Chitinase inhibitor as well as preparation and application thereof

Abstract

The invention discloses a chitinase inhibitor as well as preparation and application thereof, and belongs to the technical field of biology. The invention finds that the compound with the structure of the general formula (I) has high enzyme inhibition activity and excellent insecticidal activity on chitinase, especially has high inhibition activity on Asiatic corn borer chitinase h (OfChih), and has strong insecticidal activity on agricultural pests such as corn borer and plutella xylostella. The compound with the structure can be used for chitinase inhibitors or insecticides, has a very high pesticide research value, and has a wide application prospect in the field of agricultural pharmacology.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a chitinase inhibitor and the preparation and application thereof. Background Art

[0002] Crop diseases and insect pests can cause huge losses to agricultural production, resulting in a 20-40% reduction in global crop yields each year. The growth and development of agricultural pests are closely related to their chitin metabolism. Chitin metabolism is divided into chitin anabolism and chitin hydrolysis. In the chitin hydrolysis process, chitin is first hydrolyzed into chito-oligosaccharides by glycosyl hydrolase 18 family chitinase, and then hydrolyzed into N-acetylhexosamine by 20 family β-N-acetylhexosaminidase. Down-regulating the expression level of insect chitinase can lead to insect molting disorders, growth inhibition, abnormal pupation, and ultimately the death of insects. Therefore, 18 family chitinase is an important target for green pesticides.

[0003] Insect chitinase Chi-h from family 18 is an indispensable part of insect molting and metamorphosis, and the crystal structure of OfChi-h from Asian corn borer has been successfully resolved. It is worth mentioning that Chi-h has up to 70% homology with chitinase from bacteria, and is a chitinase obtained through horizontal gene transfer that is unique to Lepidoptera insects. Considering that Lepidoptera insects (such as diamondback moth and corn borer) are the most destructive type of crop pests, and most beneficial insects (such as parasitic wasps and bees) do not have Chi-h. Therefore, the research and development of new inhibitors targeting Chi-h, especially OfChi-h, has a good structural biology basis and important target specificity advantages. The development of new and efficient enzyme inhibitors targeting insect chitinase Chi-h, especially OfChi-h, is of great significance for the green prevention and control of agricultural pests. Summary of the invention

[0004] The purpose of the present invention is to provide a chitinase inhibitor and its preparation and application, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a chitinase inhibitor, the general structural formula of the chitinase inhibitor is shown in formula (I):

[0007]

[0008] Wherein: R1-R5 are independently selected from one of H, C1-C4 alkyl, halogen, OCH3, CN, OH, NHAc and NO2;

[0009] R6-R9 are independently selected from one of H, C1-C4 alkyl, OH, OCH3 and halogen;

[0010] R 10 -R 12 Independently selected from one of H, C1-C4 alkyl and halogen;

[0011] XY is CC or C=C;

[0012] WZ is CC or C=C.

[0013] The compound represented by formula (I) has high enzyme inhibitory activity against chitinase.

[0014] The second technical solution of the present invention: use of the above chitinase inhibitor and pharmaceutically acceptable salts thereof in the preparation of a drug for inhibiting chitinase activity.

[0015] The chitinase inhibitor represented by the above formula (I) is actually a 2-phenylisoindole-1,3-dione compound, that is, the present scheme substantially protects the use of a 2-phenylisoindole-1,3-dione compound and a pharmaceutically acceptable salt thereof in inhibiting chitinase activity.

[0016] Furthermore, the chitinase is a glycosyl hydrolase 18 family chitinase.

[0017] Furthermore, the glycosyl hydrolase 18 family chitinase is Asian corn borer chitinase h (OfChi-h).

[0018] Furthermore, the final concentration of the chitinase inhibitor or its pharmaceutically acceptable salt in the drug for inhibiting chitinase activity is not less than 0.1 μM, that is, when the chitinase inhibitor and its pharmaceutically acceptable salt are used to inhibit the activity of corn borer chitinase h, the final concentration used in the reaction system is not less than 0.1 μM.

