WO2025025716A1 - Crystal form of kasugamycin hydrochloride, kasugamycin acetamide, and composition of crystal form and kasugamycin acetamide - Google Patents

Abstract

The present invention relates to a crystal form I of an agricultural antibiotic kasugamycin hydrochloride and a composition of the crystal form I and kasugamycin acetamide. The crystal form I shows good stability in a high-temperature, high-humidity, and illumination environment, and shows an unexpected plant disease prevention and treatment effect while better meeting the requirements of pesticides in production, processing, and transportation and storage.

Description

Kasugamycin hydrochloride crystal form, kasugamycin acetamide and composition thereof

Priority information

The present invention claims priority and benefits of patent application CN202310969303.3 filed with the State Intellectual Property Office of China on August 3, 2023 and patent application CN202311103321.X filed with the State Intellectual Property Office of China on August 30, 2023, and the entire text of which is incorporated herein by reference.

Technical Field

The invention belongs to the technical field of agriculture, and in particular relates to kasugamycin hydrochloride crystal forms, kasugamycin acetamide and a composition thereof.

Background Art

5-Amino-2-methyl-6-(2,3,4,5,6-hydroxycyclohexyloxy)pyran-3-ylamino-α-imidoacetic acid (CAS 6980-18-3, kasugamycin) belongs to the aminoglycoside antibiotics. Since its discovery in the 1960s and 1970s, kasugamycin has been widely used to prevent and control various diseases of various crops including rice, potato, cabbage, melon, etc. Due to its excellent prevention and control effect and its environmentally friendly characteristics, kasugamycin is currently the main biological pesticide product for the prevention and control of crop diseases. Kasugamycin is extremely unstable in an alkaline environment, and kasugamycin is a hygroscopic compound. Considering transportation, storage and quality control, more stable and less hygroscopic kasugamycin salt products are desired by production and processing companies.

At the beginning of the discovery of kasugamycin, Ikekawa T et al. briefly investigated the crystal structure of kasugamycin hydrobromide (The Journal of Antibiotics, 01 Jan 1966, 19 (1): 49-50). CN115925475A reported the use of kasugamycin phosphate to supplement phosphorus while preventing and treating bacteria. CN108822167A reported kasugamycin hydrochloride and its white needle-shaped or flaky crystals. CN106083951B and CN109666051B reported the extraction of kasugamycin hydrochloride by adding acetone, methanol, ethanol and other solvents to water for crystallization.

Although the prior art has disclosed a method for preparing high-purity kasugamycin hydrochloride, the crystalline products prepared by different crystallization methods are often different, and their physical and chemical properties are also quite different, and the performance of the corresponding crystalline products in formulation processing is also different. Therefore, a stable crystalline product of kasugamycin salt that is conducive to formulation processing and storage is the goal pursued by biological pesticide companies.

In addition, the applicant unexpectedly discovered that the Streptomyces microaureaus strain metabolizes to produce kasugamycin and kasugaetiamide (CAS 6189-95-3 or 21256-64-4 or 38420-31-4, with a structure as shown in Formula 1), while there are no reports on the preparation process and detection method of kasugaetiamide, and its efficacy and application have not been studied and developed.

Summary of the invention

In view of the above problems existing in the prior art, the present invention provides a technical solution to the above problems.

According to one aspect of the present invention, there is provided an agricultural antibiotic kasugamycin hydrochloride crystal form I, wherein the crystal form I uses Cu-Kα radiation, and the characteristic diffraction peaks of the X-ray powder diffraction pattern expressed by 2θ value ±0.2° include 8.66, 10.11, 11.05 and 13.3, preferably, the crystal form I uses Cu-Kα radiation, and the characteristic diffraction peaks of the X-ray powder diffraction pattern expressed by 2θ value ±0.2° include 8.66, 10.11, 11.05, 13.3, 13.92 and 15.56; preferably, the characteristic diffraction peaks of the X-ray powder diffraction pattern expressed by 2θ value ±0.2° include 13.3, 13.92, 15.56, 16.47 and 17.29; more preferably, the characteristic diffraction peaks of the X-ray powder diffraction pattern expressed as 2θ values ± 0.2° include 8.66, 10.11, 11.05, 13.3, 13.92, 15.56, 16.47, 17.29, 18.59, 19.25, or include 8.66, 10.11, 11.05, 13.3, 13.92, 15.56, 16.47, 17.29, 18.59, 19.25,

Optionally, differential scanning calorimetry (DSC) shows that the kasugamycin hydrochloride form I has a continuous endothermic signal in the range of 110±2°C to 210±2°C, and an endothermic signal at 226±2°C; compared with the height of the endothermic peak (or peak valley), the endothermic process of the form I at 226±2°C is a strong endothermic process, and the endothermic process in the range of 110±2°C to 210±2°C is a weak endothermic process.

Thermogravimetric analysis (TGA) shows that the crystalline form I has a weight loss of less than 5% when heated from 100±2°C to 220±2°C, and a weight loss of less than 15.5% when heated from 220±2°C to 255±2°C.

Optionally, the crystalline form I of kasugamycin hydrochloride described in the present application is a hydrate.

In a specific embodiment, the crystal form I of the compound kasugamycin hydrochloride described in the present application is granular or rod-shaped. From the perspective of meeting the actual production, facilitating solid-liquid filtration, and facilitating the processing of subsequent pesticide solid preparations, the particle size D ₉₀ of the crystal form I is 10 μm-200 μm. Preferably, the particle size D ₉₀ of the crystal form I is 15 μm-150 μm, 20 μm-120 μm, for example, the particle size D ₉₀ of the crystal form I can also be 10, 15, 25, 35, 45, 55, 65, 75, 85, 95, 100, 110, 130, 140, 150, 160, 170, 180, 190 or 200 μm.

In one embodiment, the crystalline solid can be prepared by dissolution back drop method, liquid vapor diffusion method, single solvent room temperature suspension method, single solvent high temperature suspension method, binary solvent forward drop method, single solvent cooling method, binary solvent cooling method and the like.

Preferably, Form I is prepared by a dissolution back-drip method, a liquid vapor diffusion method, and the like, and is not limited by the examples of the preparation examples of the present application. The kasugamycin hydrochloride Form I described in the present invention is prepared by other crystallization methods such as a single solvent room temperature suspension method, a single solvent high temperature suspension method, a binary solvent positive drop method, a single solvent cooling method, a binary solvent cooling method, and the like, and also falls within the scope of protection of the present invention.

Preferably, the amorphous material is prepared by freeze drying, and the freeze drying can be selected from circulating freezer drying or medium freeze drying (such as dry ice freeze drying or nitrogen freeze drying).

Preferably, the elution back-titration method comprises the following steps: dropping an aqueous solution containing kasugamycin hydrochloride into a poor solvent, suspending for 10-120 minutes after precipitation, and filtering to obtain a solid. The dropping process may be stirred or not stirred, and the stirring rate is 20-120 rpm; the poor solvent is dioxane or ethylene glycol dimethyl ether.

Preferably, the liquid vapor diffusion method comprises the following steps: weighing a certain amount of kasugamycin hydrochloride solution dissolved in a good solvent, placing the clear solution in a poor solvent atmosphere, standing at room temperature until solids precipitate and then filtering. The good solvent is water or formamide, and the poor solvent is trifluoroethanol.

According to another aspect of the present invention, the present invention provides an amorphous form of kasugamycin hydrochloride.

Optionally, the amorphous material has an X-ray powder diffraction peak as shown in FIG6 .

DSC showed that the amorphous substance had endothermic signals at 73±2°C and 187±2°C;

TGA results show that the amorphous material has a weight loss of less than 9% when heated to 175±2°C, and has a weight loss of less than 15% when heated from 175±2°C to 255±2°C.

Optionally, the amorphous kasugamycin hydrochloride is a sheet-like structure.

Optionally, the amorphous material D ₉₀ is 10 μm-200 μm, preferably 15 μm-150 μm, more preferably 20 μm-120 μm.

According to another aspect of the present invention, a technical drug, a finished drug, a pesticide preparation or a pesticide composition is provided, wherein the technical drug, the finished drug, the pesticide preparation or the pesticide composition contains the above-mentioned crystal form I and/or amorphous material.

