Carbonic anhydrase inhibitors. inhibition of cytosolic isozymes I and II and transmembrane, cancer-associated isozyme IX with anions

Except for sulfonamides, metal complexing anions represent the second class of inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The first inhibition study of the transmembrane, tumor-associated isozyme CA IX with anions is reported here. Inhibition data of the cytosolic isozymes CA I and CA II with a large number of anionic species such as halides, pseudohalides, bicarbonate, nitrate, hydrosulfide, arsenate, etc., are also provided for comparison. Isozyme IX has an inhibition profile by anions different in some aspects from those of CA I and CA II, that may have interesting physiological consequences.     

Phenylethynylbenzenesulfonamide regioisomers strongly and selectively inhibit the transmembrane, tumor-associated carbonic anhydrase isoforms IX and XII over the cytosolic isoforms I and II

A series of compounds incorporating regioisomeric phenylethynylbenzenesulfonamide moieties has been investigated for the inhibition of four human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, hCA I, II, IX and XII. Inhibition between the low nanomolar to the milliomolar range has been observed against them, with several low nanomolar and tumor-CA selective inhibitors detected. The position of the sulfamoyl group with respect to the alkyne functionality, and the nature of the moieties substituting the second aromatic ring were the principal structural features influencing CA inhibition. The para-sulfamoyl-substituted derivatives were effective inhibitors of CA IX and XII, the meta-substituted regioisomers of CA I, IX and XII, whereas the ortho-substituted sulfonamides were weak inhibitors of CA I, II and IX, but inhibited significantly CA XII.     

Carbonic Anhydrase Inhibitors. Inhibition of Cytosolic Isozymes I and II and Transmembrane,Cancer-associated Isozyme IX with Lipophilic Sulfonamides

A series of new compounds was obtained by reaction of aromatic/heterocyclic sulfonamides incorporating amino groups with N,N-diphenylcarbamoyl chloride and diphenylacetyl chloride. These sulfonamides were assayed for the inhibition of three carbonic anhydrase (CA, EC 4.2.1.1) isozymes: the cytosolic CA I and CA II, and the transmembrane, cancer-associated isozyme CA IX. Good inhibitors against all these isoforms were detected, and the inhibition profile of the newly investigated isozyme IX was observed to be different from that of the cytosolic isozymes, I and II. This may lead to the development of novel anticancer therapies based on the selective inhibition of CA IX.     

Synthesis and biological evaluation of coumarin-1,3,4-oxadiazole hybrids as selective carbonic anhydrase IX and XII inhibitors

With an aim to develop novel heterocyclic hybrids as potent anticancer agents, we synthesized a series of coumarin-1,3,4-oxadiazole hybrids (7a-t) and evaluated for their inhibitory activity against the four physiologically relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms CA I, CA II, CA IX and CA XII. The CA inhibition results clearly indicated that the coumarin-1,3,4-oxadiazole derivatives (7a-t) exhibited selective inhibition of the tumor associated isoforms, CA IX and CA XII over CA I and II isoforms. Among all, compound 7b, exhibited significant inhibition in lower micromolar potency against hCA XII, with a K_i of 0.16 µM and compound 7n, exhibited significant inhibition in lower micromolar potency against hCA IX, with a K_i of 2.34 µM respectively. Therefore, compound 7b and 7n could be the potential leads for development of selective anticancer agents by exhibiting a novel mechanism of action through hCA IX and XII inhibition.     

Carbonic anhydrase inhibitors. Inhibition of transmembrane isozymes XII (cancer-associated) and XIV with anions

Metal complexing anions represent an important class of inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The first inhibition study of the transmembrane isozymes CA XII (tumor-associated) and XIV with anions is reported. These isozymes showed inhibition profiles with physiologic/non-physiologic anions quite distinct from any other cytosolic (CA I and II) or transmembrane isoforms (e.g., CA IX) investigated earlier. hCA XII has a good affinity for fluoride and bicarbonate but is not inhibited by heavier halides, perchlorate, nitrate, and nitrite. The best hCA XII inhibitors were cyanide (K(I) of 1 microM) and azide (K(I) of 80 microM). hCA XIV was on the other hand weakly inhibited by fluoride and not at all inhibited by perchlorate, but showed good affinity for most other anions investigated here. Chloride and bicarbonate showed K(I)s in the range of 0.75-0.77 mM for this isoform. The best hCA XIV anion inhibitors were sulfate, phenylarsonic, and phenylboronic acid (K(I) in the range of 10-92 microM).     

