Research on Novel Laccase Inhibitors with Thiourea as the Core Skeleton As Potential Fungicides

Developing active linkers is an important strategy for discovering molecules with high biological activity. To develop novel green fungicides with high efficiency and low toxicity, we synthesized three series of compounds by replacing the amide bond with three types of linkers: urea, thiourea, and squaramide. We then determined their in vitro antifungal activities against eight plant pathogenic fungi and oomycetes and obtained a series of thiourea derivatives with high antifungal and antioomycete activities. Notably, compound F18 demonstrated superior inhibitory activity against Valsa mali (EC₅₀ = 0.59 mg/L), against Sclerotinia sclerotiorum (EC₅₀ = 0.59 mg/L), against Rhizoctonia solani (EC₅₀ = 1.09 mg/L), and against Phytophthora capsici (EC₅₀ = 0.67 mg/L), indicating its remarkable in vitro antifungal/oomycete activities. Furthermore, the laccase activity test revealed that F18 had significant laccase inhibitory activity (IC₅₀ = 0.097 mmol/L), surpassing that of l-cysteine (IC₅₀ = 0.458 mmol/L). F18 is an excellent laccase inhibitor that can be further validated through transcriptomic analysis. The results of molecular docking indicate that F18 binds very tightly to the active cavity of laccase, and the SEM test shows that F18 can affect the normal morphology of the fungi. Additionally, the relationship between the antifungal efficacy of the compounds and their structures was analyzed by density functional theory analysis. The acute oral toxicity test on Apis mellifera Linnaeus demonstrated that compound F18 had low toxicity to Apis mellifera Linnaeus (LD₅₀ > 1000 mg/L). Overall, the discovery of these thiourea derivatives offers promising prospects for the development of new laccase inhibitors and highly efficient, low-toxicity fungicides.