[0019] Furthermore, the final concentration of the chitinase inhibitor or a pharmaceutically acceptable salt thereof in the drug for inhibiting the activity of chitinase is preferably 0.1 μM to 10 mM.

[0020] The third technical solution of the present invention: the use of the above chitinase inhibitor and its pharmaceutically acceptable salt in the preparation of drugs for controlling agricultural pests. This solution essentially protects the use of the compound represented by formula (I) and its pharmaceutically acceptable salt in controlling agricultural pests.

[0021] Furthermore, the agricultural pest is Plutella xylostella or Ostrinia nubilalis.

[0022] Furthermore, the final concentration of the chitinase inhibitor or its pharmaceutically acceptable salt in the drug for controlling agricultural pests is not less than 0.1 μM, that is, when the chitinase inhibitor and its pharmaceutically acceptable salt are used for controlling agricultural pests, the final concentration used in the reaction system is not less than 0.1 μM.

[0023] Furthermore, the final concentration of the chitinase inhibitor or a pharmaceutically acceptable salt thereof in the drug for controlling agricultural pests is preferably 0.1 μM to 10 mM.

[0024] Technical solution 4 of the present invention: The preparation method of the above chitinase inhibitor comprises the following steps:

[0025] The compound represented by formula (II), the compound represented by formula (III) and solvent 1 are mixed, and reacted at a temperature of 120° C. to 170° C. for 0.2 to 15 hours to obtain a compound represented by formula (IV);

[0026] The compound represented by formula (IV), the compound represented by formula (V), a base and solvent 2 are mixed, and reacted at a temperature of -20°C to 100°C for 0.5 to 24 hours to obtain the compound represented by formula (I), which is the chitinase inhibitor;

[0027]

[0028] Wherein: R1-R5 are independently selected from one of H, C1-C4 alkyl, halogen, OCH3, CN, OH, NHAc or NO2;

[0029] R6-R9 are independently selected from one of H, C1-C4 alkyl, OH, OCH3 or halogen;

[0030] R 10 -R 12 Independently selected from one of H, C1-C4 alkyl or halogen;

[0031] XY is CC or C=C;

[0032] WZ is CC or C=C.

[0033] Furthermore, the solvent 1 is one of DMF, xylene and DMSO; the base is one or two of sodium hydroxide, potassium fluoride, sodium fluoride, sodium bicarbonate, sodium acetate, sodium carbonate, potassium carbonate, potassium acetate and ammonium acetate; and the solvent 2 is one of acetonitrile, dichloromethane and DMF.

[0034] Furthermore, in terms of molar ratio, the compound represented by formula (II): the compound represented by formula (III) = 1:1-2; the compound represented by formula (IV): the compound represented by formula (V): the base = 1:0.5-2:0.5-30.

[0035] Furthermore, the amount ratio of the compound represented by formula (II) to solvent 1 is 0.01-5 mmol:1 mL; the amount ratio of the compound represented by formula (IV) to solvent 2 is 0.01-5 mmol:1 mL.

[0036] The present invention discloses the following technical effects:

[0037] The present invention finds that the compound with the general formula (I) has high enzyme inhibition activity and excellent insecticidal activity against chitinase, especially high inhibition activity against Asian corn borer chitinase h (OfChi-h), and strong insecticidal activity against agricultural pests corn borer and diamondback moth. The compound with the structure can be used as a chitinase inhibitor or insecticide, has high pesticide research value, and has broad application prospects in the field of pesticide science. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0039] Figure 1 is the H NMR spectrum of compound HAU-IIO15;

[0040] Figure 2 is the IC of compound HAU-IIO1 against OfChi-h 50 Test results. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0043] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0044] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0045] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0046] The present invention provides a chitinase inhibitor, the general structural formula of the chitinase inhibitor is shown in formula (I):

[0047]

[0048] Wherein: R1-R5 are independently selected from one of H, C1-C4 alkyl, halogen, OCH3, CN, OH, NHAc or NO2;

[0049] R6-R9 are independently selected from one of H, C1-C4 alkyl, OH, OCH3 or halogen;

[0050] R 10 -R 12 Independently selected from one of H, C1-C4 alkyl or halogen;

[0051] XY is CC or C=C;

[0052] WZ is CC or C=C.

