CN105543319A - Method for preparing steroidanti-inflammatory drugintermediatetetraene acetate using enzyme process - Google Patents
Method for preparing steroidanti-inflammatory drugintermediatetetraene acetate using enzyme process Download PDFInfo
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- CN105543319A CN105543319A CN201511020171.1A CN201511020171A CN105543319A CN 105543319 A CN105543319 A CN 105543319A CN 201511020171 A CN201511020171 A CN 201511020171A CN 105543319 A CN105543319 A CN 105543319A
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
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Abstract
The invention discloses a method for preparing steroidanti-inflammatory drugintermediatetetraene acetate using an enzyme process. In the method, a compound II serves as a reaction substrate and experiences reactions in a buffer solution with pH 6.0-8.0 at 15-30 DEG EC under the effect of steroid 1,2-dehydrogenase to generate a steroidanti-inflammatory drugintermediate I. The method solves the problem that preparation of a steroidanti-inflammatory drugintermediate using an enzyme process is unavailable in the prior art; and compared with traditional methods, the method has the advantages of high substrate concentration, low enzyme consumption, simple preparation steps, environmental friendliness and low production cost and is suitable for large-scale production.
Description
Technical field
The present invention relates to bio-pharmaceuticals and technical field of biochemical industry, particularly relate to a kind of method that enzyme process prepares steroidal antiphlogistic drug intermediate 21-hydroxy-1,4,9(11),16-pregnatetraene-3,20-dione-21-acetate.
Background technology
21-hydroxy-1,4,9(11),16-pregnatetraene-3,20-dione-21-acetate (Compound I has another name called teraene, 3TR), its structural formula is:
21-hydroxy-1,4,9(11),16-pregnatetraene-3,20-dione-21-acetate can prepare the multiple steroidal compounds with good antiinflammation as intermediate, as Triamcinolone Acetonide (TriamcinoloneAcetonide), prednisolone (trade(brand)name Donison) and dexamethasone (Dexamethasone) etc., these medicines have extensive and a large amount of application (Bioorganic & MedicinalChemistry21 (2013) 2241 – 2249, Steroids78 (2013) 1281 – 1287 etc.) in clinical.
Patent CN201210038066,200910070841 and document Steroids78 (2013) 1281 – 1287 in disclose several chemical processes preparing Compound I, usually need polystep reaction to be prepared, and need to use a large amount of organic solvent and metal catalyst in reaction process, and document Steroids68 (2003) 415 – 421 discloses the method that fermentation method prepares Compound I analogue, but reaction substrate concentration low (5g/L), product separation complexity (cell concentration is 4 times of substrate, is difficult to transform completely).In sum, there is no the biological method of High-efficient Production Compound I at present.
Summary of the invention
The object of this invention is to provide a kind of method that enzyme process prepares steroidal antiphlogistic drug intermediate 21-hydroxy-1,4,9(11),16-pregnatetraene-3,20-dione-21-acetate, overcome in prior art by this method the unfriendly problem of environment that the step adopting chemical process to prepare to exist in steroidal antiphlogistic drug intermediate process is complicated, use a large amount of organic solvents and metal catalyst to bring; Overcome simultaneously and adopt in fermentation method preparation process, the problem that reaction substrate concentration is low, product separation is complicated.
For achieving the above object, the technical solution used in the present invention is: a kind of enzyme process prepares the method for steroidal antiphlogistic drug intermediate 21-hydroxy-1,4,9(11),16-pregnatetraene-3,20-dione-21-acetate, take compound ii as reaction substrate, make it at steroidal 1, under the effect of 2 desaturases, temperature be 15 ~ 30 DEG C, pH is in the buffered soln of 6.0 ~ 8.0, reaction generates steroidal antiphlogistic drug intermediate I
The structural formula of described compound ii is:
The structural formula of described steroidal antiphlogistic drug intermediate I is:
Preferably, the pH of described buffered soln is 7.0 ~ 8.0.
Preferably, under temperature 20 ~ 30 DEG C of conditions, described reaction is carried out.
Further preferably, under temperature 25 ~ 30 DEG C of conditions, described reaction is carried out.
Preferably, described steroidal 1,2 desaturase is be steroidal 1,2 desaturase of EW1145 purchased from the trade mark of Suzhou Chinese biotechnology of enzymes company limited.
Further preferably, the quality that feeds intake of described steroidal 1,2 desaturase is that described compound ii feeds intake 0.1% ~ 100% of quality.
Still more preferably, the quality that feeds intake of described steroidal 1,2 desaturase is that described compound ii feeds intake 1% ~ 10% of quality.
Preferably, described buffered soln is phosphate buffer soln.
Further preferably, specific implementation process is as follows: by described compound ii and steroidal 1,2 desaturases join in described buffered soln, control temperature of reaction between 15 ~ 30 DEG C, concussion reaction, utilizes HPLC detection reaction process, after reaction terminates, centrifugally operated is carried out to reaction system, abandons supernatant liquor, washing of precipitate, filter, revolve steam obtain chemical compounds I.
Due to the utilization of technique scheme, the present invention compared with prior art has following advantages:
1) solve employing chemical method to prepare in 21-hydroxy-1,4,9(11),16-pregnatetraene-3,20-dione-21-acetate process, step is many, and in preparation process, use a large amount of organic solvents and metal catalyst, to the disagreeableness problem of environment;
2) solving employing fermentation method prepares in 21-hydroxy-1,4,9(11),16-pregnatetraene-3,20-dione-21-acetate process, the problem that reaction substrate concentration is low, product separation is complicated;
3) the invention solves the problem preparing 21-hydroxy-1,4,9(11),16-pregnatetraene-3,20-dione-21-acetate in prior art without enzyme process, and preparation process of the present invention is simple, environmentally friendly, concentration of substrate is high, enzyme dosage is few and production cost is low.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described in detail, but the present invention is not limited to following examples.The implementation condition adopted in embodiment can require to do further adjustment according to the concrete difference used, and not marked implementation condition is the condition in normal experiment.
The screening of embodiment 1 steroidal 1,2 desaturase
Join in the phosphate buffer soln of 2mLpH7.0 by 10mg compound ii, then add steroidal 1, the 2 dehydrogenase 10 mg of the different trades mark produced purchased from Suzhou Chinese biotechnology of enzymes company limited respectively, in 30 DEG C of concussion 2h, the transformation efficiency of detection substrate is as table 1.
Table 1
| Enzyme | EW1142 | EW1144 | EW1145 | EW1146 | EW1147 | EW1148 |
| Transformation efficiency % | 9.61 | 34.48 | 100 | 11.93 | 18.47 | 73.2 |
As can be seen from Table 1, when other conditions are identical, adopt the trade mark be steroidal 1,2 desaturase of EW1145 as steroidal 1, during 2 desaturase, substrate conversion efficiency is the highest.
Substrate conversion efficiency situation under embodiment 2 condition of different temperatures
Join in the phosphate buffer soln of 2mLpH7.0 by 10mg compound ii, add steroidal 1,2 desaturases are (purchased from Suzhou Chinese biotechnology of enzymes company limited, the trade mark is EW1145, lower same) 1mg, under condition of different temperatures, concussion reaction 12h, the transformation efficiency of detection substrate is as table 2.
Table 2
| Temperature DEG C | 15 | 20 | 25 | 30 | 35 |
| Transformation efficiency % | 48.0 | 56.1 | 58.5 | 74.3 | 37.3 |
As can be seen from Table 2, when other conditions are identical, temperature of reaction controls 30 DEG C time, and the transformation efficiency of compound ii is the highest.
Substrate conversion efficiency situation under embodiment 3 condition of different pH
Joined by 10mg compound ii in the phosphate buffer soln of the different pH of 2mL, add steroidal 1,2 dehydrogenase 1 mg, in 30 DEG C of concussion reaction 12h, the transformation efficiency of detection substrate is as table 3.
Table 3
| pH | 6.0 | 7.0 | 8.0 |
| Transformation efficiency % | 75 | 85 | 80 |
As can be seen from Table 3, when other conditions are identical, be carry out above-mentioned reaction in the buffered soln of 7.0 at pH, the transformation efficiency of substrate is the highest.
Embodiment 4 iodine
Join in the phosphate buffer soln of 500mLpH7.0 by 20g compound ii, add steroidal 1,2 dehydrogenase 1 g, in 30 DEG C of concussion 12h, detecting transformation efficiency is 100%.Then carry out the centrifugal 10min of 10000g to product, abandon supernatant, add the washing precipitation of 50mL trichloromethane, filter, revolve steaming and obtain acetic acid product tetraene thing 19.8g, its purity is 98%, and content is 99%.
Here, the analytical procedure of transformation efficiency is specific as follows: adopt HPLC (Shimadzu LC-2010) to be equipped with C18 chromatographic column (4.6*50mm, 5 μm), moving phase: 0-5min, 40% acetonitrile, 50% concentration is 0.1% aqueous formic acid, flow velocity 0.3mL/min, determined wavelength 254nm, column temperature 30 DEG C, appearance time is: substrate 3.2min, product 3.8min.
Above-described embodiment is only for illustrating technical conceive of the present invention and feature; its object is to person skilled in the art can be understood content of the present invention and be implemented; can not limit the scope of the invention with this; all equivalences done according to spirit of the present invention change or modify, and all should be encompassed in protection scope of the present invention.
Claims (9)
1. enzyme process prepares a method for steroidal antiphlogistic drug intermediate 21-hydroxy-1,4,9(11),16-pregnatetraene-3,20-dione-21-acetate, it is characterized in that, take compound ii as reaction substrate, make it at steroidal 1, under the effect of 2 desaturases, temperature be 15 ~ 30 DEG C, pH is in the buffered soln of 6.0 ~ 8.0, reaction generates steroidal antiphlogistic drug intermediate I
The structural formula of described compound ii is:
The structural formula of described steroidal antiphlogistic drug intermediate I is:
2. enzyme process according to claim 1 prepares the method for steroidal antiphlogistic drug intermediate 21-hydroxy-1,4,9(11),16-pregnatetraene-3,20-dione-21-acetate, it is characterized in that: the pH of described buffered soln is 7.0 ~ 8.0.
