A difluoromethyl pyrimidine substituted 1,3,4-oxadiazole derivative for preventing and treating edible mushroom pathogenic bacteria, and a preparation method and application thereof

Abstract

This invention discloses a difluoromethylpyrimidine-substituted 1,3,4-oxadiazole derivative for the prevention and control of pathogenic fungi in edible fungi, its preparation method, and its application. It relates to the field of biochemistry, and the key technical points are: through rational molecular design, a class of thioether derivatives (6a-6t) with difluoromethylpyrimidine as the parent nucleus and 1,3,4-oxadiazole as the active terminal group were constructed, and their inhibitory activity against *Trichoderma* and *Mucor* was systematically studied. The results show that some compounds exhibit superior EC50 activity compared to the commercially available fungicide pyrimethanil. 50 It has high value, and the synthesis process is simple and controllable, showing good development prospects.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, and more specifically, to a difluoromethylpyrimidine-substituted 1,3,4-oxadiazole derivative for the prevention and control of pathogens in edible fungi, its preparation method, and its application. Background Technology

[0002] In recent years, the edible fungi industry, as an important component of high-efficiency agriculture, has faced severe challenges from fungal diseases in improving its yield and quality. Trichoderma spp. and Mucor spp. are major pathogens affecting edible fungi cultivation, causing problems such as inhibited mycelial growth and fruiting body rot, resulting in significant economic losses. Currently, chemical control remains the primary means of controlling these diseases, but traditional fungicides have drawbacks such as insufficient efficacy, easy induction of drug resistance in pathogens, and high residue risks. Therefore, developing highly effective, low-toxicity antifungal agents with novel mechanisms of action is of great significance for ensuring the safe production of edible fungi.

[0003] Studies have shown that fluorinated heterocyclic compounds have become a hot area in pesticide development due to their unique biological activity and metabolic stability. 1,3,4-oxadiazole and pyrimidine ring systems, as dominant structural units, can synergistically enhance affinity for pathogenic targets. However, the design of existing fluorinated pyrimidine-oxadiazole derivatives mainly focuses on mammalian targets (such as kinase inhibitors), with limited research on specific regulatory mechanisms against plant pathogenic fungi. Furthermore, the antibacterial activity and industrial production potential of reported compounds still have room for improvement.

[0004] Therefore, there is an urgent need to explore novel fluoropyrimidine-oxadiazole derivatives to meet the demand of green agriculture for safe and efficient fungicides. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a difluoromethylpyrimidine-substituted 1,3,4-oxadiazole derivative for the prevention and control of pathogenic fungi in edible fungi, along with its preparation method and application. A class of thioether derivatives (6a-6t) with difluoromethylpyrimidine as the parent nucleus and 1,3,4-oxadiazole as the active end group were constructed, and their inhibitory activity against Trichoderma and Mucor was systematically studied.

[0006] The above-mentioned technical objective of the present invention is achieved through the following technical solution:

[0007] In a first aspect, a difluoromethylpyrimidine-substituted 1,3,4-oxadiazole derivative or a pharmaceutically acceptable salt thereof for the prevention and control of pathogenic fungi in edible fungi is provided, the general structural formula of said derivative being shown in Formula I:

[0008] Formula I;

[0009] Wherein, R1 is benzyl chloride with different substituents.

[0010] Preferably, R1 is selected from one of the following structures:

[0011] .

[0012] Preferably, the derivative is selected from the following compounds:

[0013] 6c: N-((5-(benzylthio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidin-4-amine;

[0014] 6d: 6-(difluoromethyl)-N-((5-((4-fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)pyrimidin-4-amine;

[0015] 6e: N-((5-((2-chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidin-4-amine;

[0016] 6f: N-((5-((3-chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidin-4-amine;

[0017] 6p: N-((5-((4-chloro-2-fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidin-4-amine;

[0018] 6r: N-((5-((2,3-dichlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidin-4-amine.

[0019] In a second aspect, a method for preparing a difluoromethylpyrimidine-substituted 1,3,4-oxadiazole derivative for the prevention and control of pathogenic fungi in edible fungi, as described in any one of the first aspects, is provided, comprising the following steps:

[0020] S1: Sodium methoxide and formamidin hydrochloride are reacted in methanol, and then condensed with ethyl difluoroacetoacetate to obtain intermediate 1;

[0021] S2: Under ice bath conditions, intermediate 1 is chlorinated with phosphorus oxychloride to obtain intermediate 2;

[0022] S3: Intermediate 2 is condensed with glycine methyl ester in isopropanol to obtain intermediate 3;

[0023] S4: Intermediate 3 is refluxed with hydrazine hydrate in ethanol to obtain intermediate 4;

[0024] S5: Intermediate 4 is reacted with carbon disulfide under reflux in ethanol to obtain intermediate 5;

[0025] S6: Intermediate 5 is condensed with substituted benzyl chloride in an alkaline aqueous solution, and the resulting compound is purified to obtain the target compound.

[0026] Preferably, intermediate 1 is 4-hydroxy-6-difluoromethylpyrimidine;

[0027] The intermediate 2 is 4-chloro-6-difluoromethylpyrimidine;

[0028] The intermediate 3 is (6-(difluoromethyl)pyrimidin-4-yl)glycine methyl ester;

[0029] The intermediate 4 is 2-((6-(difluoromethyl)pyrimidin-4-yl)amino)acetylhydrazine;

[0030] And, the intermediate 5 is 5-(((6-(difluoromethyl)pyrimidin-4-yl)amino)methyl)-1,3,4-oxadiazole-2-thiol.

