Application of small molecule compound ZINC19765938 in pest control

Abstract

The invention belongs to the technical field of pest control, discloses an application of a small molecule compound ZINC19765938 in pest control, and discloses an application of a compound (such as ZINC19765938) shown in a formula (I) or a derivative thereof in pest control for the first time, and the compound or the derivative thereof can be used for controlling pests by inhibiting CYP321A8 enzyme activity of the pests. The purposes of preventing and controlling the pests, improving the sensitivity of the pests to the insecticide, improving the insecticidal efficiency of the insecticide and inhibiting the growth of the pests are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of pest control, and particularly relates to the application of the small molecule compound ZINC19765938 in pest control. Background Art

[0002] At present, Spodoptera frugiperda, also known as fall armyworm, belongs to the family Noctuidae of the order Lepidoptera, is a world-wide migratory agricultural pest, and has serious cross-resistance. In agriculture, pesticides with different action mechanisms are used alternately. However, due to the existence of cross-resistance, whether developing new drugs or replacing pesticides, a large number of research reports have proved that detoxifying enzymes such as cytochrome P450 (CYP) in pests metabolize pesticides, enabling pests to quickly develop resistance. Therefore, there is an urgent need to find a method to solve the problem of cross-resistance. Finding inhibitors that inhibit the activity of all P450 enzymes is one of the methods. At present, there is less research on insect P450 inhibitors. Piperonyl butoxide (PBO) is the only widely recognized P450 enzyme inhibitor, which has broad-spectrum activity and is also a pesticide synergist recognized worldwide. However, PBO does not have species specificity, has high toxicity and general efficiency, and is difficult to be applied to field pest control. Summary of the Invention

[0003] The purpose of the first aspect of the present invention is to provide the application of the compound or its derivative represented by formula (I) in pest control.

[0004] The purpose of the second aspect of the present invention is to provide the application of the compound or its derivative represented by formula (I) in inhibiting the activity of cytochrome P450 or preparing a product for inhibiting the activity of cytochrome P450.

[0005] The purpose of the third aspect of the present invention is to provide a product.

[0006] The purpose of the fourth aspect of the present invention is to provide a method for pest control.

[0007] The purpose of the fifth aspect of the present invention is to provide a method for inhibiting the activity of CYP321A8 enzyme and thus inhibiting the cross-resistance of pests.

[0008] In order to achieve the above purposes, the technical solutions adopted by the present invention are as follows:

[0009] The first aspect of the present invention provides the application of the compound or its derivative represented by formula (I) in any one of (1) to (8):

[0010] (1) Pest control;

[0011] (2) Preparing a product for pest control;

[0012] (3) Improve the sensitivity of pests to pesticides;

[0013] (4) Prepare a product for improving the sensitivity of pests to pesticides;

[0014] (5) Improve the insecticidal efficiency of pesticides;

[0015] (6) Prepare a product for improving the insecticidal efficiency of pesticides;

[0016] (7) Inhibit the growth of pests;

[0017] (8) Prepare a product for inhibiting the growth of pests;

[0018]

[0019] In formula (I), R1 is selected from hydrogen or amino, and R2 is selected from hydrogen or an alkyl group of C 1～5 of.

[0020] In some embodiments of the present invention, the structural formula of the compound of formula (I) is

[0021]

[0022] In some embodiments of the present invention, the derivatives include pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, tautomers and prodrugs.

[0023] In some embodiments of the present invention, the pharmaceutically acceptable salts include at least one of metal salts, ammonium salts, salts formed with inorganic acids, salts formed with organic bases, salts formed with organic acids, salts formed with basic amino acids, and salts formed with acidic amino acids.

[0024] In some embodiments of the present invention, the metal salts include alkali metal salts and alkaline earth metal salts.

[0025] In some embodiments of the present invention, the alkali metal salts include at least one of sodium salts and potassium salts.

[0026] In some embodiments of the present invention, the alkaline earth metal salts include at least one of calcium salts, magnesium salts, barium salts and aluminum salts.

[0027] In some embodiments of the present invention, the salts formed with organic bases include salts formed with the following organic bases: at least one of trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0028] In some embodiments of the present invention, the salts formed with inorganic acids include salts formed with at least one of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid.

