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Mechanisms of action of carbonic anhydrase inhibitors
C H A P T E R 12
Mechanisms of action of carbonic anhydrase inhibitors: compounds that bind “out of the binding site” and compounds with an unknown mechanism of action Simone Carradori1, Paolo Guglielmi2 1 2
Department of Pharmacy, “G. D’Annunzio” University of Chieti-Pescara, Chieti, Italy; Department of Drug Chemistry and Technologies, Sapienza University of Rome, Rome, Italy
12.1 Introduction Despite the large plethora of carbonic anhydrase (CA) inhibitors published so far, in-depth crystallographic studies demonstrating the exhaustive mechanism of inhibition of each chemical class have been validated and reported only for few of them [1,2]. They usually coordinate the catalytic Zn(II) ion in the active site (e.g., deprotonated primary and secondary sulfonamides and their bioisosteres, dithiocarbamates, small inorganic anions) in tetrahedral geometries (albeit some of them also show distorted tetrahedral or trigonal bipyramidal coordination geometries), the water molecules network around the zinc ion (phenols, polyamines), or they occlude the entrance of the active site ([thio]coumarins and related compounds). By means of a classic medicinal chemistry approach, the rational design prompted all the efforts toward the catalytic site of the enzyme trying to block the carbon dioxide hydration. Conversely, the production of CA activators was strictly oriented against the proton shuttle (His64) responsible for the “boosting” of the catalytic activity during the slow enzymatic restoring of the active site. In 2015, a serendipitous discovery opened a new scenario for CA inhibition with the proposal of a new chemical scaffold endowed with an innovative mechanism of inhibition: “out of the binding site.” Moreover in the last years, starting from the classic and potent sulfonamide and sulfamate compounds, new chemical entities were synthesized as CA inhibitors without presenting the general features as zinc binders. The absence of deprotonatable moieties led to an unexpected biological activity with isoform selectivity,
Carbonic Anhydrases. https://doi.org/10.1016/B978-0-12-816476-1.00012-5 Copyright © 2019 Elsevier Inc. All rights reserved.
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258 Chapter 12 which warranted investigation. Currently, the absence of crystallographic data does not allow drawing information on the rational design of new inhibitors.
12.2 “Out of the binding site” inhibitors Among the various classes of inhibitors, carboxylic acids can efficiently display different binding modes both coordinating the Zn(II) ion (formate, acetate, some phenolic acids) and anchoring to the zinc-bound water molecule (some phenolic acids). Surprisingly, two phenolic acids (1 and 2) were reported in hCA I and II adducts to be present not only in the active site but also to establish pivotal interactions with Tyr7, Asn11, and water molecules in an external region of the enzyme close to the proton shuttle His64 [3]. With the aim to enlarge the knowledge of this scaffold, an ortho-substituted benzoic acid, namely 2-benzylsulfinylbenzoic acid (3), was synthesized as racemic mixture by means of sulfur oxidation of compound 4 [4]. The starting idea was to add an additional functional group (sulfoxide in its zwitterionic form), rarely investigated as inhibitor of metalloenzymes, with the concurrent presence of COOH, thus inferring an ambidentate character. The presence of a steric-hindered benzyl group should have limited the interaction with active site with respect to 1 and 2. Moreover, the introduction of a chiral center could determine a selective chiral recognition of the enzyme (Fig. 12.1). The inhibition data of four CA isoforms (I, II, IX, and XII) revealed an unusual behavior. Whereas compound 4 was almost inactive against all of them, 3 displayed a higher selectivity and a nanomolar activity against hCA II. The X-ray crystallographic analysis revealed, accordingly to the results for compounds 1 and 2, the presence of the
Figure 12.1 “Out of the binding site” inhibitors.
“Out of the binding site” and unknown mechanisms of action 259
Figure 12.2 (A) Solvent accessible surface of hCA II in its complex with 3. Residues delimiting the cavity where the inhibitor is bound are highlighted in gold. The catalytic zinc ion is represented as a blue sphere at the bottom of the active site cavity. (B) Cartoon view of the binding mode of 3 into hCA II accessory pocket. Hydrogen bonds are represented as black dashed lines.
˚ far away from the inhibitor in a cavity on the protein surface, at a distance of about 14 A Zn(II) ion and surrounded by Gly6, Tyr7, Gly8, Asn11, His64, Phe231, Asn232, and Glu239 (Fig. 12.2). Differently from compounds 1 and 2, derivative 3 was not found in the active site, despite a structural superposition showed that the phenyl rings of the three inhibitors were largely superimposable. The proximity with His64 prompted the investigation of the putative role exerted by 3 in the limitation of the rate-determining step of CA-catalyzed reaction (Fig. 12.2B). His64 is well known in most of the highly efficient CA isoforms as a proton shuttle, and it can exist in two opposite conformations, namely “in” and “out.” In the former, the histidine, oriented toward the active site, is prone to accept a proton to regenerate the nucleophilic species, whereas in the latter, this residue is oriented toward the exterior of the active site to release the proton to the bulk solvent. This flexible movement is responsible for the restoration of the catalytic activity of the active site. Compound 3 established numerous polar interactions with two amino acids (Tyr7 and Asn11) and with water molecules bound to Trp5 and His64, and strong van der Waals contacts (Fig. 12.2). Collectively acting on the protein surface and not entering the active site, these interactions froze His64 in a rigid and not functional “out” conformation. To further demonstrate this atypical enzymatic decrement, compound 3 was coincubated with imidazole as proton acceptor and a well-known CA activator. The addition of solutions with increasing amounts of imidazole counterbalanced the inhibition exhibited by 3. These results should be more investigated as different amino acid residues can delimit this cavity
260 Chapter 12 in each hCA isozyme, thus explaining the isoform selectivity of this compound and revealing the structural requirements for future drug design studies of selective CA inhibitors.
