Isoforms hCA Research Articles

Novel 6- and 7-Substituted Coumarins with Inhibitory Action against Lipoxygenase and Tumor-Associated Carbonic Anhydrase IX.

A series of carboxamide derivatives of 6- and 7-substituted coumarins have been prepared by an original procedure starting from the corresponding 6- or 7-hydroxycoumarins which were alkylated with ethyl iodoacetate, and the obtained ester was converted to the corresponding carboxylic acids which were thereafter reacted with a series of aromatic/aliphatic/heterocyclic amines leading to the desired amides. The new derivatives were investigated as inhibitors of two enzymes, human carbonic anhydrases (hCAs) and soy bean lipoxygenase (LOX). Compounds 4a and 4b were potent LOX inhibitors, whereas many effective hCA IX inhibitors (KIs in the range of 30.2–30.5 nM) were detected in this study. Two compounds, 4b and 5b, showed the phenomenon of dual inhibition. Furthermore, these coumarins did not significantly inhibit the widespread cytosolic isoforms hCA I and II, whereas they were weak hCA IV inhibitors, making them hCA IX-selective inhibitors. As hCA IX and LOX are validated antitumor targets, these results are promising for the investigation of novel drug targets involved in tumorigenesis.

Iodine-mediated one-pot intramolecular decarboxylation domino reaction for accessing functionalised 2-(1,3,4-oxadiazol-2-yl)anilines with carbonic anhydrase inhibitory action

A practical and transition metal-free one-pot domino synthesis of diversified (1,3,4-oxadiazol-2-yl)anilines has been developed employing isatins and hydrazides as the starting materials, in the presence of molecular iodine. The prominent feature of this domino process involves consecutive condensation, hydrolytic ring cleavage, and an intramolecular decarboxylation, in a one-pot process that leads to the oxidative formation of a C–O bond. Fluorescence properties of some of the representative molecules obtained in this way were studied. The synthesised 2-(1,3,4-oxadiazolo-2-yl)aniline-benzene sulphonamides (8a–o) were screened for their carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity. Most of the compounds exhibited low micromolar to nanomolar activity against human (h) isoforms hCA I, hCA II, hCA IV, and XII, with some compounds displaying selective CA inhibitory activity towards hCA II with KIs of 6.4–17.6 nM.

Evaluation of sulphonamide derivatives acting as inhibitors of human carbonic anhydrase isoforms I, II and Mycobacterium tuberculosis β-class enzyme Rv3273

A series of novel sulphonamide derivatives was obtained from sulphanilamide which was N4-alkylated with ethyl bromoacetate followed by reaction with hydrazine hydrate. The hydrazide obtained was further reacted with various aromatic aldehydes. The novel sulphonamides were characterised by infrared, mass spectrometry, 1H- and 13C-NMR and purity was determined by high-performance liquid chromatography (HPLC). Human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I and II and Mycobacterium tuberculosis β-CA encoded by the gene Rv3273 (mtCA 3) inhibition activity was investigated with the synthesised compounds which showed promising inhibition. The KIs were in the range of 54.6 nM–1.8 µM against hCA I, in the range of 32.1 nM–5.5 µM against hCA II and of 127 nM–2.12 µM against mtCA 3.

Inhibition of α-, β-, γ-, and δ-carbonic anhydrases from bacteria and diatoms with N′-aryl-N-hydroxy-ureas

The inhibition of α-, β-, γ-, and δ-class carbonic anhydrases (CAs, EC 4.2.1.1) from bacteria (Vibrio cholerae and Porphyromonas gingivalis) and diatoms (Thalassiosira weissflogii) with a panel of N’-aryl-N-hydroxy-ureas is reported. The α-/β-CAs from V. cholerae (VchCAα and VchCAβ) were effectively inhibited by some of these derivatives, with KIs in the range of 97.5 nM – 7.26 µM and 52.5 nM – 1.81 µM, respectively, whereas the γ-class enzyme VchCAγ was less sensitive to inhibition (KIs of 4.75 – 8.87 µM). The β-CA from the pathogenic bacterium Porphyromonas gingivalis (PgiCAβ) was not inhibited by these compounds (KIs > 10 µM) whereas the corresponding γ-class enzyme (PgiCAγ) was effectively inhibited (KIs of 59.8 nM – 6.42 µM). The δ-CA from the diatom Thalassiosira weissflogii (TweCAδ) showed effective inhibition with these derivatives (KIs of 33.3 nM – 8.74 µM). As most of these N-hydroxyureas are also ineffective as inhibitors of the human (h) widespread isoforms hCA I and II (KIs > 10 µM), this class of derivatives may lead to the development of CA inhibitors selective for bacterial/diatom enzymes over their human counterparts and thus to anti-infectives or agents with environmental applications.

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 Kis 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 KI-s ranged between 50.8 and 9268.5 nM. The structure–activity relationship (SAR) with these newly synthesised metanilamide derivatives are discussed in detail.

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

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

Steroids interfere with human carbonic anhydrase activity by using alternative binding mechanisms

Bile acids have been shown to inhibit human (h) carbonic anhydrases (CA, EC 4.2.1.1) along the gastrointestinal tract, including hCA II. The elucidation of the hormonal inhibition mechanism of the bile acid cholate to hCA II was provided in 2014 by X-ray crystallography. Herein, we extend the inhibition study to a wealth of steroids against four relevant hCA isoforms. Steroids displaying pendants and functional groups of the carboxylate, phenolic or sulfonate types appended at the tetracyclic ring were shown to inhibit the cytosolic CA II and the tumor-associated, transmembrane CA IX in a medium micromolar range (38.9–89.9 µM). Docking studies displayed the different chemotypes CA inhibition mechanisms. Molecular dynamics (MD) gave insights on the stability over time of hyocholic acid binding to CA II.

Activation studies of the α- and β-carbonic anhydrases from the pathogenic bacterium Vibrio cholerae with amines and amino acids

The α- and β-class carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic bacterium Vibrio cholerae, VchCAα, and VchCAβ, were investigated for their activation with natural and non-natural amino acids and amines. The most effective VchCAα activators were L-tyrosine, histamine, serotonin, and 4-aminoethyl-morpholine, which had KAs in the range of 8.21–12.0 µM. The most effective VchCAβ activators were D-tyrosine, dopamine, serotonin, 2-pyridyl-methylamine, 2-aminoethylpyridine, and 2-aminoethylpiperazine, which had KAs in the submicromolar – low micromolar range (0.18–1.37 µM). The two bacterial enzymes had very different activation profiles with these compounds, between each other, and in comparison to the human isoforms hCA I and II. Some amines were selective activators of VchCAβ, including 2-pyridylmethylamine (KA of 180 nm for VchCAβ, and more than 20 µM for VchCAα and hCA I/II). The activation of CAs from bacteria, such as VchCAα/β has not been considered previously for possible biomedical applications. It would be of interest to study in more detail the extent that CA activators are implicated in the virulence and colonisation of the host by such pathogenic bacteria, which for Vibrio cholerae, is highly dependent on the bicarbonate concentration and pH in the surrounding tissue.

First evaluation of organotellurium derivatives as carbonic anhydrase I, II, IV, VII and IX inhibitors

A series of tellurides was evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors against the human (h) carbonic anhydrase isoforms hCA I, II, IV, VII and IX, involved in a variety of diseases, including glaucoma, retinitis pigmentosa, epilepsy, arthritis and tumors. These compounds, which are the first tellurium-containing derivatives acting as inhibitors of carbonic anhydrase enzymes, showed effective inhibition against all isoforms investigated and some of them were selective for inhibiting the cytosolic or the membrane-bound CAs. Thus, these carbonic anhydrase inhibitors are interesting leads for the development of isoform-selective inhibitors.

