CN115477658A - Crystallization method for effectively reducing ansamitocin P-3 impurity - Google Patents
Crystallization method for effectively reducing ansamitocin P-3 impurity Download PDFInfo
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- 238000002425 crystallisation Methods 0.000 title claims abstract description 19
- 239000012535 impurity Substances 0.000 title claims abstract description 18
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- OPQNCARIZFLNLF-JBHFWYGFSA-N ansamitocin P3 Chemical compound CO[C@@H]([C@@]1(O)C[C@H](OC(=O)N1)[C@@H](C)[C@@H]1O[C@@]1(C)[C@@H](OC(=O)C(C)C)CC(=O)N1C)\C=C\C=C(C)\CC2=CC(OC)=C(Cl)C1=C2 OPQNCARIZFLNLF-JBHFWYGFSA-N 0.000 title claims abstract description 15
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- ZWBTYMGEBZUQTK-PVLSIAFMSA-N [(7S,9E,11S,12R,13S,14R,15R,16R,17S,18S,19E,21Z)-2,15,17,32-tetrahydroxy-11-methoxy-3,7,12,14,16,18,22-heptamethyl-1'-(2-methylpropyl)-6,23-dioxospiro[8,33-dioxa-24,27,29-triazapentacyclo[23.6.1.14,7.05,31.026,30]tritriaconta-1(32),2,4,9,19,21,24,26,30-nonaene-28,4'-piperidine]-13-yl] acetate Chemical compound CO[C@H]1\C=C\O[C@@]2(C)Oc3c(C2=O)c2c4NC5(CCN(CC(C)C)CC5)N=c4c(=NC(=O)\C(C)=C/C=C/[C@H](C)[C@H](O)[C@@H](C)[C@@H](O)[C@@H](C)[C@H](OC(C)=O)[C@@H]1C)c(O)c2c(O)c3C ZWBTYMGEBZUQTK-PVLSIAFMSA-N 0.000 description 1
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Images
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
Abstract
The invention discloses a crystallization method for effectively reducing ansamitocin P-3 impurities, which is characterized in that butanone and isopropyl ether are used for refining and crystallizing AP-3, the purity of the refined AP-3 is ensured, a solvent can be recycled, the method is environment-friendly, resources and cost can be saved, the product quality is improved, the requirements on process operation and equipment are reduced to a greater extent, and the method is suitable for industrial production.
Description
Technical Field
The invention relates to the technical field of industrial microorganisms, in particular to a crystallization method for effectively reducing ansamitocin P-3 impurities.
Background
In 1972, may Po money and the like were first isolated from Maytenus ovata collected in Africa (with a content of two ten million) to obtain maytansine, the mother nucleus of which is 19-C macrolactam or type I polyketide, and it was later found that Actinosynnemia aurantiaca (Actinosynnema depressum) can also produce various products similar to the maytansine, which is called maytansinoid antibiotic, and ansamicin P-3 (AP-3) is the main component under limited culture conditions. Because it has significant curative effects on various tumors, such as L-1210, P-388 leukemia, S-180, W-256, lewis lung cancer and in vitro nasopharyngeal carcinoma, the application prospect is huge, for example, the clinical test of treating malignant tumor by using AP-3 as immunotoxin binder at present enters the final stage. Therefore, the demand of AP-3 in the future market will be large, so that efficient mass production of AP-3 has become one of the research hotspots.
At present, most of the documents about ansamitocins are reported in fermentation processes, and the methods in a few documents about separation and purification are fuzzy or have too complicated processes, are biased to small-amount sample preparation, and are not suitable for large-scale production. Methods such as described in CN102731526A, CN102732581a and US7432088 require the use of normal phase silica gel chromatography or medium pressure reverse phase silica gel chromatography; however, the extraction of the whole fermentation liquor adopted by US6573074B2 and US20020015984 consumes too much solvent, and the fermentation liquor needs to be heated to 75 ℃ before extraction, which causes a great deal of volatilization of the extraction solvent to pollute the environment and increase the operation risk.
Therefore, there is an urgent need in the art to find a purification method that improves the yield and purity of the product, reduces the production cost, has simple operation steps, and is environmentally friendly, so as to overcome the above-mentioned defects in the prior art and meet the demand of industrial production.