12.3 Carbonic anhydrase inhibitors with unknown mechanism of action 12.3.1 Acyclic tertiary sulfonamides Primary and secondary sulfonamide inhibitors of CA were widely investigated and clinically used due to their ability to lose a proton and act as a zinc-binding group (ZBG) in their deprotonated form [5e7]. Conversely, tertiary sulfonamides, lacking this depronatable moiety, were recently proposed as alternative chemotypes for the significant inhibition of a large number of CA isoforms. A large plethora of newly synthesized derivatives can be clustered in different chemical scaffolds as reported in Fig. 12.3. The first series comprised tosyl pyrroles and benzenesulfonamides (a) with moderate inhibitory potency against hCA I and II (off-target isoforms). Changes in the substitution pattern did not alter the biological activity and selectivity [8,9]. The second series comprehended halogenated tertiary benzenesulfonamides (b) tested against hCA I, II, IX, and XII and obtained through an innovative synthetic process. With respect to the chlorinated derivative (KI hCA II/KI hCA IX, or XII w65), the introduction of fluorine atom in the aliphatic chain of the scaffold led to an improvement (KI hCA II/KI hCA IX, or XII >1000) of the selective inhibition against the tumor-associated isoforms (IX and XII). The results were in the low nanomolar range despite the different R and R1 substituents. The authors confirmed a pivotal role of the strong electron-withdrawing fluorine for these nitrogen-containing compounds leading to a modification of the nitrogen basicity and molecule conformation via fluorine “gauche” effect and electrostatic interactions. Moreover, the interaction with the Zn(II) ion could be limited due to steric impairment [9,10]. In addition, some of these potent inhibitors were investigated for the possibility to be labeled with 18F for imaging purposes in CA IX overexpressing tumors. Synthesis and pharmacokinetic data of these radiotracers were described along with positron emission tomography (PET) images in NSG mice bearing HT-29 human colorectal tumor xenografts [11]. The third series (c) dealt with fluorinated and N-substituted pyrrolidines and halogenated N-nosylpiperidines endowed with a moderate hCA II inhibition and selectivity with respect to hCA I, IX, and XII. Regarding structureeactivity relationship (SAR) studies, it was possible to note that the introduction of the fluorine atom in the b-position of the nitrogen atom increased the inhibitory potency. The biological results also confirmed the impact of the geometrical shape of the molecule on the hCA IIeselective inhibition, reinforcing the hypothesis of a nonezinc(II)-binding mode of action within this scaffold and a pivotal role
“Out of the binding site” and unknown mechanisms of action 261
Figure 12.3 Acyclic and cyclic tertiary sulfonamides as alternative hCA inhibitors.
262 Chapter 12 of the structural shape on the inhibitory efficiency. Moreover, no relationship between chirality and biological activity was demonstrated [12]. The fourth series (d) collected hybrid molecules connecting a pyridine and an N-substituted indole through a chalcone linker. The compounds were tested against hCA II and IX in vitro by measuring the esterase activity, evaluated as antiproliferative and apoptotic agents against MCF-7 and HepG-2 cells, and submitted to molecular modeling assays to unravel the structural requirements useful for the CA IX inhibition [13]. Starting from these results on tertiary sulfonamide compounds, D’Ascenzio et al. also took advantage of such structural similarity present in the uricosuric agent probenecid, 4-(N,N-dipropylsulfamoyl)benzoic acid. From a biological point of view, it was shown to inhibit hCA II and IX in the nanomolar range, hCA XII in the micromolar range, and to be inactive against hCA I. In its structure, a tertiary sulfonamide is present and characterized by a striking steric hindrance. An additional COOH probably could participate to the CA inhibitory activity. To better demonstrate this issue, 24 novel Probenecid derivatives were designed by changing this functionality to an N-substituted amide (Fig. 12.4A). The in vitro results against four hCA isoforms (I, II, IX, and XII) showed that this chemical modification, along with the introduction of aliphatic, cycloaliphatic, and (hetero)aromatic tails, oriented the inhibitory activity toward hCA IX and XII isoforms limiting the interaction against the off-target hCA I and II isoforms [14,15]. Moreover, the same research group evaluated the addition of L-amino acid tails improving the aqueous solubility of the previous series. These new derivatives displayed inhibitory activity in the nanomolar range and strong selectivity against hCA IX and XII [16]. All these Probenecid-based compounds were analyzed in silico to unravel the putative interaction with the target (Fig. 12.4B). Surprisingly, they did prefer not to orient the tertiary sulfonamide toward the Zn(II) ion, but they were able to establish additional interactions by using the opposite portion of the molecule (amide fragment) properly functionalized. The Zn(II) ion could be a part of this complex network without being the main player as reported for ZBG-possessing inhibitors. Important enzyme amino acids involved in these interactions were Asn62, Gln92, Trp5, His64, Pro202, His94, Gln67, and Leu91 for hCA IX and Lys67, Thr91, Asn62, Thr200, His94, Gln92, Ser132, Trp5, and Thr199 for hCA XII. Unfortunately, so far, no crystallographic information is available to further support these in silico hypotheses.