[0029] In some embodiments of the present invention, the salts formed with organic acids include salts formed with at least one of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid.

[0030] In some embodiments of the present invention, the salts formed with basic amino acids include salts formed with at least one of the following basic amino acids: arginine, lysine, ornithine.

[0031] In some embodiments of the present invention, the salts formed with acidic amino acids include salts formed with at least one of the following acidic amino acids: aspartic acid, glutamic acid.

[0032] In some embodiments of the present invention, the pests include Lepidoptera insects.

[0033] In some embodiments of the present invention, the pests include at least one of Spodoptera frugiperda, Spodoptera litura, Spodoptera exigua, Spodoptera littoralis, Mythimna separata, Helicoverpa zea, Helicoverpa armigera, Manduca sexta, Plutella maculipennis, Plusia ni (Hubner), Sparganothis pilleriana Denis et Schiffermüller, Sitotroga cerealella, Ilattia octo (Guenée).

[0034] In some embodiments of the present invention, the pests are polyphagous insects of the family Noctuidae in the order Lepidoptera.

[0035] In some embodiments of the present invention, the pests are Spodoptera frugiperda, Spodoptera litura, Spodoptera exigua, Spodoptera littoralis, Mythimna separata, Helicoverpa zea, Helicoverpa armigera and / or Manduca sexta.

[0036] In some embodiments of the present invention, the pests are Spodoptera frugiperda and Spodoptera litura.

[0037] In some embodiments of the present invention, the product includes insecticides, pesticidal drugs, and insecticidal synergists.

[0038] In some embodiments of the present invention, the product can be prepared into conventional dosage forms for use, such as dry powder, wettable powder, emulsifiable concentrate, microemulsion, paste, granule, or suspension.

[0039] In some embodiments of the present invention, the compound or its derivative of formula (I) achieves the purposes of controlling field pests, increasing the sensitivity of pests to insecticides, improving the insecticidal efficiency of insecticides, and inhibiting the growth of pests by inhibiting the activity of CYP321A8 enzyme in resistant pest larvae and eggs.

[0040] In some embodiments of the present invention, the effective dose of the compound or its derivative of formula (I) is 1 - 4 nmol.

[0041] The second aspect of the present invention provides the use of the compound or its derivative of formula (I) in inhibiting cytochrome P450 activity or in preparing a product for inhibiting cytochrome P450 activity:

[0042]

[0043] In formula (I), R1 is selected from hydrogen or amino, and R2 is selected from hydrogen or C 1～5 alkyl.

[0044] In some embodiments of the present invention, the structural formula of the compound of formula (I) is

[0045] (denoted as ZINC19765938) or

[0046]

[0047] In some embodiments of the present invention, the derivatives include pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, tautomers, and prodrugs.

[0048] In some embodiments of the present invention, the pharmaceutically acceptable salts include at least one of metal salts, ammonium salts, salts formed with inorganic acids, salts formed with organic bases, salts formed with organic acids, salts formed with basic amino acids, and salts formed with acidic amino acids.

[0049] In some embodiments of the present invention, the metal salts include alkali metal salts and alkaline earth metal salts.

[0050] In some embodiments of the present invention, the alkali metal salts include at least one of sodium salts and potassium salts.

[0051] In some embodiments of the present invention, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt, and aluminum salt.

[0052] In some embodiments of the present invention, the salt formed with the organic base includes salts formed with the following organic bases: at least one of trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0053] In some embodiments of the present invention, the salt formed with the inorganic acid includes salts formed with the following inorganic acids: at least one of hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0054] In some embodiments of the present invention, the salt formed with the organic acid includes salts formed with the following organic acids: at least one of formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0055] In some embodiments of the present invention, the salt formed with the basic amino acid includes salts formed with the following basic amino acids: at least one of arginine, lysine, and ornithine.

[0056] In some embodiments of the present invention, the salt formed with the acidic amino acid includes salts formed with the following acidic amino acids: at least one of aspartic acid and glutamic acid.

[0057] In some embodiments of the present invention, the cytochrome P450 includes the CYP321A8 enzyme.

