Method for preparing pregna-5-ene-3[beta], 21-diol by resting cell method

A resting cell, resting cell transformation technology, applied in the field of preparation of pregna-5-ene-3β,21-diol by resting cell method, can solve the problems of cumbersome steps, easy pollution, low yield, etc. Achieve the effect of single nutrition, save the reaction steps and post-processing steps, and high yield

Inactive Publication Date: 2020-10-16
HUNAN NORCHEM PHARMACEUTICAL CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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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 ...

Method used

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  • Method for preparing pregna-5-ene-3[beta], 21-diol by resting cell method
  • Method for preparing pregna-5-ene-3[beta], 21-diol by resting cell method
  • Method for preparing pregna-5-ene-3[beta], 21-diol by resting cell method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 strain mutagenesis

[0029] Starting strain: Mycobacterium sp.B-NRRL 3683

[0030] (1) Strain culture:

[0031] 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, pH=7.5-8.0).

[0032] Liquid seed medium: M1. (M1 reference is not clear, 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, pH=7.5-8.0).

[0033] 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.

[0034] (2...

Embodiment 2

[0041] Embodiment 2 Seed culture

[0042] Strain name: Mycobacterium sp.B-NRRL 3683 mutagenic strain

[0043] (1) Incline cultivation

[0044] Formula: peptone 0.1-10g / L, yeast extract 0.1-10g / L, glucose 0.1-10g / L, disodium hydrogen phosphate 0.1-10g / L, disodium hydrogen phosphate 0.1-10g / L, agar 20g / L, pH =7.5-8.0;

[0045] Sterilize at 121°C for 30 minutes. After solidification and molding, inoculate under sterile conditions.

[0046] After inoculation, culture at 30°C for 4 days, and store in a refrigerator at 4°C for no more than 1 month.

[0047] (2) Shake flask seed culture

[0048] Formula: peptone 0.1-10g / L, yeast extract 0.1-10g / L, glucose 0.1-10g / L, disodium hydrogen phosphate 0.1-10g / L, pH=7.5-8.0.

[0049] Sterilize at 121°C for 30 minutes. Cool to room temperature.

[0050] 1. Primary seed culture

[0051] Inoculate under sterile conditions, inoculum volume: scrape 1 ring per 100ml. After inoculation, culture at 30°C and 200rpm shaker for 48h.

[0052] 2....

Embodiment 3

[0059] Embodiment 3 Phytosterol 3-position etherification protection

[0060] 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.

[0061] 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 during the addition process not to exceed 30°C, around 25°C Stir for 1-1.5 hours. Thin-layer chromatography detects that the reaction is complete. Raise the temperature 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 117.1 g, and the crude phytosterol etherified product was obtained.

[0062] The obtained etherified crude product, 2 times t...

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Abstract

The invention relates to a production method of a steroid drug intermediate, in particular to a method for preparing pregna-5-ene-3[beta], 21-diol by a resting cell method. The method comprises the steps of (1) 3-site protection of phytosterol, (2) transformation of resting cells, (3) extraction, (4) hydrolysis and (5) refining. The phytosterol is used as a raw material to produce the pregnene-5-ene-3[beta], 21-diol, the raw material is easy to obtain, and the production cost is reduced; functional group protection is carried out on 3-hydroxyl of the phytosterol by adopting a protective agent,the pregn-5-ene-3[beta], 21-diol can be directly prepared through hydrolysis after fermentation, few byproducts are generated, the yield is higher, a reaction route is shorter, and many reaction steps and post-treatment steps required in a traditional preparation method are omitted.

Description

technical field [0001] The invention relates to a production method of a steroid drug intermediate, in particular to a method for preparing pregna-5-ene-3β,21-diol by a resting 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. Co...

Claims

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Application Information

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IPC IPC(8): C12P33/00C12N1/20C12N15/01C07J9/00C12R1/32
CPCC07J9/00C12N1/20C12N15/01C12P33/00
Inventor 孟浩刘喜荣杨芳曾春玲赵小娟
Owner HUNAN NORCHEM PHARMACEUTICAL CO LTD
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