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.     

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.     

Design,synthesis and biological evaluation of coumarin-3-carboxamides as selective carbonic anhydrase IX and XII inhibitors

A series of novel 7-hydroxycoumarin-3-carboxamides was synthesized by the reaction of 7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid with various substituted aromatic amines. The newly synthesized compounds were 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 show that the newly synthesized 7-hydroxycoumarin-3-carboxamides (4a-n) exhibited selective inhibition of the tumor associated isoforms, CA IX and CA XII over CA I and II isoforms. The inhibition constants ranged from sub micromolar to low micromolar. Amongst all the compounds tested, compound 4m was the most effective inhibitor exhibiting sub micromolar potency against both hCA IX and hCA XII, with a K_i of 0.2 µM. Therefore, it can be anticipated that compound 4m can serve as a lead for development of anticancer therapy by exhibiting a novel mechanism of action. The binding modes of the most potent compounds within hCA IX and XII catalytic clefts were investigated by docking studies.     

Novel sulfonamides incorporating 1,3,5-triazine and amino acid structural motifs as inhibitors of the physiological carbonic anhydrase isozymes I,II and IV and tumor-associated isozyme IX

A new series of thirty s-triazinyl-substituted aminoalkylbenzenesulfonamides, incorporating a symmetric pair of amino acid moieties, is reported, together with inhibition studies of physiologically relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms. Specifically, against the cytosolic hCA I, II, transmembrane hCA IV and the tumor-associated, membrane-bound hCA IX.The compounds were prepared by nucleophilic substitution of chlorine atoms from cyanuric chloride (2,4,6-trichloro-1,3,5-triazine) using environmentally friendly water-based synthetic conditions. The products yields ranged in the interval of 43–97%. Purity of the products was verified by the HPLC-DAD-ESI-Q-TOF MS method. Identity of the products was confirmed by the same method plus NMR and IR.The products showed weak inhibition of the cytosolic, off-target isozyme hCA II, but some of them were low nanomolar (i.e. strong) inhibitors of the tumor-associated hCA IX. The series offered representatives selective towards isozymes hCA I, IV and IX. 2,2′-((6-((4-sulfamoylphenethyl)amino)-1,3,5-triazine-2,4-diyl)bis(imino))disuccinic acid demonstrated highest selectivity to the tumor-associated isoform hCA IX over off-target isozymes, with impressive K_I ratio (hCA II/hCA IX) 213.9 and inhibition constant equal to acetazolamide (K_I = 25.8 nM). Although the selectivities of some other products, e.g. those conjugating Leu and Glu, were a bit lower (188.7 and 84.3, respectively) their inhibition constants were similar to acetazolamide too (24.0 and 27.1, respectively).The selected most impressive results from the inhibition study were interpreted via molecular modeling experiment (docking in Glide) revealing different inter-molecular enzyme-substrate interaction of 2,2′-((6-((4-sulfamoylphenethyl)amino)-1,3,5-triazine-2,4-diyl)bis(imino))disuccinic acid within specific hCA IX and hCA II microregions. Therefore, several selected compounds from this study can be considered as highly effective and selective inhibitors of hCA IX, worthy to further (preclinical) investigation.     

Synthesis,biological evaluation and computational studies of novel iminothiazolidinone benzenesulfonamides as potent carbonic anhydrase II and IX inhibitors

A series of iminothiazolidinone-sulfonamide hybrids (2a-k) was synthesized by heterocyclization of sulfanilamide thioureas with methyl bromoacetate and characterized by spectroscopic techniques, mass and elemental analysis. The synthesized derivatives were screened against four relevant human (h) isoforms of carbonic anydrases (CAs, EC 4.2.1.1) I, II, IV and IX. These enzymes are involved in a variety of diseases, including glaucoma, retinitis pigmentosa, epilepsy, arthritis, and tumors. Derivatives 2a-2k exhibited the best inhibitory activity against the cytosolyc hCA II (K_Is are reaching the sub-nanomolar range, 0.41–37.8 nM) and against the tumor-associated isoform hCA IX (K_Is are spanning between 24.3 and 368.3 nM). The binding mode of the reported iminothiazolidinone benzenesulfonamides within hCA II and IX catalytic clefts was investigated by docking studies.     

