Use of a carbazole derivative for controlling plant diseases

Abstract

The application discloses application of a carbazole derivative in prevention and treatment of plant diseases and relates to the technical field of medicinal chemistry. The structure of the carbazole derivative is as follows: The carbazole derivative has excellent inhibiting effect on plant pathogenic bacteria such as Xanthomonas oryzae ACCC 11602, Xanthomonas axonopodis pv.Citri, Pseudomonas soli maceraum and Erwinia aroideae, and has the advantages of simple structure, cheap raw materials and easy synthesis, and has the value of further research and development.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical chemistry, and particularly relates to application of a carbazole derivative in prevention and treatment of plant diseases. BACKGROUND

[0002] In agricultural production, the prevention and treatment of plant pathogens is an important link, which is related to the yield and quality of crops and the health of the agricultural ecosystem. In recent years, bacterial diseases have become the second most common disease in China's agricultural production after fungal diseases. However, the types of currently used bacterial prevention and treatment drugs are few, and the problems of drug resistance, environmental pollution and pesticide residues are increasingly prominent, and the effect of fungicides has been weakened. In order to prevent and treat agricultural diseases, protect food safety and agricultural product quality safety, and promote sustainable agricultural development, it is urgent to develop new agricultural fungicides.

[0003] Carbazole is fused by a central pyrrole ring and two benzene rings, and has a special structure of a three-ring aromatic basic skeleton. In 1872, Graebe and Glazer first separated carbazole from coal tar. In 1964, Chakraborty et al. separated Murrayanine (1-methoxy-3-formylcarbazole) which is a natural alkaloid with carbazole as the mother nucleus from Mwraya Spreng, and the Murrayanine has antibacterial activity. In addition, there have been many reports that natural compounds and synthetic derivatives containing carbazole structure have various biological activities, such as antibacterial, antiviral, antitumor activities, etc., so the development of carbazole derivatives has attracted widespread attention.

[0004]

[0005] In the prior art, patent CN 112624962 B discloses a class of carbazole-based isopropyl alcohol amine derivatives with a chiral center and a preparation method and application thereof. The patent synthesizes a series of isopropyl alcohol amine derivatives substituted at the 9-position of carbazole, focuses on the influence of the chirality of the hydroxyl group and the benzylamine substituent on the antibacterial activity of plants, and can be used for preventing and treating rice bacterial leaf blight, but the antibacterial activity needs to be further improved.

[0006] In summary, the present application provides application of a carbazole derivative in prevention and treatment of plant diseases. The compound and the compound reported in patent CN 112624962 B are obviously different in modification site and substituent type, and the antibacterial activity is significantly improved. SUMMARY

[0007] In view of the above, the application provides application of a carbazole derivative in prevention and treatment of plant diseases, which can effectively prevent and treat agricultural diseases caused by Xanthomonas oryzae ACCC 11602, Xanthomonas axonopodis pv. Citri, Pseudomonas sollamacearum and Erwinia aroideae, and the specific technical solutions are as follows.

[0008] A carbazole derivative, the structure of which is shown in chemical formula 1.

[0009]

[0010] Chemical formula 1.

[0011] The application also provides application of the carbazole derivative in prevention and treatment of plant diseases.

[0012] Preferably, the plant diseases are one or more of rice bacterial leaf blight, citrus canker, bacterial wilt and soft rot.

[0013] The application also provides a medicament for preventing and treating plant diseases, which contains the dicarbazole derivative and an adjuvant or an agriculturally acceptable carrier.

[0014] Preferably, the weight percentage of the carbazole derivative in the medicament is 1-99%.

[0015] The application also provides that the dosage form of the medicament is a suspension, wettable powder, emulsifiable concentrate or water dispersible granule.

[0016] Compared with the prior art, the application has the following beneficial effects:

[0017] 1. The carbazole derivative of the application has excellent inhibitory effect on plant pathogens such as Xanthomonas oryzae ACCC 11602, Xanthomonas axonopodis pv. Citri, Pseudomonas sollamacearum and Erwinia aroideae, and has simple structure, cheap raw materials and easy synthesis.