[0031] Preferably, the preparation temperature of sodium methoxide in step S1 is room temperature, the addition of phosphorus oxychloride in step S2 is carried out under ice bath conditions, and the reaction temperature of step S5 is 80°C.

[0032] Thirdly, the application of a difluoromethylpyrimidine-substituted 1,3,4-oxadiazole derivative for the prevention and control of pathogenic fungi of edible fungi, as described in any one of the first aspects, in the preparation of antifungal drugs is provided.

[0033] Preferably, the pathogenic fungus of the edible fungus is Trichoderma.

[0034] Preferably, the pathogenic fungus of the edible fungus is Mucor.

[0035] Fourthly, an antifungal pharmaceutical composition is provided, comprising a derivative as an active ingredient as described in any one of the first aspects, and a pharmaceutically acceptable carrier.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. This invention utilizes rational molecular design to construct a class of thioether derivatives (6a-6t) with difluoromethylpyrimidine as the parent nucleus and 1,3,4-oxadiazole as the active terminal group, and systematically studies their inhibitory activity against Trichoderma and Mucor. The results show that some compounds exhibit superior EC50 activity compared to the commercially available fungicide pyrimethanil. 50 It has high value, and the synthesis process is simple and controllable, showing good development prospects.

[0038] 2. This invention achieves precise control of pathogen targets by introducing the synergistic effect of the difluoromethylpyrimidine core and the active end group of 1,3,4-oxadiazole, combined with the unique physicochemical properties of the thioether bond. The introduction of fluorine atoms enhances the compound's lipid solubility, metabolic stability, and ability to penetrate fungal cell membranes, while the introduction of the oxadiazole ring strengthens its binding ability with fungal enzymes through hydrogen bonding, thus breaking through the limitation of the single mechanism of action of traditional fungicides.

[0039] 3. This invention adopts a modular synthesis strategy, using inexpensive and readily available raw materials such as sodium methoxide, phosphorus oxychloride, and carbon disulfide to construct the target compound through a five-step reaction involving condensation, chlorination, and cyclization; the reaction is carried out under ice bath conditions, which effectively controls the generation of byproducts; the purification of the target compound only requires recrystallization and silica gel column chromatography, with an overall yield of 58%-88%, making it suitable for industrial-scale production.

[0040] 4. This invention can systematically regulate the antibacterial activity spectrum of compounds by modifying benzyl chloride substituents (such as methyl, ethyl, halogen, nitro, etc.), demonstrating the potential to achieve activity optimization through simple structural modification, and providing a rich structural basis for subsequent pesticide formulation design. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. The illustrative embodiments and descriptions of this invention are only used to explain this invention and are not intended to limit this invention.

[0042] Example 1: Synthesis of intermediate 1.

[0043] Intermediate 1 is 4-hydroxy-6-difluoromethylpyrimidine, and its synthetic route is as follows:

[0044] .

[0045] The preparation method is as follows: 135 mmol of metallic sodium was weighed into a 250 mL three-necked flask and added to anhydrous methanol in small amounts several times under stirring at room temperature to obtain sodium methoxide; 108 mmol of formamidine hydrochloride was weighed and added to the above sodium methoxide solution. After reacting for 0.5 h, 90 mmol of ethyl difluoroacetoacetate was slowly added dropwise. After the reaction was completed by thin-layer chromatography (TLC), the solvent was removed under vacuum, and the solution was completely dissolved in water. When the pH was adjusted to 6 with 10% HCl solution, a large amount of solid precipitated out. The solid was filtered to obtain a white solid, which is intermediate 1, with a yield of 27%.

[0046] Example 2: Synthesis of intermediate 2.

[0047] Intermediate 2 is 4-chloro-6-difluoromethylpyrimidine, and its synthetic route is as follows:

[0048] ;

[0049] The preparation method is as follows: Weigh 167 mmol of compound 1 into a 100 mL three-necked flask, add 25 mL of acetonitrile solution to dissolve it, add 1.7 mmol of N,N-diisopropylethylamine dropwise, and slowly add 41.7 mmol of phosphorus oxychloride dropwise under ice bath conditions. After the addition is complete, heat to 90 °C and reflux (treat the tail gas with 10% NaOH solution). After the reaction is complete, remove the solvent under vacuum, add 10 mL of ice water under ice bath conditions, and use... The pH was adjusted to 7, and the mixture was extracted with dichloromethane, washed with water, and the organic layer was concentrated under reduced pressure to obtain a dark brown liquid, compound 2, with a yield of 57%.

[0050] Example 3: Synthesis of intermediate 3.

[0051] Intermediate 3 is (6-(difluoromethyl)pyrimidin-4-yl)glycine methyl ester, and its synthetic route is as follows:

[0052] ;

[0053] The preparation method is as follows: Compound 2 (39.5 mmol), 20 mL of isopropanol, and N,N-diisopropylethylamine (79.0 mmol) were added to a 100 mL round-bottom flask. After stirring at room temperature for 5 min, glycine methyl ester (51.4 mmol) was added. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain a yellow oily substance. This substance was dissolved in water and extracted with dichloromethane. The organic layer was concentrated under reduced pressure to obtain a white solid, compound 3, with a yield of 74%. mp 71.0-73.2°C; 1H NMR (600 MHz, DMSO-d6) δ 8.52(s, 1H,Pyrimidine-H), 8.26(t, 1H, J = NH-H), 6.86(s, 1H, Pyrimidine-H), 6.85(t, 1H,J = 54.6 Hz,-CHF2), 4.17(d, 2H, J = 6.0 Hz, -CH2), 3.65(s, 3H, -CH3).13C NMR(150 MHz, DMSO-d6) δ 170.94, 162.91, 158.98, 157.42(t, J = 23.85 Hz), 114.73(t, J = 237.3 (Hz), 102.62, 52.26, 42.23.

