CN113907079A

CN113907079A - Application of chloralkanoyl fluoroquinolone in preparing medicine for preventing and treating citrus canker

Info

Publication number: CN113907079A
Application number: CN202111325076.8A
Authority: CN
Inventors: 杨大成; 钱坤; 胡军华; 范莉; 李洋; 谢文文; 李永清; 陈娜; 赵晨晨; 吴玉珠; 杨旎; 潘建芳; 罗鹏; 任艳会; 李文粒
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2021-11-10
Filing date: 2021-11-10
Publication date: 2022-01-11

Abstract

Citrus canker is the second most intractable disease of citrus worldwide, causing great damage to citrus production. The invention belongs to the technical field of plant disease control, and particularly relates to application of chloralkanoyl fluoroquinolone in preparation of a drug for controlling citrus canker. The medicine comprises a compound shown as a formula (I),

in the formula, R₁Is composed of

Or

R₂is-H, -Cl or-OCH₃，R₃Is composed of

Or

Description

Application of chloralkanoyl fluoroquinolone in preparing medicine for preventing and treating citrus canker

Technical Field

The invention belongs to the technical field of disease control, and particularly relates to application of chloralkanoyl fluoroquinolone in preparation of a drug for controlling citrus canker.

Background

Citrus, as the first fruit in the world, has yield and economic value that determine its dominance throughout the fruit tree industry. In recent years, the citrus industry in China has developed rapidly, with growing areas and annual yields exceeding those in the United states and Brazil, becoming the first major citrus producing country in the world; meanwhile, the citrus industry has gradually become an important post industry for promoting rural economic development and guaranteeing farmer income. However, citrus fruits are often susceptible to infestation by pests, particularly citrus canker, during growth, storage, transport, and the like.

The citrus canker is an important bacterial disease caused by Xanthomonas carpi orange pathogenic varieties (Xac) and damaging the citrus planting industry all over the world, and mainly infects aerial parts of leaves, tree tips, fruits, skin thorns, trunks and the like of Rutaceae citrus, Hovenia and kumquata plants, so that canker surrounded by withered yellow rings and necrosis of the surfaces of the fruits or the leaves, water-immersed lesions on the leaves, wooden embolism of the leaves and the like can be caused; in addition to leaf symptoms, it can also lead to fruit abscission, fruit tree aging, and loss of commodity value of infected fruits. At present, citrus canker occurs in each main citrus producing area in China, which causes serious loss of citrus industry.

The cost for preventing and eliminating citrus canker is very high, the elimination effect is not obvious, the prevention is mainly performed at home and abroad at present, and the spread of the canker is prevented and reduced by seedling culture, copper preparation and agricultural antibiotics spraying in a non-citrus canker area. However, the copper preparation has disadvantages of easily generating drug resistance, causing heavy metal tolerance by horizontal transfer between germs, accumulating copper ions in soil as potential phytotoxin and affecting the environment, and is also a hazard to fruits, especially in dry and hot environments. Recently, the use of streptomycin for agricultural use has been prohibited, and the control agents effective against ulcer germs are seriously lacking, and new agents are urgently required for replacement.

Disclosure of Invention

In view of the above, the first objective of the present invention is to provide an application of chloralkanoyl fluoroquinolone in preparing a drug for preventing and treating citrus canker.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the application of the compound shown in the formula (I) in preparing the medicine for preventing and treating citrus canker disease:

in the formula, R₁Is composed of

R₂is-H, -Cl or-OCH₃，R₃Is composed of

The second object of the present invention is also to provide a medicament for controlling citrus canker, which comprises a compound represented by the formula (I):

in the formula, R₁Is composed of

R₂is-H, -Cl or-OCH₃，R₃Is composed of

The drug is water dispersible granules and comprises 10 wt% of the compound shown in the formula (I), 2 wt% -6 wt% of a wetting agent, 7% -15% of a dispersing agent, 4 wt% -11 wt% of a disintegrating agent and the balance of filler.

Or, the medicament is wettable powder and comprises 10 wt% of the compound shown in the formula (I), 2 wt% -6 wt% of a wetting agent, 7% -15% of a dispersing agent and the balance of a filling material.

