Carbonic anhydrase inhibitors: the first selective, membrane-impermeant inhibitors targeting the tumor-associated isozyme IX

The inhibition of the tumor-associated transmembrane carbonic anhydrase IX (CA IX) isozyme possessing an extracellular active site has been investigated with a series of positively-charged, pyridinium derivatives of sulfanilamide, homosulfanilamide and 4-aminoethylbenzenesulfonamide. Inhibition data for the physiologically relevant isozymes I and II (cytosolic forms) and IV (membrane-bound) were also provided for comparison. A very interesting inhibition profile against CA IX with these sulfonamides has been observed. Several nanomolar (K(i)'s in the range of 6-54 nM) CA IX inhibitors have also been detected. Because CA IX is a highly active isozyme predominantly expressed in tumor tissues with bad prognosis of disease progression, this finding is very promising for the potential design of CA IX-specific inhibitors with applications as anti-tumor agents. This is the first report of inhibitors that may selectively target CA IX, due to their membrane-impermeability and high affinity for this clinically relevant isozyme.     

Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides incorporating 1,2,4-triazine moieties

A series of benzenesulfonamide derivatives incorporating triazine moieties in their molecules was obtained by reaction of cyanuric chloride with sulfanilamide, homosulfanilamide, or 4-aminoethylbenzenesulfonamide. The dichlorotriazinyl-benzenesulfonamides intermediates were subsequently derivatized by reaction with various nucleophiles, such as water, methylamine, or aliphatic alcohols (methanol and ethanol). The library of sulfonamides incorporating triazinyl moieties was tested for the inhibition of three physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic hCA I and II, and the transmembrane, tumor-associated hCA IX. The new compounds reported here inhibited hCA I with K(I)s in the range of 75-136nM, hCA II with K(I)s in the range of 13-278nM, and hCA IX with K(I)s in the range of 0.12-549nM. The first hCA IX-selective inhibitors were thus detected, as the chlorotriazinyl-sulfanilamide and the bis-ethoxytriazinyl derivatives of sulfanilamide/homosulfanilamide showed selectivity ratios for CA IX over CA II inhibition in the range of 166-706. Furthermore, some of these compounds have subnanomolar affinity for hCA IX, with K(I)s in the range 0.12-0.34nM. These derivatives are interesting candidates for the development of novel unconventional anticancer strategies targeting the hypoxic areas of tumors. Clear renal cell carcinoma, which is the most lethal urologic malignancy and is both characterized by very high CA IX expression and chemotherapy unresponsiveness, could be the leading candidate of such novel therapies.     

Carbonic anhydrase inhibitors: inhibition of the human transmembrane isozyme XIV with a library of aromatic/heterocyclic sulfonamides

The first inhibition study of the transmembrane carbonic anhydrase (CA, EC 4.2.1.1) isozymes hCA XIV with a library of aromatic and heteroaromatic sulfonamides synthesized earlier is reported. Most of the inhibitors were sulfanilamide, homosulfanilamide and 4-aminoethyl-benzenesulfonamide derivatives, to which tails that would induce diverse physicochemical properties have been attached at the amino moiety. Several of these compounds were metanilamide, benzene-1,3-disulfonamide or the 1,3,4-thiadiazole/thiadiazoline-2-sulfonamide derivatives. The tails incorporated in these molecules were of the alkyl/aryl-carboxamido/ sulfonamido-, ureido- or thioureido-types. The sulfanilamides acylated at the 4-amino group with short aliphatic/aromatic moieties incorporating 2-6 carbon atoms showed modest hCA XIV inhibitory activity (K(I)-s in the range of 1.25-4.2 microM) which were anyhow better than that of sulfanilamide (K(I) of 5.4 microM). Better activity showed the homosulfanilamide and 4-aminoethyl-benzenesulfonamide derivatives bearing arylsulfonamido/ureido and thioureido moieties, with K(I)'s in the range of 203-935 nM. The best activity was observed for the heteroaromatic compounds incorporating 1,3,4-thiadiazole/thiadiazoline-2-sulfonamide and 5-arylcarboxamido/sulfonamido moieties, with K(I)'s in the range of 10-85 nM. All these compounds were generally also much better inhibitors of the other two transmembrane CA isozyme, hCA IX and XII. Thus, highly potent hCA XIV inhibitors were detected, but isozyme-specific inhibitors were not discovered for the moment.     

