Chitinase inhibitor and its preparation and application

Abstract

The present invention discloses a chitinase inhibitor and its preparation and application, belonging to the field of biotechnology. The present invention finds that a compound having a general formula (I) structure has high enzyme inhibitory activity and excellent insecticidal activity against chitinase, especially high inhibitory activity against Asian corn borer chitinase h (OfChi-h), and has potent insecticidal activity against agricultural pests such as corn borer and diamondback moth. The compound of this 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.

Description

Technical Field

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

[0002] Crop pests and diseases can cause significant losses to agricultural production, resulting in a 20-40% annual global crop yield reduction. The growth and development of agricultural pests are closely linked to their chitin metabolism. Chitin metabolism is divided into chitin anabolism and chitin hydrolysis. During chitin hydrolysis, chitin is first hydrolyzed into chito-oligosaccharides by glycosyl hydrolases from family 18 chitinases and then into N-acetylglucosamine by family 20 β-N-acetylglucosaminidase. Downregulating the expression of chitinases in insects can lead to molting disorders, growth inhibition, abnormal pupation, and ultimately, death. Therefore, family 18 chitinases are important targets 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 unique to Lepidoptera insects obtained through horizontal gene transfer. 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 for 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 drugs 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, this scheme essentially 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 family 18 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 that inhibits 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 chitinase activity is preferably 0.1 μM to 10 mM.

[0020] Technical solution three of the present invention: Use of the chitinase inhibitor and its pharmaceutically acceptable salt in the preparation of a medicament 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 diamondback moth or corn borer.

[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-mentioned chitinase inhibitor comprises the following steps:

[0025] Mixing the compound represented by formula (II), the compound represented by formula (III) and solvent 1, and reacting at a temperature of 120° C. to 170° C. for 0.2 to 15 hours to obtain the 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 ratio of the compound represented by formula (II) to solvent 1 is 0.01-5 mmol:1 mL; the 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 discovers that compounds having the general formula (I) exhibit high chitinase inhibitory activity and excellent insecticidal activity, particularly against the Asian corn borer chitinase h (OfChi-h), and have potent insecticidal activity against the agricultural pests Ostrinia nubilalis and Plutella xylostella. Compounds with this structure can be used as chitinase inhibitors or insecticides, have high pesticide research value, and have broad application prospects in the field of pesticides. 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 following briefly introduces the drawings required for use in the embodiments. 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 any 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0043] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents 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 described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be 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 pharmaceutically acceptable salts thereof in the preparation of drugs for inhibiting chitinase activity.

[0054] As a preferred embodiment of the present invention, the chitinase is a glycosyl hydrolase family 18 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 a pharmaceutically acceptable salt thereof in the drug for inhibiting chitinase activity 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 pharmaceutically acceptable salts thereof in the preparation of drugs for preventing and controlling agricultural pests.

[0058] As a preferred embodiment of the present invention, the agricultural pest is diamondback moth or corn borer.

[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] Mixing the compound represented by formula (II), the compound represented by formula (III) and solvent 1, and reacting at a temperature of 120° C. to 170° C. for 0.2 to 15 hours to obtain the 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; and the solvent 2 is one of acetonitrile, dichloromethane and DMF.

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

[0071] As a preferred embodiment of the present invention, the ratio of the compound represented by formula (II) to solvent 1 is 0.01 to 5 mmol:1 mL; the ratio of the compound represented by formula (IV) to solvent 2 is 0.01 to 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 embodiments 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 flask, 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) were added. The mixture was stirred and heated to 150°C. The heating reaction was continued for 10 h. After purification by column chromatography, 19.5 mg of isoindolinonebenzoic acid (IV) was obtained in a yield of 68.4%. ESI-MS: 284.47 (MH).

[0080] (2) Isoindolebenzoic acid (14.2 mg, 0.05 mmol), a compound represented by formula (IV), bromoacetophenone (12.8 mg, 0.06 mmol), and potassium fluoride (3.5 mg, 0.06 mmol) were dissolved in DMF (1 mL) and 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 + )418.1654found 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 having 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 all obtained by nuclear magnetic resonance spectroscopy 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: Three parallel experimental groups were set up. At 30°C, 2 nmol / L chitinase h (OfChi-h), 10 μM of each compound prepared in Example 1, and 50 μM of the substrate Mu-(GlcNAc)2 were added to a 100 μL reaction system. The mixture was incubated in 20 mM phosphate buffer (pH 6.0) for 30 min. The reaction was terminated by adding 0.5 M sodium carbonate solution. The reaction solution was excited with 360 nm excitation light, and the absorbance at 450 nm was measured.

