands SR141716A as well as capsazepine (183) a TRPV1receptor antagonist were able to antagonise this effect on theglutamatergic transmission while AM251 was notSR141716A effects on CB1 knock-out mice were furtherstudied using a test of anxiety [256] Haller et al showedthat on the one hand the distribution of the CB1 receptorinduces anxiety and SR141716A has an anxiolytic effectand on the other hand SR141716A is still active in CB1

--

mice In a following paper they reported that AM-251induces anxiogenic effects in wild-type and in knock-outmice [257] Thus SR141716A in addition to CB1 bindsto a not yet identified receptor Another evidence for thisadditional receptor for SR141716A was given by Bass andco-workers [61] They conducted a structure-activityrelationship study on the stimulation of locomotor activityinduced by SR141716A and twenty analogues Nocorrelation was found between the affinity for the CB1cannabinoid receptor and the stimulation of locomotoractivity Albeit SR141716A and five of its analogues O-1803 O-1710 O-1253 O-1254 (40) O-1255 (41) behavedas inverse agonists in [35S]-GTPγ S assay none of theseanalogues stimulated the locomotor activity In conclusionthe SR141716A-induced stimulation of locomotor activity isneither due to inverse agonism nor to inhibition of anendogenous tone but to a brain receptor distinct from theCB1 receptor Di Marzo et al previously provided evidencesthat the cannabinoid-vanilloid brain receptor could mediatesome of the cannabinoid effects on locomotion [249]

4 Other Non-CB1 Non-CB2 Cannabinoid Receptors

Evidences for the existence of additional non-CB1 non-CB2 cannabinoid receptors localised in the rat isolatedmesenteric arterial bed appeared from the works of Ralevicand collaborators Several cannabinoids were evaluated intheir model of sensory neurogenic vasorelaxation evoked byelectrical field stimulation

HU210 was found to attenuate vasorelaxation in a CB1and CB2 independent way as SR141716A LY320135 andSR144528 were ineffective in inhibiting HU210 effect [259]Similar results were obtained with ∆9-THC [260]Similarly noladin ether inhibited sensory neurogenicrelaxation in a concentration-dependent manner which wasalso unaffected by SR141716A LY320135 and SR144528Moreover this effect as the ∆9-THC one was pertussistoxin sensitive suggesting the involvement of a Gio protein-coupled receptor [261]

However the inhibitory effects of CP-55940 and WIN-55212-2 were affected by the presence of SR141716A (1microM)or LY320135 (1microM) but not by the addition of SR144528(1microM) [262]

It appears that at least two receptors should be involvedin cannabinoid-mediated attenuation of sensory nerve-mediated vasorelaxation The first one is SR141716A andLY320135 sensitive while the other one is resistant toSR141716A

Some evidences for the existence of a ldquoCB2-likerdquo receptorappeared from the studies on palmitoylethanolamide (PEA)an endogenous fatty acid amide devoid of CB1 and CB2affinity [264] antinociceptive potential Calignano and co-workers showed that PEA was able to alleviate pain inseveral animal models [265 266] For instance PEA dose-dependently inhibits kaolin-evoked writhing in mice TheCB2 cannabinoid receptor inverse agonist SR144528 (02mgkg) inhibits PEA anti-nociceptive effect [266] Thus theauthors suggest that PEA acts as agonist on a SR144528-sensitive non-CB2 cannabinoid receptor (ldquoCB2-likerdquoreceptor) at which SR144528 acts as an antagonist

Further studies are needed in order to better characterisethese additional ldquocannabinoidrdquo receptors However it islikely that several therapeutic applications will soon springfor these receptors

Very recently Pistis et al published a study on theneurophysiological effects of cannabinoids on the basolateralamygdala neurons in-vivo [258] One of their findings is thatHU-210 and WIN-55212-2 did not elicit similar effects onbasal amygdala projection neurons firing rate While theformer had no effect the latter decreased the firing rateMoreover SR141716A reversed the WIN-55212-2 effectswhile AM251 had no effect The vanilloid receptorantagonist capsazepine also antagonised the WIN-55212-2effect on firing rate of these neurons Therefore the authorssuggested the presence of a novel cannabinoid-vanilloidreceptor

V CONCLUSION

Since their development cannabinoid antagonists provedto be essential tools in the understanding of cannabinoidpharmacology and biochemistry Moreover the interestshown by the pharmaceutical industry in the development ofnew cannabinoid antagonists prove if necessary the realtherapeutic potential of this class of compounds Thetreatment of eating and movement disorders memorydeficits psychosis and dependencies from various addictivedrugs are some of the most cited applications for the CB1cannabinoid receptor antagonists

From these lines it appears that in addition to theknown CB1 cannabinoid receptor it is now likely that twoother cannabinoid receptors do exist in the brain The firstone should be an anandamide brain receptor for whichSR141716A has no affinity while the second one should be

1390 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Much less wide is the knowledge on the CB2cannabinoid receptor antagonists However potentialapplications could be disorders involving the immunesystem such as inflammation or allergies

[19] Devane WA Hanus L Breuer A Pertwee RG StevensonLA Griffin G Gibson D Mandelbaum A Etinger AMechoulam R Science 1992 258 1946

[20] Mechoulam R Ben-Shabat S Hanus L Ligumsky MKaminski NE Schatz AR Gopher A Almong S MartinBR Compton DR Biochem Pharmacol 1995 50 83

Finally the growing literature on the non CB1 non CB2cannabinoid receptors should allow the development of newdrugs First examples are given by the cannabidiol andabnormal-cannabidiol derivatives described by Kunos et alfor which potential therapeutic applications on the vascularsystem are already patented

[21] Sugiura T Kondo S Sukagawa A Nakane S Shinoda AItoh K Yamashita A Waku K Biochem Biophys Res Co1995 215 89

[22] Hanus L Abu-Lafi S Fride E Breuer A Vogel Z ShalevDE Kustanovich I Mechoulam R Proc Natl Acad SciUSA 2001 98 3662

[23] Oka S Tsuchie A Tokumura A Muramatsu M Suhara YTakayama H Waku K Sugiura T J Neurochem 2003 851374It clearly appears from this paper that the antagonists of

the cannabinoid receptors are still an active research fieldfrom which should emerge new promising therapeutic toolsas well as innovative drugs in the near future

[24] Goutopoulos A and Makriyannis A Pharmacol Therapeut2002 95 103

[25] Beal JE Olson R Lefkowitz L Laubenstein L Bellman PYangco B Morales JO Murphy R Powderly W PlasseTF Mosdell KW Shepard KV J Pain Symptom Manage1997 14 7ACKNOWLEDGEMENTS [26] Notcutt W Price M Miller R Newport S Phillips CSimmons S Sansom C Anaesthesia 2004 59 440

GGM is very indebted to the ldquoFonds pour la formationagrave la Recherche dans lrsquoIndustrie et lrsquoAgriculture (FRIA)rdquo Belgium for the award of a research fellowship Theexperimental works of the authors were supported by grantsfrom the FNRS The Belgian Charcot Foundation and theUniversiteacute catholique de Louvain

[27] Ledent C Valverde O Cossu G Petitet F Aubert JFBeslot F Bohme GA Imperato A Pedrazzini T RoquesBP Vassart G Fratta W Parmentier M Science 1999 283401

[28] Zimmer A Zimmer AM Hohmann AG Herkenham MBonner TI Proc Natl Acad Sci U S A 1999 96 5780

[29] Wiley JL and Martin BR Chem Phys Lipids 2002 121 57[30] Adam J and Cowley P Expert Opin Ther Patents 2002 12

1475

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[33] Little PJ Compton DR Johnson MR Melvin LS MartinBR J Pharmacol Exp Ther 1988 247 1046

[2] Devane WA Dysarz FA Johnson MR Melvin LSHowlett AC Mol Pharmacol 1988 34 605

[34] Pertwee RG Stevenson LA Elrick DB Mechoulam RCorbett AD Brit J Pharmacol 1992 105 980

[3] Matsuda LA Lolait SJ Brownstein MJ Young ACBonner TI Nature 1990 346 561

[35] Harrison C Traynor JR Life Sci 2003 74 489-508[4] Gerard CM Molerau C Vassart G Parmentier M BiochemJ 1991 279 129 [36] Pertwee RG Fernando SR Griffin G Ryan W Razdan

RJ Compton DR Martin BR Eur J Pharmacol 1996 315195

[5] Glass M Dragunow M Faull RLM Neuroscience 1997 2299

[37] Ross RA Brockie HC Fernando SR Saha B Razdan RKPertwee RG Brit J Pharmacol 1998 125 1345

[6] Shire D Carillon C Kaghad M Calandra B Rinaldi-Carmona M LeFur G Caput D Ferrara P J Biol Chem1995 270 3726 [38] Griffin G Wray EJ Rorrer WK Crocker PJ Ryan WJ

Saha B Razdan RK Martin BR Abood ME Brit JPharmacol 1999 126 1575

[7] Ryberg E Khang Vu H Groblewski T Larsson N ElebringT Hjorth S Sjogren S Greasley PJ 2004 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 119

[39] Ross RA Gibson TM Stevenson LA Saha B Crocker PRazdan RK Pertwee RG Brit J Pharmacol 1999 128 735

[40] Martin BR Winckler JR Wiley JL Huffman JW CrockerPJ Saha B Razdan RK J Pharmacol Exp Ther 1999 2901065

[8] Rinaldi-Carmona M Calandra B Shire D Bouaboula MOustric D Barth F Casellas P Ferrara P LeFur G JPharmacol Exp Ther 1998 278 871

[41] Martin BR Stevenson LA Pertwee RG Breivogel CSWilliams A Mahadevan A Razdan RK 2002 Symposium onthe Cannabinoids Burlington Vermont International CannabinoidResearch Society page 2

[9] Munro S Thomas KL Abu-Shaar M Nature 1993 365 61[10] Howlett AC Qualy JM Khachatrian LL Mol Pharmacol

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[42] Thomas A Ross RA Saha B Mahadevan A Razdan RKPertwee RG Eur J Pharmacol 2004 487 213

[12] Calandra B Portier M Kerneacuteis A Delpech M Carillon C LeFur G Ferrara P Shire D Eur J Pharmacol 1999 374445 [43] Compton DR Gold LH Ward SJ Balster RL Martin BR

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Daum SJ Ackerman JH Gruett MD Kumar V EstepKG Olefirowicz EM Wetzel JR Alexander MD WeaverJD Haycock DA Luttinger DA Casiano FM ChippariSM Kuster JE Stevenson JI Ward SJ J Med Chem1995 38 3094

[14] Deadwyler S Hampson RE Mu J Whyte A Childers SRJ Pharmacol Exp Ther 1995 273 734

[15] Mackie K Lai Y Westenbroek R Mitchell R J Neurosci1995 15 6552

[16] Bouaboula M Poinot CC Bourrie B Canat X Calandra BRinaldi-Carmona M LeFur G Casellas P Biochem J 1995312 637 [45] Pertwee R Griffin G Fernando S Li X Hill A

Makriyannis A Life Sci 1995 56 1949[17] Bouaboula M Poinot CC Marchand J Canat X Bourrie BRinaldi-Carmona M Calandra B LeFur G Casellas P Eur JBiochem 1996 237 704

[46] Hosohata Y Quock RM Hosohata K Makriyannis AConsroe P Roeske WR Yamamura HI EurJPharmacol 1997 321 R1[18] delPulgar T Velasco G Guzman M Biochem J 2000 347

369 [47] Landsman RS Makriyannis A Deng H Consroe P RoeskeWR Yamamura HI Life Sci 1998 62 109

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[48] Ross RA Brockie HC Stevenson LA Murphy VLTempleton F Makriyannis A Pertwee RG Brit JPharmacol 1999 126 665

[81] Gatley SJ Lan R Volkow ND Pappas N King P WongCT Gifford AN Pyatt B Dewey SL Makriyannis A JNeurosci 1998 70 417

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B Congy C Martinez S Maruani J Neliat G Caput DFerrara P Soubrieacute P Breliegravere JC Le Fur G FEBS Lett1994 350 240

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[182] Lange JHM van Stuivenberg HH Coolen HKAC AdolfsT JP McCreary AC Keizer HG Walls HC Veerman WBorst AJM de Loff W Verveer PC Kruse CG J MedChem 2005 48 1823

[212] Zhang Q Ma P Wang W Cole RB Wang G J MassSpectrom 2004 39 672

[213] Gallant M Dufresne C Gareau Y Guay D Leblanc YPrasit P Rochette C Sawyer N Slipetz D M Tremblay NBioorg Med Chem Lett 1996 6 2263[183] Lange JHM Kruse CG Tipker J Hoogendorn J World

Patent Application 2002 WO02076949 [214] Melck D De Petrocellis L Orlando P Bisogno T Laezza CBifulco M Di Marzo V Endocrinology 2000 141 118[184] Lange JHM Coolen H K A C van Stuivenberg HH

Dijksman JAR Herremans AHJ Ronken E Keizer HGTipker K McCreary AC Veerman W Wals HC Stork BVerveer PC den Hartog AP de Jong NMJ Adolfs TJPHoogendoorn J Kruse CJ J Med Chem 2004 47 628

[215] Kalgutkar AS Marnett AB Crews BC Remmel RPMarnett LJ J Med Chem 2000 43 2860

[216] Klegeris A Bissonnette CJ McGeer PL Brit J Pharmacol2003 139 775

[185] httpwwwsolvaypharmaceuticalscomhtmlsciencepipelitext-html (accessed march 2004)

[217] Rinaldi-Carmona M Barth F Millan J Derocq JM CasellasP Congy C Oustric D Sarran M Bouaboula M CalandraB Portier M Shire D Breliere JC Le Fur GL JPharmacol Exp Ther 1998 284 644

[186] Wilstermann JM and Berggren AIK World PatentApplication 2003 WO03051850

[187] Berggren AIK Bostrom SJ Elebring ST Greasley PTerricabra E Wilstermann JM World Patent Application 2003WO03051851

[218] Barth F Millan J Casellas P Oustric D Sarran M RinaldiM World Patent Application 1997 WO9721682

[219] Griffin G Wray EJ Tao Q McAllister SD Rorrer WKAung MM Martin BR Abood ME Eur J Pharmacol 1999377 117

[188] Finke PE Meurer LC Debenham JS Toupence RBWalsh TE World Patent Application 2003 WO03082191

[189] Barth F Martinez S Rinaldi-Carmona M World PatentApplication 2003 WO03084930

[220] Portier M Rinaldi-Carmona M Pecceu F Combes T Poinot-Chazel C Calandra B Barth F Le Fur G Casellas P JPharmacol Exp Ther 1999 288 582[190] Barth F Martinez S Rinaldi-Carmona M World Patent

Application 2003 WO03084943 [221] Bouaboula M Desnoyer N Carayon P Combes T CasellasP Mol Pharmacol 1999 55 473[191] Kopka I E Li B Hagmann WK World Patent Application

2004 WO04029204 [222] Rhee M-H and Kim S-K J Vet Sci 2002 3 179[192] Hagmann WK Lin LS Shah SK Guthikonda RN Qi H

Chang LL Liu P Armstrong HM World Patent Application2003 WO03077847

[223] Bouaboula M Dussossoy D Casellas P J Biol Chem 1999274 20397

[224] Shire D Calandra B Bouaboula M Barth F Rinaldi-Carmona M Casellas P Ferrara P Life Sci 1999 65 627[193] Hagmann WK Lin LS Shah SK Goulet MT Jewell JP

World Patent Application 2003 WO03082190 [225] Gouldson P Calandra B Legoux P Kerneis A Rinaldi-Carmona M Barth F Le Fur G Ferrara P Shire D Eur JPharmacol 2000 401 17

[194] Castonguay LA Hagmann WK Lin LS Shah SK WorldPatent Application 2003 WO03086288

[195] Hagmann WK Lin LS Shah SK World Patent Application2003 WO03087037

[226] Feng W and Song ZH Biochem Pharmacol 2003 63 1077[227] Barth F Millan J Oustric D Rinaldi M Vernhet M World

Patent Application 2001 WO0132629[196] Berggren AIK Bostrom SJ Cheng L Elebring STGreasley P Nagard M Wilsterlmann JM Terricabras EWorld Patent Application 2004 WO04058249

[228] Mussinu JM Ruiu S Mule AC Pau A Carai MA LorigaG Murineddu G Pinna GA Bioorg Med Chem 2003 11251[197] Guba W Haap W Marty HP Narquizian R United States

Patent Application 2004 US 20040147572 [229] Xiang JN Elliott JD Atkinson ST Christensen SB WorldPatent Application 1998 WO9831227[198] Jagerovic N Hernandez Folgado L Alkorta I Goya P

Navarro M Serrano A de Fonseca FR Dannert MTDannert MT Alsasua A Suardiaz M Pascual D MartinMI J Med Chem 2004 47 2939

[230] Xiang JN Atkinson ST Wang DY Lee J Lee JCSiegfried B 216th ACS meeting Boston (MA) USA 23-27 Aug1998

[199] Jagerovic N Goya Laza MP Hernandez Folgado L AlcortaOsoro IJ Martin Fontelles MI Suardiaz Garcia MLDannert MT World Patent Application 2003 WO03082833

[231] Inaba T Kaya T Iwamura H United States Patent 2003 US6509352 B1

[232] Iwamura H Suzuki H Ueda Y Kaya T Inaba T JPharmacol Exp Ther 2001 296 420[200] Lange JHM Kruse CG Herremans AHJ VanStuivenberg

HH Dijksman JAR McCreary AC World PatentApplication 2003 WO03078413

[233] httpwwwjticojpJTI_EIR02PL20030212_Epdf (accessed02162004)

[201] Berggren AIK Bostrom SJ Elebring ST Fallefors LWilstermann JM Greasley P World Patent Application 2004WO04058255

[234] Zygmunt PM Andersson DA Houmlgestaumltt ED J Neurosci2002 22 4720

[235] Di Marzo V De Petrocellis L Fezza F Ligresti A BisognoT Prostaglandins Leukot Essent Fatty Acids 2002 66 377[202] Toupence RB Debenham JS Goulet MT Madsen-Duggan

CB Walsh TE Shah SK World Patent Application 2004WO04012671

[236] Wagner JA Varga K Jarai Z Kunos G Hypertension 199933 429

[203] Alanine A Bleicher K Guba W Haap W Kube DLuebbers T Plancher J-M Roche O Rogers-Evans MSchneider G Zuegge J World Patent Application 2004WO04013120

[237] Mukhopadhyay S Chapnick BM Howlett AC Am JPhysiol-Heart Circul Physiol 2002 282 H2046

[238] Jarai Z Wagner JA Varga K Lake KD Compton DR Martin BR Zimmer AM Bonner TI Buckley NE MezeyE Razdan RK Zimmer A Kunos G Proc Natl Acad Sci US A 1999 96 14136

[204] Griffith DA United States Patent Application 2004 US20040092520

[205] Griffith DA United States Patent Application 2004 US20040157839

[239] Kunos G Martin B Razdan R World Patent Application 2001WO0103690

[206] Griffith DA United States Patent Application 2004 US20040157838

[240] Offertaler L Mo F-M Batkai S Liu J Begg M RazdanRK Martin BR Bukoski RD Kunos G Mol Pharmacol2003 63 699[207] Bayewitch M Avidor-Reiss T Levy R Barg J Mechoulam

R Vogel Zvi FEBS letters 1995 375 143 [241] Begg M Mo F-M Offertaler L Batkai S Pacher PRazdan R K Lovinger DM Kunos G J Biol Chem 2003278 46188

[208] Bayewitch M Rhee M-H Avidor-Reiss T Breuer AMechoulam R Vogel Z J Biol Chem 1996 271 9903

[209] Govaerts SJ Hermans E Lambert DM Eur J Pharm Sci2004 23 233

[242] Ho WSV Hiley CR Brit J Pharmacol 2003 138 1320[243] Ho WSV Hiley CR J Pharm Pharmacol 2004 56 869

[210] DAmbra TE Estep KG Bell MR Eissenstat MA JosefKA Ward SJ Haycock DA Baizman ER Casiano FMBeglin NC J Med Chem 1992 35 124

[244] Mo FM Offertaler L Kunos G Eur J Pharmacol 2004489 21

[245] Batkai S Pacher P Jarai J Wagner JA Kunos G Am JPhysiol-Heart Circul Physiol 2004 287 H595[211] Showalter VM Compton DR Martin BR Abood ME J

Pharmacol Exp Ther 1996 278 989

1394 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

[246] Kunos G World Patent Application 2003 WO03015700 [257] Haller J Varga B Ledent C Freund TF Behav Pharmacol2004 15 299[247] Franklin A and Stella N Eur J Pharmacol 2003 474 195

[248] Walter L Franklin A Witting A Wade C Xie Y KunosG Mackie K Stella N J Neurosci 2003 23 1398

[258] Pistis M Perra S Pillolla G Melis M Gessa GL MuntoniAL Neuropharmacology 2004 46 115

[249] Di Marzo V Breivogel C S Tao Q Bridgen DT RazdanRK Zimmer AM Zimmer A Martin BR J Neurochem2000 75 2434

[259] Ralevic V and Kendall DA Eur J Pharmacol 2001 418 117[260] Duncan M Kendall DA Ralevic V Brit J Pharmacol 2003

138 214P[250] Breivogel CS Griffin G Di Marzo V Martin BR Mol

Pharmacol 2001 60 155[261] Duncan M Millns P Smart D Wright JE Kendall DA

Ralevic V Brit J Pharmacol 2004 142 509[251] Monory K Tzavara ET Lexime J Ledent C Parmentier

M Borsodi A Hanoune J Biochem Bioph Res Commun2002 292 231

[262] Duncan M Kendall DA Ralevic V J Pharmacol Exp Ther2004 311 411

[263] Tao Q and Abood ME J Pharmacol Exp Ther 1998 285651[252] Muthane U Ramsay KA Jiang H Jackson-Lewis V

Donaldson D Fernando S Ferreira M Przedborki S ExpNeurol 1994 126 195

[264] Lambert DM DiPaolo FG Sonveaux P Kanyonyo MGovaerts SJ Hermans E Bueb J-L Delzenne NMTschirhart EJ Biochim Biophys Acta 1999 1440 266[253] Hajos N Freund TF Chem Phys Lipids 2002 121 73

[254] Hajos N Ledent C Freund TF Neuroscience 2001 106 1 [265] Calignano A La Rana G Giuffrida A Piomelli D Nature1998 394 277[255] Hajos N Freund TF Neuropharmacology 2002 43 503

[256] Haller J Bakos N Szirmay M Ledent C Freund TF Eur JNeurosci 2002 16 1395

[266] Calignano A La Rana G Piomelli D Eur J Pharmacol 2001419 191

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1368 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Another recent study by Higgs and co-workers furtherhighlighted the role of endocannabinoids in food taste-perception and the reduction of orosensory reward of sucrosein SR141716A treated rats [101] As it was expectedSR141716A induced effects on food consumption are absentsin CB1

-- mice [102] Very recently Cota et al obtainedresults showing that endocannabinoid system modulateshomeostasis via a dual mechanism it regulates at a centrallevel food intake while it blocks at the periphery lipogeneticprocesses [103] Moreover Cani et al showed that ghrelin(an orexigenic peptide) plasma levels are significantlyreduced 45 minutes after SR141716A administration(5mgkg ip) to fasted rats in accordance with the rapiddecrease of food intake measured by the authors It istherefore likely that SR141716 effects on body weight aredue to a conjunction of central and peripheral actions [104]

cerebrospinal fluid from patients with schizophrenia [116]Later De Marchi et al measured higher amounts ofanandamide in blood of schizophrenic patients compared tocontrols [117] On the other hand Dean et al obtainedelevated [3H]-CP-55940 binding in the dorsolateralprefrontal cortex of patients suffering from schizophrenia ascompared to controls [118] Zavitsanou using [3H]-SR141716A found elevated binding in the anterior cingulatecortex of subjects with schizophrenia [119] RecentlyMeltzer and collaborators published the results of a trialconducted to evaluate the potential of four new compoundsin treating schizophrenia and schizoaffective disorders [120]SR141716A (20 mgday) was one of the compoundsevaluated during a six weeks study The authors found noeffect of the CB1 cannabinoid receptor antagonist inimproving patientrsquos schizophrenia (72 subjects)

In addition to the anti-obesity potential the ability ofSR141716A to reduce alcohol and tobacco consumption arecurrently investigated in phase III clinical trials The effect ofSR141716A on alcohol consumption in rats was reported bythe GianLuigi Gessa team [105-108] and by Gallate et al[109] The inhibition of alcohol and nicotine induceddopamine release by SR141716A (1-3 mgkg rats) wasreported by Cohen and co-workers using a brainmicrodialysis device [110] Concerning the smokingcessation preliminary results of a clinical trial (360 subjects40 mg SR141716A) showed an increased abstinence ofsmoking [111] More recent results from the STRATUS-USstudy (787 patients) were reported 362 of patientsreceiving SR141716A (20 mgday) quit smoking against206 in the placebo group [90] Recently Le Fol andGoldberg reviewed the development of cannabinoid CB1antagonists as a new class of therapeutic agents for drugsaddictions [112]

It is known since the early nineties that theadministration of cannabinoid agonists impairs memory inrodents (for a review see Castellano et al [121]) Thusadministration of a cannabinoid antagonist was expected tosomehow improve memory [122123] However dependingon the authors SR141716A when administered alone wasreported to impair (5-10 mgkg im) [124] to have no effect(1-32 mgkg ip) [125] or to improve memory (3 mgkgip) [126] In a more recent paper Wolff and Leandershowed that SR141716A (1mgkg ip) improves memoryin rats by apparently enhancing the consolidation processesof memory [127] Further they found that at higher doses (3mgkg) this effect was lost The discrepancy in the resultsreported in the literature could be ascribed either to thedifferences in the tests used or in the doses administeredFurther experiments are needed to assess whether or not acannabinoid antagonist could be helpful in memory diseases

To conclude several patents were taken by Sanoficoncerning the therapeutic applications of their leadcompound among them being anti-obesity smokingcessation neuroinflammatory diseases and anti-diarrhoeaOne of the last patents to our knowledge concerns thetreatment of sexual dysfunctions with a cannabinoidantagonist such as SR141716A [128] However da Silva etal showed that even if this compound (2 mgkg ip)enhances the effects of apomorphine (20-80 microgkg) it has noeffect alone on penile erection [129] Meanwhile Melis et al

Since the late sixties concerns exist on the associationbetween cannabis use and schizophrenia Recent studiesshowed an association between cannabis use and an increasedrisk of developing schizophrenia [113 114] Moreover apossible role of the endocannabinoid system in schizophreniahas been suggested since its pharmacological characterisation[115] Several experimental data obtained on human subjectstend to confirm such hypothesis On the one hand Lewekeet al found elevated levels of endocannabinoids in

NN

O

NH

N

Br Cl

Cl

NN

O

NH

N

Br Cl

Cl

OH

28 29

Fig (3) CB1 cannabinoid receptor antagonists 3 Diarylpyrazole derivatives The chemical structures of 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-piperidine-carboxamide (SR147778 28 ) and 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-(4-hydroxypiperidine)-carboxamide (29 ) described by Rinaldi-Carmona et al

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1369

reported an increased rate of penile erection after directinjection of SR141716A in the paraventricular nucleus of thehypothalamus [130] This effect was dose dependent and asignificant effect was obtained with a dose of 1microgkg

AM251 in mice at a same dose range (10-30 mgkg ip) thanthe anorectic effects [143] These effects are reversed byadministration of CP-55940 and are absent in CB1

-- miceproving the implication of the CB1 receptor in theantidepressant-like effect of AM251 In 2004 Liao et alreported that AM251 was able to displace the binding of[3H]-batrachotoxin A from its binding site on sodiumchannels in mice brain synaptic preparations [144] Theyobtained an IC50 value of 112 microM and a competitivemechanism of action The authors suggested that AM251 iscapable of reducing neuronal excitability through blockade ofvoltage-sensitive sodium channels in brain

From the clinical trials involving the SR141716A themost frequently reported side-effects are nausea dizziness anddiarrhoea Depression and anxiety were not higher than inthe placebo groups Regarding one of the known side effectsof SR141716A administration the enhancement of intestinalmotility Carai et al reported recently that chronicadministration of the inverse agonists to mice induced atolerance to this prokinetic effect [131] This is not the firstreport of tolerance onset after administration of SR141716A[132] however few is known on the mechanismsresponsible for that phenomenon

Wiley and collaborators reported in 2001 a studydescribing new structure-activity relationships using thepyrazole nucleus as a central scaffold [145] Starting fromSR141716A structure they alternatively substituted one ofthe four substituents while retaining the others bysubstituents known to impart agonist activity in classicalcannabinoids One of the authors expectations was todetermine which positions are responsible respectively forthe antagonism and for the affinity of SR141716A Theaffinity of thirty compounds was assessed previous to in-vivoevaluation of their function in mice using the spontaneousactivity the tail-flick and the rectal temperature assays Theauthors showed that phenyl group in position 5 is critical foraffinity as compound O-1559 (30) lacking this phenyl has adecreased affinity (Table 6) This was already shown by Lanand co-workers [136] along with the need of a substituent inpara position on the phenyl Thomas et al previouslyshowed that this substituent could be a bromine or an iodineatom [146] However an alkyl chain is also tolerated as it isthe case in compounds O-1302 (31) O-1691 (32) or O-1704(33) All these compounds antagonise the anti-nociceptiveand hypothermic effects of ∆9-THC The authors suggestedthat the 5-substituent of pyrazoles is involved in receptorrecognition and antagonism The modulations of thesubstitution pattern of the phenyl in position 1 demonstratethat the 24-di-chloro substitution is the preferred one for theaffinity as well as for the activity Several compoundssupport this assertion Thomas et al showed that additionalhalogens result in a decreased affinity as in compounds 6-I-SR141716A (34) or 4rsquo6-di-I-SR141716A (35) (Ki values of166 and 126 nM respectively) [146] Suppression of the twochlorines (O-1300 39) or replacement of these two chlorinesby an alkyl chain like in O-1254 (40) and O-1255 (41) ledto less active compounds with Ki values ranging from 150to 430 nM However O-1254 and O-1255 behaved asantagonists in a [35S]-GTPγ S assay [61] Lan et al [136]and Wiley et al [145] also modified the substitution inposition 3 Replacement of the amido piperidinyl substituentby alkyl amides as in O-1269 (42) or O-1270 (43) etherslike in O-848 (44) and O-853 (45) ketones (O-1272 46)alcohols (O-1876 47) or alkanes (O-1877 48) resultedmostly in decreased affinity (Table 6) but also with a changeof functionality in some compounds as revealed by in-vivoassays For instance replacement of the piperidinyl by apentyl (O-1269) or by an heptyl chain (O-1270) gave agonistcompounds having Ki values of 32 and 48 nM respectivelyThus the authors suggested that the 3-substituent region isinvolved in receptor recognition and agonist activity Wileyet al on the basis of their results concluded that while the3-position seems to be involved in agonism the 1- 4- and

One close analogue of SR141716A 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-piperidinecarbox-amide (SR147778 28) was described very recently byRinaldi-Carmona et al [133 134] (Fig 3) It possesses highaffinity and selectivity for the hCB1 cannabinoid receptorwith Ki values for the hCB1 and hCB2 receptors of 35 and400 nM respectively ([3H]-CP-55940 hCB1amp2-CHO cells)Further SR147778 antagonised CP-55940 effects on mousevas deferens contractions (pA2=81) and on forskolin-stimulated adenylyl cyclase activity in U373MG cells(pA2=82) but had no effects alone In-vivo SR147778 afteroral administration reversed WIN-55212-2 inducedhypothermia and analgesia As SR141716A SR147778dose-dependently reduced ethanol and sucrose solutionintake with significant effects starting at 03 mgkg (sc) and3 mgkg (po) respectively This compound is currentlyinvestigated in Phase I clinical trials for the treatment ofobesity as well as nicotine and alcohol addictions

Another derivative the 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-(4-hydroxypiperidine)-carboxamide (29) was described in a very recent patent fromSanofi [135] (Fig 3) This antagonist possesses an IC50value for the hCB1 cannabinoid receptor of 32 nM ([3H]-CP-55940 hCB1-CHO cells)

The first structure-affinity relationships for theSR141716A derivatives were reviewed by Barth andRinaldi-Carmona in 1999 [32] Since then many papersdescribing new derivatives were published In 1999 Lan etal described around thirty 15-diarylpyrazoles derivatives[136] Among them was AM251 previously reported as aradioimaging ligand for the CB1 receptor [78] and thatappeared to be more potent (Ki = 749 nM) and selective(selectivity ratio of 306) than SR141716A (Ki = 115 nMselectivity ratio of 143) They reported a Kd value of 05 nMin the mouse vas deferens model using WIN-55212 asagonist Interestingly New et al described for the samecompound an inverse agonist effect on the hCB2 receptorusing a forskolin-induced cAMP accumulation assay (EC50= 650 nM) [137] Recently the anti-obesity effects ofAM251 were reported and are not surprisingly similar tothose of SR141716A [138 139 140] Chen et aldemonstrated that there are synergistic effects on food intakesuppression between AM251 and nalmefene an opioidantagonist [141] This synergistic effect was also presentbetween SR141716A and naloxone as shown by Kirkham[142] Shearman et al found antidepressant-like effects of

1370 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

5-positions appear to be involved in antagonism The O-derivatives reported were also described in a patent alongwith their synthetic pathways which were not given in thepaper [147]

Another investigation of the SR141716Aaminopiperidine region was conducted by Francisco et al[148] They synthesised 21 analogues possessing either analkyl amide or an alkyl hydrazide substituent of various

Table 6 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structure of some of the characterised 1- 3- 4- and 5-pyrazole derivatives Binding affinities (Ki values nM) were obtained on rat brain homogenatesusing [3H]-CP-55940 [136 145 146 148 150] or [125I]-AM-251 [155]

NN

R1

R5

R3R4

Cpd ndeg R1 R3 R4 R5 [136] [145] [146] [148] [150] [155]

SR141716 14 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Cl-Ph 115 62 62 62 13 18

O-1559 30 24-di-Cl-Ph CO-NH-piperidinyl Me 1-Methylpentyl 233

O-1302 31 24-di-Cl-Ph CO-NH-piperidinyl Me 4-((CH2)5)-Ph 21 1

O-1691 32 24-di-Cl-Ph CO-NH-piperidinyl Br 4-((CH2)5)-Ph 15

O-1704 33 24-di-Cl-Ph CO-NH-piperidinyl I 4-((CH2)5)-Ph 22

6-I-SR141716 34 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-Cl-Ph 166

4rsquo6-di-I-SR141716 35 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-I-Ph 126

4rsquo-I-SR141716 36 24-di-Cl-Ph CO-NH-piperidinyl Me 4-I-Ph 75 25 6

4rsquo-Br-SR141716 37 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Br-Ph 168 3

Cpd 25 in ref [136] 38 24-di-Cl-Ph CO-NH-morpholinyl Me 4-Br-Ph 54

O-1300 39 Ph CO-NH-piperidinyl Me 4-Cl-Ph 150

O-1254 40 4-(CH2)4-Ph CO-NH-piperidinyl Me 4-Cl-Ph 226 256

O-1255 41 4-(CH2)5-Ph CO-NH-piperidinyl Me 4-Cl-Ph 433

O-1269 42 24-di-Cl-Ph CO-NH-pentyl Me 4-Cl-Ph 32 114 3

O-1270 43 24-di-Cl-Ph CO-NH-heptyl Me 4-Cl-Ph 48 462 3

O-848 44 24-di-Cl-Ph CH2-O-(CH2)2-piperidinyl

Me 4-Cl-Ph 2450 232

O-853 45 24-di-Cl-Ph CH2-O-CH2-cyclohexyl

Me 4-Cl-Ph 388 100

O-1272 46 24-di-Cl-Ph CO-heptyl Me 4-Cl-Ph 221

O-1876 47 24-di-Cl-Ph 1rsquo-OH-heptyl Me 4-Cl-Ph 657

O-1877 48 24-di-Cl-Ph Heptyl Me 4-Cl-Ph 422

MF9725-64-17 49 24-di-Cl-Ph CO-NH-butyl Me 4-Cl-Ph 134

MF9725-179-32 50 24-di-Cl-Ph CO-NH-NH-butyl Me 4-Cl-Ph 51

MF9725-66-11 51 24-di-Cl-Ph CO-NH-cyclohexyl Me 4-Cl-Ph 25

MF9725-95-31 52 24-di-Cl-Ph CO-NH-(4-hydroxybutyl)

Me 4-Cl-Ph 154

Cpd 15 [150] 53 n-pentyl CO-NH-piperidinyl Me Ph 23

Cpd 16 [150] 54 n-pentyl CO-NH-piperidinyl Me 4-Br-Ph 63

Cpd 17 [150] 55 n-hexyl CO-NH-piperidinyl Me Ph 21

Cpd 18 [150] 56 n-heptyl CO-NH-piperidinyl Me Ph 47

NIDA-41109 57 24-di-Cl-Ph CO-NH-piperidinyl Br 4-Cl-Ph 14

NIDA-41119 58 24-di-Cl-Ph CO-NH-piperidinyl H 4-Cl-Ph 9

NIDA-41057 59 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OH-Ph 104

NIDA-41020 60 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 41

NIDA-41087 61 2-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 8

NIDA-42055 62 24-di-Cl-Ph CO-NH-piperidinyl Br 4-OMe-Ph 62

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1371

lengths in position 3 of the pyrazole moiety They observedthat until five carbons the affinity increases along with thecarbon chain length This is observed with the alkylhydrazide the alkyl amide and the hydroxyalkyl amideseries Moreover the hydrazide analogues exhibit a loweraffinity for the rCB1 than the amide analogues as illustratedby compounds MF9725-64-17 (49) and MF9725-179-32(50) having Ki values of 134 and 51 nM respectively(Table 6) From their structure-activity relationships (SAR)studies the authors concluded that the pharmacophoricrequirement of the amidopiridine region is a chain not longerthan 3 Aring and that a substituent having a positive chargedensity would probably result in increased affinity andpotency The same team described in a patent several othercompounds [149] Binding affinities (Ki) against [3H]-CP-55940 [3H]-SR141716A or [3H]-WIN55212-2 on wholerat brain or on hCB1-transfected cells were given Moreoveractivity data were obtained using the [35S]-GTPγ S assaydemonstrating that these alkyl amide and hydrazide analogsact as antagonists or inverse agonists Despite the greatnumber of derivatives claimed none of them posses asignificantly greater affinity for the CB1 receptor than theSR141716A Nevertheless they showed a slightenhancement of the selectivity for the CB1 receptor over theCB2 cannabinoid receptor

affinity of these derivatives increases with the length of thealkyl chain with an optimal length of 5-6 carbons The bestcompound of this series N-(piperidin-1-yl)-5-phenyl-1-hexyl-4-methyl-1H-pyrazole-3-carboxamide (55) possesses aKi value of 21 nM determined using [3H]-CP-55940 on ratbrain membranes which is higher than the 13 nMdetermined for SR141716A in the same conditions (Table6) In the same paper the authors starting from thehypothesis that antagonism by SR141716A is caused bybinding to the same region of the receptor as do the agonists(CP-55940 and WIN-55-2122) but preventing the agonistpromoted conformational change conducted extensiveconformational analysis as well as superimposition modelsand 3D-QSAR to propose a molecular mechanismsupporting the action of SR141716A Along with theauthors the C-5 aryl substituent of SR141716A occupyinga unique region could contribute in conferring the antagonistproperties Moreover the C-3 substituent could beresponsible for the antagonist or inverse agonist propertiesdepending on the interaction with the receptor

About this topic a very interesting review dealing withthe cannabinoid receptors pharmacophores as well as withthe activationinactivation of these receptors was recentlypublished by Reggio [151]

More recently Dyck et al described seven otherderivatives varying at the amide position [152] The onlycompound possessing a higher affinity for the CB1cannabinoid receptor than SR141716A was the 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta-

The affinity for the rat CB1 receptor of seven derivativespossessing an alkyl chain in position 1 instead of the 24-dichlorophenyl substituent was reported by Shim et al in apaper describing a molecular mechanism for the antagonistand inverse agonist activity of SR141716A [150] The

NN

O

NHN

Cl Cl

Cl

NN

Cl

Cl

NON

NN

O

NH

N

Cl

ClCl

NN

O

NH

MeO Cl

CP-272871 66

63 64

65

Fig (4) CB1 cannabinoid receptor antagonists 3 Diarylpyrazole derivatives Structures of 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63 ) 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methyl-pyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]-morpholine (64 ) N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65 ) and N-phenyl-1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-pyrazole-3-carboxamide (CP-272-871 66 )

1372 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

[c]pyrrol2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63)(Fig 4) The Ki values were 5 and 12 nM respectively([3H]-CP-55940 hCB1-HEK cells)

CP-55940 rat brain) For instance N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65) (Fig 4) showed a Ki value of15 nM However no data were provided concerning thefunctionality of such compounds

Over 200 other pyrazole derivatives acting at the CB1cannabinoid receptor were claimed in a patent from Pfizer[153] The affinity for the hCB1 receptor of 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methylpyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]ndashmorpholine (64) (Fig 4)was 79 nM ([3H]-SR141716A hCB1-HEK cells) Theothers compounds affinity was between 01 and 100 nM

In an effort to develop new SR141716A analoguespossessing lower lipophilicity to be used as PET tracersKatoch-Rouse and colleagues further explored thesubstitution pattern of the two phenyls [155] Bindingassays showed that a decreased lipophilicity lead to adecreased affinity for the CB1 receptor as determined bydisplacement of [3H]-AM251 For instance compoundNIDA-41057 (59) has a lipophilicity of 42 ( expressed asElogDoct) and a Ki value of 104 nM whereas SR141716Ahas a ElogDoct value of 54 and a Ki value of 18 nM In

Seventy five pyrazole derivatives were described in a veryrecent patent from Makriyannis et al [154] The affinity ofthe compounds for the cannabinoid receptors is given TheKi values for the best compounds are lower than 6 nM ([3H]-

Table 7 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structures and affinities for the cannabinoid receptors of some representative tricyclic pyrazole derivatives Values were obtained using [3H]-CP-55940as radioligand and mouse [160 228] rat [159 227] or human [158] cannabinoid receptors Compounds 69-78 are CB1 cannabinoid receptor ligandsCompounds 79-86 are CB2 cannabinoid receptor ligands

NN

O

NHX

R3

R1

R2

n

n˚ X R1 R2 n R3 Ki CB1 (nM) Ki CB2 (nM) Selectivity References

69 -(CH2)3- Cl 24-di-Cl 0 piperidine 126 [158]

70 -(CH2)3- H 24-di-Cl 0 azepane 100 [158]

71 -(CH2)3- H 24-di-Cl 0 piperidine 398 [158]

72 -(CH2)3- Cl 24-di-Cl 0 azepane 125 [158]

73 -(CH2)3- NO2 24-di-Cl 0 azepane 63 [158]

74 -(CH2)2-O- H 24-di-Cl 0 piperidine 501 [158]

75 -CH2-S- Br 24-di-Cl 0 piperidine lt 500 gt10 [159]

76 -CH2-SO2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

77 -(CH2)2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

78 -(CH2)3- Cl 24-di-Cl 0 piperidine 000035 21 60000 [160]

79 -CH2- Cl 24-di-Cl 0 piperidine 2050 034 00002 [228]

80 -CH2- Br 24-di-Cl 0 piperidine 1570 027 00002 [228]

81 -CH2- CH3 24-di-Cl 0 piperidine 363 0037 00001 [228]

82 -CH2- Cl 4-Cl 0 piperidine 1787 09 00005 [228]

83 -CH2- H 24-di-Cl 0 piperidine 1152 0385 00003 [228]

84 -CH2- Cl 34-di-Cl 1 133-trimethylbicyclo[221]heptyl

lt500 gt10 [227]

85 -CH2- Br 24-di-Cl 1 bicyclo[321]oct-3-yl lt500 gt10 [227]

86 -CH2- Br 4-Me 1 77-dimethylbicyclo[410]hept-3-yl

lt500 gt10 [227]

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1373

another paper Katoch-Rouse described the synthesis of[18F]-NIDA-42033 (26) a ligand useful as PET radiotracerstarting from its bromo derivative [156] Recently Kumar etal [157] proposed the synthesis of a 11C derivative ofNIDA-41087 (61) a compound previously described byKatoch-Rouse (Table 5) Post-mortem binding of thiscompound to human prefrontal cortex was also investigatedInterestingly CP-272871 (66) described by Meschler as aCB1 inverse agonist (Ki=57 nM [3H]-CP-55940 rat brainhomogenates) possesses a lipophilicity similar to theNIDA-41087 one [62] However despite the presence of amethoxy function no radiolabelled derivative has beendescribed to date One explanation could be the compoundlow selectivity (2 fold) for the CB1 cannabinoid receptor

To further explore the pharmacology of this compound afunctional assay using the mouse vas deferens model wasperformed by Ruiu and colleagues enlightening thecompetitive antagonist properties of NESS (pA2= 1246against WIN-55212-2) which had no effect by itself up to1microM Moreover NESS 0327 was unable to affect basalbinding of the [35S]-GTPγ S demonstrating its lack ofnegative intrinsic activity In-vivo studies were conductedusing the hot plate and tail flick tests NESS dose-dependently abolished the antinociceptive effect of WIN-55212-2 but had no effect by itself However the authorssuggested based on the affinity and in-vivo activity of theircompound that NESS 0327 possesses a poor centralbioavailability

Several new attempts to increase the affinity of thediarylpyrazole derivatives were recently made by rigidifyingthe SR141716A structure (Table 7) Six fused ringanalogues of SR141716A obtained by fusion of the 5-(4-chlorophenyl) substituent with the central pyrazole to forman indazole ring were published by Bass et al in 2002 [61]The compound possessing the highest affinity O-1248 (67)has a Ki value of 475 nM as determined by displacement of[3H]-CP-55940 from rat brain membranes

Another example of ring-constrained biarylpyrazole wasdescribed by Herbert Seltzman team [161] Theyinvestigated a photocyclisation reaction starting from theSR141716A leading to a pyrazolo[15-f]phenanthridinestructure (Fig 5) The new compound (68) was tested on theCB1 receptor (whole rat brain) against [3H]-CP-55940[3H]-SR141716A and [3H]-WIN-55212-2 and exhibited Kivalues of 48 35 and 50 nM respectively The CB2 affinitywas assessed using [3H]-CP-55940 and was found to be 100times lower (Ki = 3340 nM) No effect on the [35S]-GTPγ Sassay (rat brain) was observed by the authors

Stoit and colleagues from Solvay Pharmaceuticalspublished a paper describing the synthesis andpharmacological characterisation of new benzocyclohepta-pyrazole derivatives as CB1 antagonists (69-74) [158] Theaffinity of these compounds was more or less one order ofmagnitude lower than the SR141716A one with pKi valuesranging from 64 to 72 compared to 76 for the SRcompound (Table 7) Compounds were shown to beantagonists with pA2 values from 70 to 89 as theyprevent CP-55940-induced cAMP accumulation Howeverthe authors investigating the bioavailability of theircompounds either po or after ip injection found negligibleblood plasma levels

NN

Cl

Cl

O

NH

N

NN

Cl

O

NHN

Cl

Cl

O-1248 67 68However just before the publication of Stoit results

Sanofi-Synthelabo took a patent describing tricyclicpyrazolecarboxylic acid amide derivatives having antagonistproperties on the CB1 cannabinoid receptor andsubmicromolar affinities (75-77) [159] Thirty examples weregiven along with their synthetic pathway but no individualpharmacological data were shown

Fig (5) CB1 cannabinoid receptor antagonists 3Diarylpyrazole derivatives Two ring-constrained diarypyrazolederivatives O-1248 (67 ) and a pyrazolo[15-f]phenanthridinederivative (68 )

It appears from these attempts to increase SR141716Aaffinity by rigidifying its structure that only a limitedincrease in the CB1 cannabinoid receptor affinity can beexpected with the apparent exception of NESS 0327Moreover rigidifying the structure often results in a morelipophilic compound in a family of compounds being yetlipophilic

More recently Ruiu and colleagues published a paperdescribing the synthesis and complete pharmacologicalcharacterisation of a very potent CB1 receptor ligandchristened NESS 0327 (78) [160] This compound has thesame structure as compound 69 published by Stoit et al(compound 20 in the reference [158]) Nevertheless Ruiu hasobtained an affinity for the CB1 cannabinoid receptor 5000times bigger than the SR141716A one with Ki values of035 pM and 18 nM respectively using mouse forebrainhomogenates and [3H]-CP-55940 as radioligandFurthermore the selectivity ratio obtained for this compoundby Ruiu is 60000 with a Ki value for the CB2 receptor of21 nM against the [3H]-CP-55940 The discrepancybetween the data obtained independently by Stoit et al andRuiu et al could hardly be explained by the differences inthe origin of the receptor used respectively hCB1-CHO andmouse forebrain

4 Phenyl Benzofuranone Derivatives

In a 1997 patent from Eli Lilly was described thesynthesis of aryl-benzothiophene and aryl-benzofuranederivatives (87-89 Fig 6) having CB1 receptor affinity andantagonist properties [162] From ten compounds having aKi value lower than 25 microM ([3H]-CP-55940 hCB1-CHOcells) one was selected and claimed to have a Ki of 170 nMCompound 88 was shown in the patent to antagonise theeffect of anandamide on cAMP accumulation with an IC50 of

1374 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

O

O

O

O

NC

S

O

O

O

O

O

O

O

NC

LY320135 8887 89

Fig (6) CB1 cannabinoid receptor antagonists 4 Phenyl benzofuranone derivatives Chemical structures of two aryl-benzofuranederivatives (87 -88 ) and one aryl-benzothiophene (89 ) derivative from Eli Lilly

500 nM and the effect of WIN-55212-2 on calcium channelsat 1microM Moreover the intra-peritoneal injection (20 mgkg)of this compound antagonised the in-vivo effect ofanandamide in the Open Field Assay a mouse behaviourmodel Later on Felder and colleagues gave a furtherdescription of this compound called LY320135 in a paperdescribing its effect on cAMP accumulation and on ioniccurrents [163] The selectivity ratio of LY320135 was 106with Ki values of 141 and 14900 nM for hCB1 and hCB2receptors ([3H]-CP-55940) respectively LY320135antagonised anandamide inhibition of forskolin-inducedcAMP accumulation with an IC50 value of 734 nMChristopoulos et al in 2001 reported for LY320135 apKb value of 527 in the inhibition of WIN-55212-2mediated response in the rat electrically-stimulated vasdeferens while the pKb value for SR141716A was 75[164]

Other patents were taken by Aventis in which somepotential applications for their compounds were describedFor instance an association between an azetidine derivativehaving CB1 antagonists properties and sibutramine aserotonin and norepinephrine reuptake inhibitor was claimed[170] as a treatment against obesity Oral administration ofcompound 90 (3 mgkg) and sibutramine (06 mgkg) to fafaZucker rats which are genetically obese rats has resulted ina decreased food intake compared to control lean Zuckerrats In another patent the association of an azetidinederivative (1-10 mgkg po ) and a D2D3 agonist likequinpirole (01 mgkg ip) was claimed to have a therapeuticpotential in the treatment of Parkinsonrsquos disease [171] Theeffect has been evidenced using an akinesia model in the ratEvidences supporting the use of CB1 antagonists as adjuvantin the treatment of Parkinsonrsquos disease begin to appear Itseems that such compounds would be helpful in thetreatment of Parkinsonism [172] and levodopa-induceddyskinesia [173] Brotchie published an interesting paperreviewing the CB1 cannabinoid receptor signaling inParkinsonrsquos disease [174] Nevertheless further clinicalstudies should be conducted to further confirm the usefulnessof CB1 antagonists in the treatment of Parkinsonrsquos disease

5 Azetidine Derivatives

The family of azetidine compounds illustrated bycompounds 90 and 91 (Fig 7) was developed at Aventis byDaniel Achard and colleagues and was described in a seriesof patents [165-168] They claimed the IC50 values of thesecompounds for the CB1 receptor to be less than or equal to100 nM These values were obtained following the proceduredescribed by Kuster et al [169] The antagonist propertywas shown using an in-vivo model the reversal ofhypothermia induced by CP-55940 in mice and the ED50obtained were lower than 50 mgkg

6 Aryl-Imidazolidine-24-Diones Derivatives

In 1999 the Didier Lambert team published a paperdescribing the affinity of 24 new 3-alkyl-55rsquo-diphenyl-imidazolidine-24-dione derivatives for the hCB1 receptor[175] The preliminary structure-activity relationships

N

SO

O

Cl

Cl

F

F

N

SO

O

Cl

NS

90 91

Fig (7) CB1 cannabinoid receptor antagonists 5 Azetidine derivatives Two examples (90 -91 ) of the azetidine derivatives developedby Aventis

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1375

Table 8 CB1 Cannabinoid Receptor Antagonists 6 Aryl-Imidazolidine-24-Diones Derivatives

Structures affinities ([3H]-SR141716A hCB1-CHO cells) and pKb values ([35 S]-GTPγS HU210 rat brain) of three aryl-imidazolidine-24-dionederivatives (92-94)

N

NH

OO

R

Br

Br

Cpd ndeg R Ki (nM) a pKb b

DML20 92 ethylmorpholine 70 611

DML21 93 1-hydroxypropyl 103 625

DML23 94 heptyl 98 574

a [175]b [177]

showed that the substitution at the nitrogen in position 3 aswell as a bromine atom in para of the phenyl rings aremandatory for the compounds affinity Later on threecompounds termed DML20 (92) DML21 (93) and DML23(94) were characterised as neutral antagonists using the [35S]-GTPγ S assay on rat cerebellum membranes [176] (Table 8)In the same model they competitively inhibited HU-210-induced [35S]-GTPγ S binding with pKB values of 611625 and 574 respectively Moreover these compoundswere proven to be quite selective for the CB1 receptor

higher than the one for the CB2 cannabinoid receptor (IC50 =300 nM) The inverse agonism of the compound wasextrapolated by inhibiting CP-55940-induced hypothermiain mice (EC50 = 18 nM) Moreover one oral dose (10mgkg) reduces the food intake in diet-induced obese ratsInterestingly food intake of CB1

-- mice was not alteredBody weight loss was maintained all over the study during achronic administration (14 days) of 10 mgkg of Cpd A

Later on Merck published a patent [180] in which someof the 45-diaryl-pyrazoles derivatives previously describedwere claimed to be useful in the treatment or the preventionof obesity in association with compounds inhibiting the11β-hydroxysteroid dehydrogenase type 1 (1β-HSD1)enzyme The 1β-HSD1 enzyme is responsible for thesynthesis of cortisol increased levels of which according tothe inventors are associated with obesity However neitherresults on appetite suppression nor on weight loss weregiven even if some indications on the affinity of thecompounds for the cannabinoid receptors were given

Very recently the functionality of these DML compoundswas explored using the [35S]-GTPγ S assay both on rat andhuman CB1 cannabinoid receptors [177] The data obtainedconfirm that the 3-alkyl-55rsquo-diphenyl-imidazolidinedionederivatives behave as neutral antagonists on rat CB1cannabinoid receptor (rat brain) However DML 20 DML21and DML23 acted as inverse agonists on the human receptor(hCB1-CHO cells) Furthermore the authors showed thatthis different functionality is not due to the hCB1 receptorlevel of expression in the recombinant cell line as whateverthe level of expression (Bmax of 44 pmolmg or 32pmolmg) DML derivatives behaved as inverse agonists ofthe hCB1 cannabinoid receptor

Another imidazole ring pattern of substitution wasproposed by Hagmann and collaborators [181] Thecompounds described 43 examples are given possess twophenyls the first one in position 1 linked to the nitrogenand the second one in position 2 The carboxamidesubstituent is in position 4 instead of in position 2 for thediarylimidazoles described by Finke These 12-diaryl-imidazoles are claimed to be CB1 cannabinoid receptorantagonists or inverse agonists but the absence of precisepharmacological values do not allow any comparisonbetween the two substitutions patterns However a samepattern of substitution was used by other researchers Amongthem Dyck et al described in 2004 ten 1-(4-chlorophenyl)-2-(24-dichlorophenyl)-imidazole derivatives varying by theircarboxamide substituent in position 4 (101-103 Table 9)[152] For instance with a 3-azabicyclo[330]octan-3-ylsubstituent (101) they obtained a Ki value of 14 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) In a [35S]-GTPγ Sassay 101 exhibited an IC50 value of 19 nM

7 Diarylimidazoles Derivatives

Finke and colleagues at Merck developed a new class ofCB1 receptor antagonists based on an imidazole nucleusOver eighty 45-diaryl-imidazoles derivatives having CB1antagonist properties were claimed in a patent published in2003 [178] Some representative compounds of the patent areillustrated in Table 9 (95-100) The affinity of the newcompounds was determined using [3H]-CP-55940 andhCB1-CHO cells Although no detailed pharmacologicaldata were given in the patent in-vitro and in-vivo propertiesof one compound termed Cpd A (100) have been describedelsewhere [179] Its affinity for the CB1 cannabinoid receptorwas shown to be nanomolar (IC50 = 4 nM) and 75 times

1376 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 9 CB1 Cannabinoid Receptor Antagonists 7 Diaryl-Imidazole Derivatives

Structure and affinity range of some 45-diaryl-imidazole derivatives for the human cannabinoid receptors determined using [3H]-CP-55940 asradioligand and hCB1amp2-CHO cells

N

NR2

R1

R4

R3

ndeg R1 R2 R3 R4 hCB1 (IC50 or Ki) hCB2 (IC50 or Ki) References

95 CH3 CO-NH-piperidinyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

96 CH3 CO-NH-piperidinyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

97 CH3 CO-NH-cyclohexyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

98 CH3 CO-NH-phenyl 24-di-Cl-Ph 4-Cl-Ph 10 ndash 100 nM a 100 ndash1000 nM a [178]

99 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

100 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph 4 nM a 300 nM a [179]

101 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

CH3 14 nM b ND [152]

102 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

H 66 nM b ND [152]

103 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 85 nM b ND [152]

104 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 23 nM b 542 nM b [182]

105 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 30 nM b 608 nM b [182]

106 4-Br-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 60 nM b 489 nM b [182]

107 4-CF3-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 29 nM b 634 nM b [182]

108 4-Cl-Ph 24-di-Cl-Ph CO-NH-morpholinyl CH3 197 nM b 3297 nM b [182]

109 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidin-4-ol CH3 172 nM b 3959 nM b [182]

110 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CN 30 nM b 1590 nM b [182]

111 4-Cl-Ph 24-di-Cl-Ph CO-NH- piperidinyl CH2F 36 nM b 906 nM b [182]

112 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH2-CH3 14 nM b 430 nM b [182]

113 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Cl 27 nM b 823 nM b [182]

114 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Br 23 nM b 746 nM b [182]

115 4-Cl-Ph 24-di-Cl-Ph CO-NH-tetrahydroisoquinoline CH3 34 nM b 696 nM b [182]

116 4-Cl-Ph 24-di-Cl-Ph CO-NH-cycloheptyl CH3 35 nM b 349 nM b [182]

117 24-di-Cl-Ph 4-Cl-Ph CO-NH-piperidinyl CH3 403 nM b 208 nM b [182]a IC50 value b Ki value

Lange and co-workers from Solvay described twenty-eight imidazole derivatives [182] The 24-dichlorophenylsubstituent at position 2 was kept constant while thesubstitutions at the other positions were explored (Table 9)Position 5 can accommodate a large range of littlesubstituents like hydrogen (104) methyl (105) ethyl (112)chlorine (113) bromine (114) fluoromethyl (111) or cyano(110) without major changes in the affinity At position 4more bulky substituents like 1234-tetraisoquinoline (115)or cycloheptyl (116) were well tolerated whereas thepresence of a more hydrophilic moiety such as morpholine(108) or piperidin-4-ol (109) was detrimental to the affinity

Interestingly position exchange between the 24-diphenyland the 4-chlorophenyl substituents led to a 13 fold loweraffinity with Ki values of 30 and 403 nM for 105 and 117respectively ([3H]-CP-55940 hCB1-CHO cells) In thefunctional assay which was inhibition of WIN-55212-2-induced [3H]-arachidonic acid release by hCB1-CHO cellsall tested compounds behaved as antagonists Compound105 showed a pA2 value of 86 which is the same theauthors obtained for SR141716A It would be interesting toassess if these compounds are true antagonists or if they actmore as inverse agonists Compound 105 when administeredto rats was able to inhibit CP-55940-induced hypotension

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1377

with an ED50 value of 24 mgkg which is close to the 3mgkg obtained for SR141716A The compound was alsoactive in another in-vivo model the WIN-55212-2-inducedhypothermia in mice Compound 104 which has nosubstituent in position 5 of the imidazole ring was lessactive in the hypotension model and inactive in thehypothermia model Thus a methyl group is the preferredsubstituent in position 5 of the imidazole moiety Otherproperties either experimental (P-glycoprotein affinitylogPHPLC) or computational (molecular volume polarsurface area) obtained for compounds 105 were similar tothose obtained for SR141716A Finally molecularmodelling studies revealed a close structural overlap betweenthe two compounds

value of 197 nM is selective for hCB1 and is as potent asSR141716A The structural modulations of 118 led to someinteresting compounds (Table 10) Six compounds possess aKi value for the hCB1 cannabinoid receptor equal to or lowerthan 25 nM antagonist properties in the inhibition of [3H]-arachidonic acid release by WIN-55212-2-stimulated hCB1-CHO cells and Ki values for the hCB2 receptor over 1microMThe diaryl-pyrazoline derivatives contained a chiral center atposition 4 The authors resolved the racemic mixture of twocompounds (121 and 130) The levorotatory enantiomersappeared to be the eutomers For compound SLV319levorotatory enantiomer of 121 the Ki and pA2 values were78 nM and 99 respectively The Ki and pA2 values ofSLV326 levorotatory enantiomer of 130 were 359 nM and9 respectively

8 34-Diaryl-Pyrazoline Derivatives Two in-vivo models CP-55940-induced hypotension inrat and WIN-55212-2-induced hypothermia in mouse wereused to assess the potential of these compounds The resultsare of the same order of magnitude than those obtained forSR141716A For instance in the hypotension modelSLV319 ((-)121) and SLV326 ((-)130) showed ED50 values

Lange and collaborators also discovered 34-diaryl-pyrazoline derivatives after a screening of compoundsresembling to SR141716A [183184] Compound 118 thatwas initially identified during the screening possesses a Ki

Table 10 CB1 Cannabinoid Receptor Antagonists 8 34-Diaryl-Pyrazolines Derivatives

Structure affinity for the hCB1 and hCB2 cannabinoid receptors (Ki nM) expressed in CHO transfected cells ([3H]-CP-55940) and potency (pA2) ofsome 34-dirylpyrazolines derivatives Table adapted from [184]

NN

N N

SO2

R3

R2

Cl R1

R4

n˚ R1 R2 R3 R4 Ki hCB1 (nM) Ki hCB2 (nM) pA2 (CB1)

118 H H H 4-CH3 197 gt 1000 84

119 H H H 246- CH3 24 gt 1000 94

120 H H H 4-Cl 16 gt 1000 95

121 H CH3 H 4-Cl 25 gt 1000 87

122 H CH3 CH3 4-Cl 280 gt 1000 85

123 H H H 4-F 53 gt 1000 9

124 H CH3 H 4-F 338 gt 1000 85

125 H CH3 CH3 4-F gt 1000 gt 1000 lt 75

126 H CH3 H 3-Cl 14 gt 1000 86

127 4-Cl CH3 H 4-Cl 255 ND ND

128 4-F CH3 H 4-Cl 584 ND ND

129 H CH3 H H 170 ND 75

130 H CH3 H 4- CF3 221 gt 1000 93

SR141716A 25 1580 86

1378 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

N

HN

O

N

Cl Cl

Cl

N

N

HN

O

Br

Br131 132

Fig (8) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Chemical structures of two 56-diaryl-pyrazine-2-amide derivatives (131-132) developed as CB1 cannabinoid receptorantagonists by AstraZeneca

of 55 and 2 mgkg (po) respectively proving their in-vivoefficacy after oral administration The authors alsodemonstrated that SLV319 is devoid of affinity for the P-glycoprotein pump and that it possessed a goodCNSplasma ratio (17) These two compounds (SLV319and SLV326) were chosen as development candidates bySolvay and entered clinical Phase I trials in 2003 [185]

132 Fig 8) useful as CB1 cannabinoid receptor antagonists[186 187] The activity of twenty derivatives wasdetermined using a [35S]-GTPγ S assay and hCB1-CHOtransfected cells The concentration required to give halfmaximal inhibition of CP-55940-induced [35S]-GTPγ Sbinding (IC50) is lower than 200 nM for the preferredcompounds No affinity data were given for thesecompounds

9 Diaryl-Pyrazine Diphenyl-Pyridine Diphenyl-Phenyland Diaryl-Pyrimidines Derivatives

The same year in a patent from Merck Finke andcollaborators described several 56-di-phenyl-pyridinederivatives as hCB1 antagonists or inverse agonists [188]The compounds have a substituent in position 3 of thepyridine core (Fig 9) This substituent could be a cyano(133-134) or a nitro group a halogen an ester or an amide(135-136) Over 150 compounds were synthesised toillustrate the invention but no pharmacological data wasdisclosed in the patent

In 2003 appeared several patents describing new familiesof compounds that bind to the CB1 cannabinoid receptorThese compounds have in common a central six atomsaromatic ring which could be a pyrazine a pyridine aphenyl or a pyrimidine and that is substituted by at leasttwo phenyl rings

The first patents were from Berggren and collaboratorsfrom AstraZeneca who published two patents describing thesynthesis of 56-diaryl-pyrazine-2-amide derivatives (131-

A few months later another patent was taken by Sanoficlaiming the antagonist properties of 56-di-phenyl-2-pyridine carboxamide derivatives [189] These compounds

N

NH

O

Cl

Cl

N

O

Cl

O NH

N

O

Cl

ClCl

NH

CN O

N

Cl

Cl

Cl

OHN

N

O

Cl

ClCl

CNF

F

133 134

135 136 137

Fig (9) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives 56-Di-phenyl-pyridine derivatives patented by Merck (133-136) and by Sanofi (137) as CB1 cannabinoid receptorsantagonists

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1379

NH

O

N

Cl

ClCl

NH

O

N

Cl

F3CCl

N

N

O

Cl

Cl Cl F138 139 140

Fig (10) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Representative structures of the diphenyl-phenyl (138-139) and 45-diphenyl-pyrimidine (140) derivatives developed ascannabinoid antagonists at Sanofi and Merck respectively

28 examples are given structurally related to the diphenyl-phenyl derivatives have their pyridine core substituted bytwo phenyl rings and by an amide moiety (137 Fig 9) TheIC50 values were lower than 100 nM but no data were givenshowing the antagonist properties although the compoundswere assayed in the adenylate cyclase inhibition assay

10 Other Derivatives

Several amide derivatives structures recently appearedamong the CB1 cannabinoid receptor antagonists In a seriesof patents taken by Merck in 2003 Hagmann and colleaguesdescribed a large amount of compounds obtained by parallelsynthesis which are claimed to be CB1 cannabinoid receptorantagonists [192-195] They were synthesised by reacting alibrary of substituted amines with a library of carboxylicacids (141-144 Fig 11) However as no value was givenfor the affinity or activity of these amides we will notfurther discuss this class of compounds

Along this line Sanofi Synthelabo developed newdiphenyl-phenyl derivatives claimed to have CB1cannabinoid receptor antagonist properties [190] They arebased on a central phenyl ring substituted by two phenylsand by an amide moiety (138-139 Fig 10) Thirteencompounds were claimed along with their synthesis TheirIC50 values were lower than 100 nM ([3H]-CP55-940hCB1-CHO cells) but no other specific data (selectivitypA2hellip) was given

Several 15-diaryl-pyrrole-3-carboxamide derivatives weresynthesised and claimed to be CB1 cannabinoid receptorantagonists by Berggren et al [196] The affinity valueswere not disclosed in the patent (145-146 Fig 12) Otherpyrrole derivatives were synthesised by Guba et al atHofmann-La Roche [197] More specifically 2-(thiazol-4-yl)pyrrole derivatives were claimed as CB1 cannabinoidreceptor antagonist (IC50lt2microM) (147-148 Fig 12)

Finally Kopka et al from Merck described more thanone hundred 45-diaryl-pyrimidine derivatives as CB1cannabinoid receptor antagonists (140 Fig10) [191]However no precise data were provided concerning theaffinity and functionality of the compounds

R2

R1N

R3

R4

R5

O

NH

O

Cl

O F

F

R2

R1NH

R3

R4

HO R5

O

NH

O

Cl

CN

NH

O

Cl

O

O

NH

O

Cl

Cl

+

141142

143 144

Fig (11) CB1 cannabinoid receptor antagonists 10 Other derivatives Retrosynthetic scheme of a CB1 cannabinoid antagonistlibrary of amide derivatives developed at Merck The structures of four representative compounds are illustrated (141-144)

1380 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

NH

O N

Cl

Cl

Cl

N

NH

O

OMe

MeO

N

NH

O

N

S

MeO

N

NH

O

N

S

MeO

145 146

147 148

Fig (12) CB1 cannabinoid receptor antagonists 10 Other derivatives Two examples of 15-diaryl-pyrrole derivatives described byBerggren et al (145-146) While the 2-(thiazol-4yl)pyrroles derivatives 147 and 148 were described by Guba et al from Hofmann-LaRoche All these pyrroles derivatives were claimed to be CB1 cannabinoid receptor antagonists

Another new structure is represented by the 124-triazolederivatives developed by Jagerovic and collaborators In arecent paper were described the synthesis andpharmacological properties of five new 15-diphenyl-3-alkyl-

triazole derivatives [198] Among these compounds onlyone (149) behaved as a CB1 antagonist inhibiting the WIN-55212-2-induced contractions in a mouse vas deferenspreparation However despite its effects in isolated tissue

Table 11 CB1 Cannabinoid Receptor Antagonists 10 Other Derivatives

Structure and affinity of diaryl-triazole derivatives

N

NN

R3

R2

R1

Cpd R1 R2 R3 Ki CB1 (nM) References

149 24-diCl 4-Cl hexyl 855 a [198]

150 24-diCl 4-Cl2-OMe CO-NH-(3-azabicyclo[330]octan-3-yl) 270 b [152]

151 24-diCl 4-Cl CO-NH-(3-azabicyclo[330]octan-3-yl) 164 b [152]

152 4-Cl 24-diCl CO-NH-(3-azabicyclo[330]octan-3-yl) 137 b [152]

153 4-Cl 24-diCl CO-NH-benzylpyrrolidin-3-yl 29 b [152]

154 4-Cl 24-diCl CO-NH-1-(4-chlorophenyl)ethyl 66 b [152]

155 4-Cl 24-diCl CO-NH-piperidinyl 356 c [182]

156 24-diCl 4-Cl CO-NH-piperidinyl 382 c [182]

a [3H]-SR141716A rat cerebellar membranesb [3H]-CP-55940 hCB1-HEK EDNA cellsc [3H]-CP-55940 hCB1-CHO cells

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1381

N

S

O

NH

Cl

Cl

Cl

F

N

S

O

NHN

ClCl

Cl

N

S

O

NH

Br

N

S

O

NHN

Cl

Cl

Cl

156 157 158 159

Fig (13) CB1 cannabinoid receptor antagonists 10 Other derivatives Structures of 45-diaryl-thiazole derivatives patented as hCB1cannabinoid receptor antagonists by Solvay Pharmaceuticals (156 157 158) or by AstraZeneca (157 158 159)

assays (mouse vas deferens and guinea pig ileum) thecompound possessed only a reduced rCB1 affinity with Kivalues of 855 and 748 nM using [3H]-SR141716A and [3H]-WIN-55-212-2 respectively (Table 11) Further four seriesof compounds differing by the nature of the substituentaround the triazole core were also described in a patent[199] Usually the compounds claimed possess two aromaticrings possibly substituted and one linear alkyl chain Theaffinity for the cannabinoid receptors was not givenNevertheless 149 was evaluated in two isolated tissuemodels the inhibition of the electrically evoked contractionsin the guinea pig ileum and in the mouse vas deferens Inthe two models compound 149 inhibited the effect of WIN-55212-2 but had no effect by itself In the vas deferensmodel the authors obtained a pA2 value of 748 to becompared with 763 obtained for AM251

Yet another five membered ring a thiazole has beenused as central moiety of a new class of cannabinoid ligandsThe thiazole ring is substituted by two aryls in position 4and 5 and by an amide (position 2) as in compounds 156-159 (Fig 13) Forty derivatives of this type weresynthesised by Lange and colleagues from SolvayPharmaceuticals and claimed in a patent as agonists orantagonists of the CB1 cannabinoid receptor [200] Nopharmacological data were provided however their activitywas assessed by measuring the cAMP in transfected hCB1-CHO cells In a recent paper from Lange et al two of thesethiazole derivatives were further described [182] Compound13 (157) the most closely related to SR141716A has a Kivalue of 227 nM while for compound 14 (158) Ki value isover 1000 nM Thus as in the imidazole series (see Table9) the aromatic substitution pattern is of great importancefor the CB1 cannabinoid receptor affinity Compound 13however was devoid of in vivo activity

Others 124-triazole derivatives were described by Dycket al compounds 150-154 unlike the Jagerovic ones havean amide moiety in position 3 [152] The highest affinitywas obtained with a 1-benzyl-pyrrolidin-3-yl substituentThe Ki value was 29 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) to be compared to 12 nM obtained for theSR141716A

Seventeen 45-diaryl-thiazole derivatives were alsosynthesised by Berggren et al and patented by AstraZeneca(157-159 Fig 13) [201]

In a patent from Merck Toupence et al described over190 substituted furo[23]pyridines claimed to be CB1cannabinoid receptor antagonists [202] The affinity (IC50 lt1microM) was measured using recombinant CHO cells and [3H]-CP-55940 and the activity using cAMP dosage (160 Fig14)

Lange et al described a triazole derivative (155) whichcan be superimposed with SR141716A Compound 155showed a Ki value of 356 nM ([3H]-CP-55940 hCB1-CHOcells) a pA2 value of 83 in the inhibition of WIN-55212-induced release of [3H]-arachidonic acid by hCB1-CHO cellsand was active in vivo in the CP-55940-inducedhypotension in rat (ED50 = 236 mgkg) [182]

Very recently Alanine from Hoffmann-LaRoche foundthat diarylbenzo[13]dioxole derivatives act as CB1

O

O

S

N

OO

F

ClCl

O

O

F O

N

OON

NH2

O

Cl

Cl

Cl

160 161 162

Fig (14) CB1 cannabinoid receptor antagonists 10 Other derivatives One example of a furo[23]pyridine derivative (160) and twoexamples of diarylbenzo[13]dioxole derivatives (161-162) described as hCB1 cannabinoid receptor antagonists by Toupence et aland Alanine et al respectively

1382 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

cannabinoid receptor antagonists (161-162 Fig 14) [203]Approximate IC50 values for twelve compounds ([3H]-CP-55940 hCB1-HEK cells) among the three hundred andeighty compounds described were given (IC50lt2microM)

et al in 1995 [207] In their hands ∆9-THC was not able toinhibit the forskolin-stimulated adenylyl cyclase activity inan assay performed on hCB2-CHO cells Subsequently theyshowed that ∆9-THC was able to antagonise HU210-inducedinhibition of adenylyl cyclase activity thus acting as anantagonist of the hCB2 cannabinoid receptor expressed inCHO cells [208] Furthermore very recently Govaerts et al[209] reported an inverse agonist effect of ∆9-THC and ∆8-THC on hCB2-CHO cells using the [35S]-GTPγ S assayThe pEC50 values were 763 and 888 respectively whilethe Emax values were ndash27 and ndash16 as compared to basal

Griffith from Pfizer described three new series of CB1cannabinoid receptor antagonists based on a bicyclic centralaromatic ring Purine pyrazoltriazines andpyrazolo[15a]pyrimidines derivatives were synthesised andinvestigated for their ability to interact with the CB1cannabinoid receptor Albeit a [35S]-GTPγ S assay wasdescribed no data were given concerning the antagonist orinverse agonist properties of such compounds Two hundredand eighty purine derivatives were synthesised [204] Theaffinities of 163 and 164 were given in the patent (28 and12 nM [3H]-SR141716A) In a following patent thesynthesis of sixty pyrazoltriazine derivatives was described(165 Fig 15) [205] The affinity of each compound was notgiven but was said to be ranging between 01 and 590 nMAnd finally more than forty pyrazolo[15a]pyrimidines weresynthesised (166 Fig 15) [206] Their affinity for the CB1cannabinoid receptor was lower than 155 nM Despite thefact that a great number of derivatives share the samesubstitution pattern around the central bicyclic aromatic ringthe comparison of these three new series of compounds ismade difficult by the absence of precise affinity data

Some other classical cannabinoids were shown to possessan antagonist activity at the CB2 receptor This is the casefor instance for the non-selective O-1184 (2) (Ki = 74 nM)which enhances the forskolin-induced cAMP production inCB2-CHO cells (EMax = 161 EC50 = 63 nM) and alsofor O-584 (3) (EMax = 246 EC50 = 138 nM) [39]

2 Indole Derivatives

Since the disclosure in 1993 of a second cannabinoidreceptor many attempts have been made to synthesise CB2cannabinoid receptor selective ligands The first compoundreported to have a somehow greater affinity for the peripheralcannabinoid receptor was WIN-55212-2 The compoundwas introduced by DrsquoAmbra and colleagues [210] back in1992 as a new cannabinoid ligand having a nanomolaraffinity and acting as an agonist In 1996 Showalter et alused a transfected cell line expressing the CB2 cannabinoidreceptor in order to identify selective ligands for thisreceptor [211] They showed that the CB2 affinity of WIN-55212-2 was 7 times greater than the CB1 with Ki valuesof 189 and 028 nM for the CB1 and CB2 cannabinoidreceptors respectively However WIN-55212-2 is anagonist of the cannabinoid receptors Interestingly WIN-55212-3 (167 Fig 16) the so-called inactive enantiomer ofWIN-55212 was recently shown to behave as a low affinity(Ki=363 microM) inverse agonist of the hCB2 cannabinoidreceptor as it decreased the [35S]-GTPγ S binding [209]

III CB2 LIGANDS RECEPTOR ANTAGONISTS ANDINVERSE AGONISTS

The compounds acting as antagonist or inverse agonistsat the CB2 cannabinoid receptor are reviewed in this thirdpart of the paper As for the CB1 cannabinoid receptorligands they are classified depending on their chemicalstructures

1 Classical Cannabinoid Derivatives

The first report of an antagonism mediated by ∆9-THC atthe peripheral cannabinoid receptor was made by Bayewitch

N N

NN

NH COOH

Cl

Cl

N N

NN

N

Cl

Cl

NH2OH

O

N

N

N

N

N

NHNH2

O

Cl

ClN

N

N

N

Cl

Cl

N

O

163 164

165 166

Fig (15) CB1 cannabinoid receptor antagonists 10 Other derivatives Examples of purine (163-164) pyrazoltriazines (165) andpyrazolo[15-a]pyrimidines (166) derivatives described as CB1 cannabinoid receptor antagonists by Griffith

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1383

The pEC50 value was 554 and the Emax value ndash26 ascompared to basal

AM630 also acts as an inverse agonist at the hCB1cannabinoid receptor (EC50 = 900 nM) as Landsman et aldemonstrated [47] Recently Zhang and colleagues reportedthe characterisation of the microsomal metabolism of AM630[212]

N

O

N

O

O

N

O

Cl

O

N

O

(S)-WIN-55212-3 167 BML-190 168

In 1996 Gallant and colleagues identified by submittinga large number of compounds to a binding assay anotherindole analog as CB2 cannabinoid receptor ligand [213] Onthe basis of this compound christened BML-190 (168 Fig16) or called indomethacin morpholinylamide theysynthesised several derivatives that possessed a selectivityratio for the CB2 cannabinoid receptor of up to 140 Thesewere the first compounds specifically designed to be CB2cannabinoid receptor ligands The first report on thepharmacological properties of BML-190 appeared onlyrecently New and colleagues highlighted in the paper theinverse agonist properties of the compound BML-190 dose-dependently increases (103 EC50= 980plusmn70 nM) theforskolin-stimulated levels of cAMP in hCB2-HEK cellswhile WIN-55212-2 decreases (44 45plusmn42nM) thisaccumulation [137]

Fig (16) CB2 cannabinoid receptor antagonists 2 Indolederivatives Chemical structures of WIN-55212-3 (167) andBML-190 (168) two indoles derivatives acting as inverseagonists at the hCB2 cannabinoid receptor Surprisingly in 2002 Melck and collaborators used

BML-190 as a CB2 receptor agonist in a cell proliferationassay [214] BML-190 was also tested as inhibitor of thecyclooxygenase-2 enzyme by Kalgutkar et al [215] Theyobtained IC50 values higher than 33 and 66 microM for theCOX-I and COX-II enzymes respectively These valueshave to be compared with the submicromolar activity ofindomethacin the parent compound Very recently Klegerisand colleagues [216] looking for an antineurotoxic action ofcannabinoids showed that BML-190 increases TNF-αsecretion by stimulated THP-1 monocytic cells but is noteffective on the IL-1β secretion

Further on the basis of WIN-54461 (12) a CB1antagonist AM630 (13) was synthesised by replacement ofthe bromine atom by an iodine one In the initial reportPertwee et al showed the antagonist effects of AM630 butalso suggested that the CB1 cannabinoid receptor may not bethe preferential receptor of AM630 [45] Later on Ross et alreported using hCB1amp2-CHO cells and [3H]-CP-55940that AM630 binds to the CB2 cannabinoid receptor (Ki =312 nM) with a selectivity ratio over 160 [48]Furthermore using a [35S]-GTPγ S assay they showed itsinverse agonist properties (EC50 = 76 nM) However

Table 12 CB2 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Reported binding affinities (Ki) and selectivity ratio (CB1CB2) of SR144528 (169) for rat and human cannabinoid receptors determined using [3H]-CP55940 as radioligand

NN

O

NH

Cl

SR144528 169

rCB2 (spleen) rCB1 (whole brain) Selectivity Ki hCB2 (transfected CHOcells)

Ki hCB1 (transfected CHOcells)

Selectivity References

038 nM 305 nM 800 06 nM 437 nM 730 [217]

067 nM 548 nM 80 [219]

56 nM gt10microM gt1780 [48]

25 nM a gt 1microM a gt400 [224]

024 nM 276 nM 115 199 nM 503 nM 25 [232]

a COS cells

1384 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

3 Diarylpyrazole Derivatives CHO cells is constitutively active and phosphorylated onSer 352 Upon treatment by SR144528 thisphosphorylation of the receptor was inhibited Moreover theSR compound was able to induce dephosphorylation ofagonist-induced CB2 phosphorylation The question whetherSR144528 induces a CB2 receptor conformational changemaking it a better phosphatase-specific substrate or ifSR144528 binding activates specific phosphatases remainsopen However this remains a nice example of inverseagonist-induced receptor resensitisation

The same Sanofi team which synthesised the CB1antagonist SR141716A reported in 1998 the synthesis andpharmacological characterisation of a CB2 cannabinoidreceptor selective antagonist the SR144528 (169) [217218] The structure of the new compound is derived fromSR141716A one SR144528 is the first selective CB2receptor antagonist reported as the inverse agonist propertiesof BML-190 were shown only recently by New et al [137]The authors reported for 169 using [3H]-CP-55940 asradioligand Ki values of 04 nM and 06 nM for the rat andthe human CB2 receptors respectively and found a CB1-CB2 selectivity ratio around 700 Table 12 sums up thebinding affinities of SR144528 for the cannabinoid receptorsobtained by different authors Interestingly while the CB2affinity is more or less constant the affinity obtained for theCB1 receptor varies by one order of magnitude [219] Theantagonist effect of SR144528 was highlighted by evaluatingits effect on cAMP production by hCB2-CHO cells exposedto 3 nM CP-55940 (EC50=10nM) The antagonism wasalso observed on the activation of the MAP kinase pathwayby CP-55940 (IC50=39nM)

The Sanofi research team working on cannabinoidsconducted investigations in order to determine the keyresidues for SR144528 interaction with the receptor (Table13) as well as the binding mode of the compound Firstlythey identified the TM4-EL2-TM5 region as a regioncontaining crucial residues for the affinity of several ligandsamong them SR144528 [224] Among these residues themutation of two cysteine of the second extracellular loop(C174 and C179) abolishes the affinity for the CB2 receptorLater on the implication of S453(161) and S457(165) inthe binding of SR144528 was evidenced by mutationalstudies In the model proposed for the docking of SR144528into the CB2 receptor the crucial contacts are comprised intothe TM4 this is the case for S453(161) and S457(165)which are proposed to form hydrogen bonds with the ligandand W464(172) proposed to have aromatic interactions withthe 4-methylbenzyl of the SR144528 [225] In 2003 Fengand Song showed that point mutations D349(130)A andA634(241)E abolished ligand binding However mutationof the arginine R350(131) member of the highly conservedDRY motif to alanine has no influence on SR144528binding [226]

Early after the report of the synthesis and characterisationof SR144528 the same authors further explored the functionof this compound and showed its inverse agonist propertiesusing CB2-CHO cells co-transfected with the luciferasereporter gene with either the CRE or the murine krox24regulatory sequence [220] In these models SR144528 notonly inhibits CP-55940 effects but also reproduces theeffects of a pertussin toxin treatment proving its inverseagonist properties SR144528 functionality was alsoexplored by [35S]-GTPγ S binding in hCB2-CHO cells(IC50=3 nM) [221] The inverse agonist function ofSR144528 was further illustrated by Rhee and Kim usingCOS cells co-transfected with hCB2 receptor and adenylylcyclase [222]

As for the CB1 cannabinoid receptor ligands Sanofideveloped tricyclic derivatives based on the 1-benzylpyrazole-3-carboxylic scaffold More than twentycompounds were disclosed in a patent [227] in 2001 (84-86Table 7) The benzyl in position 4 characteristic of the CB2diarylpyrazoles from Sanofi is conserved as well as thebicyclic residue in position 3 The length of the second linkbetween the phenyl and the pyrazole can be made of one or

Bouaboula and co-workers investigated SR144528 effectson CB2 cannabinoid receptor phosphorylation status [223]Firstly they showed that the CB2 receptor transfected in

Table 13 CB2 Cannabinoid Receptor Antagonists 3 Di-Aryl-Pyrazole Derivatives

Some of the single point mutations reported for the hCB2 cannabinoid receptor for which pharmacological data concerning the SR144528 are available

Mutant Effect References

R350(131)A no effect [226]

S453(161)A loss of affinity [225]

V456(164)I no effect [225]

S457(165)A loss of affinity [225]

C174S a loss of affinity [224 225]

C175S a loss of affinity [225]

C177S a no effect [225]

C179S a loss of affinity [224 225]

S544(193)G no effect [225]

a Second extra-cellular loop

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1385

two methylene but according to the patent one methylenebridge is preferred The Ki values of these compounds for theCB2 receptor were said to be lower than 500 nM and theirantagonist properties have been assessed using the adenylatecyclase inhibition assay

compounds by the presence of an alkyl substituent inposition 5 instead of a phenyl and of course by position 3which remains unsubstituted However except a lecture atthe 1998 ACS meeting [230] where compound 171 wasreported to have a Ki value of 835 nM and a selectivity ratioover 100 no further report on these compounds waspublished untill now

In a recent paper Mussinu and colleagues reported thesynthesis of other tricyclic derivatives starting fromSR141716A structure [228] N-piperidin-1-yl-14-di-hydroindeno[12-c]pyrazole-3-carboxamide derivatives areCB2 receptor ligands and some of them possess high affinityand selectivity for the CB2 receptor as shown in Table 7 (79-83) These tricyclic derivatives differ from the Sanofi tricyclicderivatives as they do have a phenyl ring instead of a benzylin position 1 Another difference is the nature of thesubstituent in position 3 which is not a bicycloalkyl but acyclic amine One of the compounds synthesised byMussinu et al 81 possesses the highest affinity for the CB2receptor ever reported with a Ki value of 0037 nM for themurine CB2 receptor ([3H]-CP-55940 mice spleenhomogenate) Interestingly 79 the closest SR141716Aanalog lost almost all its affinity for the CB1 receptor andincreased by four orders of magnitude its affinity for the CB2receptor Even if the structure quite similar to the SRcompounds could suggest that these derivatives behave asantagonists no pharmacological data were given in the paperto support this hypothesis

5 2-Oxoquinoline Derivatives

Inaba and colleagues from Japan Tobacco introduced in2001 a completely new structure in the cannabinoid fieldbased on a 2-oxoquinoline scaffold Over eighty derivativeswere disclosed in a patent [231] and one compound JTE-907 (173) was subsequently characterised in-vitro as well asin-vivo [232] In Table 14 are listed some of the compounds(172-178) disclosed in the patent along with their affinity forthe cannabinoid receptors These values were obtained onhuman receptors against [3H]-WIN-55212-2 For twelvecompounds the anti-inflammatory activity per os wasassessed in a carrageenin-induced paw edema model Thehighest effect reported was obtained with JTE-907 with anED50 value lower than 01 mgkg Therefore thesecompounds were claimed to be useful as anti-inflammatoryand anti-allergic agents

To further characterize JTE-907 the authors determinedits affinity for cannabinoid receptors of different species using[3H]-CP-55940 as radioligand In their hands JTE-907behaved as a very selective hCB2 ligand with Ki values of36 and 2370 nM for the CB2 and CB1 cannabinoid receptorsrespectively even more selective than the SR144528 (Kivalues of 199 and 50 nM respectively) However it has tobe mentioned that the affinities reported in the paper forJTE-907 are quite different from those reported in the patent(hCB1 Ki values of 35nM and 009 nM respectively) Italso appears that the SR144528 affinity for the CB1 receptordetermined by Iwamura et al is higher than the affinityreported by Rinaldi-Carmona et al with values of 50 and437nM respectively The authors ascribed the difference tothe variability of SR144528 binding affinities to the assayconditions The function of JTE-907 was evaluated bymeasuring the increase in cAMP production by forskolin-stimulated hCB2-CHO cells Maximum stimulation wasattained at a concentration of 1microM of JTE-907 In the sameassay WIN-55212-2 decreased the cAMP production in adose-dependent manner

Finally it is interesting to strengthen that the five-membered ring derivatives (79-86) of the diarylpyrazolefamily are reported to possess a high CB2 receptor affinitywhile the six- and seven-membered ring derivatives (69-78)are described as CB1 receptor ligands

4 Other Pyrazole Derivatives

Other CB2 ligands based on a pyrazole nucleus wereintroduced by SmithKline Beecham in a patent describingthe synthesis of about sixty new compounds [229] Theiraffinity (Ki) for the hCB2 receptor transfected in HEK293cells was reported to be ranging between 25nM and 10microMTheir structures (170-171 Fig 17) differ from the Sanofi

NN

O

N

ON

NN

N

NN

O

N

ON

NN

N

ClCl

1

5

4 3

2

170 171

Fig (17) CB2 cannabinoid receptor antagonists 4 Otherpyrazole derivatives Structure of two compounds (170-171)among those claimed by SmithKline Beecham to be hCB2antagonists

In conclusion JTE-907 was shown to be a CB2 selectiveinverse-agonist with anti-inflammatory and anti-allergicproperties This compound is currently undergoing Phase Iclinical trials as anti-allergic drug [233]

IV NON-CB1 NON-CB2 CANNABINOIDRECEPTORS LIGANDS

In the last years the possible presence of additional non-CB1 non-CB2 cannabinoid receptors was raised frompharmacological evidences

One of the putative receptors possesses an endotheliallocalisation while other ones are localized in the CNS or atperipheral nerves For instance an additional target for ∆9-

1386 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 14 CB2 Cannabinoid Receptor Antagonists 5 2-Oxoquinoline Derivatives

Structure of six compounds among the oxoquinoline derivatives patented by Japan Tobaco Affinity (Ki nM) and selectivity (CB1CB2) for the cannabinoidreceptors are given The affinity was measured by binding against [3H]-WIN-55212-2 on either hCB2-CHO or hCB1-CHO cells Adapted from [231]

Cpd ndeg Structure hCB2 hCB1 Selectivity

1-2 172

NH

MeO O

O

OMeO

0014 3671 262215

JTE-907 173

NH

O

MeO O

O

NH

O

O

0087 3436 39495

2-2 174

OH

O

NH

MeO O

O 077 3247 4215

3-27 175

NH

O

F

NH

MeO O

O0021 336 16000

3-32 176

NH

MeO O

O

NH

F

O

0036 2398 66610

3-38 177

O

O

NH

N

O

O

NMeO

0085 935 11000

3-43 178

NMeO O

O

NH

F

O

0043 864 20100

tetrahydrocannabinol was described in capsaicin-sensitivesensory nerves by Zygmunt et al [234]

[29] and by Di Marzo et al [235] Thus in the presentreview we will focus on the description of the so faridentified ligands of the non-CB1 non-CB2 receptors withan emphasis on the compounds having antagonist properties

Pharmacological evidences for the existence of additionalcannabinoid receptors were reviewed by Wiley and Martin

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1387

(Table 15) In particular the knowledge about three not yetcloned targets has grown The growing molecularpharmacology of the three putative new cannabinoidreceptors is reviewed in the three following sections Thefourth section summarises the data on the other proposedcannabinoid targets

vasodilatator activity In addition the anandamide effect ispartially sensitive to SR141716A inhibition The authorsproposed the existence of two distinct receptors responsiblefor the vasodilatatory effect of anandamide one located in theendothelium is SR141716A-sensitive while the other onelocated on the vascular smooth muscle is SR141716A-insensitive Thus SR141716A is an antagonist of anendothelial anandamide receptor Several studies wereconducted in order to understand the molecularpharmacology of the endothelial anandamide receptor It ispertussis toxin-sensitive [237] Cannabidiol acts asantagonist and abnormal-cannabidiol (Abn-cbd 179) and itsanalogue O-1602 (180) act as agonists [238] The synthesisof O-1602 and of its analogues are disclosed in a patentdescribing the vasodilatatory activity of cannabinoid

1 Endothelial Anandamide Receptor ndash Abnormal-Cannabidiol Receptor

In 1999 Wagner et al showed that anandamide couldinduce mesenteric vasodilatation through an endotheliallylocated ldquoanandamide receptorrdquo pharmacologically distinctfrom CB1 [236] Other cannabinoids such as ∆9-THC HU-210 or WIN-55212-2 were devoid of mesenteric

Table 15 Non-CB1 Non-CB2 Cannabinoid Receptors Antagonists

An overview of the putative new non-CB1 non-CB2 anandamide-receptors ligands The antagonists are highlighted in bold

NH

N

S

HO OH

Cl

OH

OH

OH OH OH OH

OMe

OMe

RR

RR

RR

RR

HO

OH

NH

O

cannabidiol (10) Abnormal-cannabidiol 179 O-1602 180

O-1918 181 capsazepine 183capsaicin 182

Compound Endothelial AEA receptor Brain AEA receptor Cannabinoid-vanilloid brain receptor

AEA agonist a agonist de

WIN-55212-2 no effecta

agonist e agonist g h

CP-55940 no effect e agonist i

SR141716A antagonist a b j no effect ([35 S]-GTPγS binding) d e f antagonist g h

AM251 no effect k no effect h i

Cannabidiol antagonist b no effect e

O-1918 antagonist c jm

Abd-cbd agonist b l

O-1602 agonist b

Capsaicin no effectj agonist i

Capsazepine no effect b antagonist h

a [236] b [238] c [240] d [249] e [250] f [251] g [254] h [258] i [ 255] k [244] j [ 243] l [ 242] m [241]

1388 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

analogues [239] More recently Offertaler et al showed thatO-1918 (181) a cannabidiol analog acts as an antagonist ofthe endothelial anandamide receptor [240] In addition O-1918 does not bind to the CB1 and CB2 cannabinoidreceptors Furthermore O-1918 unlike cannabidiol doesnot cause vasorelaxation in the mesenteric artery preparationmodel used and thus acts as a true neutral antagonistUsing O-1918 as a pharmacological tool Begg et alproposed a coupling of this new G protein-coupledendothelial receptor through a GiGo mechanism to ionchannels in primary cultured human vascular endothelialcells [241] However in their hands SR141716A wasineffective in antagonising Abd-cbd Ho and Hiley furtherdemonstrated that Abn-cbd relaxes rat small mesentericarteries through an activation of K+ channels via aSR141716A-sensitive pathway [242] Further using thesame model they characterized the vasorelaxant activity ofvirodhamine a novel endocannabinoid apparently actingthrough the same receptor [243] The virodhamine-inducedvasorelaxation is sensitive to SR141716A and O-1918 butinsensitive to AM251 SR144528 and AM630 Moreovervanilloid receptor desensitisation by capsaicin (182) had noeffect

244] Thus position 4 on the 5-phenyl could be furtherexplored to obtain selective ligands

To come to an end despite the fact that the endothelialanandamide receptor is neither identified nor cloned at leastthree antagonists are identified SR141716A cannabidioland O-1918 It is likely that a growing number of ligandsand therapeutic applications will appear in the very nearfuture

