WO2020224520A1 - 5-oxomilbemycins crystal forms and preparation method therefor - Google Patents

5-oxomilbemycins crystal forms and preparation method therefor Download PDF

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WO2020224520A1
WO2020224520A1 PCT/CN2020/088110 CN2020088110W WO2020224520A1 WO 2020224520 A1 WO2020224520 A1 WO 2020224520A1 CN 2020088110 W CN2020088110 W CN 2020088110W WO 2020224520 A1 WO2020224520 A1 WO 2020224520A1
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ketomilbemycin
crystal form
methanol
ketokimilbemycin
heptane
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PCT/CN2020/088110
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French (fr)
Chinese (zh)
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朱明新
沈敢锋
余贞
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浙江海正药业股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/22—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

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  • the invention relates to the field of pharmacy, especially the field of veterinary medicine. More specifically, the present invention relates to 5-ketomibemycin crystal form I and crystal form II and their preparation processes.
  • Milbemycins (milbemycins) is a sixteen-membered ring macrolide anthelmintic produced by Streptomyces. It was discovered by Sankyo Co., Ltd. in 1967. After nearly 20 years of improvement, it was used in 1986 with milbemycin oxime. oxime) was listed. Milbex oxime has a good effect on controlling and preventing most common parasitic diseases. It is usually used to prevent heartworm disease, control dog and cat diseases caused by nematodes and hookworms, and canine whipworm disease. Due to its high insecticidal activity and low toxicity, the LD 50 is more than 2000 times the recommended clinical dosage. At the same time, dogs that are sensitive to abamectin drugs are less toxic, so it has a good market prospect.
  • Milbex oxime is produced by fermentation and semi-synthesis.
  • the milbemycin A3 and A4 obtained by fermentation of Streptomyces are oxidized to obtain intermediate 5-ketomilbemycin A3 and A4, and then the obtained intermediate is subjected to oximation to obtain a mixture of milbemycin A3 and A4.
  • EP and USP have strict limits on the ratio of milbe oxime A3 and milbe oxime A4.
  • 5-keto milbemycin A3 and A4 are key intermediates.
  • CN108586481 discloses the extraction of the fermentation broth of 5-ketomilbemycin and the crystallization process of the mixture of 5-ketomilbemycin A3 and A4, but there is no information about 5-ketomilbemycin A3 and A4. Report on the crystalline forms of the components.
  • the prior art discloses a mixture of 5-ketomilbemycin A3 and A4. This mixture is not yet satisfactory in terms of stability, purity and quality stability of the ratio of A3 and A4. It is very necessary to prepare and study the crystal form of ketomilbemycin A3 and A4. The preparation of high-purity and stable single-component 5-ketokilmbemycin A3 and A4 will lay a solid foundation for the production of milbemycin with a stable ratio of A3 and A4 and high purity.
  • One of the objectives of the present invention is to provide two single-component crystal forms of 5-ketokimirbemycin, namely 5-ketokimirbemycin A3 crystal form I (abbreviated as crystal form I) and 5-ketokimirbemycin Bemycin A4 crystal form II (abbreviated as crystal form II).
  • the 5-ketomilbemycin A3 crystal form I of the present invention uses Cu-K ⁇ radiation, and the X-ray powder diffraction pattern expressed at 2 ⁇ angles has characteristic peaks at the following positions: 4.89 ⁇ 0.20°, 8.69 ⁇ 0.20 °, 13.64 ⁇ 0.20°, 14.71 ⁇ 0.20°, 16.66 ⁇ 0.20°, 19.85 ⁇ 0.20°, 20.90 ⁇ 0.20°.
  • the 5-ketomilbemycin A3 crystal form I of the present invention uses Cu-K ⁇ radiation, and the X-ray powder diffraction pattern expressed at 2 ⁇ angles further has characteristic peaks at the following positions: 14.45 ⁇ 0.20° , 15.33 ⁇ 0.20°, 17.08 ⁇ 0.20°, 17.82 ⁇ 0.20°, 19.24 ⁇ 0.20°, 22.29 ⁇ 0.20°, 23.01 ⁇ 0.20°, 23.51 ⁇ 0.20°.
  • the 5-ketomilbemycin A3 crystal form I of the present invention uses Cu-K ⁇ radiation, and the X-ray powder diffraction pattern expressed at 2 ⁇ angles further has characteristic peaks at the following positions: 12.16 ⁇ 0.20 °, 17.49 ⁇ 0.20°, 22.64 ⁇ 0.20°, 25.70 ⁇ 0.20°, 26.50 ⁇ 0.20°, 28.27 ⁇ 0.20°, 29.29 ⁇ 0.20°, 31.24 ⁇ 0.20°, 33.79 ⁇ 0.20°, 42.62 ⁇ 0.20°.
  • the XRPD pattern of 5-ketomilbemycin A3 crystal form I of the present invention is shown in FIG. 1.
  • the 5-ketomilbemycin A3 crystal form I of the present invention can be characterized by the infrared absorption spectrum measured by KBr tablet. According to the spectrum, it has characteristic peaks at the following positions: 2932cm -1 , 2922cm -1, 1727cm -1, 1677cm -1 , 1367cm -1, 1219cm -1, 1057cm -1, 995cm -1, 909cm -1.
  • the infrared absorption spectrum of 5-ketomilbemycin A3 crystal form I of the present invention also has characteristic peaks at the following positions: 3506cm -1 , 2966cm -1 , 2874cm -1 , 2856cm -1 , 1446cm -1 , 1379cm -1, 1276cm -1, 1180cm -1, 1167cm -1, 1035cm -1, 963cm -1, 887cm -1, 541cm -1, 483cm -1.
  • the differential scanning calorimetry (DSC) spectrum of the 5-ketomilbemycin A3 crystal form I of the present invention has two exothermic peaks at 208°C-212°C and 258°C-262°C.
  • thermogravimetric analysis (TGA) pattern of 5-ketomilbemycin A3 crystal form I of the present invention is shown in FIG. 4.
  • the 5-ketomilbemycin A4 crystal form II of the present invention uses Cu-K ⁇ radiation, and the X-ray powder diffraction pattern expressed at 2 ⁇ angles has characteristic peaks at the following positions: 5.13 ⁇ 0.20°, 9.00 ⁇ 0.20 °, 12.56 ⁇ 0.20°, 15.95 ⁇ 0.20°, 18.73 ⁇ 0.20°, 19.42 ⁇ 0.20°, 20.50 ⁇ 0.20°, 22.78 ⁇ 0.20°.
  • the 5-ketomilbemycin A4 crystal form II of the present invention uses Cu-K ⁇ radiation, and the X-ray powder diffraction pattern expressed at 2 ⁇ angles further has characteristic peaks at the following positions: 14.52 ⁇ 0.20° , 15.11 ⁇ 0.20°, 16.42 ⁇ 0.20°, 22.36 ⁇ 0.20°, 23.30 ⁇ 0.20°, 25.63 ⁇ 0.20°, 26.44 ⁇ 0.20°, 27.24 ⁇ 0.20°.
  • the 5-ketomilbemycin A4 crystal form II of the present invention uses Cu-K ⁇ radiation, and the X-ray powder diffraction pattern expressed at 2 ⁇ angles further has characteristic peaks at the following positions: 28.34 ⁇ 0.20 °, 29.40 ⁇ 0.20°, 30.35 ⁇ 0.20°, 32.15 ⁇ 0.20°, 34.70 ⁇ 0.20°, 41.64 ⁇ 0.20°.
  • the XRPD pattern of 5-ketomilbemycin A4 crystal form II of the present invention is shown in FIG. 5.
  • the 5-ketomilbemycin A4 crystal form II of the present invention can be characterized by the infrared absorption spectrum measured by KBr tablet. According to the spectrum, it has characteristic peaks at the following positions: 3510cm -1 , 2921cm -1 , 1677cm -1 , 1372cm -1 , 1243cm -1 , 1165cm -1 , 990cm -1 .
  • the infrared absorption spectrum of 5-ketomilbemycin A4 crystal form II of the present invention also has characteristic peaks at the following positions: 2956cm -1 , 2873cm -1 , 1459cm -1 , 1436cm -1 , 1334cm -1 , 1103cm -1, 1061cm -1, 964cm -1 , 948cm -1, 907cm -1, 887cm -1, 858cm -1, 820cm -1, 543cm -1, 487cm -1, 459cm -1.
  • the differential scanning calorimetry (DSC) spectrum of the 5-ketomilbemycin A4 crystal form II of the present invention has two exothermic peaks at 218°C-222°C and 258°C-262°C.
  • the DSC chart of 5-ketomilbemycin A4 crystal form II of the present invention is shown in FIG. 7.
  • Another object of the present invention is to provide a method for preparing 5-ketomilbemycin A3 crystal form I and 5-ketomilbemycin A4 crystal form II, which comprises the following steps:
  • Step (1) Load the crude extract of 5-ketomilbemycin on a reversed-phase preparation column, and elute with a methanol solution with a volume concentration of 60%-90% to obtain 5-ketomilbemycin respectively A3 collection solution and 5-ketokimilbemycin A4 collection solution;
  • Step (2) Respectively concentrate the 5-ketomilbemycin A3 and 5-ketomilbemycin A4 of step (1) to a methanol volume concentration of 45%-55% to obtain 5 -Ketomilbemycin A3 methanol concentrate and 5-ketomilbemycin A4 methanol concentrate, and then extracted with n-heptane to obtain 5-ketomilbemycin A3 n-heptane extract and 5 -Ketomilbemycin A4 n-heptane extract, which was concentrated, stirred and crystallized, filtered, and dried to obtain 5-ketokimilbemycin A3 crystal form I and 5-ketokimilbemycin A3 respectively A4 crystal form II.
  • the filler of the reverse phase preparation column in the step (1) is C18 or C8.
  • the crude extract is applied to the column, it is eluted with a methanol solution with a volume concentration of 60%-90%. Collect each fraction according to the peak condition, and then according to the HPLC analysis results of each fraction, combine the high purity parts of 5-ketomilbemycin A3 to obtain 5-ketomilbemycin A3 collection solution. The fractions with high purity of milbemycin A4 are combined to obtain 5-ketokimilbemycin A4 collection solution.