Disclosure of Invention
The invention aims to provide a crystallization method for effectively reducing ansamitocin P-3 impurities so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a crystallization method for effectively reducing ansamitocin P-3 impurities comprises the following steps:
s1, taking 1 part by mass of a refined product AP-3 with purity not up to the standard, which is obtained by a conventional method;
s2, dissolving the refined product AP-3 obtained in the S1 by using 7-8 parts by mass of butanone, and heating to 65 ℃;
s3, dropwise adding 7-9 parts by mass of isopropyl ether, and stirring at 65 ℃ after dropwise adding is completed for three hours;
s4, cooling the temperature from 65 ℃ to 45 ℃, keeping the temperature for a period of time, and then stirring;
s5, cooling the temperature from 45 ℃ to 25 ℃, keeping the temperature for a period of time, and then stirring;
s6, filtering the solution to remove crystals, soaking and washing the crystals obtained by filtering with a mixed solution of 1-2 parts by mass of butanone and 1-3 parts by mass of isopropyl ether, and filtering and drying to obtain the AP-3 product with the purity reaching the standard.
Preferably, the S3 is stirred for 60-70min at 65 ℃.
Preferably, the temperature in the S4 is reduced from 65 ℃ to 45 ℃, then the temperature is kept for 60min, and finally the temperature is kept at 45 ℃ and the stirring is carried out for 40-50min.
Preferably, the temperature in S5 is reduced from 45 ℃ to 25 ℃, then the temperature is kept for 60min, and then the temperature is kept at 25 ℃ and the stirring is carried out for 60-70min.
Preferably, the purity of the refined AP-3 product obtained by the crystallization method is not less than 98%.
Has the beneficial effects that: compared with the prior art, the invention has the beneficial effects that: the butanone and the isopropyl ether are utilized to refine and crystallize the AP-3, the purity of the refined AP-3 is ensured, the solvent can be recycled, the method is environment-friendly, the resources and the cost can be saved, the product quality is improved, the requirements on process operation and equipment are reduced to a greater extent, and the method is suitable for industrial production.
Drawings
FIG. 1 is a chromatogram before refining of AP-3;
FIG. 2 is a chromatogram after 01 batch of AP-3 refining;
FIG. 3 is a chromatogram of 02 batches of refined AP-3.
Detailed Description
In order that the above objects, features and advantages of the present invention can be more clearly understood, the present invention will be further described with reference to the following examples. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments of the present disclosure.
AP-3 extraction bench:
the fermentation small test is cultured until 424h is finished (the volume of the fermentation liquid is about 19L, and the content is 0.636 g/L).
Adjusting the pH value of the fermentation liquor to 4.5 by using a 3M sulfuric acid solution, generating larger foam in the adjusting process, supplementing a small amount of defoamer, adding 760g of perlite, stirring for 15min, and raising the pH value back to more than 5.5; adding 16L of methanol, continuing stirring for 1h, adding 1.8L of methanol, stirring for 3h, and then putting into a tank.
740g of deionized water suspended diatomite (radioactive lite # 500) was used to fill a filter press tester, and filtered by using a filter press, and the filter cake was washed with methanol water; thereby obtaining 42L of filter liquor of plate-frame filter pressing (the content of the filter liquor is 0.134g/L, the volume is 42L, and is converted into AP-3.628g, and the yield is 46.6%).
Taking 10g of a plate-frame filter cake, adding methanol to a constant volume of 100ml, and ultrasonically testing the content for 20min, namely 0.000086g/kg.
Taking 10g of a plate-and-frame filter cake, adding acetone to a constant volume of 100ml, and carrying out ultrasonic treatment for 20min to obtain a detection content of 0.00012g/kg.