According to another aspect of the present application, there is provided the use of the above-mentioned Form I, the amorphous material, the pesticide formulation or the pesticide composition pesticide formulation in the preparation of a plant disease control formulation.

According to another aspect of the present application, there is provided the use of the above-mentioned Form I, the amorphous material, the pesticide formulation or the pesticide composition pesticide formulation in plant disease control.

Furthermore, the plant disease is cucumber downy mildew or rice blast.

Furthermore, based on the total mass, the content of Form I and/or amorphous matter in the original drug is not less than 65%, such as not less than 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%; preferably, not less than 85% or 90% or 95%.

Furthermore, based on the total mass, the content of Form I and/or amorphous matter in the mother drug is not less than 5% or 10%, such as not less than 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%; preferably not less than 85% or 90% or 95%.

Further, the pesticide preparation or pesticide composition contains the above-mentioned crystalline form I and/or amorphous substance and adjuvants, and optionally, the weight percentage of the crystalline form I and/or amorphous substance in the pesticide preparation or pesticide composition is at least 0.001%, for example, the weight percentage of the crystalline form I and/or amorphous substance in the preparation is at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. Preferably, the weight percentage of the crystalline form I and/or amorphous substance in the preparation is 0.1%-10%, 0.5-7%, or 1-5%.

Optionally, the dosage form of the preparation is selected from any one of powder, granules, large granules, fine granules, microgranules, microcapsule granules, wettable powder, oil-dispersible powder, water-dispersible granules, emulsions, effervescent granules, dispersible tablets, effervescent tablets, sustained-release agents, sustained-release blocks, sustained-release tubes, sustained-release particles, soluble powders, soluble granules, soluble tablets, soluble solutions, aqueous solutions, soluble gels, oils, film-spreading oils, ultra-low volume liquids, ultra-low volume microcapsule suspensions, emulsifiable concentrates, latex, dispersible liquids, pastes, concentrated gels, emulsions in water, oil emulsions, microemulsions, ointments, suspensions, microcapsule suspensions, oil suspensions, suspoemulsions, dispersible powders for seed treatment, soluble powders for seed treatment, liquid solutions for seed treatment, emulsions for seed treatment, suspensions for seed treatment, suspended seed coatings, and microcapsule suspensions for seed treatment. Preferably, the crystal form I or amorphous solid of the present invention exhibits excellent processability and solid application control effect in pesticide solid formulations. Specifically, the crystal form I or amorphous solid of the present invention is used in wettable powders or dusts; wherein the particle size D90 of the crystal form I and/or amorphous solid in the dusts or wettable powders is 5 μm-80 μm, and the preferred particle size D90 is 10 μm-50 μm, or 15 μm-45 μm, or 20 μm, 30 μm, 40 μm, 60 μm and 70 μm.

According to another aspect of the present invention, there is provided the use of the above-mentioned Form I, the amorphous material, the technical or parent drug, or the preparation or pesticide composition in the process of plant disease prevention and control.

According to another aspect of the present application, there is provided the use of the above-mentioned Form I, the amorphous material, the technical drug or the mother drug, or the preparation or the pesticide composition in the preparation of a plant disease control preparation.

The inventors unexpectedly discovered and confirmed that kasugamycin amide has excellent bactericidal and antibacterial effects, and the effect is more obvious when combined with kasugamycin.

Therefore, another aspect of the present invention further provides a cinnamonamide composition, which contains cinnamonamide and/or a salt of cinnamonamide. Based on the total mass of the composition, the content of cinnamonamide and/or the salt of cinnamonamide is not less than 0.01%, or the content is not less than 0.03%, or not less than 0.05%, or not less than 0.1%, or not less than 0.5%, or not less than 1%, wherein the mass of the salt of cinnamonamide is calculated based on the mass of the cinnamonamide it contains.

Considering factors such as cost, the content of cinnamonamide and/or its salt is not higher than 10%, preferably not higher than 8% or not higher than 7%, based on the total mass of the composition.

Optionally, the composition is any one of a technical drug, a mother drug or a preparation; further, the dosage form of the preparation is selected from any one of powders, granules, large granules, fine granules, microgranules, microcapsule granules, wettable powders, oil-dispersible powders, water-dispersible granules, emulsions, effervescent granules, dispersible tablets, effervescent tablets, sustained-release agents, sustained-release blocks, sustained-release tubes, sustained-release particles, soluble powders, soluble granules, soluble tablets, soluble solutions, aqueous solutions, soluble gels, oils, film-spreading oils, ultra-low volume liquids, ultra-low volume microcapsule suspensions, emulsifiable concentrates, latexes, dispersible liquids, pastes, concentrated gels, emulsions in water, oil emulsions, microemulsions, ointments, suspensions, microcapsule suspensions, oil suspensions, suspoemulsions, dispersible powders for seed treatment, soluble powders for seed treatment, liquid solutions for seed treatment, emulsions for seed treatment, suspensions for seed treatment, suspended seed coatings, and microcapsule suspensions for seed treatment.

Another aspect of the present invention is to provide use of the above composition in preventing and controlling plant diseases.

Another aspect of the present invention is to provide use of the above composition in preparing a plant disease control formulation.

Optionally, the plant disease is corn Curvularia leaf spot and/or rice blast. Optionally, another aspect of the present invention provides a method for preparing high-purity kasugaetanamide, comprising the following steps: a fermentation broth purification step and an octadecylsilane bonded silica gel column separation step.

Optionally, the fermentation broth purification step comprises the following steps:

1) Acidifying and filtering the fermentation broth;

2) The filtrate is adsorbed by resin and then analyzed;

3) The analytical solution is filtered to remove impurities.

Optionally, a decolorization step is also included after the nanofiltration impurity removal.

Furthermore, the acidulant in the fermentation broth purification step 1 includes at least one of sulfuric acid, hydrochloric acid, phosphoric acid or oxalic acid.

Furthermore, the resin in the fermentation broth purification step 2 is a strongly acidic cation exchange resin.

Furthermore, in the fermentation broth purification step 2, the resin adsorption analytical agent includes at least one of ammonium chloride solution, sodium hydroxide solution or sodium chloride solution.

Optionally, in the silica gel column separation step, the eluting phase is a mixed solution of acetonitrile and sodium alkyl sulfonate;

Furthermore, the sodium alkyl sulfonate is optionally sodium hexane sulfonate or sodium dodecyl sulfonate, and acetonitrile:sodium alkyl sulfonate solution = 1%:99%-10%:90%.

The applicant unexpectedly discovered that when the ultraviolet detection wavelength of the liquid chromatography is 195nm, caesalpinia has a very sensitive response. Therefore, another aspect of the present invention is to provide a method for detecting caesalpinia, comprising the following steps:

The sample to be tested is dissolved in deionized water to prepare a solution with a concentration of 0.001-4 mg/mL. An appropriate amount of the solution to be tested is taken and injected into a liquid chromatograph. The peak position is used to determine whether kasugamycin acetamide is contained or the peak position is compared with that of the kasugamycin acetamide standard to determine whether kasugamycin acetamide is contained. The detection wavelength is 195 nm.

Optionally, the HPLC column is a reverse phase C18 filler;

Optionally, the mobile phase is acetonitrile and an aqueous solution of sodium alkyl sulfonate;

Optionally, running time: 10-20min;

Optionally, column temperature: 30 ± 2 °C;

Optionally, flow rate: 0.5-2 mL/min;

Optionally, signal bandwidth: 4nm-8nm;

Optionally, injection volume: 5 μL.

Furthermore, the sodium alkyl sulfonate is optionally sodium hexane sulfonate or sodium dodecyl sulfonate, and acetonitrile:sodium alkyl sulfonate solution = 1%:99%-10%:90%.

Those skilled in the art can determine whether caesium is contained by comparing the liquid phase data (spectrum) of the sample to be tested with the liquid phase data (spectrum) of the standard under the same liquid phase analysis conditions, in the presence of a standard, and the content (concentration) of caesium in the sample to be tested can be determined by the content (concentration) of the standard.