  • the volume ratio of the n-heptane and 5-ketomilbemycin A3 methanol concentrate used in the extraction in the step (2) is 1:1 to 2:1; the extraction in the step (2) is The volume ratio of n-heptane to 5-ketomilbemycin A4 methanol concentrate is 1:1 to 2:1.
  • the 5-ketomilbemycin A3 n-heptane extract is concentrated to a 5-ketomilbemycin A3 concentration of 50g/L ⁇ 150g/L;
  • -Ketomilbemycin A4 n-heptane extract is concentrated to a 5-ketomilbemycin A4 concentration of 50g/L ⁇ 150g/L.
  • the stirring time in the step (2) is 20-30 hours.
  • the temperature of crystallization in the step (2) is 10-30°C
  • the drying temperature in the step (2) is 30-40°C, and the drying time is 2 to 3 hours.
  • the crude 5-ketomilbemycin in step (1) is prepared by the following method:
  • the methanol or ethanol extract of kimilbemycin is concentrated to obtain a 5-ketokimilbemycin concentrate, and the concentrated solution is extracted with n-heptane to obtain a 5-ketokimilbemycin n-heptane extract;
  • the crude 5-ketomilbemycin is dissolved in a methanol aqueous solution with a volume concentration of more than 60% or an ethanol aqueous solution with a volume concentration of more than 60% to obtain a crude 5-ketomilbemycin extract, wherein the 5-
  • the crude ketomilbemycin can be prepared by a conventional synthesis method (for example, the synthesis method disclosed in CN105949217);
  • the 5-ketokimilbemycin fermentation broth in step (a) of the method 1 adopts the method disclosed in CN103789339, using Streptomyces F2-18 (see literature Huang Jun, Lin Jiatan, Zhou Min etc . Streptomyces hygroscopicus HS023 milF gene knockout constructs a genetically engineered strain producing 5-ketokimilbemycin. "Acta Microbiology", 2015, 55(1):107-113).
  • the ratio of the volume concentration of 80%-100% ethanol to mycelium is 4:1-10:1, the unit It is L/Kg, more preferably 5:1.
  • the ratio of the volume concentration of 80%-100% methanol to mycelium is 4:1-10:1, the unit It is L/Kg, more preferably 5:1.
  • the volume concentration of ethanol or methanol in the 5-ketomilbemycin concentrate in step (a) of the method 1 is 45-55%.
  • the volume ratio of n-heptane to 5-ketomilbemycin concentrate is 1:1 to 2:1.
  • the volume concentration of methanol or ethanol used in the method 2 is more than 95%.
  • the concentration of 5-ketokimilbemycin in the crude extract in the method 2 is 20-100 g/L.
  • the concentration in the present invention can be concentrated under reduced pressure, and the negative pressure is 0.08-0.1Mpa.
  • the inventors of the present invention have found that 5-ketokimirbemycin A3 crystal form I and 5-ketokimirbemycin A4 crystal form II after a lot of research fill in the prior art that there is nothing about 5-ketokimirbemycin.
  • the crystal forms I and II of the present invention have good stability, and no crystal conversion phenomenon is found when placed at room temperature for several months. The process is simple and easy to implement industrialization.
  • To produce 5-ketokimilbemycin A3 and A4 single-component crystals it is convenient to use 5-ketokimilbemycin A3 and A4 in accordance with the requirements of the A3 and A4 ratio in the quality standard of the Milbex oxime product.
  • Milbex oxime A3 is mixed in proportion and then oximated to produce a higher quality Milbex oxime with a stable proportion. It is also possible to directly produce milbexime A3 single-component or milbex oxime A4 single-component from 5-ketomilbemycin A3 and A4 single-component crystals as raw materials to adjust the proportion of the components in the Milbex oxime product. , To ensure that the ratio between Milbex oxime A3 and Milbex oxime A4 in the finished product meets the requirements.
  • the 5-ketomilbemycin in the present invention is a sixteen-membered ring macrolide compound containing 5-ketomilbemycin A3 and 5-ketomilbemycin A4, and its structure is as follows:
  • Both normal temperature and room temperature in the present invention refer to 10°C to 30°C.
  • Example 5 is the X-ray powder diffraction pattern of 5-ketomilbemycin A4 crystal form II obtained in Example 10.
  • Example 7 is the DSC chart of 5-ketomilbemycin A4 crystal form II obtained in Example 10.
  • the 5-ketomilbemycin fermentation broth used in the present invention is obtained by fermentation according to Example 1 of Chinese invention patent CN108586481.
  • the X-ray powder diffraction instrument and test conditions involved in the present invention are: X-ray diffraction instrument model Rigaku D/max-2115 Cu target; operation method: scanning speed 3°/min, scanning step width 0.01°.
  • the infrared spectrophotometer and test conditions involved in the present invention are: infrared spectrophotometer model: BRWKER VECTOR 22; operation method: KBr compression method with a scanning range of 400-4000 cm-1.
  • the DSC detector and test conditions involved in the present invention are: the model of the DSC detector is: NETZSCHDSC214; the operation method: the heating rate is 10°C/min, and the temperature range: 25°C to 300°C.
  • TGA detector and test conditions involved in the present invention are: TGA detector model: PerkinElmer TGA400; operation method: heating rate 10°C/min, temperature range: 30°C to 280°C.
  • the percentage concentration (%) of the ethanol or methanol solution in the present invention is the volume percentage concentration.
  • Example 2 Take 20L of the n-heptane extract obtained in Example 1 and concentrate it at 38°C and -0.095Mpa to an oily substance. Then add 0.6L of methanol with a volume concentration of 95% to dissolve and filter to obtain 0.7L of 5-ketokimilbemycin crude extract, which contains 5-ketokimilbemycin A3 and 5-ketokimilbemycin The total concentration of A4 is 50g/L, that is, the total mass of A3 and A4 is 35.15g.
  • Respectively concentrated under reduced pressure A3 mixture was concentrated to 6.2L, methanol content was 51%; A4 mixture was concentrated to 10.5L, methanol content was 47%.
  • a sample was taken for X-ray powder diffraction, DSC, TGA, and infrared analysis, and it was confirmed to be 5-ketomilbemycin A3 crystal form I.
  • the X-ray powder diffraction pattern is shown in Figure 1, and the infrared absorption pattern is shown in Figure 2.
  • the DSC spectrum is shown in Figure 3, and the TGA spectrum is shown in Figure 4.
  • Example 8 Take 6L of the 5-ketomilbemycin A4 n-heptane extract in Example 8 and concentrate it to 98.5mL, where the concentration of 5-ketomilbemycin A4 is 100g/L, stir and crystallize at room temperature for 25 hours, The crystalline liquid was suction filtered, and the wet crystal filter cake was rinsed with n-heptane. The wet crystals obtained were dried at 40°C and a negative pressure of 0.09Mpa for 3 hours to obtain 5.9g of 5-ketomilbemycin A4 crystals. The HPLC chromatographic purity of the crystal was 99.2%.
  • the crystal forms I and II obtained in Examples 9 and 10 were kept in 3 bags of each crystal form, and each bag was divided into 0.1 g, and they were packaged in a double-layer PE bag and an aluminum foil bag. Placed in a drug stability test box (temperature: 40°C, humidity 75%) for accelerated testing. Samples were taken at different time points of the placement cycle for 1 month, 2 months, and 3 months, and tested by XRPD and HPLC. The data are shown in Table 3.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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Abstract

The present invention provides 5-oxomilbemycins crystal form I and crystal form II. The two crystal forms can be represented by means of an X-ray powder diffraction (XRPD) pattern, a differential scanning calorimetry (DSC) pattern, an infrared (IR) absorption pattern and the like. Moreover, the present invention also provides a preparation process for the 5-oxomilbemycins crystal form I and crystal form II. The purity of the 5-oxomilbemycins crystal form I and crystal form II which are produced by means of the process in the present invention reaches up to 99.2% or above, the yield is high, and the stability is good.

Description

5-酮基米尔贝霉素晶型及其制备方法5-ketokimilbemycin crystal form and preparation method thereof
交叉引用cross reference
本申请要求发明名称为“5-酮基米尔贝霉素晶型及其制备方法”于2019年5月5日提交到中国专利局的中国专利申请201910368043.8的优先权，其内容通过引用以整体并入本文。This application claims the priority of Chinese patent application 201910368043.8 filed with the Chinese Patent Office on May 5, 2019 under the title of "5-ketokimilbemycin crystal form and its preparation method", the content of which is incorporated by reference in its entirety. Into this article.
技术领域Technical field
本发明涉及制药领域，尤其是兽药领域。更具体地，本发明涉及5-酮基米贝霉素晶型I和晶型II及它们的制备工艺。The invention relates to the field of pharmacy, especially the field of veterinary medicine. More specifically, the present invention relates to 5-ketomibemycin crystal form I and crystal form II and their preparation processes.
背景技术Background technique
米尔贝霉素(milbemycins)是由链霉菌产生的十六元环大环内酯类驱虫药物，日本三共株式会社于1967年发现，经过近20年的改良于1986年以米尔贝肟(milbemycin oxime)上市。米尔贝肟对控制和预防大部分常见寄生虫疾病都有很好的效果。通常用来预防恶丝虫病，控制线虫、钩虫引发的犬、猫疾病及犬的鞭虫病。由于米尔贝肟杀虫活性高、毒性小，LD 50是临床推荐用量的2000倍以上，同时对阿维菌素类药物敏感的犬毒性较小，所以具有很好的市场前景。 Milbemycins (milbemycins) is a sixteen-membered ring macrolide anthelmintic produced by Streptomyces. It was discovered by Sankyo Co., Ltd. in 1967. After nearly 20 years of improvement, it was used in 1986 with milbemycin oxime. oxime) was listed. Milbex oxime has a good effect on controlling and preventing most common parasitic diseases. It is usually used to prevent heartworm disease, control dog and cat diseases caused by nematodes and hookworms, and canine whipworm disease. Due to its high insecticidal activity and low toxicity, the LD 50 is more than 2000 times the recommended clinical dosage. At the same time, dogs that are sensitive to abamectin drugs are less toxic, so it has a good market prospect.
