Substituted hydroxybenzimine compound as well as preparation and application thereof

Abstract

The invention belongs to the technical field of plant protection, and particularly relates to a substituted hydroxybenzimine compound as well as preparation and application thereof. The compound as shown in the formula I is used as a chitin deacetylase inhibitor, has a certain inhibition effect on growth and pathogenicity of common plant disease fungi, can be applied to prevention and control of crop diseases, and provides a new strategy for green prevention and control of the diseases. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of plant protection, and specifically relates to substituted hydroxybenzimine compounds and preparation and application thereof, and more specifically relates to substituted hydroxybenzimine compounds and preparation and application thereof as chitin deacetylase inhibitors. Background Art

[0002] In the process of grain production, agricultural fungal diseases are one of the important factors affecting the stable and increased yield of crops. [1] According to statistics, more than half of plant diseases in the world are caused by pathogenic fungi. [2] Major crop diseases represented by wheat stripe rust, wheat head blight, rice blast, and rice sheath blight are all agricultural disasters that farmers are terrified of. They have caused large-scale grain production reductions and even crop failures countless times, and are a major concern for global agricultural production. As the world's largest grain producer, my country faces the current situation of high incidence of agricultural diseases and insect pests. Chemical control is one of the important means of preventing and controlling agricultural diseases. However, the long-term irrational use of chemical fungicides has also caused serious resistance problems and ecological and environmental problems. [3] Therefore, it is urgent to develop new fungicides with low resistance risk and safety.

[0003] Chitin deacetylase (CDA) is one of the key enzymes targeting chitin. In addition to participating in the formation of fungal cell walls, it also plays an important role in the process of fungal invasion of host plants. It is one of the important functional proteins for pathogenic fungi to exert their pathogenicity. It can infect pathogens by evading or inhibiting the occurrence of plant immunity. In addition, its lack in mammals makes fungal chitin deacetylase a candidate target for the development of new fungicides. [4,5] In recent years, the analysis of the crystal structure of fungal CDA and the elucidation of its binding mode have also laid the foundation for the design and development of new fungicides based on the CDA protein structure. So far, many CDA inhibitors have been reported. They can play a role in the activation of plant disease resistance and have a good control effect on fungal diseases of wheat, soybeans and cotton. [6-8] .

[0004] Therefore, targeting fungal chitin deacetylase to create new low-risk and eco-friendly fungicides has important practical significance for the green prevention and control of pathogenic fungi and ensuring food security.

[0005] References:

[0006] [1] Tyczewska A, E,Gracz J,et al.Towards Food Security:CurrentState and Future Prospects of Agrobiotechnology[J].Trends in Biotechnology,2018,36(12):1219-1229.

[0007] [2]Savary S,Willocquet L,Pethybridge S J,et al.The global burden ofpathogens and pests on major food crops[J].Nature Ecology&Evolution,2019,3(3):430-439.

[0008] [3]Lykogianni M,Bempelou E,Karamaouna F,et al.Do pesticides promoteor hinder sustainability in agriculture?The challenge of sustainable use ofpesticides in modern agriculture[J].Science of The Total Environment,2021,795:148625.

[0009] [4]Jiang J,Xie X,Li X.Acetyl-Proteomic Profiling ofSorghum bicolorSeedlings after Chitin Treatment Reveals the Involvement of AcetylatedChlorophyll a / b Binding Proteins in the Innate Immune Response[J].Journal ofAgricultural and Food Chemistry,2023,71(27):10438-10447.

[0010] [5]Wang Y, Pruitt RN, Nürnberger T, et al. Evasion of plant immunity by microbial pathogens[J]. Nature Reviews Microbiology, 2022, 20(8): 449-464.

[0011] [6]Liu L,Xia Y,Li Y,et al.Inhibition of chitin deacetylases toattenuate plant fungal diseases[J].Nature Communications,2023,14(1):3857.

[0012] [7]Liu Y, Ahmed S, Fang Y, et al. Discovery of Chitin DeacetylaseInhibitors through Structure-Based Virtual Screening and Biological Assays[J].2022,32(4):504-513.

