CADD-based discovery of novel heterocyclic pyrimidine-based CDK4/6 inhibitors: Design, synthesis, and anti-breast cancer activity studies

Cyclin-dependent kinase 4/6 (CDK4/6) plays a pivotal role in cell cycle regulation, and its abnormal activation is closely associated with the initiation and progression of breast Cancer.1 However, the limited number of clinically available CDK4/6 inhibitors restricts treatment options. This study focuses on the design, synthesis, and antitumor activity investigation of novel CDK4/6 inhibitors.Employing computer-aided drug design (CADD) strategies and utilizing drug-like and bioisostere principles, we innovatively introduced pyridine-2-aminopyrimidine, thieno[3,2-d]pyrimidine, and pyrazolo[1,5-a]pyrimidine as core skeletal structures to design and screen 250 compound molecules. Based on computational results, including molecular docking and MM/GBSA binding free energy, 40 potential novel CDK4/6 inhibitors were selected for synthesis.Cellular assays validated that compound 3c exhibited the strongest inhibitory activity against CDK4/6 in breast Cancer cell lines, with IC₅₀ values of 0.11 ± 0.01 μM (MCF-7), 0.21 ± 0.01 μM (4T1), and 0.12 ± 0.01 μM (MDA-MB-231). And compound 3c demonstrated favorable CDK4/6 inhibition rates and showed a certain degree of selectivity among 23 kinases. In vitro mechanistic studies revealed that 3c consistently inhibited cell colony formation and migration. Furthermore, 3c effectively arrested the MCF-7 cell cycle at the G1 phase and induced Apoptosis. In the MCF-7 xenograft model, compound 3c achieved a tumor inhibition rate of 45.20%, highlighting its significant potential as a therapeutic CDK4/6 inhibitor.

Antitumor activity; Breast cancer; CADD strategy; CDK4/6 inhibitor; Heterocyclic pyrimidine structure.