Our previous research proved that benzenesulfonylguanidine derivatives display significant cytotoxic activity against human cancer cells. Here, we describe new 1-(2-alkylthio-4-chloro-5-methylbenzenesulfonyl)guanidines with cytotoxic activity against HCT-116 and MCF-7 cells. The planned derivatives were obtained by a two-step synthesis. The starting substrates were 1-(2-alkylthio-4-chloro-5-methyl)benzenesulfonyl)-3-aminoguanidines 1-4 which were transformed into 1-(2-alkylthio-4-chloro-5-methylbenzenesulfonyl)-3-[(2-chloroacetyl)amino]guanidines 5-8 by a reaction with chloroacetyl chloride. In the next step, the derivatives 5-8 were reacted with potassium thiocyanate yielding 1-(2-alkylthio-4-chloro-5-methylbenzenesulfonyl)-3-(2-imino-4-oxothiazolidine-3-yl)guanidines 9-12.

The synthesized derivatives 5−12 were evaluated in vitro by MTT assays for their activity against three human cancer cell lines: colon cancer HCT-116, breast cancer MCF-7, and cervical cancer HeLa. The activity against non-cancerous human epidermal keratinocyte line HaCaT was also examined. The data indicate that compounds 5-8 inhibit the growth of cancer cells stronger than derivatives 9-12. The selective cytotoxic effect against HCT-116 cells was found for benzenesulfonylguanidine 6 containing 2-(trifluoromethyl)benzylthio group at position 2 of benzenesulfonyl scaffold. The IC₅₀ value was 13 μM, while IC₅₀ for HaCaT cells was 48 μM. Good selectivity was also observed for compound 7, with 2-chloromethylbenzylthio substituent, against HCT-116 and MCF-7 cells (IC₅₀ = 12 and 19 μM, respectively for HCT-116 and MCF-7 cells, IC₅₀ = 47 μM for HaCaT cells). Among compounds 9-12, only compound 9 showed moderate but selective cytotoxicity against MCF-7 cells, with IC₅₀ = 18 μM compared with IC₅₀ = 54 μM for HaCaT cells.