CN103387606B - Method for preparing echinocandin B parent nucleus - Google Patents
Method for preparing echinocandin B parent nucleus Download PDFInfo
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- CN103387606B CN103387606B CN201210146480.3A CN201210146480A CN103387606B CN 103387606 B CN103387606 B CN 103387606B CN 201210146480 A CN201210146480 A CN 201210146480A CN 103387606 B CN103387606 B CN 103387606B
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Abstract
The invention relates to a method for preparing echinocandin B parent nucleus. The method comprises the steps that: (1) when echinocandin B conversion is finished, pump filtration is carried out; a filtrate is obtained, and the filtrate is processed by using an acidic alumina column; (2) the pH of the liquid collected in the step (1) is regulated to 4-5; rotary evaporation concentration is carried out; the material is processed by using macroporous non-polar resin; salt is removed by water washing; and a methanol aqueous solution is used for gradient elution; (3) the pH of the liquid collected in the step (3) is regulated to 6-7; the material is processed by using reversed-phase filler chromatographic column; and a methanol aqueous solution is used for elution; and (4) the pH of the liquid collected in the step (3) is regulated to 4-5; rotary evaporation concentration is carried out; and lyophilization is carried out, such that echinocandin B parent nucleus is obtained. With the method, a final purity of echinocandin B parent nucleus can be improved to higher than 95%.
Description
Technical field
The present invention relates to the exploitation of antifungal drug, particularly, the present invention relates to a kind of method preparing anidulafungin precursor ECB parent nucleus.
Background technology
Due to a large amount of uses of broad-spectrum antifungal medicine, Antifungal resistance strengthens gradually.Some immunosuppressed patients comprise HIV person, organ transplantation person and those suffer from Other diseases and just carry out in the patient of immunotherapy, fungi infestation be its occurrence frequency or infect kind all constantly increasing.Therefore, in the urgent need to finding new antifungal drug safely and effectively.
The echinocandin class medicine that 20 century 70s find is one group of natural product, structure is made up of similar ring type polypeptide core and different fatty acid side chains, β-(1 is suppressed by noncompetitive mechanism of action, 3) synthesis of-D-dextran, causes the dissolving of the fixed and fungal cell of emptying, the osmotic transient of cell wall glucan and plays its antifungic action.Its mechanism of action is unique, bad should rate low, showing has a broad antifungal spectrum, active strong feature as a kind of medicine killing fungi, is the important selection medicine for the treatment of immunosuppressed patient and immune normal patient fungi infestation.Echinocandin class microbiotic mainly contains three types: B, C and D.Wherein ECB (Echinocandin B, referred to as ECB) is topmost type.ECB, due to the existence of acyl side-chain, makes it have certain hemolytic toxicity.Be that lead compound carries out structure of modification with ECB, the compound that some have clinical value can be obtained, as Caspofungin (Caspofungin, MK991), MFG (Micafungin, FK463), anidulafungin (Anidulafungin, LY303366).ECB parent nucleus (Echinocandin B Nucleus is generated after being disconnected by deacylation by the side chain of ECB; referred to as ECBN), then connect modification group formation anidulafungin, this medicine goes on the market; show outstanding clinical effectiveness, market potential is huge.
ECBN carries out deacylation by actinoplanes to ECB and obtains; document DEACYLATION OF ECHNOCANDIN B BY ACTINOPLANES UTAHENSIS (MAR 1989THE JOURNAL OF ANTIBIOTICS) describes the method for its separation and purification: first carry out initial gross separation with HP-20 macroporous adsorbent resin to conversion fluid; then refine with reversed-phase HPLC; this method can obtain highly purified ECBN; but this technique is high to equipment requirements; preparation cost is huge, is difficult to carry out large-scale production.
Summary of the invention
In view of the deficiencies in the prior art, the invention provides a kind of method preparing ECB parent nucleus newly, described method technique is simple, and equipment requirements is low, is applicable to large-scale industrial production, has good commercial value.Especially, the final purity of ECB parent nucleus can be brought up to more than 95% by the inventive method.
Therefore, the present invention relates to the method preparing ECB parent nucleus, described method comprises the steps:
1) ECB has transformed rear suction filtration, gets filtrate, peracidity alumina column;
2) by step 1) collect the pH of liquid and be adjusted to 4-5, concentrated by rotary evaporation, upper macropore non-polar resin, water-washing desalting, aqueous methanol gradient wash-out;
3) by step 2) merge and collect the pH of liquid and be adjusted to 6-7, upper reverse phase filler chromatography column, methanol aqueous solution wash-out;
4) by step 3) to collect the pH of liquid and be adjusted to 4-5, concentrated by rotary evaporation, freeze-drying obtains ECB parent nucleus.
