Novel Pyrazole Derivatives: Blocking the Self-assembly Progress and Breaking the Structure of Tobacco Mosaic Virus

A series of novel 1,5-disubstituted-4-pyrazole analogs containing 1,3,4-thiadiazole thioether moieties have been successfully developed with rational molecular design and synthesis. All the title compounds underwent comprehensive structural elucidation by ¹H, ¹³C, and ¹⁹F nuclear magnetic resonance spectroscopy complemented by high-resolution mass spectrometric analysis. Biological evaluation demonstrated that compound 5c-2 displayed superior antiviral efficacy against tobacco mosaic virus (TMV), with half-maximal effective concentrations of 284.2 µg/mL (curative activity) and 62.2 µg/mL (inaction activity). To investigate the underlying antiviral mechanism, an integrated experimental approach was employed, including complementary in vitro biochemical evaluations, transmission electron microscopy analysis, incorporating computational molecular docking simulations, and microscale thermophoresis binding assays. It was shown that 5c-2 can significantly bind to TMV-coat protein (TMV-CP), affecting their self-assembly, which breaks the integrity of the TMV rod-shaped structure and causes the virion to lose its ability to infect the plant. It provides a structural basis for the design of more efficient TMV-CP targeting agents.