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.     

Kinetic and X-ray crystallographic investigations on carbonic anhydrase isoforms I,II, IX and XII of a thioureido analog of SLC-0111

SLC-0111 (4-(4-fluorophenylureido)-benzenesulfonamide) is the first carbonic anhydrase (CA, EC 4.2.1.1) IX inhibitor to reach phase I clinical trials as an antitumor/antimetastatic agent. Here we report a kinetic and X-ray crystallographic study of a congener of SLC-0111 which incorporates a thioureido instead of ureido linker between the two aromatic rings as inhibitor of four physiologically relevant CA isoforms. Similar to SLC-0111, the thioureido derivative was a weak hCA I and II inhibitor and a potent one against hCA IX and XII. X-ray crystallography of its adduct with hCA II and comparison of the structure with that of other five hCA II—sulfonamide adducts belonging to the SLC-0111 series, afforded us to understand the particular inhibition profile of the new sulfonamide. Similar to SLC-0111, the thioureido sulfonamide primarily interacted with the hydrophobic side of the hCA II active site, with the tail participating in van der Waals interactions with Phe131 and Pro202, in addition to the coordination of the deprotonated sulfonamide to the active site metal ion. On the contrary, the tail of other sulfonamides belonging to the SLC-0111 series (2-isopropyl-phenyl; 3-nitrophenyl) were orientated towards the hydrophilic half of the active site, which was correlated with orders of magnitude better inhibitory activity against hCA II, and a loss of selectivity for the inhibition of the tumor-associated CAs.     

Effect of incorporating a thiophene tail in the scaffold of acetazolamide on the inhibition of human carbonic anhydrase isoforms I,II, IX and XII

The high resolution crystal structure of 5-(2-thienylacetamido)-1,3,4-thiadiazole-2-sulfonamide complexed to human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoform hCA II is reported. The compound binds in a similar manner with acetazolamide when the sulfamoyl–thiadiazolyl–acetamido fragment of the two compounds is considered, but the thienyl tail was positioned in the subpocket 2, rarely observed by other investigated CA inhibitors. This positioning allows interaction with amino acid residues (such as Asn67, Ile91, Gln92 and Val121 which are variable in other isoforms of medicinal chemistry interest, such as hCA I, IX and XII. Indeed, the investigated sulfonamide was a medium potency hCA I and II inhibitor but was highly effective as a hCA IX and XII inhibitor. This different behavior with respect to acetazolamide (a promiscuous inhibitor of all these isoforms) has been explained by resolving the crystal structure, and may be used to design more isoform-selective compounds.     

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 and carbonic anhydrase I,II, VII,and IX inhibition studies with a series of benzo[d]thiazole-5- and 6-sulfonamides

A series of benzo[d]thiazole-5- and 6-sulfonamides has been synthesized and investigated for the inhibition of several human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms, using ethoxzolamide (EZA) as lead molecule. 2-Amino-substituted, 2-acylamino- and halogenated (bromo-and iodo-derivatives at the heterocyclic ring) compounds led to several interesting inhibitors against the cytosolic hCA I, II and VII, as well as the transmembrane, tumor-associated hCA IX isoforms. Several subnanomolar/low nanomolar, isoform-selective sulfonamide inhibitors targeting hCA II, VII and IX were detected. The sharp structure–activity relationship for CA inhibition with this small series of derivatives, with important changes of activity observed even after minor changes in the scaffold or at the 2-amino moiety, make this class of scarcely investigated sulfonamides of particular interest for further investigations.     

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.     

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.     

Crystal structure of the human carbonic anhydrase II adduct with 1-(4-sulfamoylphenyl-ethyl)-2,4,6-triphenylpyridinium perchlorate,a membrane-impermeant,isoform selective inhibitor

Pyridinium containing sulfonamides have been largely investigated as carbonic anhydrase inhibitors (CAIs), showing interesting selectivity features. Nevertheless, only few structural studies are so far available on adducts that these compounds form with diverse CA isoforms. In this paper, we report the structural characterization of the adduct that a triphenylpyridinium derivative forms with hCA II, showing that the substitution of the pyridinium ring plays a key role in determining the conformation of the inhibitor in the active site and consequently the binding affinity to the enzyme. These findings open new perspectives on the basic structural requirements for designing sulfonamide CAIs with a selective inhibition profile.     