12.3.2 Cyclic tertiary sulfonamides and their “open” derivatives: saccharin-based compounds The sweetener saccharin was proposed and tested as hCA inhibitor due to the presence of a cyclic secondary sulfonamide group endowed with a nonselective CA inhibition profile,
“Out of the binding site” and unknown mechanisms of action 263
Figure 12.4 (A) Probenecid-based derivatives as unusual hCA inhibitors and (B) their putative interactions with the enzyme.
264 Chapter 12 although with a strong preference (>1000-fold) for the tumor-associated isoforms in the nanomolar range. hCA II-saccharin and hCA IX-mimic-saccharin adducts have determined that the cyclic secondary sulfonamide behaved as a classic ZBG (benzoic sulfinimide) displacing in the deprotonated form the OH/H2O bound to the Zn(II) ion [17,18]. The concurrent presence of other ZBGs (e.g., primary sulfonamide) also led to its direct interaction within the active site prevailing on the cyclic secondary sulfonamide moiety [19]. To investigate the potential of this chemical structure, researchers have adopted many synthetic strategies to obtain more selective derivatives. The first approach was the derivatization of the sulfonamide nitrogen leading to 52 novel tertiary cyclic sulfonamides (Fig. 12.5A). The absence of a deprotonatable sulfonamide made them interesting compounds for the further design of innovative CA inhibitors.
Figure 12.5 (A) Saccharin-based derivatives as unusual hCA inhibitors and (B) their chemical modifications.
“Out of the binding site” and unknown mechanisms of action 265 The chemical space around this position was deeply explored by the insertion of aliphatic chains with or without the presence of unsaturation, functional groups (ketone, ester, carboxylic acid, hydroxamic acid, amide), ortho-/meta-/para-substituted benzyl and benzoyl moieties, and heterocyclic ring (isoxazole, phthalimide). All the compounds were fully characterized and tested in vitro against the two off-target isoforms (I and II) and the tumor-associated transmembrane isoforms (IX and XII) achieving potent nanomolar inhibition with high selectivity toward hCA XII [20e22]. Indeed, when another substituent could act as zinc binder within the molecule, it was demonstrated that the mechanism of inhibition could be ascribed to its direct interaction with the Zn(II) ion [23]. In this case, the remaining portion (saccharin nucleus) of the molecule adapted itself to improve the stability of the adduct. SAR studies of this large series of saccharin-based compounds revealed an almost inactivity against hCA I and II (with the exception of few derivatives) and a strong inhibitory activity by all compounds toward hCA XII. The hCA IX inhibition followed a not defined profile with steep variations due to small chemical changes. To further corroborate the importance of this scaffold, the same research groups, starting from the N-substituted saccharin, aimed at opening the isothiazolone core nucleus by means of hydrolytic (NaOH) or reductive (NaBH4) agents obtaining compounds characterized by a strong nanomolar inhibitory activity and selectivity against hCA IX and XII (Fig. 12.5B). Among closed and open saccharin derivatives, there were substantial differences in terms of ZBGs, but they displayed almost the same biological profile, thus confirming the possibility for the saccharin derivatives to act through an unknown mechanism of action [22,24].
12.3.3 Cyclic sulfamate inhibitors: acesulfame derivatives Starting from the chemical structure and biological results of saccharin derivatives, another synthetic sweetener (acesulfame as an ambidentate nucleophile) was deeply investigated. As previously reported for saccharin, acesulfame behaved as a classic CA inhibitor orienting its ZBG (a cyclic secondary sulfamate) toward the Zn(II) ion in the hCA II-acesulfame and hCA IX-mimic-acesulfame adducts at 1.5 A resolution [25]. Confirming the in vitro biological data (KI hCA IX ¼ 2.41 mM, KI hCA I, II, and XII > 20 mM), acesulfame was shown to bind directly to the Zn(II) ion in hCA IX-mimic, whereas different active site binding modes were detected for hCA II. The replacement of a not deprotonatable and bioisosteric cyclic moiety (sulfamate) with that of saccharin (sulfonamide) stimulated the design and synthesis of novel 19 N- and 12 O-substituted compounds, in vitro biological evaluation and in silico validation of their potency/selectivity [21,26]. The nucleophilic sites of acesulfame were decorated with alkyl (saturated and unsaturated) chains and (substituted) benzyl and benzoyl moieties. Collectively, they displayed a low affinity for hCA I and II isoforms and a higher preference for hCA IX and XII in the low nanomolar range. Nevertheless, a proper SAR for
266 Chapter 12
Figure 12.6 Acesulfame-based derivatives as unusual hCA inhibitors and their chemical modifications.
this scaffold can be hardly extrapolated because most of them are potent and selective disregarding the substitution pattern. The introduction of such substituents on the parent compound, which was only characterized by a slight hCA IX inhibition in the micromolar range, led to an improvement of CA blockade. Moreover, O-substituted derivatives were better inhibitors than their N-counterparts against hCA XII (Fig. 12.6). Docking studies confirmed that none of the compounds could interact directly with the Zn(II) ion in the hCA IX and XII active sites due to steric clashes, leading the sulfamate and carbonyl oxygens to be solvent exposed. Additional electrostatic interactions could be ascribed to substituents (bromine, nitro) on the benzyl/benzoyl portion of the inhibitor. Unfortunately, so far, no hCA adducts were obtained for these compounds to further determine the atypical mode of inhibition.
