Carbonic anhydrase inhibitors: Synthesis of membrane-impermeant low molecular weight sulfonamides possessing in vivo selectivity for the membrane-bound versus cytosolic isozymes

Aromatic/heterocyclic sulfonamides act as strong inhibitors of the zinc enz yme carbonic anhydrase (CA; EC 4.2.1.1), but the presently available compou nds do not generally discriminate between the 14 isozymes isolated in highe r vertebrates. Thus, clinically used drugs from this class of pharmacologic al agents show many undesired side effects due to unselective inhibition of all CA isozymes present in a tissue/organ. Here we propose a new approach for the selective in vivo inhibition of membrane-bound versus cytosolic CA isozymes with a new class of positively charged, membrane-impermeant sulfon amides. This approach is based on the attachment of trisubstituted-pyridini um-methylcarboxy moieties (obtained from 2,4,6-trisubstituted-pyrylium salt s and glycine) to the molecules of classical aromatic/heterocyclic sulfonam ides possessing free amino, imino, hydrazino, or hydroxyl groups in their m olecules. Efficient in vitro inhibition (in the nanomolar range) was observ ed with some of the new derivatives against three investigated CA isozymes: i.e., hCA I, hCA II (cytosolic forms), and bCA IV (membrane-bound isozyme) (h = human isozyme; b = bovine isozyme). Due to their salt-like character, the new type of inhibitors reported here, unlike the classical, clinically used compounds (such as acetazolamide, methazolamide, and ethoxzolamide), are unable to penetrate through biological membranes, as shown by ex vivo a nd in vivo perfusion experiments in rats. The level of bicarbonate excreted into the urine of the experimental animals perfused with solutions of the new and classical inhibitors undoubtedly proved that: (i) when using the ne w type of positively charged sulfonamides, only the membrane-bound enzyme ( CA TV) was inhibited, whereas the cytosolic isozymes (CA I and II) were not affected; (ii) in the experiments in which the classical compounds (acetaz olamide, benzolamide, etc.) were used, unselective inhibition of all CA iso zymes (I, II, and IV) has been evidenced.