[0058] In some embodiments of the present invention, the product includes a reagent (such as an enzyme inhibitor) or a kit.

[0059] In a third aspect of the present invention, there is provided a product including a compound or its derivative in the first aspect or the second aspect of the present invention, and an insecticide.

[0060] In some embodiments of the present invention, the insecticide includes at least one of organophosphorus insecticides, neonicotinoid insecticides, carbamate insecticides, pyrethroid insecticides, benzoylurea insecticides, and plant-derived insecticides (such as rotenone).

[0061] In some embodiments of the present invention, the insecticide includes chlorpyrifos, deltamethrin, emamectin benzoate·chlorfenapyr, cypermethrin, imidacloprid, buprofezin, pymetrozine, thiamethoxam, isoprocarb, diafenthiuron, indoxacarb, metaflumizone, chlorantraniliprole, cyflumetofen, emamectin benzoate, and avermectin.

[0062] In some embodiments of the present invention, the drug comprises a pharmaceutically acceptable excipient, and / or any one or more other active ingredients.

[0063] In some embodiments of the present invention, the pharmaceutically acceptable excipient comprises at least one of a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic agent, an antiadhesive, a chelating agent, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foaming agent, a defoaming agent, a thickening agent, an inclusion agent, a humectant, an absorbent, a diluent, a flocculant and an anti-flocculant, a filter aid, a release retardant, a carrier.

[0064] In some embodiments of the present invention, for the convenience of administration, the active ingredient ZINC19765938 or its pharmaceutically acceptable salt can be processed into a specific dosage form with any one or several pharmaceutically acceptable excipients. These excipients can be diluents (such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, microcrystalline cellulose, etc.), absorbents (such as calcium sulfate, calcium hydrogen phosphate, light magnesium oxide, calcium carbonate, etc.), wetting agents (such as water, ethanol, etc.), binders (such as hydroxypropyl methylcellulose, povidone, starch paste, syrup, etc.), disintegrants (such as dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrant, cross-linked povidone, etc.), lubricants (such as magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol, colloidal silica, etc.), colorants (such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide, etc.), coating materials (such as acrylic resin, hydroxypropyl methylcellulose, povidone, etc.), solvents (such as water for injection, ethanol, propylene glycol, glycerol, etc.), acid-base regulators (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid, sodium tartrate, etc.), antioxidants (such as sodium sulfite, sodium metabisulfite, sodium thiosulfate, etc.), bacteriostatic agents (such as phenol, benzyl alcohol, thimerosal, etc.), and can also be isotonicity regulators (such as sodium chloride, glucose, etc.).

[0065] In some embodiments of the present invention, the product is a spraying preparation, a bait, an injection.

[0066] In the fourth aspect of the present invention, a method for controlling pests is provided, which includes the step of applying the product of the third aspect of the present invention to pests or pest habitats.

[0067] In some embodiments of the present invention, an effective dose of the product is applied to pests or pest habitats.

[0068] The present invention provides a method for controlling pests, which includes treating pests, the food of pests, the habitats or breeding grounds of pests (the soil, area, material or environment where pests are growing or can grow, or the materials, cultivated plants, plant propagation materials (such as seeds), soil, surface or space to be protected from pest attack or infestation) with a product containing the compound or its derivative of formula (I).

[0069] Generally, an "effective amount" means the amount of the active ingredient required to achieve an observable effect on growth, and the observable effects include necrosis, death, arrest, prevention and removal, destruction or reduction of the presence and activity of the target organism. For the compound or its derivative of formula (I) used in the present invention, the effective amount can vary. The effective amount of the compound or its derivative of formula (I) also varies according to main conditions, such as the desired insecticidal effect and duration, climate, target species, location, application mode, etc.

[0070] In some embodiments of the present invention, a product containing the compound or its derivative of formula (I) is applied to pests by injection.

[0071] In some embodiments of the present invention, the pests include Lepidoptera insects.

[0072] In some embodiments of the present invention, the pests include at least one of Spodoptera frugiperda, Spodoptera litura, Spodoptera exigua, cotton leafworm, Mythimna separata, Helicoverpa zea, Helicoverpa armigera, Manduca sexta, Plutella xylostella, Trichoplusia ni, Lobesia botrana, Sitotroga cerealella, and Athetis dissimilis.

[0073] In some embodiments of the present invention, the pests are polyphagous insects of the family Noctuidae in Lepidoptera.

[0074] In some embodiments of the present invention, the pests are Spodoptera frugiperda, Spodoptera litura, Spodoptera exigua, cotton leafworm, Mythimna separata, Helicoverpa zea, Helicoverpa armigera and / or Manduca sexta.

[0075] In some embodiments of the present invention, the pests are Spodoptera frugiperda and Spodoptera litura.

[0076] The fifth aspect of the present invention provides a method for inhibiting the activity of CYP321A8 enzyme, which includes the step of acting on the activity of CYP321A8 enzyme with the compound or its derivative of formula (I) in the present invention or a product containing the compound or its derivative of formula (I).

[0077] By inhibiting the activity of CYP321A8 enzyme, the cross-resistance of pests is inhibited.

[0078] The beneficial effects of the present invention are as follows:

[0079] The present invention discloses for the first time the application of the compound of formula (I) (such as ZINC19765938) or its derivatives in pest control. The compound or its derivatives can inhibit the activity of CYP321A8 enzyme in pests, so as to achieve the purposes of controlling resistant pests in the field, improving the sensitivity of pests to insecticides, enhancing the insecticidal efficiency of insecticides, and inhibiting the growth of pests.

[0080] Specifically, the present invention proves through experiments that the compound of formula (I) (ZINC19765938) or its derivatives can significantly improve the sensitivity of resistant pests to chemical insecticides, significantly increase the mortality rate of pests, slow down the development of pest bodies, and it has a better effect in enhancing the efficacy of insecticides than the commonly used pesticide synergist (PBO) at present. On the premise of not affecting the ecological balance, it can achieve the control of the quantity and growth of pests, so as to effectively control pests. At the same time, this compound hardly has an inhibitory effect on human P450 enzyme activity and has high safety. Brief Description of the Drawings

[0081] Figure 1 It is a bond-line formula diagram of the small molecule compound ZINC19765938 and its scaffold.

[0082] Figure 2 It is a diagram of the enzyme activity determination results of the small molecule compound ZINC19765938 and PBO on the CYP321A8 gene (A) of Spodoptera frugiperda and the CYP3A4 gene (B) of humans. In the figure, ns represents no significant difference, and * represents P < 0.05.

[0083] Figure 3 It is the determination result of the total P450 enzyme activity of the whole larvae of Spodoptera frugiperda chlorpyrifos-resistant strain after injection with the small molecule compound ZINC19765938 and PBO.

[0084] Figure 4 It is the analysis of the synergistic effect of the small molecule compound ZINC19765938 and PBO on the insecticides of Spodoptera frugiperda chlorpyrifos-resistant strain and field pests; among them, A is the statistical result of the survival rate of larvae after injection of the small molecule compound ZINC19765938 and PBO into Spodoptera frugiperda, B is the diagram of the larval characterization results after injection of the small molecule compound ZINC19765938 and PBO into Spodoptera frugiperda, C is the statistical result of the body weight of larvae after injection of the small molecule compound ZINC19765938 and PBO into Spodoptera frugiperda. In the figure, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001. The blue * represents the chlorpyrifos control group vs the chlorpyrifos + PBO group, the red * represents the chlorpyrifos control group vs the chlorpyrifos + ZINC19765938 group, and the black * represents the chlorpyrifos + PBO group vs the chlorpyrifos + ZINC19765938 group.

[0085] Figure 5 Analysis of the synergistic effect of the small molecule compounds ZINC19765938 and PBO on the chlorpyrifos-resistant strain of Spodoptera litura; among them, A is the statistical result of the survival rate of larvae after injecting the compounds ZINC19765938 and PBO into Spodoptera litura, B is the statistical result of the body weight of larvae after injecting the small molecule compounds ZINC19765938 and PBO into Spodoptera litura, C is the result chart of the larval characterization after injecting the compounds ZINC19765938 and PBO into Spodoptera litura. In the figure, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, **** represents P < 0.0001. The blue * represents the chlorpyrifos control group vs the chlorpyrifos + PBO group, the red * represents the chlorpyrifos control group vs the chlorpyrifos + ZINC19765938 group, and the black * represents the chlorpyrifos + PBO group vs the chlorpyrifos + ZINC19765938 group.