目前，米尔贝肟都是通过发酵半合成的方法生产。首先，将链霉菌发酵获得的米尔贝霉素A3、A4经过氧化得到中间体5-酮基米尔贝霉素A3、A4，然后所得的中间体再进行肟化得到米尔贝肟A3、A4混合物。EP和USP对米尔贝肟A3和米尔贝肟A4之间的比例都有严格的限制，在生产米尔贝肟过程中，5-酮基米尔贝霉素A3、A4是关键的中间体。因此，确保5-酮基米尔贝霉素A3和5-酮基米尔贝霉素A4间的比例也满足该比例要求，并提高该中间体的纯度和稳定性，对生产出高纯度、A3和A4比例符合要求的米尔贝肟是非常有必要的。Currently, Milbex oxime is produced by fermentation and semi-synthesis. First, the milbemycin A3 and A4 obtained by fermentation of Streptomyces are oxidized to obtain intermediate 5-ketomilbemycin A3 and A4, and then the obtained intermediate is subjected to oximation to obtain a mixture of milbemycin A3 and A4. EP and USP have strict limits on the ratio of milbe oxime A3 and milbe oxime A4. In the production of milbe oxime, 5-keto milbemycin A3 and A4 are key intermediates. Therefore, it is ensured that the ratio between 5-ketomilbemycin A3 and 5-ketokimilbemycin A4 also meets the requirements of this ratio, and the purity and stability of the intermediate are improved, which is important for the production of high purity, A3 and A3. Milbe oxime with A4 ratio meeting the requirements is very necessary.
CN108586481公开了5-酮基米尔贝霉素发酵液的提取和5-酮基米尔贝霉素A3和A4的混合物的结晶工艺，但目前还没有关于5-酮基米尔贝霉素A3、A4单组分晶型的报道。现有技术公开的均是5-酮基米尔贝霉素A3、A4的混合物，该混合物在A3、A4比例的稳定性、纯度和质量稳定性等方面还不能令人满意，因此，对5-酮基米尔贝霉素A3、A4单组分进行制备和晶型研究显得非常必要。制备出高纯度、稳定的5-酮基米尔贝霉素A3、A4单组分，将为生产A3、A4比例稳定、纯度高的米尔贝肟打下坚实的基础。CN108586481 discloses the extraction of the fermentation broth of 5-ketomilbemycin and the crystallization process of the mixture of 5-ketomilbemycin A3 and A4, but there is no information about 5-ketomilbemycin A3 and A4. Report on the crystalline forms of the components. The prior art discloses a mixture of 5-ketomilbemycin A3 and A4. This mixture is not yet satisfactory in terms of stability, purity and quality stability of the ratio of A3 and A4. It is very necessary to prepare and study the crystal form of ketomilbemycin A3 and A4. The preparation of high-purity and stable single-component 5-ketokilmbemycin A3 and A4 will lay a solid foundation for the production of milbemycin with a stable ratio of A3 and A4 and high purity.
发明内容Summary of the invention
本发明的目的之一在于提供5-酮基米尔贝霉素的两种单组分晶型即5-酮基米尔贝霉素A3晶型I(简称为晶型I)和5-酮基米尔贝霉素A4晶型II(简称为晶型II)。One of the objectives of the present invention is to provide two single-component crystal forms of 5-ketokimirbemycin, namely 5-ketokimirbemycin A3 crystal form I (abbreviated as crystal form I) and 5-ketokimirbemycin Bemycin A4 crystal form II (abbreviated as crystal form II).
本发明所述的5-酮基米尔贝霉素A3晶型I，使用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射图谱在以下位置具有特征峰：4.89±0.20°、8.69±0.20°、13.64±0.20°、14.71±0.20°、16.66±0.20°、19.85±0.20°、20.90±0.20°。The 5-ketomilbemycin A3 crystal form I of the present invention uses Cu-Kα radiation, and the X-ray powder diffraction pattern expressed at 2θ angles has characteristic peaks at the following positions: 4.89±0.20°, 8.69±0.20 °, 13.64±0.20°, 14.71±0.20°, 16.66±0.20°, 19.85±0.20°, 20.90±0.20°.
进一步地，本发明所述的5-酮基米尔贝霉素A3晶型I，使用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射图谱进一步在以下位置具有特征峰：14.45±0.20°、15.33±0.20°、17.08±0.20°、17.82±0.20°、19.24±0.20°、22.29±0.20°、23.01±0.20°、23.51±0.20°。Further, the 5-ketomilbemycin A3 crystal form I of the present invention uses Cu-Kα radiation, and the X-ray powder diffraction pattern expressed at 2θ angles further has characteristic peaks at the following positions: 14.45±0.20° , 15.33±0.20°, 17.08±0.20°, 17.82±0.20°, 19.24±0.20°, 22.29±0.20°, 23.01±0.20°, 23.51±0.20°.
更进一步地，本发明所述的5-酮基米尔贝霉素A3晶型I，使用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射图谱进一步在以下位置具有特征峰：12.16±0.20°、17.49±0.20°、22.64±0.20°、25.70±0.20°、26.50±0.20°、28.27±0.20°、29.29±0.20°、31.24±0.20°、33.79±0.20°、42.62±0.20°。Furthermore, the 5-ketomilbemycin A3 crystal form I of the present invention uses Cu-Kα radiation, and the X-ray powder diffraction pattern expressed at 2θ angles further has characteristic peaks at the following positions: 12.16±0.20 °, 17.49±0.20°, 22.64±0.20°, 25.70±0.20°, 26.50±0.20°, 28.27±0.20°, 29.29±0.20°, 31.24±0.20°, 33.79±0.20°, 42.62±0.20°.
本发明所述的5-酮基米尔贝霉素A3晶型I的2θ角度、d值和相对强度数据如下表1所示：The 2θ angle, d value and relative intensity data of 5-ketomilbemycin A3 crystal form I of the present invention are shown in Table 1 below:
表1Table 1
峰编号Peak number 20《°》20《°》 d(晶面间距)d (interplanar spacing) 相对强度(％)Relative Strength(%)
11 4.894.89 18.056818.0568 34.834.8
22 8.698.69 10.167010.1670 64.664.6
33 12.1612.16 7.27217.2721 7.27.2
44 13.6413.64 6.48666.4866 44.144.1
55 14.4514.45 6.12466.1246 34.434.4
66 14.7114.71 6.01716.0171 67.567.5
77 15.3315.33 5.77515.7751 33.733.7
88 16.6616.66 5.31695.3169 100.0100.0
99 17.0817.08 5.18715.1871 36.736.7
1010 17.4917.49 5.06655.0665 20.820.8
1111 17.8217.82 4.97334.9733 22.622.6
1212 19.2419.24 4.60934.6093 28.828.8
1313 19.8519.85 4.46914.4691 60.660.6
1414 20.9020.90 4.24684.2468 80.480.4
1515 22.2922.29 3.98503.9850 18.418.4
1616 22.6422.64 3.92433.9243 11.411.4
1717 23.0123.01 3.86193.8619 19.319.3
1818 23.5123.51 3.78103.7810 20.420.4
1919 25.7025.70 3.46353.4635 11.311.3
2020 26.5026.50 3.36063.3606 5.65.6
21twenty one 28.2728.27 3.15423.1542 6.86.8
22twenty two 29.2929.29 3.04663.0466 12.812.8
23twenty three 31.2431.24 2.86082.8608 6.36.3
24twenty four 33.7933.79 2.65052.6505 6.06.0
2525 42.6242.62 2.11962.1196 4.94.9
本发明所述的5-酮基米尔贝霉素A3晶型I的XRPD图谱如图1所示。The XRPD pattern of 5-ketomilbemycin A3 crystal form I of the present invention is shown in FIG. 1.
本发明所述的5-酮基米尔贝霉素A3晶型I，可以用KBr压片测得的红外吸收图谱来表征，根据所述图谱，其在以下位置处有特征峰：2932cm -1、2922cm -1、1727cm -1、1677cm -1、1367cm -1、1219cm -1、1057cm -1、995cm -1、909cm -1。 The 5-ketomilbemycin A3 crystal form I of the present invention can be characterized by the infrared absorption spectrum measured by KBr tablet. According to the spectrum, it has characteristic peaks at the following positions: 2932cm -1 , 2922cm -1, 1727cm -1, 1677cm -1 , 1367cm -1, 1219cm -1, 1057cm -1, 995cm -1, 909cm -1.
本发明所述的5-酮基米尔贝霉素A3晶型I的红外吸收谱图还在以下位置处有特征峰：3506cm -1、2966cm -1、2874cm -1、2856cm -1、1446cm -1、1379cm -1、1276cm -1、1180cm -1、1167cm -1、1035cm -1、963cm -1、887cm -1、541cm -1、483cm -1。 The infrared absorption spectrum of 5-ketomilbemycin A3 crystal form I of the present invention also has characteristic peaks at the following positions: 3506cm -1 , 2966cm -1 , 2874cm -1 , 2856cm -1 , 1446cm -1 , 1379cm -1, 1276cm -1, 1180cm -1, 1167cm -1, 1035cm -1, 963cm -1, 887cm -1, 541cm -1, 483cm -1.
本发明所述的5-酮基米尔贝霉素A3晶型I的红外谱图如图2所示。The infrared spectrum of 5-ketomilbemycin A3 crystal form I of the present invention is shown in FIG. 2.
本发明所述的5-酮基米尔贝霉素A3晶型I的差示扫描量热(DSC)图谱在208℃-212℃和258℃-262℃有两个放热峰。The differential scanning calorimetry (DSC) spectrum of the 5-ketomilbemycin A3 crystal form I of the present invention has two exothermic peaks at 208°C-212°C and 258°C-262°C.
本发明所述的5-酮基米尔贝霉素A3晶型I的DSC图谱如图3所示。The DSC spectrum of 5-ketomilbemycin A3 crystal form I of the present invention is shown in FIG. 3.
本发明所述的5-酮基米尔贝霉素A3晶型I的热重分析(TGA)图谱如图4所示。The thermogravimetric analysis (TGA) pattern of 5-ketomilbemycin A3 crystal form I of the present invention is shown in FIG. 4.
本发明所述的5-酮基米尔贝霉素A4晶型II，使用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射图谱在以下位置具有特征峰：5.13±0.20°、9.00±0.20°、12.56±0.20°、15.95±0.20°、18.73±0.20°、19.42±0.20°、20.50±0.20°、22.78±0.20°。The 5-ketomilbemycin A4 crystal form II of the present invention uses Cu-Kα radiation, and the X-ray powder diffraction pattern expressed at 2θ angles has characteristic peaks at the following positions: 5.13±0.20°, 9.00±0.20 °, 12.56±0.20°, 15.95±0.20°, 18.73±0.20°, 19.42±0.20°, 20.50±0.20°, 22.78±0.20°.