(R)-/(S)-10-camphorsulfonyl-substituted aromatic/heterocyclic sulfonamides selectively inhibit mitochondrial over cytosolic carbonic anhydrases

A series of aromatic and heterocyclic sulfonamides incorporating R- and S-camphorsulfonyl moieties were synthesized and investigated for the inhibition of several mammalian isoforms of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The new sulfonamides selectively inhibited the mitochondrial isozymes hCA VA and VB (h = human isoform) over the cytosolic, off-target ones hCA I and II, with inhibition constants in the low nanomolar range. The chirality and position of the groups substituting the sulfonamide scaffold greatly influenced CA inhibitory properties. These compounds are excellent leads for designing isoform-selective enzyme inhibitors targeting mitochondrial CAs involved in lipogenesis and obesity.     

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.     

Synthesis of novel acridine and bis acridine sulfonamides with effective inhibitory activity against the cytosolic carbonic anhydrase isoforms II and VII

4-Amino-N-(4-sulfamoylphenyl)benzamide was synthesized by reduction of 4-nitro-N-(4-sulfamoylphenyl)benzamide and used to synthesize novel acridine sulfonamide compounds, by a coupling reaction with cyclic-1,3-diketones and aromatic aldehydes. The new compounds were investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), and more precisely the cytosolic isoforms hCA I, II and VII. hCA I was inhibited in the micromolar range by the new compounds (K_Is of 0.16–9.64 μM) whereas hCA II and VII showed higher affinity for these compounds, with K_Is in the range of 15–96 nM for hCA II, and of 4–498 nM for hCA VII. The structure–activity relationships for the inhibition of these isoforms with the acridine–sulfonamides reported here were also elucidated.     

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.     

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.     

Molecular modeling study for the binding of zonisamide and topiramate to the human mitochondrial carbonic anhydrase isoform VA

Zonisamide and topiramate are two antiepileptic drugs known to induce weight loss in epilepsy patients. These molecules were recently shown to act as carbonic anhydrase (CA) inhibitors, being presumed that the weight loss may be due to the inhibition of the mitochondrial isozymes CA VA and CA VB involved in metabolic processes, among which lipid biosynthesis. To better understand the interaction of these compounds with CAs, here, we report a homology modeling and molecular dynamics simulations study on their adducts with human carbonic anhydrase VA (hCA VA). According to our results, in both cases the inhibitor sulfamate/sulfonamide moiety participates in the canonical interactions with the catalytic zinc ion, whereas the organic scaffold establishes a large number of van der Waals and polar interactions with the active site cleft. A structural comparison of these complexes with the corresponding homologues with human carbonic anhydrase II (hCA II) provides a rationale to the different affinities measured for these drugs toward hCA VA and hCA II. In particular, our data suggest that a narrower active site cleft, together with a different hydrogen bond network arrangement of hCA VA compared to hCA II, may account for the different Kd values of zonisamide and topiramate toward these physiologically relevant hCA isoforms. These results provide useful insights for future design of more isozyme-selective hCA inhibitors with potential use as anti-obesity drugs possessing a novel mechanism of action.     

Carbonic anhydrase inhibitors. Inhibition of the human cytosolic isozyme VII with aromatic and heterocyclic sulfonamides

The inhibition of a newly cloned human carbonic anhydrase (CA, EC 4.2.1.1), isozyme VII (hCA VII), has been investigated with a series of aromatic and heterocyclic sulfonamides, including some of the clinically used derivatives (acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide and benzolamide), as well as the sulfamate antiepileptic drug topiramate. Inhibition data for the the other physiologically relevant cytosolic isoforms hCA I, hCA II and mCA XIII are also provided for comparison. hCA VII shows a high catalytic activity for the CO(2) hydration reaction, with a k(cat) of 9.5 x 10(5)s(-1) and k(cat)/K(m) of 8.3 x 10(7)M(-1)s(-1) at pH7.5 and 20 degrees C. A very interesting inhibition profile against hCA VII with this series of 32 sulfonamides/sulfamates was observed. hCA VII shows high affinity for all the investigated compounds, with inhibition constants in the range of 0.45-210 nM. Topiramate, ethoxzolamide and benzolamide showed subnanomolar hCA VII inhibitory activity, whereas acetazolamide, methazolamide, dorzolamide and brinzolamide showed K(I)-s in the range of 2.1-3.5 nM. Dichlorophenamide was slightly less active (K(I) of 26.5 nM). A number of heterocyclic or bicyclic aromatic sulfonamides also showed excellent hCA VII inhibitory properties (K(I)-s in the range of 4.3-7.0 nM) whereas many monosubstituted or disubstituted benzenesulfonamides were less active (K(I)-s in the range of 45-89 nM). The least active hCA VII inhibitors were some substituted benzene-1,3-disulfonamides as well as some halogenated sulfanilamides (K(I)-s in the range of 100-210 nM). The inhibition profile of hCA VII is rather different of that of the other cytosolic isozymes, providing thus a possibility for the design of more selective, hCA VII-specific inhibitors. In addition, these data furnish further evidence that hCA VII is the isozyme responsible for the anticonvulsant/antiepileptic activity of sulfonamides and sulfamates.     

