part one medicinal chemistry - wiley-vchpart one medicinal chemistry 1 bioorganometallic chemistry:...

Part OneMedicinal Chemistry

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Bioorganometallic Chemistry: Applications in Drug Discovery, Biocatalysis, and Imaging, First Edition.Edited by Gérard Jaouen and Michèle Salmain. 2015 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2015 by Wiley-VCH Verlag GmbH & Co. KGaA.

1Organometallic Complexes as EnzymeInhibitors: A Conceptual OverviewPhilipp Anstaett and Gilles Gasser

1.1Introduction

Enzyme inhibitors are currently playing a crucial role in medicine. A high pro-portion of the drugs currently reaching the market are exerting their activity byinhibiting an enzyme. For example, the best-selling drug in pharmaceutical his-tory, the lowering blood cholesterol drug Atorvastatin, sold under the tradename Lipitor, is inhibiting an enzyme present in liver tissue [1]. The anticancerdrug Imatinib marketed under the trade names Gleevec and Glivec specificallytargets a tyrosine kinase. From a medicinal inorganic chemistry perspective, themechanism of action of several metal-based drugs having reached the marketcan be linked to enzyme inhibition. Examples of such compounds include theantiarthritic gold complexes, the antimony-based drugs against leishmaniasis, orthe arsenic-based drugs against syphilis, trypanosomiasis, and cancer, althoughthe exact mechanism(s) of action of these compounds have not been (yet) fullyuncovered. Due to these successful examples, several research groups around theworld are currently exploring the possibility of using organometallic compoundsto inhibit enzymes involved in diseases. This field of research has been reviewedin detail over the last years [2–6]. In this chapter, we aim to take an alternativeapproach by presenting the different concepts employed to achieve enzyme inhi-bition using organometallic complexes rather than to list all organometalliccompounds reported to date that can act as enzyme inhibitors. We will use afew concrete examples to exemplify each concept.

1.2Organometallic Compounds as Inert Structural Scaffolds for Enzyme Inhibition

Nature evolved proteins, including enzymes, with active sites of very unique andspecific shapes and electrostatic surfaces, providing unmatched specificity andcatalytic activity for the intended substrates. Obtaining a great selectivity is achallenge in the design of enzyme inhibitors. These small molecules have to

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Bioorganometallic Chemistry: Applications in Drug Discovery, Biocatalysis, and Imaging, First Edition.Edited by Gérard Jaouen and Michèle Salmain. 2015 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2015 by Wiley-VCH Verlag GmbH & Co. KGaA.

match the interaction pattern dictated by the active pocket in order to effectivelybind to and block the activity of an enzyme. For this task, the greater the chemi-cal space used in the search of an inhibitor is, the greater are the chances to finda “perfect” inhibitor. Thus, the use of metal complexes greatly increases thestructural possibilities to form enzyme inhibitors. Additional metal-specificgeometries, such as square planar or octahedral, can be indeed exploited. More-over, unique bond lengths and bond polarities, which are unavailable to purelyorganic compounds, further increase the potential associated with the use ofmetal compounds as enzyme inhibitors. There are several examples in the litera-ture describing organometallic enzyme inhibitors that have been built around aninert metal center and this topic has been previously reviewed [7,8]. The mostextensively studied organometallic complexes are undoubtedly the kinase inhibi-tors developed by the group of Meggers. In this chapter, we highlight the rangeof opportunities that has been opened up with the use of metal centers as inertscaffolds, on basis of this work.Protein kinases are enzymes that catalyze the attachment of phosphate groups

to proteins, thereby regulating their function. Hundreds of different subtypes areknown, usually phosphorylating only a few specific substrates. Consequently,protein kinases are involved in many cellular pathways, including, but not exclu-sively, ones upregulated in cancer tissues leading to their uncontrolled growth.Therefore, selective protein kinase inhibitors represent attractive targets fordrug discovery, for example, as anticancer agents (see introduction).The common phosphate source of all protein kinases is ATP (Figure 1.1a),

whose terminal phosphate group is transferred to the respective substrates. TheATP binding pocket is the main target for protein kinase inhibitors [9–11]. As

(a) (b) (c)

Figure 1.1 Structure of (a) ATP; (b) stauro-sporine; (c) staurosporine-inspired octahedralmetal complex with variable ligands L1–L4.Gray scales indicate the parts of the respectivemolecules that occupy the same spatial posi-tion in the binding pocket of the protein

kinases; the dark gray parts are involved inhydrogen bonding to corresponding aminoacids; the light gray parts occupy an areaallowing for secondary interactions potentiallyleading to subtype specificity.

4 1 Organometallic Complexes as Enzyme Inhibitors: A Conceptual Overview

there are more than 500 human kinase genes [12], which have a structurallyhighly conserved ATP binding pocket, it is essential, and particularly difficult, todesign inhibitors with high specificity for a particular kinase subtype.Staurosporine (Figure 1.1b) is a natural alkaloid that inhibits protein kinases

with low nanomolar IC50 values, but that possesses only low subtype specific-ity [13–17]. It binds to the ATP binding site of kinases, which is very similaramong the different subtypes. Within the ATP binding site, the lactam (the darkgray part in Figure 1.1b) forms two hydrogen bonds to the protein, as adeninedoes in the case of ATP [18]. The glycosyl unit in staurosporine (light gray partin Figure 1.1b) replaces the ribose part of ATP. This part of the inhibitor wasfound to be less important for the binding affinity of staurosporine to the activesite of kinases [19]. On the basis of those observations, Meggers and cow-orkers [20] designed their organometallic kinase inhibitors. They envisagedkeeping the general structure of staurosporine. However, they modified it in away that it could serve as a ligand for metal complexation (Figure 1.1c). Hence,the initial affinity toward protein kinases would be conserved. The remainingopen coordination sites of the metal ion then give the opportunity to introduceadditional ligands, inter alia, replacing the glycosyl moiety present in staurospor-ine. These ligands were later shown to influence the specificity, but also to someextend the affinity of the metal-containing inhibitors to certain kinase subtypes(see next).Accordingly, ruthenium complexes such as 1 were synthesized and their bind-

ing affinity toward protein kinases was investigated (Figure 1.2) [20]. Compound1 has been found to be over 100 times more potent against the kinase GSK-3than staurosporine and over 50 times more potent than the pyridocarbazole lig-and by itself.A cocrystal structure of the protein kinase Pim-1 and 1 showed that the com-

plex 1 binds to the ATP binding site in a homologous manner to staurosporine(Figure 1.3) [21]. Previously, a Lineweaver–Burke analysis, which is a kinetic

Figure 1.2 Isostructural ruthenium and osmium complexes with pyridocarbazole ligand.

1.2 Organometallic Compounds as Inert Structural Scaffolds for Enzyme Inhibition 5

method allowing the distinction of competitive and noncompetitive bindingmodes, was performed for the ruthenium complex 2, revealing an ATP-competitive binding mode [22]. Both findings together clearly show thatMeggers’ organometallic staurosporine derivatives, like staurosporine itself, bindto the ATP binding pocket of kinases.The cyclopentadienyl moiety and particularly the carbonyl ligand of 1 occupy

positions within the ATP binding pocket that are not accessible to purelyorganic molecules due to the specific geometric properties of the metal complex.For example, as can be seen in the cocrystal structure of 1 with Pim-1, the COligand of 1 is involved in dipolar interactions with the glycine-rich loop of Pim-1 [21]. This interaction seems to be of great significance for the subtype specific-ity toward Pim-1 and GSK3, which possess similar glycine-rich loops. Similarobservations were made for other related organometallic kinase inhibi-tors, [21,24–28] and, to date, all specific organometallic inhibitors found forthose enzymes carry this CO ligand. Worthy of note, a CO ligand, unlike anorganic carbonyl group in a molecule, is not polar [28]. Hence, the electronicproperties responsible for the favorable interactions of the CO ligand of Meg-gers’ enzyme inhibitors with the protein cannot be imitated by organic carbonylgroups. Due to their unique geometric and electronic characteristics, only otherorganometallic inhibitors are, to the best of our knowledge, known to establishcomparable interactions with the glycine-rich loop of protein kinases, introduc-ing hence this great selectivity [24].The metal center of 1 appears to be unable to directly interact with the protein

as the metal is “protected” by its ligands. In order to further confirm that the role

