3H-1,2-benzoxathiepine 2,2-dioxides: a new class of isoform-selective carbonic anhydrase inhibitors

A new chemotype with carbonic anhydrase (CA, EC 4.2.1.1) inhibitory action has been discovered, the homo-sulfocoumarins (3H-1,2-benzoxathiepine 2,2-dioxides) which have been designed considering the (sulfo)coumarins as lead molecules. An original synthetic strategy of a panel of such derivatives led to compounds with a unique inhibitory profile and very high selectivity for the inhibition of the tumour associated (CA IX/XII) over the cytosolic (CA I/II) isoforms. Although the CA inhibition mechanism with these new compounds is unknown for the moment, we hypothesize that it may be similar to that of the sulfocoumarins, i.e. hydrolysis to the corresponding sulfonic acids which thereafter anchor to the zinc-coordinated water molecule within the enzyme active site.     

7-Aryl-triazolyl-substituted sulfocoumarins are potent,selective inhibitors of the tumor-associated carbonic anhydrase IX and XII

Sulfocoumarins behave as interesting inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Here, we report a new series of 7-substituted derivatives which were obtained by the click chemistry approach from 7-propargyloxy-sulfocoumarin and aryl azides incorporating halogens, hydroxy, methoxy and carboxyl moieties in their molecules. The new compounds were screened for the inhibition on four physiologically relevant human CA (hCA) isoforms, the cytosolic hCA I and II and the transmembrane tumor-associated hCA IX and XII. The new compounds did not inhibit the cytosolic isoforms but were low nanomolar inhibitors of the tumor-associated ones hCA IX and XII.     

A novel library of saccharin and acesulfame derivatives as potent and selective inhibitors of carbonic anhydrase IX and XII isoforms

Small libraries of N-substituted saccharin and N-/O-substituted acesulfame derivatives were synthesized and tested as atypical and selective inhibitors of four different isoforms of human carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). Most of them inhibited hCA XII in the low nanomolar range, hCA IX with K_Is ranging between 19 and 2482 nM, whereas they were poorly active against hCA II (K_Is >10 μM) and hCA I (K_Is ranging between 318 nM and 50 μM). Since hCA I and II are ubiquitous off-target isoforms, whereas the cancer-related isoforms hCA IX and XII were recently validated as drug targets, these results represent an encouraging achievement in the development of new anticancer candidates. Moreover, the lack of a classical zinc binding group in the structure of these inhibitors opens innovative, yet unexplored scenarios for different mechanisms of inhibition that could explain the high inhibitory selectivity. A computational approach has been carried out to further rationalize the biological data and to characterize the binding mode of some of these inhibitors.     

Pyrazolylbenzo[d]imidazoles as new potent and selective inhibitors of carbonic anhydrase isoforms hCA IX and XII

Novel pyrazolylbenzo[d]imidazole derivatives (2a–2f) were designed, synthesized and evaluated against four human carbonic anhydrase isoforms belonging to α family comprising of two cytosolic isoforms hCA I and II as well as two transmembrane tumor associated isoforms hCA IX and XII. Starting from these derivatives that showed high potency but low selectivity in favor of tumor associated isoforms hCA IX and XII, we investigated the impact of removing the sulfonamide group. Thus, analogs 3a–3f without sulfonamide moiety were synthesized and biological assay revealed a good activity as well as an excellent selectivity as inhibitors for tumor associated hCA IX and hCA XII and the same was analyzed by molecular docking studies.     

