An indole methyl guanidine urea compound, its preparation method and application

Abstract

This invention discloses an indole-based methylguanidinourea compound and its preparation and applications. The structural formula of this class of compounds is shown in Formula I. Based on an enzyme catalytic mechanism-based method for constructing active substructures, and through computer-aided optimization, this invention designs and synthesizes a novel class of indole-based guanidinourea compounds. Activity assays show that these compounds possess chitinase inhibitory activity, and also exhibit highly efficient growth-regulating insecticidal activity against insects such as the diamondback moth and the Asian corn borer.

Description

Technical Field

[0001] This invention relates to an indole-type methylguanidinourea compound, its preparation method and application, belonging to the field of pesticide compound preparation. Background Technology

[0002] Chitin is a polymer composed of β-N-acetylglucosamine (GlcNAc) linked by β-1,4-glycosidic bonds. It is mainly found in the exoskeleton of insects, collagen in vertebrates, cell walls of fungi, and the shells of some marine organisms. The GH18 family of chitinases, OfChtI and OfChi-h, can degrade this polysaccharide polymer. Therefore, chitinases play a crucial role in insect growth and development. Consequently, chitinases have become important targets for the development of novel insecticides in recent years.

[0003] Both OfChtI and OfChi-h from the Asian corn borer possess a narrow substrate-binding slit and multiple substrate-binding sites (+2, +1, -1, -2, -3, -4, and -5), with catalytic cleavage occurring between +1 and -1, and containing highly conserved amino acids. Chitinase inhibitors can suppress chitinase activity, thus showing broad application prospects in agriculture for controlling pests and diseases. Currently, the natural chitinase inhibitors Argifin and Argadin (J. Antibiot. 53, 603–606 and Chem. Pharm. Bull. 48, 1442–1446) exhibit good inhibitory activity against chitinase, but their synthesis is difficult and costly, thus limiting their large-scale application. Summary of the Invention

[0004] The purpose of this invention is to provide a novel and highly efficient chitinase inhibitor, which has the advantages of simple structure and easy synthesis.

[0005] The specific technical solution of the present invention is as follows:

[0006] According to a first aspect of the present invention, an indole-type methylguanidinourea compound is provided, the structure of which is shown in Formula I:

[0007]

[0008] Wherein, R is selected from any one of the following groups:

[0009]

[0010] According to a second aspect of the present invention, a method for preparing the above-mentioned indole-type methylguanidinourea compound is provided, comprising the following steps:

[0011] (1) Indoleacetic acid (IAA) reacts with formula II under catalytic conditions to give formula III;

[0012]

[0013] (2) The reaction of hemisulfate isomethylthiourea type IV with ditert-butyl dicarbonate type V yields isomethylthiourea type VI protected by a single Boc.

[0014]

[0015] (3) The mono-Boc protected isomethylthiourea formula VI reacts with methylcarbamoyl chloride formula VII under alkaline conditions to obtain formula VIII;

[0016]

[0017] (4) Formula VIII and Formula IX ethanolamine react to obtain Formula X;

[0018]

[0019] (5) Formulas III and X first undergo a condensation reaction and then the Boc protecting group is removed to obtain Formula I;

[0020]

[0021] Furthermore, in step (1), the reaction solvent is N,N-dimethylformamide or tetrahydrofuran, the reaction temperature is 0-30℃, and the catalyst used in the reaction is sodium hydride or potassium hydride.

[0022] Furthermore, in step (2), the solvent used is a mixture of two solvents in a volume ratio of 1:1 to 5:1, and the two solvents are dioxane / water, tetrahydrofuran / water, or acetone / water; the base used is selected from any one of sodium bicarbonate, sodium carbonate, potassium carbonate, and sodium hydroxide.

[0023] Furthermore, the reaction temperature is 0℃-25℃, and the solvent used is dichloromethane, toluene, DMF or acetone; the base used is triethylamine, 4-DMAP or potassium carbonate.

[0024] Furthermore, the molar ratio of ethanolamine of formula IX to formula VIII is between 2:1 and 5:1, and the solvent used is tetrahydrofuran, dichloromethane, or DMF.

[0025] Furthermore, the solvent used is dichloromethane or tetrahydrofuran, the condensing agent used is EDCI, DCC or CDI, and the base used is triethylamine or 4-DMAP.

[0026] According to a third aspect of the invention, the use of the indole-type methylguanidinourea compounds described in the first aspect in insect chitinase inhibitors or in the control of agricultural pests is provided.

[0027] In specific cases, when used as an insect chitinase inhibitor, the chitinase is derived from the Asian corn borer.