Figure 1.3 Ruthenium complex 1 (purple; PDB code 2BZI) and staurosporine (green; PDB code1YHS) inside the ATP binding pocket of Pim-1 [21,23]. Both inhibitors occupy a similar spatialposition and exhibit homologous interactions to the enzyme.


of the metal is solely of structural nature, an complex isosteric to 1, namely theosmium complex 3, was prepared [26]. In contrast to their geometry, the redoxpotentials of the two metal complexes are different. Hence, if the metal has apurely structural role the bioactivity should remain unaffected, while any addi-tional influences should alter the activity profile. Cocrystal structures of Pim-1with 1 and 3 show nearly identical binding geometries and interactions for bothcomplexes (Figure 1.4). In vitro studies, such as the anticancer activity in 1205Lu melanoma cells and the activation of Wnt signaling as result of inhibition ofGSK-3β in human embryonic kidney cells (HEK293OT), also verified the almostidentical biological activity of both compounds 1 and 3, thus clearly demonstrat-ing the purely structural role of the metal center.Nevertheless, the change of the metal center in this type of inhibitors can lead

to an overall different affinity and selectivity pattern among protein kinases.Apart from ruthenium(II) [8,20,21,25,27–38] and osmium(II) [26], iridium(III), [24,39–42] platinum(II) [43], and rhodium(III) [42,44,45] have also servedas metal centers for kinase inhibitors. Such swaps can lead to several conse-quences depending on the metal ion chosen. First, due to the different electronicproperties of the metal ions, different synthetic pathways might be employed toprepare the complexes and thus ease the screening of large compound libraries(see below). Second, a swap of the metal center can, in contrast to the isostericreplacement of ruthenium with osmium discussed earlier, lead to a differentoverall complex geometry, which can alter the affinity and selectivity for certainkinases. In accordance with these points, the change from one metal center toanother is an option worth considering, as it opens up new avenues for molecu-lar diversity.Although tetrahedral half-sandwich complexes have geometries that can

hardly be mimicked by organic compounds (see above), octahedrally substitutedcomplexes are even more intriguing as they offer additional geometric complex-ity [24,25,31,38,39,41,44–46]. The consequences of this become striking if the

Figure 1.4 Superimposed cocrystal structuresof Pim-1 (green) with 1 (red; PDB code2BZI) [21] and 3 (blue; PDB code 3BWF) [26].The amino acid residues involved in the most

important interactions with the inhibitors areshown as stick models. Reproduced withpermission of the American Chemical Society.


number of possible stereoisomers is taken into consideration: a tetrahedral cen-ter can form up to two enantiomers, octahedral centers can form up to 30 ster-eoisomers. Meggers coined the term “octasporines” for this group ofcompounds, as they feature an octahedral coordination sphere and are derivedfrom staurosporine. Notably, the group of octasporines is not limited to organo-metallic compounds, but also includes classical inorganic complexes, such as 4(Figure 1.5). Compounds 4–7 (Figure 1.5) nicely demonstrate the tremendousimpact of the geometric complexity on biological systems. Despite their similargeneral arrangement, all of these complexes are highly selective inhibitors of dif-ferent protein kinase subtypes (Figures 1.5 and 1.6) [24,39]. It has to be notedthat already most of the tetrahedral metal complexes also show high subtypeselectivities that surpass many organic inhibitors.

Figure 1.6 Selectivity of 5 in a screening against 102 kinases. Figure adapted with permissionfrom Feng et al. [24]. Copyright 2011, American Chemical Society.

α

Figure 1.5 Octasporines with distinct selectivity profiles.


The great structural variability gained by the multiple ligands around themetal center can be exploited for Structure Activity Relationship (SAR) studies.The synthesis of complexes with labile ligands allows for rapid screening of dif-ferent ligands around the metal center as it is the case for the ruthenium com-plex 8 (Figure 1.7) [31,43,44]. For example, 6 is a selective Pim-1 inhibitor(IC50= 0.075 nM), while 4 is selective for DAPK1 (IC50= 2.0 nM). Replacementof the CO ligand, which is a common motif for Pim-1 selective inhibitors, with aNCS ligand changes the selectivity toward DAPK1. The switch in selectivity isdue to the interaction of the respective ligands with the glycine-rich loop of therespective enzymes. In the case of DAPK1, compared with other kinases such asPim-1, the glycine-rich loop provides more space and can therefore accommo-date the larger thiocyanato ligand.Apart from exchanging ligands around the metal center, the ligands them-

selves can also be modified as part of the screening process to obtain the bestenzyme inhibition possible. This is in line with conventional medicinal chemistryapproaches. For example, the pyridocarbazole ligand has been substituted with avariety of functional groups [34,47]. A library of 68 amides has been screened,which was synthesized from a ruthenium(II) complex carrying a NHS activeester modified cyclopentadienyl ligand [32]. More recently, even the pyridocar-bazole unit, which has been the essential part of almost all enzyme inhibitorspreviously developed by Meggers and coworkers, has been substantially modified(9–11 in Figure 1.8) [41,45,46]. In all cases, it was shown that the ligand modifi-cation leads to substantially different affinity and/or selectivity profiles. In thisregard, the complexes behave like “traditional” fully organic enzyme inhibitors.The impressive scope of the above described system can be seen by the num-

ber of different kinases that can be targeted by these various complexes. Itincludes GSK3α [22,47], GSK3β [33,49], Pim1 [31,44], Pim2 [27], PAK1 [25,46],MST1 [29], BRAF [36], PI3Kγ [37], FLT4 [39,40], TrkA [35], DAPK1 [24],MYLK [48], PKCδ [45], among others (Figure 1.9). This is a result of the exten-sive variation possibilities gained by using inert metal centers as scaffolds for

Figure 1.7 Precursor complex 8 can be used to create libraries of complexes carrying differentligands L1–L4 by substituting labile ligands at elevated temperatures [31].


Figure 1.8 Major revisions of the pyridocarbazole framework (excluding “simple” substitutionsof the pyridocarbazole) leading to the new frameworks 9 [48], 10 [45], and 11 [41,46].

Figure 1.9 Binding selectivities of selectedMeggers-type kinase inhibitors within thehuman kinase dendrogram that displays theevolutionary relationship between kinases.

Adapted with permission from Blancket al. [48]. Copyright 2011, Reproduced withpermission of the American Chemical Society.


ligands, which can be altered or exchanged – in addition to the metal itself.However, the complete biological consequences of the changes in the ligand ormetal center have to be considered. Changes in the electronic properties of themetal ion lead to different excited state energies, possibly introducing previouslyunavailable side reactivity [40,50]. Such drastic changes in complex stability cancompletely change the modes of action of the metal complexes. For example,ruthenium(II) complex 12 possesses, in addition to the protein kinase inhibition,light-activatable properties that can lead to apoptosis [40]. It was indeed recentlyfound that light irradiation of 12 leads to a ligand exchange generating complex13 (Scheme 1.1). The exact cellular pathway leading to apoptosis is not known,but kinase dependence has been ruled out. Nevertheless, metal centers can serveas inert structural elements giving access to geometries that are not available forpurely organic compounds. It is expected that the concept of metal complexes asinert scaffolds for enzyme inhibition purposes will become increasingly impor-tant in the future, leading to more agents with unprecedented affinity andselectivity.

1.3Organometallic Compounds Targeting Specific Protein Residues

One of the most used and effective methods to inhibit enzymes using organo-metallic complexes, and more generally metal complexes, is by covalent coordi-nation of a metal center to a residue of a specific amino acid involved in thecatalytic activity of the enzyme (at active or allosteric sites). This metal coordina-tion renders the enzyme inactive, usually in an irreversible manner (seeScheme 1.2 for an example with a tyrosine phosphatase and a gold organo-metallic complex inhibitor [51]). More specifically, the catalytic mechanism oftyrosine phosphatases relies on the transfer of a phosphate group from the sub-strate to a cysteine residue in the catalytic site (Scheme 1.2, path (a)). Thecovalent coordination of the gold center to the cysteine residue allows for the

Scheme 1.1 Photoinduced exchange of the SeCN ligand of 12 with a chloride leads to metalcomplex 13, which can induce apoptosis via a kinase independent pathway.