Substituted benzene sulfonamides incorporating 1,3,5-triazinyl moieties potently inhibit human carbonic anhydrases II,IX and XII

A series of benzene sulfonamides incorporating 1,3,5-triazinyl moieties were synthesized using cyanuric chloride as starting material. Inhibition studies against human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms I, II (cytosolic) and IX, XII (transmembrane, tumor-associated) isoforms were performed with the new compounds. hCA I was modestly inhibited (K_Is in the range of 87 nM–4.35 μM), hCA II was moderately inhibited by most of the new compounds (K_Is in the range of 12.5–130 nM), whereas the tumor associated isoforms were potently inhibited, with K_Is in the range of 1.2–34.1 nM against hCA IX and of 2.1–33.9 against hCA XII, respectively. Docking studies of some of the new compounds showed an effective binding mode within the enzyme active site, as demonstrated earlier by X-ray crystallography for structurally-related sulfonamides incorporating 1,3,5-triazinyl functionalities.     

7-Substituted-sulfocoumarins are isoform-selective,potent carbonic anhydrase II inhibitors

A series of 7-substituted sulfocoumarins and 3,4-dihydrosulfocoumarins was obtained by cyclization of the methanesulfonate of 2,4-dihydroxy- or 2-hydroxy-4-methoxybenzaldehyde, followed by derivatization reactions. The new compounds incorporate a range of substituents in position 7 of the heterocyclic ring (hydroxyl, methoxy, carboxylic and alkylsulfonate ester). The compounds were tested for the inhibition of the zinc enzyme human (h) carbonic anhydrase (hCA, EC 4.2.1.1). Unlike the 6-substituted sulfocoumarins which were potent hCA IX and XII inhibitors and ineffective hCA I and II inhibitors, compounds from this series showed low nanomolar hCA II inhibitory properties, and inhibited the mitochondrial isoform hCA VA with K_Is in the range of 91–9960 nM, but were ineffective as hCA I, IX and XII inhibitors. The structure activity relationship for this class of inhibitors was rather clear, with the nature of the 7-substituent strongly influencing hCA VA inhibition, whereas the nature of these groups were less relevant for hCA II inhibition (all reported compounds were highly effective hCA II inhibitors, with K_Is in the range of 1.5–8.4 nM). Since both hCA II and hCA VA are important drug targets (hCA II for antiglaucoma agents; hCA VA for antiobesity drugs), these isoform-selective inhibitors reported here may be considered of interest for various biomedical applications.     

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.     

Sulfonamides incorporating piperazine bioisosteres as potent human carbonic anhydrase I,II, IV and IX inhibitors

Starting from the molecular simplification of (R) 4-(3,4-dibenzylpiperazine-1-carbonyl)benzenesulfonamide 9a, a compound endowed with selectivity for human Carbonic Anhydrase (hCA) IV, a series of piperazines and 4-aminopiperidines carrying a 4-sulfamoylbenzamide moiety as Zn-binding group have been designed and tested on human isoforms hCA I, II, IV and IX, using a stopped flow CO₂ hydrase assay. The aim of the work was to derive structure-activity relationships useful for designing isoform selective compounds. These structural modifications changed the selectivity profile of the analogues from hCA IV to hCA I and II, and improved potency. Several of the new compounds showed subnanomolar activity on hCA II. X-ray crystallography of ligand-hCAII complexes was used to compare the binding modes of the new piperazines and the previously synthesized 2-benzyl-piperazine analogues, explaining the inhibition profiles.     

Synthesis,biological evaluation and molecular docking of novel pyrazole derivatives as potent carbonic anhydrase and acetylcholinesterase inhibitors

A series of substituted pyrazole compounds (1–8 and 9a, b) were synthesized and their structure was characterized by IR, NMR, and Mass analysis. These obtained novel pyrazole derivatives (1–8 and 9a, b) were emerged as effective inhibitors of the cytosolic carbonic anhydrase I and II isoforms (hCA I and II) and acetylcholinesterase (AChE) enzymes with K_i values in the range of 1.03 ± 0.23–22.65 ± 5.36 µM for hCA I, 1.82 ± 0.30–27.94 ± 4.74 µM for hCA II, and 48.94 ± 9.63–116.05 ± 14.95 µM for AChE, respectively. Docking studies were performed for the most active compounds, 2 and 5, and binding mode between the compounds and the receptors were determined.     

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.     

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.     

