Design, synthesis, and anticancer evaluation of new pyrazolo[3,4-d]pyrimidine-based derivatives: CDK2 inhibition, apoptosis-inducing activity, molecular modelling studies.

Three series of pyrazolo[3,4-d]pyrimidine derivatives were synthesized to assess their potential as anticancer agents through the inhibition of the CDK2 enzyme pathway. The synthesized compounds underwent anticancer activity screening at the National Cancer Institute (NCI), USA, against 60 different human cancer cell lines. Compound 3d showed the most potent antiproliferative activity at sub-micromolar concentrations across most of the tested human cancer cell lines, with GI values ranging from 0.0263 to 0.513 µM. Compound 3d also showed the highest effectiveness and selectivity against the renal A498 cell line, with GI and TGI values of 0.0263 µM and 0.0854 µM, respectively. Additionally, compound 3e demonstrated notable activity against the renal A498 cell line, with GI and TGI values of 0.237 µM and 0.877 µM, respectively. Both compounds effectively inhibited CDK2/CyclinA2, with IC values of 0.332 ± 0.018 µM and 1.133 ± 0.062 µM, compared to roscovitine, which had an IC of 0.457 ± 0.025 µM. Compound 3d also significantly downregulated total CDK2 and reduced the phosphorylation at Thr160. Furthermore, compound 3d induced apoptosis in renal A498 cell line, marked by a 26.95-fold rise in the total apoptosis percent and an 8.84-fold increase in caspase-3 levels. Cell cycle analysis revealed that derivative 3d primarily induced cell cycle arrest at the S phase. Molecular docking studies confirmed the binding pose and affinity of compound 3d within the CDK2 active site, while molecular dynamics simulations showed that 3d formed a stable complex with the protein. The lower fluctuation range throughout the simulation indicated stronger binding interactions within the protein-ligand complex.