Carbonic anhydrase inhibitors. Inhibition of cytosolic isozymes I and II and transmembrane, cancer-associated isozyme IX with lipophilic sulfonamides

A series of new compounds was obtained by reaction of aromatic/heterocyclic sulfonamides incorporating amino groups with N,N-diphenylcarbamoyl chloride and diphenylacetyl chloride. These sulfonamides were assayed for the inhibition of three carbonic anhydrase (CA, EC 4.2.1.1) isozymes: the cytosolic CA I and CA II, and the transmembrane, cancer-associated isozyme CA IX. Good inhibitors against all these isoforms were detected, and the inhibition profile of the newly investigated isozyme IX was observed to be different from that of the cytosolic isozymes, I and II. This may lead to the development of novel anticancer therapies based on the selective inhibition of CA IX.     

Carbonic anhydrase inhibitors. Interaction of isozymes I, II, IV, V, and IX with carboxylates

A detailed inhibition study of five carbonic anhydrase (CA, EC 4.2.1.1) isozymes with carboxylates including aliphatic (formate, acetate), dicarboxylic (oxalate, malonate), hydroxy/keto acids (l-lactate, l-malate, pyruvate), tricarboxylic (citrate), or aromatic (benzoate, tetrafluorobenzoate) representatives, some of which are important intermediates in the Krebs cycle, is presented. The cytosolic isozyme hCA I was strongly activated by acetate, oxalate, pyruvate, l-lactate, and citrate (K(A) around 0.1 microM), whereas formate, malonate, malate, and benzoate were weaker activators (K(A) in the range 0.1-1mM). The cytosolic isozyme hCA II was weakly inhibited by all the investigated anions, with inhibition constants in the range of 0.03-24 mM. The membrane-associated isozyme hCA IV was the most sensitive to inhibition by carboxylates, showing a K(I) of 99 nM for citrate and oxalate, of 2.8 microM for malonate and of 14.5 microM for pyruvate among others. The mitochondrial isozyme hCA V was weakly inhibited by all these carboxylates (K(I)s in the range of 1.67-25.9 mM), with the best inhibitor being citrate (K(I) of 1.67 mM), whereas this is the most resistant CA isozyme to pyruvate inhibition (K(I) of 5.5mM), which may be another proof that CA V is the isozyme involved in the transfer of acetyl groups from the mitochondrion to the cytosol for the provision of substrate(s) for de novo lipogenesis. Furthermore, the relative resistance of CA V to inhibition by pyruvate may be an evolutionary adaptation of this mitochondrial isozyme to the presence of high concentrations of this anion within this organelle. The transmembrane, tumor-associated isozyme hCA IX was similar to isozyme II in its slight inhibition by all these anions (K(I) in the range of 1.12-7.42 mM), except acetate, lactate, and benzoate, which showed a K(I)>150 mM. The lactate insensitivity of CA IX also represents an interesting finding, since it is presumed that this isozyme evolved in such a way as to show a high catalytic activity in hypoxic tumors rich in lactate, and suggests a possible metabolon in which CA IX participates together with the monocarboxylate/H(+) co-transporter in dealing with the high amounts of lactate/H(+) present in tumors.     

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 tumor-associated isozyme IX with fluorine-containing sulfonamides. The first subnanomolar CA IX inhibitor discovered

Polyfluorinated CAIs show very good inhibitory properties against different carbonic anhydrase (CA) isozymes, such as CA I, II, and IV, but such compounds have not been tested for their interaction with the transmembrane, tumor-associated isozyme CA IX. Thus, a series of such compounds has been obtained by attaching 2,3,5,6-tetrafluorobenzoyl- and 2,3,5,6-tetrafluorophenylsulfonyl- moieties to aromatic/heterocyclic sulfonamides possessing derivatizable amino moieties. Some of these compounds showed excellent CA IX inhibitory properties and also selectivity ratios favorable to CA IX over CA II, the other physiologically relevant isozyme with high affinity for sulfonamide inhibitors. The first subnanomolar and rather selective CA IX inhibitor has been discovered, as the 2,3,5,6-tetrafluorobenzoyl derivative of metanilamide showed an inhibition constant of 0.8 nM against hCA IX, and a selectivity ratio of 26.25 against CA IX over CA II. Several other low nanomolar CA IX inhibitors were detected among the new derivatives reported here. The reported derivatives constitute valuable candidates for the development of novel antitumor therapies based on the selective inhibition of tumor-associated CA isozymes.     

Kinetic and docking studies of phenol-based inhibitors of carbonic anhydrase isoforms I, II, IX and XII evidence a new binding mode within the enzyme active site

Carbonic anhydrases (CAs, EC 4.2.1.1) are inhibited by sulfonamides, inorganic anions, phenols, coumarins (acting as prodrugs) and polyamines. A novel class of CA inhibitors (CAIs), interacting with the CA isozymes I, II (cytosolic) and IX, XII (transmembrane, tumor-associated) in a different manner, is reported here. Kinetic measurements allowed us to identify hydroxy-/methoxy-substituted benzoic acids as well as di-/tri-methoxy benzenes as submicromolar-low micromolar inhibitors of the four CA isozymes. Molecular docking studies of a set of such inhibitors within CA I and II allowed us to understand the inhibition mechanism. This new class of inhibitors binds differently compared to all other classes of inhibitors known to date: they were found between the phenol-binding site and the coumarin-binding site, filling thus the middle of the enzyme cavity. They exploit different interactions with amino acid residues and water molecules from the CA active site compared to other classes of inhibitors, offering the possibility to design CAIs with an interesting inhibition profile compared to the clinically used sulfonamides/sulfamates.     