Heterocyclic periphery in the design of carbonic anhydrase inhibitors: 1,2,4-Oxadiazol-5-yl benzenesulfonamides as potent and selective inhibitors of cytosolic hCA II and membrane-bound hCA IX isoforms

A series of novel aromatic primary sulfonamides decorated with diversely substituted 1,2,4-oxadiazole periphery groups has been prepared using a parallel chemistry approach. The compounds displayed a potent inhibition of cytosolic hCA II and membrane-bound hCA IX isoforms. Due to a different cellular localization of the two target enzymes, the compounds can be viewed as selective inhibition tools for either isoform, depending on the cellular permeability profile. The SAR findings revealed in this study has been well rationalized by docking simulation of the key compounds against the crystal structures of the relevant hCA isoforms.

Sulfocoumarin-, Coumarin-, 4-Sulfamoylphenyl-Bearing Indazole-3-carboxamide Hybrids: Synthesis and Selective Inhibition of Tumor-Associated Carbonic Anhydrase Isozymes IX and XII.

A series of sulfocoumarin-, coumarin-, and 4-sulfamoylphenyl-bearing indazole-3-carboxamide hybrids were synthesized and investigated as inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms I and II (cytosolic isozymes), as well as hCA IX and XII (transmembrane, tumor-associated enzymes). Compounds 6 a-g (amide derivatives) and 7 a-h (triazoles) act as "prodrugs", and their hydrolysis products are the de facto CA inhibitors. These compounds displayed sub-micromolar to high-nanomolar inhibitory activity against hCA isoforms IX and XII, which were recently validated as antitumor drug targets. Moreover, no inhibition of the off-target hCA I and II isoforms was observed. Compounds 8 a-f (another set of triazoles) exhibited nanomolar inhibition against hCA isoforms I, II, IX and XII, among which compounds 8 c, 8 d, and 8 f were found to inhibit the tumor-associated hypoxia-induced hCA isoform IX with Ki values of 1.8, 2.3, and 2.0 nm respectively. Further exploration of these compounds could be useful for the development of novel antitumor agents with selective mechanisms of CA inhibitory action.

Acyl selenoureido benzensulfonamides show potent inhibitory activity against carbonic anhydrases from the pathogenic bacterium Vibrio cholerae

A series of acyl selenoureido benzensulfonamides was evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors against two Vibrio cholerae such enzymes (VchCAα over VchCAβ) belonging to the α- and β-classes, potential novel targets for anti-infective drugs development. These compounds showed strong inhibitory action against VchCAα over VchCAβ and excellent selectivity over the human (h) off-target isoforms hCA I and II. Identification of potent and possibly selective inhibitors of VchCAα and/or VchCAβ over the human counterparts may lead to pharmacological tools useful for understanding the physiological role(s) of these under-investigated proteins, possibly involved in the virulence of the bacterium and colonization of the host in bicarbonate rich regions of the gastro-intestinal tract.

Development of sulfonamides incorporating phenylacrylamido functionalities as carbonic anhydrase isoforms I, II, IX and XII inhibitors

A series of novel sulfonamides incorporating phenylacrylamido functionalities were synthesized and investigated for the inhibition of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The physiologically and pharmacologically relevant human (h) isoforms hCA I and II (cytosolic isozymes), as well as the transmembrane tumor-associated hCA IX and XII were included in the study. These compounds showed low nanomolar or sub-nanomolar inhibition constants against hCA II (KIs in the range of 0.50–50.5nM), hCA IX (KIs of 1.8–228.5nM), and hCA XII (KIs of 3.5–96.2nM) being less effective as inhibitors of the off target isoform hCA I. A detailed structure–activity relationship study demonstrates that the nature and position of substituents present on the aromatic part of the scaffold strongly influence the inhibition of CA isoforms. As hCA II, IX and XII are involved in pathologies such as glaucoma and hypoxic, and metastatic tumors, compounds of the type reported in this work may be useful preclinical candidates.

