Polycyclic-Fused Cytochalasins with Anti-Liver Fibrosis Activity Produced by the Endophytic Fungus Trichoderma harzianum

A molecular networking-guided investigation of extracts from the growth medium of the endophytic fungus Trichoderma harzianum MLJ-4 resulted in the isolation of eight new polycyclic-fused cytochalasins (CYTs), triharziachalasins A–H (1–8), along with six known analogues (9–14). Their structures, including absolute configurations, were elucidated by comprehensive spectroscopic analysis, NMR calculations with DP4+ analysis, theoretical ECD simulations, and single-crystal X-ray diffraction. Compounds 1–3 feature a rare 5/6/5/8-fused tetracyclic system, while 4–8 possess a novel 5/6/6/7/5-fused pentacyclic scaffold. Compound 1 is the first cytochalasin analogue containing a cis-fused 5/8 ring within the 5/6/5/8 framework. All CYTs were evaluated for antiliver fibrosis activity in TGF-β1-stimulated LX-2 cells using high-content screening assays. The most promising compound, triharziachalasin H (8), dose-dependently suppressed the expression of key fibrotic markers, including fibronectin (FN), collagen I, and α-smooth muscle actin (α-SMA). Mechanism studies revealed that its antiliver fibrosis effect is mediated via inhibition of the NF-κB signaling pathway. This work expands the structural diversity of bioactive CYTs and reveals the antiliver fibrosis activity of this novel polycyclic-fused architecture, highlighting its potential as a lead compound for antiliver fibrosis drug development.