A natural indole derivative, its preparation method and application

Abstract

The application discloses a natural indole derivative and a preparation method and application thereof. The structure of the natural indole derivative is shown as formula I. The natural indole derivative is conveniently prepared through one-pot multi-component reaction by using a substituted indole, a substituted aldehyde and a substituted amine as raw materials. The natural indole derivative has significant immune induction activity on soil-borne diseases such as pepper Phytophthora capsici. The potting prevention efficiency of a representative compound reaches 45.38% at a concentration of 100 μg / mL. In vitro bactericidal test shows that the natural indole derivative does not directly kill pathogenic bacteria, but activates the plant immune system by up-regulating the defense-related metabolic pathways such as Plant-pathogen interaction, Phenylpropanoid biosynthesis and MAPK signaling pathway-plant in the plant body. The natural indole derivative provided by the application can be used as a novel plant immune inducer and is used for the development of green ecological pesticides and the comprehensive prevention and control of agricultural diseases.

Description

Technical Field

[0001] This application relates to the field of biopesticide technology, specifically to a natural indole derivative and its preparation method and application. Background Technology

[0002] Developing green and ecological agriculture is a key measure to ensure national food security and promote ecological civilization. Ecological pesticides specifically refer to pollution-free pesticides that are safe for humans and animals, environmentally friendly, and leave no residue on crops and agricultural products. They mainly include biopesticides and highly efficient, low-risk chemical pesticides. Guided by this development direction, natural products play a core role in original innovation in pesticide creation. Many natural products themselves or their structural derivatives possess highly efficient, low-toxicity, and highly selective biological activities, providing rich molecular templates and mechanisms of action for developing new green pesticides. For example, in 2024, the global market share of glufosinate reached US$499 million, and sales of abamectin series insecticides reached US$750 million, demonstrating the huge market potential of naturally derived pesticide lead molecules. Therefore, finding pesticide lead molecules from animal, plant, and microbial secondary metabolites to create new pesticides has become one of the current hot topics in green and ecological pesticide research.

[0003] In recent years, with changes in the ecological environment, the frequency and severity of plant diseases have increased significantly, posing a serious threat to modern agricultural production and often leading to reduced crop yields or even crop failure. Against this backdrop, the development of new plant protection products that are highly efficient, low in toxicity, low in residue, and environmentally compatible has become an urgent need. Among these, immunomodulatory agents have attracted considerable attention due to their unique mechanism of action: they do not directly kill harmful organisms, but rather enhance the plant's defense against pathogens by regulating its own physiological state, stimulating or inducing systemic resistance. These agents are considered a highly promising area within the family of green pesticides.

[0004] However, while some natural products or their derivatives have been reported to possess immune-inducing activity, the overall pool of naturally sourced small molecules that can serve as highly efficient and stable immune inducers remains very limited. Reported compounds often suffer from insufficient activity, complex structures making synthesis difficult, or unstable field performance. In particular, systematic research on the structurally diverse natural indole derivatives in plant immune induction is insufficient, lacking clear structure-activity relationships and readily available candidate molecules for practical application. Therefore, developing novel, structurally sound, synthetically feasible, and significantly immune-inducing small molecule compounds based on the natural indole skeleton, and establishing their preparation methods and application technologies, are urgent technical problems that need to be solved in this field at present. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide a natural indole derivative, its preparation method, and its application. The structural formula of the natural indole derivative is shown in Formula I. Experiments show that this type of compound does not directly kill pathogens, but rather significantly enhances the plant's disease resistance by activating the plant's own immune system (such as upregulating plant-pathogen interaction, phenylpropanoid biosynthesis, MAPK signaling pathway-plant, and other defense-related metabolic pathways). The representative compound has a potted plant control efficacy of over 45% against Phytophthora capsici, demonstrating good application prospects as a novel green immune-inducing drug.

[0006] To achieve the above objectives, this application provides the following technical solution:

[0007] In a first aspect, this application provides a natural indole derivative, the structure of which is shown in Formula I below:

[0008] ;in,

[0009] In formula I, R 1 It is one of 4-F, 4-Me, 5-Br, 5-OH, 5-MeO, 6-F, 6-Cl, 7-Me, and 7-F. The number before the substituent indicates the connection position of the substituent on the indole ring, and the position number is consistent with the numbering shown in Formula I.

[0010] In formula I, R 2 For H, Ph, , , , , , , One of them;

[0011] In formula I, R 3 and R 4 Each of these components is independently one of the following: hydrogen, hydroxyl, hydroxyethyl, C1-8 alkyl, C1-8 alkoxy, C1-3 alkyl sulfonic acid, benzyl, substituted benzyl, C2-8 alkyl or C3-8 alkoxy containing an unsaturated bond, C1-8 alkyl-C3-8 alkoxy, C1-6 haloalkyl, C1-6 haloalkoxy, 2-pyridyl, and haloheterocyclic-carbonyl.