Novel 4/3-((4-oxo-5-(2-oxoindolin-3-ylidene)thiazolidin-2-ylidene)amino) benzenesulfonamides: Synthesis, carbonic anhydrase inhibitory activity, anticancer activity and molecular modelling studies

Herein we report the synthesis of two series of novel 4/3-((4-oxo-5-(2-oxoindolin-3-ylidene)thiazolidin-2-ylidene)amino)benzenesulfonamides (4a-m and 7a-g). All the newly prepared sulfonamides were in vitro investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, II, IV and IX, using a stopped-flow CO2 hydrase assay. In particular, hCA isoforms II and IX (tumor-associated) were more susceptible to inhibition by the synthesized derivatives, with KIs in the range of 2.6–598.2 nM for hCA II, and of 16.1–321 nM for hCA IX. All compounds (4a-m and 7a-g) were evaluated for their anti-proliferative activity against breast cancer MCF-7 and colorectal cancer Caco-2 cell lines. Compound 4c was found to be the most potent derivative against MCF-7 (IC50 = 3.96 ± 0.21 μM), while 4j was the most active member against Caco-2 cells (IC50 = 5.87 ± 0.37 μM). Compound 4c induced the intrinsic apoptotic mitochondrial pathway in MCF-7 cells; evidenced by the enhanced expression of the pro-apoptotic protein Bax and the reduced expression of the anti-apoptotic protein Bcl-2, and the up-regulated active caspase-9 and caspase-3 levels.

Synthesis and carbonic anhydrase inhibition of polycyclic imides incorporating N-benzenesulfonamide moieties

A series of polycyclic imides was prepared by reaction of the benzenesulfonamide with an appropriate polycyclic acid anhydride in refluxing glacial acetic acid. The synthesized mono- and bis-sulfonamides were evaluated as a carbonic anhydrase inhibitors (CA, EC 4.2.1.1), more precisely against the human (h) isoforms hCA I, II, IX and XII, some of which are involved in various pathologies, such as glaucoma, epilepsy and cancer. Several low nanomolar and isoform-selective hCA II, IX and XII inhibitors were detected, and the structure-activity relationship for CA inhibition with this class of compounds is discussed in details. Computational studies allowed us to explain the efficacy and isoform-selective behaviour for some of these enzyme inhiibtors.

Synthesis of novel acyl selenoureido benzensulfonamides as carbonic anhydrase I, II, VII and IX inhibitors

A novel series of acyl selenoureido benzensulfonamides was evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors against the human (h) isoforms hCA I, II, VII and IX, which are involved in a variety of diseases such as glaucoma, retinitis pigmentosa, epilepsy and tumors etc. These compounds showed excellent inhibitory activity for these isoforms, with several low nanomolar derivatives identified against all of them. Furthermore, the selenoureido group may provide an antioxidant activity to these enzyme inhibitors.

Sulfonamide inhibition profiles of the β-carbonic anhydrase from the pathogenic bacterium Francisella tularensis responsible of the febrile illness tularemia

A new β-class carbonic anhydrase (CA, EC 4.2.1.1) has been cloned, purified and characterized in the genome of the pathogenic bacterium Francisella tularensis responsible of the febrile illness tularemia. This enzyme, FtuβCA, showed a kcat of 9.8 ×105s−1 and a kcat/KM of 8.9 ×107M−1s−1 for the CO2 hydration, physiological reaction, being one of the most effective β-CAs known to date, with a catalytic activity only 1.68-times lower than that of the human(h) isoform hCA II. A panel of 39 simple aromatic and heterocyclic sulfonamides, as well as clinically used drugs incorporating sulfonamide/sulfamate zinc-binding groups, was used to investigate the inhibition profile of FtuβCA with these classes of derivatives. The enzyme generally showed a weaker affinity for these inhibitors compared to other α- and β-CAs investigated earlier, with only acetazolamide and its deacetylated precursor having inhibition constant <1µM. Indeed, the two compounds acetazolamide AAZ and its deacetylated precursor 13 (KIs of 655–770nM), as well as metanilamide and methazolamide (KIs of 2.53–2.92µM), were the best FtuβCA inhibitors detected so far. As the physiological role of bacterial β-CAs is poorly understood for the virulence/life cycle of these pathogens, the present study may constitute a starting point for the design of effective pathogenic bacteria CA inhibitors with potential use as antiinfectives.

