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Method for preparing pregna-5-ene-3beta,21-diol by cell growth

A technology of growing cells and diols, which is applied in the field of preparing pregna-5-ene-3β,21-diols by growing cells, and can solve the problems of easy pollution, low yield, cumbersome steps, etc.

Inactive Publication Date: 2020-10-23
HUNAN NORCHEM PHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Pregn-5-ene-3β,21-diol is an important intermediate in the synthesis of steroids. The traditional process is to use the similar structure of the 3-position ketone group to selectively reduce the 3-position ketone group to the β-hydroxyl group by chemical methods to obtain The products often contain a certain amount of 3-position α hydroxyl isomers, the yield is low and the steps are cumbersome, and the use of various organic reagents is easy to cause pollution

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 strain mutagenesis

[0027] Starting strain: Mycobacterium sp.B-NRRL 3683

[0028] (1) Strain culture:

[0029] Solid slant medium: M1+2% agar. (M1+2% agar is unclear, change to: peptone 0.1-10g / L, yeast extract 0.1-10g / L, glucose 0.1-10g / L, disodium hydrogen phosphate 0.1-10g / L, agar 20g / L L, pH=7.5-8.0)

[0030] Liquid seed medium: M1 (M1 refers to unclear, change to: peptone 0.1-10g / L, yeast extract 0.1-10g / L, glucose 0.1-10g / L, disodium hydrogen phosphate 0.1-10g / L, agar 20g / L, pH=7.5-8.0).

[0031] Cultivate the strain B-NRRL 3683 in a solid slant medium, connect a ring of well-grown slant seeds and activate in a 500ml Erlenmeyer flask containing 100ml liquid seed medium, shake and culture at 200rpm at 30°C for 48h; take the activated slant seeds 10ml of the primary liquid seeds were inserted into a 500ml Erlenmeyer flask containing 100ml of liquid seed medium for secondary seed cultivation, and cultured at 30°C and 200rpm on a shaker for 48h.

...

Embodiment 2

[0039] Embodiment 2 Seed culture

[0040] Species name: mycobacterium sp.B-NRRL 3683

[0041] (1) Incline cultivation

[0042] Formula: peptone 0.1-1.0g / L, yeast extract 0.1-1.0g / L, glucose 0.1-1.0g / L, disodium hydrogen phosphate 0.1-1.0g / L, agar: 2%, pH=7.5-8.0.

[0043] Sterilize at 121°C for 30 minutes. After solidification and molding, inoculate under sterile conditions.

[0044] After inoculation, culture at 30°C for 4 days, and store in a refrigerator at 4°C for no more than 1 month.

[0045] (2) Shake flask seed culture

[0046] Formula: peptone 0.1-1.0g / L, yeast extract 0.1-1.0g / L, glucose 0.1-1.0g / L, disodium hydrogen phosphate 0.1-1.0g / L, pH=7.5-8.0. Sterilize at 121°C for 30 minutes . Cool to room temperature.

[0047] 1. First-level training

[0048] Inoculate under sterile conditions, inoculum volume: scrape 1 ring per 100 ml. After inoculation, culture at 30°C and 200rpm shaker for 48h.

[0049] 2. Secondary training

[0050] Inoculate under sterile co...

Embodiment 3

[0051] Embodiment 3 Phytosterol 3-position etherification protection

[0052] (1) Ratio of materials: 1500g of methylal, 100g of phytosterol, 100g of diatomaceous earth, 50g of phosphorus pentoxide, 4g of sodium carbonate (used as 1% aqueous solution), 200g of water.

[0053] (2) Add phytosterol and methylal in proportion to the reaction bottle, heat up to 25°C, stir until completely dissolved, add diatomaceous earth, then slowly add phosphorus pentoxide, control the temperature not to exceed 30°C during the addition process, Stir at around 25°C for 1-1.5 hours, the reaction is complete as detected by thin-layer chromatography, heat up to above 30°C, filter while hot, wash the filter cake and reaction bottle with a small amount of water, and dry at 50°C. A light yellow solid was obtained, which was dried in an oven at 40-50° C. to a constant weight of 124 g, and the crude phytosterol etherified product was obtained.

[0054] The obtained etherified crude product, 2 times the ...

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Abstract

The invention relates to a production method of steroid drug intermediates, in particular to a method for preparing pregna-5-ene-3beta,21-diol by cell growth. The method comprises the following steps:(1) 3-site protection on phytosterol; (2) transforming of growth cells; (3) extracting; (4) hydrolyzing; and (5) refining. The preparation method disclosed by the invention is relatively low in costand shorter in route, and omits a plurality of reaction steps and post-treatment steps. The shortening of the reaction route is also beneficial to the improvement of the reaction yield, and the intermediate loss is reduced. According to the method, the step of 3-site hydroxyl protection is adopted firstly, and growth cell biological fermentation reaction is carried out later, so that the solubility of the fermentation substrate in the fermentation liquor is directly increased, the fermentation is facilitated, the yield of the product can be increased, and the generation of impurities is reduced. The preparation method provided by the invention can also reduce the use of chemical reagents and is beneficial to environmental protection.

Description

technical field [0001] The invention relates to a production method of a steroidal drug intermediate, in particular to a method for preparing pregna-5-ene-3β,21-diol by a growing cell method. Background technique [0002] Pregn-5-ene-3β,21-diol is an important intermediate in the synthesis of steroids. The traditional process is to use the similar structure of the 3-position ketone group to selectively reduce the 3-position ketone group to the β-hydroxyl group by chemical methods to obtain The products often contain a certain amount of 3-position α hydroxyl isomers, the yield is low and the steps are cumbersome, and the use of various organic reagents is easy to cause pollution. [0003] The strain Mycobacterium sp.B-NRRL 3683 is recorded in the literature of U.S. Patent No. 4755463. It is generally only used for the fermentation of phytosterols to 4-AD and ADD. The current general research is to improve the 4-AD and ADD by controlling the reaction conditions. ADD yield. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P33/00C12N1/20C12N15/01C07J5/00C12R1/32
CPCC07J5/0015C12N1/20C12N15/01C12P33/00C12P33/005
Inventor 杨芳刘喜荣孟浩曾春玲赵小娟
Owner HUNAN NORCHEM PHARMACEUTICAL CO LTD
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