4-Substituted benzenesulfonamides featuring cyclic imides moieties exhibit potent and isoform-selective carbonic anhydrase II/IX inhibition

The synthesis, characterization and biological evaluation of series of cyclic imides incorporating the 4-sulfamoylbenzamide scaffold (16–29) is disclosed. The compounds were designed by application of the “tail approach” to the aromatic sulfonamide scaffold and prepared by reacting the proper acid anhydride with 4-(hydrazinecarbonyl)benzenesulfonamide (15). Phtalimides and cyclic imides are biologically privileged scaffolds, endowed with versatile biological activity, such as an anti-proliferative action. The compounds were investigated for the inhibition of four human (h) isoforms of zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), and more specifically against the cytosolic hCA I and II and the transmembrane hCA IV and IX. Most screened sulfonamides exhibited great potency in inhibiting CA isoforms II, widely involved in glaucoma and other pathologies (K_Is in the range of 0.7–62.3 nM), and IX, that is a validated anti-tumor target (K_Is in the range of 3.0–50.9 nM), whereas interesting hydrophilicity-dependent inhibitory profiles were measured against isoform CA IV (K_Is in the range of 3.9–428.6 nM). In silico studies were carried out to assess the binding mode of selected derivatives to hCA II, IV and IX.     

Synthesis and biological evaluation of novel tris-chalcones as potent carbonic anhydrase,acetylcholinesterase, butyrylcholinesterase and α-glycosidase inhibitors

A novel class of fluoro-substituted tris-chalcones derivatives (5a-5i) was synthesized from phloroglucinol and corresponding benzaldehydes. A three step synthesis method was followed for the production of these tris-chalcone compounds. The structures of the newly synthesized compounds (5a-5i) were confirmed on the basis of IR, ¹H NMR, ¹³C NMR, and elemental analysis. The compounds’ inhibitory activities were tested against human carbonic anhydrase I and II isoenzymes (hCA I and hCA II), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α-glycosidase (α-Gly). These chalcone derivatives had K_i values in the range of 19.58–78.73 nM for hCA I, 12.23–41.70 nM for hCA II, 1.09–6.84 nM for AChE, 8.30–32.30 nM for BChE and 0.93 ± 0.20–18.53 ± 5.06 nM against α-glycosidase. These results strongly support the promising nature of the tris-chalcone scaffold as selective carbonic anhydrase, acetylcholinesterase, butyrylcholinesterase, and α-glycosidase inhibitor. Overall, due to these derivatives’ inhibitory potential on the tested enzymes, they are promising drug candidates for the treatment of diseases like glaucoma, leukemia, epilepsy; Alzheimer’s disease; type-2 diabetes mellitus that are associated with high enzymatic activity of carbonic anhydrase, acetylcholine esterase, butyrylcholinesterase, and α-glycosidase.     

Dual carbonic anhydrase/matrix metalloproteinase inhibitors incorporating bisphosphonic acid moieties targeting bone tumors

A set of bisphosphonate matrix metalloproteinase (MMP) inhibitors was investigated for inhibitory activity against several carbonic anhydrase (CA, EC 4.2.1.1) isozymes, some of which are overexpressed in hypoxic tumors. Some of the bisphosphonate revealed to be very potent inhibitors (in the low nanomolar range) of the cytosolic isoform CA II and the membrane-bound CA IX, XII and XIV isozymes, a feature useful for considering them as interesting compounds for bone resorption inhibition applications. We suggest here that it is possible to develop dual enzyme inhibitors bearing bisphosphonate moieties that may target both MMPs and CAs, two families of enzymes involved in tumor formation, growth, and metastasis.     

Design,synthesis & biological evaluation of ferulic acid-based small molecule inhibitors against tumor-associated carbonic anhydrase IX

Carbonic anhydrase IX (CAIX) is an emerging drug target for hypoxia associated cancers. To identify potent and selective inhibitors of CAIX, a small library of ferulic acid (FA) derivatives bearing triazole moiety has been designed, synthesized and evaluated against different human CA isoforms (CAII, CAVA & CAIX). Though most of the compounds showed CAIX inhibition in the micromolar range, compound 7i selectively inhibits CAIX in the nanomolar range (IC₅₀ = 24 nM). In silico analysis revealed binding of 7i with the catalytically important amino acid residues of CAIX. Further, cell-based studies indicate that 7i inhibits the activity of CAIX, decreases the epithelial to mesenchymal transitions, induces apoptosis, inhibits cell migration and colonization potential of cancer cells. Taken together, these results emphasized the use of 7i as a prospective pharmacological lead molecule in CAIX targeted anticancer therapeutics.     

