CN110157764A - A kind of preparation method of Dexamethasone Intermediate - Google Patents
A kind of preparation method of Dexamethasone Intermediate Download PDFInfo
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Abstract
The present invention obtains the preparation method of dexamethasone epoxy hydrolysate 8DM using Arthrobacter simplex Arthrobacter Simple CPCC140451 one-step fermentation, and this method mode of operation is simple, process stabilizing, is suitble to industrialized production.
Description
Technical field
The invention belongs to steroidal field of biotechnology, in particular to a kind of biofermentation synthesizes Dexamethasone Intermediate 8DM
Method.
Background technique
Dexamethasone is clinically widely used in the treatment of a variety of diseases, such as activity rheumatism, rheumatoid arthritis, tight
Weight bronchial asthma, serious dermatitis, acute leukemia etc., clinical dosage increases year by year.8DM, also referred to as dexamethasone ring
Oxygen hydrolysis object, is one of the important intermediate of steroid drugs chemistry, CAS#:24916-90-3, and structural formula is as follows:
8-DM is mainly used to synthesize dexamethasone, cuts down the glucocorticoid medicines such as clocortolone, momestasone furoate.
Initially, the technique of dexamethasone is prepared by starting material of diene derivative, and 8DM is in one of key
Mesosome.The Dioscin that diene derivative extracts in yellow ginger, Dioscin are cracked to form acetic acid gestation diene through open loop
Alcohol ketone；Through epoxy, hydrolysis, fertile formula oxide, mould dehydrogen substance then is obtained through Aspergillus ochraceus, arthrobacterium dehydrogenation；Again through C16
Iodization obtains Dexamethasone Intermediate in position methylation, side chain；8DM is obtained using reactions such as bromine hydroxyl epoxies.The production road
It is wire length, complicated for operation, it is seriously polluted, with environmental requirement upgrading and diene derivative saponin resource it is increasingly depleted, make
Be able to diene derivative route obtains 8DM rise in price, leads to the big adjustment of the steroidal industrial structure.
At this point, emerging rapidly by the dexamethasone synthetic route of raw material of phytosterol.Phytosterol is production soybean oil
By-product, cheap, abundance.Nowadays domestic that phytosterol direct fermentation is generated into 4-AD, 9 α-OH-AD is real
Existing industrialized production.Russ P RU2532902C1 is disclosed to be prepared the process route of 8DM using 9 α-OH-AD, and China is specially
Sharp CN 105440094A discloses the preparation method of Dexamethasone Intermediate；The for another example China of Publication No. CN 107417754A
Patent application discloses the preparation method of a kind of dexamethasone and betamethasone key intermediate；Three patents all specifically have studied
9 α-OH-AD arrive the process route of dexamethasone, and the route relative to original diene derivative has cost advantage, meet green
Environmental protection concept.
It can be seen that 8DM is an important intermediate in the existing synthetic method of dexamethasone.Existing report document
In, by starting material I, -17 α of 16 Alpha-Methyl, -21 acetate system of -4 alkene -9,11- epoxy -3,20- diketone of 21- dimonohydric pregnant
There are two types of modes by standby 8DM: first is that bioanalysis combination chemical method, i.e., first with microbial fermentation by starting material I, and the 1 of A ring, 2 take off
Hydrogen, then 21 acetates of D ring are hydrolyzed by chemical method, and obtain 8DM.For example Chinese patent CN 105440094A is public
The preparation method of Dexamethasone Intermediate is opened, two steps are merged into a step by this method, and wherein chemical hydrolysis can generate impurity, are led
Fine work unstable quality is caused, yield is relatively low, yield of weight about 60%；Second is that biological fermentation process, i.e., only utilize microorganism
Microbe conversion prepares 8DM；As Chinese patent CN 106520896A by Arthrobacter simplex (Arthrobacter simplex,
ATCC6946) and bacillus megaterium (Bacillus megaterium) co-fermentation, synergistic effect by dehydrogenation, hydrolyze two steps
It reacts while carrying out, directly obtain product dexamethasone epoxy hydrolysate 8DM, this method, which has, reacts mild, environmental protection, yield
High advantage, yield of weight about 70%, total recovery about 76%.Though the result is better than the 8DM of first way acquisition,
The problems such as there are strains mutually to inhibit for mixed bacteria, fermentation process not degrade by easy-regulating, material, yield reduction；Further, since two
Strain bacterium, which needs first to carry out individually to cultivate, to be remixed, and equipment investment is big, cumbersome, is increased the risk of system microbiological contamination, is not suitable for
Industrialized production.
In order to improve the product quality of 8DM, industry always searches for and establishes environmentally protective production process, high income, behaviour
Make the easy and low-cost transform mode with commercialization competitiveness.
Summary of the invention
For the defect for synthesizing dexamethasone important intermediate 8DM in the prior art, the object of the present invention is to provide one kind
The method of microbial fermentation synthesis 8DM: starting material I progress one-step fermentation is obtained into 8DM, micro- life using microorganism fungus kind
Object refers to Arthrobacter simplex Arthrobacter simplex CPCC 140451；The starting material I refers to 16 Alpha-Methyl -17a,
- 21 acetate of -4 alkene -9,11- epoxy -3,20- diketone of 21- dimonohydric pregnant, conversion such as following formula.After this zymotechnique is optimized,
The yield for the 8DM for obtaining synthesis is higher, and quality is more stable.
