CN109400621A - A kind of preparation method of high-purity mibemycin - Google Patents
A kind of preparation method of high-purity mibemycin Download PDFInfo
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- CN109400621A CN109400621A CN201811400424.1A CN201811400424A CN109400621A CN 109400621 A CN109400621 A CN 109400621A CN 201811400424 A CN201811400424 A CN 201811400424A CN 109400621 A CN109400621 A CN 109400621A
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- mibemycin
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- 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
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Abstract
The present invention provides a kind of preparation methods of high-purity mibemycin, including the filter residue of mibemycin fermentation liquid after the extraction of low-boiling alcohol, to be concentrated under reduced pressure, cooling filters off solid content through hot water；Filtrate is added the back extraction of highly polar organic solvent, and the feed liquid after back extraction washes through salt, alkali cleaning, is concentrated to dryness after washing；It is added after the dissolution of highly polar solvent and maintains certain temperature and be added with stirring a large amount of low polarity solvent, stir lower cooling, crystallization, the mibemycin that high-purity is obtained by filtration.Method of the invention uses the principle of phase transfer and solubility and polarity difference, instead of traditional macroporous resin adsorption isolation technics and silica gel column chromatography technology, this is technically simple stably and controllable, the production cycle can be shortened, save a large amount of organic solvent, a large amount of solid waste is reduced, so that having saved the energy reduces production cost.
Description
Technical field
The present invention relates to biofermentation pharmaceutical technology fields, relate in particular to a kind of method for forcing crystallization using phase inversion
Obtain high-purity mibemycin.
Background technique
Mibemycin (milbemycin) is parasite medicine inside and outside the antibody of macrolides, contains two kinds of A3, A4
Active princlple is usually used in the anthelmintic of canid.Mibemycin is to control and prevents most of common parasitic worm disease all
There is good effect.Commonly used to prevent dirofilariasis, the trichuriasis of dog, cat disease and dog that control nematode, hookworm cause.Rice
Your shellfish mycin is the intermediate for synthesizing milbemycin oxime, and the intermediate of high-purity provides efficient means for synthesis milbemycin oxime.
The purity of mibemycin has very big association to subsequent yield and cost；Mibemycin passes through at present
Purification process is that macroporous resin adsorption parsing and silica gel column chromatography are purified, and needs a large amount of organic solvent and generates very much
Solid waste.With the gradually reinforcement of environmental consciousness, the requirement of built environment friendly, conservation-minded society is responded, is pressed
According to current safety and environmental protection requirement, in mibemycin purification process, how to reduce the usage amount of organic solvent and reduce solid
Useless is the task of top priority.
Summary of the invention
It is an object of the invention to overcome technical deficiency of the prior art in terms of mibemycin extraction, provide a kind of high
The preparation method of purity mibemycin.
The ingenious thinking using phase transfer of the present invention, instead of macroporous resin adsorption and silica gel column chromatography technology, including it is following
Step:
(1) filter residue of mibemycin fermentation liquid is concentrated under reduced pressure after the extraction of low-boiling alcohol through hot water, cooling filtering
Solid content is removed, filtrate is taken；
(2) back extraction of highly polar organic solvent is added in filtrate, collects organic phase after back extraction, washes, alkali cleaning, is added after washing through salt
Filtrate is collected in adsorbent, filters pressing, and filtrate decompression is concentrated to dryness, and collects solid content；
(3) after solid content is dissolved with highly polar solvent, heating keeps temperature, is added with stirring low pole organic solvent, drops
Temperature, crystallization, centrifugal drying obtain the mibemycin of high-purity.
In the filter residue of step (1) mibemycin fermentation liquid, mibemycin chromatographic purity is 50%-55%.Specifically,
The chromatographic purity of mibemycin A3+A4 is 50%-55%.