3. enzyme process according to claim 1 prepares the method for steroidal antiphlogistic drug intermediate 21-hydroxy-1,4,9(11),16-pregnatetraene-3,20-dione-21-acetate, it is characterized in that: under temperature 20 ~ 30 DEG C of conditions, carry out described reaction.
4. enzyme process according to claim 3 prepares the method for steroidal antiphlogistic drug intermediate 21-hydroxy-1,4,9(11),16-pregnatetraene-3,20-dione-21-acetate, it is characterized in that: under temperature 25 ~ 30 DEG C of conditions, carry out described reaction.
5. enzyme process according to claim 1 prepares the method for steroidal antiphlogistic drug intermediate 21-hydroxy-1,4,9(11),16-pregnatetraene-3,20-dione-21-acetate, it is characterized in that: described steroidal 1,2 desaturase is be steroidal 1,2 desaturase of EW1145 purchased from the trade mark of Suzhou Chinese biotechnology of enzymes company limited.
6. enzyme process prepares the method for steroidal antiphlogistic drug intermediate 21-hydroxy-1,4,9(11),16-pregnatetraene-3,20-dione-21-acetate according to claim 1 or 5, it is characterized in that: the quality that feeds intake of described steroidal 1,2 desaturase is that described compound ii feeds intake 0.1% ~ 100% of quality.
7. enzyme process according to claim 6 prepares the method for steroidal antiphlogistic drug intermediate 21-hydroxy-1,4,9(11),16-pregnatetraene-3,20-dione-21-acetate, it is characterized in that: the quality that feeds intake of described steroidal 1,2 desaturase is that described compound ii feeds intake 1% ~ 10% of quality.
8. enzyme process according to claim 1 prepares the method for steroidal antiphlogistic drug intermediate 21-hydroxy-1,4,9(11),16-pregnatetraene-3,20-dione-21-acetate, it is characterized in that: described buffered soln is phosphate buffer soln.
9. enzyme process according to claim 1 prepares the method for steroidal antiphlogistic drug intermediate 21-hydroxy-1,4,9(11),16-pregnatetraene-3,20-dione-21-acetate, it is characterized in that, specific implementation process is as follows: join in described buffered soln by described compound ii and steroidal 1,2 desaturase, controls temperature of reaction between 15 ~ 30 DEG C, concussion reaction, utilize HPLC detection reaction process, after reaction terminates, centrifugally operated is carried out to reaction system, abandon supernatant liquor, washing of precipitate, filter, revolve steam obtain chemical compounds I.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101633682A (en) * | 2009-08-24 | 2010-01-27 | 朱岩安 | Refining technique of intermediate acetate arachidonic substance of triamcinolone acetonide series products |
| CN102603843A (en) * | 2012-02-20 | 2012-07-25 | 湖南诺凯生物医药有限公司 | Preparation method of dexamethasone intermediate |
| CN102863505A (en) * | 2012-10-22 | 2013-01-09 | 宝鸡康乐生物科技有限公司 | Process for synthesizing triamcinolone acetonide acetate |
| CN104263791A (en) * | 2014-09-12 | 2015-01-07 | 苏州汉酶生物技术有限公司 | Method for preparing 11A, 17A-dihydroxy-pregna-1,4-diene-3,20-dione by enzymatic method |
| CN104628808A (en) * | 2015-02-28 | 2015-05-20 | 苏州汉酶生物技术有限公司 | Synthesis method and intermediates of pregnene-1,4,9 (11),16 (17)-tetraenol-3, 20-diketone |
-
2015
- 2015-12-30 CN CN201511020171.1A patent/CN105543319A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101633682A (en) * | 2009-08-24 | 2010-01-27 | 朱岩安 | Refining technique of intermediate acetate arachidonic substance of triamcinolone acetonide series products |
| CN102603843A (en) * | 2012-02-20 | 2012-07-25 | 湖南诺凯生物医药有限公司 | Preparation method of dexamethasone intermediate |
| CN102863505A (en) * | 2012-10-22 | 2013-01-09 | 宝鸡康乐生物科技有限公司 | Process for synthesizing triamcinolone acetonide acetate |
| CN104263791A (en) * | 2014-09-12 | 2015-01-07 | 苏州汉酶生物技术有限公司 | Method for preparing 11A, 17A-dihydroxy-pregna-1,4-diene-3,20-dione by enzymatic method |
| CN104628808A (en) * | 2015-02-28 | 2015-05-20 | 苏州汉酶生物技术有限公司 | Synthesis method and intermediates of pregnene-1,4,9 (11),16 (17)-tetraenol-3, 20-diketone |
Non-Patent Citations (1)
| Title |
|---|
| 李龙: "微生物转化21-醋酸酯-孕甾-4,9(11),16(17)-三烯-3,20-二酮的研究", 《中国学位论文全文数据库（万方）》 * |
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