[0054] Example 4: Synthesis of intermediate 4.

[0055] Intermediate 4 is 2-((6-(difluoromethyl)pyrimidin-4-yl)amino)acetylhydrazine, and its synthetic route is as follows:

[0056] ;

[0057] The preparation method is as follows: add (28 mmol) of compound 3, 15 mL of anhydrous ethanol and (69 mmol) of hydrazine hydrate to a 100 mL three-necked flask, and heat to reflux at 85 °C. After the reactants reacted completely, a large amount of white solid was produced. The mixture was filtered under reduced pressure, and the filter cake was dried to obtain 5.28 g of white powdery solid, namely compound 4, yield: 88%; mp 141.8-144.6°C; ¹H NMR (600 MHz, DMSO-d6) δ 9.18(s, 1H, NH), 8.49(s, 1H, Pyrimidine-H), 8.07(s, 1H, NH), 6.84(s, 1H, Pyrimidine-H), 6.83(t, 1H, J = 54.6 Hz, -CHF2), 4.24(s, 2H, -NH2), 3.96(d, 2H, J = 5.4 Hz, -CH2). ¹³C NMR (150 MHz, DMSO-d6) δ 168.62, 162.94, 158.95, 156.97(t, J = 22.8 Hz), 114.83(t, J = 237.6 Hz), 102.95, 42.37.

[0058] Example 5: Synthesis of intermediate 5.

[0059] Intermediate 5 is 5-(((6-(difluoromethyl)pyrimidin-4-yl)amino)methyl)-1,3,4-oxadiazole-2-thiol, and its synthetic route is as follows:

[0060] ;

[0061] The preparation method is as follows: 23 mmol of compound 4 and 20 mL of anhydrous ethanol were added to a 100 mL round-bottom flask. A reflux condenser was installed, and 69 mmol of carbon disulfide was added from the top of the condenser using a pipette. The mixture was then heated to reflux at 80 °C. After the reactants had reacted completely, the mixture was concentrated under reduced pressure. Pure water was added until completely dissolved. The pH was adjusted to 5-6 with 10% hydrochloric acid solution, at which point a solid precipitated. The solid was filtered under reduced pressure, and the filter cake was dried to obtain a pale yellow solid, which was compound 5, with a yield of 63%. mp206.5 -208.8°C; 1H NMR (600MHz, DMSO-d6) δ 14.49(s, 1H, -SH), 8.57(s, 1H, NH), 8.49(s, 1H, Pyrimidine-H), 6.86(s, 1H, Pyrimidine-H), 6.77(t, 1H, J = 54.6 Hz, -CHF2), 4.70(d, 2H, J= 5.4 Hz, -CH2). 13C NMR(150 MHz, DMSO-d6) δ 178.25, 162.69, 161.87, 159.05,157.79(t, J = 27.0 Hz), 114.64(t, J = 237.6 Hz), 102.78, 35.70. HRMS(ESI)m / z[M+H]+ calcd for C8H8F2N5OS: 260.0412, found: 260.0409.

[0062] Example 6: Synthesis of the target compound.

[0063] The synthetic route for the target compound is as follows:

[0064] ;

[0065] The preparation method is as follows: 1.5 mmol of compound 5 was weighed into a 50 mL round-bottom flask. Using purified water as the solvent, sodium hydroxide solution was added to create an alkaline environment. After compound 5 was completely dissolved, benzyl chloride containing different substituents was added, and the mixture was stirred at room temperature. After the reaction was complete, the mixture was filtered under reduced pressure, and purified by recrystallization, silica gel column chromatography, and silica gel thin-layer chromatography to obtain the target compounds (6a-6t).

[0066] Its physicochemical properties and spectral data are shown below:

[0067] 6-(difluoromethyl)-N-((5-(methimero)-1,3,4-oxadiazol-2-yl)methyl)pyrimidine-4-amine (6a); white solid; yield 71.5%, mp 139.7–141.9 °C; ¹H NMR (600 MHz, DMSO-d6) 8.56 (s, 1H, Pyrimidine-H), 8.52 (s, 1H, NH), 6.86 (s, 1H, Pyrimidine-H), 6.77 (t, 1H, J = 54.6 Hz, CHF2), 4.82 (s, 2H, CH2), 2.68 (s, 3H, CH3); ¹³C NMR (150 MHz, DMSO-d6) δ 165.58, 165.06, 162.74, 159.05, 114.67(t, J = 237.9 Hz), 102.78, 35.46.HRMS(ESI)m / z [M+H]+ calcd for C9H10F2N5OS: 274.0569, found: 274.0566.

[0068] 6-(difluoromethyl)-N-((5-(ethimercapto)-1,3,4-oxadiazol-2-yl)methyl)pyrimidine-4-amine (6b); white solid; yield 75.6%, mp 106.8–108.3 °C; ¹H NMR (600 MHz, DMSO-d6) 8.56 (s, 1H, Pyrimidine-H), 8.50 (s, 1H, NH), 6.88 (t, 1H, J = 65.4 Hz, CHF2), 6.87 (s, 1H, Pyrimidine-H), 4.83 (d, 2H, J = 6.6 Hz, CH2), 3.24 (q, 2H, J = 8.4 Hz, CH2), 1.37 (t, 3H, J = 8.4 Hz, CH3); 13C NMR(150 MHz, DMSO-d6) δ 165.61, 164.09,162.78, 159.02, 115.00(t, J = 285.45 Hz), 102.79, 35.51, 27.07, 15.22. HRMS(ESI)m / z [M+H]+ calcd for C10H12F2N5OS: 274.0569, found: 274.0566.

