Coumarin derivative containing oxadiazole (thiadiazole) thioether as well as preparation method and application of coumarin derivative

Abstract

The invention provides a coumarin derivative containing oxadiazole (thiadiazole) thioether and a preparation method and application thereof, the structural general formula of the coumarin derivative containing oxadiazole (thiadiazole) thioether is shown as I, and the structural general formula of the coumarin derivative containing thiadiazole thioether is shown as II, the synthesis route of the coumarin derivative containing oxadiazole (thiadiazole) thioether is simple and practical, the preparation raw materials are easy to obtain, the cost is low, and the prepared compound has a good control effect on plant viruses including tobacco mosaic virus, cucumber mosaic virus, potato Y virus, southern rice black-streaked dwarf virus or rice stripe virus. According to the invention, a favorable lead compound is provided for the creation of a natural bionic antiviral agent; # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of synthesis of coumarin derivatives, in particular to a coumarin derivative containing oxadiazole (thiadiazole) sulfide, and a preparation method and application thereof. Background Art

[0002] Finding lead compounds from natural products is an effective strategy for the development of new pesticides. Coumarins are a class of natural products with diverse biological activities, renowned for their unique physical and chemical properties. Compounds with a coumarin skeleton possess diverse pharmacological, biological, and physiological activities, making their applications in medicine, the food industry, and agriculture of great significance. In agriculture, syringostyrin, a strobilurin fungicide, was developed using coumarin as a lead compound for the control of cucumber downy mildew and apple rot. Therefore, the development of fungicides using coumarins as lead compounds holds great promise.

[0003] 1,3,4-Oxadiazole sulfide is a five-membered nitrogen-containing heterocyclic compound with significant antibacterial, antiviral, antifungal, and insecticidal biological activities, and plays a vital role in the creation and application of new pesticides.

[0004] However, there has been no report on the introduction of 1,3,4-oxadiazole sulfide into coumarin to create coumarin derivatives containing oxadiazole sulfide and to conduct agricultural activity testing. Summary of the Invention

[0005] In view of this, the present invention proposes a coumarin derivative containing oxadiazole sulfide, a preparation method and application thereof. The present invention uses 4-hydroxycoumarin as a precursor, introduces a 1,3,4-oxadiazole sulfide structural group at the hydroxyl position of coumarin by means of substructure active splicing, and designs and synthesizes a coumarin derivative containing 1,3,4-oxadiazole sulfide, which has a good control effect on tobacco mosaic virus, cucumber mosaic virus, potato virus Y, southern rice black streaked dwarf virus or rice stripe virus.

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

[0007] A coumarin derivative containing oxadiazole sulfide, the general structural formula of the coumarin derivative containing oxadiazole sulfide is shown in I:

[0008]

[0009] The general structural formula of coumarin derivatives containing thiadiazole sulfide is shown in II:

[0010]

[0011] wherein R1 is any one of hydrogen, halogen, C1-C5 alkyl, C1-C2 haloalkyl, C1-C3 alkoxy, nitro or cyano;

[0012] R2 is any one of hydrogen, halogen, C1-C5 alkyl, C1-C2 haloalkyl, C1-C3 alkoxy, nitro or cyano.

[0013] Furthermore, the halogen is any one of fluorine, chlorine, bromine or iodine;

[0014] The C1-C5 alkyl group is any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl or neopentyl;

[0015] The C1-C2 haloalkyl group is any one of trifluoromethane, difluorodichloromethane, trichloromethane, difluoromethane, dichloromethane, monofluoromethane, 1,2-difluoroethane, 1,2-methylfluoroethane, 1,2-dichloroethane or 1,2-tetrachloroethane;

[0016] The C1-C3 alkoxy group is any one of methoxy, ethoxy or n-propoxy;

[0017] A method for preparing a coumarin derivative containing oxadiazole sulfide, wherein the coumarin derivative containing 1,3,4-oxadiazole comprises the following steps:

[0018] (1) Using substituted benzoic acid as raw material and concentrated sulfuric acid as catalyst to prepare substituted benzoic acid esters;

[0019]

[0020] (2) preparing substituted benzoyl hydrazines by using substituted benzoic acid esters and hydrazine hydrate;

[0021]

[0022] (3) Preparation of 5-phenyl-1,3,4-oxadiazole-2-thiol using substituted benzoylhydrazine, potassium hydroxide and CS2;

[0023]

[0024] (4) Using 4-OH coumarin, epibromopropane and K2CO3 as catalyst, 4-(oxiran-2-ylmethoxy)-2H-benzopyran-2-one was prepared by reaction at room temperature;

