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.     

Selective inhibition of human carbonic anhydrases by novel amide derivatives of probenecid: Synthesis,biological evaluation and molecular modelling studies

Novel amide derivatives of probenecid, a well-known uricosuric agent, were synthesized and evaluated as inhibitors of human carbonic anhydrases (hCAs, EC 4.2.1.1). The transmembrane isoforms (hCA IX and XII) were potently and selectively inhibited by some of them. The proposed chemical modification led to a complete loss of hCA II inhibition (K_is > 10,000 nM) and enhanced the inhibitory activity against the tumour-associated hCA XII (compound 4 showed a K_i value of 15.3 nM). The enzyme inhibitory data have also been validated by docking studies of the compounds within the active site of hCA XII.     

Carbonic anhydrase inhibitors: Synthesis and inhibition of the human carbonic anhydrase isoforms I,II, IX and XII with benzene sulfonamides incorporating 4- and 3-nitrophthalimide moieties

A series of 4 and 5 nitro-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1–8) was synthesized by reaction of benzenesulfonamide derivatives with 4 and 3-nitrophthalic anhydrides. 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 and II and the transmembrane, tumor-associated hCA IX and XII. Most of the novel compounds were medium potency-weak hCA I inhibitors (K_is in the range of 295–10,000 nM), but were more effective hCA II inhibitors (K_is of 1.7–887 nM). The tumor-associated hCA IX was also inhibited, with K_is in the micromolar range, whereas against hCA XII the inhibition constants were in the range of 90–3746 nM. 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 studies was performed in order to rationalize the activities reported and binding mode to different hCA as inhibitors.     

Development of 3-(4-aminosulphonyl)-phenyl-2-mercapto-3H-quinazolin-4-ones as inhibitors of carbonic anhydrase isoforms involved in tumorigenesis and glaucoma

A series of heterocyclic benzenesulfonamides incorporating 2-mercapto-3H-quinazolin-4-one tails were prepared by condensation of substituted anthranilic acids with 4-isothiocyanato-benzenesulfonamide. 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 (trans-membrane, tumor-associated enzymes). They acted as medium potency inhibitors of hCA I (K_Is of 81.0–3084 nM), being highly effective as hCA II (K_Is in the range of 0.25–10.8 nM), IX (K_Is of 3.7–50.4 nM) and XII (K_Is of 0.60–52.9 nM) inhibitors. These compounds should thus be of interest as preclinical candidates in pathologies in which the activity of these enzymes should be inhibited, such as glaucoma (CA II and XII as targets) or some tumors in which the activity of three isoforms (CA II, IX and XII) is dysregulated.     

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.     

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.     

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.     

Fluorescent sulfonamide carbonic anhydrase inhibitors incorporating 1,2,3-triazole moieties: Kinetic and X-ray crystallographic studies

Fluorescent sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) were essential for demonstrating the role played by the tumor-associated isoform CA IX in acidification of tumors, cancer progression towards metastasis and for the development of imaging and therapeutic strategies for the management of hypoxic tumors which overexpress CA IX. However, the presently available such compounds are poorly water soluble which limits their use. Here we report new fluorescent sulfonamides 7, 8 and 10 with increased water solubility. The new derivatives showed poor hCA I inhibitory properties, but were effective inhibitors against the hCA II (K_Is of 366–127 nM), CA IX (K_Is of 8.1–36.9 nM), CA XII (K_Is of 4.1–20.5 nM) and CA XIV (K_Is of 12.8–53.6 nM). A high resolution X-ray crystal structure of one of these compounds bound to hCA II revealed the factors associated with the good inhibitory properties. Furthermore, this compound showed a three-fold increase of water solubility compared to a similar derivative devoid of the triazole moiety, making it an interesting candidate for ex vivo/in vivo studies.     

o-Benzenedisulfonimido–sulfonamides are potent inhibitors of the tumor-associated carbonic anhydrase isoforms CA IX and CA XII

By using phthalimido-substituted aromatic sufonamides as lead molecules, a series of new sulfonamides incorporating ortho-benzenedisulfonimide moieties have been synthesized and tested against the human (h) cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isozymes hCA I and hCA II and the transmembrane, tumor-associated isozymes hCA IX and hCA XII. All these compounds showed K_i values lower than 100 nM and many of them showed better K_is than the reference compound acetazolamide, a clinically used sulfonamide. The tumor-associated isozymes were better inhibited than the cytosolic ones. A molecular docking within the active site of some CA isoforms, such as hCA I, explained these findings, as the benzenedisulfonimide moiety makes favorable interactions (hydrogen bonds) with amino acid residues involved in binding of inhibitors, such as Gln92, His67, and His64.     