Based on the beneficial effects of the compounding of kasugamycin and kasugamycin acetamide, the present invention provides, on the other hand, a composition containing kasugamycin and/or a salt of kasugamycin and kasugamycin acetamide and/or a salt of kasugamycin acetamide, wherein the mass ratio of kasugamycin and/or a salt of kasugamycin to kasugamycin acetamide and/or a salt of kasugamycin acetamide in the composition is 10:1-200:1, preferably 13:1-130:1, or optionally, the mass ratio of kasugamycin and/or a salt of kasugamycin to kasugamycin acetamide and/or a salt of kasugamycin acetamide is 11:1, 13:1, 14:1, 15:1, 16:1, 20:1, 25:1 , 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, any one value among 110:1, 120:1, 140:1, 150:1, 160:1, 170:1, 180:1 or 190:1, or a range value between any two values; wherein the mass of the salt of kasugamycin is measured by the mass of kasugamycin it contains, and the mass of the salt of kasugamycin is measured by the mass of kasugamycin it contains.

Optionally, the composition is any one of a raw drug, a mother drug or a preparation.

Optionally, based on the total mass of the preparation, the total content of kasugamycin and/or kasugamycin salts and kasugamycin acetamide and/or kasugamycin salts in the preparation is not less than 0.01%, or not less than 0.03%, or not less than 0.1%, or not less than 1%, wherein the mass of kasugamycin salt is calculated based on the mass of kasugamycin acetamide it contains, and the mass of kasugamycin salt is calculated based on the mass of kasugamycin it contains.

The dosage form of the preparation is selected from powders, granules, large granules, fine granules, microgranules, microcapsule granules, wettable powders, oil-dispersible powders, water-dispersible granules, emulsions, foams, Any one of effervescent granules, dispersible tablets, effervescent tablets, sustained-release agents, sustained-release blocks, sustained-release tubes, sustained-release granules, soluble powders, soluble granules, soluble tablets, soluble solutions, aqueous solutions, soluble gels, oils, film-spreading oils, ultra-low volume liquids, ultra-low volume microcapsule suspensions, emulsifiable concentrates, latexes, dispersible liquids, pastes, concentrated gels, emulsions in water, oil emulsions, microemulsions, ointments, suspensions, microcapsule suspensions, oil suspensions, suspoemulsions, dispersible powders for seed treatment, soluble powders for seed treatment, seed treatment liquids, seed treatment emulsions, seed treatment suspensions, suspended seed coatings, and microcapsule suspensions for seed treatment.

Based on the beneficial effects of kasugamycin hydrochloride crystal form I and/or its amorphous material in terms of stability and processing convenience, the salt of kasugamycin in the composition is preferably kasugamycin hydrochloride crystal form I and/or its amorphous material.

Another aspect of the present invention is to provide use of the above composition in controlling plant diseases.

Another aspect of the present invention is to provide use of the above composition in preparing a plant disease control formulation.

Optionally, the plant disease is Curvularia spp. leaf spot of corn and/or rice blast.

The positive and progressive effects of the present invention are:

The applicant believes that the crystal form I provided in this application has good stability, almost no hygroscopicity, and can remain stable under light, high temperature, high humidity, and accelerated conditions, effectively extending the shelf life of the drug storage, and can better meet the requirements of pesticides in production, processing, transportation, and storage. At the same time, the crystal form I has also achieved good technical effects in plant disease prevention and control. Unexpectedly, the amorphous material has shown the best effect in plant disease prevention and control, and the agricultural terminal application effect is more prominent.

The inventors unexpectedly discovered that kasugalacetamide has excellent bactericidal and antibacterial effects.

When kasugamycin and/or its salt and kasugamycin and/or its salt are controlled in a certain ratio, a beneficial synergistic effect is exhibited, and the synergistic effect can be better applied to the prevention and treatment of plant diseases.

The invention also provides a method for preparing high-purity kasugalacetamide. The obtained high-purity kasugalacetamide can be used as a standard product or applied in the detection of active ingredients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a hydrogen nuclear magnetic resonance analysis (1H-NMR) spectrum of crystalline form I of kasugamycin hydrochloride compound;

FIG2 is an X-ray powder diffraction (XRPD) spectrum of Form I, wherein the abscissa is 2θ (°) and the ordinate is intensity (counts);

FIG3 is a 1H-NMR spectrum of the crystal form II of the kasugamycin hydrochloride compound;

FIG4 is an X-ray powder diffraction (XRPD) spectrum of Form II, wherein the abscissa is 2θ (°) and the ordinate is intensity (counts);

FIG5 is a 1H-NMR spectrum of kasugamycin hydrochloride amorphous substance;

FIG6 is an X-ray powder diffraction (XRPD) spectrum of an amorphous material, wherein the abscissa is 2θ (°) and the ordinate is intensity (counts);

FIG7 is a differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) diagram of Form I;

FIG8 is a differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) diagram of Form II;

FIG9 is a differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) diagram of an amorphous material;

FIG10 is a polarizing light microscope analysis (PLM) spectrum of Form I;

FIG11 is a polarizing light microscope analysis (PLM) image of Form II;

FIG12 is a polarizing light microscope analysis (PLM) image of an amorphous material;

FIG13 is a particle size distribution measurement (PSD) image of Form I;

FIG14 is a particle size distribution measurement (PSD) image of Form II;

FIG15 is a dynamic moisture adsorption and desorption analysis (DVS) spectrum of Form I;

FIG16 is a stability test result of Form I;

Figure 17 shows the results of the potted antibacterial experiment, where a-1 is the front view of the blank control leaf, a-2 is the back view of the blank control leaf, b-1 is the front view of the crystal form II powder experiment leaf, b-2 is the back view of the crystal form II powder experiment leaf, c-1 is the front view of the crystal form I powder experiment leaf, c-2 is the back view of the crystal form I powder experiment leaf, Figure 17d-1 is the front view of the amorphous powder experiment leaf, d-2 is the back view of the amorphous powder experiment leaf.

FIG18 is a purity characterization diagram of caesalpinia suspensa; the retention time of caesalpinia suspensa is 10.469 min, and the purity factor is 999.843, indicating that the method of the present invention can prepare caesalpinia suspensa with high purity, and the high-purity caesalpinia suspensa can be used as a standard product.

Figure 19 is a liquid chromatography-mass spectrometry ultraviolet chromatogram (HPLC-DAD) and total ion current spectrum (TIC) of kauriacetamide;

FIG20 is a HPLC-MSD of caesalpinia cyrtonema;

FIG21 is a hydrogen nuclear magnetic spectrum (1H-NMR) of caesalpinia cypermethrin;

Figure 22 is the infrared spectrum (FTIR) of kasuga acetamide;

FIG23 is a HPLC of kasugamycin aqueous composition;

Figure 24 shows the antibacterial effect of different concentrations of kasuga acetamide on rice blast fungus, where ck is the blank control group, a is the antibacterial effect of 0.001wt% concentration, b is the antibacterial effect of 0.01wt% concentration, c is the antibacterial effect of 0.03wt% concentration, d is the antibacterial effect of 0.05wt% concentration, and e is the antibacterial effect of 0.1wt% concentration.

DETAILED DESCRIPTION OF THE INVENTION

The term "solvate" refers to those forms of the crystalline forms of the compounds described herein that are used to form complexes with molecules of solvents (e.g., water, organic solvents such as formic acid, toluene, etc.) by coordination. Hydrates are a specific form of solvates in which coordination is performed with water. For example, the solvate may be a hydrate.

The term "technical product" refers to the product obtained during the production process consisting of active ingredients and related impurities, and a small amount of additives may be added if necessary.

The term "mother drug" refers to the product obtained during the production process consisting of active ingredients and related impurities, and may contain small amounts of necessary additives and appropriate diluents.

The term "formulation" refers to a stable product made from the original pesticide (mother pesticide) and suitable adjuvants, or processed by biological fermentation, plant extraction and other methods.

The term "adjuvant" refers to any single component or multiple components added to pesticide products, in addition to the active ingredient, which does not have pesticide activity and active ingredient function itself, but can or helps to enhance or improve the physical and chemical properties of the pesticide product.

