Phenoxybenzamide derivatives containing substituted pyrazoles, methods of preparation and uses thereof

Abstract

The application discloses a substituted pyrazole-containing phenoxybenzamide derivative, a preparation method and application thereof, wherein R1 in the chemical structural general formula of the derivative is trifluoromethyl or difluoromethyl; R2 is hydrogen, methyl, methoxy, trifluoromethyl, fluorine, chlorine, bromine or a multi-substituted combination thereof at different positions; and R3 is hydrogen, fluorine, chlorine, bromine or thiocyanogen. The application organically combines a phenoxybenzoic acid structure and a substituted pyrazole fragment through an amide bond, synthesizes a new type of substituted pyrazole-containing phenoxybenzamide derivative, the compound has good bacteriostatic activity, and the bacteriostatic mechanism of the compound is preliminarily determined through molecular docking and SDH inhibition activity. The application provides a basis for developing a new bactericide of the substituted pyrazole-containing phenoxybenzamide.

Description

Technical Field

[0001] This invention relates to the technical field of pesticide chemistry, and in particular to a phenoxybenzamide derivative containing substituted pyrazole, its preparation method, and its application. Background Technology

[0002] Pesticides are essential production materials in food production, playing a crucial role in ensuring high and stable crop yields. Statistics show that plant pathogenic fungal infections reduce global crop yields by 20% annually, with an additional 10% reduction after harvest. This not only causes huge economic losses for farmers but also seriously threatens global food security. The application of fungicides is currently the most effective measure for controlling plant pathogenic fungi, saving significant losses each year. Among numerous fungicides, SDHIs (Synthetic Pathogens Inhibitors) are widely used due to their highly effective and broad-spectrum antifungal activity. Their mechanism of action involves affecting the respiratory chain electron transport system of pathogens by acting on protein complex II, hindering energy metabolism, inhibiting pathogen growth, and leading to their death, thereby preventing disease.

[0003] The most significant chemical characteristic of SDHIs (Synthetic Activated Hydroxide Inhibitors) is the presence of an amide group. Newly developed SDHIs are derived from existing ones through group substitution. From 1996 to 2022, 24 SDH inhibitors were marketed, 13 of which contain a pyrazole ring, representing 54.17% (13 / 24). Due to their broad-spectrum and highly effective antifungal activity, these fungicides have been used to control fungal diseases on many crops and have become an important class of fungicides in production. However, with the widespread use and misuse of SDHIs, coupled with their increasing years of use, many plant pathogenic fungi have developed significant resistance to existing SDHIs. Therefore, there is an urgent need to develop new and highly effective SDHIs to effectively control plant diseases and delay fungal resistance development. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0005] Therefore, this invention proposes a phenoxybenzamide derivative containing substituted pyrazole, its preparation method, and its application. It utilizes a diphenyl ether structure with a superior structure and organically combines the diphenyl ether structure with the substituted pyrazole through an amide bond to synthesize a novel class of phenoxybenzamide derivatives containing substituted pyrazole, which becomes a compound with highly efficient and broad-spectrum inhibitory activity against plant pathogens.

[0006] According to an embodiment of the present invention, a phenoxybenzamide derivative containing a substituted pyrazole is characterized in that its general chemical structural formula (I) is as follows:

[0007]

[0008] Wherein, R1 is trifluoromethyl or difluoromethyl; R2 is hydrogen, methyl, methoxy, trifluoromethyl, fluorine, chlorine, bromine, or a combination of multiple substitutions thereof, substituted at different positions; and R3 is hydrogen, fluorine, chlorine, bromine, or thiocyanate.

[0009] This invention also provides a method for preparing phenoxybenzamide derivatives containing substituted pyrazoles, wherein the phenoxybenzamide derivatives containing substituted pyrazoles are synthesized via the following synthetic route:

[0010]

[0011] Intermediate II is a substituted pyrazolamine; intermediate III is a substituted phenoxybenzoic acid.

[0012] The present invention also provides an application of a phenoxybenzamide derivative containing substituted pyrazole, wherein the phenoxybenzamide derivative containing substituted pyrazole is applied to an agricultural fungicide.

[0013] The beneficial effects of this invention are that it organically combines the phenoxybenzoic acid structure with the substituted pyrazole fragment via an amide bond to synthesize a novel class of phenoxybenzamide derivatives containing substituted pyrazoles. Common plant fungi and oomycetes, such as *Sclerotinia sclerotiorum*, *Pseudomonas aeruginosa*, *Rhizoctonia solani*, *Botrytis cinerea*, *Phytophthora capsulatum*, and *Tricholoma materia humicana*, were selected as targets for antibacterial activity assays. The results showed that these compounds exhibited broad-spectrum antibacterial activity against plant pathogenic fungi, particularly showing excellent antibacterial activity against *Pseudomonas aeruginosa* and *Sclerotinia sclerotiorum*. Structure-activity relationship analysis indicated that the fluorine substitution on the trifluoromethylpyrazole ring and the 3,5-di-Cl substitution on the benzene ring in the molecular skeleton play a crucial role in maintaining the high efficiency and broad-spectrum antibacterial activity of these compounds. These compounds possess original and novel structures, are simple to synthesize, and are completely different from existing commercial fungicides, showing promise for development into a novel, efficient, safe, economical, and environmentally friendly green fungicide.

[0014] According to one embodiment of the present invention, the phenoxybenzamide derivative I containing substituted pyrazole is a compound as follows:

[0015]

[0016] According to one embodiment of the present invention, the specific preparation steps are as follows:

[0017] Using substituted pyrazolamide (intermediate II) and substituted phenoxybenzoic acid (intermediate III) as raw materials, an amide condensation reaction was carried out under dichloromethane and alkaline conditions using tetramethylchlorourea hexafluorophosphate (TCFH) as a condensing agent to generate phenoxybenzamide derivatives containing substituted pyrazol, i.e., general formula (I). The obtained product was purified by column chromatography to obtain a pure product.

[0018] According to one embodiment of the present invention, the phenoxybenzamide derivative containing substituted pyrazole is applied to control plant pathogens such as apple rot fungus, grape gray mold fungus, wheat take-all fungus, rapeseed sclerotinia rot fungus, and rice sheath blight fungus.

[0019] According to one embodiment of the present invention, the phenoxybenzamide derivative containing substituted pyrazole is used to prepare various pesticide formulations for the prevention and control of plant diseases caused by plant pathogens.

[0020] Other features and advantages of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention are realized and obtained through the structures particularly pointed out in the description and the drawings.

[0021] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The efficacy of compound I-21 against Sclerotinia sclerotinia in rapeseed (100 mg / L);

[0024] Figure 2 The efficacy of compounds I-17 and I-21 against apple rot pathogens (100 mg / L);

[0025] Figure 3 It refers to the binding modes of compounds I-12, I-17, I-21, and fluopyram with SDH. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] The following describes in detail the phenoxybenzamide derivatives containing substituted pyrazoles, their preparation methods, and their applications according to embodiments of the present invention.

[0028] Example 1

[0029] The phenoxybenzamide derivative I-1 containing substituted pyrazole is N-(1-methyl-3-trifluoromethyl-1H-pyrazole-5-yl)-2-phenoxybenzamide.

[0030] The specific preparation method of phenoxybenzamide derivative I-1 containing substituted pyrazole in this embodiment is as follows: At room temperature, 1 mmol of intermediate pyrazoleamine II-a, 1.1 mmol of 2-phenoxybenzoic acid, 3 mL of anhydrous dichloromethane, 3.5 mmol of N-methylimidazolium, and 1.2 mmol of tetramethylchlorourea hexafluorophosphate were added sequentially to a 10 mL pressure-resistant tube. The mixture was then stirred for 10 minutes, and the reaction system was heated to 40 °C and reacted for 4 hours. The reaction was tracked by TLC. After the reaction was completed, the mixture was extracted, dried, concentrated, and column filtered to obtain a yellow solid, namely compound I-1, with a yield of 51.5% and a melting point of 137.2–140.1 °C. 1 H NMR (400MHz, DMSO-d6) δ10.66(s,1H),7.77(d,J=7.5Hz,1H),7.56(t,J=7.5Hz,1H),7.41(t,J=7.9

[0031] Hz,2H),7.30(t,J=7.6Hz,1H),7.17(t,J=7.4Hz,1H),7.08(d,J=8.0Hz,2H),7.00(d,J=8.2Hz,1H),6.67(s,1H),3.69(s,3H); 13 C NMR(126MHz,DMSO-d6)δ164.8,156.7,154.4,139.3(q,J=37.3Hz),138.3,133.2,130.6,1 30.6,127.2,124.3,124.2,122.6(q,J=62.1Hz),119.4,119.2,118.0,98.2,36.8; ESI-MS calculated for C18 H 15 F3N3O2[M+H] + ,362.1111; found 362.1118.