Although microorganism it is widely distributed the strain with dehydrogenation activity and hydrolysing activity, it is some have dehydrogenation activity
Arthrobacterium and Nocard's bacillus to starting material I, there is no stronger hydrolysises, through testing, listed strain can not be very in following table
Good realization starting material I carries out dehydrogenation simultaneously and hydrolysis obtains 8DM, and Arthrobacter simplex Arthrobacter simplex
CPCC 140451 is that can have the active single bacterium colony of stronger hydrolysis/dehydrogenation to starting material I, and the selection result is as follows:
As can be seen from the above table, Arthrobacter simplex CPCC 140451 provided by the invention is by starting material I
The zymotechnique for preparing 8DM has unique advantage.
The microorganism used: the Arthrobacter simplex Arthrobacter simplex CPCC 140451 that the present invention uses,
In in March, 2014 purchase from Chinese pharmacy Microbiological Culture Collection administrative center, the strain original preservation time is 2 months 1989
19, primary source was the second pharmaceutical factory of Henan, China Anyang, original number A-1-3.
Particularly, inventor provides the following technical solution:
(1) Spawn incubation
The Arthrobacter simplex inclined-plane of 100mL eggplant type bottle is inoculated into 100mL culture medium after the sterile water elution of 50mL
(culture Flask volume is 500mL) carries out level-one culture in shaking table, 29~35 DEG C of cultivation temperature, shaking table revolution 180rpm, trains
Bring about 18 up~for 24 hours；Here Arthrobacter simplex refers to Arthrobacter simplex CPCC 140451.
Above-mentioned first cell culture medium uses: glucose 2.0%, corn pulp 3.2%, yeast extract 1.5%, potassium dihydrogen phosphate
0.3%, GPE 0.04%, 30% sodium hydroxide adjusts pH 7.0~7.5.
(2) microbe conversion
Aseptically, first order seed is moved into secondary medium, 29~34 DEG C of cultivation temperature, culture 10~
Starting material I is micronized or solvent dissolution feeds intake, after feeding intake, coacetylase is added, adjusts microbe conversion temperature 30~36 by 13h or so
DEG C converted, the microbe conversion time in 48~76h, to 8DM HPLC content >=95.0% (HPLC area normalization method), into
85 DEG C of sterilizing 30min of row, cool to 25 DEG C, to be extracted.
Above-mentioned second order fermentation culture medium is: glucose 2.0%, corn pulp 3.2%, yeast extract 1.5%, potassium dihydrogen phosphate
0.3%, starting material I 0.2~0.5%, GPE 0.04%, 30% sodium hydroxide adjust pH7.5~8.5；
Starting material I 0.2~0.5% is added in above-mentioned second order fermentation culture medium, as fermentation inducement agent.
Above-mentioned second order fermentation culture medium adjusts pH7.5~8.5 with 30% sodium hydroxide, so that starting material I is hydrolyzed；
Above-mentioned starting material I dissolves feeding mode using solvent, and solvent is selected from: methanol, ethyl alcohol, dioxane, DMF, the third two
One of alcohol；Solvent is further preferred are as follows: methanol.
Above-mentioned starting material I feed concentrations are 30~60g/L.
Above-mentioned starting material I can also be fed intake using micronization, powder particle diameter D90≤100 μm；
It is above-mentioned feed intake after the completion of, be added coacetylase, to increase thallus ability of dehydrogenation；When micronization feeds intake 60g/L, pass through
Coacetylase is added, so that 8DM conversion ratio improves nearly 10%, shows the effect for improving thallus ability of dehydrogenation well, further
Ground, the additional amount of coacetylase are 0~0.2%；
(3) Hydrolysis kinetics
Fermentation liquid is filtered, filter cake is obtained, the mixed solvent of 20~30V methylene chloride/methanol, material is added in filter cake
The active carbon of weight 5% carries out refluxing extraction, and filtering, filtrate is concentrated under reduced pressure again, and with a certain amount of dry Extraction solvent of water band
Afterwards, 25 DEG C are cooled to, is filtered, the wet crude product of 8DM is obtained, it is dry, 8DM crude product is obtained, wait refine；
8DM crude product is added in purification bottle, is heated using 20~30V methylene chloride/acetone, after the 30min that flows back, decompression
After being concentrated into 1~3V acetone, cools to 20 DEG C and obtain wet fine work hereinafter, filter, wet fine work is dry, obtains 8DM fine work.
The mixed solvent of said extracted solvent selection methylene chloride/methanol, wherein methylene chloride and methanol volumetric mixture ratio
Example is 6: 1~2: 1；
Above-mentioned refining solvent selects methylene chloride/acetone mixed solvent, and wherein methylene chloride and acetone mixed proportion are 4
: 1~4: 3；
Inventor to microbe species, feed concentrations and mode, Spawn incubation and conversion condition used in converting etc. into
It has gone careful research, has obtained the zymotechnique for efficiently preparing 8DM.
In order to which the solution of the present invention is explained further, in embodiment 1, it is micronized feeding mode, starting material I feed concentrations
For 30g/L, conversion ratio 96.32%；
In comparative example 1, using micronization feeding mode, coacetylase, feed concentrations 60g/L are not added, conversion ratio is
78.21%；
In example 2, using micronization feeding mode, coacetylase, feed concentrations 60g/L is added, conversion ratio is
87.56%；
In embodiment 3-7, feeding mode is dissolved using solvent, coacetylase is added, feed concentrations 60g/L selects dissolution
Solvent is respectively methanol, ethyl alcohol, dioxane, DMF, propylene glycol, the amount of corresponding coacetylase is respectively 0.2%, 0.1%,
0.15%, 0.10%, 0.20%, conversion ratio is respectively 97.32%, 95.21%, 93.21%, 96.99%, 96.70%；
Compared with prior art, the beneficial effect that the present invention reaches is:
(1) using Arthrobacter simplex Arthrobacter simplex CPCC 140451 simultaneously to starting material I A ring
21 1,2 dehydrogenation, D ring acetic acid ester hydrolysis, one-step fermentation obtain 8DM.Optimized, feed concentrations are reached up to 60g/L, conversion ratio
To 93%~97%, while simplifying production technology, a yield of 8DM is up to 70%~75%.