[0086] Figure 6 Phylogenetic tree analysis of the CYP321A8 protein sequence in different species; among them, A is the result of the alignment analysis of CYP321A8 in the genomes of different species, and B is the result of the similarity analysis of CYP321A8 in polyphagous insects. Detailed implementation manners

[0087] The content of the present invention will be further described in detail below through specific examples.

[0088] It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.

[0089] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0090] The features and performance of the present invention will be further described in detail below in combination with the embodiments.

[0091] Example 1 A small molecule compound ZINC19765938

[0092] The inventors studied the resistance mechanisms of Spodoptera frugiperda in different regions and found that CYP321A8 is involved in the resistance to various types of insecticides and is a key cross-resistance gene. Moreover, it is unique to polyphagous pests in the family Noctuidae of Lepidoptera. Therefore, designing specific, safe and efficient novel small molecule inhibitors targeting CYP321A8 is expected to solve the problem of cross-resistance in Spodoptera frugiperda and is of great significance for pest control.

[0093] In the present invention, homology modeling of CYP321A8 of Spodoptera frugiperda was carried out, 241,712 small molecule compounds were downloaded from the ZINC15 database, virtual screening was performed using Autodock Vina, re-docking screening was carried out through AutoDock, and drug screening was performed using 4 pharmacokinetic analysis websites such as ADMETlab2.0, NERDD, swissadme, and admetSAR. Finally, it was screened out that the small molecule compound numbered ZINC19765938 in the ZINC15 database has the effect of an insecticide synergist. There is no research report yet that the small molecule ZINC19765938 can be used as an inhibitor in pest control.

[0094] The molecular formula of the small molecule compound ZINC19765938 is C 25 H 32 N4O3, with a molar mass of 436.556 g / mol. Its structural formula with the scaffold is as Figure 1 shown. This compound was purchased from Asinex company, with the manufacturer's number BDC24752608 and the name compound BDC24752608(SC).

[0095] The smile format of the small molecule compound ZINC19765938 is Cc1ccc([C@H](C)C(=O)N2CCC(c3ccc(C(=O)N4CCOCC4)c(N)n3)CC2)cc1, the InChI format is InChI=1S / C25H32N4O3 / c1-17-3-5-19(6-4-17)18(2)24(30)28-11-9-20(10-12-28)22-8-7-21(23(26)27-22)25(31)29-13-15-32-16-14-29 / h3-8,18,20H,9-16H2,1-2H3,(H2,26,27) / t18- / m0 / s1, and the InChI Key format is VSYUSPQDYOFVJZ-SFHVURJKSA-N.

[0096] The SMILES format of the small molecule compound ZINC19765938 scaffold is O=C(Cc1ccccc1)N1CCC(c2ccc(C(=O)N3CCOCC3)cn2)CC1, the InChI format is InChI=1S / C23H27N3O3 / c27-22(16-18-4-2-1-3-5-18)25-10-8-19(9-11-25)21-7-6-20(17-24-21)23(28)26-12-14-29-15-13-26 / h1-7,17,19H,8-16H2, and the InChI Key format is BRMWLQZWOQFHQY-UHFFFAOYSA-N.

[0097] Example 2: Determination of the inhibitory effect of the small molecule compound ZINC19765938 on the enzyme activity of the detoxifying enzyme CYP321A8

[0098] By constructing expression vectors for the Spodoptera frugiperda CYP321A8 gene (NBCI Gene ID: 118268604) and the human key P450 gene CYP3A4 gene (NBCI Gene ID: 1576), obtaining pure proteins in a prokaryotic expression system and purifying the proteins to obtain enzyme solutions, the specific process is as follows.

