阅读说明：本技术 一类柞蚕空胴病防治化合物及使用方法 (Tussah empty carcass disease prevention and treatment compound and use method thereof ) 是由 牛雄雷 杨金琛 刘铁成 陈有嗣 赫英姿 刘洪丽 王立石 曾航 于 2020-12-21 设计创作，主要内容包括：本发明提供了一类基于苯并异噻唑啉酮衍生物结构的柞蚕空胴病防治化合物及其使用方法。本发明所述的苯并异噻唑啉酮衍生物既具有体外抑菌活性,又具有蚕体内治疗活性,对柞蚕相对安全,可用于蚕业生产。(The invention provides a compound for preventing and treating tussah empty carcass disease based on a benzisothiazolinone derivative structure and a using method thereof. The benzisothiazolinone derivative has in vitro antibacterial activity and in vivo therapeutic activity on tussah, is relatively safe to tussah and can be used for silkworm production.)

1. A tussah empty carcass disease prevention and treatment compound is characterized in that the compound is one or more than two of the following compounds with the structural general formula:

in the formula: r represents one of the following:

alkyl groups: a straight or branched chain alkane having 1 to 7 carbon atoms;

substituted aromatic hydrocarbon groups on the benzene ring: 2-and/or 4-position of benzene ringRespectively exist of CH₃Or F or Cl or CH₃O or NO₂。

2. The compound according to claim 1, wherein said compound is preferably:

r is one or more than two of compounds corresponding to straight chain or branched chain alkyl with 1-5 carbon atoms.

3. A medicine for preventing and/or treating tussah empty carcass disease, which contains one or more compounds of claim 1 or 2 as active component.

4. The use of a compound of claim 1 or 2 for the prevention and/or treatment of tussah dry-carcass disease.

5. Use according to claim 4, characterized in that: it is used for preventing and/or treating tussah empty carcass disease by taking one or more than two compounds in any one of the compounds of claims 1-2 as active ingredients.

6. The use according to claim 4 or 5, characterized in that, one or more than two of the compounds of any one of claims 1 to 2 are prepared into a dispersing agent, the dispersing agent is diluted by adding water until the effective content of the compounds is 50-2000 mg/L, and the dispersing agent is administrated in a foliar feeding manner to control the streptococcus tussah in tussah bodies.

7. The use according to claim 6, wherein the water dilution with the effective content of 100-1000 mg/L of one or more compounds in any one of claims 1-2 is sprayed on the surfaces of oak leaves, and the oak leaves are used as the food of tussah silkworms, so that the tussah silks can eat the medicated leaves for more than 2 days.

Technical Field

The invention relates to the field of veterinary drugs, in particular to the field of prevention and treatment of tussah dry carcass disease.

Background

The empty disease of tussah silkworm is also called softening disease, commonly called 'shivering buttock', and is one of the main bacterial diseases damaging tussah silkworm. The disease is caused by infection of streptococcus tussah (streptococcus pernyi sp. nov.) and parasitizing the midgut of tussah. The literature indicates that besides the infection of tussah, the streptococcus tussah can infect the larvae of 21 kinds of tussah garden insects such as tenebrio molitor, oak dust moth, yellow two-star moth and the like, and the infected larva excreta and dead larva bodies carry the streptococcus tussah, which can infect the tussah. Therefore, the disease is widely distributed and occurs in China tussah production areas, wherein the annual general morbidity of the tussah areas in Liaoning province is 30-40%, the serious tussah areas can reach 70%, and even the tussah areas are in failure.

For the prevention and treatment of the streptococcus tussah, the prior method mainly prevents the egg-surface infection of bacterial diseases through a disinfectant, and the infection of the larva stage of the tussah in a tussah garden through oral ingestion is not effectively controlled all the time, so that corresponding treatment medicaments are lacked. In recent years, the occurrence frequency of the empty carcase of tussah is becoming more and more serious, and the development of a preventive and therapeutic drug for streptococcus tussah with both preventive and therapeutic significance is urgently needed in the production of silkworm industry.