[0012] Or, R 3 and R 4 Together they form -CH2-(CH2)n-CH2- or -CH2-Z(R) 5)-CH2-, where n is 0, 1, 2 or 3, and Z is a heteroatom (i.e., an atom other than carbon and hydrogen, such as nitrogen, oxygen, sulfur, phosphorus or boron); R 5 It is one of hydrogen, methyl, or ethyl.

[0013] In some embodiments, the natural indole derivative comprises at least one of the compounds shown in Formulas I-1 to I-11:

[0014] .

[0015] Secondly, this application provides a method for preparing the natural indole derivative described in the first aspect, comprising the following steps:

[0016] Starting with the substituted indole shown in Formula 1, it reacts with the substituted aldehyde shown in Formula 2 and the substituted amine shown in Formula 3 to obtain the substituted natural indole derivative shown in Formula I; Formulas 1-3 and the specific reaction formulas are as follows:

[0017] ;in,

[0018] The solvent for the reaction is one of anhydrous acetonitrile, anhydrous ethanol, tetrahydrofuran, toluene, and 1,2-dichloroethane; the catalyst for the reaction is at least one of zinc chloride, zinc sulfate, zinc nitrate, nickel chloride, aluminum chloride, zinc bromide, zinc acetate, bismuth trifluoromethanesulfonate, and scandium trifluoromethanesulfonate.

[0019] In some preferred embodiments, the reaction temperature is 60-70 °C.

[0020] Thirdly, this application provides a plant immune-inducing composition comprising the natural indole derivative described in the first aspect.

[0021] In some embodiments, the weight percentage of the natural indole derivative in the plant immune-inducing composition is 5% to 30%.

[0022] In some embodiments, the plant immune induction composition further includes at least one diluent or adjuvant selected from one or more of the following (a)-(e):

[0023] (a) Solid carriers: talc, dolomite, quartz, kaolin, bentonite, clay, diatomaceous earth, montmorillonite, activated clay, calcium carbonate, and siliceous magnesium clay;

[0024] (b) Liquid solvents: propanol, isopropanol, glycerol, toluene, xylene, chlorobenzene, 1,2-dichloroethane, 1,2-dibromoethane, methanol, ethanol, DMF, DMSO, ethyl acetate, acetone, butanone, cyclohexanone, paraffin, turpentine;

[0025] (c) Surfactants: alkyl sulfonates, alkyl sulfonates, alkyl aryl sulfonates, sorbitol polyoxyethylene esters, polyoxyethylene-fatty alcohol ethers, polyoxyethylene-fatty acid esters, aralkyl polyethylene glycol ethers, fluoroalkyl sulfonates, alkyl sulfates, lignin sulfonates;

[0026] (d) Tackifiers or stabilizers: polyvinyl alcohol, carboxymethyl cellulose, gum arabic;

[0027] (e) Other auxiliaries: inorganic dyes, organic dyes, trace nutrients.

[0028] Fourthly, this application provides the use of the natural indole derivative described in the first aspect or the plant immune-inducing composition described in the third aspect in the preparation of plant disease control drugs.

[0029] In some embodiments, the plant disease includes one or more soil-borne diseases selected from Phytophthora capsici, stem rot, root rot, Verticillium wilt, damping-off, and Fusarium wilt.

[0030] In some embodiments, the dosage form of the plant immune-inducing drug is at least one of the following: emulsion, suspension, wettable powder, or water-dispersible granules.

[0031] The active ingredient in the aforementioned plant disease control drug, namely the natural indole derivative, achieves disease control by inducing the plant's own immune system rather than directly killing pathogens; preferably, the natural indole derivative activates the plant's own immune defense by upregulating the expression of genes in the plant's plant-pathogen interaction, Phenylpropanoid biosynthesis, and / or MAPKsignaling pathway-plant metabolic pathway.

[0032] Compared with the prior art, this application has at least the following advantages and beneficial effects:

[0033] 1. Based on the natural indole skeleton, this application has obtained a series of structurally diverse and novel compounds as shown in Formula I through reasonable substituent design, which enriches the library of small molecule compounds from natural products and provides new lead molecules for the creation of green pesticides.

[0034] 2. This application employs a one-pot, multi-component reaction, using readily available substituted indoles, substituted aldehydes, and substituted amines as raw materials. Under mild conditions (60-70°C, conventional solvents such as anhydrous ethanol and acetonitrile), the target compound can be efficiently prepared by catalysis (such as bismuth trifluoromethanesulfonate, zinc nitrate, and zinc chloride). This method is concise, convenient, and yields high results, making it suitable for industrial production.

