• • Compounds 1–6 exhibited potent cytotoxicity against HepG2 cells with IC50 values ranging from 2.61 to 13.94 μmol/L, demonstrating significant anti-hepatocellular carcinoma potential. Compound 6 (japonicone X) was the most active (IC50 = 2.61 μmol/L), outperforming the positive control doxorubicin (IC50 = 0.82 μmol/L) by a factor of ~3.2, yet still within a clinically relevant range for lead optimization.
• • The new compound 1 (inujaponolide T) showed an IC50 of 3.82 ± 0.21 μmol/L, while compound 3 (inujaponolide U) had an IC50 of 13.94 ± 0.28 μmol/L. The 3.6-fold difference in potency between these two new dimers highlights the impact of structural variations, particularly the 1,10-seco modification in compound 3, on cytotoxic activity.
• • A preliminary structure-activity relationship analysis revealed a positive correlation between the number of acetoxy substituents and anti-tumor activity. This suggests that acetoxy groups contribute to the cytotoxic effects, providing a strategic handle for synthetic or semi-synthetic optimization to enhance potency.
• • Colony formation assays confirmed that compounds 1 and 3 inhibit HepG2 cell viability and reduce colony numbers in a dose-dependent manner (P < 0.001 vs. DMSO control), indicating suppression of clonogenic survival, a key metric for evaluating long-term anti-proliferative effects and potential to prevent tumor recurrence.