CN107286215A - A kind of steroidal compounds preparation method of multiple olefin groups - Google Patents
A kind of steroidal compounds preparation method of multiple olefin groups Download PDFInfo
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- CN107286215A CN107286215A CN201610204468.1A CN201610204468A CN107286215A CN 107286215 A CN107286215 A CN 107286215A CN 201610204468 A CN201610204468 A CN 201610204468A CN 107286215 A CN107286215 A CN 107286215A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J7/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms
- C07J7/0005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21
- C07J7/001—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21 substituted in position 20 by a keto group
- C07J7/0015—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21 substituted in position 20 by a keto group not substituted in position 17 alfa
- C07J7/002—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21 substituted in position 20 by a keto group not substituted in position 17 alfa not substituted in position 16
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12P33/02—Dehydrogenating; Dehydroxylating
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Abstract
The present invention relates to the preparation method of Steroid medicine intermediates, more particularly to androstane-14 alkene 3,17 diketones are that substrate prepares a kind of pregnant steroid 1 of pregna medicament intermediate, 4,9 (11), the diketone of 16 (17) tetraene 3,20, pregnant steroid 4, the method of the α hydroxyls of 93,20 diketone of (11) diene 17.
Description
Technical field：
The present invention relates to the preparation method of Steroid medicine intermediates, more particularly to a kind of pregnant steroid of pregna medicament intermediate
- Isosorbide-5-Nitrae, 9 (11), 16 (17)-tetraene -3,20- diketone, pregnant steroid -4,9 (11)-diene -3,20- diketone
- 17 alpha-hydroxy preparation methods.
Background technology：
Glucocorticoid is the sterid of adrenocortical secretion, the effect of its physiological show to sugar,
Protein, fat, the influence of water and electrolyte.It can suppress bradykinin, prostaglandin E2, serotonin,
The synthesis and release of the inflammatory mediators such as histamine, reduction vasopermeability, stable lysosome membrane, suppression phagocytosis.
Have very strong antiinflammatory action to inflammation caused by a variety of causes, the oozing out of the early stage that can reduce inflammation, oedema,
Telangiectasis, leukocyte infiltration and phagocytosis reaction, can suppress inflammation later stage capillary and fibroblast
Hyperplasia, suppresses the pathological change that immune response is produced, releases the symptom of the disease.
Pregnant steroid-Isosorbide-5-Nitrae, 9 (11), 16 (17)-tetraene -3,20- diketone, pregnant steroid -4,9 (11)-diene -3,
The Alpha-hydroxy of 20- diketones -17 is pregna medicament important intermediate, can prepare glucocorticoid, for example
Just described in CN200710061254.4 with pregnant steroid-Isosorbide-5-Nitrae, 9 (11), 16 (17)-tetraene -3,20- is double
Ketone is the method that substrate prepares betamethasone and its series of products.
In order to preferably prepare pregnant steroid-Isosorbide-5-Nitrae, 9 (11), 16 (17)-tetraene -3,20- diketone, researcher
Take a variety of methods.
CN201010278556.9 prepares 5ST by mould dehydrogen substance and is divided into two-step reaction：The first step takes off
Water obtains methyl dehydrate, and second step is through reducing to obtain 5ST (5ST).
CN201510090371.8 is disclosed with androstane-4-alkene-3, and the Alpha-hydroxy of 17- diketones -9 is substrate, by de-
Water, it is etherified, connects side chain, biological dehydrogenation and obtain pregnant steroid-Isosorbide-5-Nitrae, 9 (11), 16 (17)-tetraene -3,20-
Diketone.
The content of the invention
The present invention relates to the preparation method of Steroid medicine intermediates, more particularly to a kind of pregnant steroid of pregna medicament intermediate
- Isosorbide-5-Nitrae, 9 (11), 16 (17)-tetraene -3,20- diketone, pregnant steroid -4,9 (11)-diene -3,20- diketone
- 17 alpha-hydroxy preparation methods.
We are by constantly research, it has surprisingly been found that by optimizing reaction scheme, adjusting process, Ke Yixiong
Gona-4-ene-3,17- diketones are substrate, should obtain pregnant by cyaniding, ketal, grignard, biological oxidation, dehydration
Steroid -4,9 (the 11)-Alpha-hydroxy of diene -3,20- diketone -17, then by biological dehydrogenation, dehydration, obtain pregnant
Steroid-Isosorbide-5-Nitrae, 9 (11), 16 (17)-tetraene -3,20- diketone.
