Carbonic anhydrase inhibitors: Benzenesulfonamides incorporating cyanoacrylamide moieties are low nanomolar/subnanomolar inhibitors of the tumor-associated isoforms IX and XII

A series of benzenesulfonamides incorporating cyanoacrylamide moieties (tyrphostine analogues) have been obtained by reaction of sulfanilamide with ethylcyanoacetate followed by condensation with aromatic/heterocyclic aldehydes, isothiocyanates or diazonium salts. The new compounds have been investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4. 2.1.1), and more specifically against the cytosolic human (h) isoforms hCA I and II, as well as the transmembrane, tumor-associated ones CA IX and XII, which are validated antitumor targets. Most of the new benzenesulfonamides were low nanomolar or subnanomolar CA IX/XII inhibitors whereas they were less effective as inhibitors of CA I and II. The structure–activity relationship for this class of effective CA inhibitors is also discussed. Generally, electron donating groups in the starting aldehyde reagent favored CA IX and XII inhibition, whereas halogeno, methoxy and dimethylamino moieties led to very potent CA XII inhibitors.     

Sulfonamides incorporating boroxazolidone moieties are potent inhibitors of the transmembrane, tumor-associated carbonic anhydrase isoforms IX and XII

A new series of sulfonamides was synthesized by the reaction of the boroxazolidone complex of l-lysine with isothiocyanates incorporating sulfamoyl moieties and diverse organic scaffolds. The obtained thioureas have been investigated as inhibitors of four physiologically relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, hCA I, II, IX and XII. Inhibition between the low nanomolar to the micromolar range has been observed against them, with several low nanomolar and tumor-CA selective inhibitors detected. These boron-containing compounds might be useful for the management of hypoxic tumors overexpressing hCA IX/XII by means of boron neutron capture therapy, a technique not investigated so far with inhibitors of this enzyme.     

Carbonic anhydrase inhibitors. Inhibition of cytosolic isoforms I and II, and extracellular isoforms IV, IX, and XII with sulfamides incorporating sugar moieties

A series of glycosylated sulfamides possessing a diverse substitution pattern, with benzylated, peracetylated, and unsaturated six- and five-membered ring sugar moieties attached to the NHSO(2)NH(2) zinc binding group is reported. These derivatives were tested for the inhibition of five human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, hCA I, II, IV, IX, and XII. Against hCA I the sulfamides behaved as weak inhibitors, whereas they showed low nanomolar activity against hCA II, IX, and XII, being slightly less effective as hCA IV inhibitors. One compound showed selectivity for inhibiting the tumor-associated isoforms hCA IX and XII over the ubiquitous cytosolic hCA II. The sulfamide zinc binding group may thus indeed lead to very effective glycosylated inhibitors targeting several physiologically relevant isozymes.     

Inhibition of carbonic anhydrase isoforms I,II, IX and XII with novel Schiff bases: Identification of selective inhibitors for the tumor-associated isoforms over the cytosolic ones

A series of new Schiff bases was obtained from sulfanilamide, 3-fluorosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide and aromatic/heterocyclic aldehydes incorporating both hydrophobic and hydrophilic moieties. The obtained sulfonamides were investigated as inhibitors of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic CA I and II, as well as the transmembrane, tumor-associated CA IX and XII. Most derivatives were medium potency or weak hCA I/II inhibitors, but several of them showed nanomolar affinity for CA IX and/or XII, making them an interesting example of isoform-selective compounds. The nature of the aryl/hetaryl moiety present in the initial aldehyde was the main factor influencing potency and isoform selectivity. The best and most CA IX-selective compounds incorporated moieties such as 4-methylthiophenyl, 4-cyanophenyl-, 4-(2-pyridyl)-phenyl and the 4-aminoethylbenzenesulfonamide scaffold. The best hCA XII inhibitors, also showing selectivity for this isoform, incorporated 2-methoxy-4-nitrophenyl-, 2,3,5,6-tetrafluorophenyl and 4-(2-pyridyl)-phenyl functionalities and were also derivatives of 4-aminoethylbenzenesulfonamide. The sulfanilamide and 3-fluorosulfanilamide derived Schiff bases were less active compared to the corresponding 4-aminoethyl-benzenesulfonamide derivatives. As hCA IX/XII selective inhibition is attractive for obtaining antitumor agents/diagnostic tools with a new mechanism of action, compounds of the type described here may be considered interesting preclinical candidates.     

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.     

