Discovery of indole-carbohydrazide hybrids as novel broad-spectrum fungicidal lead compound though membrane-targeting mechanism.

The escalating prevalence of fungicide resistance has severely diminished the effectiveness of conventional antifungal agents, creating substantial challenges for fungal infection control. To combat this emerging threat, we rationally designed a series of indole-carbohydrazide hybrids, aiming to develop novel membrane-targeting antifungal agents. Of particular note, compound b6 demonstrated optimal antifungal efficacy against Colletotrichum fructicola (C. f., EC₅₀ = 3.39 μg/mL) and Gibberella zeae (G. z., EC₅₀ = 3.49 μg/mL). Comprehensive mechanistic investigations employing multiple approaches-including scanning electron microscopy (SEM), propidium iodide (PI) permeability assays, extracellular conductivity monitoring, and cytoplasmic leakage analysis-collectively revealed that b6 compromised fungal membrane integrity, leading to irreversible cellular damage via increased membrane permeability. Molecular electrostatic potential (MEP) mapping further corroborated the compound's optimal electronic properties for membrane interactions. Comprehensive ADMET analysis demonstrated favorable pharmacokinetic properties, complemented by toxicological evaluations showing exceptional biocompatibility: zebrafish models exhibited a 96-h LC₅₀ > 10 μg/mL, while cytotoxicity assays revealed low toxicity toward various human cell lines. This collective safety profile underscores b6's agricultural applicability. Furthermore, potted plant experiments revealed that compound b6 (200 μg/mL) exhibited protective and curative efficacies of 47.33 and 40.94% against C. f., respectively; these values were comparable or superior to those of the commercial fungicide chlorothalonil. Overall, these findings collectively identified b6 as a promising structural scaffold for developing new antifungal agent alternative, offering significant advantages through its unique membrane-targeting mechanism and synthetic accessibility.