[0013] [8] Zanni R, Martínez-Cruz J, Gálvez-Llompart M, et al. Rational Design of Chitin Deacetylase Inhibitors for Sustainable Agricultural Use Based on Molecular Topology [J]. Journal of Agricultural and Food Chemistry, 2022, 70(41): 13118-13131. Summary of the invention

[0014] The purpose of the present invention is to provide a substituted hydroxybenzimine compound and a preparation method thereof.

[0015] The substituted hydroxybenzimine compounds provided by the present invention have a structural formula as shown in Formula I:

[0016]

[0017] In formula I, X is CH or N;

[0018] R 1is selected from substituted or unsubstituted saturated or unsaturated five-membered or six-membered ring groups containing heteroatoms, substituted or unsubstituted phenyl groups,

[0019] Wherein, the substituents in the substituted heteroatom-containing saturated or unsaturated five-membered or six-membered ring group may be one or more, specifically one or two, and the substituents are each independently selected from thiocarbonyl, carbonyl;

[0020] The substituent in the substituted benzene ring may be an amide group (specifically an acetamide group) or may form a saturated or unsaturated five-membered or six-membered ring containing heteroatoms at adjacent positions of the benzene ring, wherein the saturated or unsaturated five-membered or six-membered ring containing heteroatoms may be substituted by a substituent selected from a thiocarbonyl group and a carbonyl group,

[0021] The heteroatom in the saturated or unsaturated five-membered or six-membered ring group containing heteroatoms may be selected from at least one of nitrogen atoms, sulfur atoms and oxygen atoms;

[0022] R 2 One or more selected from H, C1-C6 alkyl, C1-C6 alkoxy, hydroxy, carboxyl, nitro, cyano, halogen, and halogen-substituted C1-C6 alkyl.

[0023] Specifically, the substituted or unsubstituted saturated or unsaturated five-membered or six-membered ring group containing a heteroatom is Any of;

[0024] The substituted or unsubstituted phenyl group is selected from Any one of.

[0025] More specifically, the substituted hydroxybenzimine compound is any one of the following compounds:

[0026]

[0027]

[0028] The hydroxybenzimine compound represented by the above formula I is prepared by a method comprising the following steps: under the catalysis of glacial acetic acid, the compound represented by the formula II and the compound represented by the formula III undergo an aldehyde-amine condensation reaction to obtain the hydroxybenzimine compound represented by the formula I,

[0029]

[0030] In Formula II, R 1 The definition of R is the same as that of Formula Ⅰ 1 Definition of;

[0031] In Formula III, X and R 2 The definitions of X and R are the same as those of X and R in Formula I. 2 Definition of .

[0032] In the above method, the molar ratio of the compound represented by formula II to the compound represented by formula III may be 1:1-2, specifically 1:1.1;

[0033] The aldehyde-amine condensation reaction is carried out in ethanol, the temperature of the aldehyde-amine condensation reaction can be 15-75° C., specifically 55° C., and the time of the aldehyde-amine condensation reaction can be 1-8 hours, specifically 6 hours.

[0034] The use of the hydroxybenzimine compounds represented by the above formula I as chitin deacetylase inhibitors also falls within the protection scope of the present invention.

[0035] The application of the hydroxybenzimine compounds represented by the above formula I in the prevention and treatment of agricultural fungal diseases also falls within the protection scope of the present invention.

[0036] In the application, the agricultural fungal diseases include rapeseed sclerotinia, pepper phytophthora, rice sheath blight, rice blast, wheat fusarium, and tomato gray mold.

[0037] The invention also provides an agricultural fungicide.

[0038] The agricultural fungicide provided by the present invention has an active ingredient of the substituted hydroxybenzylamine compound shown in the above formula I, and its dosage form is a pharmacologically acceptable dosage form, including at least one of emulsifiable concentrate, wettable powder, suspension, dust, soluble powder, aqueous solution, water-dispersible powder, smoke agent, granule and seed coating agent.

