A succinate dehydrogenase inhibitor and its synthesis method and application

Abstract

The present invention discloses a novel succinate dehydrogenase inhibitor compound, the structural formula of which is shown in the following formula (I), and the present invention also discloses a synthesis method of the compound and its application in preventing and controlling plant fungal diseases, belonging to the field of pesticide chemistry. The compound is improved on the basis of a pyrazole amide fungicide by introducing a monofluoroolefin group into a hydrophobic structure, and the antibacterial test confirms that the biological activity of the pyrazole amide fungicide is improved.

Description

Technical Field

[0001] The invention belongs to the field of pesticide chemistry, and in particular relates to a novel succinate dehydrogenase inhibitor. The invention also relates to a synthesis method of the novel succinate dehydrogenase inhibitor and application of the novel succinate dehydrogenase inhibitor in preventing and controlling plant fungal diseases. Background Art

[0002] As the population increases, people's demand for food is also increasing; however, fungal diseases have seriously affected food production. It is estimated that fungal diseases have reduced the output of the five major food crops by 125 million tons per year worldwide. Among them, the damage caused by fungal diseases to rice, wheat and corn alone brings economic losses of US$60 billion to global agriculture each year. The effective methods to prevent plant fungal diseases mainly include biological control, the application of chemical pesticides and improving crop resistance. However, the application of chemical pesticides is widely used because of its convenience and timeliness. However, the large-scale use of chemical pesticides will cause pesticide residues, affect human health and produce pesticide resistance. This requires researchers to continuously develop pesticide varieties that are efficient, environmentally friendly, and have no cross-resistance with the current major commercial agents to meet the needs of plant protection and serve agricultural production.

[0003] Succinate dehydrogenase (SDH), also known as mitochondrial respiratory chain complex II, is an important component of the tricarboxylic acid cycle. It is connected to the mitochondrial electron transport chain and catalyzes the coupled reaction from succinate oxidation to fumarate and from ubiquinone reduction to ubiquinol. It is composed of four subunits: flavoprotein (Fp, SdhA), iron-sulfur protein (Ip, SdhB) and two other membrane-embedded proteins (SdhC and SdhD). Flavoprotein and iron-sulfur protein constitute the membrane-peripheral domain of succinate dehydrogenase, which has succinate dehydrogenase activity and can oxidize succinate to fumarate. The membrane anchoring domain composed of two membrane-embedded proteins has the main function of fixing flavoprotein and iron-sulfur protein on the inner membrane and has ubiquinone reductase activity. SDHIs fungicides cover the sites of ubiquinone, blocking the transfer of electrons from succinate to ubiquinone, affecting the respiratory chain electron transfer system of pathogens, hindering their energy metabolism, and thus inhibiting the growth of pathogens and causing their death, ultimately achieving the purpose of preventing and controlling plant diseases.

[0004] As early as 1969, the SDHI fungicide carboxin was launched on the market, which was earlier than the triazole fungicides developed in the 1970s and the strobilurins fungicides in the 1990s. However, it was not until 2009 that succinate dehydrogenase inhibitors became a separate system. In that year, the International Fungicide Resistance Action Committee (FRAC) classified them separately according to their unique fungicide mechanism. To date, 24 succinate dehydrogenase inhibitors have been commercialized, such as Boscalid developed by BASF in 2003, Fluxapyroxad developed in 2011, and Pyrazole developed by Syngenta in 2010. Isopyrazam, Sedaxane developed in 2011, Benzovindiflupyr developed in 2013, Pydiflumetofen developed in 2016, these compounds are popular because of their strong effects. Succinate dehydrogenase inhibitors have become the third largest category of fungicides after triazoles and acrylates. Now succinate dehydrogenase has become an important fungicide target that cannot be ignored. It is foreseeable that the development of low-toxic, novel, broad-spectrum and highly effective SDHIs fungicides will become the dominant fungicide in the future. Summary of the invention

[0005] The purpose of the present invention is to provide a novel succinate dehydrogenase inhibitor, a preparation method and an application thereof. The inhibitor is improved on the basis of pyrazole amide fungicides by introducing monofluoroolefins into the hydrophobic structure. The introduction of the monofluoroolefin group improves the biological activity of the pyrazole amide fungicides.