[0032] Example 2

[0033] The phenoxybenzamide derivative I-2 containing substituted pyrazole is N-(1-methyl-3-trifluoromethyl)-1H-pyrazole-5-yl)-2-o-tolylbenzamide.

[0034] The preparation method of the phenoxybenzamide derivative I-2 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(o-tolyloxy)benzoic acid is substituted for 2-phenoxybenzoic acid, and the other steps are the same as in Example 1. The resulting compound I-2 is a yellow solid with a yield of 57.2% and a melting point of 112.5–113.7 °C. 1 HNMR (400MHz, DMSO-d6) δ10.65(s,1H),7.81–7.75(m,1H),7.50(t,J=8.4Hz,1H),7.34(d,J=7.1Hz,1H),7.25(t,J=7. 4Hz,2H),7.13(t,J=7.4Hz,1H),6.99(d,J=7.9Hz,1H),6.77(d,J=8.3Hz,1H),6.71(s,1H),3.72(s,3H),2.20(s,3H); 13 C NMR(101MHz,DMSO-d6)δ164.8,155.0,154.0,139.8,139.4,139.1,138.7,138.3,133.1,132.0,130.6,129.7 ,128.0,125.9,125.8,125.1,123.3,123.1,120.4,119.9,117.8,117.4,98.3,36.7,16.2; ESI-MScalculated for C 19 H 17 F3N3O2[M+H] + ,376.1267; found376.1274.

[0035] Example 3

[0036] The phenoxybenzamide derivative I-3 containing substituted pyrazole is N-methyl-3-trifluoromethyl-1H-pyrazole-5-yl)-2-m-tolylbenzamide.

[0037] The preparation method of the phenoxybenzamide derivative I-3 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(m-tolyloxy)benzoic acid is substituted for 2-phenoxybenzoic acid; the other steps are the same as in Example 1. The resulting compound I-3 is a yellow solid with a yield of 62.3% and a melting point of 145.0–147.3 °C. 1 HNMR (400MHz, DMSO-d6) δ10.63(s,1H),7.76(d,J=7.5Hz,1H),7.55(t,J=7.8Hz,1H),7.28(t,J =7.8Hz,2H),6.99(d,J=7.9Hz,2H),6.93–6.83(m,2H),6.66(s,1H),3.69(s,3H),2.29(s,3H); 13 C NMR (101MHz, DMSO-d6) δ164.7, 156.7, 154.4, 140.3, 139.2 (d, J = 37.3Hz), 138.3, 133.2, 130.5, 130.3,127.1,125.1,124.0,120.1(d,J=71.9Hz),119.7,119.4,116.2,98.1,36.7,21.4; ESI-MS calculated for C 19 H 17 F3N3O2[M+H] + ,376.1267; found 376.1273.

[0038] Example 4

[0039] The phenoxybenzamide derivative I-4 containing substituted pyrazole is N-(1-methyl-3-trifluoromethyl)-1H-pyrazole-5-yl)-2-p-tolylbenzamide.

[0040] The preparation method of the phenoxybenzamide derivative I-4 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(p-tolyloxy)benzoic acid is substituted for 2-phenoxybenzoic acid, and the other steps are the same as in Example 1. The resulting compound I-4 is a yellow solid with a yield of 57.5% and a melting point of 148.2–151.4 °C. 1H NMR(400MHz,Chloroform-d)δ9.92(s,1H),8.32(s,1H),7.51–7.42(m,1H),7.28–7.26(m,1H),7.24(dd, J=5.6,1.8Hz,2H),7.03(d,J=8.5Hz,2H),6.87(d,J=8.3Hz,1H),6.81(s,1H),3.73(s,3H),2.39(s,3H); 13 C NMR(101MHz,Chloroform-d)δ161.9,156.4,152.1,141.2(q,J=38.2Hz),137.2,135.6,134.2,1 32.7,131.1,125.1,123.8,121.1(q,J=268.6Hz),119.8,119.8,117.6,97.2,35.9,20.8; ESI-MS calculated for C 19 H 17 F3N3O2[M+H] + ,376.1267; found376.1277.

[0041] Example 5

[0042] The phenoxybenzamide derivative I-5 containing substituted pyrazole is 2-(2-methoxyphenoxy)-N-(1-methyl-3-trifluoromethyl)pyrazole-5-ylbenzamide.

[0043] The preparation method of the phenoxybenzamide derivative I-5 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(o-methoxyphenyloxy)benzoic acid is substituted for 2-phenoxybenzoic acid, and the other steps are the same as in Example 1. The resulting compound I-5 is a yellow solid with a yield of 66.3% and a melting point of 63.2–66.0 °C. 1 HNMR(400MHz,Chloroform-d)δ10.00(s,1H),8.24(d,J=7.9Hz,1H),7.44–7.38(m,1H),7.33–7 .27(m,1H),7.23–7.18(m,2H),7.10–7.03(m,2H),6.83–6.78(m,2H),3.79(s,3H),3.76(s,3H); 13C NMR (126MHz, Chloroform-d) δ162.5, 156.6, 151.2, 142.3, 141.1 (q, J = 38.2Hz), 137.4, 133.8, 132. 5,127.1,123.3,122.8,122.7,121.5,121.2(q,J=268.5Hz),121.1,120.1,97.1,55.9,35.8; ESI-MS calculated for C 19 H 17 F3N3O3[M+H] + ,392.1217; found 392.1218.

[0044] Example 6

[0045] The phenoxybenzamide derivative I-6 containing substituted pyrazole is 2-(3-methoxyphenoxy)-N-(1-methyl-3-trifluoromethyl)pyrazole-5-ylbenzamide.

[0046] The preparation method of phenoxybenzamide derivative I-6 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(3-methoxyphenyloxy)benzoic acid is substituted for 2-phenoxybenzoic acid, and the other steps are the same as in Example 1. The resulting compound I-6 is a yellow solid with a yield of 59.2% and a melting point of 102.5–103.5 °C. 1 HNMR (400MHz, DMSO-d6) δ10.64(s,1H),7.75(d,J=7.5Hz,1H),7.50(t,J=7.8Hz,1H),7.22(t,J=7.5H z,1H),7.13(s,2H),7.00(d,J=9.0Hz,2H),6.84(d,J=8.3Hz,1H),6.73(s,1H),3.75(d,J=3.7Hz,6H); 13 C NMR (101MHz, DMSO-d6) δ164.7, 156.5, 155.9, 149.2, 139.3 (q, J = 37.4Hz), 138.3, 133.1, 130. 6,125.9,123.1,122.5,121.5,119.13(q,J=267.7Hz),117.4,115.6,98.0,55.9,36.8; ESI-MS calculated for C 19 H 17 F3N3O3[M+H] + ,392.1217; found 392.1225.

[0047] Example 7

[0048] The phenoxybenzamide derivative I-7 containing substituted pyrazole is 2-(4-methoxyphenoxy)-N-(1-methyl-3-trifluoromethyl)pyrazole-5-ylbenzamide.

[0049] The preparation method of the phenoxybenzamide derivative I-7 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(4-methoxyphenyloxy)benzoic acid is substituted for 2-phenoxybenzoic acid, and the other steps are the same as in Example 1. The resulting compound I-7 is a yellow solid with a yield of 48.1% and a melting point of 113.6–116.2 °C. 1 HNMR (400MHz, DMSO-d6) δ10.64(s,1H),7.76(d,J=7.5Hz,1H),7.56(t,J=7.8Hz,1H),7.31(t,J=8.2Hz ,2H),7.03(d,J=8.2Hz,1H),6.75(dd,J=8.0,2.1Hz,1H),6.69–6.60(m,3H),3.73(s,3H),3.70(s,3H); 13 C NMR(126MHz,DMSO-d6)δ164.7,161.2,157.8,154.2,139.3,138.3,133.2,131.1, 130.5,127.2,124.2,119.6,118.5,111.2,110.0,105.4,98.2,55.8,36.8; ESI-MS calculated for C 19 H 17 F3N3O3[M+H] + ,392.1217;found 392.1217.

[0050] Example 8

[0051] The phenoxybenzamide derivative I-8 containing substituted pyrazole is 2-(2-fluorophenoxy)-N-(1-methyl-3-trifluoromethyl)-1H-pyrazole-5-ylbenzamide.

