CN104497003A - Preparation method of high-purity milbemycins - Google Patents
Preparation method of high-purity milbemycins Download PDFInfo
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- CN104497003A CN104497003A CN201410696342.1A CN201410696342A CN104497003A CN 104497003 A CN104497003 A CN 104497003A CN 201410696342 A CN201410696342 A CN 201410696342A CN 104497003 A CN104497003 A CN 104497003A
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- preparation
- mibemycin
- acetonitrile
- methyl alcohol
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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
Abstract
The invention discloses a preparation method of high-purity milbemycins. A milbemycin crude product is enriched by a macroporous adsorption resin, and milbemycins A3 and A4 are separated out, concentrated and dried; and then the dried powder of the milbemycins A3 and A4 is dissolved with methanol or acetonitrile, and the high-purity milbemycins A3 and A4 are prepared by semi-preparative liquid chromatography. The preparation method is simple, no side products and impurity residues exist, the product separation and purification is stable, and the purity of the finished products is high and can reach up to 99.99%.
Description
Technical field
The invention belongs to chemical field, be specifically related to a kind of preparation method of macrolide antibiotics mibemycin standard substance.
Background technology
Mibemycin (milbemycin) is parasite medicine inside and outside macrolide antibody-like, mainly contains A3, A4 homologue, the conventional insect repellent doing dog.Mibemycin has good effect to Control and prevention major part common parasitic worm disease.Be commonly used to prevent dirofilariasis, control the trichuriasis of nematode, dog that hookworm causes, cat disease and dog.Because mibemycin is protected by foreign patent, the mibemycin that China uses mainly comes from external import, and expensive price makes the use of mibemycin receive huge restriction.In production technology and application, China is all well below foreign level, and does not also occur the preparation method about mibemycin in current document.The preparation method researching and developing mibemycin voluntarily will break this monopolization phenomenon, greatly can accelerate the step of China's autonomous production mibemycin, therefore how prepare highly purified mibemycin finished product, be subject to people's attention always.
Summary of the invention
The object of the invention is to solve the defect of China for mibemycin sterling technology of preparing cavity, a kind of preparation method of macrolide antibiotics mibemycin is provided, its preparation technology is simple, yield is high, compare other means, cost has obvious reduction, and separation and purification of products result is stable and finished product purity is high.
For realizing above-mentioned technical purpose, the technical scheme that the present invention takes is:
A preparation method for high purity mibemycin, comprises the steps:
1) content >=50% (mass percentage content) mibemycin crude product macroporous adsorbing resin for purification is isolated mibemycin A3 and A4, concentrated, drying obtains dry powder;
2) mibemycin A3 and A4 dry powder methyl alcohol or acetonitrile are dissolved, be then prepared into high purity mibemycin A3 and A4 with semi-preparative liquid chromatography.
Above-mentioned steps 1) resin such as HP20, HPD100, HPD300 polymeric adsorbent of enrichment.Usually, the blade diameter length ratio 1:(5-8 of resin dress post).
In specific embodiments more of the present invention, step 1) by mibemycin crude product upper prop after 60% ethanol or dissolve with methanol, then be that moving phase carries out desorb with the methanol solution of the ethanolic soln of 50%-62% (V/V) or 55%-65% (V/V), the stripping liquid of mibemycin A3 and A4 is merged, concentrates, with based organic solvent (such as ethyl acetate, butylacetate) extraction, reconcentration obtains the dry powder of mibemycin A3 and A4.
Above-mentioned steps 2) methyl alcohol used is the AG methyl alcohol that purity reaches 99.%, acetonitrile used is that purity reaches 99.% AG acetonitrile; Utilizing methyl alcohol or acetonitrile by step 1) dry powder that obtains is dissolved as the solution that concentration is 30mg/mL ~ 67mg/mL, then carries out semi-preparative liquid chromatography.
