Discovery of curcumin inspired sulfonamide derivatives as a new class of carbonic anhydrase isoforms I,II, IX,and XII inhibitors

A series of curcumin inspired sulfonamide derivatives was prepared from various chalcones and 4-sulfamoyl benzaldehyde via Claisen–Schmidt condensation. All new compounds were assayed as inhibitors of four human isoforms of the metalloenzyme carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I, II, IX and XII. Interesting inhibitory activities were observed against all these isoforms. hCA I, an isoform involved in several eye diseases was inhibited moderately with K_Is in the range of 191.8–904.2?nM, hCA II, an antiglaucoma drug target was highly inhibited by the new sulfonamides, with K_Is in the range of 0.75–8.8?nM. hCA IX, a tumor-associated isoform involved in cancer progression and metastatic spread was potently inhibited by the new sulfonamides, with K_Is in the range of 2.3–87.3?nM, whereas hCA XII, and antiglaucoma and anticancer drug target, was inhibited with K_Is in the range of 6.1–71.8?nM. It is noteworthy that one of the new compounds, 5d, was found to be almost 9 times more selective against hCA II (K_I =?0.89?nM) over hCA IX and hCA XII, whereas 5e was 3 and 70 times more selective against hCA II (K_I =?0.75?nM) over hCA IX and hCA XII, respectively.     

4-Functionalized 1,3-diarylpyrazoles bearing 6-aminosulfonylbenzothiazole moiety as potent inhibitors of carbonic anhydrase isoforms hCA I,II, IX and XII

A series of 24 novel heterocyclic compounds—functionalized at position 4 with aldehyde (5a–5f), carboxylic acid (6a–6f), nitrile (7a–7f) and oxime (8a–8f) functional groups—bearing 6-aminosulfonybenzothiazole moiety at position 1 of pyrazole has been synthesized and investigated for the inhibition of four isoforms of the α-class carbonic anhydrases (CAs, EC 4.2.1.1), comprising hCAs I and II (cytosolic, ubiquitous isozymes) and hCAs IX and XII (transmembrane, tumor associated isozymes). Against the human isozyme hCA I, compounds 6a–6f showed medium-weak inhibitory potential with K_i values in the range of 157–690 nM with 6a showing better potential than the standard drug acetazolamide (AZA). Against hCA II, all the compounds showed excellent to moderate inhibition with K_i values of compounds 5a, 5d, 5f, 6a–6f, 8d and 8f lower than 12 nM (K_i of AZA). Against hCA IX, all the compounds showed moderate inhibition with the exception of 6e which showed nearly 9 fold a better profile compared to AZA, whereas against hCA XII, four compounds 6e, 7a, 7b and 7d showed K_i in the same order as that of AZA. Carboxylic acid 6e was found to be an excellent inhibitor of both hCA IX and XII, with K_i values of 2.8 nM and 5.5 nM, respectively.     

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 biological evaluation of coumarin-1,3,4-oxadiazole hybrids as selective carbonic anhydrase IX and XII inhibitors

With an aim to develop novel heterocyclic hybrids as potent anticancer agents, we synthesized a series of coumarin-1,3,4-oxadiazole hybrids (7a-t) and evaluated for their inhibitory activity against the four physiologically relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms CA I, CA II, CA IX and CA XII. The CA inhibition results clearly indicated that the coumarin-1,3,4-oxadiazole derivatives (7a-t) exhibited selective inhibition of the tumor associated isoforms, CA IX and CA XII over CA I and II isoforms. Among all, compound 7b, exhibited significant inhibition in lower micromolar potency against hCA XII, with a K_i of 0.16 µM and compound 7n, exhibited significant inhibition in lower micromolar potency against hCA IX, with a K_i of 2.34 µM respectively. Therefore, compound 7b and 7n could be the potential leads for development of selective anticancer agents by exhibiting a novel mechanism of action through hCA IX and XII inhibition.     

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.     

