Novel Piperine derivatives containing substituted Benzamide fragments: Design, synthesis, anti-breast cancer activity, and mechanistic study on the PI3K/AKT/mTOR pathway.

Breast cancer is the most prevalent malignant tumor among women worldwide, and recurrence, metastasis, and drug resistance remain major challenges that compromise treatment efficacy and patient prognosis. Piperine has been reported to possess antitumor potential, but its drug-like properties and potency remain suboptimal. In this study, a series of piperine derivatives bearing substituted benzamide moieties were designed and synthesized using a molecular hybridization strategy. Their in vitro effects were evaluated by MTT, EdU incorporation, colony formation, cell adhesion, Transwell invasion, and wound-healing assays. Among the tested compounds, HupCi-12 showed a favorable low-micromolar antiproliferative profile against MDA-MB-231 and SKBR3 breast cancer cells, with activity comparable to Chidamide in selected cell lines. Bioinformatics-based target prediction and pathway enrichment analyses, together with molecular docking and molecular dynamics simulations, suggested that PI3K/AKT/mTOR signaling may be involved in the cellular effects of HupCi-12. Consistent with this possibility, HupCi-12 reduced the phosphorylation levels of PI3K, AKT, and mTOR and modulated apoptosis-associated proteins by decreasing BCL2 and increasing BAX. Overall, these findings identify HupCi-12 as a representative bioactive compound in this series and support its further optimization and mechanistic validation in breast cancer models.