Scaffold-hopping strategy for pyrazolo[3,4-d]pyrimidines: In vitro and in silico studies of dual c-Met/STAT3 inhibition for enhanced antitumor activity

Twenty new pyrazolo[3,4-d]pyrimidine derivatives were designed and synthesized based on scaffold hopping and linker optimizations. The pyrazolo[3,4-d]pyrimidines were assessed for their in vitro antitumor activity against a panel of NCI Cancer cell lines. Compound 22b displayed the highest mean GI% of 61.2 % in the single-dose assay and was further assessed at five different concentrations, showing noteworthy antitumor activity and selectivity toward the leukemia subpanel with subpanel mean GI₅₀, TGI, and LC₅₀ of 6.97, 17.15, and 43.56 μM, respectively. Using MTT assay, compound 22b showed two-fold more potent inhibitory activity against the HCT-116 colon Cancer cell line with a high safety profile and selectivity index compared to doxorubicin. In kinase selectivity profiling assay, compound 22b showed excellent selectivity against c-Met and STAT3 with IC₅₀ values of 210 and 670 nM, respectively. Gene expression analysis of compound 22b demonstrated significant downregulation of AKT-1 and VEGF genes by 0.59 and 0.07 folds, respectively. Moreover, compound 22b induced cell cycle arrest at the G2/M phase in HCT-116 colon Cancer and Apoptosis via a preeminent Bax/Bcl-2 ratio and activation of p53, PUMA, Cyto-C, and caspases 3, 8, and 9 by 7.79, 4.74, 4.99, 4.89, 6.1, 1.9, and 5.5-folds, respectively. In network pharmacology analysis, c-Met and STAT3 were among the top-ranked genes. Molecular modeling studies demonstrated the relevance of the pyrazolo[3,4-d]pyrimidine scaffold, together with N-benzyl-2-(piperazin-1-yl)acetamide, for Anticancer activity. Furthermore, compound 22b proved not only to have promising biological activities, but also demonstrated acceptable predicted ADME and physicochemical properties. This establishes the position of compound 22b as a promising prototype for future investigation and development.

Apoptosis; Cell cycle arrest; Linker optimization; Molecular docking; Molecular dynamics simulation; Network pharmacology; Pyrazolo[3,4-d]pyrimidine; Scaffold hopping; c-Met/STAT3 inhibition.