[0006] The technical solution of the present invention is as follows:

[0007] A succinate dehydrogenase inhibitor, the structural formula of which is shown in the following formula (I):

[0008]

[0009] In formula (I), R1 is difluoromethyl or trifluoromethyl;

[0010] R2 is H or a conventional substituent on a benzene ring, wherein the conventional substituent includes halogen, alkyl, alkoxy, halogen-substituted alkyl (such as trifluoromethyl), monosubstituted or polysubstituted;

[0011] Preferably, R1 is difluoromethyl; R2 is 3, 4, 5-trifluoro.

[0012] The present invention can adopt the following route to synthesize the above-mentioned compound:

[0013]

[0014] In the synthesis step a, the raw material I is dissolved in anhydrous dichloromethane, N,N-dimethylformamide is added as a catalyst, and then oxalyl chloride is added dropwise to react at room temperature for 2-3 hours, and the tail gas is absorbed by a saturated sodium hydroxide solution. After the reaction is completed, the reaction is extracted with dichloromethane, and the organic phase is concentrated and dried to obtain the intermediate II.

[0015] In the synthesis step b, dichloromethane is used as the reaction solvent, and 2-iodoaniline is reacted with intermediate II under the protection of N2 atmosphere. After the reaction is completed, water is added to quench the reaction, and dichloromethane is used for extraction. The organic phase is concentrated and dried, and then recrystallized with ethyl acetate and petroleum ether to obtain intermediate III.

[0016] In the synthesis step c, under the protection of N2 atmosphere, triphenylphosphine, sodium difluorochloroacetate, N,N-dimethylformamide and raw material IV are added, and the temperature is raised to 100°C for reaction. After the reaction is completed, petroleum ether is used for extraction, and the organic phases are combined and concentrated to dryness, and intermediate V is obtained after column chromatography.

[0017] In the synthesis step d, tetrahydrofuran is used as the reaction solvent and (Cy3P)2CuCl is used as the catalyst to react the intermediate V, sodium acetate and biboric acid pinacol ester. After the reaction is complete, the reaction is quenched with a saturated ammonium chloride solution, extracted with ethyl acetate, and the organic phases are combined and concentrated to obtain the intermediate VI.

[0018] In the synthesis step e, tetrahydrofuran is used as the reaction solvent and tetrakistriphenylphosphine palladium is used as the catalyst to react the intermediate VI and the intermediate III. After the reaction is complete, the reaction is quenched with a saturated ammonium chloride solution, extracted with ethyl acetate, and the organic phases are combined, concentrated and dried, and the target product VII is obtained after column chromatography.

[0019] In the above synthetic route, R1 and R2 are the same as the corresponding groups in formula (I) of the present invention.

[0020] Eight important plant pathogenic fungi in agriculture were selected: Fusarium graminearum, Botrytis cinerea, Sclerotinia sclerotiorum, Pestalotiopsis, Monilinia fructicola, Cochliobolusheterostrophus, Rhizoctonia solani, and Altermaria alternata as the supply strains. The in vitro inhibition rate of the synthesized new succinate dehydrogenase inhibitors against these fungi was tested using drug-containing culture medium. The results showed that the synthesized compounds have strong biological activity.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] (1) Strong fungicidal activity. The present invention synthesized a series of compounds and verified the antibacterial activity of these compounds. The results showed that the inhibition rate of these compounds against Rhizoctonia solani was higher than that of commercial pyrazole amide fungicides, and EC 50 The activity of the fungicide is generally lower than that of commercial pyrazole amide fungicides. The present invention is based on the pyrazole amide fungicides and is improved by introducing monofluoroolefins into the hydrophobic structure. The antibacterial test confirms that the structural improvement improves the biological activity of the fungicide.

[0023] (2) Good broad-spectrum activity. The compound synthesized in the present invention has an inhibitory effect on a variety of plant fungi, and the inhibition rate on corn leaf blight, peach brown rot, and alternaria is significantly higher than that of commercial pyrazole amide fungicides.

[0024] (3) Simple structure and easy preparation. The synthesis of the succinate dehydrogenase inhibitor of the present invention requires only 5-6 steps, the raw materials are easily available, the process is controllable, and the product yield is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The present invention provides the design concept and binding energy prediction results of the compounds.

[0026] Figure 2 The figure shows the molecular docking result of the compound of the present invention and succinate dehydrogenase.

[0027] Figure 3 The present invention provides a living protective effect of the compound on fungal diseases of rapeseed leaves.

[0028] Figure 4 This is the in vivo protective effect of the compound of the present invention on tomato fungal diseases. DETAILED DESCRIPTION

[0029] The present invention is described in detail below in conjunction with specific embodiments. In the following experiments, unless otherwise specified, the relevant reagents used are all from commercial channels, and the experimental methods used are all conventional experimental methods in the art. The following are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the following embodiments, and there can be many variations. Therefore, the modifications or improvements made by those skilled in the art on the basis of the disclosure of the present invention should all belong to the scope of protection claimed in the present invention.