[0025]

[0026] (5) Coumarin derivatives containing 1,3,4-oxadiazole were prepared by reacting N-(5-mercapto-1,3,4-oxadiazole-2-yl)benzamide, K2CO3 and 4-(oxiran-2-ylmethoxy)-2H-benzopyran-2-one;

[0027]

[0028] Furthermore, in step (1), the molar ratio of the substituted benzoic acid to concentrated sulfuric acid is 1:1; the process of preparing the substituted benzoic acid ester also includes adding an extractant for extraction, wherein the extractant is ethyl acetate and sodium chloride in a volume ratio of 3:1; in step (2), the molar ratio of the substituted benzoic acid ester to hydrazine hydrate is 1:1.5-2.0; the process of preparing the substituted benzoylhydrazine also includes adding an extractant for extraction, wherein the extractant is dichloromethane.

[0029] Furthermore, in step (3), the molar ratio of the substituted benzoylhydrazine, potassium hydroxide and CS2 is 1:1.1-1.3:1.4-1.6; in step (4), the molar ratio of the 4-OH coumarin, K2CO3 and epibromopropane is 1:3-4:4-5; in step (5), the molar ratio of the 5-phenyl-1,3,4-oxadiazole-2-thiol to 4-(oxiran-2-ylmethoxy)-2H-benzopyran-2-one and K2CO3 is 1.2-2.0:1:2.

[0030] Furthermore, the specific steps of preparing the coumarin derivatives of 1,3,4-thiadiazole include:

[0031] (1) Using thiosemicarbazide and CS2 as raw materials and K2CO3 as catalyst, 3-amino-5-mercapto-1,2,4-triazole was prepared by acid adjustment;

[0032]

[0033] (2) reacting 3-amino-5-mercapto-1,2,4-triazole, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate with N,N-diisopropylethylamine and substituted benzoic acid to prepare N-(5-mercapto-1,3,4-thiadiazol-2-yl)benzamide;

[0034]

[0035] (3) Using 4-OH coumarin, epibromopropane and K2CO3 as catalyst, 4-(oxiran-2-ylmethoxy)-2H-benzopyran-2-one was prepared by reaction at room temperature;

[0036]

[0037] (4) Coumarin derivatives containing 1,3,4-thiadiazole were prepared by reacting N-(5-mercapto-1,3,4-thiadiazol-2-yl)benzamide, K2CO3 and 4-(oxiran-2-ylmethoxy)-2H-benzopyran-2-one;

[0038]

[0039] Furthermore, in step (1), the molar ratio of the semicarbazide, CS2 and K2CO3 is 1:2-3:4-5; in step (2), the molar ratio of the 3-amino-5-mercapto-1,2,4-triazole and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine and substituted benzoic acid is 3:1.1:2:1.

[0040] Furthermore, in step (3), the molar ratio of the 4-OH coumarin, K2CO3 and epibromopropane is 1:3-4:4-5; in step (4), the molar ratio of the N-(5-mercapto-1,3,4-thiadiazol-2-yl)benzamide to 4-(oxiran-2-ylmethoxy)-2H-benzopyran-2-one and K2CO3 is 1.0-1.5:1:2.

[0041] The invention relates to an application of a coumarin derivative containing oxadiazole sulfide in resisting plant viruses.

[0042] Furthermore, the plant virus includes tobacco mosaic virus, cucumber mosaic virus, potato virus Y, southern rice black streaked dwarf virus or rice leaf stripe virus.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] (1) The synthetic route of the coumarin derivatives containing oxadiazole (thiazole) sulfide provided by the present invention is simple and practical, and the raw materials for preparation are easily available, providing a favorable lead compound for the creation of natural biomimetic antiviral agents.

[0045] (2) The compounds prepared by the present invention have strong protective, therapeutic and inactivating effects on tobacco mosaic virus. The experimental examples show that the therapeutic activity of compounds H1, H2, H6, H10, H11, Y5 and Y8 against tobacco mosaic virus is 65%-79%, which is better than the 61.3% of the control positive group Ningnanmycin. The protective activity of compounds H3, H6, H9, H11, Y1, Y5, Y8 and Y9 against tobacco mosaic virus is 55%-68%, which is also better than the 54.2% of the control positive group Ningnanmycin. Among them, compound H6 has the strongest protective and therapeutic activity, and its EC 50The values were 180.7μg / mL and 190.3μg / mL, respectively, which were better than the control positive group Ningnanmycin 247.1μg / mL.