Synthesis of 4-sulfamoylphenyl-benzylamine derivatives with inhibitory activity against human carbonic anhydrase isoforms I,II, IX and XII

Imine derivatives were obtained by condensation of sulfanilamide with substituted aromatic aldehydes. The Schiff bases were thereafter reduced with sodium borohydride, leading to the corresponding amines, derivatives of 4-sulfamoylphenyl-benzylamine. 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 (transmembrane, tumor-associated enzymes). We noted that the compounds incorporating secondary amine moieties showed a better inhibitory activity against all CA isozymes compared to the corresponding Schiff bases. Low nanomolar CA II, IX and XII inhibitors were detected, whereas the activity against hCA I was less potent. The secondary amines incorporating sulfonamide or similar zinc-binding groups, poorly investigated chemotypes for designing metalloenzyme inhibitors, may offer interesting opportunities in the field due to the facile preparation and possibility to explore a vast chemical space.     

Carbonic anhydrase inhibitors: inhibition of human cytosolic isozymes I and II and tumor-associated isozymes IX and XII with S-substituted 4-chloro-2-mercapto-5-methyl-benzenesulfonamides

A series of S-substituted 4-chloro-2-mercapto-5-methyl-benzenesulfonamides has been investigated as inhibitors of four isoforms of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), that is, the cytosolic, ubiquitous isozymes CA I and II, as well as the transmembrane, tumor-associated isozymes CA IX and XII. The new derivatives were inefficient inhibitors of isoform I (K(I)s in the range of 2.7-18.7 microM) but generally had low nanomolar affinity for the inhibition of the other three isoforms (K(I)s in the range of 2.4-214 nM against hCA II; 1.4-47.5 nM against hCA IX, and 1.7-569 nM against hCA XII, respectively). Some selectivity for the inhibition of the tumor-associated versus the cyctosolic isoform II with some of these compounds has also been evidenced. As CA IX is an important marker of tumor hypoxia and its predictive, prognostic, and druggability potentials for designing antitumor therapies were recently validated, detection of selective, potent CA IX inhibitors may be relevant in the fight against cancers overexpressing CA isozymes.     

Synthesis of novel benzenesulfonamide bearing 1,2,3-triazole linked hydroxy-trifluoromethylpyrazolines and hydrazones as selective carbonic anhydrase isoforms IX and XII inhibitors

A series of twenty four hydroxy-trifluoromethylpyrazoline-carbonyl-1,2,3-triazoles and four hydrazones bearing benzenesulfonamide moieties was obtained by condensation of carboxyhydrazides with substituted 1,3-diketones. All the newly synthesized compounds were investigated as inhibitors of physiologically and pharmacologically relevant human (h) carbonic anhydrsae (CA, EC 4.2.1.1) cytosolic isoforms hCA I and II, as well as transmembrane tumor-assosciated isoforms hCA IX and XII. These compounds exhibited excellent CA inhibitory potency against the four CA isoenzymes as compared to clinically used reference drug acetazolamide (AAZ). Some compounds bearing bulkier group at C-5′ position of 1,2,3-triazoles ring were weaker inhibitors of hCA I. Inhibition assay against hCA II indicates, that several derivatives exhibited upto 27-fold more effective inhibitory activity compared to AAZ. Five of the assayed compounds displayed low nanomolar potency (K_i ≤ 10 nM) against hCA IX, whereas five compounds were found to be endowed with excellent inhibitory potencies (K_i ≤ 5 nM) against hCA XII. The biological activity profile presented herein will be useful for designing new leads and provide candidates for preclinical investigations.     

Sulfonamides incorporating 1,3,5-triazine moieties selectively and potently inhibit carbonic anhydrase transmembrane isoforms IX, XII and XIV over cytosolic isoforms I and II: Solution and X-ray crystallographic studies

Reaction of cyanuryl chloride with d,l-amino acids and amino alcohols afforded a new series of triazinyl-substituted benzenesulfonamides incorporating amino acyl/hydroxyalkyl-amino moieties. Inhibition studies of physiologically relevant human carbonic anhydrase (CA, EC 4.2.1.1) isoforms, such as CA I, II, IX, XII and XIV with these compounds are reported. They showed moderate-weak inhibition of the cytosolic, offtarget isozymes CA I and II, but many of them were low nanomolar inhibitors of the transmembrane, tumor-associated CA IX and XII (and also of CA XIV). The X-ray crystal structure of two of these compounds in adduct with CA II allowed us to understand the features associated with this strong inhibitory properties and possibly also their selectivity. Two of these compounds were also investigated for the inhibition of other human isoforms, that is, hCA IV, VA, VB, VI, VII and XIII, as well as inhibitors of the fungal pathogenic CAs Nce103 (Candida albicans) and Can2 (Cryptococcus neoformans), showing interesting activity. The 1,3,5-triazinyl-substituted benzenesulfonamides constitute thus a class of compounds with great potential for obtaining inhibitors targeting both α-class mammalian, tumor-associated, and β-class from pathogenic organisms CAs.     

