Design,Synthesis And Biological Activity Evaluation Of Novel Pyrazole Derivatives

Plant pathogenic fungi seriously threaten global agricultural production and crop safety,and it is urgent to develop new fungicides with low toxicity and high efficiency.In this experiment,44 pyrazolo[3,4-d]pyrimidin-4-one derivatives（Experiment A,Chapter 2）and 20 pyrazolecarboxamide derivatives containing oxazole rings（Experiment B,Chapter 3）were designed and synthesized.The correct structure of the compounds was verified by means of ¹H NMR,¹³C NMR,high-resolution mass spectrometry and X single crystal diffraction.The biological evaluation of the target compounds were carried out by in vitro antifungal experiments,in vivo antifungal experiments and scanning electron microscopy.In experiment A,we designed and synthesized a series of pyrazolo[3,4-d]pyrimidin-4-one derivatives and evaluated their antifungal activities against six plant pathogenic fungi.The bioassay results showed that most of the target compounds had medium to high antifungal activity in vitro,especially compound g22 showed remarkable antifungal activity against Sclerotinia sclerotiorum,and its EC₅₀ value was1.25 mg/L,which was close to that of boscalid(EC₅₀=0.96 mg/L)and flufenpyrad(EC₅₀=1.91 mg/L).In addition,compound g22 showed remarkable in vivo protective activity against S.sclerotiorum in 24 hours（95.23%）and 48 hours（93.78%）,compared with the positive control boscalid（96.63%）;48 hours（93.23%）has the same effect.Subsequent studies showed that compound g22 might hinder the growth and reproduction of S.sclerotiorum by affecting mycelium morphology,destroying cell membrane integrity and increasing cell membrane permeability.In addition,the application of compound g22 does not affect the growth and reproduction of Italian bees.The drug resistance frequency test showed that it was unlikely to produce Sclerotinia sclerotiorum strains resistant to compound g22.Experiment A showed that compound g22 is expected to be developed as an efficient and safe agricultural fungicide.In experiment B,on the basis of previous research on SDHIs,an oxazole ring was introduced into the 4th position of the pyrazole ring and 20 derivatives of pyrazole formamide containing oxazole groups were synthesized.After testing their antifungal activity against eight agricultural pathogenic fungi including S.sclerotiorum,it was found that the antifungal activity of the compounds was greatly improved after the introduction of oxazole ring,and most of the target compounds showed good antifungal activity against the strains to be tested,especially compounds C13 and C14,And C16showed better inhibitory activity against S.sclerotiorum than the control drug boscalid(EC₅₀=0.96 mg/L),with EC₅₀ values of 0.69 mg/L,0.26 mg/L and 0.95 mg/L,respectively.The preliminary analysis of structure-activity relationship found that electron-withdrawing substituents such as chlorine and bromine,when R¹ was chlorine and R² was three,were beneficial to improve antifungal activity.Subsequent in vivo experiments showed that compounds C13 and C14 exhibited excellent protective effects on rape leaves and cucumber leaves infected by S.sclerotiorum.Scanning electron microscopy showed that the normal structure of Sclerotinia sclerotiorum hyphae was seriously damaged after being treated with compounds C13 and C14 with respective EC₅₀ values.Molecular docking study revealed that the binding mode of compounds C13 and C14 to SDH was similar to that of boscalid.This work verifies the rationality of our preliminary design,and this kind of compound is expected to be developed into a promising candidate fungicide after further modification.In order to alleviate the problems of pathogen drug resistance and poor control effect caused by abuse and unscientific use of antifungal drugs,it is urgent to develop new and effective fungicides.The pyrazolopyrimidinone derivatives g22,pyrazolecarboxamide derivatives C13 and C14,which have shown promising antifungal effect on plant diseases,can be developed into new and efficient agricultural fungicides after further structural optimization.