[0069] N-((5-(benzylthio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidine-4-amine (6c); white solid; yield 73.1%, mp 119.4–121.3 °C; ¹H NMR (600 MHz, DMSO-d6) 8.56 (s, 1H, Pyrimidine-H), 8.53 (s, 1H, NH), 7.39 (d, J = 6.6 Hz, 1H, Ph-H), 7.30 (m, 3H, Ph-H), 6.87 (t, 1H, J = 54.6 Hz, CHF2), 6.86 (s, 1H, Pyrimidine-H), 4.83 (d, 2H, J = 4.8 Hz, CH2), 4.46 (s, 2H, CH2); 13C NMR (150 MHz, DMSO-d6) δ 165.85,163.60, 162.70, 159.06, 136.96, 129.42, 128.98, 128.20, 114.68(t, J = 237.3Hz), 102.79, 36.27, 35.44. HRMS(ESI)m / z [M+H]+ calcd for C15H13F2N5OS: 372.0701, found: 372.0699.

[0070] 6-(difluoromethyl)-N-((5-(((4-fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)pyrimidine-4-amine (6d); white solid; yield 68.3%, mp 80.9–82.5 °C; ¹H NMR (600 MHz, DMSO-d6) 8.56 (s, 1H, NH), 8.52 (s, 1H, Pyrimidine-H), 7.45 (dd, 2H, J1 = 8.4 Hz, J2 = 5.4 Hz, Ph-H), 7.13 (t, 2H, J = 9 Hz, Ph-H), 6.87 (s, 1H, Pyrimidine-H), 6.86 (t, 1H, J = 54.6 Hz, CHF2), 4.83 (d, 2H, J = 5.4 Hz, CH2-), 4.46(s, 2H, CH2-); 13C NMR(150 MHz, DMSO-d6) δ 165.88, 163.54, 162.85, 162.69, 161.23, 159.05, 157.73(t, J = 23.7 Hz), 133.35(d, J = 2.55 Hz), 131.57(d, J = 9.0 Hz), 115.84,115.70, 114.67, (t, J = 237.45 Hz), 102.77, 35.43. HRMS(ESI)m / z [M+H]+ calcdfor C15H13F3N5OS: 368.0787, found: 368.0782.

[0071] N-((5-(((2-chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidine-4-amine (6e); white solid; yield 71.9%, mp 87.5–90.2 °C; ¹H NMR (600 MHz, DMSO-d6) 8.66 (s, 1H, Pyrimidine-H), 7.54 (d, J = 7.2 Hz, 1H, Ph-H), 7.40 (d, J = 7.8 Hz, 1H, Ph-H), 7.27–7.19 (m, 2H, Ph-H), 6.78 (s, 1H, NH), 6.62 (s, 1H, Pyrimidine-H), 6.50 (t, 1H, J = 55.2 Hz, CHF2). 4.90 (s, 2H, CH2), 4.56 (s, 2H, CH2); 13C NMR (150 MHz, DMSO-d6) δ 165.13, 158.75, 134.36, 133.30, 131.39, 129.79, 127.12,113.95(t, J = 240.30 Hz), 77.27(t, J = 31.80 Hz), 35.84, 34.62. HRMS(ESI)m / z[M+H]+ calcd for C15H12F2ClN5OSNa: 406.0311, found: 406.0309.

[0072] N-((5-(((3-chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidine-4-amine (6f); white solid; yield 73.5%, mp 79.8–81.9 °C; ¹H NMR (600 MHz, DMSO-d6) 8.55 (s, 1H, NH), 8.52 (s, 1H, Pyrimidine-H), 7.51 (s, 1H, Ph-H), 7.36–7.30 (m, 3H, Ph-H), 6.86 (t, 1H, J = 54.6 Hz, CHF2), 6.85 (s, 1H, Pyrimidine-H), 4.82 (d, 2H, J = 5.4 Hz, CH2), 4.47 (s, 2H, CH2); 13C NMR(150 MHz, DMSO-d6) δ 165.96, 163.46,162.68, 159.03, 157.71(t, J = 24.0 Hz), 139.74, 133.44, 130.77, 129.28,128.15, 128.12, 114.66(t, J = 237.75 Hz), 102.79, 35.45. HRMS(ESI)m / z [M+H]+calcd for C15H12F2ClN5OSNa: 406.0311, found: 406.0310.

[0073] N-((5-((4-chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidine-4-amine (6 g); white solid; yield 72.1%, mp 142.3–143.8 °C; ¹H NMR (600 MHz, DMSO-d6) 8.68 (s, 1H, Pyrimidine-H), 7.37–7.29 (m, 4H, Ph-H), 6.79 (s, 1H, Pyrimidine-H), 6.52 (t, 1H, J = 55.2 Hz, CHF2), 6.17 (s, 1H, NH), 4.90 (s, 2H, CH2-), 4.43 (s, 2H, CH2-); ¹³C NMR (150 MHz, DMSO-d6) δ 164.82, 162.13, 158.79, 134.14,133.90, 130.49, 128.99, 113.97 (t, J = 240.3 Hz), 77.26 (t, J = 31.5 Hz), 35.98. HRMS(ESI)m / z [M+H]+ calcd for C15H12F2ClN5OSNa: 406.0311, found: 406.0309.