[0028] Specifically, when used for the control of agricultural pests, the pests include Lepidoptera, Coleoptera, Orthoptera, Isoptera, Hemiptera, Hymenoptera, Diptera, and Thysanoptera; or the pests include nematodes, which include nematodes of the following genera: Cystella, Heterodermella, Root-knot Nematode, Perforator Nematode, Short-bodied Nematode, Small-cusped Nematode, Long-needle Nematode, Hairy Nematode, Fasciola, Stem Nematode, Smooth-striped Nematode, and Eel Nematode.

[0029] This invention, through structure-activity relationship analysis of Argifin and Argadin and analysis of the active pockets of OfChtI (PDB ID: 3WL1) and OfChi-h (PDB ID: 5GQB), revealed that the dimethylguanidinourea fragment can interact with the -1 catalytic site of the enzyme, and the tryptophan at the +1 binding site is crucial for the affinity of the inhibitor. Therefore, based on the active substructure construction method of the enzyme catalytic mechanism and through computer-aided optimization, researchers designed and synthesized a novel class of indole-based guanidinourea compounds. Activity assays showed that these compounds possess chitinase inhibitory activity and also exhibit highly efficient growth-regulating insecticidal activity against insects such as the diamondback moth and the Asian corn borer. This research has significant implications for agricultural pest and disease control, providing a new and efficient control method for agricultural production. Attached Figure Description

[0030] Figure 1 This is the 1H NMR spectrum of compound A1.

[0031] Figure 2 This is the carbon NMR spectrum of compound A1.

[0032] Figure 3 Chitinase IC of compound A1 50 Suppression curve. Detailed Implementation

[0033] The technical solution of this invention will be described in detail below with reference to implementation examples.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. I. Preparation and Structural Characterization of Compounds of Formula I

[0036] Example 1: Preparation process of compound A1.

[0037]

[0038] 5.71 mmol of indoleacetic acid (IAA) was dissolved in N,N-dimethylformamide. 6.85 mmol of sodium hydride was added in portions under an ice-water bath. After reacting for 30 min, Formula II was slowly added, and the reaction was carried out at room temperature for 1 hour. After the reaction was completed, the reaction was quenched with ammonium chloride solution, extracted with ethyl acetate, and the ethyl acetate phase was collected, dried over anhydrous sodium sulfate, the solvent was concentrated, and the white solid product was collected using petroleum ether / ethyl acetate = 10:1 as eluent, with a yield of 97%.

[0039] The structural verification data is as follows:

[0040] 1 H NMR (500MHz, DMSO-d6) δ12.23(s,1H),7.53(d,J=7.9Hz,1H),7.40(d,J=10.4Hz,2H),7.38–7.33( m,2H),7.20(d,J=8.5Hz,2H),7.13–7.08(m,1H),7.02(t,J=7.5Hz,1H),5.37(s,2H),3.67(s,2H).

[0041] 13 C NMR (126MHz, DMSO-d6) δ172.98,137.35,135.85,131.95,128.92,128.51,127.86,127.57,121.41,119.03,118.85,48.18,30.82.

[0042]

[0043] 0.15 mol of isomethylthiourea hemisulfate (Formula IV) and 0.15 mol of sodium bicarbonate were dissolved in 40 mL of tetrahydrofuran / water (V:V = 1:1). 0.1 mol of Boc anhydride (Formula V) was slowly added dropwise under ice-water bath and stirring. The addition was completed in 20 min, and the reaction was continued at room temperature with stirring for 1 hour. After the reaction was complete, the tetrahydrofuran was removed by concentration. The residue was extracted with dichloromethane, and the dichloromethane phase was collected, dried over anhydrous sodium sulfate, and the solvent was concentrated to give a white solid product VI in 89% yield.

[0044] The structural verification data is as follows:

[0045] 1 H NMR (500MHz, CDCl3) δ12.44(s,1H),2.39(s,3H),2.22(s,3H),1.52(s,9H).

[0046] 13 C NMR (125MHz, CDCl3) δ171.19,168.32,160.89,81.21,27.97,24.53,14.41.

[0047]

[0048] 0.1 mol of isomethylthiourea VI protected by mono-Boc anhydride and 0.2 mol of triethylamine were dissolved in 40 mL of dichloromethane. Under ice bath stirring, 0.2 mol of methylcarbamoyl chloride-dichloromethane solution was slowly added dropwise to the reaction flask. After the addition was complete, stirring was continued overnight. After the reaction was completed, water was added to extract the reaction solution, and the dichloromethane phase was collected, dried over anhydrous sodium sulfate, and the concentrated solvent was filtered through a column. The white solid product was collected using petroleum ether / ethyl acetate = 10:1 as eluent, with a yield of 95%.

[0049] The structural verification data is as follows:

[0050] 1 H NMR (500MHz, CDCl3) δ12.30 (s, 1H), 5.57 (s, 1H), 2.84 (d, J = 5.1Hz, 3H), 2.29 (s, 3H), 1.48 (s, 9H).