Wherein the wetting agent is sodium dodecyl sulfate or sodium dodecyl benzene sulfonate; and/or the dispersant is a polycondensate salt dispersant; and/or, the filler comprises kaolin and diatomaceous earth.

Preferably, the mass ratio of kaolin to diatomite is 1-1.5: 1.

Preferably, the disintegrant is sodium chloride.

The invention has the beneficial effects that:

the medicine disclosed by the invention is excellent in indoor test activity, and field evaluation also shows that the medicine has a good control effect on citrus canker.

The drug of the present invention is less likely to cause problems of tolerance.

The medicine of the invention has no potential safety hazard.

Drawings

FIG. 1 shows the NMR spectrum of Compound 1¹HNMR；

FIG. 2 is a high performance liquid chromatogram of Compound 1;

FIG. 3 is a nuclear magnetic resonance spectrum of Compound 2¹HNMR；

FIG. 4 is a nuclear magnetic resonance spectrum of Compound 3¹HNMR。

Detailed Description

The examples are provided for better illustration of the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention. In the present invention, "%" is a mass percentage, and the ratio is a mass ratio, unless otherwise specified.

In the invention, nuclear magnetic resonance hydrogen spectrum and mass spectrum are entrusted to Chongqing university for detection;

in the invention, the high performance liquid chromatography is used for detecting food and drug inspection in Chongqing.

Example (b):

this example provides the synthesis methods of the three compounds and the activity detection data thereof, and also provides the drugs prepared from the three compounds and the activity detection data of the drugs. In addition, field tests are also performed on the drugs prepared from the three compounds, and specific field test data are provided for the drug prepared from one of the compounds in the present embodiment, and the specific contents are as follows:

first, compound synthesis

1.1 Synthesis of Compound No. 223 molecule, Compound 1 (3C-CLX for short)

Adding 1mmol of Clinafloxacin (CLX) hydrochloride and 2mL of Dichloromethane (DCM) into a 100mL round-bottom flask, cooling by an ice salt bath, magnetically stirring, and pouring NaHCO₃4mmol, stirring for 20min, adding 3-chloropropionyl chloride 2.5mmol DCM solution (5mL, dropping speed about 1d/4s) dropwise through a constant pressure dropping funnel, continuously stirring in ice bath for reaction after dropping, and tracking and monitoring by Thin Layer Chromatography (TLC) until the reaction is finished. After stirring was stopped, 15mL of ice-cooled saturated brine and 20mL of DCM were added, and H2-3 was adjusted with 2mol/L HCl solution under stirring. Transferred to a separatory funnel, separated, extracted with DCM (20 mL. times.2), the organic phases were combined, washed with 0.5mol/L HCl solution (10 mL. times.3), saturated brine (20 mL. times.2), and water (10 mL. times.1) in that order, and dried over anhydrous Na₂SO₄Drying and rotary steaming. Dissolving the crude product with 5mL of Ethyl Acetate (EA), adding 10mL of Petroleum Ether (PE), separating out a large amount of white solid, performing suction filtration, and dispersing the solid by using PE + EA (the volume ratio is 1.1-1.8:1) to obtain 0.40g of a pure product with the yield of 81%.

TABLE 1 Synthesis of Compound 1 Experimental results

The results of the experiments for synthesizing this molecule after process optimization are shown in table 2.

TABLE 2 Synthesis of Compound 1 and results thereof

A total of about 230 grams of sample was obtained.

The prepared sample was subjected to nuclear magnetic hydrogen spectrometry, high performance liquid chromatography and high resolution mass spectrometry, and the results are shown in fig. 1, fig. 2 and table 3.

