Synthesis and multi-target antiproliferative evaluation of novel 1,2,4-triazole-3-thione analogues against breast cancer: <i>in silico</i> and <i>in vitro</i> mechanistic insights

Cancer remains a major global health challenge, with breast cancer representing the most commonly diagnosed malignancy in women. Heterocyclic scaffolds containing nitrogen and sulfur, such as 1,2,4-triazole-3-thione derivatives, have shown broad pharmacological utility, including notable anticancer potential. In the present study, a novel series of 4,5-disubstituted-1,2,4-triazol-3-thione derivatives was designed, synthesized, and fully characterized using NMR, IR, MS, and elemental analysis. The compounds were evaluated for their antiproliferative effects against MCF-7 (breast cancer) and HepG2 (liver cancer) cell lines. Most derivatives demonstrated notable cytotoxicity, with compound 6 exhibiting the most potent activity, achieving IC₅₀ values of 4.23 μM and 16.46 μM against MCF-7 and HepG2 cells, respectively, comparable to the reference drug vinblastine. Mechanistic investigations revealed that compound 6 acts <i>via</i> a multi-targeted pathway, significantly inhibiting α-glucosidase (IC₅₀ = 122.7 μM), tubulin-β polymerization (58.5% inhibition), and aromatase activity (31% reduction). Flow cytometry analysis confirmed that compound 6 induces pronounced S-phase cell cycle arrest and promotes both early and late apoptotic cell populations, along with a moderate increase in necrosis in MCF-7 cells. Additionally, compound 6 displayed dose-dependent antioxidant activity (DPPH IC₅₀ = 25.4 μM), comparable to trolox. Complementary <i>in silico</i> studies provided molecular-level insights into the interactions of compound 6 with its biological targets. Molecular docking showed strong binding affinities through key hydrogen bonding and hydrophobic interactions within the active sites of tubulin, α-glucosidase, and aromatase. Molecular dynamics simulations over 100 ns confirmed the stability of these interactions, especially with α-glucosidase, supported by consistent RMSD, compactness, and favorable per-residue energy contributions. Overall, these findings identify compound 6 as a promising multi-target lead for further development as an anticancer agent, combining cytotoxic, enzyme-inhibitory, and antioxidant properties.