Crystal form of kasugamycin hydrochloride, kasugamycin acetamide, and composition of crystal form and kasugamycin acetamide

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 its composition

[0001] Priority information

[0002] This invention claims priority and benefit to patent application CN202310969303.3 filed with the China National Intellectual Property Administration on August 3, 2023 and patent application CN202311103321.X filed with the China National Intellectual Property Administration on August 30, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of agricultural technology, specifically relating to the crystal form of kasugamycin hydrochloride, kasugamycin acetamide, and their compositions. Background Technology

[0004] 5-Amino-2-methyl-6-(2,3,4,5,6-hydroxycyclohexyloxo)pyran-3-ylamino-α-iminoacetic acid (CAS 6980-18-3, Kasugamycin) belongs to the aminoglycoside class of antibiotics. Since its discovery in the 1960s and 1970s, Kasugamycin has been widely used to control various diseases in a variety of crops, including rice, potatoes, cabbage, and melons. Due to its excellent control efficacy and environmentally friendly characteristics, Kasugamycin is currently a major biological pesticide product for crop disease control. Kasugamycin is extremely unstable in alkaline environments and is a hygroscopic compound. Considering transportation, storage, and quality control, more stable and less hygroscopic Kasugamycin salt products are desired by production and processing enterprises.

[0005] 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 controlling pathogens. CN108822167A reported kasugamycin hydrochloride and that it is a white needle-like or flaky crystal. CN106083951B and CN109666051B reported the extraction of kasugamycin hydrochloride by crystallization in water with solvents such as acetone, methanol, and ethanol.

[0006] Although existing technologies have disclosed methods for preparing high-purity kasugamycin hydrochloride, the crystalline products obtained by different crystallization methods often differ, with significant variations in their physicochemical properties and the corresponding performance in formulation processing. Therefore, a stable crystalline product of kasugamycin salt that is conducive to formulation processing and storage is a goal pursued by biopesticide companies.

[0007] In addition, the applicant unexpectedly discovered that the Streptomyces microaureaus strain, in addition to producing kasugamycin, also produces kasugamycin acetamide (CAS 6189-95-3 or 21256-64-4 or 38420-31-4, structure as shown in Formula 1). However, there are no reports on the preparation process and detection methods of kasugamycin acetamide, and its efficacy and application have not yet been studied and developed.

[0008] Summary of the Invention

[0009] In view of the above-mentioned problems in the prior art, the present invention provides a technical solution to solve the above problems.

[0010] According to one aspect of the present invention, a crystalline form I of the agricultural antibiotic kasugamycin hydrochloride is provided, wherein the characteristic diffraction peaks of the X-ray powder diffraction pattern of crystalline form I, expressed in 2θ ± 0.2° using Cu-Kα radiation, include 8.66, 10.11, 11.05, and 13.3; preferably, the characteristic diffraction peaks of the X-ray powder diffraction pattern of crystalline form I, expressed in 2θ ± 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 in 2θ ± 0.2° include... 8.66, 10.11, 11.05, 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 in 2θ values ​​± 0.2° include 8.66, 10.11, 11.05, 13.3, 13.92, 15.56, 16.47, 17.29, 18.59, and 19.25, or include 8.66, 10.11, 11.05, 13.3, 13.92, 15.56, 16.47, 17.29, 18.59, 19.25, 20.41, and 20.78.

[0011] Optional differential scanning calorimetry (DSC) showed that the kasugamycin hydrochloride crystal form I had a continuous endothermic signal in the range of 110±2℃ to 210±2℃, and an endothermic signal at 226±2℃; in terms of the height (or valley) of the endothermic peak, crystal form I had a strong endothermic process at 226±2℃ and a weak endothermic process in the range of 110±2℃ to 210±2℃.

[0012] Thermogravimetric analysis (TGA) showed that the crystal form I had a weight loss of less than 5% during heating from 100±2℃ to 220±2℃ and a weight loss of less than 15.5% during heating from 220±2℃ to 255±2℃.

[0013] Optionally, the kasugamycin hydrochloride crystal form I described in this application is a hydrate.

[0014] In one specific embodiment, the crystal form I of the compound kasugamycin hydrochloride described in this application is granular or rod-shaped. To meet the needs of actual production, facilitate solid-liquid filtration, and facilitate subsequent processing of pesticide solid formulations, the particle size D of crystal form I is... 90 The particle size is 10μm-200μm. Preferably, the crystal form I has a grain size D. 90 The particle size ranges from 15μm to 150μm and from 20μm to 120μm, for example, crystal form I with a particle size D. 90 It 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.

[0015] In one embodiment, the crystalline solid can be prepared by methods such as dissolution-backdipation, liquid-gas phase diffusion, single-solvent room temperature suspension, single-solvent high temperature suspension, binary solvent forward drop, single-solvent cooling, and binary solvent cooling.

[0016] Preferably, crystal form I is prepared by methods such as dissolution-back-dropping or liquid-gas phase diffusion, and is not limited to the evidence provided in the preparation examples of this application. Crystal form I of kasugamycin hydrochloride described in this invention is also prepared by other crystallization methods such as single solvent room temperature suspension, single solvent high temperature suspension, binary solvent forward drop, single solvent cooling, and binary solvent cooling.

[0017] Preferably, the amorphous material is prepared by freeze-drying, which can be selected as a circulating freeze dryer or a medium freeze dryer (such as dry ice freeze dryer or nitrogen freeze dryer).

[0018] Preferably, the dissolution-precipitation back-tipping method includes the following steps: adding an aqueous solution containing kasugamycin hydrochloride dropwise to a poor solvent, suspending the precipitate for 10-120 minutes after precipitation, and filtering to obtain the solid. The dropwise addition process may or may not involve stirring; the stirring rate is 20-120 rpm; the poor solvent is dioxane or ethylene glycol dimethyl ether.

[0019] Preferably, the liquid-gas phase diffusion method includes the following steps: weighing a certain amount of kasugamycin hydrochloride solution dissolved in a good solvent, placing the clear solution in an atmosphere of a poor solvent, allowing it to stand at room temperature until solid precipitates, and then filtering. The good solvent is water or formamide, and the poor solvent is trifluoroethanol.

[0020] According to another aspect of the present invention, an amorphous product of kasugamycin hydrochloride is provided.

[0021] Optionally, the amorphous material has X-ray powder diffraction peaks as shown in Figure 6.

[0022] DSC showed that the amorphous material had endothermic signals at 73±2℃ and 187±2℃.

[0023] TGA results showed that the amorphous material had a weight loss of less than 9% when heated to 175±2℃, and a weight loss of less than 15% when heated from 175±2℃ to 255±2℃.

[0024] Optionally, the amorphous form of kasugamycin hydrochloride has a sheet-like structure.

[0025] Optionally, the amorphous object D 90 The value is 10μm-200μm, preferably 15μm-150μm, and more preferably 20μm-120μm.

[0026] According to another aspect of the present invention, a technical material, parent material, pesticide formulation, or pesticide composition is provided, wherein the technical material, parent material, pesticide formulation, or pesticide composition contains the crystal form I and / or amorphous material described above.

[0027] According to another aspect of this application, the use of the crystal form I, the amorphous material, the pesticide formulation, or the pesticide composition described above in the preparation of plant disease control formulations is provided.

[0028] According to another aspect of this application, the application of the crystal form I, the amorphous material, the pesticide formulation or pesticide composition described above in the prevention and control of plant diseases is provided.

[0029] Furthermore, the plant disease mentioned is cucumber downy mildew or rice blast.

[0030] Furthermore, based on total mass, the content of crystal form I and / or amorphous substances in the technical grade 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%, 90%, or 95%.

[0031] Furthermore, based on total mass, the content of crystalline form I and / or amorphous substances in the parent 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%, 90%, or 95%.

[0032] Further, the pesticide formulation or pesticide composition contains the aforementioned crystalline form I and / or amorphous substances and adjuvants. Optionally, the weight percentage of the crystalline form I and / or amorphous substances in the pesticide formulation or pesticide composition is at least 0.001%, for example, the weight percentage of the crystalline form I and / or amorphous substances in the formulation 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 substances in the formulation is 0.1%-10%, 0.5-7%, or 1-5%.