[0074] N-((5-(((3-bromobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidine-4-amine (6h); white solid; yield 66.2%, mp 114.6–116.1 °C; ¹H NMR (600 MHz, DMSO-d6) 8.67 (s, 1H, Pyrimidine-H), 7.58 (s, 1H, Ph-H), 7.44 (d, 1H, J = 7.8 Hz, Ph-H), 7.36 (d, 1H, J = 7.2 Hz, Ph-H), 7.21 (t, 1H, J = 7.8 Hz, Ph-H), 6.79 (s, 1H, NH), 6.51 (t, 1H, J = 55.2 Hz, CHF2), 6.42(s, 1H, Pyrimidine-H), 4.90 (s, 2H,CH2-), 4.41 (s, 2H, CH2); 13C NMR (150 MHz, DMSO-d6) δ 164.71, 158.77,137.63, 132.04, 131.34, 130.34, 127.78, 122.67, 113.96(t, J = 240.3 Hz),77.28(t, J = 31.5 Hz), 35.95, 24.30. HRMS(ESI)m / z [M+H]+ calcd forC15H12F2BrN5OSNa: 449.9806, found: 449.9805.

[0075] N-((5-((4-bromobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidine-4-amine (6i); white solid; yield 63.7%, mp 136.9–139.1 °C; ¹H NMR (600 MHz, DMSO-d6) 8.55 (s, 1H, NH), 8.51 (s, 1H, Pyrimidine-H), 7.50–7.47 (m, 2H, Ph-H), 7.36–7.34 (m, 2H, Ph-H), 6.87 (t, 1H, J = 54.6 Hz, CHF2), 6.86 (s, 1H, Pyrimidine-H), 4.82 (d, 2H, J = 5.4 Hz, CH2). 4.44(s, 2H, CH2); 13C NMR(150 MHz, DMSO-d6) δ 165.92,163.44, 162.69, 159.05, 157.73(t, J = 24.15 Hz), 136.71, 131.85, 131.65,121.38, 114.66(t, J = 237.6 Hz), 102.76, 35.47. HRMS(ESI)m / z [M+H]+ calcd forC15H12F2BrN5OSNa: 449.9806, found: 449.9806.

[0076] 6-(difluoromethyl)-N-((5-((3-(trifluoromethyl)benzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)pyrimidin-4-amine (6j); white solid; yield 69.4%, mp 52.3–54.6 °C; ¹H NMR (600 MHz, DMSO-d6) 8.54 (s, 1H, NH), 8.51 (s, 1H, Pyrimidine-H), 7.83 (s, 1H, Ph-H), 7.72 (d, 1H, J = 7.8 Hz, Ph-H), 7.63 (d, 1H, J = 7.8 Hz, Ph-H), 7.54 (t, 1H, J = 7.8 Hz, Ph-H), 6.86 (s, 1H, NH), 1H, 2H, 2H, 3H, 4H, 4H, 5H, 6 ... Pyrimidine-H), 6.77(t, 1H, J = 54.6 Hz, CHF2), 4.83(d, 2H, J = 5.4 Hz, CH2), 4.57(s, 2H, CH2-); 13C NMR(150 MHz, DMSO-d6) δ 165.97, 163.46, 162.69, 158.99, 157.73(d, J = 24.9 Hz), 138.84,133.54, 129.95, 129.78(q, J = 31.05 Hz), 126.15(q, J = 3.45 Hz), 124.86(q, J= 3.15 Hz), 114.67(t, J = 237.45 Hz), 102.77, 35.45. HRMS(ESI)m / z [M+H]+calcd for C16H12F5N5OSNa: 440.0575, found: 440.0575.

[0077] 6-(difluoromethyl)-N-((5-(((2-nitrobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)pyrimidine-4-amine (6k); white solid; yield 77.2%, mp 103.2–105.3 °C; ¹H NMR (600 MHz, DMSO-d6) 8.68 (s, 1H, Pyrimidine-H), 8.16 (d, 1H, J = 8.4 Hz, Ph-H), 7.81 (d, 1H, J = 7.2 Hz, Ph-H), 7.62 (t, 1H, J = 7.8 Hz, Ph-H), 7.52 (t, 1H, J = 7.8 Hz, Ph-H), 7.29 (s, 1H, NH), 6.78 (s, 1H, NH). Pyrimidine-H), 6.54(t, 1H, J = 55.2 Hz,CHF2), 4.90 (s, 2H, CH2), 4.81 (s, 2H, CH2); 13C NMR (150 MHz, DMSO-d6) δ158.82, 134.02, 133.10, 132.41, 129.49, 125.69, 112.36, 77.27(t, J = 31.95Hz), 35.99, 34.28. HRMS(ESI)m / z [M+H]+ calcd for C15H12F2N6O3SNa: 417.0552, found: 417.0550.