In specific embodiments more of the present invention, step 2) semi-preparative liquid chromatography with acetonitrile: the solution of water: methyl alcohol=70:28:2 (volume ratio) is moving phase, flow velocity 15mL/min ~ 20mL/min, ultraviolet detection wavelength is 244nm, collects mould A3 and A4 of Mil shellfish respectively.
In a particular embodiment of the present invention, above-mentioned steps 2) semi-preparative liquid chromatography instrument used is Agilent 1200, semipreparative column is LP-C8 (Shanghai Yue Xu Science and Technology Ltd.), and semipreparative column is 25.01*250mm, and particle diameter is 5-8 μm.
The mibemycin A3 prepared and A4 sample carry out purity detecting by high performance liquid chromatography (HPLC), and purity all reaches 98%-100%, prepares yield >=95%.
The invention provides a kind of preparation method of macrolide antibiotics mibemycin, substantially increase mibemycin and prepare purity, mibemycin A3 and A4 product purity high (can 99.% be reached), and preparation technology is simple, yield is high, compares other means, and cost has obvious reduction, not easily produce by product, achieve the breakthrough that China is separated mibemycin high purity sterling.
Embodiment
Below by way of embodiment, the invention will be further described, but this is not limitation of the present invention, those skilled in the art, according to basic thought of the present invention, can make various amendment or improvement, but only otherwise depart from basic thought of the present invention, all within the scope of the present invention.
Embodiment 1
Get 1L HP20 resin dress post (blade diameter length ratio=1:6), get mibemycin crude product 12g (content is 56%), with 60% dissolve with ethanol to 3000mL upper prop.With the ethanol desorb resin column of 50%, detect.Isolated mibemycin A3, A4 are merged, after concentrated, water layer is 500mL, adds 500mL ethyl acetate and extracts, get ethyl acetate layer, concentrated, dry matter weight 8.09g (content is 82%).
Get the dissolve with methanol of the mibemycin A3 after 2g enrichment and A4 30mL.Lysate is entered semi-preparative liquid chromatography instrument, semi-preparative liquid chromatography instrument used is Agilent 1200, and semipreparative column is LP-C8 (Shanghai Yue Xu Science and Technology Ltd.), and 25.01*250mm, particle diameter is 5-8 μm.Every pin sample introduction 4mL, moving phase is acetonitrile: water: methyl alcohol=70:28:2 (volume ratio), flow velocity 15mL/min ~ 20mL/min, and ultraviolet detection wavelength is 244nm, and collect A3 and A4, total mass 1.92g respectively, yield is 96%.
Shimadzu liquid chromatograph LC2010 is adopted to carry out HPLC detection to collection sample, sampling volume is 20 μ l, moving phase is acetonitrile: water: triethylamine: Glacial acetic acid=3000:1000:4:4 (volume ratio), ultraviolet detection wavelength 240nm, column pressure is 8.2Mpa, and sample detection purity is 100%.
Embodiment 2:
Get 1.8L HP100 resin (Shanghai Hua Zhen resin company limited) and fill post (blade diameter length ratio=1:8), get mibemycin crude product 26.39g (content is 51.20%), with 60% dissolve with ethanol to 3000mL upper prop.With the methylene dichloride desorb resin column of 60%, gradient extracts and detects.Isolated mibemycin A3, A4 are merged, after concentrated, water layer is 810mL, adds 810mL ethyl acetate and extracts, get ethyl acetate layer, concentrated, dry matter weight 14.5g (content is 93.21%).
The acetonitrile getting the mibemycin A3 after 2g enrichment and A4 30mL dissolves.Lysate is entered semi-preparative liquid chromatography instrument, semi-preparative liquid chromatography instrument used is Agilent 1200, and semipreparative column is LP-C8 (Shanghai Yue Xu Science and Technology Ltd.), and 25.01*250mm, particle diameter is 5-8 μm.Every pin sample introduction 4mL, moving phase is acetonitrile: water: methyl alcohol=70:28:2 (volume ratio), flow velocity 15mL/min ~ 20mL/min, and ultraviolet detection wavelength is 244nm, and collect A3 and A4 respectively, total mass is 1.8g, and yield is 90%.