[0052] The preparation method of the phenoxybenzamide derivative I-8 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(o-fluorophenyloxy)benzoic acid is substituted for 2-phenoxybenzoic acid, and the other steps are the same as in Example 1. The resulting compound I-8 is a yellow solid with a yield of 39.2% and a melting point of 118.2–121.0 °C. 1HNMR (400MHz, DMSO-d6) δ10.72(s,1H),7.78(d,J=7.5Hz,1H),7.55(t,J=8.5Hz,1H),7.41(ddd,J=9.9,7.7,3. 3Hz,1H),7.30(t,J=7.6Hz,1H),7.24(dd,J=6.2,3.6Hz,3H),6.94(d,J=8.3Hz,1H),6.71(s,1H),3.76(s,3H); 13 C NMR (101MHz, DMSO-d6) δ164.6, 155.0, 154.3, 152.5, 143.2, 143.1, 139.3 (q, J = 37.5Hz), 138.3, 133.2, 130.6, 126. 3,126.2,126.2,126.0,125.9,124.1,123.1,122.2,121.8(q,J=268.0Hz),117.7,117.6,117.6,98.2,36.8; ESI-MS calculated for C 18 H 14 F4N3O2[M+H] + ,380.1017; found 380.1020.

[0053] Example 9

[0054] The phenoxybenzamide derivative I-9 containing substituted pyrazole is 2-(3-fluorophenoxy)-N-(1-methyl-3-trifluoromethyl)pyrazole-5-ylbenzamide.

[0055] The preparation method of phenoxybenzamide derivative I-9 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(3-fluorophenyloxy)benzoic acid is substituted for 2-phenoxybenzoic acid, and the other steps are the same as in Example 1. The resulting compound I-9 is a yellow solid with a yield of 64.2% and a melting point of 142.1–145.0 °C. 1 HNMR(400MHz,Chloroform-d)δ9.5(s,1H),8.3(s,1H),7.5(ddd,J=8.8,7.4,1.8Hz,1H),7.4(td,J=8.3,6.4Hz,1H),7 .3(td,J=7.6,1.0Hz,1H),7.0(td,J=8.3,2.4Hz,1H),6.9(s,2H),6.9(dt,J=9.4,2.4Hz,1H),6.8(s,1H),3.7(s,3H). 13C NMR (101MHz, Chloroform-d) δ164.9, 162.4, 161.8, 155.9 (d, J = 10.4Hz), 155.0, 141.2 (q, J = 38.5Hz), 136.8, 134.4, 132.9, 131.6 (d, J = 9. 6Hz), 124.8, 122.2, 121.0 (q, J = 268.5Hz), 118.6, 115.1 (d, J = 3.4Hz), 112.6 (d, J = 21.1Hz), 107.5 (d, J = 24.7Hz), 97.6, 36.0, 0.0; ESI-MS calculatedfor C 18 H 14 F4N3O2[M+H] + ,380.1017; found380.1018.

[0056] Example 10

[0057] The phenoxybenzamide derivative I-10 containing substituted pyrazole is 2-(4-fluorophenoxy)-N-(1-methyl-3-trifluoromethyl)pyrazole-5-ylbenzamide.

[0058] The preparation method of phenoxybenzamide derivative I-10 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(4-fluorophenyloxy)benzoic acid is substituted for 2-phenoxybenzoic acid, and the other steps are the same as in Example 1. The resulting compound I-10 is a yellow solid with a yield of 52.8% and a melting point of 149.2–150.1 °C. 1 HNMR (400MHz, DMSO-d6) δ10.69(s,1H),7.76(d,J=7.4Hz,1H),7.54(t,J=7.8Hz,1H),7.27(q ,J=8.5,7.6Hz,3H),7.17(d,J=3.9Hz,2H),6.94(d,J=8.2Hz,1H),6.70(s,1H),3.74(s,3H); 13 C NMR (101MHz, DMSO-d6) δ164.7, 160.2, 157.8, 154.9, 152.5, 139.3 (q, J = 37.6Hz), 138.3, 133.2, 130. 6,126.8,123.9,123.1,121.8(q,J=268.0Hz),121.4,120.4,118.6,117.2,117.0,98.1,36.8; ESI-MS calculated for C 18 H 14 F4N3O2[M+H]+ ,380.1017; found 380.1027.

[0059] Example 11

[0060] The phenoxybenzamide derivative I-11 containing substituted pyrazole is 2-(2-chlorophenoxy)-N-(1-methyl-3-trifluoromethyl)pyrazole-5-ylbenzamide.

[0061] The preparation method of the phenoxybenzamide derivative I-11 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(2-chlorophenyloxy)benzoic acid is substituted for 2-phenoxybenzoic acid, and the other steps are the same as in Example 1. The resulting compound I-11 is a yellow solid with a yield of 71.2% and a melting point of 124.6–125.7 °C. 1 HNMR(400MHz,Chloroform-d)δ9.65(s,1H),8.32(d,J=9.4Hz,1H),7.60–7.55(m,1H),7.51–7.44(m,1H),7.42 –7.35(m,1H),7.33–7.27(m,2H),7.20(dt,J=8.1,2.1Hz,1H),6.81(s,1H),6.75(d,J=8.3Hz,1H),3.79(s,3H); 13 C NMR(101MHz,DMSO-d6)δ164.7,158.0,153.4,139.8,139.5,139.1,138.7,138.2,134.4,1 33.3,131.9,130.6,127.8,124.9,124.1,120.4,120.3,118.8,117.6,98.2,36.8; ESI-MS calculated for C 18 H 14 ClF3N3O2[M+H] + ,396.0721;found396.0721.

[0062] Example 12

[0063] The phenoxybenzamide derivative I-12 containing substituted pyrazole is 2-(3-chlorophenoxy)-N-(1-methyl-3-trifluoromethyl)pyrazole-5-ylbenzamide.

[0064] The preparation method of the phenoxybenzamide derivative I-12 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(3-chlorophenyloxy)benzoic acid is substituted for 2-phenoxybenzoic acid, and the other steps are the same as in Example 1. The resulting compound I-12 is a yellow solid with a yield of 68.5% and a melting point of 137.1–139.3 °C. 1 HNMR (400MHz, DMSO-d6) δ10.71(s,1H),7.79(d,J=7.6Hz,1H),7.60(t,J=8.5Hz,1H),7.40(dt,J=21.9,7 .9Hz,2H),7.25–7.20(m,1H),7.15–7.09(m,2H),7.04(dd,J=8.3,2.4Hz,1H),6.66(s,1H),3.71(s,3H); 13 C NMR (101MHz, DMSO-d6) δ164.7, 158.0, 153.4, 139.3 (q, J = 37.7Hz), 138.2, 134.4, 133.3, 131. 9,130.6,127.8,124.9,124.1,121.7(q,J=268.2Hz), 120.3,118.8,117.6,98.2,36.8; ESI-MS calculated for C 18 H 14 ClF3N3O2[M+H] + ,396.0721; found 396.0729.

[0065] Example 13

[0066] The phenoxybenzamide derivative I-13 containing substituted pyrazole is 2-(4-chlorophenoxy)-N-(1-methyl-3-trifluoromethyl)pyrazole-5-ylbenzamide.

[0067] The preparation method of the phenoxybenzamide derivative I-13 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(4-chlorophenyloxy)benzoic acid is substituted for 2-phenoxybenzoic acid, and the other steps are the same as in Example 1. The resulting compound I-13 is a yellow solid with a yield of 62.3% and a melting point of 138.1–140.2 °C. 1HNMR (400MHz, DMSO-d6) δ10.69(s,1H),7.78(d,J=7.5Hz,1H),7.60(t,J=7.6Hz,1H),7.39(dt,J=21.6,7 .8Hz,2H),7.22(d,J=8.6Hz,1H),7.11(d,J=8.1Hz,2H),7.03(d,J=8.1Hz,1H),6.65(s,1H),3.70(s,3H); 13 C NMR (101MHz, DMSO-d6) δ164.7,158.0,153.4,139.9,139.5,139.1,138.7,138.2,134.4,133.3,131. 9,130.6,127.8,125.7,124.9,124.1,123.1,120.4,120.3,118.8,117.7,117.6,98.2,36.8; ESI-MS calculated forC 18 H 14 ClF3N3O2[M+H] + ,396.0721; found 396.0731.