Step (1) described low-boiling point alcohol includes but is not limited to methanol, ethyl alcohol, and the amount that low-boiling point alcohol is added is the 6-8 of filter residue
Times volume；The percent by volume of the low-boiling point alcohol is 75%-80%.
The water that the hot water of step (1) is 50~60 DEG C, being concentrated under reduced pressure into alcoholic strength 18%-23% has canescence solid content to go out
Now stop concentration.Preferably, step (1), which is concentrated under reduced pressure into alcoholic strength 20%, has canescence solid content to occur stopping concentration.
Step (1) cooling is that slow cooling keeps the temperature 10-14 hours to 8-10 DEG C after reduced pressure.Preferably,
Heat preservation 12 hours, plate-frame filtering fall solid content, collect filtrate, and the chromatographic purity of mibemycin A3+A4 is 65%- in filtrate
75%.
Highly polar organic solvent described in step (2) is methylene chloride, ethyl acetate and/or butyl acetate；The salt is washed
To be rinsed using saturated salt solution, the alkali cleaning is to be rinsed using saturated sodium bicarbonate solution；The adsorbent be active carbon,
Anhydrous sodium sulfate.
Step (3) the highly polar organic solvent is methylene chloride, toluene, ethyl acetate, butyl acetate；Low pole is organic
Solvent includes: normal heptane, n-hexane, hexahydrotoluene, petroleum ether.
Step (3) is the highly polar organic solvent that 1-1.5 times of volume of solid content is added, and is warming up to 40-60 DEG C, keeps the temperature
The low pole organic solvent of 60-80 times of volume is added in degree while stirring.
In step (3), after low pole organic solvent is added, keep temperature 1.5-2 hours according to 5-7 DEG C of cooling extent/small
When, it is cooled to 8-10 DEG C, stirring ageing 10-12 hours precipitates crystal.
The present invention provides above-mentioned preparation methods to improve the application in mibemycin purity.
By high-purity mibemycin made from the above method of the invention, the chromatographic purity in the bacteria residue that ferments is
50%-55%, chromatographic purity can achieve 95%-98% after phase transfer crystallizes, and absolute content also can achieve 93-96%
Left and right.
The method of the present invention forces crystallization to obtain the mibemycin of high-purity using phase inversion, and the usage amount of solvent is greatly
It reduces, the solvent usage amount than the prior art reduces about 70%, and the amount of solid waste greatly reduces, than the solid waste amount of the prior art
About 90% is reduced, controllable (the 60 times of volumes of net product most about needed using the method that the present invention crystallizes of safety and environmental protection risk
Organic solvent, if at least needing 600-800 times of body of net product using conventional silica gel column chromatography and macroporous resin adsorption desorption
Long-pending organic solvent；In terms of solid waste, since technique of the invention does not generate discarded silica gel and resin, pure production is only generated
The active carbon and anhydrous sodium sulfate of product amount 5% or so).The method of the present invention is chromatographed instead of macroreticular resin and silica gel column chromatography technology,
Simple to operation, at low cost, product purity is high, and mass yield is good, while while reduce production cost and cost of investment, subtracting
The pollution to environment is lacked.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Without departing substantially from spirit of that invention
In the case where essence, to modifications or substitutions made by the method for the present invention, step or condition, all belong to the scope of the present invention.
Unless otherwise specified, chemical reagent used in embodiment is conventional commercial reagent, skill used in embodiment
The conventional means that art means are well known to those skilled in the art.The filter of mibemycin fermentation liquid used in the embodiment of the present invention
In slag, mibemycin chromatographic purity is 50%-55%.