[0030] The compound synthesized by the present invention is improved on the basis of pyrazole amide fungicides, and the design concept is as follows:

[0031] The unique physical and chemical properties of fluorine have made fluorine-containing substituents an important part of the creation of new pesticides. Generally, fluorine atoms have the following characteristics in pesticide molecules:

[0032] 1. Simulation effect: The van der Waals radius of fluorine atoms is similar to that of hydrogen atoms and their volume is small, making it difficult for organisms to distinguish them. As a result, they are taken into the body as hydrides, resulting in the simulation effect.

[0033] 2. Electronic effect: Since the electronegativity of fluorine atoms is much greater than that of hydrogen atoms, when a strong electron-withdrawing fluorine-containing group (such as -F, -CF3, -CF2Cl, -CF2H, etc.) is introduced into a compound, the acidity of the compound can be doubled and the efficacy can be enhanced.

[0034] 3. Inhibition effect: The bond energy of carbon-fluorine bond is much greater than that of carbon-hydrogen bond, which makes the molecular stability of compounds containing carbon-fluorine bond better. Therefore, fluorine pesticides generally have good thermal stability and light stability, and are not easily decomposed in the body, with long-lasting efficacy, which inhibits the growth of pests and diseases.

[0035] 4. Permeation effect: The outer electrons of fluorine atoms are densely arranged and not easily affected by the outside world, which makes them exhibit good repulsion and discreteness. In addition, they are hydrophobic and fat-soluble, which makes fluorine-containing compounds have good permeability in the body.

[0036] In recent years, monofluoroolefins have received extensive attention in pharmaceutical chemistry. This is mainly because the charge distribution and dipole moment properties of monofluoroolefin skeletons are very similar to those of amide structures, and they have the following two advantages: (1) monofluoroolefins are not as easily hydrolyzed and broken by hydrolases as amide bonds; (2) monofluoroolefin structural units are highly rigid and are not as easily subject to conformational changes as amides. Therefore, monofluoroolefin structural units are often used in peptide-like protease inhibitor drugs as amide-like structures.

[0037] See also Figure 1 We quoted the classic structure of commercial succinate dehydrogenase inhibitors, using pyrazole amide as the skeleton, and introduced fluoroolefins into the hydrophobic part (biphenyl) of the succinate dehydrogenase inhibitor. In the development of molecular binding targeted pesticides, binding energy is a parameter that expresses the energy released by the binding of small molecules and proteins. The more energy released, the tighter the binding with the protein, and the better the effect may be. After calculating the binding energy, we found that its binding energy decreased from -8.2kcal / mol to -9.2kcal / mol (the software used was ChemDraw and AutoDockTool), so this skeleton may have better biological activity. See Figure 2Furthermore, through the molecular docking of the target molecule skeleton with the succinate dehydrogenase, we can see that the fluorine element on the olefin introduced will interact with the protein residues on the succinate dehydrogenase. Green represents the target small molecule, and blue represents some residues of the succinate dehydrogenase protein. The fluorine on the monofluoroolefin plays a big role in affinity, for example, there is interaction with protein residues ILE-218, HIS-216, MET-39, etc. In addition, due to the lengthening of the chain length of the compound, the π-π interaction between the other benzene ring and the protein residue is more significant. Therefore, we designed this series of compounds.

[0038] Example 1: Synthesis of (Z)-N-(2-(2-([1,1'-biphenyl]-4-yl)-1-fluorovinyl)phenyl)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide (Compound 1)

[0039]

[0040] Synthesis step a, synthesis of intermediate II 3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carbonyl chloride: under N2 atmosphere protection, commercially available 3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxylic acid (1.47 g, 10 mmol) was placed in a three-necked flask, 20 ml of anhydrous dichloromethane and two drops of anhydrous N, N-dimethylformamide were added, oxalyl chloride (1.1 mL, 12 mmol) was added dropwise to the system at 0°C, and the mixture was moved to room temperature for 2 hours. The acidic gas produced by the reaction was absorbed by saturated sodium hydroxide solution. After the reaction of the raw materials was complete, water was added to the system to quench the unreacted acyl chloride, and the mixture was extracted with dichloromethane (3×30 mL). The organic phases were combined and dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain intermediate II, which was directly used in the next step. Yield: 80%.