进一步地，本发明所述的5-酮基米尔贝霉素A4晶型II，使用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射图谱进一步在以下位置具有特征峰：14.52±0.20°、15.11±0.20°、16.42±0.20°、22.36±0.20°、23.30±0.20°、25.63±0.20°、26.44±0.20°、27.24±0.20°。Further, the 5-ketomilbemycin A4 crystal form II of the present invention uses Cu-Kα radiation, and the X-ray powder diffraction pattern expressed at 2θ angles further has characteristic peaks at the following positions: 14.52±0.20° , 15.11±0.20°, 16.42±0.20°, 22.36±0.20°, 23.30±0.20°, 25.63±0.20°, 26.44±0.20°, 27.24±0.20°.
更进一步地，本发明所述的5-酮基米尔贝霉素A4晶型II，使用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射图谱进一步在以下位置具有特征峰：28.34±0.20°、29.40±0.20°、30.35±0.20°、32.15±0.20°、34.70±0.20°、41.64±0.20°。Furthermore, the 5-ketomilbemycin A4 crystal form II of the present invention uses Cu-Kα radiation, and the X-ray powder diffraction pattern expressed at 2θ angles further has characteristic peaks at the following positions: 28.34±0.20 °, 29.40±0.20°, 30.35±0.20°, 32.15±0.20°, 34.70±0.20°, 41.64±0.20°.
本发明所述的5-酮基米尔贝霉素A4晶型II的2θ角度、d值和相对强度数据如下表2所示：The 2θ angle, d value and relative intensity data of 5-ketomilbemycin A4 crystal form II of the present invention are shown in Table 2 below:
表2Table 2
峰编号Peak number 2θ(°)2θ(°) d(晶面间距)d (interplanar spacing) 相对强度(％)Relative Strength(%)
11 5.135.13 17.212317.2123 30.330.3
22 9.009.00 9.81759.8175 44.544.5
33 12.5612.56 7.04187.0418 49.649.6
44 14.5214.52 6.09536.0953 33.333.3
55 15.1115.11 5.85865.8586 50.450.4
66 15.9515.95 5.55195.5519 100.0100.0
77 16.4216.42 5.39415.3941 39.139.1
88 17.2417.24 5.13935.1393 5.05.0
99 17.9317.93 4.94284.9428 3.33.3
1010 18.7318.73 4.73374.7337 49.749.7
1111 19.4219.42 4.56704.5670 79.879.8
1212 20.5020.50 4.32884.3288 55.555.5
1313 22.3622.36 3.97273.9727 14.914.9
1414 22.7822.78 3.90043.9004 38.638.6
1515 23.3023.30 3.81463.8146 18.518.5
1616 25.6325.63 3.47283.4728 13.313.3
1717 26.4426.44 3.36823.3682 10.510.5
1818 27.2427.24 3.27113.2711 9.49.4
1919 28.3428.34 3.14663.1466 6.16.1
2020 29.4029.40 3.03553.0355 9.89.8
21twenty one 30.3530.35 2.94262.9426 7.37.3
22twenty two 32.1532.15 2.78182.7818 9.99.9
23twenty three 34.7034.70 2.58302.5830 5.35.3
24twenty four 41.6441.64 2.16722.1672 6.06.0
本发明所述的5-酮基米尔贝霉素A4晶型II的XRPD图谱如图5所示。The XRPD pattern of 5-ketomilbemycin A4 crystal form II of the present invention is shown in FIG. 5.
本发明所述的5-酮基米尔贝霉素A4晶型II，可以用KBr压片测得的红外吸收图谱表征，根据所述图谱，其在以下位置处有特征峰：3510cm -1、2921cm -1、1677cm -1、1372cm -1、1243cm -1、1165cm -1、990cm -1。 The 5-ketomilbemycin A4 crystal form II of the present invention can be characterized by the infrared absorption spectrum measured by KBr tablet. According to the spectrum, it has characteristic peaks at the following positions: 3510cm -1 , 2921cm -1 , 1677cm -1 , 1372cm -1 , 1243cm -1 , 1165cm -1 , 990cm -1 .
本发明所述5-酮基米尔贝霉素A4晶型II的红外吸收谱图还在以下位置处有特征峰：2956cm -1、2873cm -1、1459cm -1、1436cm -1、1334cm -1、1103cm -1、1061cm -1、964cm -1、948cm -1、907cm -1、887cm -1、858cm -1、820cm -1、543cm -1、487cm -1、459cm -1。 The infrared absorption spectrum of 5-ketomilbemycin A4 crystal form II of the present invention also has characteristic peaks at the following positions: 2956cm -1 , 2873cm -1 , 1459cm -1 , 1436cm -1 , 1334cm -1 , 1103cm -1, 1061cm -1, 964cm -1 , 948cm -1, 907cm -1, 887cm -1, 858cm -1, 820cm -1, 543cm -1, 487cm -1, 459cm -1.
本发明所述的5-酮基米尔贝霉素A4晶型II的红外谱图如图6所示。The infrared spectrum of 5-ketomilbemycin A4 crystal form II of the present invention is shown in FIG. 6.
本发明所述的5-酮基米尔贝霉素A4晶型II的差示扫描量热(DSC)图谱在218℃-222℃和258℃-262℃有两个放热峰。The differential scanning calorimetry (DSC) spectrum of the 5-ketomilbemycin A4 crystal form II of the present invention has two exothermic peaks at 218°C-222°C and 258°C-262°C.
本发明所述的5-酮基米尔贝霉素A4晶型II的DSC图谱如图7所示。The DSC chart of 5-ketomilbemycin A4 crystal form II of the present invention is shown in FIG. 7.
本发明所述的5-酮基米尔贝霉素A4晶型II的TGA图谱如图8所示。The TGA spectrum of 5-ketomilbemycin A4 crystal form II of the present invention is shown in FIG. 8.
本发明另一目的还在于提供一种制备5-酮基米尔贝霉素A3晶型I和5-酮基米尔贝霉素A4晶型II的方法，该方法包括如下步骤：Another object of the present invention is to provide a method for preparing 5-ketomilbemycin A3 crystal form I and 5-ketomilbemycin A4 crystal form II, which comprises the following steps:
步骤(1)：将5-酮基米尔贝霉素粗提液加载至反相制备柱，以体积浓度为60％-90％的甲醇溶液进行洗脱，分别得到5-酮基米尔贝霉素A3收集液和5-酮基米尔贝霉素A4收集液；Step (1): Load the crude extract of 5-ketomilbemycin on a reversed-phase preparation column, and elute with a methanol solution with a volume concentration of 60%-90% to obtain 5-ketomilbemycin respectively A3 collection solution and 5-ketokimilbemycin A4 collection solution;
步骤(2)：分别将步骤(1)的5-酮基米尔贝霉素A3收集液和5-酮基米尔贝霉素A4收集液浓缩至甲醇体积浓度为45％-55％，分别得到5-酮基米尔贝霉素 A3甲醇浓缩液和5-酮基米尔贝霉素A4甲醇浓缩液，再用正庚烷萃取，分别得到5-酮基米尔贝霉素A3正庚烷萃取液和5-酮基米尔贝霉素A4正庚烷萃取液，将其分别进行浓缩，搅拌结晶，过滤，干燥，分别得到5-酮基米尔贝霉素A3晶型I和5-酮基米尔贝霉素A4晶型II。Step (2): Respectively concentrate the 5-ketomilbemycin A3 and 5-ketomilbemycin A4 of step (1) to a methanol volume concentration of 45%-55% to obtain 5 -Ketomilbemycin A3 methanol concentrate and 5-ketomilbemycin A4 methanol concentrate, and then extracted with n-heptane to obtain 5-ketomilbemycin A3 n-heptane extract and 5 -Ketomilbemycin A4 n-heptane extract, which was concentrated, stirred and crystallized, filtered, and dried to obtain 5-ketokimilbemycin A3 crystal form I and 5-ketokimilbemycin A3 respectively A4 crystal form II.
优选地，所述步骤(1)中反相制备柱的填料为C18或C8，粗提液上柱结束后，以体积浓度为60％-90％的甲醇溶液进行洗脱，根据制备液相出峰情况收集各流份，再根据各流份的HPLC分析结果，将5-酮基米尔贝霉素A3纯度高的部分合并得到5-酮基米尔贝霉素A3收集液，将5-酮基米尔贝霉素A4纯度高的部分合并得到5-酮基米尔贝霉素A4收集液。Preferably, the filler of the reverse phase preparation column in the step (1) is C18 or C8. After the crude extract is applied to the column, it is eluted with a methanol solution with a volume concentration of 60%-90%. Collect each fraction according to the peak condition, and then according to the HPLC analysis results of each fraction, combine the high purity parts of 5-ketomilbemycin A3 to obtain 5-ketomilbemycin A3 collection solution. The fractions with high purity of milbemycin A4 are combined to obtain 5-ketokimilbemycin A4 collection solution.
优选地，所述步骤(2)中萃取所用的正庚烷与5-酮基米尔贝霉素A3甲醇浓缩液的体积比为1:1～2:1；所述步骤(2)中萃取所用的正庚烷与5-酮基米尔贝霉素A4甲醇浓缩液的体积比为1:1～2:1。Preferably, the volume ratio of the n-heptane and 5-ketomilbemycin A3 methanol concentrate used in the extraction in the step (2) is 1:1 to 2:1; the extraction in the step (2) is The volume ratio of n-heptane to 5-ketomilbemycin A4 methanol concentrate is 1:1 to 2:1.
优选地，所述步骤(2)中，将5-酮基米尔贝霉素A3正庚烷萃取液浓缩至5-酮基米尔贝霉素A3的浓度为50g/L～150g/L；将5-酮基米尔贝霉素A4正庚烷萃取液浓缩至5-酮基米尔贝霉素A4的浓度为50g/L～150g/L。Preferably, in the step (2), the 5-ketomilbemycin A3 n-heptane extract is concentrated to a 5-ketomilbemycin A3 concentration of 50g/L～150g/L; -Ketomilbemycin A4 n-heptane extract is concentrated to a 5-ketomilbemycin A4 concentration of 50g/L～150g/L.
