Design,synthesis and evaluation of N-substituted saccharin derivatives as selective inhibitors of tumor-associated carbonic anhydrase XII

A series of N-alkylated saccharin derivatives were synthesized and tested for the inhibition of four different isoforms of human carbonic anhydrase (CA, EC 4. 2.1.1): the transmembrane tumor-associated CA IX and XII, and the cytosolic CA I and II. Most of the reported derivatives inhibited CA XII in the nanomolar/low micromolar range, hCA IX with K_Is ranging between 11 and 390 nM, whereas they were inactive against both CA I (K_Is >50 μM) and II (K_Is ranging between 39.1 nM and 50 μM). Since CA I and II are off-targets of antitumor carbonic anhydrase inhibitors (CAIs), the obtained results represent an encouraging achievement for the development of new anticancer candidates without the common side effects of non-selective CAIs. Moreover, the lack of an explicit zinc binding function on these inhibitors opens the way towards the exploration of novel mechanisms of inhibition that could explain the high selectivity of these compounds for the inhibition of the transmembrane, tumor-associated isoforms over the cytosolic ones.     

Novel sulfonamide incorporating piperazine,aminoalcohol and 1,3,5-triazine structural motifs with carbonic anhydrase I,II and IX inhibitory action

A new series of s-triazine derivatives incorporating sulfanilamide, homosulfanilamide, 4-aminoethyl-benzenesulfonamide and piperazine or aminoalcohol structural motifs is reported. Molecular docking was exploited to select compounds from virtual combinatorial library for synthesis and subsequent biological evaluation. The compounds were prepared by using step by step nucleophilic substitution of chlorine atoms from cyanuric chloride (2,4,6-trichloro-1,3,5-triazine). The compounds were tested as inhibitors of physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms. Specifically, against the cytosolic hCA I, II and tumor-associated hCA IX. These compounds show appreciable inhibition. hCA I was inhibited with K_Is in the range of 8.5–2679.1 nM, hCA II with K_Is in the range of 4.8–380.5 nM and hCA IX with K_Is in the range of 0.4–307.7 nM. As other similar derivatives, some of the compounds showed good or excellent selectivity ratios for inhibiting hCA IX over hCA II, of 3.5–18.5. 4-[({4-Chloro-6-[(4-hydroxyphenyl)amino]-1,3,5-triazin-2-yl}amino)methyl] benzene sulfonamide demonstrated subnanomolar affinity for hCA IX (0.4 nM) and selectivity (18.50) over the cytosolic isoforms. This series of compounds may be of interest for the development of new, unconventional anticancer drugs targeting hypoxia-induced CA isoforms such as CA IX.     

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 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.     

Triterpene Biosynthesis in the Latex of Euphorbia lathyris: Calmodulin Antagonists Are Ineffective in Whole Latex

The calmodulin antagonists chlorpromazine, fluphenazine, trifluoperazine, and 2-(pentachlorophenoxy)ethyl N,N-diethylamine are not inhibitors of acetate incorporation into triterpenols (TOH) and their fatty acid esters (TE) in whole tapped latex from Euphorbia lathyris, although prior work demonstrated that these antagonists are good inhibitors of mevalonate incorporation into TOH and TE in a centrifuged pellet from the latex. Antagonist absorption into the endogenous terpene pool is the primary reason for antagonist ineffectiveness in whole latex; changes in the utilized substrate or chemical deactivation of the antagonists were ruled out as factors. A biosynthetically inactive, latex supernatant fraction containing the endogenous terpene pool was prepared. This fraction blocks antagonist action when added to the latex pellet, and proved to be a useful tool for demonstrating that inhibition of triterpene biosynthesis by a calmodulin antagonist is partially reversible.     

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.     

