Abstract
Cyclin-dependent kinase 4, a pivotal regulator of cell cycle progression, is widely recognised as a therapeutic target for controlling tumour proliferation. Inspired by the reported CDK4 inhibitor PF-07220060, a series of novel benzoxazine derivatives were designed, synthesised, and evaluated for their biological activity. From this series, compound 8j emerged as the most potent anti-proliferative agent. Subsequent in vitro studies showed 8j effectively inhibits HCT116 cell proliferation, clonogenic survival, and migration. Mechanistic investigations revealed that 8j induces G1 phase cell cycle arrest and promotes apoptosis, which was corroborated by the observed concentration-dependent downregulation of key cell cycle regulators, including CDK4 and cyclin D1. Molecular docking and dynamics simulations substantiated the binding mode of 8jwithin the ATP-binding pocket of CDK4, revealing stable interactions characterised by key hydrogen bonds and hydrophobic contacts. Collectively, these results identify compound 8j as a promising lead candidate worthy of further structural optimisation and preclinical development.