CN102584704A - Method for separating and preparing geldanamycin by using polymer microsphere - Google Patents
Method for separating and preparing geldanamycin by using polymer microsphere Download PDFInfo
- Publication number
- CN102584704A CN102584704A CN2011100006714A CN201110000671A CN102584704A CN 102584704 A CN102584704 A CN 102584704A CN 2011100006714 A CN2011100006714 A CN 2011100006714A CN 201110000671 A CN201110000671 A CN 201110000671A CN 102584704 A CN102584704 A CN 102584704A
- Authority
- CN
- China
- Prior art keywords
- nsc
- polar solvent
- solvent
- elutriant
- liquid separation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the technical field of industrial microorganisms and especially relates to a preparation method for separating and purifying geldanamycin by using a polymer microsphere in a fermentation culture.
Description
Technical field
The invention belongs to the industrial microbial technology field, be specifically related to a kind of from fermenting culture the preparation method of separation and purification NSC 122750.
Background technology
(geldanamycin GDM) in the fermented liquid that was found in streptomyces hygroscopicus (streptomyces hygroscopicus) in 1970, belongs to benzoquinones ansamycins (benzoquinone ansamycins) to NSC 122750.Its constitutional features is that a benzoquinones part links to each other with the big ring peace of planarity Sha bridge.Have multiple biological activitys such as protozoacide, antibacterium, antimycotic, antitumor, antiviral and immunomodulatory.Being mistaken as in early days is protein tyrosine kinase inhibitor, yet gos deep into along with what study, finds just that afterwards (Heat shock protein 90, ATP-binding site HSP90) suppresses its atpase activity to the GDM specific effect in heat shock protein 90.
Heat shock protein(HSP) becomes one of the most stem-winding research focus in the oncotherapy just gradually as pharmaceutically-active target in recent years.NSC 122750 can combine and suppress the function of Hsp90 through specificity; Stimulate the degraded of multiple oncoprotein and important cycle regulatory protein, the normal function of many signal transferrins all depends on Hsp90 in the cell, and NSC 122750 is exactly the normal function through the Hsp90 that disturbs tumour cell; Stop the activation of Hsp90 substrate protein; The blocking-up in inducing cell cycle can suppress virus replication simultaneously, thereby plays tumour cell and the antiviral effect of suppressing.
The special action target spot of NSC 122750 makes that it is antitumor with other, antiviral is different; Except self having medium anti-tumor activity; Under low consistency conditions, the external cytotoxicity that can significantly improve cis-platinum, ametycin, Dx and cytosine arabinoside.GDM can significantly strengthen cis-platinum and the ametycin restraining effect to rat liver cancer H22; The cisplatin combined application of GDM and HSP90C-end suppressor factor can be worked in coordination with and suppressed children's neuroblastoma and osteosarcoma, but GDM and cisplatin combined application Synergistic killing ovarian cancer A2780cis cell also.
Since 1970 found NSC 122750, numerous chemists had synthesized the verivate of a large amount of NSC 122750s, have found that some have the verivate of greater activity.Analyze and find that 17 of NSC 122750s are best decorating sites, adopt small molecules alkyl amine group methoxyl group displacement can obtain reactive derivative.At present; To two new GDM verivate 17-AAG, 17-DMAG research at most; Just carry out the clinical II phase by the research institution of a few family of the U.S., Britain and test, be expected to become new antitumor drug, the NSC 122750 class will show good prospect in the oncotherapy field.
The disclosed NSC 122750 preparation technology of U.S. Pat 3595955A1; Need to use methylene dichloride-n-butanol mixed solvent to extract earlier, re-use propyl carbinol and carry out multiple extractionly, concentrate back recycle silicon glue chromatographic column and separate; The propyl carbinol boiling point is high; When concentrated, very easily cause the NSC 122750 degraded, and complex steps, cause whole yield lower.The disclosed NSC 122750 preparation technology of U.S. Pat 2003186394A1, after using dichloromethane extraction, concentrating, direct crystallization, filtration; Through the octane-iso washing, obtain finished product after the drying, though step is easy; But product content is lower, and related substances can not be effectively controlled.
