Synthesis Of 5-aminopyrazole-4-(1,3,4-thiadiazole) Sulfide Derivatives And Study Of Their Anti-TMV Mechanis

Tobacco mosaic virus（TMV）is a systemic plant virus that poses a serious threat to crops worldwide.The use of antiviral agents is of great significance to promote the economic development of agricultural production.In recent years,1,3,4-thiadiazole derivatives containing pyrazole structure have been found to have potential anti-TMV activity,but their mechanism of action has not been reported.Therefore,based on the broad-spectrum biological activity of pyrazole and 1,3,4-thiadiazole,two series of novel1,5-disubstituted-4-pyrazole-1,3,4-thiadiazole thioether derivatives were designed and synthesized by structural modification 1-position and 5-position of pyrazole and5-position of 1,3,4-thiadiazole.And the further mechanism against TMV of highly active compounds were studied based on the preliminary biological activity test.On the basis of the above active skeleton,27 1-substituted phenyl-4-（1,3,4-thiadiazole-5-thioether）-1H-pyrazole-5-amine derivatives（series 1,5a-5c）were synthesized by changing the substituent on the 1-position of pyrazole.In vivo antiviral activity results showed that compound 5c-2 exhibited well curative and inactive activity against TMV,and the EC₅₀value was 284.2 and 62.2μg/mL,respectively.In vitro experiments showed that at a concentration of 100μg/mL,5c-2could cause TMV rod-like structure to break and shorten.In addition,the in vitro self-assembly experiments showed that compound 5c-2 could effectively inhibit the self-assembly of TMV CP and RNA at a concentration of 100μg/mL.Based on the structure of series 1,28 compounds of series 2 were synthesized by changing the substituted phenyl to phenyl at 1-position of pyrazole.The results of biological activity test showed that the series of compounds had significant protective activity against TMV.Among them,compounds 5d-10,5d-17 and 5d-21 showed comparable activity with ningnanmycin（261.5μg/mL）,and their EC₅₀were 321.5μg/mL,259.7μg/mL and 312.7μg/mL,respectively.The compounds 5d-2 and 5d-14showed better protective activity against TMV than that of ningnanmycin,and their EC₅₀values were 203.5 and 240.8μg/mL,respectively.Further study on N.benthamiana leaves infected with TMV-GFP showed that compound 5d-2 could significantly inhibit the spread of TMV in the host.The antiviral mechanism of highly active compound 5d-2 against TMV protection activity was studied by combining histomorphology and biology.The results indicated that compound 5d-2 protected the host by inducing the differentiation of sponge tissue and palisade tissue cells and increasing chlorophyll content of leaves,which promoted the photosynthetic rate of the host to improve its ability to defense the infection of TMV.In addition,compound 5d-2 could induce stomatal closure of the leaves to prevent further viral infection through stomata during TMV infection and effectively reduce the content of peroxides in the infected plants,reducing the damage to the plants caused by oxidation.These results provide important information for the study of the mechanism of antiviral compounds.