Carbonic anhydrase inhibitors: Schiff's bases of aromatic and heterocyclic sulfonamides and their metal complexes

Schiff's bases were obtained from aromatic/heterocyclic sulfonamides and amino-sulfonamide derivatives, such as sulfanilamide, homosulfanilamide, 4-aminoethyl-benzenesulfonamide and 5-amino-1,3,4-thiadiazole-2-sulfonamide. Metal complexes of some of these Schiff's bases, incorporating Zn(II), Co(lI), Ni(II) and Cu(II) ions, were also prepared and tested as inhibitors of the zinc enzyme carbonic anhydrase (CA), and more specifically the red blood cell isozymes I and II. The Schiff's bases behaved as medium potency CA I and CA II inhibitors, whereas their metal complexes showed a highly enhanced potency, with several low nanomolar CA II inhibitors detected.     

Carbonic anhydrase inhibitors: aromatic sulfonamides and disulfonamides act as efficient tumor growth inhibitors

Aromatic/heterocyclic sulfonamides generally act as strong inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). Here we report the unexpected finding that potent aromatic sulfonamide inhibitors of CA, possessing inhibition constants in the range of 10(-8)-10(-9) M (against all the isozymes), also act as efficient in vitro tumor cell growth inhibitors, with GI50 (molarity of inhibitor producing a 50% inhibition of tumor cell growth) values of 10 nM-35 microM against several leukemia, non-small cell lung cancer, ovarian, melanoma, colon, CNS, renal, prostate and breast cancer cell lines. The investigated compounds were sulfanilyl-sulfanilamide-, 4-thioureido-benzenesulfonamide- and benzene-1,3-disulfonamide-derivatives. The mechanism of antitumor action with these sulfonamides is unknown, but it might involve either inhibition of several CA isozymes (such as CA IX, CA XII, CA XIV) predominantly present in tumor cells, a reduced provision of bicarbonate for the nucleotide synthesis (mediated by carbamoyl phosphate synthetase II), the acidification of the intracellular milieu as a consequence of CA inhibition or uncoupling of mitochondria and potent CA V inhibition among others. A combination of several such mechanisms is also plausible. Optimization of such derivatives from the SAR point of view, might lead to the development of effective novel types of anticancer agents/therapies.     

Carbonic anhydrase inhibitors. Design of anticonvulsant sulfonamides incorporating indane moieties

A series of aromatic sulfonamides incorporating indane moieties were prepared starting from commercially available 1- and 2-indanamine, and their activity as inhibitors of two carbonic anhydrase (CA, EC 4.2.1.1) isozymes, hCA I and II was studied. The new sulfonamides incorporating acetamido, 4-chloro-benzoyl, valproyl, tetra-, and pentafluorobenzoyl moieties acted as very potent inhibitors of the slow red blood cell isozyme hCA I (K(i)s in the range of 1.6-8.5 nM), which usually has a lower affinity for such inhibitors, as compared to isozyme II. Some derivatives also showed excellent hCA II inhibitory properties (K(i)s in the range of 2.3-12 nM), but the anticonvulsant activity of these sulfonamides was rather low as compared to that of other sulfonamide/sulfamate CA inhibitors, such as methazolamide. Furthermore, the 2-amino/acetamido-indane-5-sulfonic acids prepared during this work also showed interesting CA inhibitory properties, with inhibition constants in the range of 43-89 nM against the two isozymes, being among the most potent sulfonic acid CA inhibitors reported so far.     

Carbonic anhydrase inhibitors: the beta-carbonic anhydrase from Helicobacter pylori is a new target for sulfonamide and sulfamate inhibitors

DNA clones for the beta-class carbonic anhydrase (CA, EC 4.2.1.1) of Helicobactor pylori (hpbetaCA) were obtained. A recombinant hpbetaCA protein lacking the N-terminal 15-amino acid residues was produced and purified, representing a catalytically efficient CA. hpbetaCA was strongly inhibited (K(I)s in the range of 24-45 nM) by many sulfonamides/sulfamates, among which acetazolamide, ethoxzolamide, topiramate, and sulpiride, all clinically used drugs. The dual inhibition of alpha- and/or beta-class CAs of H. pylori might represent a useful alternative for the management of gastritis/gastric ulcers, as well as gastric cancer. This is also the first study showing that a bacterial beta-CA can be a drug target.     