Metronidazole-coumarin conjugates and 3-cyano-7-hydroxy-coumarin act as isoform-selective carbonic anhydrase inhibitors

Reaction of 6-/7-hydroxycoumarin with metronidazole afforded conjugates which incorporate two interesting chemotypes which may inhibit carbonic anhydrases (CAs, EC 4.2.1.1) due to the presence of the coumarin moiety and possess radiosensitizing effects due to the presence of the nitroazole. Another dual action compound, which may act both as CA inhibitor as well as monocarboxylate transporter inhibitor, is 3-cyano-7-hydroxy-coumarin. These compounds have been investigated as inhibitors of 11 human CA isoforms. Submicromolar inhibition was observed against hCA VA, hCA VB, hCA VI, hCA VII, hCA IX, hCA XII and hCA XIV, whereas isoforms hCA I, II and XIII were not inhibited by these compounds. These coumarins thus act as isoform-selective CA inhibitors with the possibility to target isoforms involved in pathologies such as obesity (CA VA/VB) or cancer (CA IX and XII) without inhibiting the physiologically dominant, highly abundant hCA I and II.     

Ureidobenzenesulfonamides as efficient inhibitors of carbonic anhydrase II

Sulfonamides represent an important class of drugs because of their inhibitory effect on carbonic anhydrases (CAs). We therefore synthesized several ureidobenzenesulfonamides and evaluated their bCA II inhibition for their potential use as anti-glaucoma gents. Since these compounds must not show cytotoxic effects, their cytotoxic potential against several human tumor cell lines and non-malignant fibroblasts was investigated. Several fluorophenyl substituted sulfonamides were efficient inhibitors of bCA II. Only one benzylphenyl substituted sulfonamide, however, showed a remarkable selectivity for HT29 colorectal carcinoma cells while being significantly less cytotoxic to non-malignant fibroblasts.     

Development of 3-(4-aminosulphonyl)-phenyl-2-mercapto-3H-quinazolin-4-ones as inhibitors of carbonic anhydrase isoforms involved in tumorigenesis and glaucoma

A series of heterocyclic benzenesulfonamides incorporating 2-mercapto-3H-quinazolin-4-one tails were prepared by condensation of substituted anthranilic acids with 4-isothiocyanato-benzenesulfonamide. These sulfonamides were investigated as inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I and II (cytosolic isozymes), as well as hCA IX and XII (trans-membrane, tumor-associated enzymes). They acted as medium potency inhibitors of hCA I (K_Is of 81.0–3084 nM), being highly effective as hCA II (K_Is in the range of 0.25–10.8 nM), IX (K_Is of 3.7–50.4 nM) and XII (K_Is of 0.60–52.9 nM) inhibitors. These compounds should thus be of interest as preclinical candidates in pathologies in which the activity of these enzymes should be inhibited, such as glaucoma (CA II and XII as targets) or some tumors in which the activity of three isoforms (CA II, IX and XII) is dysregulated.     

7-Substituted-sulfocoumarins are isoform-selective,potent carbonic anhydrase II inhibitors

A series of 7-substituted sulfocoumarins and 3,4-dihydrosulfocoumarins was obtained by cyclization of the methanesulfonate of 2,4-dihydroxy- or 2-hydroxy-4-methoxybenzaldehyde, followed by derivatization reactions. The new compounds incorporate a range of substituents in position 7 of the heterocyclic ring (hydroxyl, methoxy, carboxylic and alkylsulfonate ester). The compounds were tested for the inhibition of the zinc enzyme human (h) carbonic anhydrase (hCA, EC 4.2.1.1). Unlike the 6-substituted sulfocoumarins which were potent hCA IX and XII inhibitors and ineffective hCA I and II inhibitors, compounds from this series showed low nanomolar hCA II inhibitory properties, and inhibited the mitochondrial isoform hCA VA with K_Is in the range of 91–9960 nM, but were ineffective as hCA I, IX and XII inhibitors. The structure activity relationship for this class of inhibitors was rather clear, with the nature of the 7-substituent strongly influencing hCA VA inhibition, whereas the nature of these groups were less relevant for hCA II inhibition (all reported compounds were highly effective hCA II inhibitors, with K_Is in the range of 1.5–8.4 nM). Since both hCA II and hCA VA are important drug targets (hCA II for antiglaucoma agents; hCA VA for antiobesity drugs), these isoform-selective inhibitors reported here may be considered of interest for various biomedical applications.     

