Fluoroenesulphonamides: N-sulphonylurea isosteres showing nanomolar selective cancer-related transmembrane human carbonic anhydrase inhibition

After hydrofluorination of ynesulphonamides in superacid or in the presence of hydrofluoric acid/base reagents, a series of α-fluoroenamides has been synthesised and tested for the inhibition of carbonic anhydrase (CA, EC 4.2.1.1) isoforms. This study reveals a new, highly selective family of cancer-related transmembrane human (h) CA IX/XII inhibitors. These original fluorinated ureido isosters do not inhibit the widespread cytosolic isoforms hCA I and II and selectively inhibit the transmembrane cancer-related hCA IX and XII, offering interesting new leads for future 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.     

Synthesis and carbonic anhydrase I,II, IV and XII inhibitory properties of N-protected amino acid – sulfonamide conjugates

N-protected amino acids (Gly, Ala and Phe protected with Boc and Z groups) were reacted with sulfonamide derivatives, leading to the corresponding N-protected amino acid–sulfonamide conjugates. The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity of the new compounds was assessed against four human (h) isoforms, hCA I, hCA II, hCA IV and hCA XII. Among them, hCA II, IV and XII are antiglaucoma drug targets, being involved in aqueous humor secretion within the eye. Low nanomolar inhibition was measured against all four isoforms with the 20 reported sulfonamides, but no selective inhibitory profiles, except for some CA XII-selective derivatives, were observed. hCA I, II and XII were generally better inhibited by sulfonamides incorporating longer scaffolds and Gly/Ala, whereas the best hCA IV inhibitors were homosulfanilamide derivatives, incorporating Phe moieties. The amino acid–sulfonamide conjugates show good water solubility and effective hCA II, IV and XII inhibition, and may be considered as interesting candidates for antiglaucoma studies.     

Inhibition of membrane-associated carbonic anhydrase isozymes IX, XII and XIV with a library of glycoconjugate benzenesulfonamides

A library of glycoconjugate benzenesulfonamides that contain diverse carbohydrate-triazole tails were investigated for their ability to inhibit the enzymatic activity of the three human transmembrane carbonic anhydrase (CA) isozymes hCA IX, hCA XII and hCA XIV. These isozymes have their CA domains located extracellularly, unlike the physiologically dominant hCA II, and are of immense current interest as druggable targets. Elevated expression of isozymes IX and XII is a marker for a broad spectrum of hypoxic tumors-this physiology may facilitate a novel approach to discriminate between healthy cells and cancerous cells. Many of these glycoconjugates were potent inhibitors (low nM), but importantly exhibited different isozyme selectivity profiles. The most potent hCA IX inhibitor was the glucuronic acid derivative 20 (K(i)=23nM). This compound was uniquely hCA IX selective cf. all other isozymes (16.4-, 16.8- and 4.6-fold selective against hCA II, XII, and XIV, respectively). At hCA XII there were many inhibitors with K(i)s<10nM that also demonstrated excellent selectivity (up to 344-fold) against other isozymes. Potent hCA XIV inhibitors were also identified, several with K(i)s approximately 10nM, however no hCA XIV-selective derivatives were evidenced from this library. The sugar tails of this study have shown promise as a valuable approach to both solubilize the aromatic sulfonamide CA recognition pharmacophore and to deliver potent inhibition and isozyme differentiation of the transmembrane CAs.     

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.     

Carbonic anhydrase inhibitors: inhibition of the human isozymes I, II, VA, and IX with a library of substituted difluoromethanesulfonamides