A kind of preparation method of the compound of formula 2, it is characterized in that：
Step 1：
The alcohol and inorganic base aqueous solution of 1-4 carbon are stirred, the compound of formula 3 is put into wherein, stirring
Uniformly.The acetone cyanohydrin added more than 2 times of mol ratios, control temperature stirring reaction between 25-40 DEG C is straight
To without raw material.Reaction solution is diluted into water again, with inorganic acid and dilution is neutral to pH value；Filtering
Obtain the compound of formula 4；
Step 2：
The compound of formula 4 is put into ethylene glycol, p-methyl benzenesulfonic acid, triethyl orthoformate is sequentially added, risen
Temperature is to 30-60 DEG C of reaction until without raw material, adding appropriate organic base and neutralizing, be then diluted into reaction solution
In water, filtering obtains the compound of Betamethasone Ketal structures formula 5；
Step 3：
The ether that the dry compound of formula 5 and vinyl butyl ether are put into the 2-6 carbon containing toluenesulfonic acid is molten
In liquid, stirring reaction is extremely without raw material, the intermediate reacted；Be added dropwise to again RMgBr-methyl-magnesium-chloride/
Tetrahydrofuran, after being added dropwise to complete, is stirred at room temperature reaction to without intermediate.It is slow with saturated aqueous ammonium chloride again
Grignard reagent is quenched in dropwise addition, and the pH value for then adjusting reaction solution with inorganic acid is less than 3.Heating response liquid is extremely
40-70 DEG C is incubated 2 hours, after be cooled to 5-10 DEG C, adjust pH to neutrality with inorganic base aqueous solution, concentration,
Dilution, filtering, obtains the compound of formula 6；
Step 4
The compound of formula 6 is crushed or dissolved with the alcohol of 1-4 carbon, input is entered in the fermentation medium containing mould
Row biofermentation, obtains the compound of formula 8.
Step 5
The compound of formula 8 is added in one or more of organic solvents in ether, acetonitrile in 2-6 carbon, is cooled to
- 90 to -50 DEG C are reacted with the one or more in phosphorus trichloride, phosphorus pentachloride, POCl3, reaction to raw material
After 5%, it is diluted to in water, adjusting pH value to neutrality with inorganic base, being filtrated to get the chemical combination of formula 1
Thing.
Step 6
The compound of formula 1 is crushed or dissolved with the alcohol of 1-4 carbon, input has cultivated the fermentation of Arthrobacter simplex
Bioconversion is carried out in tank, compound 7 is obtained.
Step 7：
Compound 7 is put into acetic acid, the semicarbazide hydrochloride aqueous solution added, temperature reaction is until without original
Material, is cooled to room temperature, is diluted into water, filters, obtains compound 2；
In compound 8It is α or β to represent the oh group,
4 in the compound of formula 5,5 and the 5, dotted line of 6 represent 4,5 or 5,6 there is double bond.
A kind of preparation method of the compound of formula 1, it is characterized in that：
Step 1：
The alcohol and inorganic base aqueous solution of 1-4 carbon are stirred, the compound of formula 3 is put into wherein, stirring
Uniformly.The acetone cyanohydrin added more than 2 times of mol ratios, control temperature stirring reaction between 25-40 DEG C is straight
To without raw material.Reaction solution is diluted into water again, with inorganic acid and dilution is neutral to pH value；Filtering
Obtain the compound of formula 4；
Step 2：
The compound of formula 4 is put into ethylene glycol, p-methyl benzenesulfonic acid, triethyl orthoformate is sequentially added, risen
Temperature is to 30-60 DEG C of reaction until without raw material, adding appropriate organic base and neutralizing, be then diluted into reaction solution
In water, filtering obtains the compound of Betamethasone Ketal structures formula 5；
Step 3：
The ether that the dry compound of formula 5 and vinyl butyl ether are put into the 2-6 carbon containing toluenesulfonic acid is molten
In liquid, stirring reaction is extremely without raw material, the intermediate reacted；Be added dropwise to again RMgBr-methyl-magnesium-chloride/
Tetrahydrofuran, after being added dropwise to complete, is stirred at room temperature reaction to without intermediate.It is slow with saturated aqueous ammonium chloride again
Grignard reagent is quenched in dropwise addition, and the pH value for then adjusting reaction solution with inorganic acid is less than 3.Heating response liquid is extremely
40-70 DEG C is incubated 2 hours, after be cooled to 5-10 DEG C, adjust pH to neutrality with inorganic base aqueous solution, concentration,
Dilution, filtering, obtains the compound of formula 6；
Step 4
The compound of formula 6 is crushed or dissolved with the alcohol of 1-4 carbon, input is entered in the fermentation medium containing mould
Row biofermentation, obtains the compound of formula 8.