Carbonic anhydrase inhibitory properties of some uracil derivatives

Inhibitors of carbonic anhydrase (CA) have been carried out in many therapeutic applications, especially antiglaucoma activity. In this study, we investigated some uracil derivatives (4–12) to inhibit human CA I (hCA I) and II (hCA II) isoenzymes. The K_I values of the compounds 4–12 are in the range of 0.085–428?µM for hCA I and of 0.1715–645?µM against hCA II, respectively. It is concluded from the kinetic investigations, all compounds used in the study act as competitive inhibitors with substrate, 4-NPA. Uracil derivatives are emerging agents for the inhibiton of carbonic anhydrase which could be used in biomedicine.     

Carbonic anhydrase inhibitors: synthesis of N-morpholylthiocarbonylsulfenylamino aromatic/heterocyclic sulfonamides and their interaction with isozymes I, II and IV

Several aromatic/heterocyclic sulfonamides possessing free amino, imino or hydrazino moieties were transformed into the corresponding N-morpholylthiocarbonylsulfenyl derivatives, by reaction with N-morpholyldithiocarbamate in the presence of oxidizing agents (NaClO or iodine). These compounds showed nanomolar inhibition against three CA isozymes, and interesting in vitro tumor cell growth inhibitory properties, against several leukemia, non-small cell lung, ovarian, melanoma, colon, CNS, renal, prostate and breast cancer cell lines.     

Carbonic anhydrase inhibitors: synthesis of Schiff bases of hydroxybenzaldehydes with aromatic sulfonamides and their reactions with arylsulfonyl isocyanates

Reaction of o- or p-hydroxybenzaldehydes with sulfanilamide, homosulfanilamide and p-(2-aminoethyl)- benzene-sulfonamide afforded several new Schiff bases which were subsequently derivatized at the phenolic hydroxy moiety by reaction with arylsulfonylisocyanates. The new arylsulfonylcarbamates obtained in this way possessed interesting inhibitory properties against three carbonic anhydrase (CA) isozymes, hCA I, hCA II and bCA IV (h = human, b = bovine isozyme). All these new derivatives, the simple Schiff bases and the arylsulfonylcarbamates obtained as outlined above, were more inhibitory against all isozymes as compared to the corresponding parent sulfonamide from which they were obtained. Generally, the p-hydroxybenzaldehyde derivatives were more active than the corresponding ortho isomers. An interesting behavior was evidenced for some of the ortho-substituted arylsulfonylcarbamato-sulfonamides, which showed higher affinities for the isozyme hCA I, as compared to hCA II and bCA IV (generally hCA I is 10-1000 less sensitive to "normal" sulfonamide inhibitors, such as acetazolamide, methazolamide or dorzolamide, as compared to hCA II). This make the new derivatives attractive leads for designing isozyme I-specific inhibitors.     

Carbonic anhydrase inhibitors: cloning, characterization, and inhibition studies of the cytosolic isozyme III with sulfonamides

The cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isozyme III (hCA III) has been cloned and purified by the GST-fusion protein method. Recombinant pure hCA III had the following kinetic parameters for the CO(2) hydration reaction at 20 degrees C and pH 7.5: k(cat) of 1.3 x 10(4) s(-1) and k(cat)/K(M) of 2.5 x 10(5) M(-1) s(-1), being a slower catalyst for the physiological reaction as compared to the genetically related cytosolic isoforms hCA I and II. An inhibition study with a library of sulfonamides and one sulfamate, some which are clinically used compounds, is reported. hCA III is less prone to be inhibited by these compounds as compared to hCA I and II for which many low nanomolar inhibitors were detected earlier. The best hCA III inhibitors were prontosil, sulpiride, indisulam, benzolamide, aminobenzolamide, and 4-amino-6-chloro-benzene-1,3-disulfonamide which showed K(I)s in the range of 2.3-18.1 microM. Clinically used compounds such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, brinzolamide, topiramate, zonisamide, celecoxib, and valdecoxib were less effective hCA III inhibitors, with affinities in the range of 154-2200 microM. This is the first study in which low micromolar hCA III inhibitors are reported.     

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.     

