Sesquiterpenoid Dimers from the Roots of Two Species of the Genus Inula with Cytotoxic Activity on Hepatocellular Carcinoma Cells.

Guided by the feature-based molecular networking approach, twenty-five previously undescribed sesquiterpenoid dimers (SDs) designated Inulaenoids A-Y (1-4, 8-25, and 27-29) along with four known analogues were isolated from the roots of Inula helenium L. and Inula racemosa Hook. f. Their structures and absolute configuration were unambiguously elucidated by spectroscopic data, X-ray diffraction, and theoretical calculations. Structurally, compounds 1-19 are rare SDs featuring two eudesmane units directly constructed through C-C bonds, including C-13-C-13', C-13-C-11', C-11-C-13', and through a previously undescribed C-13-C-8' connection. Fifteen compounds (3, 4, 10, 15-18, 20-23 and 25-28) showed potent cytotoxic activity to Huh-7 and HepG2 cells with the IC50 values ranging from 1.9 ± 0.1 to 20.2 ± 0.3 μM. Among them, four compounds (3, 4, 10 and 17) displayed binding to proto-oncogenic c-Fos. Notably, compound 17 exhibited the highest binding affinity to recombinant human c-Fos, thereby activating TP53 transcription and subsequently driving BAX/Bcl2-mediated apoptosis in liver cancer cells. Taken together, this study expands the structural diversity of sesquiterpenoid dimers from genus Inula as novel c-Fos-targeting lead compounds, providing new insights for the development of natural product-based anti-hepatocellular carcinoma agents.