Discovery of curcumin inspired sulfonamide derivatives as a new class of carbonic anhydrase isoforms I,II, IX,and XII inhibitors

A series of curcumin inspired sulfonamide derivatives was prepared from various chalcones and 4-sulfamoyl benzaldehyde via Claisen–Schmidt condensation. All new compounds were assayed as inhibitors of four human isoforms of the metalloenzyme carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I, II, IX and XII. Interesting inhibitory activities were observed against all these isoforms. hCA I, an isoform involved in several eye diseases was inhibited moderately with K_Is in the range of 191.8–904.2?nM, hCA II, an antiglaucoma drug target was highly inhibited by the new sulfonamides, with K_Is in the range of 0.75–8.8?nM. hCA IX, a tumor-associated isoform involved in cancer progression and metastatic spread was potently inhibited by the new sulfonamides, with K_Is in the range of 2.3–87.3?nM, whereas hCA XII, and antiglaucoma and anticancer drug target, was inhibited with K_Is in the range of 6.1–71.8?nM. It is noteworthy that one of the new compounds, 5d, was found to be almost 9 times more selective against hCA II (K_I =?0.89?nM) over hCA IX and hCA XII, whereas 5e was 3 and 70 times more selective against hCA II (K_I =?0.75?nM) over hCA IX and hCA XII, respectively.     

2-Amino-3-cyanopyridine derivatives as carbonic anhydrase inhibitors

Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous enzymes that catalyze the hydration of CO₂ to bicarbonate and protons. Inhibition of CAs has been clinically exploited for the treatment of various classes of diseases for decades, but investigating new classes of inhibitors continues to be important. We have synthesized a series of 2-amino-3-cyano-4-heteroaryl (5a–l) compounds and characterized the structures by NMR, IR and elemental analyses. We tested the ability of these compounds to inhibit two metalloenzyme human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, hCA I and hCA II. Compounds 5d and 5b showed the best inhibition activity against hCA I (IC₅₀: 33 and 34 µM, respectively), and compound 5d showed the best activity against hCA II (IC₅₀: 56 µM).     

Arylamino bisphosphonates: Potent and selective inhibitors of the tumor-associated carbonic anhydrase XII

A set of matrix metalloproteinases (MMPs) inhibitors, containing a bisphosphonate moiety (BP), has been evaluated for the inhibitory activity of carbonic anhydrases (CAs, EC 4.2.1.1). Human (h) isoforms hCA I, II, IX, XII and XIV were included in the study due to their involvement in crucial physiologic and pathologic processes. Some of these molecules selectively inhibited CA XII in the nanomolar range, showing an attractive dual mechanism (anti-MMP and anti-CA) of action as potential antitumor agents. The BP inhibitors investigated in this study are also excellent leads for obtaining even more effective compounds able to selectively target membrane-bound CA XII and having the potential to be used as tools for understanding physiologic processes regulated by this isoform.     

Synthesis,biological evaluation and molecular docking of novel pyrazole derivatives as potent carbonic anhydrase and acetylcholinesterase inhibitors

A series of substituted pyrazole compounds (1–8 and 9a, b) were synthesized and their structure was characterized by IR, NMR, and Mass analysis. These obtained novel pyrazole derivatives (1–8 and 9a, b) were emerged as effective inhibitors of the cytosolic carbonic anhydrase I and II isoforms (hCA I and II) and acetylcholinesterase (AChE) enzymes with K_i values in the range of 1.03 ± 0.23–22.65 ± 5.36 µM for hCA I, 1.82 ± 0.30–27.94 ± 4.74 µM for hCA II, and 48.94 ± 9.63–116.05 ± 14.95 µM for AChE, respectively. Docking studies were performed for the most active compounds, 2 and 5, and binding mode between the compounds and the receptors were determined.     

