Fluorinated isoindolinone–amino acid hybrids possessing antiproliferative and antitumor activity in vitro and in vivo

) and mono-fluoro analogues. The lead compounds 9c, 9d, 9e, and 10c markedly inhibited cancer cell proliferation, clonogenic survival, and migration, while inducing apoptosis and a pronounced S-phase cell-cycle arrest. Network pharmacology identified CDK2 and GSK3B as key hub genes associated with cell-cycle regulation. Mechanistic studies demonstrated downregulation of CDK2 and PCNA, activation of p53-p21 signaling pathway, and increased γ-H2AX expression, indicating replicating-associated damage. Molecular docking predicted favorable interactions with the CDK2 catalytic pocket, which was further validated by an in vitro CDK2/CyclinA2 kinase inhibition and reduced CDK2 protein expression determined by western blot analysis, collectively implicating CDK2 as a key molecular target. Furthermore, compounds 9c, 9d, 9e and 10c exhibited significant tumor growth inhibition (TGI) values of 53.50%, 79.47%, 64.50% and 69.14% respectively, in the 4T1 murine breast cancer model without evident systemic toxicity. Collectively, these findings identify fluorinated isoindolinone-amino acid conjugates as promising anticancer leads targeting CDK2-associated cell-cycle signaling against triple-negative breast cancer.