[0039] The hydroxybenzimine compounds of the present invention are easy to synthesize and can be used as chitin deacetylase inhibitors in the prevention and treatment of crop diseases, which is of great significance for the screening and development of new fungicides with a completely new mechanism of action. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0041] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0042] Example 1. Preparation of (E)-4-((4-hydroxybenzylidene)amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione

[0043] 0.23g (2mmol) 4-amino-3-mercapto-4H-1,2,4-triazole and 6mL anhydrous ethanol were added to a 50mL single-mouth bottle, and 0.27g (2.2mmol, 1.1eq) of 4-hydroxybenzaldehyde was added under stirring at room temperature, and three drops of glacial acetic acid were added dropwise, and the temperature was raised to 55°C and reacted for 6h. After the reaction was completed, the filter cake was filtered, and the filter cake was washed with a small amount of ethanol and dried to obtain a white solid - 0.32g of the target compound, with a yield of 72.7%.

[0044]

[0045] Other substituted hydroxybenzimine compounds involved in the present invention are synthesized by referring to the method described in Example 1. The properties, molecular weight and yield of the compound represented by Formula I in the present invention are shown in Table 1.

[0046] Table 1 Properties and yields of substituted hydroxybenzimides represented by formula I

[0047]

[0048]

[0049]

[0050]

[0051] Example 2: Inhibitory activity of the compound of formula I against PstCDA (Pst_13661)

[0052] 50μL reaction system: 1μM Pst_13661 enzyme, 5μM N-acetylated chitotriose 5μL, 1μL inhibitor of different concentrations (compound shown in Formula I), and finally fill the volume of 50μL with 50mM pH 7.5 HEPES buffer. Reaction conditions: Incubate the reaction system with shaking for 20 minutes at 30℃ to allow it to react fully. Determination method: After the reaction, add 5μL pH 9.0 sodium borate buffer and 20μL fluorescent amine solution, mix and react for 10min, and measure the fluorescence intensity by microplate reader, with excitation wavelength and emission wavelength of 360nm and 460nm respectively. The fluorescence value measured in the experimental group is recorded as FE, and multiple groups of controls are set at the same time: negative control (without substrate), positive control (without inhibitor) and blank control (without substrate and inhibitor), and the corresponding fluorescence values ​​are recorded as FN, FP and FB respectively. Substituting (FE-FN) into the standard curve, the molar amount of amino groups generated by the reaction in the experimental group, ME, can be obtained. Similarly, substituting (FP-FB) into the standard curve, the molar amount of amino groups generated by the reaction in the positive control group, MP, can be obtained. The formula for calculating the inhibitory activity is as follows:

[0053] Inhibition rate (%) = (MP-ME) / MP

[0054] The inhibitory activity of substituted hydroxybenzimides against Pst13661 is shown in Table 2.

[0055] Table 2 Inhibitory activity of substituted hydroxybenzimides against Pst13661 (0.2mmol / L, 0.1mmol / L)

[0056]

[0057]

[0058] The results showed that when the concentration was 0.2mmol / L, the substituted 4-hydroxybenzimides all showed a certain inhibitory activity against Pst13661, and the inhibition rate was in the range of 11.45% to 89.25%. At the same time, some compounds still showed above-moderate inhibitory activity at 0.1mmol / L, indicating that this type of compound is an inhibitor of Pst13661.

[0059] Example 3: Inhibitory activity of the compound of formula I against plant pathogens

[0060] The compound shown in formula I was tested for its in vitro antibacterial activity by the mycelium growth rate method, and the tested bacteria were rape sclerotia, pepper phytophthora, rice sheath blight, rice blast, wheat gibberellosis, and tomato gray mold. The specific method is as follows: the compound shown in formula I and the control agent prothioconazole were weighed separately, and a mother solution with a concentration of 10000 μg / mL was prepared with dimethyl sulfoxide, and the above mother solutions were respectively drawn with a pipette, and added to a sterilized potato dextrose agar medium (PDA) cooled to about 55°C. After fully mixing, the culture medium mixed with the compound was poured into a culture dish with a diameter of 9 cm, about 15 mL per dish, and the culture medium with an equal amount of dimethyl sulfoxide was used as a blank control. The above treatments were repeated 3 times. After the culture medium in the dish was cooled and solidified, a bacterial cake with a diameter of 5 mm was prepared along the edge of the colony, and was inoculated in the center of the plates with different treatments, and the culture dish was inverted in a 25°C incubator for dark culture. After the colonies in the blank control PDA plate had grown sufficiently, the diameters of the colonies in each treatment were measured using the cross method.

