Design, synthesis, and biological evaluation of sulfonamide-functionalized pyridine carbothioamides as potent tubulin-targeting anticancer agents

Pyridine carbothioamides (PCAs) are recognized for their gastric mucosal protective effects and low <i>in vivo</i> toxicity, making them attractive scaffolds for anticancer drug development. In this study, a series of <i>N</i>-phenyl 4-substituted and 2,4-disubstituted PCAs (1-8) incorporating a sulfonamide pharmacophore were synthesized, fully characterized, and evaluated as tubulin polymerization inhibitors. The compounds were tested against four cancer cell lines (A549, MCF-7, PC-3, HepG2) with colchicine and doxorubicin as reference drugs. Among them, compounds 3 and 5 exhibited potent cytotoxicity, being 2-6-fold more active than colchicine and up to 2.5-fold stronger than doxorubicin in PC-3 cells. Importantly, both showed ∼4-fold lower toxicity toward normal HLMEC cells and displayed higher selectivity towards tested cancer cells than doxorubicin. Tubulin polymerization assays confirmed their activity, with IC₅₀ values of 1.1 μM (3) and 1.4 μM (5), outperforming colchicine (10.6 μM) and CA-4 (2.96 μM). Molecular docking revealed strong binding at the colchicine site, supported by favorable inhibition constants and free binding energies. <i>In silico</i> ADME predictions indicated that the most lipophilic compounds 3 and 5 demonstrated favorable drug-likeness, as expected from computational studies, along with excellent gastrointestinal absorption, favorable bioavailability, and low hemolytic activity. Collectively, these findings highlight compounds 3 and 5 as promising lead candidates for the development of orally active anticancer and antimitotic agents.