Summary of the invention
The objective of the invention is to overcome above-mentioned weak point, obtain the NSC 122750 preparation technology of high purity, high yield.The present invention adopts polymer microballoon separation and purification fermentation broth coarse extract first; Resolve through gradient, removed the impurity that the fermentating metabolism process produces effectively, obtain the smart powder of high-load NSC 122750; Content is greater than 98.5%; Single assorted less than 1.0%, total recovery is suitable for commercial scale prodn high purity NSC 122750 greater than 80%.
Specifically describe in the face of the present invention down:
In the methods of the invention, through filtering the NSC 122750 fermented liquid, obtain solid shape fermenting culture; In fermenting culture, add the polar solvent stirring and leaching,, obtain the NSC 122750 crude extract through filtering, concentrating, refilter; Crude extract is gone up appearance to the polymer microballoon chromatography column with polar solvent dissolving back, and the mixed solution gradient of water and polar solvent is resolved, and collects the NSC 122750 elutriant; Through concentrated, filtration, drying, obtain the smart powder of high-content NSC 122750.
Gained NSC 122750 of the present invention can supply medicine to use, and NSC 122750 content can reach more than 98.5%, and single assorted less than 1%, sample recovery rate is greater than 80%.
Particularly, the present invention relates to a kind of polymer microballoon and separate the method for preparing NSC 122750, comprise the steps:
1) under normal temperature condition, the NSC 122750 fermented liquid is carried out solid-liquid separation, obtain solid shape fermenting culture;
2) add the polar solvent stirring and leaching in the fermenting culture, lixiviate is carried out solid-liquid separation after accomplishing, and obtains the NSC 122750 vat liquor;
3) concentrating under reduced pressure vat liquor to polarity solvent proportion is less than 30%;
4) liquid concentrator is carried out solid-liquid separation, get the solid shape crude extract of NSC 122750;
5) the NSC 122750 crude extract is dissolved injection of polymer microballoon chromatography column with polar solvent;
6) use the mixed solution of polar solvent and water to carry out gradient elution, collect the NSC 122750 elutriant;
7) concentrating under reduced pressure NSC 122750 elutriant to polarity solvent proportion is less than 30%.
8) elutriant after concentrating is carried out solid-liquid separation, drying under reduced pressure gets the smart powder of NSC 122750.
Step 1), 4 wherein), 8) in solid-liquid separation can be suction filtration, press filtration, centrifugal, Gu the shape fermenting culture is a mycelium.
Step 2) the single extraction time to solid shape fermenting culture in is 20-180min, preferred 60-90min, and the lixiviate number of times is 1-3 time, the volume ratio of single extraction solvent and fermented liquid is 0.1-2.0, preferred 0.4-1.0.
Step 2), 5), 6) described polar solvent is a kind of in methyl alcohol or ethanol, Virahol, the acetone, particular methanol, ethanol, the polar solvent that each step is used can be inequality.
The polymer microballoon that uses in the step 5) is to adopt PS, polyacrylic ester and verivate preparation thereof, preferred amination polystyrene microsphere, sulphonated polystyrene microballoon and carboxylic polystyrene microsphere.
The polar solvent proportion of gradient elution can be 0-100% in the step 6), preferred 50-85%.
The present invention has following advantage: 1. use the polymer microballoon chromatography, increased substantially product purity, reduced related substances content.2. yield is high, and omnidistance total recovery reaches more than 80%.3. technology is succinct, and is quality controllable, for production pharmaceutical grade raw material provides safer technical guarantee.
Description of drawings
Accompanying drawing 1: the smart powder HPLC of NSC 122750 detects collection of illustrative plates
Embodiment
Following embodiment only is used for setting forth realization method of the present invention, should not be construed as limitation of the present invention.
NSC 122750 fermented liquid used in the present invention is that North China new drug development Ltd of pharmacy group obtains with the microorganism culturing means.Polymer microballoon is that Suzhou is received little scientific & technical corporation and produced, and reagent such as ethanol, methyl alcohol are commercially available.The high performance liquid chromatograph that the present invention uses is 996 type detectors, 515 pumps (Waters company).