The term "plant disease" refers to the phenomenon that during the growth and development of plants, due to infection by other organisms and the influence of adverse abiotic factors, its growth and development is significantly hindered, pathological changes occur in the physiology and tissue structure inside and outside the plant, and it becomes morbid or even dies, resulting in reduced yield and deterioration of quality. The "plant disease" of the present application specifically refers to the disease caused by the infection of plants by other organisms, including but not limited to fungi (such as cucumber downy mildew caused by Pseudomonas aeruginosa, maize Curvularia serrata or rice blast fungus), or bacteria (such as bacterial angular leaf spot of cucurbits), etc.

The term "salt of kasugamycin" refers to a compound formed by combining kasugamycin with an acid ion, such as kasugamycin hydrochloride, kasugamycin sulfate, kasugamycin carboxylate, kasugamycin phosphate, kasugamycin nitrate or kasugamycin carbonate.

The terms “around”, “about” and “substantially” refer to numerical variations within the normal experimental or measurement error range, such as “substantially” represents an error of no more than 15%, preferably no more than 10%.

The term "salt of kasugamycin" refers to a compound formed by the combination of kasugamycin and an acid radical ion, such as kasugamycin hydrochloride, kasugamycin sulfate, etc. In the present application, the mass of the salt of kasugamycin is calculated based on the mass of kasugamycin it contains.

The mass of the salt is measured by the mass of the kasugamycin acetamide it contains. For example, the composition contains 41.6g of kasugamycin hydrochloride and 1g of kasugamycin acetamide, then the mass ratio of kasugamycin hydrochloride to kasugamycin acetamide is 37.9:1 (calculation method: the molecular weight of kasugamycin hydrochloride is 416, and the molecular weight of kasugamycin is 379, therefore, 41.6g of kasugamycin hydrochloride contains kasugamycin 37.9, and based on the mass of kasugamycin, the mass ratio of kasugamycin hydrochloride to kasugamycin acetamide is 37.9:1).

Specific embodiment:

All commercial reagents and solvents were used without further purification.

1. Crystalline form of kasugamycin hydrochloride and its amorphous form

1. Preparation Example

Example 1-1: Preparation of Kasugamycin Hydrochloride Form I

(a) acidifying the fermentation liquid containing kasugamycin with oxalic acid and filtering;

(b) collecting the filtrate, adsorbing it with a strong acidic cation exchange resin and then analyzing it;

(c) concentrating the ammonium chloride solution through a nanofiltration membrane and further decolorizing it using activated carbon;

(d) filtering the decolorized liquid and concentrating it in vacuum to obtain a vacuum concentrated liquid;

(e) taking 10 ml of the vacuum concentrate prepared in step d, adding the concentrate dropwise to 20 times the volume of dioxane (poor solvent) at room temperature, stirring at 60 rpm, suspending for 30-40 min, and filtering to obtain a precipitate;

(f) The obtained precipitate is dried at room temperature to obtain a high-purity solid crystalline product, and the solid crystalline product is analyzed by 1H-NMR and XRPD.

1H-NMR analysis is as follows:

Several milligrams of solid sample were dissolved in dimethyl sulfoxide-d6 solvent and analyzed by NMR on Bruker AVANCE NEO 400 (Bruker, GER).

The XRPD analysis method is as follows:

The solid samples obtained in the experiment were analyzed by X-ray powder diffractometer Bruker D8 Advance (Bruker, GER). The 2θ scanning angle was from 3° to 45°, the scanning step was 0.02°, and the exposure time was 0.08 seconds. The test method was Cu target Kα1 radiation, voltage 40kV, current 40mA, and the sample pan was a zero background sample pan.

result:

The 1H-NMR of the solid crystalline product is shown in FIG1 , indicating that it is kasugamycin hydrochloride, and the XRPD is shown in FIG2 , which is called Form I.

Example 1-2: Preparation of Kasugamycin Hydrochloride Form I

A high-purity solid crystalline product was prepared according to steps a-f of Example 1-1, except that the poor solvent was replaced with ethylene glycol dimethyl ether, and the solid sample was subjected to 1H-NMR and XRPD tests by referring to the 1H-NMR and XRPD analysis methods described in Example 1-1, and the solid crystalline product was identified as kasugamycin hydrochloride Form I.

Example 1-3: Preparation of Kasugamycin Hydrochloride Form I

(a) taking 250 mg of the dried solid crystalline product of Example 1-1;

(b) dissolving the solid crystalline product in 4 ml of water;

(c) taking 1 ml of the solution in step b, placing the concentrated solution in an atmosphere of trifluoroethanol, and standing at room temperature until solid precipitates;

d) using a syringe to remove the solution in the system with solid precipitation, and performing 1H-NMR and XRPD tests on the solid sample by referring to the 1H-NMR and XRPD analysis methods described in Example 1-1. The results showed that the solid sample was Form I of kasugamycin hydrochloride.

Comparative Example 1-1: Preparation of Kasugamycin Hydrochloride Form II

A fermentation broth containing kasugamycin is provided, specifically: Streptomyces aureus is used as a producing bacterium, and the fermentation broth is obtained by multi-stage fermentation in a culture medium containing low-temperature soybean cake powder, soybean oil, yeast powder, liquid sugar and other raw materials. According to the purification and crystallization method described in the embodiment of CN106083951B, the fermentation broth is pretreated, ceramic membrane filtered, macroporous resin decolorized, nanofiltration concentrated and other steps are used for purification, and organic solvents acetone, methanol, ethanol, propanol or isopropanol are added to the crystallization process for crystallization, and 5 kinds of solid crystalline products are prepared;

The solid crystalline products were vacuum dried and then subjected to 1H-NMR and XRPD measurements. NMR showed that the solid crystalline products were all kasugamycin hydrochloride, and the 1H-NMR spectrum of the solid crystalline products obtained by crystallization with organic solvent methanol is shown in Figure 3. The solid samples were subjected to NMR and XRPD tests according to the 1H-NMR and XRPD analysis methods described in Example 1-1.

Results: 1H-NMR showed that the above solid crystalline products were all kasugamycin hydrochloride, and XRPD results showed that the above solid crystalline products were a crystal form, called crystal form II, wherein the XRPD spectrum of the solid crystalline product crystallized by organic solvent acetone is shown in Figure 4.

Comparative Example 1-2: Preparation of Kasugamycin Hydrochloride Form II

Streptomyces aureus was used as the producing bacteria, and the fermentation liquid was obtained by multi-stage fermentation in a culture medium containing low-temperature soybean cake powder, soybean oil, yeast powder, liquid sugar and other raw materials. Purification and crystallization were carried out according to the method described in the example of CN109666051B, and the crystallization method adopted was cooling crystallization or adding organic solvent acetone or ethanol crystallization, and three kinds of solid crystalline products were prepared respectively; 1H-NMR and XRPD tests were carried out on the solid samples according to the 1H-NMR and XRPD analysis methods described in Example 1-1.

Results: 1H-NMR showed that the above solid crystalline products were all kasugamycin hydrochloride, and XRPD showed that the above solid crystalline products were the same as the crystal form obtained in Comparative Example 1-1, both of which were Crystal Form II.

Example 1-4: Preparation of Kasugamycin Hydrochloride Amorphous

a) taking 100 mg of the dried solid crystalline product of Example 1-1;

b) dissolving the solid crystalline product in 2 ml of water;

c) freezing the solution in step b using dry ice and freeze drying it in a freeze dryer for 1 day;

d) The freeze-dried product was subjected to 1H-NMR and XRPD testing according to the 1H-NMR and XRPD analysis methods described in Example 1-1.

Results: 1H-NMR results showed that it was kasugamycin hydrochloride (as shown in FIG5 ), and XRPD results showed that it was an amorphous substance with no obvious characteristic peaks (as shown in FIG6 ).

Example 1-5: Powder Preparation

According to the methods of Comparative Example 1-1, Example 1-1 and Example 1-4, about 70 g of three kinds of dry solids, namely, Form II, Form I and amorphous material, were prepared respectively, with contents of 98.1%, 98.7% and 96.3%, respectively, for use as technical/mother drug of the preparation.