[0035] 3. The natural indole derivatives provided in this application exhibit significant immune-inducing activity against soil-borne plant diseases. Pot efficacy tests showed that the compounds represented by Formula I (such as I-2 and I-8) at a concentration of 100 μg / mL achieved control efficacy of 45.38% and 34.56% against Phytophthora capsici, respectively, which was superior to the control amino oligosaccharide (39.05%). Simultaneously, in vitro fungicidal activity tests showed that the representative compound I-2 had a direct inhibition rate of only -0.2% against Phytophthora capsici (virtually no inhibition), and the inhibition rate of I-8 was only 33.27%, far lower than the positive control metalaxyl (100%). This indicates that the disease resistance of these compounds does not originate from the direct killing of pathogens, but rather exerts its effect by activating the plant's own immune system.

[0036] 4. The mechanism of action of the natural indole derivative provided in this application is clearly defined, inducing disease resistance by upregulating plant defense-related metabolic pathways. Transcriptomic analysis showed that after treatment with compound I-2 of this application, the gene expression of metabolic pathways closely related to induced disease resistance, such as plant-pathogen interaction, phenylpropanoid biosynthesis, and the MAPK signaling pathway-plant, was significantly upregulated in pepper leaves. This confirms at the molecular level that the compound of this application can stimulate or induce systemic resistance in plants, shifting crop disease control from "direct killing" to "active defense," and providing a new strategy for green agriculture.

[0037] 5. The natural indole derivatives provided in this application can be compounded with various diluents (solid carriers, liquid solvents, surfactants, etc.) and adjuvants to form formulations such as emulsions, suspensions, wettable powders, or water-dispersible granules, which can be used to control various soil-borne diseases such as Phytophthora in pepper, stem rot, root rot, Verticillium wilt, damping-off, and Fusarium wilt. This is of great significance for ensuring agricultural production safety and promoting green and sustainable agricultural development. Attached Figure Description

[0038] Figure 1 Representative natural indole derivative I-2 provided in the embodiments of this application 1 1H NMR analysis results.

[0039] Figure 2 The representative natural indole derivative I-2 of this application 13 C NMR analysis results.

[0040] Figure 3 The results of greenhouse pot experiment on some active natural indole derivatives provided in the embodiments of this application.

[0041] Figure 4 The results of in vitro bactericidal activity tests of some active natural indole derivatives provided in the embodiments of this application.

[0042] Figure 5 Volcano plot of difference analysis results provided for embodiments of this application.

[0043] Figure 6 This is a schematic diagram of the KEEG metabolic pathway provided in the embodiments of this application.

[0044] Figure 7 This is a schematic diagram of the plant-pathogen interaction metabolic pathway provided in this application.

[0045] Figure 8 A schematic diagram of the Phenylpropanoid biosynthesis metabolic pathway provided in the embodiments of this application.

[0046] Figure 9 This is a schematic diagram of the MAPK signaling pathway-plant metabolic pathway provided in an embodiment of this application. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0048] The materials used in the following embodiments are not limited to those listed below, and other similar materials may be used instead. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0049] In this application, unless the context clearly indicates otherwise, the terms “including,” “comprising,” “containing,” “having,” etc., shall be understood as open-ended and mean “including but not limited to.”

[0050] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may be adjusted according to the desired performance. At a minimum, each numerical parameter should be interpreted based on the reported significant figures and by applying common rounding techniques.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this application pertains. Before a detailed description of this application, the following explanations of terminology are provided to better understand this application.

[0052] 1. Immunotherapy: This refers to activating the plant's own immune system with compounds to induce systemic resistance or induce systemic resistance, thereby enhancing the plant's defense against pathogens, rather than directly killing the pathogens.

[0053] 2. Induced systemic resistance: refers to the systemic and broad-spectrum disease resistance of a plant after being stimulated by a specific inducing factor (such as the natural indole derivative of this application), which activates its defense-related signaling pathways (such as the MAPK signaling pathway, phenylpropane metabolic pathway, etc.).

[0054] 3. Plant immune-inducing composition: refers to a composition containing the natural indole derivative of this application as the active ingredient, and optionally adding diluents, surfactants, stabilizers, adhesives and other adjuvants, for inducing disease resistance in plants.

[0055] The following are specific examples:

[0056] Example 1

[0057] This embodiment provides a natural indole derivative, which is a compound having the structure shown in Figure I:

[0058] ;in,

[0059] In formula I, R 1 It is one of 4-F, 4-Me, 5-Br, 5-OH, 5-MeO, 6-F, 6-Cl, 7-Me, and 7-F. The number before the substituent indicates the connection position of the substituent on the indole ring, and the position number is consistent with the numbering shown in Formula I.

[0060] In formula I, R 2 For H, Ph, , , , , , , One of them;

[0061] In formula I, R 3 and R 4Each of these components is independently one of the following: hydrogen, hydroxyl, hydroxyethyl, C1-8 alkyl, C1-8 alkoxy, C1-3 alkyl sulfonic acid, benzyl, substituted benzyl, C2-8 alkyl or C3-8 alkoxy containing an unsaturated bond, C1-8 alkyl-C3-8 alkoxy, C1-6 haloalkyl, C1-6 haloalkoxy, 2-pyridyl, and haloheterocyclic-carbonyl.