(2) with prior art China patent CN 106520896A report, 1,2, A ring is carried out using Arthrobacter simplex and is taken off
Hydrogen is compared with the zymotechnique that the mixed bacteria that bacillus megaterium carries out 21, D ring hydrolysis carries out production 8DM, and the present invention has
Mode of operation is simple, process stabilizing, reduce system microbiological contamination risk advantage, be suitble to industrialized production.
Detailed description of the invention
The HPLC test map of Fig. 1 starting material I.
The HPLC of Fig. 2 embodiment 3 converts map.
The HPLC of Fig. 3 embodiment 6 converts map.
The HPLC of Fig. 4 embodiment 7 converts map.
Specific embodiment
Below with reference to embodiment, the content of the present invention will be explained in more detail.
In the present invention, all equipment and raw material etc. are commercially available or the industry is common；Following implementations
Method in example is unless otherwise instructed the conventional method of this field.
Primary raw material and detection method explanation:
The present invention is with -17 α of Microbial Biotransformation starting material I:16 Alpha-Methyl, -4 alkene -9,11- of 21- dimonohydric pregnant
The mode of -21 acetate of epoxy -3,20- diketone prepares 8DM.Strain used in embodiment is Arthrobacter simplex
Arthrobacter simplex CPCC 140451 is bought in Chinese pharmacy Microbiological Culture Collection administrative center.
The measurement of product yield is measured using HPLC, splitter C18Column, Detection wavelength 254nm, 40 DEG C of column temperature, stream
Dynamic is mutually acetonitrile: pure water=26: 74, flow velocity 2.3mL/min.Product yield is calculated using area normalization method.
Note: W described in text indicates weight, and V indicates volume.When W unit is g, the Unit/mL of V；When W unit is kg, V
Unit L.
Embodiment 1
A kind of preparation method of Dexamethasone Intermediate, the Dexamethasone Intermediate refer to 8-DM, in the steps below
Operation:
(1) Spawn incubation: the preparation of first cell culture medium, glucose 2.0%, corn pulp 3.2%, yeast are carried out according to the proportion
Cream 1.5%, potassium dihydrogen phosphate 0.3%, GPE 0.04% adjust pH7.5 with 30% sodium hydroxide, and 121 DEG C, sterilize 30min, cold
But room temperature is arrived, by the inclined-plane of the Arthrobacter simplex Arthrobacter simplex CPCC 140451 of 100mL eggplant type bottle culture,
After the sterile water elution of 50mL, in the culture medium of inoculation 20% to 100mL (culture Flask volume 500mL), shaking table carries out level-one
Culture 30 DEG C of cultivation temperature, shaking speed 180rpm, cultivates 19h, carries out second level culture transferring；
(2) microbe conversion: fermentation medium preparation, glucose 2.0%, corn pulp 3.2%, yeast extract are carried out according to the proportion
1.5%, potassium dihydrogen phosphate 0.3%, starting material I 0.2%, GPE 0.04%, with 30% sodium hydroxide adjust pH8.0,121 DEG C,
Sterilize 30min, is cooled to 32 DEG C, to be seeded；Fermentation is in two stages: 1. thalli growth stage: aseptically, by level-one
Strain inoculation 30% is cultivated into secondary medium, temperature controls 32 DEG C, cultivates 12h.2. the microorganism conversion stage: feed concentrations
30g/L, i.e. micronization starting material I (D90≤ 100 μm) investment 3g, adjusting conversion temperature is 34 DEG C, transformation time 48h.Sampling
HPLC analyzes (area normalization method), 8DM content 96.32%, 8DM acetate content 2.52%, starting material I content 0.05%,
Intermediate II content 0.65%.
(4) Hydrolysis kinetics: 85 DEG C of fermentation liquid carry out sterilizing 30min, and cooling down is filtered and done to the greatest extent, fermented to 25 DEG C
Fermentation filter cake addition is slowly added to 90mL methylene chloride/methanol (3: 1) mixed solvent, while 1.5g active carbon is added by filter cake,
It is heated to 40 DEG C jointly, after dissolved clarification keeps the temperature 30min, filters while hot, filtrate is concentrated under reduced pressure into dry to the greatest extent, addition suitable quantity of water, cold
But it to after 25 DEG C, is filtered, obtains wet crude product, it is dry.8DM crude product uses 70mL methylene chloride/acetone (4: 1) mixed solvent
After dissolved clarification, carry out normal pressure be concentrated into no methylene chloride, continue to be concentrated into volume of material 1V acetone, be cooled to 20 DEG C hereinafter, filtering,
Drying obtains fine work 2.18g, and (0.5%) HPLC purity >=99.0%, single impurity are respectively less than, filtrate concentration obtains mother liquor
0.23g, a yield 72.66%, total recovery 78.70%.
