In silico studies, synthesis, and biological evaluation of novel imidazopyridine-based CYP4Z1 inhibitors targeting breast cancer stem cells.

Targeting cancer stem cells (CSCs) has emerged as a promising strategy for cancer therapy and prevention. The human cytochrome P450 enzyme CYP4Z1 has been identified as a potential therapeutic target due to its role in promoting breast cancer stemness. Aiming to develop potent and selective CYP4Z1 inhibitors, our strategy involved systematic structure-activity relationship (SAR) studies of the lead compound XD-2 (1-benzyl-1H-imidazo [4,5-c] pyridine), which led to its structural optimization. A series of derivatives were designed and synthesized to enhance drug-like properties, inhibitory activity, and selectivity. Among all the synthesized compounds, the preferred analog C8, which features an imidazo[4,5-c]pyridine core connected to a terminal butyl group via an amide-containing linker, exhibited the most potent CYP4Z1 inhibitory activity, with an IC50 value of 55.3 nM against CYP4Z1. Molecular docking studies revealed that the introduced side chain extended into the hydrophobic subpocket and the phenyl group established additional aromatic stacking interactions with Trp120. Subsequent in vitro and in vivo biological assessments confirmed that compound C8 potently diminished stemness marker expression, impeded spheroid formation, and attenuated both metastatic potential and tumor-initiating capacity in breast cancer cells. Collectively, these results underscore the promise of C8 as a leading candidate for advancing clinically viable CYP4Z1-targeted therapies in breast cancer.