Design, synthesis, antifungal activity and preliminary mechanistic study of benzimidazole-containing cinnamamide derivatives against phytopathogenic fungi.

Phytopathogenic fungi continue to pose a serious threat to crop production, while the long-term use of conventional fungicides has raised concerns regarding resistance development and environmental impact. To identify new bioinspired antifungal leads, a series of benzimidazole-containing cinnamamide derivatives were designed and synthesized based on the cinnamic acid scaffold and evaluated against representative phytopathogenic fungi. The bioassay results showed that compound V35 exhibited the most potent and broad-spectrum antifungal activity, with EC50 values of 0.28, 0.69, 1.52, and 1.81 μg/mL against Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium avenaceum, and Fusarium graminearum, respectively. In vivo assays further demonstrated that compound V35 showed excellent protective efficacy against rice sheath blight (88.1% at 200 μg/mL) and markedly reduced the trypan-blue-stained necrotic areas in the rapeseed Sclerotinia stem rot assay under both protective and curative treatment conditions at 100 μg/mL. Preliminary mechanistic studies indicated that compound V35 treatment was associated with hyphal and ultrastructural damage, induced excessive reactive oxygen species accumulation, decreased mitochondrial membrane potential, increased cell membrane permeability, and disturbance of fungal energy metabolism, as reflected by reduced malate dehydrogenase and pyruvate kinase (PK) activities. Molecular docking further suggested that compound V35 may interact with the catalytic pocket of PK through hydrogen bonding, electrostatic contacts, and hydrophobic interactions, providing a plausible structural explanation for the observed PK activity inhibition. In addition, preliminary crop safety evaluation indicated that compound V35 caused no obvious inhibition of rice seed germination or bud length under the tested seed-treatment conditions. Overall, these findings identify compound V35 as a promising antifungal lead for further fungicide development.