[0078] 6-(difluoromethyl)-N-((5-(((3-nitrobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)pyrimidine-4-amine (6l); white solid; yield 68.3%, mp 116.5–118.3 °C; ¹H NMR (600 MHz, DMSO-d6) 8.53 (s, 1H, Pyrimidine-H), 8.48 (s, 1H, NH), 8.35 (s, 1H, Ph-H), 8.13 (d, 1H, J = 8.4 Hz, Ph-H), 7.87 (d, 1H, J = 7.8 Hz, Ph-H), 7.61 (t, 1H, J = 7.8 Hz, Ph-H), 6.85 (t, 1H, J = 54.6 Hz, CHF2), 6.84 (s, 1H, Pyrimidine-H), 4.81(d, 2H, J= 4.8 Hz, CH2), 4.62(s, 2H, CH2); 13C NMR (150 MHz, DMSO-d6) δ 166.01,163.37, 162.69, 159.00, 148.16, 139.80, 136.14, 130.39, 124.21, 123.07,114.67(t, J = 237.75 Hz), 102.74, 35.45, 35.17. HRMS(ESI)m / z [M+H]+ calcd forC15H12F2N6O3SNa: 417.0552, found: 417.0550.

[0079] 6-(difluoromethyl)-N-((5-(((4-nitrobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)pyrimidin-4-amine (6m); white solid; yield 65.9%, mp 132.2–135.7 °C; ¹H NMR (600 MHz, DMSO-d6) 8.69 (s, 1H, Pyrimidine-H), 8.21 (d, 2H, J = 9.0 Hz, Ph-H), 7.65 (d, 2H, J = 9.0 Hz, Ph-H), 7.29 (s, 1H, NH), 6.79 (s, 1H, Pyrimidine-H), 6.54 (t, 1H, J = 55.2 Hz, CHF2), 4.92 (d, 2H, J = 5.4 Hz, CH2), 4.53(s, 2H, CH2); 13C NMR (150 MHz, DMSO-d6) δ 164.17, 162.02, 158.82, 147.64, 143.09, 130.10, 123.97, 112.35,35.98, 35.57. HRMS(ESI)m / z [M+H]+ calcd for C15H12F2N6O3SNa: 417.0552, found: 417.0550.

[0080] N-((5-((2,6-difluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidine-4-amine (6n); white solid; yield 81.6%, mp 118.2–119.9 °C; ¹H NMR (600 MHz, DMSO-d6) 8.64 (s, 1H, Pyrimidine-H), 7.29–7.24 (m, 1H, Ph-H), 6.97 (t, 1H, J = 5.4 Hz, Ph-H), 6.90 (t, 2H, J = 7.8 Hz, Ph-H), 6.79 (s, 1H, NH), 6.48 (t, 1H, J = 55.2 Hz, CHF2), 4.92 (s, 2H, CH2). 4.49 (s, 2H, CH2); 13C NMR (150 MHz, DMSO-d6) δ164.39, 162.31, 162.09(d, J = 6.75 Hz), 113.94 (t, J = 240.3 Hz), 111.65(d, J= 4.5 Hz), 111.52(d, J = 4.65 Hz), 77.31(t, J = 31.5 Hz), 35.81, 24.30. HRMS(ESI)m / z [M+H]+ calcd for C15H11F4N5OSNa: 408.0513, found: 408.0511.

[0081] N-((5-((3-chloro-2-fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidine-4-amine (6o); white solid; yield 77.8%, mp 96.4–97.8 °C; ¹H NMR (600 MHz, DMSO-d6) 8.64 (s, 1H, Pyrimidine-H), 7.39–7.33 (m, 2H, Ph-H), 7.03 (t, 2H, J = 7.8 Hz, Ph-H), 6.78 (s, 1H, NH), 6.76 (t, 1H, J = 6.0 Hz, Pyrimidine-H), 6.49 (t, 1H, J = 55.2 Hz, CHF2), 4.90 (s, 2H, CH2), 4.47 (s, 2H, CH2); 13C NMR (150 MHz, DMSO-d6) δ 164.62, 158.73, 130.78, 129.53, 124.70, 121.45(d, J = 17.7 Hz),113.94(t, J = 240.15 Hz), 77.31(t, J = 31.95 Hz), 35.80, 34.03. HRMS(ESI)m / z[M+H]+ calcd for C15H11F3ClN5OSNa: 424.0217, found: 424.0215.

[0082] N-((5-((4-chloro-2-fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidine-4-amine (6p); white solid; yield 78.1%, mp 118.2–119.9 °C; ¹H NMR (600 MHz, DMSO-d6) 8.67 (s, 1H, Pyrimidine-H), 7.50–7.48 (m, 1H, Ph-H), 7.27–7.24 (m, 1H, Ph-H), 7.03 (t, 1H, J = 9.0 Hz, Ph-H), 6.79 (s, 1H, NH), 6.51 (t, 1H, J = 55.2 Hz, CHF2), 6.42 (s, 1H, NH), 6.42 (t, 1H, NH), 6.51 (t, 1H, J = 55.2 Hz, CHF2), 6.42 (s ... NH), 6.51 (t, NH, J = 55.2 Hz, CHF2), 6.42 (s, NH, NH), 6.51 (t, NH, NH), 6.51 (t, NH, J = 55.2 Hz, CHF2), 6.42 (t, NH, NH), 6.51 (t, NH, NH), 6.51 Pyrimidine-H), 4.93 (s, 2H, CH2), 4.43 (s, 2H, CH2-); 13CNMR (150 MHz, DMSO-d6) δ 164.56, 158.76, 137.63, 130.96, 130.02, 129.29,124.78(d, J = 16.05 Hz), 117.07, 116.92, 113.94(t, J = 240.45 Hz), 77.27(t, J= 31.65 Hz), 35.86, 29.58. HRMS(ESI)m / z [M+H]+ calcd for C15H11F3ClN5OSNa:424.0217, found: 424.0217.