优选地，所述步骤(2)中搅拌时间为20～30小时。Preferably, the stirring time in the step (2) is 20-30 hours.
优选地，所述步骤(2)中结晶的温度为10-30℃Preferably, the temperature of crystallization in the step (2) is 10-30°C
优选地，所述步骤(2)中干燥的温度为30～40℃，干燥的时间2～3小时。Preferably, the drying temperature in the step (2) is 30-40°C, and the drying time is 2 to 3 hours.
其中，步骤(1)中所述5-酮基米尔贝霉素粗提液是通过以下方法制备得到的：Wherein, the crude 5-ketomilbemycin in step (1) is prepared by the following method:
方法1：method 1:
步骤(a)：采用板框过滤收集5-酮基米尔贝霉素发酵液中的菌丝体，用体积浓度为80％-100％的甲醇或者乙醇浸泡该菌丝体，过滤得5-酮基米尔贝霉素的甲醇或者乙醇提取液，浓缩，得到5-酮基米尔贝霉素浓缩液，用正庚烷萃取该浓缩液，得到5-酮基米尔贝霉素正庚烷萃取液；Step (a): Collect the mycelium in the 5-ketokimilbemycin fermentation broth by plate and frame filtration, soak the mycelium in methanol or ethanol with a volume concentration of 80%-100%, and filter to obtain 5-ketone The methanol or ethanol extract of kimilbemycin is concentrated to obtain a 5-ketokimilbemycin concentrate, and the concentrated solution is extracted with n-heptane to obtain a 5-ketokimilbemycin n-heptane extract;
步骤(b)：将步骤(a)的5-酮基米尔贝霉素正庚烷萃取液浓缩至油状浓缩液，再用甲醇或乙醇溶解该浓缩液、过滤得到5-酮基米尔贝霉素粗提液。Step (b): Concentrate the 5-ketomilbemycin n-heptane extract of step (a) to an oily concentrate, then dissolve the concentrate with methanol or ethanol, and filter to obtain 5-ketokimilbemycin Crude extract.
或者or
方法2：Method 2:
将5-酮基米尔贝霉素粗品溶于体积浓度为60％以上的甲醇水溶液或体积浓度为60％以上的乙醇水溶液，得到5-酮基米尔贝霉素粗提液，其中所述5-酮基米尔贝霉素粗品可采用常规的合成法(例如CN105949217公开的合成法)制备得到；The crude 5-ketomilbemycin is dissolved in a methanol aqueous solution with a volume concentration of more than 60% or an ethanol aqueous solution with a volume concentration of more than 60% to obtain a crude 5-ketomilbemycin extract, wherein the 5- The crude ketomilbemycin can be prepared by a conventional synthesis method (for example, the synthesis method disclosed in CN105949217);
优选地，所述方法1步骤(a)中所述5-酮基米尔贝霉素发酵液是采用CN103789339公开的方法，利用链霉菌F2-18(参见文献黄隽,林甲檀,周敏等.吸水链霉菌HS023 milF基因敲除构建产5-酮基米尔贝霉素基因工程菌.《微生物学报》,2015,55(1):107-113)发酵制备得到的。Preferably, the 5-ketokimilbemycin fermentation broth in step (a) of the method 1 adopts the method disclosed in CN103789339, using Streptomyces F2-18 (see literature Huang Jun, Lin Jiatan, Zhou Min etc . Streptomyces hygroscopicus HS023 milF gene knockout constructs a genetically engineered strain producing 5-ketokimilbemycin. "Acta Microbiology", 2015, 55(1):107-113).
优选地，所述方法1步骤(a)中使用体积浓度为80％-100％乙醇浸泡时，体积浓度为80％-100％乙醇与菌丝体的比为4:1～10:1，单位为L/Kg，更优选为5:1。Preferably, when the volume concentration of 80%-100% ethanol is used for soaking in step (a) of the method 1, the ratio of the volume concentration of 80%-100% ethanol to mycelium is 4:1-10:1, the unit It is L/Kg, more preferably 5:1.
优选地，所述方法1步骤(a)中使用体积浓度为80％-100％甲醇浸泡时，体积浓度为80％-100％甲醇与菌丝体的比为4:1～10:1，单位为L/Kg，更优选为5:1。Preferably, when the volume concentration of 80%-100% methanol is used for soaking in step (a) of the method 1, the ratio of the volume concentration of 80%-100% methanol to mycelium is 4:1-10:1, the unit It is L/Kg, more preferably 5:1.
优选地，所述方法1步骤(a)中所述5-酮基米尔贝霉素浓缩液中乙醇或甲醇的体积浓度为45-55％。Preferably, the volume concentration of ethanol or methanol in the 5-ketomilbemycin concentrate in step (a) of the method 1 is 45-55%.
优选地，所述方法1步骤(a)中正庚烷萃取时，正庚烷与5-酮基米尔贝霉素浓缩液的体积比为1:1～2:1。Preferably, during the n-heptane extraction in step (a) of the method 1, the volume ratio of n-heptane to 5-ketomilbemycin concentrate is 1:1 to 2:1.
优选地，所述方法2中所用的甲醇或乙醇的体积浓度为95％以上。Preferably, the volume concentration of methanol or ethanol used in the method 2 is more than 95%.
优选地，所述方法2中所述粗提液中5-酮基米尔贝霉素的浓度为20～100g/L。Preferably, the concentration of 5-ketokimilbemycin in the crude extract in the method 2 is 20-100 g/L.
优选地，所述本发明所述浓缩可采用减压浓缩，负压为0.08～0.1Mpa。Preferably, the concentration in the present invention can be concentrated under reduced pressure, and the negative pressure is 0.08-0.1Mpa.
本发明的发明人经过大量研究发现5-酮基米尔贝霉素A3晶型I和5-酮基米尔贝霉素A4晶型II，填补了现有技术中没有关于5-酮基米尔贝霉素A3、A4单组分晶型的空白。本发明的晶型I和Ⅱ稳定性好，室温下放置几个月，未发现转晶现象。工艺简单，易于实行产业化。生产出5-酮基米尔贝霉素A3、A4单组分晶体，可以方便地按照米尔贝肟成品质量标准中关于A3和A4比例的要求，先将5-酮基米尔贝霉素A3、A4按比例混合，再进行肟化，进而生产出比例稳定的更高质量的米尔贝肟。也可以直接以5-酮基米尔贝霉素A3、A4单组分晶体为原料生产米尔贝肟A3单组分或米尔贝肟A4单组分，用来调整米尔贝肟成品中组分的比例，确保成品中米尔贝肟A3和米尔贝肟A4之间的比例符合要求。The inventors of the present invention have found that 5-ketokimirbemycin A3 crystal form I and 5-ketokimirbemycin A4 crystal form II after a lot of research fill in the prior art that there is nothing about 5-ketokimirbemycin. A3, A4 single-component crystal form blank. The crystal forms I and II of the present invention have good stability, and no crystal conversion phenomenon is found when placed at room temperature for several months. The process is simple and easy to implement industrialization. To produce 5-ketokimilbemycin A3 and A4 single-component crystals, it is convenient to use 5-ketokimilbemycin A3 and A4 in accordance with the requirements of the A3 and A4 ratio in the quality standard of the Milbex oxime product. It is mixed in proportion and then oximated to produce a higher quality Milbex oxime with a stable proportion. It is also possible to directly produce milbexime A3 single-component or milbex oxime A4 single-component from 5-ketomilbemycin A3 and A4 single-component crystals as raw materials to adjust the proportion of the components in the Milbex oxime product. , To ensure that the ratio between Milbex oxime A3 and Milbex oxime A4 in the finished product meets the requirements.
本发明中所述的5-酮基米尔贝霉素为含5-酮基米尔贝霉素A3和5-酮基米尔贝霉素A4的十六元环大环内酯化合物，其结构如下：The 5-ketomilbemycin in the present invention is a sixteen-membered ring macrolide compound containing 5-ketomilbemycin A3 and 5-ketomilbemycin A4, and its structure is as follows:
Figure PCTCN2020088110-appb-000001
Figure PCTCN2020088110-appb-000001
其中，当R＝CH 3时为5-酮基米尔贝霉素A3(简称为A3)：当R＝CH 2CH 3时为5-酮基米尔贝霉素A4(简称为A4)。 Among them, when R=CH 3 , it is 5-ketomilbemycin A3 (abbreviated as A3); when R=CH 2 CH 3 , it is 5-ketomilbemycin A4 (abbreviated as A4).
本发明中的常温和室温都是指10℃～30℃。Both normal temperature and room temperature in the present invention refer to 10°C to 30°C.
附图说明Description of the drawings
图1为实施例9所得5-酮基米尔贝霉素A3晶型I的X-射线粉末衍射图谱。1 is the X-ray powder diffraction pattern of 5-ketomilbemycin A3 crystal form I obtained in Example 9.
图2为实施例9所得5-酮基米尔贝霉素A3晶型I的红外吸收图谱。2 is the infrared absorption spectrum of 5-ketomilbemycin A3 crystal form I obtained in Example 9.
图3为实施例9所得5-酮基米尔贝霉素A3晶型I的DSC图谱。3 is the DSC chart of 5-ketomilbemycin A3 crystal form I obtained in Example 9.
图4为实施例9所得5-酮基米尔贝霉素A3晶型I的TGA图谱。4 is the TGA chart of 5-ketomilbemycin A3 crystal form I obtained in Example 9.
图5为实施例10所得5-酮基米尔贝霉素A4晶型II的X-射线粉末衍射图谱。5 is the X-ray powder diffraction pattern of 5-ketomilbemycin A4 crystal form II obtained in Example 10.
图6为实施例10所得5-酮基米尔贝霉素A4晶型II的红外吸收图谱。6 is the infrared absorption spectrum of 5-ketomilbemycin A4 crystal form II obtained in Example 10.
图7为实施例10所得5-酮基米尔贝霉素A4晶型II的DSC图谱。7 is the DSC chart of 5-ketomilbemycin A4 crystal form II obtained in Example 10.
图8为实施例10所得5-酮基米尔贝霉素A4晶型II的TGA图谱。8 is the TGA spectrum of 5-ketomilbemycin A4 crystal form II obtained in Example 10.