[0068] Example 14

[0069] The phenoxybenzamide derivative I-14 containing substituted pyrazole is N-(1-methyl-3-trifluoromethyl)-1H-pyrazole-5-yl)-2-(3-trifluoromethylphenoxy)benzamide.

[0070] The preparation method of the phenoxybenzamide derivative I-14 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(3-trifluoromethylphenyloxy)benzoic acid is substituted for 2-phenoxybenzoic acid, and the other steps are the same as in Example 1. The resulting compound I-14 is a yellow solid with a yield of 34.2% and a melting point of 150.2–151.7 °C. 1 H NMR(400MHz,Chloroform-d)δ9.45(s,1H),8.36(s,1H),7.64–7.51(m,3H),7. 41(s,1H),7.38–7.29(m,2H),6.90(d,J=8.2Hz,1H),6.74(s,1H),3.74(s,3H); 13C NMR(101MHz,Chloroform-d)δ161.9,155.1,141.2(q,J=38.5Hz),136.7,134.5,133.3(q,J=33.3Hz),133.1,132 .8,131.4,124.9,124.6,122.9,122.3(q,J=12.9Hz),122.3,119.7,116.7(q,J=3.7Hz),97.9,36.0,0.0; ESI-MS calculated for C 19 H 14 F6N3O2[M+H] + ,430.0985; found430.0999.

[0071] Example 15

[0072] The phenoxybenzamide derivative I-15 containing substituted pyrazole is N-(1-methyl-3-trifluoromethyl)-1H-pyrazole-5-yl)-2-(4-trifluoromethylphenoxy)benzamide.

[0073] The preparation method of the phenoxybenzamide derivative I-15 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(4-trifluoromethylphenyloxy)benzoic acid is substituted for 2-phenoxybenzoic acid, and the other steps are the same as in Example 1. The resulting compound I-15 is a yellow solid with a yield of 29.0% and a melting point of 116.8–119.2 °C. 1 H NMR(400MHz,Chloroform-d)δ9.43(s,1H),8.33(d,J=1.8Hz,1H),7.72(d,J=8.5Hz,2H),7.60–7.51( m,1H),7.37(t,J=7.6Hz,1H),7.22(d,J=8.5Hz,2H),6.94(d,J=9.0Hz,1H),6.74(s,1H),3.72(s,3H); 13 C NMR (101MHz, Chloroform-d) δ161.8,157.7,154.6,141.1(q,J=38.5Hz), 136.7,134.5,132.9,128.1(q,J=3.7H z), 127.7 (q, J = 33.2Hz), 125.2, 125.0, 122.5, 122.3 (q, J = 2.3Hz), 119.7, 119.5 (2C), 118.9, 97.8, 36.0; ESI-MS calculated for C 19 H 14F6N3O2[M+H] + ,430.0985; found 430.0988.

[0074] Example 16

[0075] The phenoxybenzamide derivative I-16 containing substituted pyrazole is 2-(3,4-dichlorophenoxy)-N-(1-methyl-3-trifluoromethyl)pyrazole-5-ylbenzamide.

[0076] The preparation method of the phenoxybenzamide derivative I-16 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(3,4-dichlorophenyloxy)benzoic acid is substituted for 2-phenoxybenzoic acid, and the other steps are the same as in Example 1. The resulting compound I-16 is a yellow solid with a yield of 56.2% and a melting point of 140.1–141.8 °C. 1 H NMR(400MHz,Chloroform-d)δ9.41(s,1H),8.33(d,J=7.9Hz,1H),7.54(t,J=8.8Hz,2H),7.34(t,J =7.6Hz,1H),7.26(s,1H),7.01(d,J=8.8Hz,1H),6.91(d,J=8.9Hz,1H),6.75(s,1H),3.75(s,3H); 13 C NMR(101MHz,Chloroform-d)δ161.8,155.0,153.6,141.2(q,J=38.1Hz),136.7,134.5,134.3,133.0 ,132.0,129.6,125.0,122.3,121.6,121.0(q,J=268.6Hz),119.7,119.2,118.2,97.9,36.1; ESI-MS calculated for C 18 H 13 Cl2F3N3O2[M+H] + ,430.0331; found430.0337.

[0077] Example 17

[0078] The phenoxybenzamide derivative I-17 containing substituted pyrazole is 2-(3,5-dichlorophenoxy)-N-(1-methyl-3-trifluoromethyl)pyrazole-5-ylbenzamide.

[0079] The preparation method of the phenoxybenzamide derivative I-17 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(3,5-dichlorophenyloxy)benzoic acid is substituted for 2-phenoxybenzoic acid, and the other steps are the same as in Example 1. The resulting compound I-17 is a yellow solid with a yield of 49.1% and a melting point of 122.3–123.6 °C. 1 HNMR(400MHz,Chloroform-d)δ9.26(s,1H),8.33(dd,J=7.9,1.7Hz,1H),7.61–7.54(m,1H),7.41–7.35 (m,1H),7.28(t,J=1.7Hz,1H),7.03(d,J=1.7Hz,2H),6.95(d,J=8.3Hz,1H),6.73(s,1H),3.76(s,3H); 13 C NMR(101MHz,Chloroform-d)δ161.8,156.0,154.4,141.2(q,J=38.5Hz),136.7,136.5,134.6,1 33.1,125.9,125.4,122.4,119.9(q,J=221.1Hz),118.8,118.3(2C),114.6,98.1,36.1; ESI-MS calculated for C 18 H 13 Cl2F3N3O2[M+H] + ,430.0331; found 430.0334.

[0080] Example 18

[0081] The phenoxybenzamide derivative I-18 containing substituted pyrazole is 2-(3,5-dichlorophenoxy)-N-(3-difluoromethyl)-1-methylpyrazole-5-ylbenzamide.

[0082] The preparation method of the phenoxybenzamide derivative I-18 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(3,5-dichlorophenyloxy)benzoic acid is replaced with 2-phenoxybenzoic acid, and the intermediate pyrazoleamine II-f is replaced with II-a. Other steps are the same as in Example 1. The resulting compound I-18 is a yellow solid with a yield of 52.5% and a melting point of 112.5–114.3 °C. 1H NMR (400MHz, DMSO-d6) δ10.60(s,1H),7.79(d,J=7.6Hz,1H),7.62(d,J=8.8Hz,1H),7.4 5–7.37(m,2H),7.20(d,J=8.2Hz,1H),7.10(d,J=1.6Hz,2H),6.46(s,1H),3.66(s,3H); 13 C NMR (101MHz, DMSO-d6) δ164.6, 158.8, 152.6, 144.2 (t, J = 27.8Hz), 137.6, 135.3 (2C), 133.3 ,130.5,128.4,125.6,123.6,121.0,117.5(2C),110.75(t,J=234.2Hz),97.3,36.4; ESI-MS calculated for C 18 H 14 Cl2F2N3O2[M+H] + ,412.0426; found 412.0434.

[0083] Example 19

[0084] The phenoxybenzamide derivative I-19 containing substituted pyrazole is N-(4-chloro-1-methyl-3-trifluoromethyl)pyrazole-5-yl)-2-(3,5-dichlorophenoxy)benzamide.

[0085] The preparation method of the phenoxybenzamide derivative I-19 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(3,5-dichlorophenyloxy)benzoic acid is replaced with 2-phenoxybenzoic acid, and the intermediate pyrazoleamine II-c is replaced with II-a. Other steps are the same as in Example 1. The resulting compound I-19 is a yellow solid with a yield of 66.5% and a melting point of 141.1–142.3 °C. 1 H NMR(400MHz,Chloroform-d)δ8.82(s,1H),8.22(s,1H),7.56–7.49(m,1H),7. 31(t,J=7.6Hz,1H),6.96(d,J=1.7Hz,2H),6.92(d,J=8.3Hz,1H),3.75(s,3H); 13C NMR(101MHz,Chloroform-d)δ163.4,156.1,154.5,142.5(q,J=138.1Hz),136.5,134.9, 133.1,125.7,125.3,122.3,121.5(q,J=20.3Hz),120.7,119.0,118.3(2C),38.2; ESI-MS calculated for C 18 H 12 Cl3F3N3O2[M+H] + ,363.9942; found 363.9952.

[0086] Example 20

[0087] The phenoxybenzamide derivative I-20 containing substituted pyrazole is N-(4-bromo-1-methyl-3-trifluoromethyl)-1H-pyrazole-5-yl)-2-(3,5-dichlorophenoxy)benzamide.

