Design of isatin-based anticancer agents targeting CDK2, with the identification of a dual CDK1/2 inhibitory lead: a study bridging synthesis, biological profiling, and in silico exploration.

Abstract

Given their central role in cell cycle regulation, cyclin-dependent kinases (CDKs) were selected as the target of this study, with CDK1/2 prioritized over CDK4/6 due to their therapeutic advantages. Guided by structural insights into CDK2 inhibitor binding and leveraging the anticancer potential of isatin-based scaffolds, a new series of isatin-derived CDK1/2 inhibitors were synthesized. The synthesized compounds inhibited CDK2/cyclin A2 with IC50 from 0.62 to 22.74 μM, compared with roscovitine (IC50 = 0.63 μM). The N-propyl oxindole-thiosemicarbazide derivative 5a exhibited equipotent activity (IC50 = 0.62 μM). The most potent inhibitors (5a, 5b, 5j, 6a, 6b, and 6e), together with ester derivatives 5k and 5l, demonstrated significant antiproliferative activity against HCT-116 and MCF-7 cell lines. Evaluation of the inhibitory effect of 5a against relevant CDKs evidenced dual inhibition of CDK1 and CDK2 by 5a, displays >100-fold selectivity for CDK1/2 over CDK4 and 77-fold over CDK6. Cell cycle analysis revealed that 5a induced G1 and G2/M phase arrest, accompanied by an ∼11-fold increase in apoptotic and necrotic populations. Mechanistically, 5a upregulated p53 and p21 expression, enhanced pro-apoptotic BAX, suppressed anti-apoptotic Bcl-2, and induced mitochondrial apoptosis, as evidenced by a 16-fold increase in cytochrome c and a 4-fold elevation in caspase-9 activity. Molecular docking and dynamics simulations confirmed stable binding of 5a within the CDK2/cyclin A ATP-binding site, highlighting conserved isatin scaffold interactions with hinge region and hydrophobic pocket residues.

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