[0062] Or, R 3 and R 4 Together they form -CH2-(CH2)n-CH2- or -CH2-Z(R) 5 )-CH2-, where n is 0, 1, 2 or 3, and Z is a heteroatom (i.e., an atom other than carbon and hydrogen, such as nitrogen, oxygen, sulfur, phosphorus or boron); R 5 It is one of hydrogen, methyl, or ethyl.

[0063] Preferably, the natural indole derivative comprises at least one of the compounds shown in Formulas I-1 to I-11:

[0064] .

[0065] Example 2

[0066] This embodiment provides a method for synthesizing compound I-1 (3-(dimethylaminomethyl)-5-hydroxyindole) from Example 1. The specific steps are as follows:

[0067] Weigh out 0.133 g (1 mmol) of 5-hydroxyindole, 0.08 g (37%) of formaldehyde solution, and 1 mmol (40%) of dimethylamine aqueous solution, and dissolve them in 5 mL of anhydrous ethanol. Then add 0.033 g (0.05 mmol) of bismuth trifluoromethanesulfonate. Stir the reaction mixture at 60–70 °C and monitor the reaction by thin-layer chromatography (TLC) until complete (eluent: ethyl acetate / petroleum ether = 2:1, v / v). After complete reaction, elute the reaction mixture with petroleum ether / ethyl acetate eluent using silica gel column chromatography gradient elution (initial v / v ratio of petroleum ether to ethyl acetate was 1:1) to obtain 105 mg of the target compound I-1, with a yield of 55.3%.

[0068] Product characterization: MS (ESI) m / z 191.46 [M+H] + Calculated value C 11 H 14 N2O = 190.11.

[0069] The structural formula of compound I-1 is .

[0070] Example 3

[0071] This embodiment provides a method for synthesizing compound I-2 (3-(dimethylaminomethyl)-indole) from Example 1. The specific steps are as follows:

[0072] Indole (0.117 g, 1 mmol), formaldehyde solution (37%, 0.08 g, 1 mmol), and dimethylamine aqueous solution (40%, 0.113 g, 1 mmol) were weighed and dissolved in 6 mL of anhydrous acetonitrile, followed by the addition of zinc nitrate hexahydrate (0.029 g, 0.1 mmol). The reaction mixture was stirred at 60–70 °C and monitored by TLC until complete (eluent: ethyl acetate / petroleum ether = 1:1, v / v). After complete reaction, the reaction mixture was separated by silica gel column chromatography gradient elution using petroleum ether / ethyl acetate eluent (initial v / v ratio of petroleum ether to ethyl acetate was 2:1) to obtain 136 mg of the target compound I-2, with a separation yield of 78.2%.

[0073] Product characterization:

[0074] 1 H NMR (600 MHz, Chloroform-d) δ 8.46 (s, 1H), 7.70 (dd, J = 8.1, 1.1Hz, 1H), 7.33 (dt, J = 8.1, 1.0 Hz, 1H), 7.22 – 7.07 (m, 3H), 3.64 (s, 2H),2.29 (s, 6H);

[0075] 13 C NMR (151 MHz, Chloroform-d) δ 136.22, 127.91, 123.72, 121.92,119.52, 119.24, 113.18, 111.09, 54.49, 45.34;

[0076] MS (ESI) m / z 175.52 [M+H] + Calculated value C 11 H 14 N2 = 174.12.

[0077] Figure 1 and Figure 2 The proton NMR spectra of compound I-2 are shown below. 1 H NMR and carbon spectroscopy (H NMR) 13 C NMR).

[0078] The structural formula of compound I-2 is .

[0079] Example 4

[0080] This embodiment provides a method for synthesizing compound I-3 (3-(dimethylaminomethyl)-5-fluoroindole) from Example 1. The specific steps are as follows:

[0081] 5-Fluoroindole (0.27 g, 2 mmol), formaldehyde solution (37%, 0.24 g, 3 mmol), and dimethylamine aqueous solution (40%, 0.34 g, 3 mmol) were weighed and dissolved in 15 mL of anhydrous ethanol, followed by the addition of anhydrous zinc sulfate (0.029 g, 0.1 mmol). The reaction mixture was stirred at 60–70 °C and monitored by TLC until complete (eluent: ethyl acetate / petroleum ether = 2:1, v / v). After complete reaction, the reaction mixture was separated by silica gel column chromatography gradient elution using petroleum ether / ethyl acetate eluent (initial v / v ratio of petroleum ether to ethyl acetate was 2:1) to give 324 mg of the target compound I-3, with a separation yield of 84.4%.

[0082] Product characterization:

[0083] MS (ESI) m / z 193.48 [M+H] + , calcd. for C 11 H 13 FN2 = 192.11.

[0084] The structural formula of compound I-3 is .