[0033] Optionally, the dosage form of the formulation is selected from any one of the following: powder, granules, large granules, fine granules, microparticles, microcapsule granules, wettable powder, oil-dispersible powder, water-dispersible granules, emulsion granules, 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, spreading oils, ultra-low volume liquids, ultra-low volume microcapsule suspensions, emulsifiable concentrates, latexes, dispersible liquids, pastes, concentrated gels, water emulsions, oil emulsions, microemulsions, greases, suspensions, microcapsule suspensions, oil suspensions, suspension emulsions, seed-treated dispersible powders, seed-treated soluble powders, seed-treated liquids, seed-treated emulsions, seed-treated suspensions, suspension seed coating agents, and seed-treated microcapsule suspensions. Preferably, the crystalline form I or amorphous solids of the present invention exhibit excellent processability and solid application control effects in pesticide solid formulations. Specifically, the crystalline form I or amorphous solids of the present invention are used in wettable powders or powder formulations; wherein the particle size D90 of the crystalline form I and / or amorphous solids in the powder or wettable powder is 5μm-80μm, preferably 10μm-50μm, or 15μm-45μm, or 20μm, 30μm, 40μm, 60μm and 70μm.

[0034] According to another aspect of the present invention, the application of the crystal form I, the amorphous material, the technical grade or parent material, or the formulation or pesticide composition described above in the process of controlling plant diseases is provided.

[0035] According to another aspect of this application, the use of the crystal form I, the amorphous material, the technical grade or parent material, or the formulation or pesticide composition described above in the preparation of plant disease control agents is provided.

[0036] The inventors accidentally discovered and confirmed that kasugamycin has excellent bactericidal and antibacterial effects, and its effect is even more obvious when combined with kasugamycin.

[0037] Therefore, in another aspect, the present invention also provides a kasugamycin acetamide composition, wherein the kasugamycin acetamide composition contains kasugamycin acetamide and / or a salt of kasugamycin acetamide, and the content of the kasugamycin acetamide and / or the salt of kasugamycin acetamide is not less than 0.01%, or 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% based on the total mass of the composition, wherein the mass of the kasugamycin acetamide salt is based on the mass of the kasugamycin acetamide it contains.

[0038] Considering factors such as cost, the content of kasugamycin and / or the salt of kasugamycin, based on the total mass of the composition, shall not exceed 10%, preferably not exceed 8% or not exceed 7%.

[0039] Optionally, the composition is any one of the technical grade drug, parent drug, or formulation; further, the dosage form of the formulation is selected from any one of the following: powder, granules, large granules, fine granules, microparticles, microcapsule granules, wettable powder, oil-dispersible powder, water-dispersible granules, emulsion granules, effervescent granules, dispersible tablets, effervescent tablets, sustained-release agents, sustained-release blocks, sustained-release tubes, sustained-release granules, soluble powder, soluble granules, soluble tablets, soluble solutions, aqueous solutions, soluble gels, oils, spreading oils, ultra-low volume liquids, ultra-low volume microcapsule suspensions, emulsifiable concentrates, latexes, dispersible liquids, pastes, concentrated gels, water emulsions, oil emulsions, microemulsions, greases, suspensions, microcapsule suspensions, oil suspensions, suspension emulsions, seed-treated dispersible powders, seed-treated soluble powders, seed-treated liquids, seed-treated emulsions, seed-treated suspensions, suspension seed coatings, and seed-treated microcapsule suspensions.

[0040] Another aspect of the present invention is to provide the application of the above-described composition in the prevention and control of plant diseases.

[0041] Another aspect of the present invention is to provide the use of the composition described above in the preparation of plant disease control agents.

[0042] Optionally, the plant disease is Curvularia zeylans leaf spot and / or rice blast. Optionally, another aspect of the invention provides a method for preparing high-purity Kasugamycin acetamide, comprising the following steps: a fermentation broth purification step and an octadecylsilane-bonded silica gel column separation step.

[0043] Optionally, the fermentation broth purification step includes the following steps:

[0044] 1) Acidify and filter the fermentation broth;

[0045] 2) The filtrate is adsorbed by resin and then desorbed;

[0046] 3) The eluent is purified by nanofiltration.

[0047] Optionally, nanofiltration for impurity removal also includes a decolorization process.

[0048] Furthermore, the acidifying agent in step 1 of the fermentation broth purification process includes at least one of sulfuric acid, hydrochloric acid, phosphoric acid, or oxalic acid.

[0049] Furthermore, the resin used in step 2 of the fermentation broth purification process is a strongly acidic cation exchange resin.

[0050] Furthermore, in step 2 of the fermentation broth purification process, the resin adsorption and desorption agent includes at least one of ammonium chloride solution, sodium hydroxide solution, or sodium chloride solution.

[0051] Optionally, in the silica gel column separation step, the elution phase is a mixed solution of acetonitrile and sodium alkyl sulfonate;

[0052] Further, the sodium alkyl sulfonate may optionally be sodium hexane sulfonate or sodium dodecyl sulfonate, and the acetonitrile:sodium alkyl sulfonate solution = 1%:99%-10%:90%.

[0053] The applicant unexpectedly discovered that kasugamycin acetamide exhibits a highly sensitive response at a UV detection wavelength of 195 nm in liquid chromatography. Therefore, another aspect of the present invention provides a method for detecting kasugamycin acetamide, comprising the following steps:

[0054] Dissolve the sample to be tested in deionized water to prepare a solution with a concentration of 0.001–4 mg / mL. Take an appropriate amount of the solution to be tested and inject it into a liquid chromatograph. Determine whether kasugamycin acetamide is present based on the peak position, or determine whether kasugamycin acetamide is present by comparing the peak position with that of kasugamycin acetamide standard. The detection wavelength is 195 nm.

[0055] Optionally, the HPLC column is a reversed-phase C18 packing material;

[0056] Optionally, the mobile phase is an aqueous solution of acetonitrile and sodium alkyl sulfonate;

[0057] Optionally, running time: 10-20 minutes;

[0058] Optional, column temperature: 30±2℃;

[0059] Optionally, the flow rate is 0.5–2 mL / min;

[0060] Optionally, the signal bandwidth is 4nm-8nm;

[0061] Optionally, the injection volume is 5 μL.

[0062] Further, the sodium alkyl sulfonate may optionally be sodium hexane sulfonate or sodium dodecyl sulfonate, and the acetonitrile:sodium alkyl sulfonate solution = 1%:99%-10%:90%.

[0063] As can be determined by those skilled in the art, when a standard is available, the presence of kasugamycin can be determined by comparing the liquid phase data (chromatogram) of the sample to be tested with the liquid phase data (chromatogram) of the standard under the same liquid phase analysis conditions. Furthermore, the content (concentration) of kasugamycin in the sample to be tested can be determined by the content (concentration) of the standard.

[0064] Based on the beneficial effects of the combination of kasugamycin and kasugamycin acetamide, another aspect of the present invention provides a composition containing kasugamycin and / or a salt of kasugamycin and / or a salt of kasugamycin, wherein the mass ratio of kasugamycin and / or a salt of kasugamycin to the salt of kasugamycin and / or a salt of kasugamycin is 10:1-200:1, preferably 13:1-130:1, or optionally, the mass ratio of kasugamycin and / or a salt of kasugamycin to the salt of kasugamycin and / or a salt of kasugamycin is 11:1, 13:1, 14:1, 15:1, 16:1, 20:1, or 25:1. The ratio of 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, 110:1, 120:1, 140:1, 150:1, 160:1, 170:1, 180:1, or 190:1 is any one value or a range between any two values; wherein, the mass of the kasugamycin salt is based on the mass of kasugamycin it contains, and the mass of the kasugamycin salt is based on the mass of kasugamycin it contains.

[0065] Optionally, the composition is any one of a technical grade drug, a parent drug, or a formulation.

[0066] Optionally, based on the total mass of the preparation, the total content of kasugamycin and / or kasugamycin salts and kasugamycin acetamide and / or kasugamycin acetamide 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 the kasugamycin acetamide salts is based on the mass of kasugamycin contained therein, and the mass of the kasugamycin salts is based on the mass of kasugamycin contained therein.

