The development of targeted anticancer agents capable of selectively eliminating breast cancer cells while sparing normal tissues remains a critical therapeutic challenge. MCB-04, a novel dihydropyrimidinone (DHPM)-tethered piperazine derivative synthesized via a TiO₂ nanoparticle-mediated catalytic strategy, demonstrates promising anticancer potential.

This study aimed to synthesize DHPM-tethered piperazine derivatives using a TiO₂-catalyzed approach and to comprehensively evaluate the cytotoxic efficacy of the lead compound MCB-04 against human breast cancer cells, with particular emphasis on elucidating its underlying molecular mechanisms of cell death.

A library of DHPM-tethered piperazine derivatives was synthesized and characterized, and MCB-04 was identified as the lead compound. Cytotoxicity was assessed using the MTT assay in MDA-MB-231, MCF-7, BT-474, and SK-BR-3 breast cancer cell lines, as well as normal MCF-10A cells. Apoptosis and autophagy were analyzed by live/dead assays, Annexin V/PI staining, immunocytochemistry, and Western blotting. Mitochondrial dysfunction and oxidative stress were evaluated by measuring mitochondrial membrane potential (Δψm) and intracellular ROS levels using flow cytometry. The involvement of paraptosis and c-Met–mediated signaling pathways was further investigated.

MCB-04 exhibited potent and selective cytotoxicity toward breast cancer cells, with the highest sensitivity observed in MDA-MB-231 cells (IC50 = 20 µM), while exerting minimal toxicity in normal MCF-10A cells. MCB-04 treatment significantly increased intracellular ROS levels and disrupted Δψm, indicating mitochondrial dysfunction. Mechanistically, MCB-04 induced apoptosis through activation of cleaved PARP and cleaved caspase-3, an increased Bax/Bcl-2 ratio, and upregulation of p53 and phosphorylated p53. Concurrently, autophagy was evidenced by LC3-II accumulation and increased Atg5 and Beclin-1 expression. Markers of ER stress-mediated paraptosis, including ATF4 and CHOP, were also elevated with concomintant decline in Alix. Furthermore, MCB-04 markedly suppressed phosphorylated c-Met, EMT-related VEGF, MMP-9 expression and downstream PI3K/Akt/mTOR/MEK signaling pathways.

MCB-04 exerts robust anti-breast cancer activity by triggering multiple programmed cell death pathways include apoptosis, autophagy, and paraptosis primarily through ROS-mediated mitochondrial dysfunction and inhibition of c-Met-dependent oncogenic signaling. These findings position MCB-04 as a promising multi-targeted therapeutic candidate, warranting further in vivo validation and preclinical development for breast cancer treatment.