Design,Synthesis,and Antifungal Activity Of Fluorine-Containing Pyrazole Amide Derivatives

The SDH inhibitors（SDHIs）inhibit electron transfer and hinder energy production by partially or completely occupying the substrate ubiquinone binding site,thereby inhibiting fungal growth.This unique mechanism of action makes them highly effective against a wide spectrum of bacteria.However,with the widespread use and abuse of SDHIs,coupled with the increasing duration of use,many plant pathogenic fungi have developed serious resistance to existing SDHIs.Therefore,it is urgently needed to develop new and efficient SDHIs to effectively prevent plant diseases and delay fungal drug resistance.In order to develop novel SDHIs with novel structures and high efficiency,this paper used pyrazole-4-carboxamide SDHIs as leads to construct fluorinated pyrazole-5-amine intermediates.By changing the carboxylic acid on the pyrazole-5-carboxamide skeleton and modifying the pyrazole ring,a total of 216 fluorinated pyrazole amide derivatives in 6 series were designed and synthesized.The structure of the synthesized compounds was confirmed by various structural identification methods such as nuclear magnetic resonance（NMR）,high-resolution mass spectrometry（HRMS）,and X-ray single crystal diffraction.The antifungal activity of all target compounds against various plant pathogenic fungi was determined by the hyphal growth rate method.The in vivo antifungal activity of some highly active compounds was determined by pot experiment and detached tissue method.The antifungal mechanism of some highly active compounds was explored by molecular docking,SDH enzyme inhibition activity,and scanning electron microscopy.The main contents are as follows:（1）Commercial pyrazole-4-carboxamide SDHIs were used as a lead to construct fluorinated pyrazole-5-amine intermediates through skeleton transition and bioelectronics strategies.Based on this intermediate,64 4-fluoro/chloro-substituted pyrazole-5-benzamide derivatives（Series I）were designed and synthesized by introducing various benzoic acids and introducing halogens（fluorine,chlorine）at the 4-position of the pyrazole.The antifungal results showed that compound I-48 had an EC₅₀ value of 0.58 mg/L against the apple rot fungus,and its antifungal activity was significantly improved compared to pyrazole-5-benzamide derivatives without 4-halogen substitution.（2）By using strategies such as active substructure splicing,bioelectronics,and the introduction of natural product active fragments,a total of 76 compounds in 3 series were designed and synthesized by introducing various types of carboxylic acids into the pyrazole amine intermediate.These series include Series II（nicotinic acid and isonicotinic acid）,Series III（thiazole-2-carboxylic acid,isothiazole-3-carboxylic acid,thiophene-2-carboxylic acid,and furan-2-carboxylic acid）,and Series IV（quinoline-2-carboxylic acid）.Among them,compound II-14 in Series II showed the highest activity,with an EC₅₀ value of only 10.35mg/L against Sclerotinia sclerotiorum and 17.01 mg/L against Valsa mali.In Series III,compound III-6 showed an EC₅₀value of 0.44 mg/L against Sclerotinia sclerotiorum,but its activity against other fungi was poor.In Series IV,compound VI-10 showed the best activity,with EC₅₀ values of 4.92 mg/L against Valsa mali and 1.71 mg/L against wheat head blight fungus.Overall,compound I-48 showed the best antifungal activity among the compounds in Series I-IV.（3）Using compound I-48 as a lead compound,a total of 76 target compounds were designed and synthesized in 2 series,Series V（phenylacetic acid）and Series VI（diphenyl ether acid）,by using strategies to extend the carbon chain of benzoic acid and construct diphenyl ether fragments.The antifungal results showed that the activity of the target compounds with extended carbon chains decreased against Sclerotinia sclerotiorum and Valsa mali.However,after introducing a fluorine atom at the 4-position of the pyrazole ring,compounds V-42 and V-49 showed significantly increased activity against Gaeumannomyces graminis and Rhizoctonia solani,with EC₅₀ values of 1.81 mg/L and 2.23 mg/L,respectively.In Series VI,compound VI-24 showed the best activity,with EC₅₀ values of0.52 mg/L against Valsa mali,3.42 mg/L against Botrytis cinerea,and 1.46 mg/L against Gaeumannomyces graminis,which were better than those of Fluxapyroxad(EC₅₀=12.45mg/L,8.33 mg/L,1.93 mg/L,respectively).Its EC₅₀ values against Sclerotinia sclerotiorum（0.52 mg/L）and Rhizoctonia solani（1.46 mg/L）were close to those of Fluxapyroxad（0.23mg/L and 0.62 mg/L,respectively）.Furthermore,a pot experiment was conducted to measure the protective activity of the highly active compounds against Sclerotinia sclerotiorum in rapeseed pots.Compound VI-24 showed 89.3%protective activity against Sclerotinia sclerotiorum at a concentration of 100 mg/L,which was close to that of Fluxapyroxad（96.4%）.Additionally,using the detached leaf assay,the protective activity of compound VI-24 against Valsa mali in apple branches was measured,and it showed 66.7%protective activity against Valsa mali,which was significantly better than that of Fluxapyroxad（37.5%）.（4）Molecular docking results showed that the pyrazole rings of the compounds in all 6series could enter the active pocket to varying degrees and stabilize the binding mode by forming different interactions with the amino acid residues in the binding site.Similar to Fluxapyroxad,the fluorine atom at the 3-position of the pyrazole ring and the amide nitrogen or oxygen atom formed hydrogen bonds with the serine（Ser）39 residue and the tryptophan（Trp）173 residue,respectively.The pyrazole ring also formed a p-πconjugation with the arginine（Arg）43 residue.It is noteworthy that the binding energy of VI-24(-45.14kcal/mol^-1)was significantly higher than that of Fluxapyroxad(-38.38 kcal/mol^-1).The results of SDH enzyme inhibition showed that the selected compounds exhibited certain inhibitory activities against SDH,and compound I-48 had an IC₅₀ value of 10.05 mg/L,which was close to that of Fluxapyroxad(IC₅₀=5.11 mg/L).Compounds III-6(IC₅₀=6.00mg/L),V-10(IC₅₀=5.55 mg/L),VI-20(IC₅₀=3.50 mg/L),and VI-24(IC₅₀=1.22 mg/L)had better SDH inhibition activity than Fluxapyroxad(IC₅₀=7.28 mg/L,8.32 mg/L).Unfortunately,the SDH inhibition and antifungal activities of the phenylacetic acid derivatives with extended carbon chains(compound V-42,IC₅₀=18.33 mg/L)were significantly lower than those of the benzoic acid series.Overall,compound VI-24 exhibited the best SDH inhibition activity and binding energy.Additionally,scanning electron microscopy observations showed that highly active compounds could cause varying degrees of wrinkling,twisting,and even rupturing of the hyphal surface,thereby inhibiting the normal growth of the hyphae.In summary,this study synthesized a total of 216 Fluorine-containing pyrazole amide derivatives in 6 series,and after multiple derivatizations and optimizations,compound VI-24with broad-spectrum and high antifungal activity was screened out.This research provides valuable references for the design and development of Fluorine-containing pyrazole amide derivatives SDHIs in the future.