[0088] The preparation method of the phenoxybenzamide derivative I-20 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(3,5-dichlorophenyloxy)benzoic acid is replaced with 2-phenoxybenzoic acid, and the intermediate pyrazoleamine II-d is replaced with II-a. Other steps are the same as in Example 1. The resulting compound I-20 is a brown solid with a yield of 56.2% and a melting point of 151.5–153.3 °C. 1 H NMR(400MHz,Chloroform-d)δ8.92(s,1H),8.30(d,J=7.9Hz,1H),7.75–7.50(m,2H ),7.37(t,J=7.6Hz,1H),7.04(d,J=1.7Hz,2H),6.99(d,J=8.2Hz,1H),3.85(s,3H); 13 C NMR(101MHz,Chloroform-d)δ163.4,156.1,154.6,142.9(q,J=106.8Hz),136.5,136.0,134.8,1 33.0,130.9,128.8,125.7,125.3,122.3,121.5(q,J=29.2Hz),119.0,118.4,87.9,38.4; ESI-MS calculated for C 18 H 12 BrCl2F3N3O2[M+H] + ,507.9437; found507.9439.

[0089] Example 21

[0090] The phenoxybenzamide derivative I-21 containing substituted pyrazole is 2-(3,5-dichlorophenoxy)-N-(4-fluoro-1-methyl-3-trifluoromethyl)pyrazole-5-ylbenzamide.

[0091] The preparation method of the phenoxybenzamide derivative I-21 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(3,5-dichlorophenyloxy)benzoic acid is replaced with 2-phenoxybenzoic acid, and the intermediate pyrazoleamine II-b is replaced with II-a. Other steps are the same as in Example 1. The resulting compound I-21 is a white solid with a yield of 52.5% and a melting point of 113.2–114.1 °C. 1 H NMR(400MHz,Chloroform-d)δ8.68(s,1H),8.21(s,1H),7.52(s,1H),7.30(s,1H) ,7.20(t,J=1.8Hz,1H),6.96(d,J=1.7Hz,2H),6.90(d,J=8.3Hz,1H),3.67(s,3H). 13 C NMR(101MHz,Chloroform-d)δ163.6,156.0,154.6,142.7(d,J=130.6Hz),136.5,134 .8,133.1,125.8,125.2,122.6(d,J=22.1Hz),122.1,118.8,118.4(2C),37.7; ESI-MS calculated for C 18 H 11 Cl2F4N3O2[M+H] + ,448.0237; found 448.0244.

[0092] Example 22

[0093] The phenoxybenzamide derivative I-22 containing substituted pyrazole is N-(4-chloro-3-difluoromethyl)-1-methylpyrazole-5-yl)-2-(3,5-dichlorophenoxy)benzamide.

[0094] The preparation method of the phenoxybenzamide derivative I-22 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(3,5-dichlorophenyloxy)benzoic acid is replaced with 2-phenoxybenzoic acid, and the intermediate pyrazoleamine II-h is replaced with II-a. Other steps are the same as in Example 1. The resulting compound I-22 is a white solid with a yield of 61.8% and a melting point of 121.1–113.4 °C. 1H NMR(400MHz,Chloroform-d)δ8.86(s,1H),8.28(d,J=9.6Hz,1H),7.63–7.55(m,1H),7.37(t,J =7.6Hz,1H),7.25(t,J=1.7Hz,1H),7.03(d,J=1.7Hz,2H),6.99(d,J=8.1Hz,1H),3.78(s,3H); 13 C NMR(101MHz,Chloroform-d)δ163.4,156.2,154.4,140.6(t,J=26.0Hz),136.4,134.7,13 3.7,133.0,125.6,125.3,122.5,119.1,118.2,110.5(t,J=235.1Hz),103.2,37.9; ESI-MS calculated for C 18 H 12 F3N3O2[M+H] + ,376.1267; found 376.1277.

[0095] Example 23

[0096] The phenoxybenzamide derivative I-23 containing substituted pyrazole is N-(4-bromo-3-difluoromethyl)-1-methylpyrazole-5-yl)-2-(3,5-dichlorophenoxy)benzamide.

[0097] The preparation method of the phenoxybenzamide derivative I-23 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(3,5-dichlorophenyloxy)benzoic acid is replaced with 2-phenoxybenzoic acid, and the intermediate pyrazoleamine II-i is replaced with II-a. Other steps are the same as in Example 1. The resulting compound I-23 is a brown solid with a yield of 42.5% and a melting point of 132.5–134.5 °C. 1 H NMR(400MHz,Chloroform-d)δ8.88(s,1H),8.28(s,1H),7.62–7.56(m,1H),7.37(t,J=7.9Hz,1H ),7.25(d,J=1.7Hz,1H),7.04(d,J=1.7Hz,2H),6.99(d,J=8.2Hz,1H),6.60(s,1H),3.81(s,3H); 13C NMR(101MHz,Chloroform-d)δ163.3,156.2,154.5,142.1,142.1,142.0,136.4,135.5,1 34.7,133.0,125.6,125.3,122.5,119.1,118.3,113.0,110.7,108.3,87.7,38.1; ESI-MS calculated for C 18 H 13 Cl3F2N3O2[M+H] + ,446.0036; found 446.0045.

[0098] Example 24

[0099] The phenoxybenzamide derivative I-24 containing substituted pyrazole is 2-(3-chlorophenoxy)-N-(1-methyl-4-thiocyano-3-trifluoromethyl)pyrazole-5-yl)benzamide.

[0100] The preparation method of the phenoxybenzamide derivative I-24 containing substituted pyrazole in this embodiment is as follows: The method described in Example 1 is used, except that 2-(3-chlorophenyloxy)benzoic acid is replaced with 2-phenoxybenzoic acid, and the intermediate pyrazoleamine II-e is replaced with II-a. Other steps are the same as in Example 1. The resulting compound I-24 is a pale yellow solid with a yield of 82% and a melting point of 174.1–176.3 °C. 1 H NMR(400MHz,Chloroform-d)δ9.50(s,1H,CONH-H),8.29(dd,J=7.9,1.8Hz,1H,Ph-H),7.58(m,1H,Ph-H),7.39(t,J=8.1Hz,1H,Ph-H),7.33(td,J=7.6,1.1H z,1H,Ph-H),7.26(m,1H,Ph-H),7.18(t,J=2.2Hz,1H,Ph-H),7.10(ddd,J=8.3 ,2.5,1.0Hz,1H,Ph-H),6.97(dd,J=8.3,1.1Hz,1H,Ph-H),3.86(s,3H,NCH3); 13 C NMR(101MHz,Chloroform-d)δ163.5,155.8,155.4,142.2(q,J=38.1Hz),141.2,136.0,135.2,1 33.0,131.5,126.0,124.7,121.4,120.5,120.1(q,J=270.4Hz)118.6,118.3,109.0,92.0,38.5.

[0101] Example 25

[0102] The phenoxybenzamide derivatives containing substituted pyrazole structures synthesized in Examples 1 to 24, namely compounds I-1 to I-24, exhibit inhibitory effects on pathogenic fungi and oomycetes of the tested plants.

[0103] 1. Experimental subjects

[0104] The phenoxybenzamide derivatives containing substituted pyrazole structures synthesized in Examples 1 to 24 are compounds I-1 to I-24.

[0105] 2. Experimental Methods

[0106] The in vitro inhibitory activities of compounds I-1 to I-24 against six tested plant pathogens—*Sclerotinia sclerotiorum* (causal agent of rapeseed rot), *Valsa mali* (causal agent of apple rot), *Botrytiscinerea* (causal agent of grape gray mold), *Rhizoctonia solani* (causal agent of rice sheath blight), *Phytophthora capsica* (causal agent of pepper rot), and *Gaeumannomyces graminis* (causal agent of wheat take-all)—were determined using the mycelial linear growth rate method. The selected fungi and oomycetes were provided by the College of Plant Protection, Northwest A&F University.

[0107] (1) Accurately weigh 2.4 mg of the compound and the reference reagent and dissolve them in 0.1 mL of dimethyl sulfoxide (DMSO) solution, shake well and set aside;

[0108] (2) Under aseptic conditions on a clean bench, bring the sterilized culture medium to a final volume of 120 mL. Add the dissolved target compound or control agent to the culture medium, shake well, and prepare the culture medium with a drug concentration of 20 mg / L. Pour the drug-containing culture medium evenly into 15 petri dishes with a diameter of 90 mm for later use. Add an equal volume of dimethyl sulfoxide solution to the blank group.