First evaluation of organotellurium derivatives as carbonic anhydrase I,II, IV,VII and IX inhibitors

A series of tellurides was evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors against the human (h) carbonic anhydrase isoforms hCA I, II, IV, VII and IX, involved in a variety of diseases, including glaucoma, retinitis pigmentosa, epilepsy, arthritis and tumors. These compounds, which are the first tellurium-containing derivatives acting as inhibitors of carbonic anhydrase enzymes, showed effective inhibition against all isoforms investigated and some of them were selective for inhibiting the cytosolic or the membrane-bound CAs. Thus, these carbonic anhydrase inhibitors are interesting leads for the development of isoform-selective inhibitors.     

Dual carbonic anhydrase/matrix metalloproteinase inhibitors incorporating bisphosphonic acid moieties targeting bone tumors

A set of bisphosphonate matrix metalloproteinase (MMP) inhibitors was investigated for inhibitory activity against several carbonic anhydrase (CA, EC 4.2.1.1) isozymes, some of which are overexpressed in hypoxic tumors. Some of the bisphosphonate revealed to be very potent inhibitors (in the low nanomolar range) of the cytosolic isoform CA II and the membrane-bound CA IX, XII and XIV isozymes, a feature useful for considering them as interesting compounds for bone resorption inhibition applications. We suggest here that it is possible to develop dual enzyme inhibitors bearing bisphosphonate moieties that may target both MMPs and CAs, two families of enzymes involved in tumor formation, growth, and metastasis.     

Benzoxepinones: A new isoform-selective class of tumor associated carbonic anhydrase inhibitors

Benzoxepinones (“homocoumarins”) are identified as a new class of selective inhibitors for tumor associated human carbonic anhydrases (hCA, EC 4.2.1.1) isoforms IX and XII. Similar to coumarins, they do not inhibit or poorly inhibit cytosolic human (h) isoforms hCA I and II, but act as nanomolar inhibitors of the trans-membrane, tumor associated isoforms hCA IX and XII.     

Synthesis and inhibition potency of novel ureido benzenesulfonamides incorporating GABA as tumor-associated carbonic anhydrase IX and XII inhibitors

New ureido benzenesulfonamides incorporating a GABA moiety as a linker between the ureido and the sulfonamide functionalities were synthesized and their inhibition potency determined against both the predominant cytosolic (hCA I and II) and the transmembrane tumor-associated (hCA IX and XII) isoforms of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The majority of these compounds were medium potency inhibitors of the cytosolic isoform hCA I and effective hCA II inhibitors, whereas they showed strong inhibition of the two transmembrane tumor-associated isoforms hCA IX and XII, with K_Is in nanomolar range. Only one derivative had a good selectivity for inhibition of the tumor-associated hCA IX target isoform over the cytosolic and physiologically dominant off-target hCA I and II, being thus a potential tool to develop new anticancer agents.     

Discovery of arjunolic acid as a novel non-zinc binding carbonic anhydrase II inhibitor

Elevated levels of carbonic anhydrase II (CA II) have been shown to be associated with cardiac hypertrophy and heart failure. Although arjunolic acid (AA) has a diverse range of therapeutic applications including cardio-protection, there have been no reports on the effect of AA on CA II. The present study describes for the first time, the novel zinc independent inhibition of CA II by AA. The molecular docking studies of AA indicated that the hydroxyl group at C₂ of the A-ring, which hydrogen bonds with the catalytic site residues (His64, Asn62 and Asn67), along with the gem-dimethyl group at C₂₀ of the E-ring, greatly influences the inhibitory activity, independent of the catalytic zinc, unlike the inhibition observed with most CA II inhibitors. Among the triterpenoids tested viz. arjunolic acid, arjunic acid, asiatic acid, oleanolic acid and ursolic acid, AA was the most potent in inhibiting CA II in vitro with an IC₅₀ of 9 μM. It was interesting to note, that in spite of exhibiting very little differences in their structures, these triterpenoids exhibited vast differences in their inhibitory activities, with IC₅₀ values ranging from 9 μM to as high as 333 μM. Furthermore, AA also inhibited the cytosolic activity of CA in H9c2 cardiomyocytes, as reflected by the decrease in acidification of the intracellular pH (pH_i). The decreased acidification reduced the intracellular calcium levels, which further prevented the mitochondrial membrane depolarization. Thus, these studies provide a better understanding for establishing the novel molecular mechanism involved in CA II inhibition by the non-zinc binding inhibitor AA.     