Embodiment 1
The method and step that the present embodiment prepares high-purity mibemycin is as follows:
(1) mibemycin fermentation liquid filter residue is extracted using 75% ethyl alcohol, extracts 92% or more mibemycin,
Filtrate is obtained after filters pressing, mibemycin A3+A4 component chromatographic purity is 57% in the filtrate；
(2) hot water heating is used, 50-60 DEG C of water temperature, the filtrate decompression obtained through step (1) is concentrated into alcoholic strength about
20% or so has canescence solid content to occur stopping concentration, is cooled to 8-10 DEG C, keeps the temperature 12 hours, plate-frame filtering falls solid
Object collects filtrate, and obtaining mibemycin A3+A4 component chromatographic purity is 67%；
(3) filtrate for obtaining step (2) is stripped using ethyl acetate, and water phase is exhausted after extraction and collects organic phase, organic
It mutually passes sequentially through salt and washes (saturated salt solution), alkali cleaning (saturated sodium bicarbonate solution), washing, collect organic phase；
(4) organic be added to for obtaining step (3) accounts for the needle-shaped active carbon and net product 5% of net product volume 5%
Anhydrous sodium sulfate, after mixing evenly, plate compression collects clear filtrate；
(5) clear filtrate that step (4) obtains is concentrated to dryness, obtains gelatinous solid content；
(6) the ethyl acetate dissolution of its 1 times of volume of the gluey solid content for obtaining step (5), is warming up to 40 DEG C, keeps
Temperature as above is slowly added to the n-hexane of 60 times of volumes of solid content, is added while stirring, keeps the temperature 2 hours or so, after open
Chilled water slow cooling is opened, 5 DEG C/h of cooling extent, is cooled to 10 DEG C or so, stirring ageing 12 hours, crystal is precipitated, centrifugation
Machine separation, drying.The mibemycin A3+A4 component chromatographic purity for obtaining high-purity is 96.7%.
Embodiment 2
The method and step that the present embodiment prepares high-purity mibemycin is as follows:
By mibemycin fermentation liquid filter residue using 80% ethyl alcohol extract, extract 93.5% or more Mir shellfish it is mould
Element obtains filtrate after filters pressing, mibemycin A3+A4 component chromatographic purity is 57.6% in filtrate；
(2) hot water heating is used, and water temperature is no more than 55 DEG C, the filtrate decompression (1) obtained through step is concentrated into alcoholic strength about
20% or so has canescence solid content to occur stopping concentration, is cooled to 8-10 DEG C, keeps the temperature 12 hours, plate-frame filtering falls solid
Object collects filtrate, and obtaining mibemycin A3+A4 component chromatographic purity is 68%；
(3) the filtrate (2) step obtained is stripped using a certain amount of methylene chloride, and it is organic that water phase collection is exhausted after extraction
Phase, organic phase pass sequentially through salt and wash (saturated salt solution), alkali cleaning (saturated sodium bicarbonate solution), washing, collect organic phase；
(4) organic be added to (3) step obtained accounts for the nothing of the needle-shaped active carbon and net product 5% of net product volume 5%
Aqueous sodium persulfate, after mixing evenly, plate compression collects clear filtrate；(5) the clear filtrate (4) step obtained is concentrated under reduced pressure
To doing, gelatinous solid content is obtained；
(6) the butyl acetate of its 1.5 times of volumes of the gluey solid content (5) step obtained dissolves, and is warming up to 45 DEG C, keeps
Temperature as above is slowly added to the petroleum ether of 60 times of volumes of solid content, is added while stirring, keeps the temperature 2 hours or so, after open
Chilled water slow cooling is opened, 5 DEG C/h of cooling extent, is cooled to 10 DEG C or so, stirring ageing 12 hours, crystal is precipitated, centrifugation
Machine separation, the mibemycin A3+A4 component chromatographic purity for being dried to obtain high-purity are 96.1%.
