Use of an indole compound for the control of agricultural pathogens

Abstract

The application discloses an application of an indole compound in prevention and treatment of agricultural pathogenic bacteria and relates to the technical field of chemical pesticides. The indole compound of the application shows excellent inhibitory effect on plant pathogenic fungi such as rhizoctonia solani, sclerotinia sclerotiorum, botrytis cinerea, fusarium graminearum and pyricularia oryzae, and on plant pathogenic bacteria such as xanthomonas oryzae pv. oryzae, xanthomonas campestris pv. citri and pseudomonas solanacearum. Meanwhile, the indole compound of the application is easy to synthesize, simple in structure, cheap in raw material, and has the value of further research and development in the prevention and treatment of agricultural diseases.

Description

Technical Field

[0001] This invention relates to the field of chemical pesticide technology, and in particular to the use of an indole compound in the control of agricultural pathogens. Background Technology

[0002] Microbial diseases not only continue to threaten global food security but also affect human health, negatively impacting all aspects of human production and life. In agricultural production, the control of pathogens is a crucial link in ensuring crop yield and quality and maintaining the health of agricultural ecosystems. Fungal and bacterial diseases are the main causes of plant diseases.

[0003] Natural products are an important source of drug discovery, attracting much attention due to their unique structures and diverse biological activities. Carbazole, composed of a central pyrrole ring and two benzene rings, possesses a unique structure with a basic tricyclic aromatic skeleton. Furthermore, numerous reports have demonstrated that natural compounds and synthetic derivatives containing the carbazole structure exhibit a variety of biological activities, such as antibacterial, antiviral, and antitumor activities. Therefore, natural carbazole alkaloids and carbazole derivatives have received widespread attention.

[0004] In the prior art, patent CN 117397688 A discloses the application of indole compounds in the prevention and control of agricultural diseases, showing that indole compounds exhibit certain inhibitory activity against bacteria such as rice bacterial blight pathogen, citrus canker pathogen, bacterial wilt pathogen, and soft rot pathogen. At a concentration of 100 μg / mL, its in vitro antibacterial activity against plant pathogenic bacteria is over 90%, but the inhibitory concentration is relatively high, and there is still room for improvement in the antibacterial effect. Patent CN 108530335 A discloses an indole compound, its preparation method, and its application, showing that indole compounds have good inhibitory activity against rice sheath blight pathogen, but its antibacterial activity at low concentrations (10 μg / mL) is very weak, only 26.33%. Therefore, there is still an urgent need to explore indole compounds with better activity and a wider range of applications for the prevention and control of agricultural pathogens to meet the needs of agricultural production for the control of various pathogenic diseases. Summary of the Invention

[0005] To address the above shortcomings, this invention provides the use of indole compounds in the control of agricultural pathogens, specifically their use in controlling agricultural diseases caused by plant pathogenic fungi such as Rhizoctonia solani, Sclerotinia sclerotiorum, Gray mold of tomato, Fusarium graminearum, and Rice blast fungus, as well as by plant pathogenic bacteria such as Bacterium oxysporum of rice, Citrus canker, and Rhizoctonia solani. The specific technical solution is as follows: The use of an indole compound in the control of agricultural pathogens, said indole compound comprising D1-1 to D1-15 and D2-1 to D2-9, wherein compounds D1-1 to D1-15 are shown in Chemical Formula 1, and compounds D2-1 to D2-9 are shown in Chemical Formula 2: Specifically, the chemical structures of D1-1 to D1-15 and D2-1 to D2-9 are shown below: .

[0006] Furthermore, the agricultural pathogens are plant pathogenic fungi and / or plant pathogenic bacteria.

[0007] Furthermore, the plant pathogenic fungus is one or more of the following: Rhizoctonia solani (the pathogen of rice sheath blight), Sclerotinia sclerotiorum (the pathogen of rapeseed sclerotinia rot), Botrytis cinerea (the pathogen of tomato gray mold), Fusarium graminearum (the pathogen of wheat scab), and Pyrrosia oryzae (the pathogen of rice blast).

[0008] Furthermore, the plant pathogenic bacteria are one or more of the following: rice bacterial blight fungus, citrus canker fungus, and bacterial wilt fungus (Ralstonia solanacearum).