[0053] The present invention also provides the use of the chitinase inhibitor and the pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting the activity of chitinase.

[0054] As a preferred embodiment of the present invention, the chitinase is a glycosyl hydrolase 18 family chitinase.

[0055] As a preferred embodiment of the present invention, the glycosyl hydrolase 18 family chitinase is Ostrinia furnacalis chitinase h (OfChi-h).

[0056] As a preferred embodiment of the present invention, the final concentration of the chitinase inhibitor or its pharmaceutically acceptable salt in the drug for inhibiting the activity of chitinase is not less than 0.1 μM, preferably 0.1 μM to 10 mM, that is, when the chitinase inhibitor and its pharmaceutically acceptable salt are used to inhibit the activity of corn borer chitinase h, the final concentration used in the reaction system is not less than 0.1 μM, preferably 0.1 μM to 10 mM.

[0057] The present invention also provides the use of the chitinase inhibitor and its pharmaceutically acceptable salt in the preparation of medicines for preventing and controlling agricultural pests.

[0058] As a preferred embodiment of the present invention, the agricultural pest is Plutella xylostella or Ostrinia nubilalis.

[0059] As a preferred embodiment of the present invention, the final concentration of the chitinase inhibitor or its pharmaceutically acceptable salt in the drug for controlling agricultural pests is not less than 0.1 μM, preferably 0.1 μM to 10 mM, that is, when the chitinase inhibitor and its pharmaceutically acceptable salt are used for controlling agricultural pests, the final concentration used in the reaction system is not less than 0.1 μM, preferably 0.1 μM to 10 mM.

[0060] The present invention also provides a method for preparing the chitinase inhibitor, comprising the following steps:

[0061] The compound represented by formula (II), the compound represented by formula (III) and solvent 1 are mixed, and reacted at a temperature of 120° C. to 170° C. for 0.2 to 15 hours to obtain a compound represented by formula (IV);

[0062] The compound represented by formula (IV), the compound represented by formula (V), a base and solvent 2 are mixed, and reacted at a temperature of -20°C to 100°C for 0.5 to 24 hours to obtain the compound represented by formula (I), which is the chitinase inhibitor;

[0063]

[0064] Wherein: R1-R5 are independently selected from one of H, C1-C4 alkyl, halogen, OCH3, CN, OH, NHAc and NO2;

[0065] R6-R9 are independently selected from one of H, C1-C4 alkyl, OH, OCH3 and halogen;

[0066] R 10 -R 12 Independently selected from one of H, C1-C4 alkyl and halogen;

[0067] XY is CC or C=C;

[0068] WZ is CC or C=C.

[0069] As a preferred embodiment of the present invention, the solvent 1 is one of DMF, xylene and DMSO; the base is one or two of sodium hydroxide, potassium fluoride, sodium fluoride, sodium bicarbonate, sodium acetate, sodium carbonate, potassium carbonate, potassium acetate and ammonium acetate; the solvent 2 is one of acetonitrile, dichloromethane and DMF.

[0070] As a preferred embodiment of the present invention, in terms of molar ratio, the compound represented by formula (II): the compound represented by formula (III) = 1:1-2; the compound represented by formula (IV): the compound represented by formula (V): the base = 1:0.5-2:0.5-30.

[0071] As a preferred embodiment of the present invention, the amount ratio of the compound represented by formula (II) to solvent 1 is 0.01-5 mmol:1 mL; the amount ratio of the compound represented by formula (IV) to solvent 2 is 0.01-5 mmol:1 mL

[0072] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0073] All raw materials and reagents involved in the specific embodiments of the present invention are common commercially available products.

