CN117281120A - Application of tetrahydrocarbazole derivative in preventing and controlling agricultural bacterial diseases - Google Patents

Abstract

The invention discloses application of any compound of tetrahydrocarbazole derivatives KZa-1-KZb-3 in preventing and treating agricultural bacterial diseases. The biological activity test shows that the derivative has excellent inhibitory activity on bacterial blight pathogenic bacteria, citrus canker pathogenic bacteria, bacterial wilt pathogenic bacteria, soft rot pathogenic bacteria and other bacteria, and part of the compounds have good inhibitory effect on bacterial blight pathogenic bacteria and citrus canker pathogenic bacteria. The compound disclosed by the invention is low in preparation difficulty, low in cost and easy to obtain raw materials, and is expected to be developed into a novel agricultural antibacterial agent.

Description

Use of a tetrahydrocarbazole derivative in preventing and treating agricultural bacterial diseases

Technical field

The invention belongs to the field of medicinal chemistry and discloses a new use of tetrahydrocarbazole derivatives, specifically involving the use of compounds KZa-1 to KZb-3 in the prevention and treatment of rice bacterial blight pathogen Xanthomonas oryzae ACCC 11602 and citrus canker pathogen Xanthomonas It is used in agricultural diseases caused by axonopodispv.Citri, bacterial wilt pathogen Pseudo-monassollamacearum and soft rot pathogen Erwinia aroideae.

Background technique

Plant health is fundamental to agricultural production, and pathogenic microorganisms currently affect biotic stress on plants, ultimately leading to annual crop yield reductions around the world. Phytopathogenic bacteria are among the most harmful plant pathogenic microorganisms. Although the use of fungicides can inhibit the spread of pathogens in plants and increase crop yields, they can contaminate the environment and cause some health problems in humans and animals. Therefore, it is necessary to provide a new pesticide with high efficiency, low toxicity and low residue for controlling plant diseases, which has become one of the primary goals of pesticide creation.

Natural products exist widely in nature with a wide variety and complex structures. They have a variety of pharmacological and biological activities. In recent years, alkaloids have gradually attracted the attention of various researchers. As natural products, they have low toxicity, selectivity and specificity. They also have good environmental compatibility and can meet the requirements of new pesticides. In 1964, Chakraborty et al. first extracted a class of natural alkaloids with carbazole as the core from Mwraya Spreng. Carbazole is a fusion of a central pyrrole ring and two benzene rings, and has a special tricyclic aromatic group. The basic skeleton structure, in which Murrayanine has antibacterial activity, indicates that carbazole compounds are worthy of further exploration. At the same time, we also noticed that tetrahydrocarbazole (THC) is a special structural scaffold containing natural indole as the central part. This novel framework is characterized by a large number of naturally occurring pharmacological compounds and alkaloids, and exhibits significant antibacterial, antitumor, anti-inflammatory and other activities. It has long been favored by scientists due to its wide range of biological properties.

Therefore, we modified the structure of tetrahydrocarbazole alkaloids to obtain a series of tetrahydrocarbazole alkaloid derivatives, and tested their anti-phytopathogenic activity. Test results show that this type of compound has potential inhibitory effects on agricultural bacteria such as rice bacterial blight pathogen, citrus canker pathogen, bacterial wilt pathogen, and soft rot pathogen. Among them, it has better inhibitory effects on rice bacterial blight and citrus canker pathogens. The inhibitory effect is significantly better than that of the control drugs phylloxazole and copper thiazide, and it is expected to be developed into a new type of drug against plant pathogenic bacteria.

Contents of the invention

The present invention improves the antibacterial activity and broad spectrum of this type of compound by modifying and derivatizing the tetrahydrocarbazole structure. The present invention uses 1,2-cyclohexanedione and phenylhydrazine as raw materials to prepare tetrahydrocarbazolone derivatives, and further constructs tetrahydrocarbazolamine derivatives with amine compounds, and evaluates their antibacterial activity.