Embodiment 3
The method and step that the present embodiment prepares high-purity mibemycin is as follows:
(1) mibemycin fermentation liquid filter residue is used into 85% methanol extraction, extracts 96% or more mibemycin,
Filtrate is obtained after filters pressing, mibemycin A3+A4 component chromatographic purity is 64.8% in filtrate；
(2) hot water heating is used, 58 DEG C of water temperature, it is left that the filtrate decompression (1) obtained through step is concentrated into alcoholic strength about 20%
The right side has canescence solid content to occur stopping concentration, is cooled to 8-10 DEG C, keeps the temperature 12 hours, and plate-frame filtering falls solid content, collects
Filtrate, obtaining mibemycin A3+A4 component chromatographic purity is 71.6%；
(3) the filtrate (2) step obtained is stripped using methylene chloride, and water phase is exhausted after extraction and collects organic phase, organic phase
It passes sequentially through salt and washes (saturated salt solution), alkali cleaning (saturated sodium bicarbonate solution), washing, collect organic phase；
(4) organic be added to (3) step obtained accounts for the nothing of the needle-shaped active carbon and net product 5% of net product volume 5%
Aqueous sodium persulfate, after mixing evenly, plate compression collects clear filtrate；
(5) the clear filtrate (4) step obtained is concentrated to dryness, and obtains gelatinous solid content；
(6) the ethyl acetate of its 1 times of volume of the gluey solid content (5) step obtained dissolves, and is warming up to 40 DEG C, keeps such as
Upper temperature is slowly added to the normal heptane of 60 times of volumes of solid content, is added while stirring, keeps the temperature 2 hours or so, rear to open
Chilled water slow cooling, is cooled to 10 DEG C or so, stirring ageing 12 hours, crystal is precipitated, centrifuge by 5 DEG C/h of cooling extent
The mibemycin A3+A4 component chromatographic purity for separating, being dried to obtain high-purity is 98.7%.
Embodiment 4
The method and step that the present embodiment prepares high-purity mibemycin is as follows:
(1) mibemycin fermentation liquid filter residue is used into 75% methanol extraction, extracts 93% or more mibemycin,
Filtrate is obtained after filters pressing, mibemycin A3+A4 component chromatographic purity is 59% in filtrate；
(2) hot water heating is used, 56 DEG C of water temperature, it is left that the filtrate decompression (1) obtained through step is concentrated into alcoholic strength about 20%
The right side has canescence solid content to occur stopping concentration, is cooled to 8-10 DEG C, keeps the temperature 12 hours, and plate-frame filtering falls solid content, collects
Filtrate, obtaining mibemycin A3+A4 component chromatographic purity is 68%；
(3) the filtrate (2) step obtained is stripped using butyl acetate, and water phase is exhausted after extraction and collects organic phase, organic phase
It passes sequentially through salt and washes (saturated salt solution), alkali cleaning (saturated sodium bicarbonate solution), washing, collect organic phase；
(4) organic be added to (3) step obtained accounts for the nothing of the needle-shaped active carbon and net product 5% of net product volume 5%
Aqueous sodium persulfate, after mixing evenly, plate compression collects clear filtrate；
(5) the clear filtrate (4) step obtained is concentrated to dryness, and obtains gelatinous solid content；
(6) the ethyl acetate of its 1.2 times of volumes of the gluey solid content (5) step obtained dissolves, and is warming up to 43 DEG C, keeps
Temperature as above is slowly added to the normal heptane of 60 times of volumes of solid content, is added while stirring, keeps the temperature 2 hours or so, after open
Chilled water slow cooling is opened, 5 DEG C/h of cooling extent, is cooled to 10 DEG C or so, stirring ageing 12 hours, crystal is precipitated, centrifugation
Machine separation, the mibemycin A3+A4 component chromatographic purity for being dried to obtain high-purity are 97.7%.
Above embodiment be only preferred embodiments of the present invention will be described, not to the scope of the present invention into
Row limits, and without departing from the spirit of the design of the present invention, this field ordinary engineering and technical personnel is to technical side of the invention
The all variations and modifications that case is made, should fall within the scope of protection determined by the claims of the present invention.
