Chitinase inhibitors, methods for their production and agricultural use

Abstract

This invention discloses a chitinase inhibitor, its preparation method, and its agricultural application. The inhibitor is a compound of formula (I), or a pesticide-acceptable salt, hydrate, solvate, stereoisomer, or tautomer thereof. Compounds of formula (I) have a core skeleton composed of a guanidinyl-bridged structure and aromatic end groups. Representative compounds can inhibit the activity of chitinase HgCht2 from soybean cyst nematodes and homologous chitinases from lepidopteran insects in the low to sub-micromolar concentration range, and can be used to prepare pesticides or pesticide compositions for controlling chitinase-dependent agricultural pests such as plant parasitic nematodes and lepidopteran pests.

Description

Technical Field

[0001] This invention belongs to the field of pesticide chemistry and plant disease and pest control technology, specifically involving chitinase inhibitors and their agricultural applications. Background Technology

[0002] Crops are susceptible to damage from various agricultural pests, including plant-parasitic nematodes and lepidopteran insects, during their growth. Plant-parasitic nematodes secrete a variety of cell wall-degrading enzymes and effector proteins during infection, among which chitinases are closely related to nematode infection, development, and their interaction with the host. Chitinases also participate in insect molting, peritrophic membrane renewal, and growth and development. Therefore, chitinases are functional targets with developmental value in the control of agricultural pests. Existing chemical pesticides are mostly broad-spectrum killers, which easily lead to resistance and non-target risks. Selective regulation of related secreted enzymes or key developmental enzymes remains limited. Therefore, developing small molecule compounds that can inhibit or regulate chitinase activity has significant application value for the control of plant-parasitic nematodes and some chitinase-dependent agricultural pests. Summary of the Invention

[0003] This invention aims to provide a class of small molecule inhibitors targeting chitinases and their agricultural applications, providing new candidate active ingredients and technical pathways for the control of plant parasitic nematodes and some chitinase-dependent agricultural pests.

[0004] Based on the structural information of chitinase, this invention employs structure-driven small molecule design centered around the substrate binding pocket and key recognition sites, establishing a core general framework of "guanidinyl-bridging structure-aromatic end group." Systematic substitution design and screening around R1, R2, R3, X, and n yielded a series of CAAS-YL candidate compounds. Representative compounds exhibited inhibitory activity against chitinase HgCht2 derived from soybean cyst nematode in the low to sub-micromolar concentration range.

[0005] Chitinases are widely present in agricultural pests such as plant-parasitic nematodes and lepidopteran insects, and their catalytic core regions and key substrate recognition sites are somewhat conserved. Furthermore, through in vitro enzymatic verification of chitinases derived from the Asian corn borer, some representative compounds of this invention also exhibited high inhibitory activity, indicating that these compounds have the potential to be applied to the control of different chitinase-dependent agricultural pests.

[0006] Compared to existing chitinase inhibitors containing guanidine structural units, the compounds of formula (I) of this invention form a novel structural combination in terms of bridging group type, amide linkage direction, and aromatic end-group substitution mode. This structural combination facilitates the compound's adaptation to the chitinase substrate binding pocket and key recognition site, thereby achieving higher enzyme inhibitory activity and agricultural application potential. In preferred embodiments, representative compounds exhibit significantly enhanced chitinase inhibitory activity compared to known control compounds.

[0007] According to a first aspect of the present invention, a chitinase inhibitor is provided, the inhibitor being a compound of formula (I), denoted as CAAS-YL; Formula (I) R1 is selected from C1–C4 alkyl, C1–C4 alkylamino, di(C1–C4 alkyl)amino, C6–C 10 The aryl group or a 5-10 heteroaryl group containing 1-3 heteroatoms selected from N, O, and S; the aryl group or heteroaryl group may optionally be substituted by 1-3 substituents selected from halogens, C1-C4 alkyl groups, or methoxy groups; n=0 or 1, representing the number of methylene groups in the linker unit between R2 and the guanidine backbone; R2 is a divalent bridging group selected from optionally substituted C6 arylene, optionally substituted 6-membered heteroarylene, optionally substituted C3–C8 cycloalkylene, or optionally substituted 6-membered heteroalkylene; the substituent is selected from halogen, C1–C4 alkyl, or methoxy. X is selected from -C(O)NH- or -NHC(O)-; R3 is selected from C6–C 10 aryl, 5–10 heteroaryl groups containing 1–3 heteroatoms selected from N, O, and S, C6–C 10 Aryl-C1–C2 alkyl or 5–10 heteroaryl-C1–C2 alkyl; the above groups are optionally substituted by 1–3 substituents selected from halogens, C1–C4 alkyls, methoxys, methylenedioxys, or phenyls.

