A new series of 1,3,4-thiadiazole-2-thione derivatives 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 thiones, showed inhibition constants in the range of 2.55–222 lM, against hCA II in the range of 2.0–433 lM, and against hCA IX in the range of 1.25–148 lM. Compound 5c, 4-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl)-1-(5-nitro-2-oxoindolin-3-ylidene)semicarbazide showed interesting inhibition of the tumor-associated hCA IX with KI value of 1.25 lM, being the first non-sulfonamide type inhibitor of such activity. This result is rather important taking into consideration the known antitumor activity of thiones. In addition, docking of the tested compounds into CA II active site was performed in order to predict the affinity and orientation of these compounds at the isozyme active site. The results showed similar orientation of the target compounds at CA II active site compared with reported sulfonamide type CAIs with the thione group acting as a zinc-binding moiety.