Prenylated 4-Hydroxycoumarin derivatives as inducers of CDK4/6-mediated cell cycle arrest: From design and synthesis to in vitro and in vivo evaluation of their anti-breast Cancer activity.

Breast cancer is the most frequently diagnosed malignancy in women, and safer, more effective therapies are urgently needed. Inspired by the prenylated coumarin scaffolds of Ferulin C and Miliusol, we rationally designed and synthesized a series of novel 4-hydroxycoumarin derivatives (A, B, and PB series) to develop potent anti-breast cancer agents. Among them, lead compound PB1-3 (7-methoxy, geranyl-substituted) exhibited the most potent antiproliferative activity against MCF-7 (IC₅₀ = 3.13 μM) and 4 T1 (IC₅₀ = 4.28 μM) cells, with high selectivity over normal cells. Structure-activity relationship (SAR) analysis underscored that the combination of a methoxy group and an extended geranyl side chain is crucial for activity. Integrated computational studies (molecular docking, 100 ns MD simulations, and MM-PBSA) confirmed that PB1-3 establishes stable hydrogen-bond and hydrophobic interactions with CDK4/6. Mechanistically, PB1-3 functions as a dual-acting CDK4/6 pathway modulator: it not only directly binds to CDK4/6 but, notably, downregulates their total protein expression, thereby reducing Rb phosphorylation and inducing G0/G1 phase arrest. Concurrently, PB1-3 triggers a potent ROS burst, collapses mitochondrial membrane potential (MMP), upregulates the Bax/Bcl-2 ratio, and activates cleaved caspase-3, driving the intrinsic apoptotic cascade. In a 4 T1 orthotopic syngeneic model, PB1-3 (20 mg/kg, i.p.) significantly suppressed tumor growth comparable to cisplatin, while exhibiting excellent biosafety (LD₅₀ > 2000 mg/kg, no hepatotoxicity/nephrotoxicity) and acceptable oral pharmacokinetics (T₁/₂ = 3.0 h). Collectively, PB1-3 represents a promising prenylated coumarin lead that orchestrates both CDK4/6-Rb cell cycle checkpoint blockade and ROS-dependent mitochondrial apoptosis, offering a valuable scaffold for developing targeted breast cancer therapies, especially for TNBC.