Carbonic anhydrase inhibitors: inhibition of the tumor-associated isozymes IX and XII with a library of aromatic and heteroaromatic sulfonamides

The inhibition of the two transmembrane, tumor-associated isozymes of carbonic anhydrase (CA, EC 4.2.1.1) of human origin, hCA IX and XII, with a library of aromatic and heteroaromatic sulfonamides has been investigated. Most of them were sulfanilamide, homosulfanilamide, and 4-aminoethyl-benzenesulfonamide derivatives, to which tails that should induce diverse physico-chemical properties have been attached at the amino moiety, whereas several of these compounds were derived from metanilamide, benzene-1,3-disulfonamide or the 1,3,4-thiadiazole/thiadiazoline-2-sulfonamides. The tails were of the alkyl/aryl-carboxamido/sulfonamido-, ureido or thioureido type. Against hCA IX the investigated compounds showed inhibition constants in the range of 3-294 nM, whereas against hCA XII in the range of 1.9-348 nM, respectively. The best hCA IX inhibitors were ureas/thioureas incorporating 4-aminoethyl-benzenesulfonamide and metanilamide moieties. The best hCA XII inhibitors were 1,3,4-thiadiazole/thiadiazoline-2-sulfonamides incorporating 5-acylamido or 5-arylsulfonylamido moieties. These compounds also inhibited appreciably the cytosolic isozymes hCA I and II, but some selectivity for the transmembrane, tumor-associated isozymes was observed for some of them, which is an encouraging result for the design of novel therapies targeting hypoxic tumors, in which these carbonic anhydrases are highly overexpressed.     

Carbonic anhydrase inhibitors. Inhibition of cytosolic isozyme XIII with aromatic and heterocyclic sulfonamides: a novel target for the drug design

The inhibition of the newly discovered cytosolic carbonic anhydrase isozyme XIII (CA XIII) has been investigated with a series of aromatic and heterocyclic sulfonamides, including some of the clinically used derivatives, such as acetazolamide, methazolamide, dichlorophenamide, dorzolamide, and valdecoxib. Inhibition data for the physiologically relevant isozymes I and II (cytosolic forms) and the tumor associated isozyme IX (transmembrane) were also provided for comparison. A very interesting and unusual inhibition profile against CA XIII with these sulfonamides has been observed. The clinically used compounds (except valdecoxib, which was a weak CA XIII inhibitor) potently inhibit CA XIII, with Ki's in the range of 17-23 nM, whereas sulfanilamide, halogenated sulfanilamides, homosulfanilamide, 4-aminoethylbenzenesulfonamide, and orthanilamide were slightly less effective, with Ki's in the range of 32-56 nM. Several low nanomolar (Ki's in the range of 1.3-2.4 nM) CA XIII inhibitors have also been detected, all of them belonging to the sulfanilyl-sulfonamide type of inhibitors, of which aminobenzolamide is the best known representative. Because CA XIII is an active isozyme predominantly expressed in salivary glands, kidney, brain, lung, gut, uterus, and testis, where it probably plays an important role in pH regulation, its inhibition by sulfonamides may lead to novel therapeutic applications for this class of pharmacological agents.     

Carbonic anhydrase inhibitors. Synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with boron-containing sulfonamides, sulfamides, and sulfamates: toward agents for boron neutron capture therapy of hypoxic tumors

A library of boron-containing carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, including sulfonamides, sulfamides, and sulfamates is reported. The new compounds have been synthesized by derivatization reactions of 4-carboxy-/amino-/hydroxy-phenylboronic acid pinacol esters with amino/isothiocyanato-substituted aromatic/heteroaromatic sulfonamides or by sulfamoylation reactions with sulfamoyl chloride. The new derivatives have been assayed for the inhibition of three physiologically relevant CA isozymes, the cytosolic CA I and II, and the transmembrane, tumor-associated isozyme CA IX. Effective inhibitors were detected both among sulfonamides, sulfamates, and sulfamides. Against the human isozyme hCA I the new compounds showed inhibition constants in the range of 34-94nM, against hCA II in the range of 3.1-48nM, and against hCA IX in the range of 7.3-89nM, respectively. As hypoxic tumors highly overexpress CA IX, the design of boron-containing inhibitors with high affinity for the tumor-associated CA isozymes may lead to important advances in boron neutron capture therapy (BNCT) applications targeting such tumors, which are non-responsive to both classical chemo- and radiotherapy.     

Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides incorporating thioureido-sulfanilyl scaffolds

The tumor-associated transmembrane carbonic anhydrase (CA, EC 4.2.1.1) isozyme IX (CA IX) is overexpressed in hypoxic tumors and appears to be involved in acidification of the tumor microenvironment, a process correlated with cancer progression and bad prognosis. The acidification may be reduced by inhibiting the enzyme with potent sulfonamide/sulfamate CA inhibitors. A series of such aromatic sulfonamides incorporating thioureido-sulfanilyl moieties has been prepared and investigated for its interaction with the catalytic domain of the human isozyme hCA IX. The key intermediates in the synthesis were obtained by reacting sulfanilamide, homosulfanilamide, or 4-aminoethylbenzenesulfonamide with 4-acetamido-benzenesulfonyl chloride followed by deacetylation and reaction with thiophosgene. The obtained isothiocyanato sulfonamides were reacted with aliphatic or aromatic primary amines or hydrazines, leading to the corresponding thioureas. Some of these compounds showed excellent inhibitory properties against isozymes I, II, and IX, with several inhibitors also presenting selectivity for the inhibition of CA IX over that of the ubiquitous isozyme CA II. Such sulfonamides may constitute interesting candidates for the development of novel antitumor therapies based on the inhibition of the CA isozymes overexpressed in hypoxic tumors. Due to the highest expression of CA IX in clear renal cell carcinoma and its chemo/radioresistance, our efforts are first of all directed to generate effective therapeutic strategies for the cure of this malignancy.     

Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides derived from 4-isothiocyanato-benzolamide

A series of sulfonamides incorporating 4-thioureido-benzolamide moieties have been prepared from aminobenzolamide and thiophosgene followed by the reaction of the thiocyanato intermediate with aliphatic/aromatic amines or hydrazines. The new derivatives have been investigated as inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), and more precisely of the cytosolic isozymes hCA I and II, as well as the tumor-associated isozyme hCA IX (all of human origin). The new compounds showed excellent inhibitory properties against all three isozymes with inhibition constants in the range of 0.6-62 nM against hCA I, 0.5-1.7 nM against hCA II and 3.2-23 nM against hCA IX, respectively. These derivatives are interesting candidates for the development of novel therapies targeting hypoxic tumors.     

Synthesis of 4-sulfamoylphenyl-benzylamine derivatives with inhibitory activity against human carbonic anhydrase isoforms I,II, IX and XII

Imine derivatives were obtained by condensation of sulfanilamide with substituted aromatic aldehydes. The Schiff bases were thereafter reduced with sodium borohydride, leading to the corresponding amines, derivatives of 4-sulfamoylphenyl-benzylamine. These sulfonamides were investigated as inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I and II (cytosolic isozymes), as well as hCA IX and XII (transmembrane, tumor-associated enzymes). We noted that the compounds incorporating secondary amine moieties showed a better inhibitory activity against all CA isozymes compared to the corresponding Schiff bases. Low nanomolar CA II, IX and XII inhibitors were detected, whereas the activity against hCA I was less potent. The secondary amines incorporating sulfonamide or similar zinc-binding groups, poorly investigated chemotypes for designing metalloenzyme inhibitors, may offer interesting opportunities in the field due to the facile preparation and possibility to explore a vast chemical space.     