[0046] (3) The coumarin derivatives containing oxadiazole sulfide provided by the present invention have good control effects on cucumber mosaic virus, potato virus Y, southern rice black streak dwarf virus or rice stripe virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is the H NMR spectrum (A), C NMR spectrum (B), and mass spectrum (C) of compound H1;

[0048] Figure 2 is a hydrogen nuclear magnetic resonance spectrum (A), a carbon nuclear magnetic resonance spectrum (B), a fluorine nuclear magnetic resonance spectrum (C), and a mass spectrum (D) of compound H2;

[0049] Figure 3 is the H NMR spectrum (A), C NMR spectrum (B), and mass spectrum (C) of compound H3;

[0050] Figure 4 is a hydrogen nuclear magnetic resonance spectrum (A), a carbon nuclear magnetic resonance spectrum (B), and a mass spectrum (C) of compound H4;

[0051] Figure 5 is a hydrogen nuclear magnetic resonance spectrum (A), a carbon nuclear magnetic resonance spectrum (B), and a mass spectrum (C) of compound H5;

[0052] Figure 6 is a hydrogen nuclear magnetic resonance spectrum (A), a carbon nuclear magnetic resonance spectrum (B), and a mass spectrum (C) of compound H6;

[0053] Figure 7 is a hydrogen nuclear magnetic resonance spectrum (A), a carbon nuclear magnetic resonance spectrum (B), and a mass spectrum (C) of compound H7;

[0054] Figure 8 is a hydrogen nuclear magnetic resonance spectrum (A), a carbon nuclear magnetic resonance spectrum (B), and a mass spectrum (C) of compound H8;

[0055] Figure 9 is a hydrogen nuclear magnetic resonance spectrum (A), a carbon nuclear magnetic resonance spectrum (B), a fluorine nuclear magnetic resonance spectrum (C), and a mass spectrum (D) of compound H9;

[0056] Figure 10 is a hydrogen nuclear magnetic resonance spectrum (A), a carbon nuclear magnetic resonance spectrum (B), and a mass spectrum (C) of compound H10;

[0057] Figure 11 is a hydrogen nuclear magnetic resonance spectrum (A), a carbon nuclear magnetic resonance spectrum (B), and a mass spectrum (C) of compound H11;

[0058] Figure 12 is a hydrogen nuclear magnetic resonance spectrum (A), a carbon nuclear magnetic resonance spectrum (B), and a mass spectrum (C) of compound H12;

[0059] Figure 13 is a hydrogen nuclear magnetic resonance spectrum (A), a carbon nuclear magnetic resonance spectrum (B), and a mass spectrum (C) of compound Y1;

[0060] Figure 14 is a hydrogen nuclear magnetic resonance spectrum (A), a carbon nuclear magnetic resonance spectrum (B), a fluorine nuclear magnetic resonance spectrum (C), and a mass spectrum (D) of compound Y2;

[0061] Figure 15 is a hydrogen NMR spectrum (A), a carbon NMR spectrum (B), a fluorine NMR spectrum (C), and a mass spectrum (D) of compound Y3;

[0062] Figure 16 is a hydrogen nuclear magnetic resonance spectrum (A), a carbon nuclear magnetic resonance spectrum (B), and a mass spectrum (C) of compound Y4;

[0063] Figure 17 is a hydrogen nuclear magnetic resonance spectrum (A), a carbon nuclear magnetic resonance spectrum (B), and a mass spectrum (C) of compound Y5;

[0064] Figure 18 is a hydrogen nuclear magnetic resonance spectrum (A), a carbon nuclear magnetic resonance spectrum (B), and a mass spectrum (C) of compound Y6;

[0065] Figure 19 is a hydrogen nuclear magnetic resonance spectrum (A), a carbon nuclear magnetic resonance spectrum (B), and a mass spectrum (C) of compound Y7;

[0066] Figure 20 is a hydrogen nuclear magnetic resonance spectrum (A), a carbon nuclear magnetic resonance spectrum (B), and a mass spectrum (C) of compound Y8;

[0067] Figure 21 is the H NMR spectrum (A), C NMR spectrum (B), and mass spectrum (C) of compound Y9. DETAILED DESCRIPTION

[0068] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0069] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0070] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0071] The abbreviation of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate of the present invention is HATU, the abbreviation of N,N-diisopropylethylamine is DIEA, and the abbreviation of N,N-dimethylformamide is DMF.