[0094] Positive Control: Three parallel positive controls were performed. At 30°C, 2 nmol / L chitinase h (OfChi-h) and 50 μM substrate Mu-(GlcNAc)2 were added to a 100 μL reaction system and incubated in 20 mM phosphate buffer, pH 6.0, for 30 min. The reaction was terminated by adding 0.5 M sodium carbonate solution. The reaction solution was excited with 360 nm excitation light, and the absorbance at 450 nm 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 is taken). Table 2 lists the numbers of 21 representative compounds and their enzyme inhibitory activities against chitinase h (OfChi-h).

[0098] Table 2

[0099]

[0100] Example 3

[0101] The half-maximal inhibitory concentration IC of the compound on OfChi-h 50 The specific steps are as follows:

[0102] At a reaction temperature of 30°C, 2 nmol / L OfChi-h, representative compounds HAU-IIO1, HAU-IIO2 or HAU-IIO6 from each compound prepared in Example 1 with an appropriate concentration gradient (0.16 μM, 0.31 μM, 0.63 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) and 50 μM substrate Mu-(GlcNAc)2 were added to a 100 μL reaction system and incubated for 30 minutes in a 20 mM phosphate buffer solution with a pH of 6.0; 0.5 M sodium carbonate solution was added to terminate the reaction, and the reaction solution was excited with 360 nm wavelength excitation light and the absorbance at 450 nm wavelength was measured. Graphpad Prism was used to process the data and plot the graphs (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 for compound HAU-IIO1 is shown in FIG). Figure 2 As shown, the corresponding figures of other compounds are no longer listed one by one, and 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 the average of the three parallel experiments).

[0103] Table 3

[0104] Compound number <![CDATA[IC 50 (μM)]]> HAU-IIO1 2.55 HAU-IIO2 2.79 HAU-IIO6 5.57

[0105] Example 4

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

[0107] The insecticidal activity of the compounds prepared in Example 1 against corn borers and diamondback moths was tested using the insect immersion method. Healthy third-instar larvae were immersed in a 500 μg / mL (or 200 μg / mL) solution of each compound prepared in Example 1 for 5-10 seconds. The excess solution was absorbed with filter paper, and the test insects were then transferred to normal conditions for 72 hours. The mortality was examined, the mortality rate was counted, and the corrected mortality rate was calculated. Hexaflumuron was used as a positive control, and distilled water was used as a 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 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 inhibitory concentration IC 50 ) The results showed that the 21 representative compounds shown in Table 1 all exhibited good inhibitory activity against chitinase OfChi-h. In particular, compounds HAU-IIO1, HAU-IIO2, HAU-IIO3, HAU-IIO4, HAU-IIO17, HAU-IIO18, and HAU-IIO21 all exhibited inhibition rates of greater than 75% against OfChi-h at a test concentration of 10 μM. Compounds HAU-IIO1, HAU-IIO2, and HAU-IIO6 exhibited IC values ​​of 1. 50 The values ​​were 2.55 μM, 2.79 μM, and 5.57 μM, respectively.

[0115] Furthermore, the compounds represented by formula (I) of the present invention exhibit strong insecticidal activity against agricultural pests. The vast majority of the 21 representative compounds shown in Table 1 exhibit 100% insecticidal activity against Plutella xylostella and Ostrinia nubilalis at a concentration of 500 μg / mL. Compounds HAU-IIO1, HAU-IIO2, HAU-IIO4, HAU-IIO7, and HAU-IIO21 exhibit insecticidal activity against Plutella xylostella of at least 90% at a concentration of 200 μg / mL, significantly superior to the control drug hexaflumuron. Compounds HAU-IIO2, HAU-IIO4, HAU-IIO9, and HAU-IIO13 exhibit insecticidal activity against Plutella xylostella of at least 90% at a concentration of 200 μg / mL, surpassing the control drug hexaflumuron.

[0116] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A chitinase inhibitor and a pharmaceutically acceptable salt thereof for preparing a drug for inhibiting chitinase activity, 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. The use according to claim 1, characterized in that The chitinase is a glycosyl hydrolase family 18 chitinase.

3. The use according to claim 2, characterized in that The glycosyl hydrolase 18 family chitinase is Asian corn borer chitinase h.

4. The use according to claim 3, characterized in that The final concentration of the chitinase inhibitor or a pharmaceutically acceptable salt thereof in the drug for inhibiting chitinase activity is not less than 0.1 μM.

5. Use of a chitinase inhibitor and a pharmaceutically acceptable salt thereof in the preparation of a medicament for controlling agricultural pests, 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.

6. The use according to claim 5, characterized in that The agricultural pests are diamondback moth or corn borer.

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