Synthesis 4-[2-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamides with subnanomolar carbonic anhydrase II and XII inhibitory properties

Condensation of substituted anthranilic acids with 4-isothiocyanatoethyl-benzenesulfonamide led to series of heterocyclic benzenesulfonamides incorporating 2-mercapto-quinazolin-4-one tails. 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 XII (a transmembrane, tumor-associated enzyme also involved in glaucoma-genesis). The new sulfonamides acted as medium potency inhibitors of hCA I (K_Is of 28.5–2954 nM), being highly effective as hCA II (K_Is in the range of 0.62–12.4 nM) and XII (K_Is of 0.54–7.11 nM) inhibitors. All substitution patterns present in these compounds (e.g., halogens, methyl and methoxy moieties, in positions 6, 7 and/or 8 of the 2-mercapto-quinazolin-4-one ring) led to highly effective hCA II/XII inhibitors. These compounds should thus be of interest as preclinical candidates in pathologies in which the activity of these enzymes should be inhibited, such as glaucoma (CA II and XII as targets) or some tumors in which the activity of isoforms CA II and XII is dysregulated.     

Structural study of the location of the phenyl tail of benzene sulfonamides and the effect on human carbonic anhydrase inhibition

The crystal structure of 4-phenylacetamidomethyl-benzenesulfonamide (4ITP) bound to human carbonic anhydrase (hCA, EC 4.2.1.1) II is reported. 4ITP is a medium potency hCA I and II inhibitor (K_Is of 54–75 nM), a strong mitochondrial CA VA/VB inhibitor (K_Is of 8.3–8.6 nM) and a weak transmembrane CA inhibitor (K_Is of 136–212 nM against hCA IX and XII). This elongated compound binds in an extended conformation to hCA II, with its tail lying towards the hydrophobic half of the active site whereas the sulfonamide moiety coordinates the zinc ion. The present structure was compared to that of structurally related aromatic sulfonamides, such as 4-phenylacetamido-benzene-sulfonamide (3OYS), 4-(2-mercaptophenylacetamido)-benzene-sulfonamide (2HD6) and 4-(3-nitrophenyl)-ureido-benzenesulfonamide (3N2P). Homology models of the hCA I, VA, VB, IX and XII structures were build which afforded an understanding of the amino acids involved in the binding of these compounds to these isoforms. The main conclusion of the study is that the orientation of the tail moiety and the presence of flexible linkers as well polar groups in it, strongly influence the potency and the selectivity of the sulfonamides for the inhibition of cytosolic, mitochondrial or transmembrane CA isoforms.     

Synthesis of 6-tetrazolyl-substituted sulfocoumarins acting as highly potent and selective inhibitors of the tumor-associated carbonic anhydrase isoforms IX and XII

A series of 6-substituted sulfocoumarins incorporating substituted-1,2,3,4-tetrazol-5-yl moieties were synthesized by reaction of 6-iodo-sulfocoumarin and the corresponding tetrazole via the CH activation reaction. The new sulfocoumarins incorporating alkyl and substituted aryl moieties at the 1-position of the tetrazole, were investigated for the inhibition of four human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, the cytosolic hCA I and II; and the transmembrane, tumor-associated hCA IX and XII. The tetrazole-substituted sulfocoumarins did not inhibit the ubiquitous, off-target cytosolic isoforms (K_Is >10 μM) but showed effective inhibition against the two transmembrane CAs, with K_Is ranging from 6.5 to 68.6 nM against hCA IX, and between 4.3 and 59.8 nM against hCA XII. As hCA IX and XII are validated anti-tumor targets, such prodrug, isoform-selective inhibitors as the sulfocoumarins reported here, may be useful for identifying suitable drug candidates for clinical trials.     

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.     

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.     

6-Triazolyl-substituted sulfocoumarins are potent,selective inhibitors of the tumor-associated carbonic anhydrases IX and XII

A series of 6-substituted sulfocoumarins incorporating substituted-1,2,3-triazol-4-yl-/5-yl moieties were synthesized by employing click chemistry. The new sulfocoumarins incorporated cycloalkyl, tert-butyl and substituted aryl moieties at the triazole ring, and were investigated for the inhibition of four human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, the cytosolic hCA I and II; and the transmembrane, tumor-associated hCA IX and XII. The triazole-substituted sulfocoumarins did not inhibit the ubiquitous, off-target cytosolic isoforms hCA I and II (K_Is >10 μM) but showed effective inhibition against the two transmembrane CAs, with K_Is ranging from 7.2 to 10.5 nM against hCA IX, and between 5.5 and 17.7 nM against hCA XII. As hCA IX and XII are validated anti-tumor targets, such prodrug, isoform-selective inhibitors as the sulfocoumarins reported here, may be useful for identifying suitable drug candidates for clinical trials.     