[0008] Specifically, the preparation method of compound CAAS-YL shown in formula (I) includes the following steps: (1) After protecting the guanidinium precursor or its derivative shown in formula (II), react it with substituted acyl chloride, substituted carbamoyl chloride, substituted isocyanate or other reagents that can introduce R1 structural units to obtain the intermediate shown in formula (III). Equation (II) Equation (III) Formula (IV) (2) An amine intermediate containing an R2 structural unit and a carboxylic acid intermediate containing an R3 structural unit are subjected to an amidation reaction in the presence of a condensing agent, or a carboxylic acid intermediate containing an R2 structural unit and an amine intermediate containing an R3 structural unit are subjected to an amidation reaction in the presence of a condensing agent to obtain an intermediate of formula (IV) containing an R2-X-R3 structural unit. (3) The intermediate shown in formula (III) and the intermediate shown in formula (IV) are coupled in an organic solvent and then deprotected by acid to obtain the compound shown in formula (I). The organic solvent may be tetrahydrofuran, dichloromethane, N,N-dimethylformamide or a mixture thereof; the acid deprotecting agent may be hydrochloric acid, hydrochloric acid / dioxane solution or trifluoroacetic acid.

[0009] In this invention, the aryl group refers to an aromatic carbocyclic group, including phenyl, naphthyl, anthracene, phenanthrene, etc. The heteroaryl group refers to a monocyclic, bicyclic, or tricyclic group containing at least one heteroatom selected from N, O, or S, and at least one ring having aromaticity, such as furanyl, thiophene, pyridinyl, pyrimidinyl, quinoline-3-yl, isoquinoline-3-yl, etc. The aryl-C1–C2 alkyl or heteroaryl-C1–C2 alkyl group refers to a group formed by linking an aryl or heteroaryl group to the molecular skeleton via a methylene or ethylene group, such as benzyl, naphthylmethyl, pyridinylmethyl, quinoline-3-yl, etc. The divalent bridging group in R2 can be an aromatic divalent cyclic group, or a saturated or partially unsaturated divalent cyclic group, including but not limited to phenylene, pyridinyl, pyrimidinyl, cyclohexylene, piperidinyl, etc.

[0010] In a preferred embodiment of the present invention, R1 is selected from methylamino, dimethylamino, butyl, furanyl, 4-fluorophenyl, 2-pyridylmethyl, 4-methoxyphenyl, pyridin-2-yl or thiophen-2-yl; R2 is selected from 1,4-phenylene, 1,3-phenylene, 3-fluoro-1,4-phenylene, 1,4-pyridylene, 1,4-piperidinidyl or 1,4-cyclohexadiyl; R3 is selected from 4-fluorobenzyl, 3,4-methylenedioxybenzyl, quinoline-3-methyl, naphthyl, naphthylmethyl, 4-methoxybenzyl, pyridylmethyl, biphenylmethyl or benzyl.

[0011] In addition, the inhibitor may also be a pesticide-acceptable salt, hydrate, solvate, stereoisomer or tautomer of the compound shown in formula (I).

[0012] According to a second aspect of the invention, the use of the chitinase inhibitor described in the first aspect of the invention in the control of agricultural pests is provided. The agricultural pests are pests in which chitinase is involved in their infection, growth, or development, including plant parasitic nematodes and lepidopteran pests; preferably, the plant parasitic nematodes include soybean cyst nematodes, and the lepidopteran pests include the Asian corn borer.