[0109] (3) On a clean bench, place a 5mm diameter hole punch in the outer flame of an alcohol lamp for sterilization. After the hole punch is completely sterilized, punch holes in a culture dish containing activated fungi for later use;

[0110] (4) Sterilize the inoculation needle by heating it in the outer flame of an alcohol lamp. After sterilization, allow the inoculation needle to cool slightly, then use the inoculation needle to transfer the pre-drilled bacterial pellet to the center of the drug-containing culture dish. Cover the dish with the lid, seal it with sealing film or plastic wrap, and invert it in a 24°C biochemical incubator for cultivation. When the blank group reaches approximately two-thirds of the diameter of the culture dish, measure the diameter of each treated colony using the cross-sectional method. Perform three parallel experiments for each compound, and take the average value when compiling the results. The experimental results are calculated according to the following formula.

[0111]

[0112] (5) Select compounds with good initial screening activity and determine their EC values. 50 Value. Based on the linear regression relationship between the probability value (y) and the logarithm of the mass concentration (x) of the mycelial growth inhibition rate, the toxicity regression equation of the agent was calculated as y = a + bx, and the inhibition median concentration (EC50) was determined. 50 ) and correlation coefficient (R) 2 The concentration gradients of the compounds prepared for the assay were 10, 5, 2.5, 1.25, 0.63, 0.31 and 0.16 mg / L.

[0113] 3. Experimental Results

[0114] 3.1 Initial screening for antibacterial activity

[0115] A strategy guided by antibacterial activity results was employed to derive and modify target compounds. Initially, only target compounds I-1–I-15 were synthesized. Using the mycelial growth rate method, antibacterial activity was screened against six different plant pathogenic fungi, including *Sclerotinia sclerotinia*, at a target compound concentration of 20 mg / L. The results are shown in the figure. For *Sclerotinia sclerotinia*, five compounds showed inhibition rates exceeding 60%: compound I-1 (67.6%), compound I-4 (65.7%), compound I-5 (79.0%), compound I-9 (76.2%), and compound I-12 (81.2%). For rice sheath blight pathogens, four compounds showed inhibition rates exceeding 60%: compound I-6 (63.1%), compound I-7 (64.2%), compound I-10 (63.0%), and compound I-12 (72.1%). For apple rot pathogens, only four compounds showed inhibition rates below 60%: compound I-2 (52.3%), compound I-14 (43.4%), and compound I-15 (45.0%). The inhibition rates of the remaining compounds ranged from 60.5% to 94.4%.

[0116] For both wheat take-all and grape botrytis, only one compound showed an inhibition rate exceeding 60%, namely compound I-6 (60.8%) and compound I-7 (64.0%). Unfortunately, for pepper phytophthora, the inhibition rates of all compounds were less than 60%, with the compound with the highest activity being I-12 (41.0%).

[0117] In general, most compounds showed good inhibitory effects against *Rhizoctonia solani*, *Sclerotinia sclerotiorum*, and *Phytophthora rottensis* of apples; however, they were less effective against *Tricholoma matsutake*, *Botrytis cinerea*, and *Phytophthora capsici*. Compounds I-6, I-7, and I-12 all showed good broad-spectrum activity, with inhibition rates exceeding 60% against all three fungi. This indicates that substitution of the benzene ring with methoxy and chlorine atoms resulted in better antibacterial activity. However, when the benzene ring was substituted with difluoromethoxy, the activity against *Sclerotinia sclerotiorum* and *Rhizoctonia solani* increased, while the activity against other fungi decreased to varying degrees. This also highlights the importance of the diphenyl ether skeleton in the antibacterial activity of the target compound. Comparative analysis revealed that the antibacterial activity was significantly better when the benzene ring at position 3 was substituted than when it was substituted at position 4. Compound I-12 showed the best activity. Based on this, to consider whether multiple substitutions would enhance the antibacterial activity of the target compounds, we synthesized disubstituted target compounds such as I-16 (3,4-di-Cl) and I-17 (3,5-di-Cl). Antibacterial activity assays showed that compared with compound I-12, compounds I-16 and I-17 significantly enhanced the inhibitory effect on all fungi, with compound I-17 (3,5-di-Cl) showing the most significant improvement.

[0118] Compounds I-16 and I-17 showed improved inhibition rates against *Sclerotinia sclerotiniae* (80.2%, 90.1%), *Rhizoctonia solani* (75.2%, 79.2%), *Pseudomonas aeruginosa* (85.2%, 100%), *Tricholoma materia velutipes* (78.21%, 83.8%), *Botrytis cinerea* (68.3%, 72.5%), and *Phytophthora capsici* (45.2%, 48.1%). Comparative analysis revealed that, except for *Phytophthora capsici*, the inhibition rates against the other five fungi were significantly improved, particularly against *Tricholoma materia velutipes* and *Botrytis cinerea*. Based on this, we believe that introducing multiple substitutions can not only significantly enhance the antifungal activity of the target compounds but also significantly broaden their control spectrum.

[0119] To investigate the effect of introducing a halogen at the 4-position of the pyrazole ring on the antibacterial activity of the target compound, we selected compounds I-17 and I-18 as lead compounds and synthesized compounds I-19–I-24 by introducing a halogen and a thiocyanate group at the 4-position of their pyrazole rings. The results showed that introducing a fluorine atom at the 4-position of the pyrazole ring enhanced the inhibition rates of compound I-21 against *Sclerotinia sclerotiorum* (96.5%), *Rhizoctonia solani* (83.8%), *Pseudomonas aeruginosa* (100%), *Tricholoma materia humicinum* (91.6%), *Botrytis cinerea* (76.1%), and *Phytophthora capsici* (58.4%) to varying degrees. Based on this, we believe that introducing a halogen at the 4-position of the pyrazole ring can significantly improve the antibacterial activity of the target compound.

[0120] Table 1. Antibacterial activity (inhibition rate %) of compound I-1–I-24 at 20 mg / L a

[0121]

[0122]

[0123] a Data are given as the mean of triplicate experiments; Ss b S. sclerotiorum; Rs c R.solani;Vm d V.mali; Gg e G. graminis; Bc f B.cinerea; Pc g P.capsici; FZ h Fluxapyroxad.

[0124] 3.2 Antibacterial activity rescreening

[0125] Based on the initial screening results, to further determine their antibacterial activity and structure-activity relationship, we selected compounds with good antibacterial activity and further measured their EC50 against *Aureobasidium canis*, *Botrytis cinerea*, *Tricholoma materia granatum*, *Tricholoma take-all*, *Sclerotinia sclerotiorum*, and *Rhizoctonia solani*. 50 The results are shown in Table 2. The results indicate that among compounds I-1–I-18, the optimal compound is I-17, which exhibits the best EC50 concentration against *Aureobasidium aureum*, *Botrytis cinerea*, *Tricholoma materia granatum*, *Sclerotinia sclerotiorum*, *Rhizoctonia solani*, and *Rhizoctonia solani*. 50The concentrations were 0.79 mg / L, 10.54 mg / L, 3.50 mg / L, 2.78 mg / L, and 3.45 mg / L, respectively. Except for *Sclerotinia sclerotinia*, its control efficacy against other fungi was superior to fluopyram. It is worth noting that although compound I-16 showed better initial screening activity against *Ailuropoda rubra* than compound I-12, its EC50 concentration against *Ailuropoda rubra* was lower. 50 The concentration was 4.79 mg / L, significantly lower than that of compound I-12 (EC). 50 =1.71 mg / L); Furthermore, the activity of compound I-16 was superior to that of compound I-12. In addition, for the apple rot pathogen, the EC50 values ​​of compounds I-4, I-6, and I-8 were... 50 The values ​​were 6.73 mg / L, 3.68 mg / L, and 1.98 mg / L, respectively, which were significantly better than those of fluopyram (12.45 mg / L). For rice sheath blight pathogens, the EC50 values ​​of compounds I-12 (R2 = 3-Cl, R3 = F), I-16, and I-17 were... 50 The values ​​were 9.74 mg / L, 4.36 mg / L and 3.45 mg / L, respectively.

[0126] Introducing fluorine and chlorine atoms at the 4-position of the pyrazole ring enhances the activity of the target compound to varying degrees, with fluorine introduction showing the best effect, while bromine introduction slightly decreases the activity. Furthermore, comparing compounds I-19 and I-22, and compounds I-20 and I-23, we found that compounds with a trifluoromethyl group at the 3-position of the pyrazole ring exhibit significantly better activity than those with a difluoromethyl group.