[0067] The dosage form of the formulation is selected from any one of the following: powder, granules, large granules, fine granules, microparticles, microcapsule granules, wettable powder, oil-dispersible powder, water-dispersible granules, emulsion granules, 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, spreading oils, ultra-low volume liquids, ultra-low volume microcapsule suspensions, emulsifiable concentrates, latexes, dispersible liquids, pastes, concentrated gels, water emulsions, oil emulsions, microemulsions, greases, suspensions, microcapsule suspensions, oil suspensions, suspension emulsions, seed-treated dispersible powders, seed-treated soluble powders, seed-treated liquids, seed-treated emulsions, seed-treated suspensions, suspension seed coating agents, and seed-treated microcapsule suspensions.

[0068] Based on the beneficial effects of kasugamycin hydrochloride crystal form I and / or its amorphous form on stability and ease of processing, the salt of kasugamycin in the composition is preferably kasugamycin hydrochloride crystal form I and / or its amorphous form.

[0069] Another aspect of the present invention is to provide the application of the composition described above in the prevention and control of plant diseases.

[0070] Another aspect of the present invention is to provide the use of the composition described above in the preparation of plant disease control agents.

[0071] Optionally, the plant disease is Curvularia micrantha leaf spot and / or rice blast.

[0072] The positive and progressive effects of this invention are as follows:

[0073] The applicant believes that the crystalline form I provided in this application possesses excellent stability, exhibits virtually no hygroscopicity, and remains stable under conditions of light, high temperature, high humidity, and accelerated processes, effectively extending the shelf life of the pesticide and better meeting the requirements for pesticide production, processing, transportation, and storage. Simultaneously, crystalline form I has also achieved good technical results in plant disease control. Unexpectedly, the amorphous form demonstrated the best effect in plant disease control, with even more outstanding results in agricultural end-use applications.

[0074] The inventors unexpectedly discovered that kasugamycin acetamide has excellent bactericidal and antibacterial effects.

[0075] When kasugamycin and / or kasugamycin salts are controlled in a certain ratio, a beneficial synergistic effect is observed, which can be better applied to the prevention and control of plant diseases.

[0076] This invention also proposes a method for preparing high-purity kasugamycin acetamide, the obtained high-purity kasugamycin acetamide can be used as a standard or applied in the detection of active ingredients. Attached Figure Description

[0077] Figure 1 shows the 1H NMR spectrum of the crystal form of kasugamycin hydrochloride compound;

[0078] Figure 2 shows the X-ray powder diffraction (XRPD) pattern of crystal form I, where the horizontal axis represents 2θ (°) and the vertical axis represents intensity (count).

[0079] Figure 3 shows the 1H-NMR spectrum of crystal form II of kasugamycin hydrochloride compound;

[0080] Figure 4 shows the X-ray powder diffraction (XRPD) pattern of crystal form II, where the horizontal axis represents 2θ (°) and the vertical axis represents intensity (count).

[0081] Figure 5 shows the 1H-NMR spectrum of the amorphous kasugamycin hydrochloride.

[0082] Figure 6 shows the X-ray powder diffraction (XRPD) pattern of the amorphous material, where the horizontal axis represents 2θ (°) and the vertical axis represents intensity (count).

[0083] Figure 7 shows the differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) spectra of crystal form I;

[0084] Figure 8 shows the differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) spectra of crystal form II;

[0085] Figure 9 shows the differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) spectra of the amorphous material;

[0086] Figure 10 shows the polarized light microscopy (PLM) analysis spectrum of crystal form I;

[0087] Figure 11 shows the polarized light microscopy (PLM) image of crystal form II;

[0088] Figure 12 shows polarized light microscopy (PLM) images of amorphous objects;

[0089] Figure 13 shows the particle size distribution (PSD) image of crystal form I;

[0090] Figure 14 shows the particle size distribution (PSD) image of crystal form II;

[0091] Figure 15 shows the dynamic water adsorption-desorption analysis (DVS) spectrum of crystal form I;

[0092] Figure 16 shows the stability test results for crystal form I;

[0093] Figure 17 shows the results of the potted antibacterial experiment. Among them, 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 leaf of the crystal form II powder experiment, b-2 is the back view of the leaf of the crystal form II powder experiment, c-1 is the front view of the leaf of the crystal form I powder experiment, c-2 is the back view of the leaf of the crystal form I powder experiment, and d-1 is the front view of the leaf of the amorphous powder experiment, d-2 is the back view of the leaf of the amorphous powder experiment.

[0094] Figure 18 shows the purity characterization of kasugamycin acetamide; the retention time of kasugamycin acetamide is 10.469 min and the purity factor is 999.843, indicating that the method described in this invention can prepare high-purity kasugamycin acetamide, and the high-purity kasugamycin acetamide can be used as a standard.

[0095] Figure 19 shows the liquid chromatography-mass spectrometry (HPLC-DAD) and total ion chromatogram (TIC) of kaempferol acetamide.

[0096] Figure 20 shows the HPLC-MSD of acetamide from Kasugamycin.

[0097] Figure 21 shows the hydrogen NMR spectrum (1H-NMR) of acetamide from Chunlei.

[0098] Figure 22 shows the Fourier Transform Infrared (FTIR) spectrum of acetamide from the spring thunder.

[0099] Figure 23 shows the HPLC of the kasugamycin-kasugamycin aqueous solution composition;

[0100] Figure 24 shows the antibacterial effect of different concentrations of kasugamycin acetamide on rice blast fungus, where ck is the blank control group, a is the antibacterial effect of 0.001 wt%, b is the antibacterial effect of 0.01 wt%, c is the antibacterial effect of 0.03 wt%, d is the antibacterial effect of 0.05 wt%, and e is the antibacterial effect of 0.1 wt%.

[0101] Detailed description of the invention

[0102] The term "solvent" refers to those crystal forms of the compounds described in this application that coordinate with solvent molecules (such as water, organic solvents such as formic acid, toluene, etc.) to form complexes. Hydrates are a specific form of solvate in which coordination with water occurs. For example, a solvate can be a hydrate.

[0103] The term "raw material" refers to the product obtained during the production process, which consists of active ingredients and related impurities, and may include a small amount of additives if necessary.

[0104] The term "mother drug" refers to the product obtained during the production process, which consists of the active ingredient and related impurities, and may contain small amounts of necessary additives and appropriate diluents.

[0105] The term "formulation" refers to a stable product made from pesticide technical (mother drug) and suitable adjuvants, or processed by methods such as bio-fermentation and plant extraction.

[0106] The term "adjuvant" refers to any single or multiple components, other than the active ingredient, added to a pesticide product that do not possess pesticide activity or the function of the active ingredient, but can or help to improve or enhance the physicochemical properties of the pesticide product.

[0107] The term "plant disease" refers to the phenomenon where, during the growth and development of a plant, its growth and development are significantly hindered due to infection by other organisms and adverse abiotic factors. This results in pathological changes both internally and externally, in terms of physiology and tissue structure, leading to illness or even death, resulting in reduced yield and deteriorated quality. In this application, "plant disease" specifically refers to diseases caused by infection of plants by other organisms, including but not limited to fungi (such as cucumber downy mildew caused by *Pseudomonas columbinis*, corn curvature fungus, or rice blast fungus), or bacteria (such as bacterial angular leaf spot in cucurbits).

[0108] The term "salts of kasugamycin" refers to compounds formed by the combination of kasugamycin with acid radicals, such as kasugamycin hydrochloride, kasugamycin sulfate, kasugamycin carboxylate, kasugamycin phosphate, kasugamycin nitrate, or kasugamycin carbonate.

[0109] The terms “approximately”, “about”, and “basically” refer to numerical variations within the normal experimental or measurement error range. For example, “basically” represents an error of no more than 15%, preferably no more than 10%.

[0110] The term "salt of kasugamycin" refers to compounds formed by the combination of kasugamycin acetamide and an acid radical, such as kasugamycin acetamide hydrochloride and kasugamycin acetamide sulfate. In this application, the mass of the kasugamycin salt is calculated based on the mass of kasugamycin it contains, and the mass of kasugamycin acetamide...

