Carbonic anhydrase inhibitors. Diazenylbenzenesulfonamides are potent and selective inhibitors of the tumor-associated isozymes IX and XII over the cytosolic isoforms I and II Fabrizio Carta, Alfonso Maresca, Andrea Scozzafava, Daniela Vullo, Claudiu T. Supuran * Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy article info Article history: Received 9 July 2009 Revised 29 August 2009 Accepted 3 September 2009 Available online 6 September 2009 Keywords: Carbonic anhydrase Tumor-associated isoforms CA IX CA XII Isoform-selective inhibitor Diazenylbenzenesulfonamides abstract A series of diazenylbenzenesulfonamides, azo-dye derivatives of sulfanilamide or metanilamide incorpo- rating phenol and amine moieties, were tested for inhibition of the tumor-associated isozymes of car- bonic 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 I s 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. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Carbonic anhydrases (CAs, EC 4.2.1.1) constitute an ubiquitous family of metallo-enzymes found in prokaryotes and eukaryotes, which catalyze the reversible hydration of carbon dioxide to the bicarbonate ion and a proton (CO 2 +H 2 O¡HCO 3 À +H + ). 1–5 These proteins are encoded by five evolutionarily unrelated gene fami- lies: the a-CAs (in vertebrates, bacteria, algae and cytoplasm of green plants), the b-CAs (predominantly in bacteria algae and chlo- roplasts), the c-CAs (in archaea and some bacteria) 1–3 and the d- and n-CAs (in marine diatoms). 2,3 There are no significant sequence homologies between representatives of the different CA families, but all members contain one Zn(II) or Cd(II) ion in their active site, which are critical for the catalytic activity of the enzymes. 1–3 a-CA isozymes are widely distributed in many tissues and or- gans of vertebrates (including humans), as 16 different isoforms (only 15 are found in primates, which lack the CA XV isoform). 1 They play crucial roles in various physiological processes of such organisms, such as CO 2 /HCO 3 À transport between metabolizing tis- sues and lungs, pH and CO 2 homeostasis, electrolyte secretion, bio- synthetic reactions (gluconeogenesis, lipogenesis and ureagenesis), bone resorption and tumorigenicity. 1,6–10 As a consequence, many of these CA isoforms are drug targets of interest for the design of various pharmacological agents, such as diuretics, antiglaucoma, antiobesity, antiepileptic or antitumor drugs/diagnostic agents. 1 Among all these applications, the potential use of CA inhibitors (CAIs) for the management of solid hypoxic tumors which overex- press two isoforms, CA IX and XII, became a hot topic, with many investigations aiming to develop potent and isoform-selective CAIs targeting these two isozymes. 1,6–11 Indeed, hypoxia, through the hypoxia inducible factor (HIF) cascade, leads to a strong overex- pression of CA IX/XII in many tumors. 8 The overall consequence of these phenomena is a pH imbalance, with most hypoxic tumors having acidic pH values around 6–6.5, in contrast to normal tissue which have characteristic pH values around 7.4. 8–10 Constitutive expression of human CA IX (hCA IX) was recently shown to de- crease extracellular pH (pH e ) also in Madin-Darby canine kidney (MDCK) epithelial cells (which normally do not express CA IX) by this group. 8 CA IX potent sulfonamide inhibitors were then shown to reduce the medium acidity by inhibiting the catalytic activity of the enzyme, and thus the generation of H + ions, binding specifically only to hypoxic cells expressing CA IX. 8 Deletion of the CA active site was demonstrated to reduce the medium acidity, but sulfon- amide inhibitors did not bind to the active site of the mutant pro- tein. Therefore, tumor cells decrease their pH e both by production of lactic acid (due to the high glycolysis rates), and by CO 2 hydra- tion catalyzed by the tumor-associated CA IX/XII, possessing extra- cellular catalytic domains. 8–10 A low pH e has been associated with tumorigenic transformation, chromosomal rearrangements, 0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2009.09.003 * Corresponding author. Tel.: +39 055 457 3005; fax: +39 055 457 3385. E-mail address: claudiu.supuran@unifi.it (C.T. Supuran). Bioorganic & Medicinal Chemistry 17 (2009) 7093–7099 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc