Synthesis,Antifungal Activity And Structure-Activity Relationship Study Of Isoxazole-Containing Stilbene Derivatives Against Botrytis Cinerea

Botrytis cinerea has been recognized as one of key pathogenic fungi with such features as broad host range,diverse attack modes and high risk for fungicide-resistance development,which has posed a growingly serious threat to many vegetables and fruits cultivated in the greenhouse in recent years.Stilbenes of plant origin,such as resveratrol,exhibit good fungicidal activities against several phytopathogens,including B.cinerea,making them lead compounds for developing new drugs.Heterocyclic isoxazole,which has unique structural characteristics and physicochemical properties,shows broad-spectrum and efficient biological effects.In view of these findings,a molecular design approach was carried out in this study by introducing the active substructure 3-（substituted phenyl）isoxazole into the stilbene skeleton via molecular hybridization method.And a series of stilbene derivatives containing a 3-（substituted phenyl）isoxazole moiety were obtained.Then their antifungal activities against B.cinerea were evaluated,and a quantitative structure-activity relationship model was established.Additionally,the fungicidal mechanism of the most active compound in this series was preliminarily investigated.The main methods and results were drawn as follows:（1）Theα,β-unsaturated ketone was treated with N-hydroxyl-4-toluenesulfonamide in the presence of potassium bicarbonate to afford the intermediate 3,5-diaryl isoxazole by cyclization and elimination reaction,followed by bromination reaction with NBS,Arbuzov reaction and Wittig-Horner reaction to give twenty-one novel stilbene derivatives containing a 3-（3,4-dichlorophenyl）isoxazole moiety.The structures of the target compounds were confirmed by¹H NMR,¹³C NMR and HRMS.（2）The in vitro antifungal activities of the target compounds against B.cinerea were assessed using the mycelium growth rate method.Among them,compound 5r was found to be as effective as resveratrol.Their in vivo protective activities against gray mold was next evaluated by a tomato-fruit inoculation assay.Compound 5r displayed the highest control efficacy（56.11%）,which was comparable to those of resveratrol（50.99%）and commercial fungicide carbendazim（55.56%）.（3）On the basis of the in vivo bioassay results,a hologram QSAR model was generated using B/DA as fragment distinction parameters,3–8 as fragment size,and 61 as hologram length(q²=0.604,r²=0.941,r²_pred=0.844).A conclusion could be derived from the atomic contribution map that the ortho position of phenyl ring and the C-4 position of isoxazole ring in the 3-phenylisoxazole fragment could be employed for structural modifications to improve biological activity.（4）Compound 5r could cause hyphal morphological abnormalities of B.cinerea by optical microscope observation.Combined with the results of cell membrane permeability assay,it was speculated that compound 5r could affect the normal function of cell membrane,increase the cell membrane permeability,and lead to the leakage of cell contents,thereby exerting the antifungal activity.