[0072] Example 1 - Preparation of coumarin derivatives containing 1,3,4-thiadiazole

[0073] (1) Preparation of 3-amino-5-mercapto-1,2,4-triazole

[0074] Thiosemicarbazide (1.0 eq) and CS2 (4.0 eq) were dissolved in anhydrous ethanol, and K2CO3 (3.0 eq) was added and heated under reflux for 12 h. After the reaction was completed, the mixture was cooled, the solvent was removed under reduced pressure, water was added, and the pH was adjusted to 2-3. The solid precipitated and filtered to obtain 3-amino-5-mercapto-1,2,4-triazole;

[0075] (2) Preparation of N-(5-mercapto-1,3,4-thiadiazole-2-yl)benzamide

[0076] Substituted benzoic acid (1.0 eq), HATU (1.1 eq), and DIEA (2.0 eq) were added to DMF, and 3-amino-5-mercapto-1,2,4-triazole (3.0 eq) was added. The mixture was heated under reflux for 12 h. After the reaction was completed, the mixture was cooled, the solvent was removed under reduced pressure, and water was added and filtered to obtain N-(5-mercapto-1,3,4-thiadiazol-2-yl)benzamide.

[0077] (3) Preparation of 4-(oxiran-2-ylmethoxy)-2H-benzopyran-2-one

[0078] 4-OH coumarin (1.0 eq), K2CO3 (4.0 eq) and epibromopropane (5.0 eq) were added to DMF and reacted at room temperature. TLC was performed until the intermediate was completely reacted. Water was added to precipitate the solid, which was filtered to obtain 4-(oxiran-2-ylmethoxy)-2H-benzopyran-2-one.

[0079] (4) Preparation of coumarin derivatives containing 1,3,4-thiadiazole

[0080] First, K2CO3 (2.0eq) and 4-(oxiran-2-ylmethoxy)-2H-benzopyran-2-one (1.0eq) were added to DMF, and then N-(5-mercapto-1,3,4-thiadiazole-2-yl)benzamide (1.0eq) was added and heated under reflux. After TLC detection until the reaction was complete, saturated sodium chloride aqueous solution was added, and the precipitated solid was filtered, separated and purified to obtain a coumarin derivative containing 1,3,4-thiadiazole.

[0081] Example 2 - Preparation of coumarin derivatives containing 1,3,4-oxadiazole

[0082] (1) Preparation of substituted benzoates

[0083] Dissolve the substituted benzoic acid (1.0 eq) in methanol, slowly add 98% v / v concentrated sulfuric acid (1.0 eq) at room temperature, heat to reflux, monitor the reaction by TLC until completion, add ethyl acetate and saturated sodium chloride solution in a volume ratio of 3:1 to extract, and obtain an ethyl acetate solution containing the intermediate benzoate. Dry the solution over anhydrous sodium sulfate, and remove the solvent under reduced pressure to obtain the substituted benzoate.

[0084] (2) Preparation of substituted benzoic acid hydrazide

[0085] The substituted benzoate (1.0 eq) was dissolved in anhydrous ethanol and placed in a low-temperature constant-temperature stirring reaction bath. The system temperature was controlled below 0°C. 80 wt% hydrazine hydrate (2.0 eq) was added. After TLC monitoring until the reaction was completed, dichloromethane was added for extraction to obtain a dichloromethane solution containing the intermediate benzoic acid hydrazide. The solution was dried over anhydrous sodium sulfate and desolvated under reduced pressure to obtain the intermediate benzoic acid hydrazide.

[0086] (3) Preparation of 5-phenyl-1,3,4-oxadiazole-2-thiol

[0087] Benzoic acid hydrazide (1.0 eq) and KOH (1.2 eq) were added to anhydrous ethanol and stirred until dissolved. CS2 (1.5 eq) was added and the mixture was heated under reflux. TLC was used to detect the reaction until the intermediate was completely reacted. The mixture was filtered to obtain 5-phenyl-1,3,4-oxadiazole-2-thiol.

[0088] (4) Preparation of 4-(oxiran-2-ylmethoxy)-2H-benzopyran-2-one

[0089] 4-OH coumarin (1.0 eq), K2CO3 (4.0 eq) and epibromopropane (5.0 eq) were added to DMF and reacted at room temperature. TLC was performed until the intermediate was completely reacted. Water was added to precipitate the solid, which was filtered to obtain 4-(oxiran-2-ylmethoxy)-2H-benzopyran-2-one.