Step 1) terminate after, the yield of ECB parent nucleus is more than 95%, and liquid phase purity is 55-65%; Step 2) terminate after, the yield of ECB parent nucleus is more than 70%, and liquid phase purity is 85-90%; Step 3) terminate after, the yield of ECB parent nucleus is more than 90%, and liquid phase purity is more than 95%; The total recovery of aforesaid method is 60%, and the whole purity of ECB parent nucleus is more than 95%.
According to of the present invention one preferred embodiment, step 1) in containing the conversion filtrate peracidity alumina column of ECB parent nucleus, can pigment and segment polarity impurity and ECB parent nucleus can not be adsorbed by acidic alumina in active adsorption conversion fluid, alleviate the upper column pressure of subsequent step.Preferably, acidic alumina specification used is: chromatography FCP, 100 ~ 200 orders.
According to of the present invention one preferred embodiment, step 2) in by step 1) collect liquid concentrated by rotary evaporation after after upper macropore non-polar resin post, first use 2-3CV deionized water wash-out desalination, the larger impurity of polarity and residual pigment is removed afterwards with 5-10% methanol aqueous solution wash-out, with 15-20% methanol aqueous solution, ECB parent nucleus is eluted again, fraction collection in this process, merges the elutriant of ECB parent nucleus liquid phase purity more than 85%.Preferably, the 5-10% methanol-water effluent volume used in this step for 2-3CV, 15-20% methanol-water effluent volume be 3-4CV.Preferably, macropore non-polar resin is selected from framework material is styrene-divinylbenzene resinoid.
According to of the present invention one preferred embodiment, step 3) in use methanol aqueous solution wash-out, fraction collection after upper prop, the elutriant of merging purity more than 95%.Concentrated by rotary evaporation, after lyophilize, obtained white is to pale yellow powder shape ECB parent nucleus.Preferably, methanol-water effluent volume used in this step is 3-4CV, and methanol concentration is 15-25%.
According to of the present invention one preferred embodiment, in above each step, upper column flow rate all can be 1%CV/min, and elution flow rate can be 1-2%CV/min.
This method is simple to operate, is applicable to large-scale industrial production, has good commercial application value.
Embodiment
Separately point out as non-, the units of percent in the present invention is volume percent.
In the inventive method, the conversion of ECB parent nucleus is carried out by the following method:
Bacterial classification: actinoplanes utahensis (Actinoplanes utahensis NRRL12052).
Yeast culture condition:
Slant medium (g/L): yeast extract 30, Fructus Hordei Germinatus extract 30, glucose 10, agar 25, pH 7.0-7.2, cultivates 5-7 days for 30 DEG C.
Seed culture medium (g/L): sucrose 25, yeast powder 25, K
2hPO
41.0, KCl 0.5, MgSO
47H
2o 0.5, FeSO
47H
2o 0.002, pH=6.8,30 DEG C cultivates 3-4 days.
Fermention medium (g/L): sucrose 25, peanut meal powder 12, K
2hPO
41.0, MgSO
47H
2o3.0, pH=6.8,30 DEG C of fermentation 4-5 days.
Thalline conversion condition:
Conversion of substrate: with the water dissolution ECB containing 10%DMSO (dimethyl sulfoxide (DMSO)), ECB preparation technology refers to the embodiment 1 (application number: 200810042128.9) of a kind of Chinese patent " method preparing high purity anidulafungin precursor Echinocandin B ", wherein main technique is centrifugal acquisition mycelium after fermentation, crude extract is obtained with organic solvent extracting, crude extract uses macroporous adsorbent resin, aluminum oxide, reverse phase filler chromatography successively, add water after chromatographic solution evaporate to dryness dissolve with methanol crystallization, and final acquisition purity is the ECB of 95%.
Conversion condition: by the conversion thalline deionized water wash rear suction filtration for several times fermented, thalline pH6.8, concentration 0.1M phosphoric acid buffer suspend, add substrate mixing, 30 DEG C of 250r/min carry out conversion reaction 5 hours, obtain finally containing the conversion fluid of ECB parent nucleus.