Coumarinyl-substituted sulfonamides strongly inhibit several human carbonic anhydrase isoforms: solution and crystallographic investigations

We investigated a series of coumarinyl-substituted aromatic sulfonamides as inhibitors of four carbonic anhydrase (CA, EC 4.2.1.1) isoforms with medical applications, the cytosolic hCA I, and II, and the transmembrane, tumor-associated hCA IX and XII. Compounds incorporating 7-methoxy-coumarin-4-yl-acetamide-tails and benzenesulfonamide and benzene-1,3-disulfonamide scaffolds showed medium potency inhibition of hCA I (K_Is of 73–131 nM), effective hCA II inhibition (K_Is of 9.1–36 nM) and less effective hCA IX and XII inhibition (K_Is of 55–128 nM). Only one compound, the derivatized 4-amino-6-trifluoromethyl-benzene-1,3-disulfonamide with the coumarinyl tail, showed effective inhibition of the transmembrane isoforms, with K_Is of 5.9–14.2 nM, although it was less effective as hCA I and II inhibitor (K_Is of 36–120 nM). An X-ray crystal structure of hCA II in complex with 4-(7-methoxy-coumarin-4-yl-acetamido)-benzenesulfonamide (K_I of 9.1 nM against hCA II) showed the intact inhibitor coordinated to the zinc ion from the enzyme active site by the sulfonamide moiety, and participating in a edge-to-face stacking with Phe131, in addition to other hydrophobic and hydrophilic interactions with water molecules and amino acid residues from the active site. Thus, sulfonamides incorporating coumarin rings have a distinct inhibition mechanism compared to the coumarins, and may lead to compounds with interesting inhibition profiles against various α-CAs found in mammals or parasites, such as Plasmodium falciparum.     

Carbonic anhydrase inhibitors; Fluorinated phenyl sulfamates show strong inhibitory activity and selectivity for the inhibition of the tumor-associated isozymes IX and XII over the cytosolic ones I and II

A series of fluorinated-phenylsulfamates have been prepared by sulfamoylation of the corresponding phenols and the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic CA I and II (off-targets), and the transmembrane, tumor-associated CA IX and XII is investigated. Unlike the lead molecule (phenylsulfamate), a very potent CA I and II inhibitor and a modest CA IX/XII inhibitor, the fluorinated sulfamates were stronger inhibitors of CA IX (K_Is of 2.8–47 nM) and CA XII (K_Is of 1.9–35 nM) than of CA I (K_Is of 53–415 nM) and CA II (K_Is of 20–113 nM). Some of these compounds were selective CA IX over CA II inhibitors, with selectivity ratios in the range of 11.4–12.1, making them interesting candidates for targeting hypoxic tumors overexpressing CA IX and/or XII.     

Synthesis and biological evaluation of a series of benzoxazole/benzothiazole-containing 2,3-dihydrobenzo[b][1,4]dioxine derivatives as potential antidepressants

A series of benzoxazole/benzothiazole-2,3-dihydrobenzo[b][1,4]dioxine derivatives (5a–5d and 8a–8j) was synthesized. Compounds were evaluated for binding affinities at the 5-HT_1A and 5-HT_2A receptors. Antidepressant activities of the compounds were screened using the forced swimming test (FST) and the tail suspension test (TST). The results indicated that the compounds exhibited high affinities for the 5-HT_1A and 5-HT_2A receptors and showed a marked antidepressant-like activity. Compound 8g exhibited high affinities for the 5-HT_1A (K_i = 17 nM) and 5-HT_2A (K_i = 0.71 nM) receptors; it also produced a decrease of the immobility time and exhibited potent antidepressant-like effects in the FST and TST in mice.     

Carbonic anhydrase inhibitors: Two-prong versus mono-prong inhibitors of isoforms I,II, IX,and XII exemplified by photochromic cis-1,2-α-dithienylethene derivatives

We investigated the inhibition of five physiologically relevant CA isoforms with photochromic cis-1,2-α-dithienylethene-based compounds incorporating either a benzenesulfonamide and Cu(II)-iminodiacetic acid (IDA)-, bis-benzenesulfonamide-, bis-Cu(II)-IDA-, and bis-ethyleneglycol-methyl ether moieties, in both their open- and closed-ring forms. For hCA I the best inhibitors were the mono-prong bis-sulfonamide and the bis-Cu-IDA complexes (K_Is of 2–3 nM) in their open form. For hCA II, best inhibitors were the open and closed forms of the mono-prong bis-sulfonamide (K_Is of 13–18 nM). hCA IX was moderately inhibited by these compounds (K_Is of 9–376 nM) whereas hCA XII and XIV were less susceptible to inhibition (K_Is of 1.12–16.7 μM).     

Facile synthesis and characterization of novel pyrazole-sulfonamides and their inhibition effects on human carbonic anhydrase isoenzymes

In the current study, a series of pyrazole-sulfonamide derivatives (2–14) were synthesized, characterized, and the inhibition effects of the derivatives on human carbonic anhydrases (hCA I and hCA II) were investigated as in vitro. Structures of these sulfonamides were confirmed by FT-IR, ¹H NMR, ¹³C NMR and LC–MS analysis. ¹H NMR and ¹³C NMR revealed the tautomeric structures. hCA I and hCA II isozymes were purified from human erythrocytes and inhibitory effects of newly synthesized sulfonamides on esterase activities of these isoenzymes have been studied. The K_i values of compounds were 0.062–1.278 μM for hCA I and 0.012–0.379 μM for hCA II. The inhibition effects of 7 for hCA I and 4 for hCA II isozymes were almost in nanomolar concentration range.