Evaluation of antifungal activity, mechanism of action, and zebrafish toxicity of N-(3-fluorobenzyl)-N,3-dimethylbut-2-enamide

To address the urgent need for novel green fungicides in eco-friendly plant disease control, this study involved the evaluation of N-(3-fluorobenzyl)-N,3-dimethylbut-2-enamide. The work aims to provide a solid theoretical and data-driven foundation for elucidating antifungal mechanisms, and assessing the ecotoxicological risks associated with this substance. This study encompassed the bio-evaluation of the compound N-(3-fluorobenzyl)N,3-dimethylbut-2-enamide, specifically its antifungal activity (against 13 fungi via poisoned food and mycelial growth rate methods, with MIC for, Monilinia fructigena), multi-target mechanism (via ultrastructure, carbon metabolism, TCA cycle enzymes, and energy substances), and ecotoxicity in zebrafish embryos (LC50, morphological defects, and effects on hematopoiesis and cardiac development). N-(3-fluorobenzyl)-N,3-dimethylbut-2-enamide exhibited concentration-dependent antifungal activity against all 13 tested plant pathogenic fungi, showing the highest inhibition rate (79%) against M. fructigena at 20 mg/mL, with a minimum inhibitory concentration of 4.69 μL/mL. Investigation into its mode of action revealed multi-target synergistic effects: it disrupted fungal cell ultrastructure, induced carbon metabolism disorder (increasing soluble sugars by 52.1% and reducing sugars by 71.4% while decreasing soluble protein by 14.0%), inhibited key TCA cycle enzymes (reducing SDH and MDH activities by 27.2% and 37.0%, respectively), and depleted ATP while disrupting the NADH/NAD⁺ balance, collectively leading to energy metabolism collapse and cell death. In ecotoxicity assessment, the compound (denoted as compound A) demonstrated significant concentration-dependent toxicity in zebrafish embryos, with an LC₅₀ of 75.42 mg/L and 100% mortality at 96.0 mg/L. It also induced morphological abnormalities including pericardial edema, tail curvature, and axial shortening (p<0.01), suppressed hematopoietic stem cell generation, and caused linearized cardiac chamber stretching. The findings of this study provide a solid scientific foundation for the molecular optimization, in-depth investigation of antifungal mechanisms, and systematic ecological risk assessment of this class of green amide fungicides.