[0085] Example 5

[0086] This embodiment provides a method for synthesizing compound I-7 (2-(((1H-indol-3-yl)methyl)amino)ethanol) from Example 1. The specific steps are as follows:

[0087] Indole (0.117 g, 1 mmol), formaldehyde solution (37%, 0.096 g, 1.2 mmol), and ethanolamine (0.073 g, 1.2 mmol) were weighed and dissolved in 10 mL of anhydrous acetonitrile, followed by the addition of nickel chloride hexahydrate (0.024 g, 0.1 mmol). The reaction mixture was stirred at 60 °C and monitored by TLC until complete (eluent: ethyl acetate / petroleum ether = 4:1, v / v). After complete reaction, the reaction mixture was separated by silica gel column chromatography gradient elution using petroleum ether / ethyl acetate eluent (initial v / v ratio of petroleum ether to ethyl acetate was 1:3) to give 83 mg of the target compound I-7, with a separation yield of 43.7%.

[0088] Product characterization: MS (ESI) m / z 191.37 [M+H] + Calculated value C 11 H 14 N2O = 190.11.

[0089] The structural formula of compound I-7 is .

[0090] Example 6

[0091] This embodiment provides a method for synthesizing compound I-8 (3-(di(hydroxyethyl)aminomethyl)-indole) from Example 1. The specific steps are as follows:

[0092] Indole (0.234 g, 2 mmol), formaldehyde solution (37%, 0.16 g, 2 mmol), and diethanolamine (0.21 g, 2 mmol) were weighed and dissolved in 10 mL of anhydrous tetrahydrofuran, followed by the addition of zinc nitrate hexahydrate (0.058 g, 0.2 mmol). The reaction mixture was stirred and refluxed at approximately 65 °C, and TLC was monitored until complete (eluent: ethyl acetate / petroleum ether = 5:1, v / v). After complete reaction, the reaction mixture was separated by silica gel column chromatography gradient elution using petroleum ether / ethyl acetate eluent (initial v / v ratio of petroleum ether to ethyl acetate was 1:5), yielding 186 mg of the target compound I-8, with a separation yield of 39.7%.

[0093] Product characterization: MS (ESI) m / z 235.45 [M+H] + Calculated value C 13 H 18 N2O2 = 234.14.

[0094] The structural formula of compound I-8 is .

[0095] Example 7

[0096] This embodiment provides a method for synthesizing compound I-9 (4-((1H-indol-3-yl)methyl)-morpholine) from Example 1. The specific steps are as follows:

[0097] Indole (0.234 g, 2 mmol), formaldehyde solution (37%, 0.16 g, 2 mmol), and morpholine (0.174 g, 2 mmol) were weighed and dissolved in 8 mL of anhydrous ethanol, followed by the addition of zinc chloride (0.027 g, 0.2 mmol). The reaction mixture was stirred at 70 °C and monitored by TLC until complete (eluent: ethyl acetate / petroleum ether = 2:1, v / v). After complete reaction, the reaction mixture was separated by silica gel column chromatography gradient elution using petroleum ether / ethyl acetate eluent (initial v / v ratio of petroleum ether to ethyl acetate was 1:1) to obtain 336 mg of the target compound I-9, with a separation yield of 77.8%.

[0098] Product characterization: MS (ESI) m / z 217.48 [M+H] + Calculated value C 13 H 16 N2O = 216.13.

[0099] The structural formula of compound I-9 is .

[0100] Example 8

[0101] This embodiment provides a method for synthesizing other compounds (I-4, I-5, I-6, I-10, I-11), as follows: Compounds I-4, I-5, I-6, and I-10 were prepared according to the method in Example 3, with different substituted amines (diethylamine, hydroxylamine, methanesulfonic acid, N-methylpiperazine) replacing the aqueous dimethylamine solution to obtain target compounds with different substituent types; Compound I-11 was prepared according to the method in Example 7, with 2-aminopyridine and p-fluorobenzaldehyde replacing the corresponding reaction raw materials.

[0102] The final products were verified through product characterization. The structural formulas of compounds I-4, I-5, I-6, I-10, and I-11 are as follows: , , , , .

[0103] Example 9

[0104] This embodiment provides a determination of the plant immune-inducing activity (pot experiment) of the compound represented by Formula I synthesized above.

[0105] 9.1 Test Materials

[0106] Test plant: chili pepper (variety "Julong 828"), seeds purchased from Taobao.

[0107] The tested pathogen was Phytophthora capsici (isolated and preserved by the Hubei Provincial Engineering Research Center for Biological Pesticides).

[0108] Test compound: Compound I-1-I-11 prepared in the examples of this application was used as a positive control, with amino oligosaccharide (kmk1269, Kmick Biotech) as a positive control.

[0109] 9.2 Chili Seedling Raising

[0110] Chili seeds were germinated and then sown in 72-cell trays. The substrate consisted of peat moss, vermiculite, and perlite in a volume ratio of 3:1:1. The experiment was conducted when the chili peppers reached the 4-leaf stage.

