Carbonic anhydrase inhibitors. Biphenylsulfonamides with inhibitory action towards the transmembrane,tumor-associated isozymes IX possess cytotoxic activity against human colon,lung and breast cancer cell lines

Reaction of 4,4-biphenyl-disulfonyl chloride with aromatic/heterocyclic sulfonamides also incorporating a free amino group, such as 4-aminobenzenesulfonamide, 4-aminoethyl-benzenesulfonamide, 6-chloro-4-aminobenzene-1,3-disulfonamide or 5-amino-1,3,4-thiadiazole-2-sulfonamide afforded bis-sulfonamides which have been tested as inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4..2.1.1). The compounds were rather modest inhibitors of isozymes CA I and XII, but were more efficient as inhibitors of the cytosolic CA II and transmembrane, tumor-associated CA IX (inhibition constants in the range of 21–129 nM gainst hCA II, and 23–79 nM against hCA IX, respectively). The new bis-sulfonamides also showed inhibition of growth of several tumor cell lines (ex vivo), with GI₅₀ values in the range of 0.74–10.0 μg/mL against the human colon cancer cell line HCT116, the human lung cancer cell line H460 and the human breast cancer cell line MCF-7.     

Carbonic anhydrase inhibitors. N-cyanomethylsulfonamides--a new zinc binding group in the design of inhibitors targeting cytosolic and membrane-anchored isoforms

A series of N-cyanomethyl aromatic sulfonamides and bis-sulfonamides was prepared by reaction of arylsulfonyl halides with aminoacetonitrile. The obtained derivatives incorporated various aryl moieties, such as 4-halogeno/alkyl/aryl/nitro-substituted-phenyl, pentafluorophenyl or 2-naphthyl. Moderate inhibitory activity was detected for some compounds against the cytosolic human isoform II of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), hCA II, with inhibition constants of 90, 180 and 560n M for the 4-nitrophenyl-, 4-iodophenyl- and pentafluorophenyl-N-cyanomethylsulfonamides, respectively. Other derivatives acted as weak inhibitors of isoforms hCA I (KIs of 720 nM-45 microM), hCA II (KIs of 1000-9800 nM) and hCA IX (KIs of 900-10200 nM). Thus, the N-cyanomethylsulfonamide zinc binding group is less effective than the sulfonamide, sulfamate or sulfamide ones for the design of effective CA inhibitors.     

Carbonic anhydrase inhibitors. N-Cyanomethylsulfonamides—a new zinc binding group in the design of inhibitors targeting cytosolic and membrane-anchored isoforms

A series of N-cyanomethyl aromatic sulfonamides and bis-sulfonamides was prepared by reaction of arylsulfonyl halides with aminoacetonitrile. The obtained derivatives incorporated various aryl moieties, such as 4-halogeno/alkyl/aryl/nitro-substituted-phenyl, pentafluorophenyl or 2-naphthyl. Moderate inhibitory activity was detected for some compounds against the cytosolic human isoform II of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), hCA II, with inhibition constants of 90, 180 and 560 nM for the 4-nitrophenyl-, 4-iodophenyl- and pentafluorophenyl-N-cyanomethylsulfonamides, respectively. Other derivatives acted as weak inhibitors of isoforms hCA I (K_Is of 720 nM–45 μM), hCA II (K_Is of 1000–9800 nM) and hCA IX (K_Is of 900–10200 nM). Thus, the N-cyanomethylsulfonamide zinc binding group is less effective than the sulfonamide, sulfamate or sulfamide ones for the design of effective CA inhibitors.     

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.     

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. 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.     

Synthesis of 4-(thiazol-2-ylamino)-benzenesulfonamides with carbonic anhydrase I,II and IX inhibitory activity and cytotoxic effects against breast cancer cell lines

A series of 4-(thiazol-2-ylamino)-benzenesulfonamides was synthesized and screened for their carbonic anhydrase (CA, EC 4.2.1.1) inhibitory and cytotoxic activity on human breast cancer cell line MCF-7. Human (h) CA isoforms I, II and IX were included in the study. The new sulfonamides showed excellent inhibition of all three isoforms, with K_Is in the range of 0.84–702 nM against hCA I, of 0.41–288 nM against hCA II and of 5.6–29.2 against the tumor-associated hCA IX, a validated anti-tumor target, with a sulfonamide (SLC-0111) in Phase I clinical trials for the treatment of hypoxic, metastatic solid tumors overexpressing CA IX. The new compounds showed micromolar inhibition of growth efficacy against breast cancer MCF-7 cell lines.     