Carbonic anhydrase inhibitors: inhibition of the transmembrane isozyme XIV with sulfonamides

The inhibition of the last human carbonic anhydrase (CA, EC 4.2.1.1) isozyme (hCA XIV) discovered has been investigated with a series of sulfonamides, including some clinically used derivatives (acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide, and zonisamide), as well as the sulfamate antiepileptic drug topiramate. The full-length hCA XIV is an enzyme showing a medium-low catalytic activity, quite similar to that of hCA XII, with the following kinetic parameters at 20 degrees C and pH 7.5, for the CO2 hydration reaction: k(cat) = 3.12 x 10(5) s(-1) and k(cat)/K(M) = 3.9 x 10(7) M(-1) s(-1). All types of activities have been detected for the investigated compounds, with several micromolar inhibitors, including zonisamide, topiramate, and simple sulfanilamide derivatives (K(I)-s in the range of 1.46-6.50 microM). In addition, topiramate and zonisamide were observed to behave as weak hCA XII inhibitors, while zonisamide was an effective hCA IX inhibitor (K(I) of 5.1 nM). Some benzene-1,3-disulfonamide derivatives or simple five-membered heteroaromatic sulfonamides showed K(I)-s in the range of 180-680 nM against hCA XIV, whereas the most effective of such inhibitors, including 3-chloro-/bromo-sulfanilamide, benzolamide-like, ethoxzolamide-like, and acetazolamide/methazolamide-like derivatives, showed inhibition constant in the range of 13-48 nM. The best hCA XIV inhibitor was aminobenzolamide (K(I) of 13 nM), but no CA XIV-selective derivatives were evidenced. There are important differences of affinity of these sulfonamides/sulfamates for the three transmembrane CA isozymes, with CA XII showing the highest affinity, followed by CA IX, whereas CA XIV usually showed the lowest affinity for these inhibitors.     

Carbonic anhydrase inhibitors: novel sulfonamides incorporating 1,3,5-triazine moieties as inhibitors of the cytosolic and tumour-associated carbonic anhydrase isozymes I, II and IX

A new series of aromatic benzenesulfonamides incorporating 1,3,5-triazine moieties in their molecules is reported. This series was obtained by reaction of cyanuric chloride with sulfanilamide, homosulfanilamide or 4-aminoethylbenzenesulfonamide. The prepared dichlorotriazinyl-benzenesulfonamides were subsequently derivatized by reacting them with various nucleophiles, such as ammonia, hydrazine, primary and secondary amines, amino acid derivatives or phenol. The library of sulfonamides incorporating triazinyl moieties was tested for the inhibition of three physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic hCA I and II, and the transmembrane, tumour-associated hCA IX. The new compounds inhibited hCA I with inhibition constants in the range of 31-8500 nM, hCA II with inhibition constants in the range of 14-765 nM and hCA IX with inhibition constants in the range of 1.0-640 nM. Structure-activity relationship was straightforward and rather simple in this class of CA inhibitors, with the compounds incorporating compact moieties at the triazine ring (such as amino, hydrazino, ethylamino, dimethylamino or amino acyl) being the most active ones, and the derivatives incorporating such bulky moieties (n-propyl, n-butyl, diethylaminoethyl, piperazinylethyl, pyridoxal amine or phenoxy) being less effective hCA I, II and IX inhibitors. Some of the new derivatives also showed selectivity for inhibition of hCA IX over hCA II (selectivity ratios of 23.33-32.00), thus constituting excellent leads for the development of novel approaches for the management of hypoxic tumours.     

Carbonic anhydrase inhibitors: design of thioureido sulfonamides with potent isozyme II and XII inhibitory properties and intraocular pressure lowering activity in a rabbit model of glaucoma

A new series of thioureido-substituted sulfonamides were prepared by reacting 4-isothiocyanato- or 4-isothiocyanatoethyl-benzenesulfonamide with amines, hydrazines, or amino acids bearing moieties that can lead to an enhanced hydrosolubility, such as 2-dimethylamino-ethylamine, fluorine-containing aromatic amines/hydrazines, an aminodiol, heterocyclic polyamines (derivatives of morpholine and piperazine), 4-aminobenzoic acid, or natural amino acids (Gly, Cys, Asn, Arg, and Phe). The new compounds showed good inhibitory properties against three physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, with K(I)s in the range of 24-324 nM against the cytosolic isoform CA I, of 6-185 nM against the other cytosolic isozyme CA II, and of 1.5-144 nM against the transmembrane isozyme CA XII. Some of the new derivatives were also very effective in reducing elevated intraocular pressure in hypertensive rabbits as a glaucoma animal model. Considering that this is the first study in which potent CA II/CA XII inhibitors are designed and investigated in vivo, it may be assumed that the target isozymes of the antiglaucoma sulfonamides are indeed the cytosolic CA II and the transmembrane CA XII.     