[0111] 9.3 Preparation of pathogenic fungal spore suspension

[0112] Phytophthora capsici was activated from agar slants and transferred to V8 plates, where it was incubated at 28 °C for 7 days. The plates were then rinsed with sterile water and submerged in double-distilled water, followed by 10 days of light incubation to induce zoosporangium production. Mycelia and sporangia were eluted, filtered through double-layered sterile gauze, and the filtrate was transferred to a 4 °C refrigerator for 30 minutes, followed by incubation at room temperature for 30 minutes to promote zoospore release. The zoospore concentration was adjusted to 2 × 10⁻⁶ using sterile water. 4 Quantity / mL, for later use.

[0113] 9.4 Drug treatment and inoculation

[0114] The test compound (I-1-I-11) and the control agent, amino oligosaccharide, were dissolved in an appropriate amount of DMSO, then diluted with sterile water to 100 μg / mL, and 0.5% (v / v) Tween 80 was added as a wetting agent. The solution was sprayed evenly onto the pepper leaves until saturated (approximately 5 mL per plant). A blank control group (CK) was also set up, which was sprayed with an equal amount of sterile water containing 0.5% Tween 80. Each treatment consisted of 72 pepper seedlings, and the treatment was repeated 3 times (i.e., 24 seedlings per replicate).

[0115] Seven days after induction treatment, each pepper plant was inoculated with 1 mL of the above-mentioned Phytophthora capsici zoospore suspension (concentration 2×10⁻⁶). 4 (number / mL). After inoculation, the cells were cultured in a greenhouse for 12 days, and the disease index was recorded and the control effect was calculated. The disease grading criteria are shown in Table 1 below:

[0116] Table 1 Disease Grading Standards

[0117]

[0118] Disease index and prevention and control effectiveness are calculated using the following formula:

[0119] Disease index = [∑(number of plants at each level × level)] / (total number of plants surveyed × highest representative level 5) × 100;

[0120] Prevention and control effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0121] 9.5 Test Results

[0122] Multiple potted plant efficacy tests showed that only the tested compounds I-2 and I-8 exhibited good activity, as indicated by the results. Figure 3 And as shown in Table 2 below.

[0123] Table 2. Results of immune induction against Phytophthora capsici at 100 μg / mL.

[0124]

[0125] As shown in Table 2, compound I-2 exhibited a control efficacy of 45.38%, superior to the 39.05% of amino oligosaccharides; compound I-8, with a control efficacy of 34.56%, also demonstrated good immunogenic activity. Multiple pot experiments showed that some of the natural indole derivatives prepared in this application possessed significant immunogenic activity against the soil-borne disease *Phytophthora capsici* at a concentration of 100 μg / mL.

[0126] Example 10

[0127] This embodiment provides an experiment to determine the in vitro bactericidal activity (mycelial growth rate method) of the natural indole derivative prepared in Example 1.

[0128] 10.1 Test Target

[0129] Phytophthora capsici, same as in Example 9.

[0130] 10.2 Test Methods

[0131] Compounds I-1, I-2, I-7, I-8, and I-9 were dissolved in DMSO to prepare 1% EC stock solutions (i.e., emulsifiable concentrates containing 1% active ingredient) for later use. The mycelial growth rate inhibition method was used to evaluate the in vitro bactericidal activity of the test compounds against the target at a dose of 100 μg / mL. DMSO was used as a blank control (CK), and metalaxyl (Yuanye, BW10305) was used as a positive control.

[0132] Specific procedures: Weigh a certain mass of the test compound and dissolve it in 500 μL DMSO. Mix the calculated amount of drug solution with PDA medium to prepare a drug-containing plate (9 cm in diameter) with a final concentration of 100 μg / mL. Cut 2 mm diameter pieces of *Phytophthora capsici* using a punch and inoculate them in the center of the drug-containing plate. Each treatment was repeated in triplicate. After inoculation, the plates were incubated in the dark at 28 ℃. When the colony diameter of the blank control plate reached 7.5-8 cm, the colony diameter of each treatment and the control was measured using the cross-cross method. The mycelial growth inhibition rate was calculated using the following formula:

[0133] Inhibition rate (%) = (Control colony diameter - Treatment colony diameter) / Control colony diameter × 100%.

[0134] 10.3 Test Results

[0135] Results of in vitro bactericidal activity assay as follows Figure 4 And as shown in Table 3 below.

[0136] Table 3 shows the inhibition rates of the compounds against *Phytophthora capsici* at 100 μg / mL.

[0137]

[0138] As shown in Table 2, compared with the positive control metalaxyl (inhibition rate 100%), most of the compounds in this application showed no significant inhibitory effect on the mycelial growth of *Phytophthora capsici*. In particular, the representative compound I-2 showed an inhibition rate of -0.2% (which can be considered as no inhibition within the experimental error range). This result indicates that the disease resistance exhibited by the compounds in this application against *Phytophthora capsici* does not derive from direct fungicidal activity.

