Author's personal copy Inhibition of a-class cytosolic human carbonic anhydrases I, II, IX and XII, and b-class fungal enzymes by carboxylic acids and their derivatives: New isoform-I selective nanomolar inhibitors Mario Sechi a,⇑ , Alessio Innocenti b , Nicolino Pala a , Dominga Rogolino c , Mauro Carcelli c , Andrea Scozzafava b , Claudiu T. Supuran b,⇑ a Dipartimento di Chimica e Farmacia, Università di Sassari, Via Muroni 23/A, 07100 Sassari, Italy b Dipartimento di Chimica, Laboratorio di Chimica Bioinorganica, Università degli Studi di Firenze, Rm 188, Via della Lastruccia 3, Polo Scientifico, I 50019 Sesto Fiorentino (Firenze), Italy c Dipartimento di Chimica, Università di Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy article info Article history: Received 25 June 2012 Revised 23 July 2012 Accepted 25 July 2012 Available online 2 August 2012 Keywords: CA inhibitors CA I isoform Carboxylic acids/carboxylates Metal complexes 5-(1-Alyl-1H-indol-3-yl)-1H-pyrazole-3- carboxylic acids (Z)-Methyl 2-hydroxy-2-(2-oxo-1- substituted-indolin-3-ylidene)acetates abstract The members of a focused series of carboxylic acids and of their derivatives (esters, amides and metal complexes) have been investigated as inhibitors of the main cytosolic/transmembrane carbonic anhy- drase isoforms, CA I, II, IX and XII, belonging to the mammalian a-class of CAs. These enzymes are present in red blood cells in submillimolar concentration, and typical sulfonamide CA inhibitors do not selectively inhibit any of them. Among such isozymes, the isoform-I is an ‘orphan’ target that mediates hemorrhagic retinal and cerebral vascular permeability, involved in retinal and cerebral disease. In the present study, we identified the first selective CA I nanomolar inhibitors, that displayed activity against other isozymes in micromolar/millimolar concentration range. Selective CA II over CA I inhibition has also been observed with some diketo acids/metal complexes. Few diketo acid derivatives showed inhibition activities against the fungal b-class enzymes from Candida albicans and Cryptococcus neoformans in low micromolar con- centration range. Prediction of drug-like properties for the most interesting compounds suggests a favor- able bioavailability. Ó 2012 Elsevier Ltd. All rights reserved. Carbonic anhydrases (CAs, EC 4.2.1.1), as isoforms I and II, are highly abundant in red blood cells (and in gastrointestinal tract) of vertebrates, arriving at concentrations as high as 0.2 mM, and having an important role in CO 2 transport to the lungs, and in blood pH homeostasis. 1,2 These two cytosolic isoforms are the dominant ones among the 16 CAs found to date in various cell/tissues in vertebrates. 3–7 This family of metalloproteins uses Zn 2+ as metal cofactor and catalyzes the simple reaction between carbon dioxide and water with generation of bicarbonate and protons: CO 2 +H 2 O=H + +HCO 3  . 3–6 In humans, in addition to blood, CAs are present in a large variety of tissues including gastrointestinal and reproductive tracts, central nervous system, kidneys, lungs, skin and eyes. 3–6 The different isozymes are localized in various cellular compartments with CA I, CA II, CA III, CA VII and CA XIII being found in the cytosol, two isoforms (CA VA and VB) are present in mitochondria and one isoform (CA VI) is secreted in saliva and milk. Several other CA isoforms are either associated through phosphoinositol glycan anchors to the plasma membranes (CA IV and XV) or they are transmembrane proteins with extracellular ac- tive sites (CA IX, XII and XIV). 3–7 Many of the CA isozymes are important therapeutic targets, and are involved in several physio- logical/pathological processes, with the potential to be inhibited/ activated for treating a large range of disorders. 3–10 For example, CA II plays a role in bicarbonate production in the eye and it is there- fore a validated target for the therapy of glaucoma. 3,4 CA IX and CA XII are extracellularly localized mainly on hypoxic tumor cells, 8–10 where they are involved in tumorigenesis by regulating pH inside and outside the cancer cell, 5 thus interfering with phosphorylation processes or by playing a role in the cell–cell adhesion. 5,10 There- fore, they provide a target for cancer therapy because of their spec- ificity towards the hypoxic tumor cells. Concerning the isozyme CA I, that is considered an ‘orphan’ target, it has been shown that the presence of extracellular CA I inside either the blood–retinal or blood–brain barrier can induce vasogenic edema. 11 Increased reti- nal vascular permeability contributes to the pathogenesis of prolif- erative diabetic retinopathy and diabetic macular edema, leading causes of vision loss in working-age adults. Elevated levels of extra- cellular CA I in vitreous from individuals with diabetic retinopathy suggest that retinal hemorrhage and erythrocyte lysis contribute to 0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.07.094 ⇑ Corresponding authors. Tel.: +39 079 228 753; fax: +39 079 228 745 (M.S.); tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S.). E-mail addresses: mario.sechi@uniss.it (M. Sechi), claudiu.supuran@unifi.it (C.T. Supuran). Bioorganic & Medicinal Chemistry Letters 22 (2012) 5801–5806 Contents lists available at SciVerse ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl