Synthesis and In Vitro/In silico Evaluation of Novel 2‑Aryl-6-carboxamide-Substituted Benzoxazole Derivatives with...

In this study, we synthesized novel 2-aryl-6-carboxamide-substituted benzoxazole derivatives and evaluated their anticancer potential against breast cancer cell lines, with a focus on mTOR inhibitory activity. The compounds were synthesized via a three-step method and characterized by NMR analysis. In silico studiesincluding molecular docking, molecular dynamics simulations, and ADMET predictionswere conducted to predict interactions with the target protein and support the observed biological activity. IC₅0 values were determined in MCF-7 and MDA-MB-231 cells, with MCF-7 cells exhibiting greater sensitivity to the compound. The most active compounds, COH-17 and COH-19, demonstrated cytotoxicity as indicated by LDH release assays. Apoptotic effects were investigated at both molecular and cellular levels: Western blot analysis assessed key apoptotic proteins (Bcl-2, Bax, caspase-3, and p53), while RT-qPCR quantified the expression of BRCA1, BRCA2, PTEN, TP53, BCL-2, BAX, Caspase-3, PI3K, AKT, and BRAD1 genes. Morphological changes associated with apoptosis were confirmed by DAPI staining and fluorescence microscopy, and early and late apoptosis were quantified using Annexin V-FITC/PI flow cytometry. Cell cycle analysis revealed phase-specific arrest, further supporting the antiproliferative activity of the compound. Overall, COH-17 and COH-19 demonstrated potent anticancer effects through mTOR inhibition, induction of apoptosis, and cell cycle arrest, highlighting their potential as targeted therapeutic agents for breast cancer.