Discovery of novel 1,3-diaryltriazene sulfonamides as carbonic anhydrase I,II, VII,and IX inhibitors

A series of new 1,3-diaryltriazene sulfonamides was synthesised by reaction of diazonium salt of metanilamide (3-aminobenzene sulfonamide) with substituted aromatic amines. The obtained new compounds were assayed as inhibitors of four physiologically and pharmacologically relevant human (h) isoforms of carbonic anhydrases (CA, EC 4.2.1.1), specifically, hCA I, hCA II, and hCA VII (cytosolic isoforms), as well as the tumour-associated membrane-bound isoform hCA IX. All isoforms investigated here were inhibited by the newly synthesised 1,3-diaryltriazene sulfonamide derivatives from the micromolar to the nanomolar range. The cytosolic isoforms were inhibited with K_is in the range of 92.3–8371.1?nM (hCA I), 4.3–9194.0?nM (hCA II), and 15.6–9477.8?nM (hCA VII), respectively. For the membrane-bound tumour-associated isoform hCA IX, the K_I-s ranged between 50.8 and 9268.5?nM. The structure–activity relationship (SAR) with these newly synthesised metanilamide derivatives are discussed in detail.     

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 activators: an activation study of the human mitochondrial isoforms VA and VB with amino acids and amines

The mitochondrial isozymes of human carbonic anhydrase (hCA, EC 4.2.1.1), hCA VA and hCA VB, were investigated for activation with a series of amino acids and amines. D-His, L-DOPA, histamine, dopamine, and 4-(2-aminoethyl)morpholine were excellent hCA VA activators, with KAs in the range of 10-130 nM. Good hCA VB activating effects were identified for L-His, D-Phe, D-DOPA, L-Trp, L-Tyr, serotonin, and 2-(2-aminoethyl)-pyridine, with KAs in the range of 44-110 nM. All these activators enhanced kcat, having no effect on KM, favoring thus the rate-determining step in the catalytic cycle, the proton transfer reactions between the active site and environment. The activation pattern of the two mitochondrial isoforms is very different from each other and as compared to those of the cytosolic isoforms hCA I and II.     

Carbonic anhydrase inhibitors. Aromatic/heterocyclic sulfonamides incorporating phenacetyl,pyridylacetyl and thienylacetyl tails act as potent inhibitors of human mitochondrial isoforms VA and VB

A series of aromatic/heterocyclic sulfonamides incorporating phenyl(alkyl), halogenosubstituted-phenyl- or 1,3,4-thiadiazole-sulfonamide moieties and thienylacetamido; phenacetamido and pyridinylacetamido tails were prepared and assayed as inhibitors of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic human (h) hCA I and hCA II, and the mitochondrial hCA VA and hCA VB. The new compounds showed moderate inhibition of the two cytosolic isoforms (K_Is of 50–390 nM) and excellent inhibitory activity against the two mitochondrial enzymes, with many low nanomolar inhibitors detected (K_Is in the range of 5.9–10.2 nM). All substitution patterns explored here lead to effective hCA VA/VB inhibitors. Some hCA VA/VB selective inhibitors were also detected, with selectivity ratios for inhibiting the mitochondrial over the cytosolic isozymes of around 55.5–56.9. As hCA VA/VB are involved in several biosynthetic processes catalyzed by pyruvate carboxylase, acetyl CoA carboxylase, and carbamoyl phosphate synthetases I and II, providing the bicarbonate substrate to these carboxylating enzymes involved in fatty acid biosynthesis, their selective inhibition may lead to the development of antiobesity agents possessing a new mechanism of action.     

Carbonic anhydrase activators: Activation of the human cytosolic isozyme III and membrane-associated isoform IV with amino acids and amines

An activation study of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA III (cytosolic) and IV (membrane-associated) with a series of natural and non-natural amino acids and aromatic/heterocyclic amines is reported. hCA III was efficiently activated by d-His, serotonin, pyridyl-alkylamines, and aminoethyl-piperazine/morpholine (K_As of 91nM–1.12 μM), whereas the best hCA IV activators were 4-amino-phenylalanine, serotonin, and 4-(2-aminoethyl)-morpholine (K_As of 79 nM–3.14 μM). Precise steric and electronic requirements are needed to be present in the molecules of effective CA III/IV activators, in order to assure an adequate fit within the enzyme active site for the formation of the enzyme-activator complex, and for efficient proton transfer processes between the active site and the reaction medium. The activation profiles of CA III and IV are distinct from those of all other mammalian CA isoforms investigated so far for their interaction with amino acids and amines.     

