CN107722038A - A kind of method of purification of the epimer of tacrolimus 8 - Google Patents
A kind of method of purification of the epimer of tacrolimus 8 Download PDFInfo
- Publication number
- CN107722038A CN107722038A CN201711050000.2A CN201711050000A CN107722038A CN 107722038 A CN107722038 A CN 107722038A CN 201711050000 A CN201711050000 A CN 201711050000A CN 107722038 A CN107722038 A CN 107722038A
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- CN
- China
- Prior art keywords
- tacrolimus
- epimer
- solution
- purification according
- epimers
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
- C07D498/18—Bridged systems
-
- 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
- C07B2200/07—Optical isomers
Abstract
The invention provides a kind of method of purification of the epimer of tacrolimus 8, comprise the following steps：(1) crude tacrolimus is dissolved using organic solvent, membrane filtration, obtains sample solution；(2) isolated and purified using liquid-phase chromatographic column, obtain refined solution；(3) refined solution is concentrated, crystallisation by cooling, is filtrated to get the epimer crude product of tacrolimus 8；(4) epimer of tacrolimus 8 is dissolved using organic solvent, membrane filtration, obtains the epimer sterling solution of tacrolimus 8；(5) after the epimer sterling solution of tacrolimus 8 is concentrated, crystallisation by cooling, the epimer sterling of tacrolimus 8 is obtained.A kind of method of purification of the epimer of tacrolimus 8 disclosed by the present invention, realizes the realization to the epimer of tacrolimus 8 in crude tacrolimus and efficiently separates, so as to significantly increase the purity of tacrolimus.
Description
Technical field
The present invention relates to biopharmaceutical technology, more particularly to a kind of purification side of tacrolimus 8- epimers
Method.
Background technology
Tacrolimus (Tacrolimus) also known as FK506, it is from streptomyces (streptomyces tsukubaensis)
In the tunning isolated, its chemical constitution belongs to 23 membered macrolide antibiotic.Suppress for a kind of novel immune of strength
Agent, mainly by suppressing proleulzin (L-2) release, suppress the effect of T lymphocytes comprehensively, it is strong by 100 compared with cyclosporine (CsA)
Times.In recent years, as liver, the fiest-tire medication of kidney transplant, in 14 country's listings such as Japan, U.S..Clinical trial shows, its
Being applied in the transplanting such as the heart, lung, intestines, marrow has the effect of fine.FK506 is in treatment atopic dermatitis (AD), systematicness simultaneously
Positive effect is also played in the autoimmune diseases such as lupus erythematosus (SLE), Autoimmune ophthalmopathy.
Tacrolimus is a kind of macrolide immunosuppressants of novel potent, the entitled general pleasure of Japan's exploitation in 1991
Tacrolimus capsules agent and injection official listing can be answered, the rejection for Primary Hepatic transplanting and kidney transplant is treated, after
Bone-marrow transplantation is approved for again.In China, Japan, U.S. etc., more than 80 countries list tacrolimus formulations at present, extensively
Applied to the anti-repelling treatment after the solid organ transplantations such as liver, pancreas, kidney, heart and lung.Tacrolimus paste in 1999
Japanese market is launched, for treating the atopic dermatitis of adult.
Separation and Extraction obtains tacrolimus generally from zymotic fluid.In order to ensure drug safety, imitation medicine development and
In production, detection research should be carried out to impurity of the drug, so as to improve drug quality, reduce adverse drug reaction.Due to he gram
The production do not taken charge of can have many impurity mainly by fermentation, and tacrolimus 8- epimers are in zymotic fluid
One of major impurity, chemical formula C44H69NO12。
At present, isolation and purification is carried out to preparing the zymotic fluid obtained by tacrolimus using fermentation method, with to Ta Kemo
Department's 8- epimers are separated into for the research topic of hot topic.
The skill such as crude tacrolimus purifying disclosed in the prior arts such as Chinese invention patent CN101712685A, refined
Art means can not be solved to separate tacrolimus 8- epimers in zymotic fluid and asked with the technology of purification of tacrolimus
Topic.
In view of this, it is necessary to in the prior art to separating tacrolimus 8- epimers in crude tacrolimus
Technology is improved, to solve the above problems.
The content of the invention
It is an object of the invention to disclose a kind of method of purification of tacrolimus 8- epimers, to realize to he gram
The tacrolimus 8- epimers do not taken charge of in crude product efficiently separate, to improve the purity of tacrolimus, to improve Ta Kemo
The drug quality of department, and reduce adverse reaction caused by tacrolimus.
