Despite the crucial role of CDK2 in tumorigenesis, few inhibitors reached clinical trials for managing lung cancer, the leading cause of cancer death. Herein, we report combinatorial stereoselective synthesis of rationally designed spiroindeno[1,2-
b
]quinoxaline-based CDK2 inhibitors for NSCLC therapy. The design relied on merging pharmacophoric motifs and biomimetic scaffold hopping into this privileged skeleton
via
cost‐effective one-pot multicomponent [3 + 2] cycloaddition reaction. Absolute configuration was assigned by single crystal x-ray diffraction analysis and reaction mechanism was studied by Molecular Electron Density Theory. Initial MTT screening of the series against A549 cells and normal lung fibroblasts Wi-38 elected
6b
as the study hit regarding potency (IC
50
= 54 nM) and safety (SI = 6.64).
In vitro
CDK2 inhibition assay revealed that
6b
(IC
50
= 177 nM) was comparable to roscovitine (IC
50
= 141 nM). Docking and molecular dynamic simulations suggested that
6b
was stabilised into CDK2 cavity by hydrophobic interactions with key aminoacids.