Synthesis,biological evaluation and in silico studies with 4-benzylidene-2-phenyl-5(4H)-imidazolone-based benzenesulfonamides as novel selective carbonic anhydrase IX inhibitors endowed with anticancer activity

In the presented work, we report the synthesis of a series of 4-benzylidene-2-phenyl-5(4H)-imidazolone-based benzenesulfonamides 7a-f via the Erlenmeyer–Plöchl reaction. All the prepared imidazolones 7a-f were evaluated as inhibitors of human (h) carbonic anhydrases (CA, EC 4.2.1.1) cytosolic isoforms hCA I and II, as well as transmembrane tumor-associated isoforms hCA IX and XII. All the tested hCA isoforms were inhibited by the prepared imidazolones 7a-f in variable degrees with the following K_Is ranges: 673.2–8169 nM for hCA I, 61.2–592.1 nM for hCA II, 23–155.4 nM for hCA XI, and 21.8–179.6 nM for hCA XII. In particular, imidazolones 7a, 7e, and 7f exhibited good selectivity towards the tumor-associated isoforms (CAs IX and XII) over the off-target cytosolic (CAs I and II) with selectivity index (SI) in the range of 6.2–19.4 and 3.3–8, respectively. Moreover, imidazolones 7a-f were screened for their anticancer activity in one dose (10⁻⁵ M) assay against a panel of 60 cancer cell lines according to US-NCI protocol. Furthermore, 7a, 7e and 7f were evaluated for their anti-proliferative activity against colorectal cancer HCT-116 and breast cancer MCF-7 cell lines. Furthermore, 7e and 7f were screened for cell cycle disturbance and apoptosis induction in HCT-116 cells. Finally, a molecular docking study was carried out to rationalize the obtained results.     

Carbonic anhydrase inhibitors: Synthesis and inhibition of the human carbonic anhydrase isoforms I,II, IX and XII with benzene sulfonamides incorporating 4- and 3-nitrophthalimide moieties

A series of 4 and 5 nitro-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1–8) was synthesized by reaction of benzenesulfonamide derivatives with 4 and 3-nitrophthalic anhydrides. These new sulfonamides were investigated as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmembrane, tumor-associated hCA IX and XII. Most of the novel compounds were medium potency-weak hCA I inhibitors (K_is in the range of 295–10,000 nM), but were more effective hCA II inhibitors (K_is of 1.7–887 nM). The tumor-associated hCA IX was also inhibited, with K_is in the micromolar range, whereas against hCA XII the inhibition constants were in the range of 90–3746 nM. The structure–activity relationship (SAR) with this series of sulfonamides is straightforward, with the main features leading to good activity for each isoforms being established. The high sequence hCA alignment homology and molecular docking studies was performed in order to rationalize the activities reported and binding mode to different hCA as inhibitors.     

Inhibition of tumor-associated human carbonic anhydrase isozymes IX and XII by a new class of substituted-phenylacetamido aromatic sulfonamides

Here, we investigate 28 structurally new sulfonamides and their subsequent testing for enzyme inhibition of cytosolic and tumor-associated carbonic anhydrases (CAs, EC 4.2.1.1). The compounds showed very potent inhibition of four physiologically relevant human (h) CA isoforms, namely hCA I, II, IX and XII. Interestingly, the K_I values were in the nanomolar range for the tumor-associated hCA IX and hCA XII. Docking studies have revealed details regarding the very favorable interactions between the scaffolds of this new class of inhibitors and the active sites of the investigated CA isoforms. As there are reported cases of tumors overexpressing both CA II and IX, such potent inhibitors for the two isoforms as those detected in this work, may have applications for targeting more than one CA present in tumors.     

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.     