[0139] Example 11 Validation of the immune induction mechanism of active compound I-2 (transcriptomics analysis)

[0140] 11.1 Test Materials and Processing

[0141] The tested chili pepper variety was "Julong 828," planted in 32-cell seed trays. The substrate was peat moss:vermiculite:perlite = 3:1:1 (volume ratio). Treatment was administered when the peppers reached the 4-leaf stage. Compound I-2 was dissolved in DMSO, diluted with sterile water to 100 μg / mL, and sprayed evenly onto the leaves until saturated. The control group was sprayed with the same amount of sterile water.

[0142] 11.2 Sample Collection

[0143] Seven days after treatment, leaves from the same leaf position were collected from both the treatment and control groups. Twelve leaves were collected each time, with five replicates per group (a total of 60 leaves). The collected leaves were immediately flash-frozen in liquid nitrogen and then stored at -80°C. The samples were transported on dry ice to a sequencing company (Wuhan Zhenyue Biotechnology Co., Ltd.) for transcriptome sequencing.

[0144] 11.3 Analysis results of transcriptome sequencing

[0145] Figure 5 The volcano plot shows the results of the differential analysis. As can be seen from the figure, compared with the control group (sprayed with sterile water), after 7 days of treatment with compound I-2 (100 μg / mL), the expression of 1402 genes was significantly upregulated and the expression of 215 genes was significantly downregulated in pepper leaves.

[0146] KEGG (Kyoto Encyclopedia of Genes and Genomes) metabolic pathway enrichment analysis was performed on differentially expressed genes, and the results are as follows: Figure 6 As shown in the figure. The results indicate that multiple pathways closely related to plant immune responses were significantly enriched.

[0147] Further analysis revealed that the following three metabolic pathways directly related to the induction of disease resistance were significantly upregulated:

[0148] Figure 7 This is a pathway diagram of plant-pathogen interaction, which describes how plants recognize pathogens and activate defense responses, and how pathogens attempt to suppress these defenses. Core processes include:

[0149] 1. Pathogen recognition: Plants recognize pathogen-related molecular patterns through pattern recognition receptors on the cell membrane, or recognize effectors secreted by pathogens through intracellular receptors, thereby triggering an immune response.

[0150] 2. Signal transduction: Recognizing a signal activates a series of signal cascade reactions. Key signaling molecules include calcium ions, reactive oxygen species, nitric oxide, and plant hormones (such as salicylic acid, jasmonic acid, and ethylene).

[0151] 3. Defense expression: Ultimately, it activates transcription factors, regulates the expression of defense genes, produces antimicrobial substances, strengthens the cell wall, or induces programmed cell death (i.e., allergic reaction) at the site of infection to limit the spread of pathogens.

[0152] The genes marked in red in the figure are upregulated. As can be seen from the figure, after treatment with compound I-2, the expression of multiple key node genes in this pathway (such as WRKY9 and the pathogenesis-related protein gene PR1) is upregulated, indicating that the plant's recognition of pathogens and early defense response are activated.

[0153] Figure 8 This diagram illustrates the phenylpropanoid biosynthesis pathway; it is one of the core pathways of plant secondary metabolism, providing the framework for lignin, flavonoids, coumarins, stilbenes, and various phenolic compounds. Its core function is to generate phenylpropanoid products with multiple functions, including structural support, antioxidant activity, antibacterial properties, pollinator attraction, and UV protection, starting from phenylalanine (or tyrosine) and through a series of enzymatic reactions. The genes encoding the enzymes marked in red in the diagram (PAL1, PAL4) were upregulated in the treatment group. Activation of this pathway helps enhance the mechanical strength of plant cell walls (lignin deposition) and synthesize phenolic substances with antibacterial activity, thereby indirectly improving the plant's resistance to pathogens.

[0154] Figure 9 This is a diagram of the MAPK signaling pathway-plant. The MAPK signaling pathway-plant is a highly conserved three-level phosphorylation cascade signaling module in plants, responsible for transmitting external signals (pathogens, stress, hormones, developmental signals) sensed by the cell membrane to the nucleus step by step, regulating gene expression and physiological responses. It is the core hub connecting upstream receptors and downstream defense / developmental responses. Genes marked with red boxes in the diagram (such as the pathogenesis-related protein gene PR1 and the reactive oxygen species gene OXII) were upregulated in the treatment group, indicating that compound I-2 can activate the plant's MAPK signaling cascade, thereby initiating systemic resistance acquisition or inducing systemic resistance.

[0155] The combined results of Example 9 (potted resistance induction activity), Example 10 (in vitro bactericidal activity), and this example (transcriptomics analysis) show that compound I-2 at a concentration of 100 μg / mL achieved a potted resistance efficacy of 45.38% against Phytophthora capsici, significantly superior to amino oligosaccharide (39.05%). Compound I-2 at the same concentration showed an in vitro mycelial growth inhibition rate of only -0.2% against Phytophthora capsici, indicating almost no direct bactericidal effect. After treatment with compound I-2, 1402 genes were upregulated and 215 genes were downregulated in pepper leaves, with significant upregulation of genes in the three metabolic pathways closely related to induced resistance: plant-pathogen interaction, phenylpropanoid biosynthesis, and MAPK signaling pathway-plant.