Comparative example 1
A kind of preparation method of Dexamethasone Intermediate, the Dexamethasone Intermediate refer to 8-DM, in the steps below
Operation:
(1) Spawn incubation: the preparation of first cell culture medium, glucose 2.0%, corn pulp 3.2%, yeast are carried out according to the proportion
Cream 1.5%, potassium dihydrogen phosphate 0.3%, GPE 0.04% adjust pH7.2 with 30% sodium hydroxide, and 121 DEG C, sterilize 30min, cold
But room temperature is arrived, by the inclined-plane of the Arthrobacter simplex Arthrobacter simplex CPCC 140451 of 100mL eggplant type bottle culture,
After the sterile water elution of 50mL, in the culture medium of inoculation 20% to 100mL (culture Flask volume 500mL), shaking table carries out level-one
Culture 30 DEG C of cultivation temperature, shaking speed 180rpm, cultivates 18h, carries out second level culture transferring；
(2) microbe conversion: fermentation medium preparation, glucose 2.0%, corn pulp 3.2%, yeast extract are carried out according to the proportion
1.5%, potassium dihydrogen phosphate 0.3%, starting material I 0.2%, GPE 0.04%, with 30% sodium hydroxide adjust pH7.5,121 DEG C,
Sterilize 30min, is cooled to 33 DEG C, to be seeded；Fermentation is in two stages: 1. thalli growth stage: aseptically, by level-one
Strain inoculation 30% is cultivated into secondary medium, temperature controls 32 DEG C, cultivates 10h.2. the microorganism conversion stage: feed concentrations
60g/L, i.e. micronization starting material I (D90≤ 100 μm) investment 6g, conversion temperature is 34 DEG C, transformation time 76h.Sample HPLC
It analyzes (area normalization method), 8DM content 78.21%, 8DM acetate content 1.92%, starting material I content 19.11%, it is intermediate
Body II content 0.65%；Continue to extend transformation time 12h, sampling HPLC analysis (area normalization method), 8DM content 79.89%.
(4) Hydrolysis kinetics: 85 DEG C of fermentation liquid carry out sterilizing 30min, and cooling down is filtered and done to the greatest extent, fermented to 25 DEG C
Fermentation filter cake addition is slowly added to 180mL methylene chloride/methanol (6: 1) mixed solvent, while 3g active carbon is added by filter cake,
It is heated to 40 DEG C jointly, after dissolved clarification keeps the temperature 30min, filters while hot, filtrate is concentrated under reduced pressure into dry to the greatest extent, addition suitable quantity of water, cold
But it to after 25 DEG C, is filtered, obtains wet crude product, wet crude product drying；8DM crude product uses 150mL methylene chloride/acetone (4: 3)
After mixed solvent dissolved clarification, carry out normal pressure be concentrated into no methylene chloride, continue to be concentrated into volume of material 3V acetone, be cooled to 20 DEG C with
Under, filtering, the repetition purification step is primary, and drying obtains fine work 2.98g, and (HPLC purity >=99.0%, single impurity are respectively less than
0.5%), filtrate concentration obtains mother liquor 1.92g, a yield 49.67%, total recovery 73.03%.
Embodiment 2
A kind of preparation method of Dexamethasone Intermediate, the Dexamethasone Intermediate refer to 8-DM, in the steps below
Operation:
(1) Spawn incubation: the preparation of first cell culture medium, glucose 2.0%, corn pulp 3.2%, yeast are carried out according to the proportion
Cream 1.5%, potassium dihydrogen phosphate 0.3%, GPE 0.04% adjust pH7.2 with 30% sodium hydroxide, and 121 DEG C, sterilize 30min, cold
But room temperature is arrived, by the inclined-plane of the Arthrobacter simplex Arthrobacter simplex CPCC140451 of 100mL eggplant type bottle culture,
After the sterile water elution of 50mL, in the culture medium of inoculation 20% to 100mL (culture Flask volume 500mL), shaking table carries out level-one
Culture 30 DEG C of cultivation temperature, shaking speed 180rpm, cultivates 18h, carries out second level culture transferring；
(2) microbe conversion: fermentation medium preparation, glucose 2.0%, corn pulp 3.2%, yeast extract are carried out according to the proportion
1.5%, potassium dihydrogen phosphate 0.3%, starting material I 0.2%, GPE 0.04%, with 30% sodium hydroxide adjust pH7.5,121 DEG C,
Sterilize 30min, is cooled to 33 DEG C, to be seeded；Fermentation is in two stages: 1. thalli growth stage: aseptically, by level-one
Strain inoculation 30% is cultivated into secondary medium, temperature controls 32 DEG C, cultivates 10h.2. the microorganism conversion stage: feed concentrations
60g/L, i.e. micronization starting material I (D90≤ 100 μm) investment 6g, while coacetylase amount 0.2% is added, conversion temperature is 34 DEG C, is turned
The change time is 76h.HPLC analysis (area normalization method), 8DM content 87.56% are sampled, 8DM acetate content 2.12% rises
Beginning object I content 10.61%, intermediate II content 0.55%.
(4) Hydrolysis kinetics: 85 DEG C of fermentation liquid carry out sterilizing 30min, and cooling down is filtered and done to the greatest extent, fermented to 25 DEG C
Fermentation filter cake addition is slowly added to 180mL methylene chloride/methanol (3: 1) mixed solvent, while 3g active carbon is added by filter cake,
It is heated to 40 DEG C jointly, after dissolved clarification keeps the temperature 30min, filters while hot, filtrate is concentrated under reduced pressure into dry to the greatest extent, addition suitable quantity of water, cold
But it to after 25 DEG C, is filtered, obtains wet crude product, wet crude product drying；8DM crude product uses 150mL methylene chloride/acetone (4: 3)
After mixed solvent dissolved clarification, carry out normal pressure be concentrated into no methylene chloride, continue to be concentrated into volume of material 3V acetone, be cooled to 20 DEG C with
Under, filtering, the repetition purification step is primary, and drying obtains fine work 4.03g, and (HPLC purity >=99.0%, single impurity are respectively less than
0.5%), filtrate concentration obtains mother liquor 0.92g, a yield 67.16%, total recovery 79.8%.
