Novel anticancer alkyl pyridinium derivative exhibits antiproliferative activity via G0/G1 cell cycle arrest and apoptosis activation in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) remains one of the most aggressive and treatment-resistant subtypes of breast cancer, urgently demanding novel therapeutic strategies. In this study, we report the synthesis, structure, physicochemical characterization, and anticancer evaluation of three novel 4-amino-1-alkylpyridinium derivatives bearing decyl (Compound 1), tetradecyl (Compound 2), and octadecyl (Compound 3) chains. Compound 1 and 3 were crystallographically characterized. Inspired by membrane-phospholipid, these cationic amphiphiles exhibited chain-length-dependent cytotoxicity in MDA-MB-231 TNBC cells, with Compound 3 displaying the highest potency (CC₅₀ = 5 μM), followed by Compound 2 (CC₅₀ = 9 μM) and Compound 1 (CC₅₀ = 35 μM). Mechanistic studies focused on Compound 2 due to its potent activity and favorable aqueous solubility profile. Flow cytometry assays confirmed that Compound 2 induces apoptosis as the primary mode of cell death, characterized by caspase-3/7 activation, elevated reactive oxygen species (ROS), and mitochondrial membrane depolarization. Additionally, Compound 2 treatment led to a dose-dependent G0/G1 cell cycle arrest and a marked increase in the sub-G0/G1 population, indicative of apoptotic DNA fragmentation. Dynamic light scattering analysis revealed that longer alkyl chains promote self-assembly into smaller, more uniform nanoaggregates, correlating with enhanced bioactivity. Collectively, these findings identify Compound 2 as a promising anticancer agent that activates the apoptotic pathway, causes DNA fragmentation, and demonstrates antiproliferative activity, as shown by G0/G1 phase arrest in TNBC cells.