Inhibition of carbonic anhydrase isoforms I,II, IV,VII and XII with carboxylates and sulfonamides incorporating phthalimide/phthalic anhydride scaffolds

We report a panel of carboxylates and sulfonamides incorporating phthalic anhydride and phthalimide moieties in their structure and their interaction with the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). They were synthesized from substituted anthranilic acids and trimellitic anhydride chloride, followed by reaction with primary amines and were tested for the inhibition of five physiologically relevant CA isoforms, the human (h) hCA I, II, IV, VII and XII, some of which are involved in serious pathologies (CA II, IV and XII in glaucoma; CA VII in epilepsy; CA XII in some solid tumors). The carboxylic acids were generally poor inhibitors of isoforms hCA I, II and IV but were highly effective, low nanomolar inhibitors of hCA VII and XII. The sulfonamides inhibited all isoforms significantly, and some of them were sub-nanomolar hCA VII inhibitors, although their isoform selectivity was lower compared to the carboxylates. This study proves that carboxylic acids incorporating a phthalic anhydride/phthalimide based scaffold may lead to isoform-selective inhibitors by applying the tail approach, mostly used up until now for obtaining sulfonamide, sulfamide and sulfamate CA inhibitors.     

Carbonic anhydrase inhibitors. Interaction of 2-(hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide with 12 mammalian isoforms: kinetic and X-ray crystallographic studies

2-(Hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide was tested for its interaction with 12 carbonic anhydrase (CA, EC 4.2.1.1) isoforms in the search of compounds with good inhibitory activity against isozymes with medicinal chemistry applications, such as CA I, II, VA, VB, VII, IX, and XII among others. This sulfonamide is a potent inhibitor of CA I and II (K(I)s of 7.2-7.5 nM), a medium potency inhibitor of CA VII, IX, XII, and XIV, and a weak inhibitor against the other ubiquitous isoforms, making it thus a very interesting clinical candidate for situations in which a strong inhibition of CA I and II is needed. The crystal structure of the hCA II adduct of this sulfonamide revealed many favorable interactions between the inhibitor and the enzyme which explain its strong low nanomolar affinity for this isoform but may also be exploited for the design of effective inhibitors incorporating bicyclic moieties.     

Novel sulfonamide bearing coumarin scaffolds as selective inhibitors of tumor associated carbonic anhydrase isoforms IX and XII

Four novel scaffolds consisting of total 24 compounds (1a–1o, 2a–2c, 3a–3c and 4a–4c) bearing aromatic sulfonamide and coumarin moieties connected through various linkers were synthesized in order to synergize the inhibition potential of both the moieties against four selected human carbonic anhydrase isoforms (hCA I, II, IX & XII). All compounds were found to be potent inhibitors of tumor associated hCA IX & XII while at the same time required large amounts to inhibit off-targeted housekeeping hCA I & II. Selectivity was more pronounced against hCA II over I, and hCA XII over IX. Results were compared with antitumor drug acetazolamide. One derivative 2b of series 2 was found to be a better selective inhibitor of 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.     

Isocoumarins: a new class of selective carbonic anhydrase IX and XII inhibitors

Isocoumarins, isomeric to comarins which act as effective carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, were investigated for the first time as inhibitors of this enzyme. A series of 3-substituted and 3,4-disubstituted isocoumarins incorporating phenylhydrazone, 1-phenyl-pyrazole and pyrazolo-substituted pyrimidine trione/thioxo-pyrimidine dione moieties were investigated for their interaction with four human (h) CA isoforms, hCA I, II, IX and XII, known to be important drug targets. hCA I and II were not inhibited by these compounds, whereas hCA IX and XII were inhibited in the low micromolar range by the less bulky derivatives. The inhibition constants ranged between 2.7–78.9 µM against hCA IX and of 1.2–66.5 µM against hCA XII. As for the coumarins, we hypothesise that the isocoumarins are hydrolysed by the esterase activity of the enzyme with formation of 2-carboxy-phenylacetic aldehydes which act as CA inhibitors. Isocoumarins represent a new class of CA inhibitors.     