[0018] 2. The bactericidal activity of the present application is better than that of existing agents, and the inhibition rate of the present application on the test strains of rice bacterial leaf blight pathogen, citrus canker pathogen, bacterial wilt pathogen, and soft rot pathogen is greater than 95% at 100 μg / mL. After reducing the concentration, it is found that the inhibition rate of the present application on the test strains of rice bacterial leaf blight pathogen and citrus canker pathogen is still greater than 90% at 3.12 μg / mL, and the inhibition rate on the test strain of citrus canker pathogen is still greater than 90% at 1.56 μg / mL, which has further research and development value. DETAILED DESCRIPTION

[0019] The specific embodiments of the present application are described in detail below, but it should be understood that the protection scope of the present application is not limited by the specific embodiments.

[0020] Example 1 Synthesis of the carbazole derivative:

[0021]

[0022] The synthesis method of the compound described in the present application is carried out according to the synthesis method reported in the literature J. Med. Chem. 2020, 63, 9284−9299, and the structure is identified and confirmed by nuclear magnetic resonance and mass spectrometry. The main synthesis route is as follows:

[0023]

[0024] Experimental method:

[0025] Synthesis of compound 2: Potassium tert-butoxide (2.83 g, 25.20 mmol) and potassium iodide (2.09 g, 12.60 mmol) were added to a solution of 2 (2.01 g, 6.54 mmol) in DMF (30 mL). Then, 3,3-dimethylallyl bromide (2.89 mL, 25.20 mmol) was slowly added dropwise to the mixture. The reaction mixture was stirred at 50 °C for 30 minutes. After the reaction was completed, the mixture was diluted with ethyl acetate and extracted with water three times. The organic phase was removed. The obtained crude product was purified by silica gel chromatography (ethyl acetate / petroleum ether) to obtain white solid 2.

[0026] Synthesis of compound 3: Ammonium hydroxide (16 mL) was added to a solution of compound 2 (300 mg, 1.31 mmol) in methanol (10 mL). The mixture was refluxed for 6 h. After the reaction was completed, the solvent was evaporated under reduced pressure. The obtained crude product was purified by silica gel chromatography (dichloromethane / methanol) to obtain light yellow solid 3.

[0027] Synthesis of the target compound 4: 1H-pyrazole-1-carboxamidine hydrochloride (286.34 mg, 1.95 mmol) and DIPEA (340.26 μL, 1.95 mol) were added to a solution of compound 3 (200 mg, 0.81 mmol) in DMF (20 mL). The mixture was stirred at room temperature overnight. After the reaction was completed, the mixture was extracted with 1-butanol and water three times. The combined organic phase was evaporated in vacuum. The resulting crude product was purified by neutral alumina chromatography (dichloromethane / methanol) to give the target compound 4 as a white solid.

[0028] A carbazole derivative: white solid; yield 65%; 1 H NMR (400 MHz, DMSO- d 6) δ 8.25 (d, J = 7.7 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H), 7.44 – 7.35 (m, 2H), 7.23 – 7.11 (m,2H), 6.75 (d, J = 8.0 Hz, 1H), 5.40 (s, 1H), 5.17 (t, J = 6.7 Hz, 1H), 4.97 (d, J =6.6 Hz, 2H), 4.22 – 4.12 (m, 3H), 3.52 – 3.33 (m, 2H), 1.90 (d, J = 1.3 Hz,3H), 1.68 – 1.63 (m, 3H). MS-ESI m / z: calcd for C 21 H 26 N4O2[M+H] + : 367.2089;found: 367.2097.

[0029] Example 2: Assay of the carbazole derivative against agricultural pathogenic bacteria and the results thereof

[0030] The strains used in the experiment were the strains stored in the laboratory at -80°C with 30% glycerol. The frozen strains were taken out and streaked on NB solid medium (beef extract: 3 g, peptone: 5 g, yeast powder: 1 g, sucrose: 10 g, agar: 15 g, distilled water: 1 L, pH 7.0; sterilized at 121°C for 20 min), and incubated at 28°C until single colonies grew. Single colonies on the solid medium were picked into NB liquid medium (beef extract: 3 g, peptone: 5 g, yeast powder: 1 g, sucrose: 10 g, distilled water: 1 L; sterilized at 121°C for 20 min) and incubated in a constant temperature shaker at 28°C and 180 rpm until the logarithmic growth phase. The strains in the logarithmic growth phase were diluted to about 10 6 CFU / mL with NB liquid medium. The compounds were dissolved in DMSO, added to the liquid medium, mixed uniformly, and prepared into liquid medium containing the compounds at a concentration of 200 μg / mL. 50 μL of the liquid medium containing the compounds and the same volume of bacterial culture containing about 10 6 CFU / mL were added to the wells of a 96-well plate, and the final concentration of the drug was 100 μg / mL. The same concentration of 100 μL of bacterial liquid containing an equal amount of DMSO was used as a control. The 96-well plate was incubated in a constant temperature incubator at 28°C for 24-48 h until the control bacterial liquid grew, and the OD value of the bacterial liquid in the well was determined on an enzyme marker (OD 600 ). And another 100 μL of liquid medium and 100 μg / mL of the drug were measured for OD value, and the OD value of the medium and the drug itself was corrected. The correction formula for the OD value and the inhibition rate is as follows:

[0031] Corrected OD value = OD value of bacterial culture medium - OD value of sterile culture medium

[0032] Inhibition rate = (corrected OD value of control medium - corrected OD value of drug-containing medium) / corrected OD value of control medium × 100%

[0033] The drug-containing liquid medium of the compound was diluted in the 96-well plate by the double dilution method to obtain 50 μL of drug-containing medium at a series of concentrations, and then the inhibition rate corresponding to the series of concentrations was determined according to the same test method described above. All experiments were set in triplicate.

[0034] The carbazole derivatives described in the present patent and the positive control drug fosetyl-Al (purity 20%, purchased from Zhejiang Longwan Chemical Co., Ltd.) were selected for activity testing against common pathogenic bacteria in agriculture, such as rice bacterial leaf blight, citrus canker, bacterial wilt, and soft rot.

[0035] The MIC 90The values (minimum drug concentration to inhibit 90% of bacterial growth) are shown in Table 1.

[0036] Table 1. Antimicrobial activity of carbazole derivatives against plant pathogenic bacteria

[0037]

[0038] Note: "-" means that the antimicrobial activity of the compound was not determined

[0039] As can be seen from the results of the activity test in Table 1, compared with existing agents (thiabendazole, carvedilol), the carbazole derivatives involved in the present application all showed excellent inhibitory effect on the test strains, and the inhibition rate of the carbazole derivatives on the test strains of rice bacterial leaf blight pathogen, citrus canker pathogen, bacterial wilt pathogen and soft rot pathogen was greater than 95% at 100 μg / mL. After reducing the concentration, it was found that the inhibition rate of the carbazole derivatives on the test strains of rice bacterial leaf blight pathogen and citrus canker pathogen was still greater than 90% at 3.12 μg / mL, and the inhibition rate of the test strain of citrus canker pathogen was still greater than 90% at 1.56 μg / mL.

[0040] I. Preparation Example of Pesticide Formulation

[0041] Formulation Preparation Example 1

[0042] 36% carbazole derivative suspension

[0043]

[0044] The laboratory preparation method of the suspension is as follows: the wetting agent, dispersing agent, antifreeze agent, preservative, defoaming agent and deionized water are weighed according to the ratio, mixed uniformly in a beaker, then the carbazole derivative synthesized in Example 1 is added into the beaker, and then transferred to a sand mill. Zirconium oxide beads are added in the sand mill and ground for 2-3 h. The thickening agent xanthan gum and magnesium aluminum silicate are added and ground for another 0.5 h to control the particle size D95 below 5 microns. The zirconium oxide beads are removed by filtration, and the suspension is obtained.

[0045] Formulation Preparation Example 2

[0046] 50% carbazole derivative wettable powder

[0047]

[0048] The laboratory preparation method of the wettable powder is as follows: the carbazole derivative synthesized in Example 1, wetting agent, dispersing agent and filler are weighed according to the ratio, mixed uniformly in a food processor, and then fed into an air jet mill for crushing to obtain the wettable powder.

[0049] Formulation Preparation Example 3

[0050] 40% carbazole derivative water dispersible granules

[0051]

[0052] The laboratory preparation method of the water dispersible granule is as follows: the active ingredient (the carbazole derivative synthesized in Example 1), wetting agent, dispersing agent, disintegrating agent, binder, and filler are weighed according to the proportion, mixed uniformly in a food processor, pulverized in an air flow pulverizer, mixed again in a food processor, and then extruded and granulated in a granulator after adding an appropriate amount of water to obtain the water dispersible granule.