An inhibition study of the human cytosolic isozymes I, and II, the mitochondrial isoform VA, and the tumor-associated, transmembrane isozyme IX of carbonic anhydrase (CA, EC 4.2.1.1) with a library of aromatic/heteroaromatic/polycyclic difluoromethanesulfonamides is reported. Most of the inhibitors were derivatives of benzenedifluoromethanesulfonamide incorporating substituted-phenyl moieties, or were methylsulfonamide and difluoromethyl-sulfonamide derivatives of the sulfamates COUMATE and EMATE, respectively. Except for the methylsulfonamide-COUMATE derivative which behaved as a potent CA II inhibitor (K(I) of 32nM), these sulfonamides were moderate inhibitors of all isozymes, with inhibition constants in the range of 96-5200nM against hCA I, of 80-670nM against hCA II, and of 195-9280nM against hCA IX, respectively. Remarkably, some derivatives, such as 3-bromophenyl-difluoromethanesulfonamide, showed a trend to selectively inhibit the mitochondrial isoform CA VA, showing selectivity ratios for inhibiting CA VA over CA II of 3.53; over CA I of 6.84 and over CA IX of 9.34, respectively, although it is a moderate inhibitor (K(I) of 160nM). Some of these derivatives may be considered as leads for the design of isozyme selective CA inhibitors targeting the mitochondrial isozyme CA VA, with potential use as anti-obesity agents.     

A class of carbonic anhydrase IX/XII – selective carboxylate inhibitors

Abstract

A small series of 2,4-dioxothiazolidinyl acetic acids was prepared from thiourea, chloroacetic acid, aromatic aldehydes, and ethyl-2-bromoacetate. They were assayed for the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms of human (h) origin, the cytosolic hCA I and II, and the transmembrane hCA IX and XII, involved among others in tumorigenesis (hCA IX and XII) and glaucoma (hCA II and XII). The two cytosolic isoforms were not inhibited by these carboxylates, which were also rather ineffective as hCA IX inhibitors. On the other hand, they showed submicromolar hCA XII inhibition, with K_Is in the range of 0.30–0.93?µM, making them highly CA XII-selective inhibitors.     

Carbonic anhydrase inhibitors: inhibition of human cytosolic isozyme II and mitochondrial isozyme V with a series of benzene sulfonamide derivatives

Among the 14 human isozymes of carbonic anhydrase (CA, EC 4.2.1.1) presently known, the cytosolic hCA II is the most active and plays a host of physiological functions, whereas the mitochondrial hCA V is unique due to its role in several biosynthetic reactions. An inhibition study of these isozymes with a series of sulfonamides is reported here, with the scope to detect lead molecules for the design of isozyme-specific CA inhibitors (CAIs) targeting the mitochondrial isoform. Indeed, recently it has been shown that CA V is a novel target for the drug design of anti-obesity agents among others. Compounds included in this study were mainly ortho-, meta-, and para-substituted-benzenesulfonamides, together with several halogeno-substituted sulfanilamides and disubstituted-benzene-1,3-disulfonamide derivatives. Isozyme V showed an inhibition profile with these sulfonamides different of that of hCA II. Thus, IC(50) values in the range of 80 nM to 74 microM against hCA II, and 0.78-63.7 microM against hCA V with these derivatives have been obtained. Only one compound, 2-carboxymethyl-benzenesulfonamide, was more active against hCA V over hCA II (selectivity ratio of 1.39), whereas all other derivatives investigated here were much better hCA II inhibitors (selectivity ratios CA II/CA V in the range of 0.0008-0.73) than hCA V inhibitors.     

Quinazoline–sulfonamides with potent inhibitory activity against the α-carbonic anhydrase from Vibrio cholerae

Thirteen novel sulfonamide derivatives incorporating the quinazoline scaffold were synthesized by simple, eco-friendly procedures. These compounds were tested for their ability to inhibit the α-carbonic anhydrases (CA, EC 4.2.1.1) from Vibrio cholerae (VchCA) as well as the human α-CA isoforms, hCA I and hCA II. Nine compounds were highly effective, nanomolar inhibitors of the pathogenic enzyme VchCA. Three of them were also highly effective sub-nanomolar inhibitors of the cytosolic isoform II. The best VchCA inhibitor had a K_I of 2.7 nM. Many of these developed compounds showed high selectivity for inhibition of the bacterial over the mammalian CA isoforms, with one compound possessing selectivity ratios as high as 97.9 against hCA I and 9.7 against hCA II. Compound 9d was another highly effective VchCA inhibitor presenting a selectivity ratio of 99.1 and 8.1 against hCA I and hCA II, respectively. These results suggest that sulfonamides with quinazoline backbone could be considered suitable tools to better understand the role of bacterial CAs in pathogenesis.     