Embodiment 3
A kind of preparation method of Dexamethasone Intermediate, the Dexamethasone Intermediate refer to 8-DM, in the steps below
Operation:
(1) Spawn incubation: the preparation of first cell culture medium, glucose 2.0%, corn pulp 3.2%, yeast are carried out according to the proportion
Cream 1.5%, potassium dihydrogen phosphate 0.3%, GPE 0.04% adjust pH7.5 with 30% sodium hydroxide, and 121 DEG C, sterilize 30min, cold
But room temperature is arrived, by the inclined-plane of the Arthrobacter simplex Arthrobacter simplex CPCC 140451 of 100mL eggplant type bottle culture,
After the sterile water elution of 50mL, in the culture medium of inoculation 20% to 100mL (culture Flask volume 500mL), shaking table carries out level-one
Culture 30 DEG C of cultivation temperature, shaking speed 180rpm, cultivates 19h, carries out second level culture transferring；
(2) microbe conversion: fermentation medium preparation, glucose 2.0%, corn pulp 3.2%, yeast extract are carried out according to the proportion
1.5%, potassium dihydrogen phosphate 0.3%, starting material I0.2%, GPE 0.04%, with 30% sodium hydroxide adjust pH8.0,121 DEG C,
Sterilize 30min, is cooled to 32 DEG C, to be seeded；Fermentation is in two stages: 1. thalli growth stage: aseptically, by level-one
Strain inoculation 30% is cultivated into secondary medium, temperature controls 32 DEG C, cultivates 12h.2. the microorganism conversion stage: feed concentrations
60g/L, i.e. dry powder put into 6g, while methanol solvate 4mL is added, coacetylase amount 0.2%, and conversion temperature is 34 DEG C, and transformation time is
72h.Sample HPLC analysis (area normalization method), 8DM content 97.32%, 8DM acetate content 1.54%, starting material I content
0.08%, intermediate II content 0.76%.
(4) Hydrolysis kinetics: 85 DEG C of fermentation liquid carry out sterilizing 30min, and cooling down is filtered and done to the greatest extent, fermented to 25 DEG C
Fermentation filter cake addition is slowly added to 120mL methylene chloride/methanol (5: 1) mixed solvent, while 3g active carbon is added by filter cake,
It is heated to 40 DEG C jointly, after dissolved clarification keeps the temperature 30min, filters while hot, filtrate is concentrated under reduced pressure into dry to the greatest extent, addition suitable quantity of water, cold
But it to after 25 DEG C, is filtered, obtains wet crude product, 8DM crude product is mixed using 100mL methylene chloride/acetone (4: 1) after dry crude product
After bonding solvent dissolved clarification, carry out normal pressure be concentrated into no methylene chloride, continue to be concentrated into volume of material 1V acetone, be cooled to 20 DEG C with
Under, filtering, drying obtains fine work 4.52g, and (0.5%) HPLC purity >=99.0%, single impurity are respectively less than, filtrate concentration obtains
Mother liquor 0.38g, a yield 75.33%, total recovery 80.42%.
Embodiment 4
A kind of preparation method of Dexamethasone Intermediate, the Dexamethasone Intermediate refer to 8-DM, in the steps below
Operation:
(1) Spawn incubation: the preparation of first cell culture medium, glucose 2.0%, corn pulp 3.2%, yeast are carried out according to the proportion
Cream 1.5%, potassium dihydrogen phosphate 0.3%, GPE 0.04% adjust pH7.0 with 30% sodium hydroxide, and 121 DEG C, sterilize 30min, cold
But room temperature is arrived, by the inclined-plane of the Arthrobacter simplex Arthrobacter simplex CPCC 140451 of 100mL eggplant type bottle culture,
After the sterile water elution of 50mL, in the culture medium of inoculation 20% to 100mL (culture Flask volume 500mL), shaking table carries out level-one
Culture 31 DEG C of cultivation temperature, shaking speed 180rpm, cultivates 21h, carries out second level culture transferring；
(2) microbe conversion: fermentation medium preparation, glucose 2.0%, corn pulp 3.2%, yeast extract are carried out according to the proportion
1.5%, potassium dihydrogen phosphate 0.3%, starting material I 0.5%, GPE 0.04%, with 30% sodium hydroxide adjust pH8.5,121 DEG C,
Sterilize 30min, is cooled to 32 DEG C, to be seeded；Fermentation is in two stages: 1. thalli growth stage: aseptically, by level-one
Strain inoculation 30% is cultivated into secondary medium, temperature controls 33 DEG C, cultivates 13h.2. the microorganism conversion stage: feed concentrations
60g/L, i.e. dry powder put into 6g, while alcohol solvent 4mL is added, coacetylase amount 0.10%, and conversion temperature is 33 DEG C, transformation time
For 76h.Sampling HPLC analysis (area normalization method), 8DM content 95.21%, 8DM acetate content 1.82%, starting material I contain
Amount 0.05%, intermediate II content 1.75%.
