Design, synthesis and biological evaluation of pyrazolo[3,4- <i>b</i> ]pyridine derivatives as dual CDK2/PIM1 inhibitors with potent anti-cancer activity and selectivity

The discovery of novel, selective inhibitors targeting CDK2 and PIM1 kinases, which regulate cell survival, proliferation, and treatment resistance, is crucial for advancing cancer therapy. This study reports the design, synthesis, and biological evaluation of three novel pyrazolo[3,4-<i>b</i>]pyridine derivatives (<b>6a-c</b>), confirmed <i>via</i> spectral analyses. These compounds were assessed for anti-cancer activity against breast, colon, liver, and cervical cancers using the MTT assay. Among the tested compounds, <b>6b</b> exhibited superior efficacy, with higher selectivity indices for HCT-116 (15.05) and HepG2 (9.88) compared to the reference drug staurosporine. Mechanistic studies revealed that <b>6b</b> induced apoptosis (63.04-fold increase) and arrested the cell cycle at the G0-G1 phase, highlighting its anti-proliferative effects. In an <i>in-vivo</i> solid Ehrlich carcinoma (SEC) mouse model, compound <b>6b</b> significantly reduced tumor weight and volume, exceeding the efficacy of doxorubicin. Additionally, <b>6b</b> potently inhibited CDK2 and PIM1 kinases (IC₅₀: 0.27 and 0.67 µM, respectively) and reduced tumor-promoting TNF-alpha expression, as confirmed by histopathological and immunohistochemical studies. Computational analyses, including molecular docking, molecular dynamics simulations, and DFT calculations, provided insights into the binding stability and interaction mechanisms of <b>6b</b> with CDK2 and PIM1, while <i>in-silico</i> pharmacokinetic and toxicity evaluations confirmed its favorable drug-like profile and safety. This study highlights compound <b>6b</b> as a promising dual CDK2/PIM1 inhibitor with potent anti-cancer activity and selectivity, paving the way for its further optimization and development as a lead molecule in cancer therapy.