TABLE 3 high resolution Mass Spectrometry data for Compound 1

1.2 Synthesis of Compound No. 221, Compound 2 (4C-SAR for short)

To a 100mL round bottom flask was added 2mmol Sarafloxacin (SAR) hydrochloride and 2mL DCM, cooled in an ice salt bath, magnetically stirred, and added 8mmol NaHCO₃After 20min, 5.0mmol of 4-chlorobutyryl chloride in DCM (the dropping speed is about 1d/4s) is dropped into the constant pressure dropping funnel, the reaction is continuously stirred in ice bath after the dropping is finished, and TLC tracking monitoring is carried out until the reaction is finished. Stirring was stopped, 15mL of ice-cold saturated brine and 20mL of DCM were added, the pH was adjusted to 2-3 with 2mol/L HCl solution under stirring, emulsified solid was present, the mixture was left to stand, filtered, the cake was washed with DCM 3 times, the filtrate was transferred to a separatory funnel, separated, the aqueous phase was extracted with DCM (20mL × 2), the organic phases were combined, washed with saturated brine (20mL × 2) and a small amount of water (10mL × 1), the organic phase was collected, and anhydrous Na was added₂SO₄Drying, rotary steaming, dispersing the crude product with PE/EA solution to obtain white solid, vacuum filtering to obtain white solid, dispersing with PE-EA for 2 times, and vacuum drying to obtain 0.78g pure white solid with yield of 85%. The results of the synthesis experiments are shown in Table 4.

TABLE 4 Synthesis of Compound 2 Experimental results

The nuclear magnetic hydrogen spectrum detection was performed on the obtained sample, and the result is shown in fig. 3.

1.3 Synthesis of Compound 3 (2C-GAT), molecule No. 204

Add Gatifloxacin (GAT), DCM to a 100mL round bottom flask, stir magnetically, add NaHCO₃Transferring the powder into a low-temperature reactor at the temperature of-10 ℃. After 20mi, chloroacetyl chloride solution in DCM is added dropwise through a constant pressure dropping funnel, and the dropping speed is controlled to be about 1d/4-7 s. After the dropwise addition, the reaction was stirred at-10 ℃ and monitored by TLC until the reaction was complete. If gatifloxacin does not react completely, the chloroacetyl chloride solution in DCM is added. After the reaction, stirring was stopped, ice-cold saturated brine and DCM were added, pH was adjusted to 4-5 with 2mol/L HCl solution under stirring, the mixture was allowed to stand, liquid was separated in a separatory funnel, washed with saturated brine and a little water, the organic phase was collected, and anhydrous Na was added₂SO₄Drying, rotary steaming to obtain yellow oily liquid, adding DCM and petroleum ether, pulping, refrigerating, rotary drying to obtain light yellow solid, dispersing with ethyl acetate and petroleum ether to obtain white solid, vacuum filtering to obtain white solid, and vacuum drying to obtain pure product. The results of the synthesis experiments are shown in Table 5.

TABLE 5 Synthesis of Compound 3 Experimental results

The results of nuclear magnetic hydrogen spectrum detection of the sample prepared in this example are shown in fig. 4.

TABLE 6 first batch sample information

TABLE 7 second batch of sample information

Note: pure product B in tables 6 and 7 means that the purity of the compound was 98.5% or more.

II, detection of Activity of Compound

The activity of compounds 1 to 3 against Flavobacterium carpropium pathogenic citrus variety (Xanthomonas axonopodis pv. citri, Xac) was examined and the results are shown in tables 8 and 9.

The activity test method for inhibiting citrus canker pathogenic bacteria by the compound comprises the following steps:

materials and test strains:

citrus canker bacterial strain RL (Xanthomonas campestris pv. citri, RL strain, stored in the integrated control project group of citrus institute, university in south west).

Preparation of a culture medium: LB liquid culture medium was selected for the experiment.

Preparation of bacterial suspension: activating a citrus canker bacteria RL strain on a PDA culture medium, culturing for 3 days at 28 ℃, lightly scraping a small amount of bacterial colonies in sterile water on a superclean bench by using a blue gun head pipe orifice, and performing shake culture for 2 hours at 28 ℃ and 200r on a shaking table to prepare bacterial suspension.

Control agents used agricultural benziothiazolinone (SMT), quinolinone (KLT) or streptomycin (LMS). Preparation of preparation and positive control benziothiazolinone mother liquor: accurately weighing the compound, and dissolving the compound in sterile water or an organic solvent to prepare a mother solution of 1mg/mL for later use.