[0009] The present invention also provides a drug for preventing and controlling agricultural plant pathogens, wherein the drug contains an indole compound as described in claim 1.

[0010] Compared with the prior art, the beneficial effects of the present invention are: 1. The indole compounds of the present invention exhibit excellent inhibitory effects on plant pathogenic fungi such as Rhizoctonia solani, Sclerotinia sclerotiorum var. sclerotiorum var. sclerotiorum var. tomato, Fusarium wilt pathogen of wheat, and rice blast fungus, as well as plant pathogenic bacteria such as Bacterial blight pathogen of rice, Citrus canker pathogen, and Rhizoctonia solani. Furthermore, the indole compounds of the present invention are easy to synthesize, have simple structures, and use inexpensive raw materials.

[0011] 2. The indole compounds of this invention exhibit superior bactericidal activity compared to existing agents. At 50 μg / mL, compounds D1-1 to D1-15 showed inhibition rates exceeding 90% against the test strains of *Ralstonia solanacearum*, *Ralstonia citrus*, and *Ralstonia solanacearum*. Compounds D1-1 to D1-4, D1-7, D1-9, D1-11 to D1-13, and D1-15 showed good antibacterial activity against *Ralstonia solanacearum*, with a MIC value of 12.5 μg / mL. Compounds D1-7, D1-9, and D1-15 all had MIC values ​​of 12.5 μg / mL against *Ralstonia solanacearum*. The MIC of the compound D2-6 against *Ralstonia solanacearum* was 25 μg / mL, which was superior to the positive control agents thiabendazole and tebuconazole. Furthermore, at 10 μg / mL, compounds D2-6, D2-7, and D2-9 all showed 100% inhibition against fungi such as *Rhizoctonia solani* and *Bacillus oryzae*, indicating their potential for further research and development. Detailed Implementation

[0012] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise defined, all technical terms used below have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in the present invention are commercially available or can be prepared by existing methods.

[0013] Example 1: Synthesis method of target compounds D1-1 to D1-15 and D2-1 to D2-9 The synthetic routes for the target compounds D1-1 to D1-15 are as follows: Step 1: At -10°C, POCl3 (6.3 g, 41.17 mmol) was slowly added dropwise to DMF and stirred for 0.5 h. Then, a DMF solution of indole (3.0 g, 22.97 mmol) was slowly added, the temperature was raised to 40°C, and the reaction continued until the starting material was completely consumed. The reaction solution was poured into ice water, the pH was adjusted to 8.0, and then filtered and dried to obtain a grayish-white solid indole-3-carboxaldehyde (2.7 g, yield: 81%), which required no further processing. The 5-chloro and 5-bromosubstituted indole-3-carboxaldehyde intermediates were synthesized in a similar manner.

[0014] Step 2: Indole-3-carboxaldehyde (3.0 g, 22.97 mmol) was dissolved in THF (100 mL) and stirred at 0°C. Lithium aluminum hydride (2.6 g, 68.91 mmol) was then slowly added. After reacting overnight, the reaction was quenched with water. The insoluble matter was filtered off, and the filtrate was retained. Extraction was then performed with dichloromethane / water, and the organic phase was concentrated under reduced pressure to give a white solid 3-methylindole (2.2 g, yield: 73%), requiring no further processing. The 5-chloro and 5-bromosubstituted indole-3-methyl intermediates were synthesized using a similar method.

[0015] Step 3: Cyclopropylamine (261.19 mg, 4.57 mmol) and concentrated hydrochloric acid (0.38 mL, 4.57 mmol) were added to THF (20 mL), and the mixture was stirred until homogeneous. Then, 3-methylindole (400 mg, 3.05 mmol) and formaldehyde solution (6 mL, 15.25 mmol) were added. The reaction was allowed to proceed at room temperature for 15 min, then heated to 45°C until the reaction was complete. The mixture was quenched with a saturated sodium bicarbonate aqueous solution. The quenched mixture was transferred to a separatory funnel and extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to give compound D1-1 as a white solid (450.8 mg, yield: 61%). The remaining target compounds were synthesized using a similar method.