After the plate-frame filtrate is subjected to secondary filtration, HP20 resin is used for adsorption, gradient elution is carried out, the collected solution is about 300 ml/bottle, and the detection results are shown in table 1:
table 1: detection results of content and purity of AP-3 in each bottle
| Bottle number | Content (wt.) | Purity of | Bottle number | Content (wt.) | Purity of |
| 10 | 0.232 | 37.87 | 16 | 3.01 | 85.24 |
| 11 | 0.561 | 54.14 | 17 | 2.68 | 84.86 |
| 12 | 0.928 | 73.28 | 18 | 2.11 | 87.07 |
| 13 | 0.911 | 74.37 | 19 | 1.54 | 81.64 |
| 14 | 1.36 | 78.73 | 20 | 0.687 | 70.96 |
| 15 | 1.91 | 84.59 | 21 | 0.196 | 44.24 |
The collected liquids from bottles 15 to 19 (total about 1.5L, about 3.36g in terms of AP-3, single-step yield 59.7%) were combined, 6.5g of activated carbon (aigrette Z) was added, and the mixture was stirred for 1 hour to decolorize, and the filtrate was filtered to obtain a single decolorized filtrate (about 1.5L, content 1.775g/L, purity 91.34%, about 2.663g in terms of AP-3, yield of the decolorization step 79.26%, total yield 22.05%).
The raw material fermentation liquor is post-treated to obtain 2.9L of decolorized filtrate with the content of 0.306g/L and the purity of 91.27% and the yield of 21.76% (about 20L of 001 fermentation liquor with the content of 0.0726g/L; about 26L of 002 batch fermentation liquor with the content of 0.101g/L, which is equivalent to about 4.078g of AP-3).
Stirring the residual activated carbon obtained after the two decolorization and filtration processes for 20min by using about 500ml of 70% acetone aqueous solution, and filtering to obtain about 500ml of washing liquid (the content is 1.197g/L, the purity is 90.57%, and the amount is about 0.599g compared with AP-3); about 500ml of pure acetone was used again for the second washing as above, and 200ml of acetone was used to rinse the filter cake, giving about 700ml of washing solution (content 1.188g/L, purity 88.12%, equivalent to about 0.832g of AP-3).
Mixing 002 batches of decolorized filtrate and two times of activated carbon washing filtrates, vacuum concentrating at 35 deg.C until the filtrate is turbid to obtain about 2000ml of concentrated solution, adding ethyl acetate 2000ml and NaCl 40g, stirring for 10min, transferring into a separating funnel, standing for layering, and separating organic phase and extraction water phase (content is 0.0005 g/L);
adding 600ml of 0.05M NaOH solution into the organic phase, stirring and washing, standing and layering, and separating the organic phase and the NaOH washing solution (the content is 0.0121g/L, the color is light yellow, and the organic phase is nearly colorless);
adding 600ml deionized water into the organic phase, stirring and washing, standing and layering, and separating the organic phase and the washing water phase (the content is 0.0073 g/L) to obtain about 2L of extraction washing organic phase.
Adding 10g of activated carbon (aigret Z), stirring for 30min, adding 80g of anhydrous sodium sulfate, stirring for 10min, filtering, wherein due to incomplete dehydration, sodium sulfate is agglomerated and a small amount of sodium sulfate is deposited at the bottom of the filtrate, adding a small amount of anhydrous sodium sulfate again, performing secondary dehydration, and filtering to obtain about 2.1L of decolored dehydrated filtrate, the content of which is 1.045g/L, and the purity of which is 92.21%.
Vacuum concentrating at 38 deg.C to about 80ml without turbidity, adding methanol 2.3ml, adding ethyl acetate 23ml, stirring at 40 deg.C, dropwise adding n-heptane 46ml until turbidity appears, stopping stirring, removing turbidity, allowing a little yellowish oily substance to appear at the bottom, and stirring.
Concentrating the crystal liquid to dryness, adding 30ml of ethyl acetate, heating without heating, dissolving to be clear, adding 0.85ml of methanol, adding 8.5ml of ethyl acetate, dropwise adding 17ml of n-heptane, collecting mother liquor, checking (the content is 7.002 g/L), and preserving in a freezer.
The crystal was concentrated to dryness, 47ml of ethyl acetate was added, temperature was not raised, the solution was clear, 1.0ml of methanol was added, 21ml of n-heptane was added dropwise, no solid was precipitated, 3.7ml of methanol was further added (ethyl acetate: methanol = 10).