[0013] According to a third aspect of the present invention, a composition for controlling agricultural pests is provided, comprising the chitinase inhibitor described in the first aspect of the present invention and an agronomically acceptable carrier or adjuvant.

[0014] In specific cases, the formulation of the composition is an agronomically acceptable formulation; the formulation may be a suspension, wettable powder, water-dispersible granule, soluble concentrate, microemulsion, emulsion, seed dressing agent or granule, and is suitable for the prevention and control of underground nematode diseases and agricultural pests such as lepidopterans.

[0015] For example, a wettable powder is composed of the following substances in weight percentages: 15-50% of the above-mentioned chitinase inhibitor, 10-20% of surfactant (such as surfactant NNO), and the balance being silica or other inert filler.

[0016] For example, a soluble or water-based formulation consists of the following substances in weight percentages: 1-10% of the above-mentioned chitinase inhibitor or its water-soluble salt, 5-15% of surfactant, 0.1-1% of penetrant, and the balance being water or a mixture of water and cosolvent.

[0017] For example, a seed coating agent, by weight percentage, contains 1% to 5% of the aforementioned chitinase inhibitor and 95% to 99% of the seed coating adjuvant. After being applied to the seed surface, the seed coating agent forms a uniform coating layer to protect the plant roots.

[0018] The beneficial effects of this invention are as follows: This invention provides a class of compounds of formula (I) with a core skeleton of "guanidinyl-bridging structure-aromatic end group". These compounds differ from existing chitinase inhibitors in terms of bridging groups, amide linkage direction, and aromatic end group substitution mode. Representative compounds can inhibit the enzymatic activity of chitinase HgCht2 derived from soybean cyst nematodes in the low micromolar to submicromolar concentration range, and can be used as candidate active ingredients for the control of plant parasitic nematodes.

[0019] Furthermore, the representative compounds of this invention also exhibit inhibitory activity against chitinases derived from lepidopteran insects, indicating that these compounds have the potential to be applied to the control of other chitinase-dependent agricultural pests. The compounds can be prepared into pesticide formulations such as wettable powders, suspensions, water-dispersible granules, and seed coatings, suitable for application in the control of agricultural pests and diseases. Attached Figure Description

[0020] Figure 1 This is the structural formula of the compound shown in formula (I) of this invention.

[0021] Figure 2 The image shows the 1H NMR spectrum of compound CAAS-YL-A1.

[0022] Figure 3 This is the carbon NMR spectrum of compound CAAS-YL-A1. Detailed Implementation

[0023] The present invention will be further described below through specific embodiments, but the present invention is not limited to the following embodiments. Experimental methods not specifically described in the following embodiments are conventional methods in the art; reagents and materials used, unless otherwise specified, can be obtained commercially. The test biological materials include: soybean cyst nematode (… Heterodera glycines ), Rhizobium ( Bradyrhizobium japonicum The soybean cyst nematode was provided by the research group of Guo Xiaoli at Huazhong Agricultural University, and the soybean was provided by the research group of Li Xidong at China Agricultural University. Lead compounds 1516b [ZL202310143552.7] and CAUZL-C-02 [ZL202311236373.4] were used as control compounds for the activity control experiment. The Asian corn borer was purchased from Jiyuan Baiyun Industrial Co., Ltd., Henan Province. The chitinases HgCht2 from the soybean cyst nematode and the chitinase from the Asian corn borer were tested. Of Cht I was purified and prepared in our laboratory.

[0024] Example 1 Preparation and structural identification of compound CAAS-YL-A1. In formula (I), CAAS-YL-A1 is NHCH3, n is 1, R2 is 1,4-phenylene, X is -C(O)NH-, and R3 is 4-fluorobenzyl.