[0074] The chitinase OfChi-h used in the specific embodiment of the present invention is given in the following references: Liu, T.; Chen, L.; Zhou, Y.; Jiang, X.; Duan, Y.; Yang, Q., Structure, Catalysis, and Inhibition of OfChi-h, the Lepidoptera-exclusive Insect Chitinase. J. Biol. Chem. 2017, 292 (6), 2080-2088.

[0075] Example 1

[0076] 1. Compound HAU-IIO15 (R1=R2=R4=R5=R6=R7=R8=R9=R 11 =H, R3 = R 10 =CH3, XY is CC, WZ is CC, the specific structural formula is ), the reaction route is:

[0077]

[0078] The specific preparation steps are as follows:

[0079] (1) In a 5 mL reaction bottle, add 3-methyltetrahydrophthalic anhydride (16.6 mg, 0.1 mmol) of the compound represented by formula (II), 3-aminobenzoic acid (16.4 mg, 0.12 mmol) of the compound represented by formula (III) and DMF (1 mL), stir and heat the mixture to 150°C, and continue heating and reacting for 10 hours. Purify by column chromatography to obtain 19.5 mg of isoindolinonebenzoic acid represented by formula (IV), with a yield of 68.4%. ESI-MS: 284.47 (MH).

[0080] (2) The compound represented by formula (IV) isoindolinone benzoic acid (14.2 mg, 0.05 mmol), the compound represented by formula (V) bromoacetophenone (12.8 mg, 0.06 mmol), and potassium fluoride (3.5 mg, 0.06 mmol) were dissolved in DMF (1 mL), and the mixture was stirred at 70° C. for 5 h. After purification by column chromatography, the compound represented by formula (I) HAU-IIO15 was obtained with a yield of 82.3% (17.2 mg).

[0081] Figure 1 This is the H NMR spectrum (400 MHz, DMSO-d6) of compound HAU-IIO15. The analytical data are as follows: 1 H NMR(400MHz,DMSO-d6)δ8.19-7.78(m,4H),7.69-7.26(m,4H),6.09-5.77(m,2H),5.61(s,2 H),3.42-3.16(m,2H),2.71-2.55(m,4H),2.23-2.05(m,1H),1.41(d,3H); HRMS(ESI)calcd forC 25 H 24 NO5(M+H + )418.1654 found 418.1661.

[0082] 2. Preparation of other compounds

[0083] The general reaction route is:

[0084]

[0085] Through the general reaction route and the preparation steps of the above-mentioned compound HAU-IIO15, according to the specific structure of the compound to be obtained, the compounds represented by formula (II), formula (III), formula (IV) and formula (V) are replaced with compounds with corresponding substituents (replaced in equimolar amounts) to obtain multiple compounds represented by formula (I) with different substituents, as shown in Table 1 (the target compounds were obtained by hydrogen nuclear magnetic spectrum detection).

[0086] Table 1

[0087]

[0088]

[0089]

[0090]

[0091] Example 2

[0092] The inhibition rate of the compound on chitinase h (OfChi-h) of Asian corn borer was determined as follows:

[0093] Experimental group: 3 parallel experimental groups were set up. At a reaction temperature of 30°C, 2nmol / L chitinase h (OfChi-h), 10μM of each compound prepared in Example 1 and 50μM substrate Mu-(GlcNAc)2 were added to 100μL of the reaction system, and incubated in 20mM pH=6.0 phosphate buffer for 30min; 0.5M sodium carbonate solution was added to terminate the reaction, and the reaction solution was excited with 360nm wavelength excitation light and the absorbance at 450nm wavelength was measured.

[0094] Positive control: 3 sets of parallel positive controls were set up. At a reaction temperature of 30°C, 2nmol / L chitinase h (OfChi-h) and 50μM substrate Mu-(GlcNAc)2 were added to 100μL of the reaction system, and incubated in 20mM pH=6.0 phosphate buffer for 30min; 0.5M sodium carbonate solution was added to terminate the reaction, and the reaction solution was excited with 360nm wavelength excitation light and the absorbance at 450nm wavelength was measured.