2 Brain Anandamide Receptor

Following an in-depth evaluation of anandamide effects inCB1

-- mice Di Marzo and co-workers suggested that a non-CB1 non-CB2 G protein-coupled receptor might mediatesome of the actions of AEA in mice [249] Actually effectsof AEA but not those of ∆9-THC were not decreased inCB1 cannabinoid receptor knock-out mice For instance 30mgkg of AEA were still effective in hot-plate test whereas10 mgkg of ∆9-THC had no effect on anti-nociceptionMoreover in a [35S]-GTPγ S assay AEA stimulated with anEC50 value of 223 microM the [35S]-GTPγ S binding to CB1

--

mice brain membranes (Emax= 28) ∆9-THC showed noeffect in this assay The addition of SR141716A did notaffect the [35S]-GTPγ S binding to CB1

-- mice brainmembranes Shortly after other evidences for the existence ofa new G protein-coupled cannabinoid receptor with a distinctdistribution in the central nervous system were published[250] From a set of 24 commonly used cannabinoids (AEAWIN-55212-2 JWH-030 THChellip) only AEA and WIN-55212-2 produced an increased binding of [35S]-GTPγ S onCB1

-- mice brain membranes Interestingly in this studySR141716A alone produced a significant inhibition of [35S]-GTPγ S binding similar to the inhibition obtained usingCB1

++ mice brain membranes (IC50 values of 57 microM and47 microM respectively) However SR141716A did not affectthe anandamide-induced [35S]-GTPγ S binding in CB1

--

mice brain membranes Moreover [3H]-SR141716Aexhibited significant binding in some brain regions of CB1

--

mice which are different from those exhibiting stimulation of[35S]-GTPγ S binding by WIN-55212-2 and AEA Thusthe authors proposed that the SR141716A effects on CB1

--

mice are mediated neither by the CB1 cannabinoid receptornor by the putative new anandamide brain receptor Monoryand co-workers using CB1

-- mice described a novel nonadenylyl cyclase-coupled cannabinoid binding site in micecerebellum [251] Two agonists AEA and WIN-55212-2but not ∆9-THC or HU210 were found to bind to thisreceptor as they enhanced [35S]-GTPγ S binding NeitherSR141716A nor SR144528 were able to affect the WIN-55212-2 induced [35S]-GTPγ S binding in CB1

-- mice asfound by Breivogel et al [250] However the distributionpattern of the receptor described by Monory et al differs fromthe distribution found by Breivogel et al It has to bementioned that the knock-out strains used were different Themice used by Monory were from a CD-1 strain while theone used by Breivogel were from a C57BL61 strainInterestingly Muthane et al detected differences in nigralneurons number and sensitivity to MPTP in the two mousestrains used [252]

More recently Mo et al obtained results showing thatthe Abnormal-cannabidiol receptor is also responsible for anincreased endothelial cell migration [244] Abn-cbd enhancedhuman umbilical vein endothelial cell migration while O-1918 antagonised Abn-cbd effect The CB1 cannabinoidreceptor inverse agonist SR141716A partially inhibitedAbd-cbd action whereas AM251 and SR144528 had noeffect

In another paper from George Kunos team Batkai et alreported that SR141716A is able to inhibit the endotoxichypotension by a cardiac mechanism involving neither theCB1 nor the CB2 cannabinoid receptors [245] Theinvolvement of a non-CB1 non-CB2 site of action wasshown using CB1

-- and CB1--CB2

-- knock-out miceFurther studies are needed to identify the SR141716Asensitive myocardial site of action

In a recent patent Kunos described O-1918 and itsanalogues as vasoconstrictor agents useful to reversepathological vasodilatation of blood vessels [246] Theeffects of O-1918 on mouse blood pressure on LPS-inducedhypotension in mouse as well as the reversal of hypotensionby O-1918 injection are described Furthermore thesynthesis of O-1918 based antagonists is described startingfrom p-metha-28-dien-1-ol and the appropriate resorcinols

Interestingly the Abd-cbd receptor which was firstdetected in the vascular system also seems to be present inthe CNS [247] Indeed Walter et al found that microglialcells (BV-2) migration induced by 1 microM 2-AG is inhibitedby cannabidiol (300 nM) and O-1918 (1 microM) SR141716Awas found ineffective in this assay Thus 2-AG is an agonistof the Abd-cbd receptor Moreover Abd-cbd is able to elicita dose-dependent cell migration in the same model (EC50 =600nM) [248]

From a more structural point of view SR141716Abehaves as an antagonist while on the opposite AM251(20) a close analogue which differs from the former only bythe nature of one halogen has no effect on this receptor [242

To conclude to our knowledge no compounds havingantagonist or inverse agonist properties at this receptor havebeen identified so far

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1389

3 Cannabinoid-Vanilloid Brain Receptor a cannabinoid-vanilloid receptor at which SR141716A andcapsazepine behave as antagonists

Evidences for the presence of an additional ldquocannabinoidrdquoreceptor differing from the CB1 receptor and from the brainanandamide receptor appeared from studies focused on thehippocampus (for a review see Hajos et al [253]) Hajos etal studied the cannabinoid actions on GABAergic andglutamatergic transmission using the whole-cell patch-clamp technique [254] WIN-55212-2 induced a dose-dependent reduction in glutamatergic transmission reversedby SR141716A (1microM) Thus the authors concluded that thecannabinoid actions on the glutamatergic transmission aremediated by a non-CB1 but SR141716A-sensitive receptorIn another paper Hajos and Freund further characterised thisputative receptor [255] They showed that in addition toWIN-55212-2 CP-55940 and capsaicin (182) a TRPV1receptor agonist also act as agonists Furthermore in theirhands SR141716A as well as capsazepine (183) a TRPV1receptor antagonist were able to antagonise this effect on theglutamatergic transmission while AM251 was notSR141716A effects on CB1 knock-out mice were furtherstudied using a test of anxiety [256] Haller et al showedthat on the one hand the distribution of the CB1 receptorinduces anxiety and SR141716A has an anxiolytic effectand on the other hand SR141716A is still active in CB1

--

mice In a following paper they reported that AM-251induces anxiogenic effects in wild-type and in knock-outmice [257] Thus SR141716A in addition to CB1 bindsto a not yet identified receptor Another evidence for thisadditional receptor for SR141716A was given by Bass andco-workers [61] They conducted a structure-activityrelationship study on the stimulation of locomotor activityinduced by SR141716A and twenty analogues Nocorrelation was found between the affinity for the CB1cannabinoid receptor and the stimulation of locomotoractivity Albeit SR141716A and five of its analogues O-1803 O-1710 O-1253 O-1254 (40) O-1255 (41) behavedas inverse agonists in [35S]-GTPγ S assay none of theseanalogues stimulated the locomotor activity In conclusionthe SR141716A-induced stimulation of locomotor activity isneither due to inverse agonism nor to inhibition of anendogenous tone but to a brain receptor distinct from theCB1 receptor Di Marzo et al previously provided evidencesthat the cannabinoid-vanilloid brain receptor could mediatesome of the cannabinoid effects on locomotion [249]

4 Other Non-CB1 Non-CB2 Cannabinoid Receptors

Evidences for the existence of additional non-CB1 non-CB2 cannabinoid receptors localised in the rat isolatedmesenteric arterial bed appeared from the works of Ralevicand collaborators Several cannabinoids were evaluated intheir model of sensory neurogenic vasorelaxation evoked byelectrical field stimulation

HU210 was found to attenuate vasorelaxation in a CB1and CB2 independent way as SR141716A LY320135 andSR144528 were ineffective in inhibiting HU210 effect [259]Similar results were obtained with ∆9-THC [260]Similarly noladin ether inhibited sensory neurogenicrelaxation in a concentration-dependent manner which wasalso unaffected by SR141716A LY320135 and SR144528Moreover this effect as the ∆9-THC one was pertussistoxin sensitive suggesting the involvement of a Gio protein-coupled receptor [261]

However the inhibitory effects of CP-55940 and WIN-55212-2 were affected by the presence of SR141716A (1microM)or LY320135 (1microM) but not by the addition of SR144528(1microM) [262]

It appears that at least two receptors should be involvedin cannabinoid-mediated attenuation of sensory nerve-mediated vasorelaxation The first one is SR141716A andLY320135 sensitive while the other one is resistant toSR141716A

Some evidences for the existence of a ldquoCB2-likerdquo receptorappeared from the studies on palmitoylethanolamide (PEA)an endogenous fatty acid amide devoid of CB1 and CB2affinity [264] antinociceptive potential Calignano and co-workers showed that PEA was able to alleviate pain inseveral animal models [265 266] For instance PEA dose-dependently inhibits kaolin-evoked writhing in mice TheCB2 cannabinoid receptor inverse agonist SR144528 (02mgkg) inhibits PEA anti-nociceptive effect [266] Thus theauthors suggest that PEA acts as agonist on a SR144528-sensitive non-CB2 cannabinoid receptor (ldquoCB2-likerdquoreceptor) at which SR144528 acts as an antagonist

Further studies are needed in order to better characterisethese additional ldquocannabinoidrdquo receptors However it islikely that several therapeutic applications will soon springfor these receptors

Very recently Pistis et al published a study on theneurophysiological effects of cannabinoids on the basolateralamygdala neurons in-vivo [258] One of their findings is thatHU-210 and WIN-55212-2 did not elicit similar effects onbasal amygdala projection neurons firing rate While theformer had no effect the latter decreased the firing rateMoreover SR141716A reversed the WIN-55212-2 effectswhile AM251 had no effect The vanilloid receptorantagonist capsazepine also antagonised the WIN-55212-2effect on firing rate of these neurons Therefore the authorssuggested the presence of a novel cannabinoid-vanilloidreceptor

V CONCLUSION

Since their development cannabinoid antagonists provedto be essential tools in the understanding of cannabinoidpharmacology and biochemistry Moreover the interestshown by the pharmaceutical industry in the development ofnew cannabinoid antagonists prove if necessary the realtherapeutic potential of this class of compounds Thetreatment of eating and movement disorders memorydeficits psychosis and dependencies from various addictivedrugs are some of the most cited applications for the CB1cannabinoid receptor antagonists

From these lines it appears that in addition to theknown CB1 cannabinoid receptor it is now likely that twoother cannabinoid receptors do exist in the brain The firstone should be an anandamide brain receptor for whichSR141716A has no affinity while the second one should be

1390 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Much less wide is the knowledge on the CB2cannabinoid receptor antagonists However potentialapplications could be disorders involving the immunesystem such as inflammation or allergies

[19] Devane WA Hanus L Breuer A Pertwee RG StevensonLA Griffin G Gibson D Mandelbaum A Etinger AMechoulam R Science 1992 258 1946

[20] Mechoulam R Ben-Shabat S Hanus L Ligumsky MKaminski NE Schatz AR Gopher A Almong S MartinBR Compton DR Biochem Pharmacol 1995 50 83

Finally the growing literature on the non CB1 non CB2cannabinoid receptors should allow the development of newdrugs First examples are given by the cannabidiol andabnormal-cannabidiol derivatives described by Kunos et alfor which potential therapeutic applications on the vascularsystem are already patented

[21] Sugiura T Kondo S Sukagawa A Nakane S Shinoda AItoh K Yamashita A Waku K Biochem Biophys Res Co1995 215 89

[22] Hanus L Abu-Lafi S Fride E Breuer A Vogel Z ShalevDE Kustanovich I Mechoulam R Proc Natl Acad SciUSA 2001 98 3662

[23] Oka S Tsuchie A Tokumura A Muramatsu M Suhara YTakayama H Waku K Sugiura T J Neurochem 2003 851374It clearly appears from this paper that the antagonists of

the cannabinoid receptors are still an active research fieldfrom which should emerge new promising therapeutic toolsas well as innovative drugs in the near future

[24] Goutopoulos A and Makriyannis A Pharmacol Therapeut2002 95 103

[25] Beal JE Olson R Lefkowitz L Laubenstein L Bellman PYangco B Morales JO Murphy R Powderly W PlasseTF Mosdell KW Shepard KV J Pain Symptom Manage1997 14 7ACKNOWLEDGEMENTS [26] Notcutt W Price M Miller R Newport S Phillips CSimmons S Sansom C Anaesthesia 2004 59 440

GGM is very indebted to the ldquoFonds pour la formationagrave la Recherche dans lrsquoIndustrie et lrsquoAgriculture (FRIA)rdquo Belgium for the award of a research fellowship Theexperimental works of the authors were supported by grantsfrom the FNRS The Belgian Charcot Foundation and theUniversiteacute catholique de Louvain

[27] Ledent C Valverde O Cossu G Petitet F Aubert JFBeslot F Bohme GA Imperato A Pedrazzini T RoquesBP Vassart G Fratta W Parmentier M Science 1999 283401

[28] Zimmer A Zimmer AM Hohmann AG Herkenham MBonner TI Proc Natl Acad Sci U S A 1999 96 5780

[29] Wiley JL and Martin BR Chem Phys Lipids 2002 121 57[30] Adam J and Cowley P Expert Opin Ther Patents 2002 12

1475

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[32] Barth F and Rinaldi-Carmona M Curr Med Chem 1999 6745

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[33] Little PJ Compton DR Johnson MR Melvin LS MartinBR J Pharmacol Exp Ther 1988 247 1046

[2] Devane WA Dysarz FA Johnson MR Melvin LSHowlett AC Mol Pharmacol 1988 34 605

[34] Pertwee RG Stevenson LA Elrick DB Mechoulam RCorbett AD Brit J Pharmacol 1992 105 980

[3] Matsuda LA Lolait SJ Brownstein MJ Young ACBonner TI Nature 1990 346 561

[35] Harrison C Traynor JR Life Sci 2003 74 489-508[4] Gerard CM Molerau C Vassart G Parmentier M BiochemJ 1991 279 129 [36] Pertwee RG Fernando SR Griffin G Ryan W Razdan

RJ Compton DR Martin BR Eur J Pharmacol 1996 315195

[5] Glass M Dragunow M Faull RLM Neuroscience 1997 2299

[37] Ross RA Brockie HC Fernando SR Saha B Razdan RKPertwee RG Brit J Pharmacol 1998 125 1345

[6] Shire D Carillon C Kaghad M Calandra B Rinaldi-Carmona M LeFur G Caput D Ferrara P J Biol Chem1995 270 3726 [38] Griffin G Wray EJ Rorrer WK Crocker PJ Ryan WJ

Saha B Razdan RK Martin BR Abood ME Brit JPharmacol 1999 126 1575

[7] Ryberg E Khang Vu H Groblewski T Larsson N ElebringT Hjorth S Sjogren S Greasley PJ 2004 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 119

[39] Ross RA Gibson TM Stevenson LA Saha B Crocker PRazdan RK Pertwee RG Brit J Pharmacol 1999 128 735

[40] Martin BR Winckler JR Wiley JL Huffman JW CrockerPJ Saha B Razdan RK J Pharmacol Exp Ther 1999 2901065

[8] Rinaldi-Carmona M Calandra B Shire D Bouaboula MOustric D Barth F Casellas P Ferrara P LeFur G JPharmacol Exp Ther 1998 278 871

[41] Martin BR Stevenson LA Pertwee RG Breivogel CSWilliams A Mahadevan A Razdan RK 2002 Symposium onthe Cannabinoids Burlington Vermont International CannabinoidResearch Society page 2

[9] Munro S Thomas KL Abu-Shaar M Nature 1993 365 61[10] Howlett AC Qualy JM Khachatrian LL Mol Pharmacol

1986 29 307[11] Glass M Felder CC J Neurosci 1997 17 5327

[42] Thomas A Ross RA Saha B Mahadevan A Razdan RKPertwee RG Eur J Pharmacol 2004 487 213

[12] Calandra B Portier M Kerneacuteis A Delpech M Carillon C LeFur G Ferrara P Shire D Eur J Pharmacol 1999 374445 [43] Compton DR Gold LH Ward SJ Balster RL Martin BR

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Daum SJ Ackerman JH Gruett MD Kumar V EstepKG Olefirowicz EM Wetzel JR Alexander MD WeaverJD Haycock DA Luttinger DA Casiano FM ChippariSM Kuster JE Stevenson JI Ward SJ J Med Chem1995 38 3094

[14] Deadwyler S Hampson RE Mu J Whyte A Childers SRJ Pharmacol Exp Ther 1995 273 734

[15] Mackie K Lai Y Westenbroek R Mitchell R J Neurosci1995 15 6552

[16] Bouaboula M Poinot CC Bourrie B Canat X Calandra BRinaldi-Carmona M LeFur G Casellas P Biochem J 1995312 637 [45] Pertwee R Griffin G Fernando S Li X Hill A

Makriyannis A Life Sci 1995 56 1949[17] Bouaboula M Poinot CC Marchand J Canat X Bourrie BRinaldi-Carmona M Calandra B LeFur G Casellas P Eur JBiochem 1996 237 704

[46] Hosohata Y Quock RM Hosohata K Makriyannis AConsroe P Roeske WR Yamamura HI EurJPharmacol 1997 321 R1[18] delPulgar T Velasco G Guzman M Biochem J 2000 347

369 [47] Landsman RS Makriyannis A Deng H Consroe P RoeskeWR Yamamura HI Life Sci 1998 62 109

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[48] Ross RA Brockie HC Stevenson LA Murphy VLTempleton F Makriyannis A Pertwee RG Brit JPharmacol 1999 126 665

[81] Gatley SJ Lan R Volkow ND Pappas N King P WongCT Gifford AN Pyatt B Dewey SL Makriyannis A JNeurosci 1998 70 417

[49] Huffmann JW Curr Med Chem 1999 6 705 [82] Lan R Lu Q Fan P Gatley J Volkow ND Fernando SRPertwee R Makriyannis A AAPS Pharmsci 1999 1 article 4[50] Rinaldi-Carmona M Barth F Heaulme M Shire D Calandra

B Congy C Martinez S Maruani J Neliat G Caput DFerrara P Soubrieacute P Breliegravere JC Le Fur G FEBS Lett1994 350 240

[83] Mathews WB Ravert HT Musachio JL Franck RARinaldi-Carmona M Barth F Dannals RF J Labelled CompdRad 1999 42 589

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GA Psychopharmacology 2003 167 103[68] Reggio P Umijiego U Hurst D Seltzman H Hyatt SRoche M McAllister S Fleisher D 2004 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 2

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[102] Ravinet Trillou C Delgorge C Menet M Arnone M SoubrieP Int J Obesity 2004 28 640[69] Song ZH and Bonner TI Mol Pharmacol 1996 49 891

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[103] Cota D Marsicano G Tschoumlp M Gruumlbler Y FlachskammC Schubert M Auer D Yassouridis A Thoumlne-Reineke COrtmann S Tomassoni F Cervino C Nisoli E LinthorstACE Pasquali R Lutz B Stalla GK Pagotto U J ClinInvest 2003 112 423

[71] McAllister SD Tao Q Barnett-Norris J Buehner K HurstDP Guarnieri F Reggio PH Nowell Harmon KW CabralGA Abood ME Biochem Pharmacol 2002 63 2121

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[104] Cani PD Lasa Montoya M Neyrinck AM Delzenne NMLambert DM Br J Nutr 2004 92 757

[73] Murphy JW and Kendall DA Biochem Pharmacol 2003 651623

[105] Colombo G Agabio R Fa M Guano L Lobina C LocheA Reali R Gessa GL Alcohol Alcohol 1998 33 126

[74] McAllister SD Rizvi G Anavi-Goffer S Hurst DP Barnett-Norris J Lynch DL Reggio PH Abood ME J Med Chem2003 46 5139

[106] Serra S Carai MA Brunetti G Gomez R Melis S VaccaG Colombo G Gessa GL Eur J Pharmacol 2001 430 369

[107] Serra S Brunetti G Pani M Vacca G Carai MAM GessaGL Colombo G Eur J Pharmacol 2002 443 95[75] McAllister SD Hurst DP Barnett-Norris J Lynch D

Reggio PH Abood ME J Biol Chem 2004 279 48024 [108] Colombo G Vacca G Serra S Carai MAM Gessa GLEur J Pharmacol 2004 498 119[76] Salo OM Lahtela-Kakkonen M Gynther J Jarvinen T

Poso A J Med Chem 2004 47 3048 [109] Gallate JE and McGregor IS Psychopharmacology 1999 142302[77] Rinaldi-Carmona M Pialot F Congy C Redon E Barth F

Bachy A Breliere JC Soubrie P Le Fur G Life Sci 199658 1239

[110] Cohen C Perrault G Voltz C Steinberg R Soubrie P BehavPharmacol 2002 13 451

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[111] Le Fur G Cohen C Steinberg R Soubrie P 2003 Symposiumon the Cannabinoids Burlington Vermont InternationalCannabinoid Research Society page 55[79] Gatley SJ Lan R Volkow ND Pappas N King P Wong

CT Gifford AN Pyatt B Dewey SL Makriyannis A JNeurochem 1998 70 417

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Behav 1997 56 803 [153] Dow RL Hammond M World Patent Application 2004WO04052864[116] Leweke FM Giuffrida A Wurster U Emrich HM Piomelli

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F Di Marzo V Lipids Health Dis 2003 2 5 [155] Katoch-Rouse R Pavlova OA Caulder T Hoffman AFMukhin AG Horti AG J Med Chem 2003 46 642[118] Dean B Sundram S Bradbury R Scarr E Copolov D

Neuroscience 2001 103 9 [156] Katoch-Rouse R and Horti AG J Labelelled Compd Rad2003 46 93[119] Zavitsanou K Garrick T Huang XF Prog Neuro-

Psychopharmacol Biol Psychiatry 2004 28 355 [157] Kumar JSD Prabhakaran J Arango V Parsey RV Underwood MD Simpson NR Kassir SA Majo VJ VanHeertum RL Mann JJ Bioorg Med Chem Lett 2004 142393

[120] Meltzer HY Arvanitis L Bauer D Rein W Am JPsychiatry 2004 161 975

[121] Castellano C Rossi-Arnaud C Cestari V Constanzi M CurrDrug Targets 2003 2 61 [158] Stoit AR Lange JHM den Hartog AP Ronken E Tipker

K van Stuivenberg HH Dijksman JAR Wals HC KruseCG Chem Pharm Bull 2002 50 1109

[122] Terranova JP Storme JJ Lafon N Perio A Rinaldi-Carmona M Le Fur G Soubrie P Psychopharmacology 1996126 165 [159] Barth F Congy C Martinez S Rinaldi M World Patent

Application 2001 WO0132663[123] Mallet PE Beninger RJ Psychopharmacology 1998 140 11[124] Nakamura-Palacios EM Winsaure PJ Moerschbaecher JM

Behav Pharmacol 2000 11 377[160] Ruiu S Pinna GA Marchese G Mussinu J-M Saba P

Tambaro S Casti P Vargiu R Pani L J Pharmacol ExpTher 2003 306 363[125] Brodkin J Moerschbaecher JM J Pharmacol Exp Ther

1997 282 1526 [161] Francisco MEY Burgess JP George C Bailey GSGilliam AF Seltzman HH Thomas BF Magn Reson Chem2003 41 265

[126] Lichtman AH Eur J Pharmacol 2000 404 175[127] Wolff MC Leander JD Eur J Pharmacol 2003 477 213[128] Arnone M World Patent Application 2003 WO03082256 [162] Cullinan GJ Fahey KJ Koppel GA United States Patent

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Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1369

reported an increased rate of penile erection after directinjection of SR141716A in the paraventricular nucleus of thehypothalamus [130] This effect was dose dependent and asignificant effect was obtained with a dose of 1microgkg

AM251 in mice at a same dose range (10-30 mgkg ip) thanthe anorectic effects [143] These effects are reversed byadministration of CP-55940 and are absent in CB1

-- miceproving the implication of the CB1 receptor in theantidepressant-like effect of AM251 In 2004 Liao et alreported that AM251 was able to displace the binding of[3H]-batrachotoxin A from its binding site on sodiumchannels in mice brain synaptic preparations [144] Theyobtained an IC50 value of 112 microM and a competitivemechanism of action The authors suggested that AM251 iscapable of reducing neuronal excitability through blockade ofvoltage-sensitive sodium channels in brain

From the clinical trials involving the SR141716A themost frequently reported side-effects are nausea dizziness anddiarrhoea Depression and anxiety were not higher than inthe placebo groups Regarding one of the known side effectsof SR141716A administration the enhancement of intestinalmotility Carai et al reported recently that chronicadministration of the inverse agonists to mice induced atolerance to this prokinetic effect [131] This is not the firstreport of tolerance onset after administration of SR141716A[132] however few is known on the mechanismsresponsible for that phenomenon

Wiley and collaborators reported in 2001 a studydescribing new structure-activity relationships using thepyrazole nucleus as a central scaffold [145] Starting fromSR141716A structure they alternatively substituted one ofthe four substituents while retaining the others bysubstituents known to impart agonist activity in classicalcannabinoids One of the authors expectations was todetermine which positions are responsible respectively forthe antagonism and for the affinity of SR141716A Theaffinity of thirty compounds was assessed previous to in-vivoevaluation of their function in mice using the spontaneousactivity the tail-flick and the rectal temperature assays Theauthors showed that phenyl group in position 5 is critical foraffinity as compound O-1559 (30) lacking this phenyl has adecreased affinity (Table 6) This was already shown by Lanand co-workers [136] along with the need of a substituent inpara position on the phenyl Thomas et al previouslyshowed that this substituent could be a bromine or an iodineatom [146] However an alkyl chain is also tolerated as it isthe case in compounds O-1302 (31) O-1691 (32) or O-1704(33) All these compounds antagonise the anti-nociceptiveand hypothermic effects of ∆9-THC The authors suggestedthat the 5-substituent of pyrazoles is involved in receptorrecognition and antagonism The modulations of thesubstitution pattern of the phenyl in position 1 demonstratethat the 24-di-chloro substitution is the preferred one for theaffinity as well as for the activity Several compoundssupport this assertion Thomas et al showed that additionalhalogens result in a decreased affinity as in compounds 6-I-SR141716A (34) or 4rsquo6-di-I-SR141716A (35) (Ki values of166 and 126 nM respectively) [146] Suppression of the twochlorines (O-1300 39) or replacement of these two chlorinesby an alkyl chain like in O-1254 (40) and O-1255 (41) ledto less active compounds with Ki values ranging from 150to 430 nM However O-1254 and O-1255 behaved asantagonists in a [35S]-GTPγ S assay [61] Lan et al [136]and Wiley et al [145] also modified the substitution inposition 3 Replacement of the amido piperidinyl substituentby alkyl amides as in O-1269 (42) or O-1270 (43) etherslike in O-848 (44) and O-853 (45) ketones (O-1272 46)alcohols (O-1876 47) or alkanes (O-1877 48) resultedmostly in decreased affinity (Table 6) but also with a changeof functionality in some compounds as revealed by in-vivoassays For instance replacement of the piperidinyl by apentyl (O-1269) or by an heptyl chain (O-1270) gave agonistcompounds having Ki values of 32 and 48 nM respectivelyThus the authors suggested that the 3-substituent region isinvolved in receptor recognition and agonist activity Wileyet al on the basis of their results concluded that while the3-position seems to be involved in agonism the 1- 4- and

One close analogue of SR141716A 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-piperidinecarbox-amide (SR147778 28) was described very recently byRinaldi-Carmona et al [133 134] (Fig 3) It possesses highaffinity and selectivity for the hCB1 cannabinoid receptorwith Ki values for the hCB1 and hCB2 receptors of 35 and400 nM respectively ([3H]-CP-55940 hCB1amp2-CHO cells)Further SR147778 antagonised CP-55940 effects on mousevas deferens contractions (pA2=81) and on forskolin-stimulated adenylyl cyclase activity in U373MG cells(pA2=82) but had no effects alone In-vivo SR147778 afteroral administration reversed WIN-55212-2 inducedhypothermia and analgesia As SR141716A SR147778dose-dependently reduced ethanol and sucrose solutionintake with significant effects starting at 03 mgkg (sc) and3 mgkg (po) respectively This compound is currentlyinvestigated in Phase I clinical trials for the treatment ofobesity as well as nicotine and alcohol addictions

Another derivative the 5-(4-bromophenyl)-1-(24-dichlorophenyl)-4-ethylpyrazole-3-(4-hydroxypiperidine)-carboxamide (29) was described in a very recent patent fromSanofi [135] (Fig 3) This antagonist possesses an IC50value for the hCB1 cannabinoid receptor of 32 nM ([3H]-CP-55940 hCB1-CHO cells)

The first structure-affinity relationships for theSR141716A derivatives were reviewed by Barth andRinaldi-Carmona in 1999 [32] Since then many papersdescribing new derivatives were published In 1999 Lan etal described around thirty 15-diarylpyrazoles derivatives[136] Among them was AM251 previously reported as aradioimaging ligand for the CB1 receptor [78] and thatappeared to be more potent (Ki = 749 nM) and selective(selectivity ratio of 306) than SR141716A (Ki = 115 nMselectivity ratio of 143) They reported a Kd value of 05 nMin the mouse vas deferens model using WIN-55212 asagonist Interestingly New et al described for the samecompound an inverse agonist effect on the hCB2 receptorusing a forskolin-induced cAMP accumulation assay (EC50= 650 nM) [137] Recently the anti-obesity effects ofAM251 were reported and are not surprisingly similar tothose of SR141716A [138 139 140] Chen et aldemonstrated that there are synergistic effects on food intakesuppression between AM251 and nalmefene an opioidantagonist [141] This synergistic effect was also presentbetween SR141716A and naloxone as shown by Kirkham[142] Shearman et al found antidepressant-like effects of

1370 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

5-positions appear to be involved in antagonism The O-derivatives reported were also described in a patent alongwith their synthetic pathways which were not given in thepaper [147]

Another investigation of the SR141716Aaminopiperidine region was conducted by Francisco et al[148] They synthesised 21 analogues possessing either analkyl amide or an alkyl hydrazide substituent of various

Table 6 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structure of some of the characterised 1- 3- 4- and 5-pyrazole derivatives Binding affinities (Ki values nM) were obtained on rat brain homogenatesusing [3H]-CP-55940 [136 145 146 148 150] or [125I]-AM-251 [155]

NN

R1

R5

R3R4

Cpd ndeg R1 R3 R4 R5 [136] [145] [146] [148] [150] [155]

SR141716 14 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Cl-Ph 115 62 62 62 13 18

O-1559 30 24-di-Cl-Ph CO-NH-piperidinyl Me 1-Methylpentyl 233

O-1302 31 24-di-Cl-Ph CO-NH-piperidinyl Me 4-((CH2)5)-Ph 21 1

O-1691 32 24-di-Cl-Ph CO-NH-piperidinyl Br 4-((CH2)5)-Ph 15

O-1704 33 24-di-Cl-Ph CO-NH-piperidinyl I 4-((CH2)5)-Ph 22

6-I-SR141716 34 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-Cl-Ph 166

4rsquo6-di-I-SR141716 35 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-I-Ph 126

4rsquo-I-SR141716 36 24-di-Cl-Ph CO-NH-piperidinyl Me 4-I-Ph 75 25 6

4rsquo-Br-SR141716 37 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Br-Ph 168 3

Cpd 25 in ref [136] 38 24-di-Cl-Ph CO-NH-morpholinyl Me 4-Br-Ph 54

O-1300 39 Ph CO-NH-piperidinyl Me 4-Cl-Ph 150

O-1254 40 4-(CH2)4-Ph CO-NH-piperidinyl Me 4-Cl-Ph 226 256

O-1255 41 4-(CH2)5-Ph CO-NH-piperidinyl Me 4-Cl-Ph 433

O-1269 42 24-di-Cl-Ph CO-NH-pentyl Me 4-Cl-Ph 32 114 3

O-1270 43 24-di-Cl-Ph CO-NH-heptyl Me 4-Cl-Ph 48 462 3

O-848 44 24-di-Cl-Ph CH2-O-(CH2)2-piperidinyl

Me 4-Cl-Ph 2450 232

O-853 45 24-di-Cl-Ph CH2-O-CH2-cyclohexyl

Me 4-Cl-Ph 388 100

O-1272 46 24-di-Cl-Ph CO-heptyl Me 4-Cl-Ph 221

O-1876 47 24-di-Cl-Ph 1rsquo-OH-heptyl Me 4-Cl-Ph 657

O-1877 48 24-di-Cl-Ph Heptyl Me 4-Cl-Ph 422

MF9725-64-17 49 24-di-Cl-Ph CO-NH-butyl Me 4-Cl-Ph 134

MF9725-179-32 50 24-di-Cl-Ph CO-NH-NH-butyl Me 4-Cl-Ph 51

MF9725-66-11 51 24-di-Cl-Ph CO-NH-cyclohexyl Me 4-Cl-Ph 25

MF9725-95-31 52 24-di-Cl-Ph CO-NH-(4-hydroxybutyl)

Me 4-Cl-Ph 154

Cpd 15 [150] 53 n-pentyl CO-NH-piperidinyl Me Ph 23

Cpd 16 [150] 54 n-pentyl CO-NH-piperidinyl Me 4-Br-Ph 63

Cpd 17 [150] 55 n-hexyl CO-NH-piperidinyl Me Ph 21

Cpd 18 [150] 56 n-heptyl CO-NH-piperidinyl Me Ph 47

NIDA-41109 57 24-di-Cl-Ph CO-NH-piperidinyl Br 4-Cl-Ph 14

NIDA-41119 58 24-di-Cl-Ph CO-NH-piperidinyl H 4-Cl-Ph 9

NIDA-41057 59 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OH-Ph 104

NIDA-41020 60 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 41

NIDA-41087 61 2-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 8

NIDA-42055 62 24-di-Cl-Ph CO-NH-piperidinyl Br 4-OMe-Ph 62

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1371

lengths in position 3 of the pyrazole moiety They observedthat until five carbons the affinity increases along with thecarbon chain length This is observed with the alkylhydrazide the alkyl amide and the hydroxyalkyl amideseries Moreover the hydrazide analogues exhibit a loweraffinity for the rCB1 than the amide analogues as illustratedby compounds MF9725-64-17 (49) and MF9725-179-32(50) having Ki values of 134 and 51 nM respectively(Table 6) From their structure-activity relationships (SAR)studies the authors concluded that the pharmacophoricrequirement of the amidopiridine region is a chain not longerthan 3 Aring and that a substituent having a positive chargedensity would probably result in increased affinity andpotency The same team described in a patent several othercompounds [149] Binding affinities (Ki) against [3H]-CP-55940 [3H]-SR141716A or [3H]-WIN55212-2 on wholerat brain or on hCB1-transfected cells were given Moreoveractivity data were obtained using the [35S]-GTPγ S assaydemonstrating that these alkyl amide and hydrazide analogsact as antagonists or inverse agonists Despite the greatnumber of derivatives claimed none of them posses asignificantly greater affinity for the CB1 receptor than theSR141716A Nevertheless they showed a slightenhancement of the selectivity for the CB1 receptor over theCB2 cannabinoid receptor

affinity of these derivatives increases with the length of thealkyl chain with an optimal length of 5-6 carbons The bestcompound of this series N-(piperidin-1-yl)-5-phenyl-1-hexyl-4-methyl-1H-pyrazole-3-carboxamide (55) possesses aKi value of 21 nM determined using [3H]-CP-55940 on ratbrain membranes which is higher than the 13 nMdetermined for SR141716A in the same conditions (Table6) In the same paper the authors starting from thehypothesis that antagonism by SR141716A is caused bybinding to the same region of the receptor as do the agonists(CP-55940 and WIN-55-2122) but preventing the agonistpromoted conformational change conducted extensiveconformational analysis as well as superimposition modelsand 3D-QSAR to propose a molecular mechanismsupporting the action of SR141716A Along with theauthors the C-5 aryl substituent of SR141716A occupyinga unique region could contribute in conferring the antagonistproperties Moreover the C-3 substituent could beresponsible for the antagonist or inverse agonist propertiesdepending on the interaction with the receptor

About this topic a very interesting review dealing withthe cannabinoid receptors pharmacophores as well as withthe activationinactivation of these receptors was recentlypublished by Reggio [151]

More recently Dyck et al described seven otherderivatives varying at the amide position [152] The onlycompound possessing a higher affinity for the CB1cannabinoid receptor than SR141716A was the 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta-

The affinity for the rat CB1 receptor of seven derivativespossessing an alkyl chain in position 1 instead of the 24-dichlorophenyl substituent was reported by Shim et al in apaper describing a molecular mechanism for the antagonistand inverse agonist activity of SR141716A [150] The

NN

O

NHN

Cl Cl

Cl

NN

Cl

Cl

NON

NN

O

NH

N

Cl

ClCl

NN

O

NH

MeO Cl

CP-272871 66

63 64

65

Fig (4) CB1 cannabinoid receptor antagonists 3 Diarylpyrazole derivatives Structures of 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63 ) 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methyl-pyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]-morpholine (64 ) N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65 ) and N-phenyl-1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-pyrazole-3-carboxamide (CP-272-871 66 )

1372 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

[c]pyrrol2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63)(Fig 4) The Ki values were 5 and 12 nM respectively([3H]-CP-55940 hCB1-HEK cells)

CP-55940 rat brain) For instance N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65) (Fig 4) showed a Ki value of15 nM However no data were provided concerning thefunctionality of such compounds

Over 200 other pyrazole derivatives acting at the CB1cannabinoid receptor were claimed in a patent from Pfizer[153] The affinity for the hCB1 receptor of 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methylpyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]ndashmorpholine (64) (Fig 4)was 79 nM ([3H]-SR141716A hCB1-HEK cells) Theothers compounds affinity was between 01 and 100 nM

In an effort to develop new SR141716A analoguespossessing lower lipophilicity to be used as PET tracersKatoch-Rouse and colleagues further explored thesubstitution pattern of the two phenyls [155] Bindingassays showed that a decreased lipophilicity lead to adecreased affinity for the CB1 receptor as determined bydisplacement of [3H]-AM251 For instance compoundNIDA-41057 (59) has a lipophilicity of 42 ( expressed asElogDoct) and a Ki value of 104 nM whereas SR141716Ahas a ElogDoct value of 54 and a Ki value of 18 nM In

Seventy five pyrazole derivatives were described in a veryrecent patent from Makriyannis et al [154] The affinity ofthe compounds for the cannabinoid receptors is given TheKi values for the best compounds are lower than 6 nM ([3H]-

Table 7 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structures and affinities for the cannabinoid receptors of some representative tricyclic pyrazole derivatives Values were obtained using [3H]-CP-55940as radioligand and mouse [160 228] rat [159 227] or human [158] cannabinoid receptors Compounds 69-78 are CB1 cannabinoid receptor ligandsCompounds 79-86 are CB2 cannabinoid receptor ligands

NN

O

NHX

R3

R1

R2

n

n˚ X R1 R2 n R3 Ki CB1 (nM) Ki CB2 (nM) Selectivity References

69 -(CH2)3- Cl 24-di-Cl 0 piperidine 126 [158]

70 -(CH2)3- H 24-di-Cl 0 azepane 100 [158]

71 -(CH2)3- H 24-di-Cl 0 piperidine 398 [158]

72 -(CH2)3- Cl 24-di-Cl 0 azepane 125 [158]

73 -(CH2)3- NO2 24-di-Cl 0 azepane 63 [158]

74 -(CH2)2-O- H 24-di-Cl 0 piperidine 501 [158]

75 -CH2-S- Br 24-di-Cl 0 piperidine lt 500 gt10 [159]

76 -CH2-SO2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

77 -(CH2)2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

78 -(CH2)3- Cl 24-di-Cl 0 piperidine 000035 21 60000 [160]

79 -CH2- Cl 24-di-Cl 0 piperidine 2050 034 00002 [228]

80 -CH2- Br 24-di-Cl 0 piperidine 1570 027 00002 [228]

81 -CH2- CH3 24-di-Cl 0 piperidine 363 0037 00001 [228]

82 -CH2- Cl 4-Cl 0 piperidine 1787 09 00005 [228]

83 -CH2- H 24-di-Cl 0 piperidine 1152 0385 00003 [228]

84 -CH2- Cl 34-di-Cl 1 133-trimethylbicyclo[221]heptyl

lt500 gt10 [227]

85 -CH2- Br 24-di-Cl 1 bicyclo[321]oct-3-yl lt500 gt10 [227]

86 -CH2- Br 4-Me 1 77-dimethylbicyclo[410]hept-3-yl

lt500 gt10 [227]

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1373

another paper Katoch-Rouse described the synthesis of[18F]-NIDA-42033 (26) a ligand useful as PET radiotracerstarting from its bromo derivative [156] Recently Kumar etal [157] proposed the synthesis of a 11C derivative ofNIDA-41087 (61) a compound previously described byKatoch-Rouse (Table 5) Post-mortem binding of thiscompound to human prefrontal cortex was also investigatedInterestingly CP-272871 (66) described by Meschler as aCB1 inverse agonist (Ki=57 nM [3H]-CP-55940 rat brainhomogenates) possesses a lipophilicity similar to theNIDA-41087 one [62] However despite the presence of amethoxy function no radiolabelled derivative has beendescribed to date One explanation could be the compoundlow selectivity (2 fold) for the CB1 cannabinoid receptor

To further explore the pharmacology of this compound afunctional assay using the mouse vas deferens model wasperformed by Ruiu and colleagues enlightening thecompetitive antagonist properties of NESS (pA2= 1246against WIN-55212-2) which had no effect by itself up to1microM Moreover NESS 0327 was unable to affect basalbinding of the [35S]-GTPγ S demonstrating its lack ofnegative intrinsic activity In-vivo studies were conductedusing the hot plate and tail flick tests NESS dose-dependently abolished the antinociceptive effect of WIN-55212-2 but had no effect by itself However the authorssuggested based on the affinity and in-vivo activity of theircompound that NESS 0327 possesses a poor centralbioavailability

Several new attempts to increase the affinity of thediarylpyrazole derivatives were recently made by rigidifyingthe SR141716A structure (Table 7) Six fused ringanalogues of SR141716A obtained by fusion of the 5-(4-chlorophenyl) substituent with the central pyrazole to forman indazole ring were published by Bass et al in 2002 [61]The compound possessing the highest affinity O-1248 (67)has a Ki value of 475 nM as determined by displacement of[3H]-CP-55940 from rat brain membranes

Another example of ring-constrained biarylpyrazole wasdescribed by Herbert Seltzman team [161] Theyinvestigated a photocyclisation reaction starting from theSR141716A leading to a pyrazolo[15-f]phenanthridinestructure (Fig 5) The new compound (68) was tested on theCB1 receptor (whole rat brain) against [3H]-CP-55940[3H]-SR141716A and [3H]-WIN-55212-2 and exhibited Kivalues of 48 35 and 50 nM respectively The CB2 affinitywas assessed using [3H]-CP-55940 and was found to be 100times lower (Ki = 3340 nM) No effect on the [35S]-GTPγ Sassay (rat brain) was observed by the authors

Stoit and colleagues from Solvay Pharmaceuticalspublished a paper describing the synthesis andpharmacological characterisation of new benzocyclohepta-pyrazole derivatives as CB1 antagonists (69-74) [158] Theaffinity of these compounds was more or less one order ofmagnitude lower than the SR141716A one with pKi valuesranging from 64 to 72 compared to 76 for the SRcompound (Table 7) Compounds were shown to beantagonists with pA2 values from 70 to 89 as theyprevent CP-55940-induced cAMP accumulation Howeverthe authors investigating the bioavailability of theircompounds either po or after ip injection found negligibleblood plasma levels

NN

Cl

Cl

O

NH

N

NN

Cl

O

NHN

Cl

Cl

O-1248 67 68However just before the publication of Stoit results

Sanofi-Synthelabo took a patent describing tricyclicpyrazolecarboxylic acid amide derivatives having antagonistproperties on the CB1 cannabinoid receptor andsubmicromolar affinities (75-77) [159] Thirty examples weregiven along with their synthetic pathway but no individualpharmacological data were shown

Fig (5) CB1 cannabinoid receptor antagonists 3Diarylpyrazole derivatives Two ring-constrained diarypyrazolederivatives O-1248 (67 ) and a pyrazolo[15-f]phenanthridinederivative (68 )

It appears from these attempts to increase SR141716Aaffinity by rigidifying its structure that only a limitedincrease in the CB1 cannabinoid receptor affinity can beexpected with the apparent exception of NESS 0327Moreover rigidifying the structure often results in a morelipophilic compound in a family of compounds being yetlipophilic

More recently Ruiu and colleagues published a paperdescribing the synthesis and complete pharmacologicalcharacterisation of a very potent CB1 receptor ligandchristened NESS 0327 (78) [160] This compound has thesame structure as compound 69 published by Stoit et al(compound 20 in the reference [158]) Nevertheless Ruiu hasobtained an affinity for the CB1 cannabinoid receptor 5000times bigger than the SR141716A one with Ki values of035 pM and 18 nM respectively using mouse forebrainhomogenates and [3H]-CP-55940 as radioligandFurthermore the selectivity ratio obtained for this compoundby Ruiu is 60000 with a Ki value for the CB2 receptor of21 nM against the [3H]-CP-55940 The discrepancybetween the data obtained independently by Stoit et al andRuiu et al could hardly be explained by the differences inthe origin of the receptor used respectively hCB1-CHO andmouse forebrain

4 Phenyl Benzofuranone Derivatives

In a 1997 patent from Eli Lilly was described thesynthesis of aryl-benzothiophene and aryl-benzofuranederivatives (87-89 Fig 6) having CB1 receptor affinity andantagonist properties [162] From ten compounds having aKi value lower than 25 microM ([3H]-CP-55940 hCB1-CHOcells) one was selected and claimed to have a Ki of 170 nMCompound 88 was shown in the patent to antagonise theeffect of anandamide on cAMP accumulation with an IC50 of

1374 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

O

O

O

O

NC

S

O

O

O

O

O

O

O

NC

LY320135 8887 89

Fig (6) CB1 cannabinoid receptor antagonists 4 Phenyl benzofuranone derivatives Chemical structures of two aryl-benzofuranederivatives (87 -88 ) and one aryl-benzothiophene (89 ) derivative from Eli Lilly

500 nM and the effect of WIN-55212-2 on calcium channelsat 1microM Moreover the intra-peritoneal injection (20 mgkg)of this compound antagonised the in-vivo effect ofanandamide in the Open Field Assay a mouse behaviourmodel Later on Felder and colleagues gave a furtherdescription of this compound called LY320135 in a paperdescribing its effect on cAMP accumulation and on ioniccurrents [163] The selectivity ratio of LY320135 was 106with Ki values of 141 and 14900 nM for hCB1 and hCB2receptors ([3H]-CP-55940) respectively LY320135antagonised anandamide inhibition of forskolin-inducedcAMP accumulation with an IC50 value of 734 nMChristopoulos et al in 2001 reported for LY320135 apKb value of 527 in the inhibition of WIN-55212-2mediated response in the rat electrically-stimulated vasdeferens while the pKb value for SR141716A was 75[164]

Other patents were taken by Aventis in which somepotential applications for their compounds were describedFor instance an association between an azetidine derivativehaving CB1 antagonists properties and sibutramine aserotonin and norepinephrine reuptake inhibitor was claimed[170] as a treatment against obesity Oral administration ofcompound 90 (3 mgkg) and sibutramine (06 mgkg) to fafaZucker rats which are genetically obese rats has resulted ina decreased food intake compared to control lean Zuckerrats In another patent the association of an azetidinederivative (1-10 mgkg po ) and a D2D3 agonist likequinpirole (01 mgkg ip) was claimed to have a therapeuticpotential in the treatment of Parkinsonrsquos disease [171] Theeffect has been evidenced using an akinesia model in the ratEvidences supporting the use of CB1 antagonists as adjuvantin the treatment of Parkinsonrsquos disease begin to appear Itseems that such compounds would be helpful in thetreatment of Parkinsonism [172] and levodopa-induceddyskinesia [173] Brotchie published an interesting paperreviewing the CB1 cannabinoid receptor signaling inParkinsonrsquos disease [174] Nevertheless further clinicalstudies should be conducted to further confirm the usefulnessof CB1 antagonists in the treatment of Parkinsonrsquos disease

5 Azetidine Derivatives

The family of azetidine compounds illustrated bycompounds 90 and 91 (Fig 7) was developed at Aventis byDaniel Achard and colleagues and was described in a seriesof patents [165-168] They claimed the IC50 values of thesecompounds for the CB1 receptor to be less than or equal to100 nM These values were obtained following the proceduredescribed by Kuster et al [169] The antagonist propertywas shown using an in-vivo model the reversal ofhypothermia induced by CP-55940 in mice and the ED50obtained were lower than 50 mgkg

6 Aryl-Imidazolidine-24-Diones Derivatives

In 1999 the Didier Lambert team published a paperdescribing the affinity of 24 new 3-alkyl-55rsquo-diphenyl-imidazolidine-24-dione derivatives for the hCB1 receptor[175] The preliminary structure-activity relationships

N

SO

O

Cl

Cl

F

F

N

SO

O

Cl

NS

90 91

Fig (7) CB1 cannabinoid receptor antagonists 5 Azetidine derivatives Two examples (90 -91 ) of the azetidine derivatives developedby Aventis

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1375

Table 8 CB1 Cannabinoid Receptor Antagonists 6 Aryl-Imidazolidine-24-Diones Derivatives

Structures affinities ([3H]-SR141716A hCB1-CHO cells) and pKb values ([35 S]-GTPγS HU210 rat brain) of three aryl-imidazolidine-24-dionederivatives (92-94)

N

NH

OO

R

Br

Br

Cpd ndeg R Ki (nM) a pKb b

DML20 92 ethylmorpholine 70 611

DML21 93 1-hydroxypropyl 103 625

DML23 94 heptyl 98 574

a [175]b [177]

showed that the substitution at the nitrogen in position 3 aswell as a bromine atom in para of the phenyl rings aremandatory for the compounds affinity Later on threecompounds termed DML20 (92) DML21 (93) and DML23(94) were characterised as neutral antagonists using the [35S]-GTPγ S assay on rat cerebellum membranes [176] (Table 8)In the same model they competitively inhibited HU-210-induced [35S]-GTPγ S binding with pKB values of 611625 and 574 respectively Moreover these compoundswere proven to be quite selective for the CB1 receptor

higher than the one for the CB2 cannabinoid receptor (IC50 =300 nM) The inverse agonism of the compound wasextrapolated by inhibiting CP-55940-induced hypothermiain mice (EC50 = 18 nM) Moreover one oral dose (10mgkg) reduces the food intake in diet-induced obese ratsInterestingly food intake of CB1

-- mice was not alteredBody weight loss was maintained all over the study during achronic administration (14 days) of 10 mgkg of Cpd A

Later on Merck published a patent [180] in which someof the 45-diaryl-pyrazoles derivatives previously describedwere claimed to be useful in the treatment or the preventionof obesity in association with compounds inhibiting the11β-hydroxysteroid dehydrogenase type 1 (1β-HSD1)enzyme The 1β-HSD1 enzyme is responsible for thesynthesis of cortisol increased levels of which according tothe inventors are associated with obesity However neitherresults on appetite suppression nor on weight loss weregiven even if some indications on the affinity of thecompounds for the cannabinoid receptors were given

Very recently the functionality of these DML compoundswas explored using the [35S]-GTPγ S assay both on rat andhuman CB1 cannabinoid receptors [177] The data obtainedconfirm that the 3-alkyl-55rsquo-diphenyl-imidazolidinedionederivatives behave as neutral antagonists on rat CB1cannabinoid receptor (rat brain) However DML 20 DML21and DML23 acted as inverse agonists on the human receptor(hCB1-CHO cells) Furthermore the authors showed thatthis different functionality is not due to the hCB1 receptorlevel of expression in the recombinant cell line as whateverthe level of expression (Bmax of 44 pmolmg or 32pmolmg) DML derivatives behaved as inverse agonists ofthe hCB1 cannabinoid receptor

Another imidazole ring pattern of substitution wasproposed by Hagmann and collaborators [181] Thecompounds described 43 examples are given possess twophenyls the first one in position 1 linked to the nitrogenand the second one in position 2 The carboxamidesubstituent is in position 4 instead of in position 2 for thediarylimidazoles described by Finke These 12-diaryl-imidazoles are claimed to be CB1 cannabinoid receptorantagonists or inverse agonists but the absence of precisepharmacological values do not allow any comparisonbetween the two substitutions patterns However a samepattern of substitution was used by other researchers Amongthem Dyck et al described in 2004 ten 1-(4-chlorophenyl)-2-(24-dichlorophenyl)-imidazole derivatives varying by theircarboxamide substituent in position 4 (101-103 Table 9)[152] For instance with a 3-azabicyclo[330]octan-3-ylsubstituent (101) they obtained a Ki value of 14 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) In a [35S]-GTPγ Sassay 101 exhibited an IC50 value of 19 nM

7 Diarylimidazoles Derivatives

Finke and colleagues at Merck developed a new class ofCB1 receptor antagonists based on an imidazole nucleusOver eighty 45-diaryl-imidazoles derivatives having CB1antagonist properties were claimed in a patent published in2003 [178] Some representative compounds of the patent areillustrated in Table 9 (95-100) The affinity of the newcompounds was determined using [3H]-CP-55940 andhCB1-CHO cells Although no detailed pharmacologicaldata were given in the patent in-vitro and in-vivo propertiesof one compound termed Cpd A (100) have been describedelsewhere [179] Its affinity for the CB1 cannabinoid receptorwas shown to be nanomolar (IC50 = 4 nM) and 75 times

1376 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 9 CB1 Cannabinoid Receptor Antagonists 7 Diaryl-Imidazole Derivatives

Structure and affinity range of some 45-diaryl-imidazole derivatives for the human cannabinoid receptors determined using [3H]-CP-55940 asradioligand and hCB1amp2-CHO cells

N

NR2

R1

R4

R3

ndeg R1 R2 R3 R4 hCB1 (IC50 or Ki) hCB2 (IC50 or Ki) References

95 CH3 CO-NH-piperidinyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

96 CH3 CO-NH-piperidinyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

97 CH3 CO-NH-cyclohexyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

98 CH3 CO-NH-phenyl 24-di-Cl-Ph 4-Cl-Ph 10 ndash 100 nM a 100 ndash1000 nM a [178]

99 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

100 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph 4 nM a 300 nM a [179]

101 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

CH3 14 nM b ND [152]

102 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

H 66 nM b ND [152]

103 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 85 nM b ND [152]

104 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 23 nM b 542 nM b [182]

105 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 30 nM b 608 nM b [182]

106 4-Br-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 60 nM b 489 nM b [182]

107 4-CF3-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 29 nM b 634 nM b [182]

108 4-Cl-Ph 24-di-Cl-Ph CO-NH-morpholinyl CH3 197 nM b 3297 nM b [182]

109 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidin-4-ol CH3 172 nM b 3959 nM b [182]

110 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CN 30 nM b 1590 nM b [182]

111 4-Cl-Ph 24-di-Cl-Ph CO-NH- piperidinyl CH2F 36 nM b 906 nM b [182]

112 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH2-CH3 14 nM b 430 nM b [182]

113 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Cl 27 nM b 823 nM b [182]

114 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Br 23 nM b 746 nM b [182]

115 4-Cl-Ph 24-di-Cl-Ph CO-NH-tetrahydroisoquinoline CH3 34 nM b 696 nM b [182]

116 4-Cl-Ph 24-di-Cl-Ph CO-NH-cycloheptyl CH3 35 nM b 349 nM b [182]

117 24-di-Cl-Ph 4-Cl-Ph CO-NH-piperidinyl CH3 403 nM b 208 nM b [182]a IC50 value b Ki value

Lange and co-workers from Solvay described twenty-eight imidazole derivatives [182] The 24-dichlorophenylsubstituent at position 2 was kept constant while thesubstitutions at the other positions were explored (Table 9)Position 5 can accommodate a large range of littlesubstituents like hydrogen (104) methyl (105) ethyl (112)chlorine (113) bromine (114) fluoromethyl (111) or cyano(110) without major changes in the affinity At position 4more bulky substituents like 1234-tetraisoquinoline (115)or cycloheptyl (116) were well tolerated whereas thepresence of a more hydrophilic moiety such as morpholine(108) or piperidin-4-ol (109) was detrimental to the affinity

Interestingly position exchange between the 24-diphenyland the 4-chlorophenyl substituents led to a 13 fold loweraffinity with Ki values of 30 and 403 nM for 105 and 117respectively ([3H]-CP-55940 hCB1-CHO cells) In thefunctional assay which was inhibition of WIN-55212-2-induced [3H]-arachidonic acid release by hCB1-CHO cellsall tested compounds behaved as antagonists Compound105 showed a pA2 value of 86 which is the same theauthors obtained for SR141716A It would be interesting toassess if these compounds are true antagonists or if they actmore as inverse agonists Compound 105 when administeredto rats was able to inhibit CP-55940-induced hypotension

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1377

with an ED50 value of 24 mgkg which is close to the 3mgkg obtained for SR141716A The compound was alsoactive in another in-vivo model the WIN-55212-2-inducedhypothermia in mice Compound 104 which has nosubstituent in position 5 of the imidazole ring was lessactive in the hypotension model and inactive in thehypothermia model Thus a methyl group is the preferredsubstituent in position 5 of the imidazole moiety Otherproperties either experimental (P-glycoprotein affinitylogPHPLC) or computational (molecular volume polarsurface area) obtained for compounds 105 were similar tothose obtained for SR141716A Finally molecularmodelling studies revealed a close structural overlap betweenthe two compounds

value of 197 nM is selective for hCB1 and is as potent asSR141716A The structural modulations of 118 led to someinteresting compounds (Table 10) Six compounds possess aKi value for the hCB1 cannabinoid receptor equal to or lowerthan 25 nM antagonist properties in the inhibition of [3H]-arachidonic acid release by WIN-55212-2-stimulated hCB1-CHO cells and Ki values for the hCB2 receptor over 1microMThe diaryl-pyrazoline derivatives contained a chiral center atposition 4 The authors resolved the racemic mixture of twocompounds (121 and 130) The levorotatory enantiomersappeared to be the eutomers For compound SLV319levorotatory enantiomer of 121 the Ki and pA2 values were78 nM and 99 respectively The Ki and pA2 values ofSLV326 levorotatory enantiomer of 130 were 359 nM and9 respectively

8 34-Diaryl-Pyrazoline Derivatives Two in-vivo models CP-55940-induced hypotension inrat and WIN-55212-2-induced hypothermia in mouse wereused to assess the potential of these compounds The resultsare of the same order of magnitude than those obtained forSR141716A For instance in the hypotension modelSLV319 ((-)121) and SLV326 ((-)130) showed ED50 values

Lange and collaborators also discovered 34-diaryl-pyrazoline derivatives after a screening of compoundsresembling to SR141716A [183184] Compound 118 thatwas initially identified during the screening possesses a Ki

Table 10 CB1 Cannabinoid Receptor Antagonists 8 34-Diaryl-Pyrazolines Derivatives

Structure affinity for the hCB1 and hCB2 cannabinoid receptors (Ki nM) expressed in CHO transfected cells ([3H]-CP-55940) and potency (pA2) ofsome 34-dirylpyrazolines derivatives Table adapted from [184]

NN

N N

SO2

R3

R2

Cl R1

R4

n˚ R1 R2 R3 R4 Ki hCB1 (nM) Ki hCB2 (nM) pA2 (CB1)

118 H H H 4-CH3 197 gt 1000 84

119 H H H 246- CH3 24 gt 1000 94

120 H H H 4-Cl 16 gt 1000 95

121 H CH3 H 4-Cl 25 gt 1000 87

122 H CH3 CH3 4-Cl 280 gt 1000 85

123 H H H 4-F 53 gt 1000 9

124 H CH3 H 4-F 338 gt 1000 85

125 H CH3 CH3 4-F gt 1000 gt 1000 lt 75

126 H CH3 H 3-Cl 14 gt 1000 86

127 4-Cl CH3 H 4-Cl 255 ND ND

128 4-F CH3 H 4-Cl 584 ND ND

129 H CH3 H H 170 ND 75

130 H CH3 H 4- CF3 221 gt 1000 93

SR141716A 25 1580 86

1378 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

N

HN

O

N

Cl Cl

Cl

N

N

HN

O

Br

Br131 132

Fig (8) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Chemical structures of two 56-diaryl-pyrazine-2-amide derivatives (131-132) developed as CB1 cannabinoid receptorantagonists by AstraZeneca

of 55 and 2 mgkg (po) respectively proving their in-vivoefficacy after oral administration The authors alsodemonstrated that SLV319 is devoid of affinity for the P-glycoprotein pump and that it possessed a goodCNSplasma ratio (17) These two compounds (SLV319and SLV326) were chosen as development candidates bySolvay and entered clinical Phase I trials in 2003 [185]

132 Fig 8) useful as CB1 cannabinoid receptor antagonists[186 187] The activity of twenty derivatives wasdetermined using a [35S]-GTPγ S assay and hCB1-CHOtransfected cells The concentration required to give halfmaximal inhibition of CP-55940-induced [35S]-GTPγ Sbinding (IC50) is lower than 200 nM for the preferredcompounds No affinity data were given for thesecompounds

9 Diaryl-Pyrazine Diphenyl-Pyridine Diphenyl-Phenyland Diaryl-Pyrimidines Derivatives

The same year in a patent from Merck Finke andcollaborators described several 56-di-phenyl-pyridinederivatives as hCB1 antagonists or inverse agonists [188]The compounds have a substituent in position 3 of thepyridine core (Fig 9) This substituent could be a cyano(133-134) or a nitro group a halogen an ester or an amide(135-136) Over 150 compounds were synthesised toillustrate the invention but no pharmacological data wasdisclosed in the patent

In 2003 appeared several patents describing new familiesof compounds that bind to the CB1 cannabinoid receptorThese compounds have in common a central six atomsaromatic ring which could be a pyrazine a pyridine aphenyl or a pyrimidine and that is substituted by at leasttwo phenyl rings

The first patents were from Berggren and collaboratorsfrom AstraZeneca who published two patents describing thesynthesis of 56-diaryl-pyrazine-2-amide derivatives (131-

A few months later another patent was taken by Sanoficlaiming the antagonist properties of 56-di-phenyl-2-pyridine carboxamide derivatives [189] These compounds

N

NH

O

Cl

Cl

N

O

Cl

O NH

N

O

Cl

ClCl

NH

CN O

N

Cl

Cl

Cl

OHN

N

O

Cl

ClCl

CNF

F

133 134

135 136 137

Fig (9) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives 56-Di-phenyl-pyridine derivatives patented by Merck (133-136) and by Sanofi (137) as CB1 cannabinoid receptorsantagonists

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1379

NH

O

N

Cl

ClCl

NH

O

N

Cl

F3CCl

N

N

O

Cl

Cl Cl F138 139 140

Fig (10) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Representative structures of the diphenyl-phenyl (138-139) and 45-diphenyl-pyrimidine (140) derivatives developed ascannabinoid antagonists at Sanofi and Merck respectively

28 examples are given structurally related to the diphenyl-phenyl derivatives have their pyridine core substituted bytwo phenyl rings and by an amide moiety (137 Fig 9) TheIC50 values were lower than 100 nM but no data were givenshowing the antagonist properties although the compoundswere assayed in the adenylate cyclase inhibition assay

10 Other Derivatives

Several amide derivatives structures recently appearedamong the CB1 cannabinoid receptor antagonists In a seriesof patents taken by Merck in 2003 Hagmann and colleaguesdescribed a large amount of compounds obtained by parallelsynthesis which are claimed to be CB1 cannabinoid receptorantagonists [192-195] They were synthesised by reacting alibrary of substituted amines with a library of carboxylicacids (141-144 Fig 11) However as no value was givenfor the affinity or activity of these amides we will notfurther discuss this class of compounds

Along this line Sanofi Synthelabo developed newdiphenyl-phenyl derivatives claimed to have CB1cannabinoid receptor antagonist properties [190] They arebased on a central phenyl ring substituted by two phenylsand by an amide moiety (138-139 Fig 10) Thirteencompounds were claimed along with their synthesis TheirIC50 values were lower than 100 nM ([3H]-CP55-940hCB1-CHO cells) but no other specific data (selectivitypA2hellip) was given

Several 15-diaryl-pyrrole-3-carboxamide derivatives weresynthesised and claimed to be CB1 cannabinoid receptorantagonists by Berggren et al [196] The affinity valueswere not disclosed in the patent (145-146 Fig 12) Otherpyrrole derivatives were synthesised by Guba et al atHofmann-La Roche [197] More specifically 2-(thiazol-4-yl)pyrrole derivatives were claimed as CB1 cannabinoidreceptor antagonist (IC50lt2microM) (147-148 Fig 12)

Finally Kopka et al from Merck described more thanone hundred 45-diaryl-pyrimidine derivatives as CB1cannabinoid receptor antagonists (140 Fig10) [191]However no precise data were provided concerning theaffinity and functionality of the compounds

R2

R1N

R3

R4

R5

O

NH

O

Cl

O F

F

R2

R1NH

R3

R4

HO R5

O

NH

O

Cl

CN

NH

O

Cl

O

O

NH

O

Cl

Cl

+

141142

143 144

Fig (11) CB1 cannabinoid receptor antagonists 10 Other derivatives Retrosynthetic scheme of a CB1 cannabinoid antagonistlibrary of amide derivatives developed at Merck The structures of four representative compounds are illustrated (141-144)

1380 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

NH

O N

Cl

Cl

Cl

N

NH

O

OMe

MeO

N

NH

O

N

S

MeO

N

NH

O

N

S

MeO

145 146

147 148

Fig (12) CB1 cannabinoid receptor antagonists 10 Other derivatives Two examples of 15-diaryl-pyrrole derivatives described byBerggren et al (145-146) While the 2-(thiazol-4yl)pyrroles derivatives 147 and 148 were described by Guba et al from Hofmann-LaRoche All these pyrroles derivatives were claimed to be CB1 cannabinoid receptor antagonists

Another new structure is represented by the 124-triazolederivatives developed by Jagerovic and collaborators In arecent paper were described the synthesis andpharmacological properties of five new 15-diphenyl-3-alkyl-

triazole derivatives [198] Among these compounds onlyone (149) behaved as a CB1 antagonist inhibiting the WIN-55212-2-induced contractions in a mouse vas deferenspreparation However despite its effects in isolated tissue

Table 11 CB1 Cannabinoid Receptor Antagonists 10 Other Derivatives

Structure and affinity of diaryl-triazole derivatives

N

NN

R3

R2

R1

Cpd R1 R2 R3 Ki CB1 (nM) References

149 24-diCl 4-Cl hexyl 855 a [198]

150 24-diCl 4-Cl2-OMe CO-NH-(3-azabicyclo[330]octan-3-yl) 270 b [152]

151 24-diCl 4-Cl CO-NH-(3-azabicyclo[330]octan-3-yl) 164 b [152]

152 4-Cl 24-diCl CO-NH-(3-azabicyclo[330]octan-3-yl) 137 b [152]

153 4-Cl 24-diCl CO-NH-benzylpyrrolidin-3-yl 29 b [152]

154 4-Cl 24-diCl CO-NH-1-(4-chlorophenyl)ethyl 66 b [152]

155 4-Cl 24-diCl CO-NH-piperidinyl 356 c [182]

156 24-diCl 4-Cl CO-NH-piperidinyl 382 c [182]

a [3H]-SR141716A rat cerebellar membranesb [3H]-CP-55940 hCB1-HEK EDNA cellsc [3H]-CP-55940 hCB1-CHO cells

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1381

N

S

O

NH

Cl

Cl

Cl

F

N

S

O

NHN

ClCl

Cl

N

S

O

NH

Br

N

S

O

NHN

Cl

Cl

Cl

156 157 158 159

Fig (13) CB1 cannabinoid receptor antagonists 10 Other derivatives Structures of 45-diaryl-thiazole derivatives patented as hCB1cannabinoid receptor antagonists by Solvay Pharmaceuticals (156 157 158) or by AstraZeneca (157 158 159)

assays (mouse vas deferens and guinea pig ileum) thecompound possessed only a reduced rCB1 affinity with Kivalues of 855 and 748 nM using [3H]-SR141716A and [3H]-WIN-55-212-2 respectively (Table 11) Further four seriesof compounds differing by the nature of the substituentaround the triazole core were also described in a patent[199] Usually the compounds claimed possess two aromaticrings possibly substituted and one linear alkyl chain Theaffinity for the cannabinoid receptors was not givenNevertheless 149 was evaluated in two isolated tissuemodels the inhibition of the electrically evoked contractionsin the guinea pig ileum and in the mouse vas deferens Inthe two models compound 149 inhibited the effect of WIN-55212-2 but had no effect by itself In the vas deferensmodel the authors obtained a pA2 value of 748 to becompared with 763 obtained for AM251

Yet another five membered ring a thiazole has beenused as central moiety of a new class of cannabinoid ligandsThe thiazole ring is substituted by two aryls in position 4and 5 and by an amide (position 2) as in compounds 156-159 (Fig 13) Forty derivatives of this type weresynthesised by Lange and colleagues from SolvayPharmaceuticals and claimed in a patent as agonists orantagonists of the CB1 cannabinoid receptor [200] Nopharmacological data were provided however their activitywas assessed by measuring the cAMP in transfected hCB1-CHO cells In a recent paper from Lange et al two of thesethiazole derivatives were further described [182] Compound13 (157) the most closely related to SR141716A has a Kivalue of 227 nM while for compound 14 (158) Ki value isover 1000 nM Thus as in the imidazole series (see Table9) the aromatic substitution pattern is of great importancefor the CB1 cannabinoid receptor affinity Compound 13however was devoid of in vivo activity

Others 124-triazole derivatives were described by Dycket al compounds 150-154 unlike the Jagerovic ones havean amide moiety in position 3 [152] The highest affinitywas obtained with a 1-benzyl-pyrrolidin-3-yl substituentThe Ki value was 29 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) to be compared to 12 nM obtained for theSR141716A

Seventeen 45-diaryl-thiazole derivatives were alsosynthesised by Berggren et al and patented by AstraZeneca(157-159 Fig 13) [201]

In a patent from Merck Toupence et al described over190 substituted furo[23]pyridines claimed to be CB1cannabinoid receptor antagonists [202] The affinity (IC50 lt1microM) was measured using recombinant CHO cells and [3H]-CP-55940 and the activity using cAMP dosage (160 Fig14)

Lange et al described a triazole derivative (155) whichcan be superimposed with SR141716A Compound 155showed a Ki value of 356 nM ([3H]-CP-55940 hCB1-CHOcells) a pA2 value of 83 in the inhibition of WIN-55212-induced release of [3H]-arachidonic acid by hCB1-CHO cellsand was active in vivo in the CP-55940-inducedhypotension in rat (ED50 = 236 mgkg) [182]

Very recently Alanine from Hoffmann-LaRoche foundthat diarylbenzo[13]dioxole derivatives act as CB1

O

O

S

N

OO

F

ClCl

O

O

F O

N

OON

NH2

O

Cl

Cl

Cl

160 161 162

Fig (14) CB1 cannabinoid receptor antagonists 10 Other derivatives One example of a furo[23]pyridine derivative (160) and twoexamples of diarylbenzo[13]dioxole derivatives (161-162) described as hCB1 cannabinoid receptor antagonists by Toupence et aland Alanine et al respectively

1382 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

cannabinoid receptor antagonists (161-162 Fig 14) [203]Approximate IC50 values for twelve compounds ([3H]-CP-55940 hCB1-HEK cells) among the three hundred andeighty compounds described were given (IC50lt2microM)

et al in 1995 [207] In their hands ∆9-THC was not able toinhibit the forskolin-stimulated adenylyl cyclase activity inan assay performed on hCB2-CHO cells Subsequently theyshowed that ∆9-THC was able to antagonise HU210-inducedinhibition of adenylyl cyclase activity thus acting as anantagonist of the hCB2 cannabinoid receptor expressed inCHO cells [208] Furthermore very recently Govaerts et al[209] reported an inverse agonist effect of ∆9-THC and ∆8-THC on hCB2-CHO cells using the [35S]-GTPγ S assayThe pEC50 values were 763 and 888 respectively whilethe Emax values were ndash27 and ndash16 as compared to basal

Griffith from Pfizer described three new series of CB1cannabinoid receptor antagonists based on a bicyclic centralaromatic ring Purine pyrazoltriazines andpyrazolo[15a]pyrimidines derivatives were synthesised andinvestigated for their ability to interact with the CB1cannabinoid receptor Albeit a [35S]-GTPγ S assay wasdescribed no data were given concerning the antagonist orinverse agonist properties of such compounds Two hundredand eighty purine derivatives were synthesised [204] Theaffinities of 163 and 164 were given in the patent (28 and12 nM [3H]-SR141716A) In a following patent thesynthesis of sixty pyrazoltriazine derivatives was described(165 Fig 15) [205] The affinity of each compound was notgiven but was said to be ranging between 01 and 590 nMAnd finally more than forty pyrazolo[15a]pyrimidines weresynthesised (166 Fig 15) [206] Their affinity for the CB1cannabinoid receptor was lower than 155 nM Despite thefact that a great number of derivatives share the samesubstitution pattern around the central bicyclic aromatic ringthe comparison of these three new series of compounds ismade difficult by the absence of precise affinity data

Some other classical cannabinoids were shown to possessan antagonist activity at the CB2 receptor This is the casefor instance for the non-selective O-1184 (2) (Ki = 74 nM)which enhances the forskolin-induced cAMP production inCB2-CHO cells (EMax = 161 EC50 = 63 nM) and alsofor O-584 (3) (EMax = 246 EC50 = 138 nM) [39]

2 Indole Derivatives

Since the disclosure in 1993 of a second cannabinoidreceptor many attempts have been made to synthesise CB2cannabinoid receptor selective ligands The first compoundreported to have a somehow greater affinity for the peripheralcannabinoid receptor was WIN-55212-2 The compoundwas introduced by DrsquoAmbra and colleagues [210] back in1992 as a new cannabinoid ligand having a nanomolaraffinity and acting as an agonist In 1996 Showalter et alused a transfected cell line expressing the CB2 cannabinoidreceptor in order to identify selective ligands for thisreceptor [211] They showed that the CB2 affinity of WIN-55212-2 was 7 times greater than the CB1 with Ki valuesof 189 and 028 nM for the CB1 and CB2 cannabinoidreceptors respectively However WIN-55212-2 is anagonist of the cannabinoid receptors Interestingly WIN-55212-3 (167 Fig 16) the so-called inactive enantiomer ofWIN-55212 was recently shown to behave as a low affinity(Ki=363 microM) inverse agonist of the hCB2 cannabinoidreceptor as it decreased the [35S]-GTPγ S binding [209]

III CB2 LIGANDS RECEPTOR ANTAGONISTS ANDINVERSE AGONISTS

The compounds acting as antagonist or inverse agonistsat the CB2 cannabinoid receptor are reviewed in this thirdpart of the paper As for the CB1 cannabinoid receptorligands they are classified depending on their chemicalstructures

1 Classical Cannabinoid Derivatives

The first report of an antagonism mediated by ∆9-THC atthe peripheral cannabinoid receptor was made by Bayewitch

N N

NN

NH COOH

Cl

Cl

N N

NN

N

Cl

Cl

NH2OH

O

N

N

N

N

N

NHNH2

O

Cl

ClN

N

N

N

Cl

Cl

N

O

163 164

165 166

Fig (15) CB1 cannabinoid receptor antagonists 10 Other derivatives Examples of purine (163-164) pyrazoltriazines (165) andpyrazolo[15-a]pyrimidines (166) derivatives described as CB1 cannabinoid receptor antagonists by Griffith

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1383

The pEC50 value was 554 and the Emax value ndash26 ascompared to basal

AM630 also acts as an inverse agonist at the hCB1cannabinoid receptor (EC50 = 900 nM) as Landsman et aldemonstrated [47] Recently Zhang and colleagues reportedthe characterisation of the microsomal metabolism of AM630[212]

N

O

N

O

O

N

O

Cl

O

N

O

(S)-WIN-55212-3 167 BML-190 168

In 1996 Gallant and colleagues identified by submittinga large number of compounds to a binding assay anotherindole analog as CB2 cannabinoid receptor ligand [213] Onthe basis of this compound christened BML-190 (168 Fig16) or called indomethacin morpholinylamide theysynthesised several derivatives that possessed a selectivityratio for the CB2 cannabinoid receptor of up to 140 Thesewere the first compounds specifically designed to be CB2cannabinoid receptor ligands The first report on thepharmacological properties of BML-190 appeared onlyrecently New and colleagues highlighted in the paper theinverse agonist properties of the compound BML-190 dose-dependently increases (103 EC50= 980plusmn70 nM) theforskolin-stimulated levels of cAMP in hCB2-HEK cellswhile WIN-55212-2 decreases (44 45plusmn42nM) thisaccumulation [137]

Fig (16) CB2 cannabinoid receptor antagonists 2 Indolederivatives Chemical structures of WIN-55212-3 (167) andBML-190 (168) two indoles derivatives acting as inverseagonists at the hCB2 cannabinoid receptor Surprisingly in 2002 Melck and collaborators used

BML-190 as a CB2 receptor agonist in a cell proliferationassay [214] BML-190 was also tested as inhibitor of thecyclooxygenase-2 enzyme by Kalgutkar et al [215] Theyobtained IC50 values higher than 33 and 66 microM for theCOX-I and COX-II enzymes respectively These valueshave to be compared with the submicromolar activity ofindomethacin the parent compound Very recently Klegerisand colleagues [216] looking for an antineurotoxic action ofcannabinoids showed that BML-190 increases TNF-αsecretion by stimulated THP-1 monocytic cells but is noteffective on the IL-1β secretion

Further on the basis of WIN-54461 (12) a CB1antagonist AM630 (13) was synthesised by replacement ofthe bromine atom by an iodine one In the initial reportPertwee et al showed the antagonist effects of AM630 butalso suggested that the CB1 cannabinoid receptor may not bethe preferential receptor of AM630 [45] Later on Ross et alreported using hCB1amp2-CHO cells and [3H]-CP-55940that AM630 binds to the CB2 cannabinoid receptor (Ki =312 nM) with a selectivity ratio over 160 [48]Furthermore using a [35S]-GTPγ S assay they showed itsinverse agonist properties (EC50 = 76 nM) However

Table 12 CB2 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Reported binding affinities (Ki) and selectivity ratio (CB1CB2) of SR144528 (169) for rat and human cannabinoid receptors determined using [3H]-CP55940 as radioligand

NN

O

NH

Cl

SR144528 169

rCB2 (spleen) rCB1 (whole brain) Selectivity Ki hCB2 (transfected CHOcells)

Ki hCB1 (transfected CHOcells)

Selectivity References

038 nM 305 nM 800 06 nM 437 nM 730 [217]

067 nM 548 nM 80 [219]

56 nM gt10microM gt1780 [48]

25 nM a gt 1microM a gt400 [224]

024 nM 276 nM 115 199 nM 503 nM 25 [232]

a COS cells

1384 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

3 Diarylpyrazole Derivatives CHO cells is constitutively active and phosphorylated onSer 352 Upon treatment by SR144528 thisphosphorylation of the receptor was inhibited Moreover theSR compound was able to induce dephosphorylation ofagonist-induced CB2 phosphorylation The question whetherSR144528 induces a CB2 receptor conformational changemaking it a better phosphatase-specific substrate or ifSR144528 binding activates specific phosphatases remainsopen However this remains a nice example of inverseagonist-induced receptor resensitisation

The same Sanofi team which synthesised the CB1antagonist SR141716A reported in 1998 the synthesis andpharmacological characterisation of a CB2 cannabinoidreceptor selective antagonist the SR144528 (169) [217218] The structure of the new compound is derived fromSR141716A one SR144528 is the first selective CB2receptor antagonist reported as the inverse agonist propertiesof BML-190 were shown only recently by New et al [137]The authors reported for 169 using [3H]-CP-55940 asradioligand Ki values of 04 nM and 06 nM for the rat andthe human CB2 receptors respectively and found a CB1-CB2 selectivity ratio around 700 Table 12 sums up thebinding affinities of SR144528 for the cannabinoid receptorsobtained by different authors Interestingly while the CB2affinity is more or less constant the affinity obtained for theCB1 receptor varies by one order of magnitude [219] Theantagonist effect of SR144528 was highlighted by evaluatingits effect on cAMP production by hCB2-CHO cells exposedto 3 nM CP-55940 (EC50=10nM) The antagonism wasalso observed on the activation of the MAP kinase pathwayby CP-55940 (IC50=39nM)

The Sanofi research team working on cannabinoidsconducted investigations in order to determine the keyresidues for SR144528 interaction with the receptor (Table13) as well as the binding mode of the compound Firstlythey identified the TM4-EL2-TM5 region as a regioncontaining crucial residues for the affinity of several ligandsamong them SR144528 [224] Among these residues themutation of two cysteine of the second extracellular loop(C174 and C179) abolishes the affinity for the CB2 receptorLater on the implication of S453(161) and S457(165) inthe binding of SR144528 was evidenced by mutationalstudies In the model proposed for the docking of SR144528into the CB2 receptor the crucial contacts are comprised intothe TM4 this is the case for S453(161) and S457(165)which are proposed to form hydrogen bonds with the ligandand W464(172) proposed to have aromatic interactions withthe 4-methylbenzyl of the SR144528 [225] In 2003 Fengand Song showed that point mutations D349(130)A andA634(241)E abolished ligand binding However mutationof the arginine R350(131) member of the highly conservedDRY motif to alanine has no influence on SR144528binding [226]

Early after the report of the synthesis and characterisationof SR144528 the same authors further explored the functionof this compound and showed its inverse agonist propertiesusing CB2-CHO cells co-transfected with the luciferasereporter gene with either the CRE or the murine krox24regulatory sequence [220] In these models SR144528 notonly inhibits CP-55940 effects but also reproduces theeffects of a pertussin toxin treatment proving its inverseagonist properties SR144528 functionality was alsoexplored by [35S]-GTPγ S binding in hCB2-CHO cells(IC50=3 nM) [221] The inverse agonist function ofSR144528 was further illustrated by Rhee and Kim usingCOS cells co-transfected with hCB2 receptor and adenylylcyclase [222]

As for the CB1 cannabinoid receptor ligands Sanofideveloped tricyclic derivatives based on the 1-benzylpyrazole-3-carboxylic scaffold More than twentycompounds were disclosed in a patent [227] in 2001 (84-86Table 7) The benzyl in position 4 characteristic of the CB2diarylpyrazoles from Sanofi is conserved as well as thebicyclic residue in position 3 The length of the second linkbetween the phenyl and the pyrazole can be made of one or

Bouaboula and co-workers investigated SR144528 effectson CB2 cannabinoid receptor phosphorylation status [223]Firstly they showed that the CB2 receptor transfected in

Table 13 CB2 Cannabinoid Receptor Antagonists 3 Di-Aryl-Pyrazole Derivatives

Some of the single point mutations reported for the hCB2 cannabinoid receptor for which pharmacological data concerning the SR144528 are available

Mutant Effect References

R350(131)A no effect [226]

S453(161)A loss of affinity [225]

V456(164)I no effect [225]

S457(165)A loss of affinity [225]

C174S a loss of affinity [224 225]

C175S a loss of affinity [225]

C177S a no effect [225]

C179S a loss of affinity [224 225]

S544(193)G no effect [225]

a Second extra-cellular loop

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1385

two methylene but according to the patent one methylenebridge is preferred The Ki values of these compounds for theCB2 receptor were said to be lower than 500 nM and theirantagonist properties have been assessed using the adenylatecyclase inhibition assay

compounds by the presence of an alkyl substituent inposition 5 instead of a phenyl and of course by position 3which remains unsubstituted However except a lecture atthe 1998 ACS meeting [230] where compound 171 wasreported to have a Ki value of 835 nM and a selectivity ratioover 100 no further report on these compounds waspublished untill now

In a recent paper Mussinu and colleagues reported thesynthesis of other tricyclic derivatives starting fromSR141716A structure [228] N-piperidin-1-yl-14-di-hydroindeno[12-c]pyrazole-3-carboxamide derivatives areCB2 receptor ligands and some of them possess high affinityand selectivity for the CB2 receptor as shown in Table 7 (79-83) These tricyclic derivatives differ from the Sanofi tricyclicderivatives as they do have a phenyl ring instead of a benzylin position 1 Another difference is the nature of thesubstituent in position 3 which is not a bicycloalkyl but acyclic amine One of the compounds synthesised byMussinu et al 81 possesses the highest affinity for the CB2receptor ever reported with a Ki value of 0037 nM for themurine CB2 receptor ([3H]-CP-55940 mice spleenhomogenate) Interestingly 79 the closest SR141716Aanalog lost almost all its affinity for the CB1 receptor andincreased by four orders of magnitude its affinity for the CB2receptor Even if the structure quite similar to the SRcompounds could suggest that these derivatives behave asantagonists no pharmacological data were given in the paperto support this hypothesis

5 2-Oxoquinoline Derivatives

Inaba and colleagues from Japan Tobacco introduced in2001 a completely new structure in the cannabinoid fieldbased on a 2-oxoquinoline scaffold Over eighty derivativeswere disclosed in a patent [231] and one compound JTE-907 (173) was subsequently characterised in-vitro as well asin-vivo [232] In Table 14 are listed some of the compounds(172-178) disclosed in the patent along with their affinity forthe cannabinoid receptors These values were obtained onhuman receptors against [3H]-WIN-55212-2 For twelvecompounds the anti-inflammatory activity per os wasassessed in a carrageenin-induced paw edema model Thehighest effect reported was obtained with JTE-907 with anED50 value lower than 01 mgkg Therefore thesecompounds were claimed to be useful as anti-inflammatoryand anti-allergic agents

To further characterize JTE-907 the authors determinedits affinity for cannabinoid receptors of different species using[3H]-CP-55940 as radioligand In their hands JTE-907behaved as a very selective hCB2 ligand with Ki values of36 and 2370 nM for the CB2 and CB1 cannabinoid receptorsrespectively even more selective than the SR144528 (Kivalues of 199 and 50 nM respectively) However it has tobe mentioned that the affinities reported in the paper forJTE-907 are quite different from those reported in the patent(hCB1 Ki values of 35nM and 009 nM respectively) Italso appears that the SR144528 affinity for the CB1 receptordetermined by Iwamura et al is higher than the affinityreported by Rinaldi-Carmona et al with values of 50 and437nM respectively The authors ascribed the difference tothe variability of SR144528 binding affinities to the assayconditions The function of JTE-907 was evaluated bymeasuring the increase in cAMP production by forskolin-stimulated hCB2-CHO cells Maximum stimulation wasattained at a concentration of 1microM of JTE-907 In the sameassay WIN-55212-2 decreased the cAMP production in adose-dependent manner

Finally it is interesting to strengthen that the five-membered ring derivatives (79-86) of the diarylpyrazolefamily are reported to possess a high CB2 receptor affinitywhile the six- and seven-membered ring derivatives (69-78)are described as CB1 receptor ligands

4 Other Pyrazole Derivatives

Other CB2 ligands based on a pyrazole nucleus wereintroduced by SmithKline Beecham in a patent describingthe synthesis of about sixty new compounds [229] Theiraffinity (Ki) for the hCB2 receptor transfected in HEK293cells was reported to be ranging between 25nM and 10microMTheir structures (170-171 Fig 17) differ from the Sanofi

NN

O

N

ON

NN

N

NN

O

N

ON

NN

N

ClCl

1

5

4 3

2

170 171

Fig (17) CB2 cannabinoid receptor antagonists 4 Otherpyrazole derivatives Structure of two compounds (170-171)among those claimed by SmithKline Beecham to be hCB2antagonists

In conclusion JTE-907 was shown to be a CB2 selectiveinverse-agonist with anti-inflammatory and anti-allergicproperties This compound is currently undergoing Phase Iclinical trials as anti-allergic drug [233]

IV NON-CB1 NON-CB2 CANNABINOIDRECEPTORS LIGANDS

In the last years the possible presence of additional non-CB1 non-CB2 cannabinoid receptors was raised frompharmacological evidences

One of the putative receptors possesses an endotheliallocalisation while other ones are localized in the CNS or atperipheral nerves For instance an additional target for ∆9-

1386 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 14 CB2 Cannabinoid Receptor Antagonists 5 2-Oxoquinoline Derivatives

Structure of six compounds among the oxoquinoline derivatives patented by Japan Tobaco Affinity (Ki nM) and selectivity (CB1CB2) for the cannabinoidreceptors are given The affinity was measured by binding against [3H]-WIN-55212-2 on either hCB2-CHO or hCB1-CHO cells Adapted from [231]

Cpd ndeg Structure hCB2 hCB1 Selectivity

1-2 172

NH

MeO O

O

OMeO

0014 3671 262215

JTE-907 173

NH

O

MeO O

O

NH

O

O

0087 3436 39495

2-2 174

OH

O

NH

MeO O

O 077 3247 4215

3-27 175

NH

O

F

NH

MeO O

O0021 336 16000

3-32 176

NH

MeO O

O

NH

F

O

0036 2398 66610

3-38 177

O

O

NH

N

O

O

NMeO

0085 935 11000

3-43 178

NMeO O

O

NH

F

O

0043 864 20100

tetrahydrocannabinol was described in capsaicin-sensitivesensory nerves by Zygmunt et al [234]

[29] and by Di Marzo et al [235] Thus in the presentreview we will focus on the description of the so faridentified ligands of the non-CB1 non-CB2 receptors withan emphasis on the compounds having antagonist properties

Pharmacological evidences for the existence of additionalcannabinoid receptors were reviewed by Wiley and Martin

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1387

(Table 15) In particular the knowledge about three not yetcloned targets has grown The growing molecularpharmacology of the three putative new cannabinoidreceptors is reviewed in the three following sections Thefourth section summarises the data on the other proposedcannabinoid targets

vasodilatator activity In addition the anandamide effect ispartially sensitive to SR141716A inhibition The authorsproposed the existence of two distinct receptors responsiblefor the vasodilatatory effect of anandamide one located in theendothelium is SR141716A-sensitive while the other onelocated on the vascular smooth muscle is SR141716A-insensitive Thus SR141716A is an antagonist of anendothelial anandamide receptor Several studies wereconducted in order to understand the molecularpharmacology of the endothelial anandamide receptor It ispertussis toxin-sensitive [237] Cannabidiol acts asantagonist and abnormal-cannabidiol (Abn-cbd 179) and itsanalogue O-1602 (180) act as agonists [238] The synthesisof O-1602 and of its analogues are disclosed in a patentdescribing the vasodilatatory activity of cannabinoid

1 Endothelial Anandamide Receptor ndash Abnormal-Cannabidiol Receptor

In 1999 Wagner et al showed that anandamide couldinduce mesenteric vasodilatation through an endotheliallylocated ldquoanandamide receptorrdquo pharmacologically distinctfrom CB1 [236] Other cannabinoids such as ∆9-THC HU-210 or WIN-55212-2 were devoid of mesenteric

Table 15 Non-CB1 Non-CB2 Cannabinoid Receptors Antagonists

An overview of the putative new non-CB1 non-CB2 anandamide-receptors ligands The antagonists are highlighted in bold

NH

N

S

HO OH

Cl

OH

OH

OH OH OH OH

OMe

OMe

RR

RR

RR

RR

HO

OH

NH

O

cannabidiol (10) Abnormal-cannabidiol 179 O-1602 180

O-1918 181 capsazepine 183capsaicin 182

Compound Endothelial AEA receptor Brain AEA receptor Cannabinoid-vanilloid brain receptor

AEA agonist a agonist de

WIN-55212-2 no effecta

agonist e agonist g h

CP-55940 no effect e agonist i

SR141716A antagonist a b j no effect ([35 S]-GTPγS binding) d e f antagonist g h

AM251 no effect k no effect h i

Cannabidiol antagonist b no effect e

O-1918 antagonist c jm

Abd-cbd agonist b l

O-1602 agonist b

Capsaicin no effectj agonist i

Capsazepine no effect b antagonist h

a [236] b [238] c [240] d [249] e [250] f [251] g [254] h [258] i [ 255] k [244] j [ 243] l [ 242] m [241]

1388 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

analogues [239] More recently Offertaler et al showed thatO-1918 (181) a cannabidiol analog acts as an antagonist ofthe endothelial anandamide receptor [240] In addition O-1918 does not bind to the CB1 and CB2 cannabinoidreceptors Furthermore O-1918 unlike cannabidiol doesnot cause vasorelaxation in the mesenteric artery preparationmodel used and thus acts as a true neutral antagonistUsing O-1918 as a pharmacological tool Begg et alproposed a coupling of this new G protein-coupledendothelial receptor through a GiGo mechanism to ionchannels in primary cultured human vascular endothelialcells [241] However in their hands SR141716A wasineffective in antagonising Abd-cbd Ho and Hiley furtherdemonstrated that Abn-cbd relaxes rat small mesentericarteries through an activation of K+ channels via aSR141716A-sensitive pathway [242] Further using thesame model they characterized the vasorelaxant activity ofvirodhamine a novel endocannabinoid apparently actingthrough the same receptor [243] The virodhamine-inducedvasorelaxation is sensitive to SR141716A and O-1918 butinsensitive to AM251 SR144528 and AM630 Moreovervanilloid receptor desensitisation by capsaicin (182) had noeffect

244] Thus position 4 on the 5-phenyl could be furtherexplored to obtain selective ligands

To come to an end despite the fact that the endothelialanandamide receptor is neither identified nor cloned at leastthree antagonists are identified SR141716A cannabidioland O-1918 It is likely that a growing number of ligandsand therapeutic applications will appear in the very nearfuture

2 Brain Anandamide Receptor

Following an in-depth evaluation of anandamide effects inCB1

-- mice Di Marzo and co-workers suggested that a non-CB1 non-CB2 G protein-coupled receptor might mediatesome of the actions of AEA in mice [249] Actually effectsof AEA but not those of ∆9-THC were not decreased inCB1 cannabinoid receptor knock-out mice For instance 30mgkg of AEA were still effective in hot-plate test whereas10 mgkg of ∆9-THC had no effect on anti-nociceptionMoreover in a [35S]-GTPγ S assay AEA stimulated with anEC50 value of 223 microM the [35S]-GTPγ S binding to CB1

--

mice brain membranes (Emax= 28) ∆9-THC showed noeffect in this assay The addition of SR141716A did notaffect the [35S]-GTPγ S binding to CB1

-- mice brainmembranes Shortly after other evidences for the existence ofa new G protein-coupled cannabinoid receptor with a distinctdistribution in the central nervous system were published[250] From a set of 24 commonly used cannabinoids (AEAWIN-55212-2 JWH-030 THChellip) only AEA and WIN-55212-2 produced an increased binding of [35S]-GTPγ S onCB1

-- mice brain membranes Interestingly in this studySR141716A alone produced a significant inhibition of [35S]-GTPγ S binding similar to the inhibition obtained usingCB1

++ mice brain membranes (IC50 values of 57 microM and47 microM respectively) However SR141716A did not affectthe anandamide-induced [35S]-GTPγ S binding in CB1

--

mice brain membranes Moreover [3H]-SR141716Aexhibited significant binding in some brain regions of CB1

--

mice which are different from those exhibiting stimulation of[35S]-GTPγ S binding by WIN-55212-2 and AEA Thusthe authors proposed that the SR141716A effects on CB1

--

mice are mediated neither by the CB1 cannabinoid receptornor by the putative new anandamide brain receptor Monoryand co-workers using CB1

-- mice described a novel nonadenylyl cyclase-coupled cannabinoid binding site in micecerebellum [251] Two agonists AEA and WIN-55212-2but not ∆9-THC or HU210 were found to bind to thisreceptor as they enhanced [35S]-GTPγ S binding NeitherSR141716A nor SR144528 were able to affect the WIN-55212-2 induced [35S]-GTPγ S binding in CB1

-- mice asfound by Breivogel et al [250] However the distributionpattern of the receptor described by Monory et al differs fromthe distribution found by Breivogel et al It has to bementioned that the knock-out strains used were different Themice used by Monory were from a CD-1 strain while theone used by Breivogel were from a C57BL61 strainInterestingly Muthane et al detected differences in nigralneurons number and sensitivity to MPTP in the two mousestrains used [252]

More recently Mo et al obtained results showing thatthe Abnormal-cannabidiol receptor is also responsible for anincreased endothelial cell migration [244] Abn-cbd enhancedhuman umbilical vein endothelial cell migration while O-1918 antagonised Abn-cbd effect The CB1 cannabinoidreceptor inverse agonist SR141716A partially inhibitedAbd-cbd action whereas AM251 and SR144528 had noeffect

In another paper from George Kunos team Batkai et alreported that SR141716A is able to inhibit the endotoxichypotension by a cardiac mechanism involving neither theCB1 nor the CB2 cannabinoid receptors [245] Theinvolvement of a non-CB1 non-CB2 site of action wasshown using CB1

-- and CB1--CB2

-- knock-out miceFurther studies are needed to identify the SR141716Asensitive myocardial site of action

In a recent patent Kunos described O-1918 and itsanalogues as vasoconstrictor agents useful to reversepathological vasodilatation of blood vessels [246] Theeffects of O-1918 on mouse blood pressure on LPS-inducedhypotension in mouse as well as the reversal of hypotensionby O-1918 injection are described Furthermore thesynthesis of O-1918 based antagonists is described startingfrom p-metha-28-dien-1-ol and the appropriate resorcinols