Carbonic anhydrase inhibitors: aromatic and heterocyclic sulfonamides incorporating adamantyl moieties with strong anticonvulsant activity

A series of aromatic/heterocyclic sulfonamides incorporating adamantyl moieties were prepared by reaction of aromatic/heterocyclic aminosulfonamides with the acyl chlorides derived from adamantyl-1-carboxylic acid and 1-adamantyl-acetic acid. Related derivatives were obtained from the above-mentioned aminosulfonamides with adamantyl isocyanate and adamantyl isothiocyanate, respectively. Some of these derivatives showed good inhibitory potency against two human CA isozymes involved in important physiological processes, CA I, and CA II, of the same order of magnitude as the clinically used drugs acetazolamide and methazolamide. The lipophilicity of the best CA inhibitors was determined and expressed as their experimental log k' IAM and theoretical ClogP value. Their lipophilicity was propitious with the crossing of the blood-brain barrier (log k' > IAM > 1.35). The anticonvulsant activity of some of the best CA inhibitors reported here has been evaluated in a MES test in mice. After intraperitoneal injection (30 mg kg(-1)), compounds A8 and A9 exhibited a high protection against electrically induced convulsions (> 90%). Their ED50 was 3.5 and 2.6 mg kg(-1), respectively.     

Carbonic anhydrase inhibitors. Inhibition studies of a coral secretory isoform by sulfonamides

The inhibition of a newly cloned coral carbonic anhydrase (CA, EC 4.2.1.1) has been investigated with a series of sulfonamides, including some clinically used derivatives (acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide, and sulpiride, or indisulam, a compound in clinical development as antitumor drug), as well as the sulfamate antiepileptic topiramate. Some simple amino-/hydrazine-/hydroxy-substituted aromatic/heterocyclic sulfonamides have also been included in the study. All types of activity have been detected, with low potency inhibitors (K_Is in the range of 163–770 nM), or with medium potency inhibitors (K_Is in the range of 75.1–105 nM), whereas ethoxzolamide, several clinically used sulfonamides and heterocyclic compounds showed stronger potency, with K_Is in the range of 16–48.2 nM. These inhibitors may be useful to better understand the physiological role of the Stylophora pistillata CA (STPCA) in corals and its involvement in biomineralisation in this era of global warming.     

A quantitative structure-activity relationship study on some aromatic/heterocyclic sulfonamides and their charged derivatives acting as carbonic anhydrase inhibitors

A quantitative structure-activity relationship (QSAR) study is made on a series of aromatic/heterocyclic sulfonamides and their charged derivatives acting as carbonic anhydrase (CA) inhibitors. These compounds were studied by Scozzafava et al. (J. Med. Chem. 2000; 43: 292) for the selective inhibition of CAs--sulfonamides generally do not discriminate between different CA isozymes and hence exhibit many undesirable side effects when used as drugs against a particular disease. In this communication, an attempt has been made to investigate the physicochemical and structural properties that can make them selective for a given CA isozyme. Based on in vitro data reported by Scozzafava et al. against two cytosolic isozymes and one membrane-bound isozyme, the QSAR study has shown that uncharged compounds cannot be made selective for cytosolic or membrane-bound isozyme since in both the cases the compounds appear to follow the same mechanism of inhibition. However, for the charged compounds the polarizability of the molecule seems to greatly favor the inhibition of the membrane-bound enzyme, and hence they can be made selective for this enzyme by enhancing their polarizability, which is found to play no role in the inhibition of cytosolic enzymes.     

Carbonic anhydrase VII is S-glutathionylated without loss of catalytic activity and affinity for sulfonamide inhibitors

Human carbonic anhydrase (CA, EC 4.2.1.1) VII is a cytosolic enzyme with high carbon dioxide hydration activity. Here we report an unexpected S-glutathionylation of hCA VII which has also been observed earlier in vivo for hCA III, another cytosolic isoform. Cys183 and Cys217 were found to be the residues involved in reaction with glutathione for hCA VII. The two reactive cysteines were then mutated and the corresponding variant (C183S/C217S) expressed. The native enzyme, the variant and the S-glutathionylated adduct (sgCA VII) as well as hCA III were fully characterized for their CO(2) hydration, esterase/phosphatase activities, and inhibition with sulfonamides. Our findings suggest that hCA VII could use the in vivo S-glutathionylation to function as an oxygen radical scavenger for protecting cells from oxidative damage, as the activity and affinity for inhibitors of the modified enzyme are similar to those of the wild type.     