[0061] The mycelial growth inhibition rate was calculated using the following formula:

[0062] Inhibition rate (%) = [(control colony diameter - treated colony diameter) / (control colony diameter - 5 mm)] × 100%

[0063] The in vitro antibacterial activity data (25 mg / L) of some compounds represented by Formula I are shown in Table 3.

[0064] Table 3 In vitro antibacterial activity data of some compounds shown in formula I (25 mg / L)

[0065]

[0066]

[0067] The results showed that at a concentration of 25 mg / L, substituted 4-hydroxybenzimine compounds had a certain inhibitory effect on the mycelial growth of five common plant pathogens. Some compounds in this series showed moderate and above inhibitory activity against rice sheath blight and rapeseed sclerotinia. The above experiments proved that the compounds provided by the present invention can be used as potential fungicidal active molecules. Since this series of compounds has good enzyme inhibitory activity against PstCDA (Pst13661), and the fungal CDA functions in the process of plant-pathogen interaction, it can stimulate plant native immunity and is a virulence factor of pathogens, which mainly affects the pathogenicity of fungi. Therefore, it is speculated that this series of compounds may have better preventive effects at the active level.

[0068] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Substituted hydroxybenzimine compounds shown in formula I: In formula I, X is CH or N; R 1 is selected from substituted or unsubstituted saturated or unsaturated five-membered or six-membered ring groups containing heteroatoms, substituted or unsubstituted phenyl groups, in, The substituents in the substituted heteroatom-containing saturated or unsaturated five-membered or six-membered ring group are one or more, and the substituents are each independently selected from thiocarbonyl, carbonyl; The substituent in the substituted benzene ring is an amide group or forms a saturated or unsaturated five-membered or six-membered ring containing heteroatoms at adjacent positions of the benzene ring, and the saturated or unsaturated five-membered or six-membered ring containing heteroatoms may be substituted by a substituent selected from thiocarbonyl and carbonyl, The heteroatom in the saturated or unsaturated five-membered or six-membered ring group containing a heteroatom is selected from at least one of a nitrogen atom, a sulfur atom and an oxygen atom; R 2 One or more selected from H, C1-C6 alkyl, C1-C6 alkoxy, hydroxy, carboxyl, nitro, cyano, halogen, and halogen-substituted C1-C6 alkyl.

2. The compound according to claim 1, characterized in that The substituted or unsubstituted saturated or unsaturated five-membered or six-membered ring group containing a heteroatom is Any of; The substituted or unsubstituted phenyl group is selected from Any one of.

3. A method for preparing a substituted hydroxybenzimine compound of formula I in claim 1 or 2, comprising the following steps: reacting a compound of formula II with a compound of formula III to produce an aldehyde-amine condensation reaction under the catalysis of glacial acetic acid to obtain a hydroxybenzimine compound of formula I, In Formula II, R 1 The definition of R is the same as that of Formula Ⅰ 1 Definition of; In Formula III, X and R 2 The definitions of X and R are the same as those of X and R in Formula I. 2 Definition of .

4. The method according to claim 3, characterized in that The molar ratio of the compound represented by formula II to the compound represented by formula III is 1:1-2; The aldehyde-amine condensation reaction is carried out in ethanol, the temperature of the aldehyde-amine condensation reaction is 15-75° C., and the time of the aldehyde-amine condensation reaction is 1-8 hours.

5. Use of the substituted hydroxybenzimides represented by formula I in claim 1 or 2 as chitin deacetylase inhibitors.

6. Use of the substituted hydroxybenzimine compounds represented by formula I in claim 1 or 2 in the prevention and treatment of agricultural fungal diseases.

7. The use according to claim 6, characterized in that: In the application, the agricultural fungal diseases include rapeseed sclerotinia, pepper phytophthora, rice sheath blight, rice blast, wheat fusarium, and tomato gray mold.

8. An agricultural fungicide, the active ingredient of which is a substituted hydroxybenzimine compound represented by formula I in claim 1 or 2.

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