1.3 Organometallic Compounds Targeting Specific Protein Residues 11

irreversible inhibition of the tyrosine phosphatase (Scheme 1.2, path (b)). At thisstage, we also invite the interested readers to look at Chapter 4, which is specifi-cally dedicated to the use of organometallic gold complexes in medicine.To achieve such an inhibition, the organometallic compound should ideally

have several features:

1) The complex has to be designed in a way that a ligand exchange is possibleas for the gold complex in Scheme 1.2 where a chloride ligand is exchangedby the sulfur of the cysteine residue.

2) The complex must be specific for an amino acid residue. This means thatthe metal ion has to be carefully selected. For example, if a cysteine residueis targeted, a (relatively) soft metal ion such as Au(I) or Pt(II) has to bechosen (see also point 3).

3) The complex has to be stable enough to reach its anticipated target. Inother words, the ligand must stabilize the complex but has to be labileenough! This “ideal” reactivity can be achieved by fine-tuning of themetal–ligand interaction. As an example of this improved kinetic stability,Berners-Price and coworkers demonstrated that Au(I) N-heterocyclic car-bene (NHC) complexes such as 14 shown in Figure 1.10 could display ahigh kinetic stability in presence of thiols [52]. Interestingly, the authorsshowed that their complexes were much more reactive (rate constants20–80-fold higher) toward selenocysteine (Sec) than to cysteine (Cys) [52].

Catalytic Site ofthe Enzyme

S

OP

O

O

O

OOH Phosphate Substrate Catalytic Site of

the Enzyme

S

OO

OP

O

O


S

OOH

N

NAuCl

N N

Au

INACTIVE

ACTIVE

Organometallic Inhibitor

(a)

(b)


S

OOH

Cysteine

Aspartic Acid

Scheme 1.2 Simplified depiction of the catalytic mechanism (path (a)) and covalent inhibition(path (b)) of a tyrosine phosphatase.


This difference was explained by the difference in pKa values of the twoamino acids (Cys= 8.5 [53]; Sec= 5.2 [54]). At pH= 7.2, the selenol is fullyionized, while the thiol is not, facilitating the attack of the selenium to thegold cation [52]. Importantly, these lipophilic, cationic Au(I) complexesselectively induced apoptosis in cancer cells but not in normal cells [52].

4) The complex should be selective for a specific enzyme. To date, as men-tioned by Kilpin and Dyson in a recent review, this endeavor has neverbeen achieved [6]. The metal complexes are targeting several enzymesinstead of a single enzyme. In the case of Au(I) complexes, in additionto the targeted enzymes thioredoxin reductases (TrxRs), cysteine prote-ases, kinases, and glutathione S-transferases (GST P1-1), these com-pounds were also shown to inhibit other cysteine-containing proteinssuch as serum albumin [6]. However, the quest of highly selectiveenzyme inhibitor is a topic of intensive investigations and researchersfrom all around the globe are coming closer and closer to achieve thisimportant aim. For example, Ott et al. have recently demonstrated thatboth the enzymatic inhibition and enzyme selectivity of NHC–Au–Lcomplexes could be tuned by subtle ligand modifications (L=Cl (15),PPh3 (16), or NHC (17); see Figure 1.10 for the structures) [55]. Morespecifically, the authors showed that the more stable the Au-L bondwas (15<16<17), the less reactive the complex was toward enzyme inhi-bition. However, a higher selectivity could be achieved when the Au-Lbond is extremely stable [55]. For example, the most stable complex 17was much more selective to TrxR than for glutathione reductase (GR)and glutathione peroxidase (GPx) [55]. These findings on gold com-pounds, although obtained on isolated enzymes, are extremely promis-ing and it can be anticipated that even more selective organometallic-based inhibitors will be discovered in the near future.

We will not review all organometallic complexes that inhibit enzymes bycovalent coordination of a metal center to an amino acid residue. We advise thereader to specific reviews and book chapters [3,4,6,56–62]. We will rather high-light an extension of the concept presented in this section. As already mentioned(point 4), ideally, an organometallic complex should be selective for an enzymeand hence not inhibit several enzymes. As a first step toward solving this

Figure 1.10 Structure of Au(I) N-heterocyclic carbene complexes.

1.3 Organometallic Compounds Targeting Specific Protein Residues 13

problem, Metzler-Nolte and coworkers recently reported the preparation oforganometallic Au(I)–peptide bioconjugates that act as TrxR inhibitors [63].More specifically, the authors envisaged to selectively bring different organo-metallic Au(I) complexes to a specific cellular organelle, namely mitochondria,which are known to host the targeted enzyme. For this purpose, they coupled,using a [3+ 2] cycloaddition reaction, tetrapeptides that have been previouslyshown by Szeto [64] and Kelley and coworkers [65] to cross cell membranes andaccumulate in mitochondria [63]. Examples of Au(I) peptide conjugates pre-pared in their work are presented in Figure 1.11. As desired, the metal conju-gates 18–20 displayed prolonged stability in the presence of one equivalent ofcysteine [63]. Worthy of note, the authors could demonstrate an interestingselectivity of their metal-containing bioconjugates for the selenocysteine-containing TrxR over the cysteine-containing GR with EC50 values for TrxRlower by at least one order of magnitude compared with the EC50 values for GR.The inhibition of TrxR is of high relevance since this enzyme plays an importantrole in the cellular and mitochondrial redox system [63]. It has been describedthat TrxR was responsible for the antiproliferative activity of gold compounds(see also Chapter 4) [66,67].

1.4The Bioisosteric Substitution

Although still not fully obvious for part of the drug design community, manyorganometallic complexes can be handled in presence of oxygen and water justlike “normal” organic compounds. Therefore, in addition to screening the substi-tution on a drug candidate with organic groups (e.g., methyl, ethyl, isopropyl, or

Figure 1.11 Structures of organometallic Au(I)-peptide bioconjugates. Prepared by Metzler-Nolte and coworkers [63].


phenyl), organometallic moieties like ferrocenyl or ruthenocenyl could (should?)be employed during this screening as well. This would allow the exploration ofadditional structures that can possibly occupy the binding pocket of an enzymein a more efficient manner than purely organic inhibitors. The idea of addingorganometallic moieties to enhance the activity of enzyme inhibitors is not new.Already in the 1970s, Edwards et al. prepared ferrocenyl-substituted penicillinand cephalosporin derivatives, which showed high antibacterial activity [68–71].In this section, we will explain, on the basis of examples from recent literature,how to design organometallic enzyme inhibitors starting from known bioactiveorganic molecules.Starting from preexisting organic enzyme inhibitors, a straight-forward man-

ner to introduce organometallic moieties is to make use of so-called bioisostericsubstitutions. Bioisosteric groups exhibit similar biological effects as a result oftheir structural resemblance. Most prominently in terms of organometallicchemistry, ferrocenyl moieties are, despite their three-dimensional rather thanflat structure, classified as bioisosteric to phenyl. Ferrocene has therefore beenextensively used in medicinal chemistry [72–75]. Ferrocene derivatives havebeen tested for their toxicity against various cancer cell lines as well as for theirantimicrobial activity. However, in most cases, the exact mechanism of actionhas not been identified. In this section, we will focus on examples in which themechanism clearly involves inhibition of an enzyme.Histones are proteins that are responsible for packing and ordering DNA in

the nucleus. They are inherently basic and therefore positively charged at physi-ological pH. The charge state of histones controls their binding strength to DNAand can be altered by changing their degree of acetylation. Histone deacetylases(HDACs) catalyze the removal of negatively charged acetyl groups from theϵ-lysine tails of histone proteins utilizing a catalytic zinc ion [76,77]. Negativelycharged DNA then tightly wraps around the positively charged histone coreleading to transcriptional silencing of genes. Different subtypes of HDACscontrol the acetylation of different histones leading to different changes in thetranscription. Class I HDACs, comprising HDAC1, 2, 3, and 8, are often overex-pressed in cancer tissues and are linked to circumvention of apoptosis and pro-gression of cancer [78]. Subtype specific HDAC inhibitors are therefore subjectof ongoing research as anticancer agents [79]. A number of HDAC inhibitorshave been found to feature an aryl “cap” combined with a zinc binding motif,such as hydroxamic acid. Vorinostat 21 (Suberoylanilide hydroxamic acid,SAHA) is a representative of this class and is marketed as Zolinza for the treat-ment of cutaneous T-cell lymphoma (Scheme 1.3) [80,81].Making use of the bioisosteric substitution paradigm, Spencer et al. designed