ECB Nucleus HPLC testing conditions
Chromatographic column: ODS-C18 (5 μm, 4.6 × 250mm);
Determined wavelength: 222nm;
Moving phase: ammonium acetate buffer: acetonitrile=94: 6
Flow velocity: 0.8ml/min
Column temperature: 40 DEG C
In embodiment, chemical reagent used and chromatography acidic alumina are all purchased from Chemical Reagent Co., Ltd., Sinopharm Group, and macropore non-polar resin and reversed phase chromatography filler are purchased from calm moral bio tech ltd, Shanghai
Embodiment 1
After thalline has transformed, suction filtration, obtains 1.5L conversion fluid, peracidity alumina column, rushes post, all flow out to make ECB parent nucleus in post after crossing post with 1CV deionized water.Adjust the pH to 4.0 collecting liquid, concentrated by rotary evaporation is to 54ml, upper macropore non-polar resin XAD18 post, 2CV deionization water-washing desalting, 3CV 10% methanol aqueous solution wash-out removes the larger impurity of polarity and residual pigment, with 3CV 20% methanol aqueous solution, ECB parent nucleus is eluted again, fraction collection in this process, merge the elutriant of ECB parent nucleus liquid phase purity > 85%.Adjustment merges the pH of collection liquid to 6, upper reverse phase filler chromatography column CG161c, and with 4CV 20% methanol aqueous solution wash-out after upper prop, fraction collection, merges the collection liquid of purity > 95%.Adjust the pH to 4.5 collecting liquid, concentrated by rotary evaporation, freeze-drying obtains white ECB parent nucleus dry powder, and purity is 96.2%, total recovery 64%.
Embodiment 2
After thalline has transformed, suction filtration, obtains 1.8L conversion fluid, peracidity alumina column, rushes post, all flow out to make ECB parent nucleus in post after crossing post with 1.5CV deionized water.Adjust the pH to 5.0 collecting liquid, concentrated by rotary evaporation is to 50ml, upper macropore non-polar resin XAD18 post, 2.5CV deionization water-washing desalting, 2.5CV 5% methanol aqueous solution wash-out removes the larger impurity of polarity and residual pigment, with 3.5CV 20% methanol aqueous solution, ECB parent nucleus is eluted again, fraction collection in this process, merge the elutriant of ECB parent nucleus liquid phase purity > 85%.Adjustment merges the pH of collection liquid to 6.7, upper reverse phase filler chromatography column CG161c, and with 3CV 15% methanol aqueous solution wash-out after upper prop, fraction collection, merges the collection liquid of purity > 95%.Adjust the pH to 4.0 collecting liquid, concentrated by rotary evaporation, freeze-drying obtains white ECB parent nucleus dry powder, and purity is 96.2%, total recovery 65%.
Embodiment 3
After thalline has transformed, suction filtration, obtains 1.2L conversion fluid, peracidity alumina column, rushes post, all flow out to make ECB parent nucleus in post after crossing post with 1.2CV deionized water.Adjust the pH to 4.0 collecting liquid, concentrated by rotary evaporation is to 50ml, upper macropore non-polar resin XAD18 post, 2CV deionization water-washing desalting, 3CV 8% methanol aqueous solution wash-out removes the larger impurity of polarity and residual pigment, with 3CV 15% methanol aqueous solution, ECB parent nucleus is eluted again, fraction collection in this process, merge the elutriant of ECB parent nucleus liquid phase purity > 85%.Adjustment merges the pH of collection liquid to 6.7, upper reverse phase filler chromatography column CG161c, and with 4CV 18% methanol aqueous solution wash-out after upper prop, fraction collection, merges the collection liquid of purity > 95%.Adjust the pH to 4.7 collecting liquid, concentrated by rotary evaporation, freeze-drying obtains white ECB parent nucleus dry powder, and purity is 97.1%, total recovery 64%.
Embodiment 4
After thalline has transformed, suction filtration, obtains 1.5L conversion fluid, peracidity alumina column, rushes post, all flow out to make ECB parent nucleus in post after crossing post with 1CV deionized water.Adjust the pH to 4.5 collecting liquid, concentrated by rotary evaporation is to 58ml, upper macropore non-polar resin XAD18 post, 3CV deionization water-washing desalting, 3CV 8% methanol aqueous solution wash-out removes the larger impurity of polarity and residual pigment, with 3CV 17% methanol aqueous solution, ECB parent nucleus is eluted again, fraction collection in this process, merge the elutriant of ECB parent nucleus liquid phase purity > 85%.Adjustment merges the pH of collection liquid to 6.8, upper reverse phase filler chromatography column CG161c, and with 4CV 16% methanol aqueous solution wash-out after upper prop, fraction collection, merges the collection liquid of purity > 95%.Adjust the pH to 5.0 collecting liquid, concentrated by rotary evaporation, freeze-drying obtains white ECB parent nucleus dry powder, and purity is 95.6%, total recovery 72%.