The above-mentioned technical/concentrated drug was crushed by a small vertical sand mill and zirconium beads (φ1.0-1.2 mm, Zhimo (Shanghai) New Material Technology Co., Ltd.), wherein the amount of zirconium beads was 40 g, the sand mill speed was 1500 r/min, the grinding time was 30 min, and the zirconium beads and fine powder were separated after grinding.

PSD analysis showed that the particle size D90 of the three technical/concentrated drug fine powders was 36.6μm-40.2μm.

4 g of each of the three technical/mother drug fine powders were taken, and the three fine powders were sieved with 96 g of diatomaceous earth through 200 meshes and then fully mixed to prepare three powders.

Example 1-6: Preparation of wettable powder

The dosage of each component of wettable powder is shown in the following table:

The wettable powder is prepared by the following steps:

1) According to the above table, the crystal form I and amorphous original drug/mother drug fine powders prepared in Examples 1-5 are respectively taken and set aside;

2) fully mixing copper oxychloride, sodium lauryl sulfate, sodium lignin sulfonate, white carbon black and diatomaceous earth, and grinding them by an ultra-fine grinder to obtain an additive powder;

3) The fine powder of the original drug/concentrated drug is fully mixed with the auxiliary agent powder prepared in step 2) to obtain a wettable powder.

2. Test analysis

(1) XRPD diffraction peak data analysis

The XRPD diffraction peak data of the crystalline form I of the compound kasugamycin hydrochloride described in the present application are shown in Table 1; the XRPD diffraction peak data of the crystalline form II are shown in Table 2.

Table 1 XRPD diffraction peak data of Form I

As can be seen from Table 1, the main characteristic diffraction peaks of the X-ray powder diffraction pattern of Form I using Cu-Kα radiation, represented by 2θ values ±0.2°, include 8.66, 10.11, 11.05 and 13.3, and also include any one or more characteristic diffraction peaks of 13.92, 15.56, 16.47 and 17.29, specifically including 8.66, 10.11, 11.05, 13.3, 13.92 and 15.56; or including 8.66, 10.11, 11.05, 13.3, 16.47 and 17.29; or including 8.66, 10.11, 11.05, 13.3, 13.92, 15.56, 16.47 and 17.29.

Table 2 XRPD diffraction peak data of Form II

As can be seen from Table 2, the main characteristic diffraction peaks of the X-ray powder diffraction pattern of Form II using Cu-Kα radiation and represented by 2θ values ±0.2° include 8.91, 10.03, 10.51, 12.12, 15.01, 16.40, 17.14 and 26.68; compared with Form I, the characteristic diffraction peaks of Form II do not contain characteristic diffraction peaks such as 8.66, 11.05 and 13.30, but contain characteristic diffraction peaks such as 8.91, 10.51 and 12.12.

As shown in FIG6 , the freeze-drying method produced an amorphous substance having no significant diffraction peak.

In summary, three solid products are prepared by the preparation method of the present invention, namely, Form I, Form II and amorphous matter. By comparing the XRPD spectra and diffraction peak data of Form I and Form II, it can be seen that the two belong to different crystal forms.

(2) Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA)

The DSC analysis method is as follows: The model of the differential scanning calorimeter is TA Discovery 2500 (TA, US). 1-2 mg of sample is accurately weighed and placed in a DSC Tzero sample pan with holes, and heated to the final temperature at a rate of 10 °C/min, and the nitrogen purge rate in the furnace is 50 mL/min.

The TGA analysis method is as follows: The model of the thermogravimetric analyzer is TA Discovery 55 (TA, US). 2-5 mg of sample is placed in a balanced open aluminum sample pan and automatically weighed in the TGA heating furnace. The sample is heated to the final temperature at a rate of 10 ° C / min, and the nitrogen purge rate at the sample is 60 mL / min, and the nitrogen purge rate at the balance is 40 mL / min.

Results: The DSC spectrum and TGA spectrum of the crystal form I described in the present application are shown in Figure 7; the DSC spectrum and TGA spectrum of the crystal form II are shown in Figure 8.

DSC shows that the crystal form I has a continuous endothermic signal in the range of 110±2°C to 210±2°C, and an endothermic signal at 226±2°C; compared with the height of the endothermic peak (or peak and valley), the crystal form I has a strong endothermic process at 226±2°C, and the endothermic process in the range is a weak endothermic process.

TGA shows that the crystalline form I has a weight loss of about 0.2% when heated to 100±2°C, a weight loss of less than 5% when heated from 100±2°C to 220±2°C, and a weight loss of less than 15.5% when heated from 220±2°C to 255C±2°C.

DSC shows that the crystal form II has a weak endothermic signal at 147±2°C and a strong endothermic signal at 215±2°C; compared with the height of the endothermic peak (or peak valley), the crystal form I has a strong endothermic process at 215±2°C and a weak endothermic process at 147±2°C.

TGA shows that the crystalline form II has a weight loss of less than 6% when heated to 195±2°C, and has a weight loss of less than 15% when heated from 220±2°C to 250±2°C.

DSC showed that the amorphous substance had endothermic signals at 73±2°C and 187±2°C;

TGA results show that the amorphous phase has a weight loss of less than 9% when heated to 175±2°C, and a weight loss of substantially less than 15% when heated from 175±2°C to 255±2°C.

(3) Polarized light microscopy (PLM) and particle size distribution (PSD)

The PLM analysis method is as follows: The laser particle size analyzer model is Mastersizer 3000 (Malvern Panalytical, UK). Take 20 mg of sample and disperse it in 8 mL of dispersant. Add sample dispersion unit until the shading degree is 10 20% and start measuring. Stirring speed is 2000 rpm, duration is 10 s, dispersant is ethanol, scattering model is Mie, and analysis model is universal.

The PSD analysis method is as follows: The laser particle size analyzer model is Mastersizer 3000 (Malvern Panalytical, UK). Take 20 mg of sample and disperse it in 8 mL of dispersant. Add sample dispersion unit until the shading degree is 10 20% and start measuring. Stirring speed is 2000 rpm, duration is 10 s, dispersant is ethanol, scattering model is Mie, and analysis model is universal.

The PLM image shows that Form I is mainly granular (as shown in FIG10 ); the particle size distribution analysis shows that the particle size D ₅₀ of Form I is 64.2 μm and the D ₉₀ is 119 μm (as shown in FIG13 ).

The PLM image shows that Form II is mainly needle-shaped (as shown in FIG11 ); the particle size distribution analysis shows that the particle size D ₅₀ of Form II is 24.7 μm and the D ₉₀ is 70.1 μm (as shown in FIG14 ).

The PLM image showed that the amorphous material was in the form of flakes (as shown in FIG. 12 ) and had a particle size D ₅₀ of 30.2 μm.

(4) Powder fluidity

In order to verify the processing properties of different solid products, the fluidity of the three fine powders prepared in Examples 1-5 was tested, and the fluidity of the fine powder was measured according to the "angle of repose" method in the "Guidelines for Determination of Powder Flowability" issued by the National Pharmacopoeia Commission on December 19, 2022. Among them, the test was carried out in a fixed funnel height mode (10 cm), the experimental temperature was room temperature, the humidity was about 5%, and the experimental fine powder dosage was 60 g. The experimental results are shown in Table 3.

Table 3 Powder fluidity evaluation

As can be seen from Table 3, the crystalline form I and the amorphous material after crushing under the same conditions have better powder fluidity than the crystalline form II, indicating that the crystalline form I and the amorphous form of kasugamycin hydrochloride are more conducive to the processing and utilization of subsequent mixtures and preparations, among which the kasugamycin hydrochloride with the crystalline form I structure shows the best effect in processing. The hygroscopicity and stability of the crystalline form I, the crystalline form II and the amorphous material will be further investigated in the future.

(5) DVS analysis

As shown in FIG15 , Form I only gained 1.57% weight at 95% humidity, gained 0.40% weight at 80% humidity, and lost 1.02% weight at 0% humidity, indicating that Form I has basically no hygroscopicity, indicating that Form I is also a stable solid product in a high humidity environment.

In comparison, Form II gained 2.2% weight by absorbing moisture under 95% high humidity and 0.74% weight by 80% humidity, showing weak hygroscopicity under high humidity; the amorphous material showed the common hygroscopic characteristics of amorphous materials.