A novel library of saccharin and acesulfame derivatives as potent and selective inhibitors of carbonic anhydrase IX and XII isoforms

Small libraries of N-substituted saccharin and N-/O-substituted acesulfame derivatives were synthesized and tested as atypical and selective inhibitors of four different isoforms of human carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). Most of them inhibited hCA XII in the low nanomolar range, hCA IX with K_Is ranging between 19 and 2482 nM, whereas they were poorly active against hCA II (K_Is >10 μM) and hCA I (K_Is ranging between 318 nM and 50 μM). Since hCA I and II are ubiquitous off-target isoforms, whereas the cancer-related isoforms hCA IX and XII were recently validated as drug targets, these results represent an encouraging achievement in the development of new anticancer candidates. Moreover, the lack of a classical zinc binding group in the structure of these inhibitors opens innovative, yet unexplored scenarios for different mechanisms of inhibition that could explain the high inhibitory selectivity. A computational approach has been carried out to further rationalize the biological data and to characterize the binding mode of some of these inhibitors.     

Carbonic anhydrase inhibitors: Synthesis,molecular docking,cytotoxic and inhibition of the human carbonic anhydrase isoforms I,II, IX,XII with novel benzenesulfonamides incorporating pyrrole,pyrrolopyrimidine and fused pyrrolopyrimidine moieties

A series of novel pyrroles, pyrrolopyrimidines, pyrazolopyrrolopyrimidine, triazolopyrrolopyrimidines, tetrazolopyrrolopyrimidine, triazinopyrrolopyrimidines and pyrrolopyrimidotriazepines bearing the biologically active benzenesulfonamide moiety were synthesized by using pyrrole-o-amino-carbonitrile as key intermediate. All the synthesized compounds were evaluated for their in vitro carbonic anhydrase (CA, EC 4.2.1.1) inhibitory effects against the human (h) isoforms hCA I, II, IX and XII. Among the tested derivatives, compounds 16, 18 and 20–24 showed potent activity as inhibitors for the tumor associated transmembrane isoforms (hCA IX and XII) in the nanomolar and subnanomolar range, with high selectivity. All compounds underwent cytotoxic activity assays on human breast cancer cell line (MCF-7) showing effective activity, comparable to that of the clinically used drug doxorubicin.     

Arylamino bisphosphonates: Potent and selective inhibitors of the tumor-associated carbonic anhydrase XII

A set of matrix metalloproteinases (MMPs) inhibitors, containing a bisphosphonate moiety (BP), has been evaluated for the inhibitory activity of carbonic anhydrases (CAs, EC 4.2.1.1). Human (h) isoforms hCA I, II, IX, XII and XIV were included in the study due to their involvement in crucial physiologic and pathologic processes. Some of these molecules selectively inhibited CA XII in the nanomolar range, showing an attractive dual mechanism (anti-MMP and anti-CA) of action as potential antitumor agents. The BP inhibitors investigated in this study are also excellent leads for obtaining even more effective compounds able to selectively target membrane-bound CA XII and having the potential to be used as tools for understanding physiologic processes regulated by this isoform.     

Synthesis and evaluation of 18F-labeled tertiary benzenesulfonamides for imaging carbonic anhydrase IX expression in tumours with positron emission tomography

Three tertiary benzenesulfonamide inhibitors 4a–c were radiolabeled with ¹⁸F and evaluated for imaging carbonic anhydrase IX (CA IX) expression with positron emission tomography. All three inhibitors exhibit <10 nM affinity for CA IX with no measurable affinity for CA II. Despite good affinity/selectivity to CA IX and excellent stability in plasma, uptake of [¹⁸F]4a–c in CA IX-expressing HT-29 tumours was low without significant contrast. [¹⁸F]4a,b were excreted rapidly, while [¹⁸F]4c exhibited significant in vivo defluorination leading to high bone uptake. Due to minimal uptake in HT-29 tumours compared to normal organs/tissues, ¹⁸F-labeled benzenesulfonamides [¹⁸F]4a–c are not suitable as CA IX imaging agents.