[0025]

[0026] S-methylisothiourea hemisulfate (1.5 mmol) was added to a 500 mL round-bottom flask containing 50 mL of pure water. Then, 50 mL of tetrahydrofuran containing Boc anhydride (2.4 mmol) was added dropwise, and the mixture was stirred overnight at 0 °C. After the tetrahydrofuran was evaporated to dryness, the solution was extracted three times with 50 mL of dichloromethane (DCM). The organic phase was collected, washed with water and brine, dried over anhydrous Na₂SO₄, and concentrated. The residue was purified by silica gel column chromatography using a petroleum ether / ethyl acetate gradient of 50 / 1 to give Boc-protected S-methylisothiourea (3) in 71.2% yield.

[0027] At 0 °C, methylcarbamoyl chloride (1 mmol) and triethylamine (1.2 mmol) were dissolved in 50 mL of DCM containing Boc-protected S-methylisothiourea (3), and the mixture was gradually brought back to room temperature while stirring for 2 h. After the reaction was complete, the solution was washed with water and brine, and the organic phases were collected, combined, dried over anhydrous Na2SO4, and concentrated. The concentrated oil was purified by silica gel column chromatography with a petroleum ether / ethyl acetate gradient of 10 / 1 to give the intermediate (4).

[0028] 4-F-benzylamine (5, 30 mmol) and Boc-protected p-carboxybenzylamine (6, 33 mmol) were dissolved in dichloromethane (200 mL), and EDCI (45 mmol) and DMAP (6 mmol) were added. The mixture was reacted in a 500 mL round-bottom flask at room temperature for 3 hours. The reaction was confirmed by TLC (5:1 petroleum ether / ethyl acetate). The mixture was washed with water, dried, filtered, recrystallized from ethyl acetate, and deprotected with trifluoroacetic acid to give intermediate (7).

[0029] Intermediate (4) (10 mmol), intermediate (7) (10 mmol), and Et3N (15 mmol) were dissolved in 20 mL of tetrahydrofuran, and the mixture was stirred at room temperature for 5 hours. After the reaction was complete, the mixture was evaporated to dryness and dissolved in 10 mL of dichloromethane. Dilute hydrochloric acid was slowly added to the resulting organic phase, and the mixture was stirred at room temperature for 2 hours to remove protection. After the reaction was complete, the organic solvent was removed under reduced pressure, and the residue was concentrated twice by azeotropic distillation with toluene. The residue was extracted with saturated sodium bicarbonate solution and dichloromethane, and the organic phase was collected, washed with brine, dried, and concentrated. The residue was purified by silica gel column chromatography, eluting with a 1 / 1 gradient of petroleum ether / ethyl acetate to give the target compound as a white solid.

[0030] Other compounds in the CAAS-YL series can be prepared by referring to the above method or by appropriately adjusting the condensation, coupling, and deprotection conditions according to the properties of the substituents. Their compound numbers, corresponding substituents, appearance, and yields are shown in Table 1. The piperidine dimethyl group participates in bonding through corresponding linking sites in the piperidine ring; the specific linking methods are as shown in Table 1 and the corresponding structural formulas. The structure of compound CAAS-YL-A1 was confirmed by 1H NMR and 1C NMR spectroscopy, and the spectra are shown in Table 1. Figure 2 and Figure 3 .

[0031] Table 1. Numbering, substituents, and preparation results of some compounds in the CAAS-YL series.

[0032]

[0033]

[0034] Example 2: Preparation of wettable powder of compound CAAS-YL-A1 The wettable powder comprises, by weight percentage, 15%–50% of the compound CAAS-YL-A1, 10%–20% of a surfactant, and the balance being silica or other inert filler. The components are weighed according to the above proportions, mixed evenly, and pulverized to obtain a wettable powder containing 15%–50% of the compound CAAS-YL-A1.

[0035] Other wettable powders with the general formula CAAS-YL can be prepared according to the above method.

[0036] Example 3: Formulation of seed coating agent of compound CAAS-YL-A1 The seed coating agent comprises, by weight percentage, 1%–5% of the compound CAAS-YL-A1 and 95%–99% of a seed coating adjuvant. The components are weighed according to the above proportions, mixed thoroughly, and then coated onto the surface of crop seeds to form a uniform coating layer. After drying, seeds treated with the seed coating agent containing 1%–5% of the compound CAAS-YL-A1 are obtained.

