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.     

New coumarin/sulfocoumarin linked phenylacrylamides as selective transmembrane carbonic anhydrase inhibitors: Synthesis and in-vitro biological evaluation

Two novel series of phenylacrylamide linked coumarins and sulfocoumarins (6a-p, 8a-i, and 14a-g) were synthesized and evaluated against four physiologically relevant human carbonic anhydrases (hCAs, EC 4.2.1.1), isoforms hCA I, hCA II, hCA IX and hCA XII for their inhibitory action. All new compounds when screened for carbonic anhydrase inhibitory activity have shown selective inhibition towards the tumor associated isoforms hCA IX and XII over CA I and II, with inhibition constants in the submicromolar to low nanomolar range. Compound 6b and 14g exhibited significant inhibition with low nanomolar potency against hCA IX, whereas 6k was effective against hCA XII. Compounds 6b, 14g and 6k may be considered as lead molecules for future development of cancer therapeutics based on a novel mechanism of action.     

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

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

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 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.     

New thiopyrimidine-benzenesulfonamide conjugates as selective carbonic anhydrase II inhibitors: synthesis,in vitro biological evaluation,and molecular docking studies

In the present work, a new series of thiopyrimidine-benzenesulfonamide conjugates was designed, synthesized and tested as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. Our design strategy was based on the molecular hybridization of the benzenesulfonamide moiety as a zinc binding group (ZBG), an alkylated thiopyrimidine moiety as a spacer and (un)substituted phenyl moieties with various electronic and hydrophobic environments as a tail. The designed and synthesized compounds were evaluated against four human (h) CA isoforms hCA I, hCA II, hCA IX and hCA XII. Series 6 showed promising activity and selectivity toward the cytosolic isoforms hCA I and hCA II versus the membrane bound isoforms hCA IX and hCA XII. Compounds 6e and 6f showed K_i of 0.04 µM against hCA II with a selectivity of 15.8- to 980-fold towards hCA II over hCA I, hCA IX, hCA XII isoforms. Molecular docking in the hCA II active site attributed the promising inhibitory activity of series 6 to the interaction of their sulfonamide moiety with the active site Zn²⁺ ion as well as its hydrogen bonding with the key amino acids Thr199 and Thr200. Through hydrophobic interaction, the benzenesulfonamide and the thiopyrimidine moieties interact with the hydrophobic side chains of the amino acids Val121/Leu198 and Ile91/Phe131, respectively. These results indicated that the designed and synthesized series is an interesting scaffold that can be further optimized for the development of selective antiglaucoma drugs.     

Pyrazolylbenzo[d]imidazoles as new potent and selective inhibitors of carbonic anhydrase isoforms hCA IX and XII

Novel pyrazolylbenzo[d]imidazole derivatives (2a–2f) were designed, synthesized and evaluated against four human carbonic anhydrase isoforms belonging to α family comprising of two cytosolic isoforms hCA I and II as well as two transmembrane tumor associated isoforms hCA IX and XII. Starting from these derivatives that showed high potency but low selectivity in favor of tumor associated isoforms hCA IX and XII, we investigated the impact of removing the sulfonamide group. Thus, analogs 3a–3f without sulfonamide moiety were synthesized and biological assay revealed a good activity as well as an excellent selectivity as inhibitors for tumor associated hCA IX and hCA XII and the same was analyzed by molecular docking studies.     

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.     

Carbonic anhydrase inhibitors. Selective inhibition of human tumor-associated isozymes IX and XII and cytosolic isozymes I and II with some substituted-2-mercapto-benzenesulfonamides

A series of 2-mercapto-substituted-benzenesulfonamides has been prepared by a unique two-step procedure starting from the corresponding 2-chloro-substituted benzenesulfonamides. Compounds bearing an unsubstituted mercapto group and the corresponding S-benzoyl derivatives were investigated as inhibitors of four isoforms of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), i.e., the cytosolic, ubiquitous isozymes CA I and II, as well as the transmembrane, tumor associated isozymes CA IX and XII. These derivatives were medium potency hCA I inhibitors (K_Is in the range of 1.5–5.7 μM), two derivatives were strong hCA II inhibitors (K_Is in the range of 15–16 nM), whereas the others showed weak activity. These compounds inhibited hCA IX with inhibition constants in the range 160–1950 nM and hCA XII with inhibition constants in the range 1.2–413 nM. Some of these derivatives showed a certain degree of selectivity for inhibition of the tumor-associated over the cytosolic isoforms, being thus interesting leads for the development of potentially novel applications in the management of hypoxic tumors which overexpress CA IX and XII.     