(6) Stability determination

The stability of Form I and Form II was studied under high temperature (60°C), high humidity (25°C/92.5% RH), light (25°C/4500Lux) and accelerated (40°C/75% RH) conditions. Samples were taken at 7 days and 15 days, respectively, and the appearance, XRPD characterization and HPLC test were observed, and the results were compared with the original samples. The results showed that Form I was stable under high temperature, high humidity, light (25°C/4500Lux) and accelerated (40°C/75% RH) conditions. It remains stable under both illumination and accelerated stabilization conditions, specifically: the appearance remains unchanged, no crystal form transformation occurs, and the chemical purity does not change significantly (as shown in Table 4 and Figure 16), indicating that Form I is a stable solid crystalline product.

The stability results of Form II showed that the appearance of Form II remained stable under 15 days of high temperature and high humidity test conditions. After 7 days of light exposure test, the appearance changed from white to yellow, and after 15 days, the color changed to light brown, indicating that Form II was less stable under continuous light exposure.

Table 4 Experimental results of different environmental factors affecting Form I

(7) Antibacterial effect of potted plants

(7-1) Preparation of cucumber downy mildew spore suspension

Cucumber downy mildew bacteria were collected from naturally diseased leaves in Weinan Ecological Agriculture Demonstration Park. The mold layer on the back of the diseased leaves was removed with a brush dipped in distilled water. The leaves were placed in an artificial climate box at a temperature of 25°C and a relative humidity of 80% for dark culture for 16 hours to produce fresh sporangia. The fresh sporangia were then brushed into a culture dish filled with distilled water with a brush, and a spore suspension (concentration of 2×10 ⁵ -4×10 ⁵ /mL) was prepared after two filtrations for later use.

(7-2) Cucumber Indoor Cultivation

Cucumbers (Xinnong 58, 20 pots in total, 1 plant/pot) were planted in plastic pots with a diameter of 5 cm. The plants were cultured at a temperature of 20°C-28°C (day and night), a relative humidity of (90±5)%, and a fluorescent light for 12 h until they were about 1 meter tall. Twelve cucumber seedlings with similar growth potential were selected as samples for subsequent experiments.

(7-3) Preventive foliar application

Two leaves with the same height (calculated from the soil surface) and a leaf width of about 5-8 cm were selected from each of the 12 cucumber seedlings. The three mixed powders prepared in Examples 1-5 were respectively loaded in the same specification powder spraying bottles, and preventive application was carried out according to the powder pesticide application method: two powder sprays were carried out at a height of about 40 cm from the leaf surface, and each powder was sprayed on three plants and 6 leaves as a parallel experiment, and the blank group was sprayed with diatomaceous earth powder only. The leaves were marked and recorded accordingly (Group A was a blank group, Group B was a group containing crystalline form II powder, Group C was a group containing crystalline form I powder, and Group D was a group containing amorphous powder), and cucumber downy mildew was inoculated after continuing to cultivate for 48 hours according to the previous planting conditions.

(7-4) Cucumber downy mildew inoculation and curative application

The prepared spore suspension was poured into a watering can, sprayed twice on the leaves from a height of about 20 cm from the selected leaves, and immediately covered with a transparent plastic bag and fastened with the bag buckle after spraying, so as to complete the downy mildew inoculation of 24 leaves on 12 cucumber seedlings. After inoculation, the transparent plastic bag was removed at 2h, 6h and 10h respectively, the inoculated leaves were moistened with pure water by spraying, and then the bagging process was continued. 24h after the completion of inoculation, the preventive application method was used for therapeutic application (Group A was sprayed with diatomaceous earth powder, Group B was sprayed with powder containing crystal form II, Group C was sprayed with powder containing crystal form I, and Group D was sprayed with powder containing amorphous substance), and the leaves were cleaned 15 days after the therapeutic application, and the experimental leaf surface conditions were observed and recorded, and statistical analysis was performed.

The disease is graded according to the area of the lesions. The grading standards are:

Level 0: No lesions on leaves;

Level 1: The lesion area accounts for less than 5% of the entire leaf area;

Level 3: The lesion area accounts for 6-10% of the entire leaf area;

Level 5: The lesion area accounts for 11-25% of the entire leaf area;

Level 7: The lesion area accounts for 26-50% of the entire leaf area;

Level 9: The lesion area accounts for more than 50% of the entire leaf area;

Disease index = {[∑(number of diseased leaves at each level of each treatment × corresponding level value)]/total number of leaves investigated × 9} × 100;

Control effect (%) = [(blank control disease index - drug treatment disease index) / blank control disease index] × 100;

The results of the four groups of experiments are shown in Table 5, and some of the results are shown in Figure 17.

Table 5 Foliar control effect

According to the plant disease index and control effect, surprisingly, there are significant differences in the plant disease control effects between crystal form I, crystal form II and amorphous, among which the plant disease control effect of the powder containing amorphous is the best, followed by the powder containing crystal form I. This shows that different crystal forms and amorphous forms of kasugamycin hydrochloride may have differences in drug release, plant absorption and antibacterial ability, and the plant disease control effects of the amorphous form and crystal form I of kasugamycin hydrochloride are better than those of crystal form II.

2. Kasuga acetamide and its composition

1. Preparation Example

Example 2-1: Preparation of caesalpinia ceramide

(1) A high-yield Streptomyces microaureaus strain MKL-2 was selected as the fermentation bacteria, and the seed liquid after being cultured in the seed culture medium was inoculated into a 50L fermentation tank for fermentation, wherein the culture medium in the fermentation tank was composed of: 4.0% soybean cake powder, 0.07% potassium dihydrogen phosphate, 0.3% sodium chloride, 0.72% soybean oil, 1.1% corn steep liquor powder, 0.01% polyoxypropylene polyoxyethylene glycerol ether GPE 0.01%, 0.4% ammonia water, and 0.1% sodium citrate; the fermentation temperature was 30°C, the rotation speed was 60rpm, ammonia water was dripped into the fermentation process, and the pH of the fermentation system was controlled to be 7.1±0.1 to facilitate the production of caesalpinamide, and the culture was carried out for 120h;

(2) taking 20 L of caesalpinia oxyphylla fermentation broth, acidifying the fermentation broth with oxalic acid, and filtering the fermentation broth with a ceramic membrane;

(3) collecting the filtrate, adsorbing it with a strong acidic cation exchange resin, and then analyzing it with an ammonium chloride solution;

(4) filtering the analytical solution through a nanofiltration membrane and then concentrating it under vacuum, and collecting the concentrated filtrate;

(5) decolorizing the concentrated filtrate using activated carbon;

(6) adding the decolorized filtrate to an octadecylsilane bonded silica gel column and performing separation and purification using a mixture of 10% acetonitrile and 90% sodium hexanesulfonate aqueous solution as an eluent;

(7) The separated and purified liquid was dried to obtain about 6 g of caesalpinia ceramide powder.

The caesalpinia oxycarbazide powder can be directly used as the original pesticide or the mother pesticide of the pesticide.

Example 2-2: Preparation of caesalpinia acetamide aqueous solution

100 mg of the kasugalacetamide original drug prepared in Example 2-1 was added to 10 mL of deionized water to prepare a 1% kasugalacetamide aqueous solution, and an appropriate amount of the 1% aqueous solution was further diluted and prepared into 4 different kasugalacetamide aqueous solutions with mass fractions of 0.01%, 0.1%, 0.3% and 0.5%.

Example 2-3: Preparation of caesalpinia acetamide wettable powder

The following components are available:

1 g of caesalpinia acetamide technical prepared in Example 2-1;

Sodium lauryl sulfate (wetting agent) 4g;

Sodium lignin sulfonate (dispersant) 5g;

White carbon black (filler) 10g;

Diatomaceous earth (filler) 80g;

The above components are fully mixed according to the formula weight, and crushed by an ultrafine grinder to obtain 1% caesalpinia acetamide wettable powder.

Example 2-4: Preparation of Kasugamycin Aqueous Solution

85 mg of kasugamycin hydrochloride technical drug (provided by Xi'an Masidi Bioengineering Co., Ltd.) and 1.5 mg of kasugamycin technical drug prepared in Example 2-1 were dissolved in 50 ml of deionized water to prepare kasugamycin acetamide·kasugamycin aqueous solution.