[0090] (5) Preparation of coumarin derivatives containing 1,3,4-oxadiazole

[0091] K2CO3 (2.0 eq) and 4-(oxirane-2-ylmethoxy)-2H-benzopyran-2-one (1.0 eq) were dissolved in an organic solvent, and then 5-phenyl-1,3,4-oxadiazole-2-thiol (2.0 eq) was added and heated under reflux. After TLC detection until the reaction was complete, a saturated sodium chloride aqueous solution was added to the system, and after the precipitated solid was filtered, separated and purified to obtain the coumarin derivative containing 1,3,4-oxadiazole.

[0092] Example 3

[0093] Based on the preparation methods of 1,3,4-oxadiazole coumarin derivatives of Example 1 and Example 2, compounds H1-H12 and compounds Y1-Y9 were prepared, as shown in Table 1-2.

[0094] Table 1

[0095]

[0096]

[0097]

[0098] Table 2

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106] Test Example 1

[0107] (1) Anti-tobacco mosaic virus activity test

[0108] A. Virus extraction: Take common tobacco leaves that have been infected with the virus for more than 3 weeks, remove the veins, cut the leaves into pieces, place them in a mortar, add liquid nitrogen and grind them into a fine powder, and weigh them; add 0.2 mol / L phosphate buffer (pH = 7.2, containing 1% mercaptoethanol) at a solid-liquid ratio of 1:1 g / mL, stir in an ice bath for 10 minutes, filter through double-layer gauze, retain and measure the volume of the filtrate (primary extract); add 8% by volume of n-butanol to the primary extract, stir in an ice bath for 10 minutes, filter through double-layer gauze , retain and measure the volume of the filtrate (primary extract); add 8% n-butanol by volume to the primary extract, stir in an ice bath for 20 minutes, centrifuge for 20 minutes (4°C, 8000rpm), collect the supernatant and measure its volume; add 4% NaCl and n-butanol by volume of the supernatant, collect the supernatant and measure its volume, add 4% NaCl and polyethylene glycol by volume of the supernatant, centrifuge, suspend the precipitate with phosphate buffer, centrifuge and collect the supernatant, repeat 3 times, and combine the supernatants to obtain the purified virus.

[0109] B. Solution preparation of the test compound: Accurately weigh 2 mg of the target compound (H1-H12 and Y1-Y9) and place it in a 15 mL centrifuge tube. Add 30 μL of DMSO to dissolve it, and then use 1% Tween-80 to prepare a target compound solution of the desired concentration (500 μg / mL).

[0110] C. Study of the therapeutic activity of compounds (H1-H12 and Y1-Y9) against tobacco mosaic virus: Select heartleaf tobacco plants of the same growth period. After topping, retain 3-5 healthy leaves. Evenly sprinkle a small amount of corundum on each leaf. Use a brush dipped in a 500-fold diluted TMV solution to inoculate all leaves with the virus. After 30 minutes of infection, rinse the leaves of the adamantine from the surface. Air-dry the leaves naturally. Use a brush to apply a 500 μg / mL solution evenly to the right side of the leaf. Apply dimethyl sulfoxide to the left side of the leaf as a blank control. Place the treated tobacco plants in an artificially intelligent climate greenhouse for 2-3 days. Once spots appear on the leaves, count the number of spots on both sides and calculate the inhibition rate. Experiments were repeated three times.

[0111] Inhibition rate (%) = (number of black spots on the left half of the leaf - number of black spots on the right half of the leaf) / number of black spots on the left half of the leaf × 100%

[0112] D. Study of the protective activity of compounds (H1-H12 and Y1-Y9) against tobacco mosaic virus: Select heartleaf tobacco plants of the same growth stage. After topping, retain 3-5 healthy leaves. Apply a 500 μg / mL solution of the drug evenly to the right leaf with a brush, and apply DMSO to the left leaf as a blank control. After 24 hours, sprinkle corundum evenly on both the left and right leaves. Inoculate both leaves with a 500-fold dilution of the virus using a brush. Wait 30 minutes, rinse off the corundum, and incubate in an artificially intelligent climate chamber for 2-3 days. After the appearance of spots on the leaves, count the number of spots on both sides, and calculate the inhibition rate (using the same formula as in C). The experiment was repeated three times.

[0113] E. Study on the inactivation activity of compounds (H1-H12 and Y1-Y9) against tobacco mosaic virus: Select heartleaf tobacco plants of the same growth stage. After topping, retain 3-5 healthy leaves. Sprinkle a small amount of corundum evenly on each leaf. Add 1 mL of a 250-fold diluted virus solution to 1 mL of a 500 μg / mL solution and inactivate for 0.5 h. Inoculate a row of pencils on the right side of the leaf and a 500-fold diluted virus solution on the left side. Wait 30 minutes before rinsing off the corundum. Incubate in an artificial climate greenhouse for 2-3 days. After lesions appear on the leaves, count the number of lesions on both sides and calculate the inhibition rate (using the same formula as in C). The assay is repeated three times.