Carbonic anhydrase inhibitors: antisecretory and cytoprotective properties

Experimental studies located carbonic anhydrase (CA) in the parietal cells close to secretory canaliculi, in superficial epithelial cells and gastric microvasculature. The role of CA is CO2 hydration resulting H+ for acid secretion and conversion of OH into HCO3-. Our studies showed that the physiological secretagogue histamine, acetylcholine and gastrin are all CA activators, achieving potentiating interactions. Catecholamines are also strong enzymatic activators. Beside sulfonamides, other CA inhibitors are anticholinergics, PGE and PGI2, some calcium channel blockers, alpha 2- and beta 1-adrenoceptor blockers and Zn2+. Cytoprotective properties of CA inhibitors gained experimental evidence in the past years. These effects could be based on increase of gastric mucosal blood flow, proved experimentally, which might be mediated by increase of endogenous prostaglandin synthesis and sulfhydryls and, respectively, motility changes. The unique combination of strong antisecretory effect with the cytoprotective action explain the outstanding clinical efficacy of CA inhibitors in the healing of gastric and duodenal ulcers.     

Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides derived from 4-isothiocyanato-benzolamide

A series of sulfonamides incorporating 4-thioureido-benzolamide moieties have been prepared from aminobenzolamide and thiophosgene followed by the reaction of the thiocyanato intermediate with aliphatic/aromatic amines or hydrazines. The new derivatives have been investigated as inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), and more precisely of the cytosolic isozymes hCA I and II, as well as the tumor-associated isozyme hCA IX (all of human origin). The new compounds showed excellent inhibitory properties against all three isozymes with inhibition constants in the range of 0.6-62 nM against hCA I, 0.5-1.7 nM against hCA II and 3.2-23 nM against hCA IX, respectively. These derivatives are interesting candidates for the development of novel therapies targeting hypoxic tumors.     

Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides incorporating 1,2,4-triazine moieties

A series of benzenesulfonamide derivatives incorporating triazine moieties in their molecules was obtained by reaction of cyanuric chloride with sulfanilamide, homosulfanilamide, or 4-aminoethylbenzenesulfonamide. The dichlorotriazinyl-benzenesulfonamides intermediates were subsequently derivatized by reaction with various nucleophiles, such as water, methylamine, or aliphatic alcohols (methanol and ethanol). 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, tumor-associated hCA IX. The new compounds reported here inhibited hCA I with K(I)s in the range of 75-136nM, hCA II with K(I)s in the range of 13-278nM, and hCA IX with K(I)s in the range of 0.12-549nM. The first hCA IX-selective inhibitors were thus detected, as the chlorotriazinyl-sulfanilamide and the bis-ethoxytriazinyl derivatives of sulfanilamide/homosulfanilamide showed selectivity ratios for CA IX over CA II inhibition in the range of 166-706. Furthermore, some of these compounds have subnanomolar affinity for hCA IX, with K(I)s in the range 0.12-0.34nM. These derivatives are interesting candidates for the development of novel unconventional anticancer strategies targeting the hypoxic areas of tumors. Clear renal cell carcinoma, which is the most lethal urologic malignancy and is both characterized by very high CA IX expression and chemotherapy unresponsiveness, could be the leading candidate of such novel therapies.     

Carbonic anhydrase inhibitors: Inhibition of the tumor-associated isozymes IX and XII with polyfluorinated aromatic/heterocyclic sulfonamides

The tumor-associated transmembrane carbonic anhydrase (CA, EC 4.2.1.1) isozymes IX (CA IX) and XII (CA XII) are involved in acidification of hypoxic tumors, a process correlated with poor prognosis and clinical outcome of patients harboring such tumors. This process may be reversed by inhibiting these enzymes with potent sulfonamide/sulfamate inhibitors. A series of such aromatic/heterocyclic sulfonamides incorporating 2,3,5,6-tetrafluorobenzoyl-, 2,3,5,6-tetrafluorophenylsulfonyl- and pentafluorophenylureido moieties has been investigated for its interaction with the catalytic domain of the human isozymes hCA IX and hCA XII. Some of these compounds showed excellent inhibitory properties against both isozymes IX and XII, with several subnanomolar inhibitors detected for the first time. These sulfonamides may constitute valuable candidates for the development of novel antitumor therapies based on the inhibition of such tumor-associated CA isozymes.     