(4) Hydrolysis kinetics: 85 DEG C of fermentation liquid carry out sterilizing 30min, and cooling down is filtered and done to the greatest extent, fermented to 25 DEG C
Fermentation filter cake addition is slowly added to 160mL methylene chloride/methanol (2: 1) mixed solvent, while 3g active carbon is added by filter cake,
It is heated to 40 DEG C jointly, after dissolved clarification keeps the temperature 30min, filters while hot, filtrate is concentrated under reduced pressure into dry to the greatest extent, addition suitable quantity of water, cold
But it to after 25 DEG C, is filtered, obtains wet crude product, dry crude product.8DM crude product is mixed using 130mL methylene chloride/acetone (4: 2)
After bonding solvent dissolved clarification, carry out normal pressure be concentrated into no methylene chloride, continue to be concentrated into volume of material 2V acetone, be cooled to 20 DEG C with
Under, filtering, drying obtains fine work 4.30g, and (0.5%) HPLC purity >=99.0%, single impurity are respectively less than, filtrate concentration obtains
Mother liquor 0.58g, a yield 71.67%, total recovery 79.33%.
Embodiment 5
A kind of preparation method of Dexamethasone Intermediate, the Dexamethasone Intermediate refer to 8-DM, in the steps below
Operation:
(1) Spawn incubation: the preparation of first cell culture medium, glucose 2.0%, corn pulp 3.2%, yeast are carried out according to the proportion
Cream 1.5%, potassium dihydrogen phosphate 0.3%, GPE 0.04% adjust pH7.2 with 30% sodium hydroxide, and 121 DEG C, sterilize 30min, cold
But room temperature is arrived, by the inclined-plane of the Arthrobacter simplex Arthrobacter simplex CPCC140451 of 100mL eggplant type bottle culture,
After the sterile water elution of 50mL, in the culture medium of inoculation 20% to 100mL (culture Flask volume 500mL), shaking table carries out level-one
Culture, 35 DEG C of cultivation temperature, shaking speed 180rpm, culture for 24 hours, carries out second level culture transferring；
(2) microbe conversion: fermentation medium preparation, glucose 2.0%, corn pulp 3.2%, yeast extract are carried out according to the proportion
1.5%, potassium dihydrogen phosphate 0.3%, starting material I0.5%, GPE 0.04%, with 30% sodium hydroxide adjust pH8.5,121 DEG C,
Sterilize 30min, is cooled to 32 DEG C, to be seeded；Fermentation is in two stages: 1. thalli growth stage: aseptically, by level-one
Strain inoculation 30% is cultivated into secondary medium, temperature controls 33 DEG C, cultivates 12h.2. the microorganism conversion stage: feed concentrations
60g/L, i.e. dry powder put into 6g, while dioxane solvent 3mL is added, coacetylase amount 0.15%, and conversion temperature is 35 DEG C, conversion
Time is 76h.Sample HPLC analysis (area normalization method), 8DM content 93.21%, 8DM acetate content 2.82%, starting
Object I content 0.08%, intermediate II content 2.51%.
(4) Hydrolysis kinetics: 85 DEG C of fermentation liquid carry out sterilizing 30min, and cooling down is filtered and done to the greatest extent, fermented to 25 DEG C
Fermentation filter cake addition is slowly added to 180mL methylene chloride/methanol (4: 1) mixed solvent, while 3g active carbon is added by filter cake,
It is heated to 40 DEG C jointly, after dissolved clarification keeps the temperature 30min, filters while hot, filtrate is concentrated under reduced pressure into dry to the greatest extent, addition suitable quantity of water, cold
But it to after 25 DEG C, is filtered, obtains wet crude product, 8DM crude product is mixed using 140mL methylene chloride/acetone (4: 3) after dry crude product
After bonding solvent dissolved clarification, carry out normal pressure be concentrated into no methylene chloride, continue to be concentrated into volume of material 3V acetone, be cooled to 20 DEG C with
Under, filtering, drying obtains fine work 4.25g, and (0.5%) HPLC purity >=99.0%, single impurity are respectively less than, filtrate concentration obtains
Mother liquor 0.63g, a yield 70.83%, total recovery 79.29%.
Embodiment 6
A kind of preparation method of Dexamethasone Intermediate, the Dexamethasone Intermediate refer to 8-DM, in the steps below
Operation:
(1) Spawn incubation: the preparation of first cell culture medium, glucose 2.0%, corn pulp 3.2%, yeast are carried out according to the proportion
Cream 1.5%, potassium dihydrogen phosphate 0.3%, GPE 0.04% adjust pH7.5 with 30% sodium hydroxide, and 121 DEG C, sterilize 30min, cold
But room temperature is arrived, by the inclined-plane of the Arthrobacter simplex Arthrobacter simplex CPCC 140451 of 100mL eggplant type bottle culture,
After the sterile water elution of 50mL, in the culture medium of inoculation 20% to 100mL (culture Flask volume 500mL), shaking table carries out level-one
Culture 34 DEG C of cultivation temperature, shaking speed 180rpm, cultivates 22h, carries out second level culture transferring；
(2) microbe conversion: fermentation medium preparation, glucose 2.0%, corn pulp 3.2%, yeast extract are carried out according to the proportion
1.5%, potassium dihydrogen phosphate 0.3%, starting material I 0.4%, GPE 0.04%, with 30% sodium hydroxide adjust pH8.0,121 DEG C,
Sterilize 30min, is cooled to 32 DEG C, to be seeded；Fermentation is in two stages: 1. thalli growth stage: aseptically, by level-one
Strain inoculation 30% is cultivated into secondary medium, temperature controls 33 DEG C, cultivates 12h.2. the microorganism conversion stage: feed concentrations
60g/L, i.e. dry powder put into 6g, while DMF solvent 3mL is added, coacetylase amount 0.10%, and conversion temperature is 33 DEG C, and transformation time is
76h.Sample HPLC analysis (area normalization method), 8DM content 96.99%, 8DM acetate content 1.57%, starting material I content
0.05%, intermediate II content 0.94%.
