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    Published: June 23, 2011

    r 2011 American Chemical Society 5562| J. Med. Chem. 2011, 54, 55625575


    Design, Synthesis, and Biological Evaluation of 3,4-Dihydroquinolin-2(1H)-one and 1,2,3,4-Tetrahydroquinoline-Based Selective Human

    Neuronal Nitric Oxide Synthase (nNOS) InhibitorsJailall Ramnauth,*,Joanne Speed, Shawn P. Maddaford, Peter Dove, Subhash C. Annedi, Paul Renton,

    Suman Rakhit,John Andrews, Sarah Silverman, Gabriela Mladenova, Salvatore Zinghini, Sheela Nair,

    Concettina Catalano, David K.H. Lee, Milena De Felice, and Frank Porreca

    NeurAxon Inc., 2395 Speakman Drive, Suite 1001, Mississauga, Ontario L5K 1B3, CanadaNoAb BioDiscoveries Inc., 2820 Argentia Road, Suite 8, Mississauga, Ontario L5N 8G4, CanadaDepartment of Pharmacology, University of Arizona, 1501 North Campbell Avenue, Tucson, Arizona 85724, United States

    bS Supporting Information

    INTRODUCTIONNitric oxide (NO) is a reactive free radical gas with diverse

    physiological roles, ranging from blood pressure regulation toneurotransmission.1 In most cases, this essential signaling mole-cule functions by activating soluble guanylate cyclase (sGC), anenzyme that catalyzes the conversion of guanosine-5 0-triphos-phate (GTP) to the intramolecular second messenger 3,5-cyclicguanosine monophosphate (cGMP).2 However, due to itsreactivity, nitric oxide can participate in responses that are notmediated by the NO/cGMP signaling pathway.3

    Three distinct isoforms of nitric oxide synthase (NOS)catalyze the five electron oxidation of L-arginine to L-citrullineto produce NO.4 These include the neuronal or brain NOS

    (nNOS or NOS1) and endothelial NOS (eNOS or NOS3),which are constitutively expressed, and the inducible isoform(iNOS or NOS2), which is expressed under conditions of stressor upon the release of inflammatory mediators (IM) such asTNFR, IL-1, or lipopolysaccharides (LPS). The NOS enzymesare homodimeric proteins, consisting of a C-terminal reductasedomain that transfers electrons from NADPH through twoprosthetic groups, FAD and FMN, to the N-terminal oxygenasedomain, which binds L-arginine, (10R,20S,6R)-5,6,7,8-tetrahydro-

    biopterin (BH4), and heme.5 While the two constitutive iso-forms are activated by increases in Ca2+ concentration and

    binding of a Ca2+/calmodulin complex, the activity of iNOSappears to be independent of Ca2+ concentration due to a tight

    binding of the complex at the dimer interface.6 The activedimeric form of NOS is stabilized by a structural zinc bindingat the oxygenase dimer interface. The reduction of BH4 andheme iron allows the activation of O2 followed by the oxidationofL-arginine to N-hydroxy-L-arginine and finally to L-citrulline,ultimately releasing NO.

    The overproduction of NO by each isoform has been asso-ciated with many disease states. In particular, excess NO in thecentral nervous system by nNOS has been associated with thespinal transmission of pain, migraine and chronic tension-typeheadaches, neurodegeneration during stroke, Parkinsons dis-ease, and Alzheimers disease.711 Consequently, the inhibitionof nNOS has the potential to be therapeutic in these diseases.The selective inhibition of nNOS is essential because of theimportant functions of the other NOS isoforms; for example,eNOS plays a crucial role in the regulation of blood pressure,12

    and the inhibition of eNOS by a nonselective inhibitor has beenshown to cause vasoconstriction in humans.13 Accordingly,selectively inhibiting nNOS has been the goal of many researchgroups in the last two decades.1416 Although there have beenmany different classes of compounds reported in the literature,the most common mode of inhibiting NOS is to mimic thenatural substrate L-arginine.

    Received: May 20, 2011

    ABSTRACT: Neuronal nitric oxide synthase (nNOS) inhibitors are effective in

    preclinicalmodelsof many neurological disorders. In this study, tworelated seriesof compounds, 3,4-dihydroquinolin-2(1H)-one and 1,2,3,4-tetrahydroquinoline,containing a 6-substituted thiophene amidine group were synthesized andevaluated as inhibitors of human nitric oxide synthase (NOS). A structureactivity relationship (SAR) study led to the identification of a number of potentand selective nNOS inhibitors. Furthermore, a few representative compounds were shown to possess druglike properties, featuresthat are often difficult to achieve when designing nNOS inhibitors. Compound (S)-35, with excellent potency and selectivity fornNOS, was shownto fully reverse thermal hyperalgesia when given to rats at a dose of 30 mg/kg intraperitonieally (ip) in the L5/L6spinal nerve ligation model of neuropathic pain (Chung model). In addition, this compound reduced tactile hyperesthesia(allodynia) after oral administration (30 mg/kg) in a rat model of dural inflammation relevant to migraine pain.
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    5563 | J. Med. Chem. 2011, 54, 55625575