N1-hydroxy-N8-ferrocenyloctanediamide 22 (Jay Amin hydroxamic acid, abbre-viated JAHA) along with other similar derivatives (Scheme 1.3) [82,83]. Dockingstudies with 22, starting from a cocrystal structure of Vorinostat 21 withHDAC8 [84], supported the expectation of homologous binding interactions [82].The hydroxylamines of both 21 and 22 interact with the catalytic zinc of theenzyme and the amides of 21 and 22 form a hydrogen bond with an aspartic

1.4 The Bioisosteric Substitution 15

acid of HDAC8. The ferrocene unit of 22 occupies a part of the binding pocketof HDAC8, which is known to be malleable. Indeed, 22 inhibits class I HDACswith nanomolar affinity (in vitro assays). The docking results are in agreementwith the experimentally obtained IC50 values for 21 and 22 with HDACs(including HDAC8). The series of different ferrocene-containing derivatives allshowed selectivity for class I and IIb over class IIa HDACs. The IC50-obtainedvalues surpass the affinity of Vorinostat for certain HDAC subtypes with inhibi-tion levels in the subnanomolar range for the class IIb HDAC6. Cytotoxicityinvestigations further showed that 22 is inhibiting growth of MCF7 breast cancercells. Triazole derivatives such as 23 were also shown to inhibit HDACs with lownanomolar IC50 values. However, the biological potential of these compounds, asevaluated in vivo in Xenopus laevis, falls short of this finding [83]. This wasattributed to their inferior cellular uptake due to their poor membrane perme-ability caused by the ferrocene and triazole moieties present in 23.Compounds 22 and 24 were also tested for their ability to inhibit cell growth

of triple-negative breast cancer cell line MDA-MB231 in vitro [85]. These cellslack receptors for estrogen, progesterone, and epidermal growth factor. They arevery aggressive and have a high malignant potential [86–88]. Despite the similaractivity profiles against the pure HDACs, out of 22 and 24, only 22 shows signif-icant activity against MDA-MB231. The mechanisms involved in the cytotoxicity

Scheme 1.3 Structures of the clinical HDAC inhibitor Vorinostat and of ferrocene derivativesthat have been designed by bioisosteric substitution of the aromatic moiety.


of 22 toward MDA-MB231 cells were further investigated. Unfortunately, Vori-nostat 21 was not tested in the same study, hence complicating direct compari-son with 22. Nevertheless, the cytotoxic effects found for 22 are similar to thoseobserved for 21 in other studies, although partially on different cancer tissuescells [89–96].It has to be noted that the replacement of phenyl derivatives in known drugs

with ferrocene can also have nondesired effects going up to the abolition of theactivity of the parent compound. For example, the fungicide fluconazole hasbeen modified to incorporate a ferrocene unit instead of the difluorophenyl moi-ety (Scheme 1.4) [97]. Compound 25 does not inhibit fungal growth, neither onfluconazole resistant nor on nonresistant yeast stems – it even stimulates growthunder certain conditions.

In contrast to the examples highlighted above, in the following examples,organometallic moieties were introduced into enzyme inhibitors without replac-ing a specific bioisosteric group, such as a phenyl moiety. Carbonic anhydrase(CA) is an enzyme that catalyzes decomposition of carbonic acid to water andcarbon dioxide: H2CO3 �H2O � CO2 [98,99]. There are several CA isozymesinvolved in different cellular processes. Inhibitors of CAs are in clinical use asdiuretics, for gastric and duodenal ulcers, neurological disorders, osteoporosis,and others [100]. In order to control the biological effect of these inhibitors, sub-type specificity is essential. In this regard, subtype hCA IX is of interest as it isoverexpressed in cancer tissues and only very little expressed in normaltissues [101].Benzenesulfonamides, or more generally arylsulfonamides, are a class of car-

bonic anhydrase inhibitors that consist of a sulfonamide attached to an aromaticmoiety, classically a benzyl ring [98–100]. The deprotonated sulfonamide coor-dinates the catalytically active Zn2+ in the substrate binding pocket of theenzyme (Figure 1.12). Many different additional substituents are tolerated on thearomatic system, as can be seen by the broad range of structures of carbonicanhydrase inhibitors in clinical use including dichlorophenamide, brinzolamide,and acetazolamide (Figure 1.13).Poulsen and coworkers have studied the effect of ferrocene and ruthenocene

as substituents on benzenesulfonamides (Figure 1.13) [103]. Metallocene deriva-tives 26–29 showed good binding affinity toward CAs. Ruthenocene derivative27 was the most active compound against the relevant carbonic anhydrase hCA

Scheme 1.4 Fungicide Fluconazole and its ferrocene derivative 25.

1.4 The Bioisosteric Substitution 17

IX, surpassing the clinically available sulfonamides. Four cocrystal structures of26–29 with hCA II showed the expected coordination of the sulfonamide groupto the catalytic Zn2+ center of the enzyme (see Figure 1.12 with 27 as an exam-ple) [102]. The metallocenes are oriented toward the exterior of the bindingpocket and form hydrophobic interactions with the surrounding amino acid resi-dues. Notably, the metal centers are not directly interacting with the enzyme. Anobserved difference in binding strength between ruthenocene and ferrocenederivatives is, therefore, thought to be due to the different size of the metals,which makes the Cp–Cp distances slightly longer for the ruthenocenes.

Figure 1.12 Cocrystal structure of sulfona-mide ruthenocene derivative 27 bound to car-bonic anhydrase hCAII (PDB code: 3P44) [102].Only amino acid residues close enough forinteractions with the ligand and Zn2+ are

shown. Hydrogen bonds are depicted as pinkdotted lines, carbon in white, oxygen in red,nitrogen in blue, and metals in gray. Protonsare omitted for clarity.


Interestingly, organometallic fragments have also been directly attached to thearomatic pharmacophore (30 in Figure 1.13) [104]. Presumably for steric rea-sons, these compounds were less active than their benzenesulfonamide ligands.

1.5Novel Mechanisms of Enzyme Inhibition with Organometallic Compounds

The structural modification of a known organic drug with an organometallicfragment can dramatically alter its mode of action. A prominent example todemonstrate this fact is ferrocifen. Ferrocifen is a ferrocenyl derivative of theanticancer drug tamoxifen [105,106]. It was found to have an additional mode ofaction compared with its parent organic compound, which is based on theoxidation of the iron present in ferrocifen. Next, we will focus our attention tothe case of hexacarbonyl[2-propyn-1-ylacetylsalicylate]dicobalt (Co-AAS, 31)(Figure 1.14).More than 15 years ago, Jung et al. reported the synthesis of the dicobalthex-

acarbonyl derivative 31 (Co-ASS) of the well-known analgesic aspirin (acetylsali-cylic acid (ASS)) [107]. Non-steroidal anti-inflammatory drugs (NSAIDs) such asASS are known to bind to cyclooxygenases (COXs). This family of enzymes isresponsible for the formation of prostaglandin H2 from arachidonic acid. Threesubtypes of COX are known, namely COX-1, COX-2, and COX-3, which is asplice variant of COX-1. COX-1 is constitutionally expressed, while COX-2 isoverexpressed in many cancer cell lines. ASS is a selective inhibitor of the COX-1 subtype and its analgetic and antiaggregating effects stem from this interaction.

