Targeting tubulin protein to combat fungal disease: Design, synthesis, and its new mechanistic insights of benzimidazole hydrazone derivatives.

As the vital the biomacromolecule in eukaryotic cells, tubulin protein is essential for preserving cell shape, facilitating cell division, and cell viability. Tubulin has been approved as promising target for anticancer, and antifungal therapy. However, there are still many gaps in tubulin-targeted fungicidal discovery. To expand the structural diversity of benzimidazoles and achieve the distinct interaction model, a series of novel benzimidazole hydrazone derivatives were therefore synthesized. Antifungal results showed that compound A was the most effective, achieving an EC value of 2.88 μg/mL in vitro against Colletotrichum sublineola. In vivo assay, compound A displayed encouraging efficacy, outperforming the reference agents ferimzone and tetramethylthiuram disulfide. Interestingly, mechanistic studies indicated that, compared with carbendazim, compound A might form stronger interactions with tubulin, exemplified by the presence of multiple hydrogen bonds and π-π interactions, leading to intracellular microtubule aggregation in compound A-treated cells. The significantly different interactions models between A-tubulin and carbendazim-tubulin complexes may endow to produce the low resistance risk. Additionally, compound A possessed low phytotoxicity and satisfactory ADME properties. This study not only provides a structural basis for the development of benzimidazole-based fungicides targeting tubulin but also offers new insights into the use of immunofluorescence assays in tubulin-targeting studies.