Interestingly the Abd-cbd receptor which was firstdetected in the vascular system also seems to be present inthe CNS [247] Indeed Walter et al found that microglialcells (BV-2) migration induced by 1 microM 2-AG is inhibitedby cannabidiol (300 nM) and O-1918 (1 microM) SR141716Awas found ineffective in this assay Thus 2-AG is an agonistof the Abd-cbd receptor Moreover Abd-cbd is able to elicita dose-dependent cell migration in the same model (EC50 =600nM) [248]

From a more structural point of view SR141716Abehaves as an antagonist while on the opposite AM251(20) a close analogue which differs from the former only bythe nature of one halogen has no effect on this receptor [242

To conclude to our knowledge no compounds havingantagonist or inverse agonist properties at this receptor havebeen identified so far

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1389

3 Cannabinoid-Vanilloid Brain Receptor a cannabinoid-vanilloid receptor at which SR141716A andcapsazepine behave as antagonists

Evidences for the presence of an additional ldquocannabinoidrdquoreceptor differing from the CB1 receptor and from the brainanandamide receptor appeared from studies focused on thehippocampus (for a review see Hajos et al [253]) Hajos etal studied the cannabinoid actions on GABAergic andglutamatergic transmission using the whole-cell patch-clamp technique [254] WIN-55212-2 induced a dose-dependent reduction in glutamatergic transmission reversedby SR141716A (1microM) Thus the authors concluded that thecannabinoid actions on the glutamatergic transmission aremediated by a non-CB1 but SR141716A-sensitive receptorIn another paper Hajos and Freund further characterised thisputative receptor [255] They showed that in addition toWIN-55212-2 CP-55940 and capsaicin (182) a TRPV1receptor agonist also act as agonists Furthermore in theirhands SR141716A as well as capsazepine (183) a TRPV1receptor antagonist were able to antagonise this effect on theglutamatergic transmission while AM251 was notSR141716A effects on CB1 knock-out mice were furtherstudied using a test of anxiety [256] Haller et al showedthat on the one hand the distribution of the CB1 receptorinduces anxiety and SR141716A has an anxiolytic effectand on the other hand SR141716A is still active in CB1

--

mice In a following paper they reported that AM-251induces anxiogenic effects in wild-type and in knock-outmice [257] Thus SR141716A in addition to CB1 bindsto a not yet identified receptor Another evidence for thisadditional receptor for SR141716A was given by Bass andco-workers [61] They conducted a structure-activityrelationship study on the stimulation of locomotor activityinduced by SR141716A and twenty analogues Nocorrelation was found between the affinity for the CB1cannabinoid receptor and the stimulation of locomotoractivity Albeit SR141716A and five of its analogues O-1803 O-1710 O-1253 O-1254 (40) O-1255 (41) behavedas inverse agonists in [35S]-GTPγ S assay none of theseanalogues stimulated the locomotor activity In conclusionthe SR141716A-induced stimulation of locomotor activity isneither due to inverse agonism nor to inhibition of anendogenous tone but to a brain receptor distinct from theCB1 receptor Di Marzo et al previously provided evidencesthat the cannabinoid-vanilloid brain receptor could mediatesome of the cannabinoid effects on locomotion [249]

4 Other Non-CB1 Non-CB2 Cannabinoid Receptors

Evidences for the existence of additional non-CB1 non-CB2 cannabinoid receptors localised in the rat isolatedmesenteric arterial bed appeared from the works of Ralevicand collaborators Several cannabinoids were evaluated intheir model of sensory neurogenic vasorelaxation evoked byelectrical field stimulation

HU210 was found to attenuate vasorelaxation in a CB1and CB2 independent way as SR141716A LY320135 andSR144528 were ineffective in inhibiting HU210 effect [259]Similar results were obtained with ∆9-THC [260]Similarly noladin ether inhibited sensory neurogenicrelaxation in a concentration-dependent manner which wasalso unaffected by SR141716A LY320135 and SR144528Moreover this effect as the ∆9-THC one was pertussistoxin sensitive suggesting the involvement of a Gio protein-coupled receptor [261]

However the inhibitory effects of CP-55940 and WIN-55212-2 were affected by the presence of SR141716A (1microM)or LY320135 (1microM) but not by the addition of SR144528(1microM) [262]

It appears that at least two receptors should be involvedin cannabinoid-mediated attenuation of sensory nerve-mediated vasorelaxation The first one is SR141716A andLY320135 sensitive while the other one is resistant toSR141716A

Some evidences for the existence of a ldquoCB2-likerdquo receptorappeared from the studies on palmitoylethanolamide (PEA)an endogenous fatty acid amide devoid of CB1 and CB2affinity [264] antinociceptive potential Calignano and co-workers showed that PEA was able to alleviate pain inseveral animal models [265 266] For instance PEA dose-dependently inhibits kaolin-evoked writhing in mice TheCB2 cannabinoid receptor inverse agonist SR144528 (02mgkg) inhibits PEA anti-nociceptive effect [266] Thus theauthors suggest that PEA acts as agonist on a SR144528-sensitive non-CB2 cannabinoid receptor (ldquoCB2-likerdquoreceptor) at which SR144528 acts as an antagonist

Further studies are needed in order to better characterisethese additional ldquocannabinoidrdquo receptors However it islikely that several therapeutic applications will soon springfor these receptors

Very recently Pistis et al published a study on theneurophysiological effects of cannabinoids on the basolateralamygdala neurons in-vivo [258] One of their findings is thatHU-210 and WIN-55212-2 did not elicit similar effects onbasal amygdala projection neurons firing rate While theformer had no effect the latter decreased the firing rateMoreover SR141716A reversed the WIN-55212-2 effectswhile AM251 had no effect The vanilloid receptorantagonist capsazepine also antagonised the WIN-55212-2effect on firing rate of these neurons Therefore the authorssuggested the presence of a novel cannabinoid-vanilloidreceptor

V CONCLUSION

Since their development cannabinoid antagonists provedto be essential tools in the understanding of cannabinoidpharmacology and biochemistry Moreover the interestshown by the pharmaceutical industry in the development ofnew cannabinoid antagonists prove if necessary the realtherapeutic potential of this class of compounds Thetreatment of eating and movement disorders memorydeficits psychosis and dependencies from various addictivedrugs are some of the most cited applications for the CB1cannabinoid receptor antagonists

From these lines it appears that in addition to theknown CB1 cannabinoid receptor it is now likely that twoother cannabinoid receptors do exist in the brain The firstone should be an anandamide brain receptor for whichSR141716A has no affinity while the second one should be

1390 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Much less wide is the knowledge on the CB2cannabinoid receptor antagonists However potentialapplications could be disorders involving the immunesystem such as inflammation or allergies

[19] Devane WA Hanus L Breuer A Pertwee RG StevensonLA Griffin G Gibson D Mandelbaum A Etinger AMechoulam R Science 1992 258 1946

[20] Mechoulam R Ben-Shabat S Hanus L Ligumsky MKaminski NE Schatz AR Gopher A Almong S MartinBR Compton DR Biochem Pharmacol 1995 50 83

Finally the growing literature on the non CB1 non CB2cannabinoid receptors should allow the development of newdrugs First examples are given by the cannabidiol andabnormal-cannabidiol derivatives described by Kunos et alfor which potential therapeutic applications on the vascularsystem are already patented

[21] Sugiura T Kondo S Sukagawa A Nakane S Shinoda AItoh K Yamashita A Waku K Biochem Biophys Res Co1995 215 89

[22] Hanus L Abu-Lafi S Fride E Breuer A Vogel Z ShalevDE Kustanovich I Mechoulam R Proc Natl Acad SciUSA 2001 98 3662

[23] Oka S Tsuchie A Tokumura A Muramatsu M Suhara YTakayama H Waku K Sugiura T J Neurochem 2003 851374It clearly appears from this paper that the antagonists of

the cannabinoid receptors are still an active research fieldfrom which should emerge new promising therapeutic toolsas well as innovative drugs in the near future

[24] Goutopoulos A and Makriyannis A Pharmacol Therapeut2002 95 103

[25] Beal JE Olson R Lefkowitz L Laubenstein L Bellman PYangco B Morales JO Murphy R Powderly W PlasseTF Mosdell KW Shepard KV J Pain Symptom Manage1997 14 7ACKNOWLEDGEMENTS [26] Notcutt W Price M Miller R Newport S Phillips CSimmons S Sansom C Anaesthesia 2004 59 440

GGM is very indebted to the ldquoFonds pour la formationagrave la Recherche dans lrsquoIndustrie et lrsquoAgriculture (FRIA)rdquo Belgium for the award of a research fellowship Theexperimental works of the authors were supported by grantsfrom the FNRS The Belgian Charcot Foundation and theUniversiteacute catholique de Louvain

[27] Ledent C Valverde O Cossu G Petitet F Aubert JFBeslot F Bohme GA Imperato A Pedrazzini T RoquesBP Vassart G Fratta W Parmentier M Science 1999 283401

[28] Zimmer A Zimmer AM Hohmann AG Herkenham MBonner TI Proc Natl Acad Sci U S A 1999 96 5780

[29] Wiley JL and Martin BR Chem Phys Lipids 2002 121 57[30] Adam J and Cowley P Expert Opin Ther Patents 2002 12

1475

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[32] Barth F and Rinaldi-Carmona M Curr Med Chem 1999 6745

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[33] Little PJ Compton DR Johnson MR Melvin LS MartinBR J Pharmacol Exp Ther 1988 247 1046

[2] Devane WA Dysarz FA Johnson MR Melvin LSHowlett AC Mol Pharmacol 1988 34 605

[34] Pertwee RG Stevenson LA Elrick DB Mechoulam RCorbett AD Brit J Pharmacol 1992 105 980

[3] Matsuda LA Lolait SJ Brownstein MJ Young ACBonner TI Nature 1990 346 561

[35] Harrison C Traynor JR Life Sci 2003 74 489-508[4] Gerard CM Molerau C Vassart G Parmentier M BiochemJ 1991 279 129 [36] Pertwee RG Fernando SR Griffin G Ryan W Razdan

RJ Compton DR Martin BR Eur J Pharmacol 1996 315195

[5] Glass M Dragunow M Faull RLM Neuroscience 1997 2299

[37] Ross RA Brockie HC Fernando SR Saha B Razdan RKPertwee RG Brit J Pharmacol 1998 125 1345

[6] Shire D Carillon C Kaghad M Calandra B Rinaldi-Carmona M LeFur G Caput D Ferrara P J Biol Chem1995 270 3726 [38] Griffin G Wray EJ Rorrer WK Crocker PJ Ryan WJ

Saha B Razdan RK Martin BR Abood ME Brit JPharmacol 1999 126 1575

[7] Ryberg E Khang Vu H Groblewski T Larsson N ElebringT Hjorth S Sjogren S Greasley PJ 2004 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 119

[39] Ross RA Gibson TM Stevenson LA Saha B Crocker PRazdan RK Pertwee RG Brit J Pharmacol 1999 128 735

[40] Martin BR Winckler JR Wiley JL Huffman JW CrockerPJ Saha B Razdan RK J Pharmacol Exp Ther 1999 2901065

[8] Rinaldi-Carmona M Calandra B Shire D Bouaboula MOustric D Barth F Casellas P Ferrara P LeFur G JPharmacol Exp Ther 1998 278 871

[41] Martin BR Stevenson LA Pertwee RG Breivogel CSWilliams A Mahadevan A Razdan RK 2002 Symposium onthe Cannabinoids Burlington Vermont International CannabinoidResearch Society page 2

[9] Munro S Thomas KL Abu-Shaar M Nature 1993 365 61[10] Howlett AC Qualy JM Khachatrian LL Mol Pharmacol

1986 29 307[11] Glass M Felder CC J Neurosci 1997 17 5327

[42] Thomas A Ross RA Saha B Mahadevan A Razdan RKPertwee RG Eur J Pharmacol 2004 487 213

[12] Calandra B Portier M Kerneacuteis A Delpech M Carillon C LeFur G Ferrara P Shire D Eur J Pharmacol 1999 374445 [43] Compton DR Gold LH Ward SJ Balster RL Martin BR

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Daum SJ Ackerman JH Gruett MD Kumar V EstepKG Olefirowicz EM Wetzel JR Alexander MD WeaverJD Haycock DA Luttinger DA Casiano FM ChippariSM Kuster JE Stevenson JI Ward SJ J Med Chem1995 38 3094

[14] Deadwyler S Hampson RE Mu J Whyte A Childers SRJ Pharmacol Exp Ther 1995 273 734

[15] Mackie K Lai Y Westenbroek R Mitchell R J Neurosci1995 15 6552

[16] Bouaboula M Poinot CC Bourrie B Canat X Calandra BRinaldi-Carmona M LeFur G Casellas P Biochem J 1995312 637 [45] Pertwee R Griffin G Fernando S Li X Hill A

Makriyannis A Life Sci 1995 56 1949[17] Bouaboula M Poinot CC Marchand J Canat X Bourrie BRinaldi-Carmona M Calandra B LeFur G Casellas P Eur JBiochem 1996 237 704

[46] Hosohata Y Quock RM Hosohata K Makriyannis AConsroe P Roeske WR Yamamura HI EurJPharmacol 1997 321 R1[18] delPulgar T Velasco G Guzman M Biochem J 2000 347

369 [47] Landsman RS Makriyannis A Deng H Consroe P RoeskeWR Yamamura HI Life Sci 1998 62 109

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[48] Ross RA Brockie HC Stevenson LA Murphy VLTempleton F Makriyannis A Pertwee RG Brit JPharmacol 1999 126 665

[81] Gatley SJ Lan R Volkow ND Pappas N King P WongCT Gifford AN Pyatt B Dewey SL Makriyannis A JNeurosci 1998 70 417

[49] Huffmann JW Curr Med Chem 1999 6 705 [82] Lan R Lu Q Fan P Gatley J Volkow ND Fernando SRPertwee R Makriyannis A AAPS Pharmsci 1999 1 article 4[50] Rinaldi-Carmona M Barth F Heaulme M Shire D Calandra

B Congy C Martinez S Maruani J Neliat G Caput DFerrara P Soubrieacute P Breliegravere JC Le Fur G FEBS Lett1994 350 240

[83] Mathews WB Ravert HT Musachio JL Franck RARinaldi-Carmona M Barth F Dannals RF J Labelled CompdRad 1999 42 589

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GA Psychopharmacology 2003 167 103[68] Reggio P Umijiego U Hurst D Seltzman H Hyatt SRoche M McAllister S Fleisher D 2004 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 2

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[102] Ravinet Trillou C Delgorge C Menet M Arnone M SoubrieP Int J Obesity 2004 28 640[69] Song ZH and Bonner TI Mol Pharmacol 1996 49 891

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[103] Cota D Marsicano G Tschoumlp M Gruumlbler Y FlachskammC Schubert M Auer D Yassouridis A Thoumlne-Reineke COrtmann S Tomassoni F Cervino C Nisoli E LinthorstACE Pasquali R Lutz B Stalla GK Pagotto U J ClinInvest 2003 112 423

[71] McAllister SD Tao Q Barnett-Norris J Buehner K HurstDP Guarnieri F Reggio PH Nowell Harmon KW CabralGA Abood ME Biochem Pharmacol 2002 63 2121

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[104] Cani PD Lasa Montoya M Neyrinck AM Delzenne NMLambert DM Br J Nutr 2004 92 757

[73] Murphy JW and Kendall DA Biochem Pharmacol 2003 651623

[105] Colombo G Agabio R Fa M Guano L Lobina C LocheA Reali R Gessa GL Alcohol Alcohol 1998 33 126

[74] McAllister SD Rizvi G Anavi-Goffer S Hurst DP Barnett-Norris J Lynch DL Reggio PH Abood ME J Med Chem2003 46 5139

[106] Serra S Carai MA Brunetti G Gomez R Melis S VaccaG Colombo G Gessa GL Eur J Pharmacol 2001 430 369

[107] Serra S Brunetti G Pani M Vacca G Carai MAM GessaGL Colombo G Eur J Pharmacol 2002 443 95[75] McAllister SD Hurst DP Barnett-Norris J Lynch D

Reggio PH Abood ME J Biol Chem 2004 279 48024 [108] Colombo G Vacca G Serra S Carai MAM Gessa GLEur J Pharmacol 2004 498 119[76] Salo OM Lahtela-Kakkonen M Gynther J Jarvinen T

Poso A J Med Chem 2004 47 3048 [109] Gallate JE and McGregor IS Psychopharmacology 1999 142302[77] Rinaldi-Carmona M Pialot F Congy C Redon E Barth F

Bachy A Breliere JC Soubrie P Le Fur G Life Sci 199658 1239

[110] Cohen C Perrault G Voltz C Steinberg R Soubrie P BehavPharmacol 2002 13 451

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[111] Le Fur G Cohen C Steinberg R Soubrie P 2003 Symposiumon the Cannabinoids Burlington Vermont InternationalCannabinoid Research Society page 55[79] Gatley SJ Lan R Volkow ND Pappas N King P Wong

CT Gifford AN Pyatt B Dewey SL Makriyannis A JNeurochem 1998 70 417

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Behav 1997 56 803 [153] Dow RL Hammond M World Patent Application 2004WO04052864[116] Leweke FM Giuffrida A Wurster U Emrich HM Piomelli

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F Di Marzo V Lipids Health Dis 2003 2 5 [155] Katoch-Rouse R Pavlova OA Caulder T Hoffman AFMukhin AG Horti AG J Med Chem 2003 46 642[118] Dean B Sundram S Bradbury R Scarr E Copolov D

Neuroscience 2001 103 9 [156] Katoch-Rouse R and Horti AG J Labelelled Compd Rad2003 46 93[119] Zavitsanou K Garrick T Huang XF Prog Neuro-

Psychopharmacol Biol Psychiatry 2004 28 355 [157] Kumar JSD Prabhakaran J Arango V Parsey RV Underwood MD Simpson NR Kassir SA Majo VJ VanHeertum RL Mann JJ Bioorg Med Chem Lett 2004 142393

[120] Meltzer HY Arvanitis L Bauer D Rein W Am JPsychiatry 2004 161 975

[121] Castellano C Rossi-Arnaud C Cestari V Constanzi M CurrDrug Targets 2003 2 61 [158] Stoit AR Lange JHM den Hartog AP Ronken E Tipker

K van Stuivenberg HH Dijksman JAR Wals HC KruseCG Chem Pharm Bull 2002 50 1109

[122] Terranova JP Storme JJ Lafon N Perio A Rinaldi-Carmona M Le Fur G Soubrie P Psychopharmacology 1996126 165 [159] Barth F Congy C Martinez S Rinaldi M World Patent

Application 2001 WO0132663[123] Mallet PE Beninger RJ Psychopharmacology 1998 140 11[124] Nakamura-Palacios EM Winsaure PJ Moerschbaecher JM

Behav Pharmacol 2000 11 377[160] Ruiu S Pinna GA Marchese G Mussinu J-M Saba P

Tambaro S Casti P Vargiu R Pani L J Pharmacol ExpTher 2003 306 363[125] Brodkin J Moerschbaecher JM J Pharmacol Exp Ther

1997 282 1526 [161] Francisco MEY Burgess JP George C Bailey GSGilliam AF Seltzman HH Thomas BF Magn Reson Chem2003 41 265

[126] Lichtman AH Eur J Pharmacol 2000 404 175[127] Wolff MC Leander JD Eur J Pharmacol 2003 477 213[128] Arnone M World Patent Application 2003 WO03082256 [162] Cullinan GJ Fahey KJ Koppel GA United States Patent

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[246] Kunos G World Patent Application 2003 WO03015700 [257] Haller J Varga B Ledent C Freund TF Behav Pharmacol2004 15 299[247] Franklin A and Stella N Eur J Pharmacol 2003 474 195

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[263] Tao Q and Abood ME J Pharmacol Exp Ther 1998 285651[252] Muthane U Ramsay KA Jiang H Jackson-Lewis V

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1370 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

5-positions appear to be involved in antagonism The O-derivatives reported were also described in a patent alongwith their synthetic pathways which were not given in thepaper [147]

Another investigation of the SR141716Aaminopiperidine region was conducted by Francisco et al[148] They synthesised 21 analogues possessing either analkyl amide or an alkyl hydrazide substituent of various

Table 6 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structure of some of the characterised 1- 3- 4- and 5-pyrazole derivatives Binding affinities (Ki values nM) were obtained on rat brain homogenatesusing [3H]-CP-55940 [136 145 146 148 150] or [125I]-AM-251 [155]

NN

R1

R5

R3R4

Cpd ndeg R1 R3 R4 R5 [136] [145] [146] [148] [150] [155]

SR141716 14 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Cl-Ph 115 62 62 62 13 18

O-1559 30 24-di-Cl-Ph CO-NH-piperidinyl Me 1-Methylpentyl 233

O-1302 31 24-di-Cl-Ph CO-NH-piperidinyl Me 4-((CH2)5)-Ph 21 1

O-1691 32 24-di-Cl-Ph CO-NH-piperidinyl Br 4-((CH2)5)-Ph 15

O-1704 33 24-di-Cl-Ph CO-NH-piperidinyl I 4-((CH2)5)-Ph 22

6-I-SR141716 34 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-Cl-Ph 166

4rsquo6-di-I-SR141716 35 24-di-Cl-6-I-Ph CO-NH-piperidinyl Me 4-I-Ph 126

4rsquo-I-SR141716 36 24-di-Cl-Ph CO-NH-piperidinyl Me 4-I-Ph 75 25 6

4rsquo-Br-SR141716 37 24-di-Cl-Ph CO-NH-piperidinyl Me 4-Br-Ph 168 3

Cpd 25 in ref [136] 38 24-di-Cl-Ph CO-NH-morpholinyl Me 4-Br-Ph 54

O-1300 39 Ph CO-NH-piperidinyl Me 4-Cl-Ph 150

O-1254 40 4-(CH2)4-Ph CO-NH-piperidinyl Me 4-Cl-Ph 226 256

O-1255 41 4-(CH2)5-Ph CO-NH-piperidinyl Me 4-Cl-Ph 433

O-1269 42 24-di-Cl-Ph CO-NH-pentyl Me 4-Cl-Ph 32 114 3

O-1270 43 24-di-Cl-Ph CO-NH-heptyl Me 4-Cl-Ph 48 462 3

O-848 44 24-di-Cl-Ph CH2-O-(CH2)2-piperidinyl

Me 4-Cl-Ph 2450 232

O-853 45 24-di-Cl-Ph CH2-O-CH2-cyclohexyl

Me 4-Cl-Ph 388 100

O-1272 46 24-di-Cl-Ph CO-heptyl Me 4-Cl-Ph 221

O-1876 47 24-di-Cl-Ph 1rsquo-OH-heptyl Me 4-Cl-Ph 657

O-1877 48 24-di-Cl-Ph Heptyl Me 4-Cl-Ph 422

MF9725-64-17 49 24-di-Cl-Ph CO-NH-butyl Me 4-Cl-Ph 134

MF9725-179-32 50 24-di-Cl-Ph CO-NH-NH-butyl Me 4-Cl-Ph 51

MF9725-66-11 51 24-di-Cl-Ph CO-NH-cyclohexyl Me 4-Cl-Ph 25

MF9725-95-31 52 24-di-Cl-Ph CO-NH-(4-hydroxybutyl)

Me 4-Cl-Ph 154

Cpd 15 [150] 53 n-pentyl CO-NH-piperidinyl Me Ph 23

Cpd 16 [150] 54 n-pentyl CO-NH-piperidinyl Me 4-Br-Ph 63

Cpd 17 [150] 55 n-hexyl CO-NH-piperidinyl Me Ph 21

Cpd 18 [150] 56 n-heptyl CO-NH-piperidinyl Me Ph 47

NIDA-41109 57 24-di-Cl-Ph CO-NH-piperidinyl Br 4-Cl-Ph 14

NIDA-41119 58 24-di-Cl-Ph CO-NH-piperidinyl H 4-Cl-Ph 9

NIDA-41057 59 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OH-Ph 104

NIDA-41020 60 24-di-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 41

NIDA-41087 61 2-Cl-Ph CO-NH-piperidinyl Me 4-OMe-Ph 8

NIDA-42055 62 24-di-Cl-Ph CO-NH-piperidinyl Br 4-OMe-Ph 62

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1371

lengths in position 3 of the pyrazole moiety They observedthat until five carbons the affinity increases along with thecarbon chain length This is observed with the alkylhydrazide the alkyl amide and the hydroxyalkyl amideseries Moreover the hydrazide analogues exhibit a loweraffinity for the rCB1 than the amide analogues as illustratedby compounds MF9725-64-17 (49) and MF9725-179-32(50) having Ki values of 134 and 51 nM respectively(Table 6) From their structure-activity relationships (SAR)studies the authors concluded that the pharmacophoricrequirement of the amidopiridine region is a chain not longerthan 3 Aring and that a substituent having a positive chargedensity would probably result in increased affinity andpotency The same team described in a patent several othercompounds [149] Binding affinities (Ki) against [3H]-CP-55940 [3H]-SR141716A or [3H]-WIN55212-2 on wholerat brain or on hCB1-transfected cells were given Moreoveractivity data were obtained using the [35S]-GTPγ S assaydemonstrating that these alkyl amide and hydrazide analogsact as antagonists or inverse agonists Despite the greatnumber of derivatives claimed none of them posses asignificantly greater affinity for the CB1 receptor than theSR141716A Nevertheless they showed a slightenhancement of the selectivity for the CB1 receptor over theCB2 cannabinoid receptor

affinity of these derivatives increases with the length of thealkyl chain with an optimal length of 5-6 carbons The bestcompound of this series N-(piperidin-1-yl)-5-phenyl-1-hexyl-4-methyl-1H-pyrazole-3-carboxamide (55) possesses aKi value of 21 nM determined using [3H]-CP-55940 on ratbrain membranes which is higher than the 13 nMdetermined for SR141716A in the same conditions (Table6) In the same paper the authors starting from thehypothesis that antagonism by SR141716A is caused bybinding to the same region of the receptor as do the agonists(CP-55940 and WIN-55-2122) but preventing the agonistpromoted conformational change conducted extensiveconformational analysis as well as superimposition modelsand 3D-QSAR to propose a molecular mechanismsupporting the action of SR141716A Along with theauthors the C-5 aryl substituent of SR141716A occupyinga unique region could contribute in conferring the antagonistproperties Moreover the C-3 substituent could beresponsible for the antagonist or inverse agonist propertiesdepending on the interaction with the receptor

About this topic a very interesting review dealing withthe cannabinoid receptors pharmacophores as well as withthe activationinactivation of these receptors was recentlypublished by Reggio [151]

More recently Dyck et al described seven otherderivatives varying at the amide position [152] The onlycompound possessing a higher affinity for the CB1cannabinoid receptor than SR141716A was the 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta-

The affinity for the rat CB1 receptor of seven derivativespossessing an alkyl chain in position 1 instead of the 24-dichlorophenyl substituent was reported by Shim et al in apaper describing a molecular mechanism for the antagonistand inverse agonist activity of SR141716A [150] The

NN

O

NHN

Cl Cl

Cl

NN

Cl

Cl

NON

NN

O

NH

N

Cl

ClCl

NN

O

NH

MeO Cl

CP-272871 66

63 64

65

Fig (4) CB1 cannabinoid receptor antagonists 3 Diarylpyrazole derivatives Structures of 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63 ) 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methyl-pyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]-morpholine (64 ) N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65 ) and N-phenyl-1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-pyrazole-3-carboxamide (CP-272-871 66 )

1372 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

[c]pyrrol2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63)(Fig 4) The Ki values were 5 and 12 nM respectively([3H]-CP-55940 hCB1-HEK cells)

CP-55940 rat brain) For instance N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65) (Fig 4) showed a Ki value of15 nM However no data were provided concerning thefunctionality of such compounds

Over 200 other pyrazole derivatives acting at the CB1cannabinoid receptor were claimed in a patent from Pfizer[153] The affinity for the hCB1 receptor of 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methylpyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]ndashmorpholine (64) (Fig 4)was 79 nM ([3H]-SR141716A hCB1-HEK cells) Theothers compounds affinity was between 01 and 100 nM

In an effort to develop new SR141716A analoguespossessing lower lipophilicity to be used as PET tracersKatoch-Rouse and colleagues further explored thesubstitution pattern of the two phenyls [155] Bindingassays showed that a decreased lipophilicity lead to adecreased affinity for the CB1 receptor as determined bydisplacement of [3H]-AM251 For instance compoundNIDA-41057 (59) has a lipophilicity of 42 ( expressed asElogDoct) and a Ki value of 104 nM whereas SR141716Ahas a ElogDoct value of 54 and a Ki value of 18 nM In

Seventy five pyrazole derivatives were described in a veryrecent patent from Makriyannis et al [154] The affinity ofthe compounds for the cannabinoid receptors is given TheKi values for the best compounds are lower than 6 nM ([3H]-

Table 7 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structures and affinities for the cannabinoid receptors of some representative tricyclic pyrazole derivatives Values were obtained using [3H]-CP-55940as radioligand and mouse [160 228] rat [159 227] or human [158] cannabinoid receptors Compounds 69-78 are CB1 cannabinoid receptor ligandsCompounds 79-86 are CB2 cannabinoid receptor ligands

NN

O

NHX

R3

R1

R2

n

n˚ X R1 R2 n R3 Ki CB1 (nM) Ki CB2 (nM) Selectivity References

69 -(CH2)3- Cl 24-di-Cl 0 piperidine 126 [158]

70 -(CH2)3- H 24-di-Cl 0 azepane 100 [158]

71 -(CH2)3- H 24-di-Cl 0 piperidine 398 [158]

72 -(CH2)3- Cl 24-di-Cl 0 azepane 125 [158]

73 -(CH2)3- NO2 24-di-Cl 0 azepane 63 [158]

74 -(CH2)2-O- H 24-di-Cl 0 piperidine 501 [158]

75 -CH2-S- Br 24-di-Cl 0 piperidine lt 500 gt10 [159]

76 -CH2-SO2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

77 -(CH2)2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

78 -(CH2)3- Cl 24-di-Cl 0 piperidine 000035 21 60000 [160]

79 -CH2- Cl 24-di-Cl 0 piperidine 2050 034 00002 [228]

80 -CH2- Br 24-di-Cl 0 piperidine 1570 027 00002 [228]

81 -CH2- CH3 24-di-Cl 0 piperidine 363 0037 00001 [228]

82 -CH2- Cl 4-Cl 0 piperidine 1787 09 00005 [228]

83 -CH2- H 24-di-Cl 0 piperidine 1152 0385 00003 [228]

84 -CH2- Cl 34-di-Cl 1 133-trimethylbicyclo[221]heptyl

lt500 gt10 [227]

85 -CH2- Br 24-di-Cl 1 bicyclo[321]oct-3-yl lt500 gt10 [227]

86 -CH2- Br 4-Me 1 77-dimethylbicyclo[410]hept-3-yl

lt500 gt10 [227]

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1373

another paper Katoch-Rouse described the synthesis of[18F]-NIDA-42033 (26) a ligand useful as PET radiotracerstarting from its bromo derivative [156] Recently Kumar etal [157] proposed the synthesis of a 11C derivative ofNIDA-41087 (61) a compound previously described byKatoch-Rouse (Table 5) Post-mortem binding of thiscompound to human prefrontal cortex was also investigatedInterestingly CP-272871 (66) described by Meschler as aCB1 inverse agonist (Ki=57 nM [3H]-CP-55940 rat brainhomogenates) possesses a lipophilicity similar to theNIDA-41087 one [62] However despite the presence of amethoxy function no radiolabelled derivative has beendescribed to date One explanation could be the compoundlow selectivity (2 fold) for the CB1 cannabinoid receptor

To further explore the pharmacology of this compound afunctional assay using the mouse vas deferens model wasperformed by Ruiu and colleagues enlightening thecompetitive antagonist properties of NESS (pA2= 1246against WIN-55212-2) which had no effect by itself up to1microM Moreover NESS 0327 was unable to affect basalbinding of the [35S]-GTPγ S demonstrating its lack ofnegative intrinsic activity In-vivo studies were conductedusing the hot plate and tail flick tests NESS dose-dependently abolished the antinociceptive effect of WIN-55212-2 but had no effect by itself However the authorssuggested based on the affinity and in-vivo activity of theircompound that NESS 0327 possesses a poor centralbioavailability

Several new attempts to increase the affinity of thediarylpyrazole derivatives were recently made by rigidifyingthe SR141716A structure (Table 7) Six fused ringanalogues of SR141716A obtained by fusion of the 5-(4-chlorophenyl) substituent with the central pyrazole to forman indazole ring were published by Bass et al in 2002 [61]The compound possessing the highest affinity O-1248 (67)has a Ki value of 475 nM as determined by displacement of[3H]-CP-55940 from rat brain membranes

Another example of ring-constrained biarylpyrazole wasdescribed by Herbert Seltzman team [161] Theyinvestigated a photocyclisation reaction starting from theSR141716A leading to a pyrazolo[15-f]phenanthridinestructure (Fig 5) The new compound (68) was tested on theCB1 receptor (whole rat brain) against [3H]-CP-55940[3H]-SR141716A and [3H]-WIN-55212-2 and exhibited Kivalues of 48 35 and 50 nM respectively The CB2 affinitywas assessed using [3H]-CP-55940 and was found to be 100times lower (Ki = 3340 nM) No effect on the [35S]-GTPγ Sassay (rat brain) was observed by the authors

Stoit and colleagues from Solvay Pharmaceuticalspublished a paper describing the synthesis andpharmacological characterisation of new benzocyclohepta-pyrazole derivatives as CB1 antagonists (69-74) [158] Theaffinity of these compounds was more or less one order ofmagnitude lower than the SR141716A one with pKi valuesranging from 64 to 72 compared to 76 for the SRcompound (Table 7) Compounds were shown to beantagonists with pA2 values from 70 to 89 as theyprevent CP-55940-induced cAMP accumulation Howeverthe authors investigating the bioavailability of theircompounds either po or after ip injection found negligibleblood plasma levels

NN

Cl

Cl

O

NH

N

NN

Cl

O

NHN

Cl

Cl

O-1248 67 68However just before the publication of Stoit results

Sanofi-Synthelabo took a patent describing tricyclicpyrazolecarboxylic acid amide derivatives having antagonistproperties on the CB1 cannabinoid receptor andsubmicromolar affinities (75-77) [159] Thirty examples weregiven along with their synthetic pathway but no individualpharmacological data were shown

Fig (5) CB1 cannabinoid receptor antagonists 3Diarylpyrazole derivatives Two ring-constrained diarypyrazolederivatives O-1248 (67 ) and a pyrazolo[15-f]phenanthridinederivative (68 )

It appears from these attempts to increase SR141716Aaffinity by rigidifying its structure that only a limitedincrease in the CB1 cannabinoid receptor affinity can beexpected with the apparent exception of NESS 0327Moreover rigidifying the structure often results in a morelipophilic compound in a family of compounds being yetlipophilic

More recently Ruiu and colleagues published a paperdescribing the synthesis and complete pharmacologicalcharacterisation of a very potent CB1 receptor ligandchristened NESS 0327 (78) [160] This compound has thesame structure as compound 69 published by Stoit et al(compound 20 in the reference [158]) Nevertheless Ruiu hasobtained an affinity for the CB1 cannabinoid receptor 5000times bigger than the SR141716A one with Ki values of035 pM and 18 nM respectively using mouse forebrainhomogenates and [3H]-CP-55940 as radioligandFurthermore the selectivity ratio obtained for this compoundby Ruiu is 60000 with a Ki value for the CB2 receptor of21 nM against the [3H]-CP-55940 The discrepancybetween the data obtained independently by Stoit et al andRuiu et al could hardly be explained by the differences inthe origin of the receptor used respectively hCB1-CHO andmouse forebrain

4 Phenyl Benzofuranone Derivatives

In a 1997 patent from Eli Lilly was described thesynthesis of aryl-benzothiophene and aryl-benzofuranederivatives (87-89 Fig 6) having CB1 receptor affinity andantagonist properties [162] From ten compounds having aKi value lower than 25 microM ([3H]-CP-55940 hCB1-CHOcells) one was selected and claimed to have a Ki of 170 nMCompound 88 was shown in the patent to antagonise theeffect of anandamide on cAMP accumulation with an IC50 of

1374 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

O

O

O

O

NC

S

O

O

O

O

O

O

O

NC

LY320135 8887 89

Fig (6) CB1 cannabinoid receptor antagonists 4 Phenyl benzofuranone derivatives Chemical structures of two aryl-benzofuranederivatives (87 -88 ) and one aryl-benzothiophene (89 ) derivative from Eli Lilly

500 nM and the effect of WIN-55212-2 on calcium channelsat 1microM Moreover the intra-peritoneal injection (20 mgkg)of this compound antagonised the in-vivo effect ofanandamide in the Open Field Assay a mouse behaviourmodel Later on Felder and colleagues gave a furtherdescription of this compound called LY320135 in a paperdescribing its effect on cAMP accumulation and on ioniccurrents [163] The selectivity ratio of LY320135 was 106with Ki values of 141 and 14900 nM for hCB1 and hCB2receptors ([3H]-CP-55940) respectively LY320135antagonised anandamide inhibition of forskolin-inducedcAMP accumulation with an IC50 value of 734 nMChristopoulos et al in 2001 reported for LY320135 apKb value of 527 in the inhibition of WIN-55212-2mediated response in the rat electrically-stimulated vasdeferens while the pKb value for SR141716A was 75[164]

Other patents were taken by Aventis in which somepotential applications for their compounds were describedFor instance an association between an azetidine derivativehaving CB1 antagonists properties and sibutramine aserotonin and norepinephrine reuptake inhibitor was claimed[170] as a treatment against obesity Oral administration ofcompound 90 (3 mgkg) and sibutramine (06 mgkg) to fafaZucker rats which are genetically obese rats has resulted ina decreased food intake compared to control lean Zuckerrats In another patent the association of an azetidinederivative (1-10 mgkg po ) and a D2D3 agonist likequinpirole (01 mgkg ip) was claimed to have a therapeuticpotential in the treatment of Parkinsonrsquos disease [171] Theeffect has been evidenced using an akinesia model in the ratEvidences supporting the use of CB1 antagonists as adjuvantin the treatment of Parkinsonrsquos disease begin to appear Itseems that such compounds would be helpful in thetreatment of Parkinsonism [172] and levodopa-induceddyskinesia [173] Brotchie published an interesting paperreviewing the CB1 cannabinoid receptor signaling inParkinsonrsquos disease [174] Nevertheless further clinicalstudies should be conducted to further confirm the usefulnessof CB1 antagonists in the treatment of Parkinsonrsquos disease

5 Azetidine Derivatives

The family of azetidine compounds illustrated bycompounds 90 and 91 (Fig 7) was developed at Aventis byDaniel Achard and colleagues and was described in a seriesof patents [165-168] They claimed the IC50 values of thesecompounds for the CB1 receptor to be less than or equal to100 nM These values were obtained following the proceduredescribed by Kuster et al [169] The antagonist propertywas shown using an in-vivo model the reversal ofhypothermia induced by CP-55940 in mice and the ED50obtained were lower than 50 mgkg

6 Aryl-Imidazolidine-24-Diones Derivatives

In 1999 the Didier Lambert team published a paperdescribing the affinity of 24 new 3-alkyl-55rsquo-diphenyl-imidazolidine-24-dione derivatives for the hCB1 receptor[175] The preliminary structure-activity relationships

N

SO

O

Cl

Cl

F

F

N

SO

O

Cl

NS

90 91

Fig (7) CB1 cannabinoid receptor antagonists 5 Azetidine derivatives Two examples (90 -91 ) of the azetidine derivatives developedby Aventis

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1375

Table 8 CB1 Cannabinoid Receptor Antagonists 6 Aryl-Imidazolidine-24-Diones Derivatives

Structures affinities ([3H]-SR141716A hCB1-CHO cells) and pKb values ([35 S]-GTPγS HU210 rat brain) of three aryl-imidazolidine-24-dionederivatives (92-94)

N

NH

OO

R

Br

Br

Cpd ndeg R Ki (nM) a pKb b

DML20 92 ethylmorpholine 70 611

DML21 93 1-hydroxypropyl 103 625

DML23 94 heptyl 98 574

a [175]b [177]

showed that the substitution at the nitrogen in position 3 aswell as a bromine atom in para of the phenyl rings aremandatory for the compounds affinity Later on threecompounds termed DML20 (92) DML21 (93) and DML23(94) were characterised as neutral antagonists using the [35S]-GTPγ S assay on rat cerebellum membranes [176] (Table 8)In the same model they competitively inhibited HU-210-induced [35S]-GTPγ S binding with pKB values of 611625 and 574 respectively Moreover these compoundswere proven to be quite selective for the CB1 receptor

higher than the one for the CB2 cannabinoid receptor (IC50 =300 nM) The inverse agonism of the compound wasextrapolated by inhibiting CP-55940-induced hypothermiain mice (EC50 = 18 nM) Moreover one oral dose (10mgkg) reduces the food intake in diet-induced obese ratsInterestingly food intake of CB1

-- mice was not alteredBody weight loss was maintained all over the study during achronic administration (14 days) of 10 mgkg of Cpd A

Later on Merck published a patent [180] in which someof the 45-diaryl-pyrazoles derivatives previously describedwere claimed to be useful in the treatment or the preventionof obesity in association with compounds inhibiting the11β-hydroxysteroid dehydrogenase type 1 (1β-HSD1)enzyme The 1β-HSD1 enzyme is responsible for thesynthesis of cortisol increased levels of which according tothe inventors are associated with obesity However neitherresults on appetite suppression nor on weight loss weregiven even if some indications on the affinity of thecompounds for the cannabinoid receptors were given

Very recently the functionality of these DML compoundswas explored using the [35S]-GTPγ S assay both on rat andhuman CB1 cannabinoid receptors [177] The data obtainedconfirm that the 3-alkyl-55rsquo-diphenyl-imidazolidinedionederivatives behave as neutral antagonists on rat CB1cannabinoid receptor (rat brain) However DML 20 DML21and DML23 acted as inverse agonists on the human receptor(hCB1-CHO cells) Furthermore the authors showed thatthis different functionality is not due to the hCB1 receptorlevel of expression in the recombinant cell line as whateverthe level of expression (Bmax of 44 pmolmg or 32pmolmg) DML derivatives behaved as inverse agonists ofthe hCB1 cannabinoid receptor

Another imidazole ring pattern of substitution wasproposed by Hagmann and collaborators [181] Thecompounds described 43 examples are given possess twophenyls the first one in position 1 linked to the nitrogenand the second one in position 2 The carboxamidesubstituent is in position 4 instead of in position 2 for thediarylimidazoles described by Finke These 12-diaryl-imidazoles are claimed to be CB1 cannabinoid receptorantagonists or inverse agonists but the absence of precisepharmacological values do not allow any comparisonbetween the two substitutions patterns However a samepattern of substitution was used by other researchers Amongthem Dyck et al described in 2004 ten 1-(4-chlorophenyl)-2-(24-dichlorophenyl)-imidazole derivatives varying by theircarboxamide substituent in position 4 (101-103 Table 9)[152] For instance with a 3-azabicyclo[330]octan-3-ylsubstituent (101) they obtained a Ki value of 14 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) In a [35S]-GTPγ Sassay 101 exhibited an IC50 value of 19 nM

7 Diarylimidazoles Derivatives

Finke and colleagues at Merck developed a new class ofCB1 receptor antagonists based on an imidazole nucleusOver eighty 45-diaryl-imidazoles derivatives having CB1antagonist properties were claimed in a patent published in2003 [178] Some representative compounds of the patent areillustrated in Table 9 (95-100) The affinity of the newcompounds was determined using [3H]-CP-55940 andhCB1-CHO cells Although no detailed pharmacologicaldata were given in the patent in-vitro and in-vivo propertiesof one compound termed Cpd A (100) have been describedelsewhere [179] Its affinity for the CB1 cannabinoid receptorwas shown to be nanomolar (IC50 = 4 nM) and 75 times

1376 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 9 CB1 Cannabinoid Receptor Antagonists 7 Diaryl-Imidazole Derivatives

Structure and affinity range of some 45-diaryl-imidazole derivatives for the human cannabinoid receptors determined using [3H]-CP-55940 asradioligand and hCB1amp2-CHO cells

N

NR2

R1

R4

R3

ndeg R1 R2 R3 R4 hCB1 (IC50 or Ki) hCB2 (IC50 or Ki) References

95 CH3 CO-NH-piperidinyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

96 CH3 CO-NH-piperidinyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

97 CH3 CO-NH-cyclohexyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

98 CH3 CO-NH-phenyl 24-di-Cl-Ph 4-Cl-Ph 10 ndash 100 nM a 100 ndash1000 nM a [178]

99 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

100 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph 4 nM a 300 nM a [179]

101 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

CH3 14 nM b ND [152]

102 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

H 66 nM b ND [152]

103 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 85 nM b ND [152]

104 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 23 nM b 542 nM b [182]

105 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 30 nM b 608 nM b [182]

106 4-Br-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 60 nM b 489 nM b [182]

107 4-CF3-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 29 nM b 634 nM b [182]

108 4-Cl-Ph 24-di-Cl-Ph CO-NH-morpholinyl CH3 197 nM b 3297 nM b [182]

109 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidin-4-ol CH3 172 nM b 3959 nM b [182]

110 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CN 30 nM b 1590 nM b [182]

111 4-Cl-Ph 24-di-Cl-Ph CO-NH- piperidinyl CH2F 36 nM b 906 nM b [182]

112 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH2-CH3 14 nM b 430 nM b [182]

113 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Cl 27 nM b 823 nM b [182]

114 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Br 23 nM b 746 nM b [182]

115 4-Cl-Ph 24-di-Cl-Ph CO-NH-tetrahydroisoquinoline CH3 34 nM b 696 nM b [182]

116 4-Cl-Ph 24-di-Cl-Ph CO-NH-cycloheptyl CH3 35 nM b 349 nM b [182]

117 24-di-Cl-Ph 4-Cl-Ph CO-NH-piperidinyl CH3 403 nM b 208 nM b [182]a IC50 value b Ki value

Lange and co-workers from Solvay described twenty-eight imidazole derivatives [182] The 24-dichlorophenylsubstituent at position 2 was kept constant while thesubstitutions at the other positions were explored (Table 9)Position 5 can accommodate a large range of littlesubstituents like hydrogen (104) methyl (105) ethyl (112)chlorine (113) bromine (114) fluoromethyl (111) or cyano(110) without major changes in the affinity At position 4more bulky substituents like 1234-tetraisoquinoline (115)or cycloheptyl (116) were well tolerated whereas thepresence of a more hydrophilic moiety such as morpholine(108) or piperidin-4-ol (109) was detrimental to the affinity

Interestingly position exchange between the 24-diphenyland the 4-chlorophenyl substituents led to a 13 fold loweraffinity with Ki values of 30 and 403 nM for 105 and 117respectively ([3H]-CP-55940 hCB1-CHO cells) In thefunctional assay which was inhibition of WIN-55212-2-induced [3H]-arachidonic acid release by hCB1-CHO cellsall tested compounds behaved as antagonists Compound105 showed a pA2 value of 86 which is the same theauthors obtained for SR141716A It would be interesting toassess if these compounds are true antagonists or if they actmore as inverse agonists Compound 105 when administeredto rats was able to inhibit CP-55940-induced hypotension

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1377

with an ED50 value of 24 mgkg which is close to the 3mgkg obtained for SR141716A The compound was alsoactive in another in-vivo model the WIN-55212-2-inducedhypothermia in mice Compound 104 which has nosubstituent in position 5 of the imidazole ring was lessactive in the hypotension model and inactive in thehypothermia model Thus a methyl group is the preferredsubstituent in position 5 of the imidazole moiety Otherproperties either experimental (P-glycoprotein affinitylogPHPLC) or computational (molecular volume polarsurface area) obtained for compounds 105 were similar tothose obtained for SR141716A Finally molecularmodelling studies revealed a close structural overlap betweenthe two compounds

value of 197 nM is selective for hCB1 and is as potent asSR141716A The structural modulations of 118 led to someinteresting compounds (Table 10) Six compounds possess aKi value for the hCB1 cannabinoid receptor equal to or lowerthan 25 nM antagonist properties in the inhibition of [3H]-arachidonic acid release by WIN-55212-2-stimulated hCB1-CHO cells and Ki values for the hCB2 receptor over 1microMThe diaryl-pyrazoline derivatives contained a chiral center atposition 4 The authors resolved the racemic mixture of twocompounds (121 and 130) The levorotatory enantiomersappeared to be the eutomers For compound SLV319levorotatory enantiomer of 121 the Ki and pA2 values were78 nM and 99 respectively The Ki and pA2 values ofSLV326 levorotatory enantiomer of 130 were 359 nM and9 respectively

8 34-Diaryl-Pyrazoline Derivatives Two in-vivo models CP-55940-induced hypotension inrat and WIN-55212-2-induced hypothermia in mouse wereused to assess the potential of these compounds The resultsare of the same order of magnitude than those obtained forSR141716A For instance in the hypotension modelSLV319 ((-)121) and SLV326 ((-)130) showed ED50 values

Lange and collaborators also discovered 34-diaryl-pyrazoline derivatives after a screening of compoundsresembling to SR141716A [183184] Compound 118 thatwas initially identified during the screening possesses a Ki

Table 10 CB1 Cannabinoid Receptor Antagonists 8 34-Diaryl-Pyrazolines Derivatives

Structure affinity for the hCB1 and hCB2 cannabinoid receptors (Ki nM) expressed in CHO transfected cells ([3H]-CP-55940) and potency (pA2) ofsome 34-dirylpyrazolines derivatives Table adapted from [184]

NN

N N

SO2

R3

R2

Cl R1

R4

n˚ R1 R2 R3 R4 Ki hCB1 (nM) Ki hCB2 (nM) pA2 (CB1)

118 H H H 4-CH3 197 gt 1000 84

119 H H H 246- CH3 24 gt 1000 94

120 H H H 4-Cl 16 gt 1000 95

121 H CH3 H 4-Cl 25 gt 1000 87

122 H CH3 CH3 4-Cl 280 gt 1000 85

123 H H H 4-F 53 gt 1000 9

124 H CH3 H 4-F 338 gt 1000 85

125 H CH3 CH3 4-F gt 1000 gt 1000 lt 75

126 H CH3 H 3-Cl 14 gt 1000 86

127 4-Cl CH3 H 4-Cl 255 ND ND

128 4-F CH3 H 4-Cl 584 ND ND

129 H CH3 H H 170 ND 75

130 H CH3 H 4- CF3 221 gt 1000 93

SR141716A 25 1580 86

1378 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

N

HN

O

N

Cl Cl

Cl

N

N

HN

O

Br

Br131 132

Fig (8) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Chemical structures of two 56-diaryl-pyrazine-2-amide derivatives (131-132) developed as CB1 cannabinoid receptorantagonists by AstraZeneca

of 55 and 2 mgkg (po) respectively proving their in-vivoefficacy after oral administration The authors alsodemonstrated that SLV319 is devoid of affinity for the P-glycoprotein pump and that it possessed a goodCNSplasma ratio (17) These two compounds (SLV319and SLV326) were chosen as development candidates bySolvay and entered clinical Phase I trials in 2003 [185]

132 Fig 8) useful as CB1 cannabinoid receptor antagonists[186 187] The activity of twenty derivatives wasdetermined using a [35S]-GTPγ S assay and hCB1-CHOtransfected cells The concentration required to give halfmaximal inhibition of CP-55940-induced [35S]-GTPγ Sbinding (IC50) is lower than 200 nM for the preferredcompounds No affinity data were given for thesecompounds

9 Diaryl-Pyrazine Diphenyl-Pyridine Diphenyl-Phenyland Diaryl-Pyrimidines Derivatives

The same year in a patent from Merck Finke andcollaborators described several 56-di-phenyl-pyridinederivatives as hCB1 antagonists or inverse agonists [188]The compounds have a substituent in position 3 of thepyridine core (Fig 9) This substituent could be a cyano(133-134) or a nitro group a halogen an ester or an amide(135-136) Over 150 compounds were synthesised toillustrate the invention but no pharmacological data wasdisclosed in the patent

In 2003 appeared several patents describing new familiesof compounds that bind to the CB1 cannabinoid receptorThese compounds have in common a central six atomsaromatic ring which could be a pyrazine a pyridine aphenyl or a pyrimidine and that is substituted by at leasttwo phenyl rings

The first patents were from Berggren and collaboratorsfrom AstraZeneca who published two patents describing thesynthesis of 56-diaryl-pyrazine-2-amide derivatives (131-

A few months later another patent was taken by Sanoficlaiming the antagonist properties of 56-di-phenyl-2-pyridine carboxamide derivatives [189] These compounds

N

NH

O

Cl

Cl

N

O

Cl

O NH

N

O

Cl

ClCl

NH

CN O

N

Cl

Cl

Cl

OHN

N

O

Cl

ClCl

CNF

F

133 134

135 136 137

Fig (9) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives 56-Di-phenyl-pyridine derivatives patented by Merck (133-136) and by Sanofi (137) as CB1 cannabinoid receptorsantagonists

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1379

NH

O

N

Cl

ClCl

NH

O

N

Cl

F3CCl

N

N

O

Cl

Cl Cl F138 139 140

Fig (10) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Representative structures of the diphenyl-phenyl (138-139) and 45-diphenyl-pyrimidine (140) derivatives developed ascannabinoid antagonists at Sanofi and Merck respectively

28 examples are given structurally related to the diphenyl-phenyl derivatives have their pyridine core substituted bytwo phenyl rings and by an amide moiety (137 Fig 9) TheIC50 values were lower than 100 nM but no data were givenshowing the antagonist properties although the compoundswere assayed in the adenylate cyclase inhibition assay

10 Other Derivatives

Several amide derivatives structures recently appearedamong the CB1 cannabinoid receptor antagonists In a seriesof patents taken by Merck in 2003 Hagmann and colleaguesdescribed a large amount of compounds obtained by parallelsynthesis which are claimed to be CB1 cannabinoid receptorantagonists [192-195] They were synthesised by reacting alibrary of substituted amines with a library of carboxylicacids (141-144 Fig 11) However as no value was givenfor the affinity or activity of these amides we will notfurther discuss this class of compounds

Along this line Sanofi Synthelabo developed newdiphenyl-phenyl derivatives claimed to have CB1cannabinoid receptor antagonist properties [190] They arebased on a central phenyl ring substituted by two phenylsand by an amide moiety (138-139 Fig 10) Thirteencompounds were claimed along with their synthesis TheirIC50 values were lower than 100 nM ([3H]-CP55-940hCB1-CHO cells) but no other specific data (selectivitypA2hellip) was given

Several 15-diaryl-pyrrole-3-carboxamide derivatives weresynthesised and claimed to be CB1 cannabinoid receptorantagonists by Berggren et al [196] The affinity valueswere not disclosed in the patent (145-146 Fig 12) Otherpyrrole derivatives were synthesised by Guba et al atHofmann-La Roche [197] More specifically 2-(thiazol-4-yl)pyrrole derivatives were claimed as CB1 cannabinoidreceptor antagonist (IC50lt2microM) (147-148 Fig 12)

Finally Kopka et al from Merck described more thanone hundred 45-diaryl-pyrimidine derivatives as CB1cannabinoid receptor antagonists (140 Fig10) [191]However no precise data were provided concerning theaffinity and functionality of the compounds

R2

R1N

R3

R4

R5

O

NH

O

Cl

O F

F

R2

R1NH

R3

R4

HO R5

O

NH

O

Cl

CN

NH

O

Cl

O

O

NH

O

Cl

Cl

+

141142

143 144

Fig (11) CB1 cannabinoid receptor antagonists 10 Other derivatives Retrosynthetic scheme of a CB1 cannabinoid antagonistlibrary of amide derivatives developed at Merck The structures of four representative compounds are illustrated (141-144)

1380 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

NH

O N

Cl

Cl

Cl

N

NH

O

OMe

MeO

N

NH

O

N

S

MeO

N

NH

O

N

S

MeO

145 146

147 148

Fig (12) CB1 cannabinoid receptor antagonists 10 Other derivatives Two examples of 15-diaryl-pyrrole derivatives described byBerggren et al (145-146) While the 2-(thiazol-4yl)pyrroles derivatives 147 and 148 were described by Guba et al from Hofmann-LaRoche All these pyrroles derivatives were claimed to be CB1 cannabinoid receptor antagonists

Another new structure is represented by the 124-triazolederivatives developed by Jagerovic and collaborators In arecent paper were described the synthesis andpharmacological properties of five new 15-diphenyl-3-alkyl-

triazole derivatives [198] Among these compounds onlyone (149) behaved as a CB1 antagonist inhibiting the WIN-55212-2-induced contractions in a mouse vas deferenspreparation However despite its effects in isolated tissue

Table 11 CB1 Cannabinoid Receptor Antagonists 10 Other Derivatives

Structure and affinity of diaryl-triazole derivatives

N

NN

R3

R2

R1

Cpd R1 R2 R3 Ki CB1 (nM) References

149 24-diCl 4-Cl hexyl 855 a [198]

150 24-diCl 4-Cl2-OMe CO-NH-(3-azabicyclo[330]octan-3-yl) 270 b [152]

151 24-diCl 4-Cl CO-NH-(3-azabicyclo[330]octan-3-yl) 164 b [152]

152 4-Cl 24-diCl CO-NH-(3-azabicyclo[330]octan-3-yl) 137 b [152]

153 4-Cl 24-diCl CO-NH-benzylpyrrolidin-3-yl 29 b [152]

154 4-Cl 24-diCl CO-NH-1-(4-chlorophenyl)ethyl 66 b [152]

155 4-Cl 24-diCl CO-NH-piperidinyl 356 c [182]

156 24-diCl 4-Cl CO-NH-piperidinyl 382 c [182]

a [3H]-SR141716A rat cerebellar membranesb [3H]-CP-55940 hCB1-HEK EDNA cellsc [3H]-CP-55940 hCB1-CHO cells

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1381

N

S

O

NH

Cl

Cl

Cl

F

N

S

O

NHN

ClCl

Cl

N

S

O

NH

Br

N

S

O

NHN

Cl

Cl

Cl

156 157 158 159

Fig (13) CB1 cannabinoid receptor antagonists 10 Other derivatives Structures of 45-diaryl-thiazole derivatives patented as hCB1cannabinoid receptor antagonists by Solvay Pharmaceuticals (156 157 158) or by AstraZeneca (157 158 159)

assays (mouse vas deferens and guinea pig ileum) thecompound possessed only a reduced rCB1 affinity with Kivalues of 855 and 748 nM using [3H]-SR141716A and [3H]-WIN-55-212-2 respectively (Table 11) Further four seriesof compounds differing by the nature of the substituentaround the triazole core were also described in a patent[199] Usually the compounds claimed possess two aromaticrings possibly substituted and one linear alkyl chain Theaffinity for the cannabinoid receptors was not givenNevertheless 149 was evaluated in two isolated tissuemodels the inhibition of the electrically evoked contractionsin the guinea pig ileum and in the mouse vas deferens Inthe two models compound 149 inhibited the effect of WIN-55212-2 but had no effect by itself In the vas deferensmodel the authors obtained a pA2 value of 748 to becompared with 763 obtained for AM251

Yet another five membered ring a thiazole has beenused as central moiety of a new class of cannabinoid ligandsThe thiazole ring is substituted by two aryls in position 4and 5 and by an amide (position 2) as in compounds 156-159 (Fig 13) Forty derivatives of this type weresynthesised by Lange and colleagues from SolvayPharmaceuticals and claimed in a patent as agonists orantagonists of the CB1 cannabinoid receptor [200] Nopharmacological data were provided however their activitywas assessed by measuring the cAMP in transfected hCB1-CHO cells In a recent paper from Lange et al two of thesethiazole derivatives were further described [182] Compound13 (157) the most closely related to SR141716A has a Kivalue of 227 nM while for compound 14 (158) Ki value isover 1000 nM Thus as in the imidazole series (see Table9) the aromatic substitution pattern is of great importancefor the CB1 cannabinoid receptor affinity Compound 13however was devoid of in vivo activity

Others 124-triazole derivatives were described by Dycket al compounds 150-154 unlike the Jagerovic ones havean amide moiety in position 3 [152] The highest affinitywas obtained with a 1-benzyl-pyrrolidin-3-yl substituentThe Ki value was 29 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) to be compared to 12 nM obtained for theSR141716A

Seventeen 45-diaryl-thiazole derivatives were alsosynthesised by Berggren et al and patented by AstraZeneca(157-159 Fig 13) [201]

In a patent from Merck Toupence et al described over190 substituted furo[23]pyridines claimed to be CB1cannabinoid receptor antagonists [202] The affinity (IC50 lt1microM) was measured using recombinant CHO cells and [3H]-CP-55940 and the activity using cAMP dosage (160 Fig14)

Lange et al described a triazole derivative (155) whichcan be superimposed with SR141716A Compound 155showed a Ki value of 356 nM ([3H]-CP-55940 hCB1-CHOcells) a pA2 value of 83 in the inhibition of WIN-55212-induced release of [3H]-arachidonic acid by hCB1-CHO cellsand was active in vivo in the CP-55940-inducedhypotension in rat (ED50 = 236 mgkg) [182]

Very recently Alanine from Hoffmann-LaRoche foundthat diarylbenzo[13]dioxole derivatives act as CB1

O

O

S

N

OO

F

ClCl

O

O

F O

N

OON

NH2

O

Cl

Cl

Cl

160 161 162

Fig (14) CB1 cannabinoid receptor antagonists 10 Other derivatives One example of a furo[23]pyridine derivative (160) and twoexamples of diarylbenzo[13]dioxole derivatives (161-162) described as hCB1 cannabinoid receptor antagonists by Toupence et aland Alanine et al respectively

1382 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

cannabinoid receptor antagonists (161-162 Fig 14) [203]Approximate IC50 values for twelve compounds ([3H]-CP-55940 hCB1-HEK cells) among the three hundred andeighty compounds described were given (IC50lt2microM)

et al in 1995 [207] In their hands ∆9-THC was not able toinhibit the forskolin-stimulated adenylyl cyclase activity inan assay performed on hCB2-CHO cells Subsequently theyshowed that ∆9-THC was able to antagonise HU210-inducedinhibition of adenylyl cyclase activity thus acting as anantagonist of the hCB2 cannabinoid receptor expressed inCHO cells [208] Furthermore very recently Govaerts et al[209] reported an inverse agonist effect of ∆9-THC and ∆8-THC on hCB2-CHO cells using the [35S]-GTPγ S assayThe pEC50 values were 763 and 888 respectively whilethe Emax values were ndash27 and ndash16 as compared to basal

Griffith from Pfizer described three new series of CB1cannabinoid receptor antagonists based on a bicyclic centralaromatic ring Purine pyrazoltriazines andpyrazolo[15a]pyrimidines derivatives were synthesised andinvestigated for their ability to interact with the CB1cannabinoid receptor Albeit a [35S]-GTPγ S assay wasdescribed no data were given concerning the antagonist orinverse agonist properties of such compounds Two hundredand eighty purine derivatives were synthesised [204] Theaffinities of 163 and 164 were given in the patent (28 and12 nM [3H]-SR141716A) In a following patent thesynthesis of sixty pyrazoltriazine derivatives was described(165 Fig 15) [205] The affinity of each compound was notgiven but was said to be ranging between 01 and 590 nMAnd finally more than forty pyrazolo[15a]pyrimidines weresynthesised (166 Fig 15) [206] Their affinity for the CB1cannabinoid receptor was lower than 155 nM Despite thefact that a great number of derivatives share the samesubstitution pattern around the central bicyclic aromatic ringthe comparison of these three new series of compounds ismade difficult by the absence of precise affinity data

Some other classical cannabinoids were shown to possessan antagonist activity at the CB2 receptor This is the casefor instance for the non-selective O-1184 (2) (Ki = 74 nM)which enhances the forskolin-induced cAMP production inCB2-CHO cells (EMax = 161 EC50 = 63 nM) and alsofor O-584 (3) (EMax = 246 EC50 = 138 nM) [39]

2 Indole Derivatives

Since the disclosure in 1993 of a second cannabinoidreceptor many attempts have been made to synthesise CB2cannabinoid receptor selective ligands The first compoundreported to have a somehow greater affinity for the peripheralcannabinoid receptor was WIN-55212-2 The compoundwas introduced by DrsquoAmbra and colleagues [210] back in1992 as a new cannabinoid ligand having a nanomolaraffinity and acting as an agonist In 1996 Showalter et alused a transfected cell line expressing the CB2 cannabinoidreceptor in order to identify selective ligands for thisreceptor [211] They showed that the CB2 affinity of WIN-55212-2 was 7 times greater than the CB1 with Ki valuesof 189 and 028 nM for the CB1 and CB2 cannabinoidreceptors respectively However WIN-55212-2 is anagonist of the cannabinoid receptors Interestingly WIN-55212-3 (167 Fig 16) the so-called inactive enantiomer ofWIN-55212 was recently shown to behave as a low affinity(Ki=363 microM) inverse agonist of the hCB2 cannabinoidreceptor as it decreased the [35S]-GTPγ S binding [209]

III CB2 LIGANDS RECEPTOR ANTAGONISTS ANDINVERSE AGONISTS

The compounds acting as antagonist or inverse agonistsat the CB2 cannabinoid receptor are reviewed in this thirdpart of the paper As for the CB1 cannabinoid receptorligands they are classified depending on their chemicalstructures

1 Classical Cannabinoid Derivatives

The first report of an antagonism mediated by ∆9-THC atthe peripheral cannabinoid receptor was made by Bayewitch

N N

NN

NH COOH

Cl

Cl

N N

NN

N

Cl

Cl

NH2OH

O

N

N

N

N

N

NHNH2

O

Cl

ClN

N

N

N

Cl

Cl

N

O

163 164

165 166

Fig (15) CB1 cannabinoid receptor antagonists 10 Other derivatives Examples of purine (163-164) pyrazoltriazines (165) andpyrazolo[15-a]pyrimidines (166) derivatives described as CB1 cannabinoid receptor antagonists by Griffith

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1383

The pEC50 value was 554 and the Emax value ndash26 ascompared to basal

AM630 also acts as an inverse agonist at the hCB1cannabinoid receptor (EC50 = 900 nM) as Landsman et aldemonstrated [47] Recently Zhang and colleagues reportedthe characterisation of the microsomal metabolism of AM630[212]

N

O

N

O

O

N

O

Cl

O

N

O

(S)-WIN-55212-3 167 BML-190 168

In 1996 Gallant and colleagues identified by submittinga large number of compounds to a binding assay anotherindole analog as CB2 cannabinoid receptor ligand [213] Onthe basis of this compound christened BML-190 (168 Fig16) or called indomethacin morpholinylamide theysynthesised several derivatives that possessed a selectivityratio for the CB2 cannabinoid receptor of up to 140 Thesewere the first compounds specifically designed to be CB2cannabinoid receptor ligands The first report on thepharmacological properties of BML-190 appeared onlyrecently New and colleagues highlighted in the paper theinverse agonist properties of the compound BML-190 dose-dependently increases (103 EC50= 980plusmn70 nM) theforskolin-stimulated levels of cAMP in hCB2-HEK cellswhile WIN-55212-2 decreases (44 45plusmn42nM) thisaccumulation [137]

Fig (16) CB2 cannabinoid receptor antagonists 2 Indolederivatives Chemical structures of WIN-55212-3 (167) andBML-190 (168) two indoles derivatives acting as inverseagonists at the hCB2 cannabinoid receptor Surprisingly in 2002 Melck and collaborators used

BML-190 as a CB2 receptor agonist in a cell proliferationassay [214] BML-190 was also tested as inhibitor of thecyclooxygenase-2 enzyme by Kalgutkar et al [215] Theyobtained IC50 values higher than 33 and 66 microM for theCOX-I and COX-II enzymes respectively These valueshave to be compared with the submicromolar activity ofindomethacin the parent compound Very recently Klegerisand colleagues [216] looking for an antineurotoxic action ofcannabinoids showed that BML-190 increases TNF-αsecretion by stimulated THP-1 monocytic cells but is noteffective on the IL-1β secretion

Further on the basis of WIN-54461 (12) a CB1antagonist AM630 (13) was synthesised by replacement ofthe bromine atom by an iodine one In the initial reportPertwee et al showed the antagonist effects of AM630 butalso suggested that the CB1 cannabinoid receptor may not bethe preferential receptor of AM630 [45] Later on Ross et alreported using hCB1amp2-CHO cells and [3H]-CP-55940that AM630 binds to the CB2 cannabinoid receptor (Ki =312 nM) with a selectivity ratio over 160 [48]Furthermore using a [35S]-GTPγ S assay they showed itsinverse agonist properties (EC50 = 76 nM) However

Table 12 CB2 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Reported binding affinities (Ki) and selectivity ratio (CB1CB2) of SR144528 (169) for rat and human cannabinoid receptors determined using [3H]-CP55940 as radioligand

NN

O

NH

Cl

SR144528 169

rCB2 (spleen) rCB1 (whole brain) Selectivity Ki hCB2 (transfected CHOcells)

Ki hCB1 (transfected CHOcells)

Selectivity References

038 nM 305 nM 800 06 nM 437 nM 730 [217]

067 nM 548 nM 80 [219]

56 nM gt10microM gt1780 [48]

25 nM a gt 1microM a gt400 [224]

024 nM 276 nM 115 199 nM 503 nM 25 [232]

a COS cells

1384 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

3 Diarylpyrazole Derivatives CHO cells is constitutively active and phosphorylated onSer 352 Upon treatment by SR144528 thisphosphorylation of the receptor was inhibited Moreover theSR compound was able to induce dephosphorylation ofagonist-induced CB2 phosphorylation The question whetherSR144528 induces a CB2 receptor conformational changemaking it a better phosphatase-specific substrate or ifSR144528 binding activates specific phosphatases remainsopen However this remains a nice example of inverseagonist-induced receptor resensitisation

The same Sanofi team which synthesised the CB1antagonist SR141716A reported in 1998 the synthesis andpharmacological characterisation of a CB2 cannabinoidreceptor selective antagonist the SR144528 (169) [217218] The structure of the new compound is derived fromSR141716A one SR144528 is the first selective CB2receptor antagonist reported as the inverse agonist propertiesof BML-190 were shown only recently by New et al [137]The authors reported for 169 using [3H]-CP-55940 asradioligand Ki values of 04 nM and 06 nM for the rat andthe human CB2 receptors respectively and found a CB1-CB2 selectivity ratio around 700 Table 12 sums up thebinding affinities of SR144528 for the cannabinoid receptorsobtained by different authors Interestingly while the CB2affinity is more or less constant the affinity obtained for theCB1 receptor varies by one order of magnitude [219] Theantagonist effect of SR144528 was highlighted by evaluatingits effect on cAMP production by hCB2-CHO cells exposedto 3 nM CP-55940 (EC50=10nM) The antagonism wasalso observed on the activation of the MAP kinase pathwayby CP-55940 (IC50=39nM)

The Sanofi research team working on cannabinoidsconducted investigations in order to determine the keyresidues for SR144528 interaction with the receptor (Table13) as well as the binding mode of the compound Firstlythey identified the TM4-EL2-TM5 region as a regioncontaining crucial residues for the affinity of several ligandsamong them SR144528 [224] Among these residues themutation of two cysteine of the second extracellular loop(C174 and C179) abolishes the affinity for the CB2 receptorLater on the implication of S453(161) and S457(165) inthe binding of SR144528 was evidenced by mutationalstudies In the model proposed for the docking of SR144528into the CB2 receptor the crucial contacts are comprised intothe TM4 this is the case for S453(161) and S457(165)which are proposed to form hydrogen bonds with the ligandand W464(172) proposed to have aromatic interactions withthe 4-methylbenzyl of the SR144528 [225] In 2003 Fengand Song showed that point mutations D349(130)A andA634(241)E abolished ligand binding However mutationof the arginine R350(131) member of the highly conservedDRY motif to alanine has no influence on SR144528binding [226]

Early after the report of the synthesis and characterisationof SR144528 the same authors further explored the functionof this compound and showed its inverse agonist propertiesusing CB2-CHO cells co-transfected with the luciferasereporter gene with either the CRE or the murine krox24regulatory sequence [220] In these models SR144528 notonly inhibits CP-55940 effects but also reproduces theeffects of a pertussin toxin treatment proving its inverseagonist properties SR144528 functionality was alsoexplored by [35S]-GTPγ S binding in hCB2-CHO cells(IC50=3 nM) [221] The inverse agonist function ofSR144528 was further illustrated by Rhee and Kim usingCOS cells co-transfected with hCB2 receptor and adenylylcyclase [222]

As for the CB1 cannabinoid receptor ligands Sanofideveloped tricyclic derivatives based on the 1-benzylpyrazole-3-carboxylic scaffold More than twentycompounds were disclosed in a patent [227] in 2001 (84-86Table 7) The benzyl in position 4 characteristic of the CB2diarylpyrazoles from Sanofi is conserved as well as thebicyclic residue in position 3 The length of the second linkbetween the phenyl and the pyrazole can be made of one or

Bouaboula and co-workers investigated SR144528 effectson CB2 cannabinoid receptor phosphorylation status [223]Firstly they showed that the CB2 receptor transfected in

Table 13 CB2 Cannabinoid Receptor Antagonists 3 Di-Aryl-Pyrazole Derivatives

Some of the single point mutations reported for the hCB2 cannabinoid receptor for which pharmacological data concerning the SR144528 are available

Mutant Effect References

R350(131)A no effect [226]

S453(161)A loss of affinity [225]

V456(164)I no effect [225]

S457(165)A loss of affinity [225]

C174S a loss of affinity [224 225]

C175S a loss of affinity [225]

C177S a no effect [225]

C179S a loss of affinity [224 225]

S544(193)G no effect [225]

a Second extra-cellular loop

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1385

two methylene but according to the patent one methylenebridge is preferred The Ki values of these compounds for theCB2 receptor were said to be lower than 500 nM and theirantagonist properties have been assessed using the adenylatecyclase inhibition assay

compounds by the presence of an alkyl substituent inposition 5 instead of a phenyl and of course by position 3which remains unsubstituted However except a lecture atthe 1998 ACS meeting [230] where compound 171 wasreported to have a Ki value of 835 nM and a selectivity ratioover 100 no further report on these compounds waspublished untill now

In a recent paper Mussinu and colleagues reported thesynthesis of other tricyclic derivatives starting fromSR141716A structure [228] N-piperidin-1-yl-14-di-hydroindeno[12-c]pyrazole-3-carboxamide derivatives areCB2 receptor ligands and some of them possess high affinityand selectivity for the CB2 receptor as shown in Table 7 (79-83) These tricyclic derivatives differ from the Sanofi tricyclicderivatives as they do have a phenyl ring instead of a benzylin position 1 Another difference is the nature of thesubstituent in position 3 which is not a bicycloalkyl but acyclic amine One of the compounds synthesised byMussinu et al 81 possesses the highest affinity for the CB2receptor ever reported with a Ki value of 0037 nM for themurine CB2 receptor ([3H]-CP-55940 mice spleenhomogenate) Interestingly 79 the closest SR141716Aanalog lost almost all its affinity for the CB1 receptor andincreased by four orders of magnitude its affinity for the CB2receptor Even if the structure quite similar to the SRcompounds could suggest that these derivatives behave asantagonists no pharmacological data were given in the paperto support this hypothesis

5 2-Oxoquinoline Derivatives

Inaba and colleagues from Japan Tobacco introduced in2001 a completely new structure in the cannabinoid fieldbased on a 2-oxoquinoline scaffold Over eighty derivativeswere disclosed in a patent [231] and one compound JTE-907 (173) was subsequently characterised in-vitro as well asin-vivo [232] In Table 14 are listed some of the compounds(172-178) disclosed in the patent along with their affinity forthe cannabinoid receptors These values were obtained onhuman receptors against [3H]-WIN-55212-2 For twelvecompounds the anti-inflammatory activity per os wasassessed in a carrageenin-induced paw edema model Thehighest effect reported was obtained with JTE-907 with anED50 value lower than 01 mgkg Therefore thesecompounds were claimed to be useful as anti-inflammatoryand anti-allergic agents

To further characterize JTE-907 the authors determinedits affinity for cannabinoid receptors of different species using[3H]-CP-55940 as radioligand In their hands JTE-907behaved as a very selective hCB2 ligand with Ki values of36 and 2370 nM for the CB2 and CB1 cannabinoid receptorsrespectively even more selective than the SR144528 (Kivalues of 199 and 50 nM respectively) However it has tobe mentioned that the affinities reported in the paper forJTE-907 are quite different from those reported in the patent(hCB1 Ki values of 35nM and 009 nM respectively) Italso appears that the SR144528 affinity for the CB1 receptordetermined by Iwamura et al is higher than the affinityreported by Rinaldi-Carmona et al with values of 50 and437nM respectively The authors ascribed the difference tothe variability of SR144528 binding affinities to the assayconditions The function of JTE-907 was evaluated bymeasuring the increase in cAMP production by forskolin-stimulated hCB2-CHO cells Maximum stimulation wasattained at a concentration of 1microM of JTE-907 In the sameassay WIN-55212-2 decreased the cAMP production in adose-dependent manner

Finally it is interesting to strengthen that the five-membered ring derivatives (79-86) of the diarylpyrazolefamily are reported to possess a high CB2 receptor affinitywhile the six- and seven-membered ring derivatives (69-78)are described as CB1 receptor ligands

4 Other Pyrazole Derivatives

Other CB2 ligands based on a pyrazole nucleus wereintroduced by SmithKline Beecham in a patent describingthe synthesis of about sixty new compounds [229] Theiraffinity (Ki) for the hCB2 receptor transfected in HEK293cells was reported to be ranging between 25nM and 10microMTheir structures (170-171 Fig 17) differ from the Sanofi

NN

O

N

ON

NN

N

NN

O

N

ON

NN

N

ClCl

1

5

4 3

2

170 171

Fig (17) CB2 cannabinoid receptor antagonists 4 Otherpyrazole derivatives Structure of two compounds (170-171)among those claimed by SmithKline Beecham to be hCB2antagonists

In conclusion JTE-907 was shown to be a CB2 selectiveinverse-agonist with anti-inflammatory and anti-allergicproperties This compound is currently undergoing Phase Iclinical trials as anti-allergic drug [233]

IV NON-CB1 NON-CB2 CANNABINOIDRECEPTORS LIGANDS

In the last years the possible presence of additional non-CB1 non-CB2 cannabinoid receptors was raised frompharmacological evidences

One of the putative receptors possesses an endotheliallocalisation while other ones are localized in the CNS or atperipheral nerves For instance an additional target for ∆9-

1386 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 14 CB2 Cannabinoid Receptor Antagonists 5 2-Oxoquinoline Derivatives

Structure of six compounds among the oxoquinoline derivatives patented by Japan Tobaco Affinity (Ki nM) and selectivity (CB1CB2) for the cannabinoidreceptors are given The affinity was measured by binding against [3H]-WIN-55212-2 on either hCB2-CHO or hCB1-CHO cells Adapted from [231]

Cpd ndeg Structure hCB2 hCB1 Selectivity

1-2 172

NH

MeO O

O

OMeO

0014 3671 262215

JTE-907 173

NH

O

MeO O

O

NH

O

O

0087 3436 39495

2-2 174

OH

O

NH

MeO O

O 077 3247 4215

3-27 175

NH

O

F

NH

MeO O

O0021 336 16000

3-32 176

NH

MeO O

O

NH

F

O

0036 2398 66610

3-38 177

O

O

NH

N

O

O

NMeO

0085 935 11000

3-43 178

NMeO O

O

NH

F

O

0043 864 20100

tetrahydrocannabinol was described in capsaicin-sensitivesensory nerves by Zygmunt et al [234]

[29] and by Di Marzo et al [235] Thus in the presentreview we will focus on the description of the so faridentified ligands of the non-CB1 non-CB2 receptors withan emphasis on the compounds having antagonist properties

Pharmacological evidences for the existence of additionalcannabinoid receptors were reviewed by Wiley and Martin

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1387

(Table 15) In particular the knowledge about three not yetcloned targets has grown The growing molecularpharmacology of the three putative new cannabinoidreceptors is reviewed in the three following sections Thefourth section summarises the data on the other proposedcannabinoid targets

vasodilatator activity In addition the anandamide effect ispartially sensitive to SR141716A inhibition The authorsproposed the existence of two distinct receptors responsiblefor the vasodilatatory effect of anandamide one located in theendothelium is SR141716A-sensitive while the other onelocated on the vascular smooth muscle is SR141716A-insensitive Thus SR141716A is an antagonist of anendothelial anandamide receptor Several studies wereconducted in order to understand the molecularpharmacology of the endothelial anandamide receptor It ispertussis toxin-sensitive [237] Cannabidiol acts asantagonist and abnormal-cannabidiol (Abn-cbd 179) and itsanalogue O-1602 (180) act as agonists [238] The synthesisof O-1602 and of its analogues are disclosed in a patentdescribing the vasodilatatory activity of cannabinoid

1 Endothelial Anandamide Receptor ndash Abnormal-Cannabidiol Receptor

In 1999 Wagner et al showed that anandamide couldinduce mesenteric vasodilatation through an endotheliallylocated ldquoanandamide receptorrdquo pharmacologically distinctfrom CB1 [236] Other cannabinoids such as ∆9-THC HU-210 or WIN-55212-2 were devoid of mesenteric

Table 15 Non-CB1 Non-CB2 Cannabinoid Receptors Antagonists

An overview of the putative new non-CB1 non-CB2 anandamide-receptors ligands The antagonists are highlighted in bold

NH

N

S

HO OH

Cl

OH

OH

OH OH OH OH

OMe

OMe

RR

RR

RR

RR

HO

OH

NH

O

cannabidiol (10) Abnormal-cannabidiol 179 O-1602 180

O-1918 181 capsazepine 183capsaicin 182

Compound Endothelial AEA receptor Brain AEA receptor Cannabinoid-vanilloid brain receptor

AEA agonist a agonist de

WIN-55212-2 no effecta

agonist e agonist g h

CP-55940 no effect e agonist i

SR141716A antagonist a b j no effect ([35 S]-GTPγS binding) d e f antagonist g h

AM251 no effect k no effect h i

Cannabidiol antagonist b no effect e

O-1918 antagonist c jm

Abd-cbd agonist b l

O-1602 agonist b

Capsaicin no effectj agonist i

Capsazepine no effect b antagonist h

a [236] b [238] c [240] d [249] e [250] f [251] g [254] h [258] i [ 255] k [244] j [ 243] l [ 242] m [241]

1388 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

analogues [239] More recently Offertaler et al showed thatO-1918 (181) a cannabidiol analog acts as an antagonist ofthe endothelial anandamide receptor [240] In addition O-1918 does not bind to the CB1 and CB2 cannabinoidreceptors Furthermore O-1918 unlike cannabidiol doesnot cause vasorelaxation in the mesenteric artery preparationmodel used and thus acts as a true neutral antagonistUsing O-1918 as a pharmacological tool Begg et alproposed a coupling of this new G protein-coupledendothelial receptor through a GiGo mechanism to ionchannels in primary cultured human vascular endothelialcells [241] However in their hands SR141716A wasineffective in antagonising Abd-cbd Ho and Hiley furtherdemonstrated that Abn-cbd relaxes rat small mesentericarteries through an activation of K+ channels via aSR141716A-sensitive pathway [242] Further using thesame model they characterized the vasorelaxant activity ofvirodhamine a novel endocannabinoid apparently actingthrough the same receptor [243] The virodhamine-inducedvasorelaxation is sensitive to SR141716A and O-1918 butinsensitive to AM251 SR144528 and AM630 Moreovervanilloid receptor desensitisation by capsaicin (182) had noeffect

244] Thus position 4 on the 5-phenyl could be furtherexplored to obtain selective ligands

To come to an end despite the fact that the endothelialanandamide receptor is neither identified nor cloned at leastthree antagonists are identified SR141716A cannabidioland O-1918 It is likely that a growing number of ligandsand therapeutic applications will appear in the very nearfuture

2 Brain Anandamide Receptor

Following an in-depth evaluation of anandamide effects inCB1

-- mice Di Marzo and co-workers suggested that a non-CB1 non-CB2 G protein-coupled receptor might mediatesome of the actions of AEA in mice [249] Actually effectsof AEA but not those of ∆9-THC were not decreased inCB1 cannabinoid receptor knock-out mice For instance 30mgkg of AEA were still effective in hot-plate test whereas10 mgkg of ∆9-THC had no effect on anti-nociceptionMoreover in a [35S]-GTPγ S assay AEA stimulated with anEC50 value of 223 microM the [35S]-GTPγ S binding to CB1

--

mice brain membranes (Emax= 28) ∆9-THC showed noeffect in this assay The addition of SR141716A did notaffect the [35S]-GTPγ S binding to CB1

-- mice brainmembranes Shortly after other evidences for the existence ofa new G protein-coupled cannabinoid receptor with a distinctdistribution in the central nervous system were published[250] From a set of 24 commonly used cannabinoids (AEAWIN-55212-2 JWH-030 THChellip) only AEA and WIN-55212-2 produced an increased binding of [35S]-GTPγ S onCB1

-- mice brain membranes Interestingly in this studySR141716A alone produced a significant inhibition of [35S]-GTPγ S binding similar to the inhibition obtained usingCB1

++ mice brain membranes (IC50 values of 57 microM and47 microM respectively) However SR141716A did not affectthe anandamide-induced [35S]-GTPγ S binding in CB1

--

mice brain membranes Moreover [3H]-SR141716Aexhibited significant binding in some brain regions of CB1

--

mice which are different from those exhibiting stimulation of[35S]-GTPγ S binding by WIN-55212-2 and AEA Thusthe authors proposed that the SR141716A effects on CB1

--

mice are mediated neither by the CB1 cannabinoid receptornor by the putative new anandamide brain receptor Monoryand co-workers using CB1

-- mice described a novel nonadenylyl cyclase-coupled cannabinoid binding site in micecerebellum [251] Two agonists AEA and WIN-55212-2but not ∆9-THC or HU210 were found to bind to thisreceptor as they enhanced [35S]-GTPγ S binding NeitherSR141716A nor SR144528 were able to affect the WIN-55212-2 induced [35S]-GTPγ S binding in CB1

-- mice asfound by Breivogel et al [250] However the distributionpattern of the receptor described by Monory et al differs fromthe distribution found by Breivogel et al It has to bementioned that the knock-out strains used were different Themice used by Monory were from a CD-1 strain while theone used by Breivogel were from a C57BL61 strainInterestingly Muthane et al detected differences in nigralneurons number and sensitivity to MPTP in the two mousestrains used [252]

More recently Mo et al obtained results showing thatthe Abnormal-cannabidiol receptor is also responsible for anincreased endothelial cell migration [244] Abn-cbd enhancedhuman umbilical vein endothelial cell migration while O-1918 antagonised Abn-cbd effect The CB1 cannabinoidreceptor inverse agonist SR141716A partially inhibitedAbd-cbd action whereas AM251 and SR144528 had noeffect

In another paper from George Kunos team Batkai et alreported that SR141716A is able to inhibit the endotoxichypotension by a cardiac mechanism involving neither theCB1 nor the CB2 cannabinoid receptors [245] Theinvolvement of a non-CB1 non-CB2 site of action wasshown using CB1

-- and CB1--CB2

-- knock-out miceFurther studies are needed to identify the SR141716Asensitive myocardial site of action

In a recent patent Kunos described O-1918 and itsanalogues as vasoconstrictor agents useful to reversepathological vasodilatation of blood vessels [246] Theeffects of O-1918 on mouse blood pressure on LPS-inducedhypotension in mouse as well as the reversal of hypotensionby O-1918 injection are described Furthermore thesynthesis of O-1918 based antagonists is described startingfrom p-metha-28-dien-1-ol and the appropriate resorcinols

Interestingly the Abd-cbd receptor which was firstdetected in the vascular system also seems to be present inthe CNS [247] Indeed Walter et al found that microglialcells (BV-2) migration induced by 1 microM 2-AG is inhibitedby cannabidiol (300 nM) and O-1918 (1 microM) SR141716Awas found ineffective in this assay Thus 2-AG is an agonistof the Abd-cbd receptor Moreover Abd-cbd is able to elicita dose-dependent cell migration in the same model (EC50 =600nM) [248]

From a more structural point of view SR141716Abehaves as an antagonist while on the opposite AM251(20) a close analogue which differs from the former only bythe nature of one halogen has no effect on this receptor [242

To conclude to our knowledge no compounds havingantagonist or inverse agonist properties at this receptor havebeen identified so far

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1389

3 Cannabinoid-Vanilloid Brain Receptor a cannabinoid-vanilloid receptor at which SR141716A andcapsazepine behave as antagonists

Evidences for the presence of an additional ldquocannabinoidrdquoreceptor differing from the CB1 receptor and from the brainanandamide receptor appeared from studies focused on thehippocampus (for a review see Hajos et al [253]) Hajos etal studied the cannabinoid actions on GABAergic andglutamatergic transmission using the whole-cell patch-clamp technique [254] WIN-55212-2 induced a dose-dependent reduction in glutamatergic transmission reversedby SR141716A (1microM) Thus the authors concluded that thecannabinoid actions on the glutamatergic transmission aremediated by a non-CB1 but SR141716A-sensitive receptorIn another paper Hajos and Freund further characterised thisputative receptor [255] They showed that in addition toWIN-55212-2 CP-55940 and capsaicin (182) a TRPV1receptor agonist also act as agonists Furthermore in theirhands SR141716A as well as capsazepine (183) a TRPV1receptor antagonist were able to antagonise this effect on theglutamatergic transmission while AM251 was notSR141716A effects on CB1 knock-out mice were furtherstudied using a test of anxiety [256] Haller et al showedthat on the one hand the distribution of the CB1 receptorinduces anxiety and SR141716A has an anxiolytic effectand on the other hand SR141716A is still active in CB1

--

mice In a following paper they reported that AM-251induces anxiogenic effects in wild-type and in knock-outmice [257] Thus SR141716A in addition to CB1 bindsto a not yet identified receptor Another evidence for thisadditional receptor for SR141716A was given by Bass andco-workers [61] They conducted a structure-activityrelationship study on the stimulation of locomotor activityinduced by SR141716A and twenty analogues Nocorrelation was found between the affinity for the CB1cannabinoid receptor and the stimulation of locomotoractivity Albeit SR141716A and five of its analogues O-1803 O-1710 O-1253 O-1254 (40) O-1255 (41) behavedas inverse agonists in [35S]-GTPγ S assay none of theseanalogues stimulated the locomotor activity In conclusionthe SR141716A-induced stimulation of locomotor activity isneither due to inverse agonism nor to inhibition of anendogenous tone but to a brain receptor distinct from theCB1 receptor Di Marzo et al previously provided evidencesthat the cannabinoid-vanilloid brain receptor could mediatesome of the cannabinoid effects on locomotion [249]

4 Other Non-CB1 Non-CB2 Cannabinoid Receptors

Evidences for the existence of additional non-CB1 non-CB2 cannabinoid receptors localised in the rat isolatedmesenteric arterial bed appeared from the works of Ralevicand collaborators Several cannabinoids were evaluated intheir model of sensory neurogenic vasorelaxation evoked byelectrical field stimulation

HU210 was found to attenuate vasorelaxation in a CB1and CB2 independent way as SR141716A LY320135 andSR144528 were ineffective in inhibiting HU210 effect [259]Similar results were obtained with ∆9-THC [260]Similarly noladin ether inhibited sensory neurogenicrelaxation in a concentration-dependent manner which wasalso unaffected by SR141716A LY320135 and SR144528Moreover this effect as the ∆9-THC one was pertussistoxin sensitive suggesting the involvement of a Gio protein-coupled receptor [261]

However the inhibitory effects of CP-55940 and WIN-55212-2 were affected by the presence of SR141716A (1microM)or LY320135 (1microM) but not by the addition of SR144528(1microM) [262]

It appears that at least two receptors should be involvedin cannabinoid-mediated attenuation of sensory nerve-mediated vasorelaxation The first one is SR141716A andLY320135 sensitive while the other one is resistant toSR141716A

Some evidences for the existence of a ldquoCB2-likerdquo receptorappeared from the studies on palmitoylethanolamide (PEA)an endogenous fatty acid amide devoid of CB1 and CB2affinity [264] antinociceptive potential Calignano and co-workers showed that PEA was able to alleviate pain inseveral animal models [265 266] For instance PEA dose-dependently inhibits kaolin-evoked writhing in mice TheCB2 cannabinoid receptor inverse agonist SR144528 (02mgkg) inhibits PEA anti-nociceptive effect [266] Thus theauthors suggest that PEA acts as agonist on a SR144528-sensitive non-CB2 cannabinoid receptor (ldquoCB2-likerdquoreceptor) at which SR144528 acts as an antagonist

Further studies are needed in order to better characterisethese additional ldquocannabinoidrdquo receptors However it islikely that several therapeutic applications will soon springfor these receptors

Very recently Pistis et al published a study on theneurophysiological effects of cannabinoids on the basolateralamygdala neurons in-vivo [258] One of their findings is thatHU-210 and WIN-55212-2 did not elicit similar effects onbasal amygdala projection neurons firing rate While theformer had no effect the latter decreased the firing rateMoreover SR141716A reversed the WIN-55212-2 effectswhile AM251 had no effect The vanilloid receptorantagonist capsazepine also antagonised the WIN-55212-2effect on firing rate of these neurons Therefore the authorssuggested the presence of a novel cannabinoid-vanilloidreceptor

V CONCLUSION

Since their development cannabinoid antagonists provedto be essential tools in the understanding of cannabinoidpharmacology and biochemistry Moreover the interestshown by the pharmaceutical industry in the development ofnew cannabinoid antagonists prove if necessary the realtherapeutic potential of this class of compounds Thetreatment of eating and movement disorders memorydeficits psychosis and dependencies from various addictivedrugs are some of the most cited applications for the CB1cannabinoid receptor antagonists

From these lines it appears that in addition to theknown CB1 cannabinoid receptor it is now likely that twoother cannabinoid receptors do exist in the brain The firstone should be an anandamide brain receptor for whichSR141716A has no affinity while the second one should be

1390 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Much less wide is the knowledge on the CB2cannabinoid receptor antagonists However potentialapplications could be disorders involving the immunesystem such as inflammation or allergies

[19] Devane WA Hanus L Breuer A Pertwee RG StevensonLA Griffin G Gibson D Mandelbaum A Etinger AMechoulam R Science 1992 258 1946

[20] Mechoulam R Ben-Shabat S Hanus L Ligumsky MKaminski NE Schatz AR Gopher A Almong S MartinBR Compton DR Biochem Pharmacol 1995 50 83

Finally the growing literature on the non CB1 non CB2cannabinoid receptors should allow the development of newdrugs First examples are given by the cannabidiol andabnormal-cannabidiol derivatives described by Kunos et alfor which potential therapeutic applications on the vascularsystem are already patented

[21] Sugiura T Kondo S Sukagawa A Nakane S Shinoda AItoh K Yamashita A Waku K Biochem Biophys Res Co1995 215 89

[22] Hanus L Abu-Lafi S Fride E Breuer A Vogel Z ShalevDE Kustanovich I Mechoulam R Proc Natl Acad SciUSA 2001 98 3662

[23] Oka S Tsuchie A Tokumura A Muramatsu M Suhara YTakayama H Waku K Sugiura T J Neurochem 2003 851374It clearly appears from this paper that the antagonists of

the cannabinoid receptors are still an active research fieldfrom which should emerge new promising therapeutic toolsas well as innovative drugs in the near future

[24] Goutopoulos A and Makriyannis A Pharmacol Therapeut2002 95 103

[25] Beal JE Olson R Lefkowitz L Laubenstein L Bellman PYangco B Morales JO Murphy R Powderly W PlasseTF Mosdell KW Shepard KV J Pain Symptom Manage1997 14 7ACKNOWLEDGEMENTS [26] Notcutt W Price M Miller R Newport S Phillips CSimmons S Sansom C Anaesthesia 2004 59 440

GGM is very indebted to the ldquoFonds pour la formationagrave la Recherche dans lrsquoIndustrie et lrsquoAgriculture (FRIA)rdquo Belgium for the award of a research fellowship Theexperimental works of the authors were supported by grantsfrom the FNRS The Belgian Charcot Foundation and theUniversiteacute catholique de Louvain

[27] Ledent C Valverde O Cossu G Petitet F Aubert JFBeslot F Bohme GA Imperato A Pedrazzini T RoquesBP Vassart G Fratta W Parmentier M Science 1999 283401

[28] Zimmer A Zimmer AM Hohmann AG Herkenham MBonner TI Proc Natl Acad Sci U S A 1999 96 5780

[29] Wiley JL and Martin BR Chem Phys Lipids 2002 121 57[30] Adam J and Cowley P Expert Opin Ther Patents 2002 12

1475

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[33] Little PJ Compton DR Johnson MR Melvin LS MartinBR J Pharmacol Exp Ther 1988 247 1046

[2] Devane WA Dysarz FA Johnson MR Melvin LSHowlett AC Mol Pharmacol 1988 34 605

[34] Pertwee RG Stevenson LA Elrick DB Mechoulam RCorbett AD Brit J Pharmacol 1992 105 980

[3] Matsuda LA Lolait SJ Brownstein MJ Young ACBonner TI Nature 1990 346 561

[35] Harrison C Traynor JR Life Sci 2003 74 489-508[4] Gerard CM Molerau C Vassart G Parmentier M BiochemJ 1991 279 129 [36] Pertwee RG Fernando SR Griffin G Ryan W Razdan

