To enhance the efficacy of hecogenin (HCG) against breast cancer cells, we designed and synthesized two series of new HCG‑nitrogen mustard hybrids (4a-4f and 5a-5f) by linking benzoic acid mustard or chlorambucil to HCG via amino acid residues. The derivatives were screened to assess their anti-proliferative activity against three human breast cancer cell lines (MDA-MB-231, MDA-MB-468, and MCF-7), and one normal human breast MCF-10A cell line. Among the synthesized compounds, hybrid 5d exhibited the most potent anti-proliferative activity against the triple-negative breast cancer cell line MDA-MB-231, with an IC50 value of 2.2 μM. This represents a 27.2-fold increase in potency compared to the parent compound HCG (IC50 = 59.8 μM). Furthermore, hybrid 5d exhibited low toxicity toward MCF-10A cells (IC50 > 100 μM), indicating certain selectivity. Notably, the transwell migration assay revealed that hybrid 5d significantly inhibited the invasion of MDA-MB-231 cells. Preliminary mechanism studies indicated that hybrid 5d induced G2/M phase arrest and apoptosis via the mitochondria-related apoptotic pathway, as well as caused DNA damage. Collectively, these results suggest that hybrid 5d is a promising lead compound for anti-breast cancer research worthy of further investigation.