[0041] Synthesis step b, synthesis of intermediate III 3-(difluoromethyl)-N-(2-iodophenyl)-1-methyl-1H-pyrazole-4-carboxamide: Under N2 atmosphere protection, 2-iodoaniline (2.19g, 10mmol) was placed in a three-necked flask, 20ml of anhydrous dichloromethane was added, triethylamine (1.52g, 15mmol) was added, and the intermediate II was dissolved with 10ml of anhydrous dichloromethane. The anhydrous dichloromethane solution containing the intermediate II was added dropwise to the three-necked flask under ice bath conditions, and the mixture was slowly heated to room temperature and reacted for 2 hours. After the reaction of the raw materials was completed by TLC monitoring, 5ml of water was added to quench the reaction, and the mixture was extracted three times with dichloromethane (3×20ml), the organic phases were combined and dried with anhydrous sodium sulfate, and the crude intermediate III was obtained by concentrating under reduced pressure, and the white solid intermediate I was obtained by recrystallization with ethyl acetate and petroleum ether (1:2). Yield: 75%.

[0042] Synthesis step c, synthesis of intermediate V 4-(2,2-difluorovinyl)-1,1'-biphenyl: under N2 atmosphere protection, triphenylphosphine (3.94 g, 15 mmol), sodium difluorochloroacetate (1.85 g, 12 mmol) were added to a three-necked round-bottom flask, 10 ml of anhydrous N, N-dimethylformamide was added to the three-necked round-bottom flask, and then intermediate IV p-phenylbenzaldehyde (1.82 g, 10 mmol) was added, and the temperature was raised to 100°C for reaction for 1 h. After the reaction of intermediate IV p-phenylbenzaldehyde was completed by TCL monitoring, 5 ml of water was added to the reaction system to quench the reaction, and petroleum ether was used for extraction (3×50 mL), the organic phases were combined and dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain product V by column chromatography (petroleum ether). Yield: 70%.

[0043] Synthesis step d, synthesis of intermediate VI (Z)-2-(2-([1,1'-biphenyl]-4-yl)-1-fluorovinyl)-4,4,5,5-tetramethyl-1,3-dioxolane: Preparation of (Cy3P)2CuCl catalyst: In a glove box, weigh CuCl (99 mg, 1 mmol) and Cy3P (560.86 mg, 2 mmol) in a round-bottom flask, add 10 ml of tetrahydrofuran and stir for 1 hour for use. In a glove box, weigh intermediate V (620 mg, 2 mmol), 2 ml of tetrahydrofuran solution of (Cy3P)2CuCl, sodium acetate (463.2 mg, 2.4 mmol), and biboronic acid pinacol ester (Bpin)2 (761.82 mg, 3 mmol) into a Shrek tube, add 10 ml of anhydrous tetrahydrofuran, seal and take out of the glove box and move to an oil pan at 40 ° C to react overnight. After monitoring the complete reaction of intermediate V by TCL, add 5 ml of saturated ammonium chloride solution to the reaction system to quench the reaction, and extract with ethyl acetate (3×20 ml). Combine the organic phases, dry over anhydrous sodium sulfate and evaporate to obtain intermediate VI, which is directly used in the next step.

[0044] Synthesis step e, synthesis of target product VII (Z) -N- (2- (2- ([1,1'-biphenyl] -4-yl) -1-fluorovinyl) phenyl) -3- (difluoromethyl) -1-methyl -1H- pyrazole -4- carboxamide: weigh intermediate VI (318mg, 1mmol), intermediate III (300mg, 0.8mmol), tetrakis triphenylphosphine palladium (57.78mg, 5%) in a glove box and add them to a round-bottom flask, seal it well and take it out of the glove box, add 10ml of tetrahydrofuran bubbled with nitrogen, move to 45°C for reaction, monitor the reaction of intermediate V by TCL, add 5ml of saturated ammonium chloride solution to the reaction system to quench the reaction, and extract with ethyl acetate (3×20ml). Combine the organic phases, dry with anhydrous sodium sulfate, and chromatograph on a rotary evaporation column (petroleum ether: ethyl acetate = 2:1) to obtain the target product VII. Yield: 65%.

[0045] 1 H NMR(600MHz,Chloroform-d)δ8.42(s,1H),8.22(d,J=8.2Hz,1H),7.92(s,1H),7.68(d,J=8.3Hz,2H),7.63(s,4H),7.51(d,J=7.7Hz ,1H),7.48–7.39(m,3H),7.37(t,J=7.4Hz,1H),7.22(t,J=7.6Hz,1H),6.94(t,J=54.1Hz,1H),6.12(d,J=39.5Hz,1H),3.93(s,3H).