[0111] The mass of the salt is based on the mass of kasugamycin acetamide it contains. For example, if 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 37.9g ​​of kasugamycin. Based on the mass of kasugamycin, the mass ratio of kasugamycin hydrochloride to kasugamycin acetamide is 37.9:1). Specific implementation examples:

[0112] All commercially available reagents and solvents were not further purified.

[0113] I. Crystal forms and amorphous compounds of kasugamycin hydrochloride

[0114] 1. Preparation Examples

[0115] Example 1-1: Preparation of Kasugamycin Hydrochloride Crystal Form I

[0116] (a) The fermentation broth containing kasugamycin was acidified with oxalic acid and then filtered;

[0117] (b) Collect the filtrate, adsorb it with a strong acid cation exchange resin, and then elute it.

[0118] (c) The ammonium chloride eluent is concentrated by nanofiltration and then further decolorized using activated carbon;

[0119] (d) The decolorized liquid is filtered and then concentrated under vacuum to obtain a vacuum concentrate;

[0120] (e) Take 10 ml of the vacuum concentrate prepared in step d, add the concentrate dropwise to 20 times the volume of dioxane (a poor solvent) at room temperature, stir at 60 rpm, suspend for 30-40 min, and filter to obtain the precipitate;

[0121] (f) The precipitate was dried at room temperature to obtain a high-purity solid crystal product. The solid crystal product was analyzed by 1H-NMR and XRPD.

[0122] The 1H-NMR analysis is as follows:

[0123] Several milligrams of solid sample were dissolved in dimethyl sulfoxide-d6 solvent and analyzed by nuclear magnetic resonance on a Bruker AVANCE NEO 400 (Bruker, GER).

[0124] The XRPD analysis method is as follows:

[0125] The solid samples obtained in the experiment were analyzed using a Bruker D8 Advance X-ray powder diffractometer (Bruker, GER). The 2θ scanning angle ranged from 3° to 45°, the scanning step size was 0.02°, and the exposure time was 0.08 seconds. The testing method was Cu target Kα1 radiation, voltage 40 kV, current 40 mA, and a zero-background sample disk.

[0126] result:

[0127] The 1H-NMR of the solid crystal product is shown in Figure 1, indicating that it is kasugamycin hydrochloride. The XRPD is shown in Figure 2, which is called crystal form I.

[0128] Examples 1-2: Preparation of Kasugamycin Hydrochloride Crystal Form I

[0129] A high-purity solid crystalline product was prepared according to step af of Example 1-1, except that the unsuitable solvent was replaced with ethylene glycol dimethyl ether. The solid sample was tested by 1H-NMR and XRPD according to the analytical methods described in Example 1-1, and the solid crystalline product was identified as kasugamycin hydrochloride crystal form I.

[0130] Examples 1-3: Preparation of Kasugamycin Hydrochloride Crystal Form I

[0131] (a) Take 250 mg of the dried solid crystalline product from Example 1-1;

[0132] (b) After dissolving the solid crystalline product in 4 ml of water;

[0133] (c) Take 1 ml of the solution from step b, place the concentrate in a trifluoroethanol atmosphere, and let it stand at room temperature until a solid precipitates out.

[0134] d) Remove the solution from the system with solid precipitate using a syringe, and perform 1H-NMR and XRPD tests on the solid sample according to the 1H-NMR and XRPD analysis methods described in Example 1-1. The results show that the solid sample is crystal form I of kasugamycin hydrochloride.

[0135] Comparative Example 1-1: Preparation of Kasugamycin Hydrochloride Crystal Form II

[0136] A fermentation broth containing kasugamycin is provided, specifically: using *Streptomyces aureus* as the producing strain, the fermentation broth is obtained through multi-stage fermentation in a culture medium containing low-temperature soybean meal, soybean oil, yeast powder, liquid sugar, and other raw materials. Following the purification and crystallization method described in the example of CN106083951B, purification is carried out through steps such as fermentation broth pretreatment, ceramic membrane filtration, macroporous resin decolorization, and nanofiltration concentration. During the crystallization process, organic solvents such as acetone, methanol, ethanol, propanol, or isopropanol are added to induce crystallization, resulting in five solid crystalline products.

[0137] The above-mentioned solid crystalline products were vacuum dried and then subjected to 1H-NMR and XRPD analysis. NMR analysis showed that the solid crystalline products were all kasugamycin hydrochloride. The 1H-NMR spectrum of the solid crystalline product obtained by crystallization with the organic solvent methanol is shown in Figure 3. The solid samples were analyzed using the 1H-NMR and XRPD methods described in Examples 1-1.

[0138] Results: 1H-NMR showed that the above solid crystalline products were all kasugamycin hydrochloride. XRPD results showed that the above solid crystalline products were of a crystal form, called crystal form II. The XRPD pattern of the solid crystalline product crystallized by the organic solvent acetone is shown in Figure 4.

[0139] Comparative Examples 1-2: Preparation of Kasugamycin Hydrochloride Crystal Form II

[0140] Using *Streptomyces simonii* as the producing strain, a fermentation broth was obtained through multi-stage fermentation in a culture medium containing low-temperature soybean meal, soybean oil, yeast powder, and liquid sugar. The broth was purified and crystallized according to the method described in the example of CN109666051B. Crystallization was performed by cooling crystallization or crystallization with the addition of organic solvents acetone or ethanol, resulting in three solid crystalline products. The solid samples were analyzed by 1H-NMR and XRPD using the methods described in Examples 1-1.

[0141] Results: 1H-NMR showed that the above solid crystalline products were all kasugamycin hydrochloride, and XRPD showed that the above solid crystalline products had the same crystal form as those obtained in Comparative Example 1-1, which were all crystal form II.

[0142] Examples 1-4: Preparation of amorphous kasugamycin hydrochloride

[0143] a) Take 100 mg of the dried solid crystalline product from Example 1-1;

[0144] b) Dissolve the solid crystalline product in 2 ml of water;

[0145] c) Freeze the solution from step b using dry ice and freeze-dry it in a freeze dryer for 1 day;

[0146] d) The freeze-dried material was subjected to 1H-NMR and XRPD tests according to the 1H-NMR and XRPD analysis methods described in Example 1-1.

[0147] Results: 1H-NMR results showed that it was kasugamycin hydrochloride (as shown in Figure 5), and XRPD results showed that it was an amorphous substance without obvious characteristic peaks (as shown in Figure 6).

[0148] Examples 1-5: Powder Preparation

[0149] Approximately 70g of three dry solids—crystal form II, crystal form I, and amorphous solid—were prepared according to the methods of Comparative Example 1-1, Example 1-1, and Example 1-4, with contents of 98.1%, 98.7%, and 96.3%, respectively, and were used as technical grade / parent drug for the formulation.

[0150] The above-mentioned raw materials / master materials were pulverized using a small vertical sand mill and zirconium beads (φ1.0~1.2mm, Zhimo (Shanghai) New Material Technology Co., Ltd.). The amount of zirconium beads used was 40g, the speed of the sand mill was 1500r / min, and the grinding time was 30min. After grinding, the zirconium beads and fine powder were separated.

[0151] PSD analysis showed that the particle size D90 of the three technical grade / master powders ranged from 36.6 μm to 40.2 μm.

[0152] Take 4g of each of the three raw materials / parent materials powders mentioned above, and mix the three powders with 96g of diatomaceous earth through a 200-mesh sieve before thoroughly mixing to prepare three powder formulations.

[0153] Examples 1-6: Preparation of wettable powders

[0154] The dosage of each component in the wettable powder is shown in the table below:

[0155] The wettable powder is prepared by the following steps:

[0156] 1) Take the fine powders of crystal form I and amorphous active ingredients / master powders prepared in Examples 1-5 according to the table above, and set them aside;

[0157] 2) Thoroughly mix copper hydroxide, sodium dodecyl sulfate, sodium lignosulfonate, silica, and diatomaceous earth, and then pulverize them using an ultrafine pulverizer to obtain the additive powder;

[0158] 3) Thoroughly mix the fine powder of the original drug / parent drug with the adjuvant powder prepared in step 2) to obtain a wettable powder.