Metronidazole-coumarin conjugates and 3-cyano-7-hydroxy-coumarin act as isoform-selective carbonic anhydrase inhibitors

Reaction of 6-/7-hydroxycoumarin with metronidazole afforded conjugates which incorporate two interesting chemotypes which may inhibit carbonic anhydrases (CAs, EC 4.2.1.1) due to the presence of the coumarin moiety and possess radiosensitizing effects due to the presence of the nitroazole. Another dual action compound, which may act both as CA inhibitor as well as monocarboxylate transporter inhibitor, is 3-cyano-7-hydroxy-coumarin. These compounds have been investigated as inhibitors of 11 human CA isoforms. Submicromolar inhibition was observed against hCA VA, hCA VB, hCA VI, hCA VII, hCA IX, hCA XII and hCA XIV, whereas isoforms hCA I, II and XIII were not inhibited by these compounds. These coumarins thus act as isoform-selective CA inhibitors with the possibility to target isoforms involved in pathologies such as obesity (CA VA/VB) or cancer (CA IX and XII) without inhibiting the physiologically dominant, highly abundant hCA I and II.     

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

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

A class of carbonic anhydrase IX/XII – selective carboxylate inhibitors

Abstract

A small series of 2,4-dioxothiazolidinyl acetic acids was prepared from thiourea, chloroacetic acid, aromatic aldehydes, and ethyl-2-bromoacetate. They were assayed for the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms of human (h) origin, the cytosolic hCA I and II, and the transmembrane hCA IX and XII, involved among others in tumorigenesis (hCA IX and XII) and glaucoma (hCA II and XII). The two cytosolic isoforms were not inhibited by these carboxylates, which were also rather ineffective as hCA IX inhibitors. On the other hand, they showed submicromolar hCA XII inhibition, with K_Is in the range of 0.30–0.93?µM, making them highly CA XII-selective inhibitors.     

Synthesis of new 3-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-benzenesulfonamides with strong inhibition properties against the tumor associated carbonic anhydrases IX and XII

We report a series of novel metanilamide-based derivatives 3a–q bearing the 2-mercapto-4-oxo-4H-quinazolin-3-yl moiety as tail. All compounds were synthesized by means of straightforward condensation procedures and were investigated in vitro for their inhibition potency against the human (h) carbonic anhydrase (CA; EC 4.2.1.1.1) isoforms I, II, IX and XII. Among all compounds tested the 6-iodo 3g and the 7-fluoro 3i derivatives were the most potent inhibitors against the tumor associated CA IX and XII isoform (K_Is 1.5 and 2.7 nM respectively for the hCA IX and K_Is 0.57 and 1.9 nM respectively for the hCA XII).The kinetic data reported here strongly support compounds of this type for their future development as radiotracers in tumor pathologies which are strictly dependent on the enzymatic activity of the hCA IX and XII isoforms.     

Synthesis and comparative carbonic anhydrase inhibition of new Schiff’s bases incorporating benzenesulfonamide,methanesulfonamide, and methylsulfonylbenzene scaffolds