Carbonic anhydrase inhibitors: Inhibition of the tumor-associated isozymes IX and XII with polyfluorinated aromatic/heterocyclic sulfonamides

The tumor-associated transmembrane carbonic anhydrase (CA, EC 4.2.1.1) isozymes IX (CA IX) and XII (CA XII) are involved in acidification of hypoxic tumors, a process correlated with poor prognosis and clinical outcome of patients harboring such tumors. This process may be reversed by inhibiting these enzymes with potent sulfonamide/sulfamate inhibitors. A series of such aromatic/heterocyclic sulfonamides incorporating 2,3,5,6-tetrafluorobenzoyl-, 2,3,5,6-tetrafluoro- phenylsulfonyl- and pentafluorophenylureido moieties has been investigated for its interaction with the catalytic domain of the human isozymes hCA IX and hCA XII. Some of these compounds showed excellent inhibitory properties against both isozymes IX and XII, with several subnanomolar inhibitors detected for the first time. These sulfonamides may constitute valuable candidates for the development of novel antitumor therapies based on the inhibition of such tumor-associated CA isozymes.     

Carbonic anhydrase inhibitors. Inhibition of the prokariotic beta and gamma-class enzymes from Archaea with sulfonamides

A detailed inhibition study of carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the beta- and gamma-families from Archaea with sulfonamides has been performed. Compounds included in this study were the clinically used sulfonamide CA inhibitors, such as acetazolamide, methazolamide, ethoxzolamide, topiramate, valdecoxib, celecoxib, dorzolamide, sulfanilamide, dichlorophanamide, as well as sulfanilamide analogs, halogenated sulfanilamides, and some 1,3-benzenedisulfonamide derivatives. The two gamma-CAs from Methanosarcina thermophila (Zn-Cam and Co-Cam) showed very different inhibitory properties with these compounds, as compared to the alpha-CA isozymes hCA I, II, and IX, and the beta-CA from Methanobacterium thermoautotrophicum (Cab). The best Zn-Cam inhibitors were sulfamic acid and acetazolamide, with inhibition constants in the range of 63-96 nM, whereas other investigated aromatic/heterocylic sulfonamides showed a rather levelled behavior, with KIs in the range of 0.12-1.70 microM. The best Co-Cam inhibitors were topiramate and p-aminoethyl-benzenesulfonamide, with KIs in the range of 0.12-0.13 microM, whereas the worst one was homosulfanilamide (KI of 8.50 microM). In the case of Cab, the inhibitory power of these compounds varied to a much larger extent, with sulfamic acid and sulfamide showing millimolar affinities (KIs in the range of 44-103 mM), whereas the best inhibitor was ethoxzolamide, with a KI of 5.35 microM. Most of these sulfonamides showed inhibition constants in the range of 12-100 microM against Cab. Thus, the three CA families investigated up to now possess a very diverse affinity for sulfonamides, the inhibitors with important medicinal, and environmental applications.     

Synthesis and characterization of novel dioxoacridine sulfonamide derivatives as new carbonic anhydrase inhibitors

Novel dioxoacridine sulfonamide compounds were synthesized from reaction of cyclic 1,3-diketones, sulfanilamide (4-amino benzene sulfonamide) and aromatic aldehydes. The structures of these compounds were confirmed by using spectral analysis (IR, H-NMR, (13)C-NMR, and mass). Human carbonic anhydrase isoenzymes (hCA I and hCA II) were purified from erythrocyte cells by affinity chromatography. The inhibitory effects of sulfanilamide, acetazolamide (AAZ), and newly synthesized sulfonamides on hydratase and esterase activities of these isoenzymes have been studied in vitro. The IC(50) values of compounds for esterase activity are 0.71-0.11 μM for hCA I and 0.45-0.12 μM for hCA II, respectively. The K(i) values of these inhibitors were determined as 0,38-0,008 μM for hCA I and 0,19-0,001 μM for hCA II, respectively.