[0016] The synthetic routes for the target compounds D2-1 to D2-5 are as follows: p-Toluenesulfonic acid (1074.8 mg, 6.24 mmol) was added to an ethanolic solution of tryptamine (1000 mg, 6.24 mmol) and indigo (1102.0 mg, 7.49 mmol), and heated to 100°C. After the starting material was completely consumed, the solvent was removed under reduced pressure, and the mixture was extracted with ethyl acetate / water. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 2) to give a brown solid, compound D2-1 (1.39 g, yield: 92%). The remaining target compounds were synthesized using a similar method.

[0017] The synthetic route for the target compounds D2-6 to D2-8 is as follows: Step 1: At -10°C, POCl3 (813.7 mg, 5.31 mmol) was slowly added dropwise to DMF and stirred for 0.5 h. Then, a DMF solution of 5-fluoro-6-chloroindole (500 mg, 2.94 mmol) was slowly added, the temperature was raised to 40°C, and the reaction was continued for 1 h. The reaction solution was poured into ice water, the pH was adjusted to 8.0, and the mixture was extracted with ethyl acetate / water and concentrated under reduced pressure to obtain a brown solid, 5-fluoro-6-chloroindole-3-carboxaldehyde (350 mg, yield: 60%), which required no further processing. The intermediate 5,6-difluoroindole-3-carboxaldehyde was synthesized using a similar method.

[0018] Step 2: In a round-bottom flask, add 5-fluoro-6-chloroindole-3-carboxaldehyde (500 mg, 1.77 mmol), ammonium acetate (300.4 mg, 3.90 mmol), and nitrobenzene (15 mL), and heat to 110°C. After the reaction is complete, remove the solvent under reduced pressure, extract with ethyl acetate / water, and concentrate the organic phase under reduced pressure to give a brownish-yellow solid 6-chloro-5-fluoro-3-(2-nitro-1-propen-1-yl)-indole (430 mg, yield: 90%), which requires no further processing. The intermediate 5,6-difluoro-3-(2-nitro-1-propen-1-yl)-indole is synthesized by a similar method.

[0019] Step 3: Under nitrogen protection, lithium aluminum hydride (2.3 mL, 2.5 M, 5.65 mmol) was added to a two-necked flask, and a THF solution of 5-fluoro-6-chloro-3-(2-nitro-1-propen-1-yl)-indole (360 mg, 1.41 mmol) was slowly added at 0°C. The reaction was then carried out at 70°C for 24 h. After the reaction was complete, the temperature was lowered to 0°C, and a 15% NaOH solution was slowly added. The insoluble matter was removed by filtration, the solvent was removed under reduced pressure, and the mixture was extracted with ethyl acetate / water. The organic phase was concentrated under reduced pressure to give a brown solid 1-(6-chloro-5-fluoro-1H-indole-3-yl)prop-2-amine (207.8 mg, yield: 65%), which required no further processing. 1-(5,6-difluoro-1H-indole-3-yl)prop-2-amine was synthesized by a similar method.

[0020] Step 4: Toluenesulfonic acid (411.3 mg, 2.39 mmol) was added to an ethanolic solution of 5-fluoro-6-chlorotryptamine (540 mg, 2.39 mmol) and 5-chloroindigo (520.5 mg, 2.87 mmol), and heated to 100°C. After 6 h, the solvent was removed under reduced pressure and the mixture was extracted with ethyl acetate / water. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate = 1 / 2) to give compound D2-6 as a white solid (802.1 mg, yield: 86%). The remaining target compounds were synthesized using a similar method.

[0021] The synthetic route for the target compound D2-9 is as follows: Step 1: At 0°C, indigo (5.0 g, 33.98 mmol) and KOH (3.81 g, 67.96 mmol) were stirred in DMF (30 mL) for 30 min, followed by dropwise addition of 1-bromo-3-methyl-2-butene (12.66 g, 84.95 mmol). After the reaction was complete, the system was diluted with an appropriate amount of ethyl acetate, transferred to a separatory funnel, and repeatedly extracted and washed with distilled water. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an orange-red solid 1-(3-methyl-2-buten-1-yl)indoline-2,3-dione (7.13 g, yield: 97%). The methoxy-substituted 1-(3-methyl-2-buten-1-yl)indoline-2,3-dione intermediate was synthesized using a similar method.