[0037] Seed coating agents for other compounds with the general formula CAAS-YL can be prepared according to the above method.

[0038] Example 4: Determination of enzyme inhibitory activity of compounds with the general formula CAAS-YL Enzyme inhibitory activity assay: Chitinase was mixed with buffer (20 mM NaH2PO4, pH 6.0) in a 96-well plate, with a premixed volume of 59 μL. 1 μL of the inhibitor solution of different concentrations was added to each well, and the mixture was incubated at room temperature for 10 min. An equal volume of DMSO was used as a solvent control, and 1516b was used as a positive control.

[0039] The reaction was then initiated with 40 μL of 10 μM 4MU-(GlcNAc)2 (CAS No.: 53643-12-2) fluorescent substrate solution, with a final reaction volume of 100 μL. The reaction was carried out at 30 ℃ for 25 min. After the reaction was completed, 100 μL of 0.5 M Na2CO3 solution was added to terminate the reaction. The fluorescence intensity was measured at an excitation wavelength of 360 nm and an emission wavelength of 450 nm.

[0040] Concentration-inhibition rate curves were plotted based on the enzyme activity inhibition rate under various concentration conditions, and IC50 was calculated using a nonlinear regression method. 50 Value (half-maximal inhibitory concentration). The enzyme inhibitory activities of some compounds are shown in Table 2 below.

[0041] Table 2. Inhibitory activities of some compounds in the CAAS-YL series against two chitinases

[0042] Example 5: Determination of the infection activity of compounds with the general formula CAAS-YL against cyst nematodes. Prepare a 1 mM stock solution of CAAS-YL compound or its water-soluble salt in DMSO (10 mM for reference compound 1516b), store at -20 ℃ protected from light, and warm to room temperature before use. Inoculate each plant with approximately 300 nematodes. To avoid non-specific effects of the solvent on roots and microorganisms, the final DMSO volume fraction for each treatment was kept consistent and ≤0.5%, and a corresponding DMSO control was set up. Healthy Williams 82 soybean seedlings (5-7 days after germination) were used as test subjects. The soybean cyst nematode suspension and the prepared inhibitor treatment solution were simultaneously applied to the sterile sandy substrate in which the soybean seedlings were cultivated. At the same time, the seedlings were cultured to OD... 600 =0.8~1.2 and diluted to OD 600 =0.001 of rhizobia ( Bradyrhizobium japonicum USDA110 was inoculated into soybean roots; a solvent control group was established with no inhibitor but with an equal volume fraction of DMSO, and a positive control group was established with 100 μM 1516b. The final volume fraction of DMSO in each treatment group was ≤0.5%. After treatment, soybean seedlings were cultured under suitable conditions for 14 days, and the number of root nodules was counted, and the rhizobium recovery rate was calculated (recovery rate = number of rhizobium nodules in the treatment group / number of rhizobium nodules in the normal nodulation group × 100%). The normal nodulation group was the control group of soybean seedlings inoculated only with rhizobium, without nematode inoculation and without inhibitor treatment.

[0043] The effect of the inhibitor on the symbiotic recovery of root nodules in leguminous plants under cyst nematode infection conditions was evaluated by comparing the recovery rates of rhizobia in different concentration treatment groups, solvent control, and positive control group. The activity data are shown in Table 3.

[0044] Table 3. Results of determination of the symbiotic restoration activity of representative compounds in the CAAS-YL series against rhizobia in a cyst nematode infection model.

[0045] Example 6: Determination of the in vivo toxicity of CAAS-YL formula compounds to lepidopteran insects. Asian corn borers were hatched and reared in an artificial climate incubator under the following conditions: temperature 27 ℃ and light / dark cycle of 14 h / 10 h. The insecticidal activity of the target compounds was evaluated using an artificial feed treatment method. A negative control group containing an equal volume fraction of solvent was included in the experiment, and the chitinase inhibitor CAUZL-C-02 for Asian corn borers reported in patent [ZL202311236373.4] was used as a positive control. 150 third-instar larvae were randomly divided into three groups and placed in artificial feed culture dishes containing the treated compounds for continuous feeding for 72 h. After the experiment, the number of surviving larvae was counted and the mortality rate was calculated; larvae that did not react to light touch of a brush were considered dead. The insecticidal activity results of representative compounds are listed in Table 4.