Claims (10)
1. a kind of preparation method of high-purity mibemycin, which comprises the following steps:
(1) filter residue of mibemycin fermentation liquid is concentrated under reduced pressure after the extraction of low-boiling alcohol through hot water, cooling filtering removal
Solid content takes filtrate；
(2) back extraction of highly polar organic solvent is added in filtrate, collects organic phase after back extraction, washes through salt, alkali cleaning, absorption is added after washing
Filtrate is collected in agent, filters pressing, and filtrate decompression is concentrated to dryness, and collects solid content；
(3) after solid content is dissolved with highly polar solvent, heating keeps temperature, is added with stirring low pole organic solvent, cool down,
Crystallization, centrifugal drying obtain the mibemycin of high-purity.
2. preparation method according to claim 1, which is characterized in that in the filter residue of step (1) mibemycin fermentation liquid,
Mibemycin chromatographic purity is 50%-55%.
3. preparation method according to claim 1, which is characterized in that step (1) described low-boiling point alcohol includes but is not limited to
Methanol, ethyl alcohol, the amount that low-boiling point alcohol is added is 6-8 times of volume of filter residue；The percent by volume of the low-boiling point alcohol is 75%-
80%.
4. preparation method according to claim 1, which is characterized in that the water that the hot water of step (1) is 50~60 DEG C, decompression
It is concentrated into alcoholic strength 18%-23% and stops concentration.
5. preparation method according to claim 1, which is characterized in that step (1) cooling is after reduced pressure
Slow cooling keeps the temperature 10-14 hours to 8-10 DEG C.
6. preparation method according to claim 1, which is characterized in that highly polar organic solvent described in step (2) is two
Chloromethanes, ethyl acetate and/or butyl acetate；The salt is washed to be rinsed using saturated salt solution, and the alkali cleaning is using saturation
Sodium bicarbonate solution rinses；The adsorbent is active carbon, anhydrous sodium sulfate.
7. preparation method according to claim 1, which is characterized in that step (3) the highly polar organic solvent is dichloro
Methane, toluene, ethyl acetate, butyl acetate；Low pole organic solvent includes: normal heptane, n-hexane, hexahydrotoluene, petroleum
Ether.
8. -7 any preparation method according to claim 1, which is characterized in that step (3) is to be added 1-1.5 times of solid content
The highly polar organic solvent of volume, is warming up to 40-60 DEG C, keeps the temperature, and 60-80 times of volume of solid content is added while stirring
Low pole organic solvent.
9. preparation method according to claim 8, which is characterized in that in step (3), after low pole organic solvent is added,
It is kept for temperature 1.5-2.0 hours according to 5-7 DEG C/h of cooling extent, is cooled to 8-10 DEG C, stirring ageing 10-12 hours is precipitated
Crystal.
10. any preparation method of claim 1-9 is improving the application in mibemycin purity.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103772458A (en) * | 2012-10-25 | 2014-05-07 | 北大方正集团有限公司 | Purification method for Nemadectin |
| CN106432264A (en) * | 2016-09-06 | 2017-02-22 | 东北农业大学 | Two kinds of macrolide compound and preparation method and application thereof |
| CN106946905A (en) * | 2017-04-19 | 2017-07-14 | 丽珠集团福州福兴医药有限公司 | A kind of production method of mibemycin |
-
2018
- 2018-11-22 CN CN201811400424.1A patent/CN109400621B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103772458A (en) * | 2012-10-25 | 2014-05-07 | 北大方正集团有限公司 | Purification method for Nemadectin |
| CN106432264A (en) * | 2016-09-06 | 2017-02-22 | 东北农业大学 | Two kinds of macrolide compound and preparation method and application thereof |
| CN106946905A (en) * | 2017-04-19 | 2017-07-14 | 丽珠集团福州福兴医药有限公司 | A kind of production method of mibemycin |
Non-Patent Citations (1)
| Title |
|---|
| 谢捷，等: "大孔树脂分离纯化米尔贝霉素工艺研究", 《浙江工业大学学报》 * |
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