[0022] Step 2: p-Toluenesulfonic acid (438.8 mg, 2.55 mmol) was added to an ethanolic solution of 5-methoxytryptamine hydrochloride (577.7 mg, 2.55 mmol) and 5-methoxy-1-(3-methylbut-2-en-1-yl)indoline-2,3-dione (750 mg, 3.06 mmol), and heated to 100°C. After 6 h, the solvent was removed under reduced pressure and the mixture was extracted with ethyl acetate / water. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 2) to obtain a light brown solid, compound D2-9 (777.2 mg, yield: 73%). The remaining target compounds were synthesized using a similar method.

[0023] Structural characterization parameters of target compounds D1-1 ~ D2-9: see Table 1 Table 1. Structural characterization parameters of target compounds D1-1 ~ D2-21 Example 2: Determination of activity against plant pathogenic fungi 1) Test reagents: D1-1 to D1-15 and D2-1 to D2-9 prepared in Example 1.

[0024] 2) Test strains: Plant pathogenic fungi Rhizoctonia solani, Sclerotinia sclerotiorum var. spp., Gray mold pathogen of tomato, Fusarium graminearum pathogen of wheat, and rice blast fungus.

[0025] 3) Testing method: The agricultural pathogens used in this experiment were strains preserved at 4℃ in the laboratory, and the culture medium used was potato agar-dextrose medium (PDA). PDA medium formula: 200g potato (peeled), 20g glucose, 15g agar, 1000mL distilled water, natural pH.

[0026] PDA culture medium preparation method: Wash and peel potatoes, weigh 200g and cut into small pieces. Boil in distilled water for about 20 minutes (until the potato pieces are soft but not mushy). Filter through eight layers of gauze, add distilled water to make up to 1000mL, add 15g agar and 20g glucose, stir to dissolve completely, dispense into Erlenmeyer flasks, sterilize at 121℃ for 20 minutes, and cool before use. Indoor activity determination uses the mycelial growth rate method.

[0027] Strain activation: Incubate agricultural pathogens on PDA plates at 25°C for 3-6 days.

[0028] Preparation of drug-coated plates: Heat and melt PDA culture medium, cool to 45-50℃, and add different concentrations of mixtures to prepare drug-coated plates.

[0029] Inoculation and cultivation: In a clean bench, use a punch to make a 5mm diameter mycelium cake at the edge of the mycelium after 3-6 days of cultivation (with the growth conditions as uniform as possible), then use an inoculation needle to pick it up and place it in the center of the plate. Finally, incubate it upside down in an incubator (25℃).

[0030] Results determination: After the hyphae in the blank control group were fully grown, the growth diameter of the hyphae in the drug-treated group was measured by the cross-cross method, and the inhibition rate was calculated.

[0031] Inhibition rate (%) = (control mycelium diameter - treated mycelium diameter) / (control mycelium diameter - mycelium cake diameter) × 100. Three parallel experiments were set up for each concentration. The inhibition rate of the compounds was measured and is shown in Table 2.

[0032] Table 2. Inhibition rates (%) of D1-1~D1-15 and D2-1~D2-9 against plant pathogenic fungi at 10 μg / mL Note: Only compounds with inhibition rates of 80% or higher are listed. As shown in Table 2, the D1 series compounds at 10 μg / mL did not achieve an inhibition rate of 80% against any of the five plant pathogenic fungi. Among the D2 series compounds, D2-6, D2-7, and D2-9 achieved inhibition rates exceeding 80% against some plant pathogenic fungi. At 10 μg / mL, compounds D2-6, D2-7, and D2-9 all achieved 100% inhibition against *Rhizoctonia solani* and *Bacillus oryzae*; compound D2-9 achieved 100% inhibition against *Sclerotinia sclerotiorum*, and also achieved inhibition rates exceeding 80% against *Botrytis cinerea* and *Fusarium graminearum*. In conclusion, the compounds described in this invention exhibit certain antifungal activity against *Rhizoctonia solani*, *Sclerotinia sclerotiorum*, *Botrytis cinerea*, *Fusarium graminearum*, and *Bacillus oryzae*, and warrant further investigation in the control of agricultural fungi.

[0033] Example 3: Determination of activity against plant pathogenic bacteria 1) Test reagents: D1-1 to D1-15 and D2-1 to D2-9 prepared in Example 1.

[0034] 2) Test strains: plant pathogens and bacteria such as rice bacterial blight, citrus canker, and Ralstonia solanacearum.