4-Functionalized 1,3-diarylpyrazoles bearing 6-aminosulfonylbenzothiazole moiety as potent inhibitors of carbonic anhydrase isoforms hCA I,II, IX and XII

A series of 24 novel heterocyclic compounds—functionalized at position 4 with aldehyde (5a–5f), carboxylic acid (6a–6f), nitrile (7a–7f) and oxime (8a–8f) functional groups—bearing 6-aminosulfonybenzothiazole moiety at position 1 of pyrazole has been synthesized and investigated for the inhibition of four isoforms 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 isozyme hCA I, compounds 6a–6f showed medium-weak inhibitory potential with K_i values in the range of 157–690 nM with 6a showing better potential than the standard drug acetazolamide (AZA). Against hCA II, all the compounds showed excellent to moderate inhibition with K_i values of compounds 5a, 5d, 5f, 6a–6f, 8d and 8f lower than 12 nM (K_i of AZA). Against hCA IX, all the compounds showed moderate inhibition with the exception of 6e which showed nearly 9 fold a better profile compared to AZA, whereas against hCA XII, four compounds 6e, 7a, 7b and 7d showed K_i in the same order as that of AZA. Carboxylic acid 6e was found to be an excellent inhibitor of both hCA IX and XII, with K_i values of 2.8 nM and 5.5 nM, respectively.     

N-β-glycosyl sulfamides are selective inhibitors of the cancer associated carbonic anhydrase isoforms IX and XII

The transmembrane isoforms of carbonic anhydrase (CA IX and XII) have been shown to be linked to carcinogenesis and their inhibition to arrest primary tumor and metastases growth. In this Letter, we present a series of peracetylated and deprotected N-β-glycosyl sulfamides that were tested for the inhibition of 4 carbonic anhydrase isoforms: the cytosolic hCA I and hCA II and transmembrane tumor-associated IX and XII. Compounds 1-4 and 6-8 selectively target cancer-associated CAs (IX and XII) with K_Is in the low nanomolar range.     

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.     

Substituted benzene sulfonamides incorporating 1,3,5-triazinyl moieties potently inhibit human carbonic anhydrases II,IX and XII

A series of benzene sulfonamides incorporating 1,3,5-triazinyl moieties were synthesized using cyanuric chloride as starting material. Inhibition studies against human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms I, II (cytosolic) and IX, XII (transmembrane, tumor-associated) isoforms were performed with the new compounds. hCA I was modestly inhibited (K_Is in the range of 87 nM–4.35 μM), hCA II was moderately inhibited by most of the new compounds (K_Is in the range of 12.5–130 nM), whereas the tumor associated isoforms were potently inhibited, with K_Is in the range of 1.2–34.1 nM against hCA IX and of 2.1–33.9 against hCA XII, respectively. Docking studies of some of the new compounds showed an effective binding mode within the enzyme active site, as demonstrated earlier by X-ray crystallography for structurally-related sulfonamides incorporating 1,3,5-triazinyl functionalities.     

Carbonic anhydrase inhibitors: Synthesis and inhibition of the human carbonic anhydrase isoforms I,II, VII,IX and XII with benzene sulfonamides incorporating 4,5,6,7-tetrabromophthalimide moiety

A series of 4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1–8) was synthesized by reaction of benzene sulfonamide derivatives with 4,5,6,7-tetrabromophthalic anhydride moiety. These new sulfonamides were investigated as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more specifically against the human (h) cytosolic isoforms hCA I, II and VII and the transmembrane tumor-associated isoform hCA IX and XII. The new compounds were good hCA I inhibitors (Kis in the range of 143 to >10,000 nM), but were moderately effective, as hCA II inhibitors (Kis of 47–190 nM) and poor hCA VII inhibitors (Kis in the range of 54–175 nM) compared to acetazolamide. The tumor-associated hCA IX was effectively inhibited with Kis ranging between 8.5 and 234 nM and hCA XII with inhibition constants in the range of 6.1–197 nM with high selectivity ratio. The structure–activity relationship (SAR) with this series of sulfonamides is straightforward, with the main features leading to good activity for each isoforms being established. The high sequence hCA alignment homology and molecular docking study of compounds was performed to rationalize the SAR reported over here.     