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. Inhibition of the cytosolic and tumor-associated carbonic anhydrase isozymes I, II, and IX with a series of 1,3,4-thiadiazole- and 1,2,4-triazole-thiols

A series of heterocyclic mercaptans incorporating 1,3,4-thiadiazole- and 1,2,4-triazole rings have been prepared and assayed for the inhibition of three physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic human isozymes I and II, and the transmembrane, tumor-associated hCA IX. Against hCA I the investigated thiols showed inhibition constants in the range of 97 nM to 548 microM, against hCA II in the range of 7.9-618 microM, and against hCA IX in the range of 9.3-772 microM. Thiadiazoles were generally more active than triazoles against all investigated isozymes. Generally, the best inhibitors were the simple derivative 5-amino-1,3,4-thiadiazole-2-thiol and its N-acetylated derivative, which were anyhow at least two orders of magnitude less effective inhibitors when compared to the corresponding sulfonamides, acetazolamide, and its deacetylated derivative. An exception was constituted by 5-(2-pyridylcarboxamido)-1,3,4-thiadiazole-2-thiol, which is the first hCA I-selective inhibitor ever reported, possessing an inhibition constant of 97 nM against isozyme I, and being a 105 times less effective hCA II inhibitor, and 3154 times less effective hCA IX inhibitor. Thus, the thiol moiety may lead to effective CA inhibitors targeting isozyme I, whereas it is a less effective zinc-binding function for the design of CA II and CA IX inhibitors over the sulfonamide group.     

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: 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.     

Design,synthesis, in vitro inhibition and toxicological evaluation of human carbonic anhydrases I,II and IX inhibitors in 5-nitroimidazole series

Abstract

With the aim to obtain novel compounds possessing both strong affinity against human carbonic anhydrases and low toxicity, we synthesised novel thiourea and sulphonamide derivatives 3, 4 and 10, and studied their in vitro inhibitory properties against human CA I, CA II and CA IX. We also evaluated the toxicity of these compounds using zebrafish larvae. Among the three compounds, derivative 4 showed efficient inhibition against hCA II (KI = 58.6?nM). Compound 10 showed moderate inhibition against hCA II (KI = 199.2?nM) and hCA IX (KI = 147.3?nM), whereas it inhibited hCA I less weakly at micromolar concentrations (KI = 6428.4?nM). All other inhibition constants for these compounds were in the submicromolar range. The toxicity evaluation studies showed no adverse effects on the zebrafish larvae. Our study suggests that these compounds are suitable for further preclinical characterisation as potential inhibitors of hCA I, II and IX.     

Synthesis,biological evaluation and in silico modelling studies of 1,3,5-trisubstituted pyrazoles carrying benzenesulfonamide as potential anticancer agents and selective cancer-associated hCA IX isoenzyme inhibitors

Inhibition of carbonic anhydrases (CAs, EC 4.2.1.1) has clinical importance for the treatment of several diseases. They participate in crucial regulatory mechanisms for balancing intracellular and extracellular pH of the cells. Among CA isoforms, selective inhibition of hCA IX has been linked to decreasing of cell growth for both primary tumors and metastases. The discovery of novel CA inhibitors as anticancer drug candidates is a current topic in medicinal chemistry. 1,3,5-Trisubstituted pyrazoles carrying benzenesulfonamide were evaluated against physiologically abundant cytosolic hCA I and hCA II and trans-membrane, tumor-associated hCA IX isoforms by a stopped-flow CO₂ hydrase method. Their in vitro cytotoxicities were screened against human oral squamous cell carcinoma (OSCC) cell lines (HSC-2) and human mesenchymal normal oral cells (HGF) via 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) test. Compounds 6, 8, 9, 11, and 12 showed low nanomolar hCA II inhibitory potency with Ki < 10 nM, whereas compounds 9 and 12 displayed Ki < 10 nM against hCA IX isoenzyme when compared with reference Acetazolamide (AZA). Compound 9, 4-(3-(hydrazinecarbonyl)-5-(4-nitrophenyl)-1H-pyrazol-1-yl)benzenesulfonamide, can be considered as the most selective hCA IX inhibitor over off-target cytosolic isoenzymes hCA I and hCA II with the lowest Ki value of 2.3 nM and selectivity ratios of 3217 (hCA I/hCA IX) and 3.9 (hCA II/hCA IX). Isoform selectivity profiles were also discussed using in silico modelling. Cytotoxicity results pointed out that compounds 5 (CC₅₀ = 37.7 μM) and 11 (CC₅₀ = 58.1 μM) can be considered as lead cytotoxic compounds since they were more cytotoxic than 5-Fluorouracil (5-FU) and Methotrexate (MTX).     