具体实施方式Detailed ways
本发明所用的5-酮基米尔贝霉素发酵液是按照中国发明专利CN108586481中的实施例1发酵得到。The 5-ketomilbemycin fermentation broth used in the present invention is obtained by fermentation according to Example 1 of Chinese invention patent CN108586481.
本发明所涉及的X-射线粉末衍射仪器及测试条件为：X-衍射仪器型号Rigaku D/max-2115 Cu靶；操作方法：扫描速度3°/min，扫描步宽0.01°。The X-ray powder diffraction instrument and test conditions involved in the present invention are: X-ray diffraction instrument model Rigaku D/max-2115 Cu target; operation method: scanning speed 3°/min, scanning step width 0.01°.
本发明所涉及的红外分光光度仪及测试条件为：红外分光光度仪型号：BRWKER VECTOR 22；操作方法：采用KBr压片法，扫描范围400～4000cm-1。The infrared spectrophotometer and test conditions involved in the present invention are: infrared spectrophotometer model: BRWKER VECTOR 22; operation method: KBr compression method with a scanning range of 400-4000 cm-1.
本发明涉及的DSC检测仪及测试条件为：DSC检测仪型号为：NETZSCHDSC214；操作方法：升温速率10℃/min，温度范围：25℃～300℃。The DSC detector and test conditions involved in the present invention are: the model of the DSC detector is: NETZSCHDSC214; the operation method: the heating rate is 10°C/min, and the temperature range: 25°C to 300°C.
本发明涉及的TGA检测仪及测试条件为：TGA检测仪型号：PerkinElmerTGA400；操作方法：升温速率10℃/min，温度范围：30℃～280℃。The TGA detector and test conditions involved in the present invention are: TGA detector model: PerkinElmer TGA400; operation method: heating rate 10°C/min, temperature range: 30°C to 280°C.
本发明涉及的液相(HPLC)测试条件为：色谱柱为：Hypersil OSD2-C18，4.6mm×150mm；流动相为乙腈：水＝85:15；检测波长：250nm；流速：1.0ml/min；柱温：室温；进样量：10μl。The liquid phase (HPLC) test conditions involved in the present invention are: the chromatographic column is: Hypersil OSD2-C18, 4.6mm×150mm; the mobile phase is acetonitrile: water=85:15; detection wavelength: 250nm; flow rate: 1.0ml/min; Column temperature: room temperature; injection volume: 10μl.
本发明所述乙醇或甲醇溶液的百分浓度(％)为体积百分比浓度。The percentage concentration (%) of the ethanol or methanol solution in the present invention is the volume percentage concentration.
实施例1：正庚烷萃取液的制备Example 1: Preparation of n-heptane extract
将5-酮基米尔贝霉素发酵液(按照专利CN108586481中的实施例1发酵得到)35L通过板框过滤后，得到14.56Kg菌丝体，菌丝体再用70L体积浓度为95％的乙醇进行浸泡，板框过滤后，滤液负压0.09Mpa浓缩，至乙醇体积浓度为50％，此时体积约50L。再用50L正庚烷萃取，得正庚烷萃取液40L。After 35L of 5-ketokimirbemycin fermentation broth (obtained by fermentation according to Example 1 in patent CN108586481) was filtered through a plate and frame, 14.56Kg of mycelium was obtained, and the mycelium was then used 70L of ethanol with a volume concentration of 95% After soaking and frame filtration, the filtrate is concentrated under a negative pressure of 0.09Mpa until the ethanol volume concentration is 50%, at which time the volume is about 50L. Then extract with 50L of n-heptane to obtain 40L of n-heptane extract.
实施例2：5-酮基米尔贝霉素粗提液的制备Example 2: Preparation of 5-ketokimilbemycin crude extract
取实施例1所得正庚烷萃取液20L于38℃，-0.09Mpa进行浓缩，浓缩至油状物。再加入0.3L体积浓度为95％的甲醇进行溶解、过滤得5-酮基米尔贝霉素粗提液0.37L，其中含5-酮基米尔贝霉素A3和5-酮基米尔贝霉素A4总浓度为95g/L，即A3和A4总质量为35.15g。Take 20L of the n-heptane extract obtained in Example 1 and concentrate at 38°C and -0.09Mpa, and concentrate to an oily substance. Then add 0.3L of methanol with a volume concentration of 95% to dissolve and filter to obtain 0.37L of 5-ketokimirbemycin crude extract, which contains 5-ketokimilbemycin A3 and 5-ketokimirbemycin The total concentration of A4 is 95g/L, that is, the total mass of A3 and A4 is 35.15g.
实施例3：5-酮基米尔贝霉素粗提液的制备Example 3: Preparation of 5-ketomilbemycin crude extract
取实施例1所得正庚烷萃取液20L于38℃，-0.095Mpa进行浓缩，浓缩至油状物。再加入0.6L体积浓度为95％的甲醇进行溶解、过滤得5-酮基米尔贝霉素粗提液0.7L，其中含5-酮基米尔贝霉素A3和5-酮基米尔贝霉素A4总浓度为50g/L，即A3和A4总质量为35.15g。Take 20L of the n-heptane extract obtained in Example 1 and concentrate it at 38°C and -0.095Mpa to an oily substance. Then add 0.6L of methanol with a volume concentration of 95% to dissolve and filter to obtain 0.7L of 5-ketokimilbemycin crude extract, which contains 5-ketokimilbemycin A3 and 5-ketokimilbemycin The total concentration of A4 is 50g/L, that is, the total mass of A3 and A4 is 35.15g.
实施例4：5-酮基米尔贝霉素粗提液的制备Example 4: Preparation of crude 5-ketomilbemycin
取化学合成法制得的5-酮基米尔贝霉素粗品(按照专利CN105949217实施例2的化学合成方法制得)30g，采用1.5L体积浓度为80％的甲醇水溶液溶解， 得到5-酮基米尔贝霉素粗提液1.5L，其中5-酮基米尔贝霉素A3和5-酮基米尔贝霉素A4总浓度约为20g/L。Take 30 g of crude 5-ketokimilbemycin prepared by the chemical synthesis method (prepared in accordance with the chemical synthesis method in Example 2 of Patent CN105949217), and dissolve it with 1.5L of a methanol aqueous solution with a volume concentration of 80% to obtain 5-ketokimir 1.5L of crude perimycin, in which the total concentration of 5-ketomilbemycin A3 and 5-ketomilbemycin A4 is about 20g/L.
实施例5：5-酮基米尔贝霉素A3、A4单组分的制备Example 5: Preparation of 5-ketomilbemycin A3 and A4 single components
取实施例2中所得的5-酮基米尔贝霉素粗提液100mL(其中5-酮基米尔贝霉素A3和5-酮基米尔贝霉素A4的总量共计9.5g)，加载到C18制备柱(100*250mm)，采用体积浓度90％甲醇进行洗脱，根据洗脱液紫外出峰情况分瓶收集，再根据各流分的HPLC结果确定5-酮基米尔贝霉素A3、A4单组分收集液。得到5-酮基米尔贝霉素A3收集液2.4L，其中5-酮基米尔贝霉素A3的浓度为0.534g/L，5-酮基米尔贝霉素A3的质量为1.28g；和5-酮基米尔贝霉素A4收集液6.8L，其中5-酮基米尔贝霉素A4的浓度为0.97g/L，5-酮基米尔贝霉素A4的质量为6.6g。Take 100 mL of the crude 5-ketomilbemycin obtained in Example 2 (the total amount of 5-ketomilbemycin A3 and 5-ketokimilbemycin A4 totals 9.5g), and load it into C18 preparative column (100*250mm), eluted with 90% methanol by volume concentration, collected in bottles according to the UV peak of the eluent, and then determined 5-ketokimirbemycin A3, A4 single component collection solution. Obtain 2.4L of 5-ketokimilbemycin A3 collection solution, wherein the concentration of 5-ketokimilbemycin A3 is 0.534g/L, and the mass of 5-ketokimilbemycin A3 is 1.28g; and 5 -6.8L of the ketomilbemycin A4 collection solution, in which the concentration of 5-ketomilbemycin A4 is 0.97g/L, and the mass of 5-ketokimilbemycin A4 is 6.6g.
实施例6：5-酮基米尔贝霉素A3、A4单组分的制备Example 6: Preparation of 5-ketomilbemycin A3 and A4 single components
取实施例3中的5-酮基米尔贝霉素粗提液200mL，(其中5-酮基米尔贝霉素A3和5-酮基米尔贝霉素A4的总量共计10g)，加载到C18制备柱(100*250mm)，采用体积浓度70％甲醇进行洗脱，根据洗脱液紫外出峰情况分瓶收集，再根据各流分的HPLC结果确定5-酮基米尔贝霉素A3、A4单组分收集液。得到5-酮基米尔贝霉素A3收集液3.8L，其中5-酮基米尔贝霉素A3的浓度为0.43g/L，5-酮基米尔贝霉素A3的质量为1.63g；和5-酮基米尔贝霉素A4收集液7L，其中5-酮基米尔贝霉素A4的浓度为0.93g/L，5-酮基米尔贝霉素A4的质量为6.5g。Take 200 mL of the crude extract of 5-ketomilbemycin in Example 3, (the total amount of 5-ketomilbemycin A3 and 5-ketokimilbemycin A4 is 10g), and load it into C18 Preparative column (100*250mm), eluted with 70% methanol by volume concentration, collected in bottles according to the UV peak of the eluent, and then determined 5-ketokimirbemycin A3 and A4 according to the HPLC results of each fraction Single component collection solution. Obtain 3.8L of 5-ketokimilbemycin A3 collection solution, wherein the concentration of 5-ketokimilbemycin A3 is 0.43g/L and the mass of 5-ketokimilbemycin A3 is 1.63g; and 5 -7L of ketomilbemycin A4 collection solution, in which the concentration of 5-ketomilbemycin A4 is 0.93g/L, and the mass of 5-ketomilbemycin A4 is 6.5g.