[0159] 2. Test Analysis

[0160] (1) XRPD diffraction peak data analysis

[0161] The XRPD diffraction peak data of kasugamycin hydrochloride crystal form I described in this application are shown in Table 1; the XRPD diffraction peak data of crystal form II are shown in Table 2.

[0162] Table 1. XRPD diffraction peak data for crystal form I.

[0163] As shown in Table 1, the main characteristic diffraction peaks of the X-ray powder diffraction pattern of crystal form I using Cu-Kα radiation, expressed as 2θ values ​​± 0.2°, include 8.66, 10.11, 11.05, and 13.3, as well as any one or more characteristic diffraction peaks among 13.92, 15.56, 16.47, and 17.29. Specifically, they may include 8.66, 10.11, 11.05, 13.3, 13.92, and 15.56; or 8.66, 10.11, 11.05, 13.3, 16.47, and 17.29; or 8.66, 10.11, 11.05, 13.3, 13.92, 15.56, 16.47, and 17.29.

[0164] Table 2 XRPD diffraction peak data for crystal form II

[0165] As shown in Table 2, the main characteristic diffraction peaks of the X-ray powder diffraction pattern of crystal form II using Cu-Kα radiation, expressed as 2θ values ​​± 0.2°, include 8.91, 10.03, 10.51, 12.12, 15.01, 16.40, 17.14, and 26.68. Compared with crystal form I, crystal form II does not contain characteristic diffraction peaks such as 8.66, 11.05, and 13.30, but contains characteristic diffraction peaks such as 8.91, 10.51, and 12.12.

[0166] As shown in Figure 6, the amorphous material with no significant diffraction peaks was obtained by freeze-drying.

[0167] In summary, the preparation method of this invention yielded three solid products, namely crystal form I, crystal form II, and amorphous product. By comparing the XRPD patterns and diffraction peak data of crystal form I and crystal form II, it can be seen that they belong to different crystal forms.

[0168] (2) Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA)

[0169] The DSC analysis method is as follows: The differential scanning calorimeter is a TA Discovery 2500 (TA, US). 1-2 mg of sample is accurately weighed and placed in a perforated DSC Tzero sample pan. It is heated to the final temperature at a rate of 10 °C / min, with nitrogen purging at a rate of 50 mL / min.

[0170] The TGA analysis method is as follows: The thermogravimetric analyzer is a TA Discovery 55 (TA, US). 2-5 mg of sample is placed in a pre-equilibrated open aluminum sample pan and automatically weighed inside the TGA furnace. The sample is heated to the final temperature at a rate of 10 °C / min, with nitrogen purging at the sample at 60 mL / min and at the balance at 40 mL / min.

[0171] Results: The DSC and TGA spectra of crystal form I described in this application are shown in Figure 7; the DSC and TGA spectra of crystal form II are shown in Figure 8.

[0172] The DSC display shows that crystal form I has a continuous endothermic signal in the range of 110±2℃ to 210±2℃, and an endothermic signal at 226±2℃; in terms of the height (or valley) of the endothermic peak, crystal form I has a strong endothermic process at 226±2℃ and a weak endothermic process in the range.

[0173] TGA shows that the crystal 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 255°C±2°C.

[0174] The DSC analysis showed that crystal form II had a weak endothermic signal at 147±2℃ and a strong endothermic signal at 215±2℃. In terms of the endothermic peak height (or peak-valley), crystal form I had a strong endothermic process at 215±2℃ and a weak endothermic process at 147±2℃.

[0175] TGA shows that the crystal form II exhibits a weight loss of less than 6% during heating to 195±2°C and a weight loss of less than 15% during heating from 220±2°C to 250±2°C.

[0176] DSC showed that the amorphous material had endothermic signals at 73±2℃ and 187±2℃;

[0177] TGA results showed that the amorphous material had a weight loss of less than 9% when heated to 175±2℃, and a weight loss of essentially less than 15% when heated from 175±2℃ to 255±2℃.

[0178] (3) Polarizing microscopy (PLM) analysis and particle size distribution determination (PSD)

[0179] The PLM analysis method is as follows: The laser particle size analyzer is a Mastersizer 3000 (Malvern Panalytical, UK). 20 mg of sample is dispersed in 8 mL of dispersant. Sample dispersion units are added until the opacity reaches 10-20%, at which point measurement begins. The stirring speed is 2000 rpm for 10 seconds. The dispersant is ethanol, the scattering model is Mie, and the analysis model is general.

[0180] The PSD analysis method is as follows: The laser particle size analyzer is a Mastersizer 3000 (Malvern Panalytical, UK). 20 mg of sample is dispersed in 8 mL of dispersant. Sample dispersion units are added until the opacity reaches 10-20%, at which point measurement begins. The stirring speed is 2000 rpm for 10 seconds. The dispersant is ethanol, the scattering model is Mie, and the analysis model is general.

[0181] PLM images show that crystal form I is predominantly granular (as shown in Figure 10); particle size distribution analysis indicates that the particle size D of crystal form I is... 50 64.2μm, D 90 It is 119 μm (as shown in Figure 13).

[0182] PLM images show that crystal form II is predominantly needle-like (as shown in Figure 11); particle size distribution analysis indicates that the particle size D of crystal form II is... 50 24.7μm, D 90 It is 70.1 μm (as shown in Figure 14).

[0183] PLM images show that the amorphous material is flaky (as shown in Figure 12), with a particle size D. 50 It is 30.2 μm.

[0184] (4) Powder flowability

[0185] To verify the processing properties of different solid products, the flowability of the three fine powders prepared in Examples 1-5 was tested. The flowability of the fine powders was determined 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. The test was conducted with a fixed funnel height (10 cm), at room temperature and with a humidity of approximately 5%. The amount of fine powder used was 60 g. The experimental results are shown in Table 3.

[0186] Table 3 Evaluation of Powder Flowability

[0187] As shown in Table 3, under the same conditions, the pulverized crystalline form I and amorphous form have better powder flowability than crystalline form II, indicating that kasugamycin hydrochloride in crystalline form I and amorphous form is more conducive to the subsequent processing and utilization of mixtures and formulations. Among them, kasugamycin hydrochloride in crystalline form I showed the best performance in processing. Further investigation will be conducted on the hygroscopicity and stability of crystalline form I, crystalline form II and amorphous form.

[0188] (5) DVS Analysis

[0189] As shown in Figure 15, crystal 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 crystal form I has virtually no hygroscopic properties and is a stable solid product even in high humidity environments.

[0190] In comparison, crystal form II showed a 2.2% weight gain due to moisture absorption at 95% humidity and a 0.74% weight gain at 80% humidity, exhibiting weak hygroscopicity under high humidity conditions; while amorphous materials showed the general hygroscopic characteristics of amorphous materials.

[0191] (6) Stability determination

[0192] Stability studies were conducted on crystal forms I and II under high temperature (60℃), high humidity (25℃ / 92.5% RH), light exposure (25℃ / 4500 Lux), and accelerated stabilization (40℃ / 75% RH) conditions. Samples were taken at 7 and 15 days to observe appearance, XRPD characterization, and HPLC analysis, and the results were compared with those of the original samples. The results showed that crystal form I remained stable under high temperature, high humidity, light exposure, and accelerated stabilization conditions. Specifically, its appearance remained unchanged, no crystal form transformation occurred, and its chemical purity did not change significantly (as shown in Table 4 and Figure 16), indicating that crystal form I is a stable solid crystalline product.

[0193] The stability results of crystal form II showed that crystal form II maintained a stable appearance under 15 days of high temperature and high humidity testing. After 7 days of light exposure, the appearance changed from white to yellow, and after 15 days, the color changed to light brown, indicating that crystal form II is relatively unstable under continuous light exposure.

[0194] Table 4. Experimental results of different environmental influencing factors for crystal form I.

[0195] (7) Antibacterial effect of potted plants

[0196] (7-1) Preparation of cucumber downy mildew spore suspension

[0197] The fungus causing cucumber downy mildew was collected from naturally infected leaves in the Weinan Ecological Agriculture Demonstration Park. The collected diseased leaves were cleaned of the moldy layer on the underside of the lesions using a brush dipped in distilled water. The leaves were then placed in a dark incubator at 25℃ and 80% relative humidity for 16 hours to induce the production of fresh sporangia. The fresh sporangia were then brushed onto petri dishes containing distilled water and filtered twice to prepare a spore suspension (concentration 2×10⁻⁶). 5 -4×10 5 (pcs / mL), for later use.