[0051] 13 C NMR (126MHz, CDCl3) δ167.16,162.55,151.12,82.53,28.01,26.70,14.21.

[0052]

[0053] 0.05 mol of compound VIII and 0.15 mol of ethanolamine IX were dissolved in 15 mL of tetrahydrofuran solvent and stirred at room temperature for 1.5 hours. After the reaction was completed, the reaction solution was concentrated and passed through a column to collect the white solid product X, with a yield of 96%.

[0054] The structural verification data is as follows:

[0055] 1 H NMR (500MHz, CDCl3) δ12.19(s,1H),8.42(s,1H),5.47(s,1H),4.91(s,1H),3 .78–3.72(m,2H),3.50(d,J=4.4Hz,2H),2.77(d,J=4.6Hz,3H),1.48(s,9H).

[0056] 13C NMR (126MHz, CDCl3) δ164.51(s), 155.74(s), 153.17(s), 82.69(s), 63.12(s), 43.83(s), 27.97(s), 26.60(s).

[0057]

[0058] (a) In a round-bottom flask containing 10 mL of solvent DCM, add 1 mmol of compound III containing carboxylic acid, 1.2 mmol of condensing agent EDCI, and 0.1 mmol of 4-DMAP. Then add 1 mmol of compound X. Stir the reaction at room temperature for 30 min. After the reaction is complete, concentrate the reaction solution, extract the reactants with ethyl acetate and water, collect the ethyl acetate phase, dry it with anhydrous sodium sulfate, concentrate the solvent and pass it through a column to collect the colorless oily product.

[0059] (b) Trifluoroacetic acid was added to the collected product and stirred at room temperature for 30 minutes. After the reaction was completed, the trifluoroacetic acid was concentrated, the remaining trifluoroacetic acid was neutralized with saturated sodium bicarbonate aqueous solution, the reactants were extracted with dichloromethane, dried with anhydrous sodium sulfate, the solvent was concentrated, and the product was dried under vacuum. The yield was 85%.

[0060] The structural verification data is as follows:

[0061] 1 H NMR (500MHz, CDCl3) δ12.52(s,1H),9.84(s,1H),7.58(d,J=7.8Hz,1H),7.24–7.08(m,4H),6.68(tt,J=8.8,2.4Hz,1H),6.58(h,J =4.9Hz, 2H), 6.22 (q, J = 4.8Hz, 1H), 5.29 (s, 2H), 4.22 (t, J = 5.4Hz, 2H), 3.86 (s, 2H), 3.40 (d, J = 6.2Hz, 2H), 2.79 (d, J = 4.7Hz, 3H).

[0062] 13 C NMR (126MHz, CDCl3) δ171.92,164.46,164.36,162.47,162.37,155.82,155.14,141.68,136.48,127.84,127.38,122.79,12 0.22,119.00,110.00,109.72,109.67,109.56,109.51,107.38,103.47,103.27,103.07,62.78,49.31,40.19,31.18,26.49.

[0063] Other compounds of formula I can be prepared by the above method. The compound numbers, structures, and physicochemical data are shown in Table 1, and the structural identification data (H1N, C1N, and mass spectrometry) are shown in Table 2.

[0064] Table 1. Structures and physicochemical properties of some compounds

[0065]

[0066]

[0067]

[0068] Table 2. NMR characterization and high-resolution data of some compounds

[0069]

[0070]

[0071]

[0072] II. Determination of enzyme inhibitory activity of compounds with general formula I

[0073] Enzyme activity assay: When measuring the inhibitory activity of OfChtI and OfChi-h, 4-methylumbelliferyl N,N'-diacetyl-β-D-chitobioside was used as the substrate. The enzyme was mixed with buffer solution (20 mM NaH2PO4, pH 6.5) in a 96-well plate to a final volume of 90 μL. 10 μL of 40 μM substrate pNP-β-GlcNAc was added to start the reaction. The reaction was incubated at 25 °C for 5 min. 100 μL of 0.5 M sodium carbonate was added to terminate the reaction. The absorbance was measured at 405 nm.

[0074] Method for determining the inhibitory activity of compounds: Samples were dissolved in DMSO and diluted to multiple concentration gradients ranging from 0.001 to 100 μM. In a 96-well plate, 2 μL of inhibitor, 88 μL of enzyme solution, and 10 μL of substrate were added to each well, making the total solution volume 100 μL per well. The plate was incubated at 30°C with shaking for 20 min. Then, 100 μL of stop solution was added to each well, and the fluorescence intensity was measured using a microplate reader. The excitation wavelength was set to 350 nm, and the emission wavelength to 450 nm. The inhibition rates of representative compounds at a concentration of 20 μM are shown in Table 3.