    Journal of Medicinal Chemistry ARTICLE

    The general pharmacophore model for competitively inhibit-ing NOS at the substrate binding site is depicted by 1, whichcontainsa guanidine isosteric group and a basic group attached toa central linker.17 It should be noted, however, that someinhibitors only contain a guanidine isostere attached to a centrallinker. By varying the guanidine isostere, the central linker, or the

    basic group, potent and selective nNOS inhibitors have been

    obtained (for example, compounds 2

    6) (Chart 1).16

    Werecently reported a series of potent and selective small molecule2-aminobenzothiazole-based thiophene amidine inhibitors ofnNOS exemplified by compound 6.17 In continuation of our

    work on designing selective nNOS inhibitors for treating CNSdisorders, we now describe the synthesis and structureactivityrelationships (SAR) of 3,4-dihydroquinolin-2(1H)-one and1,2,3,4-tetrahydroquinoline-based nNOS inhibitors that led tothe identification of compound (S)-35. This compound is apotent and selective nNOS inhibitor with druglike properties,and it is shown to be efficacious in two rat pain models, one of

    which employs an oral dosing protocol.


    Chemistry. Compounds 2631 , which are from the 3,4-dihydroquinolin-2(1H)-one series, were prepared according toScheme 1. The alkylation of 7 with a series of commerciallyavailable chloroalkylamine hydrochloride salts 913 in DMF

    with potassium carbonate at room temperature gave compounds1418. In a similar fashion, fluoro compound 8 was alkylated

    with 9 to give 19. The nitro groups of1419were reduced to thecorresponding anilines 2025with either catalytic Pd on carbonunder a H2 atmosphere or catalytic Raney nickel using hydrazinehydrate. These amines were converted to the products 2631bycoupling with thiophene-2-carbimidothioate hydroiodide undermild conditions in ethanol at room temperature.

    Two compounds (41 and 42) with a 3-carbon linker to the

    basic amine in the 3,4-dihydroquinolin-2(1H)-one series wereprepared as outlined in Scheme 2. Treatment of 7 with NaH inDMF followed by reaction with chloroiodopropane provided thechloropropyl compound 36. Nucleophilic displacement of thechloride with dimethylamine and pyrrolidine in aqueous acet-onitrile using a catalytic amount of potassium iodide at 60 Cgave 37 and 38, respectively. These compounds were reducedand then coupled with the 2-thiophene thioimidate reagent togive the final compounds.

    We have utilized two methods for the synthesis of compoundsin the 1,2,3,4-tetrahydroquinoline series with an acyclic sidechain. In the first method (Scheme 3), anilines 20 and 24 werereduced with LiAlH4 in THF to give compounds 32 and 33.Coupling of these compounds with the 2-thiophene thioimidate

    provided compounds 34 and 35. Compound 35 was easilyseparated into its enantiomers by chiral column chromatography.

    Chart 1. Pharmacophore Model and Literature Examples ofSelective nNOS Inhibitors

    Scheme 1a

    a Reagents and conditions: (a) K2CO3, DMF, 25 C, 24 h; (b) Pd/C, H2, EtOH, 317 h or Raney Ni, NH2NH2 3H2O, MeOH, reflux, 15 min;(c) thiophene-2-carbimidothioate HI, EtOH, 24 h.

    Scheme 2a

    a Reagents and conditions: (a) Cl(CH2)3I, NaH, DMF, 25 C, 5 h;(b)R1R2NH,K2CO3,KI,ACN,H2O,60 C,16h;(c)Pd/C,H2, EtOH,317 h or Raney Ni, NH2NH2.H2O, MeOH, reflux, 15 min;(d) thiophene-2-carbimidothioate HI, EtOH, 24 h.
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    5564 | J. Med. Chem. 2011, 54, 55625575

    Journal of Medicinal Chemistry ARTICLE

    To confirm the stereochemistry of compound (S)-35 , it wassynthesized using the known optically pure alkylating agent (S)-13,18 which was obtained from the commercially availableoptically pure (S)-N-Boc-L-homoproline according to Scheme 4.Scheme 5 depicts a second method for preparing compounds inthe 1,2,3,4-tetrahydroquinoline series. This method is potentially

    broader in scope because it uses a milder reducing agent that iscompatible with many functional groups. Reduction of amides

    15, 19, 37, and 38with 1 M borane in THF at room temperaturegave compounds 4346 , which were converted to products5053 in a similar fashion as described for all preceding targetcompounds.

    To synthesize compounds with a cyclic side chain in the1,2,3,4-tetrahydroquinoline ser