Design, synthesis and molecular docking of Pyrazolo[3,4-b]pyridine derivatives as potential CDK2 pathway inhibitors in colorectal cancer cells.

Within the framework of discovering novel colorectal cancer chemotherapeutic agents with improved efficacy and safety profiles, efforts were directed towards advancing this area of research. In this study, new pyrazolo[3,4-b]pyridine series were designed as Cyclin Dependent Kinase 2 (CDK2) inhibitors, synthesized, and biologically evaluated. All chemical structures were docked into the active site of CDK2 crystalline structure (1HCK). Binding energies and receptor interactions were elucidated. Antiproliferative activities against human colorectal cancer (CRC) cell lines HCT-116, HT-29 and related cytotoxicity on non-tumorigenic human colorectal cell line NCM-460D were studied by MTT assays. Compounds 6, 9c, 10, and 14 possessed notable activity against HCT-116 and HT-29 cells with IC values ranging from 11.11 to 62.61 μM. Compounds 6, 10, and 14 exhibited low cytotoxicity on NCM-460D, promoting them as promising chemotherapeutic agents. Structure-Activity Relationship of synthesized compounds was established, highlighting the influence of extended planarity, aromatic environments, and presence of electron donor-acceptor groups. Compounds 6 and 14 were selected for molecular investigations. They were not considered pro-apoptotic and showed non-significant influence on CDK2 protein expression. However, they displayed a dose-dependent inhibition of CDK2 kinase activity in in-vitro ADP-Glo™ assay with IC values of 23.47 and 82.04 nM, respectively, compared to 0.51 and 700 nM for Dinaciclib and Roscovitine, respectively. Compound 6 downregulated CDK2 protein targets involved in DNA replication process; Polα, MCM7, ORC2, and ORC4 in CRC cell lines. Subjected to cell cycle analysis, HCT-116 and HT-29 treated with compound 6 demonstrated pre-G1 phase arrest with no similar observation in S phase.