(4) Hydrolysis kinetics: 85 DEG C of fermentation liquid carry out sterilizing 30min, and cooling down is filtered and done to the greatest extent, fermented to 25 DEG C
Fermentation filter cake addition is slowly added to 180mL methylene chloride/methanol (4: 1) mixed solvent, while 3g active carbon is added by filter cake,
It is heated to 40 DEG C jointly, after dissolved clarification keeps the temperature 30min, filters while hot, filtrate is concentrated under reduced pressure into dry to the greatest extent, addition suitable quantity of water, cold
But it to after 25 DEG C, is filtered, obtains wet crude product, it is dry；8DM crude product is molten using 140mL methylene chloride/acetone (4: 3) mixing
After agent dissolved clarification, carries out normal pressure and be concentrated into no methylene chloride, continue to be concentrated into volume of material 3V acetone, be cooled to 20 DEG C hereinafter, mistake
Filter, drying obtains fine work 4.38g, and (0.5%) HPLC purity >=99.0%, single impurity are respectively less than, filtrate concentration obtains mother liquor
0.50g, a yield 73.00%, total recovery 79.78%.
Embodiment 7
A kind of preparation method of Dexamethasone Intermediate, the Dexamethasone Intermediate refer to 8-DM, in the steps below
Operation:
(1) Spawn incubation: the preparation of first cell culture medium, glucose 2.0%, corn pulp 3.2%, yeast are carried out according to the proportion
Cream 1.5%, potassium dihydrogen phosphate 0.3%, GPE 0.04% adjust pH7.5 with 30% sodium hydroxide, and 121 DEG C, sterilize 30min, cold
But room temperature is arrived, by the inclined-plane of the Arthrobacter simplex Arthrobacter simplex CPCC 140451 of 100mL eggplant type bottle culture,
After the sterile water elution of 50mL, in the culture medium of inoculation 20% to 100mL (culture Flask volume 500mL), shaking table carries out level-one
Culture 33 DEG C of cultivation temperature, shaking speed 180rpm, cultivates 21h, carries out second level culture transferring；
(2) microbe conversion: fermentation medium preparation, glucose 2.0%, corn pulp 3.2%, yeast extract are carried out according to the proportion
1.5%, potassium dihydrogen phosphate 0.3%, starting material I0.3%, GPE 0.04%, with 30% sodium hydroxide adjust pH8.0,121 DEG C,
Sterilize 30min, is cooled to 32 DEG C, to be seeded；Fermentation is in two stages: 1. thalli growth stage: aseptically, by level-one
Strain inoculation 30% is cultivated into secondary medium, temperature controls 33 DEG C, cultivates 12h.2. the microorganism conversion stage: feed concentrations
60g/L, i.e. dry powder put into 6g, while propylene glycol solvent 4mL are added, coacetylase amount 0.20%, and conversion temperature is 33 DEG C, when conversion
Between be 76h.Sample HPLC analysis (area normalization method), 8DM content 96.70%, 8DM acetate content 1.09%, starting material I
Content 0.05%, intermediate II content 1.72%.
(4) Hydrolysis kinetics: 85 DEG C of fermentation liquid carry out sterilizing 30min, and cooling down is filtered and done to the greatest extent, fermented to 25 DEG C
Fermentation filter cake addition is slowly added to 170mL methylene chloride/methanol (4: 1) mixed solvent, while 3g active carbon is added by filter cake,
It is heated to 40 DEG C jointly, after dissolved clarification keeps the temperature 30min, filters while hot, filtrate is concentrated under reduced pressure into dry to the greatest extent, addition suitable quantity of water, cold
But it to after 25 DEG C, is filtered, obtains wet crude product, 8DM crude product is mixed using 130mL methylene chloride/acetone (4: 2) after dry crude product
After bonding solvent dissolved clarification, carry out normal pressure be concentrated into no methylene chloride, continue to be concentrated into volume of material 1.5v acetone, be cooled to 20 DEG C with
Under, filtering, drying obtains fine work 4.47g, and (0.5%) HPLC purity >=99.0%, single impurity are respectively less than, filtrate concentration obtains
Mother liquor 0.46g, a yield 74.50%, total recovery 80.68%.
Claims (10)
1. a kind of preparation method of Dexamethasone Intermediate 8DM, which is characterized in that preparation method includes: to utilize simple pole
Mono- step of starting material I is converted 8DM by bacterium, and the Arthrobacter simplex refers to, Arthrobacter Simplex deposit number is
CPCC 140451, the conversion process such as following formula:
2. the preparation method of any one Dexamethasone Intermediate 8DM according to claim 1, feed concentrations are 30~60g/L.
3. the preparation method of Dexamethasone Intermediate 8DM according to claim 2, which is characterized in that starting material I uses micro mist
Change feeds intake, and powder particle diameter D90≤100 μm.
4. the preparation method of Dexamethasone Intermediate 8DM according to claim 3, which is characterized in that after the completion of feeding intake, be added
Concentration is 0~0.2% coacetylase, and adjusting microbe conversion temperature is 30~36 DEG C, and the microbe conversion time is 48~76h.
5. the preparation method of Dexamethasone Intermediate 8DM according to claim 2, which is characterized in that starting material I uses solvent
Change feeds intake, and the solvent is selected from: one of methanol, ethyl alcohol, dioxane, DMF, propylene glycol.