[0046] 13 C NMR (150MHz, Chloroform-d) δ159.91, 156.70 (d, J = 259.7Hz), 144.06 (t, J = 27.7Hz), 140.76, 140.73 (d, J = 2.3Hz), 135.59, 134.87, 132.44 (d, J = 3.6Hz), 130.78,129.83–129.59(m),129.19,127.84,127.56,127.28,125.29–125.0 4(m),123.87,117.24,111.44(d,J=10.6Hz),111.33(t,J=234.2Hz),39.97.

[0047] 19 F NMR(565MHz,Chloroform-d)δ-100.36,-110.41.

[0048] Example 2: Synthesis of (Z)-3-(difluoromethyl)-N-(2-(1-fluoro-2-(4-fluorophenyl)vinyl)phenyl)-1-methyl-1H-pyrazole-4-carboxamide (Compound 2)

[0049]

[0050] The synthesis steps a and b are the same as those in Example 1.

[0051] Synthesis step c, synthesis of intermediate V1-(2,2-difluorovinyl)-4-fluorobenzene: under N2 atmosphere protection, triphenylphosphine (3.94 g, 15 mmol), sodium difluorochloroacetate (1.85 g, 12 mmol) were added to a three-necked round-bottom flask, and 10 ml of anhydrous N, N-dimethylformamide was added to the three-necked round-bottom flask, followed by intermediate IV p-fluorobenzaldehyde (1.24 mg, 10 mmol), and the temperature was raised to 100°C for reaction for 1 hour. After the reaction of intermediate IV p-fluorobenzaldehyde was completed by TCL monitoring, 5 ml of water was added to the reaction system to quench the reaction, and petroleum ether was used for extraction (3×50 mL), the organic phases were combined and dried with anhydrous sodium sulfate, and the product V1-(2,2-difluorovinyl)-4-fluorobenzene was obtained by column chromatography (petroleum ether) under reduced pressure, with a yield of 70%.

[0052] Synthesis step d: Synthesis of intermediate VI (Z) -2- (1-fluoro-2- (4-fluorophenyl) vinyl) -4,4,5,5-tetramethyl-1,3-dioxolane: Preparation of chlorobenzene (tricyclohexylphosphine) copper (-) (Cy3P) 2CuCl catalyst: In a glove box, weigh CuCl (99 mg, 1 mmol) and Cy3P (560.86 mg, 2 mmol) in a round-bottom flask, add 10 ml of tetrahydrofuran and stir for 1 hour for use. In the glove box, weigh intermediate V (620 mg, 2 mmol), 2 ml (Cy3P)2CuCl tetrahydrofuran solution, sodium acetate (463.2 mg, 2.4 mmol), and biboric acid pinacol ester (761.82 mg, 3 mmol) into a Shrek tube, add 10 ml of anhydrous tetrahydrofuran, seal and take out of the glove box and move to an oil pan at 40 ° C to react overnight. After monitoring the reaction of intermediate V by TCL, add 5 ml of saturated ammonium chloride solution to the reaction system to quench the reaction, and extract with ethyl acetate (3×20 ml). Combine the organic phases, dry with anhydrous sodium sulfate, and evaporate to obtain intermediate VI, which is directly used in the next step.

[0053] Synthesis step e: Synthesis of target product VII (Z) -3- (difluoromethyl) -N- (2- (1-fluoro-2- (4-fluorophenyl) vinyl) phenyl) -1-methyl -1H- pyrazole -4- carboxamide: In a glove box, weigh intermediate VI (268mg, 1mmol), intermediate III (300mg, 0.8mmol), tetrakis triphenylphosphine palladium (57.78mg, 5%) into a round-bottom flask, seal it well, take it out of the glove box, add 10ml of tetrahydrofuran bubbled with nitrogen, move to 45°C for reaction, monitor the reaction of intermediate V by TCL, add 5ml of saturated ammonium chloride solution to the reaction system to quench the reaction, and extract with ethyl acetate (3×20ml). Combine the organic phases, dry and chromatograph on a rotary evaporation column (petroleum ether: ethyl acetate = 2:1) to obtain the target product VII. Yield: 45%.

[0054] 1 H NMR(600MHz,Chloroform-d)δ8.38(s,1H),8.21(d,J=8.3Hz,1H),7.93(s,2H),7.52(d,J=7.7Hz,1H),7.45(t,J=7 .9Hz,1H),7.35–7.14(m,3H),7.07(t,J=9.4Hz,1H),6.93(t,J=54.1Hz,1H),6.34(d,J=39.1Hz,1H),3.93(s,3H).