Step 5
The compound of formula 8 is added in one or more of organic solvents in ether, acetonitrile in 2-6 carbon, is cooled to
- 90 to -50 DEG C are reacted with the one or more in phosphorus trichloride, phosphorus pentachloride, POCl3, reaction to raw material
After 5%, it is diluted to in water, adjusting pH value to neutrality with inorganic base, being filtrated to get the chemical combination of formula 1
Thing,
4 in the compound of formula 5,5 and the 5, dotted line of 6 represent 4,5 or 5,6 there is double bond,
In compound 8It is α or β to represent the oh group.
Preparation method as described above, it is characterized in that the alcohol of the 1-4 carbon is methanol or ethanol.
Preparation method as described above, it is characterized in that inorganic base is one in NaOH, KOH in the step 1
Plant or several, inorganic base aqueous solution concentration is 30-60%, the compound of formula 4 and inorganic base aqueous solution weighing body
Product is than being 1:0.01-0.1.
Preparation method as described above, it is characterized in that p-methyl benzenesulfonic acid is catalytic amount, formula 5 in the step 2
The w/v of compound and triethyl orthoformate is 1:One in 0.7-2, the non-pyridine of organic base or triethylamine
Kind.
Preparation method as described above, it is characterized in that the compound of step 3 Chinese style 6 and vinyl butyl ether
W/v be 1:0.5-1, the ethereal solution concentration of 2-6 carbon of toluenesulfonic acid is 0.01-1%, 2-6
The ether of carbon is ether, tetrahydrofuran, 1,2- dioxane, ethyl methyl ether, dimethyl ether, and inorganic acid is hydrochloric acid, nothing
Machine alkali is NaOH, KOH, Na2CO3、NaHCO3、K2CO3、KHCO3In one or more.
Preparation method as described above, it is characterized in that mould is rhizopus niger, green stiff mould, wood in step 4
One or more in mould, Aspergillus ochraceus bacterium, Aspergillus flavus.
Preparation method as described above, it is characterized in that mould is rhizopus niger in step 4.
Preparation method as described above, it is characterized in that mould is China General Microbiological culture presevation in step 4
Administrative center CGMCC numberings 3.235,3.904 rhizopus niger.
Preparation method as described above, it is characterized in that g/L w/vs are phosphorus in slant medium in step 4
Sour hydrogen dipotassium 0.05, magnesium sulfate 0.05, ferric citrate 0.005, citric acid 2, ammonium nitrate 2, calcium carbonate
10, glycerine 20, glucose 5, agar 15.
Preparation method as described above, it is characterized in that g/L w/vs are jade in fermentation medium in step 4
Ground rice 3~8, glucose 10~25, potassium chloride 0.02~0.15, potassium nitrate 0.5~3.0, potassium dihydrogen phosphate
0.5~2.5, dipotassium hydrogen phosphate 1.0~4.0, borate 0.1~10.
Preparation method as described above, it is characterized in that the compound of step 5 Chinese style 8 and phosphorus trichloride, phosphoric
The weight ratio of phosphorus or POCl3 is 1:0.8-2.
The preparation method of the compound of formula 2 as described above, it is characterized in that in step 6 compound of dissolution type 1 solvent
One or more in methanol, ethanol, tetrahydrofuran, dioxane, DMF,
Arthrobacter simplex for AS 1.754, AS 1.94*.
The preparation method of the compound of formula 2 as described above, it is characterized in that slant medium is adopted in step 6 bioconversion
With following proportioning：The distilled water of glucose 1.3%, yeast extract 1.3%, agar 2.0%, and surplus, with nothing
Machine aqueous slkali is adjusted to pH7.0-7.2, for inclined-plane culture.
The preparation method of the compound of formula 2 as described above, it is characterized in that slant medium is adopted in step 6 bioconversion
With following proportioning：Yeast extract 0.5%, peptone 0.5%, grape wards off 1%, and agar 2%, running water is prepared,
PH to 7.0-7.2 is adjusted to inorganic alkali solution, for inclined-plane culture.
The preparation method of the compound of formula 2 as described above, it is characterized in that fermentation medium is in step 6 bioconversion
Match below：Glucose 1.0, yeast extract 0.16%, KH2PO40.25%, corn steep liquor 0.1%, and surplus
Distilled water, pH 7.0-7.2 are adjusted to inorganic alkali solution.
The preparation method of the compound of formula 2 as described above, it is characterized in that the water-soluble liquid hold-up of semicarbazide hydrochloride in step 7
For 3-10%, temperature reaction temperature is more than 60 DEG C.
Described bio-fermentation process can also such as be referred to using any known bio-fermentation process method《It is biological
Synthetic pharmacology》(Chemical Industry Press, publishes for 2000, Chu Zhiyi chief editors；Page 666~675) in it is public
The bio-fermentation process opened.
