Thymol-based derivatives as anticancer agents: design, synthesis, biological evaluation, and molecular docking studies

Seventeen thymol-based derivatives (TM1-7, TBr1-2, TCl1-4, and TF1-4) were designed and synthesized by esterification with halo- and non-halogenated cinnamoyl molecules employing a key-step coupling reaction. All derivatives were chemically characterized (¹H NMR, ¹³C NMR, HR-MS, and HPLC analysis) and evaluated for their potential anticancer properties. The anti-proliferative effectiveness was assessed using an MTT assay against four cancer cell lines (U87MG, SHSY-5Y, MDA-MB-231, and JIMT1) and human dermal fibroblasts (HDFa) compared to Triton X-100. Among the thymol-based derivatives, TM7 and TBr1 showed the highest cytotoxic activity against U87MG cells showing IC₅₀ equal to 0.014 μM and 0.054 μM, respectively. Compounds TBr1 and TBr2 exhibited selective cytotoxic activity toward neuroblastoma (SH-SY5Y) cancer cells displaying IC₅₀ of 5.123 μM and 1.103 μM, respectively. On the other hand, TM7, TBr1, and TBr2 showed IC₅₀ higher than 200 μM toward MDA-MB-231 and JIMT1 cells. Highest anti-proliferative effect was detected with TBr2 in both breast cancer cell lines and TBr1 and TBr2 induced apoptosis more rapidly than TM7, especially in MDA-MB-231 cells. Cells used in this study depend on FAK and PI3Kα signaling for survival and proliferation, and inhibition of these pathways reduces viability and induces apoptosis. To support their potential as molecular targets, in silico studies including docking, 100 ns MD simulations, and ADMET analysis were performed. TBr1 and TBr2 showed stronger predicted binding and more stable in silico interactions with FAK and PI3Kα, suggesting that these kinases may represent candidate molecular partners involved in their anticancer activity, whereas TM7 displayed a more favorable predicted pharmacokinetic profile. These computational findings are hypothesis-generating and have not yet been confirmed by biochemical target-engagement assays.