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Method for preparing intermediate by using resting cell biological fermentation of ergosterol etherate

A technology of ergosterol and biological fermentation, applied in biochemical equipment and methods, microorganism-based methods, treatment of microorganisms with electricity/wave energy, etc. Problems such as low yield, to achieve the effect of improved reaction yield, low cost and short route

Inactive Publication Date: 2020-04-03
HUNAN NORCHEM PHARMACEUTICAL CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of the above technical problems, the object of the present invention is to provide a method for preparing the intermediate pregna-5,7diene-3β,21-diol by biologically fermenting ergosterol etherate with resting cells, which can solve the intermediate synthesis process The steps are complex, easy to produce 3-position α-hydroxy isomer impurities, and the cost is high and the yield is low

Method used

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  • Method for preparing intermediate by using resting cell biological fermentation of ergosterol etherate
  • Method for preparing intermediate by using resting cell biological fermentation of ergosterol etherate
  • Method for preparing intermediate by using resting cell biological fermentation of ergosterol etherate

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1 strain mutagenesis

[0036] Starting strain: Mycobacterium sp.B-NRRL 3683

[0037] (1) Strain culture:

[0038] Solid slant medium: M1+2% agar.

[0039] Liquid seed medium: M1.

[0040] 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.

[0041] (2) Bacterial suspension preparation

[0042] Put the grown secondary seeds into a 10ml centrifuge tube and centrifuge at 10000rpm for 5min, discard the supernatant, collect the thallus, wash twice with the potassium phosphate buffer solution of pH6.0, and then use sterile potassium phosphate buffer solution (pH6 .0) ...

Embodiment 2

[0048] Embodiment 2 Seed culture

[0049] Strain name: Mycobacterium sp.B-NRRL 3683 mutagenic strain

[0050] (1) Incline cultivation

[0051] Formula: 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.

[0052] Sterilize at 121°C for 30 minutes. After solidification and molding, inoculate under sterile conditions.

[0053] After inoculation, culture at 30°C for 4 days, and store in a refrigerator at 4°C for no more than 1 month.

[0054] (2) Shake flask seed culture

[0055] 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.

[0056] Sterilize at 121°C for 30 minutes. Cool to room temperature.

[0057] 1. Primary seed culture

[0058] Inoculate under sterile conditions, inoculum volume: scrape 1 ring per 100ml. After inoculation, culture at 30°C and 200rpm shaker for 48h.

[0059] 2. Secondary seed culture

[0060] Inocu...

Embodiment 3

[0065] Embodiment 3 Ergosterol 3-position etherification protection

[0066] Ratio of materials: 1500g of methylal, 100g of ergosterol, 100g of diatomaceous earth, 50g of phosphorus pentoxide, 4g of sodium carbonate (used as 1% aqueous solution), 200g of water.

[0067] Add ergosterol 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 1-1.5h, 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 118.4 g, thus obtaining the crude ergosterol ether compound.

[0068] Add 2 times the volume of acetone to the crude etherified product obtained,...

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Abstract

The invention belongs to production methods of steroidal drug intermediates, and in particular relates to a method for preparing an intermediate by using resting cell biological fermentation of an ergosterol etherate. The method comprises the following steps: (1) 3-position protection; (2) resting cell transformation; (3) extraction; (4) hydrolysis; and (5) refining. The preparation method of theinvention has relatively low cost and relatively short route, and saves many reaction steps and post-treatment steps, and the shortening of the reaction route is also beneficial to improvement of thereaction yield and reduction of intermediate loss. According to the method, the step of 3-hydroxyl protection is performed first, and then a biological fermentation reaction of growing cells is performed, so that the solubility of a fermentation substrate in a fermentation liquid is directly increased, fermentation is conducive to performing, the yield of a product can be improved, and the production of impurities is reduced. The preparation method of the invention can also reduce the use of chemical reagents, and is beneficial to environmental protection.

Description

technical field [0001] The invention belongs to a production method of a steroid drug intermediate, and in particular relates to a method for preparing an intermediate by biologically fermenting ergosterol etherification with resting cells. Background technique [0002] The structural formula of the intermediate pregna-5,7-diene-3β,21-diol is shown below. This product is an important intermediate in the synthesis of steroids. In the traditional preparation method, a similar structure of the 3-position keto group is used. And use chemical methods to selectively reduce the 3-position ketone group to β-hydroxyl, but the products obtained by chemical methods often have a certain amount of 3-position α-hydroxyl isomers, and need to be completed by multi-step chemical reactions, which require multiple This kind of reagent is expensive and not conducive to environmental protection, the cost is higher, and the yield is low simultaneously, and the product quality is poor. [0003] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P33/00C12N13/00C12N1/20C07J5/00C12R1/32
CPCC07J5/0015C12N1/20C12N13/00C12P33/00
Inventor 赵小娟刘喜荣孟浩曾春玲杨芳
Owner HUNAN NORCHEM PHARMACEUTICAL CO LTD
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