[0114] The test results are shown in Table 3.

[0115] Table 3

[0116]

[0117]

[0118] The activity of the synthesized target compounds against tobacco mosaic virus was determined using the half-leaf spot assay. As shown in Table 3, when the concentration of the agent was 500 μg / mL, the therapeutic activities of compounds H1, H2, H6, H10, H11, Y5, and Y8 against tobacco mosaic virus were 65.4%, 68.5%, 79.6%, 68.0%, 66.8%, 76.2%, and 72.7%, respectively, which were superior to the positive control Ningnanmycin (61.3%). Next, the protective activities of compounds H3, H6, H9, H11, Y1, Y5, Y8, and Y9 against tobacco mosaic virus were 59.5%, 68.3%, 54.7%, 58.8%, 58.0%, 65.2%, 64.7%, and 55.1%, respectively, all superior to the positive control Ningnanmycin (54.2%).

[0119] (2) Virulence determination against tobacco mosaic virus

[0120] The target compound was prepared into concentrations of 500, 250, 125, 62.5 and 31.25 μg / mL in sequence. The drug was applied and the virus was inoculated according to the method in (1). The number of lesions was counted and the inhibition rate was calculated. The corresponding EC 50 The results are shown in Table 4.

[0121] Table 4

[0122]

[0123] As can be seen from Table 4, the EC of H1, H2, H6, H10, H11, Y5 and Y8 50 The EC values of H6, Y5 and Y8 were 281.9, 248.8, 180.7, 258.6, 266.2, 21.8 and 233.2 μg / mL, respectively, which were better than ningnanmycin 284.1 μg / mL. 50 The values were 190.3, 218.6 and 244.8 μg / mL, respectively, which were all better than Ningnanmycin 247.1 μg / mL. Among them, compound H6 had the best antiviral activity for treatment and protection.

[0124] Test Example 2

[0125] Potato virus Y activity test

[0126] A. Virus purification

[0127] Using the method of Zhou Xue et al. (Zhou XP, Xu ZX, Xu J, Li D BJ South Chin. Agric. Univ. 1995, 16, 74-79), upper leaves of Nicotiana tabacum plants, inoculated for at least three weeks and systemically infected with PVY, were homogenized in phosphate buffer at 4°C, filtered through double gauze, and centrifuged at 8000 rpm. The precipitate was treated with polyethylene glycol twice and then centrifuged. The precipitate was suspended in phosphate buffer to obtain a PVY extract. The absorbance at 260 nm was measured using an ultraviolet spectrophotometer, and the viral concentration was calculated according to the formula.

[0128] Virus concentration (mg / mL) = (A 260 × dilution factor) / E 0.1％ 1cm 260nm

[0129] Where E represents the extinction coefficient, which is the light absorption value of a suspension with a concentration of 0.1% (1 mg / mL) at a wavelength of 260 nm and a light path of 1 cm. 0.1％ 1cm 260nm It is 5.0.

[0130] B. In vivo therapeutic effects of compounds on PVY

[0131] Select the Amaranth quinoa at the 5-6 leaf stage and grow uniformly, sprinkle emery on the whole leaf, and use a brush to dip the virus juice (6×10 -3 mg / mL) whole leaves were inoculated with the virus, allowed to air dry, and then rinsed with clean water until dry. The left half of the leaf was lightly coated with the agent using a brush, and the right half of the leaf was coated with a solvent of the corresponding solvent concentration as a control. After 3-4 days, the number of dead spots was recorded and the inhibition rate was calculated.

[0132] Inhibition rate (%) = (average number of necrosis spots on half leaf not treated with pesticide - average number of necrosis spots on half leaf treated with pesticide) / average number of necrosis spots on half leaf not treated with pesticide × 100%

[0133] Among them, the average number of necrotic spots on the half leaf without pesticide application and the average number of necrotic spots on the half leaf with pesticide application were the average values of three repetitions.

[0134] C. In vivo protective effect of compounds on PVY

[0135] Select the 5-6 leaf stage of Amaranth quinoa and top it. Use a brush to lightly apply the pesticide on the left half of the leaf, and apply the solvent of the corresponding concentration on the right half of the leaf as a control. After 24 hours, sprinkle emery on the whole leaf, and use a brush to dip the virus juice (6×10 -3 mg / mL) whole leaves were inoculated with the virus and then rinsed with clean water. After 3-4 days, the number of dead spots was recorded and the inhibition rate was calculated. The inhibition rate calculation formula was the same as that in B.

