Design, synthesis and antifungal evaluation of perillaldehyde derivatives as potential laccase inhibitors

The development of novel fungicides has still been a hot topic in the field of pesticide research. In this study, three series of new perillaldehyde hydrazide, amide and acylthiourea derivatives (3a-3n, 4a-4f, and 6a-6f) were designed and synthesized. The in vitro antifungal activity of the title compounds against seven plant pathogens was evaluated. The results displayed that several hydrazide derivatives showed significant antifungal activity. Especially, compound 3b exhibited the most potent inhibitory activity against Monilinia fructicola (EC₅₀ = 0.142 mg/L), outperforming the commercial fungicides bixafen and carbendazim. In vivo experiments further confirmed that 3b presented superior protective and curative effects on pear fruits infected by M. fructicola compared to bixafen. Mechanism studies revealed that 3b could damage the mycelial morphology and cell membrane integrity, increase cell membrane permeability, reduce mycelial dry weight and exocellular polysaccharide content, and increase intracellular ROS content of M. fructicola. Additionally, 3b exhibited significant laccase inhibitory activity (IC₅₀ = 4.87 μM), suggesting that laccase could be a key target for its antifungal action. Molecular docking studies further confirmed the strong binding affinity of 3b with the active sites of laccase. This study highlighted the potential of this perillaldehyde hydrazide derivative as a promising lead for the development of novel fungicides controlling the brown rot caused by M. fructicola.