12.4 Conclusions Despite the large number of newly synthesized derivatives and natural products endowed with a high inhibitory potential against hCAs, few of them are characterized by a promising selectivity profile. Sulfonamides and other zinc binders are well known to interact with a strongly conserved portion of the active site within the 15 hCA isoforms. To overcome this problem, medicinal chemists have elegantly explored new chemotypes without the possibility to act as zinc binders (absence of depronatable protons). Along with saccharin-, acesulfameand probenecid-based compounds, several tertiary sulfonamides and sulfamates were designed, synthesized, and tested. The results suggested a promising in vitro selectivity profile especially oriented toward the two tumor-associated cancer isoforms (hCA IX and XII) in the nanomolar range. Lastly, one carboxylic acid derivative (3, Fig. 12.1) was serendipitously shown to interact with the proton shuttle His64 leading to a catalytically “frozen” enzyme. This completely different adduct opened a new scenario in the field of hCA inhibitors being involved a not conserved region of the enzyme.
“Out of the binding site” and unknown mechanisms of action 267
References [1] Supuran CT. How many carbonic anhydrase inhibition mechanisms exist? J Enzym Inhib Med Chem 2016;31:345e60. [2] Lomelino CL, Supuran CT, McKenna R. Non-classical inhibition of carbonic anhydrase. Int J Mol Sci 2016;17:1150. [3] Martin DP, Cohen SM. Nucleophile recognition as an alternative inhibition mode for benzoic acid based carbonic anhydrase inhibitors. Chem Commun 2012;48:5259e61. [4] D’Ambrosio K, Carradori S, Monti SM, Secci D, Vullo D, Supuran CT, et al. Out of the active site binding pocket for carbonic anhydrase inhibitors. Chem Commun 2015;51:302e5. [5] De Simone G, Langella E, Esposito D, Supuran CT, Monti SM, Winum JY, et al. Insights into the binding mode of sulphamates and sulphamides to hCA II: crystallographic studies and binding free energy calculations. J Enzym Inhib Med Chem 2017;32:1002e11. [6] Supuran CT. Advances in structure-based drug discovery of carbonic anhydrase inhibitors. Expert Opin Drug Discov 2017;12:61e88. [7] D’Ambrosio K, Smaine FZ, Carta F, De Simone G, Winum JY, Supuran CT. Development of potent carbonic anhydrase inhibitors incorporating both sulfonamide and sulfamide groups. J Med Chem 2012;55:6776e83. [8] Alp C, Maresca A, Alp NA, Gu¨ltekin MS, Ekinci D, Scozzafava A, et al. Secondary/tertiary benzenesulfonamides with inhibitory action against the cytosolic human carbonic anhydrase isoforms I and II. J Enzym Inhib Med Chem 2013;28:294e8. [9] Me´tayer B, Martin-Mingot A, Vullo D, Supuran CT, Thibaudeau S. Superacid synthesized tertiary benzenesulfonamides and benzofuzed sultams act as selective hCA IX inhibitors: toward understanding a new mode of inhibition by tertiary sulfonamides. Org Biomol Chem 2013;11:7540e9. [10] Me´tayer B, Mingot A, Vullo D, Supuran CT, Thibaudeau S. New superacid synthesized (fluorinated) tertiary benzenesulfonamides acting as selective hCA IX inhibitors: toward a new mode of carbonic anhydrase inhibition by sulfonamides. Chem Commun 2013;49:6015e7. [11] Lau J, Pan J, Zhang Z, Hundal-Jabal N, Liu Z, Benard F, Lin K-S. Synthesis and evaluation of 18Flabeled tertiary benzenesulfonamides for imaging carbonic anhydrase IX expression in tumours with positron emission tomography. Bioorg Med Chem Lett 2014;24:3064e8. [12] Le Darz A, Mingot A, Bouazza F, Castelli U, Karam O, Tanc M, et al. Fluorinated pyrrolidines and piperidines incorporating tertiary benzenesulfonamide moieties are selective carbonic anhydrase II inhibitors. J Enzym Inhib Med Chem 2015;30:737e45. [13] Peerzada MN, Khan P, Ahmad K, Hassan MI, Azam A. Synthesis, characterization and biological evaluation of tertiary sulfonamide derivatives of pyridyl-indole based heteroaryl chalcone as potential carbonic anhydrase IX inhibitors and anticancer agents. Eur J Med Chem 2018;155:13e23. [14] Carradori S, Mollica A, Ceruso M, D’Ascenzio M, De Monte C, Chimenti P, et al. New amide derivatives of Probenecid as selective inhibitors of carbonic anhydrase IX and XII: biological evaluation and molecular modelling studies. Bioorg Med Chem 2015;23:2975e81. [15] D’Ascenzio M, Carradori S, Secci D, Vullo D, Ceruso M, Akdemir A, et al. Selective inhibition of human carbonic anhydrases by novel amide derivatives of probenecid: synthesis, biological evaluation and molecular modelling studies. Bioorg Med Chem 2014;22:3982e8. [16] Mollica A, Costante R, Akdemir A, Carradori S, Stefanucci A, Macedonio G, et al. Exploring new Probenecid-based carbonic anhydrase inhibitors: synthesis, biological evaluation and docking studies. Bioorg Med Chem 2015;23:5311e8. [17] Mahon BP, Hendon AM, Driscoll JM, Rankin GM, Poulsen S-A, Supuran CT, et al. Saccharin: a lead compound for structure-based drug design of carbonic anhydrase IX inhibitors. Bioorg Med Chem 2015;23:849e54. [18] Moeker J, Peat TS, Bornaghi LF, Vullo D, Supuran CT, Poulsen SA. Cyclic secondary sulfonamides: unusually good inhibitors of cancer-related carbonic anhydrase enzymes. J Med Chem 2014;57:3522e31.