[0075] Table 3. Inhibition rates of some compounds against chitinases OfChtⅠ and OfChi-h at 20 μM concentration

[0076]

[0077]

[0078] The inhibition rate for each sample concentration was calculated, and IC50 was fitted using the software GraphPad Prism. 50 The values ​​and chitinase inhibitory activities of some compounds are shown in Table 4.

[0079] Table 4. Chitinase half-maximal inhibitory concentrations (IC50) of some compounds 50 )

[0080] A1 6.17 1.08 A2 10.96 9.99 A3 0.96 2.59 A4 5.56 3.89 A5 3.3 1.64 A6 0.96 2.94 A7 0.7 1.41 A8 6.81 1.42 A9 9.19 1.86 A10 2.41 1.45 A11 12.97 9.25

[0081] III. Insecticidal activity determination of compounds of formula I in this invention

[0082] Assay method: Insects were treated using a spot method at concentrations of 500 mg / L, 200 mg / L, and 50 mg / L. The corrected mortality rate (%) was calculated during the pupal stage; insects that failed to emerge were considered dead. The toxicity was determined by comparing the insecticide with the control drug diflubenzuron. Insecticidal activity data for some compounds are shown in Table 5.

[0083] The following test targets, the diamondback moth (Plutella xylostella) and the Asian corn borer (Ostriniafurnacalis), were commercially available. They were fed indoors with appropriate feed under the following conditions: room temperature (27±1)℃, humidity 40%, light intensity 2000 lux, and light duration 12 hours per day. Under these indoor rearing conditions, third-instar larvae of uniform age, weight, and physiological condition were used for pesticide activity screening. All insects in the test groups were fed until pupation, and the lethality of the compounds was recorded based on the emergence rate.

[0084] Table 5. Insecticidal activity of some compounds (500 mg / L, 200 mg / L, 50 mg / L)

[0085]

[0086]

[0087] Compounds A3 and A11 exhibited good lethality against diamondback moth. Therefore, the samples were diluted into multiple different concentration gradients to test the lethality of compounds A3 and A11 against diamondback moth, as shown in Table 6. LC-15 of compounds A3 and A11 against diamondback moth. 50The values ​​were 9.165 mg·L. -1 and 0.794 mg·L -1 .

[0088] Table 6. Lethality of compounds A3 and A11 at different concentrations against diamondback moth

[0089]

[0090] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. An indole-type methylguanidinourea compound, characterized in that, Its structure is shown in Equation I: Wherein, R is selected from any one of the following groups: 。 2. A method for preparing an indole-type methylguanidinourea compound according to claim 1, characterized in that, Includes the following steps: (1) Indoleacetic acid (IAA) reacts with formula II under catalytic conditions to give formula III; (2) The reaction of hemisulfate isomethylthiourea type IV with ditert-butyl dicarbonate type V yields mono-Boc protected isomethylthiourea type VI; (3) The mono-Boc protected isomethylthiourea of ​​formula VI reacts with methylcarbamoyl chloride of formula VII under alkaline conditions to obtain formula VIII; (4) Formula VIII and Formula IX ethanolamine react to obtain Formula X; (5) Formulas III and X first undergo a condensation reaction and then the Boc protecting group is removed to obtain Formula I; 。 3. The preparation method according to claim 2, characterized in that, In step (1), the reaction solvent is N,N-dimethylformamide or tetrahydrofuran, the reaction temperature is 0-30℃, and the catalyst used in the reaction is sodium hydride or potassium hydride.

4. The preparation method according to claim 2, characterized in that, In step (2), the solvent used is a mixture of two solvents in a volume ratio of 1:1 to 5:

1. The two solvents are dioxane / water, tetrahydrofuran / water, or acetone / water. The base used is selected from any one of sodium bicarbonate, sodium carbonate, potassium carbonate, and sodium hydroxide.

5. The preparation method according to claim 2, characterized in that, In step (3), the reaction temperature is 0℃-25℃, and the solvent used is dichloromethane, toluene, DMF or acetone; the base used is triethylamine, 4-DMAP or potassium carbonate.

6. The preparation method according to claim 2, characterized in that, In step (4), the molar ratio of ethanolamine formula IX to formula VIII is 2:1 to 5:1, and the solvent used is tetrahydrofuran, dichloromethane or DMF.

7. The preparation method according to claim 2, characterized in that, In step (5), the solvent used is dichloromethane or tetrahydrofuran, the condensing agent used is EDCI, DCC or CDI, and the base used is triethylamine or 4-DMAP.

8. The use of the indole-type methylguanidinourea compound according to claim 1 in the preparation of insect chitinase inhibitors.

9. The application according to claim 8, wherein the insect is a diamondback moth or an Asian corn borer.

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