实施例7：5-酮基米尔贝霉素A3、A4单组分的制备Example 7: Preparation of 5-ketomilbemycin A3 and A4 single components
取实施例4中的5-酮基米尔贝霉素粗提液500mL(其中5-酮基米尔贝霉素A3和5-酮基米尔贝霉素A4的总量共计10g)，加载到C18制备柱(100*250mm)，采用体积浓度60％甲醇进行洗脱，根据洗脱液紫外出峰情况分瓶收集，再根据各流分的HPLC结果确定5-酮基米尔贝霉素A3、A4单组分收集液。得到5-酮基米尔贝霉素A3收集液4.9L，其中5-酮基米尔贝霉素A3的浓度为0.34g/L，5-酮基米尔贝霉素A3的质量为1.67g；和5-酮基米尔贝霉素A4收集液8.6L，其中5-酮基米尔贝霉素A4的浓度为0.77g/L，5-酮基米尔贝霉素A4的质量为6.68g。Take 500 mL of the crude extract of 5-ketomilbemycin in Example 4 (the total amount of 5-ketomilbemycin A3 and 5-ketomilbemycin A4 totals 10g) and load it into C18 for preparation Column (100*250mm), eluted with 60% methanol in volume concentration, and collected in bottles according to the UV peak of the eluent, and then determined the 5-ketomilbemycin A3 and A4 according to the HPLC results of each fraction. Fraction collection liquid. Obtain 4.9L of 5-ketokimirbemycin A3 collection solution, wherein the concentration of 5-ketokimilbemycin A3 is 0.34g/L, and the mass of 5-ketokimilbemycin A3 is 1.67g; and 5 -8.6L of ketomilbemycin A4 collection solution, in which the concentration of 5-ketomilbemycin A4 is 0.77g/L, and the mass of 5-ketomilbemycin A4 is 6.68g.
实施例8：5-酮基米尔贝霉素A3、A4单组分正庚烷萃取液的制备Example 8: Preparation of 5-ketomilbemycin A3, A4 single-component n-heptane extract
将实施例5、6、7中得到的5-酮基米尔贝霉素A3收集液混合，混合液体积为11.1L，其中5-酮基米尔贝霉素A3质量为4.58g，HPLC色谱纯度92.8％；将实施例5、6、7中得到的5-酮基米尔贝霉素A4收集液混合，混合液体积为22.4L，其中5-酮基米尔贝霉素A4质量为19.78g，HPLC色谱纯度88.3％。分别减压浓缩，A3混合液浓缩至6.2L，甲醇度51％；A4混合液浓缩至10.5L，甲醇度47％。各加入正庚烷6.5L和11L分别进行萃取，分别得到5-酮基米尔贝霉素A3正庚烷萃取液6.0L，其中5-酮基米尔贝霉素A3为3.89g；5-酮基米尔贝霉素A4正庚烷萃取液10.6L，其中5-酮基米尔贝霉素A4为17.41g。The 5-ketomilbemycin A3 collected in Examples 5, 6, and 7 were mixed, and the volume of the mixed solution was 11.1L. The mass of 5-ketomilbemycin A3 was 4.58g and the HPLC purity was 92.8. %; The 5-ketomilbemycin A4 collected in Examples 5, 6, and 7 were mixed, and the volume of the mixed solution was 22.4L, of which the mass of 5-ketomilbemycin A4 was 19.78g, and the HPLC chromatography The purity is 88.3%. Respectively concentrated under reduced pressure, A3 mixture was concentrated to 6.2L, methanol content was 51%; A4 mixture was concentrated to 10.5L, methanol content was 47%. 6.5L and 11L of n-heptane were added to extract respectively to obtain 6.0L of 5-ketomilbemycin A3 n-heptane extract, of which 5-ketomilbemycin A3 was 3.89g; 5-keto Milbemycin A4 n-heptane extract 10.6L, of which 5-ketokimilbemycin A4 is 17.41g.
实施例9：5-酮基米尔贝霉素A3晶型I的制备Example 9: Preparation of 5-ketomilbemycin A3 crystal form I
取实施例8中5-酮基米尔贝霉素A3正庚烷萃取液6L，浓缩至26mL，其中5-酮基米尔贝霉素A3的浓度为150g/L，常温下搅拌结晶20小时，结晶液再进行抽滤，并用正庚烷淋洗湿晶体滤饼，所得湿晶体再于40℃、负压0.09Mpa下烘干3小时，得到2.18g 5-酮基米尔贝霉素A3晶体，该晶体HPLC色谱纯度为99.6％。取样进行X-射线粉末衍射、DSC、TGA、红外分析，确认为5-酮基米尔贝霉素A3晶型I，其X-射线粉末衍射图谱如图1所示，红外吸收图谱如图2所示，DSC图谱如图3所示，TGA图谱如图4所示。Take 6L of the 5-ketomilbemycin A3 n-heptane extract in Example 8 and concentrate it to 26mL. The concentration of 5-ketomilbemycin A3 is 150g/L. Stir and crystallize at room temperature for 20 hours. The liquid was suction filtered, and the wet crystal filter cake was rinsed with n-heptane. The wet crystals obtained were dried at 40°C and a negative pressure of 0.09Mpa for 3 hours to obtain 2.18g 5-ketomilbemycin A3 crystals. The purity of the crystal HPLC chromatography is 99.6%. A sample was taken for X-ray powder diffraction, DSC, TGA, and infrared analysis, and it was confirmed to be 5-ketomilbemycin A3 crystal form I. The X-ray powder diffraction pattern is shown in Figure 1, and the infrared absorption pattern is shown in Figure 2. As shown, the DSC spectrum is shown in Figure 3, and the TGA spectrum is shown in Figure 4.
实施例10：5-酮基米尔贝霉素A4晶型II的制备Example 10: Preparation of 5-ketomilbemycin A4 crystal form II
取实施例8中5-酮基米尔贝霉素A4正庚烷萃取液6L，浓缩至98.5mL，其中5-酮基米尔贝霉素A4的浓度为100g/L，常温下搅拌结晶25小时，结晶液再进行抽滤，并用正庚烷淋洗湿晶体滤饼，所得湿晶体再于40℃、负压0.09Mpa下烘干3小时，得到5.9g 5-酮基米尔贝霉素A4晶体，该晶体HPLC色谱纯度为99.2％。取样进行X-射线粉末衍射、DSC、TGA、红外分析，确认为5-酮基米尔贝霉素A4晶型II，其X-射线粉末衍射图谱如图5所示，红外吸收图谱如图6所示，DSC图谱如图7所示，TGA图谱如图8所示。Take 6L of the 5-ketomilbemycin A4 n-heptane extract in Example 8 and concentrate it to 98.5mL, where the concentration of 5-ketomilbemycin A4 is 100g/L, stir and crystallize at room temperature for 25 hours, The crystalline liquid was suction filtered, and the wet crystal filter cake was rinsed with n-heptane. The wet crystals obtained were dried at 40°C and a negative pressure of 0.09Mpa for 3 hours to obtain 5.9g of 5-ketomilbemycin A4 crystals. The HPLC chromatographic purity of the crystal was 99.2%. Sampling for X-ray powder diffraction, DSC, TGA, and infrared analysis confirmed that it is 5-ketomilbemycin A4 crystal form II. The X-ray powder diffraction pattern is shown in Figure 5, and the infrared absorption pattern is shown in Figure 6. As shown, the DSC spectrum is shown in Figure 7 and the TGA spectrum is shown in Figure 8.
实施例11：5-酮基米尔贝霉素A4晶型II的制备Example 11: Preparation of 5-ketomilbemycin A4 crystal form II
取实施例8中5-酮基米尔贝霉素A4正庚烷萃取液4.5L，浓缩至148mL，其中5-酮基米尔贝霉素A4的浓度为50g/L，常温下搅拌结晶30小时，结晶液再进行抽滤，并用正庚烷淋洗湿晶体滤饼，所得湿晶体再于40℃、负压0.095Mpa下烘干3小时，得到3.9g 5-酮基米尔贝霉素A4晶体，该晶体HPLC色谱纯度为 99.5％。经X-射线粉末衍射、DSC、TGA、分析确认为5-酮基米尔贝霉素A4晶型II。Take 4.5L of the 5-ketomilbemycin A4 n-heptane extract in Example 8 and concentrate it to 148mL. The concentration of 5-ketomilbemycin A4 is 50g/L. Stir and crystallize at room temperature for 30 hours. The crystalline liquid was suction filtered, and the wet crystal filter cake was rinsed with n-heptane. The obtained wet crystals were dried at 40°C and a negative pressure of 0.095Mpa for 3 hours to obtain 3.9g 5-ketomilbemycin A4 crystals. The HPLC chromatographic purity of the crystal was 99.5%. It was confirmed by X-ray powder diffraction, DSC, TGA, and analysis to be 5-ketokimilbemycin A4 crystal form II.
实施例12：晶型I和II的稳定性试验Example 12: Stability test of crystal forms I and II
将实施例9、10得到的晶型I和II，每种晶型留样3袋，每袋0.1g分装，都采用双层PE袋加一层铝箔袋封装。放置于药品稳定性试验箱(温度：40℃，湿度75％)进行加速试验。分别于1个月、2个月、3个月不同放置周期时间点取样，经XRPD、HPLC检测，数据见表3。The crystal forms I and II obtained in Examples 9 and 10 were kept in 3 bags of each crystal form, and each bag was divided into 0.1 g, and they were packaged in a double-layer PE bag and an aluminum foil bag. Placed in a drug stability test box (temperature: 40°C, humidity 75%) for accelerated testing. Samples were taken at different time points of the placement cycle for 1 month, 2 months, and 3 months, and tested by XRPD and HPLC. The data are shown in Table 3.
表3table 3
Figure PCTCN2020088110-appb-000002
Figure PCTCN2020088110-appb-000002
通过上述试验数据可以得出，通过本发明工艺制备的5-酮基米尔贝霉素A3晶型I和5-酮基米尔贝霉素A4晶型II具有较高的纯度，且稳定性很好。From the above test data, it can be concluded that the 5-ketomilbemycin A3 crystal form I and 5-ketomilbemycin A4 crystal form II prepared by the process of the present invention have high purity and good stability. .
以上所述仅是本发明的优选实施方案，本发明的保护范围不局限于上述实施例，凡本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰也应视为本发明的保护范围。The above are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments. All technical solutions under the idea of the present invention belong to the protection scope of the present invention. It should be pointed out that for those of ordinary skill in the art, several improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims (12)

  1. 一种5-酮基米尔贝霉素A3晶型I，其特征在于，使用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射图谱在以下位置具有特征峰：4.89±0.20°、8.69±0.20°、13.64±0.20°、14.71±0.20°、16.66±0.20°、19.85±0.20°、20.90±0.20°。A 5-ketokimirbemycin A3 crystal form I, characterized in that, using Cu-Kα radiation, the X-ray powder diffraction pattern expressed in 2θ angles has characteristic peaks at the following positions: 4.89±0.20°, 8.69± 0.20°, 13.64±0.20°, 14.71±0.20°, 16.66±0.20°, 19.85±0.20°, 20.90±0.20°.