(b)

(a)

Figure 1.13 Therapeutic (a) and organometallic (b) arylsulfonamide carbonic anhydraseinhibitors.

1.5 Novel Mechanisms of Enzyme Inhibition with Organometallic Compounds 19

Co-ASS 31 has been shown to be toxic toward several cancer cell lines, includ-ing 3677 human melanoma and H2981 lung adenocarcinoma cancer celllines [107], LAMA-84 and K-562 leukemia cells [108], as well as MCF-7 andMDA-BM 231 human breast cancer cells [109]. For classical NSAIDs includingaspirin, there are still debates whether they are effective against cancer, and if so,by which mechanism(s) [110–113]. However, ASS has been clearly proven to beineffective in all screenings against the cancer cell lines in which 31 has shownto be active. There has been great interest in understanding the molecular mech-anism(s) responsible for the cytotoxicity of 31. Next, we discuss the reasons forthe spectacular changes in cytotoxic activity between Co-ASS 31 and ASS.By analogy to the known effects of ASS, the inhibition efficiency of both COX

subtypes has been evaluated for a series of dicobalthexacarbonyl complexes. Theinhibition constants of 31 for COX-1 and COX-2 are lower than those forASS [109]. Interestingly, 31 binds equally strongly to COX-2 and COX-1, whileASS is known to bind strongly only to COX-1. As the binding pocket of COX-2is bigger than the one of COX-1, the bulkiness of the Co2(CO)6 group couldexplain this shift in selectivity. Importantly, comparison of the COX inhibitionamong dicobalt hexacarbonyl complexes 31–34 (Figure 1.15) with their order ofcytotoxic strength revealed that a higher inhibition strength toward COX corre-lates with a higher cytotoxicity. COX inhibition is therefore thought to be (partof) the mode of action of this class of compounds.ASS is known to deactivate COX by irreversibly acetylating serine 530 in the

active site of COX-1 [116], and correspondingly serine 516 in COX-2 [117].Hence, Ott et al. were interested in the potential of Co-ASS 31 to acetylateCOX [117]. The exact acetylation sites of COX-2 after incubation with Co-ASSwere examined by LC-ESI tandem mass spectrometry. In contrast to ASS, incu-bation with Co-ASS did not lead to acetylation of serine 516, but to acetylationof several lysine residues, namely lysine 166, lysine 346, lysine 432, and lysine598 (Figure 1.16). Lysine 346 is part of the entrance channel of the active siteand thus acetylation could hinder the access of the substrate. Lysine 166 andlysine 432 are close to the heme prosthetic group, and therefore acetylation ofthese residues could abolish the function of the protein. In analogy to ASS, thesemodifications can be assumed to be irreversible.

Figure 1.14 Antiproliferative compound hexacarbonyl[2-propyn-1-ylacetylsalicylate]dicobalt(Co-ASS, 31) and its parent drug, aspirin (ASS).


In summary, Co-ASS 31 leads to acetylation of COX, however, at differentsites than ASS. Nevertheless, these acetylations are certainly involved in theinactivation of the COX enzymes.Interestingly, 32, which lacks the acetyl moiety present in Co-ASS 31 and ASS

and, thus, cannot acetylate COX, is still inhibiting COX [118]. However, thepresence of the organometallic moiety in Co-ASS enables to postulate severalalternative modes of COX inhibition. It has been shown that both the alkyneligand and the Co cluster by themselves are not effective antiproliferative

Figure 1.16 Acetylation sites of COX-2 due to incubation with Co-ASS 31. Figure reproducedwith permission from Ott et al. [117]. Copyright 2009 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim.

R = OAc 31OH 32

H 33F 35

Figure 1.15 Co-ASS 31 and derivatives Co-Prop 34 [114], Co-Diph 38 [114], and the salicylates32, 33, 35, 36 [109], and 44 [115].


agents [118]. However, if the salicylate ligand acts as a carrier to bring theorganometallic cluster to the COX binding pocket, the Co2(CO)6 moiety couldexert an enzyme inhibition activity as previously demonstrated by Jaouen andcoworkers with the interaction of the Co2(CO)6 motif with an amino acid resi-due inside a protein binding pocket [119]. In this work, the authors designed theorganometallic hexacarbonyl[ethinylestradiol]dicobalt 37, a derivative of 17α-ethinylestradiol, to bind to estrogen hormone receptors to deactivate them(Scheme 1.5) [119]. Indeed, the Co2(CO)6 moiety of 37 could react with a close-by nucleophilic cysteine moiety inside the binding pocket of the estrogen recep-tor in a Nicholas-type reaction via formation of a reactive carbocation bindingcovalently to the receptor [120]. In analogy, a similar reactivity pattern has to beconsidered as mode of action of Co-ASS 31. However, the overall structure of 31is significantly less favorable for the formation of a carbocation than the struc-ture of 37 [119,121–123]. Furthermore, the structurally related cobalt complex38 (Figure 1.15), which is cytotoxic, does not contain any methylene groups ena-bling the formation of a carbocation species [108]. Another indicator against thereactive carbocation hypothesis is the absence of Nicholas-like reactions whenincubating 31 with nucleophilic thiols [124]. In summary, a COX inhibitionmechanism based on the formation of a reactive carbocation species seemsextremely unlikely for 31.

Depending on the substituents on the alkynyl ligand, Co2(CO)6 moieties canrelease carbon monoxide [125]. The COX enzymes contain a heme prostheticgroup that is essential for electron transfer between the protein and the physio-logical substrate arachidonic acid. Since the heme group is known to bind CO, a

Scheme 1.5 Hexacarbonyl[ethinylestradiol]dicobalt 37, its potential formation of a carboca-tionic species, and subsequent reaction with a thiolate [119,122].


CO-dependent mechanism seems appealing. The influence of the alkyne substit-uents on the CO release of dicobalt hexacarbonyl alkyne complexes is signifi-cant, leading either to compounds that are stable or to compounds with rapidCO release rates (t1/2= 1.1min) [125]. Indeed, decomposition of the Co2(CO)6moiety of 31 has been observed after treatment with thiols [124]. On the otherhand, UV/vis and HPLC analysis of 31 incubated in EMEM cell culture mediumcontaining fetal calf serum did not clearly show any decomposition of 31 within24 h [124]. Notably, other metal-based ASS derivatives without CO ligands,namely copper and silver derivatives, were also shown to have antiproliferativeproperties [126]. Based on these findings, CO release could potentially play arole in the activity of Co-ASS 31 although there is no clear evidence to date.As already presented, an interaction between 31 and COX enzymes seems

likely to be involved in the cytotoxic activity observed. The higher inhibition effi-ciency of COX and the altered subtype specificity of 31 compared with its parentdrug ASS could explain the differences in activity on cancer cell lines. Further-more, the aforementioned molecular mechanisms could be amplified by agreater cellular uptake and accumulation of the compound in cells. The lipophi-licity of the Co2(CO)6 moiety is typically higher than the lipophilicity of thealkyne ligand by itself [109,127]. This might play an important role in the bio-activity of 31 since a greater lipophilicity often leads to a better cellular uptakethrough the hydrophobic cellular membrane. Indeed, for the Co2(CO)6-modified5-alkynyl-2´-deoxyuridines 39 and 40 (Figure 1.17), a correlation between cellu-lar uptake that was determined by measuring the intracellular cobalt content viaatomic absorption spectroscopy and their cytotoxicity in MCF-7 and MDA-MB231 breast cancer cells has been demonstrated [127].A similar trend has been observed for the only moderately cytotoxic fructopyr-

anose derivatives 41–43 (Figure 1.18) [128]. For these compounds, it was foundthat the cytotoxicity depends on the degree of acetalization of the hydroxylgroups of the sugar, which is directly responsible for the lipophilicity of the com-pounds. The cellular uptake was found to correlate with both cytotoxicity andlipophilicity.

Figure 1.17 Dicobalt hexacarbonyl derivatives of 5-alkynyl-2´-deoxyuridine [127].