Carbonic anhydrase activators: activation of the human tumor-associated isozymes IX and XII with amino acids and amines

The first activation study of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms associated to tumors, hCA IX and XII, with a small library of natural and non-natural amino acids as well as aromatic/heterocyclic amines is reported. hCA IX was activated efficiently by dopamine, adrenaline and heterocyclic amines possessing aminoethyl-/aminomethyl-moieties (K(A)s of 9 nM-1.07 microM), whereas the best hCA XII activators were serotonin, L-adrenaline, 4-(2-aminoethyl)-morpholine and d-Phe (K(A) of 0.24-0.41 microM). Precise steric and electronic requirements are needed to be present in the molecules of effective hCA IX/hCA XII activators, in order to assure an adequate fit within the enzyme active site cavity for the formation of the enzyme-activator complex, and for an efficient proton transfer process within this complex, leading to the release of a proton and formation of the catalytically active, zinc-hydroxide species of the enzyme. Selective activation of these CA isoforms might be useful to develop pharmacologic tools or to understand whether some of these biogenic amines/amino acids may influence the progression of tumors overexpressing CA IX and/or CA 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.     

Mono- and di-halogenated histamine,histidine and carnosine derivatives are potent carbonic anhydrase I,II, VII,XII and XIV activators

Mono- and di-halogenated histamines, l-histidine methyl ester derivatives and carnosine derivatives incorporating chlorine, bromine and iodine were prepared and investigated as activators of five carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic hCA I, II and VII, and the transmembrane hCA XII and XIV. All of them were activated in a diverse manner by the investigated compounds, with a distinct activation profile.     

Microwave assisted synthesis of novel tetrazole/sulfonamide derivatives based on octahydroacridine,xanthene and chromene skeletons as inhibitors of the carbonic anhydrases isoforms I,II, IV and VII

The synthesis of novel tetrazole/sulfonamide derivatives based on octahydroacridine, xanthene and chromene scaffold by using microwave (MW) assisted techniques is reported in this study. These synthesized hybrid compounds were assayed for the inhibition of carbonic anhydrase (CA, EC 4.2.1.1). The inhibitory activities were determined against three cytosolic human isoforms (hCA I, II and VII) and one membrane-associated (hCA IV) isoform. Some of the newly synthesized sulfonamides showed micromolar to nanomolar inhibitory activity against these enzymes.     

Synthesis and biological evaluation of novel N,N′-diaryl cyanoguanidines acting as potent and selective carbonic anhydrase II inhibitors

A series of novel N,N′′-diaryl cyanoguanidines were synthesized by reacting diphenyl N-cyanocarbonimidate with sulfanilamide followed by treatment of the obtained cyano-O-phenylisourea with substituted aromatic amines. The newly prepared N,N′′-diaryl cyanoguanidines showed a very interesting inhibition profile against four selected human carbonic anhydrase (CA, EC 4.2.1.1) isoforms, hCA I and hCA II (cytosolic), hCA IV (membrane-bound), and hCA IX (transmembrane). All these compounds showed a potent inhibition against isoform hCA II,with inhibition constants in the low nanomolar range, as well as a high selectivity for hCA II over hCA I, IV and IX. Since hCA II is an important drug target for antiglaucoma agents, these isoform-selective inhibitors may be considered of interest for further medicinal/pharmacologic studies.     