The feed concentrations of substrate are≤4% (relative to zymotic fluid volume) in step 4,6；It is preferred that feed concentrations
For 1%~3%, 30~34 DEG C of fermentation temperature, preferably chaotropic agent addition are 2~5% (v/v).Biology is de-
30~72 hours hydrogen reaction time.
Described biological respinse can use following zymotechnique：
Inclined-plane culture-one-level culture-one-level culture-addition compound (1) fermentation carries out biological de-
Hydrogen reaction-terminating reaction.
Step 4,6 biological respinses terminate rear terminating reaction, it is preferred to use zymotic fluid is warming up to 70~90 DEG C and
Dehydrogenation is extracted after the method for inactivating thalline, terminating reaction with the form for being preferred to use solvent extraction zymotic fluid to produce
Thing.The extraction solvent ethyl acetate, dichloromethane, chloroform.
The mould that step 4 biological oxidation is used be rhizopus niger (Rhizopus nigricans), green stiff mould,
Trichoderma, Aspergillus ochraceus bacterium, Aspergillus flavus.In the preferred China General Microbiological culture presevation management of rhizopus niger
Heart CGMCC numberings 3.235,3.904.
Culture medium used in the biological oxidation of the step 4 is preferred to use following proportioning：
G/L w/vs are dipotassium hydrogen phosphate 0.05, magnesium sulfate 0.05, lemon in slant medium in step 4
Lemon acid iron ammonium 0.005, citric acid 2, ammonium nitrate 2, calcium carbonate 10, glycerine 20, glucose 5, agar 15.
G/L w/vs are corn flour 3~8, glucose 10~25, chlorination in fermentation medium in step 4
Potassium 0.02~0.15, potassium nitrate 0.5~3.0, potassium dihydrogen phosphate 0.5~2.5, dipotassium hydrogen phosphate 1.0~4.0, boron
Hydrochlorate 0.1~10.
Step 6 biological dehydrogenation uses Arthrobacter simplex (Arthrobacter simplex) preferably following several
Plant bacterial strain：AS 1.754, AS 1.94* (being provided by Institute of Microorganism, Academia Sinica).
Culture medium used in the biological dehydrogenation of the step 6 is preferred to use following proportioning：
Slant medium (%)：The distilled water of glucose 1.3, yeast extract 1.3, agar 2.0, and surplus, pH7.0-7.2,
For inclined-plane culture
Fermentation medium (%)：Glucose 1.0, yeast extract 0.16, KH2PO40.25, corn steep liquor 0.1, Yi Jiyu
The distilled water of amount, pH 7.0-7.2, in one-level, two grades of cultures and final biological dehydrogenation reaction.
Embodiment
HPLC：High performance liquid chromatography
Embodiment 1
Embodiment 1-1
The compound of 10g formulas 3 is put into 30ml methanol and the 1ml 60%KOH aqueous solution, stirred.Again plus
Enter 8ml acetone cyanohydrin, stirring reaction to raw material point disappears.Reaction solution is diluted into 150ml water again, used
With dilution to neutrality in watery hydrochloric acid.Filtering, washing.Dry, obtain the compound 9.7g of formula 4.
Embodiment 1-2
The compound of 10g formulas 3 is put into 20ml methanol and the 0.5ml 50%KOH aqueous solution, stirred.Again
15ml acetone cyanohydrin is added, stirring reaction to raw material point disappears.Reaction solution is diluted into 300ml water again,
With in watery hydrochloric acid and dilution is to neutrality.Filtering, washing.Dry, obtain the compound 10.4g of formula 4.
Embodiment 1-3
The compound of 10g formulas 3 is put into 15ml methanol and the sodium hydrate aqueous solutions of 0.2ml 40%, stirred.
20ml acetone cyanohydrin is added, stirring reaction to raw material point disappears.Reaction solution is diluted into 300ml water again,
With in watery hydrochloric acid and dilution is to neutrality.Filtering, washing.Dry, obtain the compound 10.3g of formula 4.
Embodiment 1-4
The compound of 10g formulas 3 is put into 10ml methanol and the sodium hydrate aqueous solutions of 0.1ml 30%, stirred.
25ml acetone cyanohydrin is added, stirring reaction to raw material point disappears.Reaction solution is diluted into 200ml water again,
With in watery hydrochloric acid and dilution is to neutrality.Filtering, washing.Dry, obtain the compound 10.1g of formula 4.
Embodiment 2
Embodiment 2-1 (this step reaction yield is relatively low, may be set to 105%-110%)
The compound of 10g formulas 4 is put into 15ml ethylene glycol, 0.1g p-methyl benzenesulfonic acid, 20ml is sequentially added
Triethyl orthoformate, is warming up to 55-60 DEG C of reaction to raw material point and disappears.Appropriate pyridine is added to neutralize.Then will
Reaction solution is diluted into 100ml water, is filtered, washing.Dry, obtain the compound 10.4g of formula 5.
