Antifungal Activity and Mechanism of Novel Benzimidazole-Containing Flavonol Derivatives as Potential Tubulin Polymerization Inhibitors against Botrytis cinerea

Microtubules are essential components of cells, contributing to the maintenance of cell shape, intracellular transport, cell division, signal transduction, and various other functions. To discover novel tubulin-targeting fungicides, two series of benzimidazole-containing flavonol derivatives were designed, synthesized, and evaluated for their antifungal activity. Compound A23 exhibited optimal antifungal activity against Botrytis cinerea (EC₅₀ = 0.338 μg/mL), which was superior to those of boscalid (EC₅₀ = 0.870 μg/mL) and carbendazim (EC₅₀ = 0.625 μg/mL). In vivo experiments demonstrated that compound A23 effectively inhibited B. cinerea infection on tomato fruits at a concentration of 200 μg/mL. Further microscopic observations revealed that compound A23 significantly altered the normal morphology of the mycelia. Immunofluorescence staining experiments revealed that treatment with compound A23 caused significant changes in the structure of intracellular microtubules, consistent with the effects observed for the positive control carbendazim. Moreover, molecular dynamics (MD) simulations, docking experiments, and binding free energy calculations further demonstrated the mechanism of action and binding mode of compound A23 with β-tubulin. The analysis results indicated that compound A23 exhibited a stronger binding affinity for β-tubulin than carbendazim. Ecological and environmental risks of the target compounds were predicted using an online AI-based platform, and the overall profiles indicated a relatively low potential impact. Overall, our study provides a valuable reference for the development of novel tubulin-targeting fungicides.