[0055] 13 C NMR 151(MHz,Chloroform-d)δ159.87(d,J=249.3Hz),159.69,158.79–

[0056] 155.67(m),143.87(t,J=27.8Hz),135.40,134.67,130.77,130.21(dd,J=13. 3,2.5Hz),129.59(d,J=4.3Hz),129.41(d,J=8.3Hz),124.98,124.72(d,J=24 .1Hz),124.36(d,J=3.6Hz),123.67,121.03(dd,J=11.8,3.6Hz),117.01,115 .41(d,J=22.2Hz), 111.12(t,J=234.2Hz), 102.88(dd,J=10.0,7.2Hz), 39.73.

[0057] 19 F NMR(565MHz,Chloroform-d)δ-98.87,-110.57,-116.32.

[0058] According to the above synthetic route, we synthesized a series of compounds. These compounds are improved by introducing fluoroolefins on the basis of pyrazole amide succinate dehydrogenase inhibitors. Some representative compound structures are as follows:

[0059]

[0060] Example 3: Antibacterial activity of novel succinate dehydrogenase inhibitors against Rhizoctonia solani

[0061] The plant-derived fungus Rhizoctonia solani was inoculated on a PDA medium, and cultured in a constant temperature incubator at 25±0.1°C for 3-6 days. The mycelium was grown and then used for later use.

[0062] 1. Determination of the inhibition rate of compounds on Rhizoctonia solani

[0063] The in vitro inhibitory activity was determined by the mycelial growth rate method. The test compound or control agent was prepared into a 40mM solution with dimethyl sulfoxide (DMSO), and then the calculated amount of the solution was mixed with PDA medium to a test concentration of 1μM. The medium was prepared into a plate in a 9cm culture dish, and the mycelium on the 0.15mm Rhizoctonia solani block was scraped and placed in the middle of the culture medium. After inoculation, it was placed in a constant temperature incubator at 25±0.1℃ for 24 hours. After the colony diameter in the blank control culture dish reached 7.5-8cm, the colony diameter of each treatment and control was measured by the cross method, and the inhibition rate was calculated. There were 3 replicates in each group. DMSO was used as the blank control, and Boscalid and Fluxapyroxad were used as positive controls.

[0064] Mycelial growth inhibition rate (%) = [blank colony growth diameter (mm) - colony growth diameter on drug-containing medium (mm)] / control colony growth diameter (mm) × 100.

[0065] The inhibition rate of the synthesized succinate dehydrogenase inhibitor on Rhizoctonia solani is shown in the following table:

[0066] Inhibitors Inhibition rate (%) Inhibitors Inhibition rate (%) Compound 1 90.82 Compound 8 91.62 Compound 2 88.40 Compound 9 87.76 Compound 3 91.26 Compound 10 92.43 Compound 4 86.63 Compound 11 89.21 Compound 5 87.44 Compound 12 89.05 Compound 6 90.34 Compound 13 92.11 Compound 7 84.54 Compound 14 90.82 Boscalid 42.97 Fluopyrab 84.38

[0067] 2. EC50 determination of compounds inhibiting Rhizoctonia solani

[0068] In order to further determine the fungicidal activity of these new compounds, the linear regression relationship between the probability value (y) and the logarithm of the mass concentration (x) was performed according to the mycelial growth inhibition rate. SPSS Statistics software was used to analyze the data and the toxicity regression equation y = a + bx, the inhibition concentration (EC50) and the correlation coefficient (R 2 ).

[0069] Test compounds: Based on the initial screening, the compounds with better effects were selected for rescreening, with pyraclostrobin and fluopyrabdo as positive controls and DMSO as blank control.

[0070] Experimental method: For Rhizoctonia solani, six concentration gradients with PDA medium were set at 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 μM, and the concentration was diluted twice. The experiment set up PDA medium without drugs. PDA with DMSO was used as the blank control. The culture medium was prepared into a plate in a 9 cm culture dish. The hyphae on the 0.15 mm Rhizoctonia solani block were scraped and placed in the middle of the culture medium. After inoculation, it was placed in a constant temperature incubator at 25±0.1℃ for 24 hours. When the colony diameter in the blank control culture dish was 7.5-8 cm, the colony diameter of each treatment and control was measured by the cross method. Each test group and blank control were repeated three times.