[0099] The cDNA of the Spodoptera frugiperda CYP321A8 gene was from the whole Spodoptera frugiperda samples in the laboratory. After extracting RNA using Trizol (AG RNAex Pro Reagent), a reverse transcription experiment was carried out using the reverse transcription kit Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) produced by Yeasen Biotech Co., Ltd.; the cDNA of the human CYP3A4 gene was purchased from Youbao Biotech Co., Ltd., product number G119802. Using cDNA as a template, forward and reverse PCR primers were designed based on the CDS region of the gene, and the primer sequences are shown in Table 1 below.

[0100] Table 1 Primer sequences

[0101]

[0102] The target gene fragment was obtained by performing a PCR experiment using the Taq enzyme kit from TAKARA. The PCR product was detected by 1.5% agarose gel electrophoresis and then recovered and purified using the Hipure Gel Pure DNA Kits from Magen Biotechnology Co., Ltd. T-vector ligation was carried out according to the product manual of TAKARApMD18-T vector, and the transformation experiment was performed using the competent cells DH5α from Kangti Co., Ltd. A single well-grown colony was selected and added to 500 μL of resistant liquid culture medium in a 1.5 mL EP tube and cultured for 1 h. Then, 2 μL of the bacterial solution was taken as a template for the PCR experiment. After the PCR product was detected by agarose gel electrophoresis, colonies with target band sizes, bright and single bands were identified. The plasmid was extracted using the plasmid extraction kit from Tiangen Biochemical Technology Co., Ltd. 12 h after inoculation with the identified colony. Referring to the TAKARA QuickCut double digestion system, the CYP321A8 gene was double-digested with the restriction enzymes EcoRI and Not I, and the CYP3A4 gene was double-digested with the restriction enzymes EcoR V and Xho I. After the enzyme digestion products were detected by agarose gel electrophoresis and gel recovery, according to the product description of T4 DNA ligase (Promega, USA), the CYP321A8 fragment and the pGEX-4T-1 vector, and the CYP3A4 fragment and the pET-32a-c vector were ligated at 16°C for more than 3 h. The same method was used for the transformation experiment with DH5α, followed by colony monoclonalization and then bacterial solution PCR. After agarose gel electrophoresis, suitable colonies were identified. After amplification, the plasmid was extracted. After the extracted plasmid was verified to be correct by double digestion again, the BL21(DE3) chemically competent cells from Yeasen Biotech Co., Ltd. were used for the transformation experiment. The same method was used, followed by colony monoclonalization and then bacterial solution PCR. After agarose gel electrophoresis, suitable colonies were identified, and the construction of the two protein expression vectors, CYP321A8-pGEX-4T-1 and CYP3A4-pET-32a-c, was completed.

[0103] 1% of the bacterial solution obtained in the previous step was inoculated into the resistant medium for resuscitation and amplification for 12 h, and then inoculated into the resistant medium at 1% and cultured for 2 h. Then, 0.1% IPTG was added and induced overnight at 16°C for 16 h, and then the bacterial solution was collected. The CYP321A8 enzyme solution was purified using the GST-tagged protein purification kit from Beyotime Biotechnology Co., Ltd. according to the instructions. The CYP3A4 enzyme solution was purified using the Ni-NTA Superflow Column (1.5 mL) produced by Qiagen according to the His-tagged protein purification method.

[0104] Dissolve p-nitroanisole (p-NA) in ethanol to prepare a 500 mmol / L stock solution. Dilute the p-NA stock solution to 2 mmol / L with 0.1 mol / L sodium phosphate buffer (pH 7.8) preheated to 37°C. Add 250 μmol of piperonyl butoxide (PBO) and the small molecule ZINC19765938 to the purified enzyme solution. The inhibitor concentration in the enzyme solution is 277.78 mol / L. Add 100 μL of 2 mmol / L p-NA and 90 μL of the enzyme solution containing the inhibitor to a 96-well plate in sequence. After preheating at 30°C for 5 min, quickly add 10 μL of 9.6 mmol / L NADPH to start the reaction. The inhibitor concentration is 250 μmol / well. Record every 0.5 - 10 min and record the OD value at 405 nm by a microplate reader within 1 h for statistical analysis.