Carbonic anhydrase inhibitors: novel sulfonamides incorporating 1,3,5-triazine moieties as inhibitors of the cytosolic and tumour-associated carbonic anhydrase isozymes I, II and IX

A new series of aromatic benzenesulfonamides incorporating 1,3,5-triazine moieties in their molecules is reported. This series was obtained by reaction of cyanuric chloride with sulfanilamide, homosulfanilamide or 4-aminoethylbenzenesulfonamide. The prepared dichlorotriazinyl-benzenesulfonamides were subsequently derivatized by reacting them with various nucleophiles, such as ammonia, hydrazine, primary and secondary amines, amino acid derivatives or phenol. The library of sulfonamides incorporating triazinyl moieties was tested for the inhibition of three physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic hCA I and II, and the transmembrane, tumour-associated hCA IX. The new compounds inhibited hCA I with inhibition constants in the range of 31-8500 nM, hCA II with inhibition constants in the range of 14-765 nM and hCA IX with inhibition constants in the range of 1.0-640 nM. Structure-activity relationship was straightforward and rather simple in this class of CA inhibitors, with the compounds incorporating compact moieties at the triazine ring (such as amino, hydrazino, ethylamino, dimethylamino or amino acyl) being the most active ones, and the derivatives incorporating such bulky moieties (n-propyl, n-butyl, diethylaminoethyl, piperazinylethyl, pyridoxal amine or phenoxy) being less effective hCA I, II and IX inhibitors. Some of the new derivatives also showed selectivity for inhibition of hCA IX over hCA II (selectivity ratios of 23.33-32.00), thus constituting excellent leads for the development of novel approaches for the management of hypoxic tumours.     

Design,synthesis and biological evaluation of coumarin-3-carboxamides as selective carbonic anhydrase IX and XII inhibitors

A series of novel 7-hydroxycoumarin-3-carboxamides was synthesized by the reaction of 7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid with various substituted aromatic amines. The newly synthesized compounds were evaluated for their inhibitory activity against the four physiologically relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms CA I, CA II, CA IX and CA XII. The CA inhibition results show that the newly synthesized 7-hydroxycoumarin-3-carboxamides (4a-n) exhibited selective inhibition of the tumor associated isoforms, CA IX and CA XII over CA I and II isoforms. The inhibition constants ranged from sub micromolar to low micromolar. Amongst all the compounds tested, compound 4m was the most effective inhibitor exhibiting sub micromolar potency against both hCA IX and hCA XII, with a K_i of 0.2 µM. Therefore, it can be anticipated that compound 4m can serve as a lead for development of anticancer therapy by exhibiting a novel mechanism of action. The binding modes of the most potent compounds within hCA IX and XII catalytic clefts were investigated by docking studies.     

Carbonic anhydrase inhibitors. Inhibition of the cytosolic and tumor-associated carbonic anhydrase isozymes I, II, and IX with a series of 1,3,4-thiadiazole- and 1,2,4-triazole-thiols

A series of heterocyclic mercaptans incorporating 1,3,4-thiadiazole- and 1,2,4-triazole rings have been prepared and assayed for the inhibition of three physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic human isozymes I and II, and the transmembrane, tumor-associated hCA IX. Against hCA I the investigated thiols showed inhibition constants in the range of 97 nM to 548 microM, against hCA II in the range of 7.9-618 microM, and against hCA IX in the range of 9.3-772 microM. Thiadiazoles were generally more active than triazoles against all investigated isozymes. Generally, the best inhibitors were the simple derivative 5-amino-1,3,4-thiadiazole-2-thiol and its N-acetylated derivative, which were anyhow at least two orders of magnitude less effective inhibitors when compared to the corresponding sulfonamides, acetazolamide, and its deacetylated derivative. An exception was constituted by 5-(2-pyridylcarboxamido)-1,3,4-thiadiazole-2-thiol, which is the first hCA I-selective inhibitor ever reported, possessing an inhibition constant of 97 nM against isozyme I, and being a 105 times less effective hCA II inhibitor, and 3154 times less effective hCA IX inhibitor. Thus, the thiol moiety may lead to effective CA inhibitors targeting isozyme I, whereas it is a less effective zinc-binding function for the design of CA II and CA IX inhibitors over the sulfonamide group.     