[0127] In summary, compound I-21 exhibits the best activity against the apple rot pathogen (EC). 50 =0.52 mg / L), Grape Botrytis cinerea (EC) 50 =3.42 mg / L), wheat take-all pathogen (EC) 50 =1.46 mg / L) has better activity than fluopyram (EC). 50 =12.45 mg / L, EC 50 =8.33 mg / L, EC 50 =1.93 mg / L), which is effective against Sclerotinia sclerotinia, the causal agent of rapeseed disease (EC). 50 =0.82 mg / L) and rice sheath blight pathogen (EC) 50 EC = 1.46 mg / L 50 Approaching fluopyram (EC) 50 =0.23 mg / L, EC 50 =0.62 mg / L).

[0128] Table 2 EC values ​​of the selected compounds 50Value (mg / L)

[0129]

[0130]

[0131]

[0132] Example 26

[0133] The control effect of compound I-21 synthesized in Example 21 on potted seedlings

[0134] 1. Experimental subjects

[0135] Compound 21 synthesized in Example 21

[0136] 2. Experimental Methods

[0137] Preparation of rapeseed seedlings in pots: Select 200 seeds of Shaanxi Oil 19 type rapeseed, place the rapeseed seeds in a 90mm petri dish, add 20mL of pure water, and place it in a 4℃ refrigerator for about 2-3 days. Then, take out the petri dish containing the rapeseed seeds and place it at room temperature for about 12-24 hours. After the rapeseed seeds show signs of sprouting, select 3 rapeseed seeds that have sprouted and plant them in a pot containing nutrient soil and vermiculite in a ratio of about 3:1. Place the planted pots in a greenhouse for cultivation. Cultivate a total of 50 pots of rapeseed seedlings for later use.

[0138] Preparation of mycelial blocks of Sclerotinia sclerotiorum causal agent in rapeseed: Use a sterile punch to prepare mycelial blocks with a diameter of 5 mm from agar medium covered with Sclerotinia sclerotiorum mycelia.

[0139] Preparation of moisturizing cotton: Cut the absorbent cotton into squares with a side length of 1 cm, place them in an Erlenmeyer flask, add distilled water to 150 mL, seal the flask opening with sealing film, place it in an autoclave, sterilize at 121℃ for 25 min, and then take it out for use.

[0140] Compound 21, exhibiting the best in vitro antibacterial activity, was selected to determine its in vivo antibacterial activity against *Sclerotinia sclerotinia*, the causal agent of rapeseed rot. Healthy, disease-free rapeseed plants of uniform size were selected, and one leaf was inoculated from each plant. The leaves were treated with 75% ethanol, rinsed with sterile water, and allowed to dry. The leaves were then punctured with a sterilized needle. A 20 mg / L solution of the compound and a control agent was prepared and sprayed evenly onto the leaves. After the leaves dried again, a mycelial-side of the mycelial cake was applied to the wound using a sterilized inoculation needle. A piece of absorbent cotton was then placed over the mycelial cake and secured with plastic wrap. Each treatment was repeated in triplicate, with water containing DMSO as a blank. After culturing at 25°C for 7 days, the infection and rot of *Sclerotinia sclerotinia* on the rapeseed leaf surface were observed. Based on the results of the disease observation in the blank control, the diameter of the lesions was measured using the cross-sectional method. The experimental results were calculated using the following formula.

[0141]

[0142] 3. Experimental Results

[0143] The antibacterial activity of all target compounds against *Sclerotinia sclerotinia* causal agent was determined. The results showed that compound I-21 exhibited EC50-95% activity against *Sclerotinia sclerotinia* causal agent. 50 =0.82 mg / L. Based on this, compound I-21 was selected to conduct an in vivo experiment on the activity of compound I-21 against Sclerotinia sclerotinia in rapeseed leaves. The results showed that the protective activities of compound I-21 and fluopyram were 89.3% and 96.4%, respectively. In the rapeseed Sclerotinia sclerotinia control experiment, there was a significant difference between the untreated group and the treated group ( Figure 1 (and Table 3).

[0144] Table 3. Control efficacy of compound I-21 against Sclerotinia sclerotinia in rapeseed (100 mg / L)

[0145]

[0146] Example 27

[0147] The control effects of compounds I-17 and I-21 synthesized in Examples 17 and 21 on apple branches

[0148] 1. Experimental subjects

[0149] Compounds I-17 and I-21 synthesized in Examples 17 and 21

[0150] 2. Experimental Methods

[0151] Apple branches: collected from the experimental garden of the North Campus of Northwest A&F University

[0152] Select apple branches of roughly the same thickness and length. First, wash the surface of the branches with purified water to remove dust. After drying, soak them in a 0.06% sodium hypochlorite solution for 10 minutes for disinfection. After disinfection, remove the branches, let them dry, and seal the top and bottom ends of the branches with melted paraffin wax. Place the hole punch over the outer flame of an alcohol lamp and heat it. After the temperature drops, punch a hole in the middle of the branch for later use.

[0153] For the protective activity assay, the compound and control agent were prepared into solutions with a concentration of 50 mg / L. Healthy apples were sprayed with the target compound and control agent, respectively. After the apple branches dried, a piece of apple rot fungus (d = 5 mm) was inoculated into the center of each apple branch. Each treatment was repeated in triplicate, with water containing DMSO as a blank. After incubation at 25°C for 7 days, the infection and rot status of the apple rot fungus on the apple surface were observed. Based on the results of the blank control disease incidence survey, the diameter of the lesions was measured using the cross-sectional method. The experimental results were calculated according to the following formula.

[0154]

[0155] 3. Experimental Results

[0156] Preliminary studies were conducted on the activity of all target compounds against various plant pathogenic fungi. The results showed that compound I-21 exhibited the highest activity against *Pseudomonas aeruginosa*, the causal agent of apple rot, with an EC50 value of [missing information]. 50 The concentration was 0.52 mg / L. Based on this, compound I-21 was selected for in vivo antifungal activity against apple rot pathogens on apple branches. The protective activities of compounds I-17, I-21, and fluopyram were 37.5%, 66.7%, and 29.2%, respectively. Compound I-21 showed better inhibitory activity than fluopyram. In the apple disease control experiment, there was a significant difference between the untreated and treated groups. Figure 2 (and Table 4).

[0157] Table 4. The efficacy of compounds I-17 and I-21 against apple rot pathogens (100 mg / L)

[0158]

[0159] Example 28

[0160] Investigation of the interaction mechanism of compounds I-12, I-17 and I-21 synthesized in Examples 12, 17 and 21 with SDH protein

[0161] 1. Experimental subjects

[0162] Compounds I-12, I-17, and I-21 synthesized in Examples 12, 17, and 21

[0163] 2. Experimental Methods

[0164] The CDOCKER docking system in Discovery Studio 2019 software was used. Compounds with excellent activity were used as ligands. Ligand structures were drawn using Chem Bio Draw Ultra 14.0 and imported into Chem Bio3D Ultra 14.0. A local low-energy conformation (Minmize) search method was employed to perform charge loading and field optimization on the ligand molecules to obtain their appropriate low-energy conformations. Subsequently, the processed small molecules were opened in Discovery Studio 2019, and the ligand preparation module was run to complete the ligand preparation process.

[0165] The three-dimensional structure of the SDH protein crystal was obtained from the RCSB Protein Data Bank (http: / / www.rcsb.org / pdb / home / home.do), PDB number: 2FBW. The protein was imported into Discovery Studio 2019. Since 2FBW is a dimer, the duplicate half was first removed, followed by the removal of water and some non-essential substructures from the crystal. Then, the co-crystallized ligand, oxychloride, was extracted from 2FBW. The protein crystal was then hydrogenated, and the protein preparation module was run. After protein preparation, the prepared protein was defined as the receptor, and the original position of oxychloride was defined as the binding site. The receptor SDH and the ligand molecule were then imported into Discovery Studio 4.0 software for simulated docking experiments to analyze the interaction mechanism between the molecular target and the target compound.

[0166] 3. Experimental Results

[0167] To investigate the binding modes of compounds I-12, I-17, and I-21 to SDH, they were coupled to the active site of SDH with fluopyram; from Figure 3 It can be seen that the pyrazole rings of all four compounds are deeply embedded in the active pocket, forming a good shape complementarity with the active pocket. It can also be seen that the compound I-21 has the best degree of binding.