Embodiment 1
Get NSC 122750 fermented liquid 10.0L, fermentation unit 2.11g/L, vacuum filtration obtains the 2.03kg mycelium.Adding 6L concentration is 95% ethanol in mycelium, and 1 hour final vacuum suction filtration of stirring at normal temperature is collected filtrating.Under 40 ℃ filtrate decompression is concentrated into alcohol concn less than 5%, is cooled to 20 ℃, vacuum filtration; Get NSC 122750 crude extract 43.2g, add absolute ethyl alcohol 64.8mL dissolving, inject length 50cm; Diameter is 5.5cm, and PSA30 type polymer microballoon loading amount is the chromatography column of 1000ml, uses 65% concentration ethanol to wash 20min as moving phase; 75% concentration ethanol is washed to the NSC 122750 wash-out as moving phase and is finished, and flow velocity is 1500mL/h, collects elutriant according to the HPLC detected result; 40 ℃ of concentrating under reduced pressure elutriant to alcohol concn are 5%, are cooled to 20 ℃, filter to obtain the NSC 122750 wet-milling; With the NSC 122750 wet-milling 45 ℃ of following vacuum-dryings, until moisture less than 5%.Obtain the smart powder of the light yellow NSC 122750 of 17.6g at last, yield is 82.43%.Smart powder HPLC content is 98.82%, wherein maximum single assorted＜1.0%.Its HPLC detects collection of illustrative plates referring to accompanying drawing 1.
Get NSC 122750 fermented liquid 5.0L, fermentation unit 2.23g/L, vacuum filtration obtains the 1.13kg mycelium.Adding 2L concentration is 99% methyl alcohol in mycelium, and 2 hours final vacuum suction filtrations of stirring at normal temperature are collected filtrating, and adding 1L concentration is 99% methyl alcohol in the filter cake, and 2 hours final vacuum suction filtrations of stirring at normal temperature merge filtered liq.Under 40 ℃ filtrate decompression is concentrated into methanol concentration less than 5%, is cooled to 20 ℃, vacuum filtration; Get NSC 122750 crude extract 21.6g, add methyl alcohol 30mL dissolving, inject length 50cm; Diameter is 5.5cm, and PS30 type polymer microballoon loading amount is the chromatography column of 500ml, uses 70% concentration methyl alcohol to wash 20min as moving phase; 85% concentration methyl alcohol washs to the NSC 122750 wash-out as moving phase and finishes, and flow velocity is 750mL/h, collects elutriant according to the HPLC detected result; 40 ℃ of concentrating under reduced pressure elutriant to methanol concentrations are cooled to 20 ℃ less than 5%, filter to obtain the NSC 122750 wet-milling; With the NSC 122750 wet-milling 45 ℃ of following vacuum-dryings, until moisture less than 5%.Obtain the smart powder of the light yellow NSC 122750 of 9.37g at last, yield is 83.06%.Smart powder HPLC content is 98.85%, wherein maximum single assorted＜1.0%.
Embodiment 3
Get NSC 122750 fermented liquid 5.0L, fermentation unit 2.23g/L, 3200 rev/mins are centrifugal, and the supernatant that inclines obtains the 1.35kg mycelium.Adding 3L concentration is 99% methyl alcohol in mycelium, and 40 ℃ are stirred 1 hour final vacuum suction filtration, collect filtrating.Under 40 ℃ filtrate decompression is concentrated into methanol concentration less than 5%, is cooled to 20 ℃, vacuum filtration; Get NSC 122750 crude extract 22.4g, add methyl alcohol 30mL dissolving, inject length 25cm; Diameter is 5.5cm, and PS30 type polymer microballoon loading amount is the chromatography column of 500ml, uses 70% concentration methyl alcohol to wash 20min as moving phase; 85% concentration methyl alcohol washs to the NSC 122750 wash-out as moving phase and finishes, and flow velocity is 750mL/h, collects elutriant according to the HPLC detected result; 40 ℃ of concentrating under reduced pressure elutriant to methanol concentrations are cooled to 20 ℃ less than 5%, filter to obtain the NSC 122750 wet-milling; With the NSC 122750 wet-milling 45 ℃ of following vacuum-dryings, until moisture less than 5%.Obtain the smart powder of the light yellow NSC 122750 of 9.65g at last, yield is 85.37%.Smart powder HPLC content is 98.65%, wherein maximum single assorted＜1.0%.
