New hybrid pyridine–1,2,4-triazole scaffolds: synthesis, in vitro evaluation of anticancer and antimicrobial activity, and in silico insights

Abstract 1,2,4-Triazole-3-thiones bearing 2-amino-5-chloropyridine or 2-amino-3,5-dibromopyridine moieties were S -alkylated with substituted bromoacetamides to obtain target 1,2,4-triazole-3-thiols. Anticancer effects of the synthesized compounds were evaluated in vitro using cell monolayer (MTT and ‘wound healing’ assays) and 3D spheroid models against human lung adenocarcinoma (A549), triple-negative breast cancer (MDA-MB-231), and melanoma (IGR39) cell lines. In the initial screening, compounds incorporating the 3,5-dibromopyridyl fragment were generally the most cytotoxic and compound 34 (5-chloropyridinyl derivative) was the most potent, with EC 50 values below 5 µM for all tested cancer cell lines. Although cytotoxic, they did not markedly inhibit cancer cell migration. In 3D spheroid cultures, 34 significantly inhibited the growth of MDA-MB-231 and IGR39 spheroids, while compounds 24 (phenyl) and 26 ( p -tolyl) decreased spheroid viability without altering their size. Compound 26 also demonstrated notable antimicrobial properties, exhibiting stronger antifungal activity against C. tenuis (7.8 µg/mL) than nystatin (15.6 µg/mL) and matching the antibacterial potency of vancomycin against M. luteum (7.8 µg/mL). Molecular docking suggested possible MEK/BRAF-related interactions within the mitogen-activated protein kinase pathway. Compound 34 showed the most favourable predicted binding profile for MEK (-11.134 kcal/mol) and BRAF (–10.336 kcal/mol). However, these in silico findings require to be confirmed experimentally.