[0198] (7-2) Indoor cultivation of cucumbers

[0199] Cucumbers (Xinong No. 58, 20 pots in total, 1 plant per pot) were planted in plastic pots with a diameter of 5cm. They were cultivated to a height of about 1 meter under the conditions of temperature of 20℃-28℃ (day and night), relative humidity of (90±5)%, and fluorescent light for 12 hours. Twelve cucumber seedlings with similar growth were selected as samples for subsequent experiments.

[0200] (7-3) Foliar preventative application

[0201] Two leaves of similar height (measured from the soil surface) and approximately 5-8 cm wide were selected from each of 12 cucumber seedlings. The three mixed powders prepared in Examples 1-5 were packaged into identical spray bottles and applied preventatively as powdered pesticides: two sprays were performed at a height of approximately 40 cm above the leaves. Each powder was sprayed on three seedlings and six leaves as a parallel experiment; the control group was sprayed only with diatomaceous earth powder. The leaves were marked and recorded accordingly (Group A was the control group, Group B contained crystalline form II powder, Group C contained crystalline form I powder, and Group D contained amorphous powder). After 48 hours of cultivation under the previous planting conditions, cucumbers were inoculated against downy mildew.

[0202] (7-4) Inoculation and therapeutic application for cucumber downy mildew

[0203] The prepared spore suspension was poured into a spray bottle and sprayed twice onto the selected leaves from a height of about 20 cm. Immediately after spraying, a transparent plastic bag was placed over the leaves and the bag was tied tightly. This process was repeated for inoculating 24 leaves on 12 cucumber seedlings with downy mildew. The transparent plastic bags were removed at 2, 6, and 10 hours post-inoculation, and the inoculated leaves were moistened with pure water via spraying. The bags were then reapplied. 24 hours after inoculation, a therapeutic application was performed using the same methods as preventative treatment (Group A: diatomaceous earth powder; Group B: powder containing crystal form II; Group C: powder containing crystal form I; Group D: powder containing amorphous substances). Fifteen days after the therapeutic application, the leaves were washed, and the condition of the experimental leaves was observed, recorded, and statistically analyzed.

[0204] The disease is graded based on the area of ​​the lesions, and the grading criteria are as follows:

[0205] Grade 0: Leaves show no disease spots;

[0206] Grade 1: The lesion area accounts for less than 5% of the total leaf area;

[0207] Grade 3: Lesions cover 6-10% of the total leaf area;

[0208] Level 5: Lesions cover 11-25% of the total leaf area;

[0209] Level 7: Lesions cover 26-50% of the total leaf area;

[0210] Level 9: The lesion area accounts for more than 50% of the total leaf area;

[0211] Disease index = {[∑(number of diseased leaves at each treatment level × corresponding level value)] / total number of leaves surveyed × 9} × 100;

[0212] Prevention and control effect (%) = [(disease index of blank control - disease index of drug treatment) / disease index of blank control group] × 100;

[0213] The results of the four sets of experiments are shown in Table 5, and some of the effects are shown in Figure 17.

[0214] Table 5. Effects of foliar control

[0215] Surprisingly, based on plant disease index and control efficacy, there were significant differences in the control effects of crystalline form I, crystalline form II, and amorphous formulations on plant diseases. The powder containing the amorphous formulation showed the best control effect, followed by the powder containing crystalline form I. This indicates that different crystalline and amorphous forms of kasugamycin hydrochloride may differ in terms of drug release, plant absorption, and antibacterial ability. The amorphous form and crystalline form I of kasugamycin hydrochloride showed better plant disease control effects than crystalline form II.

[0216] II. Kasugamycin acetamide and its compositions

[0217] 1. Preparation Examples

[0218] Example 2-1: Preparation of Kasugamycin Acetamide

[0219] (1) The high-yielding Streptomyces microaureaus strain MKL-2 was selected as the fermentation strain. The seed liquid after being cultured in the seed culture medium was inoculated into a 50L fermenter for fermentation. The culture medium in the fermenter consisted of: 4.0% soybean meal, 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), 0.4% ammonia, and 0.1% sodium citrate. The fermentation temperature was 30℃, the rotation speed was 60 rpm, ammonia was added dropwise during the fermentation process, and the pH of the fermentation system was controlled at 7.1±0.1 to facilitate the production of kasugamycin acetamide. The fermentation time was 120 h.

[0220] (2) Take 20L of Chunlei acetamide fermentation broth, acidify the fermentation broth with oxalic acid, and then filter it with a ceramic membrane;

[0221] (3) Collect the filtrate, adsorb it with a strong acid cation exchange resin, and then elute it with ammonium chloride solution;

[0222] (4) After filtering the eluent with a nanofiltration membrane, the solution is concentrated under vacuum and the concentrated filtrate is collected.

[0223] (5) Decolorize the concentrated filtrate using activated carbon;

[0224] (6) The decolorized filtrate was added to an octadecylsilane-bonded silica column and separated and purified using a mixture of 10% acetonitrile and 90% sodium hexanesulfonate aqueous solution as the eluent.

[0225] (7) After drying the separation and purification solution, about 6g of kasugamycin acetamide powder was obtained.

[0226] This acetamide powder can be used directly as a technical grade or parent material for pesticides.

[0227] Example 2-2: Preparation of Chunlei Acetamide Aqueous Solution

[0228] Take 100 mg of the kasugamycin technical material prepared in Example 2-1, add it to 10 mL of deionized water, and prepare a kasugamycin aqueous solution with a content of 1%. Take an appropriate amount of the 1% aqueous solution and further dilute and prepare four kasugamycin aqueous solutions with mass fractions of 0.01%, 0.1%, 0.3% and 0.5%.

[0229] Examples 2-3: Preparation of Chunlei Acetamide Wettable Powder

[0230] The following components are provided:

[0231] 1g of kasugamycin acetamide technical material was prepared in Example 2-1;

[0232] Sodium dodecyl sulfate (wetting agent) 4g;

[0233] Sodium lignosulfonate (dispersant) 5g;

[0234] 10g of white carbon black (filler);

[0235] 80g of diatomaceous earth (filler);

[0236] Mix all the above components thoroughly according to the formula weight, and then pulverize them using an ultrafine pulverizer to obtain 1% kasugamycin wettable powder.

[0237] Examples 2-4: Preparation of Kasugamycin-Acetamide Aqueous Solution

[0238] Take 85 mg of kasugamycin hydrochloride technical grade (provided by Xi'an Masdi Biotechnology Co., Ltd.) and 1.5 mg of kasugamycin acetamide technical grade prepared in Example 2-1, dissolve them in 50 ml of deionized water to prepare kasugamycin acetamide·kasugamycin aqueous solution.

[0239] Examples 2-5: Preparation of Kasugamycin-Acetamide Wettable Powder

[0240] The following components are provided:

[0241] 1g of kasugamycin acetamide technical material was prepared in Example 2-1;

[0242] Kasugamycin hydrochloride technical grade 13g (provided by Xi'an Masdi Bioengineering Co., Ltd.);

[0243] Sodium dodecyl sulfate (wetting agent) 3g;

[0244] Sodium lignosulfonate (dispersant) 5g;

[0245] 10g of white carbon black (filler);

[0246] 68g of diatomaceous earth (filler);

[0247] Mix the above components thoroughly according to the weight of the formula, and then pulverize them using an ultrafine pulverizer to obtain kasugamycin acetamide-kasugamycin wettable powder.

[0248] 2. Test Analysis

[0249] (1) Purity determination of kasugamycin acetamide

[0250] Accurately weigh 38 mg of the kasugamycin acetamide prepared in Example 2-1, dissolve and dilute it with water to 25 mL to prepare a kasugamycin acetamide standard stock solution; accurately transfer 1.25 mL of the above standard stock solution into a 25 mL volumetric flask, dilute it with water to the mark and mix well to complete the preparation of the test solution.