[0095] The enzyme inhibitory activity of the compounds was calculated according to the following formula:

[0096] Inhibition percentage = (positive control - experimental group) / positive control × 100

[0097] The results are shown in Table 2 (the average value of three parallel experiments), which lists the sequences of 21 representative compounds and their enzyme inhibitory activities against chitinase h (OfChi-h).

[0098] Table 2

[0099]

[0100] Example 3

[0101] IC50% inhibitory concentration of compounds on OfChi-h 50 The specific steps are as follows:

[0102] At a reaction temperature of 30°C, 2nmol / L OfChi-h, representative compounds HAU-IIO1, HAU-IIO2 or HAU-IIO6 prepared in Example 1 with appropriate concentration gradients (0.16μM, 0.31μM, 0.63μM, 1.25μM, 2.5μM, 5μM, 10μM, 20μM) and 50μM substrate Mu-(GlcNAc)2 were added to a 100μL reaction system, and incubated in a 20mM phosphate buffer at pH=6.0 for 30min; 0.5M sodium carbonate solution was added to terminate the reaction, and the reaction solution was excited with an excitation light of 360nm wavelength and then the absorbance value at 450nm wavelength was measured. Graphpad Prism was used to process the data and plot the graph (the horizontal axis is the logarithm of the compound concentration, the vertical axis is the percentage of inhibition of the compound on OfChi-h, and the corresponding graph of compound HAU-IIO1 is as shown in the figure below). Figure 2 As shown, the corresponding figures of other compounds are no longer listed one by one, only the specific IC 50 value), and calculate IC 50 The results are shown in Table 3 (three parallel experiments were set for each compound, and the results were taken as the average value of the three parallel experiments).

[0103] Table 3

[0104] Compound No. <![CDATA[IC 50 (μM)]]> HAU-IIO1 2.55 HAU-IIO2 2.79 HAU-IIO6 5.57

[0105] Example 4

[0106] Determination of insecticidal activity of compounds. The specific steps are as follows:

[0107] The insecticidal activity of the compound prepared in Example 1 against corn borer and diamondback moth was tested by the insect immersion method. Healthy third-instar larvae were immersed in a solution of each compound prepared in Example 1 at a concentration of 500 μg / mL (or 200 ug / mL) for 5-10 seconds, and the excess solution was absorbed with filter paper. The test insects were then transferred to normal conditions for 72 hours, and the mortality was checked. The mortality rate was counted and the corrected mortality rate was calculated. Hexaflumuron was selected as the positive control and distilled water was used as the blank control (three parallel groups were set for each concentration of each compound, and three parallel groups were also set for the positive control and blank control groups. The results were taken as the average of the three parallel experiments).

[0108] The adjusted mortality rate was calculated according to the following formula:

[0109] Corrected mortality rate (%) = (sample mortality rate - blank control mortality rate) / (1 - blank control mortality rate) × 100.

[0110] The insecticidal activity test results of the compounds are shown in Table 4:

[0111] Table 4

[0112]

[0113]

[0114] From the above test results, it can be seen that the compound of the structure represented by formula (I) of the present invention has excellent enzyme inhibitory activity against chitinase OfChi-h. The inhibitory activity data of chitinase OfChi-h (including the inhibition rate of the inhibitor, the half inhibition concentration IC 50 ) The results showed that the 21 representative compounds shown in Table 1 all showed good inhibitory activity against chitinase OfChi-h. In particular, the inhibition rates of compounds HAU-IIO1, HAU-IIO2, HAU-IIO3, HAU-IIO4, HAU-IIO17, HAU-IIO18, and HAU-IIO21 against OfChi-h were all greater than 75% at a test concentration of 10 μM. The IC values ​​of compounds HAU-IIO1, HAU-IIO2, and HAU-IIO6 against OfChi-h were 50 The values ​​are 2.55μM, 2.79μM, and 5.57μM, respectively.