Synthesis and exploration of 2-morpholino-4-phenylthiazol-5-yl acrylamide derivatives for their effects against carbonic anhydrase I,II, IX and XII isoforms as a non-sulfonamide class of inhibitors

Novel series of 2-morpholino-4-phenylthiazol-5-yl acrylamide derivatives (8a–s) have been synthesized and explored as a non-sulfonamide class of carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The newly synthesized molecules were evaluated for their CA inhibitory potency against four isoforms: the cytosolic isozyme hCA I, II as well as trans-membrane tumor associated isoform hCA IX and hCA XII taking acetazolamide (AAZ) as standard drug. The results revealed that most of the compounds showed good activity against hCA II, IX, and XII whereas none of them were active against hCA I (K_i >100 μM). It is observed that the physiologically most important cytosolic isoform hCA II was inhibited by these molecules in the range of K_i 9.3–77.7 μM. It is also found the both the transmembrane isoforms hCA IX and XII were also inhibited with K_is ranging between 54.7–96.7 μM and 4.6–8.8 μM, respectively. The binding modes of the active compounds within the catalytic pockets of hCA II, IX and XII were evaluated by docking studies. This new non-sulfonamide class of selective inhibitors of hCA II, IX and XII over the hCA I isoform may be used for further understanding the physiological roles of some of these isoforms in various pathologies.     

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.     

Discovery of new ureido benzenesulfonamides incorporating 1,3,5-triazine moieties as carbonic anhydrase I,II, IX and XII inhibitors

A series of twenty novel ureido benzenesulfonamides incorporating 1,3,5-triazine moieties substituted on one side with aromatic amines and on the other side with dimethylamine, morpholine and piperidine is reported. The compounds were synthesized from the 4-(3-(4,6-dichloro-1,3,5-triazin-2-yl)ureido)benzensulfonamide (1) by using stepwise nucleophilic substitution of the chlorine atoms of cyanuric chloride. The intermediates 2(a-e) and final compounds 3(a-o) were tested for their efficiency as carbonic anhydrase (CA) inhibitors against four selected physiologically relevant human carbonic anhydrase (CA, EC 4.2.1.1) isoforms, namely, the cytosolic ones hCA I and II, and the transmembrane, tumor associated ones hCA IX, and XII. The compounds 2a, 2e and 3m showed the highest activity for hCA IX with K_is in the range of 11.8–14.6?nM. Most of the compounds showed high hCA IX selectivity over the abundant off-target isoforms hCA I and II. Since hCA IX is a validated drug target for anticancer/antimetastatic agents, these isoform-selective and potent inhibitors may be considered of interest for further medicinal/pharmacologic studies.     

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. Identification of selective inhibitors of the human mitochondrial isozymes VA and VB over the cytosolic isozymes I and II from a natural product-based phenolic library

We have investigated the enzyme inhibition characteristics of a natural product (NP)-based phenolic library against a panel of human carbonic anhydrases (hCAs, EC 4.2.1.1) which included hCAs I and II (cytosolic) and hCA VA/VB (mitochondrial isoforms). Most of these compounds were weak, micromolar inhibitors of the two cytosolic hCAs (K_Is >10 μM) but showed good hCA VA/VB inhibitory activity with inhibition constants in the range of 70–125 nM. The selectivity ratios for inhibiting the mitochondrial over the cytosolic isoforms for these phenol derivatives were in the range of 120–3800, making them the most isoform-selective compounds for inhibiting hCA VA/VB known to date. The CA VA/VB enzymes are involved in biosynthetic processes such as gluconeogenesis, lipogenesis and ureagenesis, and no pharmacological inhibitors with good selectivity are currently available. Thus the NP inhibitors identified during these studies are excellent leads for obtaining even more effective compounds that selectively target mitochondrial hCAs, and also have the potential to be used as tools for understanding the physiological processes that are regulated by the two mitochondrial CA isoforms.     

Synthesis and biological evaluation of histamine Schiff bases as carbonic anhydrase I,II, IV,VII, and IX activators

A series of 20 histamine Schiff base was synthesised by reaction of histamine, a well known carbonic anhydrase (CA, E.C 4.2.2.1.) activator pharmacophore, with substituted aldehydes. The obtained histamine Schiff bases were assayed as activators of five selected human (h) CA isozymes, the cytosolic hCA I, hCA II, and hCA VII, the membrane-anchored hCA IV and transmembrane hCA IX. Some of these compounds showed efficient activity (in the nanomolar range) against the cytosolic isoform hCA VII, which is a key CA enzyme involved in brain metabolism. Moderate activity was observed against hCA I and hCA IV (in the nanomolar to low micromolar range). The structure–activity relationship for activation of these isoforms with the new histamine Schiff bases is discussed in detail based on the nature of the aliphatic, aromatic, or heterocyclic moiety present in the aldehyde fragment of the molecule, which may participate in diverse interactions with amino acid residues at the entrance of the active site, where activators bind, and which is the most variable part among the different CA isoforms.     

