Structural Diversity of Glycosylated Cytochalasins and Congeners from the Endophytic Fungus Trichoderma harzianum MLJ-4 with Anti-NSCLC Activity

The endophytic fungus Trichoderma harzianum MLJ-4, a proven agricultural biocontrol agent with untapped biosynthetic potential, yielded 11 new (1–11) and 22 known (12–33) macrocyclic cytochalasins (CYTs) via rice solid culture. Structural elucidation, including absolute configurations, employed HRESIMS, NMR, NMR calculations with DP4+ probability analysis, chemical methods, ECD calculations, and X-ray crystallography. Trichoharzchalasins A–C (1–3) represent the first reported glycosylated CYTs, and trichoharzchalasin D (4) is a rare example of 12-nor-cytochalasin. Cytotoxicity screening against non-small-cell lung cancer (NSCLC) H1975 cells identified five active compounds (IC₅₀ = 4.04–14.87 μM), with trichoharzchalasin H (8) showing superior potency (IC₅₀ = 4.04 μM) to gefitinib (IC₅₀ = 11.33 μM) and comparable efficacy to cisplatin (IC₅₀ = 4.34 μM). Mechanistically, 8 induced G2/M phase arrest and mitochondrial-mediated apoptosis. These findings underscore the biosynthetic potential of T. harzianum and highlight CYTs as promising NSCLC drug leads.