Phlecarinatones H-N: Abietane-type diterpenoids from Phlegmariurus carinatus with proliferative inhibitory effect on U251 glioblastoma cells.

Thirteen abietane-type diterpenoids, including seven previously undescribed compounds and six known analogs, were isolated from the root and aerial parts of Phlegmariurus carinatus. Their structures were elucidated by comprehensive spectroscopic data analysis (UV, IR, NMR, and HRESIMS) and quantum chemical calculations (calculated ECD or 13C NMR). Notably, these compounds exhibited high structural diversity. Compounds 1-8 possessed six distinct fused ring systems. Phlecarinatones I (2) and J (3) were identified as rare 6,7-seco-abietane diterpenoids featuring a five-membered lactone ring B. Compounds (1-12) were evaluated for their anti-proliferative activities in the U251 cell line. In particular, phlecarinatone I (2) exhibited potential inhibitory effects on U251 cell proliferation, with an IC50 value of 13.88 ± 1.82 μM. To elucidate the underlying molecular mechanism, p53 signal pathway was enriched from our RNA-seq data. Further investigations using western blot and immunofluorescence assays confirmed that p53 expression was up-regulated in a concentration-independent manner. Additionally, molecular docking studies suggested 2 likely binds to the MDM2-p53 binding region. Finally, an shRNA-mediated MDM2 knockdown experiment provided definitive evidence that 2 acts as a potent inhibitor of glioma proliferation, operating via the modulation of the MDM2-p53 pathway. These findings suggest that 2 might be a valuable of lead compound with anti-proliferative activity.