The crystals of 1.64gAP-3 were again purified, 17ml of ethyl acetate and 1.7ml of methanol were added to the solution, 34ml of n-heptane was added dropwise thereto, and after crystal growth, 1.41g of wet AP-3 (purity 95.156%) was obtained by filtration.
Crystallization and refining were carried out as described above to obtain 1.41g of sample (purity > 95%)
Impurity removal and refining: the purity of the refined product obtained from the two batches of materials does not reach the standard (98 percent), the two impurities of AP-4 and AP-4' are large, and the refined product is refined by using resin without removing effect. Therefore, the crystallization method for reducing the ansamitocin P-3 impurity provided by the invention is adopted to remove the related impurities: butanone and isopropyl ether were used for purification and crystallization.
Example 1
The crystallization method for effectively reducing the ansamitocin P-3 impurity provided by the embodiment comprises the following steps:
s1, taking 1 part by mass of a refined product AP-3 with purity not up to the standard, which is obtained by a conventional method;
s2, dissolving the refined product AP-3 obtained in the S1 by using 7 parts by mass of butanone, and heating to 65 ℃;
s3, dropwise adding 7 parts by mass of isopropyl ether, and after dropping for three hours, keeping the temperature of 65 ℃ and stirring for 60min;
s4, reducing the temperature from 65 ℃ to 45 ℃, keeping the temperature for 60min, and finally keeping the temperature at 45 ℃ and stirring for 40min;
s5, reducing the temperature from 45 ℃ to 25 ℃, keeping the temperature for 60min, keeping the temperature at 25 ℃ and stirring for 60min;
and S6, filtering the solution to remove crystals, soaking and washing the crystals obtained by filtering by using a mixed solution of 1 part by mass of butanone and 1 part by mass of isopropyl ether, and filtering and drying to obtain the AP-3 product with the purity reaching the standard.
Example 2
S1, taking 1 part by mass of a refined product AP-3 with purity not up to the standard, which is obtained by a conventional method;
s2, dissolving the refined product AP-3 obtained in the S1 by using 7.5 parts by mass of butanone, and heating to 65 ℃;
s3, dropwise adding 8 parts by mass of isopropyl ether, and stirring for 65min at 65 ℃ after dropwise adding for three hours;
s4, cooling the temperature from 65 ℃ to 45 ℃, keeping the temperature for 60min, and finally keeping the temperature at 45 ℃ and stirring for 45min;
s5, reducing the temperature from 45 ℃ to 25 ℃, keeping the temperature for 60min, keeping the temperature at 25 ℃, and stirring for 65min;
s6, filtering the solution to remove crystals, soaking and washing the crystals obtained by filtering with a mixed solution of 1.5 parts by mass of butanone and 2 parts by mass of isopropyl ether, and filtering and drying to obtain the AP-3 product with the purity reaching the standard.
Example 3
S1, taking 1 part by mass of a refined product AP-3 with purity not up to the standard, which is obtained by a conventional method;
s2, dissolving the refined product AP-3 obtained in the S1 by using 8 parts by mass of butanone, and heating to 65 ℃;
s3, dropwise adding 9 parts by mass of isopropyl ether, and stirring for 70min at 65 ℃ after dropwise adding for three hours;
s4, reducing the temperature from 65 ℃ to 45 ℃, keeping the temperature for 60min, and finally keeping the temperature at 45 ℃ and stirring for 50min;
s5, reducing the temperature from 45 ℃ to 25 ℃, keeping the temperature for 60min, keeping the temperature at 25 ℃ and stirring for 70min;
s6, filtering the solution to remove crystals, soaking and washing the crystals obtained by filtering with a mixed solution of 2 parts by mass of butanone and 3 parts by mass of isopropyl ether, and filtering and drying to obtain the AP-3 product with the purity reaching the standard.
The purity of the refined AP-3 product obtained by the crystallization method for reducing the ansamitocin P-3 impurity provided by the embodiment is not less than 98%.
According to the crystallization method for effectively reducing the ansamitocin P-3 impurities, the butanone and the isopropyl ether are used for refining and crystallizing the AP-3, the purity of the refined AP-3 is ensured, the solvent can be recycled, the method is environment-friendly, the resources and the cost can be saved, the product quality is improved, the requirements of process operation and equipment are reduced to a greater extent, and the method is suitable for industrial production.
