Design,Synthesis And Antifungal Activity Of 1,5-disubstituted-4-pyrazole Carboxamide Derivatives

Plant fungal diseases pose a great threat to human survival and production,and fungicides became one of the important means to protect agricultural production.Succinate dehydrogenase inhibitors,an important part of fungicides,exhoboted good selectivity,high efficiency and low toxicity,which made them become one of the research hotspots in recent years.In this work,a series of novel 1,5-disubstituted-4-pyrazole carboxamide derivatives were designed and synthesized by changing the substituents at the 1-and 5-positions of the pyrazole ring,base on the potential succinate dehydrogenase inhibitor skeleton of 1-phenyl-5-trifluoromethyl-4-pyrazolamide that found in our previous work.The structure-activity relationship analysis（SAR）was performed based on the results of bioactivity resluts,combined with molecular docking and 3D-QSAR.The preliminary antifungal mechanism studies of the highly active compounds were also carried out to lay the foundation for further studies on the antifungal mechanism of pyrazole carboxamides.Based on the potential succinate dehydrogenase inhibitor"1-substituted-5-trifluoromethyl-4-pyrazolamide"skeleton obtained by the group,we changed the substituted group on 1-position of pyrazole ring,and explored the effect of 1-substituted group on the activities of the compound by bioactivity testing and molecular docking for further SAR analysis.A total of twenty 1-substituted-5-trifluoromethyl-4-pyrazole carboxamide derivatives were synthesized,and the bioactivity assay results showed that the EC₅₀ values of Y₁₃ against G.zeae,B.dothidea,F.prolifeatum and F.oxysporum were13.1,14.4,13.3 and 21.4 mg/L,respectively.In vivo protective activity of Y₁₃ against G.zeae was 50.65%at 100 mg/L.SAR analysis revealed that the phenyl group at the 1-position of pyrazole ring was important to enhance the activity of the tsarget compounds,and the total score of ligands Y₁₃ and fluopyram docking to SDH protein（PDB:2FBW）were 6.3 and 7.1,which indicated that the compound bound well to the SDH receptor protein.Secondly,based on the SAR analysis,the benzene ring substituent at the 1-position of pyrazole ring was retained and the substituent at the 5-position of pyrazole ring was changed.The antifungal activities of the compounds were tested by mycelial growth rate method to verify the conclusions of the first part,and 3D-QSAR models were builded to predict and provide a reference for further structural optimization.A total of forty-eight1,5-disubstituted-4-pyrazole carboxamide derivatives were synthesized,and the biological activities results showed that the EC₅₀ values of Y₆₈ against G.zeae,N.oryzae,T.cucumeris and V.dahliae were 5.5,9.8,12.8 and 17.6 mg/L,respectively.In vivo test results reveled that Y₆₈ exhibited certain protective activity（57.18%）and therapeutic activity（44.71%）at 100 mg/L.Based on the 3D-QSAR analysis of Co MFA and Co MSIA models,it found that the trends of the electrostatic fields of Co MFA model and Co MSA model were similar,and introducting the large steric hindrance and electronegative groups in the 5-position region of pyrazole ring,the neighboring substitution of the benzene ring at the 1-position substituent of the pyrazole ring is a hydrophobic group,and the neighboring position of the pyridine ring is an electronegative,hydrogen bond donor group both increased the inhibitory activity of the compounds against G.zeae.Preliminary antifungal mechanism studies of Y₁₃ and Y₆₈ against G.zeae suggested that this series of compounds,potential succinate dehydrogenase inhibitors,not only bind well to SDH receptor proteins,have some inhibitory ability on succinate dehydrogenase activity,but also can disrupt the cell membrane of mycelium by increasing the content of malondialdehyde,thus inhibiting the growth of the fungus.The results of these mechanism studies were inconsistent with those of traditional amide compounds,and might provide a new perspective for the deep mechanism study of this series of pyrazole carboxamide derivatives.In genernal,we firstly investigated the substitution at the 1-and 5-positions of pyrazole ring by molecular docking and 3D-QSAR,and found that the fungal inhibitory activity of the compounds could be improved when the 1-position of pyrazole ring was a benzene ring substituent and the 5-position was a substituent with high positional resistance and electronegativity.Secondly,we screened the highly active compounds Y₁₃and Y₆₈,and initially investigated the mechanism of action of 1,5-disubstituted-4-pyrazolamide derivatives by morphological and biological crossover.The antifungal mechanism of the 1,5-disubstituted-4-pyrazolamide derivatives was initially investigated by morphological and biological intersection,and the possible inhibition of succinate dehydrogenase activity and disruption of fungal cell membranes of these compounds were found,providing a new perspective and theoretical basis for the in-depth study of the mechanism of action of this series of derivatives.