Dereplication of Sesquiterpene-Proline Conjugates and Phenalenones from sp. CNUFC-EML-48 Using Molecular Networking and Cytotoxicity Profiling.

The use of feature-based molecular networking (FBMN) for metabolomic analysis of the fungal strain sp. CNUFC-EML-48 resulted in the isolation of 17 secondary metabolites. These included four new sesquiterpene-proline conjugates, aculenamides A-D (-), and six new phenalenones (-). The chemical structures of the isolated compounds were elucidated through extensive spectroscopic analyses, using 1D and 2D NMR, electronic circular dichroism (ECD), and MS/MS fragmentation analysis. Compounds - possessed a rare sesquiterpene-amino acid scaffold incorporating a proline moiety, and the natural occurrence of this structural motif was confirmed through the semisynthesis of compound . The cytotoxic effects of compounds - were evaluated in MC38 murine colorectal cancer cells. Among them, compounds - and significantly inhibited cancer cell proliferation. Notably, compound showed an antiproliferative effect by inducing S-phase cell cycle arrest, which was accompanied by upregulation of the tumor suppressor proteins p53 and p27. Furthermore, treatment with compound markedly increased intracellular reactive oxygen species (ROS) levels, suggesting that ROS-mediated oxidative stress may contribute to the induction of cell cycle arrest in MC38 cells. Collectively, these findings support the potential of compound as a cell cycle-modulating scaffold for the treatment of colorectal cancer.