268 Chapter 12 [19] Alterio V, Tanc M, Ivanova J, Zalubovskis R, Vozny I, Monti SM, et al. X-ray crystallographic and kinetic investigationsof 6-sulfamoyl-saccharin as a carbonic anhydrase inhibitor. Org Biomol Chem 2015;13:4064e9. [20] D’Ascenzio M, Carradori S, De Monte C, Secci D, Ceruso M, Supuran CT. Design, synthesis and evaluation of N-substituted saccharin derivatives as selective inhibitors of tumor-associated carbonic anhydrase XII. Bioorg Med Chem 2014;22:1821e31. [21] Carradori S, Secci D, De Monte C, Mollica A, Ceruso M, Akdemir A, et al. A novel library of saccharin and acesulfame derivatives as potent and selective inhibitors of carbonic anhydrase IX and XII isoforms. Bioorg Med Chem 2016;24:1095e105. [22] Ivanova J, Carta F, Vullo D, Leitans J, Kazaks A, Tars K, et al. N-Substituted and ring opened saccharin derivatives selectively inhibit transmembrane, tumor-associated carbonic anhydrases IX and XII. Bioorg Med Chem 2017;25:3583e9. [23] Langella E, D’Ambrosio K, D’Ascenzio M, Carradori S, Monti SM, Supuran CT, et al. A combined crystallographic and theoretical study explains the capability of carboxylic acids to adopt multiple binding modes within carbonic anhydrase active site. Chem Eur J 2016;22:97e100. [24] D’Ascenzio M, Guglielmi P, Carradori S, Secci D, Florio R, Mollica A, et al. Open saccharin-based secondary sulfonamides as potent and selective inhibitors of cancer-related carbonic anhydrase IX and XII isoforms. J Enzym Inhib Med Chem 2017;32:51e9. [25] Murray AB, Lomelino CL, Supuran CT, McKenna R. “Seriously sweet”: acesulfame K exhibits selective inhibition using alternative binding modes in carbonic anhydrase isoforms. J Med Chem 2018;61:1176e81. [26] De Monte C, Carradori S, Secci D, D’Ascenzio M, Vullo D, Ceruso M, et al. Cyclic tertiary sulfamates: selective inhibition of the tumor-associated carbonic anhydrases IX and XII by N- and O-substituted acesulfame derivatives. Eur J Med Chem 2014;84:240e6.
Mechanisms of action of carbonic anhydrase inhibitors: compounds that bind “out of the binding site” and compounds with an unknown mechanism of action Simone Carradori1, Paolo Guglielmi2 1 2
Department of Pharmacy, “G. D’Annunzio” University of Chieti-Pescara, Chieti, Italy; Department of Drug Chemistry and Technologies, Sapienza University of Rome, Rome, Italy
12.1 Introduction Despite the large plethora of carbonic anhydrase (CA) inhibitors published so far, in-depth crystallographic studies demonstrating the exhaustive mechanism of inhibition of each chemical class have been validated and reported only for few of them [1,2]. They usually coordinate the catalytic Zn(II) ion in the active site (e.g., deprotonated primary and secondary sulfonamides and their bioisosteres, dithiocarbamates, small inorganic anions) in tetrahedral geometries (albeit some of them also show distorted tetrahedral or trigonal bipyramidal coordination geometries), the water molecules network around the zinc ion (phenols, polyamines), or they occlude the entrance of the active site ([thio]coumarins and related compounds). By means of a classic medicinal chemistry approach, the rational design prompted all the efforts toward the catalytic site of the enzyme trying to block the carbon dioxide hydration. Conversely, the production of CA activators was strictly oriented against the proton shuttle (His64) responsible for the “boosting” of the catalytic activity during the slow enzymatic restoring of the active site. In 2015, a serendipitous discovery opened a new scenario for CA inhibition with the proposal of a new chemical scaffold endowed with an innovative mechanism of inhibition: “out of the binding site.” Moreover in the last years, starting from the classic and potent sulfonamide and sulfamate compounds, new chemical entities were synthesized as CA inhibitors without presenting the general features as zinc binders. The absence of deprotonatable moieties led to an unexpected biological activity with isoform selectivity,
Carbonic Anhydrases. https://doi.org/10.1016/B978-0-12-816476-1.00012-5 Copyright © 2019 Elsevier Inc. All rights reserved.