Superacid synthesis of halogen containing N-substituted-4-aminobenzene sulfonamides: New selective tumor-associated carbonic anhydrase inhibitors

A series of new, halogen containing N-substituted 4-aminobenzenesulfonamides were synthesized by using superacid HF/SbF₅ chemistry and investigated as inhibitors of several human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, that is, the cytosolic hCA I and II and, the tumor-associated transmembrane isoforms hCA IX and XII. Despite the substitution of the sulfonamide function, the presence of fluorine atom(s) in β position of the sulfonamide function strongly favors hCA inhibition. A similar effect of the β-fluorinated alkyl substitution on the amino function has been also observed. Among the tested compounds, several chlorinated derivatives have been identified as selective nanomolar, tumor-associated isoforms inhibitors. These non-primary sulfonamides probably bind in the coumarin-binding site, at the entrance of the cavity, and not to the metal ion as the primary sulfonamide inhibitors.     

Chromone containing sulfonamides as potent carbonic anhydrase inhibitors

A series of sulfonamide derivatives incorporating substituted 3-formylchromone moieties were investigated for the inhibition of three human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, hCA I, II, and VI. All these compounds, together with the clinically used sulfonamide acetazolamide, were investigated as inhibitors of the physiologically relevant isozymes I, II (cytosolic), and VI (secreted isoform). These sulfonamides showed effective inhibition against all these isoforms with K(I)'s in the range of 0.228 to 118 μM. Such molecules can be used as leads for discovery of novel effective CA inhibitors against other isoforms with medicinal chemistry applications.     

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.     

Chromone containing sulfonamides as potent carbonic anhydrase inhibitors

A series of sulfonamide derivatives incorporating substituted 3-formylchromone moieties were investigated for the inhibition of three human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, hCA I, II, and VI. All these compounds, together with the clinically used sulfonamide acetazolamide, were investigated as inhibitors of the physiologically relevant isozymes I, II (cytosolic), and VI (secreted isoform). These sulfonamides showed effective inhibition against all these isoforms with K_I’s in the range of 0.228 to 118 µM. Such molecules can be used as leads for discovery of novel effective CA inhibitors against other isoforms with medicinal chemistry applications.     

Carbonic anhydrases: current state of the art, therapeutic applications and future prospects

Carbonic anhydrases (CAs, EC 4.2.1.1) are wide-spread enzymes, present in mammals in at least 14 different isoforms. Some of these isozymes are cytosolic (CA I, CA II, CA III, CA VII, CA XIII), others are membrane-bound (CA IV, CA IX, CA XII and CA XIV), CA V is mitochondrial and CA VI is secreted in the saliva and milk. Three cytosolic acatalytic forms are also known (CARP VIII, CARP X and CARP XI). The catalytically active isoforms, which play important physiological and patho-physiological functions, are strongly inhibited by aromatic and heterocyclic sulfonamides. The catalytic and inhibition mechanisms of these enzymes are understood in great detail, and this greatly helped the design of potent inhibitors, some of which possess important clinical applications. The use of such CA inhibitors (CAIs) as antiglaucoma drugs are discussed in detail, together with the recent developments that led to isozyme-specific and organ-selective inhibitors. A recent discovery is connected with the involvement of CAs and their sulfonamide inhibitors in cancer: many potent CAIs were shown to inhibit the growth of several tumor cell lines in vitro and in vivo, thus constituting interesting leads for developing novel antitumor therapies. Future prospects for drug design of inhibitors of these ubiquitous enzymes are dealt with. Although activation of CAs has been a controversial issue for some time, recent kinetic, spectroscopic and X-ray crystallographic experiments offered an explanation of this phenomenon, based on the catalytic mechanism. It has been demonstrated recently, that molecules that act as carbonic anhydrase activators (CAAs) bind at the entrance of the enzyme active site participating in facilitated proton transfer processes between the active site and the reaction medium. In addition to CA II-activator adducts, X-ray crystallographic studies have been also reported for ternary complexes of this isozyme with activators and anion (azide) inhibitors. Structure-activity correlations for diverse classes of activators is discussed for the isozymes for which the phenomenon has been studied, i.e., CA I, II, III and IV. The possible physiological relevance of CA activation/inhibition is also addressed, together with recent pharmacological/ biomedical applications of such compounds in different fields of life sciences.