[0168] Table 5. Binding energies of compounds I-12, I-17, I-21 and fluopyram with SDH

[0169]

[0170] As can be seen from Table 5, compound I-12 (-36.50 kcal / mol) -1 The binding energy of ) is lower than that of fluopyram (-38.38 kcal / mol)-1 In addition, compound I-17 (-43.26 kcal / mol) -1 ) and compound I-21 (-45.14 kcal / mol) -1 The binding energy of [a specific substance] is significantly better than that of fluopyram. For example... Figure 3 As shown in (E, F, G, H), the tested compounds form different interactions with the amino acid residues at their binding sites, thus stabilizing the binding mode. Similarly, the amide bonds of both compounds and fluopyram undergo tautomerism, and the nitrogen atom of the amide bond in both compounds forms a hydrogen bond with residue 173 of tryptophan (Trp); the fluorine atom of the trifluoromethyl group in both compounds forms a hydrogen bond with residue 39 of serine (Ser), while in fluopyram, the fluorine atom of the difluoromethyl group forms a hydrogen bond with residue 39 of serine; furthermore, the pyrazole ring in all four compounds forms a p-π interaction with residue 43 of arginine (Arg). In contrast, the fluorine atom of the trifluoromethyl group in compounds I-17 and I-21 each forms two hydrogen bonds with residue 39 of serine (Ser). Additionally, in the structure of compound I-21, the fluorine atom at position 4 of the pyrazole group also forms a hydrogen bond with residue 39 of serine (Ser). This may explain why compound I-21 exhibits significantly better binding energy and antibacterial activity than compounds I-17 and I-12. The benzene ring of fluopyram forms a pi-pi interaction with residue 173 of tryptophan (Trp) and residue 58 of tyrosine (Tyr), respectively, and also forms a hydrogen bond with residue 43 of arginine (Arg). In general, compounds I-12, I-17, and I-21 share some similar binding modes with fluopyram, but compound I-21 shows better binding to SDH.

[0171] Table 6 details the hydrogen bonding interactions between the selected compounds and SDH.

[0172]

[0173] Example 29

[0174] Investigation of the SDH inhibitory activity of compounds I-17 and I-21 synthesized in Examples 17 and 21

[0175] 1. Experimental subjects

[0176] Compounds I-17 and I-21 synthesized in Examples 17 and 21

[0177] 2. Experimental Methods

[0178] (1) Preparation of test strains

[0179] First, under aseptic conditions, holes were punched in the culture medium containing *Sclerotinia sclerotiorum*, using a sterilized 5mm diameter punch. Then, using a sterilized inoculation needle, the holes were transferred to Erlenmeyer flasks containing 200mL of PDB medium. The flasks were sealed with sealing film and placed in a shaker at 25℃ and 140rpm for 72 hours to allow rapid growth of the seed culture. After cultivation, 1mL of the seed culture was transferred to Erlenmeyer flasks pre-filled with 200mL of PDB medium using a sterile pipette. After mixing thoroughly, the flasks were incubated under the same conditions for 24 hours. Then, 0.5mL of different concentrations of the drug solution were added to the culture medium, resulting in drug concentrations of 0.2, 0.5, 1.0, 5.0, and 10.0 mg / L, respectively. After mixing thoroughly, the flasks were incubated for another 72 hours, and mycelia were collected. Each treatment group was replicated in triplicate, with 0.5mL of acetone used as a blank control.

[0180] (2) Preparation of fermentation broth

[0181] Weigh 2g of mycelium and place it in a pre-cooled mortar. Grind it rapidly into powder under liquid nitrogen at low temperature. Quickly transfer the powdered mycelium to a pre-cooled centrifuge tube containing physiological saline solution. The ratio of mycelium to physiological saline is 1:9 (weight (g):volume (mL)). Centrifuge the mixture at 3000 rpm for 10 min at 4℃. Transfer the supernatant to a clean centrifuge tube. Centrifuge the tube at 1000 rpm for 15 min. Collect the supernatant and dissolve it in 200 μL of physiological saline to prepare a suspension. Store the suspension at -20℃ for later use. All reagents and instruments used in the experiment must be pre-cooled to prevent enzyme inactivation.

[0182] (3) Assay of succinate dehydrogenase activity

[0183] Strictly follow the instructions for the succinate dehydrogenase (SDH) kit, and store the prepared reagents in the dark and refrigerated. Under dark conditions, take 2.6 mL of the working solution and place it on a 1 cm optical path. Measure the absorbance at 600 nm. Record the absorbance OD1 value after 5 seconds and the absorbance OD2 value after 65 seconds. The enzyme activity calculation formula is as follows:

[0184]

[0185] 3. Experimental Results

[0186] This series selected target compounds I-17 and I-21, which exhibited good activity and a certain broad spectrum, for SDH inhibitory activity assays. Their in vitro inhibitory activity against SDH extracted from *Pseudomonas aeruginosa* was determined. Table 7 shows the IC50 values ​​of compounds I-17 and I-21 against SDH. 50 The concentrations were 3.50 mg / L and 1.22 mg / L, respectively, indicating good inhibitory effects on SDH, significantly superior to fluopyram (IC50). 50 =8.32 mg / L). This fully demonstrates that the antibacterial mechanism of compounds I-17 and I-21 may be to inhibit fungal growth by inhibiting SDH activity.

[0187] Table 7 SDH inhibitory activity (IC50) 50 )

[0188]

[0189] In summary, all 24 pyrazole amide compounds prepared by chemical synthesis in this application exhibit certain antibacterial activities. Compound I-21, in particular, demonstrates broad-spectrum antibacterial activity against *Pseudomonas aeruginosa*, *Botrytis cinerea*, *Tricholoma matsutake*, *Sclerotinia sclerotiorum*, and *Rhizoctonia solani*. 50 The concentrations were 0.52 mg / L, 3.42 mg / L, 1.46 mg / L, 0.82 mg / L, and 1.86 mg / L, respectively. The antibacterial mechanism of compound I-21 was investigated by combining molecular docking and SDH inhibitory activity. The results showed that compound I-21 (-45.140 kcal / mol) -1 The binding energy between ) and SDH is significantly better than that of fluopyram (-38.38 kcal / mol). -1 ), and compound I-21 (IC 50 The SDH inhibitory activity of 1.22 mg / L was significantly better than that of fluopyram (IC50). 50 =8.32 mg / L). The above results fully demonstrate that the antifungal mechanism of compound I-21 may be through inhibiting the activity of SDH, thereby inhibiting the normal growth of fungi. This provides a direction for further research on the antifungal activity of different compounds and lays the foundation for the preparation of fungicides with pyrazole amide derivatives as the main antifungal active ingredients.

[0190] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A phenoxybenzamide derivative containing substituted pyrazole, characterized in that: Its general chemical structural formula (I) is as follows: Wherein, R1 is trifluoromethyl or difluoromethyl; R2 is hydrogen, methyl, methoxy, trifluoromethyl, fluorine, chlorine, bromine, or a combination of multiple substitutions thereof, substituted at different positions; R3 is hydrogen, fluorine, chlorine, bromine, or thiocyanate. The phenoxybenzamide derivatives containing substituted pyrazoles were synthesized via the following synthetic route: Intermediate II is a substituted pyrazolamine; Intermediate III is a substituted phenoxybenzoic acid. The specific preparation steps are as follows: Using substituted pyrazolamide and substituted phenoxybenzoic acid as raw materials, an amide condensation reaction is carried out under dichloromethane and alkaline conditions using tetramethylchlorourea hexafluorophosphate (TCFH) and N-methylimidazole (NMI) as condensing agents to generate phenoxybenzamide derivatives containing substituted pyrazol, i.e., general formula (I). The obtained product is purified by column chromatography to obtain pure product.

2. The phenoxybenzamide derivative containing substituted pyrazole according to claim 1, characterized in that, The phenoxybenzamide derivative I containing substituted pyrazole is the following compound:

3. The application of a phenoxybenzamide derivative containing a substituted pyrazole as described in any one of claims 1 to 2, characterized in that: The phenoxybenzamide derivatives containing substituted pyrazoles are applied to agricultural fungicides.

4. The application of the phenoxybenzamide derivative containing substituted pyrazole according to claim 3, characterized in that: The phenoxybenzamide derivatives containing substituted pyrazoles are used to control apple rot fungus, grape gray mold fungus, wheat take-all fungus, rapeseed sclerotinia rot fungus and rice sheath blight fungus.

5. The application of the phenoxybenzamide derivative containing substituted pyrazole according to claim 3, characterized in that: The phenoxybenzamide derivatives containing substituted pyrazoles are used to prepare pesticide formulations for the prevention and control of plant diseases caused by plant pathogens.

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