Sulfonamides incorporating heteropolycyclic scaffolds show potent inhibitory action against carbonic anhydrase isoforms I,II, IX and XII

Three series of polycyclic compounds possessing either primary sulfonamide or carboxylic acid moieties as zinc-binding groups were investigated as inhibitors of four physiologically relevant CA isoforms, the cytosolic hCA I and II, as well as the transmembrane hCA IX and XII. Most of the new sulfonamides reported here showed excellent inhibitory effects against isoforms hCA II, IX and XII, but no highly isoform-selective inhibition profiles. On the other hand, the carboxylates selectively inhibited hCA IX (K_Is ranging between 40.8 and 92.7 nM) without inhibiting significantly the other isoforms. Sulfonamides/carboxylates incorporating polycyclic ring systems such as benzothiopyranopyrimidine, pyridothiopyranopyrimidine or dihydrobenzothiopyrano[4,3-c]pyrazole may be considered as interesting candidates for exploring the design of isoform-selective CAIs with various pharmacologic applications.     

The human carbonic anhydrase isoenzymes I and II inhibitory effects of some hydroperoxides,alcohols, and acetates

The carbonic anhydrases (CAs, EC 4.2.1.1) represent a superfamily of widespread enzymes, which catalyze a crucial biochemical reaction, the reversible hydration of carbon dioxide to bicarbonate and protons. Human CA isoenzymes I and II (hCA I and hCA II) are ubiquitous cytosolic isoforms. In this study, a series of hydroperoxides, alcohols, and acetates were tested for the inhibition of the cytosolic hCA I and II isoenzymes. These compounds inhibited both hCA isozymes in the low nanomolar ranges. These compounds were good hCA I inhibitors (K_is in the range of 24.93–97.99?nM) and hCA II inhibitors (K_is in the range of 26.04–68.56?nM) compared to acetazolamide as CA inhibitor (K_i: 34.50?nM for hCA I and K_i: 28.93?nM for hCA II).     

Carbonic anhydrase activators: activation of the human isoforms VII (cytosolic) and XIV (transmembrane) with amino acids and amines

An activation study of the human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes VII and XIV using a small library of natural/non-natural amino acids and aromatic/heterocyclic amines is reported. hCA VII was efficiently activated by L-/D-His, dopamine and serotonin (K(A)s of 0.71-0.93 microM). The best hCA XIV activators were histamine (K(A) of 10 nM), L-Phe, L-/D-His and 4-amino-L-Phe (K(A)s of 0.24-2.90 microM). In view of the significant expression levels of CA VII and CA XIV in the brain, selective activation of these isoforms may be useful when developing pharmacologic agents for the management of major disorders such as epilepsy and Alzheimer's disease.     

Inhibition of human carbonic anhydrase isoforms I–XIV with sulfonamides incorporating fluorine and 1,3,5-triazine moieties

Reaction of cyanuryl fluoride with sulfanilamide or 4-aminoethylbenzenesulfonamide afforded triazinyl-substituted benzenesulfonamides incorporating fluorine, which were further derivatized by reaction with amines, amino alcohols, amino acids or amino acid esters. Inhibition studies of all the human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms, hCA I–XIV with these compounds revealed that they show moderate-weak inhibition of hCA III, IV, VA and XIII, rather moderate inhibition against hCA I, VI, and IX, and excellent inhibition of the physiologically relevant hCA II, VII and XII. The inhibition profile of these fluorine containing triazinyl sulfonamides is thus very different from the corresponding analogs incorporating chlorine, which were previously investigated as inhibitors of some of these enzymes.