Embodiment 2-2
The compound of 10g formulas 4 is put into 20ml ethylene glycol, 0.2g p-methyl benzenesulfonic acid, 15ml is sequentially added
Triethyl orthoformate, is warming up to 50-55 DEG C of reaction to raw material point and disappears.Appropriate pyridine is added to neutralize.Then will
Reaction solution is diluted into 150ml water, is filtered, washing.Dry, obtain the compound 10.5g of formula 5.
Embodiment 2-3
The compound of 10g formulas 4 is put into 25ml ethylene glycol, 0.2g p-methyl benzenesulfonic acid, 10ml is sequentially added
Triethyl orthoformate, is warming up to 40-45 DEG C of reaction to raw material point and disappears.Appropriate pyridine is added to neutralize.Then will
Reaction solution is diluted into 200ml water, is filtered, washing.Dry, obtain the compound 10.8g of formula 5.
Embodiment 2-4
The compound of 10g formulas 4 is put into 30ml ethylene glycol, 0.3g p-methyl benzenesulfonic acid, 20ml is sequentially added
Triethyl orthoformate, is warming up to 30-35 DEG C of reaction to raw material point and disappears.Appropriate pyridine is added to neutralize.Then will
Reaction solution is diluted into 200ml water, is filtered, washing.Dry, obtain the compound 10.7g of formula 5.
Embodiment 3
Embodiment 3-1
Formula 5 compound 10g and 5ml vinyl butyl ether are put into 50ml and contain 0.01% pair of toluene sulphur
In the tetrahydrofuran solution of acid, stirring reaction is extremely without raw material, the intermediate reacted.
It is added dropwise in 30ml 3M RMgBr methyl-magnesium-chloride/tetrahydrofuran, after being added dropwise to complete, stirs again
Reaction is mixed to without intermediate.It is slowly added dropwise again with saturated aqueous ammonium chloride and RMgBr is quenched, then uses dense salt
Acid adjusts reaction solution pH to 1-2.Heating response liquid is incubated 1 hour to 60-70 DEG C, then is cooled to 15-20 DEG C,
Adjust pH to neutrality with 10% sodium hydrate aqueous solution, concentration removes tetrahydrofuran.It will be diluted at concentration residual night
In 100ml water.Filtering, washing.DEG C dry, obtain the compound 8.3g of formula 6.
Embodiment 3-2
Formula 5 compound 10g and 7.5ml vinyl butyl ether are put into 40ml and contain 0.1% pair of toluene sulphur
In the diethyl ether solution of acid, stirring reaction is extremely without raw material, the intermediate reacted.
It is added dropwise in 35ml 3M RMgBr methyl-magnesium-chloride/tetrahydrofuran, after being added dropwise to complete, stirs again
Reaction is mixed to without intermediate.It is slowly added dropwise again with saturated aqueous ammonium chloride and RMgBr is quenched, then uses dense salt
Acid adjusts reaction solution pH to 1-2.Heating response liquid is incubated 2 hours to 50-60 DEG C, then is cooled to 10-15 DEG C,
Adjust pH to neutrality with 20% wet chemical, concentration removes tetrahydrofuran.Concentration raffinate is diluted into 150ml
In water.Filtering, washing.DEG C dry, obtain the compound 8.6g of formula 6.
Embodiment 3-3
Formula 5 compound 10g and 9ml vinyl butyl ether are put into 30ml and contain 0.1% pair of toluene sulphur
In 1, the 2- dioxane solutions of acid, stirring reaction is extremely without raw material, the intermediate reacted.
It is added dropwise in 40ml 3M RMgBr methyl-magnesium-chloride/tetrahydrofuran, after being added dropwise to complete, stirs again
Reaction is mixed to without intermediate.It is slowly added dropwise again with saturated aqueous ammonium chloride and RMgBr is quenched, then uses dense salt
Acid adjusts reaction solution pH to 1-2.Heating response liquid is incubated 4 hours to 45-55 DEG C, then is cooled to 15-20 DEG C,
Adjust pH to neutrality with 30% sodium bicarbonate aqueous solution, concentration removes tetrahydrofuran.It will be diluted at concentration residual night
In 200ml water.Filtering, washing.DEG C dry, obtain the compound 8.4g of formula 6.