Embodiment 5
After thalline has transformed, suction filtration, obtains 2.2L conversion fluid, peracidity alumina column, rushes post, all flow out to make ECB parent nucleus in post after crossing post with 1.3CV deionized water.Adjust the pH to 4.2 collecting liquid, concentrated by rotary evaporation is to 55ml, upper macropore non-polar resin XAD18 post, 2.5CV deionization water-washing desalting, 2CV 5% methanol aqueous solution wash-out removes the larger impurity of polarity and residual pigment, with 3.5CV 20% methanol aqueous solution, ECB parent nucleus is eluted again, fraction collection in this process, merge the elutriant of ECB parent nucleus liquid phase purity > 85%.Adjustment merges the pH of collection liquid to 6.4, upper reverse phase filler chromatography column CG161c, and with 4CV 15% methanol aqueous solution wash-out after upper prop, fraction collection, merges the collection liquid of purity > 95%.Adjust the pH to 4.5 collecting liquid, concentrated by rotary evaporation, freeze-drying obtains white ECB parent nucleus dry powder, and purity is 95.6%, total recovery 76%.
Claims (9)
1. prepare the method for ECB parent nucleus, described method comprises the steps:
1) ECB has transformed rear suction filtration, gets filtrate, peracidity alumina column;
2) by step 1) collect the pH of liquid and be adjusted to 4-5, concentrated by rotary evaporation, upper macropore non-polar resin, water-washing desalting, aqueous methanol gradient wash-out;
3) by step 2) collect the pH of liquid and be adjusted to 6-7, upper reverse phase filler chromatography column, methanol aqueous solution wash-out;
4) by step 3) to collect the pH of liquid and be adjusted to 4-5, concentrated by rotary evaporation, freeze-drying obtains ECB parent nucleus.
2. the method for claim 1, wherein step 2) in, described macropore non-polar resin is selected from the resin that framework material is styrene-divinylbenzene class.
3. the method for claim 1, wherein step 2) in, described water-washing desalting carries out according to the following procedure: with 2-3CV deionized water wash-out desalination.
4. the method for claim 1, wherein step 2) in, described aqueous methanol gradient wash-out carries out according to the following procedure: remove impurity with 5-10% methanol aqueous solution wash-out, then use 15-20% methanol aqueous solution wash-out ECB parent nucleus.
5. method as claimed in claim 4, wherein said 5-10% methanol-water effluent volume is 2-3CV, and described 15-20% methanol-water effluent volume is 3-4CV.
6. the method for claim 1, wherein step 2) in, carry out fraction collection after aqueous methanol gradient wash-out, merge the elutriant of liquid phase purity more than 85%.
7. the method for claim 1, wherein step 3) in, fraction collection after methanol aqueous solution wash-out, merges the elutriant of purity more than 95%.
8. method as claimed in claim 7, wherein said methanol-water effluent volume is 3-4CV, and methanol concentration is 15-25%.
9. the method for claim 1, wherein in each step, upper column flow rate is 0.5%-1%CV/min, and elution flow rate is 1-2%CV/min.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105669839A (en) * | 2014-12-05 | 2016-06-15 | 重庆乾泰生物医药有限公司 | Hydrate of echinocandin B mother nucleus or salt thereof, preparation method and application thereof |
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| CN103910783B (en) * | 2014-04-23 | 2016-07-06 | 华北制药集团新药研究开发有限责任公司 | A kind of preparation method of high-purity echinocandin B parent nucleus |
| CN105648000B (en) * | 2014-11-19 | 2019-08-13 | 重庆乾泰生物医药有限公司 | A kind of echinocandin B microbial enzyme method for transformation |
| CN107778359B (en) * | 2016-08-27 | 2020-08-28 | 鲁南制药集团股份有限公司 | Method for purifying echinocandin B mother nucleus |
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| DE102004060750A1 (en) * | 2004-12-15 | 2006-07-13 | Sanofi-Aventis Deutschland Gmbh | Process for the deacylation of lipopeptides |
| HUE029147T2 (en) * | 2009-08-14 | 2017-02-28 | Xellia Pharmaceuticals Aps | Separation and/or purification of pneumocandin b0 from c0 |
| CN102443560A (en) * | 2010-10-13 | 2012-05-09 | 上海医药工业研究院 | Gene engineering bacterium for high efficiency conversion of echinocandin B and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105669839A (en) * | 2014-12-05 | 2016-06-15 | 重庆乾泰生物医药有限公司 | Hydrate of echinocandin B mother nucleus or salt thereof, preparation method and application thereof |
| CN105669839B (en) * | 2014-12-05 | 2019-08-13 | 重庆乾泰生物医药有限公司 | The hydrate and preparation method and purposes of a kind of echinocandin B parent nucleus or its salt |
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