The test method comprises the following steps: the experiment was performed on a 96-well plate, each crude drug was set to 6 dilution concentrations, see table 8, diluted according to the standard gradient dilution method; mixing completely, and culturing in 28 deg.C constant temperature incubator for 48 hr; determination of OD Using Spectrophotometer₆₀₀The light absorption value is calculated according to the following formula:

bacteria inhibition rate%₆₀₀- (Compound OD)₆₀₀The compound CK1 OD₆₀₀)/CKOD₆₀₀*100

Data analysis LC50 and 95% confidence limits were calculated using a regression equation for determining virulence.

TABLE 8 inhibitory Activity of Compounds on Sclerotinia citrea (%)

TABLE 9 rescreening data for inhibition of citrus canker pathogen by compounds

Preparation of medicine

The embodiment provides preparation methods of two dosage forms of the medicine, namely wettable powder and water dispersible granules respectively, which specifically comprise the following steps:

3.1 preparation of Water dispersible granules

0.5 g of each raw drug sample is taken, and water dispersible granules are prepared according to the formula shown in table 10.

TABLE 10 formulation formula

WG is short for water dispersible granule. The preparation method of the water dispersible granule comprises the following steps: uniformly mixing the auxiliary agent and the original medicine according to the formula, crushing the mixture in a universal crusher for 30s, taking out the crushed mixture, and sufficiently and uniformly mixing the crushed mixture again; adding a proper amount of water (the water amount is the best water amount which can just extrude particles) into the crushed and uniformly mixed sample, and then extruding and granulating the sample in an extrusion granulator; and drying, finishing and screening the sample obtained by granulation to prepare the water dispersible granule.

3.2 preparation of wettable powder

0.5 g of each crude drug sample was used to prepare wettable powders according to the formulation shown in Table 11.

TABLE 11 formulation

Wherein WP is short for wettable powder. The preparation method of the wettable powder comprises the steps of weighing the original medicine, the wetting agent, the dispersing agent and the filler according to the formula, premixing and coarsely crushing in a universal crusher, then finely crushing according to the crushing flow of a jet mill, and screening to obtain the wettable powder.

Fourth, the activity detection of the medicine

The activity of the formulations described in tables 10 and 11 on Xanthomonas carpi citrobacter var citri (Xanthomonas axonopodis pv. citri, xaca) was examined and the results are shown in tables 12-15;

the method for detecting the activity of the preparation on citrus canker pathogenic bacteria comprises the following steps:

4.1 materials and test strains

Citrus canker bacteria RL strain (Xanthomonas campestris pv. citri, RL strain, stored in the comprehensive control subject group of citrus institute, university in south west);

4.2 test methods

Experiments were performed on 96-well plates, with 6 doses per drug (2000, 4000, 8000, 16000, 32000, and 64000 dilutions, i.e., 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0.015625mg/kg concentrations, respectively), repeated 5 times for each concentration, with different concentrations corresponding to control CK1 without added suspension (thereby excluding the effect of the color of the compound itself), and with no drug added only (CK). Sequentially diluting the sample solution by adopting a standard multiple dilution method, and finally preparing a mixed bacterial solution with the total volume of 200 mu L so that the final concentrations of the samples are respectively A (0.5 mu g/mL), B (0.25 mu g/mL), C (0.125 mu g/mL), D (0.0625 mu g/mL), E (0.03125 mu g/mL) and F (0.015625 mu g/mL); replacing the volume of the bacterial suspension in CK1 with 20 μ L LB culture medium, and supplementing the volume of the compound in CK with sterilized water; mixing completely, and culturing in 28 deg.C constant temperature incubator for 48 hr;

determination of OD Using Spectrophotometer₆₀₀The light absorption value of the time and the bacteriostasis rate are calculated according to the following formula;

TABLE 12 summary table of the average bacterial growth of wettable powder with different concentrations (2019-02-19 data)

TABLE 13 regression equation of toxicity of wettable powder (2019-02-19 data)