Benzenesulfonamide bearing 1,2,4-triazole scaffolds as potent inhibitors of tumor associated carbonic anhydrase isoforms hCA IX and hCA XII

Three series of novel heterocyclic compounds (3a–3g, 4a–4g and 5a–5g) containing benzenesulfonamide moiety and incorporating a 1,2,4-triazole ring, have been synthesized and investigated as inhibitors against four isomers of the α-class carbonic anhydrases (CAs, EC 4.2.1.1), comprising hCAs I and II (cytosolic, ubiquitous isozymes) and hCAs IX and XII (transmembrane, tumor associated isozymes). Against the human isozymes hCA I and II, compounds of two series (3a–3g and 4a–4g) showed K_i values in the range of 84–868 nM and 5.6–390 nM, respectively whereas compounds of series 5a–5g were found to be poor inhibitors (K_i values exceeding 10,000 nM in some cases). Against hCA IX and XII, all the tested compounds exhibited excellent to moderate inhibitory potential with K_i values in the range of 2.8–431 nM and 1.3–63 nM, respectively. Compounds 3d, 3f and 4f exhibited excellent inhibitory potential against all of the four isozymes hCA I, II, IX and XII, even better than the standard drug acetazolamide (AZA) whereas compound of the series 5a–5g were comparatively less potent but more selective towards hCA IX and XII.     

Multicomponent chemistry in the synthesis of carbonic anhydrase inhibitors

Abstract

Carbonic anhydrase inhibitors (CAIs) are of growing interest since various isoforms of the enzyme are identified as promising drug targets for treatment of disease. The principal drawback of the clinically used CAIs is the lack of isoform selectivity, which may lead to observable side effects. Studies aiming at the design of isoform-selective CAIs entail generation and biological testing of arrays of compounds, which is a resource- and time-consuming process. Employment of multicomponent reactions is an efficient synthetic strategy in terms of gaining convenient and speedy access to a range of scaffolds with a high degree of molecular diversity. However, this powerful tool appears to be underutilized for the discovery of novel CAIs. A number of studies employing multicomponent reactions in CAI synthesis have been reported in literature. Some of these reports provide inspiring examples of successful use of multicomponent chemistry to construct novel potent and often isoform-selective inhibitors. On critical reading of several publications, however, it becomes apparent that for some chemical series designed as CAIs, the desired inhibitory properties are only assumed and never tested for. In these cases, the biological profile is reported based on the results of phenotypical cellular assays, with no correlation with the intended on-target activity. Present review aims at critically assessing the current literature on the multicomponent chemistry in the CAI design.     

Synthesis and evaluation of sulfonamide-bearing thiazole as carbonic anhydrase isoforms hCA I and hCA II

Sulfonamide-bearing thiazole compounds were synthesized and their inhibitory effects on the activity of purified human carbonic anhydrase I and II were evaluated. Human carbonic anhydrase isoenzymes (hCA-I and hCA-II) were purified from erythrocyte cells by affinity chromatography. The inhibitory effects of the 12 synthesized sulfonamide (5a–l) on the hydratase and esterase activities of these isoenzymes (hCA-I and hCA-II) were studied in vitro. In relation to these activities, the inhibition equilibrium constants (Ki) were determined. The results showed that all the synthesized compounds inhibited the CA isoenzyme activity. Among them 5b was found to be the most active (IC_50?=_?0.35?μM; Ki: 0.33?μM) for hCA I and hCA II.