RJ Compton DR Martin BR Eur J Pharmacol 1996 315195

[5] Glass M Dragunow M Faull RLM Neuroscience 1997 2299

[37] Ross RA Brockie HC Fernando SR Saha B Razdan RKPertwee RG Brit J Pharmacol 1998 125 1345

[6] Shire D Carillon C Kaghad M Calandra B Rinaldi-Carmona M LeFur G Caput D Ferrara P J Biol Chem1995 270 3726 [38] Griffin G Wray EJ Rorrer WK Crocker PJ Ryan WJ

Saha B Razdan RK Martin BR Abood ME Brit JPharmacol 1999 126 1575

[7] Ryberg E Khang Vu H Groblewski T Larsson N ElebringT Hjorth S Sjogren S Greasley PJ 2004 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 119

[39] Ross RA Gibson TM Stevenson LA Saha B Crocker PRazdan RK Pertwee RG Brit J Pharmacol 1999 128 735

[40] Martin BR Winckler JR Wiley JL Huffman JW CrockerPJ Saha B Razdan RK J Pharmacol Exp Ther 1999 2901065

[8] Rinaldi-Carmona M Calandra B Shire D Bouaboula MOustric D Barth F Casellas P Ferrara P LeFur G JPharmacol Exp Ther 1998 278 871

[41] Martin BR Stevenson LA Pertwee RG Breivogel CSWilliams A Mahadevan A Razdan RK 2002 Symposium onthe Cannabinoids Burlington Vermont International CannabinoidResearch Society page 2

[9] Munro S Thomas KL Abu-Shaar M Nature 1993 365 61[10] Howlett AC Qualy JM Khachatrian LL Mol Pharmacol

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[42] Thomas A Ross RA Saha B Mahadevan A Razdan RKPertwee RG Eur J Pharmacol 2004 487 213

[12] Calandra B Portier M Kerneacuteis A Delpech M Carillon C LeFur G Ferrara P Shire D Eur J Pharmacol 1999 374445 [43] Compton DR Gold LH Ward SJ Balster RL Martin BR

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Daum SJ Ackerman JH Gruett MD Kumar V EstepKG Olefirowicz EM Wetzel JR Alexander MD WeaverJD Haycock DA Luttinger DA Casiano FM ChippariSM Kuster JE Stevenson JI Ward SJ J Med Chem1995 38 3094

[14] Deadwyler S Hampson RE Mu J Whyte A Childers SRJ Pharmacol Exp Ther 1995 273 734

[15] Mackie K Lai Y Westenbroek R Mitchell R J Neurosci1995 15 6552

[16] Bouaboula M Poinot CC Bourrie B Canat X Calandra BRinaldi-Carmona M LeFur G Casellas P Biochem J 1995312 637 [45] Pertwee R Griffin G Fernando S Li X Hill A

Makriyannis A Life Sci 1995 56 1949[17] Bouaboula M Poinot CC Marchand J Canat X Bourrie BRinaldi-Carmona M Calandra B LeFur G Casellas P Eur JBiochem 1996 237 704

[46] Hosohata Y Quock RM Hosohata K Makriyannis AConsroe P Roeske WR Yamamura HI EurJPharmacol 1997 321 R1[18] delPulgar T Velasco G Guzman M Biochem J 2000 347

369 [47] Landsman RS Makriyannis A Deng H Consroe P RoeskeWR Yamamura HI Life Sci 1998 62 109

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[48] Ross RA Brockie HC Stevenson LA Murphy VLTempleton F Makriyannis A Pertwee RG Brit JPharmacol 1999 126 665

[81] Gatley SJ Lan R Volkow ND Pappas N King P WongCT Gifford AN Pyatt B Dewey SL Makriyannis A JNeurosci 1998 70 417

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B Congy C Martinez S Maruani J Neliat G Caput DFerrara P Soubrieacute P Breliegravere JC Le Fur G FEBS Lett1994 350 240

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[213] Gallant M Dufresne C Gareau Y Guay D Leblanc YPrasit P Rochette C Sawyer N Slipetz D M Tremblay NBioorg Med Chem Lett 1996 6 2263[183] Lange JHM Kruse CG Tipker J Hoogendorn J World

Patent Application 2002 WO02076949 [214] Melck D De Petrocellis L Orlando P Bisogno T Laezza CBifulco M Di Marzo V Endocrinology 2000 141 118[184] Lange JHM Coolen H K A C van Stuivenberg HH

Dijksman JAR Herremans AHJ Ronken E Keizer HGTipker K McCreary AC Veerman W Wals HC Stork BVerveer PC den Hartog AP de Jong NMJ Adolfs TJPHoogendoorn J Kruse CJ J Med Chem 2004 47 628

[215] Kalgutkar AS Marnett AB Crews BC Remmel RPMarnett LJ J Med Chem 2000 43 2860

[216] Klegeris A Bissonnette CJ McGeer PL Brit J Pharmacol2003 139 775

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[217] Rinaldi-Carmona M Barth F Millan J Derocq JM CasellasP Congy C Oustric D Sarran M Bouaboula M CalandraB Portier M Shire D Breliere JC Le Fur GL JPharmacol Exp Ther 1998 284 644

[186] Wilstermann JM and Berggren AIK World PatentApplication 2003 WO03051850

[187] Berggren AIK Bostrom SJ Elebring ST Greasley PTerricabra E Wilstermann JM World Patent Application 2003WO03051851

[218] Barth F Millan J Casellas P Oustric D Sarran M RinaldiM World Patent Application 1997 WO9721682

[219] Griffin G Wray EJ Tao Q McAllister SD Rorrer WKAung MM Martin BR Abood ME Eur J Pharmacol 1999377 117

[188] Finke PE Meurer LC Debenham JS Toupence RBWalsh TE World Patent Application 2003 WO03082191

[189] Barth F Martinez S Rinaldi-Carmona M World PatentApplication 2003 WO03084930

[220] Portier M Rinaldi-Carmona M Pecceu F Combes T Poinot-Chazel C Calandra B Barth F Le Fur G Casellas P JPharmacol Exp Ther 1999 288 582[190] Barth F Martinez S Rinaldi-Carmona M World Patent

Application 2003 WO03084943 [221] Bouaboula M Desnoyer N Carayon P Combes T CasellasP Mol Pharmacol 1999 55 473[191] Kopka I E Li B Hagmann WK World Patent Application

2004 WO04029204 [222] Rhee M-H and Kim S-K J Vet Sci 2002 3 179[192] Hagmann WK Lin LS Shah SK Guthikonda RN Qi H

Chang LL Liu P Armstrong HM World Patent Application2003 WO03077847

[223] Bouaboula M Dussossoy D Casellas P J Biol Chem 1999274 20397

[224] Shire D Calandra B Bouaboula M Barth F Rinaldi-Carmona M Casellas P Ferrara P Life Sci 1999 65 627[193] Hagmann WK Lin LS Shah SK Goulet MT Jewell JP

World Patent Application 2003 WO03082190 [225] Gouldson P Calandra B Legoux P Kerneis A Rinaldi-Carmona M Barth F Le Fur G Ferrara P Shire D Eur JPharmacol 2000 401 17

[194] Castonguay LA Hagmann WK Lin LS Shah SK WorldPatent Application 2003 WO03086288

[195] Hagmann WK Lin LS Shah SK World Patent Application2003 WO03087037

[226] Feng W and Song ZH Biochem Pharmacol 2003 63 1077[227] Barth F Millan J Oustric D Rinaldi M Vernhet M World

Patent Application 2001 WO0132629[196] Berggren AIK Bostrom SJ Cheng L Elebring STGreasley P Nagard M Wilsterlmann JM Terricabras EWorld Patent Application 2004 WO04058249

[228] Mussinu JM Ruiu S Mule AC Pau A Carai MA LorigaG Murineddu G Pinna GA Bioorg Med Chem 2003 11251[197] Guba W Haap W Marty HP Narquizian R United States

Patent Application 2004 US 20040147572 [229] Xiang JN Elliott JD Atkinson ST Christensen SB WorldPatent Application 1998 WO9831227[198] Jagerovic N Hernandez Folgado L Alkorta I Goya P

Navarro M Serrano A de Fonseca FR Dannert MTDannert MT Alsasua A Suardiaz M Pascual D MartinMI J Med Chem 2004 47 2939

[230] Xiang JN Atkinson ST Wang DY Lee J Lee JCSiegfried B 216th ACS meeting Boston (MA) USA 23-27 Aug1998

[199] Jagerovic N Goya Laza MP Hernandez Folgado L AlcortaOsoro IJ Martin Fontelles MI Suardiaz Garcia MLDannert MT World Patent Application 2003 WO03082833

[231] Inaba T Kaya T Iwamura H United States Patent 2003 US6509352 B1

[232] Iwamura H Suzuki H Ueda Y Kaya T Inaba T JPharmacol Exp Ther 2001 296 420[200] Lange JHM Kruse CG Herremans AHJ VanStuivenberg

HH Dijksman JAR McCreary AC World PatentApplication 2003 WO03078413

[233] httpwwwjticojpJTI_EIR02PL20030212_Epdf (accessed02162004)

[201] Berggren AIK Bostrom SJ Elebring ST Fallefors LWilstermann JM Greasley P World Patent Application 2004WO04058255

[234] Zygmunt PM Andersson DA Houmlgestaumltt ED J Neurosci2002 22 4720

[235] Di Marzo V De Petrocellis L Fezza F Ligresti A BisognoT Prostaglandins Leukot Essent Fatty Acids 2002 66 377[202] Toupence RB Debenham JS Goulet MT Madsen-Duggan

CB Walsh TE Shah SK World Patent Application 2004WO04012671

[236] Wagner JA Varga K Jarai Z Kunos G Hypertension 199933 429

[203] Alanine A Bleicher K Guba W Haap W Kube DLuebbers T Plancher J-M Roche O Rogers-Evans MSchneider G Zuegge J World Patent Application 2004WO04013120

[237] Mukhopadhyay S Chapnick BM Howlett AC Am JPhysiol-Heart Circul Physiol 2002 282 H2046

[238] Jarai Z Wagner JA Varga K Lake KD Compton DR Martin BR Zimmer AM Bonner TI Buckley NE MezeyE Razdan RK Zimmer A Kunos G Proc Natl Acad Sci US A 1999 96 14136

[204] Griffith DA United States Patent Application 2004 US20040092520

[205] Griffith DA United States Patent Application 2004 US20040157839

[239] Kunos G Martin B Razdan R World Patent Application 2001WO0103690

[206] Griffith DA United States Patent Application 2004 US20040157838

[240] Offertaler L Mo F-M Batkai S Liu J Begg M RazdanRK Martin BR Bukoski RD Kunos G Mol Pharmacol2003 63 699[207] Bayewitch M Avidor-Reiss T Levy R Barg J Mechoulam

R Vogel Zvi FEBS letters 1995 375 143 [241] Begg M Mo F-M Offertaler L Batkai S Pacher PRazdan R K Lovinger DM Kunos G J Biol Chem 2003278 46188

[208] Bayewitch M Rhee M-H Avidor-Reiss T Breuer AMechoulam R Vogel Z J Biol Chem 1996 271 9903

[209] Govaerts SJ Hermans E Lambert DM Eur J Pharm Sci2004 23 233

[242] Ho WSV Hiley CR Brit J Pharmacol 2003 138 1320[243] Ho WSV Hiley CR J Pharm Pharmacol 2004 56 869

[210] DAmbra TE Estep KG Bell MR Eissenstat MA JosefKA Ward SJ Haycock DA Baizman ER Casiano FMBeglin NC J Med Chem 1992 35 124

[244] Mo FM Offertaler L Kunos G Eur J Pharmacol 2004489 21

[245] Batkai S Pacher P Jarai J Wagner JA Kunos G Am JPhysiol-Heart Circul Physiol 2004 287 H595[211] Showalter VM Compton DR Martin BR Abood ME J

Pharmacol Exp Ther 1996 278 989

1394 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

[246] Kunos G World Patent Application 2003 WO03015700 [257] Haller J Varga B Ledent C Freund TF Behav Pharmacol2004 15 299[247] Franklin A and Stella N Eur J Pharmacol 2003 474 195

[248] Walter L Franklin A Witting A Wade C Xie Y KunosG Mackie K Stella N J Neurosci 2003 23 1398

[258] Pistis M Perra S Pillolla G Melis M Gessa GL MuntoniAL Neuropharmacology 2004 46 115

[249] Di Marzo V Breivogel C S Tao Q Bridgen DT RazdanRK Zimmer AM Zimmer A Martin BR J Neurochem2000 75 2434

[259] Ralevic V and Kendall DA Eur J Pharmacol 2001 418 117[260] Duncan M Kendall DA Ralevic V Brit J Pharmacol 2003

138 214P[250] Breivogel CS Griffin G Di Marzo V Martin BR Mol

Pharmacol 2001 60 155[261] Duncan M Millns P Smart D Wright JE Kendall DA

Ralevic V Brit J Pharmacol 2004 142 509[251] Monory K Tzavara ET Lexime J Ledent C Parmentier

M Borsodi A Hanoune J Biochem Bioph Res Commun2002 292 231

[262] Duncan M Kendall DA Ralevic V J Pharmacol Exp Ther2004 311 411

[263] Tao Q and Abood ME J Pharmacol Exp Ther 1998 285651[252] Muthane U Ramsay KA Jiang H Jackson-Lewis V

Donaldson D Fernando S Ferreira M Przedborki S ExpNeurol 1994 126 195

[264] Lambert DM DiPaolo FG Sonveaux P Kanyonyo MGovaerts SJ Hermans E Bueb J-L Delzenne NMTschirhart EJ Biochim Biophys Acta 1999 1440 266[253] Hajos N Freund TF Chem Phys Lipids 2002 121 73

[254] Hajos N Ledent C Freund TF Neuroscience 2001 106 1 [265] Calignano A La Rana G Giuffrida A Piomelli D Nature1998 394 277[255] Hajos N Freund TF Neuropharmacology 2002 43 503

[256] Haller J Bakos N Szirmay M Ledent C Freund TF Eur JNeurosci 2002 16 1395

[266] Calignano A La Rana G Piomelli D Eur J Pharmacol 2001419 191

Page 11: Current Knowledge on the Antagonists and Inverse Agonists of

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1371

lengths in position 3 of the pyrazole moiety They observedthat until five carbons the affinity increases along with thecarbon chain length This is observed with the alkylhydrazide the alkyl amide and the hydroxyalkyl amideseries Moreover the hydrazide analogues exhibit a loweraffinity for the rCB1 than the amide analogues as illustratedby compounds MF9725-64-17 (49) and MF9725-179-32(50) having Ki values of 134 and 51 nM respectively(Table 6) From their structure-activity relationships (SAR)studies the authors concluded that the pharmacophoricrequirement of the amidopiridine region is a chain not longerthan 3 Aring and that a substituent having a positive chargedensity would probably result in increased affinity andpotency The same team described in a patent several othercompounds [149] Binding affinities (Ki) against [3H]-CP-55940 [3H]-SR141716A or [3H]-WIN55212-2 on wholerat brain or on hCB1-transfected cells were given Moreoveractivity data were obtained using the [35S]-GTPγ S assaydemonstrating that these alkyl amide and hydrazide analogsact as antagonists or inverse agonists Despite the greatnumber of derivatives claimed none of them posses asignificantly greater affinity for the CB1 receptor than theSR141716A Nevertheless they showed a slightenhancement of the selectivity for the CB1 receptor over theCB2 cannabinoid receptor

affinity of these derivatives increases with the length of thealkyl chain with an optimal length of 5-6 carbons The bestcompound of this series N-(piperidin-1-yl)-5-phenyl-1-hexyl-4-methyl-1H-pyrazole-3-carboxamide (55) possesses aKi value of 21 nM determined using [3H]-CP-55940 on ratbrain membranes which is higher than the 13 nMdetermined for SR141716A in the same conditions (Table6) In the same paper the authors starting from thehypothesis that antagonism by SR141716A is caused bybinding to the same region of the receptor as do the agonists(CP-55940 and WIN-55-2122) but preventing the agonistpromoted conformational change conducted extensiveconformational analysis as well as superimposition modelsand 3D-QSAR to propose a molecular mechanismsupporting the action of SR141716A Along with theauthors the C-5 aryl substituent of SR141716A occupyinga unique region could contribute in conferring the antagonistproperties Moreover the C-3 substituent could beresponsible for the antagonist or inverse agonist propertiesdepending on the interaction with the receptor

About this topic a very interesting review dealing withthe cannabinoid receptors pharmacophores as well as withthe activationinactivation of these receptors was recentlypublished by Reggio [151]

More recently Dyck et al described seven otherderivatives varying at the amide position [152] The onlycompound possessing a higher affinity for the CB1cannabinoid receptor than SR141716A was the 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta-

The affinity for the rat CB1 receptor of seven derivativespossessing an alkyl chain in position 1 instead of the 24-dichlorophenyl substituent was reported by Shim et al in apaper describing a molecular mechanism for the antagonistand inverse agonist activity of SR141716A [150] The

NN

O

NHN

Cl Cl

Cl

NN

Cl

Cl

NON

NN

O

NH

N

Cl

ClCl

NN

O

NH

MeO Cl

CP-272871 66

63 64

65

Fig (4) CB1 cannabinoid receptor antagonists 3 Diarylpyrazole derivatives Structures of 5-(4-chlorophenyl)-1-(24-dichlorophenyl)-N-(hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63 ) 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methyl-pyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]-morpholine (64 ) N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65 ) and N-phenyl-1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-pyrazole-3-carboxamide (CP-272-871 66 )

1372 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

[c]pyrrol2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63)(Fig 4) The Ki values were 5 and 12 nM respectively([3H]-CP-55940 hCB1-HEK cells)

CP-55940 rat brain) For instance N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65) (Fig 4) showed a Ki value of15 nM However no data were provided concerning thefunctionality of such compounds

Over 200 other pyrazole derivatives acting at the CB1cannabinoid receptor were claimed in a patent from Pfizer[153] The affinity for the hCB1 receptor of 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methylpyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]ndashmorpholine (64) (Fig 4)was 79 nM ([3H]-SR141716A hCB1-HEK cells) Theothers compounds affinity was between 01 and 100 nM

In an effort to develop new SR141716A analoguespossessing lower lipophilicity to be used as PET tracersKatoch-Rouse and colleagues further explored thesubstitution pattern of the two phenyls [155] Bindingassays showed that a decreased lipophilicity lead to adecreased affinity for the CB1 receptor as determined bydisplacement of [3H]-AM251 For instance compoundNIDA-41057 (59) has a lipophilicity of 42 ( expressed asElogDoct) and a Ki value of 104 nM whereas SR141716Ahas a ElogDoct value of 54 and a Ki value of 18 nM In

Seventy five pyrazole derivatives were described in a veryrecent patent from Makriyannis et al [154] The affinity ofthe compounds for the cannabinoid receptors is given TheKi values for the best compounds are lower than 6 nM ([3H]-

Table 7 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structures and affinities for the cannabinoid receptors of some representative tricyclic pyrazole derivatives Values were obtained using [3H]-CP-55940as radioligand and mouse [160 228] rat [159 227] or human [158] cannabinoid receptors Compounds 69-78 are CB1 cannabinoid receptor ligandsCompounds 79-86 are CB2 cannabinoid receptor ligands

NN

O

NHX

R3

R1

R2

n

n˚ X R1 R2 n R3 Ki CB1 (nM) Ki CB2 (nM) Selectivity References

69 -(CH2)3- Cl 24-di-Cl 0 piperidine 126 [158]

70 -(CH2)3- H 24-di-Cl 0 azepane 100 [158]

71 -(CH2)3- H 24-di-Cl 0 piperidine 398 [158]

72 -(CH2)3- Cl 24-di-Cl 0 azepane 125 [158]

73 -(CH2)3- NO2 24-di-Cl 0 azepane 63 [158]

74 -(CH2)2-O- H 24-di-Cl 0 piperidine 501 [158]

75 -CH2-S- Br 24-di-Cl 0 piperidine lt 500 gt10 [159]

76 -CH2-SO2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

77 -(CH2)2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

78 -(CH2)3- Cl 24-di-Cl 0 piperidine 000035 21 60000 [160]

79 -CH2- Cl 24-di-Cl 0 piperidine 2050 034 00002 [228]

80 -CH2- Br 24-di-Cl 0 piperidine 1570 027 00002 [228]

81 -CH2- CH3 24-di-Cl 0 piperidine 363 0037 00001 [228]

82 -CH2- Cl 4-Cl 0 piperidine 1787 09 00005 [228]

83 -CH2- H 24-di-Cl 0 piperidine 1152 0385 00003 [228]

84 -CH2- Cl 34-di-Cl 1 133-trimethylbicyclo[221]heptyl

lt500 gt10 [227]

85 -CH2- Br 24-di-Cl 1 bicyclo[321]oct-3-yl lt500 gt10 [227]

86 -CH2- Br 4-Me 1 77-dimethylbicyclo[410]hept-3-yl

lt500 gt10 [227]

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1373

another paper Katoch-Rouse described the synthesis of[18F]-NIDA-42033 (26) a ligand useful as PET radiotracerstarting from its bromo derivative [156] Recently Kumar etal [157] proposed the synthesis of a 11C derivative ofNIDA-41087 (61) a compound previously described byKatoch-Rouse (Table 5) Post-mortem binding of thiscompound to human prefrontal cortex was also investigatedInterestingly CP-272871 (66) described by Meschler as aCB1 inverse agonist (Ki=57 nM [3H]-CP-55940 rat brainhomogenates) possesses a lipophilicity similar to theNIDA-41087 one [62] However despite the presence of amethoxy function no radiolabelled derivative has beendescribed to date One explanation could be the compoundlow selectivity (2 fold) for the CB1 cannabinoid receptor

To further explore the pharmacology of this compound afunctional assay using the mouse vas deferens model wasperformed by Ruiu and colleagues enlightening thecompetitive antagonist properties of NESS (pA2= 1246against WIN-55212-2) which had no effect by itself up to1microM Moreover NESS 0327 was unable to affect basalbinding of the [35S]-GTPγ S demonstrating its lack ofnegative intrinsic activity In-vivo studies were conductedusing the hot plate and tail flick tests NESS dose-dependently abolished the antinociceptive effect of WIN-55212-2 but had no effect by itself However the authorssuggested based on the affinity and in-vivo activity of theircompound that NESS 0327 possesses a poor centralbioavailability

Several new attempts to increase the affinity of thediarylpyrazole derivatives were recently made by rigidifyingthe SR141716A structure (Table 7) Six fused ringanalogues of SR141716A obtained by fusion of the 5-(4-chlorophenyl) substituent with the central pyrazole to forman indazole ring were published by Bass et al in 2002 [61]The compound possessing the highest affinity O-1248 (67)has a Ki value of 475 nM as determined by displacement of[3H]-CP-55940 from rat brain membranes

Another example of ring-constrained biarylpyrazole wasdescribed by Herbert Seltzman team [161] Theyinvestigated a photocyclisation reaction starting from theSR141716A leading to a pyrazolo[15-f]phenanthridinestructure (Fig 5) The new compound (68) was tested on theCB1 receptor (whole rat brain) against [3H]-CP-55940[3H]-SR141716A and [3H]-WIN-55212-2 and exhibited Kivalues of 48 35 and 50 nM respectively The CB2 affinitywas assessed using [3H]-CP-55940 and was found to be 100times lower (Ki = 3340 nM) No effect on the [35S]-GTPγ Sassay (rat brain) was observed by the authors

Stoit and colleagues from Solvay Pharmaceuticalspublished a paper describing the synthesis andpharmacological characterisation of new benzocyclohepta-pyrazole derivatives as CB1 antagonists (69-74) [158] Theaffinity of these compounds was more or less one order ofmagnitude lower than the SR141716A one with pKi valuesranging from 64 to 72 compared to 76 for the SRcompound (Table 7) Compounds were shown to beantagonists with pA2 values from 70 to 89 as theyprevent CP-55940-induced cAMP accumulation Howeverthe authors investigating the bioavailability of theircompounds either po or after ip injection found negligibleblood plasma levels

NN

Cl

Cl

O

NH

N

NN

Cl

O

NHN

Cl

Cl

O-1248 67 68However just before the publication of Stoit results

Sanofi-Synthelabo took a patent describing tricyclicpyrazolecarboxylic acid amide derivatives having antagonistproperties on the CB1 cannabinoid receptor andsubmicromolar affinities (75-77) [159] Thirty examples weregiven along with their synthetic pathway but no individualpharmacological data were shown

Fig (5) CB1 cannabinoid receptor antagonists 3Diarylpyrazole derivatives Two ring-constrained diarypyrazolederivatives O-1248 (67 ) and a pyrazolo[15-f]phenanthridinederivative (68 )

It appears from these attempts to increase SR141716Aaffinity by rigidifying its structure that only a limitedincrease in the CB1 cannabinoid receptor affinity can beexpected with the apparent exception of NESS 0327Moreover rigidifying the structure often results in a morelipophilic compound in a family of compounds being yetlipophilic

More recently Ruiu and colleagues published a paperdescribing the synthesis and complete pharmacologicalcharacterisation of a very potent CB1 receptor ligandchristened NESS 0327 (78) [160] This compound has thesame structure as compound 69 published by Stoit et al(compound 20 in the reference [158]) Nevertheless Ruiu hasobtained an affinity for the CB1 cannabinoid receptor 5000times bigger than the SR141716A one with Ki values of035 pM and 18 nM respectively using mouse forebrainhomogenates and [3H]-CP-55940 as radioligandFurthermore the selectivity ratio obtained for this compoundby Ruiu is 60000 with a Ki value for the CB2 receptor of21 nM against the [3H]-CP-55940 The discrepancybetween the data obtained independently by Stoit et al andRuiu et al could hardly be explained by the differences inthe origin of the receptor used respectively hCB1-CHO andmouse forebrain

4 Phenyl Benzofuranone Derivatives

In a 1997 patent from Eli Lilly was described thesynthesis of aryl-benzothiophene and aryl-benzofuranederivatives (87-89 Fig 6) having CB1 receptor affinity andantagonist properties [162] From ten compounds having aKi value lower than 25 microM ([3H]-CP-55940 hCB1-CHOcells) one was selected and claimed to have a Ki of 170 nMCompound 88 was shown in the patent to antagonise theeffect of anandamide on cAMP accumulation with an IC50 of

1374 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

O

O

O

O

NC

S

O

O

O

O

O

O

O

NC

LY320135 8887 89

Fig (6) CB1 cannabinoid receptor antagonists 4 Phenyl benzofuranone derivatives Chemical structures of two aryl-benzofuranederivatives (87 -88 ) and one aryl-benzothiophene (89 ) derivative from Eli Lilly

500 nM and the effect of WIN-55212-2 on calcium channelsat 1microM Moreover the intra-peritoneal injection (20 mgkg)of this compound antagonised the in-vivo effect ofanandamide in the Open Field Assay a mouse behaviourmodel Later on Felder and colleagues gave a furtherdescription of this compound called LY320135 in a paperdescribing its effect on cAMP accumulation and on ioniccurrents [163] The selectivity ratio of LY320135 was 106with Ki values of 141 and 14900 nM for hCB1 and hCB2receptors ([3H]-CP-55940) respectively LY320135antagonised anandamide inhibition of forskolin-inducedcAMP accumulation with an IC50 value of 734 nMChristopoulos et al in 2001 reported for LY320135 apKb value of 527 in the inhibition of WIN-55212-2mediated response in the rat electrically-stimulated vasdeferens while the pKb value for SR141716A was 75[164]

Other patents were taken by Aventis in which somepotential applications for their compounds were describedFor instance an association between an azetidine derivativehaving CB1 antagonists properties and sibutramine aserotonin and norepinephrine reuptake inhibitor was claimed[170] as a treatment against obesity Oral administration ofcompound 90 (3 mgkg) and sibutramine (06 mgkg) to fafaZucker rats which are genetically obese rats has resulted ina decreased food intake compared to control lean Zuckerrats In another patent the association of an azetidinederivative (1-10 mgkg po ) and a D2D3 agonist likequinpirole (01 mgkg ip) was claimed to have a therapeuticpotential in the treatment of Parkinsonrsquos disease [171] Theeffect has been evidenced using an akinesia model in the ratEvidences supporting the use of CB1 antagonists as adjuvantin the treatment of Parkinsonrsquos disease begin to appear Itseems that such compounds would be helpful in thetreatment of Parkinsonism [172] and levodopa-induceddyskinesia [173] Brotchie published an interesting paperreviewing the CB1 cannabinoid receptor signaling inParkinsonrsquos disease [174] Nevertheless further clinicalstudies should be conducted to further confirm the usefulnessof CB1 antagonists in the treatment of Parkinsonrsquos disease

5 Azetidine Derivatives

The family of azetidine compounds illustrated bycompounds 90 and 91 (Fig 7) was developed at Aventis byDaniel Achard and colleagues and was described in a seriesof patents [165-168] They claimed the IC50 values of thesecompounds for the CB1 receptor to be less than or equal to100 nM These values were obtained following the proceduredescribed by Kuster et al [169] The antagonist propertywas shown using an in-vivo model the reversal ofhypothermia induced by CP-55940 in mice and the ED50obtained were lower than 50 mgkg

6 Aryl-Imidazolidine-24-Diones Derivatives

In 1999 the Didier Lambert team published a paperdescribing the affinity of 24 new 3-alkyl-55rsquo-diphenyl-imidazolidine-24-dione derivatives for the hCB1 receptor[175] The preliminary structure-activity relationships

N

SO

O

Cl

Cl

F

F

N

SO

O

Cl

NS

90 91

Fig (7) CB1 cannabinoid receptor antagonists 5 Azetidine derivatives Two examples (90 -91 ) of the azetidine derivatives developedby Aventis

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1375

Table 8 CB1 Cannabinoid Receptor Antagonists 6 Aryl-Imidazolidine-24-Diones Derivatives

Structures affinities ([3H]-SR141716A hCB1-CHO cells) and pKb values ([35 S]-GTPγS HU210 rat brain) of three aryl-imidazolidine-24-dionederivatives (92-94)

N

NH

OO

R

Br

Br

Cpd ndeg R Ki (nM) a pKb b

DML20 92 ethylmorpholine 70 611

DML21 93 1-hydroxypropyl 103 625

DML23 94 heptyl 98 574

a [175]b [177]

showed that the substitution at the nitrogen in position 3 aswell as a bromine atom in para of the phenyl rings aremandatory for the compounds affinity Later on threecompounds termed DML20 (92) DML21 (93) and DML23(94) were characterised as neutral antagonists using the [35S]-GTPγ S assay on rat cerebellum membranes [176] (Table 8)In the same model they competitively inhibited HU-210-induced [35S]-GTPγ S binding with pKB values of 611625 and 574 respectively Moreover these compoundswere proven to be quite selective for the CB1 receptor

higher than the one for the CB2 cannabinoid receptor (IC50 =300 nM) The inverse agonism of the compound wasextrapolated by inhibiting CP-55940-induced hypothermiain mice (EC50 = 18 nM) Moreover one oral dose (10mgkg) reduces the food intake in diet-induced obese ratsInterestingly food intake of CB1

-- mice was not alteredBody weight loss was maintained all over the study during achronic administration (14 days) of 10 mgkg of Cpd A

Later on Merck published a patent [180] in which someof the 45-diaryl-pyrazoles derivatives previously describedwere claimed to be useful in the treatment or the preventionof obesity in association with compounds inhibiting the11β-hydroxysteroid dehydrogenase type 1 (1β-HSD1)enzyme The 1β-HSD1 enzyme is responsible for thesynthesis of cortisol increased levels of which according tothe inventors are associated with obesity However neitherresults on appetite suppression nor on weight loss weregiven even if some indications on the affinity of thecompounds for the cannabinoid receptors were given

Very recently the functionality of these DML compoundswas explored using the [35S]-GTPγ S assay both on rat andhuman CB1 cannabinoid receptors [177] The data obtainedconfirm that the 3-alkyl-55rsquo-diphenyl-imidazolidinedionederivatives behave as neutral antagonists on rat CB1cannabinoid receptor (rat brain) However DML 20 DML21and DML23 acted as inverse agonists on the human receptor(hCB1-CHO cells) Furthermore the authors showed thatthis different functionality is not due to the hCB1 receptorlevel of expression in the recombinant cell line as whateverthe level of expression (Bmax of 44 pmolmg or 32pmolmg) DML derivatives behaved as inverse agonists ofthe hCB1 cannabinoid receptor

Another imidazole ring pattern of substitution wasproposed by Hagmann and collaborators [181] Thecompounds described 43 examples are given possess twophenyls the first one in position 1 linked to the nitrogenand the second one in position 2 The carboxamidesubstituent is in position 4 instead of in position 2 for thediarylimidazoles described by Finke These 12-diaryl-imidazoles are claimed to be CB1 cannabinoid receptorantagonists or inverse agonists but the absence of precisepharmacological values do not allow any comparisonbetween the two substitutions patterns However a samepattern of substitution was used by other researchers Amongthem Dyck et al described in 2004 ten 1-(4-chlorophenyl)-2-(24-dichlorophenyl)-imidazole derivatives varying by theircarboxamide substituent in position 4 (101-103 Table 9)[152] For instance with a 3-azabicyclo[330]octan-3-ylsubstituent (101) they obtained a Ki value of 14 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) In a [35S]-GTPγ Sassay 101 exhibited an IC50 value of 19 nM

7 Diarylimidazoles Derivatives

Finke and colleagues at Merck developed a new class ofCB1 receptor antagonists based on an imidazole nucleusOver eighty 45-diaryl-imidazoles derivatives having CB1antagonist properties were claimed in a patent published in2003 [178] Some representative compounds of the patent areillustrated in Table 9 (95-100) The affinity of the newcompounds was determined using [3H]-CP-55940 andhCB1-CHO cells Although no detailed pharmacologicaldata were given in the patent in-vitro and in-vivo propertiesof one compound termed Cpd A (100) have been describedelsewhere [179] Its affinity for the CB1 cannabinoid receptorwas shown to be nanomolar (IC50 = 4 nM) and 75 times

1376 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 9 CB1 Cannabinoid Receptor Antagonists 7 Diaryl-Imidazole Derivatives

Structure and affinity range of some 45-diaryl-imidazole derivatives for the human cannabinoid receptors determined using [3H]-CP-55940 asradioligand and hCB1amp2-CHO cells

N

NR2

R1

R4

R3

ndeg R1 R2 R3 R4 hCB1 (IC50 or Ki) hCB2 (IC50 or Ki) References

95 CH3 CO-NH-piperidinyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

96 CH3 CO-NH-piperidinyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

97 CH3 CO-NH-cyclohexyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

98 CH3 CO-NH-phenyl 24-di-Cl-Ph 4-Cl-Ph 10 ndash 100 nM a 100 ndash1000 nM a [178]

99 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

100 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph 4 nM a 300 nM a [179]

101 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

CH3 14 nM b ND [152]

102 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

H 66 nM b ND [152]

103 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 85 nM b ND [152]

104 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 23 nM b 542 nM b [182]

105 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 30 nM b 608 nM b [182]

106 4-Br-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 60 nM b 489 nM b [182]

107 4-CF3-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 29 nM b 634 nM b [182]

108 4-Cl-Ph 24-di-Cl-Ph CO-NH-morpholinyl CH3 197 nM b 3297 nM b [182]

109 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidin-4-ol CH3 172 nM b 3959 nM b [182]

110 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CN 30 nM b 1590 nM b [182]

111 4-Cl-Ph 24-di-Cl-Ph CO-NH- piperidinyl CH2F 36 nM b 906 nM b [182]

112 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH2-CH3 14 nM b 430 nM b [182]

113 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Cl 27 nM b 823 nM b [182]

114 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Br 23 nM b 746 nM b [182]

115 4-Cl-Ph 24-di-Cl-Ph CO-NH-tetrahydroisoquinoline CH3 34 nM b 696 nM b [182]

116 4-Cl-Ph 24-di-Cl-Ph CO-NH-cycloheptyl CH3 35 nM b 349 nM b [182]

117 24-di-Cl-Ph 4-Cl-Ph CO-NH-piperidinyl CH3 403 nM b 208 nM b [182]a IC50 value b Ki value

Lange and co-workers from Solvay described twenty-eight imidazole derivatives [182] The 24-dichlorophenylsubstituent at position 2 was kept constant while thesubstitutions at the other positions were explored (Table 9)Position 5 can accommodate a large range of littlesubstituents like hydrogen (104) methyl (105) ethyl (112)chlorine (113) bromine (114) fluoromethyl (111) or cyano(110) without major changes in the affinity At position 4more bulky substituents like 1234-tetraisoquinoline (115)or cycloheptyl (116) were well tolerated whereas thepresence of a more hydrophilic moiety such as morpholine(108) or piperidin-4-ol (109) was detrimental to the affinity

Interestingly position exchange between the 24-diphenyland the 4-chlorophenyl substituents led to a 13 fold loweraffinity with Ki values of 30 and 403 nM for 105 and 117respectively ([3H]-CP-55940 hCB1-CHO cells) In thefunctional assay which was inhibition of WIN-55212-2-induced [3H]-arachidonic acid release by hCB1-CHO cellsall tested compounds behaved as antagonists Compound105 showed a pA2 value of 86 which is the same theauthors obtained for SR141716A It would be interesting toassess if these compounds are true antagonists or if they actmore as inverse agonists Compound 105 when administeredto rats was able to inhibit CP-55940-induced hypotension

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1377

with an ED50 value of 24 mgkg which is close to the 3mgkg obtained for SR141716A The compound was alsoactive in another in-vivo model the WIN-55212-2-inducedhypothermia in mice Compound 104 which has nosubstituent in position 5 of the imidazole ring was lessactive in the hypotension model and inactive in thehypothermia model Thus a methyl group is the preferredsubstituent in position 5 of the imidazole moiety Otherproperties either experimental (P-glycoprotein affinitylogPHPLC) or computational (molecular volume polarsurface area) obtained for compounds 105 were similar tothose obtained for SR141716A Finally molecularmodelling studies revealed a close structural overlap betweenthe two compounds

value of 197 nM is selective for hCB1 and is as potent asSR141716A The structural modulations of 118 led to someinteresting compounds (Table 10) Six compounds possess aKi value for the hCB1 cannabinoid receptor equal to or lowerthan 25 nM antagonist properties in the inhibition of [3H]-arachidonic acid release by WIN-55212-2-stimulated hCB1-CHO cells and Ki values for the hCB2 receptor over 1microMThe diaryl-pyrazoline derivatives contained a chiral center atposition 4 The authors resolved the racemic mixture of twocompounds (121 and 130) The levorotatory enantiomersappeared to be the eutomers For compound SLV319levorotatory enantiomer of 121 the Ki and pA2 values were78 nM and 99 respectively The Ki and pA2 values ofSLV326 levorotatory enantiomer of 130 were 359 nM and9 respectively

8 34-Diaryl-Pyrazoline Derivatives Two in-vivo models CP-55940-induced hypotension inrat and WIN-55212-2-induced hypothermia in mouse wereused to assess the potential of these compounds The resultsare of the same order of magnitude than those obtained forSR141716A For instance in the hypotension modelSLV319 ((-)121) and SLV326 ((-)130) showed ED50 values

Lange and collaborators also discovered 34-diaryl-pyrazoline derivatives after a screening of compoundsresembling to SR141716A [183184] Compound 118 thatwas initially identified during the screening possesses a Ki

Table 10 CB1 Cannabinoid Receptor Antagonists 8 34-Diaryl-Pyrazolines Derivatives

Structure affinity for the hCB1 and hCB2 cannabinoid receptors (Ki nM) expressed in CHO transfected cells ([3H]-CP-55940) and potency (pA2) ofsome 34-dirylpyrazolines derivatives Table adapted from [184]

NN

N N

SO2

R3

R2

Cl R1

R4

n˚ R1 R2 R3 R4 Ki hCB1 (nM) Ki hCB2 (nM) pA2 (CB1)

118 H H H 4-CH3 197 gt 1000 84

119 H H H 246- CH3 24 gt 1000 94

120 H H H 4-Cl 16 gt 1000 95

121 H CH3 H 4-Cl 25 gt 1000 87

122 H CH3 CH3 4-Cl 280 gt 1000 85

123 H H H 4-F 53 gt 1000 9

124 H CH3 H 4-F 338 gt 1000 85

125 H CH3 CH3 4-F gt 1000 gt 1000 lt 75

126 H CH3 H 3-Cl 14 gt 1000 86

127 4-Cl CH3 H 4-Cl 255 ND ND

128 4-F CH3 H 4-Cl 584 ND ND

129 H CH3 H H 170 ND 75

130 H CH3 H 4- CF3 221 gt 1000 93

SR141716A 25 1580 86

1378 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

N

HN

O

N

Cl Cl

Cl

N

N

HN

O

Br

Br131 132

Fig (8) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Chemical structures of two 56-diaryl-pyrazine-2-amide derivatives (131-132) developed as CB1 cannabinoid receptorantagonists by AstraZeneca

of 55 and 2 mgkg (po) respectively proving their in-vivoefficacy after oral administration The authors alsodemonstrated that SLV319 is devoid of affinity for the P-glycoprotein pump and that it possessed a goodCNSplasma ratio (17) These two compounds (SLV319and SLV326) were chosen as development candidates bySolvay and entered clinical Phase I trials in 2003 [185]

132 Fig 8) useful as CB1 cannabinoid receptor antagonists[186 187] The activity of twenty derivatives wasdetermined using a [35S]-GTPγ S assay and hCB1-CHOtransfected cells The concentration required to give halfmaximal inhibition of CP-55940-induced [35S]-GTPγ Sbinding (IC50) is lower than 200 nM for the preferredcompounds No affinity data were given for thesecompounds

9 Diaryl-Pyrazine Diphenyl-Pyridine Diphenyl-Phenyland Diaryl-Pyrimidines Derivatives

The same year in a patent from Merck Finke andcollaborators described several 56-di-phenyl-pyridinederivatives as hCB1 antagonists or inverse agonists [188]The compounds have a substituent in position 3 of thepyridine core (Fig 9) This substituent could be a cyano(133-134) or a nitro group a halogen an ester or an amide(135-136) Over 150 compounds were synthesised toillustrate the invention but no pharmacological data wasdisclosed in the patent

In 2003 appeared several patents describing new familiesof compounds that bind to the CB1 cannabinoid receptorThese compounds have in common a central six atomsaromatic ring which could be a pyrazine a pyridine aphenyl or a pyrimidine and that is substituted by at leasttwo phenyl rings

The first patents were from Berggren and collaboratorsfrom AstraZeneca who published two patents describing thesynthesis of 56-diaryl-pyrazine-2-amide derivatives (131-

A few months later another patent was taken by Sanoficlaiming the antagonist properties of 56-di-phenyl-2-pyridine carboxamide derivatives [189] These compounds

N

NH

O

Cl

Cl

N

O

Cl

O NH

N

O

Cl

ClCl

NH

CN O

N

Cl

Cl

Cl

OHN

N

O

Cl

ClCl

CNF

F

133 134

135 136 137

Fig (9) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives 56-Di-phenyl-pyridine derivatives patented by Merck (133-136) and by Sanofi (137) as CB1 cannabinoid receptorsantagonists

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1379

NH

O

N

Cl

ClCl

NH

O

N

Cl

F3CCl

N

N

O

Cl

Cl Cl F138 139 140

Fig (10) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Representative structures of the diphenyl-phenyl (138-139) and 45-diphenyl-pyrimidine (140) derivatives developed ascannabinoid antagonists at Sanofi and Merck respectively

28 examples are given structurally related to the diphenyl-phenyl derivatives have their pyridine core substituted bytwo phenyl rings and by an amide moiety (137 Fig 9) TheIC50 values were lower than 100 nM but no data were givenshowing the antagonist properties although the compoundswere assayed in the adenylate cyclase inhibition assay

10 Other Derivatives

Several amide derivatives structures recently appearedamong the CB1 cannabinoid receptor antagonists In a seriesof patents taken by Merck in 2003 Hagmann and colleaguesdescribed a large amount of compounds obtained by parallelsynthesis which are claimed to be CB1 cannabinoid receptorantagonists [192-195] They were synthesised by reacting alibrary of substituted amines with a library of carboxylicacids (141-144 Fig 11) However as no value was givenfor the affinity or activity of these amides we will notfurther discuss this class of compounds

Along this line Sanofi Synthelabo developed newdiphenyl-phenyl derivatives claimed to have CB1cannabinoid receptor antagonist properties [190] They arebased on a central phenyl ring substituted by two phenylsand by an amide moiety (138-139 Fig 10) Thirteencompounds were claimed along with their synthesis TheirIC50 values were lower than 100 nM ([3H]-CP55-940hCB1-CHO cells) but no other specific data (selectivitypA2hellip) was given

Several 15-diaryl-pyrrole-3-carboxamide derivatives weresynthesised and claimed to be CB1 cannabinoid receptorantagonists by Berggren et al [196] The affinity valueswere not disclosed in the patent (145-146 Fig 12) Otherpyrrole derivatives were synthesised by Guba et al atHofmann-La Roche [197] More specifically 2-(thiazol-4-yl)pyrrole derivatives were claimed as CB1 cannabinoidreceptor antagonist (IC50lt2microM) (147-148 Fig 12)

Finally Kopka et al from Merck described more thanone hundred 45-diaryl-pyrimidine derivatives as CB1cannabinoid receptor antagonists (140 Fig10) [191]However no precise data were provided concerning theaffinity and functionality of the compounds

R2

R1N

R3

R4

R5

O

NH

O

Cl

O F

F

R2

R1NH

R3

R4

HO R5

O

NH

O

Cl

CN

NH

O

Cl

O

O

NH

O

Cl

Cl

+

141142

143 144

Fig (11) CB1 cannabinoid receptor antagonists 10 Other derivatives Retrosynthetic scheme of a CB1 cannabinoid antagonistlibrary of amide derivatives developed at Merck The structures of four representative compounds are illustrated (141-144)

1380 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

NH

O N

Cl

Cl

Cl

N

NH

O

OMe

MeO

N

NH

O

N

S

MeO

N

NH

O

N

S

MeO

145 146

147 148

Fig (12) CB1 cannabinoid receptor antagonists 10 Other derivatives Two examples of 15-diaryl-pyrrole derivatives described byBerggren et al (145-146) While the 2-(thiazol-4yl)pyrroles derivatives 147 and 148 were described by Guba et al from Hofmann-LaRoche All these pyrroles derivatives were claimed to be CB1 cannabinoid receptor antagonists

Another new structure is represented by the 124-triazolederivatives developed by Jagerovic and collaborators In arecent paper were described the synthesis andpharmacological properties of five new 15-diphenyl-3-alkyl-

triazole derivatives [198] Among these compounds onlyone (149) behaved as a CB1 antagonist inhibiting the WIN-55212-2-induced contractions in a mouse vas deferenspreparation However despite its effects in isolated tissue

Table 11 CB1 Cannabinoid Receptor Antagonists 10 Other Derivatives

Structure and affinity of diaryl-triazole derivatives

N

NN

R3

R2

R1

Cpd R1 R2 R3 Ki CB1 (nM) References

149 24-diCl 4-Cl hexyl 855 a [198]

150 24-diCl 4-Cl2-OMe CO-NH-(3-azabicyclo[330]octan-3-yl) 270 b [152]

151 24-diCl 4-Cl CO-NH-(3-azabicyclo[330]octan-3-yl) 164 b [152]

152 4-Cl 24-diCl CO-NH-(3-azabicyclo[330]octan-3-yl) 137 b [152]

153 4-Cl 24-diCl CO-NH-benzylpyrrolidin-3-yl 29 b [152]

154 4-Cl 24-diCl CO-NH-1-(4-chlorophenyl)ethyl 66 b [152]

155 4-Cl 24-diCl CO-NH-piperidinyl 356 c [182]

156 24-diCl 4-Cl CO-NH-piperidinyl 382 c [182]

a [3H]-SR141716A rat cerebellar membranesb [3H]-CP-55940 hCB1-HEK EDNA cellsc [3H]-CP-55940 hCB1-CHO cells

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1381

N

S

O

NH

Cl

Cl

Cl

F

N

S

O

NHN

ClCl

Cl

N

S

O

NH

Br

N

S

O

NHN

Cl

Cl

Cl

156 157 158 159

Fig (13) CB1 cannabinoid receptor antagonists 10 Other derivatives Structures of 45-diaryl-thiazole derivatives patented as hCB1cannabinoid receptor antagonists by Solvay Pharmaceuticals (156 157 158) or by AstraZeneca (157 158 159)

assays (mouse vas deferens and guinea pig ileum) thecompound possessed only a reduced rCB1 affinity with Kivalues of 855 and 748 nM using [3H]-SR141716A and [3H]-WIN-55-212-2 respectively (Table 11) Further four seriesof compounds differing by the nature of the substituentaround the triazole core were also described in a patent[199] Usually the compounds claimed possess two aromaticrings possibly substituted and one linear alkyl chain Theaffinity for the cannabinoid receptors was not givenNevertheless 149 was evaluated in two isolated tissuemodels the inhibition of the electrically evoked contractionsin the guinea pig ileum and in the mouse vas deferens Inthe two models compound 149 inhibited the effect of WIN-55212-2 but had no effect by itself In the vas deferensmodel the authors obtained a pA2 value of 748 to becompared with 763 obtained for AM251

Yet another five membered ring a thiazole has beenused as central moiety of a new class of cannabinoid ligandsThe thiazole ring is substituted by two aryls in position 4and 5 and by an amide (position 2) as in compounds 156-159 (Fig 13) Forty derivatives of this type weresynthesised by Lange and colleagues from SolvayPharmaceuticals and claimed in a patent as agonists orantagonists of the CB1 cannabinoid receptor [200] Nopharmacological data were provided however their activitywas assessed by measuring the cAMP in transfected hCB1-CHO cells In a recent paper from Lange et al two of thesethiazole derivatives were further described [182] Compound13 (157) the most closely related to SR141716A has a Kivalue of 227 nM while for compound 14 (158) Ki value isover 1000 nM Thus as in the imidazole series (see Table9) the aromatic substitution pattern is of great importancefor the CB1 cannabinoid receptor affinity Compound 13however was devoid of in vivo activity

Others 124-triazole derivatives were described by Dycket al compounds 150-154 unlike the Jagerovic ones havean amide moiety in position 3 [152] The highest affinitywas obtained with a 1-benzyl-pyrrolidin-3-yl substituentThe Ki value was 29 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) to be compared to 12 nM obtained for theSR141716A

Seventeen 45-diaryl-thiazole derivatives were alsosynthesised by Berggren et al and patented by AstraZeneca(157-159 Fig 13) [201]

In a patent from Merck Toupence et al described over190 substituted furo[23]pyridines claimed to be CB1cannabinoid receptor antagonists [202] The affinity (IC50 lt1microM) was measured using recombinant CHO cells and [3H]-CP-55940 and the activity using cAMP dosage (160 Fig14)

Lange et al described a triazole derivative (155) whichcan be superimposed with SR141716A Compound 155showed a Ki value of 356 nM ([3H]-CP-55940 hCB1-CHOcells) a pA2 value of 83 in the inhibition of WIN-55212-induced release of [3H]-arachidonic acid by hCB1-CHO cellsand was active in vivo in the CP-55940-inducedhypotension in rat (ED50 = 236 mgkg) [182]

Very recently Alanine from Hoffmann-LaRoche foundthat diarylbenzo[13]dioxole derivatives act as CB1

O

O

S

N

OO

F

ClCl

O

O

F O

N

OON

NH2

O

Cl

Cl

Cl

160 161 162

Fig (14) CB1 cannabinoid receptor antagonists 10 Other derivatives One example of a furo[23]pyridine derivative (160) and twoexamples of diarylbenzo[13]dioxole derivatives (161-162) described as hCB1 cannabinoid receptor antagonists by Toupence et aland Alanine et al respectively

1382 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

cannabinoid receptor antagonists (161-162 Fig 14) [203]Approximate IC50 values for twelve compounds ([3H]-CP-55940 hCB1-HEK cells) among the three hundred andeighty compounds described were given (IC50lt2microM)

et al in 1995 [207] In their hands ∆9-THC was not able toinhibit the forskolin-stimulated adenylyl cyclase activity inan assay performed on hCB2-CHO cells Subsequently theyshowed that ∆9-THC was able to antagonise HU210-inducedinhibition of adenylyl cyclase activity thus acting as anantagonist of the hCB2 cannabinoid receptor expressed inCHO cells [208] Furthermore very recently Govaerts et al[209] reported an inverse agonist effect of ∆9-THC and ∆8-THC on hCB2-CHO cells using the [35S]-GTPγ S assayThe pEC50 values were 763 and 888 respectively whilethe Emax values were ndash27 and ndash16 as compared to basal

Griffith from Pfizer described three new series of CB1cannabinoid receptor antagonists based on a bicyclic centralaromatic ring Purine pyrazoltriazines andpyrazolo[15a]pyrimidines derivatives were synthesised andinvestigated for their ability to interact with the CB1cannabinoid receptor Albeit a [35S]-GTPγ S assay wasdescribed no data were given concerning the antagonist orinverse agonist properties of such compounds Two hundredand eighty purine derivatives were synthesised [204] Theaffinities of 163 and 164 were given in the patent (28 and12 nM [3H]-SR141716A) In a following patent thesynthesis of sixty pyrazoltriazine derivatives was described(165 Fig 15) [205] The affinity of each compound was notgiven but was said to be ranging between 01 and 590 nMAnd finally more than forty pyrazolo[15a]pyrimidines weresynthesised (166 Fig 15) [206] Their affinity for the CB1cannabinoid receptor was lower than 155 nM Despite thefact that a great number of derivatives share the samesubstitution pattern around the central bicyclic aromatic ringthe comparison of these three new series of compounds ismade difficult by the absence of precise affinity data

Some other classical cannabinoids were shown to possessan antagonist activity at the CB2 receptor This is the casefor instance for the non-selective O-1184 (2) (Ki = 74 nM)which enhances the forskolin-induced cAMP production inCB2-CHO cells (EMax = 161 EC50 = 63 nM) and alsofor O-584 (3) (EMax = 246 EC50 = 138 nM) [39]

2 Indole Derivatives

Since the disclosure in 1993 of a second cannabinoidreceptor many attempts have been made to synthesise CB2cannabinoid receptor selective ligands The first compoundreported to have a somehow greater affinity for the peripheralcannabinoid receptor was WIN-55212-2 The compoundwas introduced by DrsquoAmbra and colleagues [210] back in1992 as a new cannabinoid ligand having a nanomolaraffinity and acting as an agonist In 1996 Showalter et alused a transfected cell line expressing the CB2 cannabinoidreceptor in order to identify selective ligands for thisreceptor [211] They showed that the CB2 affinity of WIN-55212-2 was 7 times greater than the CB1 with Ki valuesof 189 and 028 nM for the CB1 and CB2 cannabinoidreceptors respectively However WIN-55212-2 is anagonist of the cannabinoid receptors Interestingly WIN-55212-3 (167 Fig 16) the so-called inactive enantiomer ofWIN-55212 was recently shown to behave as a low affinity(Ki=363 microM) inverse agonist of the hCB2 cannabinoidreceptor as it decreased the [35S]-GTPγ S binding [209]

III CB2 LIGANDS RECEPTOR ANTAGONISTS ANDINVERSE AGONISTS

The compounds acting as antagonist or inverse agonistsat the CB2 cannabinoid receptor are reviewed in this thirdpart of the paper As for the CB1 cannabinoid receptorligands they are classified depending on their chemicalstructures

1 Classical Cannabinoid Derivatives

The first report of an antagonism mediated by ∆9-THC atthe peripheral cannabinoid receptor was made by Bayewitch

N N

NN

NH COOH

Cl

Cl

N N

NN

N

Cl

Cl

NH2OH

O

N

N

N

N

N

NHNH2

O

Cl

ClN

N

N

N

Cl

Cl

N

O

163 164

165 166

Fig (15) CB1 cannabinoid receptor antagonists 10 Other derivatives Examples of purine (163-164) pyrazoltriazines (165) andpyrazolo[15-a]pyrimidines (166) derivatives described as CB1 cannabinoid receptor antagonists by Griffith

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1383

The pEC50 value was 554 and the Emax value ndash26 ascompared to basal

AM630 also acts as an inverse agonist at the hCB1cannabinoid receptor (EC50 = 900 nM) as Landsman et aldemonstrated [47] Recently Zhang and colleagues reportedthe characterisation of the microsomal metabolism of AM630[212]

N

O

N

O

O

N

O

Cl

O

N

O

(S)-WIN-55212-3 167 BML-190 168

In 1996 Gallant and colleagues identified by submittinga large number of compounds to a binding assay anotherindole analog as CB2 cannabinoid receptor ligand [213] Onthe basis of this compound christened BML-190 (168 Fig16) or called indomethacin morpholinylamide theysynthesised several derivatives that possessed a selectivityratio for the CB2 cannabinoid receptor of up to 140 Thesewere the first compounds specifically designed to be CB2cannabinoid receptor ligands The first report on thepharmacological properties of BML-190 appeared onlyrecently New and colleagues highlighted in the paper theinverse agonist properties of the compound BML-190 dose-dependently increases (103 EC50= 980plusmn70 nM) theforskolin-stimulated levels of cAMP in hCB2-HEK cellswhile WIN-55212-2 decreases (44 45plusmn42nM) thisaccumulation [137]

Fig (16) CB2 cannabinoid receptor antagonists 2 Indolederivatives Chemical structures of WIN-55212-3 (167) andBML-190 (168) two indoles derivatives acting as inverseagonists at the hCB2 cannabinoid receptor Surprisingly in 2002 Melck and collaborators used

BML-190 as a CB2 receptor agonist in a cell proliferationassay [214] BML-190 was also tested as inhibitor of thecyclooxygenase-2 enzyme by Kalgutkar et al [215] Theyobtained IC50 values higher than 33 and 66 microM for theCOX-I and COX-II enzymes respectively These valueshave to be compared with the submicromolar activity ofindomethacin the parent compound Very recently Klegerisand colleagues [216] looking for an antineurotoxic action ofcannabinoids showed that BML-190 increases TNF-αsecretion by stimulated THP-1 monocytic cells but is noteffective on the IL-1β secretion

Further on the basis of WIN-54461 (12) a CB1antagonist AM630 (13) was synthesised by replacement ofthe bromine atom by an iodine one In the initial reportPertwee et al showed the antagonist effects of AM630 butalso suggested that the CB1 cannabinoid receptor may not bethe preferential receptor of AM630 [45] Later on Ross et alreported using hCB1amp2-CHO cells and [3H]-CP-55940that AM630 binds to the CB2 cannabinoid receptor (Ki =312 nM) with a selectivity ratio over 160 [48]Furthermore using a [35S]-GTPγ S assay they showed itsinverse agonist properties (EC50 = 76 nM) However

Table 12 CB2 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Reported binding affinities (Ki) and selectivity ratio (CB1CB2) of SR144528 (169) for rat and human cannabinoid receptors determined using [3H]-CP55940 as radioligand

NN

O

NH

Cl

SR144528 169

rCB2 (spleen) rCB1 (whole brain) Selectivity Ki hCB2 (transfected CHOcells)

Ki hCB1 (transfected CHOcells)

Selectivity References

038 nM 305 nM 800 06 nM 437 nM 730 [217]

067 nM 548 nM 80 [219]

56 nM gt10microM gt1780 [48]

25 nM a gt 1microM a gt400 [224]

024 nM 276 nM 115 199 nM 503 nM 25 [232]

a COS cells

1384 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

3 Diarylpyrazole Derivatives CHO cells is constitutively active and phosphorylated onSer 352 Upon treatment by SR144528 thisphosphorylation of the receptor was inhibited Moreover theSR compound was able to induce dephosphorylation ofagonist-induced CB2 phosphorylation The question whetherSR144528 induces a CB2 receptor conformational changemaking it a better phosphatase-specific substrate or ifSR144528 binding activates specific phosphatases remainsopen However this remains a nice example of inverseagonist-induced receptor resensitisation

The same Sanofi team which synthesised the CB1antagonist SR141716A reported in 1998 the synthesis andpharmacological characterisation of a CB2 cannabinoidreceptor selective antagonist the SR144528 (169) [217218] The structure of the new compound is derived fromSR141716A one SR144528 is the first selective CB2receptor antagonist reported as the inverse agonist propertiesof BML-190 were shown only recently by New et al [137]The authors reported for 169 using [3H]-CP-55940 asradioligand Ki values of 04 nM and 06 nM for the rat andthe human CB2 receptors respectively and found a CB1-CB2 selectivity ratio around 700 Table 12 sums up thebinding affinities of SR144528 for the cannabinoid receptorsobtained by different authors Interestingly while the CB2affinity is more or less constant the affinity obtained for theCB1 receptor varies by one order of magnitude [219] Theantagonist effect of SR144528 was highlighted by evaluatingits effect on cAMP production by hCB2-CHO cells exposedto 3 nM CP-55940 (EC50=10nM) The antagonism wasalso observed on the activation of the MAP kinase pathwayby CP-55940 (IC50=39nM)

The Sanofi research team working on cannabinoidsconducted investigations in order to determine the keyresidues for SR144528 interaction with the receptor (Table13) as well as the binding mode of the compound Firstlythey identified the TM4-EL2-TM5 region as a regioncontaining crucial residues for the affinity of several ligandsamong them SR144528 [224] Among these residues themutation of two cysteine of the second extracellular loop(C174 and C179) abolishes the affinity for the CB2 receptorLater on the implication of S453(161) and S457(165) inthe binding of SR144528 was evidenced by mutationalstudies In the model proposed for the docking of SR144528into the CB2 receptor the crucial contacts are comprised intothe TM4 this is the case for S453(161) and S457(165)which are proposed to form hydrogen bonds with the ligandand W464(172) proposed to have aromatic interactions withthe 4-methylbenzyl of the SR144528 [225] In 2003 Fengand Song showed that point mutations D349(130)A andA634(241)E abolished ligand binding However mutationof the arginine R350(131) member of the highly conservedDRY motif to alanine has no influence on SR144528binding [226]

Early after the report of the synthesis and characterisationof SR144528 the same authors further explored the functionof this compound and showed its inverse agonist propertiesusing CB2-CHO cells co-transfected with the luciferasereporter gene with either the CRE or the murine krox24regulatory sequence [220] In these models SR144528 notonly inhibits CP-55940 effects but also reproduces theeffects of a pertussin toxin treatment proving its inverseagonist properties SR144528 functionality was alsoexplored by [35S]-GTPγ S binding in hCB2-CHO cells(IC50=3 nM) [221] The inverse agonist function ofSR144528 was further illustrated by Rhee and Kim usingCOS cells co-transfected with hCB2 receptor and adenylylcyclase [222]

As for the CB1 cannabinoid receptor ligands Sanofideveloped tricyclic derivatives based on the 1-benzylpyrazole-3-carboxylic scaffold More than twentycompounds were disclosed in a patent [227] in 2001 (84-86Table 7) The benzyl in position 4 characteristic of the CB2diarylpyrazoles from Sanofi is conserved as well as thebicyclic residue in position 3 The length of the second linkbetween the phenyl and the pyrazole can be made of one or

Bouaboula and co-workers investigated SR144528 effectson CB2 cannabinoid receptor phosphorylation status [223]Firstly they showed that the CB2 receptor transfected in

Table 13 CB2 Cannabinoid Receptor Antagonists 3 Di-Aryl-Pyrazole Derivatives

Some of the single point mutations reported for the hCB2 cannabinoid receptor for which pharmacological data concerning the SR144528 are available

Mutant Effect References

R350(131)A no effect [226]

S453(161)A loss of affinity [225]

V456(164)I no effect [225]

S457(165)A loss of affinity [225]

C174S a loss of affinity [224 225]

C175S a loss of affinity [225]

C177S a no effect [225]

C179S a loss of affinity [224 225]

S544(193)G no effect [225]

a Second extra-cellular loop

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1385

two methylene but according to the patent one methylenebridge is preferred The Ki values of these compounds for theCB2 receptor were said to be lower than 500 nM and theirantagonist properties have been assessed using the adenylatecyclase inhibition assay

compounds by the presence of an alkyl substituent inposition 5 instead of a phenyl and of course by position 3which remains unsubstituted However except a lecture atthe 1998 ACS meeting [230] where compound 171 wasreported to have a Ki value of 835 nM and a selectivity ratioover 100 no further report on these compounds waspublished untill now

In a recent paper Mussinu and colleagues reported thesynthesis of other tricyclic derivatives starting fromSR141716A structure [228] N-piperidin-1-yl-14-di-hydroindeno[12-c]pyrazole-3-carboxamide derivatives areCB2 receptor ligands and some of them possess high affinityand selectivity for the CB2 receptor as shown in Table 7 (79-83) These tricyclic derivatives differ from the Sanofi tricyclicderivatives as they do have a phenyl ring instead of a benzylin position 1 Another difference is the nature of thesubstituent in position 3 which is not a bicycloalkyl but acyclic amine One of the compounds synthesised byMussinu et al 81 possesses the highest affinity for the CB2receptor ever reported with a Ki value of 0037 nM for themurine CB2 receptor ([3H]-CP-55940 mice spleenhomogenate) Interestingly 79 the closest SR141716Aanalog lost almost all its affinity for the CB1 receptor andincreased by four orders of magnitude its affinity for the CB2receptor Even if the structure quite similar to the SRcompounds could suggest that these derivatives behave asantagonists no pharmacological data were given in the paperto support this hypothesis

5 2-Oxoquinoline Derivatives

Inaba and colleagues from Japan Tobacco introduced in2001 a completely new structure in the cannabinoid fieldbased on a 2-oxoquinoline scaffold Over eighty derivativeswere disclosed in a patent [231] and one compound JTE-907 (173) was subsequently characterised in-vitro as well asin-vivo [232] In Table 14 are listed some of the compounds(172-178) disclosed in the patent along with their affinity forthe cannabinoid receptors These values were obtained onhuman receptors against [3H]-WIN-55212-2 For twelvecompounds the anti-inflammatory activity per os wasassessed in a carrageenin-induced paw edema model Thehighest effect reported was obtained with JTE-907 with anED50 value lower than 01 mgkg Therefore thesecompounds were claimed to be useful as anti-inflammatoryand anti-allergic agents

To further characterize JTE-907 the authors determinedits affinity for cannabinoid receptors of different species using[3H]-CP-55940 as radioligand In their hands JTE-907behaved as a very selective hCB2 ligand with Ki values of36 and 2370 nM for the CB2 and CB1 cannabinoid receptorsrespectively even more selective than the SR144528 (Kivalues of 199 and 50 nM respectively) However it has tobe mentioned that the affinities reported in the paper forJTE-907 are quite different from those reported in the patent(hCB1 Ki values of 35nM and 009 nM respectively) Italso appears that the SR144528 affinity for the CB1 receptordetermined by Iwamura et al is higher than the affinityreported by Rinaldi-Carmona et al with values of 50 and437nM respectively The authors ascribed the difference tothe variability of SR144528 binding affinities to the assayconditions The function of JTE-907 was evaluated bymeasuring the increase in cAMP production by forskolin-stimulated hCB2-CHO cells Maximum stimulation wasattained at a concentration of 1microM of JTE-907 In the sameassay WIN-55212-2 decreased the cAMP production in adose-dependent manner

Finally it is interesting to strengthen that the five-membered ring derivatives (79-86) of the diarylpyrazolefamily are reported to possess a high CB2 receptor affinitywhile the six- and seven-membered ring derivatives (69-78)are described as CB1 receptor ligands

4 Other Pyrazole Derivatives

Other CB2 ligands based on a pyrazole nucleus wereintroduced by SmithKline Beecham in a patent describingthe synthesis of about sixty new compounds [229] Theiraffinity (Ki) for the hCB2 receptor transfected in HEK293cells was reported to be ranging between 25nM and 10microMTheir structures (170-171 Fig 17) differ from the Sanofi

NN

O

N

ON

NN

N

NN

O

N

ON

NN

N

ClCl

1

5

4 3

2

170 171

Fig (17) CB2 cannabinoid receptor antagonists 4 Otherpyrazole derivatives Structure of two compounds (170-171)among those claimed by SmithKline Beecham to be hCB2antagonists

In conclusion JTE-907 was shown to be a CB2 selectiveinverse-agonist with anti-inflammatory and anti-allergicproperties This compound is currently undergoing Phase Iclinical trials as anti-allergic drug [233]

IV NON-CB1 NON-CB2 CANNABINOIDRECEPTORS LIGANDS

In the last years the possible presence of additional non-CB1 non-CB2 cannabinoid receptors was raised frompharmacological evidences

One of the putative receptors possesses an endotheliallocalisation while other ones are localized in the CNS or atperipheral nerves For instance an additional target for ∆9-

1386 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 14 CB2 Cannabinoid Receptor Antagonists 5 2-Oxoquinoline Derivatives

Structure of six compounds among the oxoquinoline derivatives patented by Japan Tobaco Affinity (Ki nM) and selectivity (CB1CB2) for the cannabinoidreceptors are given The affinity was measured by binding against [3H]-WIN-55212-2 on either hCB2-CHO or hCB1-CHO cells Adapted from [231]

Cpd ndeg Structure hCB2 hCB1 Selectivity

1-2 172

NH

MeO O

O

OMeO

0014 3671 262215

JTE-907 173

NH

O

MeO O

O

NH

O

O

0087 3436 39495

2-2 174

OH

O

NH

MeO O

O 077 3247 4215

3-27 175

NH

O

F

NH

MeO O

O0021 336 16000

3-32 176

NH

MeO O

O

NH

F

O

0036 2398 66610

3-38 177

O

O

NH

N

O

O

NMeO

0085 935 11000

3-43 178

NMeO O

O

NH

F

O

0043 864 20100

tetrahydrocannabinol was described in capsaicin-sensitivesensory nerves by Zygmunt et al [234]

[29] and by Di Marzo et al [235] Thus in the presentreview we will focus on the description of the so faridentified ligands of the non-CB1 non-CB2 receptors withan emphasis on the compounds having antagonist properties

Pharmacological evidences for the existence of additionalcannabinoid receptors were reviewed by Wiley and Martin

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1387

(Table 15) In particular the knowledge about three not yetcloned targets has grown The growing molecularpharmacology of the three putative new cannabinoidreceptors is reviewed in the three following sections Thefourth section summarises the data on the other proposedcannabinoid targets

vasodilatator activity In addition the anandamide effect ispartially sensitive to SR141716A inhibition The authorsproposed the existence of two distinct receptors responsiblefor the vasodilatatory effect of anandamide one located in theendothelium is SR141716A-sensitive while the other onelocated on the vascular smooth muscle is SR141716A-insensitive Thus SR141716A is an antagonist of anendothelial anandamide receptor Several studies wereconducted in order to understand the molecularpharmacology of the endothelial anandamide receptor It ispertussis toxin-sensitive [237] Cannabidiol acts asantagonist and abnormal-cannabidiol (Abn-cbd 179) and itsanalogue O-1602 (180) act as agonists [238] The synthesisof O-1602 and of its analogues are disclosed in a patentdescribing the vasodilatatory activity of cannabinoid

1 Endothelial Anandamide Receptor ndash Abnormal-Cannabidiol Receptor

In 1999 Wagner et al showed that anandamide couldinduce mesenteric vasodilatation through an endotheliallylocated ldquoanandamide receptorrdquo pharmacologically distinctfrom CB1 [236] Other cannabinoids such as ∆9-THC HU-210 or WIN-55212-2 were devoid of mesenteric

Table 15 Non-CB1 Non-CB2 Cannabinoid Receptors Antagonists

An overview of the putative new non-CB1 non-CB2 anandamide-receptors ligands The antagonists are highlighted in bold

NH

N

S

HO OH

Cl

OH

OH

OH OH OH OH

OMe

OMe

RR

RR

RR

RR

HO

OH

NH

O

cannabidiol (10) Abnormal-cannabidiol 179 O-1602 180

O-1918 181 capsazepine 183capsaicin 182

Compound Endothelial AEA receptor Brain AEA receptor Cannabinoid-vanilloid brain receptor

AEA agonist a agonist de

WIN-55212-2 no effecta

agonist e agonist g h

CP-55940 no effect e agonist i

SR141716A antagonist a b j no effect ([35 S]-GTPγS binding) d e f antagonist g h

AM251 no effect k no effect h i

Cannabidiol antagonist b no effect e

O-1918 antagonist c jm

Abd-cbd agonist b l

O-1602 agonist b

Capsaicin no effectj agonist i

Capsazepine no effect b antagonist h

a [236] b [238] c [240] d [249] e [250] f [251] g [254] h [258] i [ 255] k [244] j [ 243] l [ 242] m [241]

1388 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

analogues [239] More recently Offertaler et al showed thatO-1918 (181) a cannabidiol analog acts as an antagonist ofthe endothelial anandamide receptor [240] In addition O-1918 does not bind to the CB1 and CB2 cannabinoidreceptors Furthermore O-1918 unlike cannabidiol doesnot cause vasorelaxation in the mesenteric artery preparationmodel used and thus acts as a true neutral antagonistUsing O-1918 as a pharmacological tool Begg et alproposed a coupling of this new G protein-coupledendothelial receptor through a GiGo mechanism to ionchannels in primary cultured human vascular endothelialcells [241] However in their hands SR141716A wasineffective in antagonising Abd-cbd Ho and Hiley furtherdemonstrated that Abn-cbd relaxes rat small mesentericarteries through an activation of K+ channels via aSR141716A-sensitive pathway [242] Further using thesame model they characterized the vasorelaxant activity ofvirodhamine a novel endocannabinoid apparently actingthrough the same receptor [243] The virodhamine-inducedvasorelaxation is sensitive to SR141716A and O-1918 butinsensitive to AM251 SR144528 and AM630 Moreovervanilloid receptor desensitisation by capsaicin (182) had noeffect

244] Thus position 4 on the 5-phenyl could be furtherexplored to obtain selective ligands

To come to an end despite the fact that the endothelialanandamide receptor is neither identified nor cloned at leastthree antagonists are identified SR141716A cannabidioland O-1918 It is likely that a growing number of ligandsand therapeutic applications will appear in the very nearfuture

2 Brain Anandamide Receptor

Following an in-depth evaluation of anandamide effects inCB1

-- mice Di Marzo and co-workers suggested that a non-CB1 non-CB2 G protein-coupled receptor might mediatesome of the actions of AEA in mice [249] Actually effectsof AEA but not those of ∆9-THC were not decreased inCB1 cannabinoid receptor knock-out mice For instance 30mgkg of AEA were still effective in hot-plate test whereas10 mgkg of ∆9-THC had no effect on anti-nociceptionMoreover in a [35S]-GTPγ S assay AEA stimulated with anEC50 value of 223 microM the [35S]-GTPγ S binding to CB1

--

mice brain membranes (Emax= 28) ∆9-THC showed noeffect in this assay The addition of SR141716A did notaffect the [35S]-GTPγ S binding to CB1

-- mice brainmembranes Shortly after other evidences for the existence ofa new G protein-coupled cannabinoid receptor with a distinctdistribution in the central nervous system were published[250] From a set of 24 commonly used cannabinoids (AEAWIN-55212-2 JWH-030 THChellip) only AEA and WIN-55212-2 produced an increased binding of [35S]-GTPγ S onCB1

-- mice brain membranes Interestingly in this studySR141716A alone produced a significant inhibition of [35S]-GTPγ S binding similar to the inhibition obtained usingCB1

++ mice brain membranes (IC50 values of 57 microM and47 microM respectively) However SR141716A did not affectthe anandamide-induced [35S]-GTPγ S binding in CB1

--

mice brain membranes Moreover [3H]-SR141716Aexhibited significant binding in some brain regions of CB1

--

mice which are different from those exhibiting stimulation of[35S]-GTPγ S binding by WIN-55212-2 and AEA Thusthe authors proposed that the SR141716A effects on CB1

--

mice are mediated neither by the CB1 cannabinoid receptornor by the putative new anandamide brain receptor Monoryand co-workers using CB1

-- mice described a novel nonadenylyl cyclase-coupled cannabinoid binding site in micecerebellum [251] Two agonists AEA and WIN-55212-2but not ∆9-THC or HU210 were found to bind to thisreceptor as they enhanced [35S]-GTPγ S binding NeitherSR141716A nor SR144528 were able to affect the WIN-55212-2 induced [35S]-GTPγ S binding in CB1

-- mice asfound by Breivogel et al [250] However the distributionpattern of the receptor described by Monory et al differs fromthe distribution found by Breivogel et al It has to bementioned that the knock-out strains used were different Themice used by Monory were from a CD-1 strain while theone used by Breivogel were from a C57BL61 strainInterestingly Muthane et al detected differences in nigralneurons number and sensitivity to MPTP in the two mousestrains used [252]

More recently Mo et al obtained results showing thatthe Abnormal-cannabidiol receptor is also responsible for anincreased endothelial cell migration [244] Abn-cbd enhancedhuman umbilical vein endothelial cell migration while O-1918 antagonised Abn-cbd effect The CB1 cannabinoidreceptor inverse agonist SR141716A partially inhibitedAbd-cbd action whereas AM251 and SR144528 had noeffect

In another paper from George Kunos team Batkai et alreported that SR141716A is able to inhibit the endotoxichypotension by a cardiac mechanism involving neither theCB1 nor the CB2 cannabinoid receptors [245] Theinvolvement of a non-CB1 non-CB2 site of action wasshown using CB1

-- and CB1--CB2

-- knock-out miceFurther studies are needed to identify the SR141716Asensitive myocardial site of action

In a recent patent Kunos described O-1918 and itsanalogues as vasoconstrictor agents useful to reversepathological vasodilatation of blood vessels [246] Theeffects of O-1918 on mouse blood pressure on LPS-inducedhypotension in mouse as well as the reversal of hypotensionby O-1918 injection are described Furthermore thesynthesis of O-1918 based antagonists is described startingfrom p-metha-28-dien-1-ol and the appropriate resorcinols

Interestingly the Abd-cbd receptor which was firstdetected in the vascular system also seems to be present inthe CNS [247] Indeed Walter et al found that microglialcells (BV-2) migration induced by 1 microM 2-AG is inhibitedby cannabidiol (300 nM) and O-1918 (1 microM) SR141716Awas found ineffective in this assay Thus 2-AG is an agonistof the Abd-cbd receptor Moreover Abd-cbd is able to elicita dose-dependent cell migration in the same model (EC50 =600nM) [248]

From a more structural point of view SR141716Abehaves as an antagonist while on the opposite AM251(20) a close analogue which differs from the former only bythe nature of one halogen has no effect on this receptor [242

To conclude to our knowledge no compounds havingantagonist or inverse agonist properties at this receptor havebeen identified so far

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1389

3 Cannabinoid-Vanilloid Brain Receptor a cannabinoid-vanilloid receptor at which SR141716A andcapsazepine behave as antagonists

Evidences for the presence of an additional ldquocannabinoidrdquoreceptor differing from the CB1 receptor and from the brainanandamide receptor appeared from studies focused on thehippocampus (for a review see Hajos et al [253]) Hajos etal studied the cannabinoid actions on GABAergic andglutamatergic transmission using the whole-cell patch-clamp technique [254] WIN-55212-2 induced a dose-dependent reduction in glutamatergic transmission reversedby SR141716A (1microM) Thus the authors concluded that thecannabinoid actions on the glutamatergic transmission aremediated by a non-CB1 but SR141716A-sensitive receptorIn another paper Hajos and Freund further characterised thisputative receptor [255] They showed that in addition toWIN-55212-2 CP-55940 and capsaicin (182) a TRPV1receptor agonist also act as agonists Furthermore in theirhands SR141716A as well as capsazepine (183) a TRPV1receptor antagonist were able to antagonise this effect on theglutamatergic transmission while AM251 was notSR141716A effects on CB1 knock-out mice were furtherstudied using a test of anxiety [256] Haller et al showedthat on the one hand the distribution of the CB1 receptorinduces anxiety and SR141716A has an anxiolytic effectand on the other hand SR141716A is still active in CB1

--

mice In a following paper they reported that AM-251induces anxiogenic effects in wild-type and in knock-outmice [257] Thus SR141716A in addition to CB1 bindsto a not yet identified receptor Another evidence for thisadditional receptor for SR141716A was given by Bass andco-workers [61] They conducted a structure-activityrelationship study on the stimulation of locomotor activityinduced by SR141716A and twenty analogues Nocorrelation was found between the affinity for the CB1cannabinoid receptor and the stimulation of locomotoractivity Albeit SR141716A and five of its analogues O-1803 O-1710 O-1253 O-1254 (40) O-1255 (41) behavedas inverse agonists in [35S]-GTPγ S assay none of theseanalogues stimulated the locomotor activity In conclusionthe SR141716A-induced stimulation of locomotor activity isneither due to inverse agonism nor to inhibition of anendogenous tone but to a brain receptor distinct from theCB1 receptor Di Marzo et al previously provided evidencesthat the cannabinoid-vanilloid brain receptor could mediatesome of the cannabinoid effects on locomotion [249]

4 Other Non-CB1 Non-CB2 Cannabinoid Receptors

Evidences for the existence of additional non-CB1 non-CB2 cannabinoid receptors localised in the rat isolatedmesenteric arterial bed appeared from the works of Ralevicand collaborators Several cannabinoids were evaluated intheir model of sensory neurogenic vasorelaxation evoked byelectrical field stimulation

HU210 was found to attenuate vasorelaxation in a CB1and CB2 independent way as SR141716A LY320135 andSR144528 were ineffective in inhibiting HU210 effect [259]Similar results were obtained with ∆9-THC [260]Similarly noladin ether inhibited sensory neurogenicrelaxation in a concentration-dependent manner which wasalso unaffected by SR141716A LY320135 and SR144528Moreover this effect as the ∆9-THC one was pertussistoxin sensitive suggesting the involvement of a Gio protein-coupled receptor [261]

However the inhibitory effects of CP-55940 and WIN-55212-2 were affected by the presence of SR141716A (1microM)or LY320135 (1microM) but not by the addition of SR144528(1microM) [262]

It appears that at least two receptors should be involvedin cannabinoid-mediated attenuation of sensory nerve-mediated vasorelaxation The first one is SR141716A andLY320135 sensitive while the other one is resistant toSR141716A

Some evidences for the existence of a ldquoCB2-likerdquo receptorappeared from the studies on palmitoylethanolamide (PEA)an endogenous fatty acid amide devoid of CB1 and CB2affinity [264] antinociceptive potential Calignano and co-workers showed that PEA was able to alleviate pain inseveral animal models [265 266] For instance PEA dose-dependently inhibits kaolin-evoked writhing in mice TheCB2 cannabinoid receptor inverse agonist SR144528 (02mgkg) inhibits PEA anti-nociceptive effect [266] Thus theauthors suggest that PEA acts as agonist on a SR144528-sensitive non-CB2 cannabinoid receptor (ldquoCB2-likerdquoreceptor) at which SR144528 acts as an antagonist

Further studies are needed in order to better characterisethese additional ldquocannabinoidrdquo receptors However it islikely that several therapeutic applications will soon springfor these receptors

Very recently Pistis et al published a study on theneurophysiological effects of cannabinoids on the basolateralamygdala neurons in-vivo [258] One of their findings is thatHU-210 and WIN-55212-2 did not elicit similar effects onbasal amygdala projection neurons firing rate While theformer had no effect the latter decreased the firing rateMoreover SR141716A reversed the WIN-55212-2 effectswhile AM251 had no effect The vanilloid receptorantagonist capsazepine also antagonised the WIN-55212-2effect on firing rate of these neurons Therefore the authorssuggested the presence of a novel cannabinoid-vanilloidreceptor

V CONCLUSION

Since their development cannabinoid antagonists provedto be essential tools in the understanding of cannabinoidpharmacology and biochemistry Moreover the interestshown by the pharmaceutical industry in the development ofnew cannabinoid antagonists prove if necessary the realtherapeutic potential of this class of compounds Thetreatment of eating and movement disorders memorydeficits psychosis and dependencies from various addictivedrugs are some of the most cited applications for the CB1cannabinoid receptor antagonists

From these lines it appears that in addition to theknown CB1 cannabinoid receptor it is now likely that twoother cannabinoid receptors do exist in the brain The firstone should be an anandamide brain receptor for whichSR141716A has no affinity while the second one should be

1390 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Much less wide is the knowledge on the CB2cannabinoid receptor antagonists However potentialapplications could be disorders involving the immunesystem such as inflammation or allergies

[19] Devane WA Hanus L Breuer A Pertwee RG StevensonLA Griffin G Gibson D Mandelbaum A Etinger AMechoulam R Science 1992 258 1946

[20] Mechoulam R Ben-Shabat S Hanus L Ligumsky MKaminski NE Schatz AR Gopher A Almong S MartinBR Compton DR Biochem Pharmacol 1995 50 83

Finally the growing literature on the non CB1 non CB2cannabinoid receptors should allow the development of newdrugs First examples are given by the cannabidiol andabnormal-cannabidiol derivatives described by Kunos et alfor which potential therapeutic applications on the vascularsystem are already patented

[21] Sugiura T Kondo S Sukagawa A Nakane S Shinoda AItoh K Yamashita A Waku K Biochem Biophys Res Co1995 215 89

[22] Hanus L Abu-Lafi S Fride E Breuer A Vogel Z ShalevDE Kustanovich I Mechoulam R Proc Natl Acad SciUSA 2001 98 3662

[23] Oka S Tsuchie A Tokumura A Muramatsu M Suhara YTakayama H Waku K Sugiura T J Neurochem 2003 851374It clearly appears from this paper that the antagonists of

the cannabinoid receptors are still an active research fieldfrom which should emerge new promising therapeutic toolsas well as innovative drugs in the near future

[24] Goutopoulos A and Makriyannis A Pharmacol Therapeut2002 95 103

[25] Beal JE Olson R Lefkowitz L Laubenstein L Bellman PYangco B Morales JO Murphy R Powderly W PlasseTF Mosdell KW Shepard KV J Pain Symptom Manage1997 14 7ACKNOWLEDGEMENTS [26] Notcutt W Price M Miller R Newport S Phillips CSimmons S Sansom C Anaesthesia 2004 59 440

GGM is very indebted to the ldquoFonds pour la formationagrave la Recherche dans lrsquoIndustrie et lrsquoAgriculture (FRIA)rdquo Belgium for the award of a research fellowship Theexperimental works of the authors were supported by grantsfrom the FNRS The Belgian Charcot Foundation and theUniversiteacute catholique de Louvain

[27] Ledent C Valverde O Cossu G Petitet F Aubert JFBeslot F Bohme GA Imperato A Pedrazzini T RoquesBP Vassart G Fratta W Parmentier M Science 1999 283401

[28] Zimmer A Zimmer AM Hohmann AG Herkenham MBonner TI Proc Natl Acad Sci U S A 1999 96 5780

[29] Wiley JL and Martin BR Chem Phys Lipids 2002 121 57[30] Adam J and Cowley P Expert Opin Ther Patents 2002 12

1475

REFERENCES [31] Razdan RK and Mahadevan A Chem Phys Lipids 2002 12121

[32] Barth F and Rinaldi-Carmona M Curr Med Chem 1999 6745

[1] Hillard CJ Harris RE Bloom AS J Pharmacol Exp Ther1985 232 579

[33] Little PJ Compton DR Johnson MR Melvin LS MartinBR J Pharmacol Exp Ther 1988 247 1046

[2] Devane WA Dysarz FA Johnson MR Melvin LSHowlett AC Mol Pharmacol 1988 34 605

[34] Pertwee RG Stevenson LA Elrick DB Mechoulam RCorbett AD Brit J Pharmacol 1992 105 980

[3] Matsuda LA Lolait SJ Brownstein MJ Young ACBonner TI Nature 1990 346 561

[35] Harrison C Traynor JR Life Sci 2003 74 489-508[4] Gerard CM Molerau C Vassart G Parmentier M BiochemJ 1991 279 129 [36] Pertwee RG Fernando SR Griffin G Ryan W Razdan

RJ Compton DR Martin BR Eur J Pharmacol 1996 315195

[5] Glass M Dragunow M Faull RLM Neuroscience 1997 2299

[37] Ross RA Brockie HC Fernando SR Saha B Razdan RKPertwee RG Brit J Pharmacol 1998 125 1345

[6] Shire D Carillon C Kaghad M Calandra B Rinaldi-Carmona M LeFur G Caput D Ferrara P J Biol Chem1995 270 3726 [38] Griffin G Wray EJ Rorrer WK Crocker PJ Ryan WJ

Saha B Razdan RK Martin BR Abood ME Brit JPharmacol 1999 126 1575

[7] Ryberg E Khang Vu H Groblewski T Larsson N ElebringT Hjorth S Sjogren S Greasley PJ 2004 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 119

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1372 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

[c]pyrrol2(1H)-yl)-4-methyl-pyrazole-3-carboxamide (63)(Fig 4) The Ki values were 5 and 12 nM respectively([3H]-CP-55940 hCB1-HEK cells)

CP-55940 rat brain) For instance N-(piperidin-1-yl)-5-(3-chloro-biphenyl-4-yl)-1-(24-dichloro-phenyl)-4-methyl-pyrazole-3-carboxamide (65) (Fig 4) showed a Ki value of15 nM However no data were provided concerning thefunctionality of such compounds

Over 200 other pyrazole derivatives acting at the CB1cannabinoid receptor were claimed in a patent from Pfizer[153] The affinity for the hCB1 receptor of 2-[1-(2-chlorophenyl)-5-(4-chlorophenyl)-4-methylpyrazol-3-yl]-4-[(6-methyl-2-pyridinyl)methyl]ndashmorpholine (64) (Fig 4)was 79 nM ([3H]-SR141716A hCB1-HEK cells) Theothers compounds affinity was between 01 and 100 nM

In an effort to develop new SR141716A analoguespossessing lower lipophilicity to be used as PET tracersKatoch-Rouse and colleagues further explored thesubstitution pattern of the two phenyls [155] Bindingassays showed that a decreased lipophilicity lead to adecreased affinity for the CB1 receptor as determined bydisplacement of [3H]-AM251 For instance compoundNIDA-41057 (59) has a lipophilicity of 42 ( expressed asElogDoct) and a Ki value of 104 nM whereas SR141716Ahas a ElogDoct value of 54 and a Ki value of 18 nM In

Seventy five pyrazole derivatives were described in a veryrecent patent from Makriyannis et al [154] The affinity ofthe compounds for the cannabinoid receptors is given TheKi values for the best compounds are lower than 6 nM ([3H]-

Table 7 CB1 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Structures and affinities for the cannabinoid receptors of some representative tricyclic pyrazole derivatives Values were obtained using [3H]-CP-55940as radioligand and mouse [160 228] rat [159 227] or human [158] cannabinoid receptors Compounds 69-78 are CB1 cannabinoid receptor ligandsCompounds 79-86 are CB2 cannabinoid receptor ligands

NN

O

NHX

R3

R1

R2

n

n˚ X R1 R2 n R3 Ki CB1 (nM) Ki CB2 (nM) Selectivity References

69 -(CH2)3- Cl 24-di-Cl 0 piperidine 126 [158]

70 -(CH2)3- H 24-di-Cl 0 azepane 100 [158]

71 -(CH2)3- H 24-di-Cl 0 piperidine 398 [158]

72 -(CH2)3- Cl 24-di-Cl 0 azepane 125 [158]

73 -(CH2)3- NO2 24-di-Cl 0 azepane 63 [158]

74 -(CH2)2-O- H 24-di-Cl 0 piperidine 501 [158]

75 -CH2-S- Br 24-di-Cl 0 piperidine lt 500 gt10 [159]

76 -CH2-SO2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

77 -(CH2)2- Cl 24-di-Cl 0 piperidine lt 500 gt10 [159]

78 -(CH2)3- Cl 24-di-Cl 0 piperidine 000035 21 60000 [160]

79 -CH2- Cl 24-di-Cl 0 piperidine 2050 034 00002 [228]

80 -CH2- Br 24-di-Cl 0 piperidine 1570 027 00002 [228]

81 -CH2- CH3 24-di-Cl 0 piperidine 363 0037 00001 [228]

82 -CH2- Cl 4-Cl 0 piperidine 1787 09 00005 [228]

83 -CH2- H 24-di-Cl 0 piperidine 1152 0385 00003 [228]

84 -CH2- Cl 34-di-Cl 1 133-trimethylbicyclo[221]heptyl

lt500 gt10 [227]

85 -CH2- Br 24-di-Cl 1 bicyclo[321]oct-3-yl lt500 gt10 [227]

86 -CH2- Br 4-Me 1 77-dimethylbicyclo[410]hept-3-yl

lt500 gt10 [227]

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1373

another paper Katoch-Rouse described the synthesis of[18F]-NIDA-42033 (26) a ligand useful as PET radiotracerstarting from its bromo derivative [156] Recently Kumar etal [157] proposed the synthesis of a 11C derivative ofNIDA-41087 (61) a compound previously described byKatoch-Rouse (Table 5) Post-mortem binding of thiscompound to human prefrontal cortex was also investigatedInterestingly CP-272871 (66) described by Meschler as aCB1 inverse agonist (Ki=57 nM [3H]-CP-55940 rat brainhomogenates) possesses a lipophilicity similar to theNIDA-41087 one [62] However despite the presence of amethoxy function no radiolabelled derivative has beendescribed to date One explanation could be the compoundlow selectivity (2 fold) for the CB1 cannabinoid receptor

To further explore the pharmacology of this compound afunctional assay using the mouse vas deferens model wasperformed by Ruiu and colleagues enlightening thecompetitive antagonist properties of NESS (pA2= 1246against WIN-55212-2) which had no effect by itself up to1microM Moreover NESS 0327 was unable to affect basalbinding of the [35S]-GTPγ S demonstrating its lack ofnegative intrinsic activity In-vivo studies were conductedusing the hot plate and tail flick tests NESS dose-dependently abolished the antinociceptive effect of WIN-55212-2 but had no effect by itself However the authorssuggested based on the affinity and in-vivo activity of theircompound that NESS 0327 possesses a poor centralbioavailability

Several new attempts to increase the affinity of thediarylpyrazole derivatives were recently made by rigidifyingthe SR141716A structure (Table 7) Six fused ringanalogues of SR141716A obtained by fusion of the 5-(4-chlorophenyl) substituent with the central pyrazole to forman indazole ring were published by Bass et al in 2002 [61]The compound possessing the highest affinity O-1248 (67)has a Ki value of 475 nM as determined by displacement of[3H]-CP-55940 from rat brain membranes

Another example of ring-constrained biarylpyrazole wasdescribed by Herbert Seltzman team [161] Theyinvestigated a photocyclisation reaction starting from theSR141716A leading to a pyrazolo[15-f]phenanthridinestructure (Fig 5) The new compound (68) was tested on theCB1 receptor (whole rat brain) against [3H]-CP-55940[3H]-SR141716A and [3H]-WIN-55212-2 and exhibited Kivalues of 48 35 and 50 nM respectively The CB2 affinitywas assessed using [3H]-CP-55940 and was found to be 100times lower (Ki = 3340 nM) No effect on the [35S]-GTPγ Sassay (rat brain) was observed by the authors

Stoit and colleagues from Solvay Pharmaceuticalspublished a paper describing the synthesis andpharmacological characterisation of new benzocyclohepta-pyrazole derivatives as CB1 antagonists (69-74) [158] Theaffinity of these compounds was more or less one order ofmagnitude lower than the SR141716A one with pKi valuesranging from 64 to 72 compared to 76 for the SRcompound (Table 7) Compounds were shown to beantagonists with pA2 values from 70 to 89 as theyprevent CP-55940-induced cAMP accumulation Howeverthe authors investigating the bioavailability of theircompounds either po or after ip injection found negligibleblood plasma levels

NN

Cl

Cl

O

NH

N

NN

Cl

O

NHN

Cl

Cl

O-1248 67 68However just before the publication of Stoit results

Sanofi-Synthelabo took a patent describing tricyclicpyrazolecarboxylic acid amide derivatives having antagonistproperties on the CB1 cannabinoid receptor andsubmicromolar affinities (75-77) [159] Thirty examples weregiven along with their synthetic pathway but no individualpharmacological data were shown

Fig (5) CB1 cannabinoid receptor antagonists 3Diarylpyrazole derivatives Two ring-constrained diarypyrazolederivatives O-1248 (67 ) and a pyrazolo[15-f]phenanthridinederivative (68 )

It appears from these attempts to increase SR141716Aaffinity by rigidifying its structure that only a limitedincrease in the CB1 cannabinoid receptor affinity can beexpected with the apparent exception of NESS 0327Moreover rigidifying the structure often results in a morelipophilic compound in a family of compounds being yetlipophilic

More recently Ruiu and colleagues published a paperdescribing the synthesis and complete pharmacologicalcharacterisation of a very potent CB1 receptor ligandchristened NESS 0327 (78) [160] This compound has thesame structure as compound 69 published by Stoit et al(compound 20 in the reference [158]) Nevertheless Ruiu hasobtained an affinity for the CB1 cannabinoid receptor 5000times bigger than the SR141716A one with Ki values of035 pM and 18 nM respectively using mouse forebrainhomogenates and [3H]-CP-55940 as radioligandFurthermore the selectivity ratio obtained for this compoundby Ruiu is 60000 with a Ki value for the CB2 receptor of21 nM against the [3H]-CP-55940 The discrepancybetween the data obtained independently by Stoit et al andRuiu et al could hardly be explained by the differences inthe origin of the receptor used respectively hCB1-CHO andmouse forebrain