For achieving the above object, the invention provides a kind of method of purification of tacrolimus 8- epimers, bag
Include following steps：
(1) crude tacrolimus is dissolved using organic solvent, membrane filtration, obtains sample solution；
(2) isolated and purified using liquid-phase chromatographic column, obtain refined solution；
(3) refined solution is concentrated, crystallisation by cooling, is filtrated to get tacrolimus 8- epimer crude products；
(4) tacrolimus 8- epimers are dissolved using organic solvent, membrane filtration, it is poor obtains tacrolimus 8-
To isomers sterling solution；
(5) after tacrolimus 8- epimer sterlings solution is concentrated, crystallisation by cooling, tacrolimus 8- epimerisms are obtained
Body sterling.
As a further improvement on the present invention, the step (1) is selected from methanol, ethanol with the organic solvent in step (4)
Or the mixture of one or two kinds of any of the above ratio in acetonitrile.
As a further improvement on the present invention, the concentration of the sample solution in the step (1) is 100~200mg/ml.
As a further improvement on the present invention, the chromatographic condition of liquid-phase chromatographic column is in the step (2)：
Applied sample amount：5~10 ‰；
Mobile phase：35~45% acetonitrile or 35~45% methanol；
Detection wavelength：220nm；
Pillar height：250~300mm；
Flow velocity：1350~2250ml/min；
Filler：Silica filler；Wherein,
10 μm of the particle diameter of the silica filler, carbon content 4~7%, aperture
As a further improvement on the present invention, the thickening temperature in the step (3) is 30~40 DEG C, crystallization temperature 5~
10℃。
As a further improvement on the present invention, the thickening temperature in the step (5) is 30~40 DEG C, crystallization temperature 5~
10℃。
As a further improvement on the present invention, the step (1) is selected from ceramic membrane, polytetrafluoro with the filter membrane in step (4)
Vinyl film or composite fibre miillpore filter.
As a further improvement on the present invention, the pore size filter of the filter membrane is 0.22 μm.
Compared with prior art, the beneficial effects of the invention are as follows：By a kind of tacrolimus 8- differences for disclosing of the present invention to
The method of purification of isomers, efficiently separating to the tacrolimus 8- epimers in crude tacrolimus is realized, so as to
The purity of tacrolimus is considerably improved, and reduces adverse reaction caused by tacrolimus.
Brief description of the drawings
Fig. 1 is the high-efficient liquid phase chromatogram of the crude tacrolimus in embodiment one；
Fig. 2 is the high-efficient liquid phase chromatogram by the preparation-obtained tacrolimus 8- epimers of embodiment one.
Embodiment
The present invention is described in detail for shown each embodiment below in conjunction with the accompanying drawings, but it should explanation, these
Embodiment is not limitation of the present invention, those of ordinary skill in the art according to these embodiment institute work energy, method,
Or equivalent transformation or replacement in structure, belong within protection scope of the present invention.
Embodiment 1：
30g tacrolimus raw material is dissolved using 300ml acetonitriles, the use of pore size filter is 0.22 μm of membrane filtration, is prepared
Into 100mg/ml tacrolimus solution (i.e. sample solution).In embodiment, the filter membrane Ceramics film.Then, using liquid
Phase chromatographic column isolates and purifies to sample solution, obtains refined solution.
The chromatographic condition of liquid-phase chromatographic column is as follows：
Collect the impurity peaks part behind tacrolimus peak, collection liquid concentration, 30 DEG C of thickening temperature, 5 DEG C of crystallization temperature.
Filtering for crystallizing solution, it is filtrated to get tacrolimus 8- epimer crude products.Tacrolimus 8- epimers are thick
Product are dissolved using acetonitrile, the use of pore size filter are 0.22 μm of membrane filtration, filter membrane is ceramic membrane.Filtrate concentrates, and obtains tacrolimus
8- epimer sterling solution.After tacrolimus 8- epimer sterlings solution is concentrated, crystallisation by cooling.Thickening temperature 30
DEG C, 5 DEG C of crystallization temperature, filtering for crystallizing solution, obtain tacrolimus 8- epimer sterlings.After testing, tacrolimus 8- differences to
The liquid phase purity of isomers is up to 99.9%.
Join shown in Fig. 1, in the present embodiment, the sample solution configured by tacrolimus raw material quilt in liquid-phase chromatographic column
Sequentially eluting, wherein, peak value residing for the 25th minute or so is the peak value of tacrolimus, and the 32nd minute or so is that tacrolimus 8- is poor
To the peak value of isomers.Thus, tacrolimus 8- epimers are this most important in the presence of tacrolimus raw material
Impurity is eluted and separated in performance liquid chromatographic column.Join shown in Fig. 2, based on a kind of tacrolimus disclosed in the present embodiment
The purity of 8- epimers is up to 99.9%.