TABLE 14 data on the inhibition of citrus canker by Water dispersible granules (2021-03-17 data)

TABLE 15 toxicity equation of Water dispersible granules (2021-03-17 data)

Fifthly, field test prevention effect evaluation of wettable powder prepared from compound 1

Detecting the field control effect of the wettable powder, which comprises the following steps:

5.1 test conditions

5.1.1 selection of test subjects, crops and varieties

Citrus canker Xanthomonas campestris

Citrus (blood orange of Taruo)

5.1.2 crop cultivation and environmental conditions

The orchard is a sloping field. The management level of the fertilizer and the water is basically consistent, and the illumination condition is good. The Citrus aurantium rootstock is 6 years old, the row spacing of the plants is 3 multiplied by 4m, 55 plants are planted per mu, the average plant height is 1.8m, and the crown width is 1.2 m. In recent years, citrus canker is more severe and sporadically occurs at the beginning of the test. Neither fertilizer nor irrigation was applied during the test period.

5.2 test design and arrangement

5.2.1 Agents

Test agent

The preparation method of the 10% chloropropyl clinafloxacin wettable powder comprises the following specific steps;

according to the mixture ratio of 34.5 percent of diatomite, 34.5 percent of kaolin, 6 percent of wetting agent SDS, 9 percent of polycondensate salt, 6 percent of NaCl and 10 percent of original drug, 255.3g of diatomite, 255.3g of kaolin, 44.4g of wetting agent SDS, 66.6g of polycondensate salt and 44.4g of NaCl are weighed, added into a machine, stirred and ground uniformly, and then compound 1, namely 3C-CLX 74g is added; then the mixture is finely crushed according to the crushing flow of a jet mill, and about 695g of wettable powder is obtained.

Control agent

Fine brake (3% benziothiazolinone wettable powder, produced by Dahuate in Shaanxi);

3000 (46% copper hydroxide water dispersible granule, manufactured by DuPont corporation, USA) can be obtained;

5.2.2 dosage and treatment number

TABLE 16 test design for test agents

5.2.3 cell arrangements

Random block permutation.

Cell area and repetition

Cell area (or number of plants): each cell is 8 trees, the repetition is carried out for 4 times, and a protection tree is left among the cells.

5.3 methods of application

5.3.1 methods of use

And (4) spraying. Firstly, adding half of the required water consumption into the electric sprayer; the medicament is directly measured by a measuring cup and directly poured into a sprayer; adding water to the required water consumption, shaking up, and spraying the citrus trees uniformly.

5.3.2 applicator devices

The test was carried out using a manual sprayer HD-400. The sprayer can ensure accurate dosage and uniform distribution.

5.3.3 application time and frequency

The first test is carried out in spring shoot period of 13 days in 4 months and when the shoot length is 5-10cm, the leaves of the new shoots are sporadically attacked. Spraying the pesticide for the second time in the young fruit period of 5 months and 8 days.

5.3.4 Capacity of use

Diluting 10% chloropropyl clinafloxacin wettable powder by 400 times, 800 times and 1600 times; diluting 3% benziothiazolinone wettable powder (control) 1000 times; the 46% copper hydroxide water dispersible granule is diluted by 1500 times. The amount of the sprayed liquid was 1L per tree, as shown in Table 16.

5.4 investigation, recording and measurement methods

5.4.1 Meteorological and soil data

Weather data

TABLE 17 weather conditions

TABLE 18 weather conditions table for the day of drug administration (2021)

Soil data

The soil is red and yellow soil, the pH value is 7.0, and the soil fertility is moderate (the organic value content is 0.85 g/kg).

5.4.2 investigation methods, times and frequency

Investigation time and number of times

Before the medicine, the young leaves are sporadically attacked, and the disease condition base is not investigated. On 25 days 6 months, the summer shoots were investigated in stages. The total number of surveys was 1.

Investigation method

Each district investigates 10 trees, each tree is sampled according to 5 points of east, south, west, north and middle, each point investigates 4 total leaves on the current-year summer shoots in a grading way, and 10 trees examine 200 shoots in total.