[0053] Preparation of the dosage form in Example 4

[0054] 20% carbazole derivative emulsion

[0055]

[0056] The laboratory preparation method of the emulsion is as follows: the active ingredient (the carbazole derivative synthesized in Example 1), emulsifier, and carrier are weighed according to the proportion, mixed uniformly in a mixing kettle to obtain the emulsion.

[0057] II. The efficacy of preventing and treating citrus canker

[0058] Test agent: 36% carbazole derivative suspension concentrate, 40% carbazole derivative water dispersible granule;

[0059] Control agent: 20% thiacopper suspension concentrate, registration certificate PD20086024, Zhejiang Longwan Chemical Co., Ltd., purchased from the market. 46% copper hydroxide water dispersible granule, registration certificate PD20211431, Guangdong Zhenge Biological Technology Co., Ltd., purchased from the market.

[0060] Blank control: water

[0061] Test base conditions and citrus varieties: the test base is located in the Valencia orange base in the suburban area of Qiaojian Town, Long'an County, Nanning City, Guangxi, and the soil fertility is good. The test variety is Valencia orange, 2 years old, with good growth.

[0062] Test design and test method: from May 2, 2023 to May 17, 2023, a field efficacy test of Valencia orange canker was carried out in the Valencia orange field in Qiaojian Town, Long'an County, Nanning City, Guangxi. Spray at the spring shoot stage, focusing on the leaves, evenly spray both sides of the leaves. The test set up 6 treatment plots, each treatment plot randomly arranged in groups, with 10 Valencia orange trees per plot and a sign, repeated 3 times. Easy spray (capacity 16 kg) was used for spraying, the first spraying time was May 2, each batch of shoots was sprayed 2 times, and the spraying interval was 15 days. During the spraying period, it was mostly cloudy. The blank control group did not carry out canker disease prevention and treatment.

[0063] Investigation method: observe and record the occurrence of leaf canker disease on the 7th and 10th day after each spraying, investigate the control effect on the 10th day after the last spraying, and record the relevant data. The diagonal line sampling method is adopted, 5 points are fixedly investigated, 1 citrus tree is investigated at each point, 5 points are sampled according to east, south, west, north and center, 1 branch is investigated at each point, 12 new leaves of each branch are investigated, and the number of diseased leaves at each level is recorded.

[0064] The canker disease grade of the Valencia orange is divided according to the number of single leaf spots, and the grading standard is as follows:

[0065] 0 level: no disease;

[0066] 1 level: 1-5 spots per leaf;

[0067] 3 level: 6-10 spots per leaf;

[0068] 5 level: 11-15 spots per leaf;

[0069] 7 level: 16-20 spots per leaf;

[0070] 9 level: more than 21 spots per leaf.

[0071] The disease index and the control effect are respectively calculated according to formula (1)-(2):

[0072] Disease index (%) = [∑ (number of diseased leaves at each level x representative value at each level) / (total number of investigated leaves x highest representative value)] x 100.

[0073] Relative control effect (%) = (disease index after spraying in the blank control area-disease index after spraying in the treatment area) / disease index after spraying in the blank control area x 100.

[0074] Table 2-1 results of field efficacy test for preventing and treating canker disease

[0075]

[0076] From the above table, it can be seen that the control effect of the carbazole derivative for preventing and treating canker disease is mostly higher than that of the 46% copper hydroxide water dispersible granule and the 20% thioconazole copper suspension agent commonly used by farmers. When the carbazole derivative is diluted by 1500 times, the control effect for preventing and treating canker disease is still higher than that of the 46% copper hydroxide water dispersible granule commonly used by farmers, and it has a good application prospect to be developed as an agricultural fungicide.

[0077] In summary, the carbazole derivative described in the application shows excellent inhibitory effect on plant pathogenic bacteria, and the activity is obviously stronger than that of the commercial positive drug thioconazole copper, and has the value of further research and development.

[0078] The foregoing description of specific exemplary embodiments of the application has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the application to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. It is intended that the scope of the application be limited not with this detailed description, but rather by the claims appended hereto.

Claims

1. A use of a carbazole derivative in preventing and controlling plant diseases, characterized in that: The structure of the carbazole derivative is shown in Chemical Formula 1: Chemical formula 1.

2. The use of a carbazole derivative in preventing and controlling plant diseases according to claim 1, characterized in that: The plant diseases are one or more of rice bacterial blight, citrus canker, bacterial wilt, and soft rot.

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