Carbonic anhydrase inhibitors: 2-substituted-1,3,4-thiadiazole-5-sulfamides act as powerful and selective inhibitors of the mitochondrial isozymes VA and VB over the cytosolic and membrane-associated carbonic anhydrases I, II and IV

A series of 2-substituted-1,3,4-thiadiazole-5-sulfamides was prepared and assayed as inhibitors of several carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic CA I and II, the membrane-associated CA IV and the mitochondrial CA VA and VB. The new compounds showed weak inhibitory activity against hCA I (K(I)s of 102 nM-7.42 microM), hCA II (K(I)s of 0.54-7.42 microM) and hCA IV (K(I)s of 4.32-10.05 microM) but were low nanomolar inhibitors of hCA VA and hCA VB, with inhibition constants in the range of 4.2-32 nM and 1.3-74 nM, respectively. Furthermore, the selectivity ratios for inhibiting the mitochondrial enzymes over CA II were in the range of 67.5-415, making these sulfamides the first selective CA VA/VB inhibitors.     

Effects of dopaminergic compounds on carbonic anhydrase isozymes I, II, and VI

Studies on carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have increased due to several therapeutic applications while there are few investigations on activators. Here we investigated CA inhibitory and activatory capacities of a series of dopaminergic compounds on human carbonic anhydrase (hCA) isozymes I, II, and VI. 2-Amino-1,2,3,4-tetrahydronaphthalene-6,7-diol hydrobromide and 2-amino-1,2,3,4-tetrahydronaphthalene-5,6-diol hydrobromide were found to show effective inhibitory action on hCA I and II whereas 2-amino-5,6-dibromoindan hydrobromide and 2-amino-5-bromoindan hydrobromide exhibited only moderate inhibition against both isoforms, being more effective inhibitors of hCA VI. K(i) values of the molecules 3-6 were in the range of 41.12-363 μM against hCA I, of 0.381-470 μM against hCA II and of 0.578-1.152 μM against hCA VI, respectively. Compound 7 behaved as a CA activator with K(A) values of 27.3 μM against hCA I, of 18.4 μM against hCA II and of 8.73 μM against hCA VI, respectively.     

Acipimox inhibits human carbonic anhydrases

Acipimox, a nicotinic acid derivative in clinical use for the treatment of hyperlipidaemia, incorporates a free carboxylic acid and an N-oxide moiety, functionalities known to interact with the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and inhibit its activity. Herein we report that acipimox acts as a low micromolar CA inhibitor (CAI) against most human (h) isoforms possessing catalytic activity, hCA I – XIV. By using computational techniques (docking and molecular dynamics simulations), we propose that acipimox coordinates through its carboxylate group to the zinc ion from the enzyme active site cavity, whereas the N-oxide group is hydrogen-bonded to the proton shuttle His residue in some isoforms (hCA I) or to active site Thr or Gln residues in other isoforms (hCA II, III, IV, VII, etc). As some CA isoforms are involved in lipogenesis, these data may be useful for the design of more effective CAIs with antiobesity activity.     

Carbonic anhydrase inhibitors: in vitro inhibition of α isoforms (hCA I,hCA II,bCA III,hCA IV) by flavonoids

A series of flavonoids, such as quercetin, catechin, apigenin, luteolin, morin, were investigated for their inhibitory effects against the metalloenzyme carbonic anhydrase (CA). The compounds were tested against four α-CA isozymes purified from human and bovine (hCA I, hCA II, bCA III, hCA IV) tissues. The four isozymes showed quite diverse inhibition profiles with these compounds. The flavonoids inhibited hCA I with K_I-s in the range of 2.2–12.8 μM, hCA II with K_I-s in the range of 0.74–6.2 μM, bCA III with K_I-s in the range of 2.2–21.3 μM, and hCA IV with inhibition constants in the range of 4.4–15.7, with an esterase assay using 4-nitrophenyl acetate as substrate. Some simple phenols/sulfonamides were also investigated as standard inhibitors. The flavonoids incorporate phenol moieties which inhibit these CAs through a diverse, not yet determined inhibition mechanism, compared to classic inhibitors such as the sulfonamide/sulfamate ones.     

