Optimization of Pyrazolo[3,4‑b]pyridine Analogues: Synthesis, In Vitro Anticancer Evaluation and In Vivo Pharmacokinetics.

In this study, we designed and synthesized a series of novel pyrazolo-[3,4-b]-pyridine analogues, based on a lead compound, previously identified by our group, namely 4-(2-(cyclohexylamino)-ethoxy)-N,1-diphenyl-1H-pyrazolo-[3,4-b]-pyridin-6-amine. The new derivatives were prepared through conversion of phenylhydrazine to 5-amino-1-phenylpyrazole, which served as an intermediate for the preparation of the pyrazolopyridine scaffold. Introduction of selected substituents resulted in the series of target compounds. PC-3 and MCF-7 cell lines were used as in vitro models of prostate and breast cancer, to investigate the anticancer potential of these new derivatives. Cytotoxicity was evaluated in these models, while Human Vein Endothelial Cells (HUVEC) were included to assess effects on healthy cells. To further characterize the compounds, cellular uptake studies were performed in PC-3 cells to explore whether the level of intracellular accumulation of selected compound contributes to their antiproliferative activity. In addition, in vivo pharmacokinetic (PK) analysis was conducted in mice for the most promising compound 17. Following administration, concentration-time profiles were obtained by LC-MS/MS analysis, allowing determination of key PK parameters. Measurable systemic exposure was confirmed and provided insight into the compound's bioavailability, establishing a translational connection between structural modifications, in vitro activity, and in vivo pharmacokinetics. The results of this study suggest that compound 17 may serve as a candidate for further development as an anticancer agent.