Carbonic anhydrase inhibitory activity of sulfonamides and carboxylic acids incorporating cyclic imide scaffolds

A series of sulfonamides incorporating cyclic imide moieties were investigated as inhibitors of several human α-carbonic anhydrase (hCA, EC 4.2.1.1) isoforms. Several carboxylic acids possessing the same scaffolds as the sulfonamides were also included in the study, since the sulfonamidate and the carboxylate are among the frequently used zinc-binding groups (ZBGs) for obtaining zinc enzymes inhibitors. The cytosolic isoform hCA I was moderately inhibited by most of the 30 investigated derivatives; many low nanomolar hCA II inhibitors were detected, whereas some of these compounds were low nanomolar/subnanomolar inhibitors of the transmembrane, tumor-associated isoforms hCA IX and XII. In this series of compounds the SO₂NH⁻ and the COO⁻ ZBGs showed similar efficacy for obtaining potent inhibitors, although some carboxylates had isoform-selective inhibition profiles for the transmembrane CAs.     

Coumarinyl-substituted sulfonamides strongly inhibit several human carbonic anhydrase isoforms: solution and crystallographic investigations

We investigated a series of coumarinyl-substituted aromatic sulfonamides as inhibitors of four carbonic anhydrase (CA, EC 4.2.1.1) isoforms with medical applications, the cytosolic hCA I, and II, and the transmembrane, tumor-associated hCA IX and XII. Compounds incorporating 7-methoxy-coumarin-4-yl-acetamide-tails and benzenesulfonamide and benzene-1,3-disulfonamide scaffolds showed medium potency inhibition of hCA I (K_Is of 73–131 nM), effective hCA II inhibition (K_Is of 9.1–36 nM) and less effective hCA IX and XII inhibition (K_Is of 55–128 nM). Only one compound, the derivatized 4-amino-6-trifluoromethyl-benzene-1,3-disulfonamide with the coumarinyl tail, showed effective inhibition of the transmembrane isoforms, with K_Is of 5.9–14.2 nM, although it was less effective as hCA I and II inhibitor (K_Is of 36–120 nM). An X-ray crystal structure of hCA II in complex with 4-(7-methoxy-coumarin-4-yl-acetamido)-benzenesulfonamide (K_I of 9.1 nM against hCA II) showed the intact inhibitor coordinated to the zinc ion from the enzyme active site by the sulfonamide moiety, and participating in a edge-to-face stacking with Phe131, in addition to other hydrophobic and hydrophilic interactions with water molecules and amino acid residues from the active site. Thus, sulfonamides incorporating coumarin rings have a distinct inhibition mechanism compared to the coumarins, and may lead to compounds with interesting inhibition profiles against various α-CAs found in mammals or parasites, such as Plasmodium falciparum.     

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.     

Syntesis of thio- and seleno-acetamides bearing benzenesulfonamide as potent inhibitors of human carbonic anhydrase II and XII

A novel series of thio- and seleno-acetamides bearing benzenesulfonamide were synthetized and tested as human carbonic anhydrase inhibitors. These compounds were tested for the inhibition of four human (h) isoforms, hCA I, II, IX, and XII, involved in pathologies such as glaucoma (CA II and XII) or cancer (CA IX/XII). Several derivatives showed potent inhibition activity in low nanomolar range such as 3a, 4a, 7a and 8a. Furthermore, based on the tail approach we explain the interesting and selective inhibition profile of compound such as 5a and 9a, which were more selective for hCA I, 9b which was selective for hCA II, 3f selective for hCA IX and finally, 3e and 4b selective for hCA XII, over the other three isoforms. They are interesting leads for the development of more effective and isoform-selective inhibitors.     

5-Substituted-(1,2,3-triazol-4-yl)thiophene-2-sulfonamides strongly inhibit human carbonic anhydrases I,II, IX and XII: Solution and X-ray crystallographic studies

