Synthesis and discovery of guaianolide - eudesmanolide heterodimers as CDK2 inhibitors for the treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related death, creating an urgent need for novel therapeutic agents with unique mechanisms. Inspired by the antiHCC properties of natural sesquiterpenoid dimers and to overcome their limited structural diversity and availability, 26 new guaianolide-eudesmanolide heterodimers were synthesized via Diels-Alder reactions. AntiHCC assay suggested 19 analogues showed better inhibitory activity than sorafenib on HepG2, SK-Hep-1, and Huh7 cells. Surprisingly, chlorinated dimer 10 showed exceptional activity with IC values 3.9-6.1 fold superior to sorafenib, inhibited migration and invasion, and induced apoptosis. CDK2 was identified as the target of compound 10 as confirmed by CETSA, DARTS, and functional knockdown assays. Molecular docking and molecular dynamics simulations predicted that compound 10 acted as a novel non-ATP-competitive inhibitor of CDK2. Compound 10 inhibited CDK2/Cyclin A2 with an IC value of 236.7 nM, disrupted their interaction, promoted CDK2 degradation via the lysosomal pathway, and ultimately induced G0/G1 phase cell cycle arrest and cellular senescence. In vivo, dimer 10 at 30 and 60 mg/kg inhibited tumor weight up to 64 % and 69 % without detectable toxicity, and IHC analysis confirmed in vivo target engagement. This study identified compound 10 as a potential antiHCC agent targeting CDK2, and warrants further investigation.