[0083] N-((5-((3-chloro-4-fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidine-4-amine (6q); white solid; yield 63.3%, mp 106.1–107.9 °C; ¹H NMR (600 MHz, DMSO-d6) 8.70 (s, 1H, NH), 7.51 (d, 1H, J = 6.6 Hz, Ph-H), 7.29 (s, 1H, , Pyrimidine-H), 7.12 (t, 1H, J = 8.4 Hz, Ph-H), 6.80 (s, 1H, , Pyrimidine-H), 6.54 (t, 1H, J = 54.6 Hz, CHF2), 5.77 (s, 1H, Ph-H), 4.92(d, 2H, J = 5.4 Hz, CH2), 4.41(s,2H, CH2); 13C NMR(150 MHz, DMSO-d6) δ 164.53, 162.06, 158.83, 157.03, 132.66,131.31, 129.04, 128.99, 121.26, 116.94, 116.80, 113.97(t, J = 237.3 Hz),35.99, 35.42. HRMS(ESI)m / z [M+H]+ calcd for C15H11F3ClN5OSNa: 424.0217, found: 424.0215.

[0084] N-((5-((2,3-dichlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidine-4-amine (6r); white solid; yield 61.5%, mp 99.6–101.8 °C; ¹H NMR (600 MHz, DMSO-d6) 8.55 (s, 1H, NH), 8.51 (s, 1H, Pyrimidine-H), 7.60 (d, 1H, J = 7.8 Hz, Ph-H), 7.50 (d, 1H, J = 7.8 Hz, Ph-H), 7.30 (t, 1H, J = 7.8 Hz, Ph-H), 6.86 (s, 1H, Pyrimidine-H), 6.77 (t, 1H, J = 7.8 Hz, Ph-H), 6.86 (s, 1H, Pyrimidine-H), 6.77 (t, 1H, J = 7.8 Hz, Ph-H). 54.6 Hz, CHF2), 4.83(d, 2H, J = 5.4 Hz, CH2),4.59(s, 2H, CH2); 13C NMR(150 MHz, DMSO-d6) δ 166.21, 162.95, 162.71, 159.03,136.96, 132.62, 161.83, 130.73, 130.49, 128.65, 114.68(t, J = 237.3 Hz),102.79, 35.40. HRMS(ESI)m / z [M+H]+ calcd for C15H11F2Cl2N5OSNa: 439.9922, found: 439.9920.

[0085] N-((5-((2,4-dichlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidine-4-amine (6s); white solid; yield 58.9%, 83.3–85.8 °C; ¹H NMR (600 MHz, DMSO-d6) 8.55(s, 1H, NH), 8.52(s, 1H, Pyrimidine-H), 7.63(d, 1H, J = 1.8 Hz, Ph-H), 7.55(d, 1H, J = 7.8 Hz, Ph-H), 7.37(dd, 1H, J1 = 8.4 Hz, J2 = 1.8 Hz, Ph-H), 6.86(s, 1H, Pyrimidine-H), 6.78(t, 1H, J = 54.6 Hz, CHF2), 4.83(d, 2H, J = 5.4 Hz,CH2), 4.52(s, 2H, CH2); 13C NMR(150 MHz, DMSO-d6) δ 166.19, 162.96, 162.69,159.03, 157.73(t, J = 24.0 Hz), 134.74, 134.03, 133.51, 133.16, 129.54,127.95, 114.67(t, J = 237.3 Hz), 102.76, 35.48, 34.04. HRMS(ESI)m / z [M+H]+calcd for C15H11F2Cl2N5OSNa: 439.9922, found: 439.9922.

[0086] N-((5-((3,4-dichlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidine-4-amine (6t); white solid; yield 63.8%, mp 119.7–121.6 °C; ¹H NMR (600 MHz, DMSO-d6) 8.55 (s, 1H, NH), 8.51 (s, 1H, Pyrimidine-H), 7.71 (s, 1H, Ph-H), 7.56 (d, 1H, J = 8.4 Hz, Ph-H), 7.41 (dd, 1H, J 1 = 8.4 Hz, J 2 = 2.4 Hz, Ph-H), 6.85 (s, 1H, Pyrimidine-H), 6.77 (t, 1H, J = 54.6 Hz, CHF2), 4.82(d, 2H, J = 5.4 Hz, CH2),4.47(s, 2H, CH2); 13C NMR(150 MHz, DMSO-d6) δ 165.99, 163.37, 162.68, 159.03,157.71(t, J = 21.3 Hz), 138.6, 131.47, 131.39, 131.05, 130.82, 129.81, 114.66(t, J = 237.15 Hz), 102.78, 35.46, 34.82.HRMS(ESI)m / z [M+H]+ calcd forC15H11F2Cl2N5OSNa: 439.9922, found: 439.9923.

[0087] Table 1. Antifungal activity of target compound 6a−6t at a concentration of 50 μg / mL

[0088] As shown in Table 1, some compounds exhibited certain antibacterial activity against plant pathogens at a drug concentration of 50 μg / mL. Among them, compounds 6c, 6d, 6e, 6f, 6p, and 6r showed inhibition rates of 52.17%, 58.94%, 83.25%, 55.38%, 77.12%, and 82.05% against Trichoderma, respectively, which were superior to the control drug pyrimethanil (51.83%). Compounds 6c, 6e, and 6r showed inhibition rates of 80.72%, 85.14%, and 80.97% against Mucor, respectively, which were also superior to the control drug pyrimethanil (74.36%).