Gust and coworkers showed by analysis of HPLC retention times that thecoordination of the Co2(CO)6 cluster fragment to ASS derivatives indeedincreases their lipophilicity [109]. Additionally, it was found that the accumula-tion grade of 31, that is, the resulting concentration of 31 inside cells, is sixtimes higher for 31 than for cisplatin. However, comparison of Co-ASS 31 withthe potential metabolite Co-Prop 34 and the pseudo-dimer 38 (Figure 1.15)revealed a lack of correlation between lipophilicity, cellular uptake, and cyto-toxicity in leukemia cells [108]. A more extensive study with other salicylatecobalt derivatives confirmed this finding [109]. Thus, the lipophilicity and cellu-lar uptake of cobalthexacarbonyl derivatives of ASS seem unrelated to theircytotoxicity.Some doubt about a pathway based purely on COX inhibition came from a

recent report on other metal cluster ASS derivatives. Compounds with struc-tures corresponding to 31 with Co4(CO)10 or Ru3(CO)9 clusters showed lowerCOX-inhibition efficiencies than 31, but similar inhibition of cell growth [129].Simple COX inhibition seems not to be exclusively the reason for the activity of31. Further investigations for alternative mechanisms of action for the cyto-toxicity of 31 are therefore discussed next.Since the great success of the inorganic anticancer drug cisplatin, metal-based

drug candidates are suspected to interfere with DNA, for example, by directcoordination. Incubation of salmon testes DNA with 31–36 showed efficientDNA association for all compounds [109]. Indeed, the attachment was more effi-cient than for cisplatin. Even more effective was the precursor Co2(CO)8. Thissuggests that the interaction with DNA results from the cobalt–alkyne moiety.However, a correlation between the efficiency of DNA association and cyto-toxicity could not be found. Moreover, after incubation with cobalt salicylates,the nuclei of both MCF-7 and MDA-MB 231 cells did not contain significantamounts of cobalt, as determined by atomic absorption spectroscopy [109]. Cell

Figure 1.18 Dicobalt hexacarbonyl derivatives of fructopyranose 41, 42, and 43. The degreeof acetylation determines the lipophilicity and correlates with their cytotoxicity in MCF-7 breastcancer cells [128].


cycle analysis of MDA-MB 231 cells after incubation with 44 (Figure 1.15)revealed an increased amount of cells in the S phase, and less in the G2/Mphase [115]. This means that the cells underwent DNA duplication but did notdivide afterward. Since DNA synthesis appears to function normally, an interac-tion of 44 with the DNA is very unlikely. Overall, the in vitro studies excludeDNA binding to be part of the mechanism of action of dicobalt hexacarbonylsalicylates, including 31.Of note, due to the high cytotoxicity against the human breast cancer cell lines

MCF-7 and MDA-MB-231 with IC50 values <2 μM, the possibility of an interac-tion of 31 with the estrogen receptor was also investigated [109]. The bindingefficiency was very low and the hypothesis was dismissed.Furthermore, the redox system of the cell could be influenced by the cobalt

cluster. Compound 31 has been shown to form disulfides when treated with thi-ols such as glutathione or cysteine [124]. The activity of glutathione reductase isa very sensitive indicator for the redox state within cells. It has been shown thatglutathione reductase activity was not influenced by 34, 31, ASS, or the alkyneligand of 31 [109]. A mechanism of action based on changing the redox state ofthe cell can therefore be excluded.Overall, the combination of the results presented above suggests that several

mechanisms are responsible for the cytotoxicity observed for 31. The inhibitionof the COX enzymes is most likely one of them, but both the NSAID as well asthe organometallic moiety could have additional activities. The cytotoxicity ofcelecoxib, a COX-2-specific NSAID, in cancer cell lines that lack COX [113], aswell as the cytotoxicity of Co2(CO)6-modified peptides that clearly do not inter-act with COX [130], underline that additional mechanisms of action are likely.The case as a whole illustrates that the search for the exact mechanism of actionof a complex can be very tedious and the use of organometallic complexes bringseven more possibilities for modes of action. This is not a disadvantage though!As shown in the case of the ferrocifens or the antimalarial drug candidate ferro-quine (Chapter 5), the additional activities gained with organometallic com-pounds can be used to circumvent resistances of purely organic enzymeinhibitors, or even establish an inhibitory activity.

1.6Organometallic Compounds as Cargo Delivers of Enzyme Inhibitors

A very recent and innovative concept using organometallic-based assemblies forenzyme inhibition purposes has been described by Smith and Therrien. In theirwork, the authors employed water-soluble organometallic cages to deliver bio-logically active compounds to cancer cells [131–133]. An example of one of theirconstructs is shown in Figure 1.19. These cages present two main interests in thefield of medicinal chemistry: (1) Despite containing several aromatic moieties,these cages are water soluble as they are usually highly charged, which offers theopportunity to bring very lipophilic compounds into cells; (2) They allow a

1.6 Organometallic Compounds as Cargo Delivers of Enzyme Inhibitors 25

selective increase of the cellular uptake of the biologically active compounds intocancer cells, thank to the enhanced permeability and retention effect (EPReffect). This effect discovered by Matsumura and Maeda in 1986 describes theability of large molecules to accumulate much more in solid tumors than inhealthy tissues [134].Diverse types of compounds were encapsulated into the organometallic cages

including photosensitizers [135], antiproliferative agents [136,137], fluorescentagents [138,139], dendrimers [140,141], and recently enzyme inhibitors [142,143].For the latter, two different kinds of enzyme inhibitors were encapsulated intothe organometallic cages, namely: (1) synthetic nucleoside derivatives such asthe FDA-approved anticancer drug used against colorectal cancer Floxuridine,which is an irreversible inhibitor of thymidylate synthase and (2) antiproliferativeruthenium arene complexes from the so-called RAPTA family (see Figure 1.20

6+

NN

N

N

N

N

Ru

Ru

N

N

N

N

N

Ru

Ru

Ru

Ru

N

O O

OO

O O

OO

OO

O O

LipophilicGuest

Figure 1.19 Structure of a water-soluble organometallic cage that can be used to deliver lipo-philic guests.

Figure 1.20 Structures of the anticancer drug candidate RAPTA-C and of the FDA approvedanticancer drug Floxuridine.


for the chemical structures of these compounds). More specifically about thisclass of compounds, the organometallic RAPTA complexes from the Dysongroup are very promising anticancer drug candidates. RAPTA stands forRuthenium Arene PTA whereby PTA is the abbreviation for the ligand 1,3,4-triaza-7-phosphatricyclo-[3.3.1.1]decane present in all complexes of this family.RAPTA-C (Figure 1.20) is one of the most famous examples of this family. Theexact mechanism of action of RAPTA-C, and in general from all this class ofcompounds, is not understood in a definitive manner. However, it is clear thatthese compounds have a fully different behavior compared with cisplatin thattargets DNA. At this stage of the research, enzyme binding is the most probableexplanation for the activity of the RAPTA complexes [58]. It was indeed demon-strated that such compounds form adducts with proteins [144] and that thereactivity of RAPTA-C and cisplatin in the presence of proteins was muchdifferent [145].To allow a stable encapsulation of these two types of enzyme inhibitors into

their organometallic cages, Therrien and coworkers connected them to pyrenederivatives (Figure 1.21). The encapsulation of the pyrenyl group into the cage isdriven by hydrophobicity and π–π interactions. Of note, the successful encapsu-lation of the compounds was confirmed by diverse NMR, X-ray crystallography,and ESI-MS techniques. In the case of the Ru arene complexes, an increase in

Figure 1.21 Structures of the encapsulated pyrenyl-arene ruthenium complexes and ofpyrenyl-Floxuridine of Therrien and coworkers.