Shimadzu liquid chromatograph LC2010 is adopted to carry out HPLC detection to collection sample, sampling volume is 20 μ l, moving phase is acetonitrile: water: triethylamine: Glacial acetic acid=3000:1000:4:4 (volume ratio), ultraviolet detection wavelength 240nm, column pressure is 8.2Mpa, and sample detection purity is 99.8%.
Embodiment 3
Get 2L HP300 macroporous adsorbent resin dress post (blade diameter length ratio=1:7), get mibemycin crude product 14.94g (content is 53.21%), with 60% dissolve with ethanol to 3000mL upper prop.With the ethanol desorb resin column of 55%, gradient extracts and detects.Isolated mibemycin A3, A4 are merged, after concentrated, water layer is 450mL, adds 450mL ethyl acetate and extracts, get ethyl acetate layer, concentrated, dry matter weight 8.91g (content is 89.23%).
The acetonitrile getting the mibemycin A3 after 2g enrichment and A4 30mL dissolves.Lysate is entered semi-preparative liquid chromatography instrument, semi-preparative liquid chromatography instrument used is Agilent 1200, and semipreparative column is LP-C8 (Shanghai Yue Xu Science and Technology Ltd.), and 25.01*250mm, particle diameter is 5-8 μm.Every pin sample introduction 4mL, moving phase is acetonitrile: water: methyl alcohol=70:28:2, flow velocity 15mL/min ~ 20mL/min, and ultraviolet detection wavelength is 244nm, and collect A3 and A4 respectively, total mass is 1.96g, and yield is 98%.
Shimadzu liquid chromatograph LC2010 is adopted to carry out HPLC detection to collection sample, sampling volume is 20 μ l, moving phase is acetonitrile: water: triethylamine: Glacial acetic acid=3000:1000:4:4 (volume ratio), ultraviolet detection wavelength 240nm, column pressure is 8.2Mpa, and sample detection purity is 100%.
Claims (10)
1. a preparation method for high purity mibemycin, comprises the steps:
1) the mibemycin crude product macroporous adsorbing resin for purification of content >=50% is isolated mibemycin A3 and A4, concentrated, dry dry powder;
2) by step 1) the mibemycin A3 that obtains and A4 dry powder methyl alcohol or acetonitrile dissolve, and is prepared into high purity mibemycin A3 and A4 with semi-preparative liquid chromatography.
2. preparation method as claimed in claim 1, is characterized in that, step 1) resin of enrichment is selected from one of resin of following model: HP20, HPD100 and HPD300.
3. preparation method as claimed in claim 1, is characterized in that, step 1) blade diameter length ratio of resin column used is 1: (5 ~ 8).
4. preparation method as claimed in claim 1, it is characterized in that, step 1) specifically: by mibemycin crude product upper prop after 60% ethanol or 60% dissolve with methanol, then with the methanol solution of the ethanolic soln of 50%-62% or 55%-65% for moving phase carries out desorb, the stripping liquid of mibemycin A3 and A4 is merged, concentrates, with based organic solvent extraction, reconcentration, obtain the dry powder of mibemycin A3 and A4.
5. preparation method as claimed in claim 4, it is characterized in that, described based organic solvent is ethyl acetate or butylacetate.
6. preparation method as claimed in claim 1, is characterized in that, step 2) carry out semi-preparative liquid chromatography with the solution that dry powder to be dissolved as concentration 30mg/mL ~ 67mg/mL by methyl alcohol or acetonitrile.
7. preparation method as claimed in claim 1, is characterized in that, step 2) methyl alcohol used is the AG methyl alcohol that purity reaches 99%, acetonitrile used is that purity reaches 99.% AG acetonitrile.
8. preparation method as claimed in claim 1, it is characterized in that, step 2) semi-preparative liquid chromatography with volume ratio acetonitrile: water: the solution of methyl alcohol=70:28:2 is moving phase, flow velocity 15mL/min ~ 20mL/min, ultraviolet detection wavelength is 244nm, collects mould A3 and A4 of Mil shellfish respectively.
