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Novel amide derivatives of Probenecid were synthesized and discovered to act as potent and selective inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) transmembrane isoforms hCA IX and XII. The proposed chemical transformation of the carboxylic acid into an amide group led to a complete loss of hCA I and II inhibition (Kis >10,000nM) and enhanced the inhibitory activity against hCA IX and XII, with respect to the parent compound (incorporating a COOH function). These promising biological results have been corroborated by molecular modelling studies within the active sites of the four studied human carbonic anhydrases, which enabled us to rationalize both the isoform selectivity and high activity against the tumor-associated isoforms hCA IX/XII.

Novel amide derivatives of probenecid, a well-known uricosuric agent, were synthesized and evaluated as inhibitors of human carbonic anhydrases (hCAs, EC 4.2.1.1). The transmembrane isoforms (hCA IX and XII) were potently and selectively inhibited by some of them. The proposed chemical modification led to a complete loss of hCA II inhibition (K(i)s>10,000 nM) and enhanced the inhibitory activity against the tumour-associated hCA XII (compound 4 showed a K(i) value of 15.3 nM). The enzyme inhibitory data have also been validated by docking studies of the compounds within the active site of hCA XII.

Exploring the effect that substituents on the cycloaliphatic ring had on the inhibitory activity against human monoamine oxidase B of a series of 4-aryl-2-cycloalkylidenhydrazinylthiazoles led to the synthesis of a new series of 2-methylcyclopentyl and 3-methylcyclopentyl derivatives which were tested in vitro as mixtures of diastereoisomers. In fact, due to the presence of a chiral center on the cycloaliphatic ring and a trisubstituted CN bond, they exist as four diastereoisomers ((E)-(R), (E)-(S), (Z)-(R), (Z)-(S)). 4-(2,4- Difluorophenyl)-2-(2-(3-methylcyclopentylidene)hydrazinyl)thiazole was chosen as a model to investigate the influence of stereochemical requirements on the inhibitory activity against hMAO-B of these derivatives after a stereoconservative synthesis and semi-preparative HPLC diastereoseparation. (R)-(Z) isomer of this compound was endowed with a potent and selective hMAO-B inhibition higher than that of reference drugs as also corroborated by molecular modeling studies.

A series of N-alkylated saccharin derivatives were synthesized and tested for the inhibition of four different isoforms of human carbonic anhydrase (CA, EC 4. 2.1.1): the transmembrane tumor-associated CA IX and XII, and the cytosolic CA I and II. Most of the reported derivatives inhibited CA XII in the nanomolar/low micromolar range, hCA IX with KIs ranging between 11 and 390 nM, whereas they were inactive against both CA I (KIs >50 μM) and II (K(I)s ranging between 39.1 nM and 50 μM). Since CA I and II are off-targets of antitumor carbonic anhydrase inhibitors (CAIs), the obtained results represent an encouraging achievement for the development of new anticancer candidates without the common side effects of non-selective CAIs. Moreover, the lack of an explicit zinc binding function on these inhibitors opens the way towards the exploration of novel mechanisms of inhibition that could explain the high selectivity of these compounds for the inhibition of the transmembrane, tumor-associated isoforms over the cytosolic ones.