4 Phenyl Benzofuranone Derivatives

In a 1997 patent from Eli Lilly was described thesynthesis of aryl-benzothiophene and aryl-benzofuranederivatives (87-89 Fig 6) having CB1 receptor affinity andantagonist properties [162] From ten compounds having aKi value lower than 25 microM ([3H]-CP-55940 hCB1-CHOcells) one was selected and claimed to have a Ki of 170 nMCompound 88 was shown in the patent to antagonise theeffect of anandamide on cAMP accumulation with an IC50 of

1374 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

O

O

O

O

NC

S

O

O

O

O

O

O

O

NC

LY320135 8887 89

Fig (6) CB1 cannabinoid receptor antagonists 4 Phenyl benzofuranone derivatives Chemical structures of two aryl-benzofuranederivatives (87 -88 ) and one aryl-benzothiophene (89 ) derivative from Eli Lilly

500 nM and the effect of WIN-55212-2 on calcium channelsat 1microM Moreover the intra-peritoneal injection (20 mgkg)of this compound antagonised the in-vivo effect ofanandamide in the Open Field Assay a mouse behaviourmodel Later on Felder and colleagues gave a furtherdescription of this compound called LY320135 in a paperdescribing its effect on cAMP accumulation and on ioniccurrents [163] The selectivity ratio of LY320135 was 106with Ki values of 141 and 14900 nM for hCB1 and hCB2receptors ([3H]-CP-55940) respectively LY320135antagonised anandamide inhibition of forskolin-inducedcAMP accumulation with an IC50 value of 734 nMChristopoulos et al in 2001 reported for LY320135 apKb value of 527 in the inhibition of WIN-55212-2mediated response in the rat electrically-stimulated vasdeferens while the pKb value for SR141716A was 75[164]

Other patents were taken by Aventis in which somepotential applications for their compounds were describedFor instance an association between an azetidine derivativehaving CB1 antagonists properties and sibutramine aserotonin and norepinephrine reuptake inhibitor was claimed[170] as a treatment against obesity Oral administration ofcompound 90 (3 mgkg) and sibutramine (06 mgkg) to fafaZucker rats which are genetically obese rats has resulted ina decreased food intake compared to control lean Zuckerrats In another patent the association of an azetidinederivative (1-10 mgkg po ) and a D2D3 agonist likequinpirole (01 mgkg ip) was claimed to have a therapeuticpotential in the treatment of Parkinsonrsquos disease [171] Theeffect has been evidenced using an akinesia model in the ratEvidences supporting the use of CB1 antagonists as adjuvantin the treatment of Parkinsonrsquos disease begin to appear Itseems that such compounds would be helpful in thetreatment of Parkinsonism [172] and levodopa-induceddyskinesia [173] Brotchie published an interesting paperreviewing the CB1 cannabinoid receptor signaling inParkinsonrsquos disease [174] Nevertheless further clinicalstudies should be conducted to further confirm the usefulnessof CB1 antagonists in the treatment of Parkinsonrsquos disease

5 Azetidine Derivatives

The family of azetidine compounds illustrated bycompounds 90 and 91 (Fig 7) was developed at Aventis byDaniel Achard and colleagues and was described in a seriesof patents [165-168] They claimed the IC50 values of thesecompounds for the CB1 receptor to be less than or equal to100 nM These values were obtained following the proceduredescribed by Kuster et al [169] The antagonist propertywas shown using an in-vivo model the reversal ofhypothermia induced by CP-55940 in mice and the ED50obtained were lower than 50 mgkg

6 Aryl-Imidazolidine-24-Diones Derivatives

In 1999 the Didier Lambert team published a paperdescribing the affinity of 24 new 3-alkyl-55rsquo-diphenyl-imidazolidine-24-dione derivatives for the hCB1 receptor[175] The preliminary structure-activity relationships

N

SO

O

Cl

Cl

F

F

N

SO

O

Cl

NS

90 91

Fig (7) CB1 cannabinoid receptor antagonists 5 Azetidine derivatives Two examples (90 -91 ) of the azetidine derivatives developedby Aventis

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1375

Table 8 CB1 Cannabinoid Receptor Antagonists 6 Aryl-Imidazolidine-24-Diones Derivatives

Structures affinities ([3H]-SR141716A hCB1-CHO cells) and pKb values ([35 S]-GTPγS HU210 rat brain) of three aryl-imidazolidine-24-dionederivatives (92-94)

N

NH

OO

R

Br

Br

Cpd ndeg R Ki (nM) a pKb b

DML20 92 ethylmorpholine 70 611

DML21 93 1-hydroxypropyl 103 625

DML23 94 heptyl 98 574

a [175]b [177]

showed that the substitution at the nitrogen in position 3 aswell as a bromine atom in para of the phenyl rings aremandatory for the compounds affinity Later on threecompounds termed DML20 (92) DML21 (93) and DML23(94) were characterised as neutral antagonists using the [35S]-GTPγ S assay on rat cerebellum membranes [176] (Table 8)In the same model they competitively inhibited HU-210-induced [35S]-GTPγ S binding with pKB values of 611625 and 574 respectively Moreover these compoundswere proven to be quite selective for the CB1 receptor

higher than the one for the CB2 cannabinoid receptor (IC50 =300 nM) The inverse agonism of the compound wasextrapolated by inhibiting CP-55940-induced hypothermiain mice (EC50 = 18 nM) Moreover one oral dose (10mgkg) reduces the food intake in diet-induced obese ratsInterestingly food intake of CB1

-- mice was not alteredBody weight loss was maintained all over the study during achronic administration (14 days) of 10 mgkg of Cpd A

Later on Merck published a patent [180] in which someof the 45-diaryl-pyrazoles derivatives previously describedwere claimed to be useful in the treatment or the preventionof obesity in association with compounds inhibiting the11β-hydroxysteroid dehydrogenase type 1 (1β-HSD1)enzyme The 1β-HSD1 enzyme is responsible for thesynthesis of cortisol increased levels of which according tothe inventors are associated with obesity However neitherresults on appetite suppression nor on weight loss weregiven even if some indications on the affinity of thecompounds for the cannabinoid receptors were given

Very recently the functionality of these DML compoundswas explored using the [35S]-GTPγ S assay both on rat andhuman CB1 cannabinoid receptors [177] The data obtainedconfirm that the 3-alkyl-55rsquo-diphenyl-imidazolidinedionederivatives behave as neutral antagonists on rat CB1cannabinoid receptor (rat brain) However DML 20 DML21and DML23 acted as inverse agonists on the human receptor(hCB1-CHO cells) Furthermore the authors showed thatthis different functionality is not due to the hCB1 receptorlevel of expression in the recombinant cell line as whateverthe level of expression (Bmax of 44 pmolmg or 32pmolmg) DML derivatives behaved as inverse agonists ofthe hCB1 cannabinoid receptor

Another imidazole ring pattern of substitution wasproposed by Hagmann and collaborators [181] Thecompounds described 43 examples are given possess twophenyls the first one in position 1 linked to the nitrogenand the second one in position 2 The carboxamidesubstituent is in position 4 instead of in position 2 for thediarylimidazoles described by Finke These 12-diaryl-imidazoles are claimed to be CB1 cannabinoid receptorantagonists or inverse agonists but the absence of precisepharmacological values do not allow any comparisonbetween the two substitutions patterns However a samepattern of substitution was used by other researchers Amongthem Dyck et al described in 2004 ten 1-(4-chlorophenyl)-2-(24-dichlorophenyl)-imidazole derivatives varying by theircarboxamide substituent in position 4 (101-103 Table 9)[152] For instance with a 3-azabicyclo[330]octan-3-ylsubstituent (101) they obtained a Ki value of 14 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) In a [35S]-GTPγ Sassay 101 exhibited an IC50 value of 19 nM

7 Diarylimidazoles Derivatives

Finke and colleagues at Merck developed a new class ofCB1 receptor antagonists based on an imidazole nucleusOver eighty 45-diaryl-imidazoles derivatives having CB1antagonist properties were claimed in a patent published in2003 [178] Some representative compounds of the patent areillustrated in Table 9 (95-100) The affinity of the newcompounds was determined using [3H]-CP-55940 andhCB1-CHO cells Although no detailed pharmacologicaldata were given in the patent in-vitro and in-vivo propertiesof one compound termed Cpd A (100) have been describedelsewhere [179] Its affinity for the CB1 cannabinoid receptorwas shown to be nanomolar (IC50 = 4 nM) and 75 times

1376 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 9 CB1 Cannabinoid Receptor Antagonists 7 Diaryl-Imidazole Derivatives

Structure and affinity range of some 45-diaryl-imidazole derivatives for the human cannabinoid receptors determined using [3H]-CP-55940 asradioligand and hCB1amp2-CHO cells

N

NR2

R1

R4

R3

ndeg R1 R2 R3 R4 hCB1 (IC50 or Ki) hCB2 (IC50 or Ki) References

95 CH3 CO-NH-piperidinyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

96 CH3 CO-NH-piperidinyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

97 CH3 CO-NH-cyclohexyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

98 CH3 CO-NH-phenyl 24-di-Cl-Ph 4-Cl-Ph 10 ndash 100 nM a 100 ndash1000 nM a [178]

99 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

100 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph 4 nM a 300 nM a [179]

101 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

CH3 14 nM b ND [152]

102 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

H 66 nM b ND [152]

103 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 85 nM b ND [152]

104 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 23 nM b 542 nM b [182]

105 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 30 nM b 608 nM b [182]

106 4-Br-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 60 nM b 489 nM b [182]

107 4-CF3-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 29 nM b 634 nM b [182]

108 4-Cl-Ph 24-di-Cl-Ph CO-NH-morpholinyl CH3 197 nM b 3297 nM b [182]

109 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidin-4-ol CH3 172 nM b 3959 nM b [182]

110 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CN 30 nM b 1590 nM b [182]

111 4-Cl-Ph 24-di-Cl-Ph CO-NH- piperidinyl CH2F 36 nM b 906 nM b [182]

112 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH2-CH3 14 nM b 430 nM b [182]

113 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Cl 27 nM b 823 nM b [182]

114 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Br 23 nM b 746 nM b [182]

115 4-Cl-Ph 24-di-Cl-Ph CO-NH-tetrahydroisoquinoline CH3 34 nM b 696 nM b [182]

116 4-Cl-Ph 24-di-Cl-Ph CO-NH-cycloheptyl CH3 35 nM b 349 nM b [182]

117 24-di-Cl-Ph 4-Cl-Ph CO-NH-piperidinyl CH3 403 nM b 208 nM b [182]a IC50 value b Ki value

Lange and co-workers from Solvay described twenty-eight imidazole derivatives [182] The 24-dichlorophenylsubstituent at position 2 was kept constant while thesubstitutions at the other positions were explored (Table 9)Position 5 can accommodate a large range of littlesubstituents like hydrogen (104) methyl (105) ethyl (112)chlorine (113) bromine (114) fluoromethyl (111) or cyano(110) without major changes in the affinity At position 4more bulky substituents like 1234-tetraisoquinoline (115)or cycloheptyl (116) were well tolerated whereas thepresence of a more hydrophilic moiety such as morpholine(108) or piperidin-4-ol (109) was detrimental to the affinity

Interestingly position exchange between the 24-diphenyland the 4-chlorophenyl substituents led to a 13 fold loweraffinity with Ki values of 30 and 403 nM for 105 and 117respectively ([3H]-CP-55940 hCB1-CHO cells) In thefunctional assay which was inhibition of WIN-55212-2-induced [3H]-arachidonic acid release by hCB1-CHO cellsall tested compounds behaved as antagonists Compound105 showed a pA2 value of 86 which is the same theauthors obtained for SR141716A It would be interesting toassess if these compounds are true antagonists or if they actmore as inverse agonists Compound 105 when administeredto rats was able to inhibit CP-55940-induced hypotension

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1377

with an ED50 value of 24 mgkg which is close to the 3mgkg obtained for SR141716A The compound was alsoactive in another in-vivo model the WIN-55212-2-inducedhypothermia in mice Compound 104 which has nosubstituent in position 5 of the imidazole ring was lessactive in the hypotension model and inactive in thehypothermia model Thus a methyl group is the preferredsubstituent in position 5 of the imidazole moiety Otherproperties either experimental (P-glycoprotein affinitylogPHPLC) or computational (molecular volume polarsurface area) obtained for compounds 105 were similar tothose obtained for SR141716A Finally molecularmodelling studies revealed a close structural overlap betweenthe two compounds

value of 197 nM is selective for hCB1 and is as potent asSR141716A The structural modulations of 118 led to someinteresting compounds (Table 10) Six compounds possess aKi value for the hCB1 cannabinoid receptor equal to or lowerthan 25 nM antagonist properties in the inhibition of [3H]-arachidonic acid release by WIN-55212-2-stimulated hCB1-CHO cells and Ki values for the hCB2 receptor over 1microMThe diaryl-pyrazoline derivatives contained a chiral center atposition 4 The authors resolved the racemic mixture of twocompounds (121 and 130) The levorotatory enantiomersappeared to be the eutomers For compound SLV319levorotatory enantiomer of 121 the Ki and pA2 values were78 nM and 99 respectively The Ki and pA2 values ofSLV326 levorotatory enantiomer of 130 were 359 nM and9 respectively

8 34-Diaryl-Pyrazoline Derivatives Two in-vivo models CP-55940-induced hypotension inrat and WIN-55212-2-induced hypothermia in mouse wereused to assess the potential of these compounds The resultsare of the same order of magnitude than those obtained forSR141716A For instance in the hypotension modelSLV319 ((-)121) and SLV326 ((-)130) showed ED50 values

Lange and collaborators also discovered 34-diaryl-pyrazoline derivatives after a screening of compoundsresembling to SR141716A [183184] Compound 118 thatwas initially identified during the screening possesses a Ki

Table 10 CB1 Cannabinoid Receptor Antagonists 8 34-Diaryl-Pyrazolines Derivatives

Structure affinity for the hCB1 and hCB2 cannabinoid receptors (Ki nM) expressed in CHO transfected cells ([3H]-CP-55940) and potency (pA2) ofsome 34-dirylpyrazolines derivatives Table adapted from [184]

NN

N N

SO2

R3

R2

Cl R1

R4

n˚ R1 R2 R3 R4 Ki hCB1 (nM) Ki hCB2 (nM) pA2 (CB1)

118 H H H 4-CH3 197 gt 1000 84

119 H H H 246- CH3 24 gt 1000 94

120 H H H 4-Cl 16 gt 1000 95

121 H CH3 H 4-Cl 25 gt 1000 87

122 H CH3 CH3 4-Cl 280 gt 1000 85

123 H H H 4-F 53 gt 1000 9

124 H CH3 H 4-F 338 gt 1000 85

125 H CH3 CH3 4-F gt 1000 gt 1000 lt 75

126 H CH3 H 3-Cl 14 gt 1000 86

127 4-Cl CH3 H 4-Cl 255 ND ND

128 4-F CH3 H 4-Cl 584 ND ND

129 H CH3 H H 170 ND 75

130 H CH3 H 4- CF3 221 gt 1000 93

SR141716A 25 1580 86

1378 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

N

HN

O

N

Cl Cl

Cl

N

N

HN

O

Br

Br131 132

Fig (8) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Chemical structures of two 56-diaryl-pyrazine-2-amide derivatives (131-132) developed as CB1 cannabinoid receptorantagonists by AstraZeneca

of 55 and 2 mgkg (po) respectively proving their in-vivoefficacy after oral administration The authors alsodemonstrated that SLV319 is devoid of affinity for the P-glycoprotein pump and that it possessed a goodCNSplasma ratio (17) These two compounds (SLV319and SLV326) were chosen as development candidates bySolvay and entered clinical Phase I trials in 2003 [185]

132 Fig 8) useful as CB1 cannabinoid receptor antagonists[186 187] The activity of twenty derivatives wasdetermined using a [35S]-GTPγ S assay and hCB1-CHOtransfected cells The concentration required to give halfmaximal inhibition of CP-55940-induced [35S]-GTPγ Sbinding (IC50) is lower than 200 nM for the preferredcompounds No affinity data were given for thesecompounds

9 Diaryl-Pyrazine Diphenyl-Pyridine Diphenyl-Phenyland Diaryl-Pyrimidines Derivatives

The same year in a patent from Merck Finke andcollaborators described several 56-di-phenyl-pyridinederivatives as hCB1 antagonists or inverse agonists [188]The compounds have a substituent in position 3 of thepyridine core (Fig 9) This substituent could be a cyano(133-134) or a nitro group a halogen an ester or an amide(135-136) Over 150 compounds were synthesised toillustrate the invention but no pharmacological data wasdisclosed in the patent

In 2003 appeared several patents describing new familiesof compounds that bind to the CB1 cannabinoid receptorThese compounds have in common a central six atomsaromatic ring which could be a pyrazine a pyridine aphenyl or a pyrimidine and that is substituted by at leasttwo phenyl rings

The first patents were from Berggren and collaboratorsfrom AstraZeneca who published two patents describing thesynthesis of 56-diaryl-pyrazine-2-amide derivatives (131-

A few months later another patent was taken by Sanoficlaiming the antagonist properties of 56-di-phenyl-2-pyridine carboxamide derivatives [189] These compounds

N

NH

O

Cl

Cl

N

O

Cl

O NH

N

O

Cl

ClCl

NH

CN O

N

Cl

Cl

Cl

OHN

N

O

Cl

ClCl

CNF

F

133 134

135 136 137

Fig (9) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives 56-Di-phenyl-pyridine derivatives patented by Merck (133-136) and by Sanofi (137) as CB1 cannabinoid receptorsantagonists

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1379

NH

O

N

Cl

ClCl

NH

O

N

Cl

F3CCl

N

N

O

Cl

Cl Cl F138 139 140

Fig (10) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Representative structures of the diphenyl-phenyl (138-139) and 45-diphenyl-pyrimidine (140) derivatives developed ascannabinoid antagonists at Sanofi and Merck respectively

28 examples are given structurally related to the diphenyl-phenyl derivatives have their pyridine core substituted bytwo phenyl rings and by an amide moiety (137 Fig 9) TheIC50 values were lower than 100 nM but no data were givenshowing the antagonist properties although the compoundswere assayed in the adenylate cyclase inhibition assay

10 Other Derivatives

Several amide derivatives structures recently appearedamong the CB1 cannabinoid receptor antagonists In a seriesof patents taken by Merck in 2003 Hagmann and colleaguesdescribed a large amount of compounds obtained by parallelsynthesis which are claimed to be CB1 cannabinoid receptorantagonists [192-195] They were synthesised by reacting alibrary of substituted amines with a library of carboxylicacids (141-144 Fig 11) However as no value was givenfor the affinity or activity of these amides we will notfurther discuss this class of compounds

Along this line Sanofi Synthelabo developed newdiphenyl-phenyl derivatives claimed to have CB1cannabinoid receptor antagonist properties [190] They arebased on a central phenyl ring substituted by two phenylsand by an amide moiety (138-139 Fig 10) Thirteencompounds were claimed along with their synthesis TheirIC50 values were lower than 100 nM ([3H]-CP55-940hCB1-CHO cells) but no other specific data (selectivitypA2hellip) was given

Several 15-diaryl-pyrrole-3-carboxamide derivatives weresynthesised and claimed to be CB1 cannabinoid receptorantagonists by Berggren et al [196] The affinity valueswere not disclosed in the patent (145-146 Fig 12) Otherpyrrole derivatives were synthesised by Guba et al atHofmann-La Roche [197] More specifically 2-(thiazol-4-yl)pyrrole derivatives were claimed as CB1 cannabinoidreceptor antagonist (IC50lt2microM) (147-148 Fig 12)

Finally Kopka et al from Merck described more thanone hundred 45-diaryl-pyrimidine derivatives as CB1cannabinoid receptor antagonists (140 Fig10) [191]However no precise data were provided concerning theaffinity and functionality of the compounds

R2

R1N

R3

R4

R5

O

NH

O

Cl

O F

F

R2

R1NH

R3

R4

HO R5

O

NH

O

Cl

CN

NH

O

Cl

O

O

NH

O

Cl

Cl

+

141142

143 144

Fig (11) CB1 cannabinoid receptor antagonists 10 Other derivatives Retrosynthetic scheme of a CB1 cannabinoid antagonistlibrary of amide derivatives developed at Merck The structures of four representative compounds are illustrated (141-144)

1380 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

NH

O N

Cl

Cl

Cl

N

NH

O

OMe

MeO

N

NH

O

N

S

MeO

N

NH

O

N

S

MeO

145 146

147 148

Fig (12) CB1 cannabinoid receptor antagonists 10 Other derivatives Two examples of 15-diaryl-pyrrole derivatives described byBerggren et al (145-146) While the 2-(thiazol-4yl)pyrroles derivatives 147 and 148 were described by Guba et al from Hofmann-LaRoche All these pyrroles derivatives were claimed to be CB1 cannabinoid receptor antagonists

Another new structure is represented by the 124-triazolederivatives developed by Jagerovic and collaborators In arecent paper were described the synthesis andpharmacological properties of five new 15-diphenyl-3-alkyl-

triazole derivatives [198] Among these compounds onlyone (149) behaved as a CB1 antagonist inhibiting the WIN-55212-2-induced contractions in a mouse vas deferenspreparation However despite its effects in isolated tissue

Table 11 CB1 Cannabinoid Receptor Antagonists 10 Other Derivatives

Structure and affinity of diaryl-triazole derivatives

N

NN

R3

R2

R1

Cpd R1 R2 R3 Ki CB1 (nM) References

149 24-diCl 4-Cl hexyl 855 a [198]

150 24-diCl 4-Cl2-OMe CO-NH-(3-azabicyclo[330]octan-3-yl) 270 b [152]

151 24-diCl 4-Cl CO-NH-(3-azabicyclo[330]octan-3-yl) 164 b [152]

152 4-Cl 24-diCl CO-NH-(3-azabicyclo[330]octan-3-yl) 137 b [152]

153 4-Cl 24-diCl CO-NH-benzylpyrrolidin-3-yl 29 b [152]

154 4-Cl 24-diCl CO-NH-1-(4-chlorophenyl)ethyl 66 b [152]

155 4-Cl 24-diCl CO-NH-piperidinyl 356 c [182]

156 24-diCl 4-Cl CO-NH-piperidinyl 382 c [182]

a [3H]-SR141716A rat cerebellar membranesb [3H]-CP-55940 hCB1-HEK EDNA cellsc [3H]-CP-55940 hCB1-CHO cells

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1381

N

S

O

NH

Cl

Cl

Cl

F

N

S

O

NHN

ClCl

Cl

N

S

O

NH

Br

N

S

O

NHN

Cl

Cl

Cl

156 157 158 159

Fig (13) CB1 cannabinoid receptor antagonists 10 Other derivatives Structures of 45-diaryl-thiazole derivatives patented as hCB1cannabinoid receptor antagonists by Solvay Pharmaceuticals (156 157 158) or by AstraZeneca (157 158 159)

assays (mouse vas deferens and guinea pig ileum) thecompound possessed only a reduced rCB1 affinity with Kivalues of 855 and 748 nM using [3H]-SR141716A and [3H]-WIN-55-212-2 respectively (Table 11) Further four seriesof compounds differing by the nature of the substituentaround the triazole core were also described in a patent[199] Usually the compounds claimed possess two aromaticrings possibly substituted and one linear alkyl chain Theaffinity for the cannabinoid receptors was not givenNevertheless 149 was evaluated in two isolated tissuemodels the inhibition of the electrically evoked contractionsin the guinea pig ileum and in the mouse vas deferens Inthe two models compound 149 inhibited the effect of WIN-55212-2 but had no effect by itself In the vas deferensmodel the authors obtained a pA2 value of 748 to becompared with 763 obtained for AM251

Yet another five membered ring a thiazole has beenused as central moiety of a new class of cannabinoid ligandsThe thiazole ring is substituted by two aryls in position 4and 5 and by an amide (position 2) as in compounds 156-159 (Fig 13) Forty derivatives of this type weresynthesised by Lange and colleagues from SolvayPharmaceuticals and claimed in a patent as agonists orantagonists of the CB1 cannabinoid receptor [200] Nopharmacological data were provided however their activitywas assessed by measuring the cAMP in transfected hCB1-CHO cells In a recent paper from Lange et al two of thesethiazole derivatives were further described [182] Compound13 (157) the most closely related to SR141716A has a Kivalue of 227 nM while for compound 14 (158) Ki value isover 1000 nM Thus as in the imidazole series (see Table9) the aromatic substitution pattern is of great importancefor the CB1 cannabinoid receptor affinity Compound 13however was devoid of in vivo activity

Others 124-triazole derivatives were described by Dycket al compounds 150-154 unlike the Jagerovic ones havean amide moiety in position 3 [152] The highest affinitywas obtained with a 1-benzyl-pyrrolidin-3-yl substituentThe Ki value was 29 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) to be compared to 12 nM obtained for theSR141716A

Seventeen 45-diaryl-thiazole derivatives were alsosynthesised by Berggren et al and patented by AstraZeneca(157-159 Fig 13) [201]

In a patent from Merck Toupence et al described over190 substituted furo[23]pyridines claimed to be CB1cannabinoid receptor antagonists [202] The affinity (IC50 lt1microM) was measured using recombinant CHO cells and [3H]-CP-55940 and the activity using cAMP dosage (160 Fig14)

Lange et al described a triazole derivative (155) whichcan be superimposed with SR141716A Compound 155showed a Ki value of 356 nM ([3H]-CP-55940 hCB1-CHOcells) a pA2 value of 83 in the inhibition of WIN-55212-induced release of [3H]-arachidonic acid by hCB1-CHO cellsand was active in vivo in the CP-55940-inducedhypotension in rat (ED50 = 236 mgkg) [182]

Very recently Alanine from Hoffmann-LaRoche foundthat diarylbenzo[13]dioxole derivatives act as CB1

O

O

S

N

OO

F

ClCl

O

O

F O

N

OON

NH2

O

Cl

Cl

Cl

160 161 162

Fig (14) CB1 cannabinoid receptor antagonists 10 Other derivatives One example of a furo[23]pyridine derivative (160) and twoexamples of diarylbenzo[13]dioxole derivatives (161-162) described as hCB1 cannabinoid receptor antagonists by Toupence et aland Alanine et al respectively

1382 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

cannabinoid receptor antagonists (161-162 Fig 14) [203]Approximate IC50 values for twelve compounds ([3H]-CP-55940 hCB1-HEK cells) among the three hundred andeighty compounds described were given (IC50lt2microM)

et al in 1995 [207] In their hands ∆9-THC was not able toinhibit the forskolin-stimulated adenylyl cyclase activity inan assay performed on hCB2-CHO cells Subsequently theyshowed that ∆9-THC was able to antagonise HU210-inducedinhibition of adenylyl cyclase activity thus acting as anantagonist of the hCB2 cannabinoid receptor expressed inCHO cells [208] Furthermore very recently Govaerts et al[209] reported an inverse agonist effect of ∆9-THC and ∆8-THC on hCB2-CHO cells using the [35S]-GTPγ S assayThe pEC50 values were 763 and 888 respectively whilethe Emax values were ndash27 and ndash16 as compared to basal

Griffith from Pfizer described three new series of CB1cannabinoid receptor antagonists based on a bicyclic centralaromatic ring Purine pyrazoltriazines andpyrazolo[15a]pyrimidines derivatives were synthesised andinvestigated for their ability to interact with the CB1cannabinoid receptor Albeit a [35S]-GTPγ S assay wasdescribed no data were given concerning the antagonist orinverse agonist properties of such compounds Two hundredand eighty purine derivatives were synthesised [204] Theaffinities of 163 and 164 were given in the patent (28 and12 nM [3H]-SR141716A) In a following patent thesynthesis of sixty pyrazoltriazine derivatives was described(165 Fig 15) [205] The affinity of each compound was notgiven but was said to be ranging between 01 and 590 nMAnd finally more than forty pyrazolo[15a]pyrimidines weresynthesised (166 Fig 15) [206] Their affinity for the CB1cannabinoid receptor was lower than 155 nM Despite thefact that a great number of derivatives share the samesubstitution pattern around the central bicyclic aromatic ringthe comparison of these three new series of compounds ismade difficult by the absence of precise affinity data

Some other classical cannabinoids were shown to possessan antagonist activity at the CB2 receptor This is the casefor instance for the non-selective O-1184 (2) (Ki = 74 nM)which enhances the forskolin-induced cAMP production inCB2-CHO cells (EMax = 161 EC50 = 63 nM) and alsofor O-584 (3) (EMax = 246 EC50 = 138 nM) [39]

2 Indole Derivatives

Since the disclosure in 1993 of a second cannabinoidreceptor many attempts have been made to synthesise CB2cannabinoid receptor selective ligands The first compoundreported to have a somehow greater affinity for the peripheralcannabinoid receptor was WIN-55212-2 The compoundwas introduced by DrsquoAmbra and colleagues [210] back in1992 as a new cannabinoid ligand having a nanomolaraffinity and acting as an agonist In 1996 Showalter et alused a transfected cell line expressing the CB2 cannabinoidreceptor in order to identify selective ligands for thisreceptor [211] They showed that the CB2 affinity of WIN-55212-2 was 7 times greater than the CB1 with Ki valuesof 189 and 028 nM for the CB1 and CB2 cannabinoidreceptors respectively However WIN-55212-2 is anagonist of the cannabinoid receptors Interestingly WIN-55212-3 (167 Fig 16) the so-called inactive enantiomer ofWIN-55212 was recently shown to behave as a low affinity(Ki=363 microM) inverse agonist of the hCB2 cannabinoidreceptor as it decreased the [35S]-GTPγ S binding [209]

III CB2 LIGANDS RECEPTOR ANTAGONISTS ANDINVERSE AGONISTS

The compounds acting as antagonist or inverse agonistsat the CB2 cannabinoid receptor are reviewed in this thirdpart of the paper As for the CB1 cannabinoid receptorligands they are classified depending on their chemicalstructures

1 Classical Cannabinoid Derivatives

The first report of an antagonism mediated by ∆9-THC atthe peripheral cannabinoid receptor was made by Bayewitch

N N

NN

NH COOH

Cl

Cl

N N

NN

N

Cl

Cl

NH2OH

O

N

N

N

N

N

NHNH2

O

Cl

ClN

N

N

N

Cl

Cl

N

O

163 164

165 166

Fig (15) CB1 cannabinoid receptor antagonists 10 Other derivatives Examples of purine (163-164) pyrazoltriazines (165) andpyrazolo[15-a]pyrimidines (166) derivatives described as CB1 cannabinoid receptor antagonists by Griffith

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1383

The pEC50 value was 554 and the Emax value ndash26 ascompared to basal

AM630 also acts as an inverse agonist at the hCB1cannabinoid receptor (EC50 = 900 nM) as Landsman et aldemonstrated [47] Recently Zhang and colleagues reportedthe characterisation of the microsomal metabolism of AM630[212]

N

O

N

O

O

N

O

Cl

O

N

O

(S)-WIN-55212-3 167 BML-190 168

In 1996 Gallant and colleagues identified by submittinga large number of compounds to a binding assay anotherindole analog as CB2 cannabinoid receptor ligand [213] Onthe basis of this compound christened BML-190 (168 Fig16) or called indomethacin morpholinylamide theysynthesised several derivatives that possessed a selectivityratio for the CB2 cannabinoid receptor of up to 140 Thesewere the first compounds specifically designed to be CB2cannabinoid receptor ligands The first report on thepharmacological properties of BML-190 appeared onlyrecently New and colleagues highlighted in the paper theinverse agonist properties of the compound BML-190 dose-dependently increases (103 EC50= 980plusmn70 nM) theforskolin-stimulated levels of cAMP in hCB2-HEK cellswhile WIN-55212-2 decreases (44 45plusmn42nM) thisaccumulation [137]

Fig (16) CB2 cannabinoid receptor antagonists 2 Indolederivatives Chemical structures of WIN-55212-3 (167) andBML-190 (168) two indoles derivatives acting as inverseagonists at the hCB2 cannabinoid receptor Surprisingly in 2002 Melck and collaborators used

BML-190 as a CB2 receptor agonist in a cell proliferationassay [214] BML-190 was also tested as inhibitor of thecyclooxygenase-2 enzyme by Kalgutkar et al [215] Theyobtained IC50 values higher than 33 and 66 microM for theCOX-I and COX-II enzymes respectively These valueshave to be compared with the submicromolar activity ofindomethacin the parent compound Very recently Klegerisand colleagues [216] looking for an antineurotoxic action ofcannabinoids showed that BML-190 increases TNF-αsecretion by stimulated THP-1 monocytic cells but is noteffective on the IL-1β secretion

Further on the basis of WIN-54461 (12) a CB1antagonist AM630 (13) was synthesised by replacement ofthe bromine atom by an iodine one In the initial reportPertwee et al showed the antagonist effects of AM630 butalso suggested that the CB1 cannabinoid receptor may not bethe preferential receptor of AM630 [45] Later on Ross et alreported using hCB1amp2-CHO cells and [3H]-CP-55940that AM630 binds to the CB2 cannabinoid receptor (Ki =312 nM) with a selectivity ratio over 160 [48]Furthermore using a [35S]-GTPγ S assay they showed itsinverse agonist properties (EC50 = 76 nM) However

Table 12 CB2 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Reported binding affinities (Ki) and selectivity ratio (CB1CB2) of SR144528 (169) for rat and human cannabinoid receptors determined using [3H]-CP55940 as radioligand

NN

O

NH

Cl

SR144528 169

rCB2 (spleen) rCB1 (whole brain) Selectivity Ki hCB2 (transfected CHOcells)

Ki hCB1 (transfected CHOcells)

Selectivity References

038 nM 305 nM 800 06 nM 437 nM 730 [217]

067 nM 548 nM 80 [219]

56 nM gt10microM gt1780 [48]

25 nM a gt 1microM a gt400 [224]

024 nM 276 nM 115 199 nM 503 nM 25 [232]

a COS cells

1384 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

3 Diarylpyrazole Derivatives CHO cells is constitutively active and phosphorylated onSer 352 Upon treatment by SR144528 thisphosphorylation of the receptor was inhibited Moreover theSR compound was able to induce dephosphorylation ofagonist-induced CB2 phosphorylation The question whetherSR144528 induces a CB2 receptor conformational changemaking it a better phosphatase-specific substrate or ifSR144528 binding activates specific phosphatases remainsopen However this remains a nice example of inverseagonist-induced receptor resensitisation

The same Sanofi team which synthesised the CB1antagonist SR141716A reported in 1998 the synthesis andpharmacological characterisation of a CB2 cannabinoidreceptor selective antagonist the SR144528 (169) [217218] The structure of the new compound is derived fromSR141716A one SR144528 is the first selective CB2receptor antagonist reported as the inverse agonist propertiesof BML-190 were shown only recently by New et al [137]The authors reported for 169 using [3H]-CP-55940 asradioligand Ki values of 04 nM and 06 nM for the rat andthe human CB2 receptors respectively and found a CB1-CB2 selectivity ratio around 700 Table 12 sums up thebinding affinities of SR144528 for the cannabinoid receptorsobtained by different authors Interestingly while the CB2affinity is more or less constant the affinity obtained for theCB1 receptor varies by one order of magnitude [219] Theantagonist effect of SR144528 was highlighted by evaluatingits effect on cAMP production by hCB2-CHO cells exposedto 3 nM CP-55940 (EC50=10nM) The antagonism wasalso observed on the activation of the MAP kinase pathwayby CP-55940 (IC50=39nM)

The Sanofi research team working on cannabinoidsconducted investigations in order to determine the keyresidues for SR144528 interaction with the receptor (Table13) as well as the binding mode of the compound Firstlythey identified the TM4-EL2-TM5 region as a regioncontaining crucial residues for the affinity of several ligandsamong them SR144528 [224] Among these residues themutation of two cysteine of the second extracellular loop(C174 and C179) abolishes the affinity for the CB2 receptorLater on the implication of S453(161) and S457(165) inthe binding of SR144528 was evidenced by mutationalstudies In the model proposed for the docking of SR144528into the CB2 receptor the crucial contacts are comprised intothe TM4 this is the case for S453(161) and S457(165)which are proposed to form hydrogen bonds with the ligandand W464(172) proposed to have aromatic interactions withthe 4-methylbenzyl of the SR144528 [225] In 2003 Fengand Song showed that point mutations D349(130)A andA634(241)E abolished ligand binding However mutationof the arginine R350(131) member of the highly conservedDRY motif to alanine has no influence on SR144528binding [226]

Early after the report of the synthesis and characterisationof SR144528 the same authors further explored the functionof this compound and showed its inverse agonist propertiesusing CB2-CHO cells co-transfected with the luciferasereporter gene with either the CRE or the murine krox24regulatory sequence [220] In these models SR144528 notonly inhibits CP-55940 effects but also reproduces theeffects of a pertussin toxin treatment proving its inverseagonist properties SR144528 functionality was alsoexplored by [35S]-GTPγ S binding in hCB2-CHO cells(IC50=3 nM) [221] The inverse agonist function ofSR144528 was further illustrated by Rhee and Kim usingCOS cells co-transfected with hCB2 receptor and adenylylcyclase [222]

As for the CB1 cannabinoid receptor ligands Sanofideveloped tricyclic derivatives based on the 1-benzylpyrazole-3-carboxylic scaffold More than twentycompounds were disclosed in a patent [227] in 2001 (84-86Table 7) The benzyl in position 4 characteristic of the CB2diarylpyrazoles from Sanofi is conserved as well as thebicyclic residue in position 3 The length of the second linkbetween the phenyl and the pyrazole can be made of one or

Bouaboula and co-workers investigated SR144528 effectson CB2 cannabinoid receptor phosphorylation status [223]Firstly they showed that the CB2 receptor transfected in

Table 13 CB2 Cannabinoid Receptor Antagonists 3 Di-Aryl-Pyrazole Derivatives

Some of the single point mutations reported for the hCB2 cannabinoid receptor for which pharmacological data concerning the SR144528 are available

Mutant Effect References

R350(131)A no effect [226]

S453(161)A loss of affinity [225]

V456(164)I no effect [225]

S457(165)A loss of affinity [225]

C174S a loss of affinity [224 225]

C175S a loss of affinity [225]

C177S a no effect [225]

C179S a loss of affinity [224 225]

S544(193)G no effect [225]

a Second extra-cellular loop

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1385

two methylene but according to the patent one methylenebridge is preferred The Ki values of these compounds for theCB2 receptor were said to be lower than 500 nM and theirantagonist properties have been assessed using the adenylatecyclase inhibition assay

compounds by the presence of an alkyl substituent inposition 5 instead of a phenyl and of course by position 3which remains unsubstituted However except a lecture atthe 1998 ACS meeting [230] where compound 171 wasreported to have a Ki value of 835 nM and a selectivity ratioover 100 no further report on these compounds waspublished untill now

In a recent paper Mussinu and colleagues reported thesynthesis of other tricyclic derivatives starting fromSR141716A structure [228] N-piperidin-1-yl-14-di-hydroindeno[12-c]pyrazole-3-carboxamide derivatives areCB2 receptor ligands and some of them possess high affinityand selectivity for the CB2 receptor as shown in Table 7 (79-83) These tricyclic derivatives differ from the Sanofi tricyclicderivatives as they do have a phenyl ring instead of a benzylin position 1 Another difference is the nature of thesubstituent in position 3 which is not a bicycloalkyl but acyclic amine One of the compounds synthesised byMussinu et al 81 possesses the highest affinity for the CB2receptor ever reported with a Ki value of 0037 nM for themurine CB2 receptor ([3H]-CP-55940 mice spleenhomogenate) Interestingly 79 the closest SR141716Aanalog lost almost all its affinity for the CB1 receptor andincreased by four orders of magnitude its affinity for the CB2receptor Even if the structure quite similar to the SRcompounds could suggest that these derivatives behave asantagonists no pharmacological data were given in the paperto support this hypothesis

5 2-Oxoquinoline Derivatives

Inaba and colleagues from Japan Tobacco introduced in2001 a completely new structure in the cannabinoid fieldbased on a 2-oxoquinoline scaffold Over eighty derivativeswere disclosed in a patent [231] and one compound JTE-907 (173) was subsequently characterised in-vitro as well asin-vivo [232] In Table 14 are listed some of the compounds(172-178) disclosed in the patent along with their affinity forthe cannabinoid receptors These values were obtained onhuman receptors against [3H]-WIN-55212-2 For twelvecompounds the anti-inflammatory activity per os wasassessed in a carrageenin-induced paw edema model Thehighest effect reported was obtained with JTE-907 with anED50 value lower than 01 mgkg Therefore thesecompounds were claimed to be useful as anti-inflammatoryand anti-allergic agents

To further characterize JTE-907 the authors determinedits affinity for cannabinoid receptors of different species using[3H]-CP-55940 as radioligand In their hands JTE-907behaved as a very selective hCB2 ligand with Ki values of36 and 2370 nM for the CB2 and CB1 cannabinoid receptorsrespectively even more selective than the SR144528 (Kivalues of 199 and 50 nM respectively) However it has tobe mentioned that the affinities reported in the paper forJTE-907 are quite different from those reported in the patent(hCB1 Ki values of 35nM and 009 nM respectively) Italso appears that the SR144528 affinity for the CB1 receptordetermined by Iwamura et al is higher than the affinityreported by Rinaldi-Carmona et al with values of 50 and437nM respectively The authors ascribed the difference tothe variability of SR144528 binding affinities to the assayconditions The function of JTE-907 was evaluated bymeasuring the increase in cAMP production by forskolin-stimulated hCB2-CHO cells Maximum stimulation wasattained at a concentration of 1microM of JTE-907 In the sameassay WIN-55212-2 decreased the cAMP production in adose-dependent manner

Finally it is interesting to strengthen that the five-membered ring derivatives (79-86) of the diarylpyrazolefamily are reported to possess a high CB2 receptor affinitywhile the six- and seven-membered ring derivatives (69-78)are described as CB1 receptor ligands

4 Other Pyrazole Derivatives

Other CB2 ligands based on a pyrazole nucleus wereintroduced by SmithKline Beecham in a patent describingthe synthesis of about sixty new compounds [229] Theiraffinity (Ki) for the hCB2 receptor transfected in HEK293cells was reported to be ranging between 25nM and 10microMTheir structures (170-171 Fig 17) differ from the Sanofi

NN

O

N

ON

NN

N

NN

O

N

ON

NN

N

ClCl

1

5

4 3

2

170 171

Fig (17) CB2 cannabinoid receptor antagonists 4 Otherpyrazole derivatives Structure of two compounds (170-171)among those claimed by SmithKline Beecham to be hCB2antagonists

In conclusion JTE-907 was shown to be a CB2 selectiveinverse-agonist with anti-inflammatory and anti-allergicproperties This compound is currently undergoing Phase Iclinical trials as anti-allergic drug [233]

IV NON-CB1 NON-CB2 CANNABINOIDRECEPTORS LIGANDS

In the last years the possible presence of additional non-CB1 non-CB2 cannabinoid receptors was raised frompharmacological evidences

One of the putative receptors possesses an endotheliallocalisation while other ones are localized in the CNS or atperipheral nerves For instance an additional target for ∆9-

1386 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 14 CB2 Cannabinoid Receptor Antagonists 5 2-Oxoquinoline Derivatives

Structure of six compounds among the oxoquinoline derivatives patented by Japan Tobaco Affinity (Ki nM) and selectivity (CB1CB2) for the cannabinoidreceptors are given The affinity was measured by binding against [3H]-WIN-55212-2 on either hCB2-CHO or hCB1-CHO cells Adapted from [231]

Cpd ndeg Structure hCB2 hCB1 Selectivity

1-2 172

NH

MeO O

O

OMeO

0014 3671 262215

JTE-907 173

NH

O

MeO O

O

NH

O

O

0087 3436 39495

2-2 174

OH

O

NH

MeO O

O 077 3247 4215

3-27 175

NH

O

F

NH

MeO O

O0021 336 16000

3-32 176

NH

MeO O

O

NH

F

O

0036 2398 66610

3-38 177

O

O

NH

N

O

O

NMeO

0085 935 11000

3-43 178

NMeO O

O

NH

F

O

0043 864 20100

tetrahydrocannabinol was described in capsaicin-sensitivesensory nerves by Zygmunt et al [234]

[29] and by Di Marzo et al [235] Thus in the presentreview we will focus on the description of the so faridentified ligands of the non-CB1 non-CB2 receptors withan emphasis on the compounds having antagonist properties

Pharmacological evidences for the existence of additionalcannabinoid receptors were reviewed by Wiley and Martin

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1387

(Table 15) In particular the knowledge about three not yetcloned targets has grown The growing molecularpharmacology of the three putative new cannabinoidreceptors is reviewed in the three following sections Thefourth section summarises the data on the other proposedcannabinoid targets

vasodilatator activity In addition the anandamide effect ispartially sensitive to SR141716A inhibition The authorsproposed the existence of two distinct receptors responsiblefor the vasodilatatory effect of anandamide one located in theendothelium is SR141716A-sensitive while the other onelocated on the vascular smooth muscle is SR141716A-insensitive Thus SR141716A is an antagonist of anendothelial anandamide receptor Several studies wereconducted in order to understand the molecularpharmacology of the endothelial anandamide receptor It ispertussis toxin-sensitive [237] Cannabidiol acts asantagonist and abnormal-cannabidiol (Abn-cbd 179) and itsanalogue O-1602 (180) act as agonists [238] The synthesisof O-1602 and of its analogues are disclosed in a patentdescribing the vasodilatatory activity of cannabinoid

1 Endothelial Anandamide Receptor ndash Abnormal-Cannabidiol Receptor

In 1999 Wagner et al showed that anandamide couldinduce mesenteric vasodilatation through an endotheliallylocated ldquoanandamide receptorrdquo pharmacologically distinctfrom CB1 [236] Other cannabinoids such as ∆9-THC HU-210 or WIN-55212-2 were devoid of mesenteric

Table 15 Non-CB1 Non-CB2 Cannabinoid Receptors Antagonists

An overview of the putative new non-CB1 non-CB2 anandamide-receptors ligands The antagonists are highlighted in bold

NH

N

S

HO OH

Cl

OH

OH

OH OH OH OH

OMe

OMe

RR

RR

RR

RR

HO

OH

NH

O

cannabidiol (10) Abnormal-cannabidiol 179 O-1602 180

O-1918 181 capsazepine 183capsaicin 182

Compound Endothelial AEA receptor Brain AEA receptor Cannabinoid-vanilloid brain receptor

AEA agonist a agonist de

WIN-55212-2 no effecta

agonist e agonist g h

CP-55940 no effect e agonist i

SR141716A antagonist a b j no effect ([35 S]-GTPγS binding) d e f antagonist g h

AM251 no effect k no effect h i

Cannabidiol antagonist b no effect e

O-1918 antagonist c jm

Abd-cbd agonist b l

O-1602 agonist b

Capsaicin no effectj agonist i

Capsazepine no effect b antagonist h

a [236] b [238] c [240] d [249] e [250] f [251] g [254] h [258] i [ 255] k [244] j [ 243] l [ 242] m [241]

1388 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

analogues [239] More recently Offertaler et al showed thatO-1918 (181) a cannabidiol analog acts as an antagonist ofthe endothelial anandamide receptor [240] In addition O-1918 does not bind to the CB1 and CB2 cannabinoidreceptors Furthermore O-1918 unlike cannabidiol doesnot cause vasorelaxation in the mesenteric artery preparationmodel used and thus acts as a true neutral antagonistUsing O-1918 as a pharmacological tool Begg et alproposed a coupling of this new G protein-coupledendothelial receptor through a GiGo mechanism to ionchannels in primary cultured human vascular endothelialcells [241] However in their hands SR141716A wasineffective in antagonising Abd-cbd Ho and Hiley furtherdemonstrated that Abn-cbd relaxes rat small mesentericarteries through an activation of K+ channels via aSR141716A-sensitive pathway [242] Further using thesame model they characterized the vasorelaxant activity ofvirodhamine a novel endocannabinoid apparently actingthrough the same receptor [243] The virodhamine-inducedvasorelaxation is sensitive to SR141716A and O-1918 butinsensitive to AM251 SR144528 and AM630 Moreovervanilloid receptor desensitisation by capsaicin (182) had noeffect

244] Thus position 4 on the 5-phenyl could be furtherexplored to obtain selective ligands

To come to an end despite the fact that the endothelialanandamide receptor is neither identified nor cloned at leastthree antagonists are identified SR141716A cannabidioland O-1918 It is likely that a growing number of ligandsand therapeutic applications will appear in the very nearfuture

2 Brain Anandamide Receptor

Following an in-depth evaluation of anandamide effects inCB1

-- mice Di Marzo and co-workers suggested that a non-CB1 non-CB2 G protein-coupled receptor might mediatesome of the actions of AEA in mice [249] Actually effectsof AEA but not those of ∆9-THC were not decreased inCB1 cannabinoid receptor knock-out mice For instance 30mgkg of AEA were still effective in hot-plate test whereas10 mgkg of ∆9-THC had no effect on anti-nociceptionMoreover in a [35S]-GTPγ S assay AEA stimulated with anEC50 value of 223 microM the [35S]-GTPγ S binding to CB1

--

mice brain membranes (Emax= 28) ∆9-THC showed noeffect in this assay The addition of SR141716A did notaffect the [35S]-GTPγ S binding to CB1

-- mice brainmembranes Shortly after other evidences for the existence ofa new G protein-coupled cannabinoid receptor with a distinctdistribution in the central nervous system were published[250] From a set of 24 commonly used cannabinoids (AEAWIN-55212-2 JWH-030 THChellip) only AEA and WIN-55212-2 produced an increased binding of [35S]-GTPγ S onCB1

-- mice brain membranes Interestingly in this studySR141716A alone produced a significant inhibition of [35S]-GTPγ S binding similar to the inhibition obtained usingCB1

++ mice brain membranes (IC50 values of 57 microM and47 microM respectively) However SR141716A did not affectthe anandamide-induced [35S]-GTPγ S binding in CB1

--

mice brain membranes Moreover [3H]-SR141716Aexhibited significant binding in some brain regions of CB1

--

mice which are different from those exhibiting stimulation of[35S]-GTPγ S binding by WIN-55212-2 and AEA Thusthe authors proposed that the SR141716A effects on CB1

--

mice are mediated neither by the CB1 cannabinoid receptornor by the putative new anandamide brain receptor Monoryand co-workers using CB1

-- mice described a novel nonadenylyl cyclase-coupled cannabinoid binding site in micecerebellum [251] Two agonists AEA and WIN-55212-2but not ∆9-THC or HU210 were found to bind to thisreceptor as they enhanced [35S]-GTPγ S binding NeitherSR141716A nor SR144528 were able to affect the WIN-55212-2 induced [35S]-GTPγ S binding in CB1

-- mice asfound by Breivogel et al [250] However the distributionpattern of the receptor described by Monory et al differs fromthe distribution found by Breivogel et al It has to bementioned that the knock-out strains used were different Themice used by Monory were from a CD-1 strain while theone used by Breivogel were from a C57BL61 strainInterestingly Muthane et al detected differences in nigralneurons number and sensitivity to MPTP in the two mousestrains used [252]

More recently Mo et al obtained results showing thatthe Abnormal-cannabidiol receptor is also responsible for anincreased endothelial cell migration [244] Abn-cbd enhancedhuman umbilical vein endothelial cell migration while O-1918 antagonised Abn-cbd effect The CB1 cannabinoidreceptor inverse agonist SR141716A partially inhibitedAbd-cbd action whereas AM251 and SR144528 had noeffect

In another paper from George Kunos team Batkai et alreported that SR141716A is able to inhibit the endotoxichypotension by a cardiac mechanism involving neither theCB1 nor the CB2 cannabinoid receptors [245] Theinvolvement of a non-CB1 non-CB2 site of action wasshown using CB1

-- and CB1--CB2

-- knock-out miceFurther studies are needed to identify the SR141716Asensitive myocardial site of action

In a recent patent Kunos described O-1918 and itsanalogues as vasoconstrictor agents useful to reversepathological vasodilatation of blood vessels [246] Theeffects of O-1918 on mouse blood pressure on LPS-inducedhypotension in mouse as well as the reversal of hypotensionby O-1918 injection are described Furthermore thesynthesis of O-1918 based antagonists is described startingfrom p-metha-28-dien-1-ol and the appropriate resorcinols

Interestingly the Abd-cbd receptor which was firstdetected in the vascular system also seems to be present inthe CNS [247] Indeed Walter et al found that microglialcells (BV-2) migration induced by 1 microM 2-AG is inhibitedby cannabidiol (300 nM) and O-1918 (1 microM) SR141716Awas found ineffective in this assay Thus 2-AG is an agonistof the Abd-cbd receptor Moreover Abd-cbd is able to elicita dose-dependent cell migration in the same model (EC50 =600nM) [248]

From a more structural point of view SR141716Abehaves as an antagonist while on the opposite AM251(20) a close analogue which differs from the former only bythe nature of one halogen has no effect on this receptor [242

To conclude to our knowledge no compounds havingantagonist or inverse agonist properties at this receptor havebeen identified so far

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1389

3 Cannabinoid-Vanilloid Brain Receptor a cannabinoid-vanilloid receptor at which SR141716A andcapsazepine behave as antagonists

Evidences for the presence of an additional ldquocannabinoidrdquoreceptor differing from the CB1 receptor and from the brainanandamide receptor appeared from studies focused on thehippocampus (for a review see Hajos et al [253]) Hajos etal studied the cannabinoid actions on GABAergic andglutamatergic transmission using the whole-cell patch-clamp technique [254] WIN-55212-2 induced a dose-dependent reduction in glutamatergic transmission reversedby SR141716A (1microM) Thus the authors concluded that thecannabinoid actions on the glutamatergic transmission aremediated by a non-CB1 but SR141716A-sensitive receptorIn another paper Hajos and Freund further characterised thisputative receptor [255] They showed that in addition toWIN-55212-2 CP-55940 and capsaicin (182) a TRPV1receptor agonist also act as agonists Furthermore in theirhands SR141716A as well as capsazepine (183) a TRPV1receptor antagonist were able to antagonise this effect on theglutamatergic transmission while AM251 was notSR141716A effects on CB1 knock-out mice were furtherstudied using a test of anxiety [256] Haller et al showedthat on the one hand the distribution of the CB1 receptorinduces anxiety and SR141716A has an anxiolytic effectand on the other hand SR141716A is still active in CB1

--

mice In a following paper they reported that AM-251induces anxiogenic effects in wild-type and in knock-outmice [257] Thus SR141716A in addition to CB1 bindsto a not yet identified receptor Another evidence for thisadditional receptor for SR141716A was given by Bass andco-workers [61] They conducted a structure-activityrelationship study on the stimulation of locomotor activityinduced by SR141716A and twenty analogues Nocorrelation was found between the affinity for the CB1cannabinoid receptor and the stimulation of locomotoractivity Albeit SR141716A and five of its analogues O-1803 O-1710 O-1253 O-1254 (40) O-1255 (41) behavedas inverse agonists in [35S]-GTPγ S assay none of theseanalogues stimulated the locomotor activity In conclusionthe SR141716A-induced stimulation of locomotor activity isneither due to inverse agonism nor to inhibition of anendogenous tone but to a brain receptor distinct from theCB1 receptor Di Marzo et al previously provided evidencesthat the cannabinoid-vanilloid brain receptor could mediatesome of the cannabinoid effects on locomotion [249]

4 Other Non-CB1 Non-CB2 Cannabinoid Receptors

Evidences for the existence of additional non-CB1 non-CB2 cannabinoid receptors localised in the rat isolatedmesenteric arterial bed appeared from the works of Ralevicand collaborators Several cannabinoids were evaluated intheir model of sensory neurogenic vasorelaxation evoked byelectrical field stimulation

HU210 was found to attenuate vasorelaxation in a CB1and CB2 independent way as SR141716A LY320135 andSR144528 were ineffective in inhibiting HU210 effect [259]Similar results were obtained with ∆9-THC [260]Similarly noladin ether inhibited sensory neurogenicrelaxation in a concentration-dependent manner which wasalso unaffected by SR141716A LY320135 and SR144528Moreover this effect as the ∆9-THC one was pertussistoxin sensitive suggesting the involvement of a Gio protein-coupled receptor [261]

However the inhibitory effects of CP-55940 and WIN-55212-2 were affected by the presence of SR141716A (1microM)or LY320135 (1microM) but not by the addition of SR144528(1microM) [262]

It appears that at least two receptors should be involvedin cannabinoid-mediated attenuation of sensory nerve-mediated vasorelaxation The first one is SR141716A andLY320135 sensitive while the other one is resistant toSR141716A

Some evidences for the existence of a ldquoCB2-likerdquo receptorappeared from the studies on palmitoylethanolamide (PEA)an endogenous fatty acid amide devoid of CB1 and CB2affinity [264] antinociceptive potential Calignano and co-workers showed that PEA was able to alleviate pain inseveral animal models [265 266] For instance PEA dose-dependently inhibits kaolin-evoked writhing in mice TheCB2 cannabinoid receptor inverse agonist SR144528 (02mgkg) inhibits PEA anti-nociceptive effect [266] Thus theauthors suggest that PEA acts as agonist on a SR144528-sensitive non-CB2 cannabinoid receptor (ldquoCB2-likerdquoreceptor) at which SR144528 acts as an antagonist

Further studies are needed in order to better characterisethese additional ldquocannabinoidrdquo receptors However it islikely that several therapeutic applications will soon springfor these receptors

Very recently Pistis et al published a study on theneurophysiological effects of cannabinoids on the basolateralamygdala neurons in-vivo [258] One of their findings is thatHU-210 and WIN-55212-2 did not elicit similar effects onbasal amygdala projection neurons firing rate While theformer had no effect the latter decreased the firing rateMoreover SR141716A reversed the WIN-55212-2 effectswhile AM251 had no effect The vanilloid receptorantagonist capsazepine also antagonised the WIN-55212-2effect on firing rate of these neurons Therefore the authorssuggested the presence of a novel cannabinoid-vanilloidreceptor

V CONCLUSION

Since their development cannabinoid antagonists provedto be essential tools in the understanding of cannabinoidpharmacology and biochemistry Moreover the interestshown by the pharmaceutical industry in the development ofnew cannabinoid antagonists prove if necessary the realtherapeutic potential of this class of compounds Thetreatment of eating and movement disorders memorydeficits psychosis and dependencies from various addictivedrugs are some of the most cited applications for the CB1cannabinoid receptor antagonists

From these lines it appears that in addition to theknown CB1 cannabinoid receptor it is now likely that twoother cannabinoid receptors do exist in the brain The firstone should be an anandamide brain receptor for whichSR141716A has no affinity while the second one should be

1390 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Much less wide is the knowledge on the CB2cannabinoid receptor antagonists However potentialapplications could be disorders involving the immunesystem such as inflammation or allergies

[19] Devane WA Hanus L Breuer A Pertwee RG StevensonLA Griffin G Gibson D Mandelbaum A Etinger AMechoulam R Science 1992 258 1946

[20] Mechoulam R Ben-Shabat S Hanus L Ligumsky MKaminski NE Schatz AR Gopher A Almong S MartinBR Compton DR Biochem Pharmacol 1995 50 83

Finally the growing literature on the non CB1 non CB2cannabinoid receptors should allow the development of newdrugs First examples are given by the cannabidiol andabnormal-cannabidiol derivatives described by Kunos et alfor which potential therapeutic applications on the vascularsystem are already patented

[21] Sugiura T Kondo S Sukagawa A Nakane S Shinoda AItoh K Yamashita A Waku K Biochem Biophys Res Co1995 215 89

[22] Hanus L Abu-Lafi S Fride E Breuer A Vogel Z ShalevDE Kustanovich I Mechoulam R Proc Natl Acad SciUSA 2001 98 3662

[23] Oka S Tsuchie A Tokumura A Muramatsu M Suhara YTakayama H Waku K Sugiura T J Neurochem 2003 851374It clearly appears from this paper that the antagonists of

the cannabinoid receptors are still an active research fieldfrom which should emerge new promising therapeutic toolsas well as innovative drugs in the near future

[24] Goutopoulos A and Makriyannis A Pharmacol Therapeut2002 95 103

[25] Beal JE Olson R Lefkowitz L Laubenstein L Bellman PYangco B Morales JO Murphy R Powderly W PlasseTF Mosdell KW Shepard KV J Pain Symptom Manage1997 14 7ACKNOWLEDGEMENTS [26] Notcutt W Price M Miller R Newport S Phillips CSimmons S Sansom C Anaesthesia 2004 59 440

GGM is very indebted to the ldquoFonds pour la formationagrave la Recherche dans lrsquoIndustrie et lrsquoAgriculture (FRIA)rdquo Belgium for the award of a research fellowship Theexperimental works of the authors were supported by grantsfrom the FNRS The Belgian Charcot Foundation and theUniversiteacute catholique de Louvain

[27] Ledent C Valverde O Cossu G Petitet F Aubert JFBeslot F Bohme GA Imperato A Pedrazzini T RoquesBP Vassart G Fratta W Parmentier M Science 1999 283401

[28] Zimmer A Zimmer AM Hohmann AG Herkenham MBonner TI Proc Natl Acad Sci U S A 1999 96 5780

[29] Wiley JL and Martin BR Chem Phys Lipids 2002 121 57[30] Adam J and Cowley P Expert Opin Ther Patents 2002 12

1475

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[32] Barth F and Rinaldi-Carmona M Curr Med Chem 1999 6745

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[33] Little PJ Compton DR Johnson MR Melvin LS MartinBR J Pharmacol Exp Ther 1988 247 1046

[2] Devane WA Dysarz FA Johnson MR Melvin LSHowlett AC Mol Pharmacol 1988 34 605

[34] Pertwee RG Stevenson LA Elrick DB Mechoulam RCorbett AD Brit J Pharmacol 1992 105 980

[3] Matsuda LA Lolait SJ Brownstein MJ Young ACBonner TI Nature 1990 346 561

[35] Harrison C Traynor JR Life Sci 2003 74 489-508[4] Gerard CM Molerau C Vassart G Parmentier M BiochemJ 1991 279 129 [36] Pertwee RG Fernando SR Griffin G Ryan W Razdan

RJ Compton DR Martin BR Eur J Pharmacol 1996 315195

[5] Glass M Dragunow M Faull RLM Neuroscience 1997 2299

[37] Ross RA Brockie HC Fernando SR Saha B Razdan RKPertwee RG Brit J Pharmacol 1998 125 1345

[6] Shire D Carillon C Kaghad M Calandra B Rinaldi-Carmona M LeFur G Caput D Ferrara P J Biol Chem1995 270 3726 [38] Griffin G Wray EJ Rorrer WK Crocker PJ Ryan WJ

Saha B Razdan RK Martin BR Abood ME Brit JPharmacol 1999 126 1575

[7] Ryberg E Khang Vu H Groblewski T Larsson N ElebringT Hjorth S Sjogren S Greasley PJ 2004 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 119

[39] Ross RA Gibson TM Stevenson LA Saha B Crocker PRazdan RK Pertwee RG Brit J Pharmacol 1999 128 735

[40] Martin BR Winckler JR Wiley JL Huffman JW CrockerPJ Saha B Razdan RK J Pharmacol Exp Ther 1999 2901065

[8] Rinaldi-Carmona M Calandra B Shire D Bouaboula MOustric D Barth F Casellas P Ferrara P LeFur G JPharmacol Exp Ther 1998 278 871

[41] Martin BR Stevenson LA Pertwee RG Breivogel CSWilliams A Mahadevan A Razdan RK 2002 Symposium onthe Cannabinoids Burlington Vermont International CannabinoidResearch Society page 2

[9] Munro S Thomas KL Abu-Shaar M Nature 1993 365 61[10] Howlett AC Qualy JM Khachatrian LL Mol Pharmacol

1986 29 307[11] Glass M Felder CC J Neurosci 1997 17 5327

[42] Thomas A Ross RA Saha B Mahadevan A Razdan RKPertwee RG Eur J Pharmacol 2004 487 213

[12] Calandra B Portier M Kerneacuteis A Delpech M Carillon C LeFur G Ferrara P Shire D Eur J Pharmacol 1999 374445 [43] Compton DR Gold LH Ward SJ Balster RL Martin BR

J Pharmacol Exp Ther 1992 263 1118[13] Howlett AC Life Sci 1984 35 1803[44] Eissenstat MA Bell MR DAmbra TE Alexander EJ

Daum SJ Ackerman JH Gruett MD Kumar V EstepKG Olefirowicz EM Wetzel JR Alexander MD WeaverJD Haycock DA Luttinger DA Casiano FM ChippariSM Kuster JE Stevenson JI Ward SJ J Med Chem1995 38 3094

[14] Deadwyler S Hampson RE Mu J Whyte A Childers SRJ Pharmacol Exp Ther 1995 273 734

[15] Mackie K Lai Y Westenbroek R Mitchell R J Neurosci1995 15 6552

[16] Bouaboula M Poinot CC Bourrie B Canat X Calandra BRinaldi-Carmona M LeFur G Casellas P Biochem J 1995312 637 [45] Pertwee R Griffin G Fernando S Li X Hill A

Makriyannis A Life Sci 1995 56 1949[17] Bouaboula M Poinot CC Marchand J Canat X Bourrie BRinaldi-Carmona M Calandra B LeFur G Casellas P Eur JBiochem 1996 237 704

[46] Hosohata Y Quock RM Hosohata K Makriyannis AConsroe P Roeske WR Yamamura HI EurJPharmacol 1997 321 R1[18] delPulgar T Velasco G Guzman M Biochem J 2000 347

369 [47] Landsman RS Makriyannis A Deng H Consroe P RoeskeWR Yamamura HI Life Sci 1998 62 109

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[48] Ross RA Brockie HC Stevenson LA Murphy VLTempleton F Makriyannis A Pertwee RG Brit JPharmacol 1999 126 665

[81] Gatley SJ Lan R Volkow ND Pappas N King P WongCT Gifford AN Pyatt B Dewey SL Makriyannis A JNeurosci 1998 70 417

[49] Huffmann JW Curr Med Chem 1999 6 705 [82] Lan R Lu Q Fan P Gatley J Volkow ND Fernando SRPertwee R Makriyannis A AAPS Pharmsci 1999 1 article 4[50] Rinaldi-Carmona M Barth F Heaulme M Shire D Calandra

B Congy C Martinez S Maruani J Neliat G Caput DFerrara P Soubrieacute P Breliegravere JC Le Fur G FEBS Lett1994 350 240

[83] Mathews WB Ravert HT Musachio JL Franck RARinaldi-Carmona M Barth F Dannals RF J Labelled CompdRad 1999 42 589

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Aberman J Tardif DJ Betz AJ Ishiwari K MakriyannisA Salamone JD Behav Pharmacol 2003 14 583

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[171] Benavides J Boccio D Henin Y Piot-Grosjean O WorldPatent Application 2003 WO03020314

[141] Chen RZ Huang R-RC Shen C-P MacNeil DJ FongTM Brain Res 2004 999 227

[172] Di Marzo V Hill MP Bisogno T Crossman AR BrotchieJM FASEB J 2000 14 1432

[142] Kirkham TC and Williams CM Psychopharmacology 2001153 267

[173] Segovia G Mora F Crossman AR Brotchie JM MovementDisord 2003 18 138

[143] Shearman LP Rosko KM Fleischer R Wang J Xu STong XS Rocha BA Behav Pharmacol 2003 14 573

[174] Brotchie JM Curr Opin Pharmacol 2003 3 54[175] Kanyonyo M Govaerts SJ Hermans E Poupaert JH

Lambert DM Bioorg Med Chem Lett 1999 9 2233[144] Liao C Zheng J David LS Nicholson RA Basic ClinPharmacol Toxicol 2004 94 73 [176] Ooms F Wouters J Oscari O Happaerts T Bouchard G

Carrupt PA Testa B Lambert DM J Med Chem 2002 451748

[145] Wiley JL Jefferson RG Grier MC Mahadevan ARazdan RJ Martin BR J Pharmacol Exp Ther 2001 2961013 [177] Govaerts SJ Muccioli GG Hermans E Lambert DM Eur

J Pharmacol 2004 495 43[146] Thomas BF Gillian AF Burch DF Roche MJ SeltzmanHH J Pharmacol Exp Ther 1998 285 285 [178] Finke PE Mills SG Plummer CW Shah SK Truong QT

World Patent Application 2003 WO03007887[147] Martin BR Razdan AK Mahadevan A United States Patent2003 US6509367 [179] Shearman LP Stribling DS Camacho RE Rosko KM

Feng Y Marsh DJ MacNeil DJ Fong TM Goulet MHagmann W Plummer C Finke P Mills S Shah S TruongQ MacIntyre DE Strack AM 2003 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 150

[148] Francisco ME Seltzman HH Gilliam AF Mitchell RARider SL Pertwee RG Stevenson LA Thomas BF J MedChem 2002 45 2708

[149] Thomas B Seltzman HH Francisco MEE World PatentApplication 2003 WO03088968

[150] Shim JY Welsh WJ Cartier E Edwards JL Howlett ACJ Med Chem 2002 45 1447

[180] Fong TM Van Der Ploeg LH World Patent Application 2003WO03075660

[151] Reggio PH Curr Pharm Design 2003 9 1607 [181] Hagmann WK Qi H Shah SK World Patent Application2003 WO03063781

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1393

[182] Lange JHM van Stuivenberg HH Coolen HKAC AdolfsT JP McCreary AC Keizer HG Walls HC Veerman WBorst AJM de Loff W Verveer PC Kruse CG J MedChem 2005 48 1823

[212] Zhang Q Ma P Wang W Cole RB Wang G J MassSpectrom 2004 39 672

[213] Gallant M Dufresne C Gareau Y Guay D Leblanc YPrasit P Rochette C Sawyer N Slipetz D M Tremblay NBioorg Med Chem Lett 1996 6 2263[183] Lange JHM Kruse CG Tipker J Hoogendorn J World

Patent Application 2002 WO02076949 [214] Melck D De Petrocellis L Orlando P Bisogno T Laezza CBifulco M Di Marzo V Endocrinology 2000 141 118[184] Lange JHM Coolen H K A C van Stuivenberg HH

Dijksman JAR Herremans AHJ Ronken E Keizer HGTipker K McCreary AC Veerman W Wals HC Stork BVerveer PC den Hartog AP de Jong NMJ Adolfs TJPHoogendoorn J Kruse CJ J Med Chem 2004 47 628

[215] Kalgutkar AS Marnett AB Crews BC Remmel RPMarnett LJ J Med Chem 2000 43 2860

[216] Klegeris A Bissonnette CJ McGeer PL Brit J Pharmacol2003 139 775

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[217] Rinaldi-Carmona M Barth F Millan J Derocq JM CasellasP Congy C Oustric D Sarran M Bouaboula M CalandraB Portier M Shire D Breliere JC Le Fur GL JPharmacol Exp Ther 1998 284 644

[186] Wilstermann JM and Berggren AIK World PatentApplication 2003 WO03051850

[187] Berggren AIK Bostrom SJ Elebring ST Greasley PTerricabra E Wilstermann JM World Patent Application 2003WO03051851

[218] Barth F Millan J Casellas P Oustric D Sarran M RinaldiM World Patent Application 1997 WO9721682

[219] Griffin G Wray EJ Tao Q McAllister SD Rorrer WKAung MM Martin BR Abood ME Eur J Pharmacol 1999377 117

[188] Finke PE Meurer LC Debenham JS Toupence RBWalsh TE World Patent Application 2003 WO03082191

[189] Barth F Martinez S Rinaldi-Carmona M World PatentApplication 2003 WO03084930

[220] Portier M Rinaldi-Carmona M Pecceu F Combes T Poinot-Chazel C Calandra B Barth F Le Fur G Casellas P JPharmacol Exp Ther 1999 288 582[190] Barth F Martinez S Rinaldi-Carmona M World Patent

Application 2003 WO03084943 [221] Bouaboula M Desnoyer N Carayon P Combes T CasellasP Mol Pharmacol 1999 55 473[191] Kopka I E Li B Hagmann WK World Patent Application

2004 WO04029204 [222] Rhee M-H and Kim S-K J Vet Sci 2002 3 179[192] Hagmann WK Lin LS Shah SK Guthikonda RN Qi H

Chang LL Liu P Armstrong HM World Patent Application2003 WO03077847

[223] Bouaboula M Dussossoy D Casellas P J Biol Chem 1999274 20397

[224] Shire D Calandra B Bouaboula M Barth F Rinaldi-Carmona M Casellas P Ferrara P Life Sci 1999 65 627[193] Hagmann WK Lin LS Shah SK Goulet MT Jewell JP

World Patent Application 2003 WO03082190 [225] Gouldson P Calandra B Legoux P Kerneis A Rinaldi-Carmona M Barth F Le Fur G Ferrara P Shire D Eur JPharmacol 2000 401 17

[194] Castonguay LA Hagmann WK Lin LS Shah SK WorldPatent Application 2003 WO03086288

[195] Hagmann WK Lin LS Shah SK World Patent Application2003 WO03087037

[226] Feng W and Song ZH Biochem Pharmacol 2003 63 1077[227] Barth F Millan J Oustric D Rinaldi M Vernhet M World

Patent Application 2001 WO0132629[196] Berggren AIK Bostrom SJ Cheng L Elebring STGreasley P Nagard M Wilsterlmann JM Terricabras EWorld Patent Application 2004 WO04058249

[228] Mussinu JM Ruiu S Mule AC Pau A Carai MA LorigaG Murineddu G Pinna GA Bioorg Med Chem 2003 11251[197] Guba W Haap W Marty HP Narquizian R United States

Patent Application 2004 US 20040147572 [229] Xiang JN Elliott JD Atkinson ST Christensen SB WorldPatent Application 1998 WO9831227[198] Jagerovic N Hernandez Folgado L Alkorta I Goya P

Navarro M Serrano A de Fonseca FR Dannert MTDannert MT Alsasua A Suardiaz M Pascual D MartinMI J Med Chem 2004 47 2939

[230] Xiang JN Atkinson ST Wang DY Lee J Lee JCSiegfried B 216th ACS meeting Boston (MA) USA 23-27 Aug1998

[199] Jagerovic N Goya Laza MP Hernandez Folgado L AlcortaOsoro IJ Martin Fontelles MI Suardiaz Garcia MLDannert MT World Patent Application 2003 WO03082833

[231] Inaba T Kaya T Iwamura H United States Patent 2003 US6509352 B1

[232] Iwamura H Suzuki H Ueda Y Kaya T Inaba T JPharmacol Exp Ther 2001 296 420[200] Lange JHM Kruse CG Herremans AHJ VanStuivenberg

HH Dijksman JAR McCreary AC World PatentApplication 2003 WO03078413

[233] httpwwwjticojpJTI_EIR02PL20030212_Epdf (accessed02162004)

[201] Berggren AIK Bostrom SJ Elebring ST Fallefors LWilstermann JM Greasley P World Patent Application 2004WO04058255

[234] Zygmunt PM Andersson DA Houmlgestaumltt ED J Neurosci2002 22 4720

[235] Di Marzo V De Petrocellis L Fezza F Ligresti A BisognoT Prostaglandins Leukot Essent Fatty Acids 2002 66 377[202] Toupence RB Debenham JS Goulet MT Madsen-Duggan

CB Walsh TE Shah SK World Patent Application 2004WO04012671

[236] Wagner JA Varga K Jarai Z Kunos G Hypertension 199933 429

[203] Alanine A Bleicher K Guba W Haap W Kube DLuebbers T Plancher J-M Roche O Rogers-Evans MSchneider G Zuegge J World Patent Application 2004WO04013120

[237] Mukhopadhyay S Chapnick BM Howlett AC Am JPhysiol-Heart Circul Physiol 2002 282 H2046

[238] Jarai Z Wagner JA Varga K Lake KD Compton DR Martin BR Zimmer AM Bonner TI Buckley NE MezeyE Razdan RK Zimmer A Kunos G Proc Natl Acad Sci US A 1999 96 14136

[204] Griffith DA United States Patent Application 2004 US20040092520

[205] Griffith DA United States Patent Application 2004 US20040157839

[239] Kunos G Martin B Razdan R World Patent Application 2001WO0103690

[206] Griffith DA United States Patent Application 2004 US20040157838

[240] Offertaler L Mo F-M Batkai S Liu J Begg M RazdanRK Martin BR Bukoski RD Kunos G Mol Pharmacol2003 63 699[207] Bayewitch M Avidor-Reiss T Levy R Barg J Mechoulam

R Vogel Zvi FEBS letters 1995 375 143 [241] Begg M Mo F-M Offertaler L Batkai S Pacher PRazdan R K Lovinger DM Kunos G J Biol Chem 2003278 46188

[208] Bayewitch M Rhee M-H Avidor-Reiss T Breuer AMechoulam R Vogel Z J Biol Chem 1996 271 9903

[209] Govaerts SJ Hermans E Lambert DM Eur J Pharm Sci2004 23 233

[242] Ho WSV Hiley CR Brit J Pharmacol 2003 138 1320[243] Ho WSV Hiley CR J Pharm Pharmacol 2004 56 869

[210] DAmbra TE Estep KG Bell MR Eissenstat MA JosefKA Ward SJ Haycock DA Baizman ER Casiano FMBeglin NC J Med Chem 1992 35 124

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[245] Batkai S Pacher P Jarai J Wagner JA Kunos G Am JPhysiol-Heart Circul Physiol 2004 287 H595[211] Showalter VM Compton DR Martin BR Abood ME J

Pharmacol Exp Ther 1996 278 989

1394 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

[246] Kunos G World Patent Application 2003 WO03015700 [257] Haller J Varga B Ledent C Freund TF Behav Pharmacol2004 15 299[247] Franklin A and Stella N Eur J Pharmacol 2003 474 195

[248] Walter L Franklin A Witting A Wade C Xie Y KunosG Mackie K Stella N J Neurosci 2003 23 1398

[258] Pistis M Perra S Pillolla G Melis M Gessa GL MuntoniAL Neuropharmacology 2004 46 115

[249] Di Marzo V Breivogel C S Tao Q Bridgen DT RazdanRK Zimmer AM Zimmer A Martin BR J Neurochem2000 75 2434

[259] Ralevic V and Kendall DA Eur J Pharmacol 2001 418 117[260] Duncan M Kendall DA Ralevic V Brit J Pharmacol 2003

138 214P[250] Breivogel CS Griffin G Di Marzo V Martin BR Mol

Pharmacol 2001 60 155[261] Duncan M Millns P Smart D Wright JE Kendall DA

Ralevic V Brit J Pharmacol 2004 142 509[251] Monory K Tzavara ET Lexime J Ledent C Parmentier

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[263] Tao Q and Abood ME J Pharmacol Exp Ther 1998 285651[252] Muthane U Ramsay KA Jiang H Jackson-Lewis V

Donaldson D Fernando S Ferreira M Przedborki S ExpNeurol 1994 126 195

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[254] Hajos N Ledent C Freund TF Neuroscience 2001 106 1 [265] Calignano A La Rana G Giuffrida A Piomelli D Nature1998 394 277[255] Hajos N Freund TF Neuropharmacology 2002 43 503

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[266] Calignano A La Rana G Piomelli D Eur J Pharmacol 2001419 191

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Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1373

another paper Katoch-Rouse described the synthesis of[18F]-NIDA-42033 (26) a ligand useful as PET radiotracerstarting from its bromo derivative [156] Recently Kumar etal [157] proposed the synthesis of a 11C derivative ofNIDA-41087 (61) a compound previously described byKatoch-Rouse (Table 5) Post-mortem binding of thiscompound to human prefrontal cortex was also investigatedInterestingly CP-272871 (66) described by Meschler as aCB1 inverse agonist (Ki=57 nM [3H]-CP-55940 rat brainhomogenates) possesses a lipophilicity similar to theNIDA-41087 one [62] However despite the presence of amethoxy function no radiolabelled derivative has beendescribed to date One explanation could be the compoundlow selectivity (2 fold) for the CB1 cannabinoid receptor

To further explore the pharmacology of this compound afunctional assay using the mouse vas deferens model wasperformed by Ruiu and colleagues enlightening thecompetitive antagonist properties of NESS (pA2= 1246against WIN-55212-2) which had no effect by itself up to1microM Moreover NESS 0327 was unable to affect basalbinding of the [35S]-GTPγ S demonstrating its lack ofnegative intrinsic activity In-vivo studies were conductedusing the hot plate and tail flick tests NESS dose-dependently abolished the antinociceptive effect of WIN-55212-2 but had no effect by itself However the authorssuggested based on the affinity and in-vivo activity of theircompound that NESS 0327 possesses a poor centralbioavailability

Several new attempts to increase the affinity of thediarylpyrazole derivatives were recently made by rigidifyingthe SR141716A structure (Table 7) Six fused ringanalogues of SR141716A obtained by fusion of the 5-(4-chlorophenyl) substituent with the central pyrazole to forman indazole ring were published by Bass et al in 2002 [61]The compound possessing the highest affinity O-1248 (67)has a Ki value of 475 nM as determined by displacement of[3H]-CP-55940 from rat brain membranes

Another example of ring-constrained biarylpyrazole wasdescribed by Herbert Seltzman team [161] Theyinvestigated a photocyclisation reaction starting from theSR141716A leading to a pyrazolo[15-f]phenanthridinestructure (Fig 5) The new compound (68) was tested on theCB1 receptor (whole rat brain) against [3H]-CP-55940[3H]-SR141716A and [3H]-WIN-55212-2 and exhibited Kivalues of 48 35 and 50 nM respectively The CB2 affinitywas assessed using [3H]-CP-55940 and was found to be 100times lower (Ki = 3340 nM) No effect on the [35S]-GTPγ Sassay (rat brain) was observed by the authors

Stoit and colleagues from Solvay Pharmaceuticalspublished a paper describing the synthesis andpharmacological characterisation of new benzocyclohepta-pyrazole derivatives as CB1 antagonists (69-74) [158] Theaffinity of these compounds was more or less one order ofmagnitude lower than the SR141716A one with pKi valuesranging from 64 to 72 compared to 76 for the SRcompound (Table 7) Compounds were shown to beantagonists with pA2 values from 70 to 89 as theyprevent CP-55940-induced cAMP accumulation Howeverthe authors investigating the bioavailability of theircompounds either po or after ip injection found negligibleblood plasma levels

NN

Cl

Cl

O

NH

N

NN

Cl

O

NHN

Cl

Cl

O-1248 67 68However just before the publication of Stoit results

Sanofi-Synthelabo took a patent describing tricyclicpyrazolecarboxylic acid amide derivatives having antagonistproperties on the CB1 cannabinoid receptor andsubmicromolar affinities (75-77) [159] Thirty examples weregiven along with their synthetic pathway but no individualpharmacological data were shown

Fig (5) CB1 cannabinoid receptor antagonists 3Diarylpyrazole derivatives Two ring-constrained diarypyrazolederivatives O-1248 (67 ) and a pyrazolo[15-f]phenanthridinederivative (68 )

It appears from these attempts to increase SR141716Aaffinity by rigidifying its structure that only a limitedincrease in the CB1 cannabinoid receptor affinity can beexpected with the apparent exception of NESS 0327Moreover rigidifying the structure often results in a morelipophilic compound in a family of compounds being yetlipophilic

More recently Ruiu and colleagues published a paperdescribing the synthesis and complete pharmacologicalcharacterisation of a very potent CB1 receptor ligandchristened NESS 0327 (78) [160] This compound has thesame structure as compound 69 published by Stoit et al(compound 20 in the reference [158]) Nevertheless Ruiu hasobtained an affinity for the CB1 cannabinoid receptor 5000times bigger than the SR141716A one with Ki values of035 pM and 18 nM respectively using mouse forebrainhomogenates and [3H]-CP-55940 as radioligandFurthermore the selectivity ratio obtained for this compoundby Ruiu is 60000 with a Ki value for the CB2 receptor of21 nM against the [3H]-CP-55940 The discrepancybetween the data obtained independently by Stoit et al andRuiu et al could hardly be explained by the differences inthe origin of the receptor used respectively hCB1-CHO andmouse forebrain

4 Phenyl Benzofuranone Derivatives

In a 1997 patent from Eli Lilly was described thesynthesis of aryl-benzothiophene and aryl-benzofuranederivatives (87-89 Fig 6) having CB1 receptor affinity andantagonist properties [162] From ten compounds having aKi value lower than 25 microM ([3H]-CP-55940 hCB1-CHOcells) one was selected and claimed to have a Ki of 170 nMCompound 88 was shown in the patent to antagonise theeffect of anandamide on cAMP accumulation with an IC50 of

1374 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

O

O

O

O

NC

S

O

O

O

O

O

O

O

NC

LY320135 8887 89

Fig (6) CB1 cannabinoid receptor antagonists 4 Phenyl benzofuranone derivatives Chemical structures of two aryl-benzofuranederivatives (87 -88 ) and one aryl-benzothiophene (89 ) derivative from Eli Lilly

500 nM and the effect of WIN-55212-2 on calcium channelsat 1microM Moreover the intra-peritoneal injection (20 mgkg)of this compound antagonised the in-vivo effect ofanandamide in the Open Field Assay a mouse behaviourmodel Later on Felder and colleagues gave a furtherdescription of this compound called LY320135 in a paperdescribing its effect on cAMP accumulation and on ioniccurrents [163] The selectivity ratio of LY320135 was 106with Ki values of 141 and 14900 nM for hCB1 and hCB2receptors ([3H]-CP-55940) respectively LY320135antagonised anandamide inhibition of forskolin-inducedcAMP accumulation with an IC50 value of 734 nMChristopoulos et al in 2001 reported for LY320135 apKb value of 527 in the inhibition of WIN-55212-2mediated response in the rat electrically-stimulated vasdeferens while the pKb value for SR141716A was 75[164]

Other patents were taken by Aventis in which somepotential applications for their compounds were describedFor instance an association between an azetidine derivativehaving CB1 antagonists properties and sibutramine aserotonin and norepinephrine reuptake inhibitor was claimed[170] as a treatment against obesity Oral administration ofcompound 90 (3 mgkg) and sibutramine (06 mgkg) to fafaZucker rats which are genetically obese rats has resulted ina decreased food intake compared to control lean Zuckerrats In another patent the association of an azetidinederivative (1-10 mgkg po ) and a D2D3 agonist likequinpirole (01 mgkg ip) was claimed to have a therapeuticpotential in the treatment of Parkinsonrsquos disease [171] Theeffect has been evidenced using an akinesia model in the ratEvidences supporting the use of CB1 antagonists as adjuvantin the treatment of Parkinsonrsquos disease begin to appear Itseems that such compounds would be helpful in thetreatment of Parkinsonism [172] and levodopa-induceddyskinesia [173] Brotchie published an interesting paperreviewing the CB1 cannabinoid receptor signaling inParkinsonrsquos disease [174] Nevertheless further clinicalstudies should be conducted to further confirm the usefulnessof CB1 antagonists in the treatment of Parkinsonrsquos disease

5 Azetidine Derivatives

The family of azetidine compounds illustrated bycompounds 90 and 91 (Fig 7) was developed at Aventis byDaniel Achard and colleagues and was described in a seriesof patents [165-168] They claimed the IC50 values of thesecompounds for the CB1 receptor to be less than or equal to100 nM These values were obtained following the proceduredescribed by Kuster et al [169] The antagonist propertywas shown using an in-vivo model the reversal ofhypothermia induced by CP-55940 in mice and the ED50obtained were lower than 50 mgkg

6 Aryl-Imidazolidine-24-Diones Derivatives

In 1999 the Didier Lambert team published a paperdescribing the affinity of 24 new 3-alkyl-55rsquo-diphenyl-imidazolidine-24-dione derivatives for the hCB1 receptor[175] The preliminary structure-activity relationships

N

SO

O

Cl

Cl

F

F

N

SO

O

Cl

NS

90 91

Fig (7) CB1 cannabinoid receptor antagonists 5 Azetidine derivatives Two examples (90 -91 ) of the azetidine derivatives developedby Aventis

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1375

Table 8 CB1 Cannabinoid Receptor Antagonists 6 Aryl-Imidazolidine-24-Diones Derivatives

Structures affinities ([3H]-SR141716A hCB1-CHO cells) and pKb values ([35 S]-GTPγS HU210 rat brain) of three aryl-imidazolidine-24-dionederivatives (92-94)

N

NH

OO

R

Br

Br

Cpd ndeg R Ki (nM) a pKb b

DML20 92 ethylmorpholine 70 611

DML21 93 1-hydroxypropyl 103 625

DML23 94 heptyl 98 574

a [175]b [177]

showed that the substitution at the nitrogen in position 3 aswell as a bromine atom in para of the phenyl rings aremandatory for the compounds affinity Later on threecompounds termed DML20 (92) DML21 (93) and DML23(94) were characterised as neutral antagonists using the [35S]-GTPγ S assay on rat cerebellum membranes [176] (Table 8)In the same model they competitively inhibited HU-210-induced [35S]-GTPγ S binding with pKB values of 611625 and 574 respectively Moreover these compoundswere proven to be quite selective for the CB1 receptor

higher than the one for the CB2 cannabinoid receptor (IC50 =300 nM) The inverse agonism of the compound wasextrapolated by inhibiting CP-55940-induced hypothermiain mice (EC50 = 18 nM) Moreover one oral dose (10mgkg) reduces the food intake in diet-induced obese ratsInterestingly food intake of CB1

-- mice was not alteredBody weight loss was maintained all over the study during achronic administration (14 days) of 10 mgkg of Cpd A

Later on Merck published a patent [180] in which someof the 45-diaryl-pyrazoles derivatives previously describedwere claimed to be useful in the treatment or the preventionof obesity in association with compounds inhibiting the11β-hydroxysteroid dehydrogenase type 1 (1β-HSD1)enzyme The 1β-HSD1 enzyme is responsible for thesynthesis of cortisol increased levels of which according tothe inventors are associated with obesity However neitherresults on appetite suppression nor on weight loss weregiven even if some indications on the affinity of thecompounds for the cannabinoid receptors were given

Very recently the functionality of these DML compoundswas explored using the [35S]-GTPγ S assay both on rat andhuman CB1 cannabinoid receptors [177] The data obtainedconfirm that the 3-alkyl-55rsquo-diphenyl-imidazolidinedionederivatives behave as neutral antagonists on rat CB1cannabinoid receptor (rat brain) However DML 20 DML21and DML23 acted as inverse agonists on the human receptor(hCB1-CHO cells) Furthermore the authors showed thatthis different functionality is not due to the hCB1 receptorlevel of expression in the recombinant cell line as whateverthe level of expression (Bmax of 44 pmolmg or 32pmolmg) DML derivatives behaved as inverse agonists ofthe hCB1 cannabinoid receptor

Another imidazole ring pattern of substitution wasproposed by Hagmann and collaborators [181] Thecompounds described 43 examples are given possess twophenyls the first one in position 1 linked to the nitrogenand the second one in position 2 The carboxamidesubstituent is in position 4 instead of in position 2 for thediarylimidazoles described by Finke These 12-diaryl-imidazoles are claimed to be CB1 cannabinoid receptorantagonists or inverse agonists but the absence of precisepharmacological values do not allow any comparisonbetween the two substitutions patterns However a samepattern of substitution was used by other researchers Amongthem Dyck et al described in 2004 ten 1-(4-chlorophenyl)-2-(24-dichlorophenyl)-imidazole derivatives varying by theircarboxamide substituent in position 4 (101-103 Table 9)[152] For instance with a 3-azabicyclo[330]octan-3-ylsubstituent (101) they obtained a Ki value of 14 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) In a [35S]-GTPγ Sassay 101 exhibited an IC50 value of 19 nM

7 Diarylimidazoles Derivatives

Finke and colleagues at Merck developed a new class ofCB1 receptor antagonists based on an imidazole nucleusOver eighty 45-diaryl-imidazoles derivatives having CB1antagonist properties were claimed in a patent published in2003 [178] Some representative compounds of the patent areillustrated in Table 9 (95-100) The affinity of the newcompounds was determined using [3H]-CP-55940 andhCB1-CHO cells Although no detailed pharmacologicaldata were given in the patent in-vitro and in-vivo propertiesof one compound termed Cpd A (100) have been describedelsewhere [179] Its affinity for the CB1 cannabinoid receptorwas shown to be nanomolar (IC50 = 4 nM) and 75 times

1376 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 9 CB1 Cannabinoid Receptor Antagonists 7 Diaryl-Imidazole Derivatives

Structure and affinity range of some 45-diaryl-imidazole derivatives for the human cannabinoid receptors determined using [3H]-CP-55940 asradioligand and hCB1amp2-CHO cells

N

NR2

R1

R4

R3

ndeg R1 R2 R3 R4 hCB1 (IC50 or Ki) hCB2 (IC50 or Ki) References

95 CH3 CO-NH-piperidinyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

96 CH3 CO-NH-piperidinyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

97 CH3 CO-NH-cyclohexyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

98 CH3 CO-NH-phenyl 24-di-Cl-Ph 4-Cl-Ph 10 ndash 100 nM a 100 ndash1000 nM a [178]

99 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

100 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph 4 nM a 300 nM a [179]

101 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

CH3 14 nM b ND [152]

102 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

H 66 nM b ND [152]

103 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 85 nM b ND [152]

104 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 23 nM b 542 nM b [182]

105 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 30 nM b 608 nM b [182]

106 4-Br-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 60 nM b 489 nM b [182]

107 4-CF3-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 29 nM b 634 nM b [182]

108 4-Cl-Ph 24-di-Cl-Ph CO-NH-morpholinyl CH3 197 nM b 3297 nM b [182]

109 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidin-4-ol CH3 172 nM b 3959 nM b [182]

110 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CN 30 nM b 1590 nM b [182]

111 4-Cl-Ph 24-di-Cl-Ph CO-NH- piperidinyl CH2F 36 nM b 906 nM b [182]

112 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH2-CH3 14 nM b 430 nM b [182]

113 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Cl 27 nM b 823 nM b [182]

114 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Br 23 nM b 746 nM b [182]

115 4-Cl-Ph 24-di-Cl-Ph CO-NH-tetrahydroisoquinoline CH3 34 nM b 696 nM b [182]

116 4-Cl-Ph 24-di-Cl-Ph CO-NH-cycloheptyl CH3 35 nM b 349 nM b [182]

117 24-di-Cl-Ph 4-Cl-Ph CO-NH-piperidinyl CH3 403 nM b 208 nM b [182]a IC50 value b Ki value

Lange and co-workers from Solvay described twenty-eight imidazole derivatives [182] The 24-dichlorophenylsubstituent at position 2 was kept constant while thesubstitutions at the other positions were explored (Table 9)Position 5 can accommodate a large range of littlesubstituents like hydrogen (104) methyl (105) ethyl (112)chlorine (113) bromine (114) fluoromethyl (111) or cyano(110) without major changes in the affinity At position 4more bulky substituents like 1234-tetraisoquinoline (115)or cycloheptyl (116) were well tolerated whereas thepresence of a more hydrophilic moiety such as morpholine(108) or piperidin-4-ol (109) was detrimental to the affinity

Interestingly position exchange between the 24-diphenyland the 4-chlorophenyl substituents led to a 13 fold loweraffinity with Ki values of 30 and 403 nM for 105 and 117respectively ([3H]-CP-55940 hCB1-CHO cells) In thefunctional assay which was inhibition of WIN-55212-2-induced [3H]-arachidonic acid release by hCB1-CHO cellsall tested compounds behaved as antagonists Compound105 showed a pA2 value of 86 which is the same theauthors obtained for SR141716A It would be interesting toassess if these compounds are true antagonists or if they actmore as inverse agonists Compound 105 when administeredto rats was able to inhibit CP-55940-induced hypotension

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1377

with an ED50 value of 24 mgkg which is close to the 3mgkg obtained for SR141716A The compound was alsoactive in another in-vivo model the WIN-55212-2-inducedhypothermia in mice Compound 104 which has nosubstituent in position 5 of the imidazole ring was lessactive in the hypotension model and inactive in thehypothermia model Thus a methyl group is the preferredsubstituent in position 5 of the imidazole moiety Otherproperties either experimental (P-glycoprotein affinitylogPHPLC) or computational (molecular volume polarsurface area) obtained for compounds 105 were similar tothose obtained for SR141716A Finally molecularmodelling studies revealed a close structural overlap betweenthe two compounds

value of 197 nM is selective for hCB1 and is as potent asSR141716A The structural modulations of 118 led to someinteresting compounds (Table 10) Six compounds possess aKi value for the hCB1 cannabinoid receptor equal to or lowerthan 25 nM antagonist properties in the inhibition of [3H]-arachidonic acid release by WIN-55212-2-stimulated hCB1-CHO cells and Ki values for the hCB2 receptor over 1microMThe diaryl-pyrazoline derivatives contained a chiral center atposition 4 The authors resolved the racemic mixture of twocompounds (121 and 130) The levorotatory enantiomersappeared to be the eutomers For compound SLV319levorotatory enantiomer of 121 the Ki and pA2 values were78 nM and 99 respectively The Ki and pA2 values ofSLV326 levorotatory enantiomer of 130 were 359 nM and9 respectively

8 34-Diaryl-Pyrazoline Derivatives Two in-vivo models CP-55940-induced hypotension inrat and WIN-55212-2-induced hypothermia in mouse wereused to assess the potential of these compounds The resultsare of the same order of magnitude than those obtained forSR141716A For instance in the hypotension modelSLV319 ((-)121) and SLV326 ((-)130) showed ED50 values

Lange and collaborators also discovered 34-diaryl-pyrazoline derivatives after a screening of compoundsresembling to SR141716A [183184] Compound 118 thatwas initially identified during the screening possesses a Ki

Table 10 CB1 Cannabinoid Receptor Antagonists 8 34-Diaryl-Pyrazolines Derivatives

Structure affinity for the hCB1 and hCB2 cannabinoid receptors (Ki nM) expressed in CHO transfected cells ([3H]-CP-55940) and potency (pA2) ofsome 34-dirylpyrazolines derivatives Table adapted from [184]

NN

N N

SO2

R3

R2

Cl R1

R4

n˚ R1 R2 R3 R4 Ki hCB1 (nM) Ki hCB2 (nM) pA2 (CB1)

118 H H H 4-CH3 197 gt 1000 84

119 H H H 246- CH3 24 gt 1000 94

120 H H H 4-Cl 16 gt 1000 95

121 H CH3 H 4-Cl 25 gt 1000 87

122 H CH3 CH3 4-Cl 280 gt 1000 85

123 H H H 4-F 53 gt 1000 9

124 H CH3 H 4-F 338 gt 1000 85

125 H CH3 CH3 4-F gt 1000 gt 1000 lt 75

126 H CH3 H 3-Cl 14 gt 1000 86

127 4-Cl CH3 H 4-Cl 255 ND ND

128 4-F CH3 H 4-Cl 584 ND ND

129 H CH3 H H 170 ND 75

130 H CH3 H 4- CF3 221 gt 1000 93

SR141716A 25 1580 86

1378 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

N

HN

O

N

Cl Cl

Cl

N

N

HN

O

Br

Br131 132

Fig (8) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Chemical structures of two 56-diaryl-pyrazine-2-amide derivatives (131-132) developed as CB1 cannabinoid receptorantagonists by AstraZeneca

of 55 and 2 mgkg (po) respectively proving their in-vivoefficacy after oral administration The authors alsodemonstrated that SLV319 is devoid of affinity for the P-glycoprotein pump and that it possessed a goodCNSplasma ratio (17) These two compounds (SLV319and SLV326) were chosen as development candidates bySolvay and entered clinical Phase I trials in 2003 [185]

132 Fig 8) useful as CB1 cannabinoid receptor antagonists[186 187] The activity of twenty derivatives wasdetermined using a [35S]-GTPγ S assay and hCB1-CHOtransfected cells The concentration required to give halfmaximal inhibition of CP-55940-induced [35S]-GTPγ Sbinding (IC50) is lower than 200 nM for the preferredcompounds No affinity data were given for thesecompounds

9 Diaryl-Pyrazine Diphenyl-Pyridine Diphenyl-Phenyland Diaryl-Pyrimidines Derivatives

The same year in a patent from Merck Finke andcollaborators described several 56-di-phenyl-pyridinederivatives as hCB1 antagonists or inverse agonists [188]The compounds have a substituent in position 3 of thepyridine core (Fig 9) This substituent could be a cyano(133-134) or a nitro group a halogen an ester or an amide(135-136) Over 150 compounds were synthesised toillustrate the invention but no pharmacological data wasdisclosed in the patent

In 2003 appeared several patents describing new familiesof compounds that bind to the CB1 cannabinoid receptorThese compounds have in common a central six atomsaromatic ring which could be a pyrazine a pyridine aphenyl or a pyrimidine and that is substituted by at leasttwo phenyl rings

The first patents were from Berggren and collaboratorsfrom AstraZeneca who published two patents describing thesynthesis of 56-diaryl-pyrazine-2-amide derivatives (131-

A few months later another patent was taken by Sanoficlaiming the antagonist properties of 56-di-phenyl-2-pyridine carboxamide derivatives [189] These compounds

N

NH

O

Cl

Cl

N

O

Cl

O NH

N

O

Cl

ClCl

NH

CN O

N

Cl

Cl

Cl

OHN

N

O

Cl

ClCl

CNF

F

133 134

135 136 137

Fig (9) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives 56-Di-phenyl-pyridine derivatives patented by Merck (133-136) and by Sanofi (137) as CB1 cannabinoid receptorsantagonists

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1379

NH

O

N

Cl

ClCl

NH

O

N

Cl

F3CCl

N

N

O

Cl

Cl Cl F138 139 140

Fig (10) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Representative structures of the diphenyl-phenyl (138-139) and 45-diphenyl-pyrimidine (140) derivatives developed ascannabinoid antagonists at Sanofi and Merck respectively

28 examples are given structurally related to the diphenyl-phenyl derivatives have their pyridine core substituted bytwo phenyl rings and by an amide moiety (137 Fig 9) TheIC50 values were lower than 100 nM but no data were givenshowing the antagonist properties although the compoundswere assayed in the adenylate cyclase inhibition assay

10 Other Derivatives

Several amide derivatives structures recently appearedamong the CB1 cannabinoid receptor antagonists In a seriesof patents taken by Merck in 2003 Hagmann and colleaguesdescribed a large amount of compounds obtained by parallelsynthesis which are claimed to be CB1 cannabinoid receptorantagonists [192-195] They were synthesised by reacting alibrary of substituted amines with a library of carboxylicacids (141-144 Fig 11) However as no value was givenfor the affinity or activity of these amides we will notfurther discuss this class of compounds

Along this line Sanofi Synthelabo developed newdiphenyl-phenyl derivatives claimed to have CB1cannabinoid receptor antagonist properties [190] They arebased on a central phenyl ring substituted by two phenylsand by an amide moiety (138-139 Fig 10) Thirteencompounds were claimed along with their synthesis TheirIC50 values were lower than 100 nM ([3H]-CP55-940hCB1-CHO cells) but no other specific data (selectivitypA2hellip) was given

Several 15-diaryl-pyrrole-3-carboxamide derivatives weresynthesised and claimed to be CB1 cannabinoid receptorantagonists by Berggren et al [196] The affinity valueswere not disclosed in the patent (145-146 Fig 12) Otherpyrrole derivatives were synthesised by Guba et al atHofmann-La Roche [197] More specifically 2-(thiazol-4-yl)pyrrole derivatives were claimed as CB1 cannabinoidreceptor antagonist (IC50lt2microM) (147-148 Fig 12)

Finally Kopka et al from Merck described more thanone hundred 45-diaryl-pyrimidine derivatives as CB1cannabinoid receptor antagonists (140 Fig10) [191]However no precise data were provided concerning theaffinity and functionality of the compounds

R2

R1N

R3

R4

R5

O

NH

O

Cl

O F

F

R2

R1NH

R3

R4

HO R5

O

NH

O

Cl

CN

NH

O

Cl

O

O

NH

O

Cl

Cl

+

141142

143 144

Fig (11) CB1 cannabinoid receptor antagonists 10 Other derivatives Retrosynthetic scheme of a CB1 cannabinoid antagonistlibrary of amide derivatives developed at Merck The structures of four representative compounds are illustrated (141-144)

1380 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

NH

O N

Cl

Cl

Cl

N

NH

O

OMe

MeO

N

NH

O

N

S

MeO

N

NH

O

N

S

MeO

145 146

147 148

Fig (12) CB1 cannabinoid receptor antagonists 10 Other derivatives Two examples of 15-diaryl-pyrrole derivatives described byBerggren et al (145-146) While the 2-(thiazol-4yl)pyrroles derivatives 147 and 148 were described by Guba et al from Hofmann-LaRoche All these pyrroles derivatives were claimed to be CB1 cannabinoid receptor antagonists

Another new structure is represented by the 124-triazolederivatives developed by Jagerovic and collaborators In arecent paper were described the synthesis andpharmacological properties of five new 15-diphenyl-3-alkyl-

triazole derivatives [198] Among these compounds onlyone (149) behaved as a CB1 antagonist inhibiting the WIN-55212-2-induced contractions in a mouse vas deferenspreparation However despite its effects in isolated tissue

Table 11 CB1 Cannabinoid Receptor Antagonists 10 Other Derivatives

Structure and affinity of diaryl-triazole derivatives

N

NN

R3

R2

R1

Cpd R1 R2 R3 Ki CB1 (nM) References

149 24-diCl 4-Cl hexyl 855 a [198]

150 24-diCl 4-Cl2-OMe CO-NH-(3-azabicyclo[330]octan-3-yl) 270 b [152]

151 24-diCl 4-Cl CO-NH-(3-azabicyclo[330]octan-3-yl) 164 b [152]

152 4-Cl 24-diCl CO-NH-(3-azabicyclo[330]octan-3-yl) 137 b [152]

153 4-Cl 24-diCl CO-NH-benzylpyrrolidin-3-yl 29 b [152]

154 4-Cl 24-diCl CO-NH-1-(4-chlorophenyl)ethyl 66 b [152]

155 4-Cl 24-diCl CO-NH-piperidinyl 356 c [182]

156 24-diCl 4-Cl CO-NH-piperidinyl 382 c [182]

a [3H]-SR141716A rat cerebellar membranesb [3H]-CP-55940 hCB1-HEK EDNA cellsc [3H]-CP-55940 hCB1-CHO cells

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1381

N

S

O

NH

Cl

Cl

Cl

F

N

S

O

NHN

ClCl

Cl

N

S

O

NH

Br

N

S

O

NHN

Cl

Cl

Cl

156 157 158 159

Fig (13) CB1 cannabinoid receptor antagonists 10 Other derivatives Structures of 45-diaryl-thiazole derivatives patented as hCB1cannabinoid receptor antagonists by Solvay Pharmaceuticals (156 157 158) or by AstraZeneca (157 158 159)

assays (mouse vas deferens and guinea pig ileum) thecompound possessed only a reduced rCB1 affinity with Kivalues of 855 and 748 nM using [3H]-SR141716A and [3H]-WIN-55-212-2 respectively (Table 11) Further four seriesof compounds differing by the nature of the substituentaround the triazole core were also described in a patent[199] Usually the compounds claimed possess two aromaticrings possibly substituted and one linear alkyl chain Theaffinity for the cannabinoid receptors was not givenNevertheless 149 was evaluated in two isolated tissuemodels the inhibition of the electrically evoked contractionsin the guinea pig ileum and in the mouse vas deferens Inthe two models compound 149 inhibited the effect of WIN-55212-2 but had no effect by itself In the vas deferensmodel the authors obtained a pA2 value of 748 to becompared with 763 obtained for AM251

Yet another five membered ring a thiazole has beenused as central moiety of a new class of cannabinoid ligandsThe thiazole ring is substituted by two aryls in position 4and 5 and by an amide (position 2) as in compounds 156-159 (Fig 13) Forty derivatives of this type weresynthesised by Lange and colleagues from SolvayPharmaceuticals and claimed in a patent as agonists orantagonists of the CB1 cannabinoid receptor [200] Nopharmacological data were provided however their activitywas assessed by measuring the cAMP in transfected hCB1-CHO cells In a recent paper from Lange et al two of thesethiazole derivatives were further described [182] Compound13 (157) the most closely related to SR141716A has a Kivalue of 227 nM while for compound 14 (158) Ki value isover 1000 nM Thus as in the imidazole series (see Table9) the aromatic substitution pattern is of great importancefor the CB1 cannabinoid receptor affinity Compound 13however was devoid of in vivo activity

Others 124-triazole derivatives were described by Dycket al compounds 150-154 unlike the Jagerovic ones havean amide moiety in position 3 [152] The highest affinitywas obtained with a 1-benzyl-pyrrolidin-3-yl substituentThe Ki value was 29 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) to be compared to 12 nM obtained for theSR141716A

Seventeen 45-diaryl-thiazole derivatives were alsosynthesised by Berggren et al and patented by AstraZeneca(157-159 Fig 13) [201]

In a patent from Merck Toupence et al described over190 substituted furo[23]pyridines claimed to be CB1cannabinoid receptor antagonists [202] The affinity (IC50 lt1microM) was measured using recombinant CHO cells and [3H]-CP-55940 and the activity using cAMP dosage (160 Fig14)

Lange et al described a triazole derivative (155) whichcan be superimposed with SR141716A Compound 155showed a Ki value of 356 nM ([3H]-CP-55940 hCB1-CHOcells) a pA2 value of 83 in the inhibition of WIN-55212-induced release of [3H]-arachidonic acid by hCB1-CHO cellsand was active in vivo in the CP-55940-inducedhypotension in rat (ED50 = 236 mgkg) [182]

Very recently Alanine from Hoffmann-LaRoche foundthat diarylbenzo[13]dioxole derivatives act as CB1

O

O

S

N

OO

F

ClCl

O

O

F O

N

OON

NH2

O

Cl

Cl

Cl

160 161 162

Fig (14) CB1 cannabinoid receptor antagonists 10 Other derivatives One example of a furo[23]pyridine derivative (160) and twoexamples of diarylbenzo[13]dioxole derivatives (161-162) described as hCB1 cannabinoid receptor antagonists by Toupence et aland Alanine et al respectively

1382 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

cannabinoid receptor antagonists (161-162 Fig 14) [203]Approximate IC50 values for twelve compounds ([3H]-CP-55940 hCB1-HEK cells) among the three hundred andeighty compounds described were given (IC50lt2microM)

et al in 1995 [207] In their hands ∆9-THC was not able toinhibit the forskolin-stimulated adenylyl cyclase activity inan assay performed on hCB2-CHO cells Subsequently theyshowed that ∆9-THC was able to antagonise HU210-inducedinhibition of adenylyl cyclase activity thus acting as anantagonist of the hCB2 cannabinoid receptor expressed inCHO cells [208] Furthermore very recently Govaerts et al[209] reported an inverse agonist effect of ∆9-THC and ∆8-THC on hCB2-CHO cells using the [35S]-GTPγ S assayThe pEC50 values were 763 and 888 respectively whilethe Emax values were ndash27 and ndash16 as compared to basal

Griffith from Pfizer described three new series of CB1cannabinoid receptor antagonists based on a bicyclic centralaromatic ring Purine pyrazoltriazines andpyrazolo[15a]pyrimidines derivatives were synthesised andinvestigated for their ability to interact with the CB1cannabinoid receptor Albeit a [35S]-GTPγ S assay wasdescribed no data were given concerning the antagonist orinverse agonist properties of such compounds Two hundredand eighty purine derivatives were synthesised [204] Theaffinities of 163 and 164 were given in the patent (28 and12 nM [3H]-SR141716A) In a following patent thesynthesis of sixty pyrazoltriazine derivatives was described(165 Fig 15) [205] The affinity of each compound was notgiven but was said to be ranging between 01 and 590 nMAnd finally more than forty pyrazolo[15a]pyrimidines weresynthesised (166 Fig 15) [206] Their affinity for the CB1cannabinoid receptor was lower than 155 nM Despite thefact that a great number of derivatives share the samesubstitution pattern around the central bicyclic aromatic ringthe comparison of these three new series of compounds ismade difficult by the absence of precise affinity data

Some other classical cannabinoids were shown to possessan antagonist activity at the CB2 receptor This is the casefor instance for the non-selective O-1184 (2) (Ki = 74 nM)which enhances the forskolin-induced cAMP production inCB2-CHO cells (EMax = 161 EC50 = 63 nM) and alsofor O-584 (3) (EMax = 246 EC50 = 138 nM) [39]

2 Indole Derivatives

Since the disclosure in 1993 of a second cannabinoidreceptor many attempts have been made to synthesise CB2cannabinoid receptor selective ligands The first compoundreported to have a somehow greater affinity for the peripheralcannabinoid receptor was WIN-55212-2 The compoundwas introduced by DrsquoAmbra and colleagues [210] back in1992 as a new cannabinoid ligand having a nanomolaraffinity and acting as an agonist In 1996 Showalter et alused a transfected cell line expressing the CB2 cannabinoidreceptor in order to identify selective ligands for thisreceptor [211] They showed that the CB2 affinity of WIN-55212-2 was 7 times greater than the CB1 with Ki valuesof 189 and 028 nM for the CB1 and CB2 cannabinoidreceptors respectively However WIN-55212-2 is anagonist of the cannabinoid receptors Interestingly WIN-55212-3 (167 Fig 16) the so-called inactive enantiomer ofWIN-55212 was recently shown to behave as a low affinity(Ki=363 microM) inverse agonist of the hCB2 cannabinoidreceptor as it decreased the [35S]-GTPγ S binding [209]

III CB2 LIGANDS RECEPTOR ANTAGONISTS ANDINVERSE AGONISTS

The compounds acting as antagonist or inverse agonistsat the CB2 cannabinoid receptor are reviewed in this thirdpart of the paper As for the CB1 cannabinoid receptorligands they are classified depending on their chemicalstructures

1 Classical Cannabinoid Derivatives

The first report of an antagonism mediated by ∆9-THC atthe peripheral cannabinoid receptor was made by Bayewitch

N N

NN

NH COOH

Cl

Cl

N N

NN

N

Cl

Cl

NH2OH

O

N

N

N

N

N

NHNH2

O

Cl

ClN

N

N

N

Cl

Cl

N

O

163 164

165 166

Fig (15) CB1 cannabinoid receptor antagonists 10 Other derivatives Examples of purine (163-164) pyrazoltriazines (165) andpyrazolo[15-a]pyrimidines (166) derivatives described as CB1 cannabinoid receptor antagonists by Griffith

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1383

The pEC50 value was 554 and the Emax value ndash26 ascompared to basal

AM630 also acts as an inverse agonist at the hCB1cannabinoid receptor (EC50 = 900 nM) as Landsman et aldemonstrated [47] Recently Zhang and colleagues reportedthe characterisation of the microsomal metabolism of AM630[212]

N

O

N

O

O

N

O

Cl

O

N

O

(S)-WIN-55212-3 167 BML-190 168

In 1996 Gallant and colleagues identified by submittinga large number of compounds to a binding assay anotherindole analog as CB2 cannabinoid receptor ligand [213] Onthe basis of this compound christened BML-190 (168 Fig16) or called indomethacin morpholinylamide theysynthesised several derivatives that possessed a selectivityratio for the CB2 cannabinoid receptor of up to 140 Thesewere the first compounds specifically designed to be CB2cannabinoid receptor ligands The first report on thepharmacological properties of BML-190 appeared onlyrecently New and colleagues highlighted in the paper theinverse agonist properties of the compound BML-190 dose-dependently increases (103 EC50= 980plusmn70 nM) theforskolin-stimulated levels of cAMP in hCB2-HEK cellswhile WIN-55212-2 decreases (44 45plusmn42nM) thisaccumulation [137]

Fig (16) CB2 cannabinoid receptor antagonists 2 Indolederivatives Chemical structures of WIN-55212-3 (167) andBML-190 (168) two indoles derivatives acting as inverseagonists at the hCB2 cannabinoid receptor Surprisingly in 2002 Melck and collaborators used

BML-190 as a CB2 receptor agonist in a cell proliferationassay [214] BML-190 was also tested as inhibitor of thecyclooxygenase-2 enzyme by Kalgutkar et al [215] Theyobtained IC50 values higher than 33 and 66 microM for theCOX-I and COX-II enzymes respectively These valueshave to be compared with the submicromolar activity ofindomethacin the parent compound Very recently Klegerisand colleagues [216] looking for an antineurotoxic action ofcannabinoids showed that BML-190 increases TNF-αsecretion by stimulated THP-1 monocytic cells but is noteffective on the IL-1β secretion

Further on the basis of WIN-54461 (12) a CB1antagonist AM630 (13) was synthesised by replacement ofthe bromine atom by an iodine one In the initial reportPertwee et al showed the antagonist effects of AM630 butalso suggested that the CB1 cannabinoid receptor may not bethe preferential receptor of AM630 [45] Later on Ross et alreported using hCB1amp2-CHO cells and [3H]-CP-55940that AM630 binds to the CB2 cannabinoid receptor (Ki =312 nM) with a selectivity ratio over 160 [48]Furthermore using a [35S]-GTPγ S assay they showed itsinverse agonist properties (EC50 = 76 nM) However

Table 12 CB2 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Reported binding affinities (Ki) and selectivity ratio (CB1CB2) of SR144528 (169) for rat and human cannabinoid receptors determined using [3H]-CP55940 as radioligand

NN

O

NH

Cl

SR144528 169

rCB2 (spleen) rCB1 (whole brain) Selectivity Ki hCB2 (transfected CHOcells)

Ki hCB1 (transfected CHOcells)

Selectivity References

038 nM 305 nM 800 06 nM 437 nM 730 [217]

067 nM 548 nM 80 [219]

56 nM gt10microM gt1780 [48]

25 nM a gt 1microM a gt400 [224]

024 nM 276 nM 115 199 nM 503 nM 25 [232]

a COS cells

1384 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

3 Diarylpyrazole Derivatives CHO cells is constitutively active and phosphorylated onSer 352 Upon treatment by SR144528 thisphosphorylation of the receptor was inhibited Moreover theSR compound was able to induce dephosphorylation ofagonist-induced CB2 phosphorylation The question whetherSR144528 induces a CB2 receptor conformational changemaking it a better phosphatase-specific substrate or ifSR144528 binding activates specific phosphatases remainsopen However this remains a nice example of inverseagonist-induced receptor resensitisation

The same Sanofi team which synthesised the CB1antagonist SR141716A reported in 1998 the synthesis andpharmacological characterisation of a CB2 cannabinoidreceptor selective antagonist the SR144528 (169) [217218] The structure of the new compound is derived fromSR141716A one SR144528 is the first selective CB2receptor antagonist reported as the inverse agonist propertiesof BML-190 were shown only recently by New et al [137]The authors reported for 169 using [3H]-CP-55940 asradioligand Ki values of 04 nM and 06 nM for the rat andthe human CB2 receptors respectively and found a CB1-CB2 selectivity ratio around 700 Table 12 sums up thebinding affinities of SR144528 for the cannabinoid receptorsobtained by different authors Interestingly while the CB2affinity is more or less constant the affinity obtained for theCB1 receptor varies by one order of magnitude [219] Theantagonist effect of SR144528 was highlighted by evaluatingits effect on cAMP production by hCB2-CHO cells exposedto 3 nM CP-55940 (EC50=10nM) The antagonism wasalso observed on the activation of the MAP kinase pathwayby CP-55940 (IC50=39nM)

The Sanofi research team working on cannabinoidsconducted investigations in order to determine the keyresidues for SR144528 interaction with the receptor (Table13) as well as the binding mode of the compound Firstlythey identified the TM4-EL2-TM5 region as a regioncontaining crucial residues for the affinity of several ligandsamong them SR144528 [224] Among these residues themutation of two cysteine of the second extracellular loop(C174 and C179) abolishes the affinity for the CB2 receptorLater on the implication of S453(161) and S457(165) inthe binding of SR144528 was evidenced by mutationalstudies In the model proposed for the docking of SR144528into the CB2 receptor the crucial contacts are comprised intothe TM4 this is the case for S453(161) and S457(165)which are proposed to form hydrogen bonds with the ligandand W464(172) proposed to have aromatic interactions withthe 4-methylbenzyl of the SR144528 [225] In 2003 Fengand Song showed that point mutations D349(130)A andA634(241)E abolished ligand binding However mutationof the arginine R350(131) member of the highly conservedDRY motif to alanine has no influence on SR144528binding [226]

Early after the report of the synthesis and characterisationof SR144528 the same authors further explored the functionof this compound and showed its inverse agonist propertiesusing CB2-CHO cells co-transfected with the luciferasereporter gene with either the CRE or the murine krox24regulatory sequence [220] In these models SR144528 notonly inhibits CP-55940 effects but also reproduces theeffects of a pertussin toxin treatment proving its inverseagonist properties SR144528 functionality was alsoexplored by [35S]-GTPγ S binding in hCB2-CHO cells(IC50=3 nM) [221] The inverse agonist function ofSR144528 was further illustrated by Rhee and Kim usingCOS cells co-transfected with hCB2 receptor and adenylylcyclase [222]

As for the CB1 cannabinoid receptor ligands Sanofideveloped tricyclic derivatives based on the 1-benzylpyrazole-3-carboxylic scaffold More than twentycompounds were disclosed in a patent [227] in 2001 (84-86Table 7) The benzyl in position 4 characteristic of the CB2diarylpyrazoles from Sanofi is conserved as well as thebicyclic residue in position 3 The length of the second linkbetween the phenyl and the pyrazole can be made of one or

Bouaboula and co-workers investigated SR144528 effectson CB2 cannabinoid receptor phosphorylation status [223]Firstly they showed that the CB2 receptor transfected in

Table 13 CB2 Cannabinoid Receptor Antagonists 3 Di-Aryl-Pyrazole Derivatives

Some of the single point mutations reported for the hCB2 cannabinoid receptor for which pharmacological data concerning the SR144528 are available

Mutant Effect References

R350(131)A no effect [226]

S453(161)A loss of affinity [225]

V456(164)I no effect [225]

S457(165)A loss of affinity [225]

C174S a loss of affinity [224 225]

C175S a loss of affinity [225]

C177S a no effect [225]

C179S a loss of affinity [224 225]

S544(193)G no effect [225]

a Second extra-cellular loop

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1385

two methylene but according to the patent one methylenebridge is preferred The Ki values of these compounds for theCB2 receptor were said to be lower than 500 nM and theirantagonist properties have been assessed using the adenylatecyclase inhibition assay

compounds by the presence of an alkyl substituent inposition 5 instead of a phenyl and of course by position 3which remains unsubstituted However except a lecture atthe 1998 ACS meeting [230] where compound 171 wasreported to have a Ki value of 835 nM and a selectivity ratioover 100 no further report on these compounds waspublished untill now

In a recent paper Mussinu and colleagues reported thesynthesis of other tricyclic derivatives starting fromSR141716A structure [228] N-piperidin-1-yl-14-di-hydroindeno[12-c]pyrazole-3-carboxamide derivatives areCB2 receptor ligands and some of them possess high affinityand selectivity for the CB2 receptor as shown in Table 7 (79-83) These tricyclic derivatives differ from the Sanofi tricyclicderivatives as they do have a phenyl ring instead of a benzylin position 1 Another difference is the nature of thesubstituent in position 3 which is not a bicycloalkyl but acyclic amine One of the compounds synthesised byMussinu et al 81 possesses the highest affinity for the CB2receptor ever reported with a Ki value of 0037 nM for themurine CB2 receptor ([3H]-CP-55940 mice spleenhomogenate) Interestingly 79 the closest SR141716Aanalog lost almost all its affinity for the CB1 receptor andincreased by four orders of magnitude its affinity for the CB2receptor Even if the structure quite similar to the SRcompounds could suggest that these derivatives behave asantagonists no pharmacological data were given in the paperto support this hypothesis

5 2-Oxoquinoline Derivatives

Inaba and colleagues from Japan Tobacco introduced in2001 a completely new structure in the cannabinoid fieldbased on a 2-oxoquinoline scaffold Over eighty derivativeswere disclosed in a patent [231] and one compound JTE-907 (173) was subsequently characterised in-vitro as well asin-vivo [232] In Table 14 are listed some of the compounds(172-178) disclosed in the patent along with their affinity forthe cannabinoid receptors These values were obtained onhuman receptors against [3H]-WIN-55212-2 For twelvecompounds the anti-inflammatory activity per os wasassessed in a carrageenin-induced paw edema model Thehighest effect reported was obtained with JTE-907 with anED50 value lower than 01 mgkg Therefore thesecompounds were claimed to be useful as anti-inflammatoryand anti-allergic agents

To further characterize JTE-907 the authors determinedits affinity for cannabinoid receptors of different species using[3H]-CP-55940 as radioligand In their hands JTE-907behaved as a very selective hCB2 ligand with Ki values of36 and 2370 nM for the CB2 and CB1 cannabinoid receptorsrespectively even more selective than the SR144528 (Kivalues of 199 and 50 nM respectively) However it has tobe mentioned that the affinities reported in the paper forJTE-907 are quite different from those reported in the patent(hCB1 Ki values of 35nM and 009 nM respectively) Italso appears that the SR144528 affinity for the CB1 receptordetermined by Iwamura et al is higher than the affinityreported by Rinaldi-Carmona et al with values of 50 and437nM respectively The authors ascribed the difference tothe variability of SR144528 binding affinities to the assayconditions The function of JTE-907 was evaluated bymeasuring the increase in cAMP production by forskolin-stimulated hCB2-CHO cells Maximum stimulation wasattained at a concentration of 1microM of JTE-907 In the sameassay WIN-55212-2 decreased the cAMP production in adose-dependent manner

Finally it is interesting to strengthen that the five-membered ring derivatives (79-86) of the diarylpyrazolefamily are reported to possess a high CB2 receptor affinitywhile the six- and seven-membered ring derivatives (69-78)are described as CB1 receptor ligands

4 Other Pyrazole Derivatives

Other CB2 ligands based on a pyrazole nucleus wereintroduced by SmithKline Beecham in a patent describingthe synthesis of about sixty new compounds [229] Theiraffinity (Ki) for the hCB2 receptor transfected in HEK293cells was reported to be ranging between 25nM and 10microMTheir structures (170-171 Fig 17) differ from the Sanofi

NN

O

N

ON

NN

N

NN

O

N

ON

NN

N

ClCl

1

5

4 3

2

170 171

Fig (17) CB2 cannabinoid receptor antagonists 4 Otherpyrazole derivatives Structure of two compounds (170-171)among those claimed by SmithKline Beecham to be hCB2antagonists

In conclusion JTE-907 was shown to be a CB2 selectiveinverse-agonist with anti-inflammatory and anti-allergicproperties This compound is currently undergoing Phase Iclinical trials as anti-allergic drug [233]

IV NON-CB1 NON-CB2 CANNABINOIDRECEPTORS LIGANDS

In the last years the possible presence of additional non-CB1 non-CB2 cannabinoid receptors was raised frompharmacological evidences

One of the putative receptors possesses an endotheliallocalisation while other ones are localized in the CNS or atperipheral nerves For instance an additional target for ∆9-

1386 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 14 CB2 Cannabinoid Receptor Antagonists 5 2-Oxoquinoline Derivatives

Structure of six compounds among the oxoquinoline derivatives patented by Japan Tobaco Affinity (Ki nM) and selectivity (CB1CB2) for the cannabinoidreceptors are given The affinity was measured by binding against [3H]-WIN-55212-2 on either hCB2-CHO or hCB1-CHO cells Adapted from [231]

Cpd ndeg Structure hCB2 hCB1 Selectivity

1-2 172

NH

MeO O

O

OMeO

0014 3671 262215

JTE-907 173

NH

O

MeO O

O

NH

O

O

0087 3436 39495

2-2 174

OH

O

NH

MeO O

O 077 3247 4215

3-27 175

NH

O

F

NH

MeO O

O0021 336 16000

3-32 176

NH

MeO O

O

NH

F

O

0036 2398 66610

3-38 177

O

O

NH

N

O

O

NMeO

0085 935 11000

3-43 178

NMeO O

O

NH

F

O

0043 864 20100

tetrahydrocannabinol was described in capsaicin-sensitivesensory nerves by Zygmunt et al [234]

[29] and by Di Marzo et al [235] Thus in the presentreview we will focus on the description of the so faridentified ligands of the non-CB1 non-CB2 receptors withan emphasis on the compounds having antagonist properties

Pharmacological evidences for the existence of additionalcannabinoid receptors were reviewed by Wiley and Martin

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1387

(Table 15) In particular the knowledge about three not yetcloned targets has grown The growing molecularpharmacology of the three putative new cannabinoidreceptors is reviewed in the three following sections Thefourth section summarises the data on the other proposedcannabinoid targets

vasodilatator activity In addition the anandamide effect ispartially sensitive to SR141716A inhibition The authorsproposed the existence of two distinct receptors responsiblefor the vasodilatatory effect of anandamide one located in theendothelium is SR141716A-sensitive while the other onelocated on the vascular smooth muscle is SR141716A-insensitive Thus SR141716A is an antagonist of anendothelial anandamide receptor Several studies wereconducted in order to understand the molecularpharmacology of the endothelial anandamide receptor It ispertussis toxin-sensitive [237] Cannabidiol acts asantagonist and abnormal-cannabidiol (Abn-cbd 179) and itsanalogue O-1602 (180) act as agonists [238] The synthesisof O-1602 and of its analogues are disclosed in a patentdescribing the vasodilatatory activity of cannabinoid

1 Endothelial Anandamide Receptor ndash Abnormal-Cannabidiol Receptor

In 1999 Wagner et al showed that anandamide couldinduce mesenteric vasodilatation through an endotheliallylocated ldquoanandamide receptorrdquo pharmacologically distinctfrom CB1 [236] Other cannabinoids such as ∆9-THC HU-210 or WIN-55212-2 were devoid of mesenteric

Table 15 Non-CB1 Non-CB2 Cannabinoid Receptors Antagonists

An overview of the putative new non-CB1 non-CB2 anandamide-receptors ligands The antagonists are highlighted in bold

NH

N

S

HO OH

Cl

OH

OH

OH OH OH OH

OMe

OMe

RR

RR

RR

RR

HO

OH

NH

O

cannabidiol (10) Abnormal-cannabidiol 179 O-1602 180

O-1918 181 capsazepine 183capsaicin 182

Compound Endothelial AEA receptor Brain AEA receptor Cannabinoid-vanilloid brain receptor

AEA agonist a agonist de

WIN-55212-2 no effecta

agonist e agonist g h

CP-55940 no effect e agonist i

SR141716A antagonist a b j no effect ([35 S]-GTPγS binding) d e f antagonist g h

AM251 no effect k no effect h i

Cannabidiol antagonist b no effect e

O-1918 antagonist c jm

Abd-cbd agonist b l

O-1602 agonist b

Capsaicin no effectj agonist i

Capsazepine no effect b antagonist h

a [236] b [238] c [240] d [249] e [250] f [251] g [254] h [258] i [ 255] k [244] j [ 243] l [ 242] m [241]

1388 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

analogues [239] More recently Offertaler et al showed thatO-1918 (181) a cannabidiol analog acts as an antagonist ofthe endothelial anandamide receptor [240] In addition O-1918 does not bind to the CB1 and CB2 cannabinoidreceptors Furthermore O-1918 unlike cannabidiol doesnot cause vasorelaxation in the mesenteric artery preparationmodel used and thus acts as a true neutral antagonistUsing O-1918 as a pharmacological tool Begg et alproposed a coupling of this new G protein-coupledendothelial receptor through a GiGo mechanism to ionchannels in primary cultured human vascular endothelialcells [241] However in their hands SR141716A wasineffective in antagonising Abd-cbd Ho and Hiley furtherdemonstrated that Abn-cbd relaxes rat small mesentericarteries through an activation of K+ channels via aSR141716A-sensitive pathway [242] Further using thesame model they characterized the vasorelaxant activity ofvirodhamine a novel endocannabinoid apparently actingthrough the same receptor [243] The virodhamine-inducedvasorelaxation is sensitive to SR141716A and O-1918 butinsensitive to AM251 SR144528 and AM630 Moreovervanilloid receptor desensitisation by capsaicin (182) had noeffect

244] Thus position 4 on the 5-phenyl could be furtherexplored to obtain selective ligands

To come to an end despite the fact that the endothelialanandamide receptor is neither identified nor cloned at leastthree antagonists are identified SR141716A cannabidioland O-1918 It is likely that a growing number of ligandsand therapeutic applications will appear in the very nearfuture

2 Brain Anandamide Receptor

Following an in-depth evaluation of anandamide effects inCB1

-- mice Di Marzo and co-workers suggested that a non-CB1 non-CB2 G protein-coupled receptor might mediatesome of the actions of AEA in mice [249] Actually effectsof AEA but not those of ∆9-THC were not decreased inCB1 cannabinoid receptor knock-out mice For instance 30mgkg of AEA were still effective in hot-plate test whereas10 mgkg of ∆9-THC had no effect on anti-nociceptionMoreover in a [35S]-GTPγ S assay AEA stimulated with anEC50 value of 223 microM the [35S]-GTPγ S binding to CB1

--

mice brain membranes (Emax= 28) ∆9-THC showed noeffect in this assay The addition of SR141716A did notaffect the [35S]-GTPγ S binding to CB1

-- mice brainmembranes Shortly after other evidences for the existence ofa new G protein-coupled cannabinoid receptor with a distinctdistribution in the central nervous system were published[250] From a set of 24 commonly used cannabinoids (AEAWIN-55212-2 JWH-030 THChellip) only AEA and WIN-55212-2 produced an increased binding of [35S]-GTPγ S onCB1

-- mice brain membranes Interestingly in this studySR141716A alone produced a significant inhibition of [35S]-GTPγ S binding similar to the inhibition obtained usingCB1

++ mice brain membranes (IC50 values of 57 microM and47 microM respectively) However SR141716A did not affectthe anandamide-induced [35S]-GTPγ S binding in CB1

--

mice brain membranes Moreover [3H]-SR141716Aexhibited significant binding in some brain regions of CB1

--

mice which are different from those exhibiting stimulation of[35S]-GTPγ S binding by WIN-55212-2 and AEA Thusthe authors proposed that the SR141716A effects on CB1

--

mice are mediated neither by the CB1 cannabinoid receptornor by the putative new anandamide brain receptor Monoryand co-workers using CB1

-- mice described a novel nonadenylyl cyclase-coupled cannabinoid binding site in micecerebellum [251] Two agonists AEA and WIN-55212-2but not ∆9-THC or HU210 were found to bind to thisreceptor as they enhanced [35S]-GTPγ S binding NeitherSR141716A nor SR144528 were able to affect the WIN-55212-2 induced [35S]-GTPγ S binding in CB1

-- mice asfound by Breivogel et al [250] However the distributionpattern of the receptor described by Monory et al differs fromthe distribution found by Breivogel et al It has to bementioned that the knock-out strains used were different Themice used by Monory were from a CD-1 strain while theone used by Breivogel were from a C57BL61 strainInterestingly Muthane et al detected differences in nigralneurons number and sensitivity to MPTP in the two mousestrains used [252]

More recently Mo et al obtained results showing thatthe Abnormal-cannabidiol receptor is also responsible for anincreased endothelial cell migration [244] Abn-cbd enhancedhuman umbilical vein endothelial cell migration while O-1918 antagonised Abn-cbd effect The CB1 cannabinoidreceptor inverse agonist SR141716A partially inhibitedAbd-cbd action whereas AM251 and SR144528 had noeffect

In another paper from George Kunos team Batkai et alreported that SR141716A is able to inhibit the endotoxichypotension by a cardiac mechanism involving neither theCB1 nor the CB2 cannabinoid receptors [245] Theinvolvement of a non-CB1 non-CB2 site of action wasshown using CB1

-- and CB1--CB2

-- knock-out miceFurther studies are needed to identify the SR141716Asensitive myocardial site of action

In a recent patent Kunos described O-1918 and itsanalogues as vasoconstrictor agents useful to reversepathological vasodilatation of blood vessels [246] Theeffects of O-1918 on mouse blood pressure on LPS-inducedhypotension in mouse as well as the reversal of hypotensionby O-1918 injection are described Furthermore thesynthesis of O-1918 based antagonists is described startingfrom p-metha-28-dien-1-ol and the appropriate resorcinols

Interestingly the Abd-cbd receptor which was firstdetected in the vascular system also seems to be present inthe CNS [247] Indeed Walter et al found that microglialcells (BV-2) migration induced by 1 microM 2-AG is inhibitedby cannabidiol (300 nM) and O-1918 (1 microM) SR141716Awas found ineffective in this assay Thus 2-AG is an agonistof the Abd-cbd receptor Moreover Abd-cbd is able to elicita dose-dependent cell migration in the same model (EC50 =600nM) [248]

From a more structural point of view SR141716Abehaves as an antagonist while on the opposite AM251(20) a close analogue which differs from the former only bythe nature of one halogen has no effect on this receptor [242

To conclude to our knowledge no compounds havingantagonist or inverse agonist properties at this receptor havebeen identified so far

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1389

3 Cannabinoid-Vanilloid Brain Receptor a cannabinoid-vanilloid receptor at which SR141716A andcapsazepine behave as antagonists

Evidences for the presence of an additional ldquocannabinoidrdquoreceptor differing from the CB1 receptor and from the brainanandamide receptor appeared from studies focused on thehippocampus (for a review see Hajos et al [253]) Hajos etal studied the cannabinoid actions on GABAergic andglutamatergic transmission using the whole-cell patch-clamp technique [254] WIN-55212-2 induced a dose-dependent reduction in glutamatergic transmission reversedby SR141716A (1microM) Thus the authors concluded that thecannabinoid actions on the glutamatergic transmission aremediated by a non-CB1 but SR141716A-sensitive receptorIn another paper Hajos and Freund further characterised thisputative receptor [255] They showed that in addition toWIN-55212-2 CP-55940 and capsaicin (182) a TRPV1receptor agonist also act as agonists Furthermore in theirhands SR141716A as well as capsazepine (183) a TRPV1receptor antagonist were able to antagonise this effect on theglutamatergic transmission while AM251 was notSR141716A effects on CB1 knock-out mice were furtherstudied using a test of anxiety [256] Haller et al showedthat on the one hand the distribution of the CB1 receptorinduces anxiety and SR141716A has an anxiolytic effectand on the other hand SR141716A is still active in CB1

--

mice In a following paper they reported that AM-251induces anxiogenic effects in wild-type and in knock-outmice [257] Thus SR141716A in addition to CB1 bindsto a not yet identified receptor Another evidence for thisadditional receptor for SR141716A was given by Bass andco-workers [61] They conducted a structure-activityrelationship study on the stimulation of locomotor activityinduced by SR141716A and twenty analogues Nocorrelation was found between the affinity for the CB1cannabinoid receptor and the stimulation of locomotoractivity Albeit SR141716A and five of its analogues O-1803 O-1710 O-1253 O-1254 (40) O-1255 (41) behavedas inverse agonists in [35S]-GTPγ S assay none of theseanalogues stimulated the locomotor activity In conclusionthe SR141716A-induced stimulation of locomotor activity isneither due to inverse agonism nor to inhibition of anendogenous tone but to a brain receptor distinct from theCB1 receptor Di Marzo et al previously provided evidencesthat the cannabinoid-vanilloid brain receptor could mediatesome of the cannabinoid effects on locomotion [249]

4 Other Non-CB1 Non-CB2 Cannabinoid Receptors

Evidences for the existence of additional non-CB1 non-CB2 cannabinoid receptors localised in the rat isolatedmesenteric arterial bed appeared from the works of Ralevicand collaborators Several cannabinoids were evaluated intheir model of sensory neurogenic vasorelaxation evoked byelectrical field stimulation

HU210 was found to attenuate vasorelaxation in a CB1and CB2 independent way as SR141716A LY320135 andSR144528 were ineffective in inhibiting HU210 effect [259]Similar results were obtained with ∆9-THC [260]Similarly noladin ether inhibited sensory neurogenicrelaxation in a concentration-dependent manner which wasalso unaffected by SR141716A LY320135 and SR144528Moreover this effect as the ∆9-THC one was pertussistoxin sensitive suggesting the involvement of a Gio protein-coupled receptor [261]

However the inhibitory effects of CP-55940 and WIN-55212-2 were affected by the presence of SR141716A (1microM)or LY320135 (1microM) but not by the addition of SR144528(1microM) [262]

It appears that at least two receptors should be involvedin cannabinoid-mediated attenuation of sensory nerve-mediated vasorelaxation The first one is SR141716A andLY320135 sensitive while the other one is resistant toSR141716A

Some evidences for the existence of a ldquoCB2-likerdquo receptorappeared from the studies on palmitoylethanolamide (PEA)an endogenous fatty acid amide devoid of CB1 and CB2affinity [264] antinociceptive potential Calignano and co-workers showed that PEA was able to alleviate pain inseveral animal models [265 266] For instance PEA dose-dependently inhibits kaolin-evoked writhing in mice TheCB2 cannabinoid receptor inverse agonist SR144528 (02mgkg) inhibits PEA anti-nociceptive effect [266] Thus theauthors suggest that PEA acts as agonist on a SR144528-sensitive non-CB2 cannabinoid receptor (ldquoCB2-likerdquoreceptor) at which SR144528 acts as an antagonist

Further studies are needed in order to better characterisethese additional ldquocannabinoidrdquo receptors However it islikely that several therapeutic applications will soon springfor these receptors

Very recently Pistis et al published a study on theneurophysiological effects of cannabinoids on the basolateralamygdala neurons in-vivo [258] One of their findings is thatHU-210 and WIN-55212-2 did not elicit similar effects onbasal amygdala projection neurons firing rate While theformer had no effect the latter decreased the firing rateMoreover SR141716A reversed the WIN-55212-2 effectswhile AM251 had no effect The vanilloid receptorantagonist capsazepine also antagonised the WIN-55212-2effect on firing rate of these neurons Therefore the authorssuggested the presence of a novel cannabinoid-vanilloidreceptor

V CONCLUSION

Since their development cannabinoid antagonists provedto be essential tools in the understanding of cannabinoidpharmacology and biochemistry Moreover the interestshown by the pharmaceutical industry in the development ofnew cannabinoid antagonists prove if necessary the realtherapeutic potential of this class of compounds Thetreatment of eating and movement disorders memorydeficits psychosis and dependencies from various addictivedrugs are some of the most cited applications for the CB1cannabinoid receptor antagonists

From these lines it appears that in addition to theknown CB1 cannabinoid receptor it is now likely that twoother cannabinoid receptors do exist in the brain The firstone should be an anandamide brain receptor for whichSR141716A has no affinity while the second one should be

1390 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Much less wide is the knowledge on the CB2cannabinoid receptor antagonists However potentialapplications could be disorders involving the immunesystem such as inflammation or allergies

[19] Devane WA Hanus L Breuer A Pertwee RG StevensonLA Griffin G Gibson D Mandelbaum A Etinger AMechoulam R Science 1992 258 1946

[20] Mechoulam R Ben-Shabat S Hanus L Ligumsky MKaminski NE Schatz AR Gopher A Almong S MartinBR Compton DR Biochem Pharmacol 1995 50 83

Finally the growing literature on the non CB1 non CB2cannabinoid receptors should allow the development of newdrugs First examples are given by the cannabidiol andabnormal-cannabidiol derivatives described by Kunos et alfor which potential therapeutic applications on the vascularsystem are already patented

[21] Sugiura T Kondo S Sukagawa A Nakane S Shinoda AItoh K Yamashita A Waku K Biochem Biophys Res Co1995 215 89

[22] Hanus L Abu-Lafi S Fride E Breuer A Vogel Z ShalevDE Kustanovich I Mechoulam R Proc Natl Acad SciUSA 2001 98 3662

[23] Oka S Tsuchie A Tokumura A Muramatsu M Suhara YTakayama H Waku K Sugiura T J Neurochem 2003 851374It clearly appears from this paper that the antagonists of

the cannabinoid receptors are still an active research fieldfrom which should emerge new promising therapeutic toolsas well as innovative drugs in the near future

[24] Goutopoulos A and Makriyannis A Pharmacol Therapeut2002 95 103

[25] Beal JE Olson R Lefkowitz L Laubenstein L Bellman PYangco B Morales JO Murphy R Powderly W PlasseTF Mosdell KW Shepard KV J Pain Symptom Manage1997 14 7ACKNOWLEDGEMENTS [26] Notcutt W Price M Miller R Newport S Phillips CSimmons S Sansom C Anaesthesia 2004 59 440

GGM is very indebted to the ldquoFonds pour la formationagrave la Recherche dans lrsquoIndustrie et lrsquoAgriculture (FRIA)rdquo Belgium for the award of a research fellowship Theexperimental works of the authors were supported by grantsfrom the FNRS The Belgian Charcot Foundation and theUniversiteacute catholique de Louvain

[27] Ledent C Valverde O Cossu G Petitet F Aubert JFBeslot F Bohme GA Imperato A Pedrazzini T RoquesBP Vassart G Fratta W Parmentier M Science 1999 283401

[28] Zimmer A Zimmer AM Hohmann AG Herkenham MBonner TI Proc Natl Acad Sci U S A 1999 96 5780

[29] Wiley JL and Martin BR Chem Phys Lipids 2002 121 57[30] Adam J and Cowley P Expert Opin Ther Patents 2002 12

1475

REFERENCES [31] Razdan RK and Mahadevan A Chem Phys Lipids 2002 12121

[32] Barth F and Rinaldi-Carmona M Curr Med Chem 1999 6745

[1] Hillard CJ Harris RE Bloom AS J Pharmacol Exp Ther1985 232 579

[33] Little PJ Compton DR Johnson MR Melvin LS MartinBR J Pharmacol Exp Ther 1988 247 1046

[2] Devane WA Dysarz FA Johnson MR Melvin LSHowlett AC Mol Pharmacol 1988 34 605

[34] Pertwee RG Stevenson LA Elrick DB Mechoulam RCorbett AD Brit J Pharmacol 1992 105 980

[3] Matsuda LA Lolait SJ Brownstein MJ Young ACBonner TI Nature 1990 346 561

[35] Harrison C Traynor JR Life Sci 2003 74 489-508[4] Gerard CM Molerau C Vassart G Parmentier M BiochemJ 1991 279 129 [36] Pertwee RG Fernando SR Griffin G Ryan W Razdan

RJ Compton DR Martin BR Eur J Pharmacol 1996 315195

[5] Glass M Dragunow M Faull RLM Neuroscience 1997 2299

[37] Ross RA Brockie HC Fernando SR Saha B Razdan RKPertwee RG Brit J Pharmacol 1998 125 1345

[6] Shire D Carillon C Kaghad M Calandra B Rinaldi-Carmona M LeFur G Caput D Ferrara P J Biol Chem1995 270 3726 [38] Griffin G Wray EJ Rorrer WK Crocker PJ Ryan WJ

Saha B Razdan RK Martin BR Abood ME Brit JPharmacol 1999 126 1575

[7] Ryberg E Khang Vu H Groblewski T Larsson N ElebringT Hjorth S Sjogren S Greasley PJ 2004 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 119

[39] Ross RA Gibson TM Stevenson LA Saha B Crocker PRazdan RK Pertwee RG Brit J Pharmacol 1999 128 735

[40] Martin BR Winckler JR Wiley JL Huffman JW CrockerPJ Saha B Razdan RK J Pharmacol Exp Ther 1999 2901065

[8] Rinaldi-Carmona M Calandra B Shire D Bouaboula MOustric D Barth F Casellas P Ferrara P LeFur G JPharmacol Exp Ther 1998 278 871

[41] Martin BR Stevenson LA Pertwee RG Breivogel CSWilliams A Mahadevan A Razdan RK 2002 Symposium onthe Cannabinoids Burlington Vermont International CannabinoidResearch Society page 2

[9] Munro S Thomas KL Abu-Shaar M Nature 1993 365 61[10] Howlett AC Qualy JM Khachatrian LL Mol Pharmacol

1986 29 307[11] Glass M Felder CC J Neurosci 1997 17 5327

[42] Thomas A Ross RA Saha B Mahadevan A Razdan RKPertwee RG Eur J Pharmacol 2004 487 213

[12] Calandra B Portier M Kerneacuteis A Delpech M Carillon C LeFur G Ferrara P Shire D Eur J Pharmacol 1999 374445 [43] Compton DR Gold LH Ward SJ Balster RL Martin BR

J Pharmacol Exp Ther 1992 263 1118[13] Howlett AC Life Sci 1984 35 1803[44] Eissenstat MA Bell MR DAmbra TE Alexander EJ

Daum SJ Ackerman JH Gruett MD Kumar V EstepKG Olefirowicz EM Wetzel JR Alexander MD WeaverJD Haycock DA Luttinger DA Casiano FM ChippariSM Kuster JE Stevenson JI Ward SJ J Med Chem1995 38 3094

[14] Deadwyler S Hampson RE Mu J Whyte A Childers SRJ Pharmacol Exp Ther 1995 273 734

[15] Mackie K Lai Y Westenbroek R Mitchell R J Neurosci1995 15 6552

[16] Bouaboula M Poinot CC Bourrie B Canat X Calandra BRinaldi-Carmona M LeFur G Casellas P Biochem J 1995312 637 [45] Pertwee R Griffin G Fernando S Li X Hill A

Makriyannis A Life Sci 1995 56 1949[17] Bouaboula M Poinot CC Marchand J Canat X Bourrie BRinaldi-Carmona M Calandra B LeFur G Casellas P Eur JBiochem 1996 237 704

[46] Hosohata Y Quock RM Hosohata K Makriyannis AConsroe P Roeske WR Yamamura HI EurJPharmacol 1997 321 R1[18] delPulgar T Velasco G Guzman M Biochem J 2000 347

369 [47] Landsman RS Makriyannis A Deng H Consroe P RoeskeWR Yamamura HI Life Sci 1998 62 109

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[48] Ross RA Brockie HC Stevenson LA Murphy VLTempleton F Makriyannis A Pertwee RG Brit JPharmacol 1999 126 665

[81] Gatley SJ Lan R Volkow ND Pappas N King P WongCT Gifford AN Pyatt B Dewey SL Makriyannis A JNeurosci 1998 70 417

[49] Huffmann JW Curr Med Chem 1999 6 705 [82] Lan R Lu Q Fan P Gatley J Volkow ND Fernando SRPertwee R Makriyannis A AAPS Pharmsci 1999 1 article 4[50] Rinaldi-Carmona M Barth F Heaulme M Shire D Calandra

B Congy C Martinez S Maruani J Neliat G Caput DFerrara P Soubrieacute P Breliegravere JC Le Fur G FEBS Lett1994 350 240

[83] Mathews WB Ravert HT Musachio JL Franck RARinaldi-Carmona M Barth F Dannals RF J Labelled CompdRad 1999 42 589

[51] Rinaldi-Carmona M Barth F Heaulme M Alonso R ShireD Congy C Soubrie P Breliere JC Le Fur G Life Sci1995 56 1941

[84] Mathews WB Scheffel U Finley P Ravert HT FrankRA Rinaldi-Carmona M Barth F Dannals RF Nucl MedBiol 2000 27 757

[52] Jarai Z Wagner JA Goparaju SK Wang L Razdan RKSugiura T Zimmer AM Bonner TI Zimmer A Kunos GHypertension 2000 35 679

[85] Berding G Muumlller-Vahl K Schneider U Gielow P FitschenJ Stuhrmann M Harke H Buchert R Donnerstag FHofmann M Knoop BO Brooks DJ Emrich HM KnappWH Biol Psychiat 2004 55 904[53] Fox A Kesingland A Gentry C McNair K Patel S Urban

L James I Pain 2001 92 91 [86] Mathews WB Scheffel U Rauseo PA Ravert HT FrankRA Ellames GJ Herbert JM Barth F Rinaldi-Carmona MDannals RF Nucl Med Biol 2002 29 671

[54] Costa B Vailati S Colleoni M Behav Pharmacol 1999 10327

[55] Compton DR Aceto MD Lowe J Martin BR J PharmacolExp Ther 1996 277 586

[87] Bays HE Obes Res 2004 12 1197[88] Comment in Nat Rev Drug Discov 2004 3 288

[56] Petitet F Jeantaud B Capet M Doble A BiochemPharmacol 1997 54 1267

[89] LeFur G 2004 Symposium on the Cannabinoids BurlingtonVermont International Cannabinoid Research Society page 67

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[189] Barth F Martinez S Rinaldi-Carmona M World PatentApplication 2003 WO03084930

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CB Walsh TE Shah SK World Patent Application 2004WO04012671

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R Vogel Zvi FEBS letters 1995 375 143 [241] Begg M Mo F-M Offertaler L Batkai S Pacher PRazdan R K Lovinger DM Kunos G J Biol Chem 2003278 46188

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Pharmacol Exp Ther 1996 278 989

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[246] Kunos G World Patent Application 2003 WO03015700 [257] Haller J Varga B Ledent C Freund TF Behav Pharmacol2004 15 299[247] Franklin A and Stella N Eur J Pharmacol 2003 474 195

[248] Walter L Franklin A Witting A Wade C Xie Y KunosG Mackie K Stella N J Neurosci 2003 23 1398

[258] Pistis M Perra S Pillolla G Melis M Gessa GL MuntoniAL Neuropharmacology 2004 46 115

[249] Di Marzo V Breivogel C S Tao Q Bridgen DT RazdanRK Zimmer AM Zimmer A Martin BR J Neurochem2000 75 2434

[259] Ralevic V and Kendall DA Eur J Pharmacol 2001 418 117[260] Duncan M Kendall DA Ralevic V Brit J Pharmacol 2003

138 214P[250] Breivogel CS Griffin G Di Marzo V Martin BR Mol

Pharmacol 2001 60 155[261] Duncan M Millns P Smart D Wright JE Kendall DA

Ralevic V Brit J Pharmacol 2004 142 509[251] Monory K Tzavara ET Lexime J Ledent C Parmentier

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[263] Tao Q and Abood ME J Pharmacol Exp Ther 1998 285651[252] Muthane U Ramsay KA Jiang H Jackson-Lewis V

Donaldson D Fernando S Ferreira M Przedborki S ExpNeurol 1994 126 195

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[266] Calignano A La Rana G Piomelli D Eur J Pharmacol 2001419 191

Page 14: Current Knowledge on the Antagonists and Inverse Agonists of

1374 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

O

O

O

O

NC

S

O

O

O

O

O

O

O

NC

LY320135 8887 89

Fig (6) CB1 cannabinoid receptor antagonists 4 Phenyl benzofuranone derivatives Chemical structures of two aryl-benzofuranederivatives (87 -88 ) and one aryl-benzothiophene (89 ) derivative from Eli Lilly

500 nM and the effect of WIN-55212-2 on calcium channelsat 1microM Moreover the intra-peritoneal injection (20 mgkg)of this compound antagonised the in-vivo effect ofanandamide in the Open Field Assay a mouse behaviourmodel Later on Felder and colleagues gave a furtherdescription of this compound called LY320135 in a paperdescribing its effect on cAMP accumulation and on ioniccurrents [163] The selectivity ratio of LY320135 was 106with Ki values of 141 and 14900 nM for hCB1 and hCB2receptors ([3H]-CP-55940) respectively LY320135antagonised anandamide inhibition of forskolin-inducedcAMP accumulation with an IC50 value of 734 nMChristopoulos et al in 2001 reported for LY320135 apKb value of 527 in the inhibition of WIN-55212-2mediated response in the rat electrically-stimulated vasdeferens while the pKb value for SR141716A was 75[164]

Other patents were taken by Aventis in which somepotential applications for their compounds were describedFor instance an association between an azetidine derivativehaving CB1 antagonists properties and sibutramine aserotonin and norepinephrine reuptake inhibitor was claimed[170] as a treatment against obesity Oral administration ofcompound 90 (3 mgkg) and sibutramine (06 mgkg) to fafaZucker rats which are genetically obese rats has resulted ina decreased food intake compared to control lean Zuckerrats In another patent the association of an azetidinederivative (1-10 mgkg po ) and a D2D3 agonist likequinpirole (01 mgkg ip) was claimed to have a therapeuticpotential in the treatment of Parkinsonrsquos disease [171] Theeffect has been evidenced using an akinesia model in the ratEvidences supporting the use of CB1 antagonists as adjuvantin the treatment of Parkinsonrsquos disease begin to appear Itseems that such compounds would be helpful in thetreatment of Parkinsonism [172] and levodopa-induceddyskinesia [173] Brotchie published an interesting paperreviewing the CB1 cannabinoid receptor signaling inParkinsonrsquos disease [174] Nevertheless further clinicalstudies should be conducted to further confirm the usefulnessof CB1 antagonists in the treatment of Parkinsonrsquos disease

5 Azetidine Derivatives

The family of azetidine compounds illustrated bycompounds 90 and 91 (Fig 7) was developed at Aventis byDaniel Achard and colleagues and was described in a seriesof patents [165-168] They claimed the IC50 values of thesecompounds for the CB1 receptor to be less than or equal to100 nM These values were obtained following the proceduredescribed by Kuster et al [169] The antagonist propertywas shown using an in-vivo model the reversal ofhypothermia induced by CP-55940 in mice and the ED50obtained were lower than 50 mgkg

6 Aryl-Imidazolidine-24-Diones Derivatives

In 1999 the Didier Lambert team published a paperdescribing the affinity of 24 new 3-alkyl-55rsquo-diphenyl-imidazolidine-24-dione derivatives for the hCB1 receptor[175] The preliminary structure-activity relationships

N

SO

O

Cl

Cl

F

F

N

SO

O

Cl

NS

90 91

Fig (7) CB1 cannabinoid receptor antagonists 5 Azetidine derivatives Two examples (90 -91 ) of the azetidine derivatives developedby Aventis

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1375

Table 8 CB1 Cannabinoid Receptor Antagonists 6 Aryl-Imidazolidine-24-Diones Derivatives

Structures affinities ([3H]-SR141716A hCB1-CHO cells) and pKb values ([35 S]-GTPγS HU210 rat brain) of three aryl-imidazolidine-24-dionederivatives (92-94)

N

NH

OO

R

Br

Br

Cpd ndeg R Ki (nM) a pKb b

DML20 92 ethylmorpholine 70 611

DML21 93 1-hydroxypropyl 103 625

DML23 94 heptyl 98 574

a [175]b [177]

showed that the substitution at the nitrogen in position 3 aswell as a bromine atom in para of the phenyl rings aremandatory for the compounds affinity Later on threecompounds termed DML20 (92) DML21 (93) and DML23(94) were characterised as neutral antagonists using the [35S]-GTPγ S assay on rat cerebellum membranes [176] (Table 8)In the same model they competitively inhibited HU-210-induced [35S]-GTPγ S binding with pKB values of 611625 and 574 respectively Moreover these compoundswere proven to be quite selective for the CB1 receptor

higher than the one for the CB2 cannabinoid receptor (IC50 =300 nM) The inverse agonism of the compound wasextrapolated by inhibiting CP-55940-induced hypothermiain mice (EC50 = 18 nM) Moreover one oral dose (10mgkg) reduces the food intake in diet-induced obese ratsInterestingly food intake of CB1

-- mice was not alteredBody weight loss was maintained all over the study during achronic administration (14 days) of 10 mgkg of Cpd A

Later on Merck published a patent [180] in which someof the 45-diaryl-pyrazoles derivatives previously describedwere claimed to be useful in the treatment or the preventionof obesity in association with compounds inhibiting the11β-hydroxysteroid dehydrogenase type 1 (1β-HSD1)enzyme The 1β-HSD1 enzyme is responsible for thesynthesis of cortisol increased levels of which according tothe inventors are associated with obesity However neitherresults on appetite suppression nor on weight loss weregiven even if some indications on the affinity of thecompounds for the cannabinoid receptors were given

Very recently the functionality of these DML compoundswas explored using the [35S]-GTPγ S assay both on rat andhuman CB1 cannabinoid receptors [177] The data obtainedconfirm that the 3-alkyl-55rsquo-diphenyl-imidazolidinedionederivatives behave as neutral antagonists on rat CB1cannabinoid receptor (rat brain) However DML 20 DML21and DML23 acted as inverse agonists on the human receptor(hCB1-CHO cells) Furthermore the authors showed thatthis different functionality is not due to the hCB1 receptorlevel of expression in the recombinant cell line as whateverthe level of expression (Bmax of 44 pmolmg or 32pmolmg) DML derivatives behaved as inverse agonists ofthe hCB1 cannabinoid receptor

Another imidazole ring pattern of substitution wasproposed by Hagmann and collaborators [181] Thecompounds described 43 examples are given possess twophenyls the first one in position 1 linked to the nitrogenand the second one in position 2 The carboxamidesubstituent is in position 4 instead of in position 2 for thediarylimidazoles described by Finke These 12-diaryl-imidazoles are claimed to be CB1 cannabinoid receptorantagonists or inverse agonists but the absence of precisepharmacological values do not allow any comparisonbetween the two substitutions patterns However a samepattern of substitution was used by other researchers Amongthem Dyck et al described in 2004 ten 1-(4-chlorophenyl)-2-(24-dichlorophenyl)-imidazole derivatives varying by theircarboxamide substituent in position 4 (101-103 Table 9)[152] For instance with a 3-azabicyclo[330]octan-3-ylsubstituent (101) they obtained a Ki value of 14 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) In a [35S]-GTPγ Sassay 101 exhibited an IC50 value of 19 nM

7 Diarylimidazoles Derivatives

Finke and colleagues at Merck developed a new class ofCB1 receptor antagonists based on an imidazole nucleusOver eighty 45-diaryl-imidazoles derivatives having CB1antagonist properties were claimed in a patent published in2003 [178] Some representative compounds of the patent areillustrated in Table 9 (95-100) The affinity of the newcompounds was determined using [3H]-CP-55940 andhCB1-CHO cells Although no detailed pharmacologicaldata were given in the patent in-vitro and in-vivo propertiesof one compound termed Cpd A (100) have been describedelsewhere [179] Its affinity for the CB1 cannabinoid receptorwas shown to be nanomolar (IC50 = 4 nM) and 75 times

1376 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 9 CB1 Cannabinoid Receptor Antagonists 7 Diaryl-Imidazole Derivatives

Structure and affinity range of some 45-diaryl-imidazole derivatives for the human cannabinoid receptors determined using [3H]-CP-55940 asradioligand and hCB1amp2-CHO cells

N

NR2

R1

R4

R3

ndeg R1 R2 R3 R4 hCB1 (IC50 or Ki) hCB2 (IC50 or Ki) References

95 CH3 CO-NH-piperidinyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

96 CH3 CO-NH-piperidinyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

97 CH3 CO-NH-cyclohexyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

98 CH3 CO-NH-phenyl 24-di-Cl-Ph 4-Cl-Ph 10 ndash 100 nM a 100 ndash1000 nM a [178]

99 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

100 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph 4 nM a 300 nM a [179]

101 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

CH3 14 nM b ND [152]

102 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

H 66 nM b ND [152]

103 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 85 nM b ND [152]

104 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 23 nM b 542 nM b [182]

105 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 30 nM b 608 nM b [182]

106 4-Br-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 60 nM b 489 nM b [182]

107 4-CF3-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 29 nM b 634 nM b [182]

108 4-Cl-Ph 24-di-Cl-Ph CO-NH-morpholinyl CH3 197 nM b 3297 nM b [182]

109 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidin-4-ol CH3 172 nM b 3959 nM b [182]

110 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CN 30 nM b 1590 nM b [182]

111 4-Cl-Ph 24-di-Cl-Ph CO-NH- piperidinyl CH2F 36 nM b 906 nM b [182]

112 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH2-CH3 14 nM b 430 nM b [182]

113 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Cl 27 nM b 823 nM b [182]

114 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Br 23 nM b 746 nM b [182]

115 4-Cl-Ph 24-di-Cl-Ph CO-NH-tetrahydroisoquinoline CH3 34 nM b 696 nM b [182]

116 4-Cl-Ph 24-di-Cl-Ph CO-NH-cycloheptyl CH3 35 nM b 349 nM b [182]

117 24-di-Cl-Ph 4-Cl-Ph CO-NH-piperidinyl CH3 403 nM b 208 nM b [182]a IC50 value b Ki value

Lange and co-workers from Solvay described twenty-eight imidazole derivatives [182] The 24-dichlorophenylsubstituent at position 2 was kept constant while thesubstitutions at the other positions were explored (Table 9)Position 5 can accommodate a large range of littlesubstituents like hydrogen (104) methyl (105) ethyl (112)chlorine (113) bromine (114) fluoromethyl (111) or cyano(110) without major changes in the affinity At position 4more bulky substituents like 1234-tetraisoquinoline (115)or cycloheptyl (116) were well tolerated whereas thepresence of a more hydrophilic moiety such as morpholine(108) or piperidin-4-ol (109) was detrimental to the affinity

Interestingly position exchange between the 24-diphenyland the 4-chlorophenyl substituents led to a 13 fold loweraffinity with Ki values of 30 and 403 nM for 105 and 117respectively ([3H]-CP-55940 hCB1-CHO cells) In thefunctional assay which was inhibition of WIN-55212-2-induced [3H]-arachidonic acid release by hCB1-CHO cellsall tested compounds behaved as antagonists Compound105 showed a pA2 value of 86 which is the same theauthors obtained for SR141716A It would be interesting toassess if these compounds are true antagonists or if they actmore as inverse agonists Compound 105 when administeredto rats was able to inhibit CP-55940-induced hypotension

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1377

with an ED50 value of 24 mgkg which is close to the 3mgkg obtained for SR141716A The compound was alsoactive in another in-vivo model the WIN-55212-2-inducedhypothermia in mice Compound 104 which has nosubstituent in position 5 of the imidazole ring was lessactive in the hypotension model and inactive in thehypothermia model Thus a methyl group is the preferredsubstituent in position 5 of the imidazole moiety Otherproperties either experimental (P-glycoprotein affinitylogPHPLC) or computational (molecular volume polarsurface area) obtained for compounds 105 were similar tothose obtained for SR141716A Finally molecularmodelling studies revealed a close structural overlap betweenthe two compounds

value of 197 nM is selective for hCB1 and is as potent asSR141716A The structural modulations of 118 led to someinteresting compounds (Table 10) Six compounds possess aKi value for the hCB1 cannabinoid receptor equal to or lowerthan 25 nM antagonist properties in the inhibition of [3H]-arachidonic acid release by WIN-55212-2-stimulated hCB1-CHO cells and Ki values for the hCB2 receptor over 1microMThe diaryl-pyrazoline derivatives contained a chiral center atposition 4 The authors resolved the racemic mixture of twocompounds (121 and 130) The levorotatory enantiomersappeared to be the eutomers For compound SLV319levorotatory enantiomer of 121 the Ki and pA2 values were78 nM and 99 respectively The Ki and pA2 values ofSLV326 levorotatory enantiomer of 130 were 359 nM and9 respectively

8 34-Diaryl-Pyrazoline Derivatives Two in-vivo models CP-55940-induced hypotension inrat and WIN-55212-2-induced hypothermia in mouse wereused to assess the potential of these compounds The resultsare of the same order of magnitude than those obtained forSR141716A For instance in the hypotension modelSLV319 ((-)121) and SLV326 ((-)130) showed ED50 values

Lange and collaborators also discovered 34-diaryl-pyrazoline derivatives after a screening of compoundsresembling to SR141716A [183184] Compound 118 thatwas initially identified during the screening possesses a Ki

Table 10 CB1 Cannabinoid Receptor Antagonists 8 34-Diaryl-Pyrazolines Derivatives

Structure affinity for the hCB1 and hCB2 cannabinoid receptors (Ki nM) expressed in CHO transfected cells ([3H]-CP-55940) and potency (pA2) ofsome 34-dirylpyrazolines derivatives Table adapted from [184]

NN

N N

SO2

R3

R2

Cl R1

R4

n˚ R1 R2 R3 R4 Ki hCB1 (nM) Ki hCB2 (nM) pA2 (CB1)

118 H H H 4-CH3 197 gt 1000 84

119 H H H 246- CH3 24 gt 1000 94

120 H H H 4-Cl 16 gt 1000 95

121 H CH3 H 4-Cl 25 gt 1000 87

122 H CH3 CH3 4-Cl 280 gt 1000 85

123 H H H 4-F 53 gt 1000 9

124 H CH3 H 4-F 338 gt 1000 85

125 H CH3 CH3 4-F gt 1000 gt 1000 lt 75

126 H CH3 H 3-Cl 14 gt 1000 86

127 4-Cl CH3 H 4-Cl 255 ND ND

128 4-F CH3 H 4-Cl 584 ND ND

129 H CH3 H H 170 ND 75

130 H CH3 H 4- CF3 221 gt 1000 93

SR141716A 25 1580 86

1378 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

N

HN

O

N

Cl Cl

Cl

N

N

HN

O

Br

Br131 132

Fig (8) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Chemical structures of two 56-diaryl-pyrazine-2-amide derivatives (131-132) developed as CB1 cannabinoid receptorantagonists by AstraZeneca

of 55 and 2 mgkg (po) respectively proving their in-vivoefficacy after oral administration The authors alsodemonstrated that SLV319 is devoid of affinity for the P-glycoprotein pump and that it possessed a goodCNSplasma ratio (17) These two compounds (SLV319and SLV326) were chosen as development candidates bySolvay and entered clinical Phase I trials in 2003 [185]

132 Fig 8) useful as CB1 cannabinoid receptor antagonists[186 187] The activity of twenty derivatives wasdetermined using a [35S]-GTPγ S assay and hCB1-CHOtransfected cells The concentration required to give halfmaximal inhibition of CP-55940-induced [35S]-GTPγ Sbinding (IC50) is lower than 200 nM for the preferredcompounds No affinity data were given for thesecompounds

9 Diaryl-Pyrazine Diphenyl-Pyridine Diphenyl-Phenyland Diaryl-Pyrimidines Derivatives

The same year in a patent from Merck Finke andcollaborators described several 56-di-phenyl-pyridinederivatives as hCB1 antagonists or inverse agonists [188]The compounds have a substituent in position 3 of thepyridine core (Fig 9) This substituent could be a cyano(133-134) or a nitro group a halogen an ester or an amide(135-136) Over 150 compounds were synthesised toillustrate the invention but no pharmacological data wasdisclosed in the patent

In 2003 appeared several patents describing new familiesof compounds that bind to the CB1 cannabinoid receptorThese compounds have in common a central six atomsaromatic ring which could be a pyrazine a pyridine aphenyl or a pyrimidine and that is substituted by at leasttwo phenyl rings

The first patents were from Berggren and collaboratorsfrom AstraZeneca who published two patents describing thesynthesis of 56-diaryl-pyrazine-2-amide derivatives (131-

A few months later another patent was taken by Sanoficlaiming the antagonist properties of 56-di-phenyl-2-pyridine carboxamide derivatives [189] These compounds

N

NH

O

Cl

Cl

N

O

Cl

O NH

N

O

Cl

ClCl

NH

CN O

N

Cl

Cl

Cl

OHN

N

O

Cl

ClCl

CNF

F

133 134

135 136 137

Fig (9) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives 56-Di-phenyl-pyridine derivatives patented by Merck (133-136) and by Sanofi (137) as CB1 cannabinoid receptorsantagonists

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1379

NH

O

N

Cl

ClCl

NH

O

N

Cl

F3CCl

N

N

O

Cl

Cl Cl F138 139 140

Fig (10) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Representative structures of the diphenyl-phenyl (138-139) and 45-diphenyl-pyrimidine (140) derivatives developed ascannabinoid antagonists at Sanofi and Merck respectively

28 examples are given structurally related to the diphenyl-phenyl derivatives have their pyridine core substituted bytwo phenyl rings and by an amide moiety (137 Fig 9) TheIC50 values were lower than 100 nM but no data were givenshowing the antagonist properties although the compoundswere assayed in the adenylate cyclase inhibition assay

10 Other Derivatives

Several amide derivatives structures recently appearedamong the CB1 cannabinoid receptor antagonists In a seriesof patents taken by Merck in 2003 Hagmann and colleaguesdescribed a large amount of compounds obtained by parallelsynthesis which are claimed to be CB1 cannabinoid receptorantagonists [192-195] They were synthesised by reacting alibrary of substituted amines with a library of carboxylicacids (141-144 Fig 11) However as no value was givenfor the affinity or activity of these amides we will notfurther discuss this class of compounds

Along this line Sanofi Synthelabo developed newdiphenyl-phenyl derivatives claimed to have CB1cannabinoid receptor antagonist properties [190] They arebased on a central phenyl ring substituted by two phenylsand by an amide moiety (138-139 Fig 10) Thirteencompounds were claimed along with their synthesis TheirIC50 values were lower than 100 nM ([3H]-CP55-940hCB1-CHO cells) but no other specific data (selectivitypA2hellip) was given

Several 15-diaryl-pyrrole-3-carboxamide derivatives weresynthesised and claimed to be CB1 cannabinoid receptorantagonists by Berggren et al [196] The affinity valueswere not disclosed in the patent (145-146 Fig 12) Otherpyrrole derivatives were synthesised by Guba et al atHofmann-La Roche [197] More specifically 2-(thiazol-4-yl)pyrrole derivatives were claimed as CB1 cannabinoidreceptor antagonist (IC50lt2microM) (147-148 Fig 12)

Finally Kopka et al from Merck described more thanone hundred 45-diaryl-pyrimidine derivatives as CB1cannabinoid receptor antagonists (140 Fig10) [191]However no precise data were provided concerning theaffinity and functionality of the compounds

R2

R1N

R3

R4

R5

O

NH

O

Cl

O F

F

R2

R1NH

R3

R4

HO R5

O

NH

O

Cl

CN

NH

O

Cl

O

O

NH

O

Cl

Cl

+

141142

143 144

Fig (11) CB1 cannabinoid receptor antagonists 10 Other derivatives Retrosynthetic scheme of a CB1 cannabinoid antagonistlibrary of amide derivatives developed at Merck The structures of four representative compounds are illustrated (141-144)

1380 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

NH

O N

Cl

Cl

Cl

N

NH

O

OMe

MeO

N

NH

O

N

S

MeO

N

NH

O

N

S

MeO

145 146

147 148

Fig (12) CB1 cannabinoid receptor antagonists 10 Other derivatives Two examples of 15-diaryl-pyrrole derivatives described byBerggren et al (145-146) While the 2-(thiazol-4yl)pyrroles derivatives 147 and 148 were described by Guba et al from Hofmann-LaRoche All these pyrroles derivatives were claimed to be CB1 cannabinoid receptor antagonists

Another new structure is represented by the 124-triazolederivatives developed by Jagerovic and collaborators In arecent paper were described the synthesis andpharmacological properties of five new 15-diphenyl-3-alkyl-

triazole derivatives [198] Among these compounds onlyone (149) behaved as a CB1 antagonist inhibiting the WIN-55212-2-induced contractions in a mouse vas deferenspreparation However despite its effects in isolated tissue

Table 11 CB1 Cannabinoid Receptor Antagonists 10 Other Derivatives

Structure and affinity of diaryl-triazole derivatives

N

NN

R3

R2

R1

Cpd R1 R2 R3 Ki CB1 (nM) References

149 24-diCl 4-Cl hexyl 855 a [198]

150 24-diCl 4-Cl2-OMe CO-NH-(3-azabicyclo[330]octan-3-yl) 270 b [152]

151 24-diCl 4-Cl CO-NH-(3-azabicyclo[330]octan-3-yl) 164 b [152]

152 4-Cl 24-diCl CO-NH-(3-azabicyclo[330]octan-3-yl) 137 b [152]

153 4-Cl 24-diCl CO-NH-benzylpyrrolidin-3-yl 29 b [152]

154 4-Cl 24-diCl CO-NH-1-(4-chlorophenyl)ethyl 66 b [152]

155 4-Cl 24-diCl CO-NH-piperidinyl 356 c [182]

156 24-diCl 4-Cl CO-NH-piperidinyl 382 c [182]

a [3H]-SR141716A rat cerebellar membranesb [3H]-CP-55940 hCB1-HEK EDNA cellsc [3H]-CP-55940 hCB1-CHO cells

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1381

N

S

O

NH

Cl

Cl

Cl

F

N

S

O

NHN

ClCl

Cl

N

S

O

NH

Br

N

S

O

NHN

Cl

Cl

Cl

156 157 158 159

Fig (13) CB1 cannabinoid receptor antagonists 10 Other derivatives Structures of 45-diaryl-thiazole derivatives patented as hCB1cannabinoid receptor antagonists by Solvay Pharmaceuticals (156 157 158) or by AstraZeneca (157 158 159)

assays (mouse vas deferens and guinea pig ileum) thecompound possessed only a reduced rCB1 affinity with Kivalues of 855 and 748 nM using [3H]-SR141716A and [3H]-WIN-55-212-2 respectively (Table 11) Further four seriesof compounds differing by the nature of the substituentaround the triazole core were also described in a patent[199] Usually the compounds claimed possess two aromaticrings possibly substituted and one linear alkyl chain Theaffinity for the cannabinoid receptors was not givenNevertheless 149 was evaluated in two isolated tissuemodels the inhibition of the electrically evoked contractionsin the guinea pig ileum and in the mouse vas deferens Inthe two models compound 149 inhibited the effect of WIN-55212-2 but had no effect by itself In the vas deferensmodel the authors obtained a pA2 value of 748 to becompared with 763 obtained for AM251

Yet another five membered ring a thiazole has beenused as central moiety of a new class of cannabinoid ligandsThe thiazole ring is substituted by two aryls in position 4and 5 and by an amide (position 2) as in compounds 156-159 (Fig 13) Forty derivatives of this type weresynthesised by Lange and colleagues from SolvayPharmaceuticals and claimed in a patent as agonists orantagonists of the CB1 cannabinoid receptor [200] Nopharmacological data were provided however their activitywas assessed by measuring the cAMP in transfected hCB1-CHO cells In a recent paper from Lange et al two of thesethiazole derivatives were further described [182] Compound13 (157) the most closely related to SR141716A has a Kivalue of 227 nM while for compound 14 (158) Ki value isover 1000 nM Thus as in the imidazole series (see Table9) the aromatic substitution pattern is of great importancefor the CB1 cannabinoid receptor affinity Compound 13however was devoid of in vivo activity

Others 124-triazole derivatives were described by Dycket al compounds 150-154 unlike the Jagerovic ones havean amide moiety in position 3 [152] The highest affinitywas obtained with a 1-benzyl-pyrrolidin-3-yl substituentThe Ki value was 29 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) to be compared to 12 nM obtained for theSR141716A

Seventeen 45-diaryl-thiazole derivatives were alsosynthesised by Berggren et al and patented by AstraZeneca(157-159 Fig 13) [201]

In a patent from Merck Toupence et al described over190 substituted furo[23]pyridines claimed to be CB1cannabinoid receptor antagonists [202] The affinity (IC50 lt1microM) was measured using recombinant CHO cells and [3H]-CP-55940 and the activity using cAMP dosage (160 Fig14)

Lange et al described a triazole derivative (155) whichcan be superimposed with SR141716A Compound 155showed a Ki value of 356 nM ([3H]-CP-55940 hCB1-CHOcells) a pA2 value of 83 in the inhibition of WIN-55212-induced release of [3H]-arachidonic acid by hCB1-CHO cellsand was active in vivo in the CP-55940-inducedhypotension in rat (ED50 = 236 mgkg) [182]

Very recently Alanine from Hoffmann-LaRoche foundthat diarylbenzo[13]dioxole derivatives act as CB1

O

O

S

N

OO

F

ClCl

O

O

F O

N

OON

NH2

O

Cl

Cl

Cl

160 161 162

Fig (14) CB1 cannabinoid receptor antagonists 10 Other derivatives One example of a furo[23]pyridine derivative (160) and twoexamples of diarylbenzo[13]dioxole derivatives (161-162) described as hCB1 cannabinoid receptor antagonists by Toupence et aland Alanine et al respectively

1382 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

cannabinoid receptor antagonists (161-162 Fig 14) [203]Approximate IC50 values for twelve compounds ([3H]-CP-55940 hCB1-HEK cells) among the three hundred andeighty compounds described were given (IC50lt2microM)

et al in 1995 [207] In their hands ∆9-THC was not able toinhibit the forskolin-stimulated adenylyl cyclase activity inan assay performed on hCB2-CHO cells Subsequently theyshowed that ∆9-THC was able to antagonise HU210-inducedinhibition of adenylyl cyclase activity thus acting as anantagonist of the hCB2 cannabinoid receptor expressed inCHO cells [208] Furthermore very recently Govaerts et al[209] reported an inverse agonist effect of ∆9-THC and ∆8-THC on hCB2-CHO cells using the [35S]-GTPγ S assayThe pEC50 values were 763 and 888 respectively whilethe Emax values were ndash27 and ndash16 as compared to basal

Griffith from Pfizer described three new series of CB1cannabinoid receptor antagonists based on a bicyclic centralaromatic ring Purine pyrazoltriazines andpyrazolo[15a]pyrimidines derivatives were synthesised andinvestigated for their ability to interact with the CB1cannabinoid receptor Albeit a [35S]-GTPγ S assay wasdescribed no data were given concerning the antagonist orinverse agonist properties of such compounds Two hundredand eighty purine derivatives were synthesised [204] Theaffinities of 163 and 164 were given in the patent (28 and12 nM [3H]-SR141716A) In a following patent thesynthesis of sixty pyrazoltriazine derivatives was described(165 Fig 15) [205] The affinity of each compound was notgiven but was said to be ranging between 01 and 590 nMAnd finally more than forty pyrazolo[15a]pyrimidines weresynthesised (166 Fig 15) [206] Their affinity for the CB1cannabinoid receptor was lower than 155 nM Despite thefact that a great number of derivatives share the samesubstitution pattern around the central bicyclic aromatic ringthe comparison of these three new series of compounds ismade difficult by the absence of precise affinity data

Some other classical cannabinoids were shown to possessan antagonist activity at the CB2 receptor This is the casefor instance for the non-selective O-1184 (2) (Ki = 74 nM)which enhances the forskolin-induced cAMP production inCB2-CHO cells (EMax = 161 EC50 = 63 nM) and alsofor O-584 (3) (EMax = 246 EC50 = 138 nM) [39]

2 Indole Derivatives

Since the disclosure in 1993 of a second cannabinoidreceptor many attempts have been made to synthesise CB2cannabinoid receptor selective ligands The first compoundreported to have a somehow greater affinity for the peripheralcannabinoid receptor was WIN-55212-2 The compoundwas introduced by DrsquoAmbra and colleagues [210] back in1992 as a new cannabinoid ligand having a nanomolaraffinity and acting as an agonist In 1996 Showalter et alused a transfected cell line expressing the CB2 cannabinoidreceptor in order to identify selective ligands for thisreceptor [211] They showed that the CB2 affinity of WIN-55212-2 was 7 times greater than the CB1 with Ki valuesof 189 and 028 nM for the CB1 and CB2 cannabinoidreceptors respectively However WIN-55212-2 is anagonist of the cannabinoid receptors Interestingly WIN-55212-3 (167 Fig 16) the so-called inactive enantiomer ofWIN-55212 was recently shown to behave as a low affinity(Ki=363 microM) inverse agonist of the hCB2 cannabinoidreceptor as it decreased the [35S]-GTPγ S binding [209]

III CB2 LIGANDS RECEPTOR ANTAGONISTS ANDINVERSE AGONISTS

The compounds acting as antagonist or inverse agonistsat the CB2 cannabinoid receptor are reviewed in this thirdpart of the paper As for the CB1 cannabinoid receptorligands they are classified depending on their chemicalstructures

1 Classical Cannabinoid Derivatives

The first report of an antagonism mediated by ∆9-THC atthe peripheral cannabinoid receptor was made by Bayewitch

N N

NN

NH COOH

Cl

Cl

N N

NN

N

Cl

Cl

NH2OH

O

N

N

N

N

N

NHNH2

O

Cl

ClN

N

N

N

Cl

Cl

N

O

163 164

165 166

Fig (15) CB1 cannabinoid receptor antagonists 10 Other derivatives Examples of purine (163-164) pyrazoltriazines (165) andpyrazolo[15-a]pyrimidines (166) derivatives described as CB1 cannabinoid receptor antagonists by Griffith

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1383

The pEC50 value was 554 and the Emax value ndash26 ascompared to basal

AM630 also acts as an inverse agonist at the hCB1cannabinoid receptor (EC50 = 900 nM) as Landsman et aldemonstrated [47] Recently Zhang and colleagues reportedthe characterisation of the microsomal metabolism of AM630[212]

N

O

N

O

O

N

O

Cl

O

N

O

(S)-WIN-55212-3 167 BML-190 168

In 1996 Gallant and colleagues identified by submittinga large number of compounds to a binding assay anotherindole analog as CB2 cannabinoid receptor ligand [213] Onthe basis of this compound christened BML-190 (168 Fig16) or called indomethacin morpholinylamide theysynthesised several derivatives that possessed a selectivityratio for the CB2 cannabinoid receptor of up to 140 Thesewere the first compounds specifically designed to be CB2cannabinoid receptor ligands The first report on thepharmacological properties of BML-190 appeared onlyrecently New and colleagues highlighted in the paper theinverse agonist properties of the compound BML-190 dose-dependently increases (103 EC50= 980plusmn70 nM) theforskolin-stimulated levels of cAMP in hCB2-HEK cellswhile WIN-55212-2 decreases (44 45plusmn42nM) thisaccumulation [137]

Fig (16) CB2 cannabinoid receptor antagonists 2 Indolederivatives Chemical structures of WIN-55212-3 (167) andBML-190 (168) two indoles derivatives acting as inverseagonists at the hCB2 cannabinoid receptor Surprisingly in 2002 Melck and collaborators used

BML-190 as a CB2 receptor agonist in a cell proliferationassay [214] BML-190 was also tested as inhibitor of thecyclooxygenase-2 enzyme by Kalgutkar et al [215] Theyobtained IC50 values higher than 33 and 66 microM for theCOX-I and COX-II enzymes respectively These valueshave to be compared with the submicromolar activity ofindomethacin the parent compound Very recently Klegerisand colleagues [216] looking for an antineurotoxic action ofcannabinoids showed that BML-190 increases TNF-αsecretion by stimulated THP-1 monocytic cells but is noteffective on the IL-1β secretion

Further on the basis of WIN-54461 (12) a CB1antagonist AM630 (13) was synthesised by replacement ofthe bromine atom by an iodine one In the initial reportPertwee et al showed the antagonist effects of AM630 butalso suggested that the CB1 cannabinoid receptor may not bethe preferential receptor of AM630 [45] Later on Ross et alreported using hCB1amp2-CHO cells and [3H]-CP-55940that AM630 binds to the CB2 cannabinoid receptor (Ki =312 nM) with a selectivity ratio over 160 [48]Furthermore using a [35S]-GTPγ S assay they showed itsinverse agonist properties (EC50 = 76 nM) However

Table 12 CB2 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Reported binding affinities (Ki) and selectivity ratio (CB1CB2) of SR144528 (169) for rat and human cannabinoid receptors determined using [3H]-CP55940 as radioligand

NN

O

NH

Cl

SR144528 169

rCB2 (spleen) rCB1 (whole brain) Selectivity Ki hCB2 (transfected CHOcells)

Ki hCB1 (transfected CHOcells)

Selectivity References

038 nM 305 nM 800 06 nM 437 nM 730 [217]

067 nM 548 nM 80 [219]

56 nM gt10microM gt1780 [48]

25 nM a gt 1microM a gt400 [224]

024 nM 276 nM 115 199 nM 503 nM 25 [232]

a COS cells

1384 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

3 Diarylpyrazole Derivatives CHO cells is constitutively active and phosphorylated onSer 352 Upon treatment by SR144528 thisphosphorylation of the receptor was inhibited Moreover theSR compound was able to induce dephosphorylation ofagonist-induced CB2 phosphorylation The question whetherSR144528 induces a CB2 receptor conformational changemaking it a better phosphatase-specific substrate or ifSR144528 binding activates specific phosphatases remainsopen However this remains a nice example of inverseagonist-induced receptor resensitisation

The same Sanofi team which synthesised the CB1antagonist SR141716A reported in 1998 the synthesis andpharmacological characterisation of a CB2 cannabinoidreceptor selective antagonist the SR144528 (169) [217218] The structure of the new compound is derived fromSR141716A one SR144528 is the first selective CB2receptor antagonist reported as the inverse agonist propertiesof BML-190 were shown only recently by New et al [137]The authors reported for 169 using [3H]-CP-55940 asradioligand Ki values of 04 nM and 06 nM for the rat andthe human CB2 receptors respectively and found a CB1-CB2 selectivity ratio around 700 Table 12 sums up thebinding affinities of SR144528 for the cannabinoid receptorsobtained by different authors Interestingly while the CB2affinity is more or less constant the affinity obtained for theCB1 receptor varies by one order of magnitude [219] Theantagonist effect of SR144528 was highlighted by evaluatingits effect on cAMP production by hCB2-CHO cells exposedto 3 nM CP-55940 (EC50=10nM) The antagonism wasalso observed on the activation of the MAP kinase pathwayby CP-55940 (IC50=39nM)

The Sanofi research team working on cannabinoidsconducted investigations in order to determine the keyresidues for SR144528 interaction with the receptor (Table13) as well as the binding mode of the compound Firstlythey identified the TM4-EL2-TM5 region as a regioncontaining crucial residues for the affinity of several ligandsamong them SR144528 [224] Among these residues themutation of two cysteine of the second extracellular loop(C174 and C179) abolishes the affinity for the CB2 receptorLater on the implication of S453(161) and S457(165) inthe binding of SR144528 was evidenced by mutationalstudies In the model proposed for the docking of SR144528into the CB2 receptor the crucial contacts are comprised intothe TM4 this is the case for S453(161) and S457(165)which are proposed to form hydrogen bonds with the ligandand W464(172) proposed to have aromatic interactions withthe 4-methylbenzyl of the SR144528 [225] In 2003 Fengand Song showed that point mutations D349(130)A andA634(241)E abolished ligand binding However mutationof the arginine R350(131) member of the highly conservedDRY motif to alanine has no influence on SR144528binding [226]

Early after the report of the synthesis and characterisationof SR144528 the same authors further explored the functionof this compound and showed its inverse agonist propertiesusing CB2-CHO cells co-transfected with the luciferasereporter gene with either the CRE or the murine krox24regulatory sequence [220] In these models SR144528 notonly inhibits CP-55940 effects but also reproduces theeffects of a pertussin toxin treatment proving its inverseagonist properties SR144528 functionality was alsoexplored by [35S]-GTPγ S binding in hCB2-CHO cells(IC50=3 nM) [221] The inverse agonist function ofSR144528 was further illustrated by Rhee and Kim usingCOS cells co-transfected with hCB2 receptor and adenylylcyclase [222]

As for the CB1 cannabinoid receptor ligands Sanofideveloped tricyclic derivatives based on the 1-benzylpyrazole-3-carboxylic scaffold More than twentycompounds were disclosed in a patent [227] in 2001 (84-86Table 7) The benzyl in position 4 characteristic of the CB2diarylpyrazoles from Sanofi is conserved as well as thebicyclic residue in position 3 The length of the second linkbetween the phenyl and the pyrazole can be made of one or

Bouaboula and co-workers investigated SR144528 effectson CB2 cannabinoid receptor phosphorylation status [223]Firstly they showed that the CB2 receptor transfected in

Table 13 CB2 Cannabinoid Receptor Antagonists 3 Di-Aryl-Pyrazole Derivatives

Some of the single point mutations reported for the hCB2 cannabinoid receptor for which pharmacological data concerning the SR144528 are available

Mutant Effect References

R350(131)A no effect [226]

S453(161)A loss of affinity [225]

V456(164)I no effect [225]

S457(165)A loss of affinity [225]

C174S a loss of affinity [224 225]

C175S a loss of affinity [225]

C177S a no effect [225]

C179S a loss of affinity [224 225]

S544(193)G no effect [225]

a Second extra-cellular loop

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1385

two methylene but according to the patent one methylenebridge is preferred The Ki values of these compounds for theCB2 receptor were said to be lower than 500 nM and theirantagonist properties have been assessed using the adenylatecyclase inhibition assay

compounds by the presence of an alkyl substituent inposition 5 instead of a phenyl and of course by position 3which remains unsubstituted However except a lecture atthe 1998 ACS meeting [230] where compound 171 wasreported to have a Ki value of 835 nM and a selectivity ratioover 100 no further report on these compounds waspublished untill now

In a recent paper Mussinu and colleagues reported thesynthesis of other tricyclic derivatives starting fromSR141716A structure [228] N-piperidin-1-yl-14-di-hydroindeno[12-c]pyrazole-3-carboxamide derivatives areCB2 receptor ligands and some of them possess high affinityand selectivity for the CB2 receptor as shown in Table 7 (79-83) These tricyclic derivatives differ from the Sanofi tricyclicderivatives as they do have a phenyl ring instead of a benzylin position 1 Another difference is the nature of thesubstituent in position 3 which is not a bicycloalkyl but acyclic amine One of the compounds synthesised byMussinu et al 81 possesses the highest affinity for the CB2receptor ever reported with a Ki value of 0037 nM for themurine CB2 receptor ([3H]-CP-55940 mice spleenhomogenate) Interestingly 79 the closest SR141716Aanalog lost almost all its affinity for the CB1 receptor andincreased by four orders of magnitude its affinity for the CB2receptor Even if the structure quite similar to the SRcompounds could suggest that these derivatives behave asantagonists no pharmacological data were given in the paperto support this hypothesis

5 2-Oxoquinoline Derivatives

Inaba and colleagues from Japan Tobacco introduced in2001 a completely new structure in the cannabinoid fieldbased on a 2-oxoquinoline scaffold Over eighty derivativeswere disclosed in a patent [231] and one compound JTE-907 (173) was subsequently characterised in-vitro as well asin-vivo [232] In Table 14 are listed some of the compounds(172-178) disclosed in the patent along with their affinity forthe cannabinoid receptors These values were obtained onhuman receptors against [3H]-WIN-55212-2 For twelvecompounds the anti-inflammatory activity per os wasassessed in a carrageenin-induced paw edema model Thehighest effect reported was obtained with JTE-907 with anED50 value lower than 01 mgkg Therefore thesecompounds were claimed to be useful as anti-inflammatoryand anti-allergic agents