[0089] Based on the above experimental results, target compounds with high antibacterial activity were selected, and their half-maximal effective concentrations (EC50) against Trichoderma and Mucor were determined. 50The drug concentrations were set at five gradients (50, 25, 12.5, 6.25, and 3.125 μg / mL). The inhibition rate at different concentrations was calculated using the inhibition rate formula. The data were integrated to obtain the virulence regression equation, and finally, the EC50 of the tested compound against the pathogenic bacteria was calculated. 50 The values ​​are shown in Table 2.

[0090] Table 2 EC50 of some target compounds against pathogenic bacteria 50 value

[0091] As shown in Table 2, some compounds exhibited good antibacterial activity against the tested strains. Among them, compounds 6c, 6d, 6e, 6f, 6p, and 6r showed good antibacterial activity against *Trichoderma* EC50. 50 The values ​​were 24.81, 20.34, 2.83, 25.19, 4.23, 7.10, and 16.35 μg / mL, respectively, which were superior to the control drug pyrimethanil (EC). 50 =36.29 μg / mL); compounds 6c, 6e and 6r showed EC50 values ​​against Mucor. 50 The values ​​were 3.45, 3.37, 6.37, and 15.42 μg / mL, respectively, which were superior to the control drug pyrimethanil (EC). 50 =15.51μg / mL).

[0092] This invention utilizes molecular design to construct a class of thioether derivatives (6a-6t) with difluoromethylpyrimidine as the parent nucleus and 1,3,4-oxadiazole as the active terminal group, and systematically studies their inhibitory activity against Trichoderma and Mucor. The results show that some compounds exhibit superior EC50 activity compared to the commercially available fungicide pyrimethanil. 50 It has high value, and the synthesis process is simple and controllable, showing good development prospects.

[0093] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A difluoromethylpyrimidine-substituted 1,3,4-oxadiazole derivative or a pharmaceutically acceptable salt thereof for the prevention and control of pathogenic fungi in edible fungi, characterized in that, The general structural formula of the derivative is shown in Formula I: Formula I; Wherein, R1 is benzyl chloride with different substituents.

2. The difluoromethylpyrimidine-substituted 1,3,4-oxadiazole derivative for the prevention and control of pathogenic fungi in edible fungi according to claim 1, characterized in that, R1 is selected from one of the following structures: 。 3. The difluoromethylpyrimidine-substituted 1,3,4-oxadiazole derivative for controlling pathogenic fungi of edible fungi according to claim 1, characterized in that, The derivative is selected from the following compounds: 6c: N-((5-(benzylthio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidin-4-amine; 6d: 6-(difluoromethyl)-N-((5-((4-fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)pyrimidin-4-amine; 6e: N-((5-((2-chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidin-4-amine; 6f: N-((5-((3-chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidin-4-amine; 6p: N-((5-((4-chloro-2-fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidin-4-amine; 6r: N-((5-((2,3-dichlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)-6-(difluoromethyl)pyrimidine-4-amine.

4. A method for preparing a difluoromethylpyrimidine-substituted 1,3,4-oxadiazole derivative for the prevention and control of pathogenic fungi in edible fungi, as described in any one of claims 1-3, characterized in that, Includes the following steps: S1: Sodium methoxide and formamidin hydrochloride are reacted in methanol, and then condensed with ethyl difluoroacetoacetate to obtain intermediate 1; S2: Under ice bath conditions, intermediate 1 is chlorinated with phosphorus oxychloride to obtain intermediate 2; S3: Intermediate 2 is condensed with glycine methyl ester in isopropanol to obtain intermediate 3; S4: Intermediate 3 is refluxed with hydrazine hydrate in ethanol to obtain intermediate 4; S5: Intermediate 4 is reacted with carbon disulfide under reflux in ethanol to obtain intermediate 5; S6: Intermediate 5 is condensed with substituted benzyl chloride in an alkaline aqueous solution, and the target compound is obtained after purification.

5. The method for preparing a difluoromethylpyrimidine-substituted 1,3,4-oxadiazole derivative for the prevention and control of pathogenic fungi in edible fungi according to claim 4, characterized in that, The intermediate 1 is 4-hydroxy-6-difluoromethylpyrimidine; The intermediate 2 is 4-chloro-6-difluoromethylpyrimidine; The intermediate 3 is (6-(difluoromethyl)pyrimidin-4-yl)glycine methyl ester; The intermediate 4 is 2-((6-(difluoromethyl)pyrimidin-4-yl)amino)acetylhydrazine; And, the intermediate 5 is 5-(((6-(difluoromethyl)pyrimidin-4-yl)amino)methyl)-1,3,4-oxadiazole-2-thiol.

6. The method for preparing a difluoromethylpyrimidine-substituted 1,3,4-oxadiazole derivative for the prevention and control of pathogenic fungi in edible fungi according to claim 4, characterized in that, In step S1, sodium methoxide is prepared at room temperature; in step S2, phosphorus oxychloride is added dropwise under ice bath conditions; and in step S5, the reaction temperature is 80°C.

7. The use of a difluoromethylpyrimidine-substituted 1,3,4-oxadiazole derivative as described in any one of claims 1-3 in the preparation of an antifungal drug for preventing and controlling pathogenic fungi of edible fungi.

8. The application according to claim 7, characterized in that, The pathogenic fungus of the edible fungus is Trichoderma.

9. The application according to claim 7, characterized in that, The pathogen of the edible fungus is Mucor.

10. An antifungal pharmaceutical composition, characterized in that, It contains a derivative as described in any one of claims 1-3 as an active ingredient, and a pharmaceutically acceptable carrier.

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