Pinonic Acid Derivatives Containing Thiourea Motif: Promising Antifungal Lead Compound Targeting Cellular Barrier of <i>Colletotrichum fructicola</i>

In order to explore novel antifungal lead compounds from plant essential oil, thirty-two pinonic acid derivatives containing thiourea groups were designed and synthesized using α-pinene as a raw material. One of these pinonic acid derivatives compound <b>3a</b> exhibited noteworthy <i>in vitro</i> antifungal activity against <i>Colletotrichum fructicola</i> (EC₅₀ = 9.22 mg/L), which was comparable to that of the positive control kresoxim-methyl (EC₅₀ = 9.69 mg/L). Structure-activity relationship (SAR) studies demonstrated that the introduction of thiourea groups, F atoms, and Cl atoms into the structure of pinonic acid derivatives significantly improved their antifungal activity. The <i>in vivo</i> antifungal test revealed that compound <b>3a</b> could effectively control pear anthracnose. It also proved that compound <b>3a</b> showed low acute oral toxicity to honeybees (LD₅₀ > 100 μg/bee) and low or no cytotoxicity to LO2 and HEK293 cell lines. The preliminary mechanism of action studies revealed that compound <b>3a</b> caused mycelium deformity, increased cell membrane permeability, blocked the normal process of phospholipase C on the cell membrane, and reduced mycelium protein content. The results of molecular docking studies demonstrated the stable binding of compound <b>3a</b> to phospholipase C and chitin synthetase. This study suggested that compound <b>3a</b> could be used as a promising lead compound for the development of novel antifungal agents targeting the cellular barrier of <i>C. fructicola</i>.