cytotoxicity on four different human cancer cell lines was observed for 45–47(Figure 1.21) when these compounds were encapsulated. These observationsreinforce the postulate that the encapsulation increases the cellular uptake [143].This effect was clearly demonstrated by monitoring the fluorescence of the pyr-enyl moiety. An increase of uptake by a factor of two was determined for com-plex 45. Of note, the cage itself employed in this work was found to be highlycytotoxic and even in some cases more toxic than the encapsulated pyrenyl-arene ruthenium complex system itself. This intrinsic toxicity of the cage can bepotentially beneficial, provided the cage is selectively delivered to cancer cells. Inthe case of the caged Floxuridine derivatives, an important difference in cyto-toxicity between the cage and the encapsulated pyrenyl-enzyme inhibitor wasobserved. Hence, all encapsulated pyrenyl-Floxuridine systems studied werefound to be more toxic than the empty cages alone [142]. Importantly, as antici-pated by the authors, the host–guest complexes were found to be highly watersoluble contrary to the Floxuridine compounds used in the clinic [142].In a similar line of thought to what has been presented above, namely the

delivery of known enzyme inhibitors into cancer cells using organometalliccages, the Dyson group coupled, for example, the glutathione S-transferase(GST) inhibitor ethacrynic acid (EA) to two RAPTA derivatives (48 and 49 inFigure 1.22) [146,147]. In this work, in addition to the enzyme inhibition bythe known inhibitor, the authors also anticipated to engender an inhibition bythe organometallic delivery compound used to bring the enzyme inhibitor to thecancer cells (dual cytotoxic mode of action). More specifically, knowing that EA,

Figure 1.22 Structures of Ru organometallic complexes containing an EA moiety (48 and 49)or phenoxazine- (50) and anthracene-based (51) ligands.


which has been investigated as a potential anticancer drug [148], binds competi-tively to the hydrophobic cosubstrate (H-site) of GST and that the RAPTA com-pounds react with soft nucleophilic centers (e.g., thiol groups), the authorspostulated that 48 and 49 could bind to the enzyme at the H-site and could alsointeract with the two reactive cysteine residues present in GST P1-1 [146,149].As anticipated, 48 and 49 were found to bind the catalytic H-site but also tohave inhibition constants on GST P1-1 three or four times lower than EA. Thisobservation clearly demonstrates that the ruthenium centers of 48 and 49 areinvolved in the inhibition of GST P1-1. This was confirmed by X-ray crystallog-raphy and ESI-MS measurements. Hence, 49 was found to decompose, over aperiod of time, into EA and a ruthenium derivative. This cleavage is assumed tooccur by virtue of a possible allosteric effect or simply due to binding of the EAmoiety present in 49 to the H-site of GST [146].The same research group coupled phenoxazine (50) [150] and anthracene-

based (51) multidrug resistance (MDR) modulator ligands to Ru organometalliccomplexes (Figure 1.22) [151]. More specifically, the inhibition of P-glycoprotein(Pgp), which is a plasma membrane protein responsible for drug efflux from cellsand which is implicated in MDR, was studied [149,152]. The combination of theselectivity of ruthenium complexes toward cancer cells and the ability of thephenoxazine and anthracene derivatives for Pgp inhibition was found to lead toa synergistic effect. The coordination of the anthracene-based ligand to the Rucomplex (51) resulted indeed in an enhancement of the cytotoxicity as well as ofthe inhibition of Pgp, compared with the anthracene-based ligand itself [151].The other newly formed complexes in this study such as 50 were found to bealso generally more cytotoxic than their MDR modulator ligands. However, theyinhibited Pgp to a lesser extent than the original Pgp inhibitor derivatives used asligands [151]. Interestingly, it could be demonstrated with fluorescence measure-ment that 51 accumulated in cell nuclei, thanks to the presence of the fluores-cent anthracene group in 51. This observation correlates well with the resultsobtained by 3H-thymidine incorporation assay that suggests inhibition of DNAsynthesis as possible mechanism of cytotoxic action. Also, these latest resultsindicate that, although Pgp inhibition was confirmed in vitro, the main mecha-nism of action of 51 could be independent of inhibition of efflux proteins [151].Of note, Hartinger and coworkers used a similar strategy to couple ruthenium orosmium organometallic complexes to flavonoid derivatives that are known toinhibit human topoisomerases [153–155], paullone derivatives that are known toinhibit cyclin-dependent kinase (CDK) glycogen synthase kinase-3 inhibitors andmitochondrial malate dehydrogenase [156–158], 3-(1H-benzimidazol-2-yl)-1H-quinoxalin-2-one bearing pharmacophoric groups of known protein kinaseinhibitors [159] or [3-(1H-benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines (andindolo[3,2-d]benzazepines derivatives, which are known CDK inhibitors (seeFigure 1.23 for two examples of such compounds) [160–162]. It must be how-ever stated that for none of these compounds it was proven that the enzymeinhibitors were actually released in cells, although it is reasonable to anticipatethat release will ultimately happen.


1.7Organometallic Enzyme Inhibitors for Theranostic Purposes

The idea of having a drug (or two very similar drugs) that can be used for bothdiagnostic and therapeutic purposes is extremely appealing, not to say the holygrail of researchers in drug discovery! This strategy is called theranostics. Thisterm is a portmanteau of the words therapy and diagnostics. For this purpose, apotential approach is the use of a bioconjugate that contains a ligand that can becoordinated to an element that is known to have two different radioisotopes.One of them should have the right radionuclear properties to be used for diag-nosis and the other one for therapy. Hence, by designing a single bioconjugateincorporating a ligand suitable for one element, diagnosis and therapy could beundertaken [163]. Among the different elements of the periodic table, potentialdual candidates are 64Cu-67Cu, 123I � 131I, and 86Y � 90Y. Note that due to theircomparable chemical behaviors, 111In and the more “exotic” 177Lu have beenproposed as alternatives to 90Y due to the high-radiation energy of the latter.However, an important drawback of these duos is the production of the radio-isotopes. A synchrotron is indeed required. A more favorable option is the useof the 99mTc-188Re matched pair. The 99mTc radioisotope has ideal radionuclearproperties for diagnostic purposes and is available from a commercial99Mo=99mTc generator. It is the reason why this radionuclide is used in over90% of nuclear medicine scans [164]. Furthermore, its third row congener, Re,has relatively similar chemistry to that of Tc (although the kinetic behavior andstability in the higher oxidation states is somewhat different [165,166]) and hastwo particle-emitting radioisotopes (188Re and 186Re). One of those is of particu-lar interest for therapy due to its specific activity, namely 188Re (t1/2= 16.98 h,β= 2.12MeV, γ = 155 keV) [167]. Importantly, in the view of easy clinical appli-cations, 188Re can be produced conveniently using a 188W=188Re generator. Inaddition, the nonradioactive isotopes of Re (185Re and 187Re) provide a model

RuCl

O

O

Flavonoid-derivative-containing Ru complex

OBr

OsN

NCl

NH

HNBr

N

HO

OH +

Paullone-derivative-containing Os complex52 53

Figure 1.23 Structures of two Ru and Os organometallic complexes containing flavonoid(52) [153] and paullone (53) [156] derivatives.


for the chemistry of both the radioactive analogs. With this concept in mind,several groups have embarked into this field of research using the so-calledbifunctional chelator (BFC) approach (see Figure 1.24 for an example). In theBFC approach, a metal chelator is linked via a spacer to a targeting vector whoserole is to bring the radionuclide to a specific location (e.g., an overexpressedreceptor).To date, no such dual 99mTc=188Re theranostic bioconjugates have entered

into the market. Furthermore, with regard to the topic of this chapter, in mostof the radioactive conjugates currently approved for diagnostic or therapeuticpurposes, and more generally in the clinical pipeline, the target of the radiola-beled bioconjugate is not an enzyme but a specific receptor (e.g., somatostatinreceptor, epidermal growth factor receptor (EGFR), etc.). In this context, Albertoand coworkers recently developed a fully novel theranostic concept related toenzyme inhibition. They envisaged preparing, for therapeutic purposes, anenzyme inhibitor containing a cold Re atom. The diagnostic counterpart to thisRe complex was anticipated to be a 99mTc analog that could be easily synthesizedvia the �99mTc�CO�3�H2O�3�� precursor, which is generated using the Isolink

kit “Carbonyl Labeling Agent” [166]. The enzymes targeted in their study wereCAs. This family of enzymes, which holds 16 different isozymes, catalyses theformation of carbonic acid from water and CO2 (see also Section 1.3). Impor-tantly, from a drug discovery point of view, the two human CAs, namely hCA IXand hCA XII, are known to be overexpressed in a large number of hypoxictumors, mostly in the perinecrotic areas [168–170]. These two isozymes aretherefore interesting targets for both cancer diagnosis and therapy. With this inmind, Alberto and coworkers synthesized a small series of arylsulfonamide-,arylsulfamide-, and arylsulfamate-based compounds with the [(Cp-R)Re(CO)3]motif (54–57 in Figure 1.25) [171] knowing that unsubsituted sulfonamides andtheir bioisosters (sulfamates and sulfamites) bind to the Zn(II) ion of the activesite of CA by substitution of the nonproteinogenic zinc ligand to generate a tet-rahedral adduct [4,172,173] and, importantly, that metal complexes and notably

Figure 1.24 Schematic representation of the BFC approach using the peptide neurotensin asan example of biomolecule (PDB code: 2LNE).