[0105] The results showed that the small molecule ZINC19765938 could significantly inhibit the activity of CYP321A8 enzyme in Spodoptera frugiperda, and the inhibitory effect was significantly higher than that of PBO at the same dose ( Figure 2 in A); at the same time, ZINC19765938 and PBO at the same dose did not inhibit the activity of human CYP3A4 enzyme ( Figure 2 in B), indicating that it may have a lower inhibitory possibility for human P450 and has higher safety.

[0106] Example 3 Inhibition experiment of the small molecule compound ZINC19765938 on the total P450 enzyme activity of the chlorpyrifos-resistant strain of Spodoptera frugiperda and field pests

[0107] On the basis of determining that ZINC19765938 can inhibit the activity of CYP321A8 enzyme, further determine whether the inhibitory effect on this enzyme affects the total P450 enzyme activity in the insect body. The specific experiment is as follows:

[0108] The larvae of the Spodoptera frugiperda sensitive strain provided by South China Agricultural University were fed with the Spodoptera frugiperda feed containing 0.03% chlorpyrifos continuously for several generations, and a resistant strain with about 18-fold resistance was obtained. At the same time, the Spodoptera frugiperda larvae were directly captured from the farmland near Guangzhou and reared indoors for one generation. The general medium formula for Spodoptera frugiperda is 26 g of yeast powder, 100 g of soybean powder, 80 g of wheat bran, 26 g of yeast powder, 8 g of casein, 8 g of ascorbic acid, 1 g of choline chloride, 2 g of sorbic acid, 0.2 g of inositol, 0.1 g of streptomycin, 0.1 g of sodium penicillin, 26 g of agar, and 900 mL of ultrapure water. 1 μL of 2.3 mmol / L ZINC19765938 or PBO was injected into the 4th instar 1-day-old Spodoptera frugiperda larvae that had been starved for 4 h. Immediately after injection, the larvae were fed with the feed containing 0.03% chlorpyrifos for 48 h. The proteins of these two kinds of larvae were extracted at 48 h after injection for the determination of total P450 enzyme activity. The determination method was the same as that in Example 2, but no inhibitor was added to the enzyme solution.

[0109] The results showed that both PBO and the small molecule ZINC19765938 significantly inhibited the total P450 enzyme activity, and there was no obvious difference between them ( Figure 3 ).

[0110] Example 4 Analysis of the synergistic effect of the small molecule compound ZINC19765938 on the chlorpyrifos-resistant strain of Spodoptera frugiperda and field pests

[0111] To analyze the synergistic effect of ZINC19765938 on insecticides, the organophosphorus insecticide chlorpyrifos with high resistance in field pests was selected for testing. The specific experiment was as follows: 1 μL of 2.3 mmol / L ZINC19765938 and PBO were respectively injected into the 4th instar 1-day-old Spodoptera frugiperda larvae that had been starved for 4 h (including the chlorpyrifos-resistant variety (Example 3) and the Spodoptera frugiperda larvae captured from the farmland near Guangzhou). Immediately after injection, the larvae were fed with the feed containing 0.03% chlorpyrifos for one week, and the death situation of Spodoptera litura and the growth situation of Spodoptera litura larvae were observed and counted during the experiment.

[0112] The results were as Figure 4 shown. The addition of ZINC19765938 could increase the mortality of the chlorpyrifos-resistant strain and the field population by about 40% compared with no addition, while PBO could only increase by about 10% ( Figure 4 in A). At the same time, the growth of the surviving larvae was delayed and their body weight decreased ( Figure 4 in B and C), and the synergistic effect of ZINC19765938 was greater than that of PBO ( Figure 4 ). The above results proved that ZINC19765938 had higher application value than PBO.