[0115] In addition, the compounds of the structure shown in formula (I) of the present invention have strong insecticidal activity against agricultural pests. Most of the 21 representative compounds shown in Table 1 have an insecticidal activity of 100% against diamondback moth and corn borer at a concentration of 500 μg / mL; the insecticidal activity of compounds HAU-IIO1, HAU-IIO2, HAU-IIO4, HAU-IIO7, and HAU-IIO21 against diamondback moth at a concentration of 200 μg / mL is not less than 90%, which is much better than the control drug hexaflumuron; the insecticidal activity of compounds HAU-IIO2, HAU-IIO4, HAU-IIO9, and HAU-IIO13 against diamondback moth at a concentration of 200 μg / mL is not less than 90%, which is better than the control drug hexaflumuron.

[0116] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A chitinase inhibitor, characterized in that The general structural formula of the chitinase inhibitor is shown in formula (I): Wherein: R1-R5 are independently selected from one of H, C1-C4 alkyl, halogen, OCH3, CN, OH, NHAc and NO2; R6-R9 are independently selected from one of H, C1-C4 alkyl, OH, OCH3 and halogen; R 10 -R 12 Independently selected from one of H, C1-C4 alkyl and halogen; XY is CC or C=C; WZ is CC or C=C.

2. Use of the chitinase inhibitor and pharmaceutically acceptable salt thereof as claimed in claim 1 in the preparation of a drug for inhibiting chitinase activity.

3. The use according to claim 2, characterized in that The chitinase is a glycosyl hydrolase 18 family chitinase.

4. The use according to claim 3, characterized in that The glycosyl hydrolase 18 family chitinase is Ostrinia furnacalis chitinase h.

5. The use according to claim 4, characterized in that The final concentration of the chitinase inhibitor or a pharmaceutically acceptable salt thereof in the drug for inhibiting the activity of chitinase is not less than 0.1 μM.

6. Use of the chitinase inhibitor and pharmaceutically acceptable salt thereof as claimed in claim 1 in the preparation of drugs for controlling agricultural pests.

7. The use according to claim 6, characterized in that The agricultural pests are diamondback moth or corn borer.

8. The method for preparing a chitinase inhibitor according to claim 1, characterized in that: The following steps are involved: The compound represented by formula (II), the compound represented by formula (III) and solvent 1 are mixed, and reacted at a temperature of 120° C. to 170° C. for 0.2 to 15 hours to obtain a compound represented by formula (IV); The compound represented by formula (IV), the compound represented by formula (V), a base and solvent 2 are mixed, and reacted at a temperature of -20°C to 100°C for 0.5 to 24 hours to obtain the compound represented by formula (I), which is the chitinase inhibitor; Wherein: R1-R5 are independently selected from one of H, C1-C4 alkyl, halogen, OCH3, CN, OH, NHAc and NO2; R6-R9 are independently selected from one of H, C1-C4 alkyl, OH, OCH3 and halogen; R 10 -R 12 Independently selected from one of H, C1-C4 alkyl and halogen; XY is CC or C=C; WZ is CC or C=C.

9. The method for preparing a chitinase inhibitor according to claim 8, characterized in that: The solvent 1 is one of DMF, xylene and DMSO; the base is one or two of sodium hydroxide, potassium fluoride, sodium fluoride, sodium bicarbonate, sodium acetate, sodium carbonate, potassium carbonate, potassium acetate and ammonium acetate; and the solvent 2 is one of acetonitrile, dichloromethane and DMF.

10. The method for preparing a chitinase inhibitor according to claim 8, characterized in that: In terms of molar ratio, the compound represented by formula (II): the compound represented by formula (III) = 1:1-2; the compound represented by formula (IV): the compound represented by formula (V): the base = 1:0.5-2:0.5-30.

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