CDK2 has emerged as a pivotal target in cancer chemotherapy. To develop a novel CDK2 inhibitor scaffold, multiple rational, structure-based design strategies were applied to known potent CDK2 inhibitors (Fig. 1). Through retrosynthetic planning, chemical synthesis, and characterization, compounds 2–8 were generated. Initial in vitro screening using the NCI-60 cancer cell line panel, followed by accurate cytotoxicity (GI₅₀) measurements, shortlisted compounds 5, 8b, and 8d as promising candidates. These compounds exhibited GI₅₀ values as low as 0.6 µM and demonstrated favourable safety profiles, with selectivity indices reaching up to 7.98. The top two active compounds, 5 and 8b, were further evaluated against the most sensitive cell line, MDA-MB-468 (breast cancer), at their respective GI₅₀ concentrations. Flow cytometric cell cycle analysis revealed 82% and 78% G1 phase arrest for compounds 5 and 8b, respectively, suggesting effective CDK2/cyclin E targeting. Furthermore, Annexin V-FITC apoptosis assays showed robust pro-apoptotic effects, with total apoptosis induction elevated 34.5-fold and 32.4-fold over the negative control for compounds 5 and 8b, respectively. Subsequent CDK2/cyclin E1 enzymatic inhibition assays confirmed the potency of these compounds, with IC₅₀ values of 3.92 nM for 5 and 0.77 nM for 8b, compared to 1.94 nM for the reference inhibitor roscovitine (Table 4). Notably, the novel lead compound 8b exhibited approximately 2.5-fold greater potency than roscovitine. Molecular docking studies further supported the experimental findings and provided structural insights for future optimization of this promising inhibitor scaffold.