Synthesis of aminocyanopyrazoles via a multi-component reaction and anti-carbonic anhydrase inhibitory activity of their sulfamide derivatives against cytosolic and transmembrane isoforms

A convenient protocol for the multicomponent reaction (MCRs) between malononitrile with an orthoester and hydrazine derivatives, under acid catalyst is described. A series of aminocyanopyrazoles 4 was prepared, isolated and characterized. These pyrazoles reacted with sodium nitrite followed by secondary amine reagent and with formic acid to lead pyrazolotriazines 6 and pyrazolopyrimidinones 7. Some of the aminopyrazoles were converted to the corresponding sulfamides by reaction with sulfamoyl chloride. The aminopyrazoles incorporating phenyl and tosyl moieties were tested as inhibitors of four carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the human (h) hCA I, II, IX and XII. Many of them showed low micromolar or submicromolar inhibition of these enzymes. The corresponding sulfamides were low nanomolar CA inhibitors.     

Carbonic anhydrase inhibitors. Interaction of indapamide and related diuretics with 12 mammalian isozymes and X-ray crystallographic studies for the indapamide-isozyme II adduct

Diuretics such as hydrochlorothiazide, hydroflumethiazide, quinethazone, metolazone, chlorthalidone, indapamide, furosemide, and bumetanide containing primary sulfamoyl moieties were reevaluated as inhibitors of 12 human carbonic anhydrases (hCAs, EC 4.2.1.1). These drugs considerably inhibit (low nanomolar range) some CA isozymes involved in critical physiologic processes, among the 16 present in vertebrates, for example, metolazone against CA VII, XII, and XIII, chlorthalidone against CA VB, VII, IX, XII, and XIII, indapamide against CA VII, IX, XII, and XIII, furosemide against CA I, II, and XIV, and bumetanide against CA IX and XII. The X-ray crystal structure of the hCA II-indapamide adduct was also resolved at high resolution.     

Novel coumarins and 2-thioxo-coumarins as inhibitors of the tumor-associated carbonic anhydrases IX and XII

A series of coumarins incorporating tert-butyl-dimethylsilyloxy- or allyoxy- moieties in positions 4-, 6 or 7 of the heterocyclic ring have been synthesized and then converted to the corresponding 2-thioxo-coumarins. Other derivatives incorporating hydroxyethyloxy-, tosylethoxy- and 2-fluroethyloxy- moieties in position 7 of the coumarin ring were synthesized together with derivatives of 4-methyl-7-amino coumarin incorporating acetamido, 3,5-dimethylphenylureido- and tert-butyloxycarbonylamido functionalities. All these compounds were assayed as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The human (h) cytosolic isoforms hCA I and II were weakly inhibited (hCA I) or not inhibited at all (hCA II) by these (thioxo)coumarins whereas the tumor-associated transmembrane isoforms hCA IX and XII were inhibited with efficiencies from the submicromolar to the low micromolar range by many of these derivatives. The structure-activity relationship for these classes of less investigated CA inhibitors are delineated, with the potential of using them as leads to obtain isoform-selective inhibitors with excellent affinity for CA IX and XII (validated antitumor targets) which do not significantly inhibit the cytosolic offtarget isoforms hCA I and II.     

Carbonic anhydrase inhibitors: inhibition of the tumor-associated isozyme IX with fluorine-containing sulfonamides. The first subnanomolar CA IX inhibitor discovered

Polyfluorinated CAIs show very good inhibitory properties against different carbonic anhydrase (CA) isozymes, such as CA I, II, and IV, but such compounds have not been tested for their interaction with the transmembrane, tumor-associated isozyme CA IX. Thus, a series of such compounds has been obtained by attaching 2,3,5,6-tetrafluorobenzoyl- and 2,3,5,6-tetrafluorophenylsulfonyl- moieties to aromatic/heterocyclic sulfonamides possessing derivatizable amino moieties. Some of these compounds showed excellent CA IX inhibitory properties and also selectivity ratios favorable to CA IX over CA II, the other physiologically relevant isozyme with high affinity for sulfonamide inhibitors. The first subnanomolar and rather selective CA IX inhibitor has been discovered, as the 2,3,5,6-tetrafluorobenzoyl derivative of metanilamide showed an inhibition constant of 0.8 nM against hCA IX, and a selectivity ratio of 26.25 against CA IX over CA II. Several other low nanomolar CA IX inhibitors were detected among the new derivatives reported here. The reported derivatives constitute valuable candidates for the development of novel antitumor therapies based on the selective inhibition of tumor-associated CA isozymes.     

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.     

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.