6. the preparation method of Dexamethasone Intermediate 8DM according to claim 5, which is characterized in that after the completion of feeding intake, be added
Concentration is 0~0.2% coacetylase, and adjusting microbe conversion temperature is 30~36 DEG C, and the microbe conversion time is 48~76h.
7. the preparation method of Dexamethasone Intermediate 8DM according to claim 1, which is characterized in that the method uses
Primary-seed medium proportion it is as follows: glucose 2.0%, corn pulp 3.2%, yeast extract 1.5%, potassium dihydrogen phosphate 0.3%,
GPE 0.04%；PH 7.0~7.5 is adjusted with 30% sodium hydroxide, is used for seed culture, shaking table temperature is 29~35 DEG C, shaking table
Revolving speed is 180rpm, incubation time is 18~for 24 hours.
8. the preparation method of Dexamethasone Intermediate 8DM according to claim 7, which is characterized in that the method used
It is as follows to convert Medium Proportion: glucose 2.0%, corn pulp 3.2%, yeast extract 1.5%, potassium dihydrogen phosphate 0.3%, starting material
I 0.2~0.5%, GPE 0.04% adjust PH 7.5~8.5 with 30% sodium hydroxide, are used for second order fermentation culture, shaking table temperature
Degree is 29~35 DEG C, shaking speed 180rpm.
9. the preparation method of Dexamethasone Intermediate 8DM according to claim 8, which is characterized in that fermentation post-processing generates
Filter cake methylene chloride and methanol mixed solvent dissolution, 5% active carbon is added and extracts, extracting solution depressurize dense
Contracting obtains fermentation crude product, wherein the methylene chloride and methanol volume mixture ratio are 6: 1~2: 1.
10. the preparation method of Dexamethasone Intermediate 8DM according to claim 9, which is characterized in that the method includes
The refining methd of 8DM: being dissolved in the mixed solvent for 8DM crude product, is concentrated under reduced pressure and retains 1~3V volume, and cooling filtering obtains essence
Product, in which: methylene chloride and acetone volumetric mixture ratio are 4: 1~4: 3.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113980818A (en) * | 2021-09-13 | 2022-01-28 | 丽江映华生物药业有限公司 | Fungus dehydrogenation seed liquid culture method |
| CN114292892A (en) * | 2022-01-05 | 2022-04-08 | 浙江仙琚制药股份有限公司 | Method for producing androstadienedione by virtue of Arthrobacter simplex fermentation |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4431732A (en) * | 1980-12-23 | 1984-02-14 | Schering Aktiengesellschaft | Process for the preparation of 11β,21-dihydroxy-2'-methyl-5'βH-1,4-pregnadieno(16,17-D)-oxazole-3,20-dione |
| CN101210259A (en) * | 2007-12-25 | 2008-07-02 | 天津科技大学 | Method for producing prednisolone acetate |
| CN106520896A (en) * | 2016-12-21 | 2017-03-22 | 江苏远大仙乐药业有限公司 | Method for preparing dexamethasone intermediate product through one-step microbial fermentation and transformation |
| CN106893753A (en) * | 2015-12-21 | 2017-06-27 | 天津金耀集团有限公司 | A kind of method that prednisolone is prepared by the step of biofermentation one |
| CN107417754A (en) * | 2017-06-10 | 2017-12-01 | 浙江圃瑞药业有限公司 | A kind of preparation method of dexamethasone and betamethasone key intermediate |
| CN108588163A (en) * | 2018-04-28 | 2018-09-28 | 浙江仙琚制药股份有限公司 | The method for preparing prednisone acetate |
-
2019
- 2019-05-29 CN CN201910454561.1A patent/CN110157764B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4431732A (en) * | 1980-12-23 | 1984-02-14 | Schering Aktiengesellschaft | Process for the preparation of 11β,21-dihydroxy-2'-methyl-5'βH-1,4-pregnadieno(16,17-D)-oxazole-3,20-dione |
| CN101210259A (en) * | 2007-12-25 | 2008-07-02 | 天津科技大学 | Method for producing prednisolone acetate |
| CN106893753A (en) * | 2015-12-21 | 2017-06-27 | 天津金耀集团有限公司 | A kind of method that prednisolone is prepared by the step of biofermentation one |
| CN106520896A (en) * | 2016-12-21 | 2017-03-22 | 江苏远大仙乐药业有限公司 | Method for preparing dexamethasone intermediate product through one-step microbial fermentation and transformation |
| CN107417754A (en) * | 2017-06-10 | 2017-12-01 | 浙江圃瑞药业有限公司 | A kind of preparation method of dexamethasone and betamethasone key intermediate |
| CN108588163A (en) * | 2018-04-28 | 2018-09-28 | 浙江仙琚制药股份有限公司 | The method for preparing prednisone acetate |
Non-Patent Citations (2)
| Title |
|---|
| 周维善 等: "若干17β-乙酰基甾族化合物用节杆菌芳构化时的水解", 《化学学报》 * |
| 张丽青 等: "5α-△9（11）-16α-甲基-3β,17α,21-三羟基-孕甾烯-3β,21-双醋酸酯-20酮的微生物转化", 《药学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113980818A (en) * | 2021-09-13 | 2022-01-28 | 丽江映华生物药业有限公司 | Fungus dehydrogenation seed liquid culture method |
| CN114292892A (en) * | 2022-01-05 | 2022-04-08 | 浙江仙琚制药股份有限公司 | Method for producing androstadienedione by virtue of Arthrobacter simplex fermentation |
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