  2. 根据权利要求1所述的5-酮基米尔贝霉素A3晶型I，其特征在于，使用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射图谱进一步在以下位置具有特征峰：14.45±0.20°、15.33±0.20°、17.08±0.20°、17.82±0.20°、19.24±0.20°、22.29±0.20°、23.01±0.20°、23.51±0.20°。The 5-ketomilbemycin A3 crystalline form I of claim 1, wherein the X-ray powder diffraction pattern expressed at 2θ angles using Cu-Kα radiation further has characteristic peaks at the following positions: 14.45 ±0.20°, 15.33±0.20°, 17.08±0.20°, 17.82±0.20°, 19.24±0.20°, 22.29±0.20°, 23.01±0.20°, 23.51±0.20°.
  3. 根据权利要求1或2所述的5-酮基米尔贝霉素A3晶型I，其特征在于，使用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射图谱进一步在以下位置具有特征峰：12.16±0.20°、17.49±0.20°、22.64±0.20°、25.70±0.20°、26.50±0.20°、28.27±0.20°、29.29±0.20°、31.24±0.20°、33.79±0.20°、42.62±0.20°。The 5-ketomilbemycin A3 crystal form I according to claim 1 or 2, characterized in that the X-ray powder diffraction pattern expressed at 2θ angles using Cu-Kα radiation further has characteristic peaks at the following positions ：12.16±0.20°, 17.49±0.20°, 22.64±0.20°, 25.70±0.20°, 26.50±0.20°, 28.27±0.20°, 29.29±0.20°, 31.24±0.20°, 33.79±0.20°, 42.62±0.20° .
  4. 一种5-酮基米尔贝霉素A4晶型II，其特征在于，使用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射图谱在以下位置具有特征峰：5.13±0.20°、9.00±0.20°、12.56±0.20°、15.95±0.20°、18.73±0.20°、19.42±0.20°、20.50±0.20°、22.78±0.20°。A 5-ketokimirbemycin A4 crystal form II, characterized in that, using Cu-Kα radiation, the X-ray powder diffraction pattern expressed at 2θ angles has characteristic peaks at the following positions: 5.13±0.20°, 9.00± 0.20°, 12.56±0.20°, 15.95±0.20°, 18.73±0.20°, 19.42±0.20°, 20.50±0.20°, 22.78±0.20°.
  5. 根据权利要求4所述的5-酮基米尔贝霉素A4晶型II，其特征在于，使用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射图谱进一步在以下位置具有特征峰：14.52±0.20°、15.11±0.20°、16.42±0.20°、22.36±0.20°、23.30±0.20°、25.63±0.20°、26.44±0.20°、27.24±0.20°。The 5-ketomilbemycin A4 crystal form II of claim 4, characterized in that the X-ray powder diffraction pattern expressed at 2θ angles using Cu-Kα radiation further has characteristic peaks at the following positions: 14.52 ±0.20°, 15.11±0.20°, 16.42±0.20°, 22.36±0.20°, 23.30±0.20°, 25.63±0.20°, 26.44±0.20°, 27.24±0.20°.
  6. 根据权利要求4或5所述的5-酮基米尔贝霉素A4晶型II，其特征在于，使用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射图谱进一步在以下位置具有特征峰：28.34±0.20°、29.40±0.20°、30.35±0.20°、32.15±0.20°、34.70±0.20°、41.64±0.20°。The 5-ketomilbemycin A4 crystal form II of claim 4 or 5, wherein the X-ray powder diffraction pattern expressed at 2θ angles using Cu-Kα radiation further has characteristic peaks at the following positions : 28.34±0.20°, 29.40±0.20°, 30.35±0.20°, 32.15±0.20°, 34.70±0.20°, 41.64±0.20°.
  7. 一种制备根据权利要求1-3任一项所述的5-酮基米尔贝霉素A3晶型I以及根据权利要求4-6任一项所述的5-酮基米尔贝霉素A4晶型II的方法，该方法包括如下步骤：A method for preparing 5-ketomilbemycin A3 crystal form I according to any one of claims 1-3 and 5-ketomilbemycin A4 crystal according to any one of claims 4-6 Type II method, the method includes the following steps:
    步骤(1)：将5-酮基米尔贝霉素粗提液加载至反相制备柱，以体积浓度为60％-90％的甲醇溶液进行洗脱，分别得到5-酮基米尔贝霉素A3收集液和5-酮基 米尔贝霉素A4收集液；Step (1): Load the crude extract of 5-ketomilbemycin on a reversed-phase preparation column, and elute with a methanol solution with a volume concentration of 60%-90% to obtain 5-ketomilbemycin respectively A3 collection solution and 5-ketokimilbemycin A4 collection solution;
    步骤(2)：分别将步骤(1)的5-酮基米尔贝霉素A3收集液和5-酮基米尔贝霉素A4收集液浓缩至甲醇体积浓度为45％-55％，分别得到5-酮基米尔贝霉素A3甲醇浓缩液和5-酮基米尔贝霉素A4甲醇浓缩液，再用正庚烷萃取，分别得到5-酮基米尔贝霉素A3正庚烷萃取液和5-酮基米尔贝霉素A4正庚烷萃取液，将其分别进行浓缩，搅拌结晶，过滤，干燥，分别得到5-酮基米尔贝霉素A3晶型Ⅰ和5-酮基米尔贝霉素A4晶型II。Step (2): Respectively concentrate the 5-ketomilbemycin A3 and 5-ketomilbemycin A4 of step (1) to a methanol volume concentration of 45%-55% to obtain 5 -Ketomilbemycin A3 methanol concentrate and 5-ketomilbemycin A4 methanol concentrate, and then extracted with n-heptane to obtain 5-ketomilbemycin A3 n-heptane extract and 5 -Ketomilbemycin A4 n-heptane extract, which was concentrated, stirred and crystallized, filtered and dried to obtain 5-ketokimilbemycin A3 crystal form I and 5-ketokimilbemycin A3 respectively A4 crystal form II.
  8. 根据权利要求7的方法，所述步骤(1)中反相制备柱的填料为C18或C8。The method according to claim 7, wherein the packing of the reverse phase preparation column in the step (1) is C18 or C8.
  9. 根据权利要求7或8的方法，所述步骤(2)中萃取所用的正庚烷与5-酮基米尔贝霉素A3甲醇浓缩液的体积比为1:1～2:1；所述步骤(2)中萃取所用的正庚烷与5-酮基米尔贝霉素A4甲醇浓缩液的体积比为1:1～2:1。The method according to claim 7 or 8, wherein the volume ratio of the n-heptane used for extraction in the step (2) to the methanol concentrate of 5-ketomilbemycin A3 is 1:1 to 2:1; (2) The volume ratio of n-heptane and 5-ketomilbemycin A4 methanol concentrate used in extraction is 1:1 to 2:1.
  10. 根据权利要求7～9任一项的方法，所述步骤(2)中，将5-酮基米尔贝霉素A3正庚烷萃取液浓缩至5-酮基米尔贝霉素A3的浓度为50g/L～150g/L；将5-酮基米尔贝霉素A4正庚烷萃取液浓缩至5-酮基米尔贝霉素A4的浓度为50g/L～150g/L。The method according to any one of claims 7 to 9, wherein in step (2), the 5-ketomilbemycin A3 n-heptane extract is concentrated to a concentration of 5-ketomilbemycin A3 of 50g /L～150g/L; The 5-ketomilbemycin A4 n-heptane extract is concentrated to a concentration of 5-ketomilbemycin A4 of 50g/L～150g/L.
  11. 根据权利要求7～10任一项的方法，所述步骤(2)中搅拌时间为20～30小时；结晶的温度为10-30℃；干燥的温度为30～40℃，干燥的时间2～3小时。The method according to any one of claims 7 to 10, wherein the stirring time in step (2) is 20-30 hours; the crystallization temperature is 10-30°C; the drying temperature is 30-40°C, and the drying time is 2~ 3 hours.
  12. 根据权利要求7～11任一项的方法，步骤(1)中所述5-酮基米尔贝霉素粗提液是通过以下方法制备得到的：The method according to any one of claims 7 to 11, the crude 5-ketomilbemycin in step (1) is prepared by the following method:
    方法1：method 1:
    步骤(a)：采用板框过滤收集5-酮基米尔贝霉素发酵液中的菌丝体，用体积浓度为80％-100％的甲醇或者乙醇浸泡该菌丝体，过滤得5-酮基米尔贝霉素的甲醇或者乙醇提取液，浓缩，得到5-酮基米尔贝霉素浓缩液，用正庚烷萃取该浓缩液，得到5-酮基米尔贝霉素正庚烷萃取液；Step (a): Collect the mycelium in the 5-ketokimilbemycin fermentation broth by plate and frame filtration, soak the mycelium in methanol or ethanol with a volume concentration of 80%-100%, and filter to obtain 5-ketone The methanol or ethanol extract of kimilbemycin is concentrated to obtain a 5-ketokimilbemycin concentrate, and the concentrated solution is extracted with n-heptane to obtain a 5-ketokimilbemycin n-heptane extract;
    步骤(b)：将步骤(a)的5-酮基米尔贝霉素正庚烷萃取液浓缩至油状浓缩液，再用甲醇或乙醇溶解该浓缩液、过滤得到5-酮基米尔贝霉素粗提液；Step (b): Concentrate the 5-ketomilbemycin n-heptane extract of step (a) to an oily concentrate, then dissolve the concentrate with methanol or ethanol, and filter to obtain 5-ketokimilbemycin Crude extract
    或者or
    方法2：Method 2:
    将5-酮基米尔贝霉素粗品溶于体积浓度为60％以上的甲醇水溶液或体积浓度为60％以上的乙醇水溶液，得到5-酮基米尔贝霉素粗提液。The crude 5-ketomilbemycin is dissolved in a methanol aqueous solution with a volume concentration of more than 60% or an ethanol aqueous solution with a volume concentration of more than 60% to obtain a crude 5-ketokimirbemycin extract.
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