The blade grading method comprises the following steps:

level 0: no disease;

level 1: 1-5 scabs are formed on each leaf;

and 3, level: each leaf has 6-10 spots;

and 5, stage: each leaf has 11-15 scabs;

and 7, stage: each leaf has 16-20 scabs;

and 9, stage: each leaf has more than 20 scabs.

5.5 method of calculating drug efficacy

Direct effect on crops

None.

Yield and quality of the product

Need not be provided.

Influence on other organisms

Influence on other pests and diseases

No effect was found.

Effect on other non-target organisms

Is safe to natural enemies.

5.6 results and analysis

The significance determination adopts a Duncan's new double error (DMRT) method.

Table 1910% chloropropyl clinafloxacin wettable powder for preventing and treating citrus canker in field effect experiment (2021)

TABLE 2010% Chloroacyl clinafloxacin wettable powder for preventing and treating citrus canker

As can be seen from tables 19 and 20, the 10% chloropropyl clinafloxacin wettable powder has a good control effect on citrus canker, and the control effect is more than 89% in 48 days after the last application. The 10% chloropropyl clinafloxacin wettable powder can reach 90.23% of control effect after being diluted by 800 times, is equivalent to 1500 times of control effect of 46% copper hydroxide water dispersible granule, and is superior to 1000 times of control effect of 3% benziothiazolinone wettable powder. The 10% chloropropyl clinafloxacin wettable powder can reach the control effect of 89.05% diluted by 1600 times, is equivalent to 1500 times of 46% copper hydroxide water dispersible granule, is superior to 1000 times of the control effect of 3% benziothiazolinone wettable powder, and is not as good as the control effect of 800 times diluted 10% chloropropyl clinafloxacin wettable powder. In the test process, the high concentration of the pesticide is found to have no phytotoxicity on tender tips of citrus leaves and fruits and is safe to natural enemies, so that the high concentration 10% chloropropyl clinafloxacin wettable powder can be sprayed in the field to prevent and control citrus canker.

The experiment of the medicament for preventing and treating citrus canker on citrus leaves in the field proves that the medicament has better effect of preventing and treating citrus canker under high dilution concentration. In field experiments, high-concentration medicaments are recommended to be sprayed to prevent and control citrus canker. The spraying method is adopted, the dew is dried in sunny days, and the liquid medicine is uniformly sprayed on the tender-tip leaves after being diluted. Preferably, the application is carried out before the disease occurs, the application is carried out for 1 time about every 10 days, and the application is carried out for 2-3 times continuously, so that the harm of the citrus canker to young shoots and leaves can be reduced. In order to delay the generation of drug resistance, a plurality of medicaments are suggested to be alternately used.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims

1. The application of the compound shown in the formula (I) in preparing the medicine for preventing and treating citrus canker is characterized in that,

in the formula, R₁Is composed of

R₂is-H, -Cl or-OCH₃，R₃Is composed of

2. The drug for preventing and treating citrus canker is characterized by comprising a compound shown as a formula (I):

in the formula, R₁Is composed of

R₂is-H, -Cl or-OCH₃，R₃Is composed of

3. The drug of claim 2, which is a water dispersible granule comprising 10 wt% of the compound of formula (I), 2 wt% to 6 wt% of a wetting agent, 7 wt% to 15 wt% of a dispersant, 4 wt% to 11 wt% of a disintegrant, and the balance of a filler.

4. The medicament of claim 2, which is a wettable powder comprising 10 wt% of the compound of formula (I), 2 wt% to 6 wt% of a wetting agent, 7% to 15% of a dispersing agent, and the balance of a filler.

5. The medicament of claim 3 or 4, wherein the wetting agent is sodium dodecyl sulfate or sodium dodecyl benzene sulfonate; and/or the dispersant is a polycondensate salt dispersant; and/or, the filler comprises kaolin and diatomaceous earth.

6. The medicament according to claim 5, wherein the mass ratio of kaolin to diatomaceous earth is 1-1.5: 1.

7. The medicament of claim 3, wherein the disintegrant is sodium chloride.