CDK4/6 inhibitors plus endocrine therapy is a standard therapy for HR+/HER2- breast cancer. Herein, using structure-based drug design strategy, a novel series of palbociclib derivatives were designed and synthesized as CDK4/6 inhibitors, among which compound 17m exhibited more potent CDK4/6 inhibitory activity and in vitro antiproliferative activity against the phosphorylated Rb-positive cell line MDA-MB-453 than the approved drug palbociclib. Moreover, compound 17m possessed remarkable CDK4/6 selectivity over other CDK family members including CDK1, CDK2, CDK3, CDK5, CDK7 and CDK9. The potent and selective CDK4/6 inhibitory activity endowed compound 17m with robust G1 cell cycle arrest ability in MDA-MB-453 cells. The intracellular inhibition of CDK4/6 by 17m was confirmed by western blot analysis of the levels of phosphorylated Rb in MDA-MB-453 cells. With respect to the metabolic stability, compound 17m possessed longer half-life (t_1/2) in mouse liver microsome than palbociclib.