[0156] In summary, the resistance of the natural indole derivative provided in this application to *Phytophthora capsici* does not stem from the direct killing of the pathogen, but rather from activating the plant's own immune system (upregulating the expression of defense-related genes and metabolic pathways), inducing systemic resistance and thus enhancing its disease resistance. This compound with immune-inducing resistance function shifts crop disease control from "direct killing" to "active defense," providing a new strategy for green disease control in modern agriculture and playing a significant role in ensuring agricultural production safety and promoting sustainable agricultural development.

[0157] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A natural indole derivative, the structure of which is shown in Formula I below: ;in, In formula I, R 1 It is one of 4-F, 4-Me, 5-Br, 5-OH, 5-MeO, 6-F, 6-Cl, 7-Me, and 7-F; In formula I, R 2 For H, Ph, , , , , , , One of them; In formula I, R 3 and R 4 Each of these components is independently one of the following: hydrogen, hydroxyl, hydroxyethyl, C1-8 alkyl, C1-8 alkoxy, C1-3 alkyl sulfonic acid, benzyl, substituted benzyl, C2-8 alkyl or C3-8 alkoxy containing an unsaturated bond, C1-8 alkyl-C3-8 alkoxy, C1-6 haloalkyl, C1-6 haloalkoxy, 2-pyridyl, and haloheterocyclic-carbonyl. Or, R 3 and R 4 Together they form -CH2-(CH2)n-CH2- or -CH2-Z(R) 5 )-CH2-, where n is 0, 1, 2 or 3, and Z is a heteroatom (i.e., an atom other than carbon and hydrogen, such as nitrogen, oxygen, sulfur, phosphorus or boron); R 5 It is one of hydrogen, methyl, or ethyl.

2. The natural indole derivative of claim 1, comprising at least one of the compounds shown in formulas I-1 to I-11: 。 3. A method for preparing the natural indole derivative as described in claim 1 or 2, comprising the following steps: Starting with the substituted indole shown in Formula 1, it reacts with the substituted aldehyde shown in Formula 2 and the substituted amine shown in Formula 3 to obtain the substituted natural indole derivative shown in Formula I; Formulas 1-3 and the specific reaction formulas are as follows: ;in, The solvent for the reaction is one of anhydrous acetonitrile, anhydrous ethanol, tetrahydrofuran, toluene, and 1,2-dichloroethane; the catalyst for the reaction is at least one of zinc chloride, zinc sulfate, zinc nitrate, nickel chloride, aluminum chloride, zinc bromide, zinc acetate, bismuth trifluoromethanesulfonate, and scandium trifluoromethanesulfonate.

4. The preparation method according to claim 3, wherein the reaction temperature is 60~70 °C.

5. A plant immune-inducing composition comprising the natural indole derivative as described in claim 1 or 2.

6. The plant immune-inducing composition of claim 5, wherein the weight percentage of the natural indole derivative is 5% to 30%.

7. The plant immune-inducing composition of claim 5 or 6, further comprising at least one diluent or adjuvant selected from one or more of the following (a)-(e): (a) Solid carriers: talc, dolomite, quartz, kaolin, bentonite, clay, diatomaceous earth, montmorillonite, activated clay, calcium carbonate, and siliceous magnesium clay; (b) Liquid solvents: propanol, isopropanol, glycerol, toluene, xylene, chlorobenzene, 1,2-dichloroethane, 1,2-dibromoethane, methanol, ethanol, DMF, DMSO, ethyl acetate, acetone, butanone, cyclohexanone, paraffin, turpentine; (c) Surfactants: alkyl sulfonates, alkyl sulfonates, alkyl aryl sulfonates, sorbitol polyoxyethylene esters, polyoxyethylene-fatty alcohol ethers, polyoxyethylene-fatty acid esters, aralkyl polyethylene glycol ethers, fluoroalkyl sulfonates, alkyl sulfates, lignin sulfonates; (d) Tackifiers or stabilizers: polyvinyl alcohol, carboxymethyl cellulose, gum arabic; (e) Other auxiliaries: inorganic dyes, organic dyes, trace nutrients.

8. The use of the natural indole derivative of claim 1 or 2 or the plant immune-inducing composition of any one of claims 5-7 in the preparation of plant disease control drugs.

9. The application according to claim 8, wherein the plant disease includes one or more soil-borne diseases selected from Phytophthora capsici, stem rot, root rot, Verticillium wilt, damping-off, and Fusarium wilt.

10. The application according to claim 8, wherein the dosage form of the plant immune-inducing drug is at least one of an emulsion, a suspension, a wettable powder, or a water-dispersible granule.

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