Structure-based design of potent pyrazolo[1,5-a]pyrimidine CDK4/6 inhibitors: biological evaluation and computational validation

Abstract

Dysregulation of cyclin-dependent kinases (CDKs) drives uncontrolled cell cycle progression in several malignancies, making CDK4 and CDK6 appealing therapeutic targets. This paper details the rational design, synthesis, and thorough assessment of fifteen new pyrazolo[1,5-a]pyrimidine derivatives (19a–o) as cyclin-dependent kinase inhibitors. Structure–activity relationship research identified compound 19i as the primary candidate, exhibiting enhanced antiproliferative efficacy against HCT-116 colorectal cancer cells with an IC₅₀ of 1.02 μM, slightly higher than that of doxorubicin (IC₅₀, 1.08 μM). Mechanistic investigations demonstrated that 19i generates significant G0/G1 phase cell cycle arrest and substantial apoptotic cell death, with total apoptosis reaching 47.76% of the treated cells. ELISA analysis verified the activation of p53-dependent intrinsic apoptosis, evidenced by a 6.90-fold increase in p53, a 3.03-fold increase in Bax, a 0.39-fold decrease in Bcl-2, and a 9.56-fold increase in caspase-3 activity. Biochemical kinase tests revealed significant suppression of CDK4 (IC₅₀ 0.087 μM) and CDK6 (IC₅₀ 0.114 μM). Molecular docking revealed essential binding interactions, including hydrogen bonds with Lys35 and Val101, aromatic π–π stacking, and a new halogen bond with Glu94. Molecular dynamics simulations validated prolonged protein conformational stability and efficient target engagement. These findings collectively designate the pyrazolo[1,5-a]pyrimidine scaffold as a viable framework for CDK-targeted anticancer therapies.