Herein, we report the synthesis, characterization, and carbonic anhydrase (CA) inhibition of the newly synthesized Schiff’s bases 4–18 with benzenesulfonamide, methanesulfonamide, and methylsulfonylbenzene scaffolds. The compound inhibition profiles against human CA (hCA) isoforms I, II, IX, and XII were compared to those of the standard inhibitors, acetazolamide (AAZ) and SLC-0111 (a CA inhibitor in Phase II clinical trials for the treatment of hypoxic tumors). The hCA I was inhibited by compounds 4a–8a with inhibition constants (K_I) in the range 93.5–428.1 nM (AAZ and SLC-0111: K_I, 250.0 and 5080.0 nM, respectively). Compounds 4a–8a proved to be effective hCA II inhibitors, with K_I ranging from 18.2 to 133.3 nM (AAZ and SLC-0111: K_I, 12.0 and 960.0 nM, respectively). Compounds 4a–8a effectively inhibited hCA IX, with K_I in the range 8.5–24.9 nM; these values are superior or equivalent to that of AAZ and SLC-0111 (K_I, 25.0 and 45.0 nM, respectively). Compounds 4a–8a displayed effective hCA XII inhibitory activity with K_I values ranging from 8.6 to 43.2 nM (AAZ and SLC-0111: K_I, 5.7 and 4.5 nM, respectively). However, compounds 9b–13b and 14c–18c were found to be micromolar CA inhibitors. For molecular docking studies, compounds 5a, 6a, and 8a were selected.     

Inhibition of tumor-associated human carbonic anhydrase isozymes IX and XII by a new class of substituted-phenylacetamido aromatic sulfonamides

Here, we investigate 28 structurally new sulfonamides and their subsequent testing for enzyme inhibition of cytosolic and tumor-associated carbonic anhydrases (CAs, EC 4.2.1.1). The compounds showed very potent inhibition of four physiologically relevant human (h) CA isoforms, namely hCA I, II, IX and XII. Interestingly, the K_I values were in the nanomolar range for the tumor-associated hCA IX and hCA XII. Docking studies have revealed details regarding the very favorable interactions between the scaffolds of this new class of inhibitors and the active sites of the investigated CA isoforms. As there are reported cases of tumors overexpressing both CA II and IX, such potent inhibitors for the two isoforms as those detected in this work, may have applications for targeting more than one CA present in tumors.     

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.     

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.     

Synthesis and carbonic anhydrase inhibitory properties of amino acid – coumarin/quinolinone conjugates incorporating glycine,alanine and phenylalanine moieties

N-Protected amino acids (Gly, Ala and Phe) were reacted with amino substituted coumarin and quinolinone derivatives, leading to the corresponding N-protected amino acid–coumarin/quinolinone conjugates. The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity of the new compounds was assessed against various human (h) isoforms, such as hCA I, hCA II, hCA IV and hCA XII. The quinolinone conjugates were inactive as enzyme inhibitors, whereas the coumarins were ineffective hCA I/II inhibitors (K_Is?>?50?μM) but were submicromolar hCA IV and XII inhibitors, with inhibition constants ranging between 92?nM and 1.19?μM for hCA IV, and between 0.11 and 0.79?μM for hCA XII. These coumarin derivatives, as many others reported earlier, thus show an interesting selective inhibitory profile for the membrane-bound over the cytosolic CA isoforms.     

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.     

Design,synthesis, and carbonic anhydrase inhibition activity of benzenesulfonamide-linked novel pyrazoline derivatives

Carbonic anhydrases (CA, EC 4.2.1.1) are Zinc metalloenzymes and are present throughout most living organisms. Among the catalytically active isoforms are the cytosolic CA I and II, and tumor-associated CA IX and CA XII. The carbonic anhydrase (CA) inhibitory activities of newly synthesized pyrazoline-linked benzenesulfonamides 18–33 against human CA (hCA) isoforms I, II, IX, and XII were measured and compared with that of acetazolamide (AAZ), a standard inhibitor. Potent inhibitory activity against hCA I was exerted by compounds 18–25, with inhibition constant (K_I) values of 87.8–244.1 nM, which were greater than that of AAZ (K_I, 250.0 nM). Compounds 19, 21, 22, 29, 30, and 32 were proven to have inhibitory activities against hCA IX with K_I values (5.5–37.0 nM) that were more effective than or nearly equal to that of AAZ (K_I, 25.0 nM). Compounds 20–22, and 30 exerted potent inhibitory activities (K_Is, 7.1–10.1 nM) against hCA XII, in comparison with AAZ (K_I, 5.7 nM).