Reports show that isothiazolinone (MIT, CMIT) has certain prevention and treatment effects on bacteria in the body of silkworm, but is not produced and applied. Data show that isothiazolinones (MIT, CMIT) are structurally stable at pH between 3 and 8.5. The data show that the bombyx mori intestinal tract is alkaline, and the pH is lower than 9; the data show that the intestinal alkalinity of the tussah is stronger, and the pH value reaches more than 9, so that the research on controlling the tussah empty carcass disease is carried out by selecting Benzisothiazolinone (hereinafter referred to as BIT) which can still stably exist under stronger alkalinity, and the results show that the effect of the BIT directly applied to the tussah in vivo controlling the tussah streptococcus is not ideal (the control effect is less than 80%) (see example 7 and table 5), although the tussah in vitro controlling activity of the tussah streptococcus can reach more than 99% (see example 5 and table 3). The invention discloses a method for designing and synthesizing various derivatives and screening the control activity of streptococcus tussah by using BIT as a mother drug, and the method is used for finding that part of compounds in the compounds with the general formula of claim 1 show excellent control activity of streptococcus tussah in vivo, and relevant results of the invention are illustrated below.

Disclosure of Invention

The invention aims to provide a tussah empty carcass disease control compound based on a BIT hydroxymethyl esterification derivative structure and a use method thereof.

The invention adopts a derivative synthesis method to prepare a BIT hydroxymethyl esterified derivative, then the derivative is prepared into a dispersant, the in vitro antibacterial activity (the prevention effect is higher than 95%) of the dispersant on the streptococcus tussah is measured, the prevention and treatment activity of the dispersant on the streptococcus tussah in vivo is measured, and a compound with high in vivo therapeutic activity (the prevention effect is not lower than 80%) is screened.

A kind of tussah empty carcass disease prevention and cure compound, one or more than two of the following structural formula compounds:

in the formula: r represents one of the following:

alkyl groups: a straight or branched chain alkane having 1 to 7 carbon atoms;

substituted aromatic hydrocarbon groups on the benzene ring: CH is present at the 2-and/or 4-position of the benzene ring₃Or F or Cl or CH₃O or NO₂。

The compound is preferably: r is one or more than two of compounds corresponding to straight chain or branched chain alkyl with 1-5 carbon atoms.

The application of the compound in preventing and/or treating tussah empty carcass disease.

One or more of the compounds are prepared into a dispersing agent, the dispersing agent is diluted by adding water until the effective content of the compound is 50-2000 mg/L, and the spraying agent is applied in a leaf surface feeding mode to prevent and control the streptococcus tussah in tussah bodies.

One or more than two compounds with effective content of 100-1000 mg/L are sprayed on the surfaces of the oak leaves, and the oak leaves are used as food for tussah, so that the tussah can be guaranteed to take the medicated leaves for more than 2 days.

The main advantages of the invention are:

1. the compound for preventing and treating the streptococcus tussah not only can kill the streptococcus tussah in vitro, but also has the in vitro sterilization prevention effect of more than or equal to 95 percent, can kill the streptococcus tussah in vivo, has the in vivo prevention effect of more than or equal to 80 percent, and has the silkworm production application value.

2. The compound for preventing and treating the streptococcus tussah is safe to the tussah and has no adverse effect on the growth and development of the tussah.

3. The medicament has mild property, is more friendly to human bodies in the using process compared with the traditional medicament formaldehyde or organic chlorine oxidant, and has no corrosion effect on medicament application instruments.

4. The medicament serving as the BIT derivative has the green and environment-friendly property similar to that of BIT, is easy to biodegrade and is environment-friendly.

The BIT hydroxymethyl esterified derivative has the effects of preventing and treating the streptococcus tussah, is relatively safe to the tussah, and can be applied to tussah production.

The invention is further illustrated by the following examples, but it should be understood that the invention is not limited thereto. The synthesis yield of the compound is not optimized; the thermometer used for melting point detection was not temperature corrected.