Embodiment 2：
60g tacrolimus raw material is dissolved using 300ml acetonitriles, the use of pore size filter is 0.22 μm of membrane filtration, is prepared
Into 200mg/ml tacrolimus solution (i.e. sample solution).In embodiment, the filter membrane selects poly tetrafluoroethylene.Then,
Sample solution is isolated and purified using liquid-phase chromatographic column, obtains refined solution.
The chromatographic condition of liquid-phase chromatographic column is as follows：
Collect the impurity peaks part behind tacrolimus peak, collection liquid concentration, 40 DEG C of thickening temperature, 10 DEG C of crystallization temperature.
Filtering for crystallizing solution, it is filtrated to get tacrolimus 8- epimer crude products.Tacrolimus 8- epimers are thick
Product are dissolved using acetonitrile, the use of pore size filter are 0.22 μm of membrane filtration, filtrate concentration, it is pure to be obtained tacrolimus 8- epimers
Product solution.After tacrolimus 8- epimer sterlings solution is concentrated, crystallisation by cooling.40 DEG C of thickening temperature, crystallization temperature 10
DEG C, filtering for crystallizing solution, obtain tacrolimus 8- epimer sterlings.After testing, the liquid phase of tacrolimus 8- epimers
Purity is up to 99.7%.
Embodiment 3：
60g tacrolimus raw material 300ml methanol is dissolved, the use of pore size filter is 0.22 μm of membrane filtration, is configured to
200mg/ml tacrolimus solution (i.e. sample solution).In embodiment, the filter membrane selects composite fibre miillpore filter.So
Afterwards, sample solution is isolated and purified using liquid-phase chromatographic column, obtains refined solution.
The chromatographic condition of liquid-phase chromatographic column is as follows：
Collect the impurity peaks part behind tacrolimus peak, collection liquid concentration, 40 DEG C of thickening temperature, 10 DEG C of crystallization temperature.
Filtering for crystallizing solution, it is filtrated to get tacrolimus 8- epimer crude products.Tacrolimus 8- epimers are thick
Product are dissolved using methanol, the use of pore size filter are that 0.22 μm of filter membrane (concretely composite fibre miillpore filter) is filtered, filtrate is dense
Contracting, obtains tacrolimus 8- epimer sterling solution.After tacrolimus 8- epimer sterlings solution is concentrated, cooling knot
It is brilliant.40 DEG C of thickening temperature, 10 DEG C of crystallization temperature, filtering for crystallizing solution, obtain tacrolimus 8- epimer sterlings.After testing,
The liquid phase purity of tacrolimus 8- epimers is up to 99.6%.
Embodiment 4：
30g tacrolimus raw material is dissolved using 300ml acetonitriles, the use of pore size filter is that 0.22 μm of membrane filtration is (specific
For poly tetrafluoroethylene), it is configured to 100mg/ml tacrolimus solution (i.e. sample solution).Then, using liquid-phase chromatographic column
Sample solution is isolated and purified, obtains refined solution.
The chromatographic condition of liquid-phase chromatographic column is as follows：
Collect the impurity peaks part behind tacrolimus peak, collection liquid concentration, 30 DEG C of thickening temperature, 5 DEG C of crystallization temperature.
Filtering for crystallizing solution, it is filtrated to get tacrolimus 8- epimer crude products.Tacrolimus 8- epimers are thick
Product are dissolved using acetonitrile, the use of pore size filter are 0.22 μm of membrane filtration (concretely ceramic membrane), and filtrate concentrates, get Ta Kemo
Take charge of 8- epimer sterling solution.After tacrolimus 8- epimer sterlings solution is concentrated, crystallisation by cooling.Thickening temperature
30 DEG C, 5 DEG C of crystallization temperature, filtering for crystallizing solution, obtain tacrolimus 8- epimer sterlings.After testing, tacrolimus 8- is poor
To the liquid phase purity of isomers up to 99.8%.
Those listed above is a series of to be described in detail only for feasibility embodiment of the invention specifically
Bright, they simultaneously are not used to limit the scope of the invention, all equivalent implementations made without departing from skill spirit of the present invention
Or change should be included in the scope of the protection.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires rather than described above limits, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.Any reference in claim should not be considered as to the involved claim of limitation.
Moreover, it will be appreciated that although the present specification is described in terms of embodiments, not each embodiment is only wrapped
Containing an independent technical scheme, this narrating mode of specification is only that those skilled in the art should for clarity
Using specification as an entirety, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
It is appreciated that other embodiment.