[0071] The test results are shown in the table below:

[0072] Inhibitors regression equation EC50(nM) 95%confidenceinterval <![CDATA[R 2 ]]> Compound 3 Y=3.02+1.54X 91.20±1.05 65.889-119.175 0.976 Compound 6 Y=4.42+1.95X 187.9±1.71 151.543-233.703 0.988 Compound 9 Y=3.96+1.84X 172.5±2.56 137.758-217.474 0.956 Compound 10 Y=3.72+1.74X 136.6±2.31 106.558-172.453 0.982 Compound 12 Y=3.69+1.87X 92.14±2.72 70.514-115.836 0.951 Compound 13 Y=3.26+1.38X 227.1±1.67 162.208-312.048 0.919 Compound 14 Y=2.57+1.48X 54.65±1.55 14.215-101.280 0.905 Fluopyrab Y=4.84+2.07X 210.8±0.15 125.832-375.091 0.893 Boscalid Y=8.04+2.35X 2702.2±89.0 2129.482-3680.138 0.963

[0073] According to the above results, the inhibitory effects of the compounds synthesized by the present invention on Rhizoctonia solani are generally better than those of commercial succinate dehydrogenase inhibitors. The present invention is improved on the basis of pyrazoleamide fungicides by introducing monofluoroolefins into the hydrophobic structure. The antibacterial test further confirms that the monofluoroolefin structure can improve the biological activity of pyrazoleamide fungicides.

[0074] Example 4: Antifungal activity of novel succinate dehydrogenase inhibitors against other plant fungi

[0075] Taking compound 14 as an example, the inhibitory rate against other plant fungi such as Fusarium graminearum, Botrytis cinerea, Sclerotiniasclerotiorum, Pestalotiopsis, Monilinia fructicola, Altermaria alternata, Cochliobolus heterostrophus, and Altermaria alternata was further investigated at 200 μM. The test method is as follows:

[0076] Corn leaf spot pathogen (Cochliobolus heterostrophus): Prepare PDA solid culture medium, then add succinate dehydrogenase inhibitor to the culture medium with a final concentration of (200uM), take bacterial blocks of the same size and place them in the middle of the culture medium, and culture them in the dark at room temperature for 5 days. Measure the colony diameter using the cross method and calculate the inhibition rate. Repeat 3 times for each group.

[0077] Sclerotinia sclerotiorum: Prepare PDA solid culture medium, then add succinate dehydrogenase inhibitor to the culture medium, add the final concentration of (200uM), take hyphae from the same size of bacterial blocks and place them in the middle of the culture medium, culture at room temperature for 7 days, measure the colony diameter by the cross method, calculate the inhibition rate, and repeat 3 times for each group.

[0078] Tomato gray mold (Botrytis cinerea): Prepare PDA solid culture medium, then add succinate dehydrogenase inhibitor to the culture medium, add the final concentration of (200uM), take bacterial blocks of the same size and place them in the middle of the culture medium, culture at room temperature for 4 days, measure the colony diameter by the cross method, calculate the inhibition rate, and repeat 3 times for each group.

[0079] Pestalotiopsis: Prepare PDA solid culture medium, then add succinate dehydrogenase inhibitor to the medium with a final concentration of (200uM), take bacterial blocks of the same size and place them in the middle of the culture medium, culture them at room temperature for 3 days, measure the colony diameter using the cross method, calculate the inhibition rate, and repeat 3 times for each group.

[0080] Peach brown rot pathogen (Monilinia fructicola): Prepare PDA solid culture medium, then add succinate dehydrogenase inhibitor to the culture medium with a final concentration of (200uM), take bacterial blocks of the same size and place them in the middle of the culture medium, and culture them in the dark at room temperature for 5 days. Measure the colony diameter using the cross method and calculate the inhibition rate. Repeat 3 times for each group.

[0081] Fusarium graminearum: Place PDA solid culture medium, then add succinate dehydrogenase inhibitor to the medium, add the final concentration of (200uM), take bacterial blocks of the same size and place them in the middle of the culture medium, culture them in the dark at room temperature for 5 days, measure the colony diameter by the cross method, calculate the inhibition rate, and repeat 3 times for each group.

[0082] Tea tree Altermaria alternata: Place PDA solid culture medium, then add succinate dehydrogenase inhibitor to the medium, add the final concentration of (200uM), take bacterial blocks of the same size and place them in the middle of the culture medium, culture them in the dark at room temperature for 3 days, measure the colony diameter by the cross method, calculate the inhibition rate, and repeat 3 times for each group.