Detailed Description

Preparation of the Compound of example 1

Synthesis of compound BZ 1:

(1) preparing a BIT hydroxymethylation intermediate: 0.050mol of BIT, 0.055mol of paraformaldehyde (calculated as formaldehyde), 0.15mL of triethylamine and HCCl₃Adding 50mL of the product into a 100mL three-neck flask with a condenser pipe and magnetic stirring, heating the product in an oil bath to 70 ℃ for reflux reaction for 5 hours, stopping the reaction, removing the solvent by rotary evaporation, recrystallizing the solid with ethanol, and drying the solid under an infrared lamp to obtain 7.2g of BIT hydroxymethylation intermediate, wherein the yield is 79%, and the melting point is 145-148 ℃.

(2) Putting 0.010mol of BIT hydroxymethylation intermediate, 45mL of dried acetone and 0.011mol of triethylamine into a 100mL three-neck flask with a condenser pipe and magnetic stirring, and dropwise adding 0.011mol of acetyl chloride diluted by 5mL of acetone into a constant-pressure funnel under the stirring of ice-water bathIn a bottle, when smoke in the system disappears, heating an oil change bath to 65 ℃ for reflux reaction overnight, stopping the reaction, adsorbing the reaction liquid on activated silica gel, removing acetone, filling the column by a dry method, performing column chromatography (V (petroleum ether): V (ethyl acetate): 4: 1) to obtain 1.65g of a product, wherein the yield is 74%, the melting point is 122-129 ℃, and performing Bruker Avance III 400¹H NMR measurement confirmed the structure (compound No. BZ1,¹the H-NMR data are shown in Table 2).

All other compounds in table 1 were prepared according to a similar method (same procedure and conditions) as in example 1 above; the difference from example 1 is: during the reaction, raw materials of acetyl chloride are respectively replaced by propionyl chloride, butyryl chloride, valeryl chloride, hexanoyl chloride, heptanoyl chloride, benzoyl chloride, 4-fluorobenzoyl chloride, 2-fluorobenzoyl chloride, 4-methylbenzoyl chloride and 4-chlorobenzoyl chloride which are sequentially numbered as BZ 2-11.

TABLE 1 test BIT hydroxymethyl esterified derivatives (BZ1 ~ 11)

Preparation of emulsifiable concentrate dispersant of the Compound of example 2

The compound BZ1 is taken as an example to prepare an emulsifiable concentrate dispersant of the compound, which is used for measuring the in-vivo antibacterial activity of tussah (or the curative activity of tussah empty carcass disease).

According to the calculated mixture ratio, 5.05g of compound Z1 (the purity is calculated by 99%) is dissolved in 89.95g of solvent Methyl Isobutyl Ketone (MIK), 5.00g of Tween-80 is added, the mixture is stirred at high speed for 0.5h to prepare uniform missible oil with the compound BZ1 mass concentration of 5.00%, and sterile water is added to dilute the missible oil to the required concentration when the missible oil is used.

The other compounds are respectively prepared into the missible oil dispersant of the compound by adopting the same process.

Example 3 preparation of DMSO solutions of Compounds

Taking the compound BZ1 as an example to prepare a DMSO solution of the compound, and using the DMSO solution for the in vitro antibacterial activity determination of the isolated tussah streptococcus (or the in vitro antibacterial activity determination of tussah).

50.00mg of BZ1 was weighed out, transferred to a 10mL volumetric flask, added with DMSO solvent and made to a volume of 10mL to obtain 5000mg/L of a mother liquor. 2mL of mother liquor is measured by adopting a 5mL pipette, a 10mL volumetric flask is added, DMSO is metered to 10mL to obtain a 1000mg/L solution, the solution is diluted step by step to obtain a DMSO solution of BZ1 with the use concentration of 1000mg/L, 500mg/L, 250mg/L, 125mg/L, 62.5mg/L and 31.25mg/L, and the DMSO solution is placed in a refrigerator for storage.

The other compounds are prepared into DMSO solutions of the compounds respectively by adopting the same process.

Example 4 preparation of BIT amide derivative, preparation of emulsifiable concentrate and preparation of DMSO solution

Using XA1 as an example, a BIT acetylated product was prepared.