To further characterize JTE-907 the authors determinedits affinity for cannabinoid receptors of different species using[3H]-CP-55940 as radioligand In their hands JTE-907behaved as a very selective hCB2 ligand with Ki values of36 and 2370 nM for the CB2 and CB1 cannabinoid receptorsrespectively even more selective than the SR144528 (Kivalues of 199 and 50 nM respectively) However it has tobe mentioned that the affinities reported in the paper forJTE-907 are quite different from those reported in the patent(hCB1 Ki values of 35nM and 009 nM respectively) Italso appears that the SR144528 affinity for the CB1 receptordetermined by Iwamura et al is higher than the affinityreported by Rinaldi-Carmona et al with values of 50 and437nM respectively The authors ascribed the difference tothe variability of SR144528 binding affinities to the assayconditions The function of JTE-907 was evaluated bymeasuring the increase in cAMP production by forskolin-stimulated hCB2-CHO cells Maximum stimulation wasattained at a concentration of 1microM of JTE-907 In the sameassay WIN-55212-2 decreased the cAMP production in adose-dependent manner

Finally it is interesting to strengthen that the five-membered ring derivatives (79-86) of the diarylpyrazolefamily are reported to possess a high CB2 receptor affinitywhile the six- and seven-membered ring derivatives (69-78)are described as CB1 receptor ligands

4 Other Pyrazole Derivatives

Other CB2 ligands based on a pyrazole nucleus wereintroduced by SmithKline Beecham in a patent describingthe synthesis of about sixty new compounds [229] Theiraffinity (Ki) for the hCB2 receptor transfected in HEK293cells was reported to be ranging between 25nM and 10microMTheir structures (170-171 Fig 17) differ from the Sanofi

NN

O

N

ON

NN

N

NN

O

N

ON

NN

N

ClCl

1

5

4 3

2

170 171

Fig (17) CB2 cannabinoid receptor antagonists 4 Otherpyrazole derivatives Structure of two compounds (170-171)among those claimed by SmithKline Beecham to be hCB2antagonists

In conclusion JTE-907 was shown to be a CB2 selectiveinverse-agonist with anti-inflammatory and anti-allergicproperties This compound is currently undergoing Phase Iclinical trials as anti-allergic drug [233]

IV NON-CB1 NON-CB2 CANNABINOIDRECEPTORS LIGANDS

In the last years the possible presence of additional non-CB1 non-CB2 cannabinoid receptors was raised frompharmacological evidences

One of the putative receptors possesses an endotheliallocalisation while other ones are localized in the CNS or atperipheral nerves For instance an additional target for ∆9-

1386 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 14 CB2 Cannabinoid Receptor Antagonists 5 2-Oxoquinoline Derivatives

Structure of six compounds among the oxoquinoline derivatives patented by Japan Tobaco Affinity (Ki nM) and selectivity (CB1CB2) for the cannabinoidreceptors are given The affinity was measured by binding against [3H]-WIN-55212-2 on either hCB2-CHO or hCB1-CHO cells Adapted from [231]

Cpd ndeg Structure hCB2 hCB1 Selectivity

1-2 172

NH

MeO O

O

OMeO

0014 3671 262215

JTE-907 173

NH

O

MeO O

O

NH

O

O

0087 3436 39495

2-2 174

OH

O

NH

MeO O

O 077 3247 4215

3-27 175

NH

O

F

NH

MeO O

O0021 336 16000

3-32 176

NH

MeO O

O

NH

F

O

0036 2398 66610

3-38 177

O

O

NH

N

O

O

NMeO

0085 935 11000

3-43 178

NMeO O

O

NH

F

O

0043 864 20100

tetrahydrocannabinol was described in capsaicin-sensitivesensory nerves by Zygmunt et al [234]

[29] and by Di Marzo et al [235] Thus in the presentreview we will focus on the description of the so faridentified ligands of the non-CB1 non-CB2 receptors withan emphasis on the compounds having antagonist properties

Pharmacological evidences for the existence of additionalcannabinoid receptors were reviewed by Wiley and Martin

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1387

(Table 15) In particular the knowledge about three not yetcloned targets has grown The growing molecularpharmacology of the three putative new cannabinoidreceptors is reviewed in the three following sections Thefourth section summarises the data on the other proposedcannabinoid targets

vasodilatator activity In addition the anandamide effect ispartially sensitive to SR141716A inhibition The authorsproposed the existence of two distinct receptors responsiblefor the vasodilatatory effect of anandamide one located in theendothelium is SR141716A-sensitive while the other onelocated on the vascular smooth muscle is SR141716A-insensitive Thus SR141716A is an antagonist of anendothelial anandamide receptor Several studies wereconducted in order to understand the molecularpharmacology of the endothelial anandamide receptor It ispertussis toxin-sensitive [237] Cannabidiol acts asantagonist and abnormal-cannabidiol (Abn-cbd 179) and itsanalogue O-1602 (180) act as agonists [238] The synthesisof O-1602 and of its analogues are disclosed in a patentdescribing the vasodilatatory activity of cannabinoid

1 Endothelial Anandamide Receptor ndash Abnormal-Cannabidiol Receptor

In 1999 Wagner et al showed that anandamide couldinduce mesenteric vasodilatation through an endotheliallylocated ldquoanandamide receptorrdquo pharmacologically distinctfrom CB1 [236] Other cannabinoids such as ∆9-THC HU-210 or WIN-55212-2 were devoid of mesenteric

Table 15 Non-CB1 Non-CB2 Cannabinoid Receptors Antagonists

An overview of the putative new non-CB1 non-CB2 anandamide-receptors ligands The antagonists are highlighted in bold

NH

N

S

HO OH

Cl

OH

OH

OH OH OH OH

OMe

OMe

RR

RR

RR

RR

HO

OH

NH

O

cannabidiol (10) Abnormal-cannabidiol 179 O-1602 180

O-1918 181 capsazepine 183capsaicin 182

Compound Endothelial AEA receptor Brain AEA receptor Cannabinoid-vanilloid brain receptor

AEA agonist a agonist de

WIN-55212-2 no effecta

agonist e agonist g h

CP-55940 no effect e agonist i

SR141716A antagonist a b j no effect ([35 S]-GTPγS binding) d e f antagonist g h

AM251 no effect k no effect h i

Cannabidiol antagonist b no effect e

O-1918 antagonist c jm

Abd-cbd agonist b l

O-1602 agonist b

Capsaicin no effectj agonist i

Capsazepine no effect b antagonist h

a [236] b [238] c [240] d [249] e [250] f [251] g [254] h [258] i [ 255] k [244] j [ 243] l [ 242] m [241]

1388 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

analogues [239] More recently Offertaler et al showed thatO-1918 (181) a cannabidiol analog acts as an antagonist ofthe endothelial anandamide receptor [240] In addition O-1918 does not bind to the CB1 and CB2 cannabinoidreceptors Furthermore O-1918 unlike cannabidiol doesnot cause vasorelaxation in the mesenteric artery preparationmodel used and thus acts as a true neutral antagonistUsing O-1918 as a pharmacological tool Begg et alproposed a coupling of this new G protein-coupledendothelial receptor through a GiGo mechanism to ionchannels in primary cultured human vascular endothelialcells [241] However in their hands SR141716A wasineffective in antagonising Abd-cbd Ho and Hiley furtherdemonstrated that Abn-cbd relaxes rat small mesentericarteries through an activation of K+ channels via aSR141716A-sensitive pathway [242] Further using thesame model they characterized the vasorelaxant activity ofvirodhamine a novel endocannabinoid apparently actingthrough the same receptor [243] The virodhamine-inducedvasorelaxation is sensitive to SR141716A and O-1918 butinsensitive to AM251 SR144528 and AM630 Moreovervanilloid receptor desensitisation by capsaicin (182) had noeffect

244] Thus position 4 on the 5-phenyl could be furtherexplored to obtain selective ligands

To come to an end despite the fact that the endothelialanandamide receptor is neither identified nor cloned at leastthree antagonists are identified SR141716A cannabidioland O-1918 It is likely that a growing number of ligandsand therapeutic applications will appear in the very nearfuture

2 Brain Anandamide Receptor

Following an in-depth evaluation of anandamide effects inCB1

-- mice Di Marzo and co-workers suggested that a non-CB1 non-CB2 G protein-coupled receptor might mediatesome of the actions of AEA in mice [249] Actually effectsof AEA but not those of ∆9-THC were not decreased inCB1 cannabinoid receptor knock-out mice For instance 30mgkg of AEA were still effective in hot-plate test whereas10 mgkg of ∆9-THC had no effect on anti-nociceptionMoreover in a [35S]-GTPγ S assay AEA stimulated with anEC50 value of 223 microM the [35S]-GTPγ S binding to CB1

--

mice brain membranes (Emax= 28) ∆9-THC showed noeffect in this assay The addition of SR141716A did notaffect the [35S]-GTPγ S binding to CB1

-- mice brainmembranes Shortly after other evidences for the existence ofa new G protein-coupled cannabinoid receptor with a distinctdistribution in the central nervous system were published[250] From a set of 24 commonly used cannabinoids (AEAWIN-55212-2 JWH-030 THChellip) only AEA and WIN-55212-2 produced an increased binding of [35S]-GTPγ S onCB1

-- mice brain membranes Interestingly in this studySR141716A alone produced a significant inhibition of [35S]-GTPγ S binding similar to the inhibition obtained usingCB1

++ mice brain membranes (IC50 values of 57 microM and47 microM respectively) However SR141716A did not affectthe anandamide-induced [35S]-GTPγ S binding in CB1

--

mice brain membranes Moreover [3H]-SR141716Aexhibited significant binding in some brain regions of CB1

--

mice which are different from those exhibiting stimulation of[35S]-GTPγ S binding by WIN-55212-2 and AEA Thusthe authors proposed that the SR141716A effects on CB1

--

mice are mediated neither by the CB1 cannabinoid receptornor by the putative new anandamide brain receptor Monoryand co-workers using CB1

-- mice described a novel nonadenylyl cyclase-coupled cannabinoid binding site in micecerebellum [251] Two agonists AEA and WIN-55212-2but not ∆9-THC or HU210 were found to bind to thisreceptor as they enhanced [35S]-GTPγ S binding NeitherSR141716A nor SR144528 were able to affect the WIN-55212-2 induced [35S]-GTPγ S binding in CB1

-- mice asfound by Breivogel et al [250] However the distributionpattern of the receptor described by Monory et al differs fromthe distribution found by Breivogel et al It has to bementioned that the knock-out strains used were different Themice used by Monory were from a CD-1 strain while theone used by Breivogel were from a C57BL61 strainInterestingly Muthane et al detected differences in nigralneurons number and sensitivity to MPTP in the two mousestrains used [252]

More recently Mo et al obtained results showing thatthe Abnormal-cannabidiol receptor is also responsible for anincreased endothelial cell migration [244] Abn-cbd enhancedhuman umbilical vein endothelial cell migration while O-1918 antagonised Abn-cbd effect The CB1 cannabinoidreceptor inverse agonist SR141716A partially inhibitedAbd-cbd action whereas AM251 and SR144528 had noeffect

In another paper from George Kunos team Batkai et alreported that SR141716A is able to inhibit the endotoxichypotension by a cardiac mechanism involving neither theCB1 nor the CB2 cannabinoid receptors [245] Theinvolvement of a non-CB1 non-CB2 site of action wasshown using CB1

-- and CB1--CB2

-- knock-out miceFurther studies are needed to identify the SR141716Asensitive myocardial site of action

In a recent patent Kunos described O-1918 and itsanalogues as vasoconstrictor agents useful to reversepathological vasodilatation of blood vessels [246] Theeffects of O-1918 on mouse blood pressure on LPS-inducedhypotension in mouse as well as the reversal of hypotensionby O-1918 injection are described Furthermore thesynthesis of O-1918 based antagonists is described startingfrom p-metha-28-dien-1-ol and the appropriate resorcinols

Interestingly the Abd-cbd receptor which was firstdetected in the vascular system also seems to be present inthe CNS [247] Indeed Walter et al found that microglialcells (BV-2) migration induced by 1 microM 2-AG is inhibitedby cannabidiol (300 nM) and O-1918 (1 microM) SR141716Awas found ineffective in this assay Thus 2-AG is an agonistof the Abd-cbd receptor Moreover Abd-cbd is able to elicita dose-dependent cell migration in the same model (EC50 =600nM) [248]

From a more structural point of view SR141716Abehaves as an antagonist while on the opposite AM251(20) a close analogue which differs from the former only bythe nature of one halogen has no effect on this receptor [242

To conclude to our knowledge no compounds havingantagonist or inverse agonist properties at this receptor havebeen identified so far

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1389

3 Cannabinoid-Vanilloid Brain Receptor a cannabinoid-vanilloid receptor at which SR141716A andcapsazepine behave as antagonists

Evidences for the presence of an additional ldquocannabinoidrdquoreceptor differing from the CB1 receptor and from the brainanandamide receptor appeared from studies focused on thehippocampus (for a review see Hajos et al [253]) Hajos etal studied the cannabinoid actions on GABAergic andglutamatergic transmission using the whole-cell patch-clamp technique [254] WIN-55212-2 induced a dose-dependent reduction in glutamatergic transmission reversedby SR141716A (1microM) Thus the authors concluded that thecannabinoid actions on the glutamatergic transmission aremediated by a non-CB1 but SR141716A-sensitive receptorIn another paper Hajos and Freund further characterised thisputative receptor [255] They showed that in addition toWIN-55212-2 CP-55940 and capsaicin (182) a TRPV1receptor agonist also act as agonists Furthermore in theirhands SR141716A as well as capsazepine (183) a TRPV1receptor antagonist were able to antagonise this effect on theglutamatergic transmission while AM251 was notSR141716A effects on CB1 knock-out mice were furtherstudied using a test of anxiety [256] Haller et al showedthat on the one hand the distribution of the CB1 receptorinduces anxiety and SR141716A has an anxiolytic effectand on the other hand SR141716A is still active in CB1

--

mice In a following paper they reported that AM-251induces anxiogenic effects in wild-type and in knock-outmice [257] Thus SR141716A in addition to CB1 bindsto a not yet identified receptor Another evidence for thisadditional receptor for SR141716A was given by Bass andco-workers [61] They conducted a structure-activityrelationship study on the stimulation of locomotor activityinduced by SR141716A and twenty analogues Nocorrelation was found between the affinity for the CB1cannabinoid receptor and the stimulation of locomotoractivity Albeit SR141716A and five of its analogues O-1803 O-1710 O-1253 O-1254 (40) O-1255 (41) behavedas inverse agonists in [35S]-GTPγ S assay none of theseanalogues stimulated the locomotor activity In conclusionthe SR141716A-induced stimulation of locomotor activity isneither due to inverse agonism nor to inhibition of anendogenous tone but to a brain receptor distinct from theCB1 receptor Di Marzo et al previously provided evidencesthat the cannabinoid-vanilloid brain receptor could mediatesome of the cannabinoid effects on locomotion [249]

4 Other Non-CB1 Non-CB2 Cannabinoid Receptors

Evidences for the existence of additional non-CB1 non-CB2 cannabinoid receptors localised in the rat isolatedmesenteric arterial bed appeared from the works of Ralevicand collaborators Several cannabinoids were evaluated intheir model of sensory neurogenic vasorelaxation evoked byelectrical field stimulation

HU210 was found to attenuate vasorelaxation in a CB1and CB2 independent way as SR141716A LY320135 andSR144528 were ineffective in inhibiting HU210 effect [259]Similar results were obtained with ∆9-THC [260]Similarly noladin ether inhibited sensory neurogenicrelaxation in a concentration-dependent manner which wasalso unaffected by SR141716A LY320135 and SR144528Moreover this effect as the ∆9-THC one was pertussistoxin sensitive suggesting the involvement of a Gio protein-coupled receptor [261]

However the inhibitory effects of CP-55940 and WIN-55212-2 were affected by the presence of SR141716A (1microM)or LY320135 (1microM) but not by the addition of SR144528(1microM) [262]

It appears that at least two receptors should be involvedin cannabinoid-mediated attenuation of sensory nerve-mediated vasorelaxation The first one is SR141716A andLY320135 sensitive while the other one is resistant toSR141716A

Some evidences for the existence of a ldquoCB2-likerdquo receptorappeared from the studies on palmitoylethanolamide (PEA)an endogenous fatty acid amide devoid of CB1 and CB2affinity [264] antinociceptive potential Calignano and co-workers showed that PEA was able to alleviate pain inseveral animal models [265 266] For instance PEA dose-dependently inhibits kaolin-evoked writhing in mice TheCB2 cannabinoid receptor inverse agonist SR144528 (02mgkg) inhibits PEA anti-nociceptive effect [266] Thus theauthors suggest that PEA acts as agonist on a SR144528-sensitive non-CB2 cannabinoid receptor (ldquoCB2-likerdquoreceptor) at which SR144528 acts as an antagonist

Further studies are needed in order to better characterisethese additional ldquocannabinoidrdquo receptors However it islikely that several therapeutic applications will soon springfor these receptors

Very recently Pistis et al published a study on theneurophysiological effects of cannabinoids on the basolateralamygdala neurons in-vivo [258] One of their findings is thatHU-210 and WIN-55212-2 did not elicit similar effects onbasal amygdala projection neurons firing rate While theformer had no effect the latter decreased the firing rateMoreover SR141716A reversed the WIN-55212-2 effectswhile AM251 had no effect The vanilloid receptorantagonist capsazepine also antagonised the WIN-55212-2effect on firing rate of these neurons Therefore the authorssuggested the presence of a novel cannabinoid-vanilloidreceptor

V CONCLUSION

Since their development cannabinoid antagonists provedto be essential tools in the understanding of cannabinoidpharmacology and biochemistry Moreover the interestshown by the pharmaceutical industry in the development ofnew cannabinoid antagonists prove if necessary the realtherapeutic potential of this class of compounds Thetreatment of eating and movement disorders memorydeficits psychosis and dependencies from various addictivedrugs are some of the most cited applications for the CB1cannabinoid receptor antagonists

From these lines it appears that in addition to theknown CB1 cannabinoid receptor it is now likely that twoother cannabinoid receptors do exist in the brain The firstone should be an anandamide brain receptor for whichSR141716A has no affinity while the second one should be

1390 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Much less wide is the knowledge on the CB2cannabinoid receptor antagonists However potentialapplications could be disorders involving the immunesystem such as inflammation or allergies

[19] Devane WA Hanus L Breuer A Pertwee RG StevensonLA Griffin G Gibson D Mandelbaum A Etinger AMechoulam R Science 1992 258 1946

[20] Mechoulam R Ben-Shabat S Hanus L Ligumsky MKaminski NE Schatz AR Gopher A Almong S MartinBR Compton DR Biochem Pharmacol 1995 50 83

Finally the growing literature on the non CB1 non CB2cannabinoid receptors should allow the development of newdrugs First examples are given by the cannabidiol andabnormal-cannabidiol derivatives described by Kunos et alfor which potential therapeutic applications on the vascularsystem are already patented

[21] Sugiura T Kondo S Sukagawa A Nakane S Shinoda AItoh K Yamashita A Waku K Biochem Biophys Res Co1995 215 89

[22] Hanus L Abu-Lafi S Fride E Breuer A Vogel Z ShalevDE Kustanovich I Mechoulam R Proc Natl Acad SciUSA 2001 98 3662

[23] Oka S Tsuchie A Tokumura A Muramatsu M Suhara YTakayama H Waku K Sugiura T J Neurochem 2003 851374It clearly appears from this paper that the antagonists of

the cannabinoid receptors are still an active research fieldfrom which should emerge new promising therapeutic toolsas well as innovative drugs in the near future

[24] Goutopoulos A and Makriyannis A Pharmacol Therapeut2002 95 103

[25] Beal JE Olson R Lefkowitz L Laubenstein L Bellman PYangco B Morales JO Murphy R Powderly W PlasseTF Mosdell KW Shepard KV J Pain Symptom Manage1997 14 7ACKNOWLEDGEMENTS [26] Notcutt W Price M Miller R Newport S Phillips CSimmons S Sansom C Anaesthesia 2004 59 440

GGM is very indebted to the ldquoFonds pour la formationagrave la Recherche dans lrsquoIndustrie et lrsquoAgriculture (FRIA)rdquo Belgium for the award of a research fellowship Theexperimental works of the authors were supported by grantsfrom the FNRS The Belgian Charcot Foundation and theUniversiteacute catholique de Louvain

[27] Ledent C Valverde O Cossu G Petitet F Aubert JFBeslot F Bohme GA Imperato A Pedrazzini T RoquesBP Vassart G Fratta W Parmentier M Science 1999 283401

[28] Zimmer A Zimmer AM Hohmann AG Herkenham MBonner TI Proc Natl Acad Sci U S A 1999 96 5780

[29] Wiley JL and Martin BR Chem Phys Lipids 2002 121 57[30] Adam J and Cowley P Expert Opin Ther Patents 2002 12

1475

REFERENCES [31] Razdan RK and Mahadevan A Chem Phys Lipids 2002 12121

[32] Barth F and Rinaldi-Carmona M Curr Med Chem 1999 6745

[1] Hillard CJ Harris RE Bloom AS J Pharmacol Exp Ther1985 232 579

[33] Little PJ Compton DR Johnson MR Melvin LS MartinBR J Pharmacol Exp Ther 1988 247 1046

[2] Devane WA Dysarz FA Johnson MR Melvin LSHowlett AC Mol Pharmacol 1988 34 605

[34] Pertwee RG Stevenson LA Elrick DB Mechoulam RCorbett AD Brit J Pharmacol 1992 105 980

[3] Matsuda LA Lolait SJ Brownstein MJ Young ACBonner TI Nature 1990 346 561

[35] Harrison C Traynor JR Life Sci 2003 74 489-508[4] Gerard CM Molerau C Vassart G Parmentier M BiochemJ 1991 279 129 [36] Pertwee RG Fernando SR Griffin G Ryan W Razdan

RJ Compton DR Martin BR Eur J Pharmacol 1996 315195

[5] Glass M Dragunow M Faull RLM Neuroscience 1997 2299

[37] Ross RA Brockie HC Fernando SR Saha B Razdan RKPertwee RG Brit J Pharmacol 1998 125 1345

[6] Shire D Carillon C Kaghad M Calandra B Rinaldi-Carmona M LeFur G Caput D Ferrara P J Biol Chem1995 270 3726 [38] Griffin G Wray EJ Rorrer WK Crocker PJ Ryan WJ

Saha B Razdan RK Martin BR Abood ME Brit JPharmacol 1999 126 1575

[7] Ryberg E Khang Vu H Groblewski T Larsson N ElebringT Hjorth S Sjogren S Greasley PJ 2004 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 119

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Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1375

Table 8 CB1 Cannabinoid Receptor Antagonists 6 Aryl-Imidazolidine-24-Diones Derivatives

Structures affinities ([3H]-SR141716A hCB1-CHO cells) and pKb values ([35 S]-GTPγS HU210 rat brain) of three aryl-imidazolidine-24-dionederivatives (92-94)

N

NH

OO

R

Br

Br

Cpd ndeg R Ki (nM) a pKb b

DML20 92 ethylmorpholine 70 611

DML21 93 1-hydroxypropyl 103 625

DML23 94 heptyl 98 574

a [175]b [177]

showed that the substitution at the nitrogen in position 3 aswell as a bromine atom in para of the phenyl rings aremandatory for the compounds affinity Later on threecompounds termed DML20 (92) DML21 (93) and DML23(94) were characterised as neutral antagonists using the [35S]-GTPγ S assay on rat cerebellum membranes [176] (Table 8)In the same model they competitively inhibited HU-210-induced [35S]-GTPγ S binding with pKB values of 611625 and 574 respectively Moreover these compoundswere proven to be quite selective for the CB1 receptor

higher than the one for the CB2 cannabinoid receptor (IC50 =300 nM) The inverse agonism of the compound wasextrapolated by inhibiting CP-55940-induced hypothermiain mice (EC50 = 18 nM) Moreover one oral dose (10mgkg) reduces the food intake in diet-induced obese ratsInterestingly food intake of CB1

-- mice was not alteredBody weight loss was maintained all over the study during achronic administration (14 days) of 10 mgkg of Cpd A

Later on Merck published a patent [180] in which someof the 45-diaryl-pyrazoles derivatives previously describedwere claimed to be useful in the treatment or the preventionof obesity in association with compounds inhibiting the11β-hydroxysteroid dehydrogenase type 1 (1β-HSD1)enzyme The 1β-HSD1 enzyme is responsible for thesynthesis of cortisol increased levels of which according tothe inventors are associated with obesity However neitherresults on appetite suppression nor on weight loss weregiven even if some indications on the affinity of thecompounds for the cannabinoid receptors were given

Very recently the functionality of these DML compoundswas explored using the [35S]-GTPγ S assay both on rat andhuman CB1 cannabinoid receptors [177] The data obtainedconfirm that the 3-alkyl-55rsquo-diphenyl-imidazolidinedionederivatives behave as neutral antagonists on rat CB1cannabinoid receptor (rat brain) However DML 20 DML21and DML23 acted as inverse agonists on the human receptor(hCB1-CHO cells) Furthermore the authors showed thatthis different functionality is not due to the hCB1 receptorlevel of expression in the recombinant cell line as whateverthe level of expression (Bmax of 44 pmolmg or 32pmolmg) DML derivatives behaved as inverse agonists ofthe hCB1 cannabinoid receptor

Another imidazole ring pattern of substitution wasproposed by Hagmann and collaborators [181] Thecompounds described 43 examples are given possess twophenyls the first one in position 1 linked to the nitrogenand the second one in position 2 The carboxamidesubstituent is in position 4 instead of in position 2 for thediarylimidazoles described by Finke These 12-diaryl-imidazoles are claimed to be CB1 cannabinoid receptorantagonists or inverse agonists but the absence of precisepharmacological values do not allow any comparisonbetween the two substitutions patterns However a samepattern of substitution was used by other researchers Amongthem Dyck et al described in 2004 ten 1-(4-chlorophenyl)-2-(24-dichlorophenyl)-imidazole derivatives varying by theircarboxamide substituent in position 4 (101-103 Table 9)[152] For instance with a 3-azabicyclo[330]octan-3-ylsubstituent (101) they obtained a Ki value of 14 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) In a [35S]-GTPγ Sassay 101 exhibited an IC50 value of 19 nM

7 Diarylimidazoles Derivatives

Finke and colleagues at Merck developed a new class ofCB1 receptor antagonists based on an imidazole nucleusOver eighty 45-diaryl-imidazoles derivatives having CB1antagonist properties were claimed in a patent published in2003 [178] Some representative compounds of the patent areillustrated in Table 9 (95-100) The affinity of the newcompounds was determined using [3H]-CP-55940 andhCB1-CHO cells Although no detailed pharmacologicaldata were given in the patent in-vitro and in-vivo propertiesof one compound termed Cpd A (100) have been describedelsewhere [179] Its affinity for the CB1 cannabinoid receptorwas shown to be nanomolar (IC50 = 4 nM) and 75 times

1376 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 9 CB1 Cannabinoid Receptor Antagonists 7 Diaryl-Imidazole Derivatives

Structure and affinity range of some 45-diaryl-imidazole derivatives for the human cannabinoid receptors determined using [3H]-CP-55940 asradioligand and hCB1amp2-CHO cells

N

NR2

R1

R4

R3

ndeg R1 R2 R3 R4 hCB1 (IC50 or Ki) hCB2 (IC50 or Ki) References

95 CH3 CO-NH-piperidinyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

96 CH3 CO-NH-piperidinyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

97 CH3 CO-NH-cyclohexyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

98 CH3 CO-NH-phenyl 24-di-Cl-Ph 4-Cl-Ph 10 ndash 100 nM a 100 ndash1000 nM a [178]

99 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

100 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph 4 nM a 300 nM a [179]

101 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

CH3 14 nM b ND [152]

102 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

H 66 nM b ND [152]

103 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 85 nM b ND [152]

104 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 23 nM b 542 nM b [182]

105 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 30 nM b 608 nM b [182]

106 4-Br-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 60 nM b 489 nM b [182]

107 4-CF3-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 29 nM b 634 nM b [182]

108 4-Cl-Ph 24-di-Cl-Ph CO-NH-morpholinyl CH3 197 nM b 3297 nM b [182]

109 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidin-4-ol CH3 172 nM b 3959 nM b [182]

110 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CN 30 nM b 1590 nM b [182]

111 4-Cl-Ph 24-di-Cl-Ph CO-NH- piperidinyl CH2F 36 nM b 906 nM b [182]

112 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH2-CH3 14 nM b 430 nM b [182]

113 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Cl 27 nM b 823 nM b [182]

114 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Br 23 nM b 746 nM b [182]

115 4-Cl-Ph 24-di-Cl-Ph CO-NH-tetrahydroisoquinoline CH3 34 nM b 696 nM b [182]

116 4-Cl-Ph 24-di-Cl-Ph CO-NH-cycloheptyl CH3 35 nM b 349 nM b [182]

117 24-di-Cl-Ph 4-Cl-Ph CO-NH-piperidinyl CH3 403 nM b 208 nM b [182]a IC50 value b Ki value

Lange and co-workers from Solvay described twenty-eight imidazole derivatives [182] The 24-dichlorophenylsubstituent at position 2 was kept constant while thesubstitutions at the other positions were explored (Table 9)Position 5 can accommodate a large range of littlesubstituents like hydrogen (104) methyl (105) ethyl (112)chlorine (113) bromine (114) fluoromethyl (111) or cyano(110) without major changes in the affinity At position 4more bulky substituents like 1234-tetraisoquinoline (115)or cycloheptyl (116) were well tolerated whereas thepresence of a more hydrophilic moiety such as morpholine(108) or piperidin-4-ol (109) was detrimental to the affinity

Interestingly position exchange between the 24-diphenyland the 4-chlorophenyl substituents led to a 13 fold loweraffinity with Ki values of 30 and 403 nM for 105 and 117respectively ([3H]-CP-55940 hCB1-CHO cells) In thefunctional assay which was inhibition of WIN-55212-2-induced [3H]-arachidonic acid release by hCB1-CHO cellsall tested compounds behaved as antagonists Compound105 showed a pA2 value of 86 which is the same theauthors obtained for SR141716A It would be interesting toassess if these compounds are true antagonists or if they actmore as inverse agonists Compound 105 when administeredto rats was able to inhibit CP-55940-induced hypotension

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1377

with an ED50 value of 24 mgkg which is close to the 3mgkg obtained for SR141716A The compound was alsoactive in another in-vivo model the WIN-55212-2-inducedhypothermia in mice Compound 104 which has nosubstituent in position 5 of the imidazole ring was lessactive in the hypotension model and inactive in thehypothermia model Thus a methyl group is the preferredsubstituent in position 5 of the imidazole moiety Otherproperties either experimental (P-glycoprotein affinitylogPHPLC) or computational (molecular volume polarsurface area) obtained for compounds 105 were similar tothose obtained for SR141716A Finally molecularmodelling studies revealed a close structural overlap betweenthe two compounds

value of 197 nM is selective for hCB1 and is as potent asSR141716A The structural modulations of 118 led to someinteresting compounds (Table 10) Six compounds possess aKi value for the hCB1 cannabinoid receptor equal to or lowerthan 25 nM antagonist properties in the inhibition of [3H]-arachidonic acid release by WIN-55212-2-stimulated hCB1-CHO cells and Ki values for the hCB2 receptor over 1microMThe diaryl-pyrazoline derivatives contained a chiral center atposition 4 The authors resolved the racemic mixture of twocompounds (121 and 130) The levorotatory enantiomersappeared to be the eutomers For compound SLV319levorotatory enantiomer of 121 the Ki and pA2 values were78 nM and 99 respectively The Ki and pA2 values ofSLV326 levorotatory enantiomer of 130 were 359 nM and9 respectively

8 34-Diaryl-Pyrazoline Derivatives Two in-vivo models CP-55940-induced hypotension inrat and WIN-55212-2-induced hypothermia in mouse wereused to assess the potential of these compounds The resultsare of the same order of magnitude than those obtained forSR141716A For instance in the hypotension modelSLV319 ((-)121) and SLV326 ((-)130) showed ED50 values

Lange and collaborators also discovered 34-diaryl-pyrazoline derivatives after a screening of compoundsresembling to SR141716A [183184] Compound 118 thatwas initially identified during the screening possesses a Ki

Table 10 CB1 Cannabinoid Receptor Antagonists 8 34-Diaryl-Pyrazolines Derivatives

Structure affinity for the hCB1 and hCB2 cannabinoid receptors (Ki nM) expressed in CHO transfected cells ([3H]-CP-55940) and potency (pA2) ofsome 34-dirylpyrazolines derivatives Table adapted from [184]

NN

N N

SO2

R3

R2

Cl R1

R4

n˚ R1 R2 R3 R4 Ki hCB1 (nM) Ki hCB2 (nM) pA2 (CB1)

118 H H H 4-CH3 197 gt 1000 84

119 H H H 246- CH3 24 gt 1000 94

120 H H H 4-Cl 16 gt 1000 95

121 H CH3 H 4-Cl 25 gt 1000 87

122 H CH3 CH3 4-Cl 280 gt 1000 85

123 H H H 4-F 53 gt 1000 9

124 H CH3 H 4-F 338 gt 1000 85

125 H CH3 CH3 4-F gt 1000 gt 1000 lt 75

126 H CH3 H 3-Cl 14 gt 1000 86

127 4-Cl CH3 H 4-Cl 255 ND ND

128 4-F CH3 H 4-Cl 584 ND ND

129 H CH3 H H 170 ND 75

130 H CH3 H 4- CF3 221 gt 1000 93

SR141716A 25 1580 86

1378 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

N

HN

O

N

Cl Cl

Cl

N

N

HN

O

Br

Br131 132

Fig (8) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Chemical structures of two 56-diaryl-pyrazine-2-amide derivatives (131-132) developed as CB1 cannabinoid receptorantagonists by AstraZeneca

of 55 and 2 mgkg (po) respectively proving their in-vivoefficacy after oral administration The authors alsodemonstrated that SLV319 is devoid of affinity for the P-glycoprotein pump and that it possessed a goodCNSplasma ratio (17) These two compounds (SLV319and SLV326) were chosen as development candidates bySolvay and entered clinical Phase I trials in 2003 [185]

132 Fig 8) useful as CB1 cannabinoid receptor antagonists[186 187] The activity of twenty derivatives wasdetermined using a [35S]-GTPγ S assay and hCB1-CHOtransfected cells The concentration required to give halfmaximal inhibition of CP-55940-induced [35S]-GTPγ Sbinding (IC50) is lower than 200 nM for the preferredcompounds No affinity data were given for thesecompounds

9 Diaryl-Pyrazine Diphenyl-Pyridine Diphenyl-Phenyland Diaryl-Pyrimidines Derivatives

The same year in a patent from Merck Finke andcollaborators described several 56-di-phenyl-pyridinederivatives as hCB1 antagonists or inverse agonists [188]The compounds have a substituent in position 3 of thepyridine core (Fig 9) This substituent could be a cyano(133-134) or a nitro group a halogen an ester or an amide(135-136) Over 150 compounds were synthesised toillustrate the invention but no pharmacological data wasdisclosed in the patent

In 2003 appeared several patents describing new familiesof compounds that bind to the CB1 cannabinoid receptorThese compounds have in common a central six atomsaromatic ring which could be a pyrazine a pyridine aphenyl or a pyrimidine and that is substituted by at leasttwo phenyl rings

The first patents were from Berggren and collaboratorsfrom AstraZeneca who published two patents describing thesynthesis of 56-diaryl-pyrazine-2-amide derivatives (131-

A few months later another patent was taken by Sanoficlaiming the antagonist properties of 56-di-phenyl-2-pyridine carboxamide derivatives [189] These compounds

N

NH

O

Cl

Cl

N

O

Cl

O NH

N

O

Cl

ClCl

NH

CN O

N

Cl

Cl

Cl

OHN

N

O

Cl

ClCl

CNF

F

133 134

135 136 137

Fig (9) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives 56-Di-phenyl-pyridine derivatives patented by Merck (133-136) and by Sanofi (137) as CB1 cannabinoid receptorsantagonists

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1379

NH

O

N

Cl

ClCl

NH

O

N

Cl

F3CCl

N

N

O

Cl

Cl Cl F138 139 140

Fig (10) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Representative structures of the diphenyl-phenyl (138-139) and 45-diphenyl-pyrimidine (140) derivatives developed ascannabinoid antagonists at Sanofi and Merck respectively

28 examples are given structurally related to the diphenyl-phenyl derivatives have their pyridine core substituted bytwo phenyl rings and by an amide moiety (137 Fig 9) TheIC50 values were lower than 100 nM but no data were givenshowing the antagonist properties although the compoundswere assayed in the adenylate cyclase inhibition assay

10 Other Derivatives

Several amide derivatives structures recently appearedamong the CB1 cannabinoid receptor antagonists In a seriesof patents taken by Merck in 2003 Hagmann and colleaguesdescribed a large amount of compounds obtained by parallelsynthesis which are claimed to be CB1 cannabinoid receptorantagonists [192-195] They were synthesised by reacting alibrary of substituted amines with a library of carboxylicacids (141-144 Fig 11) However as no value was givenfor the affinity or activity of these amides we will notfurther discuss this class of compounds

Along this line Sanofi Synthelabo developed newdiphenyl-phenyl derivatives claimed to have CB1cannabinoid receptor antagonist properties [190] They arebased on a central phenyl ring substituted by two phenylsand by an amide moiety (138-139 Fig 10) Thirteencompounds were claimed along with their synthesis TheirIC50 values were lower than 100 nM ([3H]-CP55-940hCB1-CHO cells) but no other specific data (selectivitypA2hellip) was given

Several 15-diaryl-pyrrole-3-carboxamide derivatives weresynthesised and claimed to be CB1 cannabinoid receptorantagonists by Berggren et al [196] The affinity valueswere not disclosed in the patent (145-146 Fig 12) Otherpyrrole derivatives were synthesised by Guba et al atHofmann-La Roche [197] More specifically 2-(thiazol-4-yl)pyrrole derivatives were claimed as CB1 cannabinoidreceptor antagonist (IC50lt2microM) (147-148 Fig 12)

Finally Kopka et al from Merck described more thanone hundred 45-diaryl-pyrimidine derivatives as CB1cannabinoid receptor antagonists (140 Fig10) [191]However no precise data were provided concerning theaffinity and functionality of the compounds

R2

R1N

R3

R4

R5

O

NH

O

Cl

O F

F

R2

R1NH

R3

R4

HO R5

O

NH

O

Cl

CN

NH

O

Cl

O

O

NH

O

Cl

Cl

+

141142

143 144

Fig (11) CB1 cannabinoid receptor antagonists 10 Other derivatives Retrosynthetic scheme of a CB1 cannabinoid antagonistlibrary of amide derivatives developed at Merck The structures of four representative compounds are illustrated (141-144)

1380 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

NH

O N

Cl

Cl

Cl

N

NH

O

OMe

MeO

N

NH

O

N

S

MeO

N

NH

O

N

S

MeO

145 146

147 148

Fig (12) CB1 cannabinoid receptor antagonists 10 Other derivatives Two examples of 15-diaryl-pyrrole derivatives described byBerggren et al (145-146) While the 2-(thiazol-4yl)pyrroles derivatives 147 and 148 were described by Guba et al from Hofmann-LaRoche All these pyrroles derivatives were claimed to be CB1 cannabinoid receptor antagonists

Another new structure is represented by the 124-triazolederivatives developed by Jagerovic and collaborators In arecent paper were described the synthesis andpharmacological properties of five new 15-diphenyl-3-alkyl-

triazole derivatives [198] Among these compounds onlyone (149) behaved as a CB1 antagonist inhibiting the WIN-55212-2-induced contractions in a mouse vas deferenspreparation However despite its effects in isolated tissue

Table 11 CB1 Cannabinoid Receptor Antagonists 10 Other Derivatives

Structure and affinity of diaryl-triazole derivatives

N

NN

R3

R2

R1

Cpd R1 R2 R3 Ki CB1 (nM) References

149 24-diCl 4-Cl hexyl 855 a [198]

150 24-diCl 4-Cl2-OMe CO-NH-(3-azabicyclo[330]octan-3-yl) 270 b [152]

151 24-diCl 4-Cl CO-NH-(3-azabicyclo[330]octan-3-yl) 164 b [152]

152 4-Cl 24-diCl CO-NH-(3-azabicyclo[330]octan-3-yl) 137 b [152]

153 4-Cl 24-diCl CO-NH-benzylpyrrolidin-3-yl 29 b [152]

154 4-Cl 24-diCl CO-NH-1-(4-chlorophenyl)ethyl 66 b [152]

155 4-Cl 24-diCl CO-NH-piperidinyl 356 c [182]

156 24-diCl 4-Cl CO-NH-piperidinyl 382 c [182]

a [3H]-SR141716A rat cerebellar membranesb [3H]-CP-55940 hCB1-HEK EDNA cellsc [3H]-CP-55940 hCB1-CHO cells

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1381

N

S

O

NH

Cl

Cl

Cl

F

N

S

O

NHN

ClCl

Cl

N

S

O

NH

Br

N

S

O

NHN

Cl

Cl

Cl

156 157 158 159

Fig (13) CB1 cannabinoid receptor antagonists 10 Other derivatives Structures of 45-diaryl-thiazole derivatives patented as hCB1cannabinoid receptor antagonists by Solvay Pharmaceuticals (156 157 158) or by AstraZeneca (157 158 159)

assays (mouse vas deferens and guinea pig ileum) thecompound possessed only a reduced rCB1 affinity with Kivalues of 855 and 748 nM using [3H]-SR141716A and [3H]-WIN-55-212-2 respectively (Table 11) Further four seriesof compounds differing by the nature of the substituentaround the triazole core were also described in a patent[199] Usually the compounds claimed possess two aromaticrings possibly substituted and one linear alkyl chain Theaffinity for the cannabinoid receptors was not givenNevertheless 149 was evaluated in two isolated tissuemodels the inhibition of the electrically evoked contractionsin the guinea pig ileum and in the mouse vas deferens Inthe two models compound 149 inhibited the effect of WIN-55212-2 but had no effect by itself In the vas deferensmodel the authors obtained a pA2 value of 748 to becompared with 763 obtained for AM251

Yet another five membered ring a thiazole has beenused as central moiety of a new class of cannabinoid ligandsThe thiazole ring is substituted by two aryls in position 4and 5 and by an amide (position 2) as in compounds 156-159 (Fig 13) Forty derivatives of this type weresynthesised by Lange and colleagues from SolvayPharmaceuticals and claimed in a patent as agonists orantagonists of the CB1 cannabinoid receptor [200] Nopharmacological data were provided however their activitywas assessed by measuring the cAMP in transfected hCB1-CHO cells In a recent paper from Lange et al two of thesethiazole derivatives were further described [182] Compound13 (157) the most closely related to SR141716A has a Kivalue of 227 nM while for compound 14 (158) Ki value isover 1000 nM Thus as in the imidazole series (see Table9) the aromatic substitution pattern is of great importancefor the CB1 cannabinoid receptor affinity Compound 13however was devoid of in vivo activity

Others 124-triazole derivatives were described by Dycket al compounds 150-154 unlike the Jagerovic ones havean amide moiety in position 3 [152] The highest affinitywas obtained with a 1-benzyl-pyrrolidin-3-yl substituentThe Ki value was 29 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) to be compared to 12 nM obtained for theSR141716A

Seventeen 45-diaryl-thiazole derivatives were alsosynthesised by Berggren et al and patented by AstraZeneca(157-159 Fig 13) [201]

In a patent from Merck Toupence et al described over190 substituted furo[23]pyridines claimed to be CB1cannabinoid receptor antagonists [202] The affinity (IC50 lt1microM) was measured using recombinant CHO cells and [3H]-CP-55940 and the activity using cAMP dosage (160 Fig14)

Lange et al described a triazole derivative (155) whichcan be superimposed with SR141716A Compound 155showed a Ki value of 356 nM ([3H]-CP-55940 hCB1-CHOcells) a pA2 value of 83 in the inhibition of WIN-55212-induced release of [3H]-arachidonic acid by hCB1-CHO cellsand was active in vivo in the CP-55940-inducedhypotension in rat (ED50 = 236 mgkg) [182]

Very recently Alanine from Hoffmann-LaRoche foundthat diarylbenzo[13]dioxole derivatives act as CB1

O

O

S

N

OO

F

ClCl

O

O

F O

N

OON

NH2

O

Cl

Cl

Cl

160 161 162

Fig (14) CB1 cannabinoid receptor antagonists 10 Other derivatives One example of a furo[23]pyridine derivative (160) and twoexamples of diarylbenzo[13]dioxole derivatives (161-162) described as hCB1 cannabinoid receptor antagonists by Toupence et aland Alanine et al respectively

1382 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

cannabinoid receptor antagonists (161-162 Fig 14) [203]Approximate IC50 values for twelve compounds ([3H]-CP-55940 hCB1-HEK cells) among the three hundred andeighty compounds described were given (IC50lt2microM)

et al in 1995 [207] In their hands ∆9-THC was not able toinhibit the forskolin-stimulated adenylyl cyclase activity inan assay performed on hCB2-CHO cells Subsequently theyshowed that ∆9-THC was able to antagonise HU210-inducedinhibition of adenylyl cyclase activity thus acting as anantagonist of the hCB2 cannabinoid receptor expressed inCHO cells [208] Furthermore very recently Govaerts et al[209] reported an inverse agonist effect of ∆9-THC and ∆8-THC on hCB2-CHO cells using the [35S]-GTPγ S assayThe pEC50 values were 763 and 888 respectively whilethe Emax values were ndash27 and ndash16 as compared to basal

Griffith from Pfizer described three new series of CB1cannabinoid receptor antagonists based on a bicyclic centralaromatic ring Purine pyrazoltriazines andpyrazolo[15a]pyrimidines derivatives were synthesised andinvestigated for their ability to interact with the CB1cannabinoid receptor Albeit a [35S]-GTPγ S assay wasdescribed no data were given concerning the antagonist orinverse agonist properties of such compounds Two hundredand eighty purine derivatives were synthesised [204] Theaffinities of 163 and 164 were given in the patent (28 and12 nM [3H]-SR141716A) In a following patent thesynthesis of sixty pyrazoltriazine derivatives was described(165 Fig 15) [205] The affinity of each compound was notgiven but was said to be ranging between 01 and 590 nMAnd finally more than forty pyrazolo[15a]pyrimidines weresynthesised (166 Fig 15) [206] Their affinity for the CB1cannabinoid receptor was lower than 155 nM Despite thefact that a great number of derivatives share the samesubstitution pattern around the central bicyclic aromatic ringthe comparison of these three new series of compounds ismade difficult by the absence of precise affinity data

Some other classical cannabinoids were shown to possessan antagonist activity at the CB2 receptor This is the casefor instance for the non-selective O-1184 (2) (Ki = 74 nM)which enhances the forskolin-induced cAMP production inCB2-CHO cells (EMax = 161 EC50 = 63 nM) and alsofor O-584 (3) (EMax = 246 EC50 = 138 nM) [39]

2 Indole Derivatives

Since the disclosure in 1993 of a second cannabinoidreceptor many attempts have been made to synthesise CB2cannabinoid receptor selective ligands The first compoundreported to have a somehow greater affinity for the peripheralcannabinoid receptor was WIN-55212-2 The compoundwas introduced by DrsquoAmbra and colleagues [210] back in1992 as a new cannabinoid ligand having a nanomolaraffinity and acting as an agonist In 1996 Showalter et alused a transfected cell line expressing the CB2 cannabinoidreceptor in order to identify selective ligands for thisreceptor [211] They showed that the CB2 affinity of WIN-55212-2 was 7 times greater than the CB1 with Ki valuesof 189 and 028 nM for the CB1 and CB2 cannabinoidreceptors respectively However WIN-55212-2 is anagonist of the cannabinoid receptors Interestingly WIN-55212-3 (167 Fig 16) the so-called inactive enantiomer ofWIN-55212 was recently shown to behave as a low affinity(Ki=363 microM) inverse agonist of the hCB2 cannabinoidreceptor as it decreased the [35S]-GTPγ S binding [209]

III CB2 LIGANDS RECEPTOR ANTAGONISTS ANDINVERSE AGONISTS

The compounds acting as antagonist or inverse agonistsat the CB2 cannabinoid receptor are reviewed in this thirdpart of the paper As for the CB1 cannabinoid receptorligands they are classified depending on their chemicalstructures

1 Classical Cannabinoid Derivatives

The first report of an antagonism mediated by ∆9-THC atthe peripheral cannabinoid receptor was made by Bayewitch

N N

NN

NH COOH

Cl

Cl

N N

NN

N

Cl

Cl

NH2OH

O

N

N

N

N

N

NHNH2

O

Cl

ClN

N

N

N

Cl

Cl

N

O

163 164

165 166

Fig (15) CB1 cannabinoid receptor antagonists 10 Other derivatives Examples of purine (163-164) pyrazoltriazines (165) andpyrazolo[15-a]pyrimidines (166) derivatives described as CB1 cannabinoid receptor antagonists by Griffith

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1383

The pEC50 value was 554 and the Emax value ndash26 ascompared to basal

AM630 also acts as an inverse agonist at the hCB1cannabinoid receptor (EC50 = 900 nM) as Landsman et aldemonstrated [47] Recently Zhang and colleagues reportedthe characterisation of the microsomal metabolism of AM630[212]

N

O

N

O

O

N

O

Cl

O

N

O

(S)-WIN-55212-3 167 BML-190 168

In 1996 Gallant and colleagues identified by submittinga large number of compounds to a binding assay anotherindole analog as CB2 cannabinoid receptor ligand [213] Onthe basis of this compound christened BML-190 (168 Fig16) or called indomethacin morpholinylamide theysynthesised several derivatives that possessed a selectivityratio for the CB2 cannabinoid receptor of up to 140 Thesewere the first compounds specifically designed to be CB2cannabinoid receptor ligands The first report on thepharmacological properties of BML-190 appeared onlyrecently New and colleagues highlighted in the paper theinverse agonist properties of the compound BML-190 dose-dependently increases (103 EC50= 980plusmn70 nM) theforskolin-stimulated levels of cAMP in hCB2-HEK cellswhile WIN-55212-2 decreases (44 45plusmn42nM) thisaccumulation [137]

Fig (16) CB2 cannabinoid receptor antagonists 2 Indolederivatives Chemical structures of WIN-55212-3 (167) andBML-190 (168) two indoles derivatives acting as inverseagonists at the hCB2 cannabinoid receptor Surprisingly in 2002 Melck and collaborators used

BML-190 as a CB2 receptor agonist in a cell proliferationassay [214] BML-190 was also tested as inhibitor of thecyclooxygenase-2 enzyme by Kalgutkar et al [215] Theyobtained IC50 values higher than 33 and 66 microM for theCOX-I and COX-II enzymes respectively These valueshave to be compared with the submicromolar activity ofindomethacin the parent compound Very recently Klegerisand colleagues [216] looking for an antineurotoxic action ofcannabinoids showed that BML-190 increases TNF-αsecretion by stimulated THP-1 monocytic cells but is noteffective on the IL-1β secretion

Further on the basis of WIN-54461 (12) a CB1antagonist AM630 (13) was synthesised by replacement ofthe bromine atom by an iodine one In the initial reportPertwee et al showed the antagonist effects of AM630 butalso suggested that the CB1 cannabinoid receptor may not bethe preferential receptor of AM630 [45] Later on Ross et alreported using hCB1amp2-CHO cells and [3H]-CP-55940that AM630 binds to the CB2 cannabinoid receptor (Ki =312 nM) with a selectivity ratio over 160 [48]Furthermore using a [35S]-GTPγ S assay they showed itsinverse agonist properties (EC50 = 76 nM) However

Table 12 CB2 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Reported binding affinities (Ki) and selectivity ratio (CB1CB2) of SR144528 (169) for rat and human cannabinoid receptors determined using [3H]-CP55940 as radioligand

NN

O

NH

Cl

SR144528 169

rCB2 (spleen) rCB1 (whole brain) Selectivity Ki hCB2 (transfected CHOcells)

Ki hCB1 (transfected CHOcells)

Selectivity References

038 nM 305 nM 800 06 nM 437 nM 730 [217]

067 nM 548 nM 80 [219]

56 nM gt10microM gt1780 [48]

25 nM a gt 1microM a gt400 [224]

024 nM 276 nM 115 199 nM 503 nM 25 [232]

a COS cells

1384 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

3 Diarylpyrazole Derivatives CHO cells is constitutively active and phosphorylated onSer 352 Upon treatment by SR144528 thisphosphorylation of the receptor was inhibited Moreover theSR compound was able to induce dephosphorylation ofagonist-induced CB2 phosphorylation The question whetherSR144528 induces a CB2 receptor conformational changemaking it a better phosphatase-specific substrate or ifSR144528 binding activates specific phosphatases remainsopen However this remains a nice example of inverseagonist-induced receptor resensitisation

The same Sanofi team which synthesised the CB1antagonist SR141716A reported in 1998 the synthesis andpharmacological characterisation of a CB2 cannabinoidreceptor selective antagonist the SR144528 (169) [217218] The structure of the new compound is derived fromSR141716A one SR144528 is the first selective CB2receptor antagonist reported as the inverse agonist propertiesof BML-190 were shown only recently by New et al [137]The authors reported for 169 using [3H]-CP-55940 asradioligand Ki values of 04 nM and 06 nM for the rat andthe human CB2 receptors respectively and found a CB1-CB2 selectivity ratio around 700 Table 12 sums up thebinding affinities of SR144528 for the cannabinoid receptorsobtained by different authors Interestingly while the CB2affinity is more or less constant the affinity obtained for theCB1 receptor varies by one order of magnitude [219] Theantagonist effect of SR144528 was highlighted by evaluatingits effect on cAMP production by hCB2-CHO cells exposedto 3 nM CP-55940 (EC50=10nM) The antagonism wasalso observed on the activation of the MAP kinase pathwayby CP-55940 (IC50=39nM)

The Sanofi research team working on cannabinoidsconducted investigations in order to determine the keyresidues for SR144528 interaction with the receptor (Table13) as well as the binding mode of the compound Firstlythey identified the TM4-EL2-TM5 region as a regioncontaining crucial residues for the affinity of several ligandsamong them SR144528 [224] Among these residues themutation of two cysteine of the second extracellular loop(C174 and C179) abolishes the affinity for the CB2 receptorLater on the implication of S453(161) and S457(165) inthe binding of SR144528 was evidenced by mutationalstudies In the model proposed for the docking of SR144528into the CB2 receptor the crucial contacts are comprised intothe TM4 this is the case for S453(161) and S457(165)which are proposed to form hydrogen bonds with the ligandand W464(172) proposed to have aromatic interactions withthe 4-methylbenzyl of the SR144528 [225] In 2003 Fengand Song showed that point mutations D349(130)A andA634(241)E abolished ligand binding However mutationof the arginine R350(131) member of the highly conservedDRY motif to alanine has no influence on SR144528binding [226]

Early after the report of the synthesis and characterisationof SR144528 the same authors further explored the functionof this compound and showed its inverse agonist propertiesusing CB2-CHO cells co-transfected with the luciferasereporter gene with either the CRE or the murine krox24regulatory sequence [220] In these models SR144528 notonly inhibits CP-55940 effects but also reproduces theeffects of a pertussin toxin treatment proving its inverseagonist properties SR144528 functionality was alsoexplored by [35S]-GTPγ S binding in hCB2-CHO cells(IC50=3 nM) [221] The inverse agonist function ofSR144528 was further illustrated by Rhee and Kim usingCOS cells co-transfected with hCB2 receptor and adenylylcyclase [222]

As for the CB1 cannabinoid receptor ligands Sanofideveloped tricyclic derivatives based on the 1-benzylpyrazole-3-carboxylic scaffold More than twentycompounds were disclosed in a patent [227] in 2001 (84-86Table 7) The benzyl in position 4 characteristic of the CB2diarylpyrazoles from Sanofi is conserved as well as thebicyclic residue in position 3 The length of the second linkbetween the phenyl and the pyrazole can be made of one or

Bouaboula and co-workers investigated SR144528 effectson CB2 cannabinoid receptor phosphorylation status [223]Firstly they showed that the CB2 receptor transfected in

Table 13 CB2 Cannabinoid Receptor Antagonists 3 Di-Aryl-Pyrazole Derivatives

Some of the single point mutations reported for the hCB2 cannabinoid receptor for which pharmacological data concerning the SR144528 are available

Mutant Effect References

R350(131)A no effect [226]

S453(161)A loss of affinity [225]

V456(164)I no effect [225]

S457(165)A loss of affinity [225]

C174S a loss of affinity [224 225]

C175S a loss of affinity [225]

C177S a no effect [225]

C179S a loss of affinity [224 225]

S544(193)G no effect [225]

a Second extra-cellular loop

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1385

two methylene but according to the patent one methylenebridge is preferred The Ki values of these compounds for theCB2 receptor were said to be lower than 500 nM and theirantagonist properties have been assessed using the adenylatecyclase inhibition assay

compounds by the presence of an alkyl substituent inposition 5 instead of a phenyl and of course by position 3which remains unsubstituted However except a lecture atthe 1998 ACS meeting [230] where compound 171 wasreported to have a Ki value of 835 nM and a selectivity ratioover 100 no further report on these compounds waspublished untill now

In a recent paper Mussinu and colleagues reported thesynthesis of other tricyclic derivatives starting fromSR141716A structure [228] N-piperidin-1-yl-14-di-hydroindeno[12-c]pyrazole-3-carboxamide derivatives areCB2 receptor ligands and some of them possess high affinityand selectivity for the CB2 receptor as shown in Table 7 (79-83) These tricyclic derivatives differ from the Sanofi tricyclicderivatives as they do have a phenyl ring instead of a benzylin position 1 Another difference is the nature of thesubstituent in position 3 which is not a bicycloalkyl but acyclic amine One of the compounds synthesised byMussinu et al 81 possesses the highest affinity for the CB2receptor ever reported with a Ki value of 0037 nM for themurine CB2 receptor ([3H]-CP-55940 mice spleenhomogenate) Interestingly 79 the closest SR141716Aanalog lost almost all its affinity for the CB1 receptor andincreased by four orders of magnitude its affinity for the CB2receptor Even if the structure quite similar to the SRcompounds could suggest that these derivatives behave asantagonists no pharmacological data were given in the paperto support this hypothesis

5 2-Oxoquinoline Derivatives

Inaba and colleagues from Japan Tobacco introduced in2001 a completely new structure in the cannabinoid fieldbased on a 2-oxoquinoline scaffold Over eighty derivativeswere disclosed in a patent [231] and one compound JTE-907 (173) was subsequently characterised in-vitro as well asin-vivo [232] In Table 14 are listed some of the compounds(172-178) disclosed in the patent along with their affinity forthe cannabinoid receptors These values were obtained onhuman receptors against [3H]-WIN-55212-2 For twelvecompounds the anti-inflammatory activity per os wasassessed in a carrageenin-induced paw edema model Thehighest effect reported was obtained with JTE-907 with anED50 value lower than 01 mgkg Therefore thesecompounds were claimed to be useful as anti-inflammatoryand anti-allergic agents

To further characterize JTE-907 the authors determinedits affinity for cannabinoid receptors of different species using[3H]-CP-55940 as radioligand In their hands JTE-907behaved as a very selective hCB2 ligand with Ki values of36 and 2370 nM for the CB2 and CB1 cannabinoid receptorsrespectively even more selective than the SR144528 (Kivalues of 199 and 50 nM respectively) However it has tobe mentioned that the affinities reported in the paper forJTE-907 are quite different from those reported in the patent(hCB1 Ki values of 35nM and 009 nM respectively) Italso appears that the SR144528 affinity for the CB1 receptordetermined by Iwamura et al is higher than the affinityreported by Rinaldi-Carmona et al with values of 50 and437nM respectively The authors ascribed the difference tothe variability of SR144528 binding affinities to the assayconditions The function of JTE-907 was evaluated bymeasuring the increase in cAMP production by forskolin-stimulated hCB2-CHO cells Maximum stimulation wasattained at a concentration of 1microM of JTE-907 In the sameassay WIN-55212-2 decreased the cAMP production in adose-dependent manner

Finally it is interesting to strengthen that the five-membered ring derivatives (79-86) of the diarylpyrazolefamily are reported to possess a high CB2 receptor affinitywhile the six- and seven-membered ring derivatives (69-78)are described as CB1 receptor ligands

4 Other Pyrazole Derivatives

Other CB2 ligands based on a pyrazole nucleus wereintroduced by SmithKline Beecham in a patent describingthe synthesis of about sixty new compounds [229] Theiraffinity (Ki) for the hCB2 receptor transfected in HEK293cells was reported to be ranging between 25nM and 10microMTheir structures (170-171 Fig 17) differ from the Sanofi

NN

O

N

ON

NN

N

NN

O

N

ON

NN

N

ClCl

1

5

4 3

2

170 171

Fig (17) CB2 cannabinoid receptor antagonists 4 Otherpyrazole derivatives Structure of two compounds (170-171)among those claimed by SmithKline Beecham to be hCB2antagonists

In conclusion JTE-907 was shown to be a CB2 selectiveinverse-agonist with anti-inflammatory and anti-allergicproperties This compound is currently undergoing Phase Iclinical trials as anti-allergic drug [233]

IV NON-CB1 NON-CB2 CANNABINOIDRECEPTORS LIGANDS

In the last years the possible presence of additional non-CB1 non-CB2 cannabinoid receptors was raised frompharmacological evidences

One of the putative receptors possesses an endotheliallocalisation while other ones are localized in the CNS or atperipheral nerves For instance an additional target for ∆9-

1386 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 14 CB2 Cannabinoid Receptor Antagonists 5 2-Oxoquinoline Derivatives

Structure of six compounds among the oxoquinoline derivatives patented by Japan Tobaco Affinity (Ki nM) and selectivity (CB1CB2) for the cannabinoidreceptors are given The affinity was measured by binding against [3H]-WIN-55212-2 on either hCB2-CHO or hCB1-CHO cells Adapted from [231]

Cpd ndeg Structure hCB2 hCB1 Selectivity

1-2 172

NH

MeO O

O

OMeO

0014 3671 262215

JTE-907 173

NH

O

MeO O

O

NH

O

O

0087 3436 39495

2-2 174

OH

O

NH

MeO O

O 077 3247 4215

3-27 175

NH

O

F

NH

MeO O

O0021 336 16000

3-32 176

NH

MeO O

O

NH

F

O

0036 2398 66610

3-38 177

O

O

NH

N

O

O

NMeO

0085 935 11000

3-43 178

NMeO O

O

NH

F

O

0043 864 20100

tetrahydrocannabinol was described in capsaicin-sensitivesensory nerves by Zygmunt et al [234]

[29] and by Di Marzo et al [235] Thus in the presentreview we will focus on the description of the so faridentified ligands of the non-CB1 non-CB2 receptors withan emphasis on the compounds having antagonist properties

Pharmacological evidences for the existence of additionalcannabinoid receptors were reviewed by Wiley and Martin

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1387

(Table 15) In particular the knowledge about three not yetcloned targets has grown The growing molecularpharmacology of the three putative new cannabinoidreceptors is reviewed in the three following sections Thefourth section summarises the data on the other proposedcannabinoid targets

vasodilatator activity In addition the anandamide effect ispartially sensitive to SR141716A inhibition The authorsproposed the existence of two distinct receptors responsiblefor the vasodilatatory effect of anandamide one located in theendothelium is SR141716A-sensitive while the other onelocated on the vascular smooth muscle is SR141716A-insensitive Thus SR141716A is an antagonist of anendothelial anandamide receptor Several studies wereconducted in order to understand the molecularpharmacology of the endothelial anandamide receptor It ispertussis toxin-sensitive [237] Cannabidiol acts asantagonist and abnormal-cannabidiol (Abn-cbd 179) and itsanalogue O-1602 (180) act as agonists [238] The synthesisof O-1602 and of its analogues are disclosed in a patentdescribing the vasodilatatory activity of cannabinoid

1 Endothelial Anandamide Receptor ndash Abnormal-Cannabidiol Receptor

In 1999 Wagner et al showed that anandamide couldinduce mesenteric vasodilatation through an endotheliallylocated ldquoanandamide receptorrdquo pharmacologically distinctfrom CB1 [236] Other cannabinoids such as ∆9-THC HU-210 or WIN-55212-2 were devoid of mesenteric

Table 15 Non-CB1 Non-CB2 Cannabinoid Receptors Antagonists

An overview of the putative new non-CB1 non-CB2 anandamide-receptors ligands The antagonists are highlighted in bold

NH

N

S

HO OH

Cl

OH

OH

OH OH OH OH

OMe

OMe

RR

RR

RR

RR

HO

OH

NH

O

cannabidiol (10) Abnormal-cannabidiol 179 O-1602 180

O-1918 181 capsazepine 183capsaicin 182

Compound Endothelial AEA receptor Brain AEA receptor Cannabinoid-vanilloid brain receptor

AEA agonist a agonist de

WIN-55212-2 no effecta

agonist e agonist g h

CP-55940 no effect e agonist i

SR141716A antagonist a b j no effect ([35 S]-GTPγS binding) d e f antagonist g h

AM251 no effect k no effect h i

Cannabidiol antagonist b no effect e

O-1918 antagonist c jm

Abd-cbd agonist b l

O-1602 agonist b

Capsaicin no effectj agonist i

Capsazepine no effect b antagonist h

a [236] b [238] c [240] d [249] e [250] f [251] g [254] h [258] i [ 255] k [244] j [ 243] l [ 242] m [241]

1388 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

analogues [239] More recently Offertaler et al showed thatO-1918 (181) a cannabidiol analog acts as an antagonist ofthe endothelial anandamide receptor [240] In addition O-1918 does not bind to the CB1 and CB2 cannabinoidreceptors Furthermore O-1918 unlike cannabidiol doesnot cause vasorelaxation in the mesenteric artery preparationmodel used and thus acts as a true neutral antagonistUsing O-1918 as a pharmacological tool Begg et alproposed a coupling of this new G protein-coupledendothelial receptor through a GiGo mechanism to ionchannels in primary cultured human vascular endothelialcells [241] However in their hands SR141716A wasineffective in antagonising Abd-cbd Ho and Hiley furtherdemonstrated that Abn-cbd relaxes rat small mesentericarteries through an activation of K+ channels via aSR141716A-sensitive pathway [242] Further using thesame model they characterized the vasorelaxant activity ofvirodhamine a novel endocannabinoid apparently actingthrough the same receptor [243] The virodhamine-inducedvasorelaxation is sensitive to SR141716A and O-1918 butinsensitive to AM251 SR144528 and AM630 Moreovervanilloid receptor desensitisation by capsaicin (182) had noeffect

244] Thus position 4 on the 5-phenyl could be furtherexplored to obtain selective ligands

To come to an end despite the fact that the endothelialanandamide receptor is neither identified nor cloned at leastthree antagonists are identified SR141716A cannabidioland O-1918 It is likely that a growing number of ligandsand therapeutic applications will appear in the very nearfuture

2 Brain Anandamide Receptor

Following an in-depth evaluation of anandamide effects inCB1

-- mice Di Marzo and co-workers suggested that a non-CB1 non-CB2 G protein-coupled receptor might mediatesome of the actions of AEA in mice [249] Actually effectsof AEA but not those of ∆9-THC were not decreased inCB1 cannabinoid receptor knock-out mice For instance 30mgkg of AEA were still effective in hot-plate test whereas10 mgkg of ∆9-THC had no effect on anti-nociceptionMoreover in a [35S]-GTPγ S assay AEA stimulated with anEC50 value of 223 microM the [35S]-GTPγ S binding to CB1

--

mice brain membranes (Emax= 28) ∆9-THC showed noeffect in this assay The addition of SR141716A did notaffect the [35S]-GTPγ S binding to CB1

-- mice brainmembranes Shortly after other evidences for the existence ofa new G protein-coupled cannabinoid receptor with a distinctdistribution in the central nervous system were published[250] From a set of 24 commonly used cannabinoids (AEAWIN-55212-2 JWH-030 THChellip) only AEA and WIN-55212-2 produced an increased binding of [35S]-GTPγ S onCB1

-- mice brain membranes Interestingly in this studySR141716A alone produced a significant inhibition of [35S]-GTPγ S binding similar to the inhibition obtained usingCB1

++ mice brain membranes (IC50 values of 57 microM and47 microM respectively) However SR141716A did not affectthe anandamide-induced [35S]-GTPγ S binding in CB1

--

mice brain membranes Moreover [3H]-SR141716Aexhibited significant binding in some brain regions of CB1

--

mice which are different from those exhibiting stimulation of[35S]-GTPγ S binding by WIN-55212-2 and AEA Thusthe authors proposed that the SR141716A effects on CB1

--

mice are mediated neither by the CB1 cannabinoid receptornor by the putative new anandamide brain receptor Monoryand co-workers using CB1

-- mice described a novel nonadenylyl cyclase-coupled cannabinoid binding site in micecerebellum [251] Two agonists AEA and WIN-55212-2but not ∆9-THC or HU210 were found to bind to thisreceptor as they enhanced [35S]-GTPγ S binding NeitherSR141716A nor SR144528 were able to affect the WIN-55212-2 induced [35S]-GTPγ S binding in CB1

-- mice asfound by Breivogel et al [250] However the distributionpattern of the receptor described by Monory et al differs fromthe distribution found by Breivogel et al It has to bementioned that the knock-out strains used were different Themice used by Monory were from a CD-1 strain while theone used by Breivogel were from a C57BL61 strainInterestingly Muthane et al detected differences in nigralneurons number and sensitivity to MPTP in the two mousestrains used [252]

More recently Mo et al obtained results showing thatthe Abnormal-cannabidiol receptor is also responsible for anincreased endothelial cell migration [244] Abn-cbd enhancedhuman umbilical vein endothelial cell migration while O-1918 antagonised Abn-cbd effect The CB1 cannabinoidreceptor inverse agonist SR141716A partially inhibitedAbd-cbd action whereas AM251 and SR144528 had noeffect

In another paper from George Kunos team Batkai et alreported that SR141716A is able to inhibit the endotoxichypotension by a cardiac mechanism involving neither theCB1 nor the CB2 cannabinoid receptors [245] Theinvolvement of a non-CB1 non-CB2 site of action wasshown using CB1

-- and CB1--CB2

-- knock-out miceFurther studies are needed to identify the SR141716Asensitive myocardial site of action

In a recent patent Kunos described O-1918 and itsanalogues as vasoconstrictor agents useful to reversepathological vasodilatation of blood vessels [246] Theeffects of O-1918 on mouse blood pressure on LPS-inducedhypotension in mouse as well as the reversal of hypotensionby O-1918 injection are described Furthermore thesynthesis of O-1918 based antagonists is described startingfrom p-metha-28-dien-1-ol and the appropriate resorcinols

Interestingly the Abd-cbd receptor which was firstdetected in the vascular system also seems to be present inthe CNS [247] Indeed Walter et al found that microglialcells (BV-2) migration induced by 1 microM 2-AG is inhibitedby cannabidiol (300 nM) and O-1918 (1 microM) SR141716Awas found ineffective in this assay Thus 2-AG is an agonistof the Abd-cbd receptor Moreover Abd-cbd is able to elicita dose-dependent cell migration in the same model (EC50 =600nM) [248]

From a more structural point of view SR141716Abehaves as an antagonist while on the opposite AM251(20) a close analogue which differs from the former only bythe nature of one halogen has no effect on this receptor [242

To conclude to our knowledge no compounds havingantagonist or inverse agonist properties at this receptor havebeen identified so far

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1389

3 Cannabinoid-Vanilloid Brain Receptor a cannabinoid-vanilloid receptor at which SR141716A andcapsazepine behave as antagonists

Evidences for the presence of an additional ldquocannabinoidrdquoreceptor differing from the CB1 receptor and from the brainanandamide receptor appeared from studies focused on thehippocampus (for a review see Hajos et al [253]) Hajos etal studied the cannabinoid actions on GABAergic andglutamatergic transmission using the whole-cell patch-clamp technique [254] WIN-55212-2 induced a dose-dependent reduction in glutamatergic transmission reversedby SR141716A (1microM) Thus the authors concluded that thecannabinoid actions on the glutamatergic transmission aremediated by a non-CB1 but SR141716A-sensitive receptorIn another paper Hajos and Freund further characterised thisputative receptor [255] They showed that in addition toWIN-55212-2 CP-55940 and capsaicin (182) a TRPV1receptor agonist also act as agonists Furthermore in theirhands SR141716A as well as capsazepine (183) a TRPV1receptor antagonist were able to antagonise this effect on theglutamatergic transmission while AM251 was notSR141716A effects on CB1 knock-out mice were furtherstudied using a test of anxiety [256] Haller et al showedthat on the one hand the distribution of the CB1 receptorinduces anxiety and SR141716A has an anxiolytic effectand on the other hand SR141716A is still active in CB1

--

mice In a following paper they reported that AM-251induces anxiogenic effects in wild-type and in knock-outmice [257] Thus SR141716A in addition to CB1 bindsto a not yet identified receptor Another evidence for thisadditional receptor for SR141716A was given by Bass andco-workers [61] They conducted a structure-activityrelationship study on the stimulation of locomotor activityinduced by SR141716A and twenty analogues Nocorrelation was found between the affinity for the CB1cannabinoid receptor and the stimulation of locomotoractivity Albeit SR141716A and five of its analogues O-1803 O-1710 O-1253 O-1254 (40) O-1255 (41) behavedas inverse agonists in [35S]-GTPγ S assay none of theseanalogues stimulated the locomotor activity In conclusionthe SR141716A-induced stimulation of locomotor activity isneither due to inverse agonism nor to inhibition of anendogenous tone but to a brain receptor distinct from theCB1 receptor Di Marzo et al previously provided evidencesthat the cannabinoid-vanilloid brain receptor could mediatesome of the cannabinoid effects on locomotion [249]

4 Other Non-CB1 Non-CB2 Cannabinoid Receptors

Evidences for the existence of additional non-CB1 non-CB2 cannabinoid receptors localised in the rat isolatedmesenteric arterial bed appeared from the works of Ralevicand collaborators Several cannabinoids were evaluated intheir model of sensory neurogenic vasorelaxation evoked byelectrical field stimulation

HU210 was found to attenuate vasorelaxation in a CB1and CB2 independent way as SR141716A LY320135 andSR144528 were ineffective in inhibiting HU210 effect [259]Similar results were obtained with ∆9-THC [260]Similarly noladin ether inhibited sensory neurogenicrelaxation in a concentration-dependent manner which wasalso unaffected by SR141716A LY320135 and SR144528Moreover this effect as the ∆9-THC one was pertussistoxin sensitive suggesting the involvement of a Gio protein-coupled receptor [261]

However the inhibitory effects of CP-55940 and WIN-55212-2 were affected by the presence of SR141716A (1microM)or LY320135 (1microM) but not by the addition of SR144528(1microM) [262]

It appears that at least two receptors should be involvedin cannabinoid-mediated attenuation of sensory nerve-mediated vasorelaxation The first one is SR141716A andLY320135 sensitive while the other one is resistant toSR141716A

Some evidences for the existence of a ldquoCB2-likerdquo receptorappeared from the studies on palmitoylethanolamide (PEA)an endogenous fatty acid amide devoid of CB1 and CB2affinity [264] antinociceptive potential Calignano and co-workers showed that PEA was able to alleviate pain inseveral animal models [265 266] For instance PEA dose-dependently inhibits kaolin-evoked writhing in mice TheCB2 cannabinoid receptor inverse agonist SR144528 (02mgkg) inhibits PEA anti-nociceptive effect [266] Thus theauthors suggest that PEA acts as agonist on a SR144528-sensitive non-CB2 cannabinoid receptor (ldquoCB2-likerdquoreceptor) at which SR144528 acts as an antagonist

Further studies are needed in order to better characterisethese additional ldquocannabinoidrdquo receptors However it islikely that several therapeutic applications will soon springfor these receptors

Very recently Pistis et al published a study on theneurophysiological effects of cannabinoids on the basolateralamygdala neurons in-vivo [258] One of their findings is thatHU-210 and WIN-55212-2 did not elicit similar effects onbasal amygdala projection neurons firing rate While theformer had no effect the latter decreased the firing rateMoreover SR141716A reversed the WIN-55212-2 effectswhile AM251 had no effect The vanilloid receptorantagonist capsazepine also antagonised the WIN-55212-2effect on firing rate of these neurons Therefore the authorssuggested the presence of a novel cannabinoid-vanilloidreceptor

V CONCLUSION

Since their development cannabinoid antagonists provedto be essential tools in the understanding of cannabinoidpharmacology and biochemistry Moreover the interestshown by the pharmaceutical industry in the development ofnew cannabinoid antagonists prove if necessary the realtherapeutic potential of this class of compounds Thetreatment of eating and movement disorders memorydeficits psychosis and dependencies from various addictivedrugs are some of the most cited applications for the CB1cannabinoid receptor antagonists

From these lines it appears that in addition to theknown CB1 cannabinoid receptor it is now likely that twoother cannabinoid receptors do exist in the brain The firstone should be an anandamide brain receptor for whichSR141716A has no affinity while the second one should be

1390 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Much less wide is the knowledge on the CB2cannabinoid receptor antagonists However potentialapplications could be disorders involving the immunesystem such as inflammation or allergies

[19] Devane WA Hanus L Breuer A Pertwee RG StevensonLA Griffin G Gibson D Mandelbaum A Etinger AMechoulam R Science 1992 258 1946

[20] Mechoulam R Ben-Shabat S Hanus L Ligumsky MKaminski NE Schatz AR Gopher A Almong S MartinBR Compton DR Biochem Pharmacol 1995 50 83

Finally the growing literature on the non CB1 non CB2cannabinoid receptors should allow the development of newdrugs First examples are given by the cannabidiol andabnormal-cannabidiol derivatives described by Kunos et alfor which potential therapeutic applications on the vascularsystem are already patented

[21] Sugiura T Kondo S Sukagawa A Nakane S Shinoda AItoh K Yamashita A Waku K Biochem Biophys Res Co1995 215 89

[22] Hanus L Abu-Lafi S Fride E Breuer A Vogel Z ShalevDE Kustanovich I Mechoulam R Proc Natl Acad SciUSA 2001 98 3662

[23] Oka S Tsuchie A Tokumura A Muramatsu M Suhara YTakayama H Waku K Sugiura T J Neurochem 2003 851374It clearly appears from this paper that the antagonists of

the cannabinoid receptors are still an active research fieldfrom which should emerge new promising therapeutic toolsas well as innovative drugs in the near future

[24] Goutopoulos A and Makriyannis A Pharmacol Therapeut2002 95 103

[25] Beal JE Olson R Lefkowitz L Laubenstein L Bellman PYangco B Morales JO Murphy R Powderly W PlasseTF Mosdell KW Shepard KV J Pain Symptom Manage1997 14 7ACKNOWLEDGEMENTS [26] Notcutt W Price M Miller R Newport S Phillips CSimmons S Sansom C Anaesthesia 2004 59 440

GGM is very indebted to the ldquoFonds pour la formationagrave la Recherche dans lrsquoIndustrie et lrsquoAgriculture (FRIA)rdquo Belgium for the award of a research fellowship Theexperimental works of the authors were supported by grantsfrom the FNRS The Belgian Charcot Foundation and theUniversiteacute catholique de Louvain

[27] Ledent C Valverde O Cossu G Petitet F Aubert JFBeslot F Bohme GA Imperato A Pedrazzini T RoquesBP Vassart G Fratta W Parmentier M Science 1999 283401

[28] Zimmer A Zimmer AM Hohmann AG Herkenham MBonner TI Proc Natl Acad Sci U S A 1999 96 5780

[29] Wiley JL and Martin BR Chem Phys Lipids 2002 121 57[30] Adam J and Cowley P Expert Opin Ther Patents 2002 12

1475

REFERENCES [31] Razdan RK and Mahadevan A Chem Phys Lipids 2002 12121

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[254] Hajos N Ledent C Freund TF Neuroscience 2001 106 1 [265] Calignano A La Rana G Giuffrida A Piomelli D Nature1998 394 277[255] Hajos N Freund TF Neuropharmacology 2002 43 503

[256] Haller J Bakos N Szirmay M Ledent C Freund TF Eur JNeurosci 2002 16 1395

[266] Calignano A La Rana G Piomelli D Eur J Pharmacol 2001419 191

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1376 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 9 CB1 Cannabinoid Receptor Antagonists 7 Diaryl-Imidazole Derivatives

Structure and affinity range of some 45-diaryl-imidazole derivatives for the human cannabinoid receptors determined using [3H]-CP-55940 asradioligand and hCB1amp2-CHO cells

N

NR2

R1

R4

R3

ndeg R1 R2 R3 R4 hCB1 (IC50 or Ki) hCB2 (IC50 or Ki) References

95 CH3 CO-NH-piperidinyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

96 CH3 CO-NH-piperidinyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

97 CH3 CO-NH-cyclohexyl 4-Me-Ph 4-Me-Ph 100 ndash1000 nM a gt 1000 nM a [178]

98 CH3 CO-NH-phenyl 24-di-Cl-Ph 4-Cl-Ph 10 ndash 100 nM a 100 ndash1000 nM a [178]

99 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph le 10 nM a 100 ndash1000 nM a [178]

100 CH3 CO-NH-cyclohexyl 24-di-Cl-Ph 4-Cl-Ph 4 nM a 300 nM a [179]

101 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

CH3 14 nM b ND [152]

102 4-Cl-Ph 24-di-Cl-Ph CO-NH-(3-azabicyclo[330]octan-3-yl)

H 66 nM b ND [152]

103 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 85 nM b ND [152]

104 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl H 23 nM b 542 nM b [182]

105 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 30 nM b 608 nM b [182]

106 4-Br-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 60 nM b 489 nM b [182]

107 4-CF3-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH3 29 nM b 634 nM b [182]

108 4-Cl-Ph 24-di-Cl-Ph CO-NH-morpholinyl CH3 197 nM b 3297 nM b [182]

109 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidin-4-ol CH3 172 nM b 3959 nM b [182]

110 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CN 30 nM b 1590 nM b [182]

111 4-Cl-Ph 24-di-Cl-Ph CO-NH- piperidinyl CH2F 36 nM b 906 nM b [182]

112 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl CH2-CH3 14 nM b 430 nM b [182]

113 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Cl 27 nM b 823 nM b [182]

114 4-Cl-Ph 24-di-Cl-Ph CO-NH-piperidinyl Br 23 nM b 746 nM b [182]

115 4-Cl-Ph 24-di-Cl-Ph CO-NH-tetrahydroisoquinoline CH3 34 nM b 696 nM b [182]

116 4-Cl-Ph 24-di-Cl-Ph CO-NH-cycloheptyl CH3 35 nM b 349 nM b [182]

117 24-di-Cl-Ph 4-Cl-Ph CO-NH-piperidinyl CH3 403 nM b 208 nM b [182]a IC50 value b Ki value

Lange and co-workers from Solvay described twenty-eight imidazole derivatives [182] The 24-dichlorophenylsubstituent at position 2 was kept constant while thesubstitutions at the other positions were explored (Table 9)Position 5 can accommodate a large range of littlesubstituents like hydrogen (104) methyl (105) ethyl (112)chlorine (113) bromine (114) fluoromethyl (111) or cyano(110) without major changes in the affinity At position 4more bulky substituents like 1234-tetraisoquinoline (115)or cycloheptyl (116) were well tolerated whereas thepresence of a more hydrophilic moiety such as morpholine(108) or piperidin-4-ol (109) was detrimental to the affinity

Interestingly position exchange between the 24-diphenyland the 4-chlorophenyl substituents led to a 13 fold loweraffinity with Ki values of 30 and 403 nM for 105 and 117respectively ([3H]-CP-55940 hCB1-CHO cells) In thefunctional assay which was inhibition of WIN-55212-2-induced [3H]-arachidonic acid release by hCB1-CHO cellsall tested compounds behaved as antagonists Compound105 showed a pA2 value of 86 which is the same theauthors obtained for SR141716A It would be interesting toassess if these compounds are true antagonists or if they actmore as inverse agonists Compound 105 when administeredto rats was able to inhibit CP-55940-induced hypotension

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1377

with an ED50 value of 24 mgkg which is close to the 3mgkg obtained for SR141716A The compound was alsoactive in another in-vivo model the WIN-55212-2-inducedhypothermia in mice Compound 104 which has nosubstituent in position 5 of the imidazole ring was lessactive in the hypotension model and inactive in thehypothermia model Thus a methyl group is the preferredsubstituent in position 5 of the imidazole moiety Otherproperties either experimental (P-glycoprotein affinitylogPHPLC) or computational (molecular volume polarsurface area) obtained for compounds 105 were similar tothose obtained for SR141716A Finally molecularmodelling studies revealed a close structural overlap betweenthe two compounds

value of 197 nM is selective for hCB1 and is as potent asSR141716A The structural modulations of 118 led to someinteresting compounds (Table 10) Six compounds possess aKi value for the hCB1 cannabinoid receptor equal to or lowerthan 25 nM antagonist properties in the inhibition of [3H]-arachidonic acid release by WIN-55212-2-stimulated hCB1-CHO cells and Ki values for the hCB2 receptor over 1microMThe diaryl-pyrazoline derivatives contained a chiral center atposition 4 The authors resolved the racemic mixture of twocompounds (121 and 130) The levorotatory enantiomersappeared to be the eutomers For compound SLV319levorotatory enantiomer of 121 the Ki and pA2 values were78 nM and 99 respectively The Ki and pA2 values ofSLV326 levorotatory enantiomer of 130 were 359 nM and9 respectively

8 34-Diaryl-Pyrazoline Derivatives Two in-vivo models CP-55940-induced hypotension inrat and WIN-55212-2-induced hypothermia in mouse wereused to assess the potential of these compounds The resultsare of the same order of magnitude than those obtained forSR141716A For instance in the hypotension modelSLV319 ((-)121) and SLV326 ((-)130) showed ED50 values

Lange and collaborators also discovered 34-diaryl-pyrazoline derivatives after a screening of compoundsresembling to SR141716A [183184] Compound 118 thatwas initially identified during the screening possesses a Ki

Table 10 CB1 Cannabinoid Receptor Antagonists 8 34-Diaryl-Pyrazolines Derivatives

Structure affinity for the hCB1 and hCB2 cannabinoid receptors (Ki nM) expressed in CHO transfected cells ([3H]-CP-55940) and potency (pA2) ofsome 34-dirylpyrazolines derivatives Table adapted from [184]

NN

N N

SO2

R3

R2

Cl R1

R4

n˚ R1 R2 R3 R4 Ki hCB1 (nM) Ki hCB2 (nM) pA2 (CB1)

118 H H H 4-CH3 197 gt 1000 84

119 H H H 246- CH3 24 gt 1000 94

120 H H H 4-Cl 16 gt 1000 95

121 H CH3 H 4-Cl 25 gt 1000 87

122 H CH3 CH3 4-Cl 280 gt 1000 85

123 H H H 4-F 53 gt 1000 9

124 H CH3 H 4-F 338 gt 1000 85

125 H CH3 CH3 4-F gt 1000 gt 1000 lt 75

126 H CH3 H 3-Cl 14 gt 1000 86

127 4-Cl CH3 H 4-Cl 255 ND ND

128 4-F CH3 H 4-Cl 584 ND ND

129 H CH3 H H 170 ND 75

130 H CH3 H 4- CF3 221 gt 1000 93

SR141716A 25 1580 86

1378 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

N

HN

O

N

Cl Cl

Cl

N

N

HN

O

Br

Br131 132

Fig (8) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Chemical structures of two 56-diaryl-pyrazine-2-amide derivatives (131-132) developed as CB1 cannabinoid receptorantagonists by AstraZeneca

of 55 and 2 mgkg (po) respectively proving their in-vivoefficacy after oral administration The authors alsodemonstrated that SLV319 is devoid of affinity for the P-glycoprotein pump and that it possessed a goodCNSplasma ratio (17) These two compounds (SLV319and SLV326) were chosen as development candidates bySolvay and entered clinical Phase I trials in 2003 [185]

132 Fig 8) useful as CB1 cannabinoid receptor antagonists[186 187] The activity of twenty derivatives wasdetermined using a [35S]-GTPγ S assay and hCB1-CHOtransfected cells The concentration required to give halfmaximal inhibition of CP-55940-induced [35S]-GTPγ Sbinding (IC50) is lower than 200 nM for the preferredcompounds No affinity data were given for thesecompounds

9 Diaryl-Pyrazine Diphenyl-Pyridine Diphenyl-Phenyland Diaryl-Pyrimidines Derivatives

The same year in a patent from Merck Finke andcollaborators described several 56-di-phenyl-pyridinederivatives as hCB1 antagonists or inverse agonists [188]The compounds have a substituent in position 3 of thepyridine core (Fig 9) This substituent could be a cyano(133-134) or a nitro group a halogen an ester or an amide(135-136) Over 150 compounds were synthesised toillustrate the invention but no pharmacological data wasdisclosed in the patent

In 2003 appeared several patents describing new familiesof compounds that bind to the CB1 cannabinoid receptorThese compounds have in common a central six atomsaromatic ring which could be a pyrazine a pyridine aphenyl or a pyrimidine and that is substituted by at leasttwo phenyl rings

The first patents were from Berggren and collaboratorsfrom AstraZeneca who published two patents describing thesynthesis of 56-diaryl-pyrazine-2-amide derivatives (131-

A few months later another patent was taken by Sanoficlaiming the antagonist properties of 56-di-phenyl-2-pyridine carboxamide derivatives [189] These compounds

N

NH

O

Cl

Cl

N

O

Cl

O NH

N

O

Cl

ClCl

NH

CN O

N

Cl

Cl

Cl

OHN

N

O

Cl

ClCl

CNF

F

133 134

135 136 137

Fig (9) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives 56-Di-phenyl-pyridine derivatives patented by Merck (133-136) and by Sanofi (137) as CB1 cannabinoid receptorsantagonists

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1379

NH

O

N

Cl

ClCl

NH

O

N

Cl

F3CCl

N

N

O

Cl

Cl Cl F138 139 140

Fig (10) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Representative structures of the diphenyl-phenyl (138-139) and 45-diphenyl-pyrimidine (140) derivatives developed ascannabinoid antagonists at Sanofi and Merck respectively

28 examples are given structurally related to the diphenyl-phenyl derivatives have their pyridine core substituted bytwo phenyl rings and by an amide moiety (137 Fig 9) TheIC50 values were lower than 100 nM but no data were givenshowing the antagonist properties although the compoundswere assayed in the adenylate cyclase inhibition assay

10 Other Derivatives

Several amide derivatives structures recently appearedamong the CB1 cannabinoid receptor antagonists In a seriesof patents taken by Merck in 2003 Hagmann and colleaguesdescribed a large amount of compounds obtained by parallelsynthesis which are claimed to be CB1 cannabinoid receptorantagonists [192-195] They were synthesised by reacting alibrary of substituted amines with a library of carboxylicacids (141-144 Fig 11) However as no value was givenfor the affinity or activity of these amides we will notfurther discuss this class of compounds

Along this line Sanofi Synthelabo developed newdiphenyl-phenyl derivatives claimed to have CB1cannabinoid receptor antagonist properties [190] They arebased on a central phenyl ring substituted by two phenylsand by an amide moiety (138-139 Fig 10) Thirteencompounds were claimed along with their synthesis TheirIC50 values were lower than 100 nM ([3H]-CP55-940hCB1-CHO cells) but no other specific data (selectivitypA2hellip) was given

Several 15-diaryl-pyrrole-3-carboxamide derivatives weresynthesised and claimed to be CB1 cannabinoid receptorantagonists by Berggren et al [196] The affinity valueswere not disclosed in the patent (145-146 Fig 12) Otherpyrrole derivatives were synthesised by Guba et al atHofmann-La Roche [197] More specifically 2-(thiazol-4-yl)pyrrole derivatives were claimed as CB1 cannabinoidreceptor antagonist (IC50lt2microM) (147-148 Fig 12)

Finally Kopka et al from Merck described more thanone hundred 45-diaryl-pyrimidine derivatives as CB1cannabinoid receptor antagonists (140 Fig10) [191]However no precise data were provided concerning theaffinity and functionality of the compounds

R2

R1N

R3

R4

R5

O

NH

O

Cl

O F

F

R2

R1NH

R3

R4

HO R5

O

NH

O

Cl

CN

NH

O

Cl

O

O

NH

O

Cl

Cl

+

141142

143 144

Fig (11) CB1 cannabinoid receptor antagonists 10 Other derivatives Retrosynthetic scheme of a CB1 cannabinoid antagonistlibrary of amide derivatives developed at Merck The structures of four representative compounds are illustrated (141-144)

1380 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

NH

O N

Cl

Cl

Cl

N

NH

O

OMe

MeO

N

NH

O

N

S

MeO

N

NH

O

N

S

MeO

145 146

147 148

Fig (12) CB1 cannabinoid receptor antagonists 10 Other derivatives Two examples of 15-diaryl-pyrrole derivatives described byBerggren et al (145-146) While the 2-(thiazol-4yl)pyrroles derivatives 147 and 148 were described by Guba et al from Hofmann-LaRoche All these pyrroles derivatives were claimed to be CB1 cannabinoid receptor antagonists

Another new structure is represented by the 124-triazolederivatives developed by Jagerovic and collaborators In arecent paper were described the synthesis andpharmacological properties of five new 15-diphenyl-3-alkyl-

triazole derivatives [198] Among these compounds onlyone (149) behaved as a CB1 antagonist inhibiting the WIN-55212-2-induced contractions in a mouse vas deferenspreparation However despite its effects in isolated tissue

Table 11 CB1 Cannabinoid Receptor Antagonists 10 Other Derivatives

Structure and affinity of diaryl-triazole derivatives

N

NN

R3

R2

R1

Cpd R1 R2 R3 Ki CB1 (nM) References

149 24-diCl 4-Cl hexyl 855 a [198]

150 24-diCl 4-Cl2-OMe CO-NH-(3-azabicyclo[330]octan-3-yl) 270 b [152]

151 24-diCl 4-Cl CO-NH-(3-azabicyclo[330]octan-3-yl) 164 b [152]

152 4-Cl 24-diCl CO-NH-(3-azabicyclo[330]octan-3-yl) 137 b [152]

153 4-Cl 24-diCl CO-NH-benzylpyrrolidin-3-yl 29 b [152]

154 4-Cl 24-diCl CO-NH-1-(4-chlorophenyl)ethyl 66 b [152]

155 4-Cl 24-diCl CO-NH-piperidinyl 356 c [182]

156 24-diCl 4-Cl CO-NH-piperidinyl 382 c [182]

a [3H]-SR141716A rat cerebellar membranesb [3H]-CP-55940 hCB1-HEK EDNA cellsc [3H]-CP-55940 hCB1-CHO cells

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1381

N

S

O

NH

Cl

Cl

Cl

F

N

S

O

NHN

ClCl

Cl

N

S

O

NH

Br

N

S

O

NHN

Cl

Cl

Cl

156 157 158 159

Fig (13) CB1 cannabinoid receptor antagonists 10 Other derivatives Structures of 45-diaryl-thiazole derivatives patented as hCB1cannabinoid receptor antagonists by Solvay Pharmaceuticals (156 157 158) or by AstraZeneca (157 158 159)

assays (mouse vas deferens and guinea pig ileum) thecompound possessed only a reduced rCB1 affinity with Kivalues of 855 and 748 nM using [3H]-SR141716A and [3H]-WIN-55-212-2 respectively (Table 11) Further four seriesof compounds differing by the nature of the substituentaround the triazole core were also described in a patent[199] Usually the compounds claimed possess two aromaticrings possibly substituted and one linear alkyl chain Theaffinity for the cannabinoid receptors was not givenNevertheless 149 was evaluated in two isolated tissuemodels the inhibition of the electrically evoked contractionsin the guinea pig ileum and in the mouse vas deferens Inthe two models compound 149 inhibited the effect of WIN-55212-2 but had no effect by itself In the vas deferensmodel the authors obtained a pA2 value of 748 to becompared with 763 obtained for AM251

Yet another five membered ring a thiazole has beenused as central moiety of a new class of cannabinoid ligandsThe thiazole ring is substituted by two aryls in position 4and 5 and by an amide (position 2) as in compounds 156-159 (Fig 13) Forty derivatives of this type weresynthesised by Lange and colleagues from SolvayPharmaceuticals and claimed in a patent as agonists orantagonists of the CB1 cannabinoid receptor [200] Nopharmacological data were provided however their activitywas assessed by measuring the cAMP in transfected hCB1-CHO cells In a recent paper from Lange et al two of thesethiazole derivatives were further described [182] Compound13 (157) the most closely related to SR141716A has a Kivalue of 227 nM while for compound 14 (158) Ki value isover 1000 nM Thus as in the imidazole series (see Table9) the aromatic substitution pattern is of great importancefor the CB1 cannabinoid receptor affinity Compound 13however was devoid of in vivo activity

Others 124-triazole derivatives were described by Dycket al compounds 150-154 unlike the Jagerovic ones havean amide moiety in position 3 [152] The highest affinitywas obtained with a 1-benzyl-pyrrolidin-3-yl substituentThe Ki value was 29 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) to be compared to 12 nM obtained for theSR141716A

Seventeen 45-diaryl-thiazole derivatives were alsosynthesised by Berggren et al and patented by AstraZeneca(157-159 Fig 13) [201]

In a patent from Merck Toupence et al described over190 substituted furo[23]pyridines claimed to be CB1cannabinoid receptor antagonists [202] The affinity (IC50 lt1microM) was measured using recombinant CHO cells and [3H]-CP-55940 and the activity using cAMP dosage (160 Fig14)