We report here a series of 2-thiophene-sulfonamides incorporating 1-substituted aryl-1,2,3-triazolyl moieties, prepared by click chemistry from 5-ethynylthiophene-2-sulfonamide and substituted aryl azides. The new sulfonamides were investigated as inhibitors of the zinc metalloenzyme CA (EC 4.2.1.1), and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmembrane, tumor-associated ones hCA IX and XII: The new compounds were medium–weak hCA I inhibitors (K_Is in the range of 224–7544 nM), but were compactly, highly effective, low nanomolar hCA II inhibitors (K_Is of 2.2–7.7 nM). The tumor-associated hCA IX was inhibited with K_Is ranging between 5.4 and 811 nM, whereas hCA XII with inhibition constants in the range of 3.4–239 nM. The X-ray crystal structure of the adducts of two such compounds bound to hCA II (one incorporating 1-naphthyl, the other one 3-cyanophenyl moieties) evidenced the reasons of the high affinity for hCA II. Highly favorable, predominantly hydrophobic interactions between the sulfonamide scaffold and the hCA II active site were responsible for the binding, in addition to the coordination of the sulfamoyl moiety to the zinc ion. The tails of the two inhibitors adopted very diverse orientations when bound to the active site, with the naphthyltriazolyl moiety orientated towards the hydrophobic half of the active site, and the 3-cyanophenyl one pointing towards the hydrophilic half. These data may be used for the structure-based drug design of even more effective hCA II inhibitors, with potential use as antiglaucoma agents or as diuretics.     

6-Triazolyl-substituted sulfocoumarins are potent,selective inhibitors of the tumor-associated carbonic anhydrases IX and XII

A series of 6-substituted sulfocoumarins incorporating substituted-1,2,3-triazol-4-yl-/5-yl moieties were synthesized by employing click chemistry. The new sulfocoumarins incorporated cycloalkyl, tert-butyl and substituted aryl moieties at the triazole ring, and were investigated for the inhibition of four human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, the cytosolic hCA I and II; and the transmembrane, tumor-associated hCA IX and XII. The triazole-substituted sulfocoumarins did not inhibit the ubiquitous, off-target cytosolic isoforms hCA I and II (K_Is >10 μM) but showed effective inhibition against the two transmembrane CAs, with K_Is ranging from 7.2 to 10.5 nM against hCA IX, and between 5.5 and 17.7 nM against hCA XII. As hCA IX and XII are validated anti-tumor targets, such prodrug, isoform-selective inhibitors as the sulfocoumarins reported here, may be useful for identifying suitable drug candidates for clinical trials.     

Carbonic anhydrase inhibitors. Synthesis of 2,4,6-trimethylpyridinium derivatives of 2-(hydrazinocarbonyl)-3-aryl-1H-indole-5-sulfonamides acting as potent inhibitors of the tumor-associated isoform IX and XII

A series of 2-(hydrazinocarbonyl)-3-aryl-1H-indole-5-sulfonamides possessing various 2-, 3- or 4- substituted phenyl groups with methyl-, halogeno- and methoxy-functionalities, or a perfluorophenyl moiety, has been derivatized by reaction with 2,4,6-trimethylpyrylium perchlorate. The new sulfonamides were evaluated as inhibitors of four mammalian carbonic anhydrase (CA, EC 4.2.1.1) isoforms, that is, CA I, II (cytosolic), CA IX and XII (transmembrane, tumor-associated forms). Excellent inhibitory activity was observed against hCA IX with most of these sulfonamides, and against hCA XII with some of the new compounds. These compounds were generally less effective inhibitors of hCA II. Being membrane impermeant, these positively-charged sulfonamides are interesting candidates for targeting the tumor-associated CA IX and XII, as possible diagnostic tools or therapeutic agents.     

Structural study of the location of the phenyl tail of benzene sulfonamides and the effect on human carbonic anhydrase inhibition

The crystal structure of 4-phenylacetamidomethyl-benzenesulfonamide (4ITP) bound to human carbonic anhydrase (hCA, EC 4.2.1.1) II is reported. 4ITP is a medium potency hCA I and II inhibitor (K_Is of 54–75 nM), a strong mitochondrial CA VA/VB inhibitor (K_Is of 8.3–8.6 nM) and a weak transmembrane CA inhibitor (K_Is of 136–212 nM against hCA IX and XII). This elongated compound binds in an extended conformation to hCA II, with its tail lying towards the hydrophobic half of the active site whereas the sulfonamide moiety coordinates the zinc ion. The present structure was compared to that of structurally related aromatic sulfonamides, such as 4-phenylacetamido-benzene-sulfonamide (3OYS), 4-(2-mercaptophenylacetamido)-benzene-sulfonamide (2HD6) and 4-(3-nitrophenyl)-ureido-benzenesulfonamide (3N2P). Homology models of the hCA I, VA, VB, IX and XII structures were build which afforded an understanding of the amino acids involved in the binding of these compounds to these isoforms. The main conclusion of the study is that the orientation of the tail moiety and the presence of flexible linkers as well polar groups in it, strongly influence the potency and the selectivity of the sulfonamides for the inhibition of cytosolic, mitochondrial or transmembrane CA isoforms.     