1.7 Organometallic Enzyme Inhibitors for Theranostic Purposes 31

organometallic compounds have been previously shown to be excellent candi-dates as CA inhibitors (see also Section 1.4) [4,5,174,175].Of the four compounds prepared in their laboratories, 54 was found to be of

particular interest. Compound 54 inhibits CA II, CA XII, and CA XIV in the lownanomolar range similar to the known CA inhibitor acetazolamide (AZA), whichis an approved drug against a range of conditions including glaucoma, abnormalretention of fluids, epileptic seizures, and so on [176]. However, as can be seen inFigure 1.26, 54 has a much more pronounced selectivity pattern over the rangeof the 12 CA isozymes studied in this work compared with AZA. The enhancedselectivity of 54 was proposed to be due to a better space occupancy as previ-ously described by Meggers and coworkers [31] with kinase inhibitors or byPoulsen and coworkers [177] with CA inhibitors (see Sections 1.2 and 1.4). Of

Figure 1.25 Structures of Re-containing CA inhibitors prepared by Alberto and coworkers [171]and of the drug Acetazolamide (AZA).

0

100

200

300

400

500

600

CA II CA IV CA VA CA VB CA VI CA VII CA IX CA XII CA XIII CA XIV CA XV

Ki n

M

54

AZA

Figure 1.26 Comparison of the inhibition constants of 54 and AZA over a range of isozymesof hCAs.


great importance from a pharmaceutical point of view, 54 inhibits much morehCA IX, which is overexpressed in certain tumors, than the physiologically dom-inant hCA II [171].The authors of the study could also demonstrate by X-ray crystallography that

one of their organometallic CA inhibitors, namely 57, was indeed able to bind tohCA II. As shown in Figure 1.27, 57 binds its target in a manner similar to otherCA inhibitors by coordinating the Zn atom of the active site of hCA II throughthe deprotonated nitrogen of arylsulfonamide. As in the case of Meggers’ kinaseinhibitors and of Paulsen’s CA inhibitors, the metal ion is not playing a directrole in the binding of the inhibitor to the enzyme. The organometallic partCpRe(CO)3 is neither interacting with the protein nor with water molecules.However, a hydrophobic interaction between the CpRe(CO)3 moiety and threeamino acids (phenylalanine 131, leucine 198, and proline 202) was observed(Figure 1.27).In order to confirm the theranostic concept of their project, Alberto and

coworkers had still to demonstrate that a 99mTc analog of their Re-containingCA inhibitors could be easily prepared in water since the 99mTcO4

� is elutedfrom the 99Mo=99mTc generator as a saline solution from the generator. Towardthis aim, as presented in Scheme 1.6 with the 99mTc analog of 56 (58) as anexample, they prepared the 99mTc complex by reacting �99mTcO4�� with the

Figure 1.27 Crystal structure of complex 57 � hCA II (PDB code: 3RJ7). The Rhenium CA inhib-itor 57 coordinates the Zn atom (in magenta) of the active site of hCA II through the deproto-nated nitrogen of arylsulfonamide. The Zn atom is bound to three histidines.

1.7 Organometallic Enzyme Inhibitors for Theranostic Purposes 33

dicyclopentadienyl dimer 59 that they had initially synthesized. The authenticityof 58 was then confirmed by comparison of the HPLC retention times of 58(γ-trace) and 56 (UV trace). As mentioned by the authors, this concept can beextended to any enzyme inhibitor and holds therefore great promises.

1.8Conclusion

The increasing demand in specific enzyme inhibitors by the pharmaceuticalindustry for the development of new drugs is a fantastic opportunity for bioorga-nometallic chemists to display the full potential of their compounds in medicinalchemistry. As explained in this chapter, one important advantage of organo-metallic compounds compared with purely organic ones lies in the additionalgeometric and electrostatic profiles that can be created. This can be exploited by“simple” bioisosteric derivatization of a known enzyme inhibitor, or by designingnew inert organometallic scaffolds. As nicely demonstrated with the work ofMeggers on kinase inhibitors, by using the unique physicochemical properties ofsuch complexes, higher enzyme specificity can be obtained compared with whenpurely organic compounds are employed. Importantly, this specificity can befound to be useful not only for drug development reasons but also in the field ofchemical biology where such inhibitors are highly valued to understand cellularprocesses. For instance, two of Meggers’ Ru(II) organometallic complexes arecurrently commercially available as GSK3 and Pim-1 inhibitors. Moreover, thereactivity of metal centers of organometallic compounds as well as their electro-chemical properties can also be exploited for enzyme inhibition purposes. Forexample, metal centers can covalently bind to certain protein residues or releasebioactive compounds, allowing for modes of enzyme inhibition for which noresistances are known to date. Moving beyond therapeutic uses, the metal ionspresent in organometallic complexes can be tracked by various analytical meth-ods, making them valuable diagnostic tools. We are optimistic that the variousadvantages that organometallic compounds yield will soon lead to new excitingadvances in the field of medicinal chemistry and chemical biology.

Scheme 1.6 Synthetic procedure for the 99mTc analog of 56, namely 58; (i) Isolink kit “CarbonylLabeling Agent” �99mTcO4�� .


Acknowledgments

This work is funded by the Swiss National Science Foundation (SNSF Professor-ship PP00P2_133568 to G.G.), the Stiftung für wissenschaftliche Forschung ofthe University of Zurich, and the University of Zurich. We thank Dr. DanielCan and Prof. Roger Alberto for providing us with raw data for the preparationof Figure 1.26. We also thank Prof. Christian Hartinger, Dr. Henrik Braband,Prof. Ingo Ott, Prof. Ian Fairlamb, Prof. Bruno Therrien, Prof. Fabio Zobi, andAnita Schmitz for helpful discussions.

Abbreviations

ASS acetylsalicylic acid, aspirinATP adenosine triphosphateAZA acetazolamideBFC bifunctional chelatorCA carbonic anhydraseCDK cyclin-dependent kinaseCOX cyclooxygenaseCp cyclopentadienylCys cysteineDAPK1 death-associated protein kinase 1DNA deoxyribonucleic acidEA ethacrynic acidEGFR epidermal growth factor receptorEPR effect enhanced permeability and retention effectESI-MS electron spray ionization – mass spectrometryFDA Food and Drug AdministrationGPx glutathione peroxidaseGR glutathione reductaseGST glutathione S-transferaseHDAC histone deacetylaseHPLC high-performance liquid chromatographyIC50 inhibitory concentration 50JAHA Jay Amin hydroxamic acidL ligandLC-ESI liquid chromatography – electron spray ionization (mass

spectrometry)MDR multidrug resistanceNHC N-heterocyclic carbeneNMR nuclear magnetic resonanceNSAID Non-steroidal anti-inflammatory drugsPDB Protein Data BankPgp P-glycoprotein

Abbreviations 35

RAPTA ruthenium arene PTA whereby PTA is the ligand 1,3,4-triaza-7-phosphatricyclo-[3.3.1.1]decane

SAHA suberoylanilide hydroxamic acidSAR structure–activity relationshipSec selenocysteineTrxR thioredoxin reductaseUV/vis ultraviolet/visible light

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