257
258 Chapter 12 which warranted investigation. Currently, the absence of crystallographic data does not allow drawing information on the rational design of new inhibitors.
12.2 “Out of the binding site” inhibitors Among the various classes of inhibitors, carboxylic acids can efficiently display different binding modes both coordinating the Zn(II) ion (formate, acetate, some phenolic acids) and anchoring to the zinc-bound water molecule (some phenolic acids). Surprisingly, two phenolic acids (1 and 2) were reported in hCA I and II adducts to be present not only in the active site but also to establish pivotal interactions with Tyr7, Asn11, and water molecules in an external region of the enzyme close to the proton shuttle His64 [3]. With the aim to enlarge the knowledge of this scaffold, an ortho-substituted benzoic acid, namely 2-benzylsulfinylbenzoic acid (3), was synthesized as racemic mixture by means of sulfur oxidation of compound 4 [4]. The starting idea was to add an additional functional group (sulfoxide in its zwitterionic form), rarely investigated as inhibitor of metalloenzymes, with the concurrent presence of COOH, thus inferring an ambidentate character. The presence of a steric-hindered benzyl group should have limited the interaction with active site with respect to 1 and 2. Moreover, the introduction of a chiral center could determine a selective chiral recognition of the enzyme (Fig. 12.1). The inhibition data of four CA isoforms (I, II, IX, and XII) revealed an unusual behavior. Whereas compound 4 was almost inactive against all of them, 3 displayed a higher selectivity and a nanomolar activity against hCA II. The X-ray crystallographic analysis revealed, accordingly to the results for compounds 1 and 2, the presence of the
Figure 12.1 “Out of the binding site” inhibitors.
“Out of the binding site” and unknown mechanisms of action 259
Figure 12.2 (A) Solvent accessible surface of hCA II in its complex with 3. Residues delimiting the cavity where the inhibitor is bound are highlighted in gold. The catalytic zinc ion is represented as a blue sphere at the bottom of the active site cavity. (B) Cartoon view of the binding mode of 3 into hCA II accessory pocket. Hydrogen bonds are represented as black dashed lines.
˚ far away from the inhibitor in a cavity on the protein surface, at a distance of about 14 A Zn(II) ion and surrounded by Gly6, Tyr7, Gly8, Asn11, His64, Phe231, Asn232, and Glu239 (Fig. 12.2). Differently from compounds 1 and 2, derivative 3 was not found in the active site, despite a structural superposition showed that the phenyl rings of the three inhibitors were largely superimposable. The proximity with His64 prompted the investigation of the putative role exerted by 3 in the limitation of the rate-determining step of CA-catalyzed reaction (Fig. 12.2B). His64 is well known in most of the highly efficient CA isoforms as a proton shuttle, and it can exist in two opposite conformations, namely “in” and “out.” In the former, the histidine, oriented toward the active site, is prone to accept a proton to regenerate the nucleophilic species, whereas in the latter, this residue is oriented toward the exterior of the active site to release the proton to the bulk solvent. This flexible movement is responsible for the restoration of the catalytic activity of the active site. Compound 3 established numerous polar interactions with two amino acids (Tyr7 and Asn11) and with water molecules bound to Trp5 and His64, and strong van der Waals contacts (Fig. 12.2). Collectively acting on the protein surface and not entering the active site, these interactions froze His64 in a rigid and not functional “out” conformation. To further demonstrate this atypical enzymatic decrement, compound 3 was coincubated with imidazole as proton acceptor and a well-known CA activator. The addition of solutions with increasing amounts of imidazole counterbalanced the inhibition exhibited by 3. These results should be more investigated as different amino acid residues can delimit this cavity
260 Chapter 12 in each hCA isozyme, thus explaining the isoform selectivity of this compound and revealing the structural requirements for future drug design studies of selective CA inhibitors.