In order to achieve the above objectives, the invention adopts the following technical methods: the compound structure is shown in Chemical Formula 1. R ₁ in Chemical Formula 1 is 6-fluoro-substituted, 6-chloro-substituted, 6-bromo-substituted, 6-trifluoromethoxy, 6-trifluoromethyl, 6-methyl, 6-methoxy, 6- Cyano group, R ₂ is benzylamine, 4-chlorobenzylamine, 4-fluorobenzylamine, phenylethylamine, 4-fluorophenylethylamine, 4-chlorophenylethylamine, 4-methylphenylethylamine, 3,4 -Dimethoxyphenylethylamine, 3,4-methylenedioxyphenylethylamine, tryptamine, N-(2-aminoethyl)morpholine, 3-fluorophenylethylamine, 3-chlorophenylethylamine , 2-fluorophenylethylamine, 2-chlorophenylethylamine, 2,4-dichlorophenylethylamine, amphetamine, ethylamine, propylamine, butylamine.

The preparation method of tetrahydrocarbazole derivatives KZa-1 to KZb-3 according to the present invention is as shown in Chemical Formula 2:

The synthesis method of the tetrahydrocarbazole derivatives of the present invention is shown in the examples. The pure product is obtained through multiple separations by conventional methods such as silica gel column chromatography. Through mass spectrometry, nuclear magnetic resonance and other spectral techniques, the compounds KZa-1 to KZb- are determined. 3, its structural formula is shown in Chemical Formula 1. Activity screening results show that the compound of the present invention exhibits potential inhibitory effects on plant bacteria such as rice bacterial blight pathogen, citrus canker pathogen, bacterial wilt pathogen, and soft rot pathogen.

Detailed ways

In order to better understand the present invention, the above contents of the present invention will be further described in detail below through specific embodiments. However, this should not be construed as a limitation of the invention. The experimental methods described in the following examples are all conventional methods unless otherwise specified.

Example 1: Synthesis method of target compounds KZa-1～KZb-3

The synthesis method of the compound of the present invention is carried out according to the following reaction formula:

Synthesis of Intermediate I: To a solution of 1,2-cyclohexanedione (2243mg, 20mmol) and concentrated hydrochloric acid (13mL) in acetic acid (40mL), slowly add different phenylhydrazine hydrochloride (1586mg, 10mmol) in methanol. (25mL) solution. After addition, the mixture was heated to 60°C and stirred overnight. The solvent was evaporated and the pH was adjusted to slightly basic with saturated _NaHCO3 . The mixture was extracted with AcOEt (3*20 mL). The combined organic extracts were washed with brine, dried over _{anhydrous Na2SO4} , and concentrated. Purified by silica gel column chromatography (petroleum ether/AcOEt, 12/1 v/v), intermediate I (1235 mg, 62%) was obtained as brown powder.

Synthesis of target compounds KZa-1~KZb-3: Dissolve intermediate I (102 mg, 0.5 mmol), differently substituted amines (0.12 mL, 0.8 mmol) and catalytic p-TsOH in toluene (10 mL), using Dean -Stark manifold is refluxed at 140°C for 16 hours. The solvent was evaporated and the residue was dissolved in methanol and _NaBH4 (177 mg) was added at 0°C. The solution was heated to 80°C until TLC indicated completion of the reaction. Quench the reaction with water. Extract twice with ethyl acetate, combine the organic layers, dry over anhydrous Na ₂ SO ₄ and concentrate. Purified by silica gel (petroleum ether/AcOEt, 4/1v/v) column chromatography, compounds KZa-1 to KZa-16 (123 mg, yield: 72%) were obtained. (For the synthesis method, please refer to the literature: European Journal of Medicinal Chemistry 162(2019)203-211)

Structural characterization parameters of target compounds KZa-1～KZb-3: see Table 1

Table 1 Structural characterization parameters of target compounds KZa-1～KZb-3

Example 2: Determination of the activity of tetrahydrocarbazole derivatives against agricultural pathogenic bacteria and its results

1) Test reagents: tetrahydrocarbazole derivatives KZa-1~KZb-3.

2) Test strains: Rice bacterial blight pathogen Xanthomonas oryzae ACCC 11602, citrus canker pathogen Xanthomonas axonopodis pv.Citri, bacterial wilt pathogen Pseudo-monas sollamacearum, and soft rot pathogen Erwinia aroideae were provided by the Gansu Academy of Agricultural Sciences.