Claims (7)
1. a polymer microballoon separates the method for preparing NSC 122750, it is characterized in that this method comprises the steps:
1) under normal temperature condition, the NSC 122750 fermented liquid is carried out solid-liquid separation, obtain solid shape fermenting culture;
2) add the polar solvent stirring and leaching in the fermenting culture, lixiviate is carried out solid-liquid separation after accomplishing, and obtains the NSC 122750 vat liquor;
3) concentrating under reduced pressure vat liquor to polarity solvent proportion is less than 30%;
4) liquid concentrator is carried out solid-liquid separation, get the solid shape crude extract of NSC 122750;
5) the NSC 122750 crude extract is dissolved injection of polymer microballoon chromatography column with polar solvent;
6) use the mixed solution of polar solvent and water to carry out gradient elution, collect the NSC 122750 elutriant;
7) concentrating under reduced pressure NSC 122750 elutriant to polarity solvent proportion is less than 30%.
8) elutriant after concentrating is carried out solid-liquid separation, drying under reduced pressure gets the smart powder of NSC 122750.
2. method according to claim 1 is characterized in that: step 2) described extraction time is 20-180min.
3. method according to claim 1 is characterized in that: step 2) described lixiviate number of times is 1-3 time.
4. method according to claim 1 is characterized in that: step 2) volume ratio of described single extraction solvent and fermented liquid is 0.1-2.
5. method according to claim 1 is characterized in that: step 2), 5), 6) described polar solvent is a kind of in methyl alcohol or ethanol, Virahol, the acetone, the polar solvent that each step is used can be inequality.
6. method according to claim 1 is characterized in that: the said polymer microballoon of step 5) is to adopt PS or polyacrylic ester and verivate preparation thereof.
7. method according to claim 1 is characterized in that: the said gradient elution Semi-polarity of step 6) solvent proportion can be 0-100%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110000671.4A CN102584704B (en) | 2011-01-05 | 2011-01-05 | A kind of polymer microballoon is separated the method preparing geldanamycin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110000671.4A CN102584704B (en) | 2011-01-05 | 2011-01-05 | A kind of polymer microballoon is separated the method preparing geldanamycin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102584704A true CN102584704A (en) | 2012-07-18 |
| CN102584704B CN102584704B (en) | 2016-01-13 |
Family
ID=46474034
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110000671.4A Active CN102584704B (en) | 2011-01-05 | 2011-01-05 | A kind of polymer microballoon is separated the method preparing geldanamycin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102584704B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102936253A (en) * | 2012-11-12 | 2013-02-20 | 华北制药集团新药研究开发有限责任公司 | Preparation method of high-purity tacrolimus |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3595955A (en) * | 1969-03-26 | 1971-07-27 | Upjohn Co | Geldanamycin and process for producing same |
| US20030186394A1 (en) * | 2002-02-25 | 2003-10-02 | Short Kevin A. | Process to prepare and isolate geldanamycin |
| US6653469B1 (en) * | 2002-06-20 | 2003-11-25 | Murty Pharmaceuticals, Inc. | Antibiotic purification method |
| CN1478900A (en) * | 2002-08-30 | 2004-03-03 | 深圳市生尔易实业发展有限责任公司 | Preparation technology of geldanamycin |
| CN1478901A (en) * | 2002-08-30 | 2004-03-03 | 深圳市生尔易实业发展有限责任公司 | Preparation technology of geldanamycin and its derivative |
| CN1600306A (en) * | 2003-09-25 | 2005-03-30 | 中国医学科学院医药生物技术研究所 | Application of geldanamycin (C-3559) preparation of medicine for curing genital tract infection of condyloma accuminatum and herpes virus and on preparation of medicine for curing poliomyelitis |
| CN1788724A (en) * | 2003-09-25 | 2006-06-21 | 中国医学科学院医药生物技术研究所 | Use of geldanamycin (C-3559)in preparing drug for herpesvirus genital tract affection |
| CN1986546A (en) * | 2006-12-19 | 2007-06-27 | 中国人民解放军军事医学科学院毒物药物研究所 | Geldanamycin derivative and its preparing method and use |
| WO2009130706A1 (en) * | 2008-04-21 | 2009-10-29 | Biocon Limited | A process for isolation and purification of geldanamycin |