[0113] Analysis of the Synergistic Effect of Small Molecule Compound ZINC19765938 on Chlorpyrifos-Resistant Strains of Spodoptera litura

[0114] To analyze whether ZINC19765938 has a synergistic effect on other Lepidoptera pests as an insecticide, Spodoptera litura was selected for testing. The specific experimental process is as follows: The larvae of Spodoptera litura purchased from the Institute of Entomology, Sun Yat-sen University were fed with the feed containing 0.03% chlorpyrifos continuously for several generations to obtain a resistant strain with about 6-fold resistance. The formula of the ordinary culture medium for Spodoptera litura larvae is 40 g of yeast powder, 150 g of wheat germ, 4 g of ascorbic acid, 4 g of propylparaben, 24 g of sorbic acid, 5 g of sucrose, 14 g of agar, 2 - 3 drops of linoleic acid, and 900 mL of ultrapure water. 1 μL of 2.3 mmol / L ZINC19765938 or PBO was respectively injected into the 4th instar and 1-day-old resistant Spodoptera litura larvae that had been starved for 4 h. After injection, they were immediately fed with the feed containing 0.03% chlorpyrifos for one week, and the death situation of Spodoptera litura and the growth situation of Spodoptera litura larvae were observed and counted during the experiment.

[0115] The results showed that the addition of ZINC19765938 increased the mortality rate of the chlorpyrifos-resistant strain by about 40% compared with no addition, while PBO had almost no effect ( Figure 5 in A). At the same time, the growth of the larvae that did not die after 48 h of treatment was delayed and their weight decreased. The synergistic effect of ZINC19765938 was greater than that of PBO ( Figure 5 in B and C). The above results proved that ZINC19765938 has application value in multiple pests.

[0116] Example 6 Sequence Comparison of CYP321A8 in Different Species

[0117] To determine the range of insects that ZINC19765938 can act on, the present invention conducted a comparative analysis on the presence of CYP321A8 in the genomes of different species (data from NCBI).

[0118] The results are as Figure 6 shown. CYP321A8 only exists in polyphagous insects of the family Noctuidae in Lepidoptera and does not exist in other insects, animals, and humans ( Figure 6 in A). And the similarity of this gene in polyphagous insects is high ( Figure 6 in B). Therefore, ZINC19765938 developed based on CYP321A8 can be applied to these polyphagous agricultural pests and has a certain safety without acting on other organisms.

[0119] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the scope of knowledge possessed by those of ordinary skill in the art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of a compound of formula (I) or a derivative thereof in any one of (1) to (8): (1) Pest control; (2) Preparation of products for controlling pests; (3) Increase pests’ sensitivity to pesticides; (4) preparing products that increase the sensitivity of pests to pesticides; (5) Improve the insecticidal efficiency of pesticides; (6) preparing products that improve the insecticidal efficiency of insecticides; (7) Inhibit the growth of pests; (8) Preparation of products that inhibit the growth of pests; In formula (I), R1 is selected from hydrogen or amino, R2 is selected from hydrogen or C 1～5 of alkyl.

2. The use according to claim 1, characterized in that: The pests include insects of the order Lepidoptera; Preferably, the pests include at least one of the fall armyworm, fall armyworm, beet armyworm, cotton leafworm, armyworm, corn armyworm, cotton bollworm, tobacco hornworm, diamondback moth, cabbage looper, grape leafroller, wheat moth, and soybean armyworm.

3. Use of the compound described in formula (I) or its derivatives in inhibiting cytochrome P450 activity or preparing products inhibiting cytochrome P450 activity: In formula (I), R1 is selected from hydrogen or amino, R2 is selected from hydrogen or C 1～5 of alkyl.

4. The use according to claim 3, characterized in that: The cytochrome P450 includes the CYP321A8 enzyme.

5. The use according to any one of claims 1 to 4, characterized in that: The derivatives include pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, tautomers and prodrugs; Preferably, the pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

6. The use according to any one of claims 1 to 4, characterized in that: The structural formula of the compound described in formula (I) is 7. The use according to claim 5, characterized in that: The products include pesticides, insecticides, insecticide synergists, and enzyme inhibitors.

8. A product comprising the compound or derivative thereof as claimed in any one of claims 1 to 7, and an insecticide.

9. The product according to claim 8, characterized in that The insecticide includes at least one of organophosphorus insecticides, neonicotinoid insecticides, carbamate insecticides, pyrethroid insecticides, benzoylurea insecticides and botanical insecticides.

10. A method for controlling pests, comprising the step of applying the product according to claim 8 or 9 to pests or pest habitats.

Images