Lange et al described a triazole derivative (155) whichcan be superimposed with SR141716A Compound 155showed a Ki value of 356 nM ([3H]-CP-55940 hCB1-CHOcells) a pA2 value of 83 in the inhibition of WIN-55212-induced release of [3H]-arachidonic acid by hCB1-CHO cellsand was active in vivo in the CP-55940-inducedhypotension in rat (ED50 = 236 mgkg) [182]

Very recently Alanine from Hoffmann-LaRoche foundthat diarylbenzo[13]dioxole derivatives act as CB1

O

O

S

N

OO

F

ClCl

O

O

F O

N

OON

NH2

O

Cl

Cl

Cl

160 161 162

Fig (14) CB1 cannabinoid receptor antagonists 10 Other derivatives One example of a furo[23]pyridine derivative (160) and twoexamples of diarylbenzo[13]dioxole derivatives (161-162) described as hCB1 cannabinoid receptor antagonists by Toupence et aland Alanine et al respectively

1382 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

cannabinoid receptor antagonists (161-162 Fig 14) [203]Approximate IC50 values for twelve compounds ([3H]-CP-55940 hCB1-HEK cells) among the three hundred andeighty compounds described were given (IC50lt2microM)

et al in 1995 [207] In their hands ∆9-THC was not able toinhibit the forskolin-stimulated adenylyl cyclase activity inan assay performed on hCB2-CHO cells Subsequently theyshowed that ∆9-THC was able to antagonise HU210-inducedinhibition of adenylyl cyclase activity thus acting as anantagonist of the hCB2 cannabinoid receptor expressed inCHO cells [208] Furthermore very recently Govaerts et al[209] reported an inverse agonist effect of ∆9-THC and ∆8-THC on hCB2-CHO cells using the [35S]-GTPγ S assayThe pEC50 values were 763 and 888 respectively whilethe Emax values were ndash27 and ndash16 as compared to basal

Griffith from Pfizer described three new series of CB1cannabinoid receptor antagonists based on a bicyclic centralaromatic ring Purine pyrazoltriazines andpyrazolo[15a]pyrimidines derivatives were synthesised andinvestigated for their ability to interact with the CB1cannabinoid receptor Albeit a [35S]-GTPγ S assay wasdescribed no data were given concerning the antagonist orinverse agonist properties of such compounds Two hundredand eighty purine derivatives were synthesised [204] Theaffinities of 163 and 164 were given in the patent (28 and12 nM [3H]-SR141716A) In a following patent thesynthesis of sixty pyrazoltriazine derivatives was described(165 Fig 15) [205] The affinity of each compound was notgiven but was said to be ranging between 01 and 590 nMAnd finally more than forty pyrazolo[15a]pyrimidines weresynthesised (166 Fig 15) [206] Their affinity for the CB1cannabinoid receptor was lower than 155 nM Despite thefact that a great number of derivatives share the samesubstitution pattern around the central bicyclic aromatic ringthe comparison of these three new series of compounds ismade difficult by the absence of precise affinity data

Some other classical cannabinoids were shown to possessan antagonist activity at the CB2 receptor This is the casefor instance for the non-selective O-1184 (2) (Ki = 74 nM)which enhances the forskolin-induced cAMP production inCB2-CHO cells (EMax = 161 EC50 = 63 nM) and alsofor O-584 (3) (EMax = 246 EC50 = 138 nM) [39]

2 Indole Derivatives

Since the disclosure in 1993 of a second cannabinoidreceptor many attempts have been made to synthesise CB2cannabinoid receptor selective ligands The first compoundreported to have a somehow greater affinity for the peripheralcannabinoid receptor was WIN-55212-2 The compoundwas introduced by DrsquoAmbra and colleagues [210] back in1992 as a new cannabinoid ligand having a nanomolaraffinity and acting as an agonist In 1996 Showalter et alused a transfected cell line expressing the CB2 cannabinoidreceptor in order to identify selective ligands for thisreceptor [211] They showed that the CB2 affinity of WIN-55212-2 was 7 times greater than the CB1 with Ki valuesof 189 and 028 nM for the CB1 and CB2 cannabinoidreceptors respectively However WIN-55212-2 is anagonist of the cannabinoid receptors Interestingly WIN-55212-3 (167 Fig 16) the so-called inactive enantiomer ofWIN-55212 was recently shown to behave as a low affinity(Ki=363 microM) inverse agonist of the hCB2 cannabinoidreceptor as it decreased the [35S]-GTPγ S binding [209]

III CB2 LIGANDS RECEPTOR ANTAGONISTS ANDINVERSE AGONISTS

The compounds acting as antagonist or inverse agonistsat the CB2 cannabinoid receptor are reviewed in this thirdpart of the paper As for the CB1 cannabinoid receptorligands they are classified depending on their chemicalstructures

1 Classical Cannabinoid Derivatives

The first report of an antagonism mediated by ∆9-THC atthe peripheral cannabinoid receptor was made by Bayewitch

N N

NN

NH COOH

Cl

Cl

N N

NN

N

Cl

Cl

NH2OH

O

N

N

N

N

N

NHNH2

O

Cl

ClN

N

N

N

Cl

Cl

N

O

163 164

165 166

Fig (15) CB1 cannabinoid receptor antagonists 10 Other derivatives Examples of purine (163-164) pyrazoltriazines (165) andpyrazolo[15-a]pyrimidines (166) derivatives described as CB1 cannabinoid receptor antagonists by Griffith

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1383

The pEC50 value was 554 and the Emax value ndash26 ascompared to basal

AM630 also acts as an inverse agonist at the hCB1cannabinoid receptor (EC50 = 900 nM) as Landsman et aldemonstrated [47] Recently Zhang and colleagues reportedthe characterisation of the microsomal metabolism of AM630[212]

N

O

N

O

O

N

O

Cl

O

N

O

(S)-WIN-55212-3 167 BML-190 168

In 1996 Gallant and colleagues identified by submittinga large number of compounds to a binding assay anotherindole analog as CB2 cannabinoid receptor ligand [213] Onthe basis of this compound christened BML-190 (168 Fig16) or called indomethacin morpholinylamide theysynthesised several derivatives that possessed a selectivityratio for the CB2 cannabinoid receptor of up to 140 Thesewere the first compounds specifically designed to be CB2cannabinoid receptor ligands The first report on thepharmacological properties of BML-190 appeared onlyrecently New and colleagues highlighted in the paper theinverse agonist properties of the compound BML-190 dose-dependently increases (103 EC50= 980plusmn70 nM) theforskolin-stimulated levels of cAMP in hCB2-HEK cellswhile WIN-55212-2 decreases (44 45plusmn42nM) thisaccumulation [137]

Fig (16) CB2 cannabinoid receptor antagonists 2 Indolederivatives Chemical structures of WIN-55212-3 (167) andBML-190 (168) two indoles derivatives acting as inverseagonists at the hCB2 cannabinoid receptor Surprisingly in 2002 Melck and collaborators used

BML-190 as a CB2 receptor agonist in a cell proliferationassay [214] BML-190 was also tested as inhibitor of thecyclooxygenase-2 enzyme by Kalgutkar et al [215] Theyobtained IC50 values higher than 33 and 66 microM for theCOX-I and COX-II enzymes respectively These valueshave to be compared with the submicromolar activity ofindomethacin the parent compound Very recently Klegerisand colleagues [216] looking for an antineurotoxic action ofcannabinoids showed that BML-190 increases TNF-αsecretion by stimulated THP-1 monocytic cells but is noteffective on the IL-1β secretion

Further on the basis of WIN-54461 (12) a CB1antagonist AM630 (13) was synthesised by replacement ofthe bromine atom by an iodine one In the initial reportPertwee et al showed the antagonist effects of AM630 butalso suggested that the CB1 cannabinoid receptor may not bethe preferential receptor of AM630 [45] Later on Ross et alreported using hCB1amp2-CHO cells and [3H]-CP-55940that AM630 binds to the CB2 cannabinoid receptor (Ki =312 nM) with a selectivity ratio over 160 [48]Furthermore using a [35S]-GTPγ S assay they showed itsinverse agonist properties (EC50 = 76 nM) However

Table 12 CB2 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Reported binding affinities (Ki) and selectivity ratio (CB1CB2) of SR144528 (169) for rat and human cannabinoid receptors determined using [3H]-CP55940 as radioligand

NN

O

NH

Cl

SR144528 169

rCB2 (spleen) rCB1 (whole brain) Selectivity Ki hCB2 (transfected CHOcells)

Ki hCB1 (transfected CHOcells)

Selectivity References

038 nM 305 nM 800 06 nM 437 nM 730 [217]

067 nM 548 nM 80 [219]

56 nM gt10microM gt1780 [48]

25 nM a gt 1microM a gt400 [224]

024 nM 276 nM 115 199 nM 503 nM 25 [232]

a COS cells

1384 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

3 Diarylpyrazole Derivatives CHO cells is constitutively active and phosphorylated onSer 352 Upon treatment by SR144528 thisphosphorylation of the receptor was inhibited Moreover theSR compound was able to induce dephosphorylation ofagonist-induced CB2 phosphorylation The question whetherSR144528 induces a CB2 receptor conformational changemaking it a better phosphatase-specific substrate or ifSR144528 binding activates specific phosphatases remainsopen However this remains a nice example of inverseagonist-induced receptor resensitisation

The same Sanofi team which synthesised the CB1antagonist SR141716A reported in 1998 the synthesis andpharmacological characterisation of a CB2 cannabinoidreceptor selective antagonist the SR144528 (169) [217218] The structure of the new compound is derived fromSR141716A one SR144528 is the first selective CB2receptor antagonist reported as the inverse agonist propertiesof BML-190 were shown only recently by New et al [137]The authors reported for 169 using [3H]-CP-55940 asradioligand Ki values of 04 nM and 06 nM for the rat andthe human CB2 receptors respectively and found a CB1-CB2 selectivity ratio around 700 Table 12 sums up thebinding affinities of SR144528 for the cannabinoid receptorsobtained by different authors Interestingly while the CB2affinity is more or less constant the affinity obtained for theCB1 receptor varies by one order of magnitude [219] Theantagonist effect of SR144528 was highlighted by evaluatingits effect on cAMP production by hCB2-CHO cells exposedto 3 nM CP-55940 (EC50=10nM) The antagonism wasalso observed on the activation of the MAP kinase pathwayby CP-55940 (IC50=39nM)

The Sanofi research team working on cannabinoidsconducted investigations in order to determine the keyresidues for SR144528 interaction with the receptor (Table13) as well as the binding mode of the compound Firstlythey identified the TM4-EL2-TM5 region as a regioncontaining crucial residues for the affinity of several ligandsamong them SR144528 [224] Among these residues themutation of two cysteine of the second extracellular loop(C174 and C179) abolishes the affinity for the CB2 receptorLater on the implication of S453(161) and S457(165) inthe binding of SR144528 was evidenced by mutationalstudies In the model proposed for the docking of SR144528into the CB2 receptor the crucial contacts are comprised intothe TM4 this is the case for S453(161) and S457(165)which are proposed to form hydrogen bonds with the ligandand W464(172) proposed to have aromatic interactions withthe 4-methylbenzyl of the SR144528 [225] In 2003 Fengand Song showed that point mutations D349(130)A andA634(241)E abolished ligand binding However mutationof the arginine R350(131) member of the highly conservedDRY motif to alanine has no influence on SR144528binding [226]

Early after the report of the synthesis and characterisationof SR144528 the same authors further explored the functionof this compound and showed its inverse agonist propertiesusing CB2-CHO cells co-transfected with the luciferasereporter gene with either the CRE or the murine krox24regulatory sequence [220] In these models SR144528 notonly inhibits CP-55940 effects but also reproduces theeffects of a pertussin toxin treatment proving its inverseagonist properties SR144528 functionality was alsoexplored by [35S]-GTPγ S binding in hCB2-CHO cells(IC50=3 nM) [221] The inverse agonist function ofSR144528 was further illustrated by Rhee and Kim usingCOS cells co-transfected with hCB2 receptor and adenylylcyclase [222]

As for the CB1 cannabinoid receptor ligands Sanofideveloped tricyclic derivatives based on the 1-benzylpyrazole-3-carboxylic scaffold More than twentycompounds were disclosed in a patent [227] in 2001 (84-86Table 7) The benzyl in position 4 characteristic of the CB2diarylpyrazoles from Sanofi is conserved as well as thebicyclic residue in position 3 The length of the second linkbetween the phenyl and the pyrazole can be made of one or

Bouaboula and co-workers investigated SR144528 effectson CB2 cannabinoid receptor phosphorylation status [223]Firstly they showed that the CB2 receptor transfected in

Table 13 CB2 Cannabinoid Receptor Antagonists 3 Di-Aryl-Pyrazole Derivatives

Some of the single point mutations reported for the hCB2 cannabinoid receptor for which pharmacological data concerning the SR144528 are available

Mutant Effect References

R350(131)A no effect [226]

S453(161)A loss of affinity [225]

V456(164)I no effect [225]

S457(165)A loss of affinity [225]

C174S a loss of affinity [224 225]

C175S a loss of affinity [225]

C177S a no effect [225]

C179S a loss of affinity [224 225]

S544(193)G no effect [225]

a Second extra-cellular loop

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1385

two methylene but according to the patent one methylenebridge is preferred The Ki values of these compounds for theCB2 receptor were said to be lower than 500 nM and theirantagonist properties have been assessed using the adenylatecyclase inhibition assay

compounds by the presence of an alkyl substituent inposition 5 instead of a phenyl and of course by position 3which remains unsubstituted However except a lecture atthe 1998 ACS meeting [230] where compound 171 wasreported to have a Ki value of 835 nM and a selectivity ratioover 100 no further report on these compounds waspublished untill now

In a recent paper Mussinu and colleagues reported thesynthesis of other tricyclic derivatives starting fromSR141716A structure [228] N-piperidin-1-yl-14-di-hydroindeno[12-c]pyrazole-3-carboxamide derivatives areCB2 receptor ligands and some of them possess high affinityand selectivity for the CB2 receptor as shown in Table 7 (79-83) These tricyclic derivatives differ from the Sanofi tricyclicderivatives as they do have a phenyl ring instead of a benzylin position 1 Another difference is the nature of thesubstituent in position 3 which is not a bicycloalkyl but acyclic amine One of the compounds synthesised byMussinu et al 81 possesses the highest affinity for the CB2receptor ever reported with a Ki value of 0037 nM for themurine CB2 receptor ([3H]-CP-55940 mice spleenhomogenate) Interestingly 79 the closest SR141716Aanalog lost almost all its affinity for the CB1 receptor andincreased by four orders of magnitude its affinity for the CB2receptor Even if the structure quite similar to the SRcompounds could suggest that these derivatives behave asantagonists no pharmacological data were given in the paperto support this hypothesis

5 2-Oxoquinoline Derivatives

Inaba and colleagues from Japan Tobacco introduced in2001 a completely new structure in the cannabinoid fieldbased on a 2-oxoquinoline scaffold Over eighty derivativeswere disclosed in a patent [231] and one compound JTE-907 (173) was subsequently characterised in-vitro as well asin-vivo [232] In Table 14 are listed some of the compounds(172-178) disclosed in the patent along with their affinity forthe cannabinoid receptors These values were obtained onhuman receptors against [3H]-WIN-55212-2 For twelvecompounds the anti-inflammatory activity per os wasassessed in a carrageenin-induced paw edema model Thehighest effect reported was obtained with JTE-907 with anED50 value lower than 01 mgkg Therefore thesecompounds were claimed to be useful as anti-inflammatoryand anti-allergic agents

To further characterize JTE-907 the authors determinedits affinity for cannabinoid receptors of different species using[3H]-CP-55940 as radioligand In their hands JTE-907behaved as a very selective hCB2 ligand with Ki values of36 and 2370 nM for the CB2 and CB1 cannabinoid receptorsrespectively even more selective than the SR144528 (Kivalues of 199 and 50 nM respectively) However it has tobe mentioned that the affinities reported in the paper forJTE-907 are quite different from those reported in the patent(hCB1 Ki values of 35nM and 009 nM respectively) Italso appears that the SR144528 affinity for the CB1 receptordetermined by Iwamura et al is higher than the affinityreported by Rinaldi-Carmona et al with values of 50 and437nM respectively The authors ascribed the difference tothe variability of SR144528 binding affinities to the assayconditions The function of JTE-907 was evaluated bymeasuring the increase in cAMP production by forskolin-stimulated hCB2-CHO cells Maximum stimulation wasattained at a concentration of 1microM of JTE-907 In the sameassay WIN-55212-2 decreased the cAMP production in adose-dependent manner

Finally it is interesting to strengthen that the five-membered ring derivatives (79-86) of the diarylpyrazolefamily are reported to possess a high CB2 receptor affinitywhile the six- and seven-membered ring derivatives (69-78)are described as CB1 receptor ligands

4 Other Pyrazole Derivatives

Other CB2 ligands based on a pyrazole nucleus wereintroduced by SmithKline Beecham in a patent describingthe synthesis of about sixty new compounds [229] Theiraffinity (Ki) for the hCB2 receptor transfected in HEK293cells was reported to be ranging between 25nM and 10microMTheir structures (170-171 Fig 17) differ from the Sanofi

NN

O

N

ON

NN

N

NN

O

N

ON

NN

N

ClCl

1

5

4 3

2

170 171

Fig (17) CB2 cannabinoid receptor antagonists 4 Otherpyrazole derivatives Structure of two compounds (170-171)among those claimed by SmithKline Beecham to be hCB2antagonists

In conclusion JTE-907 was shown to be a CB2 selectiveinverse-agonist with anti-inflammatory and anti-allergicproperties This compound is currently undergoing Phase Iclinical trials as anti-allergic drug [233]

IV NON-CB1 NON-CB2 CANNABINOIDRECEPTORS LIGANDS

In the last years the possible presence of additional non-CB1 non-CB2 cannabinoid receptors was raised frompharmacological evidences

One of the putative receptors possesses an endotheliallocalisation while other ones are localized in the CNS or atperipheral nerves For instance an additional target for ∆9-

1386 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 14 CB2 Cannabinoid Receptor Antagonists 5 2-Oxoquinoline Derivatives

Structure of six compounds among the oxoquinoline derivatives patented by Japan Tobaco Affinity (Ki nM) and selectivity (CB1CB2) for the cannabinoidreceptors are given The affinity was measured by binding against [3H]-WIN-55212-2 on either hCB2-CHO or hCB1-CHO cells Adapted from [231]

Cpd ndeg Structure hCB2 hCB1 Selectivity

1-2 172

NH

MeO O

O

OMeO

0014 3671 262215

JTE-907 173

NH

O

MeO O

O

NH

O

O

0087 3436 39495

2-2 174

OH

O

NH

MeO O

O 077 3247 4215

3-27 175

NH

O

F

NH

MeO O

O0021 336 16000

3-32 176

NH

MeO O

O

NH

F

O

0036 2398 66610

3-38 177

O

O

NH

N

O

O

NMeO

0085 935 11000

3-43 178

NMeO O

O

NH

F

O

0043 864 20100

tetrahydrocannabinol was described in capsaicin-sensitivesensory nerves by Zygmunt et al [234]

[29] and by Di Marzo et al [235] Thus in the presentreview we will focus on the description of the so faridentified ligands of the non-CB1 non-CB2 receptors withan emphasis on the compounds having antagonist properties

Pharmacological evidences for the existence of additionalcannabinoid receptors were reviewed by Wiley and Martin

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1387

(Table 15) In particular the knowledge about three not yetcloned targets has grown The growing molecularpharmacology of the three putative new cannabinoidreceptors is reviewed in the three following sections Thefourth section summarises the data on the other proposedcannabinoid targets

vasodilatator activity In addition the anandamide effect ispartially sensitive to SR141716A inhibition The authorsproposed the existence of two distinct receptors responsiblefor the vasodilatatory effect of anandamide one located in theendothelium is SR141716A-sensitive while the other onelocated on the vascular smooth muscle is SR141716A-insensitive Thus SR141716A is an antagonist of anendothelial anandamide receptor Several studies wereconducted in order to understand the molecularpharmacology of the endothelial anandamide receptor It ispertussis toxin-sensitive [237] Cannabidiol acts asantagonist and abnormal-cannabidiol (Abn-cbd 179) and itsanalogue O-1602 (180) act as agonists [238] The synthesisof O-1602 and of its analogues are disclosed in a patentdescribing the vasodilatatory activity of cannabinoid

1 Endothelial Anandamide Receptor ndash Abnormal-Cannabidiol Receptor

In 1999 Wagner et al showed that anandamide couldinduce mesenteric vasodilatation through an endotheliallylocated ldquoanandamide receptorrdquo pharmacologically distinctfrom CB1 [236] Other cannabinoids such as ∆9-THC HU-210 or WIN-55212-2 were devoid of mesenteric

Table 15 Non-CB1 Non-CB2 Cannabinoid Receptors Antagonists

An overview of the putative new non-CB1 non-CB2 anandamide-receptors ligands The antagonists are highlighted in bold

NH

N

S

HO OH

Cl

OH

OH

OH OH OH OH

OMe

OMe

RR

RR

RR

RR

HO

OH

NH

O

cannabidiol (10) Abnormal-cannabidiol 179 O-1602 180

O-1918 181 capsazepine 183capsaicin 182

Compound Endothelial AEA receptor Brain AEA receptor Cannabinoid-vanilloid brain receptor

AEA agonist a agonist de

WIN-55212-2 no effecta

agonist e agonist g h

CP-55940 no effect e agonist i

SR141716A antagonist a b j no effect ([35 S]-GTPγS binding) d e f antagonist g h

AM251 no effect k no effect h i

Cannabidiol antagonist b no effect e

O-1918 antagonist c jm

Abd-cbd agonist b l

O-1602 agonist b

Capsaicin no effectj agonist i

Capsazepine no effect b antagonist h

a [236] b [238] c [240] d [249] e [250] f [251] g [254] h [258] i [ 255] k [244] j [ 243] l [ 242] m [241]

1388 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

analogues [239] More recently Offertaler et al showed thatO-1918 (181) a cannabidiol analog acts as an antagonist ofthe endothelial anandamide receptor [240] In addition O-1918 does not bind to the CB1 and CB2 cannabinoidreceptors Furthermore O-1918 unlike cannabidiol doesnot cause vasorelaxation in the mesenteric artery preparationmodel used and thus acts as a true neutral antagonistUsing O-1918 as a pharmacological tool Begg et alproposed a coupling of this new G protein-coupledendothelial receptor through a GiGo mechanism to ionchannels in primary cultured human vascular endothelialcells [241] However in their hands SR141716A wasineffective in antagonising Abd-cbd Ho and Hiley furtherdemonstrated that Abn-cbd relaxes rat small mesentericarteries through an activation of K+ channels via aSR141716A-sensitive pathway [242] Further using thesame model they characterized the vasorelaxant activity ofvirodhamine a novel endocannabinoid apparently actingthrough the same receptor [243] The virodhamine-inducedvasorelaxation is sensitive to SR141716A and O-1918 butinsensitive to AM251 SR144528 and AM630 Moreovervanilloid receptor desensitisation by capsaicin (182) had noeffect

244] Thus position 4 on the 5-phenyl could be furtherexplored to obtain selective ligands

To come to an end despite the fact that the endothelialanandamide receptor is neither identified nor cloned at leastthree antagonists are identified SR141716A cannabidioland O-1918 It is likely that a growing number of ligandsand therapeutic applications will appear in the very nearfuture

2 Brain Anandamide Receptor

Following an in-depth evaluation of anandamide effects inCB1

-- mice Di Marzo and co-workers suggested that a non-CB1 non-CB2 G protein-coupled receptor might mediatesome of the actions of AEA in mice [249] Actually effectsof AEA but not those of ∆9-THC were not decreased inCB1 cannabinoid receptor knock-out mice For instance 30mgkg of AEA were still effective in hot-plate test whereas10 mgkg of ∆9-THC had no effect on anti-nociceptionMoreover in a [35S]-GTPγ S assay AEA stimulated with anEC50 value of 223 microM the [35S]-GTPγ S binding to CB1

--

mice brain membranes (Emax= 28) ∆9-THC showed noeffect in this assay The addition of SR141716A did notaffect the [35S]-GTPγ S binding to CB1

-- mice brainmembranes Shortly after other evidences for the existence ofa new G protein-coupled cannabinoid receptor with a distinctdistribution in the central nervous system were published[250] From a set of 24 commonly used cannabinoids (AEAWIN-55212-2 JWH-030 THChellip) only AEA and WIN-55212-2 produced an increased binding of [35S]-GTPγ S onCB1

-- mice brain membranes Interestingly in this studySR141716A alone produced a significant inhibition of [35S]-GTPγ S binding similar to the inhibition obtained usingCB1

++ mice brain membranes (IC50 values of 57 microM and47 microM respectively) However SR141716A did not affectthe anandamide-induced [35S]-GTPγ S binding in CB1

--

mice brain membranes Moreover [3H]-SR141716Aexhibited significant binding in some brain regions of CB1

--

mice which are different from those exhibiting stimulation of[35S]-GTPγ S binding by WIN-55212-2 and AEA Thusthe authors proposed that the SR141716A effects on CB1

--

mice are mediated neither by the CB1 cannabinoid receptornor by the putative new anandamide brain receptor Monoryand co-workers using CB1

-- mice described a novel nonadenylyl cyclase-coupled cannabinoid binding site in micecerebellum [251] Two agonists AEA and WIN-55212-2but not ∆9-THC or HU210 were found to bind to thisreceptor as they enhanced [35S]-GTPγ S binding NeitherSR141716A nor SR144528 were able to affect the WIN-55212-2 induced [35S]-GTPγ S binding in CB1

-- mice asfound by Breivogel et al [250] However the distributionpattern of the receptor described by Monory et al differs fromthe distribution found by Breivogel et al It has to bementioned that the knock-out strains used were different Themice used by Monory were from a CD-1 strain while theone used by Breivogel were from a C57BL61 strainInterestingly Muthane et al detected differences in nigralneurons number and sensitivity to MPTP in the two mousestrains used [252]

More recently Mo et al obtained results showing thatthe Abnormal-cannabidiol receptor is also responsible for anincreased endothelial cell migration [244] Abn-cbd enhancedhuman umbilical vein endothelial cell migration while O-1918 antagonised Abn-cbd effect The CB1 cannabinoidreceptor inverse agonist SR141716A partially inhibitedAbd-cbd action whereas AM251 and SR144528 had noeffect

In another paper from George Kunos team Batkai et alreported that SR141716A is able to inhibit the endotoxichypotension by a cardiac mechanism involving neither theCB1 nor the CB2 cannabinoid receptors [245] Theinvolvement of a non-CB1 non-CB2 site of action wasshown using CB1

-- and CB1--CB2

-- knock-out miceFurther studies are needed to identify the SR141716Asensitive myocardial site of action

In a recent patent Kunos described O-1918 and itsanalogues as vasoconstrictor agents useful to reversepathological vasodilatation of blood vessels [246] Theeffects of O-1918 on mouse blood pressure on LPS-inducedhypotension in mouse as well as the reversal of hypotensionby O-1918 injection are described Furthermore thesynthesis of O-1918 based antagonists is described startingfrom p-metha-28-dien-1-ol and the appropriate resorcinols

Interestingly the Abd-cbd receptor which was firstdetected in the vascular system also seems to be present inthe CNS [247] Indeed Walter et al found that microglialcells (BV-2) migration induced by 1 microM 2-AG is inhibitedby cannabidiol (300 nM) and O-1918 (1 microM) SR141716Awas found ineffective in this assay Thus 2-AG is an agonistof the Abd-cbd receptor Moreover Abd-cbd is able to elicita dose-dependent cell migration in the same model (EC50 =600nM) [248]

From a more structural point of view SR141716Abehaves as an antagonist while on the opposite AM251(20) a close analogue which differs from the former only bythe nature of one halogen has no effect on this receptor [242

To conclude to our knowledge no compounds havingantagonist or inverse agonist properties at this receptor havebeen identified so far

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1389

3 Cannabinoid-Vanilloid Brain Receptor a cannabinoid-vanilloid receptor at which SR141716A andcapsazepine behave as antagonists

Evidences for the presence of an additional ldquocannabinoidrdquoreceptor differing from the CB1 receptor and from the brainanandamide receptor appeared from studies focused on thehippocampus (for a review see Hajos et al [253]) Hajos etal studied the cannabinoid actions on GABAergic andglutamatergic transmission using the whole-cell patch-clamp technique [254] WIN-55212-2 induced a dose-dependent reduction in glutamatergic transmission reversedby SR141716A (1microM) Thus the authors concluded that thecannabinoid actions on the glutamatergic transmission aremediated by a non-CB1 but SR141716A-sensitive receptorIn another paper Hajos and Freund further characterised thisputative receptor [255] They showed that in addition toWIN-55212-2 CP-55940 and capsaicin (182) a TRPV1receptor agonist also act as agonists Furthermore in theirhands SR141716A as well as capsazepine (183) a TRPV1receptor antagonist were able to antagonise this effect on theglutamatergic transmission while AM251 was notSR141716A effects on CB1 knock-out mice were furtherstudied using a test of anxiety [256] Haller et al showedthat on the one hand the distribution of the CB1 receptorinduces anxiety and SR141716A has an anxiolytic effectand on the other hand SR141716A is still active in CB1

--

mice In a following paper they reported that AM-251induces anxiogenic effects in wild-type and in knock-outmice [257] Thus SR141716A in addition to CB1 bindsto a not yet identified receptor Another evidence for thisadditional receptor for SR141716A was given by Bass andco-workers [61] They conducted a structure-activityrelationship study on the stimulation of locomotor activityinduced by SR141716A and twenty analogues Nocorrelation was found between the affinity for the CB1cannabinoid receptor and the stimulation of locomotoractivity Albeit SR141716A and five of its analogues O-1803 O-1710 O-1253 O-1254 (40) O-1255 (41) behavedas inverse agonists in [35S]-GTPγ S assay none of theseanalogues stimulated the locomotor activity In conclusionthe SR141716A-induced stimulation of locomotor activity isneither due to inverse agonism nor to inhibition of anendogenous tone but to a brain receptor distinct from theCB1 receptor Di Marzo et al previously provided evidencesthat the cannabinoid-vanilloid brain receptor could mediatesome of the cannabinoid effects on locomotion [249]

4 Other Non-CB1 Non-CB2 Cannabinoid Receptors

Evidences for the existence of additional non-CB1 non-CB2 cannabinoid receptors localised in the rat isolatedmesenteric arterial bed appeared from the works of Ralevicand collaborators Several cannabinoids were evaluated intheir model of sensory neurogenic vasorelaxation evoked byelectrical field stimulation

HU210 was found to attenuate vasorelaxation in a CB1and CB2 independent way as SR141716A LY320135 andSR144528 were ineffective in inhibiting HU210 effect [259]Similar results were obtained with ∆9-THC [260]Similarly noladin ether inhibited sensory neurogenicrelaxation in a concentration-dependent manner which wasalso unaffected by SR141716A LY320135 and SR144528Moreover this effect as the ∆9-THC one was pertussistoxin sensitive suggesting the involvement of a Gio protein-coupled receptor [261]

However the inhibitory effects of CP-55940 and WIN-55212-2 were affected by the presence of SR141716A (1microM)or LY320135 (1microM) but not by the addition of SR144528(1microM) [262]

It appears that at least two receptors should be involvedin cannabinoid-mediated attenuation of sensory nerve-mediated vasorelaxation The first one is SR141716A andLY320135 sensitive while the other one is resistant toSR141716A

Some evidences for the existence of a ldquoCB2-likerdquo receptorappeared from the studies on palmitoylethanolamide (PEA)an endogenous fatty acid amide devoid of CB1 and CB2affinity [264] antinociceptive potential Calignano and co-workers showed that PEA was able to alleviate pain inseveral animal models [265 266] For instance PEA dose-dependently inhibits kaolin-evoked writhing in mice TheCB2 cannabinoid receptor inverse agonist SR144528 (02mgkg) inhibits PEA anti-nociceptive effect [266] Thus theauthors suggest that PEA acts as agonist on a SR144528-sensitive non-CB2 cannabinoid receptor (ldquoCB2-likerdquoreceptor) at which SR144528 acts as an antagonist

Further studies are needed in order to better characterisethese additional ldquocannabinoidrdquo receptors However it islikely that several therapeutic applications will soon springfor these receptors

Very recently Pistis et al published a study on theneurophysiological effects of cannabinoids on the basolateralamygdala neurons in-vivo [258] One of their findings is thatHU-210 and WIN-55212-2 did not elicit similar effects onbasal amygdala projection neurons firing rate While theformer had no effect the latter decreased the firing rateMoreover SR141716A reversed the WIN-55212-2 effectswhile AM251 had no effect The vanilloid receptorantagonist capsazepine also antagonised the WIN-55212-2effect on firing rate of these neurons Therefore the authorssuggested the presence of a novel cannabinoid-vanilloidreceptor

V CONCLUSION

Since their development cannabinoid antagonists provedto be essential tools in the understanding of cannabinoidpharmacology and biochemistry Moreover the interestshown by the pharmaceutical industry in the development ofnew cannabinoid antagonists prove if necessary the realtherapeutic potential of this class of compounds Thetreatment of eating and movement disorders memorydeficits psychosis and dependencies from various addictivedrugs are some of the most cited applications for the CB1cannabinoid receptor antagonists

From these lines it appears that in addition to theknown CB1 cannabinoid receptor it is now likely that twoother cannabinoid receptors do exist in the brain The firstone should be an anandamide brain receptor for whichSR141716A has no affinity while the second one should be

1390 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Much less wide is the knowledge on the CB2cannabinoid receptor antagonists However potentialapplications could be disorders involving the immunesystem such as inflammation or allergies

[19] Devane WA Hanus L Breuer A Pertwee RG StevensonLA Griffin G Gibson D Mandelbaum A Etinger AMechoulam R Science 1992 258 1946

[20] Mechoulam R Ben-Shabat S Hanus L Ligumsky MKaminski NE Schatz AR Gopher A Almong S MartinBR Compton DR Biochem Pharmacol 1995 50 83

Finally the growing literature on the non CB1 non CB2cannabinoid receptors should allow the development of newdrugs First examples are given by the cannabidiol andabnormal-cannabidiol derivatives described by Kunos et alfor which potential therapeutic applications on the vascularsystem are already patented

[21] Sugiura T Kondo S Sukagawa A Nakane S Shinoda AItoh K Yamashita A Waku K Biochem Biophys Res Co1995 215 89

[22] Hanus L Abu-Lafi S Fride E Breuer A Vogel Z ShalevDE Kustanovich I Mechoulam R Proc Natl Acad SciUSA 2001 98 3662

[23] Oka S Tsuchie A Tokumura A Muramatsu M Suhara YTakayama H Waku K Sugiura T J Neurochem 2003 851374It clearly appears from this paper that the antagonists of

the cannabinoid receptors are still an active research fieldfrom which should emerge new promising therapeutic toolsas well as innovative drugs in the near future

[24] Goutopoulos A and Makriyannis A Pharmacol Therapeut2002 95 103

[25] Beal JE Olson R Lefkowitz L Laubenstein L Bellman PYangco B Morales JO Murphy R Powderly W PlasseTF Mosdell KW Shepard KV J Pain Symptom Manage1997 14 7ACKNOWLEDGEMENTS [26] Notcutt W Price M Miller R Newport S Phillips CSimmons S Sansom C Anaesthesia 2004 59 440

GGM is very indebted to the ldquoFonds pour la formationagrave la Recherche dans lrsquoIndustrie et lrsquoAgriculture (FRIA)rdquo Belgium for the award of a research fellowship Theexperimental works of the authors were supported by grantsfrom the FNRS The Belgian Charcot Foundation and theUniversiteacute catholique de Louvain

[27] Ledent C Valverde O Cossu G Petitet F Aubert JFBeslot F Bohme GA Imperato A Pedrazzini T RoquesBP Vassart G Fratta W Parmentier M Science 1999 283401

[28] Zimmer A Zimmer AM Hohmann AG Herkenham MBonner TI Proc Natl Acad Sci U S A 1999 96 5780

[29] Wiley JL and Martin BR Chem Phys Lipids 2002 121 57[30] Adam J and Cowley P Expert Opin Ther Patents 2002 12

1475

REFERENCES [31] Razdan RK and Mahadevan A Chem Phys Lipids 2002 12121

[32] Barth F and Rinaldi-Carmona M Curr Med Chem 1999 6745

[1] Hillard CJ Harris RE Bloom AS J Pharmacol Exp Ther1985 232 579

[33] Little PJ Compton DR Johnson MR Melvin LS MartinBR J Pharmacol Exp Ther 1988 247 1046

[2] Devane WA Dysarz FA Johnson MR Melvin LSHowlett AC Mol Pharmacol 1988 34 605

[34] Pertwee RG Stevenson LA Elrick DB Mechoulam RCorbett AD Brit J Pharmacol 1992 105 980

[3] Matsuda LA Lolait SJ Brownstein MJ Young ACBonner TI Nature 1990 346 561

[35] Harrison C Traynor JR Life Sci 2003 74 489-508[4] Gerard CM Molerau C Vassart G Parmentier M BiochemJ 1991 279 129 [36] Pertwee RG Fernando SR Griffin G Ryan W Razdan

RJ Compton DR Martin BR Eur J Pharmacol 1996 315195

[5] Glass M Dragunow M Faull RLM Neuroscience 1997 2299

[37] Ross RA Brockie HC Fernando SR Saha B Razdan RKPertwee RG Brit J Pharmacol 1998 125 1345

[6] Shire D Carillon C Kaghad M Calandra B Rinaldi-Carmona M LeFur G Caput D Ferrara P J Biol Chem1995 270 3726 [38] Griffin G Wray EJ Rorrer WK Crocker PJ Ryan WJ

Saha B Razdan RK Martin BR Abood ME Brit JPharmacol 1999 126 1575

[7] Ryberg E Khang Vu H Groblewski T Larsson N ElebringT Hjorth S Sjogren S Greasley PJ 2004 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 119

[39] Ross RA Gibson TM Stevenson LA Saha B Crocker PRazdan RK Pertwee RG Brit J Pharmacol 1999 128 735

[40] Martin BR Winckler JR Wiley JL Huffman JW CrockerPJ Saha B Razdan RK J Pharmacol Exp Ther 1999 2901065

[8] Rinaldi-Carmona M Calandra B Shire D Bouaboula MOustric D Barth F Casellas P Ferrara P LeFur G JPharmacol Exp Ther 1998 278 871

[41] Martin BR Stevenson LA Pertwee RG Breivogel CSWilliams A Mahadevan A Razdan RK 2002 Symposium onthe Cannabinoids Burlington Vermont International CannabinoidResearch Society page 2

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with an ED50 value of 24 mgkg which is close to the 3mgkg obtained for SR141716A The compound was alsoactive in another in-vivo model the WIN-55212-2-inducedhypothermia in mice Compound 104 which has nosubstituent in position 5 of the imidazole ring was lessactive in the hypotension model and inactive in thehypothermia model Thus a methyl group is the preferredsubstituent in position 5 of the imidazole moiety Otherproperties either experimental (P-glycoprotein affinitylogPHPLC) or computational (molecular volume polarsurface area) obtained for compounds 105 were similar tothose obtained for SR141716A Finally molecularmodelling studies revealed a close structural overlap betweenthe two compounds

value of 197 nM is selective for hCB1 and is as potent asSR141716A The structural modulations of 118 led to someinteresting compounds (Table 10) Six compounds possess aKi value for the hCB1 cannabinoid receptor equal to or lowerthan 25 nM antagonist properties in the inhibition of [3H]-arachidonic acid release by WIN-55212-2-stimulated hCB1-CHO cells and Ki values for the hCB2 receptor over 1microMThe diaryl-pyrazoline derivatives contained a chiral center atposition 4 The authors resolved the racemic mixture of twocompounds (121 and 130) The levorotatory enantiomersappeared to be the eutomers For compound SLV319levorotatory enantiomer of 121 the Ki and pA2 values were78 nM and 99 respectively The Ki and pA2 values ofSLV326 levorotatory enantiomer of 130 were 359 nM and9 respectively

8 34-Diaryl-Pyrazoline Derivatives Two in-vivo models CP-55940-induced hypotension inrat and WIN-55212-2-induced hypothermia in mouse wereused to assess the potential of these compounds The resultsare of the same order of magnitude than those obtained forSR141716A For instance in the hypotension modelSLV319 ((-)121) and SLV326 ((-)130) showed ED50 values

Lange and collaborators also discovered 34-diaryl-pyrazoline derivatives after a screening of compoundsresembling to SR141716A [183184] Compound 118 thatwas initially identified during the screening possesses a Ki

Table 10 CB1 Cannabinoid Receptor Antagonists 8 34-Diaryl-Pyrazolines Derivatives

Structure affinity for the hCB1 and hCB2 cannabinoid receptors (Ki nM) expressed in CHO transfected cells ([3H]-CP-55940) and potency (pA2) ofsome 34-dirylpyrazolines derivatives Table adapted from [184]

NN

N N

SO2

R3

R2

Cl R1

R4

n˚ R1 R2 R3 R4 Ki hCB1 (nM) Ki hCB2 (nM) pA2 (CB1)

118 H H H 4-CH3 197 gt 1000 84

119 H H H 246- CH3 24 gt 1000 94

120 H H H 4-Cl 16 gt 1000 95

121 H CH3 H 4-Cl 25 gt 1000 87

122 H CH3 CH3 4-Cl 280 gt 1000 85

123 H H H 4-F 53 gt 1000 9

124 H CH3 H 4-F 338 gt 1000 85

125 H CH3 CH3 4-F gt 1000 gt 1000 lt 75

126 H CH3 H 3-Cl 14 gt 1000 86

127 4-Cl CH3 H 4-Cl 255 ND ND

128 4-F CH3 H 4-Cl 584 ND ND

129 H CH3 H H 170 ND 75

130 H CH3 H 4- CF3 221 gt 1000 93

SR141716A 25 1580 86

1378 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

N

HN

O

N

Cl Cl

Cl

N

N

HN

O

Br

Br131 132

Fig (8) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Chemical structures of two 56-diaryl-pyrazine-2-amide derivatives (131-132) developed as CB1 cannabinoid receptorantagonists by AstraZeneca

of 55 and 2 mgkg (po) respectively proving their in-vivoefficacy after oral administration The authors alsodemonstrated that SLV319 is devoid of affinity for the P-glycoprotein pump and that it possessed a goodCNSplasma ratio (17) These two compounds (SLV319and SLV326) were chosen as development candidates bySolvay and entered clinical Phase I trials in 2003 [185]

132 Fig 8) useful as CB1 cannabinoid receptor antagonists[186 187] The activity of twenty derivatives wasdetermined using a [35S]-GTPγ S assay and hCB1-CHOtransfected cells The concentration required to give halfmaximal inhibition of CP-55940-induced [35S]-GTPγ Sbinding (IC50) is lower than 200 nM for the preferredcompounds No affinity data were given for thesecompounds

9 Diaryl-Pyrazine Diphenyl-Pyridine Diphenyl-Phenyland Diaryl-Pyrimidines Derivatives

The same year in a patent from Merck Finke andcollaborators described several 56-di-phenyl-pyridinederivatives as hCB1 antagonists or inverse agonists [188]The compounds have a substituent in position 3 of thepyridine core (Fig 9) This substituent could be a cyano(133-134) or a nitro group a halogen an ester or an amide(135-136) Over 150 compounds were synthesised toillustrate the invention but no pharmacological data wasdisclosed in the patent

In 2003 appeared several patents describing new familiesof compounds that bind to the CB1 cannabinoid receptorThese compounds have in common a central six atomsaromatic ring which could be a pyrazine a pyridine aphenyl or a pyrimidine and that is substituted by at leasttwo phenyl rings

The first patents were from Berggren and collaboratorsfrom AstraZeneca who published two patents describing thesynthesis of 56-diaryl-pyrazine-2-amide derivatives (131-

A few months later another patent was taken by Sanoficlaiming the antagonist properties of 56-di-phenyl-2-pyridine carboxamide derivatives [189] These compounds

N

NH

O

Cl

Cl

N

O

Cl

O NH

N

O

Cl

ClCl

NH

CN O

N

Cl

Cl

Cl

OHN

N

O

Cl

ClCl

CNF

F

133 134

135 136 137

Fig (9) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives 56-Di-phenyl-pyridine derivatives patented by Merck (133-136) and by Sanofi (137) as CB1 cannabinoid receptorsantagonists

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1379

NH

O

N

Cl

ClCl

NH

O

N

Cl

F3CCl

N

N

O

Cl

Cl Cl F138 139 140

Fig (10) CB1 cannabinoid receptor antagonists 9 Diaryl-pyrazine diphenyl-pyridine diphenyl-phenyl and diaryl-pyrimidinesderivatives Representative structures of the diphenyl-phenyl (138-139) and 45-diphenyl-pyrimidine (140) derivatives developed ascannabinoid antagonists at Sanofi and Merck respectively

28 examples are given structurally related to the diphenyl-phenyl derivatives have their pyridine core substituted bytwo phenyl rings and by an amide moiety (137 Fig 9) TheIC50 values were lower than 100 nM but no data were givenshowing the antagonist properties although the compoundswere assayed in the adenylate cyclase inhibition assay

10 Other Derivatives

Several amide derivatives structures recently appearedamong the CB1 cannabinoid receptor antagonists In a seriesof patents taken by Merck in 2003 Hagmann and colleaguesdescribed a large amount of compounds obtained by parallelsynthesis which are claimed to be CB1 cannabinoid receptorantagonists [192-195] They were synthesised by reacting alibrary of substituted amines with a library of carboxylicacids (141-144 Fig 11) However as no value was givenfor the affinity or activity of these amides we will notfurther discuss this class of compounds

Along this line Sanofi Synthelabo developed newdiphenyl-phenyl derivatives claimed to have CB1cannabinoid receptor antagonist properties [190] They arebased on a central phenyl ring substituted by two phenylsand by an amide moiety (138-139 Fig 10) Thirteencompounds were claimed along with their synthesis TheirIC50 values were lower than 100 nM ([3H]-CP55-940hCB1-CHO cells) but no other specific data (selectivitypA2hellip) was given

Several 15-diaryl-pyrrole-3-carboxamide derivatives weresynthesised and claimed to be CB1 cannabinoid receptorantagonists by Berggren et al [196] The affinity valueswere not disclosed in the patent (145-146 Fig 12) Otherpyrrole derivatives were synthesised by Guba et al atHofmann-La Roche [197] More specifically 2-(thiazol-4-yl)pyrrole derivatives were claimed as CB1 cannabinoidreceptor antagonist (IC50lt2microM) (147-148 Fig 12)

Finally Kopka et al from Merck described more thanone hundred 45-diaryl-pyrimidine derivatives as CB1cannabinoid receptor antagonists (140 Fig10) [191]However no precise data were provided concerning theaffinity and functionality of the compounds

R2

R1N

R3

R4

R5

O

NH

O

Cl

O F

F

R2

R1NH

R3

R4

HO R5

O

NH

O

Cl

CN

NH

O

Cl

O

O

NH

O

Cl

Cl

+

141142

143 144

Fig (11) CB1 cannabinoid receptor antagonists 10 Other derivatives Retrosynthetic scheme of a CB1 cannabinoid antagonistlibrary of amide derivatives developed at Merck The structures of four representative compounds are illustrated (141-144)

1380 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

N

NH

O N

Cl

Cl

Cl

N

NH

O

OMe

MeO

N

NH

O

N

S

MeO

N

NH

O

N

S

MeO

145 146

147 148

Fig (12) CB1 cannabinoid receptor antagonists 10 Other derivatives Two examples of 15-diaryl-pyrrole derivatives described byBerggren et al (145-146) While the 2-(thiazol-4yl)pyrroles derivatives 147 and 148 were described by Guba et al from Hofmann-LaRoche All these pyrroles derivatives were claimed to be CB1 cannabinoid receptor antagonists

Another new structure is represented by the 124-triazolederivatives developed by Jagerovic and collaborators In arecent paper were described the synthesis andpharmacological properties of five new 15-diphenyl-3-alkyl-

triazole derivatives [198] Among these compounds onlyone (149) behaved as a CB1 antagonist inhibiting the WIN-55212-2-induced contractions in a mouse vas deferenspreparation However despite its effects in isolated tissue

Table 11 CB1 Cannabinoid Receptor Antagonists 10 Other Derivatives

Structure and affinity of diaryl-triazole derivatives

N

NN

R3

R2

R1

Cpd R1 R2 R3 Ki CB1 (nM) References

149 24-diCl 4-Cl hexyl 855 a [198]

150 24-diCl 4-Cl2-OMe CO-NH-(3-azabicyclo[330]octan-3-yl) 270 b [152]

151 24-diCl 4-Cl CO-NH-(3-azabicyclo[330]octan-3-yl) 164 b [152]

152 4-Cl 24-diCl CO-NH-(3-azabicyclo[330]octan-3-yl) 137 b [152]

153 4-Cl 24-diCl CO-NH-benzylpyrrolidin-3-yl 29 b [152]

154 4-Cl 24-diCl CO-NH-1-(4-chlorophenyl)ethyl 66 b [152]

155 4-Cl 24-diCl CO-NH-piperidinyl 356 c [182]

156 24-diCl 4-Cl CO-NH-piperidinyl 382 c [182]

a [3H]-SR141716A rat cerebellar membranesb [3H]-CP-55940 hCB1-HEK EDNA cellsc [3H]-CP-55940 hCB1-CHO cells

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1381

N

S

O

NH

Cl

Cl

Cl

F

N

S

O

NHN

ClCl

Cl

N

S

O

NH

Br

N

S

O

NHN

Cl

Cl

Cl

156 157 158 159

Fig (13) CB1 cannabinoid receptor antagonists 10 Other derivatives Structures of 45-diaryl-thiazole derivatives patented as hCB1cannabinoid receptor antagonists by Solvay Pharmaceuticals (156 157 158) or by AstraZeneca (157 158 159)

assays (mouse vas deferens and guinea pig ileum) thecompound possessed only a reduced rCB1 affinity with Kivalues of 855 and 748 nM using [3H]-SR141716A and [3H]-WIN-55-212-2 respectively (Table 11) Further four seriesof compounds differing by the nature of the substituentaround the triazole core were also described in a patent[199] Usually the compounds claimed possess two aromaticrings possibly substituted and one linear alkyl chain Theaffinity for the cannabinoid receptors was not givenNevertheless 149 was evaluated in two isolated tissuemodels the inhibition of the electrically evoked contractionsin the guinea pig ileum and in the mouse vas deferens Inthe two models compound 149 inhibited the effect of WIN-55212-2 but had no effect by itself In the vas deferensmodel the authors obtained a pA2 value of 748 to becompared with 763 obtained for AM251

Yet another five membered ring a thiazole has beenused as central moiety of a new class of cannabinoid ligandsThe thiazole ring is substituted by two aryls in position 4and 5 and by an amide (position 2) as in compounds 156-159 (Fig 13) Forty derivatives of this type weresynthesised by Lange and colleagues from SolvayPharmaceuticals and claimed in a patent as agonists orantagonists of the CB1 cannabinoid receptor [200] Nopharmacological data were provided however their activitywas assessed by measuring the cAMP in transfected hCB1-CHO cells In a recent paper from Lange et al two of thesethiazole derivatives were further described [182] Compound13 (157) the most closely related to SR141716A has a Kivalue of 227 nM while for compound 14 (158) Ki value isover 1000 nM Thus as in the imidazole series (see Table9) the aromatic substitution pattern is of great importancefor the CB1 cannabinoid receptor affinity Compound 13however was devoid of in vivo activity

Others 124-triazole derivatives were described by Dycket al compounds 150-154 unlike the Jagerovic ones havean amide moiety in position 3 [152] The highest affinitywas obtained with a 1-benzyl-pyrrolidin-3-yl substituentThe Ki value was 29 nM ([3H]-CP-55940 hCB1-HEK-EDNA cells) to be compared to 12 nM obtained for theSR141716A

Seventeen 45-diaryl-thiazole derivatives were alsosynthesised by Berggren et al and patented by AstraZeneca(157-159 Fig 13) [201]

In a patent from Merck Toupence et al described over190 substituted furo[23]pyridines claimed to be CB1cannabinoid receptor antagonists [202] The affinity (IC50 lt1microM) was measured using recombinant CHO cells and [3H]-CP-55940 and the activity using cAMP dosage (160 Fig14)

Lange et al described a triazole derivative (155) whichcan be superimposed with SR141716A Compound 155showed a Ki value of 356 nM ([3H]-CP-55940 hCB1-CHOcells) a pA2 value of 83 in the inhibition of WIN-55212-induced release of [3H]-arachidonic acid by hCB1-CHO cellsand was active in vivo in the CP-55940-inducedhypotension in rat (ED50 = 236 mgkg) [182]

Very recently Alanine from Hoffmann-LaRoche foundthat diarylbenzo[13]dioxole derivatives act as CB1

O

O

S

N

OO

F

ClCl

O

O

F O

N

OON

NH2

O

Cl

Cl

Cl

160 161 162

Fig (14) CB1 cannabinoid receptor antagonists 10 Other derivatives One example of a furo[23]pyridine derivative (160) and twoexamples of diarylbenzo[13]dioxole derivatives (161-162) described as hCB1 cannabinoid receptor antagonists by Toupence et aland Alanine et al respectively

1382 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

cannabinoid receptor antagonists (161-162 Fig 14) [203]Approximate IC50 values for twelve compounds ([3H]-CP-55940 hCB1-HEK cells) among the three hundred andeighty compounds described were given (IC50lt2microM)

et al in 1995 [207] In their hands ∆9-THC was not able toinhibit the forskolin-stimulated adenylyl cyclase activity inan assay performed on hCB2-CHO cells Subsequently theyshowed that ∆9-THC was able to antagonise HU210-inducedinhibition of adenylyl cyclase activity thus acting as anantagonist of the hCB2 cannabinoid receptor expressed inCHO cells [208] Furthermore very recently Govaerts et al[209] reported an inverse agonist effect of ∆9-THC and ∆8-THC on hCB2-CHO cells using the [35S]-GTPγ S assayThe pEC50 values were 763 and 888 respectively whilethe Emax values were ndash27 and ndash16 as compared to basal

Griffith from Pfizer described three new series of CB1cannabinoid receptor antagonists based on a bicyclic centralaromatic ring Purine pyrazoltriazines andpyrazolo[15a]pyrimidines derivatives were synthesised andinvestigated for their ability to interact with the CB1cannabinoid receptor Albeit a [35S]-GTPγ S assay wasdescribed no data were given concerning the antagonist orinverse agonist properties of such compounds Two hundredand eighty purine derivatives were synthesised [204] Theaffinities of 163 and 164 were given in the patent (28 and12 nM [3H]-SR141716A) In a following patent thesynthesis of sixty pyrazoltriazine derivatives was described(165 Fig 15) [205] The affinity of each compound was notgiven but was said to be ranging between 01 and 590 nMAnd finally more than forty pyrazolo[15a]pyrimidines weresynthesised (166 Fig 15) [206] Their affinity for the CB1cannabinoid receptor was lower than 155 nM Despite thefact that a great number of derivatives share the samesubstitution pattern around the central bicyclic aromatic ringthe comparison of these three new series of compounds ismade difficult by the absence of precise affinity data

Some other classical cannabinoids were shown to possessan antagonist activity at the CB2 receptor This is the casefor instance for the non-selective O-1184 (2) (Ki = 74 nM)which enhances the forskolin-induced cAMP production inCB2-CHO cells (EMax = 161 EC50 = 63 nM) and alsofor O-584 (3) (EMax = 246 EC50 = 138 nM) [39]

2 Indole Derivatives

Since the disclosure in 1993 of a second cannabinoidreceptor many attempts have been made to synthesise CB2cannabinoid receptor selective ligands The first compoundreported to have a somehow greater affinity for the peripheralcannabinoid receptor was WIN-55212-2 The compoundwas introduced by DrsquoAmbra and colleagues [210] back in1992 as a new cannabinoid ligand having a nanomolaraffinity and acting as an agonist In 1996 Showalter et alused a transfected cell line expressing the CB2 cannabinoidreceptor in order to identify selective ligands for thisreceptor [211] They showed that the CB2 affinity of WIN-55212-2 was 7 times greater than the CB1 with Ki valuesof 189 and 028 nM for the CB1 and CB2 cannabinoidreceptors respectively However WIN-55212-2 is anagonist of the cannabinoid receptors Interestingly WIN-55212-3 (167 Fig 16) the so-called inactive enantiomer ofWIN-55212 was recently shown to behave as a low affinity(Ki=363 microM) inverse agonist of the hCB2 cannabinoidreceptor as it decreased the [35S]-GTPγ S binding [209]

III CB2 LIGANDS RECEPTOR ANTAGONISTS ANDINVERSE AGONISTS

The compounds acting as antagonist or inverse agonistsat the CB2 cannabinoid receptor are reviewed in this thirdpart of the paper As for the CB1 cannabinoid receptorligands they are classified depending on their chemicalstructures

1 Classical Cannabinoid Derivatives

The first report of an antagonism mediated by ∆9-THC atthe peripheral cannabinoid receptor was made by Bayewitch

N N

NN

NH COOH

Cl

Cl

N N

NN

N

Cl

Cl

NH2OH

O

N

N

N

N

N

NHNH2

O

Cl

ClN

N

N

N

Cl

Cl

N

O

163 164

165 166

Fig (15) CB1 cannabinoid receptor antagonists 10 Other derivatives Examples of purine (163-164) pyrazoltriazines (165) andpyrazolo[15-a]pyrimidines (166) derivatives described as CB1 cannabinoid receptor antagonists by Griffith

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1383

The pEC50 value was 554 and the Emax value ndash26 ascompared to basal

AM630 also acts as an inverse agonist at the hCB1cannabinoid receptor (EC50 = 900 nM) as Landsman et aldemonstrated [47] Recently Zhang and colleagues reportedthe characterisation of the microsomal metabolism of AM630[212]

N

O

N

O

O

N

O

Cl

O

N

O

(S)-WIN-55212-3 167 BML-190 168

In 1996 Gallant and colleagues identified by submittinga large number of compounds to a binding assay anotherindole analog as CB2 cannabinoid receptor ligand [213] Onthe basis of this compound christened BML-190 (168 Fig16) or called indomethacin morpholinylamide theysynthesised several derivatives that possessed a selectivityratio for the CB2 cannabinoid receptor of up to 140 Thesewere the first compounds specifically designed to be CB2cannabinoid receptor ligands The first report on thepharmacological properties of BML-190 appeared onlyrecently New and colleagues highlighted in the paper theinverse agonist properties of the compound BML-190 dose-dependently increases (103 EC50= 980plusmn70 nM) theforskolin-stimulated levels of cAMP in hCB2-HEK cellswhile WIN-55212-2 decreases (44 45plusmn42nM) thisaccumulation [137]

Fig (16) CB2 cannabinoid receptor antagonists 2 Indolederivatives Chemical structures of WIN-55212-3 (167) andBML-190 (168) two indoles derivatives acting as inverseagonists at the hCB2 cannabinoid receptor Surprisingly in 2002 Melck and collaborators used

BML-190 as a CB2 receptor agonist in a cell proliferationassay [214] BML-190 was also tested as inhibitor of thecyclooxygenase-2 enzyme by Kalgutkar et al [215] Theyobtained IC50 values higher than 33 and 66 microM for theCOX-I and COX-II enzymes respectively These valueshave to be compared with the submicromolar activity ofindomethacin the parent compound Very recently Klegerisand colleagues [216] looking for an antineurotoxic action ofcannabinoids showed that BML-190 increases TNF-αsecretion by stimulated THP-1 monocytic cells but is noteffective on the IL-1β secretion

Further on the basis of WIN-54461 (12) a CB1antagonist AM630 (13) was synthesised by replacement ofthe bromine atom by an iodine one In the initial reportPertwee et al showed the antagonist effects of AM630 butalso suggested that the CB1 cannabinoid receptor may not bethe preferential receptor of AM630 [45] Later on Ross et alreported using hCB1amp2-CHO cells and [3H]-CP-55940that AM630 binds to the CB2 cannabinoid receptor (Ki =312 nM) with a selectivity ratio over 160 [48]Furthermore using a [35S]-GTPγ S assay they showed itsinverse agonist properties (EC50 = 76 nM) However

Table 12 CB2 Cannabinoid Receptor Antagonists 3 Diaryl-Pyrazole Derivatives

Reported binding affinities (Ki) and selectivity ratio (CB1CB2) of SR144528 (169) for rat and human cannabinoid receptors determined using [3H]-CP55940 as radioligand

NN

O

NH

Cl

SR144528 169

rCB2 (spleen) rCB1 (whole brain) Selectivity Ki hCB2 (transfected CHOcells)

Ki hCB1 (transfected CHOcells)

Selectivity References

038 nM 305 nM 800 06 nM 437 nM 730 [217]

067 nM 548 nM 80 [219]

56 nM gt10microM gt1780 [48]

25 nM a gt 1microM a gt400 [224]

024 nM 276 nM 115 199 nM 503 nM 25 [232]

a COS cells

1384 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

3 Diarylpyrazole Derivatives CHO cells is constitutively active and phosphorylated onSer 352 Upon treatment by SR144528 thisphosphorylation of the receptor was inhibited Moreover theSR compound was able to induce dephosphorylation ofagonist-induced CB2 phosphorylation The question whetherSR144528 induces a CB2 receptor conformational changemaking it a better phosphatase-specific substrate or ifSR144528 binding activates specific phosphatases remainsopen However this remains a nice example of inverseagonist-induced receptor resensitisation

The same Sanofi team which synthesised the CB1antagonist SR141716A reported in 1998 the synthesis andpharmacological characterisation of a CB2 cannabinoidreceptor selective antagonist the SR144528 (169) [217218] The structure of the new compound is derived fromSR141716A one SR144528 is the first selective CB2receptor antagonist reported as the inverse agonist propertiesof BML-190 were shown only recently by New et al [137]The authors reported for 169 using [3H]-CP-55940 asradioligand Ki values of 04 nM and 06 nM for the rat andthe human CB2 receptors respectively and found a CB1-CB2 selectivity ratio around 700 Table 12 sums up thebinding affinities of SR144528 for the cannabinoid receptorsobtained by different authors Interestingly while the CB2affinity is more or less constant the affinity obtained for theCB1 receptor varies by one order of magnitude [219] Theantagonist effect of SR144528 was highlighted by evaluatingits effect on cAMP production by hCB2-CHO cells exposedto 3 nM CP-55940 (EC50=10nM) The antagonism wasalso observed on the activation of the MAP kinase pathwayby CP-55940 (IC50=39nM)

The Sanofi research team working on cannabinoidsconducted investigations in order to determine the keyresidues for SR144528 interaction with the receptor (Table13) as well as the binding mode of the compound Firstlythey identified the TM4-EL2-TM5 region as a regioncontaining crucial residues for the affinity of several ligandsamong them SR144528 [224] Among these residues themutation of two cysteine of the second extracellular loop(C174 and C179) abolishes the affinity for the CB2 receptorLater on the implication of S453(161) and S457(165) inthe binding of SR144528 was evidenced by mutationalstudies In the model proposed for the docking of SR144528into the CB2 receptor the crucial contacts are comprised intothe TM4 this is the case for S453(161) and S457(165)which are proposed to form hydrogen bonds with the ligandand W464(172) proposed to have aromatic interactions withthe 4-methylbenzyl of the SR144528 [225] In 2003 Fengand Song showed that point mutations D349(130)A andA634(241)E abolished ligand binding However mutationof the arginine R350(131) member of the highly conservedDRY motif to alanine has no influence on SR144528binding [226]

Early after the report of the synthesis and characterisationof SR144528 the same authors further explored the functionof this compound and showed its inverse agonist propertiesusing CB2-CHO cells co-transfected with the luciferasereporter gene with either the CRE or the murine krox24regulatory sequence [220] In these models SR144528 notonly inhibits CP-55940 effects but also reproduces theeffects of a pertussin toxin treatment proving its inverseagonist properties SR144528 functionality was alsoexplored by [35S]-GTPγ S binding in hCB2-CHO cells(IC50=3 nM) [221] The inverse agonist function ofSR144528 was further illustrated by Rhee and Kim usingCOS cells co-transfected with hCB2 receptor and adenylylcyclase [222]

As for the CB1 cannabinoid receptor ligands Sanofideveloped tricyclic derivatives based on the 1-benzylpyrazole-3-carboxylic scaffold More than twentycompounds were disclosed in a patent [227] in 2001 (84-86Table 7) The benzyl in position 4 characteristic of the CB2diarylpyrazoles from Sanofi is conserved as well as thebicyclic residue in position 3 The length of the second linkbetween the phenyl and the pyrazole can be made of one or

Bouaboula and co-workers investigated SR144528 effectson CB2 cannabinoid receptor phosphorylation status [223]Firstly they showed that the CB2 receptor transfected in

Table 13 CB2 Cannabinoid Receptor Antagonists 3 Di-Aryl-Pyrazole Derivatives

Some of the single point mutations reported for the hCB2 cannabinoid receptor for which pharmacological data concerning the SR144528 are available

Mutant Effect References

R350(131)A no effect [226]

S453(161)A loss of affinity [225]

V456(164)I no effect [225]

S457(165)A loss of affinity [225]

C174S a loss of affinity [224 225]

C175S a loss of affinity [225]

C177S a no effect [225]

C179S a loss of affinity [224 225]

S544(193)G no effect [225]

a Second extra-cellular loop

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1385

two methylene but according to the patent one methylenebridge is preferred The Ki values of these compounds for theCB2 receptor were said to be lower than 500 nM and theirantagonist properties have been assessed using the adenylatecyclase inhibition assay

compounds by the presence of an alkyl substituent inposition 5 instead of a phenyl and of course by position 3which remains unsubstituted However except a lecture atthe 1998 ACS meeting [230] where compound 171 wasreported to have a Ki value of 835 nM and a selectivity ratioover 100 no further report on these compounds waspublished untill now

In a recent paper Mussinu and colleagues reported thesynthesis of other tricyclic derivatives starting fromSR141716A structure [228] N-piperidin-1-yl-14-di-hydroindeno[12-c]pyrazole-3-carboxamide derivatives areCB2 receptor ligands and some of them possess high affinityand selectivity for the CB2 receptor as shown in Table 7 (79-83) These tricyclic derivatives differ from the Sanofi tricyclicderivatives as they do have a phenyl ring instead of a benzylin position 1 Another difference is the nature of thesubstituent in position 3 which is not a bicycloalkyl but acyclic amine One of the compounds synthesised byMussinu et al 81 possesses the highest affinity for the CB2receptor ever reported with a Ki value of 0037 nM for themurine CB2 receptor ([3H]-CP-55940 mice spleenhomogenate) Interestingly 79 the closest SR141716Aanalog lost almost all its affinity for the CB1 receptor andincreased by four orders of magnitude its affinity for the CB2receptor Even if the structure quite similar to the SRcompounds could suggest that these derivatives behave asantagonists no pharmacological data were given in the paperto support this hypothesis

5 2-Oxoquinoline Derivatives

Inaba and colleagues from Japan Tobacco introduced in2001 a completely new structure in the cannabinoid fieldbased on a 2-oxoquinoline scaffold Over eighty derivativeswere disclosed in a patent [231] and one compound JTE-907 (173) was subsequently characterised in-vitro as well asin-vivo [232] In Table 14 are listed some of the compounds(172-178) disclosed in the patent along with their affinity forthe cannabinoid receptors These values were obtained onhuman receptors against [3H]-WIN-55212-2 For twelvecompounds the anti-inflammatory activity per os wasassessed in a carrageenin-induced paw edema model Thehighest effect reported was obtained with JTE-907 with anED50 value lower than 01 mgkg Therefore thesecompounds were claimed to be useful as anti-inflammatoryand anti-allergic agents

To further characterize JTE-907 the authors determinedits affinity for cannabinoid receptors of different species using[3H]-CP-55940 as radioligand In their hands JTE-907behaved as a very selective hCB2 ligand with Ki values of36 and 2370 nM for the CB2 and CB1 cannabinoid receptorsrespectively even more selective than the SR144528 (Kivalues of 199 and 50 nM respectively) However it has tobe mentioned that the affinities reported in the paper forJTE-907 are quite different from those reported in the patent(hCB1 Ki values of 35nM and 009 nM respectively) Italso appears that the SR144528 affinity for the CB1 receptordetermined by Iwamura et al is higher than the affinityreported by Rinaldi-Carmona et al with values of 50 and437nM respectively The authors ascribed the difference tothe variability of SR144528 binding affinities to the assayconditions The function of JTE-907 was evaluated bymeasuring the increase in cAMP production by forskolin-stimulated hCB2-CHO cells Maximum stimulation wasattained at a concentration of 1microM of JTE-907 In the sameassay WIN-55212-2 decreased the cAMP production in adose-dependent manner

Finally it is interesting to strengthen that the five-membered ring derivatives (79-86) of the diarylpyrazolefamily are reported to possess a high CB2 receptor affinitywhile the six- and seven-membered ring derivatives (69-78)are described as CB1 receptor ligands

4 Other Pyrazole Derivatives

Other CB2 ligands based on a pyrazole nucleus wereintroduced by SmithKline Beecham in a patent describingthe synthesis of about sixty new compounds [229] Theiraffinity (Ki) for the hCB2 receptor transfected in HEK293cells was reported to be ranging between 25nM and 10microMTheir structures (170-171 Fig 17) differ from the Sanofi

NN

O

N

ON

NN

N

NN

O

N

ON

NN

N

ClCl

1

5

4 3

2

170 171

Fig (17) CB2 cannabinoid receptor antagonists 4 Otherpyrazole derivatives Structure of two compounds (170-171)among those claimed by SmithKline Beecham to be hCB2antagonists

In conclusion JTE-907 was shown to be a CB2 selectiveinverse-agonist with anti-inflammatory and anti-allergicproperties This compound is currently undergoing Phase Iclinical trials as anti-allergic drug [233]

IV NON-CB1 NON-CB2 CANNABINOIDRECEPTORS LIGANDS

In the last years the possible presence of additional non-CB1 non-CB2 cannabinoid receptors was raised frompharmacological evidences

One of the putative receptors possesses an endotheliallocalisation while other ones are localized in the CNS or atperipheral nerves For instance an additional target for ∆9-

1386 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Table 14 CB2 Cannabinoid Receptor Antagonists 5 2-Oxoquinoline Derivatives

Structure of six compounds among the oxoquinoline derivatives patented by Japan Tobaco Affinity (Ki nM) and selectivity (CB1CB2) for the cannabinoidreceptors are given The affinity was measured by binding against [3H]-WIN-55212-2 on either hCB2-CHO or hCB1-CHO cells Adapted from [231]

Cpd ndeg Structure hCB2 hCB1 Selectivity

1-2 172

NH

MeO O

O

OMeO

0014 3671 262215

JTE-907 173

NH

O

MeO O

O

NH

O

O

0087 3436 39495

2-2 174

OH

O

NH

MeO O

O 077 3247 4215

3-27 175

NH

O

F

NH

MeO O

O0021 336 16000

3-32 176

NH

MeO O

O

NH

F

O

0036 2398 66610

3-38 177

O

O

NH

N

O

O

NMeO

0085 935 11000

3-43 178

NMeO O

O

NH

F

O

0043 864 20100

tetrahydrocannabinol was described in capsaicin-sensitivesensory nerves by Zygmunt et al [234]

[29] and by Di Marzo et al [235] Thus in the presentreview we will focus on the description of the so faridentified ligands of the non-CB1 non-CB2 receptors withan emphasis on the compounds having antagonist properties

Pharmacological evidences for the existence of additionalcannabinoid receptors were reviewed by Wiley and Martin

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1387

(Table 15) In particular the knowledge about three not yetcloned targets has grown The growing molecularpharmacology of the three putative new cannabinoidreceptors is reviewed in the three following sections Thefourth section summarises the data on the other proposedcannabinoid targets

vasodilatator activity In addition the anandamide effect ispartially sensitive to SR141716A inhibition The authorsproposed the existence of two distinct receptors responsiblefor the vasodilatatory effect of anandamide one located in theendothelium is SR141716A-sensitive while the other onelocated on the vascular smooth muscle is SR141716A-insensitive Thus SR141716A is an antagonist of anendothelial anandamide receptor Several studies wereconducted in order to understand the molecularpharmacology of the endothelial anandamide receptor It ispertussis toxin-sensitive [237] Cannabidiol acts asantagonist and abnormal-cannabidiol (Abn-cbd 179) and itsanalogue O-1602 (180) act as agonists [238] The synthesisof O-1602 and of its analogues are disclosed in a patentdescribing the vasodilatatory activity of cannabinoid

1 Endothelial Anandamide Receptor ndash Abnormal-Cannabidiol Receptor

In 1999 Wagner et al showed that anandamide couldinduce mesenteric vasodilatation through an endotheliallylocated ldquoanandamide receptorrdquo pharmacologically distinctfrom CB1 [236] Other cannabinoids such as ∆9-THC HU-210 or WIN-55212-2 were devoid of mesenteric

Table 15 Non-CB1 Non-CB2 Cannabinoid Receptors Antagonists

An overview of the putative new non-CB1 non-CB2 anandamide-receptors ligands The antagonists are highlighted in bold

NH

N

S

HO OH

Cl

OH

OH

OH OH OH OH

OMe

OMe

RR

RR

RR

RR

HO

OH

NH

O

cannabidiol (10) Abnormal-cannabidiol 179 O-1602 180

O-1918 181 capsazepine 183capsaicin 182

Compound Endothelial AEA receptor Brain AEA receptor Cannabinoid-vanilloid brain receptor

AEA agonist a agonist de

WIN-55212-2 no effecta

agonist e agonist g h

CP-55940 no effect e agonist i

SR141716A antagonist a b j no effect ([35 S]-GTPγS binding) d e f antagonist g h

AM251 no effect k no effect h i

Cannabidiol antagonist b no effect e

O-1918 antagonist c jm

Abd-cbd agonist b l

O-1602 agonist b

Capsaicin no effectj agonist i

Capsazepine no effect b antagonist h

a [236] b [238] c [240] d [249] e [250] f [251] g [254] h [258] i [ 255] k [244] j [ 243] l [ 242] m [241]

1388 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

analogues [239] More recently Offertaler et al showed thatO-1918 (181) a cannabidiol analog acts as an antagonist ofthe endothelial anandamide receptor [240] In addition O-1918 does not bind to the CB1 and CB2 cannabinoidreceptors Furthermore O-1918 unlike cannabidiol doesnot cause vasorelaxation in the mesenteric artery preparationmodel used and thus acts as a true neutral antagonistUsing O-1918 as a pharmacological tool Begg et alproposed a coupling of this new G protein-coupledendothelial receptor through a GiGo mechanism to ionchannels in primary cultured human vascular endothelialcells [241] However in their hands SR141716A wasineffective in antagonising Abd-cbd Ho and Hiley furtherdemonstrated that Abn-cbd relaxes rat small mesentericarteries through an activation of K+ channels via aSR141716A-sensitive pathway [242] Further using thesame model they characterized the vasorelaxant activity ofvirodhamine a novel endocannabinoid apparently actingthrough the same receptor [243] The virodhamine-inducedvasorelaxation is sensitive to SR141716A and O-1918 butinsensitive to AM251 SR144528 and AM630 Moreovervanilloid receptor desensitisation by capsaicin (182) had noeffect

244] Thus position 4 on the 5-phenyl could be furtherexplored to obtain selective ligands

To come to an end despite the fact that the endothelialanandamide receptor is neither identified nor cloned at leastthree antagonists are identified SR141716A cannabidioland O-1918 It is likely that a growing number of ligandsand therapeutic applications will appear in the very nearfuture

2 Brain Anandamide Receptor

Following an in-depth evaluation of anandamide effects inCB1

-- mice Di Marzo and co-workers suggested that a non-CB1 non-CB2 G protein-coupled receptor might mediatesome of the actions of AEA in mice [249] Actually effectsof AEA but not those of ∆9-THC were not decreased inCB1 cannabinoid receptor knock-out mice For instance 30mgkg of AEA were still effective in hot-plate test whereas10 mgkg of ∆9-THC had no effect on anti-nociceptionMoreover in a [35S]-GTPγ S assay AEA stimulated with anEC50 value of 223 microM the [35S]-GTPγ S binding to CB1

--

mice brain membranes (Emax= 28) ∆9-THC showed noeffect in this assay The addition of SR141716A did notaffect the [35S]-GTPγ S binding to CB1

-- mice brainmembranes Shortly after other evidences for the existence ofa new G protein-coupled cannabinoid receptor with a distinctdistribution in the central nervous system were published[250] From a set of 24 commonly used cannabinoids (AEAWIN-55212-2 JWH-030 THChellip) only AEA and WIN-55212-2 produced an increased binding of [35S]-GTPγ S onCB1

-- mice brain membranes Interestingly in this studySR141716A alone produced a significant inhibition of [35S]-GTPγ S binding similar to the inhibition obtained usingCB1

++ mice brain membranes (IC50 values of 57 microM and47 microM respectively) However SR141716A did not affectthe anandamide-induced [35S]-GTPγ S binding in CB1

--

mice brain membranes Moreover [3H]-SR141716Aexhibited significant binding in some brain regions of CB1

--

mice which are different from those exhibiting stimulation of[35S]-GTPγ S binding by WIN-55212-2 and AEA Thusthe authors proposed that the SR141716A effects on CB1

--

mice are mediated neither by the CB1 cannabinoid receptornor by the putative new anandamide brain receptor Monoryand co-workers using CB1

-- mice described a novel nonadenylyl cyclase-coupled cannabinoid binding site in micecerebellum [251] Two agonists AEA and WIN-55212-2but not ∆9-THC or HU210 were found to bind to thisreceptor as they enhanced [35S]-GTPγ S binding NeitherSR141716A nor SR144528 were able to affect the WIN-55212-2 induced [35S]-GTPγ S binding in CB1

-- mice asfound by Breivogel et al [250] However the distributionpattern of the receptor described by Monory et al differs fromthe distribution found by Breivogel et al It has to bementioned that the knock-out strains used were different Themice used by Monory were from a CD-1 strain while theone used by Breivogel were from a C57BL61 strainInterestingly Muthane et al detected differences in nigralneurons number and sensitivity to MPTP in the two mousestrains used [252]

More recently Mo et al obtained results showing thatthe Abnormal-cannabidiol receptor is also responsible for anincreased endothelial cell migration [244] Abn-cbd enhancedhuman umbilical vein endothelial cell migration while O-1918 antagonised Abn-cbd effect The CB1 cannabinoidreceptor inverse agonist SR141716A partially inhibitedAbd-cbd action whereas AM251 and SR144528 had noeffect

In another paper from George Kunos team Batkai et alreported that SR141716A is able to inhibit the endotoxichypotension by a cardiac mechanism involving neither theCB1 nor the CB2 cannabinoid receptors [245] Theinvolvement of a non-CB1 non-CB2 site of action wasshown using CB1

-- and CB1--CB2

-- knock-out miceFurther studies are needed to identify the SR141716Asensitive myocardial site of action

In a recent patent Kunos described O-1918 and itsanalogues as vasoconstrictor agents useful to reversepathological vasodilatation of blood vessels [246] Theeffects of O-1918 on mouse blood pressure on LPS-inducedhypotension in mouse as well as the reversal of hypotensionby O-1918 injection are described Furthermore thesynthesis of O-1918 based antagonists is described startingfrom p-metha-28-dien-1-ol and the appropriate resorcinols

Interestingly the Abd-cbd receptor which was firstdetected in the vascular system also seems to be present inthe CNS [247] Indeed Walter et al found that microglialcells (BV-2) migration induced by 1 microM 2-AG is inhibitedby cannabidiol (300 nM) and O-1918 (1 microM) SR141716Awas found ineffective in this assay Thus 2-AG is an agonistof the Abd-cbd receptor Moreover Abd-cbd is able to elicita dose-dependent cell migration in the same model (EC50 =600nM) [248]

From a more structural point of view SR141716Abehaves as an antagonist while on the opposite AM251(20) a close analogue which differs from the former only bythe nature of one halogen has no effect on this receptor [242

To conclude to our knowledge no compounds havingantagonist or inverse agonist properties at this receptor havebeen identified so far

Current Knowledge on the Antagonists and Inverse Agonists Current Medicinal Chemistry 2005 Vol 12 No 12 1389

3 Cannabinoid-Vanilloid Brain Receptor a cannabinoid-vanilloid receptor at which SR141716A andcapsazepine behave as antagonists

Evidences for the presence of an additional ldquocannabinoidrdquoreceptor differing from the CB1 receptor and from the brainanandamide receptor appeared from studies focused on thehippocampus (for a review see Hajos et al [253]) Hajos etal studied the cannabinoid actions on GABAergic andglutamatergic transmission using the whole-cell patch-clamp technique [254] WIN-55212-2 induced a dose-dependent reduction in glutamatergic transmission reversedby SR141716A (1microM) Thus the authors concluded that thecannabinoid actions on the glutamatergic transmission aremediated by a non-CB1 but SR141716A-sensitive receptorIn another paper Hajos and Freund further characterised thisputative receptor [255] They showed that in addition toWIN-55212-2 CP-55940 and capsaicin (182) a TRPV1receptor agonist also act as agonists Furthermore in theirhands SR141716A as well as capsazepine (183) a TRPV1receptor antagonist were able to antagonise this effect on theglutamatergic transmission while AM251 was notSR141716A effects on CB1 knock-out mice were furtherstudied using a test of anxiety [256] Haller et al showedthat on the one hand the distribution of the CB1 receptorinduces anxiety and SR141716A has an anxiolytic effectand on the other hand SR141716A is still active in CB1

--

mice In a following paper they reported that AM-251induces anxiogenic effects in wild-type and in knock-outmice [257] Thus SR141716A in addition to CB1 bindsto a not yet identified receptor Another evidence for thisadditional receptor for SR141716A was given by Bass andco-workers [61] They conducted a structure-activityrelationship study on the stimulation of locomotor activityinduced by SR141716A and twenty analogues Nocorrelation was found between the affinity for the CB1cannabinoid receptor and the stimulation of locomotoractivity Albeit SR141716A and five of its analogues O-1803 O-1710 O-1253 O-1254 (40) O-1255 (41) behavedas inverse agonists in [35S]-GTPγ S assay none of theseanalogues stimulated the locomotor activity In conclusionthe SR141716A-induced stimulation of locomotor activity isneither due to inverse agonism nor to inhibition of anendogenous tone but to a brain receptor distinct from theCB1 receptor Di Marzo et al previously provided evidencesthat the cannabinoid-vanilloid brain receptor could mediatesome of the cannabinoid effects on locomotion [249]

4 Other Non-CB1 Non-CB2 Cannabinoid Receptors

Evidences for the existence of additional non-CB1 non-CB2 cannabinoid receptors localised in the rat isolatedmesenteric arterial bed appeared from the works of Ralevicand collaborators Several cannabinoids were evaluated intheir model of sensory neurogenic vasorelaxation evoked byelectrical field stimulation

HU210 was found to attenuate vasorelaxation in a CB1and CB2 independent way as SR141716A LY320135 andSR144528 were ineffective in inhibiting HU210 effect [259]Similar results were obtained with ∆9-THC [260]Similarly noladin ether inhibited sensory neurogenicrelaxation in a concentration-dependent manner which wasalso unaffected by SR141716A LY320135 and SR144528Moreover this effect as the ∆9-THC one was pertussistoxin sensitive suggesting the involvement of a Gio protein-coupled receptor [261]

However the inhibitory effects of CP-55940 and WIN-55212-2 were affected by the presence of SR141716A (1microM)or LY320135 (1microM) but not by the addition of SR144528(1microM) [262]

It appears that at least two receptors should be involvedin cannabinoid-mediated attenuation of sensory nerve-mediated vasorelaxation The first one is SR141716A andLY320135 sensitive while the other one is resistant toSR141716A

Some evidences for the existence of a ldquoCB2-likerdquo receptorappeared from the studies on palmitoylethanolamide (PEA)an endogenous fatty acid amide devoid of CB1 and CB2affinity [264] antinociceptive potential Calignano and co-workers showed that PEA was able to alleviate pain inseveral animal models [265 266] For instance PEA dose-dependently inhibits kaolin-evoked writhing in mice TheCB2 cannabinoid receptor inverse agonist SR144528 (02mgkg) inhibits PEA anti-nociceptive effect [266] Thus theauthors suggest that PEA acts as agonist on a SR144528-sensitive non-CB2 cannabinoid receptor (ldquoCB2-likerdquoreceptor) at which SR144528 acts as an antagonist

Further studies are needed in order to better characterisethese additional ldquocannabinoidrdquo receptors However it islikely that several therapeutic applications will soon springfor these receptors

Very recently Pistis et al published a study on theneurophysiological effects of cannabinoids on the basolateralamygdala neurons in-vivo [258] One of their findings is thatHU-210 and WIN-55212-2 did not elicit similar effects onbasal amygdala projection neurons firing rate While theformer had no effect the latter decreased the firing rateMoreover SR141716A reversed the WIN-55212-2 effectswhile AM251 had no effect The vanilloid receptorantagonist capsazepine also antagonised the WIN-55212-2effect on firing rate of these neurons Therefore the authorssuggested the presence of a novel cannabinoid-vanilloidreceptor

V CONCLUSION

Since their development cannabinoid antagonists provedto be essential tools in the understanding of cannabinoidpharmacology and biochemistry Moreover the interestshown by the pharmaceutical industry in the development ofnew cannabinoid antagonists prove if necessary the realtherapeutic potential of this class of compounds Thetreatment of eating and movement disorders memorydeficits psychosis and dependencies from various addictivedrugs are some of the most cited applications for the CB1cannabinoid receptor antagonists

From these lines it appears that in addition to theknown CB1 cannabinoid receptor it is now likely that twoother cannabinoid receptors do exist in the brain The firstone should be an anandamide brain receptor for whichSR141716A has no affinity while the second one should be

1390 Current Medicinal Chemistry 2005 Vol 12 No 12 Muccioli and Lambert

Much less wide is the knowledge on the CB2cannabinoid receptor antagonists However potentialapplications could be disorders involving the immunesystem such as inflammation or allergies

[19] Devane WA Hanus L Breuer A Pertwee RG StevensonLA Griffin G Gibson D Mandelbaum A Etinger AMechoulam R Science 1992 258 1946

[20] Mechoulam R Ben-Shabat S Hanus L Ligumsky MKaminski NE Schatz AR Gopher A Almong S MartinBR Compton DR Biochem Pharmacol 1995 50 83

Finally the growing literature on the non CB1 non CB2cannabinoid receptors should allow the development of newdrugs First examples are given by the cannabidiol andabnormal-cannabidiol derivatives described by Kunos et alfor which potential therapeutic applications on the vascularsystem are already patented

[21] Sugiura T Kondo S Sukagawa A Nakane S Shinoda AItoh K Yamashita A Waku K Biochem Biophys Res Co1995 215 89

[22] Hanus L Abu-Lafi S Fride E Breuer A Vogel Z ShalevDE Kustanovich I Mechoulam R Proc Natl Acad SciUSA 2001 98 3662

[23] Oka S Tsuchie A Tokumura A Muramatsu M Suhara YTakayama H Waku K Sugiura T J Neurochem 2003 851374It clearly appears from this paper that the antagonists of

the cannabinoid receptors are still an active research fieldfrom which should emerge new promising therapeutic toolsas well as innovative drugs in the near future

[24] Goutopoulos A and Makriyannis A Pharmacol Therapeut2002 95 103

[25] Beal JE Olson R Lefkowitz L Laubenstein L Bellman PYangco B Morales JO Murphy R Powderly W PlasseTF Mosdell KW Shepard KV J Pain Symptom Manage1997 14 7ACKNOWLEDGEMENTS [26] Notcutt W Price M Miller R Newport S Phillips CSimmons S Sansom C Anaesthesia 2004 59 440

GGM is very indebted to the ldquoFonds pour la formationagrave la Recherche dans lrsquoIndustrie et lrsquoAgriculture (FRIA)rdquo Belgium for the award of a research fellowship Theexperimental works of the authors were supported by grantsfrom the FNRS The Belgian Charcot Foundation and theUniversiteacute catholique de Louvain

[27] Ledent C Valverde O Cossu G Petitet F Aubert JFBeslot F Bohme GA Imperato A Pedrazzini T RoquesBP Vassart G Fratta W Parmentier M Science 1999 283401

[28] Zimmer A Zimmer AM Hohmann AG Herkenham MBonner TI Proc Natl Acad Sci U S A 1999 96 5780

[29] Wiley JL and Martin BR Chem Phys Lipids 2002 121 57[30] Adam J and Cowley P Expert Opin Ther Patents 2002 12

1475

REFERENCES [31] Razdan RK and Mahadevan A Chem Phys Lipids 2002 12121

[32] Barth F and Rinaldi-Carmona M Curr Med Chem 1999 6745

[1] Hillard CJ Harris RE Bloom AS J Pharmacol Exp Ther1985 232 579

[33] Little PJ Compton DR Johnson MR Melvin LS MartinBR J Pharmacol Exp Ther 1988 247 1046

[2] Devane WA Dysarz FA Johnson MR Melvin LSHowlett AC Mol Pharmacol 1988 34 605

[34] Pertwee RG Stevenson LA Elrick DB Mechoulam RCorbett AD Brit J Pharmacol 1992 105 980

[3] Matsuda LA Lolait SJ Brownstein MJ Young ACBonner TI Nature 1990 346 561

[35] Harrison C Traynor JR Life Sci 2003 74 489-508[4] Gerard CM Molerau C Vassart G Parmentier M BiochemJ 1991 279 129 [36] Pertwee RG Fernando SR Griffin G Ryan W Razdan

RJ Compton DR Martin BR Eur J Pharmacol 1996 315195

[5] Glass M Dragunow M Faull RLM Neuroscience 1997 2299

[37] Ross RA Brockie HC Fernando SR Saha B Razdan RKPertwee RG Brit J Pharmacol 1998 125 1345

[6] Shire D Carillon C Kaghad M Calandra B Rinaldi-Carmona M LeFur G Caput D Ferrara P J Biol Chem1995 270 3726 [38] Griffin G Wray EJ Rorrer WK Crocker PJ Ryan WJ

Saha B Razdan RK Martin BR Abood ME Brit JPharmacol 1999 126 1575

[7] Ryberg E Khang Vu H Groblewski T Larsson N ElebringT Hjorth S Sjogren S Greasley PJ 2004 Symposium on theCannabinoids Burlington Vermont International CannabinoidResearch Society page 119

[39] Ross RA Gibson TM Stevenson LA Saha B Crocker PRazdan RK Pertwee RG Brit J Pharmacol 1999 128 735

[40] Martin BR Winckler JR Wiley JL Huffman JW CrockerPJ Saha B Razdan RK J Pharmacol Exp Ther 1999 2901065

[8] Rinaldi-Carmona M Calandra B Shire D Bouaboula MOustric D Barth F Casellas P Ferrara P LeFur G JPharmacol Exp Ther 1998 278 871

[41] Martin BR Stevenson LA Pertwee RG Breivogel CSWilliams A Mahadevan A Razdan RK 2002 Symposium onthe Cannabinoids Burlington Vermont International CannabinoidResearch Society page 2

[9] Munro S Thomas KL Abu-Shaar M Nature 1993 365 61[10] Howlett AC Qualy JM Khachatrian LL Mol Pharmacol

1986 29 307[11] Glass M Felder CC J Neurosci 1997 17 5327

[42] Thomas A Ross RA Saha B Mahadevan A Razdan RKPertwee RG Eur J Pharmacol 2004 487 213

[12] Calandra B Portier M Kerneacuteis A Delpech M Carillon C LeFur G Ferrara P Shire D Eur J Pharmacol 1999 374445 [43] Compton DR Gold LH Ward SJ Balster RL Martin BR

J Pharmacol Exp Ther 1992 263 1118[13] Howlett AC Life Sci 1984 35 1803[44] Eissenstat MA Bell MR DAmbra TE Alexander EJ

Daum SJ Ackerman JH Gruett MD Kumar V EstepKG Olefirowicz EM Wetzel JR Alexander MD WeaverJD Haycock DA Luttinger DA Casiano FM ChippariSM Kuster JE Stevenson JI Ward SJ J Med Chem1995 38 3094

[14] Deadwyler S Hampson RE Mu J Whyte A Childers SRJ Pharmacol Exp Ther 1995 273 734

[15] Mackie K Lai Y Westenbroek R Mitchell R J Neurosci1995 15 6552

[16] Bouaboula M Poinot CC Bourrie B Canat X Calandra BRinaldi-Carmona M LeFur G Casellas P Biochem J 1995312 637 [45] Pertwee R Griffin G Fernando S Li X Hill A

Makriyannis A Life Sci 1995 56 1949[17] Bouaboula M Poinot CC Marchand J Canat X Bourrie BRinaldi-Carmona M Calandra B LeFur G Casellas P Eur JBiochem 1996 237 704

[46] Hosohata Y Quock RM Hosohata K Makriyannis AConsroe P Roeske WR Yamamura HI EurJPharmacol 1997 321 R1[18] delPulgar T Velasco G Guzman M Biochem J 2000 347

369 [47] Landsman RS Makriyannis A Deng H Consroe P RoeskeWR Yamamura HI Life Sci 1998 62 109

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[48] Ross RA Brockie HC Stevenson LA Murphy VLTempleton F Makriyannis A Pertwee RG Brit JPharmacol 1999 126 665

[81] Gatley SJ Lan R Volkow ND Pappas N King P WongCT Gifford AN Pyatt B Dewey SL Makriyannis A JNeurosci 1998 70 417

[49] Huffmann JW Curr Med Chem 1999 6 705 [82] Lan R Lu Q Fan P Gatley J Volkow ND Fernando SRPertwee R Makriyannis A AAPS Pharmsci 1999 1 article 4[50] Rinaldi-Carmona M Barth F Heaulme M Shire D Calandra

B Congy C Martinez S Maruani J Neliat G Caput DFerrara P Soubrieacute P Breliegravere JC Le Fur G FEBS Lett1994 350 240

[83] Mathews WB Ravert HT Musachio JL Franck RARinaldi-Carmona M Barth F Dannals RF J Labelled CompdRad 1999 42 589

[51] Rinaldi-Carmona M Barth F Heaulme M Alonso R ShireD Congy C Soubrie P Breliere JC Le Fur G Life Sci1995 56 1941

[84] Mathews WB Scheffel U Finley P Ravert HT FrankRA Rinaldi-Carmona M Barth F Dannals RF Nucl MedBiol 2000 27 757

[52] Jarai Z Wagner JA Goparaju SK Wang L Razdan RKSugiura T Zimmer AM Bonner TI Zimmer A Kunos GHypertension 2000 35 679

[85] Berding G Muumlller-Vahl K Schneider U Gielow P FitschenJ Stuhrmann M Harke H Buchert R Donnerstag FHofmann M Knoop BO Brooks DJ Emrich HM KnappWH Biol Psychiat 2004 55 904[53] Fox A Kesingland A Gentry C McNair K Patel S Urban

L James I Pain 2001 92 91 [86] Mathews WB Scheffel U Rauseo PA Ravert HT FrankRA Ellames GJ Herbert JM Barth F Rinaldi-Carmona MDannals RF Nucl Med Biol 2002 29 671

[54] Costa B Vailati S Colleoni M Behav Pharmacol 1999 10327

[55] Compton DR Aceto MD Lowe J Martin BR J PharmacolExp Ther 1996 277 586

[87] Bays HE Obes Res 2004 12 1197[88] Comment in Nat Rev Drug Discov 2004 3 288

[56] Petitet F Jeantaud B Capet M Doble A BiochemPharmacol 1997 54 1267

[89] LeFur G 2004 Symposium on the Cannabinoids BurlingtonVermont International Cannabinoid Research Society page 67

[57] Griffin G Atkinson PJ Showalter VM Martin BR AboodME J Pharmacol Exp Ther 1998 285 553

[90] Cleland JGF Ghosh J Freemantle N Kaye GC Nasir MClark AL Coletta AP Eur J Heart Fail 2004 6 501

[58] Kearn CS Greenberg MJ DiCamelli R Kurzawa KHillard CJ J Neurochem 1999 72 2379

[91] Arnone M Maruani J Chaperon F Thiebot MH PonceletM Soubrie P Le Fur G Psychopharmacology 1997 132 104

[59] MacLennanSJ Reynen PH Kwan J Bonhaus DW Brit JPharmacol 1998 124 619

[92] Colombo G Agabio R Diaz G Lobina C Reali R GessaGL Life Sci 1998 63 PL113

[60] Sim-Selley LJ Brunk LK Selley DE Eur J Pharmacol2001 414 135

[93] Simiand J Keane M Keane PE Soubrie P BehavPharmacol 1998 9 179

[61] Bass CE Griffin G Grier M Mahadevan A Razdan RKMartin BR Pharmacol Biochem Behav 2002 74 31

[94] Freedland CS Poston JS Porrino LJ Pharmacol BiochemBehav 2000 67 265

[62] Meschler JP Kraichely DM Wilken GH Howlett ACBiochem Pharmacol 2000 60 1315

[95] Rowland NE Mukherjee M Robertson KPsychopharmacology 2001 159 111

[63] Mato S Pazos A Valdizan EM Eur J Pharmacol 2002443 43

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