Example 2-5: Preparation of kasugamycin wettable powder

The following components are available:

1 g of caesalpinia acetamide technical prepared in Example 2-1;

Kasugamycin hydrochloride technical 13 g (provided by Xi'an Masidi Bioengineering Co., Ltd.);

Sodium lauryl sulfate (wetting agent) 3g;

Sodium lignin sulfonate (dispersant) 5g;

White carbon black (filler) 10g;

Diatomaceous earth (filler) 68g;

The above components are fully mixed according to the weight of the formula, and crushed by an ultrafine grinder to obtain kasugamycin acetamide wettable powder.

2. Test analysis

(1) Determination of the purity of caesalpinia ceramide

Accurately weigh 38 mg of the caesium acetamide prepared in Example 2-1, dissolve it in water and dilute it to 25 mL to prepare a caesium acetamide standard stock solution; accurately transfer 1.25 mL of the above standard stock solution to a 25 mL volumetric flask, dilute it to the scale line with water and mix it evenly to complete the preparation of the test solution.

Instrument: Agilent HPLC 1100;

HPLC column: Agilent aichrombond-AQ C18 4.6mm*250mm*5μm;

Mobile phase: acetonitrile: 20 MM sodium hexanesulfonate aqueous solution (H ₃ PO ₄ adjusted to pH 3) = 5%: 95% (v/v);

Running time: 13min;

Column temperature: 30°C;

Injection volume: 5 μL;

Detection wavelength: 195nm;

Flow rate: 1.0 mL/min;

Signal bandwidth: 4nm.

Results: As shown in Figure 18, the retention time of caesalpinia suspensa was 10.469 min, and the purity factor was 999.843, indicating that the method described in Example 2-1 of the present invention can prepare caesalpinia suspensa with high purity. The high-purity caesalpinia suspensa can be used as a standard product.

(2) Analysis of caesalpinamide by liquid chromatography-mass spectrometry (HPLC-MSD)

Accurately weigh 25.06 mg of the standard prepared in Example 2-1 into a 25 mL volumetric flask, dissolve it in water, make up to volume, and mix well to obtain a standard stock solution.

Accurately transfer 0.50 mL of the above standard stock solution into a 10 mL volumetric flask, then dilute to the mark with water and mix well. The analysis conditions are as follows:

Liquid phase conditions:

Instrument: Agilent 1100/1200HPLC;

HPLC column: Waters CORTECS HILIC, 150 x 4.6 mm, 2.7 μm;

Mobile phase: acetonitrile: 50 mmol ammonium formate solution = 75:25 (V/V);

Column temperature: 30°C;

Injection volume: 5 μL;

Running time: 12.00min;

Flow rate: 1.00 mL/min;

Detection wavelength: 205nm;

Signal bandwidth: 4nm;

Reference wavelength: off;

Reference bandwidth: off;

Spectral range: All (190nm-400nm).

MS conditions:

MS source: API-ES;

Polarity: positive;

Collision voltage: 70V;

Drying gas temperature: 350℃;

Drying gas flow rate: 12.0L/min;

Cap voltage: 3000V;

Acquisition mode: Scan;

Scanning range: 100-1000amu.

result:

The UV chromatogram (DAD) and total ion current (TIC) are shown in Figure 19, where the DAD retention time is about 6.8 min and the TIC retention time is about 7.0 min; the mass spectrum is shown in Figure 20, and the main fragment ions m/z are 171, 351, 373, and 701, which are consistent with the structural characteristics of caesalpinia oxyphylla.

(3) 1H-NMR analysis of caesalpinamide

The analysis method is as follows:

7 mg of solid sample was dissolved in heavy water solvent and analyzed by NMR on Bruker AVANCE NEO 400 (with BBO probe, Bruker, GER).

Results: The NMR data are shown in Figure 21, and the relevant features are consistent with the chemical structure characteristics of caesalpinia oxyphylla.

(4) Infrared test of pyroacetamide (FTIR)

Weigh about 2 g of spectrally pure KBr, grind it and dry it at 105°C for 2 h, then transfer the KBr to a desiccator and cool it to room temperature. Weigh 10 mg of kauriacetamide analytical standard, dry it at 105°C for 2 h, then transfer it to a desiccator and cool it to room temperature.

About 200 mg of anhydrous KBr powder was weighed into an agate mortar, ground and pressed into tablets by an infrared spectroscopy tablet press. The potassium bromide salt tablets were scanned by an infrared spectrometer.

Weigh about 2 mg of dry kauriacetamide analytical standard into about 200 mg of KBr, mix and grind evenly. Use an infrared spectroscopy tableting device to compress the mixture into a semi-transparent tablet and then scan it with an infrared spectrometer.

Results: The FTIR results are shown in Figure 22. The relevant infrared characteristic peaks are consistent with the absorption peak positions of the corresponding groups in the chemical structure of kaempferol acetamide.

(5) Detection of Kasugamycin Aqueous Composition

The kasugamycin aqueous solution was analyzed by HPLC under the following analysis conditions:

Instrument: Agilent HPLC 1100;

HPLC column: Agilent aichrombond-AQ C18 4.6mm*250mm*5μm;

Mobile phase: acetonitrile: 20 MM sodium hexanesulfonate aqueous solution (H ₃ PO ₄ adjusted to pH 3) = 5%: 95% (v/v);

Running time: 13min;

Column temperature: 30°C;

Detection wavelength: 195nm;

Flow rate: 1.0 mL/min;

Signal bandwidth: 4nm;

Injection volume: 5 μL.

Results: As shown in Figure 23, the retention times of kasugamycin and kasugamycin amide were 8.462 min and 10.464 min, respectively, indicating that the detection method of the present invention has good separation and sensitivity response to kasugamycin and kasugamycin amide with similar structures. The detection method of the present invention can effectively identify kasugamycin and kasugamycin amide with similar structures.

(6) Antibacterial test of caesalpinia cerevisiae

Raw materials: Prepare caesalpinia acetamide technical drug according to the method of Example 2-1.

Pathogenic bacteria: Curvularia zeae was collected from diseased corn leaves in a farm in Weinan City, isolated and purified by tissue separation method, and then identified; Rice blast was collected from diseased rice in the field, isolated, purified and cultivated.

Take 1 mL of caesium acetamide aqueous solution with mass fractions of 0.01%, 0.1%, 0.3%, 0.5% and 1% in Example 2-1, mix them evenly with PSA culture medium in a ratio of 1 mL to 9 mL, and pour them into culture dishes to prepare drug-containing plates with final concentrations of 0.001%, 0.01%, 0.03%, 0.05% and 0.1%, respectively, and use PSA culture medium with an equal amount of sterile water as a blank control.

The cultured pathogens were punched to prepare a 5 mm diameter bacterial cake, placed in the above plate, and cultured in an incubator at 28 ± 1 ° C. When the edge of the colony in the control group was close to the wall of the culture dish, the colony diameter of each treatment group was measured by the cross method, and the relative inhibition percentage was calculated. The calculation formula is as follows:

Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100%;

An inhibition rate of 1-24% was considered effective (△), 25%-39% was considered effective (▲), and ≥40% was considered markedly effective (▲▲).

The inhibition rate determination results and antibacterial test results are shown in Table 6 and Figure 24.

Table 6 Inhibition rate of casulacetamide on two pathogens

It can be seen from Table 6 and Figure 24 that the inhibitory effect of kasugamycin on rice blast fungus is similar to that of kasugamycin, both of which have high sensitivity. A concentration of 0.01% shows an effective effect, and a concentration of 0.03% shows a significant effect, with an inhibition rate of 41.6%. At the same time, kasugamycin also has a significant inhibitory effect on corn Curvularia serrata, which is insensitive to kasugamycin. At a concentration of 0.01%, kasugamycin is effective against corn Curvularia serrata, and at a concentration of 0.05%, it is significantly effective, with an inhibition rate of 42.2%. It can be seen from the above experimental results that kasugamycin and kasugamycin have similarities in the control effects of certain pathogens, which may be related to their similar chemical structures. However, for some pathogens, their control effects have greater This may indicate that the two have significant differences in their mechanisms of action and/or effects.