[0251] Instrument: Agilent HPLC 1100;

[0252] HPLC column: Agilent aichrombond-AQ C18 4.6mm*250mm*5μm;

[0253] Mobile phase: Acetonitrile: 20 mm sodium hexanesulfonate aqueous solution (adjusted to pH 3 with H3PO4) = 5%: 95% (v / v);

[0254] Running time: 13 minutes;

[0255] Column temperature: 30℃;

[0256] Injection volume: 5 μL;

[0257] Detection wavelength: 195nm;

[0258] Flow rate: 1.0 mL / min;

[0259] Signal bandwidth: 4nm.

[0260] Results: As shown in Figure 18, the retention time of kasugamycin acetamide was 10.469 min, and the purity factor was 999.843, indicating that the method described in Example 2-1 of this invention can prepare high-purity kasugamycin acetamide. This high-purity kasugamycin acetamide can be used as a standard.

[0261] (2) Analysis of acetamide by high-performance liquid chromatography-mass spectrometry (HPLC-MSD)

[0262] Accurately weigh 25.06 mg of the standard prepared in Example 2-1 into a 25 mL volumetric flask, dissolve it in water, dilute to volume, and mix thoroughly to obtain the standard stock solution.

[0263] 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 thoroughly. The analytical conditions are as follows:

[0264] Liquid phase conditions:

[0265] Instrument: Agilent 1100 / 1200 HPLC;

[0266] HPLC column: Waters CORTECS HILIC, 150 x 4.6 mm, 2.7 μm;

[0267] Mobile phase: Acetonitrile: 50 mmol ammonium formate solution = 75:25 (V / V);

[0268] Column temperature: 30℃;

[0269] Injection volume: 5 μL;

[0270] Running time: 12.00 min;

[0271] Flow rate: 1.00 mL / min;

[0272] Detection wavelength: 205nm;

[0273] Signal bandwidth: 4nm;

[0274] Reference wavelength: off;

[0275] Reference bandwidth: off;

[0276] Spectral range: All (190nm-400nm).

[0277] MS conditions:

[0278] MS source: API-ES;

[0279] Polarity: Positive;

[0280] Collision voltage: 70V;

[0281] Drying gas temperature: 350℃;

[0282] Drying airflow rate: 12.0 L / min;

[0283] Cap voltage: 3000V;

[0284] Data collection mode: Scan;

[0285] Scan range: 100-1000 amu.

[0286] result:

[0287] The ultraviolet chromatogram (DAD) and total ion chromatogram (TIC) are shown in Figure 19. The retention time of DAD is about 6.8 min and the retention time of TIC is about 7.0 min. The mass spectrum is shown in Figure 20. The main fragment ions have m / z values ​​of 171, 351, 373 and 701, which are consistent with the structural characteristics of kasugamycin acetamide.

[0288] (3) Acetamide NMR analysis (1H-NMR)

[0289] The analysis method is as follows:

[0290] 7 mg of solid sample was dissolved in heavy water and analyzed by NMR on a Bruker AVANCE NEO 400 (with BBO probe, Bruker, GER).

[0291] Results: The NMR data are shown in Figure 21, and the relevant characteristics are consistent with the chemical structure of kaempferol acetamide.

[0292] (4) Spring thunder acetamide infrared test (FTIR)

[0293] Weigh approximately 2g of spectroscopically pure KBr, grind it, and dry it at 105°C for 2 hours. Then, transfer the KBr to a desiccator and cool it to room temperature. Weigh 10mg of Kasugamycin acetamide analytical standard, dry it at 105°C for 2 hours, and then transfer it to a desiccator and cool it to room temperature.

[0294] Weigh approximately 200 mg of anhydrous KBr powder into an agate mortar, grind it, and then compress it into a tablet using an infrared spectroscopy tableting device. Scan the potassium bromide salt tablet with an infrared spectrometer.

[0295] Weigh approximately 2 mg of dried kasugamycin acetamide analytical standard into approximately 200 mg of KBr, mix thoroughly and grind finely. Press the mixture into a semi-transparent tablet using an infrared spectroscopy tablet press and then scan it with an infrared spectrometer.

[0296] 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.

[0297] (5) Detection of the Kasugamycin Acetamide-Kasugamycin Aqueous Composition

[0298] The aqueous solution of kasugamycin and kasugamycin was analyzed by HPLC under the following conditions:

[0299] Instrument: Agilent HPLC 1100;

[0300] HPLC column: Agilent aichrombond-AQ C18 4.6mm*250mm*5μm;

[0301] Mobile phase: Acetonitrile: 20 mm sodium hexanesulfonate aqueous solution (adjusted to pH 3 with H3PO4) = 5%: 95% (v / v);

[0302] Running time: 13 minutes;

[0303] Column temperature: 30℃;

[0304] Detection wavelength: 195nm;

[0305] Flow rate: 1.0 mL / min;

[0306] Signal bandwidth: 4nm;

[0307] Injection volume: 5 μL.

[0308] Results: As shown in Figure 23, the retention times of kasugamycin and kasugamycin acetamide were 8.462 min and 10.464 min, respectively, indicating that the detection method of the present invention has good separation and sensitivity response for kasugamycin and kasugamycin acetamide, which have similar structures. The detection method of the present invention can effectively identify kasugamycin and kasugamycin acetamide, which have similar structures.

[0309] (6) Spring thunder acetamide antibacterial test

[0310] Raw materials: The technical grade of kasugamycin acetamide was prepared according to the method in Example 2-1.

[0311] The pathogens were: Curvularia maize, which was isolated and purified from diseased maize leaves collected from a farm in Weinan City, and then identified; and rice blast, which was obtained from diseased rice in the field, after isolation, purification, and cultivation.

[0312] Take 1 mL of kasugamycin acetamide aqueous solution with a mass fraction of 0.01%, 0.1%, 0.3%, 0.5% and 1% as described in Example 2-1, mix it with PSA medium at a ratio of 1 mL to 9 mL, and pour it into petri dishes to prepare drug-containing plates with final concentrations of 0.001%, 0.01%, 0.03%, 0.05% and 0.1%, respectively. Use PSA medium with an equal amount of sterile water as a blank control.

[0313] Prepare 5mm diameter mycelial discs from the cultured pathogen using a punch, place them in the aforementioned agar plates, and incubate at 28±1℃. When the edge of the colonies in the control group approaches the plate wall, measure the colony diameter of each treatment group using the cross-sectional method, and calculate the relative inhibition percentage using the following formula:

[0314] Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - mycelial cake diameter) × 100%;

[0315] An inhibition rate of 1-24% is considered effective (△), 25%-39% is considered effective (▲), and ≥40% is considered significantly effective (▲▲).

[0316] The results of the inhibition rate determination and antibacterial experiment are shown in Table 6 and Figure 24.

[0317] Table 6. Inhibition rate of acetamide against two pathogens.

[0318] As shown in Table 6 and Figure 24, the inhibitory effect of kasugamycin on rice blast fungus is similar to that of kasugamycin, both exhibiting high sensitivity. An effective effect was observed at a concentration of 0.01%, and a significant effect was observed at a concentration of 0.03%, with an inhibition rate of 41.6%. Simultaneously, kasugamycin also showed significant inhibitory effects on corn curvature fungus, which is insensitive to kasugamycin. At a concentration of 0.01%, kasugamycin was effective against corn curvature fungus, and at a concentration of 0.05%, it was significantly effective, with an inhibition rate of 42.2%. These experimental results indicate that kasugamycin and kasugamycin have similar control effects against certain pathogens, which may be related to their similar chemical structures. However, their control effects differ significantly against certain pathogens, which may suggest significant differences in their mechanisms of action and / or efficacy.

[0319] (7) Indoor toxicity test of the kasugamycin-kasugamycin combination against rice blast

[0320] (7-1) Inhibition concentration determination (EC50)

[0321] Raw materials: Kasugamycin technical grade (prepared according to the method in Example 2-1); Kasugamycin technical grade (kasugamycin hydrochloride, provided by Xi'an Maisidi Biotechnology Co., Ltd.); rice blast virus was collected from diseased rice in the field, and obtained after isolation, purification and cultivation.