Benzenesulfonamide bearing 1,2,4-triazole scaffolds as potent inhibitors of tumor associated carbonic anhydrase isoforms hCA IX and hCA XII

Three series of novel heterocyclic compounds (3a–3g, 4a–4g and 5a–5g) containing benzenesulfonamide moiety and incorporating a 1,2,4-triazole ring, have been synthesized and investigated as inhibitors against four isomers of the α-class carbonic anhydrases (CAs, EC 4.2.1.1), comprising hCAs I and II (cytosolic, ubiquitous isozymes) and hCAs IX and XII (transmembrane, tumor associated isozymes). Against the human isozymes hCA I and II, compounds of two series (3a–3g and 4a–4g) showed K_i values in the range of 84–868 nM and 5.6–390 nM, respectively whereas compounds of series 5a–5g were found to be poor inhibitors (K_i values exceeding 10,000 nM in some cases). Against hCA IX and XII, all the tested compounds exhibited excellent to moderate inhibitory potential with K_i values in the range of 2.8–431 nM and 1.3–63 nM, respectively. Compounds 3d, 3f and 4f exhibited excellent inhibitory potential against all of the four isozymes hCA I, II, IX and XII, even better than the standard drug acetazolamide (AZA) whereas compound of the series 5a–5g were comparatively less potent but more selective towards hCA IX and XII.     

Phenylethynylbenzenesulfonamide regioisomers strongly and selectively inhibit the transmembrane, tumor-associated carbonic anhydrase isoforms IX and XII over the cytosolic isoforms I and II

A series of compounds incorporating regioisomeric phenylethynylbenzenesulfonamide moieties has been investigated for the inhibition of four human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, hCA I, II, IX and XII. Inhibition between the low nanomolar to the milliomolar range has been observed against them, with several low nanomolar and tumor-CA selective inhibitors detected. The position of the sulfamoyl group with respect to the alkyne functionality, and the nature of the moieties substituting the second aromatic ring were the principal structural features influencing CA inhibition. The para-sulfamoyl-substituted derivatives were effective inhibitors of CA IX and XII, the meta-substituted regioisomers of CA I, IX and XII, whereas the ortho-substituted sulfonamides were weak inhibitors of CA I, II and IX, but inhibited significantly CA XII.     

Synthesis,characterization and carbonic anhydrase inhibitory activity of novel benzothiazole derivatives

N-protected amino acids were reacted with substituted benzothiazoles to give the corresponding N-protected amino acid-benzothiazole conjugates (60–89%). Their structures were confirmed by proton nuclear magnetic resonance (¹H NMR), carbon-13 nuclear magnetic resonance (¹³C NMR), IR and elemental analysis. Their carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activities were determined against two cytosolic human isoforms (hCA I and hCA II), one membrane-associated (hCA IV) and one transmembrane (hCA XII) enzyme by a stopped-flow CO₂ hydrase assay method. The new compounds showed rather weak, micromolar inhibitory activity against most of these enzymes.     

Novel thiazolidinone-containing compounds,without the well-known sulphonamide zinc-binding group acting as human carbonic anhydrase IX inhibitors

A small collection of 26 structurally novel thiazolidinone-containing compounds, without the well-known sulphonamide zinc-binding group, were synthesised and tested in enzyme inhibition assays against the tumour-associated hCA IX enzyme. Inhibition constants in the lower micromolar region (K_I K_I values are relatively weak, the fact that they do not contain a sulphonamide moiety suggests that these compounds do not interact with the active site zinc ion. Therefore, docking studies and molecular dynamics simulations have been performed to suggest binding poses for these structurally novel inhibitors.     

Synthesis and biological evaluation of novel pyrazoline-based aromatic sulfamates with potent carbonic anhydrase isoforms II,IV and IX inhibitory efficacy

Herein we report the synthesis of a new series of aromatic sulfamates designed considering the sulfonamide COX-2 selective inhibitors celecoxib and valdecoxib as lead compounds. These latter were shown to possess important human carbonic anhydrase (CA, EC 4.2.1.1) inhibitory properties, with the inhibition of the tumor-associated isoform hCA IX likely being co-responsible of the celecoxib anti-tumor effects. Bioisosteric substitution of the pyrazole or isoxazole rings from these drugs with the pyrazoline one was considered owing to the multiple biological activities ascribed to this latter heterocycle and paired with the replacement of the sulfonamide of celecoxib and valdecoxib with its equally potent bioisoster sulfamate. The synthesized derivatives were screened for the inhibition of four human carbonic anhydrase isoforms, namely hCA I, II, IV, and IX. All screened sulfamates exhibited great potency enhancement in inhibiting isoform II and IV, widely involved in glaucoma (K_Is in the range of 0.4–12.4 nM and 17.7 and 43.3 nM, respectively), compared to the lead compounds, whereas they affected the tumor-associated hCA IX as potently as celecoxib.     

