Design, synthesis, antineoplastic activity of new pyrazolo[3,4- d]pyrimidine derivatives as dual CDK2/GSK3 β kinase inhibitors; molecular docking study, and ADME prediction

In the current study, novel pyrazolo[3,4-d]pyrimidine derivatives 5a-h were designed and synthesized as targeted anti-cancer agents through dual CDK2/GSK-3 beta inhibition. The designed compounds demonstrated moderate to potent activity on the evaluated cancer cell lines (MCF-7 and T-47D). Compounds 5c and 5 g showed the most promising cytotoxic activity against the tested cell lines surpassing that of the used reference standard; staurosporine. On the other hand, both compounds showed good safety and tolerability on normal fibroblast cell line (MCR5). The final compounds 5c and 5 g showed a promising dual CDK2/GSK-3 beta inhibitory activity with IC50 of 0.244 and 0.128 mu M, respectively, against CDK2, and IC50 of 0.317 and 0.160 mu M, respectively, against GSK-3 beta. Investigating the effect of compounds 5c and 5 g on CDK2 and GSK-3 beta downstream cascades showed that they reduced the relative cellular content of phosphorylated RB1 and beta-catenin compared to that in the untreated MCF-7 cells. Moreover, compounds 5c and 5 g showed a reasonable selective inhibition against the target kinases CDK2/GSK-3 beta in comparison to a set of seven off-target kinases. Furthermore, the most potent compound 5 g caused cell cycle arrest at the S phase in MCF-7 cells preventing the cells' progression to G2/M phase inducing cell apoptosis. Molecular docking studies showed that the final pyrazolo[3,4-d]pyrimidine derivatives have analogous binding modes in the target kinases interacting with the hinge region key amino acids. Molecular dynamics simulations confirmed the predicted binding mode by molecular docking. Moreover, in silico predictions indicated their favorable physicochemical and pharmacokinetic properties in addition to their promising cytotoxic activity.