[0136] Test Example 3

[0137] Anti-cucumber mosaic virus activity test

[0138] A. Virus purification

[0139] Using the method of Zhou Xue et al. (Zhou XP, Xu ZX, Xu J, Li D BJ South Chin. Agric. Univ. 1995, 16, 74-79), upper leaves of Nicotiana tabacum plants, systemically infected with CMV, that had been inoculated for at least three weeks were homogenized in phosphate buffer at 4°C, filtered through double gauze, and centrifuged at 8000 rpm. The precipitate was treated with polyethylene glycol twice and then centrifuged. The precipitate was suspended in phosphate buffer to obtain a CMV extract. The absorbance at a wavelength of 260 nm was measured using an ultraviolet spectrophotometer, and the viral concentration was calculated according to the formula.

[0140] Virus concentration (mg / mL) = (A 260 × dilution factor) / E 0.1％ 1cm 260nm

[0141] Where E represents the extinction coefficient, which is the light absorption value of a suspension with a concentration of 0.1% (1 mg / mL) at a wavelength of 260 nm and a light path length of 1 cm. 0.1％ 1cm 260nm It is 5.0.

[0142] B. In vivo therapeutic effects of the compounds on CMV

[0143] Select the Amaranth quinoa at the 5-6 leaf stage and grow uniformly, sprinkle emery on the whole leaf, and use a brush to dip the virus juice (6×10 -3 mg / mL) whole leaves were inoculated with the virus, allowed to air dry, and then rinsed with clean water until dry. The left half of the leaf was lightly coated with the agent using a brush, and the right half of the leaf was coated with a solvent of the corresponding solvent concentration as a control. After 3-4 days, the number of dead spots was recorded and the inhibition rate was calculated.

[0144] Inhibition rate (%) = (average number of necrosis spots on half leaf not treated with pesticide - average number of necrosis spots on half leaf treated with pesticide) / average number of necrosis spots on half leaf not treated with pesticide × 100%

[0145] Among them, the average number of necrotic spots on the half leaf without pesticide application and the average number of necrotic spots on the half leaf with pesticide application were the average values of three repetitions.

[0146] C. In vivo protective effect of the compound against CMV

[0147] Select the 5-6 leaf stage of Amaranth quinoa and top it. Use a brush to lightly apply the pesticide on the left half of the leaf, and apply the solvent of the corresponding concentration on the right half of the leaf as a control. After 24 hours, sprinkle emery on the whole leaf, and use a brush to dip the virus juice (6×10 -3 mg / mL) whole leaves were inoculated with the virus and then rinsed with clean water. After 3-4 days, the number of dead spots was recorded and the inhibition rate was calculated. The inhibition rate calculation formula was the same as that in B.

[0148] Compounds H6 and Y5 were tested in Experimental Example 2 and Experimental Example 3, with Ningnanmycin as the control group. The results are shown in Table 5.

[0149] Table 5

[0150]

[0151]

[0152] As can be seen from Table 5, the compound H6 of the present invention has strong protective and therapeutic effects on CMV, with the preventive and therapeutic effects being 72.94% and 63.33% respectively, which are better than the control positive group Ningnanmycin.

[0153] The compound of the present invention has good control effect on southern rice black streaked dwarf virus or rice stripe virus.

[0154] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A coumarin derivative containing an oxadiazole sulfide, characterized in that: The general structural formula of coumarin derivatives containing oxadiazole sulfide is shown in I: The general structural formula of coumarin derivatives containing thiadiazole sulfide is shown in II: wherein R1 is any one of hydrogen, halogen, C1-C5 alkyl, C1-C2 haloalkyl, C1-C3 alkoxy, nitro or cyano; R2 is any one of hydrogen, halogen, C1-C5 alkyl, C1-C2 haloalkyl, C1-C3 alkoxy, nitro or cyano.

2. A coumarin derivative containing oxadiazole sulfide according to claim 1, characterized in that: The halogen is any one of fluorine, chlorine, bromine or iodine; The C1-C5 alkyl group is any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl or neopentyl; The C1-C2 haloalkyl group is any one of trifluoromethane, difluorodichloromethane, trichloromethane, difluoromethane, dichloromethane, monofluoromethane, 1,2-difluoroethane, 1,2-methylfluoroethane, 1,2-dichloroethane or 1,2-tetrachloroethane; The C1-C3 alkoxy group is any one of methoxy, ethoxy or n-propoxy.