[0322] Experimental Methods: Following the guidelines of the People's Republic of China Agricultural Industry Standard NY / T1154.7-2006, pot cultivation was used. Rice seedlings at the three-leaf stage with uniform growth were selected, with two seedlings per pot, and five pots of seedlings were used for each treatment. The rice blast pathogen was cultured on tomato-oat agar medium. After sporulation, the spores were washed off with sterile water and prepared into 1×10⁻⁶ spores. 5A suspension of [[ID=]] spores / mL was evenly sprayed onto the tested rice seedlings. After inoculation, the seedlings were covered with black plastic bags to maintain humidity and cultured for 24 h. After 24 h of inoculation, chemical treatments were carried out. Five concentration gradients were set for each chemical. Spraying was performed using a Potter spray tower at a pressure of 50 Psi, approximately 5 mL per pot. After spraying, the rice seedlings were cultured under the conditions of 28 °C and a relative humidity of 92%. After 8 days, the disease index of the whole plant leaves was investigated according to the disease grading standard of rice blast, and the control effect was calculated.

[0323] Disease index = {[∑(number of diseased leaves at each level in each treatment × corresponding level value)] / total number of leaves investigated × 9} × 100;

[0324] Control effect (%) = [(disease index of blank control - disease index of chemical treatment) / disease index of blank control group] × 100.

[0325] The control effect was converted into the probit value (y), and the liquid concentration (μg / ml) was converted into the logarithm value (x). The virulence equation and the median inhibitory concentration EC50 were calculated by the least squares method.

[0326] (7 - 2) Co-toxicity experiment of春雷乙酰胺·春雷霉素盐酸盐 composition

[0327] According to the Sun Yunpei method, the virulence index and co-toxicity coefficient (CTC) of the春雷乙酰胺·春雷霉素 composition were calculated.

[0328] Virulence index of single agent = (EC50 of standard chemical / EC50 of tested single agent) × 100;

[0329] Actual virulence index (ATI) = (EC50 of standard chemical / EC50 of tested chemical) × 100;

[0330] Theoretical virulence index of mixture (TTI) = virulence index of agent A × percentage content of A in the mixture + virulence index of agent B × percentage content of B in the mixture;

[0331] Co-toxicity coefficient (CTC) = [actual virulence index of mixture (ATI) / theoretical virulence index of mixture (TTI)] × 100;

[0332] When CTC ≤ 80, the composition shows an antagonistic effect; when 80 < CTC < 120, the composition shows an additive effect; when CTC ≥ 120, the composition shows a synergistic effect.

[0333] The results of the co-toxicity experiment are shown in Table 7.

[0334] Table 7 Co-toxicity experiment data

[0335] The co-toxicity experiment of the kasugamycin-kasugamycin hydrochloride combination showed that kasugamycin and kasugamycin exhibited a synergistic effect within a mass ratio range of 13:1-130:1. The two, when mixed in a certain proportion, had a good synergistic effect and could improve the control effect on rice blast disease.

[0336] The experimental results above show that although kasugamycin and kasugamycin have similar chemical structures, they differ significantly in their efficacy against pathogens, exhibiting a certain degree of complementarity and synergy. While the underlying mechanism is not yet clear, it is speculated that kasugamycin's better lipophilicity compared to kasugamycin may increase its permeability to the cell walls of pathogens.

[0337] In the composition of kasugamycin acetamide and kasugamycin hydrochloride, kasugamycin hydrochloride can be kasugamycin hydrochloride crystal form I or its amorphous form.

[0338] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely 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 intent of the present invention should be included within the scope of protection of the present invention.

Claims

1. An agro-antibiotic kasugamycin hydrochloride salt crystalline form I characterized by, The X-ray powder diffraction pattern of the crystal form I has characteristic diffraction peaks including 8.66, 10.11, 11.05 and 13.3, expressed in terms of 2θ values ± 0.2° using Cu-Kα radiation.

2. Springomycin hydrochloride Form I according to claim 1, characterized in that, The X-ray powder diffraction pattern of the kasugamycin hydrochloride crystal form I has characteristic diffraction peaks further including 13.92 and 15.56, expressed in terms of 2θ values ± 0.2° using Cu-Kα radiation; preferably, the X-ray powder diffraction pattern has characteristic diffraction peaks further including 16.47 and 17.29, expressed in terms of 2θ values ± 0.2° using Cu-Kα radiation; more preferably, the X-ray powder diffraction pattern has characteristic diffraction peaks further including any one or more of 18.59, 19.25, 20.41 and 20.78, expressed in terms of 2θ values ± 0.2° using Cu-Kα radiation.

3. Springomycin hydrochloride Form I according to claim 1 or 2, characterized in that, The particle size D of crystal form I 90 The particle size is 10μm-200μm; preferably, the particle size D of crystal form I is... 90 The range is 15μm-150μm.

4. An original drug, a parent drug, a pesticide preparation or a pesticide composition, characterized by, The technical product, technical agent, pesticide preparation or pesticide composition contains the kasugamycin hydrochloride crystal form I according to any one of claims 1-3.

5. The original drug, parent drug, agricultural chemical preparation or agricultural chemical composition according to claim 4, characterized by, The weight percentage of the kasugamycin hydrochloride crystal form I in the technical product, technical agent, pesticide preparation or pesticide composition is at least 0.001%, wherein the mass of the kasugamycin hydrochloride is based on the mass of kasugamycin free base contained therein.

6. The original drug, parent drug, agricultural chemical preparation, or agricultural chemical composition according to claim 4 or 5, characterized by, The content of kasugamycin acetate and / or salt of kasugamycin acetate is not less than 0.01% based on the total mass of the technical product, technical agent, pesticide preparation or pesticide composition; preferably, the content of kasugamycin acetate and / or salt of kasugamycin acetate is not less than 0.03%; wherein the mass of the salt of kasugamycin acetate is based on the mass of kasugamycin acetate free base contained therein.

7. The original drug, parent drug, pesticide preparation or pesticide composition according to claim 6, characterized by The mass ratio of kasugamycin hydrochloride to kasugamycin acetate and / or salt of kasugamycin acetate is 10:1-200:1; more preferably 13:1-130:

1.

8. The prodrug, parent drug, pesticidal formulation or pesticidal composition according to claim 4, 5 or 7, characterized in that, The dosage form of the pesticide preparation or pesticide composition is selected from any one of powder, granule, macrogranule, fine granule, microgranule, microencapsulated granule, wettable powder, oil dispersible powder, water dispersible granule, emulsifiable granule, effervescent granule, dispersible tablet, effervescent tablet, sustained release agent, sustained release block, sustained release tube, sustained release granule, soluble powder, soluble granule, soluble tablet, soluble solution, aqueous agent, soluble gel agent, oil agent, spread film oil agent, ultra-low volume liquid agent, ultra-low volume microcapsule suspension agent, emulsion oil agent, emulsion, dispersible liquid agent, paste, thick gel agent, water emulsion agent, oil emulsion agent, microemulsion, grease, suspension agent, microcapsule suspension agent, oil suspension agent, suspoemulsion agent, seed treatment dispersible powder, seed treatment soluble powder, seed treatment liquid agent, seed treatment emulsion agent, seed treatment suspension agent, suspension seed coating agent, seed treatment microcapsule suspension agent; preferably, the pesticide preparation is powder or wettable powder.

9. The original drug, parent drug, pesticide preparation or pesticide composition according to Claim 8, characterized by wherein the particle size D of kasugamycin hydrochloride hydrate Form I in the dusts or wettable powders is from 5 μm to 80 μm. 90 from 5 μm to 80 μm.

10. Use of the kasugamycin hydrochloride crystal form I according to any one of claims 1-3, the technical product, technical agent, pesticide preparation or pesticide composition according to claims 4-9 in the control of plant diseases.

11. Use of the kasugamycin hydrochloride crystal form I according to any one of claims 1-3, the technical product, technical agent, pesticide preparation or pesticide composition according to claims 4-9 in the preparation of a plant disease control agent.

12. Use according to claim 10 or 11, characterized in that, The plant disease is cucumber downy mildew, corn cercospora leaf spot or rice blast.

Images