12.3 Carbonic anhydrase inhibitors with unknown mechanism of action 12.3.1 Acyclic tertiary sulfonamides Primary and secondary sulfonamide inhibitors of CA were widely investigated and clinically used due to their ability to lose a proton and act as a zinc-binding group (ZBG) in their deprotonated form [5e7]. Conversely, tertiary sulfonamides, lacking this depronatable moiety, were recently proposed as alternative chemotypes for the significant inhibition of a large number of CA isoforms. A large plethora of newly synthesized derivatives can be clustered in different chemical scaffolds as reported in Fig. 12.3. The first series comprised tosyl pyrroles and benzenesulfonamides (a) with moderate inhibitory potency against hCA I and II (off-target isoforms). Changes in the substitution pattern did not alter the biological activity and selectivity [8,9]. The second series comprehended halogenated tertiary benzenesulfonamides (b) tested against hCA I, II, IX, and XII and obtained through an innovative synthetic process. With respect to the chlorinated derivative (KI hCA II/KI hCA IX, or XII w65), the introduction of fluorine atom in the aliphatic chain of the scaffold led to an improvement (KI hCA II/KI hCA IX, or XII >1000) of the selective inhibition against the tumor-associated isoforms (IX and XII). The results were in the low nanomolar range despite the different R and R1 substituents. The authors confirmed a pivotal role of the strong electron-withdrawing fluorine for these nitrogen-containing compounds leading to a modification of the nitrogen basicity and molecule conformation via fluorine “gauche” effect and electrostatic interactions. Moreover, the interaction with the Zn(II) ion could be limited due to steric impairment [9,10]. In addition, some of these potent inhibitors were investigated for the possibility to be labeled with 18F for imaging purposes in CA IX overexpressing tumors. Synthesis and pharmacokinetic data of these radiotracers were described along with positron emission tomography (PET) images in NSG mice bearing HT-29 human colorectal tumor xenografts [11]. The third series (c) dealt with fluorinated and N-substituted pyrrolidines and halogenated N-nosylpiperidines endowed with a moderate hCA II inhibition and selectivity with respect to hCA I, IX, and XII. Regarding structureeactivity relationship (SAR) studies, it was possible to note that the introduction of the fluorine atom in the b-position of the nitrogen atom increased the inhibitory potency. The biological results also confirmed the impact of the geometrical shape of the molecule on the hCA IIeselective inhibition, reinforcing the hypothesis of a nonezinc(II)-binding mode of action within this scaffold and a pivotal role
“Out of the binding site” and unknown mechanisms of action 261
Figure 12.3 Acyclic and cyclic tertiary sulfonamides as alternative hCA inhibitors.
262 Chapter 12 of the structural shape on the inhibitory efficiency. Moreover, no relationship between chirality and biological activity was demonstrated [12]. The fourth series (d) collected hybrid molecules connecting a pyridine and an N-substituted indole through a chalcone linker. The compounds were tested against hCA II and IX in vitro by measuring the esterase activity, evaluated as antiproliferative and apoptotic agents against MCF-7 and HepG-2 cells, and submitted to molecular modeling assays to unravel the structural requirements useful for the CA IX inhibition [13]. Starting from these results on tertiary sulfonamide compounds, D’Ascenzio et al. also took advantage of such structural similarity present in the uricosuric agent probenecid, 4-(N,N-dipropylsulfamoyl)benzoic acid. From a biological point of view, it was shown to inhibit hCA II and IX in the nanomolar range, hCA XII in the micromolar range, and to be inactive against hCA I. In its structure, a tertiary sulfonamide is present and characterized by a striking steric hindrance. An additional COOH probably could participate to the CA inhibitory activity. To better demonstrate this issue, 24 novel Probenecid derivatives were designed by changing this functionality to an N-substituted amide (Fig. 12.4A). The in vitro results against four hCA isoforms (I, II, IX, and XII) showed that this chemical modification, along with the introduction of aliphatic, cycloaliphatic, and (hetero)aromatic tails, oriented the inhibitory activity toward hCA IX and XII isoforms limiting the interaction against the off-target hCA I and II isoforms [14,15]. Moreover, the same research group evaluated the addition of L-amino acid tails improving the aqueous solubility of the previous series. These new derivatives displayed inhibitory activity in the nanomolar range and strong selectivity against hCA IX and XII [16]. All these Probenecid-based compounds were analyzed in silico to unravel the putative interaction with the target (Fig. 12.4B). Surprisingly, they did prefer not to orient the tertiary sulfonamide toward the Zn(II) ion, but they were able to establish additional interactions by using the opposite portion of the molecule (amide fragment) properly functionalized. The Zn(II) ion could be a part of this complex network without being the main player as reported for ZBG-possessing inhibitors. Important enzyme amino acids involved in these interactions were Asn62, Gln92, Trp5, His64, Pro202, His94, Gln67, and Leu91 for hCA IX and Lys67, Thr91, Asn62, Thr200, His94, Gln92, Ser132, Trp5, and Thr199 for hCA XII. Unfortunately, so far, no crystallographic information is available to further support these in silico hypotheses.
12.3.2 Cyclic tertiary sulfonamides and their “open” derivatives: saccharin-based compounds The sweetener saccharin was proposed and tested as hCA inhibitor due to the presence of a cyclic secondary sulfonamide group endowed with a nonselective CA inhibition profile,
“Out of the binding site” and unknown mechanisms of action 263
Figure 12.4 (A) Probenecid-based derivatives as unusual hCA inhibitors and (B) their putative interactions with the enzyme.
264 Chapter 12 although with a strong preference (>1000-fold) for the tumor-associated isoforms in the nanomolar range. hCA II-saccharin and hCA IX-mimic-saccharin adducts have determined that the cyclic secondary sulfonamide behaved as a classic ZBG (benzoic sulfinimide) displacing in the deprotonated form the OH/H2O bound to the Zn(II) ion [17,18]. The concurrent presence of other ZBGs (e.g., primary sulfonamide) also led to its direct interaction within the active site prevailing on the cyclic secondary sulfonamide moiety [19]. To investigate the potential of this chemical structure, researchers have adopted many synthetic strategies to obtain more selective derivatives. The first approach was the derivatization of the sulfonamide nitrogen leading to 52 novel tertiary cyclic sulfonamides (Fig. 12.5A). The absence of a deprotonatable sulfonamide made them interesting compounds for the further design of innovative CA inhibitors.