Anticancer effects of new dibenzenesulfonamides by inducing apoptosis and autophagy pathways and their carbonic anhydrase inhibitory effects on hCA I,hCA II,hCA IX,hCA XII isoenzymes

In this study, new dibenzensulfonamides, 7–9, having the chemical structure 4,4′-(5′-chloro-3′-methyl-5-aryl-3,4-dihydro-1′H,H-[3,4′-bipyrazole]-1′,2-diyl)dibenzenesulfonamide were synthesized in five steps to develop new anticancer drug candidates. Their chemical structures were confirmed by ¹H NMR, ¹³C NMR and HRMS spectra. Cytotoxicities of the dibenzensulfonamides were investigated towards HCC1937, MCF7, HeLa, A549 as tumor cell lines and towards MRC5 and Vero as non-tumor cells. Carbonic anhydrase (CAs, EC 4.2.1.1) inhibitory effects of the dibenzensulfonamides 7–9 were also evaluated on the cytosolic human (h) hCA I and II and the tumor-associated hCA IX and XII isoenzymes. Results indicate that both 7 and 8 induced cleavage of poly (ADP ribose) polymerase (PARP), activation of caspases -3, -7 and -9 which are the hallmarks of apoptosis. Meanwhile both compounds induced autophagy in HCC1937 cells which is shown by enhanced expression of LC3 and decreased level of p62 protein. The compounds tested were also effectively inhibited tumor-associated hCA IX and hCA XII isoenzymes in the range of 20.7–28.1 nM and 4.5–9.3 nM, respectively.     

Inhibition of carbonic anhydrase isoforms I,II, IX and XII with secondary sulfonamides incorporating benzothiazole scaffolds

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the fundamental reaction of CO₂ hydration in all living organisms, being actively involved in the regulation of a plethora of patho/physiological conditions. A series of benzothiazole-based sulfonamides were synthesized and tested as possible CA inhibitors. Their inhibitory activity was assessed against the cytosolic human isoforms hCA I and hCA II and the transmembrane hCA IX and hCA XII. Several of the investigated derivatives showed interesting inhibition activity and selectivities for inhibiting hCA IX and hCA XII over the off-target ones hCA I and hCA II. Furthermore, computational procedures were used to investigate the binding mode of this class of compounds, within the active site of hCA IX.     

Synthesis of 6-tetrazolyl-substituted sulfocoumarins acting as highly potent and selective inhibitors of the tumor-associated carbonic anhydrase isoforms IX and XII

A series of 6-substituted sulfocoumarins incorporating substituted-1,2,3,4-tetrazol-5-yl moieties were synthesized by reaction of 6-iodo-sulfocoumarin and the corresponding tetrazole via the CH activation reaction. The new sulfocoumarins incorporating alkyl and substituted aryl moieties at the 1-position of the tetrazole, were investigated for the inhibition of four human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, the cytosolic hCA I and II; and the transmembrane, tumor-associated hCA IX and XII. The tetrazole-substituted sulfocoumarins did not inhibit the ubiquitous, off-target cytosolic isoforms (K_Is >10 μM) but showed effective inhibition against the two transmembrane CAs, with K_Is ranging from 6.5 to 68.6 nM against hCA IX, and between 4.3 and 59.8 nM against hCA XII. As hCA IX and XII are validated anti-tumor targets, such prodrug, isoform-selective inhibitors as the sulfocoumarins reported here, may be useful for identifying suitable drug candidates for clinical trials.