-
2011
- 2011-01-05 CN CN201110000671.4A patent/CN102584704B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3595955A (en) * | 1969-03-26 | 1971-07-27 | Upjohn Co | Geldanamycin and process for producing same |
| US20030186394A1 (en) * | 2002-02-25 | 2003-10-02 | Short Kevin A. | Process to prepare and isolate geldanamycin |
| US6653469B1 (en) * | 2002-06-20 | 2003-11-25 | Murty Pharmaceuticals, Inc. | Antibiotic purification method |
| CN1478900A (en) * | 2002-08-30 | 2004-03-03 | 深圳市生尔易实业发展有限责任公司 | Preparation technology of geldanamycin |
| CN1478901A (en) * | 2002-08-30 | 2004-03-03 | 深圳市生尔易实业发展有限责任公司 | Preparation technology of geldanamycin and its derivative |
| CN1600306A (en) * | 2003-09-25 | 2005-03-30 | 中国医学科学院医药生物技术研究所 | Application of geldanamycin (C-3559) preparation of medicine for curing genital tract infection of condyloma accuminatum and herpes virus and on preparation of medicine for curing poliomyelitis |
| CN1788724A (en) * | 2003-09-25 | 2006-06-21 | 中国医学科学院医药生物技术研究所 | Use of geldanamycin (C-3559)in preparing drug for herpesvirus genital tract affection |
| CN1986546A (en) * | 2006-12-19 | 2007-06-27 | 中国人民解放军军事医学科学院毒物药物研究所 | Geldanamycin derivative and its preparing method and use |
| WO2009130706A1 (en) * | 2008-04-21 | 2009-10-29 | Biocon Limited | A process for isolation and purification of geldanamycin |
Non-Patent Citations (1)
| Title |
|---|
| 刘涛 等: "格尔德霉素分离纯化工艺的研究", 《药物生物技术》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102936253A (en) * | 2012-11-12 | 2013-02-20 | 华北制药集团新药研究开发有限责任公司 | Preparation method of high-purity tacrolimus |
| CN102936253B (en) * | 2012-11-12 | 2016-01-06 | 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 tacrolimus |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102584704B (en) | 2016-01-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104177370B (en) | A kind of method preparing high-load sesamin from sesame seed meal | |
| CN101255180B (en) | Diphenyl ethylene glycosides derivatives | |
| CN105585471B (en) | A kind of extracting method of common rabdosia leaf active constituent | |
| CN106317148B (en) | A method of extracting cordycepin from Cordyceps militaris | |
| CN102372723B (en) | Method for extracting arglabin from artemisia myriantha | |
| CN103772460B (en) | The extraction process of cordycepin in silkworm Chinese caterpillar fungus | |
| CN102093374A (en) | Method for efficiently extracting camptothecin derivative | |
| CN109400566B (en) | Method for extracting and separating high-purity amentoflavone from Selaginella plant | |
| CN103145722B (en) | A kind of method of high speed adverse current chromatogram separating-purifying ebormycine | |
| CN102584704B (en) | A kind of polymer microballoon is separated the method preparing geldanamycin | |
| CN101481379B (en) | Compound separated from acetic acid ethyl ester extract of ginko endogenetic fungal bacterial strain fermentation liquor | |
| CN103242402A (en) | Method for quickly preparing high-purity N6-(2-ethoxy) adenosine | |
| CN103073624B (en) | A kind of preparation method of high purity cyclosporin A derivative | |
| CN110698532B (en) | Method for extracting sea cucumber saponin Cladoloside A | |
| CN109111444B (en) | Method for separating and purifying caffeine from camellia pollen | |
| CN102060889A (en) | Stilbene glycoside derivative | |
| CN108570016A (en) | A kind of method that PF1022A is isolated and purified | |
| CN103467352B (en) | A kind of method of prodigiosin extraction purification | |
| CN108516999B (en) | method for separating and preparing petunidin-3-O-galactoside | |
| CN105968150A (en) | Preparation method for 7-O-ethylmorroniside | |
| CN112079897A (en) | Two steroid compounds and preparation method and application thereof | |
| CN104892642A (en) | Method for purifying vindesine | |
| CN101671383B (en) | Monomeric 7-ethyoxyl ganoderic acid O and separating and purifying method of monomeric 7-ethyoxyl ganoderic acid T thereof | |
| CN103193748A (en) | Method for separating and preparing robinin monomer from wild chrysanthemum flowers | |
| CN107686489B (en) | A kind of high-purity moxidectin isolates and purifies method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |