Design, Synthesis, and Antifungal Activity Evaluation of Novel Hydrazide-Containing <i>L</i>-Perillaldehyde Derivatives as Potential Fungicides

In this study, 36 novel hydrazide-containing <i>L</i>-perillaldehyde derivatives were designed, synthesized, and evaluated for their antifungal activity. In the <i>in vitro</i> antifungal assays, most of the target compounds exhibited remarkable antifungal activity. Notably, compound <b>C4</b> displayed exceptional antifungal activities against <i>Rhizoctonia solani</i> (EC₅₀ = 0.260 μg/mL), <i>Fusarium graminearum</i> (EC₅₀ = 0.480 μg/mL), <i>Sclerotinia sclerotiorum</i> (EC₅₀ = 0.240 μg/mL), and <i>Valsa mali</i> (EC₅₀ = 0.512 μg/mL), outperforming carbendazim, which showed EC₅₀ values of 0.651, 0.804, 0.520, and 0.898 μg/mL, respectively. Meanwhile, <i>in vivo</i> assays against <i>R. solani</i> and <i>S. sclerotiorum</i> revealed compound <b>C4</b>'s potential as a novel agricultural antifungal agent. In the investigation of the antifungal mechanism, scanning electron microscopy and transmission electron microscopy observations revealed that compound <b>C4</b> induced significant morphological alterations in <i>R. solani</i> mycelia, including mitochondrial swelling and rupture, thereby inhibiting mycelial growth. Determination of cellular content leakage and propidium iodide (PI) staining experiments indicated that compound <b>C4</b> compromised the integrity of the <i>R. solani</i> cell membrane, leading to cytoplasmic efflux and, subsequently, inhibiting normal mycelial development. The detection of reactive oxygen species indicated that the antifungal activity of compound <b>C4</b> may stem from inducing accumulation of reactive oxygen species within cells. The mitochondrial membrane potential detection demonstrated that compound <b>C4</b> could decrease the mitochondrial membrane potential, thereby damaging mitochondria. Furthermore, compound <b>C4</b> (IC₅₀ = 14.5 μg/mL) exhibited potent succinate dehydrogenase (SDH) inhibitory activity comparable to that of boscalid (IC₅₀ = 12.6 μg/mL). Molecular dynamics simulations confirmed that compound <b>C4</b> could establish strong interactions with key residues of SDH. These results offer significant insights and guidance for the development of novel antifungal agents.