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. Inhibition of the human cytosolic isoforms I and II and transmembrane,tumor-associated isoforms IX and XII with boronic acids

A series of aromatic, arylalkenyl- and arylalkyl boronic acids were assayed as inhibitors of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic human (h) hCA I and II, and the transmembrane, tumor-associated hCA IX and XII. The best hCA I and II inhibitor was biphenyl boronic acid with, a K_I of 3.7–4.5 μM, whereas the remaining derivatives showed inhibition constants in the range of 6.0–1560 μM for hCA I and of 6.0–1050 μM for hCA II, respectively. hCA IX and XII were effectively inhibited by most of the aromatic boronic acids (K_Is of 7.6–12.3 μM) whereas the arylalkenyl and aryl–alkyl derivatives generally showed weaker inhibitory properties (K_Is of 34–531 μM). The nature of the moiety substituting the boronic acid group strongly influenced the CA inhibitory activity, with inhibitors possessing low micromolar to millimolar activity being detected in this small series of investigated compounds. This study proves that the B(OH)₂ moiety represents a new zinc-binding group for the generation of effective CA inhibitors targeting isoforms with medicinal chemistry applications. The boronic acids probably bind to the Zn(II) ion within the CA active site leading to a tetrahedral geometry of the metal ion and of the B(III) derivative.     

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.     

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 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 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.     

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.     

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).     

Interaction of wheat germ ca-dependent protein kinases with calmodulin antagonists and polyamines

The two soluble Ca²⁺-dependent protein kinases resolved from wheat (Triticum aestivum) embryo (protein kinases I and II) are inhibited by the phenothiazine-derived calmodulin antagonists trifluoperazine fluphenazine, and chlorpromazine. Protein kinases I and II are also inhibited by a variety of other calmodulin antagonists (including calmidazolium, amitriptyline, and iprindole), phosphodiesterase inhibitors (including flufenamic acid and papavarine) and by lanthanides. A number of compounds that inhibit mammalian Ca²⁺ - and phospholipid-activated protein kinase (protein kinase C) including quercetin, polymixin B sulfate, and polyamines (as well as phenothiazine derivatives) also inhibit protein kinases I and II. Poly-l-lysine and poly-l-ornithine activate both plant Ca²⁺-dependent protein kinases.     

Further characterization of the magnesium chelatase in isolated developing cucumber chloroplasts : substrate specificity, regulation, intactness, and ATP requirements

Mg-chelatase catalyzes the first step unique to the chlorophyll branch of tetrapyrrole biosynthesis, namely the insertion of Mg into protoporphyrin IX (Proto). Mg-chelatase was assayed in intact chloroplasts from semi-green cucumber (Cucumis sativus, cv Sumter) cotyledons. In the presence of Proto and MgATP, enzyme activity was linear for 50 minutes. Plastid intactness was directly related to (and necessary for) Mg-chelatase activity. Uncouplers and ionophores did not inhibit Mg-Chelatase in the presence of ATP. The nonhydrolyzable ATP analogs, β,γ-methylene ATP and adenylylimidodiphosphate, could not sustain Mg-chelatase activity alone and were inhibitory in the presence of ATP (I₅₀ 10 and 3 millimolar, respectively). Mg-chelatase was also inhibited by N-ethylmaleimide (I₅₀, 50 micromolar) and the metal ion chelators 2,2′-dipyridyl and 1, 10 phenanthroline (but not to the same degree by their nonchelating analogs). In addition to Proto, the following porphyrins acted as Mg-chelatase substrates, giving comparable specific activities: deuteroporphyrin, mesoporphyrin, 2-ethyl, 4-vinyl Proto and 2-vinyl, 4-ethyl Proto. Mg-chelatase activity and freely exchangeable heme levels increased steadily with greening, reaching a maximum and leveling off after 15 hours in the light. Exogenous protochlorophyllide, chlorophyllide, heme, and Mg-Proto had no measurable effect on Mg-chelatase activity. The potent ferrochelatase inhibitors, N-methylmesoporphyrin and N-methylprotoporphyrin, inhibited Mg-chelatase at micromolar concentrations.     

