Use of a zephycandidine class a compound in the control of agricultural pathogenic bacteria

Abstract

The application belongs to the field of natural medicinal chemistry, and discloses a new use of a Zephycandidine A compound in preventing and treating agricultural diseases caused by Xanthomonas oryzae pv. oryzae ACCC 11602, Xanthomonas axonopodis pv. Citri and Pseudomonas sollamacearum. The Zephycandidine A compound has the characteristics of novel structure, broad spectrum and high efficiency as a new type of agricultural bactericide, and has the value of further research and development into a new type of agricultural bactericide.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry and discloses the use of a Zephycandidine class A compound in the prevention and control of agricultural pathogenic bacteria. Background Technology

[0002] Alkaloids are diverse, and most possess significant biological activity, particularly in antibacterial applications. Zephycandidine A is a natural alkaloid isolated from the Amaryllidaceae plant *Zephyranthes candida*. Currently, research on the efficacy of this type of alkaloid is limited, especially regarding its effectiveness against agricultural pathogens. This invention is the first to discover that Zephycandidine A exhibits good inhibitory effects against agricultural pathogens. Therefore, studying this type of alkaloid and developing fungicides with novel targets to combat plant pathogen infection has significant scientific and practical value.

[0003] The Zephycandidine A and its derivatives described in this invention exhibit certain antibacterial activity against agricultural pathogens such as Xanthomonas oryzae pv. oryzae ACCC 11602 (bacterial blight of rice), Xanthomonas axonopodis pv. Citri (citrus canker), and Pseudomonas sollamacearum (bacterial wilt of plants), and are expected to be further developed into novel agricultural bactericides. Summary of the Invention

[0004] This invention provides a novel use of Zephycandidine A and its derivatives in combating agricultural pathogens, for the prevention and control of agricultural pathogens. The structures of Zephycandidine A and its derivatives in this invention are shown in Chemical Formula 1.

[0005]

[0006] This invention application protects the use of the above-mentioned Zephycandidine A and its derivatives in the preparation of drugs for the prevention or treatment of plant pathogenic bacteria such as rice bacterial blight, citrus canker, and Ralstonia solanacearum. Detailed Implementation

[0007] To better understand the present invention, the following specific embodiments further illustrate the above-described content of the present invention. However, this should not be construed as a limitation of the present invention. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods.

[0008] Example 1: Synthesis of Zephycandidine A and its derivatives

[0009]

[0010] The compound of the present invention is synthesized according to the method shown in the above formula.

[0011] Intermediate 1: Add 4 mmol of o-bromobenzaldehyde with different substitutions to a dry tert-butanol solution (10 mL), add 6 mmol of ethylenediamine under a nitrogen atmosphere, stir the mixture at room temperature for half an hour, and then add 4.8 mmol of iodine.

[0012] Add anhydrous potassium carbonate (12 mmol), then stir at 70 °C for 4 hours. After the reaction is complete, cool the reaction mixture with sodium thiosulfate and extract with ethyl acetate. Dry with anhydrous Na₂SO₄ and concentrate under vacuum. Purify by silica gel column chromatography to give yellow intermediate 1 (methanol / dichloromethane).

[0013] Intermediate 2: Under a nitrogen atmosphere, intermediate 1 (3 mmol), iodophenyl diacetic acid (4.5 mmol), and potassium carbonate (4.5 mol) were added to a two-necked round-bottom flask, followed by the addition of DMSO (10 mL). After stirring the solution at room temperature for 4 hours, it was quenched with water (20 mL) and extracted with ethyl acetate. The combined organic phases (Na₂SO₄) were dried. The mixture was then filtered and concentrated under reduced pressure.

[0014] Column chromatography purification yielded white intermediate 2 (ethyl acetate / petroleum ether).

[0015] Target compound: Intermediate 2 (1.5 mmol), 2-bromophenylboronic acid (2.3 mmol), palladium acetate (0.15 mmol), and NaOAc (4.5 mmol) were added to a round-bottom flask. Then, DMF (5.0 mL) was added to the flask under a nitrogen atmosphere. The mixture was stirred at 110 °C for 36 h, the reaction mixture was cooled, quenched with water (10 mL), and then treated with ethyl acetate (3 × 10 mL).

[0016] Extraction. The organic phase (Na₂SO₄) was dried, filtered, and concentrated under reduced pressure to obtain a pale yellow oil, which was purified by column chromatography (ethyl acetate / petroleum ether) to give the pale yellow final target compound. (For the synthetic method, see the literature: Eur. J. Org. Chem.)

[0017] 2022, e202101511)

[0018] Example 2: Determination of the anti-agricultural pathogenic bacteria activity of Zephycandidine A and its derivatives

[0019] The bacterial strains used in this experiment were cryopreserved in the laboratory at -80℃ with 30% glycerol. The cryopreserved strains were removed and streaked onto NB solid medium for agricultural bacteria (beef extract: 3g, peptone: 5g, yeast extract: 1g, sucrose: 10g, agar: 15g, distilled water: 1L, pH 7.0; sterilized at 121℃ for 20min), and incubated at 28℃ until single colonies appeared. Single colonies from the solid medium were then transferred to NB liquid medium for agricultural bacteria (beef extract: 3g, peptone: 5g, yeast extract: 1g, sucrose: 10g, distilled water: 1L; sterilized at 121℃ for 20min) and incubated at 28℃ with shaking at 180 rpm until the logarithmic growth phase. The strains in the logarithmic growth phase were diluted with the appropriate liquid medium to approximately 10... 6 CFU / mL is prepared for use. Dissolve the compounds separately in DMSO, add them to the liquid culture medium, mix thoroughly, and prepare a drug-containing liquid culture medium with a concentration of 200 μg / mL. Take 50 μL of the drug-containing culture medium and the same volume of approximately 10... 6 CFU / mL bacterial culture was added to the wells of a 96-well plate, resulting in a final drug concentration of 100 μg / mL. A control of 100 μL of bacterial culture containing an equal amount of DMSO was used. The 96-well plates were incubated at 28°C for 24–48 h until bacterial growth was observed in the control group. The OD value (OD) of the bacterial culture in each well was then measured using a microplate reader. 600 In addition, the OD values ​​of 100 μL of liquid culture medium and a 100 μg / mL drug concentration were measured to correct for the OD values ​​caused by the culture medium and the drug itself. The formulas for calculating the corrected OD value and inhibition rate are as follows:

[0020] Corrected OD value = OD value of bacterial culture medium - OD value of sterile culture medium;

[0021] Inhibition rate = (OD value of bacterial suspension in the control medium after correction - OD value of bacterial suspension in the drug-containing medium after correction) / OD value of bacterial suspension in the control medium after correction × 100%

[0022] Different Zephycandidine A and its derivatives were diluted in 96-well plates using a two-fold dilution method to obtain a series of concentrations of 50 μL of drug-containing medium. Then, the inhibition rate corresponding to the series of concentrations was determined according to the same experimental method described above.

[0023] All experiments were set up with three replicates.

[0024] Zephycandidine A and its derivatives as described in this patent, along with the positive control thiabendazole copper (purity 20%, purchased from Zhejiang Longwan Chemical Co., Ltd.), were selected for activity testing against common agricultural pathogenic bacteria such as rice bacterial blight, citrus canker, and Ralstonia solanacearum. The initial activity determination concentration of Zephycandidine A and its derivatives as described in this patent was 100 μg / mL, and the initial activity determination concentration of the positive control was 200 μg / mL. The activity test data are shown in Table 1.

[0025] Table 1. Activity of Zephycandidine A and its derivatives against agricultural pathogens

[0026]

[0027]

[0028] Note: "-" indicates that the antibacterial activity of the compound was not determined.

[0029] As shown in Table 1, the Zephycandidine A derivatives involved in this invention exhibited good inhibitory effects on all tested strains, with derivative Z-1 showing even better antibacterial performance. The MICs against *Bacillus oryzae* (rice bacterial blight) and *Bacillus canker* (citrus scab) were [not specified]. 90 Superior to the control drug thiabendazole copper. The derivative Z-2 showed a higher MIC against *Ralstonia solanacearum*. 90 It is superior to the positive control drug.

[0030] In summary, the Zephycandidine A and its derivatives described in this invention exhibit broad-spectrum activity and high activity against agricultural pathogenic bacteria, and are worthy of further research and development.

Claims

1. The use of a Zephycandidine A-type compound in the control of agricultural pathogenic bacteria, wherein the structural formula of the Zephycandidine A-type compound is as follows: ， Its features are, The compounds Zephycandidine A and Z-1 inhibit rice bacterial blight, Ralstonia solanacearum, and citrus canker pathogen; wherein compound Z-2 inhibits rice bacterial blight and Ralstonia solanacearum.

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