Carbonic anhydrase inhibitors: X-ray crystal structure of a benzenesulfonamide strong CA II and CA IX inhibitor bearing a pentafluorophenylaminothioureido tail in complex with isozyme II

N-1-(4-Sulfamoylphenyl)-N-4-pentafluorophenyl-thiosemicarbazide was prepared by the reaction of 4-isothiocyanato-benzenesulfonamide with pentafluorophenyl hydrazine, and proved to be an effective inhibitor of several isozymes of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), such as CA I, II, and IX. Against the physiologically relevant isozymes hCA II and hCA IX, the compound showed inhibition constants in the range of 15-19 nM, whereas it was less effective as a hCA I inhibitor (K(I) of 78 nM). The high-resolution X-ray crystal structure of its adduct with hCA II showed the inhibitor to bind within the hydrophobic half of the enzyme active site, making extensive and strong van der Waals contacts with amino acid residues Gln92, Val121, Phe131, Leu198, Thr200, Pro202, in addition to the coordination of the sulfonamide nitrogen to the Zn(II) ion of the active site, and participation of the SO(2)NH(2) group to a network of hydrogen bonds involving residues Thr199 and Glu106. These results are helpful for the design of better CA II or CA IX inhibitors based on the thioureido-benzenesulfonamide motif, with potential applications as anti-glaucoma or anti-cancer drugs.     

Salen and tetrahydrosalen derivatives act as effective inhibitors of the tumor-associated carbonic anhydrase XII—A new scaffold for designing isoform-selective inhibitors

Salen and tetrahydrosalen derivatives possess metal-chelating properties and have been used as ligands in organic synthesis and as scaffolds for developing therapeutic agents. Fourteen such compounds were synthesized in order to explore their ability to inhibit the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). Human (h) isoforms hCA I, hCA II, hCA IX and hCA XII were included in the investigation. Several aliphatic and aromatic spacers were introduced between the two chelating groups from salen/tetrahydrosalen in order to explore a diverse chemical space for designing CA inhibitors, which incorporate both phenol and polyamine fragments in their molecule. Some of these compounds showed CA inhibitory activity in the low micromolar–nanomolar range and a pronounced selectivity for inhibiting an isoform over-expressed in hypoxic tumors, hCA XII, over hCA I, II and IX.     

Carbonic anhydrase inhibitors. Synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with boron-containing sulfonamides, sulfamides, and sulfamates: toward agents for boron neutron capture therapy of hypoxic tumors

A library of boron-containing carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, including sulfonamides, sulfamides, and sulfamates is reported. The new compounds have been synthesized by derivatization reactions of 4-carboxy-/amino-/hydroxy-phenylboronic acid pinacol esters with amino/isothiocyanato-substituted aromatic/heteroaromatic sulfonamides or by sulfamoylation reactions with sulfamoyl chloride. The new derivatives have been assayed for the inhibition of three physiologically relevant CA isozymes, the cytosolic CA I and II, and the transmembrane, tumor-associated isozyme CA IX. Effective inhibitors were detected both among sulfonamides, sulfamates, and sulfamides. Against the human isozyme hCA I the new compounds showed inhibition constants in the range of 34-94nM, against hCA II in the range of 3.1-48nM, and against hCA IX in the range of 7.3-89nM, respectively. As hypoxic tumors highly overexpress CA IX, the design of boron-containing inhibitors with high affinity for the tumor-associated CA isozymes may lead to important advances in boron neutron capture therapy (BNCT) applications targeting such tumors, which are non-responsive to both classical chemo- and radiotherapy.