Embodiment 4
Embodiment 4-1
Rhizopus niger (CGMCC numberings 3.235) is carried out to inclined-plane culture, one-level culture and two grades of trainings successively
Support, cultivation temperature is 27-30 DEG C, the compound of 20g formulas 6 is dissolved in 20ml tetrahydrofurans input 5L fermentation tanks
In, feed concentrations are 2%, and reaction temperature is 28-31 DEG C.Until the compound of formula 6 residue is less than 5%, reaction
After the completion of be warming up to 70 DEG C of terminating reactions, adopt and extraction zymotic fluid be extracted with ethyl acetate, organic phase is concentrated,
Recrystallizing methanol obtains the compound 19.2g of formula 8.
Embodiment 4-2
Rhizopus niger (CGMCC numberings 3.904) is carried out to inclined-plane culture, one-level culture and two grades of trainings successively
Support, cultivation temperature is 27-30 DEG C, the compound of 20g formulas 6 be dissolved in 30ml methanol input 5L fermentation tanks,
Feed concentrations are 3%, and reaction temperature is 29-32 DEG C, until the compound of formula 6 residue is less than 5%, reaction is completed
After be warming up to 70 DEG C of terminating reactions, using chloroform extraction extract zymotic fluid, organic phase is concentrated, methanol is tied again
Crystalline substance obtains the compound 20.1g of formula 8.
Embodiment 4-3
Rhizopus niger (CGMCC numberings 3.904) is carried out to inclined-plane culture, one-level culture and two grades of trainings successively
Support, cultivation temperature is 27-30 DEG C, the compound of 20g formulas 6 is dissolved in 40mlN, dinethylformamide input
In 5L fermentation tanks, feed concentrations are 4%, and reaction temperature is 26-29 DEG C, until the compound residue of formula 6 is less than
5%, 70 DEG C of terminating reactions are warming up to after the completion of reaction, zymotic fluid is extracted using dichloromethane extraction, will be organic
Mutually concentrate, recrystallizing methanol obtains the compound 18.4g of formula 7.
Embodiment 5
Embodiment 5-1
The compound 10g of formula 8 is put into 50ml ether, -90 DEG C are cooled to, 8g phosphorus trichlorides are added,
Stirring reaction to raw material is less than after 5%, is diluted to in water, pH value is adjusted to neutrality with sodium hydroxide,
It is filtrated to get the compound 8.4g of formula 1.
Embodiment 5-2
The compound 10g of formula 8 is put into 50ml tetrahydrofurans, -70 DEG C are cooled to, 10g is added phosphoric
Phosphorus, stirring reaction to raw material is less than after 5%, is diluted to in water, pH value is adjusted into saleratus
Property, it is filtrated to get the compound 9.1g of formula 1.
Embodiment 5-3
The compound 10g of formula 8 is put into 50ml acetonitriles, -50 DEG C are cooled to, 10g POCl3s are added,
Stirring reaction to raw material is less than after 5%, is diluted to in water, pH value is adjusted to neutrality, mistake with sodium carbonate
Filter obtains the compound 8.2g of formula 1.
Embodiment 6
Embodiment 6-1
Arthrobacter simplex (AS 1.754) is carried out to inclined-plane culture, one-level culture and two grades of cultures, culture successively
Temperature is 31-33 DEG C, and the compound of 20g formulas 6 is dissolved in 20ml tetrahydrofurans input 5L fermentation tanks, fed intake
Concentration is 2%, and reaction temperature is 31 DEG C.Until the compound of formula 6 residue is less than 5%, heated up after the completion of reaction
To 70 DEG C of terminating reactions, adopt and extraction zymotic fluid is extracted with ethyl acetate, organic phase is concentrated, recrystallizing methanol
Obtain the compound 17.4g of formula 7.
Embodiment 6-2
Arthrobacter simplex (AS 1.94*) is carried out to inclined-plane culture, one-level culture and two grades of cultures, culture successively
Temperature is 29-32 DEG C, the compound of 20g formulas 6 is dissolved in 30ml methanol input 5L fermentation tanks, feed concentrations
For 3%, reaction temperature is 32 DEG C, until the compound of formula 6 residue is less than 5%, 70 DEG C are warming up to after the completion of reaction
Terminating reaction, extracts zymotic fluid using chloroform extraction, organic phase is concentrated, recrystallizing methanol obtains the chemical combination of formula 7
Thing 18.3g.
Embodiment 6-3
Arthrobacter simplex (AS 1.754) is carried out to inclined-plane culture, one-level culture and two grades of cultures, culture successively
Temperature is 30-32 DEG C, and the compound of 20g formulas 6 is dissolved in into 40mlN, dinethylformamide input 5L fermentations
In tank, feed concentrations are 4%, and reaction temperature is 32 DEG C, until the compound of formula 6 residue is less than 5%, reaction
After the completion of be warming up to 70 DEG C of terminating reactions, using dichloromethane extraction extract zymotic fluid, organic phase is concentrated,
Recrystallizing methanol obtains the compound 18.5g of formula 7.
