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

The endophytic fungus <i>Trichoderma harzianum</i> MLJ-4, a proven agricultural biocontrol agent with untapped biosynthetic potential, yielded 11 new (<b>1</b>-<b>11</b>) and 22 known (<b>12</b>-<b>33</b>) 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 (<b>1</b>-<b>3</b>) represent the first reported glycosylated CYTs, and trichoharzchalasin D (<b>4</b>) is a rare example of 12-<i>nor</i>-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 (<b>8</b>) showing superior potency (IC₅₀ = 4.04 μM) to gefitinib (IC₅₀ = 11.33 μM) and comparable efficacy to cisplatin (IC₅₀ = 4.34 μM). Mechanistically, <b>8</b> induced G2/M phase arrest and mitochondrial-mediated apoptosis. These findings underscore the biosynthetic potential of <i>T. harzianum</i> and highlight CYTs as promising NSCLC drug leads.