Claims (8)
1. a kind of method of purification of tacrolimus 8- epimers, it is characterised in that comprise the following steps：
(1) crude tacrolimus is dissolved using organic solvent, membrane filtration, obtains sample solution；
(2) isolated and purified using liquid-phase chromatographic column, obtain refined solution；
(3) refined solution is concentrated, crystallisation by cooling, is filtrated to get tacrolimus 8- epimer crude products；
(4) tacrolimus 8- epimers are dissolved using organic solvent, membrane filtration, obtains tacrolimus 8- differences to different
Structure body sterling solution；
(5) after tacrolimus 8- epimer sterlings solution is concentrated, crystallisation by cooling, it is pure that tacrolimus 8- epimers are obtained
Product.
2. method of purification according to claim 1, it is characterised in that the step (1) and the organic solvent in step (4)
The mixture of one or two kinds of any of the above ratio in methanol, ethanol or acetonitrile.
3. method of purification according to claim 1, it is characterised in that the concentration of the sample solution in the step (1) is
100~200mg/ml.
4. method of purification according to claim 1, it is characterised in that the chromatostrip of liquid-phase chromatographic column in the step (2)
Part is：
Applied sample amount：5~10 ‰；
Mobile phase：35~45% acetonitrile or 35~45% methanol；
Detection wavelength：220nm；
Pillar height：250~300mm；
Flow velocity：1350~2250ml/min；
Filler：Silica filler；Wherein,
10 μm of the particle diameter of the silica filler, carbon content 4~7%, aperture
5. method of purification according to claim 1, it is characterised in that the thickening temperature in the step (3) is 30~40
DEG C, 5~10 DEG C of crystallization temperature.
6. method of purification according to claim 1, it is characterised in that the thickening temperature in the step (5) is 30~40
DEG C, 5~10 DEG C of crystallization temperature.
7. method of purification according to claim 1, it is characterised in that the step (1) is selected from the filter membrane in step (4)
Ceramic membrane, poly tetrafluoroethylene or composite fibre miillpore filter.
8. method of purification according to claim 7, it is characterised in that the pore size filter of the filter membrane is 0.22 μm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711050000.2A CN107722038A (en) | 2017-10-31 | 2017-10-31 | A kind of method of purification of the epimer of tacrolimus 8 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711050000.2A CN107722038A (en) | 2017-10-31 | 2017-10-31 | A kind of method of purification of the epimer of tacrolimus 8 |
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| CN107722038A true CN107722038A (en) | 2018-02-23 |
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| CN201711050000.2A Pending CN107722038A (en) | 2017-10-31 | 2017-10-31 | A kind of method of purification of the epimer of tacrolimus 8 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101712685A (en) * | 2009-06-22 | 2010-05-26 | 鲁南制药集团股份有限公司 | Refining method of crude tacrolimus |
| US20110201639A1 (en) * | 2008-05-30 | 2011-08-18 | Lifecycle Pharma A/S | Stabilized tacrolimus composition |
| CN103819488A (en) * | 2014-02-20 | 2014-05-28 | 浙江万马药业有限公司 | Preparation method for known impurity-tacrolimus position isomer of tacrolimus |
| CN106755168A (en) * | 2016-11-28 | 2017-05-31 | 无锡福祈制药有限公司 | A kind of method of fermenting and producing tacrolimus fermentation |
| CN107090477A (en) * | 2017-05-04 | 2017-08-25 | 广州市微生物研究所 | A kind of fermentation process for improving tacrolimus yield |
-
2017
- 2017-10-31 CN CN201711050000.2A patent/CN107722038A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110201639A1 (en) * | 2008-05-30 | 2011-08-18 | Lifecycle Pharma A/S | Stabilized tacrolimus composition |
| CN101712685A (en) * | 2009-06-22 | 2010-05-26 | 鲁南制药集团股份有限公司 | Refining method of crude tacrolimus |
| TW201143770A (en) * | 2010-02-17 | 2011-12-16 | Lifecycle Pharma As | Stabilized tacrolimus composition |
| CN103819488A (en) * | 2014-02-20 | 2014-05-28 | 浙江万马药业有限公司 | Preparation method for known impurity-tacrolimus position isomer of tacrolimus |
| CN106755168A (en) * | 2016-11-28 | 2017-05-31 | 无锡福祈制药有限公司 | A kind of method of fermenting and producing tacrolimus fermentation |
| CN107090477A (en) * | 2017-05-04 | 2017-08-25 | 广州市微生物研究所 | A kind of fermentation process for improving tacrolimus yield |
Non-Patent Citations (2)
| Title |
|---|
| DORTHE M. SKYTTE等，: ""Synthesis and Characterization of an Epimer of Tacrolimus, an Immunosuppressive Drug"", 《J. NAT. PROD.》 * |
| 李似姣著，: "《现代色谱分析》", 30 June 2014, 国防工业出版社 * |
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Application publication date: 20180223 |