Embodiment 7
Embodiment 7-1
The compound of 10g formulas 7 is put into 50ml acetic acid, the 10% semicarbazide hydrochloride aqueous solution is added.
Reaction mixture is heated to 60-65 DEG C of reaction to without raw material point.Room temperature is cooled to, 200ml water is diluted into
In, filtering obtains the compound of 8.9g formulas 2.
Embodiment 7-2
The compound of 10g formulas 7 is put into 100ml acetic acid, the 8% semicarbazide hydrochloride aqueous solution is added.
Reaction mixture is heated to 70-75 DEG C of reaction to without raw material point.Room temperature is cooled to, 400ml water is diluted into
In, filtering obtains the compound of 9.1g formulas 2.
Embodiment 7-3
The compound of 10g formulas 7 is put into 100ml acetic acid, the 5% semicarbazide hydrochloride aqueous solution is added.
Reaction mixture is heated to 80-85 DEG C of reaction to without raw material point.Room temperature is cooled to, 400ml water is diluted into
In, filtering obtains the compound of 9.2g formulas 2.
Embodiment 7-4
The compound of 10g formulas 7 is put into 150ml acetic acid, the 3% semicarbazide hydrochloride aqueous solution is added.
Reaction mixture is heated to 85-90 DEG C of reaction to without raw material point.Room temperature is cooled to, 500ml water is diluted into
In, filtering obtains the compound of 9.2g formulas 2.
Claims (10)
1. a kind of preparation method of the compound of formula 2, it is characterized in that：
Step 1：
The alcohol and inorganic base aqueous solution of 1-4 carbon are stirred, the compound of formula 3 is put into wherein, stirring
Uniformly.The acetone cyanohydrin added more than 2 times of mol ratios, control temperature stirring reaction between 25-40 DEG C is straight
To without raw material.Reaction solution is diluted into water again, with inorganic acid and dilution is neutral to pH value；Filtering
Obtain the compound of formula 4；
Step 2：
The compound of formula 4 is put into ethylene glycol, p-methyl benzenesulfonic acid, triethyl orthoformate is sequentially added, risen
Temperature is to 30-60 DEG C of reaction until without raw material, adding appropriate organic base and neutralizing, be then diluted into reaction solution
In water, filtering obtains the compound of Betamethasone Ketal structures formula 5；
Step 3：
The ether that the dry compound of formula 5 and vinyl butyl ether are put into the 2-6 carbon containing toluenesulfonic acid is molten
In liquid, stirring reaction is extremely without raw material, the intermediate reacted；Be added dropwise to again RMgBr-methyl-magnesium-chloride/
Tetrahydrofuran, after being added dropwise to complete, is stirred at room temperature reaction to without intermediate.It is slow with saturated aqueous ammonium chloride again
Grignard reagent is quenched in dropwise addition, and the pH value for then adjusting reaction solution with inorganic acid is less than 3.Heating response liquid is extremely
40-70 DEG C is incubated 2 hours, after be cooled to 5-10 DEG C, adjust pH to neutrality with inorganic base aqueous solution, concentration,
Dilution, filtering, obtains the compound of formula 6；
Step 4
The compound of formula 6 is crushed or dissolved with the alcohol of 1-4 carbon, input is entered in the fermentation medium containing mould
Row biofermentation, obtains the compound of formula 8.
Step 5
The compound of formula 8 is added in one or more of organic solvents in ether, acetonitrile in 2-6 carbon, is cooled to
- 90 to -50 DEG C are reacted with the one or more in phosphorus trichloride, phosphorus pentachloride, POCl3, reaction to raw material
After 5%, it is diluted to in water, adjusting pH value to neutrality with inorganic base, being filtrated to get the chemical combination of formula 1
Thing.
Step 6
The compound of formula 1 is crushed or dissolved with the alcohol of 1-4 carbon, input has cultivated the fermentation of Arthrobacter simplex
Bioconversion is carried out in tank, compound 7 is obtained.
Step 7：
Compound 7 is put into acetic acid, the semicarbazide hydrochloride aqueous solution added, temperature reaction is until without original
Material, is cooled to room temperature, is diluted into water, filters, obtains compound 2；
In compound 8It is α or β to represent the oh group,
4 in the compound of formula 5,5 and the 5, dotted line of 6 represent 4,5 or 5,6 there is double bond.