[0083] The test results are shown in the following table:

[0084]

[0085] From the above results, it can be seen that the compounds synthesized in the present invention have inhibitory effects on a variety of plant fungi, among which the inhibition rate on corn leaf blight, peach brown rot fungus and alternaria is significantly higher than that of commercial pyrazole amide fungicides.

[0086] Example 5: In vivo test

[0087] Compound 14 was selected for in vivo experiments on rapeseed and tomatoes. The specific experimental methods are as follows:

[0088] Rapeseed leaves with sclerotinia: Rapeseed leaves were provided by the College of Plant Sciences of Huazhong Agricultural University. Healthy and disease-free rapeseed leaves of the same leaf age were picked, and the surface of the leaves was wiped with 75% alcohol. The leaves were then washed with sterile water, placed on sterilized filter paper to dry, and the petioles were wrapped with wet cotton balls. Compound 14 and the control agent boscalid were prepared into solutions with concentrations of 25μM, 12.5μM, 6.25μM, and 3.125μM. A 0.1cm wound was scratched at a similar position on the rapeseed leaf with a small blade, and 10μL of the target compound and the control agent were dripped into the wound of the rapeseed leaf, and then a piece of rapeseed sclerotinia cake (d=5mm) was inoculated on each leaf wound.

[0089] Fruits infected with Botrytis cinerea: Pick healthy, disease-free tomatoes of uniform size, wipe the surface with 75% alcohol, then wash the leaves with sterile water, place them on sterilized filter paper to dry for later use, use a sterilized blade to make a 2 cm long cross wound on the tomato, then drop a suspension containing compound 14 and boscalid Botrytis cinerea spores on the tomato wound, place them in a constant temperature incubator for four days, and observe the infection of the wound.

[0090] from Figure 3 and Figure 4 It can be seen that the compound synthesized in the present invention has a good in vivo protective effect on fungal diseases of rapeseed leaves and tomatoes, and has obvious dose dependence.

Claims

1. A succinate dehydrogenase inhibitor, the structural formula of which is shown in the following formula (I): The compound of formula (I) is specifically:

2. The succinate dehydrogenase inhibitor according to claim 1, characterized in that: R1 is difluoromethyl; R2 is 3,4,5-trifluoro.

3. A method for synthesizing the succinate dehydrogenase inhibitor according to claim 1 or 2, characterized in that The following steps are involved: In the synthesis step a, the raw material I is dissolved in anhydrous dichloromethane, N,N-dimethylformamide is added as a catalyst, and then oxalyl chloride is added dropwise to react at room temperature for 2-3 hours, and the tail gas is absorbed by a saturated sodium hydroxide solution. After the reaction is completed, dichloromethane is used for extraction, and the organic phase is concentrated and dried to obtain the intermediate II; In the synthesis step b, dichloromethane is used as the reaction solvent, and 2-iodoaniline is reacted with the intermediate II under the protection of N2 atmosphere. After the reaction is completed, water is added to quench the reaction, and dichloromethane is used for extraction. The organic phase is concentrated and dried, and then recrystallized with ethyl acetate and petroleum ether to obtain the intermediate III; In the synthesis step c, under the protection of N2 atmosphere, triphenylphosphine, sodium difluorochloroacetate, N,N-dimethylformamide, and raw material IV are added, and the temperature is raised to 100°C for reaction. After the reaction is completed, petroleum ether is used for extraction, and the organic phases are combined and concentrated and dried, and intermediate V is obtained after column chromatography; In the synthesis step d, tetrahydrofuran is used as a reaction solvent, (Cy3P)2CuCl is used as a catalyst, and the intermediate V, sodium acetate, and biboric acid pinacol ester are reacted. After the reaction is completed, the reaction is quenched with a saturated ammonium chloride solution, extracted with ethyl acetate, and the organic phases are combined and concentrated to obtain the intermediate VI; In the synthesis step e, tetrahydrofuran is used as the reaction solvent and tetrakis(triphenylphosphine)palladium is used as the catalyst to react the intermediate VI and the intermediate III. After the reaction is complete, the reaction is quenched with a saturated ammonium chloride solution, extracted with ethyl acetate, and the organic phases are combined, concentrated and dried, and the target product VII is obtained after column chromatography.

4. Use of the succinate dehydrogenase inhibitor according to claim 1 or 2 in preventing and controlling plant fungal diseases.

5. A fungicide for preventing and controlling plant fungal diseases, wherein the active ingredient of the fungicide comprises the succinate dehydrogenase inhibitor according to claim 1 or 2.

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