Selective inhibition of carbonic anhydrase IX by sulphonylated 1,2,3-triazole incorporated benzenesulphonamides capable of inducing apoptosis

In search of selective carbonic anhydrase (CA) IX inhibitors endowed with apoptotic inducing properties, we designed and synthesised two subsets of 4- and 3-(5-aryl-(4-phenylsulphonyl)-1H-1,2,3-triazol-1-yl)benzenesulphonamides. All compounds were assayed for human carbonic anhydrase (hCA) isoforms I, II, IV, and IX inhibition. Isoforms hCA I and hCA IV were weakly inhibited by most of the synthesised compounds. Many four-substituted benzenesulphonamides displayed low nanomolar inhibition against isoform hCA II, unlike the three-substituted analogues. All target compounds exhibited good inhibition profile with K_I values ranging from 16.4 to 66.0 nM against tumour-associated isoform hCA IX. Some selective and potent inhibitors of hCA IX were assayed for in vitro apoptotic induction in goat testicular cells. Compounds 10d and 10h showed interesting apoptotic induction potential. The present study may provide insights into a strategy for the design of novel anticancer agents based on hCA inhibitors endowed with apoptotic interference.     

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.     

Differential effects of tamoxifen-like compounds on osteoclastic bone degradation,H+-ATPase activity,calmodulin-dependent cyclic nucleotide phosphodiesterase activity,and calmodulin binding

We studied effects of calmodulin antagonists on osteoclastic activity and calmodulin-dependent HCl transport. The results were compared to effects on the calmodulin-dependent phosphodiesterase and antagonist-calmodulin binding affinity. Avian osteoclast degradation of labeled bone was inhibited ∼40% by trifluoperazine or tamoxifen with half-maximal effects at 1–3 μM. Four benzopyrans structurally resembling tamoxifen were compared: d-centchroman inhibited resorption 30%, with half-maximal effect at ∼100 nM, cischroman and CDRI 85/287 gave 15–20% inhibition, and l-centchroman was ineffective. No benzopyran inhibited cell attachment or protein synthesis below 10 μM. However, ATP-dependent membrane vesicle acridine transport showed that H⁺-ATPase activity was abolished by all compounds with 50% effects at 0.25–1 μM. All compounds also inhibited calmodulin-dependent cyclic nucleotide phosphodiesterase at micromolar calcium. Relative potency varied with assay type, but d- and l-centchroman, surprisingly, inhibited both H⁺-ATPase and phosphodiesterase activity at similar concentrations. However, d- and l-centchroman effects in either assay diverged at nanomolar calcium. Of benzopyrans tested, only the d-centchroman effects were calcium-dependent. Interaction of compounds with calmodulin at similar concentrations were confirmed by displacement of labeled calmodulin from immobilized trifluoperazine. Thus, the compounds tested all interact with calmodulin directly to varying degrees, and the observed osteoclast inhibition is consistent with calmodulin-mediated effects. However, calmodulin antagonist activity varies between specific reactions, and free calcium regulates specificity of some interactions. Effects on whole cells probably also reflect other properties, including transport into cells. J. Cell. Biochem. 66:358–369, 1997. © 1997 Wiley-Liss, Inc.     

Carbonic anhydrase inhibitors. Diazenylbenzenesulfonamides are potent and selective inhibitors of the tumor-associated isozymes IX and XII over the cytosolic isoforms I and II

A series of diazenylbenzenesulfonamides, azo-dye derivatives of sulfanilamide or metanilamide incorporating phenol and amine moieties, were tested for inhibition of the tumor-associated isozymes of carbonic anhydrase (CA, EC 4.2.1.1), CA IX and XII. These compounds showed moderate-low inhibitory activities against the cytosolic isoforms CA I and II (offtargets) and excellent, low nanomolar inhibitory activity against the transmembrane CA IX and XII (K_Is in the range of 3.5–63 nM against CA IX and 5.0–69.4 nM against CA XII, respectively). The selectivity ratio for inhibiting the tumor-associated CA IX over the offtarget CA II was in the range of 15–104 for these diazenylbenzenesulfonamides, making them among the most isoform-selective inhibitors targeting tumor-associated CAs (over the ubiquitous CA II). Since CA IX/XII were recently shown to be both therapeutic and diagnostic targets for hypoxic solid tumors overexpressing these proteins, such compounds held promise for the management of hypoxic tumors, which are largely non-responsible to classical chemo- and radio-therapy.