Novel Diaryl-Substituted Pyrazolo[3,4-d]pyrimidines as Tubulin/CDC5L Dual-Targeting Ligands: Discovery, Potent Antitumor Activity, and Good Metabolic Stability

Tubulin and cell division cycle 5-like (CDC5L) protein are both implicated in various biological processes, particularly in mitosis, and represent promising targets for developing antitumor agents. Herein, a series of novel diaryl-substituted pyrazolo[3,4-d]pyrimidines were designed, synthesized, and evaluated for their antiproliferative activities against multiple cancer cells, including drug-resistant ones. Among these, compounds 7c and 11i demonstrated potent in vitro antitumor activity with relatively low cytotoxicity toward normal cells. Mechanistic studies demonstrated that compounds 7c and 11i efficiently induced cell cycle arrest and apoptosis, elevated intracellular ROS levels, and exhibited antiangiogenic effects. Target identification and validation studies revealed that compound 11i could simultaneously target tubulin and CDC5L. Notably, compound 11i, which exhibited excellent solubility, metabolic stability, and acceptable pharmacokinetic profiles, could effectively suppress tumor growth and angiopoiesis in HCT116 xenograft models with acceptable safety profiles. This study provides the first demonstration of a tubulin/CDC5L dual-targeting agent with demonstrated in vivo therapeutic efficacy.