2. a kind of preparation method of the compound of formula 1, it is characterized in that：
Step 1：
The alcohol and inorganic base aqueous solution of 1-4 carbon are stirred, the compound of formula 3 is put into wherein, stirring
Uniformly.The acetone cyanohydrin added more than 2 times of mol ratios, control temperature stirring reaction between 25-40 DEG C is straight
To without raw material.Reaction solution is diluted into water again, with inorganic acid and dilution is neutral to pH value；Filtering
Obtain the compound of formula 4；
Step 2：
The compound of formula 4 is put into ethylene glycol, p-methyl benzenesulfonic acid, triethyl orthoformate is sequentially added, risen
Temperature is to 30-60 DEG C of reaction until without raw material, adding appropriate organic base and neutralizing, be then diluted into reaction solution
In water, filtering obtains the compound of Betamethasone Ketal structures formula 5；
Step 3：
The ether that the dry compound of formula 5 and vinyl butyl ether are put into the 2-6 carbon containing toluenesulfonic acid is molten
In liquid, stirring reaction is extremely without raw material, the intermediate reacted；Be added dropwise to again RMgBr-methyl-magnesium-chloride/
Tetrahydrofuran, after being added dropwise to complete, is stirred at room temperature reaction to without intermediate.It is slow with saturated aqueous ammonium chloride again
Grignard reagent is quenched in dropwise addition, and the pH value for then adjusting reaction solution with inorganic acid is less than 3.Heating response liquid is extremely
40-70 DEG C is incubated 2 hours, after be cooled to 5-10 DEG C, adjust pH to neutrality with inorganic base aqueous solution, concentration,
Dilution, filtering, obtains the compound of formula 6；
Step 4
The compound of formula 6 is crushed or dissolved with the alcohol of 1-4 carbon, input is entered in the fermentation medium containing mould
Row biofermentation, obtains the compound of formula 8.
Step 5
The compound of formula 8 is added in one or more of organic solvents in ether, acetonitrile in 2-6 carbon, is cooled to
- 90 to -50 DEG C are reacted with the one or more in phosphorus trichloride, phosphorus pentachloride, POCl3, reaction to raw material
After 5%, it is diluted to in water, adjusting pH value to neutrality with inorganic base, being filtrated to get the chemical combination of formula 1
Thing,
4 in the compound of formula 5,5 and the 5, dotted line of 6 represent 4,5 or 5,6 there is double bond,
In compound 8It is α or β to represent the oh group.
3. the preparation method as described in claim 1,2, it is characterized in that in the step 1 inorganic base be NaOH,
One or more in KOH, inorganic base aqueous solution concentration is 30-60%, and the compound of formula 4 and inorganic base are water-soluble
The w/v of liquid is 1:0.01-0.1.
4. the preparation method as described in claim 1,2, it is characterized in that p-methyl benzenesulfonic acid is in the step 2
The w/v of catalytic amount, the compound of formula 5 and triethyl orthoformate is 1:0.7-2, organic base is pyridine
Or one kind in triethylamine.
5. the preparation method as described in claim 1,2, it is characterized in that the compound of step 3 Chinese style 6 with
The w/v of vinyl butyl ether is 1:0.5-1, the ethereal solution concentration of 2-6 carbon of toluenesulfonic acid is
The ether of 0.01-1%, 2-6 carbon is ether, tetrahydrofuran, 1,2- dioxane, ethyl methyl ether, dimethyl ether, nothing
Machine acid is hydrochloric acid, and inorganic base is NaOH, KOH, Na2CO3、NaHCO3、K2CO3、KHCO3In
It is one or more of.
6. the preparation method as described in claim 1,2, it is characterized in that in step 4 mould be rhizopus niger,
One or more in green stiff mould, Trichoderma, Aspergillus ochraceus bacterium, Aspergillus flavus.
7. the preparation method as described in claim 1,2, it is characterized in that g/L in fermentation medium in step 4
W/v be corn flour 3~8, glucose 10~25, potassium chloride 0.02~0.15, potassium nitrate 0.5~3.0,
Potassium dihydrogen phosphate 0.5~2.5, dipotassium hydrogen phosphate 1.0~4.0, borate 0.1~10.
8. the preparation method as described in claim 1,2, it is characterized in that the compound of step 5 Chinese style 8 and trichlorine
The weight ratio for changing phosphorus, phosphorus pentachloride or POCl3 is 1:0.8-2.
9. preparation method as claimed in claim 1, it is characterized in that in step 6 compound of dissolution type 1 it is molten
One or more of the agent in methanol, ethanol, tetrahydrofuran, dioxane, DMF,
Arthrobacter simplex for AS 1.754, AS 1.94*.
10. preparation method as claimed in claim 1, it is characterized in that the semicarbazide hydrochloride aqueous solution contains in step 7
Measure as 3-10%, temperature reaction temperature is more than 60 DEG C.
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