Carbonic anhydrase inhibitors. Inhibition of red blood cell ostrich (Struthio camelus) carbonic anhydrase with a series of aromatic and heterocyclic sulfonamides

The purification of red blood cell carbonic anhydrase (CA, EC 4.2.1.1) from ostrich (scCA) blood is reported, as well as an inhibition study of this enzyme with a series of aromatic and heterocylic sulfonamides. The ostrich enzyme showed a high activity, comparable to that of the human isozyme II, with kcat, of 1.2 x 10(6) s(-1) and kcat/KM of 1.8 x 10(7) M(-1)s(-1), and an inhibition profile quite different from that of the human red blood cell cytosolic isozymes hCA I and II. scCA has generally a lower affinity for sulfonamide inhibitors as compared to hCA I and II. The only sulfonamide which behaved as a very potent inhibitor of this enzyme was ethoxzolamide (KI = 3.9 nM) whereas acetazolamide and sulfanilamide behaved as weaker inhibitors (inhibition constants in the range 303-570 nM). Several other aromatic and heterocyclic sulfonamides, mostly derivatives of sulfanilamide, homosulfanilamide, 4-aminoethylbenzenesulfonamide or 5-amino-1,3,4-thiadiazole-2-sulfonamide, showed good affinities for the ostrich enzyme, with KI values in the range 25-72 nM.     

Carbonic anhydrase inhibitors: inhibition of the human isozymes I, II, VA, and IX with a library of substituted difluoromethanesulfonamides

An inhibition study of the human cytosolic isozymes I, and II, the mitochondrial isoform VA, and the tumor-associated, transmembrane isozyme IX of carbonic anhydrase (CA, EC 4.2.1.1) with a library of aromatic/heteroaromatic/polycyclic difluoromethanesulfonamides is reported. Most of the inhibitors were derivatives of benzenedifluoromethanesulfonamide incorporating substituted-phenyl moieties, or were methylsulfonamide and difluoromethyl-sulfonamide derivatives of the sulfamates COUMATE and EMATE, respectively. Except for the methylsulfonamide-COUMATE derivative which behaved as a potent CA II inhibitor (K(I) of 32nM), these sulfonamides were moderate inhibitors of all isozymes, with inhibition constants in the range of 96-5200nM against hCA I, of 80-670nM against hCA II, and of 195-9280nM against hCA IX, respectively. Remarkably, some derivatives, such as 3-bromophenyl-difluoromethanesulfonamide, showed a trend to selectively inhibit the mitochondrial isoform CA VA, showing selectivity ratios for inhibiting CA VA over CA II of 3.53; over CA I of 6.84 and over CA IX of 9.34, respectively, although it is a moderate inhibitor (K(I) of 160nM). Some of these derivatives may be considered as leads for the design of isozyme selective CA inhibitors targeting the mitochondrial isozyme CA VA, with potential use as anti-obesity agents.     

Carbonic anhydrase inhibitors. Novel sulfanilamide/acetazolamide derivatives obtained by the tail approach and their interaction with the cytosolic isozymes I and II, and the tumor-associated isozyme IX

A series of sulfonamides has been obtained by reacting sulfanilamide or 5-amino-1,3,4-thiadiazole-2-sulfonamide with omega-chloroalkanoyl chlorides, followed by replacement of the omega-chlorine atom with secondary amines. Tails incorporating heterocyclic amines belonging to the morpholine, piperidine and piperazine ring systems have been attached to these sulfonamides, by means of an alkanoyl-carboxamido linker containing from two to five carbon atoms. The new derivatives prepared in this way were tested as inhibitors of three carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic isozymes CA I and II, and the catalytic domain of the transmembrane, tumor-associated isozyme CA IX. Several low nanomolar CA I and CA II inhibitors were detected both in the aromatic and heterocyclic sulfonamide series, whereas the best hCA IX inhibitors (inhibition constants in the range of 22-35 nM) all belonged to the acetazolamide-like derivatives.     