[0046] Table 4. Insecticidal activity (%) of representative compounds in the CAAS-YL series against Asian corn borer at 72 h.

[0047] 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. A compound of formula (I), or a pesticide-acceptable salt, hydrate, solvate, stereoisomer, or tautomer thereof: Formula (I) R1 is selected from C1–C4 alkyl, C1–C4 alkylamino, di(C1–C4 alkyl)amino, C6–C10 aryl, or a 5–10 heteroaryl containing 1–3 heteroatoms selected from N, O, and S; the aryl or heteroaryl may optionally be substituted by 1–3 substituents selected from halogens, C1–C4 alkyl, or methoxy groups; n=0 or 1, representing the number of methylene groups in the linker unit between R2 and the guanidine backbone; R2 is a divalent bridging group selected from optionally substituted C6 arylene, optionally substituted 6-membered heteroarylene, optionally substituted C3–C8 cycloalkylene, or optionally substituted 6-membered heteroalkylene; the substituent is selected from halogen, C1–C4 alkyl, or methoxy. X is selected from -C(O)NH- or -NHC(O)-; R3 is selected from C6–C10 aryl, 5–10 heteroaryl containing 1–3 heteroatoms selected from N, O, and S, C6–C10 aryl-C1–C2 alkyl, or 5–10 heteroaryl-C1–C2 alkyl; the above groups are optionally substituted by 1–3 substituents selected from halogens, C1–C4 alkyl, methoxy, methylenedioxy, or phenyl.

2. The compound according to claim 1, characterized in that, R1 is selected from methylamino, dimethylamino, butyl, furanyl, 4-fluorophenyl, 2-pyridinemethyl, 4-methoxyphenyl, pyridin-2-yl or thiophen-2-yl.

3. The compound according to claim 1, characterized in that, R2 is selected from 1,4-phenylene, 1,3-phenylene, 3-fluoro-1,4-phenylene, 1,4-pyridylene, 1,4-piperidinidyl or 1,4-cyclohexadiyl.

4. The compound according to claim 1, characterized in that, R3 is selected from 4-fluorobenzyl, 3,4-methylenedioxybenzyl, quinoline-3-yl, naphthyl, naphthylmethyl, 4-methoxybenzyl, pyridinemethyl, biphenylmethyl, or benzyl.

5. The compound according to claim 1, characterized in that, The compounds are selected from the CAAS-YL-A1 to CAAS-YL-A22 compounds shown in Table 1.

6. A method for preparing the compound according to any one of claims 1-5, characterized in that, The process includes the following steps: an amidation reaction is performed between an amine or carboxylic acid intermediate containing an R2 structural unit and a carboxylic acid or amine containing an R3 structural unit to obtain an intermediate containing an R2-X-R3 structural unit; the intermediate is coupled with a protected guanidine precursor and deprotected by acid to obtain the compound shown in formula (I).

7. The use of the compound of any one of claims 1–5 or a pesticide-acceptable salt thereof in the preparation of a pesticide for controlling agricultural pests, characterized in that, The agricultural pests mentioned are those in which chitinases are involved in their infection, growth, or development processes.

8. The use according to claim 7, characterized in that, The agricultural pests mentioned include plant parasitic nematodes or lepidopteran pests.

9. A pesticide composition for controlling agricultural pests, characterized in that, It comprises the compound of any one of claims 1–5 or a pesticide-acceptable salt thereof, and a pesticide-acceptable carrier, adjuvant, or combination thereof.

10. The pesticide composition according to claim 9, characterized in that, The pesticide composition is formulated as a suspension concentrate, wettable powder, water-dispersible granule, emulsifiable concentrate, microemulsion, water-in-oil emulsion, seed coating agent, or granule.

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