Refined structure of the acetazolamide complex of human carbonic anhydrase II at 1.9 Å

The binding of acetazolamide to human carbonic anhydrase II (HCA II) has been investigated by X-ray crystallography. The atomic positions of the enzyme inhibitor complex have been refined at 1.9 Å resolution using the least squares refinement program package PROLSQ. The crystallographic R-factor is 17.6%. The bound inhibitor is clearly resolved in the active site of the enzyme. The acetazolamide amine group is bound as a fourth ligand to the zinc ion, the other three are all histidine residues. In addition to van der Waals' interactions and the previously described binding of the sulphonamide group, the inhibitor forms a hydrogen bond from the carbonyl oxygen of the acetylamido group to the amino group of Gln 92.     

Stabilization of anionic and neutral forms of a fluorophoric ligand at the active site of human carbonic anhydrase I

We synthesized a fluorogenic dansylamide derivative (JB2-48), which fills the entire (15 Å deep) active site pocket of human carbonic anhydrase I, and investigated the contributions of sulfonamide and hydrophobic regions of the ligand structure on the spectral, kinetic, and thermodynamic properties of the enzyme–ligand complex. The steady-state and fluorescence lifetime data revealed that the deprotonation of the sulfonamide moiety of the enzyme bound ligand increases the fluorescence emission intensity as well as the lifetime of the fluorophores. This is manifested via the electrostatic interaction between the active site resident Zn²⁺ cofactor and the negatively charged sulfonamide group of the ligand, and such interaction contributes to about 2.2 kcal/mol (ΔΔG°) and 0.89 kcal/mol (ΔΔG^‡) energy in stabilizing the ground and the putative transition states, respectively. We provide evidence that the anionic and neutral forms of JB2-48 are stabilized by the complementary microscopic/conformational states of the enzyme. The implication of the mechanistic studies presented herein in rationale design of carbonic anhydrase inhibitors is discussed.     

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.     

Elucidation of stable intermediates in urea-induced unfolding pathway of human carbonic anhydrase IX

Human carbonic anhydrase IX (CAIX) has evolved as a promising biomarker for cancer prognosis, due to its overexpression in various cancers and restricted expression in normal tissue. However, limited information is available on its biophysical behavior. The unfolding of CAIX in aqueous urea solution was studied using all-atom molecular dynamics simulation approach. The results of this study revealed a stable intermediate state along the unfolding pathway of CAIX. At intermediate concentrations of urea (2.0–4.0 M), the protein displays a native-like structure with a large population of its secondary structure and hydrophobic contacts remaining intact in addition to small confined overall motions. Beyond 4.0 M urea, the unfolding is more gradual and at 8.0 M urea the structure is largely collapsed due to the solvent effect. The hydrophobic contact analysis suggests that the contact in terminal α-helices is separated initially which propagates in the loss of contacts from centrally located β-sheets. The reduction of 60–65% tertiary contacts in 7.0–8.0 M urea suggested the presence of residual structure in unfolded state and is confirmed with structural snap shot. Free energy landscape analysis suggested that unfolding of CAIX exists through the different intermediate states.     

Synthesis and biological evaluation of glycal-derived novel tetrahydrofuran 1,2,3-triazoles by ‘click’ chemistry

Thirteen new 1,2,3-triazoles (5a-e, 15a-d, 17a-b, 19, and 21) were synthesized by ‘click’ reaction of sugar-derived azides with commercially available acetylenes. The synthesized triazoles were tested in vitro for their biological activity, and compound 5b displayed both antibacterial and antifungal activities at an MIC value of 12.5 μg/mL, while compounds 15b and 19 showed antibacterial activity at an MIC value of 25 μg/mL.     