The above description is only a preferred embodiment of the present invention, and not intended to limit the present invention in other forms, and any person skilled in the art may apply the above modifications or changes to the equivalent embodiments with equivalent changes, without departing from the technical spirit of the present invention, and any simple modification, equivalent change and change made to the above embodiments according to the technical spirit of the present invention still belong to the protection scope of the technical spirit of the present invention.
Claims (5)
1. A crystallization method for effectively reducing ansamitocin P-3 impurities is characterized by comprising the following steps:
s1, taking 1 part by mass of a refined product AP-3 with purity not meeting the standard, which is obtained by a conventional method;
s2, dissolving the refined product AP-3 obtained in the S1 by using 7-8 parts by mass of butanone, and heating to 65 ℃;
s3, dropwise adding 7-9 parts by mass of isopropyl ether, and stirring at 65 ℃ after dropwise adding is completed for three hours;
s4, cooling the temperature from 65 ℃ to 45 ℃, keeping the temperature for a period of time, and then stirring;
s5, cooling the temperature from 45 ℃ to 25 ℃, keeping the temperature for a period of time, and then stirring;
s6, filtering the solution to remove crystals, soaking and washing the crystals obtained by filtering with a mixed solution of 1-2 parts by mass of butanone and 1-3 parts by mass of isopropyl ether, and filtering and drying to obtain the AP-3 product with the purity reaching the standard.
2. The crystallization method for effectively reducing ansamitocin P-3 impurities according to claim 1, wherein the method comprises the following steps: and keeping the temperature of 65 ℃ in the S3, and stirring for 60-70min.
3. The crystallization method for effectively reducing ansamitocin P-3 impurities according to claim 1, wherein the method comprises the following steps: and in the S4, the temperature is reduced from 65 ℃ to 45 ℃, then the temperature is kept for 60min, and finally the temperature is kept at 45 ℃ and the stirring is carried out for 40-50min.
4. The crystallization method for effectively reducing ansamitocin P-3 impurities according to claim 1, wherein: and (3) in the S5, reducing the temperature from 45 ℃ to 25 ℃, keeping the temperature for 60min, keeping the temperature at 25 ℃, and stirring for 60-70min.
5. A crystallization method according to any one of claims 1 to 4 effective in reducing ansamitocin P-3 impurities, characterized in that: the purity of the refined AP-3 product obtained by the crystallization method is not less than 98%.
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| CN202211250499.2A CN115477658A (en) | 2022-10-12 | 2022-10-12 | Crystallization method for effectively reducing ansamitocin P-3 impurity |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004015119A2 (en) * | 2002-08-08 | 2004-02-19 | Smithkline Beecham Corporation | Methods for the isolation and purification of ansamitocins |
| CN101103120A (en) * | 2005-01-19 | 2008-01-09 | 免疫基因公司 | Methods for the production of ansamitocins |
| CN108276427A (en) * | 2018-01-14 | 2018-07-13 | 常州大学 | The extraction of ansamitocin P-3 a kind of and isolation and purification method |
| CN113444106A (en) * | 2020-03-25 | 2021-09-28 | 重庆乾泰生物医药有限公司 | Preparation method of high-purity ansamitocin |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004015119A2 (en) * | 2002-08-08 | 2004-02-19 | Smithkline Beecham Corporation | Methods for the isolation and purification of ansamitocins |
| US20050261493A1 (en) * | 2002-08-08 | 2005-11-24 | Mark Fulston | Methods for the isolation and purification of ansamitocins |
| CN101103120A (en) * | 2005-01-19 | 2008-01-09 | 免疫基因公司 | Methods for the production of ansamitocins |
| CN108276427A (en) * | 2018-01-14 | 2018-07-13 | 常州大学 | The extraction of ansamitocin P-3 a kind of and isolation and purification method |
| CN113444106A (en) * | 2020-03-25 | 2021-09-28 | 重庆乾泰生物医药有限公司 | Preparation method of high-purity ansamitocin |
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