(7) Indoor toxicity test of kasugamycin combination against rice blast

(7-1) Inhibition of medium concentration (EC50)

Raw materials: kasugamycin technical (prepared according to the method of Example 2-1); kasugamycin technical (kasugamycin hydrochloride, provided by Xi'an Masidi Bioengineering Co., Ltd.); rice blast was collected from diseased rice in the field, separated, purified and cultivated to obtain.

Test method: refer to the "Agricultural Industry Standard of the People's Republic of China NY/Tll54.7-2006", potting method. Select three-leaf rice seedlings with consistent growth potential, 2 seedlings per pot, and 5 pots of test rice seedlings for each treatment. The rice blast fungus was cultured on tomato oat agar medium, and the spores were washed with sterile water after spore production to make a suspension of 1×10 ⁵ spores/mL, which was evenly sprayed and inoculated on the test rice seedlings. After inoculation, a black plastic bag was put on to keep moisture and culture for 24 hours. After 24 hours of inoculation, the drug treatment was carried out. Each drug was set with 5 concentration gradients, and sprayed with a Potter spray tower at a pressure of 50Psi, about 5mL per pot. After spraying, the rice seedlings were placed at 28℃ and a relative humidity of 92%. After 8 days, the disease index of the whole leaf was investigated according to the grading standard of rice blast, and the control effect was calculated.

Disease index = {[∑(number of diseased leaves at each level of each treatment × corresponding level value)]/total number of leaves investigated × 9} × 100;

The control effect (%) = [(blank control disease index - drug treatment disease index) / blank control disease index] × 100.

The control effect was converted into a probability value (y), the concentration of the drug solution (ug/ml) was converted into a logarithmic value (x), and the toxicity equation and the inhibition concentration EC50 were calculated by the least squares method.

(7-2) Co-toxicity experiment of kasugamycin hydrochloride combination

The toxicity index and co-toxicity coefficient (CTC) of the kasugamycin-acetamide combination were calculated according to the Sun Yunpei method.

Single-dose toxicity index = (standard agent EC50/test single-dose EC50) × 100;

Measured toxicity index (ATI) = (standard agent EC50/test agent EC50) × 100;

Theoretical toxicity index (TTI) of mixture = toxicity index of agent A × percentage of A in the mixture + toxicity index of agent B × percentage of B in the mixture;

Co-toxicity coefficient (CTC) = [measured toxicity index (ATI) of the mixture/theoretical toxicity index (TTI) of the mixture] × 100;

When CTC≤80, the combination exhibits an antagonistic effect; when 80<CTC<120, the combination exhibits an additive effect; and when CTC≥120, the combination exhibits a synergistic effect.

The results of the co-toxicity experiment are shown in Table 7.

Table 7 Co-toxicity experimental data

The co-toxicity experiment of the kasugamycin hydrochloride composition showed that kasugamycin and kasugamycin acetamide showed a synergistic effect within the mass ratio range of 13:1-130:1. When the two were mixed in a certain proportion, they had a good synergistic effect and could improve the control effect on rice blast.

From the above experimental results, it can be seen that although the chemical structures of kasugamycin and kasugamycin are similar, they have obvious differences in the prevention and treatment effects of pathogenic bacteria, and there is a certain complementarity and synergy between the two. Although the internal mechanism is still unclear, it is speculated that it may be because kasugamycin has better lipophilicity than kasugamycin, which improves the permeability of the substance on the cell wall of pathogenic bacteria.

In the composition of kasugamycin acetamide and kasugamycin hydrochloride, kasugamycin hydrochloride may be kasugamycin hydrochloride crystal form I or its amorphous form.

The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and purpose of the present invention should be included in the scope of protection of the present invention.

Claims (12)

An agricultural antibiotic kasugamycin hydrochloride crystal form I, characterized in that the crystal form I uses Cu-Kα radiation, and the characteristic diffraction peaks of the X-ray powder diffraction pattern expressed as 2θ values ±0.2° include 8.66, 10.11, 11.05 and 13.3. The kasugamycin hydrochloride form I according to claim 1, characterized in that the kasugamycin hydrochloride form I uses Cu-Kα radiation, and the characteristic diffraction peaks of the X-ray powder diffraction pattern expressed as 2θ values ±0.2° also include 13.92 and 15.56; preferably, the characteristic diffraction peaks of the X-ray powder diffraction pattern expressed as 2θ values ±0.2° also include 16.47 and 17.29; more preferably, the characteristic diffraction peaks of the X-ray powder diffraction pattern expressed as 2θ values ±0.2° also include any one or more of 18.59, 19.25, 20.41 and 20.78. The kasugamycin hydrochloride crystal form I according to claim 1 or 2, characterized in that the particle size D ₉₀ of the crystal form I is 10 μm-200 μm; preferably, the particle size D ₉₀ of the crystal form I is 15 μm-150 μm. A technical drug, a parent drug, a pesticide preparation or a pesticide composition, characterized in that the technical drug, the parent drug, the pesticide preparation or the pesticide composition contains the kasugamycin hydrochloride crystal form I according to any one of claims 1 to 3. The technical drug, parent drug, pesticide preparation or pesticide composition according to claim 4, characterized in that the weight percentage of the kasugamycin hydrochloride crystal form I in the technical drug, parent drug, pesticide preparation or pesticide composition is at least 0.001%, The mass of kasugamycin hydrochloride is calculated based on the mass of kasugamycin free base contained therein. The technical drug, the parent drug, the pesticide preparation or the pesticide composition according to claim 4 or 5, characterized in that, based on the total mass of the technical drug, the parent drug, the pesticide preparation or the pesticide composition, it contains casulacetamide and/or a salt of casulacetamide in an amount of not less than 0.01%; preferably, the content of casulacetamide and/or a salt of casulacetamide is not less than 0.03%; The mass of the salt of caesalpinia acetamide is calculated based on the mass of the free base of caesalpinia acetamide contained therein. The technical, parent drug, pesticide preparation or pesticide composition according to claim 6, characterized in that the mass ratio of kasugamycin hydrochloride to kasugamycin acetamide and/or the salt of kasugamycin acetamide is 10:1-200:1; more preferably 13:1-130:1. The technical drug, mother drug, pesticide preparation or pesticide composition according to claim 4, 5 or 7, characterized in that the dosage form of the pesticide preparation or pesticide composition is selected from powders, granules, large granules, fine granules, microgranules, microcapsule granules, wettable powders, oil-dispersible powders, water-dispersible granules, emulsifiable granules, effervescent granules, dispersible tablets, effervescent tablets, sustained-release agents, sustained-release blocks, sustained-release tubes, sustained-release particles, soluble powders, soluble granules, soluble tablets, soluble solutions, aqueous solutions, soluble gels, Any one of oil, film-spreading oil, ultra-low volume liquid, ultra-low volume microcapsule suspension, emulsifiable concentrate, latex, dispersible liquid, paste, concentrated gel, water emulsion, oil emulsion, microemulsion, ointment, suspension, microcapsule suspension, oil suspension, suspoemulsion, seed treatment dispersible powder, seed treatment soluble powder, seed treatment liquid, seed treatment emulsion, seed treatment suspension, suspension seed coating agent, seed treatment microcapsule suspension; preferably, the pesticide formulation is a powder or a wettable powder. The technical drug, parent drug, pesticide preparation or pesticide composition according to claim 8, characterized in that the particle size D ₉₀ of the crystalline form I of kasugamycin hydrochloride in the powder or wettable powder is 5 μm-80 μm. The use of the kasugamycin hydrochloride crystal form I according to any one of claims 1 to 3, and the technical drug, mother drug, pesticide preparation or pesticide composition according to claims 4 to 9 in plant disease control. Use of the kasugamycin hydrochloride crystal form I according to any one of claims 1 to 3, the technical drug, the mother drug, the pesticide preparation or the pesticide composition according to claims 4 to 9 in the preparation of a plant disease control preparation. The use according to claim 10 or 11, characterized in that the plant disease is cucumber downy mildew, corn Curvularia leaf spot or rice blast.