10.08g of BIT (0.067mol) was weighed and placed in a 100mL three-necked flask equipped with a stirring magneton, a thermometer and a condenser, 45mL of dichloromethane and 9.0g of anhydrous pyridine were added to the three-necked flask, and a mixed solution of 7.13g of acetyl chloride (0.090mol) and 5mL of dichloromethane was added dropwise with a constant pressure dropper while stirring in an ice water bath for half an hour, followed by reaction at room temperature for 1 hour. Pouring the reaction mixture into a separating funnel after the reaction is finished, washing the reaction mixture for 3 times by using 60mL of distilled water, drying an organic phase by using anhydrous sodium sulfate, filtering, removing the solvent by reduced pressure distillation to obtain a white solid, measuring a melting point, and adopting¹H NMR test, compound No. XA1, data are shown in Table 2.

All the compounds in Table 2 were prepared according to a similar manner (same procedure and conditions) to that of example 4 above; the difference from example 4 is: during the reaction, raw materials of acetyl chloride are respectively replaced by propionyl chloride, butyryl chloride, valeryl chloride, hexanoyl chloride, heptanoyl chloride, benzoyl chloride, 4-fluorobenzoyl chloride, 2-fluorobenzoyl chloride, 4-methylbenzoyl chloride and 4-chlorobenzoyl chloride which are sequentially numbered as XA 2-11.

TABLE 2 test BIT amide derivatives (XA1 ~ 11)

An emulsifiable concentrate of XA 1-11 compound and a DMSO solution were prepared by the methods described in examples 2 and 3, respectively.

Example 5: method for determining bacterial colony of compound tussah in vitro antibacterial activity by plate coating number

Taking a 5ml centrifuge tube, adding 3200 μ L of liquid culture medium (with the composition taking 1000ml as an example, 20g of sucrose, 20g of peptone, 5g of sodium chloride and 5g of beef powder, adding distilled water to 1000ml, adjusting the pH to 9 with sodium hydroxide, and performing moist heat sterilization at 121 ℃ for 30min) into 400 μ L of bacterial liquid and 400 μ L of DMSO solutions with different concentrations (500mg/L, 250mg/L, 125mg/L, 62.50mg/L, 31.25mg/L and 15.625mg/L) (shown in table 1 and table 2), and uniformly mixing on a vortex mixer, wherein the final concentration of the streptococcus tussah is 1.8 × 10⁷CFU/ml; the sample adding operation is completed within 2h, and the mixed solution is cultured in a shaking incubator at 37 ℃ for 24h at 130 r/min. After the culture is finished, the mixed solution is respectively diluted to 1000 times (V/V) step by step, 100 mu L of 1000 times of the diluted solution is taken by a pipette and smeared (the composition of a solid culture medium takes 1000ml as an example, 20g of cane sugar, 20g of peptone, 5g of sodium chloride and 5g of beef powder are added with distilled water to 1000ml, the PH value is adjusted to 9 by sodium hydroxide, 20g of agar is sterilized by moist heat at 121 ℃ for 30min, and the obtained product contains agar with the final mass concentration of 2 percent compared with a liquid culture medium), and the smeared plate is placed in an incubator to be cultured for 24h at 37 ℃ and then taken out to check the number of colonies. Blank control CK A plating operation was performed by replacing the drug solution (1000-fold dilution of the incubated mixture) with sterile water. The formula for calculating the bacteriostatic rate of medicament treatment is as follows:

the determination result shows that when the using concentration of the compound is not lower than 62.5mg/L, the bacteriostatic effects of BZ 1-11 and XA 1-11 are all over 99%, and no obvious difference exists. When the concentration of the compound is 31.25mg/L, the determination result is shown in Table 3, the bacteriostatic activity of BZ 1-11 can still reach more than 96%, the bacteriostatic activity of XA 1-4, XA 6-8 can reach more than 96%, and the bacteriostatic activity of XA5, XA 9-11 can not reach 95%. The results show that: the two compounds have good antibacterial activity at higher use concentration (such as more than 62.5 mg/L); when the using concentration is lower, the bacteriostatic activity of the ester derivative is better than that of the corresponding amide derivative.

TABLE 3 comparison of the bacteriostatic Activity of the ester and amide derivatives at a concentration of 31.25mg/L for the Compounds-plating method

Note: the blank colony was 3.0X 10⁵And (4) respectively. ND: indicating statistical colonies>Counting was stopped after 1500 and activity was not calculated.

Example 6: method for determining in vitro bacteriostatic activity of compound tussah-bacteriostatic zone

Preparing bacteria-containing plates (taking 1000ml as an example of solid medium composition, 20g of sucrose, 20g of peptone, 5g of sodium chloride and 5g of beef powder, adding distilled water to 1000ml, adjusting pH to 9 with sodium hydroxide, heating and dissolving agar 15 g), subpackaging the hot medium into test tubes with 12.5ml per test tube, sealing, and carrying out moist heat sterilization at 121 ℃ for 30 min. After sterilization, when the temperature of the culture medium is reduced to 55 ℃, 6.5 multiplied by 10 concentration is added into each test tube⁶125 mu L of CFU/ml tussah streptococcus liquid, quickly shaking uniformly, pouring into a culture dish of 90mm (2 test tubes are poured in each dish at the same time) for cooling, preparing a bacterium-containing plate, and storing at-4 ℃ for later use. Evenly punch 9 wells on each plate containing bacteria with a sterile punch: add 5. mu.L of compound DMSO solution of the specified concentration into each well, assay 3 wells in parallel; each compound was treated with 1000mg/L, 500mg/L, 250mg/L at different concentrations. After dosing, diffusion was carried out at room temperature for 2h, followed by incubation at 37 ℃ for 24 h. And measuring the diameter of the bacteriostatic circle by using a vernier caliper after the culture is finished. Blank control CK is to replace the drug solution with DMSO.

As can be seen from Table 4-1, when the concentration of the compound is 1000mg/L, the diameter of the BIT zone reaches 12.56mm, the diameter of the BZ 1-3 zone is slightly larger than that of BIT and BZ 4-6, the diameter of the BZ9 zone is close to 12mm, and the diameters of the amide zone except XA1(12.2mm) are all smaller than 10 mm.

When the using concentration of the compound is reduced to 500mg/L (table 4-2), the inhibition zone of the ester derivative can still be measured, wherein the diameter of the BZ 1-4 inhibition zone is equivalent to the diameter of BIT; and 6 compounds of the amide derivative have the inhibition zone disappeared.

In the above description, the antibacterial activity of the compounds and ester derivatives is generally higher than that of amide derivatives.

TABLE 4-1 comparison of the bacteriostatic Activity of ester derivatives and amide derivatives at a Compound application concentration of 1000 mg/L-bacteriostatic Loop method

TABLE 4-2 comparison of bacteriostatic Activity of ester derivatives and amide derivatives at 500mg/L Compound application-bacteriostatic Ring method

Example 7: method for testing treatment effect of compound tussah on empty carcass disease in vivo-feeding test

And 5, in the outdoor Liaoning Fengcheng city, placing sterile eggs of the variety 9906 of the tussah into the tussah for hatching to obtain ant silkworm test insects, and recording the ant silkworm test insects as test insects A. Indoor, newly picked branches with leaves are placed on a sponge base containing water, and the newly cultured, separated and purified streptococcus is diluted to 1 x 10 by adding sterile water⁸And (3) after CFU/ml, spraying the oak leaves, dispersing 1-2 days old newly-hatched silkworms in the leaves after the foliar bacterial liquid is dried, and obtaining the newly-hatched silkworms fed with streptococcus pernyi after the oak silkworms take the fungus-containing leaves for 48 hours and recording as a test worm B.

Medicament treatment: randomly selecting 30 test insects B in each treatment, and placing the test insects in a single insect breeding box; diluting the compound missible oil prepared in the example 2 or the example 4 with sterile water to a corresponding concentration in a spraying pot, spraying the compound missible oil on the oak tree leaf surface until fog drops are distributed, airing within 0.5h, picking and adding the compound missible oil into an insect breeding box, feeding by test insects for more than 48h, and repeating the treatment for 3 times; each compound was set at 3 treatment concentrations (1000mg/L, 500mg/L, 250 mg/L). After the test insects in the insect box are treated by the medicament for one time, fresh oak leaves which are not treated by the medicament are placed and fed to the fourth-instar stage. During the period, the mark is tracked, the silkworm excrement and sundries are removed, fresh oak leaves are added at proper time, the diseased or dead test silkworms are removed in time, the number of the remaining silkworms in each treatment is investigated, the total number N of the diseased or dead silkworms with empty carcass disease is accumulated, and the morbidity is calculated.

And (4) comparison treatment: placing 30 test insects B in a single insect breeding box, feeding with fresh oak leaves without medicament treatment, and repeating for 3 times to obtain empty carcass virus control CK-D; a30 test insects are normally fed in the insect-raising box, and are fed by fresh oak leaves without medicament treatment, and the feeding is repeated for 3 times to be used as a blank control CK-0 for empty carcass disease. Tracking and marking, removing silkworm excrement and impurities, adding fresh oak leaves in time, removing diseased or dead test silkworms in time, feeding to a fourth-instar stage, investigating the number of remaining silkworms in each treatment, and accumulating the total number N of the diseased or dead silkworms with empty carcass disease.

The formula for calculating the incidence and prevention effect of empty carcass disease is as follows:

the incidence rate of empty carcass disease is N/30X 100%

As can be seen from the data in Table 5, when the compound is used at a concentration of 1000mg/L, the therapeutic activity of the ester derivatives BZ 1-6 and BZ9-11 is not inferior to that of control BIT, wherein the activity of BZ 1-5 is more than 80% and is up to 92%; the amide derivatives have low therapeutic activity, which is less than 20%. To avoid the redundant data to show, compounds with a control effect of less than 75% are not listed in the results of the first order concentration test (except for the control BIT).

As shown in Table 6, when the compound is used at a concentration of 500mg/L, the BZ 1-5 is still higher than 75%, wherein the BZ1 and the BZ2 are higher than 80%, and the control effect is better than that of BIT (67.9%).

As can be seen from Table 7, when the compound is used at a concentration of 250mg/L, the control effect of the compound BZ 1-5 is more than 60%, and the control effect is better than that of BIT (50%).

TABLE 5 comparison of the therapeutic effects of the ester derivatives and the amide derivatives in tussah silkworm empty carcass disease at a concentration of 1000mg/L

Note: the incidence rate of CK-0 empty carcass disease is 10.0 percent; the incidence rate of CK-D empty carcass disease is 93.3%. Table 6 and Table 7 are the same.

TABLE 6 therapeutic effect of ester derivatives in tussah body for empty carcass disease at 500mg/L concentration

Compound (I)	Bacteriostatic activity/%)
		BZ1	85.7
BZ2	82.1
		BZ3	78.6
BZ4	78.6
		BZ5	77.8
BZ6	64.3
		BZ9	50.0
BZ10	53.6
		BZ11	53.6
CKY(BIT)	67.9

TABLE 7 therapeutic effect of ester derivative on tussah empty carcass disease at 250mg/L

Example 8 Studies on silkworm growth

In example 7, the test silkworms obtained by 1000mg/L treatment and blank treatment of the agents BZ1, BZ2, BZ3, BZ4 and BZ5 were recorded as 1000mg/L-BZ 1-5 and test silkworm A in this order, 25 silkworms of four ages were picked up in each treatment machine, and the weights thereof were weighed with a scale of 0.01g precision, and the results of statistical analysis were as follows (Table 8). The average silkworm body mass of each drug treatment is not obviously different from that of the blank treatment (p is less than 0.05), which shows that the drug has no adverse effect on the growth of the silkworm body at the use concentration.

TABLE 81000 silkworm body weight statistics table after drug treatment (n 25)

Group of test insects	Average mass
		1000mg/L-BZ1	3.67±0.086a
1000mg/L-BZ2	3.62±0.067a
		1000mg/L-BZ3	3.64±0.089a
1000mg/L-BZ4	3.61±0.076a
		1000mg/L-BZ5	3.63±0.068a
CK (insect test A)	3.61±0.062a

Note: at a p <0.05 level, the same letter represents no significant difference and different letters represent significant difference.