Discovery of thiazolin-4-one-based aromatic sulfamates as a new class of carbonic anhydrase isoforms I,II, IV,and IX inhibitors

Herein we report the synthesis of a new series of aromatic sulfamates investigated for the inhibition of four human (h) isoforms of zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), hCA I, II, IV, and IX. The reported derivatives, obtained by a sulfamoylation reaction of the corresponding phenolic precursors, bear arylthiazolin-4-one moieties as spacers between the benzenesulfamate fragment which binds the zinc ion from the active site, and the tail of the inhibitor. Thiazolin-4-ones are biologically privileged scaffolds, endowed with versatile biological activity, such as an anti-proliferative action. Phenolic precursors, also evaluated for CA inhibition, did not exhibit noteworthy efficacy in inhibiting the screened hCAs, whereas low nanomolar inhibitors were evidenced within the sulfamates subset mainly against hCA II (K_Is in the range of 28.7–84.3 nM) and IX (K_Is in the range of 17.6–73.3 nM). The variety of substituents appended at the outer aromatic portion almost generally reduced the inhibitory efficacy against isoforms II and IV, increasing instead that against the tumor-associated isoform IX.     

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.     

Inhibition of membrane-associated carbonic anhydrase isozymes IX, XII and XIV with a library of glycoconjugate benzenesulfonamides

A library of glycoconjugate benzenesulfonamides that contain diverse carbohydrate-triazole tails were investigated for their ability to inhibit the enzymatic activity of the three human transmembrane carbonic anhydrase (CA) isozymes hCA IX, hCA XII and hCA XIV. These isozymes have their CA domains located extracellularly, unlike the physiologically dominant hCA II, and are of immense current interest as druggable targets. Elevated expression of isozymes IX and XII is a marker for a broad spectrum of hypoxic tumors-this physiology may facilitate a novel approach to discriminate between healthy cells and cancerous cells. Many of these glycoconjugates were potent inhibitors (low nM), but importantly exhibited different isozyme selectivity profiles. The most potent hCA IX inhibitor was the glucuronic acid derivative 20 (K(i)=23nM). This compound was uniquely hCA IX selective cf. all other isozymes (16.4-, 16.8- and 4.6-fold selective against hCA II, XII, and XIV, respectively). At hCA XII there were many inhibitors with K(i)s<10nM that also demonstrated excellent selectivity (up to 344-fold) against other isozymes. Potent hCA XIV inhibitors were also identified, several with K(i)s approximately 10nM, however no hCA XIV-selective derivatives were evidenced from this library. The sugar tails of this study have shown promise as a valuable approach to both solubilize the aromatic sulfonamide CA recognition pharmacophore and to deliver potent inhibition and isozyme differentiation of the transmembrane CAs.     

Carbonic anhydrase inhibitors: inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with benzo[b]thiophene 1,1-dioxide sulfonamides

A series of selected benzo[b]thiophene-5- and 6-sulfonamide derivatives previously reported to show cytotoxic activity and some others newly synthesized has been tested for the interactions with several CA isozymes, some of which are known to be involved in tumorigenesis (hCA IX), whereas others are ubiquitously found in many normal tissues (the cytosolic isoforms hCA I and II). The unsubstituted sulfonamides inhibited hCA I with inhibition constants in the range of 63-138 nM, hCA II with inhibition constants in the range of 6.3-8.8 nM, and hCA IX with inhibition constants in the range of 2.8-15 nM, being thus more active than clinically used inhibitors such as acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide or indisulam (E 7070). Some of these derivatives also showed some selectivity for the inhibition of the tumor-associated (hCA IX) over the cytosolic isozyme hCA II. Although these derivatives may act on many targets other than the CAs (such as the NADH oxidase) or may induce apoptosis by accumulation of reactive oxygen species, it is quite important to try to decipher as many as possible of the potential mechanisms that lead to derivatives with potent antitumor activity in order to develop novel therapeutic strategies for the management of cancer.     

3-Aminobenzenesulfonamides incorporating acylthiourea moieties selectively inhibit the tumor-associated carbonic anhydrase isoform IX over the off-target isoforms I,II and IV

We describe the synthesis of a series of novel 1-aroyl/acyl-3-(3-aminosulfonylphenyl) thioureas (4a–k) acting as human carbonic anhydrase (hCA, EC 4.2.1.1) inhibitors. Reaction of alkyl/aryl isothiocyanates with 3-aminobenzenesulfonamide afforded a series of the title compounds incorporating a variety of short as well as highly lipophilic long tails. The newly synthesized sulfonamides were evaluated against 4 physiologically relevant CA isoforms (hCA I, II, IV, and IX). Several compounds showed interesting inhibitory activity. The tumor-associated hCA IX was the most sensitive isoform to inhibition with these compounds, with K_Is in the range of 21.5–44.0 nM and selectivity ratios over the major cytosolic isoform hCA II in the range of 3.35–37.3. The sulfonamides incorporating the phenylacetylthioureido and pentadecanoylthioureido moieties were the most hCA IX-selective inhibitors detected in this work, making them of interest for further investigations.     

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.