Discovery of Bioactive N-Substituted-3-methylbut-2-enamides: Synthesis, Crystal Structures, Antifungal Activity, Mechanism of Action, and Zebrafish Toxicity Evaluation

Plant pathogenic fungi pose a serious threat to agricultural and forestry production. Long- term reliance on chemical fungicides has led to rising pathogen resistance and ecological risks, making the discovery and development of novel, safe, and highly effective antifungal compounds of great practical significance. Three N-substituted-3-methylbut-2-enamide derivatives (compounds I–III) were synthesized and structurally characterized by single-crystal X-ray diffraction, infrared spectroscopy, and elemental analysis. The minimum inhibitory concentration (MIC) values of the three compounds were determined against six common plant pathogenic fungi, and the antifungal activity was found to be governed by the N-substituent structure. Compound III (3-methyl-N-(4-phenoxybenzyl) but-2-enamide) was the most active, with an MIC of 25–100 mg/mL, representing a 3-fold increase over the rigid fused-ring analogs I and II, which suggests that increased molecular flexibility favors antifungal activity. Preliminary mechanistic investigation of compound III revealed that it induces intracellular reactive oxygen species (ROS) accumulation, compensatory upregulation of reduced glutathione, and elevated malondialdehyde levels in fungi, thereby inhibiting fungal growth through oxidative stress-mediated damage. Zebrafish embryo toxicity assays showed that compound III exhibited concentration- and time-dependent developmental toxicity to zebrafish embryos. The median lethal concentration (LC50) was 1.032 mg/L under 72 hpf exposure conditions; sublethal exposure concentrations reduced embryo survival rate, caused developmental delay, and induced neurobehavioral abnormalities. This study revealed how structural features influence the antifungal activity of this class of enamide derivatives, preliminarily elucidated the oxidative stress-mediated antifungal effect, and completed the aquatic safety evaluation, providing an experimental basis for the further structural optimization of this lead scaffold.