[0035] 3) Testing method: The bacterial strains used in this experiment were cryopreserved in the laboratory at -80℃ with 30% glycerol. The cryopreserved strains were removed and streaked onto NB solid medium for plant bacteria (beef extract: 3 g, peptone: 5 g, yeast extract: 1 g, sucrose: 10 g, agar: 15 g, distilled water: 1 L, pH 7.0; sterilized at 121℃ for 20 min), and incubated at 28℃ (37℃) until single colonies appeared. Single colonies from the solid medium were transferred to NB liquid medium for plant bacteria (beef extract: 3 g, peptone: 5 g, yeast extract: 1 g, sucrose: 10 g, distilled water: 1 L; sterilized at 121℃ for 20 min), and incubated at 28℃ (37℃) with shaking at 180 rpm until the logarithmic growth phase. The strains in the logarithmic growth phase were diluted with the appropriate liquid medium to approximately 10... 6 CFU / mL was prepared for use. The compounds were dissolved separately in DMSO, added to liquid culture medium, and mixed thoroughly to prepare a drug-containing liquid culture medium with a concentration of 200 μg / mL. 50 μL of the drug-containing culture medium and the same volume of approximately 10... 6CFU / mL bacterial culture was added to the wells of a 96-well plate, resulting in a final drug concentration of 100 μg / mL. A control of 100 μL of bacterial culture containing an equal amount of DMSO was used. The 96-well plates were incubated at 28℃ (37℃) for 24–48 h until bacterial growth was observed in the control group. The OD value (OD) of the bacterial culture in each well was measured using a microplate reader. 600 In addition, the OD values ​​of 100 μL of liquid culture medium and a 100 μg / mL drug concentration were measured to correct for the OD values ​​caused by the culture medium and the drug itself. The formulas for calculating the corrected OD value and inhibition rate are as follows: Corrected OD value = OD value of bacterial culture medium - OD value of sterile culture medium; Inhibition rate = (OD value of bacterial suspension in control medium after correction - OD value of bacterial suspension in drug-containing medium after correction) / OD value of bacterial suspension in control medium after correction × 100% All experiments were conducted in triplicate. The drug-containing liquid culture medium containing the active compound was diluted in 96-well plates by a twofold dilution method to obtain a series of concentrations of 50 μL drug-containing culture medium. Then, the inhibition rate corresponding to the series of concentrations was determined according to the same test method as in Example 2 (Table 3).

[0036] Table 3. Inhibition rates (%) of D1-1~D1-15 and D2-1~D2-9 against plant pathogenic bacteria at 10 μg / mL As shown in Table 3, except for compounds D1-5, D1-6, and D1-8, the other compounds in the D1 series all achieved an inhibition rate of over 90% against the three plant pathogenic bacteria at 10 μg / mL.

[0037] The D2 series compounds consist of indoleone and carboline fragments. Only a few compounds showed good antibacterial activity against rice bacterial blight (D2-3, D2-4, D2-6, D2-7, D2-8, D2-9) and citrus canker (D2-3, D2-6, D2-7, D2-8, D2-9), with inhibition rates exceeding 90% at 10 μg / mL. No compound showed an inhibition rate of 90% against tomato bacterial wilt.

[0038] In summary, some of the compounds described in this invention exhibit good antibacterial activity against plant pathogens such as rice bacterial blight, citrus canker, and Ralstonia solanacearum, showing promising potential for the control of agricultural pathogens and warranting further research and development.

[0039] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. Use of an indole compound for the control of agricultural pathogenic fungi, characterized in that, The chemical structures of the indole compounds are shown below: 。 2. Use of an indole compound according to claim 1 for controlling agricultural pathogenic fungi, characterized in that, The agricultural pathogens are plant pathogenic fungi and / or plant pathogenic bacteria.

3. Use of an indole compound according to claim 2 for controlling agricultural phytopathogens, characterized in that, The plant pathogenic fungi are one or more of the following: Rhizoctonia solani, Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium graminearum, and Pyrrosia oryzae.

4. The use of an indole compound according to claim 2 in the control of agricultural pathogens, characterized in that, The plant pathogenic bacteria are one or more of the following: rice bacterial blight pathogen, citrus canker pathogen, and Ralstonia solanacearum.

5. A drug for controlling agricultural plant pathogens, characterized in that, The drug contains an indole compound as described in claim 1.

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