Diterpenes from Hypoestes forskaolii leaves and their cytotoxic activity

Hypoestes forskaolii (Acanthaceae) is a diterpene-rich species used in traditional medicine. However, while its roots have been chemically investigated, the leaf metabolome remains largely unexplored. Herein, an (UHPLC-HRMS/MS) analysis and molecular-networking-guided workflow, supported by in-silico annotation/dereplication, was applied to the non-polar leaf extract to prioritize diterpene-rich features and accelerate targeted isolation. This strategy led to the characterization of nine previously unreported diterpenes (1-9) together with seven known analogues. Structures were elucidated by 1D/2D NMR spectroscopy and HRESIMS experiments, while the relative configurations were established by density functional theory (DFT)-assisted NMR calculations and ECD analysis. Among the previously undescribed metabolites, the fusicoccane diterpenes 1-7 (wadigazalins A-G) were evaluated for cytotoxicity against the HeLa, A549, and SH-SY5Y cell lines. Wadigazalin D displayed the most interesting activity toward HeLa cells (IC₅₀ value of 3.03 ± 0.25 μM), and triggered G2/M arrest and apoptosis with cyclin B1 modulation. Overall, these findings expand the structural diversity of leaf-derived diterpenes in H. forskaolii and highlight molecular networking as an efficient driver for the discovery and prioritization of bioactive diterpenoids.