Design and Synthesis of Evolitrine Derivatives and Their Antifungal Activities <i>In Vitro</i> and <i>In vivo</i>

Guided by our previous finding of its antifungal activity, 46 derivatives of evolitrine (from Orixa japonica ) were designed and synthesized to develop new fungicide candidates. Their structures were confirmed via NMR, HRMS, and X-ray crystallography. Antifungal activity results indicated that C2 exhibited broad-spectrum activities against six plant fungi. Particularly, C2 showed remarkable antifungal activity on Verticillium dahliae (EC 50 = 2.31 μg/mL), a 7-fold improvement over evolitrine (EC 50 = 16.5 μg/mL). Mechanistic studies revealed that C2 disrupts hyphal integrity and induces lipid peroxidation, evidenced by SEM, TEM, and MDA assays. Additionally, C2 significantly downregulated genes in cyanoamino acid, starch/sucrose, and phenylalanine metabolism. Furthermore, C2 exhibited moderate control efficacy (36.4–54.4%) against verticillium wilt on eggplant and tomato at 200 μg/mL and was nontoxic to seedlings of eggplant/tomato and seeds of adzuki beans/mung beans. These findings indicated that C2 could be considered a potent novel antifungal candidate.