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

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

Carbonic anhydrase inhibitors: inhibition of human and murine mitochondrial isozymes V with anions

In addition to sulfonamides, metal complexing anions represent the second class of inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The first inhibition study of the mitochondrial isozyme CA V (of murine and human origin) with anions is reported here. Inhibition data of the cytosolic isozymes CA I and CA II as well as the membrane-bound isozyme CA IV with a large number of anionic species such as halides, pseudohalides, bicarbonate, nitrate, hydrosulfide, arsenate, sulfamate, and sulfamidate and so on, are also provided for comparison. Isozyme V has an inhibition profile by anions completely different to those of CA I and IV, but similar to that of hCA II, which may have interesting physiological consequences. Similarly to hCA II, the mitochondrial isozymes show micro-nanomolar affinity for sulfonamides such as sulfanilamide and acetazolamide.     

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. 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, 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 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.     

Carbonic anhydrase inhibitors. Interaction of isozymes I, II, IV, V, and IX with phosphates, carbamoyl phosphate, and the phosphonate antiviral drug foscarnet

A detailed inhibition study of five carbonic anhydrase (CA, EC 4.2.1.1) isozymes with inorganic phosphates, carbamoyl phosphate, the antiviral phosphonate foscarnet as well as formate is reported. The cytosolic isozyme hCA I was weakly inhibited by neutral phosphate, strongly inhibited by carbamoyl phosphate (K(I) of 9.4 microM), and activated by hydrogen- and dihydrogenphosphate, foscarnet and formate (best activator foscarnet, K(A)=12 microM). The cytosolic isozyme hCA II was weakly inhibited by all the investigated anions, with carbamoyl phosphate showing a K(I) of 0.31 mM. The membrane-associated isozyme hCA IV was the most sensitive to inhibition by phosphates/phosphonates, showing a K(I) of 84 nM for PO(4)(3-), of 9.8 microM for HPO(4)(2-), and of 9.9 microM for carbamoyl phosphate. Foscarnet was the best inhibitor of this isozyme (K(I) of 0.82 mM) highly abundant in the kidneys, which may explain some of the renal side effects of the drug. The mitochondrial isozyme hCA V was weakly inhibited by all phosphates/phosphonates, except carbamoyl phosphate, which showed a K(I) of 8.5 microM. Thus, CA V cannot be the isozyme involved in the carbamoyl phosphate synthetase I biosynthetic reaction, as hypothesized earlier. Furthermore, the relative resistance of CA V to inhibition by inorganic phosphates suggests an evolutionary adaptation of this mitochondrial isozyme to the presence of high concentrations of such anions in these energy-converting organelles, where high amounts of ATP are produced by ATP synthetase, from ADP and inorganic phosphates. The transmembrane, tumor-associated isozyme hCA IX was on the other hand slightly inhibited by all these anions.     

Synthesis of novel isoindoline-1,3-dione-based oximes and benzenesulfonamide hydrazones as selective inhibitors of the tumor-associated carbonic anhydrase IX

The synthesis, characterization and biological evaluation of a library of isoindoline-1,3-dione-based oximes and benzenesulfonamide hydrazones is disclosed. The set of hydroxyiminoethyl aromatic derivatives 10–18 was designed to assess the potentiality as zinc-binder for a feebly studied functional group in the field of carbonic anhydrase (CA, EC 4.2.1.1) inhibition. Analogue phenylphthalimmides were linked to benzenesulfonamide scaffold by hydrazone spacers in the second subset of derivatives 20–28 to further investigate the application of the “tail approach” as tool to afford CA selective inhibition profiles. The compounds were assayed for the inhibition of physiologically relevant isoforms of human carbonic anhydrases (hCA, EC 4.2.1.1), the cytosolic CA I and II, and the membrane-bound CA IV and tumor-associated CA IX. The new zinc-binders, both of the oxime and sulfonamide types, showed a striking selective activity against the target hCA IX over ubiquitous hCA I and II, with diverse inhibitory ranges and ratio differing the two subsets. With CA IX being a strongly current antitumor/antimetastatic drug target, these series of compounds may be of interest for the development of new, both conventional and unconventional anticancer drugs targeting hypoxia-induced CA isoforms such as CA IX with minimum ubiquitous CAs-related side effects.     

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

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