Figure 12.5 (A) Saccharin-based derivatives as unusual hCA inhibitors and (B) their chemical modifications.
“Out of the binding site” and unknown mechanisms of action 265 The chemical space around this position was deeply explored by the insertion of aliphatic chains with or without the presence of unsaturation, functional groups (ketone, ester, carboxylic acid, hydroxamic acid, amide), ortho-/meta-/para-substituted benzyl and benzoyl moieties, and heterocyclic ring (isoxazole, phthalimide). All the compounds were fully characterized and tested in vitro against the two off-target isoforms (I and II) and the tumor-associated transmembrane isoforms (IX and XII) achieving potent nanomolar inhibition with high selectivity toward hCA XII [20e22]. Indeed, when another substituent could act as zinc binder within the molecule, it was demonstrated that the mechanism of inhibition could be ascribed to its direct interaction with the Zn(II) ion [23]. In this case, the remaining portion (saccharin nucleus) of the molecule adapted itself to improve the stability of the adduct. SAR studies of this large series of saccharin-based compounds revealed an almost inactivity against hCA I and II (with the exception of few derivatives) and a strong inhibitory activity by all compounds toward hCA XII. The hCA IX inhibition followed a not defined profile with steep variations due to small chemical changes. To further corroborate the importance of this scaffold, the same research groups, starting from the N-substituted saccharin, aimed at opening the isothiazolone core nucleus by means of hydrolytic (NaOH) or reductive (NaBH4) agents obtaining compounds characterized by a strong nanomolar inhibitory activity and selectivity against hCA IX and XII (Fig. 12.5B). Among closed and open saccharin derivatives, there were substantial differences in terms of ZBGs, but they displayed almost the same biological profile, thus confirming the possibility for the saccharin derivatives to act through an unknown mechanism of action [22,24].
12.3.3 Cyclic sulfamate inhibitors: acesulfame derivatives Starting from the chemical structure and biological results of saccharin derivatives, another synthetic sweetener (acesulfame as an ambidentate nucleophile) was deeply investigated. As previously reported for saccharin, acesulfame behaved as a classic CA inhibitor orienting its ZBG (a cyclic secondary sulfamate) toward the Zn(II) ion in the hCA II-acesulfame and hCA IX-mimic-acesulfame adducts at 1.5 A resolution [25]. Confirming the in vitro biological data (KI hCA IX ¼ 2.41 mM, KI hCA I, II, and XII > 20 mM), acesulfame was shown to bind directly to the Zn(II) ion in hCA IX-mimic, whereas different active site binding modes were detected for hCA II. The replacement of a not deprotonatable and bioisosteric cyclic moiety (sulfamate) with that of saccharin (sulfonamide) stimulated the design and synthesis of novel 19 N- and 12 O-substituted compounds, in vitro biological evaluation and in silico validation of their potency/selectivity [21,26]. The nucleophilic sites of acesulfame were decorated with alkyl (saturated and unsaturated) chains and (substituted) benzyl and benzoyl moieties. Collectively, they displayed a low affinity for hCA I and II isoforms and a higher preference for hCA IX and XII in the low nanomolar range. Nevertheless, a proper SAR for
266 Chapter 12
Figure 12.6 Acesulfame-based derivatives as unusual hCA inhibitors and their chemical modifications.
this scaffold can be hardly extrapolated because most of them are potent and selective disregarding the substitution pattern. The introduction of such substituents on the parent compound, which was only characterized by a slight hCA IX inhibition in the micromolar range, led to an improvement of CA blockade. Moreover, O-substituted derivatives were better inhibitors than their N-counterparts against hCA XII (Fig. 12.6). Docking studies confirmed that none of the compounds could interact directly with the Zn(II) ion in the hCA IX and XII active sites due to steric clashes, leading the sulfamate and carbonyl oxygens to be solvent exposed. Additional electrostatic interactions could be ascribed to substituents (bromine, nitro) on the benzyl/benzoyl portion of the inhibitor. Unfortunately, so far, no hCA adducts were obtained for these compounds to further determine the atypical mode of inhibition.
12.4 Conclusions Despite the large number of newly synthesized derivatives and natural products endowed with a high inhibitory potential against hCAs, few of them are characterized by a promising selectivity profile. Sulfonamides and other zinc binders are well known to interact with a strongly conserved portion of the active site within the 15 hCA isoforms. To overcome this problem, medicinal chemists have elegantly explored new chemotypes without the possibility to act as zinc binders (absence of depronatable protons). Along with saccharin-, acesulfameand probenecid-based compounds, several tertiary sulfonamides and sulfamates were designed, synthesized, and tested. The results suggested a promising in vitro selectivity profile especially oriented toward the two tumor-associated cancer isoforms (hCA IX and XII) in the nanomolar range. Lastly, one carboxylic acid derivative (3, Fig. 12.1) was serendipitously shown to interact with the proton shuttle His64 leading to a catalytically “frozen” enzyme. This completely different adduct opened a new scenario in the field of hCA inhibitors being involved a not conserved region of the enzyme.
“Out of the binding site” and unknown mechanisms of action 267
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