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.     

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.     

Carbonic anhydrase inhibitors: inhibition of human cytosolic isozymes I and II and tumor-associated isozymes IX and XII with S-substituted 4-chloro-2-mercapto-5-methyl-benzenesulfonamides

A series of S-substituted 4-chloro-2-mercapto-5-methyl-benzenesulfonamides has been investigated as inhibitors of four isoforms of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), that is, the cytosolic, ubiquitous isozymes CA I and II, as well as the transmembrane, tumor-associated isozymes CA IX and XII. The new derivatives were inefficient inhibitors of isoform I (K(I)s in the range of 2.7-18.7 microM) but generally had low nanomolar affinity for the inhibition of the other three isoforms (K(I)s in the range of 2.4-214 nM against hCA II; 1.4-47.5 nM against hCA IX, and 1.7-569 nM against hCA XII, respectively). Some selectivity for the inhibition of the tumor-associated versus the cyctosolic isoform II with some of these compounds has also been evidenced. As CA IX is an important marker of tumor hypoxia and its predictive, prognostic, and druggability potentials for designing antitumor therapies were recently validated, detection of selective, potent CA IX inhibitors may be relevant in the fight against cancers overexpressing CA isozymes.     

Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, IX, and XII with N-hydroxysulfamides--a new zinc-binding function in the design of inhibitors

A small library of N-hydroxysulfamides was synthesized by an original approach in order to investigate whether this zinc-binding function is efficient for the design of inhibitors targeting the cytosolic (hCA I and II) and transmembrane, tumor-associated (hCA IX and XII) isozymes of carbonic anhydrase (CA, EC 4.2.1.1). The parent derivative, N-hydroxysulfamide was a more potent inhibitor as compared to sulfamide or sulfamic acid against all isozymes, with inhibition constants in the range of 473 nM-4.05 microM. Its substituted n-decyl-, n-dodecyl-, benzyl-, and biphenylmethyl-derivatives were less inhibitory against hCA I (K(I)s in the range of 5.8-8.2 microM) but more inhibitory against hCA II (K(I)s in the range of 50.5-473 nM). The same situation was true for the tumor-associated isozymes, with K(I)s in the range of 353-790 nM against hCA IX and 372-874 nM against hCA XII. Some sulfamides/sulfamates possessing similar substitution patterns have also been investigated for the inhibition of these isozymes, being shown that in some particular cases sulfamides are more efficient inhibitors as compared to the corresponding sulfamates. Potent CA inhibitors targeting the cytosolic or tumor-associated CA isozymes can thus be designed from various classes of sulfonamides, sulfamides, or sulfamates and their derivatives, considering the extensive interactions in which the inhibitor and the enzyme active site are engaged, based on recent X-ray crystallographic data.     

Carbonic anhydrase inhibitors: inhibition of cytosolic/tumor-associated isoforms I, II, and IX with iminodiacetic carboxylates/hydroxamates also incorporating benzenesulfonamide moieties

The synthesis of a new class of sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs), also possessing carboxylate/hydroxamate moieties in their molecule, is reported. These compounds may act on dual antitumor targets, the tumor-associated CA isozymes (CA IX) and some matrix metalloproteinases (MMPs). The compounds were prepared by an original method starting from iminodiacetic acid, and assayed as inhibitors of three isozymes, hCA I, II (cytosolic), and IX (transmembrane). The new derivatives showed weak inhibitory activity against isozyme I (K(I)s in the range of 95-8300 nM), were excellent to moderate CA II inhibitors (K(I)s in the range of 8.4-65 nM), and very good and selective CA IX inhibitors (K(I)s in the range of 3.8-26 nM). The primary sulfonamide moiety is a better zinc-binding group in the design of CAIs as compared to the carboxylate/hydroxamate one, but the presence of hydroxamate functionalities in the molecule of CAIs leads to selectivity for the tumor-associated isozyme IX over the ubiquitous, cytosolic isoform II.