Study On The Design,Synthesis And Biological Activity Of Pyrazole Amide Derivatives As Succinate Dehydrogenase Inhibitors

Fungicides are the most economical and effective means for controlling plant diseases and play an important role in agricultural production.As an important component of bactericides,succinate dehydrogenase inhibitors(SDHIs)can block the energy synthesis of pathogens by inhibiting electron transfer between mitochondrial succinic acid and ubiquinone,ultimately achieving bacteriostatic action.The novel SDHI has high-efficiency,broad-spectrum antifungal activity,and its structure is mainly composed of three parts:an amide bond,an aromatic cyclic acyl group and an aromatic cyclic amino group or a benzylamine group.In order to create novel and efficient SDHI structures,this paper uses succinate dehydrogenase(Organism:Gallus gallu)as the target,combined with the structural characteristics of SDHIs,and uses 3-difluoromethyl-pyrazole-4-carboxylic acid as the lead compound.Molecular docking technology was used to rational design and synthesize new pyrazole amide derivatives.The structures of all the target compounds were characterized by NMR and HRMS,and the single crystal structure of the representative compound was cultured and analyzed.The antifungal activity of the target compounds was also determined.The specific research contents are as follows:285 compounds prepared to be synthesized were designed based on the structural characteristics of SDHIs and the lead compound of 3-difluoromethyl-pyrazole-4-carboxylic acid.The Ar groups of the designed compound were benzene ring,naphthalene ring,thiophene,furan,pyrazole,pyrrole,and pyridine.At the same time,alkane and halogen were selected as substituents of Ar group.Alkane could be selected from methyl,ethyl,vinyl,isopropyl,tert-butyl,methoxy,phenyl,trifluoromethyl,and so on.Halogen could be selected from fluorine,chlorine,bromine,and so on.In this experiment,succinate dehydrogenase was used as the target,and molecular docking technology was used to obtain 32 target compounds with relative high affinity and stable conformation with SDH.At the same time,the 32 compounds were designed and synthesized.The results of molecular docking showed that compounds 8n and 8j had the highest affinity,indicating that they may have good bactericidal activity.8n and 8j were surrounded by amino acid residues(for example,Trp32,Trp172,Trp173,Prol69,Arg43)by van der Waals interaction,and biphenyl aromatic rings and difluoropyrazolyl groups of 8n and 8j could form ?-? bond with Trp32 and Tyr58,respectively.The ?-? stacking interaction enhanced their binding affinity with the target.The above results indicated that 8n and 8j might have the potential of highly active SDH inhibitors.The experiment used 4,4-difluoroacetoacetate and 40%methylhydrazine aqueous solution as starting materials,and further obtained the 3-difluoromethyl-pyrazole-4-acyl chloride according to the formylation reaction,oxidation reaction and acid chloride reaction;At the same time,2-bromo-5-chlorobenzaldehyde and an aromatic boronic acid were coupled based on the SUZUKI reaction,nucleophilic substituted with cyclopropylamine and further reduced to obtain a benzidine structure.After the amidation of 3-difluoromethyl-pyrazole-4-yl chloride with benzidine,32 difluoropyrazole-4-amide derivatives were finally synthesized.The structures of all target compounds were characterized by 1H NMR,13C NMR and high-resolution mass spectrometry.The single crystal of a representative compound 8s was obtained from a dichloromethane/ethyl acetate(20:1)solvent,and which was diffracted by graphite monochromated MoKa(?=0.71073 A)by a Bruker SMART 1000 CCD diffractometer The 8s single crystal diffraction data were collected by ?-? scanning at 113(2)K to further verify the accuracy of the structure of the synthesized compounds.In this experiment,the target compounds were evaluated for their fungicidal activity in vitro and in vivo by growth rate method and stem-leaf spray method.Under the condition of 100 ?g/mL in vitro,the inhibition rate of compound 8d on Fusarium oxysporum was 100%,which was equal to boscalid;compounds 8g,8j,8o-a and 8u-a had an inhibitory activity against Alternaria solani over 90%,especially 8j and 8u-a could completely inhibit the growth of A.solani,comparable to the positive control boscalid;8a-b inhibited Botrytis cinerea by up to 100%.At a concentration of 50 ?g/mL,the inhibition rate of A.solani by 8j was still 90%,and the EC50 was 3.06 ?g/mL,which was comparable to the positive control boscalid.At a concentration of 10 ?g/mL,the inhibitory effect on A.solani was 8%in tomato plants,and was equal to boscalid,thus,8j showed good fungicidal activity,which deserved further study.