9. preparation method as claimed in claim 1, is characterized in that, step 2) the semi-preparative liquid chromatography instrument that adopts is Agilent 1200, semipreparative column is LP-C8 chromatographic column.
10. preparation method as claimed in claim 9, is characterized in that, step 2) semipreparative column is 25.01*250mm, particle diameter is 5-8 μm.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105254644A (en) * | 2015-11-04 | 2016-01-20 | 湖北宏中药业股份有限公司 | Preparation method of milbemycin oxime |
| CN106083883A (en) * | 2016-07-01 | 2016-11-09 | 宁夏泰瑞制药股份有限公司 | A kind of purification process of mibemycin oxime crude product |
| CN112390810A (en) * | 2020-11-26 | 2021-02-23 | 内蒙古拜克生物有限公司 | Separation and purification method of milbemycin fermentation liquor |
| CN113588840A (en) * | 2021-08-17 | 2021-11-02 | 丽珠集团福州福兴医药有限公司 | Method for detecting content of milbemycins |
Citations (4)
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| US3950360A (en) * | 1972-06-08 | 1976-04-13 | Sankyo Company Limited | Antibiotic substances |
| JP2000281683A (en) * | 1999-03-29 | 2000-10-10 | Sankyo Co Ltd | Purification of milbemycins and avermectins |
| CN101100651A (en) * | 2007-05-28 | 2008-01-09 | 东北农业大学 | Streptomyces strain and application method thereof |
| CN103468625A (en) * | 2013-09-10 | 2013-12-25 | 东北农业大学 | Gene disruption mutant of streptomyces bingchenggensis as well as preparation method and application thereof |
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2014
- 2014-11-27 CN CN201410696342.1A patent/CN104497003A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3950360A (en) * | 1972-06-08 | 1976-04-13 | Sankyo Company Limited | Antibiotic substances |
| JP2000281683A (en) * | 1999-03-29 | 2000-10-10 | Sankyo Co Ltd | Purification of milbemycins and avermectins |
| CN101100651A (en) * | 2007-05-28 | 2008-01-09 | 东北农业大学 | Streptomyces strain and application method thereof |
| CN103468625A (en) * | 2013-09-10 | 2013-12-25 | 东北农业大学 | Gene disruption mutant of streptomyces bingchenggensis as well as preparation method and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| HAI-YANWANG ET AL.: ""Combined application of plasma mutagenesis and gene engineering leads to 5-oxomilbemycins A3/A4 as main components from Streptomyces bingchenggensis"", 《APPL MICROBIOL BIOTECHNOL》 * |
| JI ZHANG ET AL.: ""Genetic engineering of Streptomyces bingchenggensis to produce milbemycins A3/A4 as main components and eliminate the biosynthesis of nanchangmycin"", 《APPL MICROBIOL BIOTECHNOL》 * |
| XIANG-JING WANG ET AL.: ""Improvement of milbemycin-producing Streptomyces bingchenggensis by rational screening of ultraviolet-and chemically induced mutants"", 《WORLD J MICROBIOL BIOTECHNOL》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105254644A (en) * | 2015-11-04 | 2016-01-20 | 湖北宏中药业股份有限公司 | Preparation method of milbemycin oxime |
| CN106083883A (en) * | 2016-07-01 | 2016-11-09 | 宁夏泰瑞制药股份有限公司 | A kind of purification process of mibemycin oxime crude product |
| CN112390810A (en) * | 2020-11-26 | 2021-02-23 | 内蒙古拜克生物有限公司 | Separation and purification method of milbemycin fermentation liquor |
| CN112390810B (en) * | 2020-11-26 | 2021-09-28 | 内蒙古拜克生物有限公司 | Separation and purification method of milbemycin fermentation liquor |
| CN113588840A (en) * | 2021-08-17 | 2021-11-02 | 丽珠集团福州福兴医药有限公司 | Method for detecting content of milbemycins |
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Application publication date: 20150408 |