3. The method for preparing a coumarin derivative containing oxadiazole sulfide according to claim 1, wherein: The specific steps of preparing coumarin derivatives containing 1,3,4-oxadiazole include: (1) Using substituted benzoic acid as raw material and concentrated sulfuric acid as catalyst to prepare substituted benzoic acid esters; (2) preparing substituted benzoyl hydrazines by using substituted benzoic acid esters and hydrazine hydrate; (3) Preparation of 5-phenyl-1,3,4-oxadiazole-2-thiol using substituted benzoylhydrazine, potassium hydroxide and CS2; (4) Using 4-OH coumarin, epibromopropane and K2CO3 as catalyst, 4-(oxiran-2-ylmethoxy)-2H-benzopyran-2-one was prepared by reaction at room temperature; (5) Coumarin derivatives containing 1,3,4-oxadiazole were prepared by reacting N-(5-mercapto-1,3,4-oxadiazole-2-yl)benzamide, K2CO3 and 4-(oxiran-2-ylmethoxy)-2H-benzopyran-2-one; 4. The method for preparing a coumarin derivative containing oxadiazole sulfide according to claim 3, wherein: In step (1), the molar ratio of the substituted benzoic acid to concentrated sulfuric acid is 1:1; the process of preparing the substituted benzoic acid ester further includes adding an extractant for extraction, wherein the extractant is ethyl acetate and sodium chloride in a volume ratio of 3:1; in step (2), the molar ratio of the substituted benzoic acid ester to hydrazine hydrate is 1:1.5-2.0; the process of preparing the substituted benzoyl hydrazine further includes adding an extractant for extraction, wherein the extractant is dichloromethane.

5. The method for preparing a coumarin derivative containing oxadiazole sulfide according to claim 3, wherein: In step (3), the molar ratio of the substituted benzoylhydrazine, potassium hydroxide and CS2 is 1:1.1-1.3:1.4-1.6; in step (4), the molar ratio of the 4-OH coumarin, K2CO3 and epibromopropane is 1:3-4:4-5; in step (5), the molar ratio of the 5-phenyl-1,3,4-oxadiazole-2-thiol to 4-(oxiran-2-ylmethoxy)-2H-benzopyran-2-one and K2CO3 is 1.2-2.0:1:

2.

6. The method for preparing a coumarin derivative containing oxadiazole sulfide according to claim 1, wherein: The specific steps of preparing coumarin derivatives of 1,3,4-thiadiazole include: (1) Using thiosemicarbazide and CS2 as raw materials and K2CO3 as catalyst, 3-amino-5-mercapto-1,2,4-triazole was prepared by acid adjustment; (2) reacting 3-amino-5-mercapto-1,2,4-triazole, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate with N,N-diisopropylethylamine and substituted benzoic acid to prepare N-(5-mercapto-1,3,4-thiadiazol-2-yl)benzamide; (3) Using 4-OH coumarin, epibromopropane and K2CO3 as catalyst, 4-(oxiran-2-ylmethoxy)-2H-benzopyran-2-one was prepared by reaction at room temperature; (4) Coumarin derivatives containing 1,3,4-thiadiazole were prepared by reacting N-(5-mercapto-1,3,4-thiadiazol-2-yl)benzamide, K2CO3 and 4-(oxiran-2-ylmethoxy)-2H-benzopyran-2-one; 7. The method for preparing a coumarin derivative containing oxadiazole sulfide according to claim 6, wherein: In step (1), the molar ratio of the semicarbazide, CS2 and K2CO3 is 1:2-3:4-5; in step (2), the molar ratio of the 3-amino-5-mercapto-1,2,4-triazole and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine and substituted benzoic acid is 3:1.1:2:

1.

8. The method for preparing a coumarin derivative containing oxadiazole sulfide according to claim 6, wherein: In step (3), the molar ratio of the 4-OH coumarin, K2CO3 and epibromopropane is 1:3-4:4-5; in step (4), the molar ratio of the N-(5-mercapto-1,3,4-thiadiazol-2-yl)benzamide to 4-(oxiran-2-ylmethoxy)-2H-benzopyran-2-one and K2CO3 is 1.0-1.5:1:

2.

9. Use of the coumarin derivatives containing oxathiadiazole sulfide according to claim 1 in resisting plant viruses.

10. The use according to claim 9, characterized in that The plant viruses include tobacco mosaic virus, cucumber mosaic virus, potato virus Y, southern rice black streak dwarf virus or rice stripe virus.

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