Synthesis,antioxidant and carbonic anhydrase I and II inhibitory activities of novel sulphonamide-substituted coumarylthiazole derivatives

New secondary benzenesulphonamide-substituted coumarylthiazole derivatives were synthesized and their inhibitory effects on purified carbonic anhydrase I and II were evaluated using CO₂ as a substrate. The result showed that all the synthesized compounds exhibited inhibitory activity on both hCA I and hCA II with N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)naphthalene-2-sulphonamide (5f, IC₅₀ value of 5.63 and 8.48?µM, against hCA I and hCA II, respectively) as the strongest inhibitor revealed from this study. Structure–activity relationship revealed that the inhibitory activity of the synthesized compounds is related to the type of the halogen and bulky substituent on the phenyl ring. In addition, the cupric reducing antioxidant capacities (CUPRAC) and ABTS cation radical scavenging abilities of the synthesized compounds were assayed. 4-methoxy-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)benzenesulphonamide (5e) exhibited the strongest ABTS and CUPRAC activity with IC₅₀ value of 48.83?µM and A_0.50 value of 23.29?µM, respectively.     

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.     

Synthesis and carbonic anhydrase I,II, IX and XII inhibition studies of 4-N,N-disubstituted sulfanilamides incorporating 4,4,4-trifluoro-3-oxo-but-1-enyl,phenacylthiourea and imidazol-2(3H)-one/thione moieties

A series of sulfonamides incorporating the sulfanilamide (SA) scaffold were prepared. Reaction of the 4-amino moiety of SA with benzyl chlorides or substituted bromoacetophenones afforded the 4-mono-alkylated derivatives which were then reacted with 1,1,1-trifluoro-4-isobutoxybut-3-en-2-one leading to a series of 4-N,N-disubstituted SAs. The key intermediates were also reacted with ethoxycarbonyl isothiocyanate leading to thioureas or were cyclized in the presence of potassium cyanate/isothiocyanate to the corresponding imidazol-2(3H)-one/thiones. The new compounds were tested as inhibitors of four carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic CA I and II, and the transmembrane, tumor-associated CA IX and XII. These sulfonamides were ineffective CA I and II inhibitors but were nanomolar CA IX and XII inhibitors, making them of interest as clinical candidates for antitumor/antimetastasis applications.     

Intact intracellular tail is critical for proper functioning of the tumor-associated, hypoxia-regulated carbonic anhydrase IX

Carbonic anhydrase IX (CA IX) is a tumor-associated, hypoxia-induced enzyme involved in pH regulation and cell adhesion. Its catalytically active ectodomain (ECD) is linked to a transmembrane region and a short intracellular (IC) tail. Removal of the IC tail causes intracellular localization of CA IX. Mutations of basic amino acids within IC do not perturb the membrane position, but reduce shedding of the CA IX ectodomain as well as CA IX-mediated cell dissociation. Moreover, they abolish the CA IX capacity to acidify extracellular pH (pHe) and bind CA IX-selective sulfonamide inhibitor in hypoxia. These findings provide the first evidence for the critical contribution of the IC tail to the proper functioning of CA IX.

Structured summary

MINT-7293982: E-cadherin (uniprotkb:Q95LE0) and CA IX (genbank_protein_gi:223556027) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Novel coumarins and benzocoumarins acting as isoform-selective inhibitors against the tumor-associated carbonic anhydrase IX

Abstract

A series of coumarins and benzocoumarins incorporating methyl and hydroxyl moieties in the heterocyclic ring were investigated for the inhibition of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). These coumarins were very weak or ineffective as inhibitors of the house-keeping, offtarget isoforms CA I and II, but showed effective, submicromolar inhibition of the transmembrane, tumor-associated isoforms CA IX and to a slightly less extent, CA XII. The nature and position of the groups substituting the coumarin ring influenced CA inhibitory properties. 4-Methyl-5,7-dihydroydroxycoumarin showed K_Is >200?µM against CA I and II, of 0.19?µM against CA IX and of 6.4?µM against CA XII, being thus a selective, efficient inhibitor for the tumor-associated over cytosolic CA isoforms. These compounds are interesting leads for designing isoform-selective enzyme inhibitors.