Comparative Study of Conventional, Grinding, and Microwave‐Assisted Synthesis of Aminopyrazolones and Diaminopyrazoles: Exploring the Antitumor Activity, Dual CDK‐2/CA IX Inhibition Potential, and Apoptosis Induction

Embracing drug design approaches including ring variation, substituent variation, and bioisosteric modifications, aminopyrazolones 2-7 and diaminopyrazoles 9-14 were synthesized as dual potent CDK-2 and CA IX inhibitors for the first time. The eco-friendly preparation of the target analogs was performed by three procedures: conventional, grinding, and microwave-assisted methods. The synthesized congeners were estimated for their antitumor effect against breast MCF-7, hepatocellular HepG2, and colon HCT-116 cells where the aminopyrazolones 4 and 7 presented significant cytotoxicity against the examined carcinomas. Compound 4, bearing a dinitrophenyl ring at N-2 of aminopyrazolone scaffold, exhibited the greatest cytotoxicity and selectivity toward the tested cell lines. Hence, compounds 4 and 7 were selected for consecutive biological assays to determine their mode of action. The findings proposed that 4 and 7 may exert their antiproliferative activity via interaction with CDK-2 and CA IX receptors. Entity 4 exhibited promising dual inhibition of CDK-2 and CA IX with IC₅₀ at the micromolar level, which exceeded that of Roscovitine by three times and nearly half that of acetazolamide. Additionally, the superior derivative 4 stimulated MCF-7 cycle arrest at S phase through apoptotic induction which is supported by the upregulation of Bax and Caspase-8 and the downregulation of Bcl-2 and Cyclin E. The in silico studies showed acceptable predicted ADME and physicochemical properties together with the strong interaction between the superior compounds and both CDK-2 and CA IX binding sites inspiring such hybrids as potential lead dual inhibitors.