Synthesis and biological evaluation of novel aromatic and heterocyclic bis-sulfonamide Schiff bases as carbonic anhydrase I, II, VII and IX inhibitors

A series of sixteen novel aromatic and heterocyclic bis-sulfonamide Schiff bases were prepared by conjugation of well known aromatic and heterocyclic aminosulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor pharmacophores with aromatic and heterocyclic bis-aldehydes. The obtained bis-sulfonamide Schiff bases were investigated as inhibitors of four selected human (h) CA isoforms, hCA I, hCA II, hCA VII and hCA IX. Most of the newly synthesized compounds showed a good inhibitory profile against isoforms hCA II and hCA IX, also showing moderate selectivity against hCA I and VII. Several efficient lead compounds were identified among this bis-sulfonamide Schiff bases with low nanomolar to sub-nanomolar activity against hCA II (Kis ranging between 0.4 and 861.1nM) and IX (Kis between 0.5 and 933.6nM). Since hCA II and hCA IX are important drug targets (antiglaucoma and anti-tumor agents), these isoform-selective inhibitors may be considered of interest for various biomedical applications.

Sulfonamide carbonic anhydrase inhibitors: Zinc coordination and tail effects influence inhibitory efficacy and selectivity for different isoforms

The metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) is effectively inhibited by primary sulfonamides which coordinate as anions to the zinc ion from its active site. Inhibition of CAs has pharmacologic applications in the treatment of many diseases, but many sulfonamides are promiscuous inhibitors of most isoforms known to date, leading to side effects of these drugs. In a series of 4-aryl-benzenesulfonamides with effective inhibitory action against human (h) isoforms hCA I, II, IX and XII and selectivity for some of them, we demonstrate by means of X-ray crystallographic studies of enzyme-inhibitor adducts, that the tail present on the benzenesulfonamide scaffold significantly contributes to the observed inhibition/selectivity profile. This study may bring additional information for the structure-based drug design of effective/isoform-selective zinc-binding CA inhibitors.

Inhibition of Malassezia globosa carbonic anhydrase with phenols

A panel of 22 phenols was investigated as inhibitors of the β-class carbonic anhydrase (CAs, EC 4.2.1.1) from the fungal parasite Malassezia globosa (MgCA), a validated anti-dandruff drug target. The displayed inhibitory activities were compared to the ones previously reported against the off-target widely distributed human (h) isoforms hCA I and II. All tested phenols possessed a better efficacy in inhibiting MgCA than the clinically used sulfonamide acetazolamide, with KIs in the range of 2.5 and 65.0μM. A homology-built model of MgCA was also used for understanding the binding mode of phenols to the fungal enzyme. Indeed, a wide network of hydrogen bonds and hydrophobic interactions between the phenol and active site residues were evidenced. The OH moiety of the inhibitor was observed anchored to the zinc-coordinated water, also making hydrogen bonds with Ser48 and Asp49. The diverse substituents at the phenolic scaffold were observed to interact with different portions of the hydrophobic pocket according to their nature and position. Considering the effective MgCA inhibitory properties of phenols, beside to the rather low inhibition against the off-target hCA I and II, this class of compounds might be of considerable interest in the cosmetics field as potential anti-dandruff drugs.