3) Antibacterial activity test:

The strains used in this experiment were strains frozen in the laboratory at -80°C with 30% glycerol. Take out the frozen strains and place them on NB solid medium (beef extract: 3g, peptone: 5g, yeast powder: 1g, sucrose: 10g, agar: 15g, distilled water: 1L, pH7.0; sterilized at 121°C for 20min) Streak and incubate at 28°C until a single colony grows. Pick single colonies from the solid culture medium and add them to NB liquid culture medium (beef extract: 3g, peptone: 5g, yeast powder: 1g, sucrose: 10g, distilled water: 1L; sterilized at 121°C for 20 minutes), and place on a constant temperature shaker with Culture at 28°C, shaking at 180rpm to the logarithmic growth phase. The strain in the logarithmic growth phase was diluted to about 10 ⁶ CFU/mL with NB liquid medium for later use. Dissolve the compounds in DMSO respectively, add them to the liquid culture medium, mix evenly, and prepare a drug-containing liquid culture medium with a concentration of 200 μg/mL. Take 50 μL of drug-containing culture medium and the same volume of bacterial culture containing approximately 10 ⁶ CFU/mL and add it to the wells of a 96-well plate. The final dosage concentration is 100 μg/mL. 100 μL bacterial solution of the same concentration containing an equal amount of DMSO was used as a control. The 96-well plate was cultured in a constant temperature incubator at 28°C for 24-48 hours until the bacterial liquid in the control group grew out, and the OD value (OD ₆₀₀ ) of the bacterial liquid in the wells was measured on a microplate reader. In addition, the OD value of 100 μL liquid culture medium and the drug with a concentration of 100 μg/mL was measured, and the OD value caused by the culture medium and the drug itself was corrected. The calculation formulas for corrected OD value and inhibition rate are as follows:

Corrected OD value = OD value of bacteria-containing culture medium - OD value of sterile culture;

Inhibition rate = (corrected control culture medium bacterial liquid OD value - corrected drug-containing culture medium OD value) / corrected control culture medium bacterial liquid OD value × 100%

The drug-containing liquid culture medium of the compound was diluted in a 96-well plate by a two-fold dilution method to obtain 50 μL of drug-containing culture medium with serial concentrations, and then the inhibition rates corresponding to the serial concentrations were measured according to the same test method as above. All experiments were repeated in triplicate, and the MIC ₉₀ values of the compounds (the lowest drug concentration that inhibited 90% of bacterial growth) were determined and shown in Table 2.

Table 2 In vitro MIC ₉₀ (μg/mL) of tetrahydrocarbazole derivatives KZa-1～KZb-3 against plant pathogenic bacteria

From the activity test results in Table 2, it can be seen that the tetrahydrocarbazole derivatives KZa-1 to KZb-3 prepared in the present invention show varying degrees of inhibitory activity against four kinds of plant pathogenic bacteria, and are effective against rice bacterial blight and citrus canker. The inhibitory activity is significantly better than that of other pathogens. Among them, the MIC ₉₀ value of KZa-6 against rice bacterial blight and citrus canker can reach 1.56 μg/mL, which is significantly better than the inhibitory activity of commercial agents phylloxazole and thiobacterium. Therefore, this type of compound has the value of further development and is expected to be developed into a new agricultural antibacterial agent.

Claims (3)

1. The present invention relates to the use of tetrahydrocarbazole derivatives KZa-1 to KZb-3 in drugs against agricultural pathogenic bacteria. 2. Tetrahydrocarbazole derivatives KZa-1～KZb-3 according to claim 1 have the following molecular structural characteristics: R ₁ in Chemical Formula 1 is 6-fluoro-substituted, 6-chloro-substituted, 6-bromo-substituted, 6-trifluoromethoxy, 6-trifluoromethyl, 6-methyl, 6-methoxy, 6- Cyano group, R ₂ is benzylamine, 4-chlorobenzylamine, 4-fluorobenzylamine, phenylethylamine, 4-fluorophenylethylamine, 4-chlorophenylethylamine, 4-methylphenylethylamine, 3,4 -Dimethoxyphenylethylamine, 3,4-methylenedioxyphenylethylamine, tryptamine, N-(2-aminoethyl)morpholine, 3-fluorophenylethylamine, 3-chlorophenylethylamine , 2-fluorophenylethylamine, 2-chlorophenylethylamine, 2,4-dichlorophenylethylamine, amphetamine, ethylamine, propylamine, butylamine. 3. Any compound of the tetrahydrocarbazole derivatives KZa-1 to KZb-3 according to claim 2 is used in the preparation of medicines for preventing and treating plant bacteria such as rice bacterial blight, citrus canker, bacterial wilt, and soft rot fungi. uses in.