CN107338281A - The method for preparing methylprednisolone - Google Patents
The method for preparing methylprednisolone Download PDFInfo
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Abstract
The invention discloses a kind of method for preparing methylprednisolone; using hydrocortisone as raw material; hydroxyl is protected by ketal reaction; again 6 methyl are introduced through methylenation and catalytic hydrogenation; hydrolysis deprotection obtains 6 α hydrogenated methyl cortisones; biological dehydrogenation obtains methylprednisolone, and reaction equation is as follows：
Description
Technical field
The present invention relates to a kind of preparation method of chemicals, and in particular to a kind of method for preparing methylprednisolone.
Background technology
Methylprednisolone belongs to glucocorticoids medicine, the first aid for critical illness, it may also be used for endocrinopathy,
Rheumatic disease, collagen venereal disease, disease of skin, allergic reaction, ophthalmology disease, enterogastric diseases, hematologic disease, leukaemia, stop
Gram, encephaledema, polyneuritis, myelitis and prevent vomiting etc. caused by cancer chemotherapy.Clinically it is mainly used at present dirty
Device is transplanted.
Patent of invention CN 1763066A are disclosed using the pregnant steroid diene alcohol ketone of acetic acid as initiation material, anti-through epoxidation, walsh
Should, mould reaction, Pu Shi reactions, upper debrominate, upper iodization, 11 reduction reactions, 1-2 dehydrogenation reactions, 6 methylate, hydrolysis
Methylprednisolone is obtained, reaction equation is as follows：
Patent of invention CN 101230084A are disclosed using mold oxide as initiation material, reacted successively through Pu Shi, upper debrominate, 6
Precedence methylates, 6 methylate, ketal protection, 11 reduction, 1-2 dehydrogenation reactions, upper iodizations, hydrolysis obtains the bold and vigorous Buddhist nun of methyl
Song Long, reaction equation are as follows：
Patent of invention CN 101418029A are disclosed the β of 6 Alpha-Methyl -11, the pregnant steroid -1,4- diene -3,20- two of 17 alpha-dihydroxys
Ketone in a solvent, reacts, then separation of solid and liquid with brominated reagent under ammonium salt in catalysis agent catalysis, collects liquid phase, fling to solvent, so
Afterwards by it in solvent, react in the presence of the phase transfer catalyst of acetate and catalytic amount, then collected from reaction solution
Product, then in the presence of a basic, hydrolysis obtains methylprednisolone, and reaction equation is as follows：
Starting material in existing route, all it is that expensive, technique is multiple using mould dehydrogen substance as raw material or key intermediate
It is miscellaneous, complex steps.
The content of the invention
Present invention aim to address the production technology of current methylprednisolone be all using mould dehydrogen substance as raw material or
Key intermediate, expensive, complex process, the technical problem of complex steps.
In order to solve the above technical problems, the present invention provides a kind of method for preparing methylprednisolone, comprise the following steps：
（1）Ketal protection reaction：Hydrocortisone 1 is dissolved in dichloromethane, stirs lower addition formalin, slowly drop
Enriching hydrochloric acid, the stirring reaction certain time under certain temperature, TLC detect no raw material point, stop reaction, separate organic phase,
Aqueous phase is extracted with dichloromethane, merges organic phase；Organic phase is washed with saturated sodium bicarbonate aqueous solution, uses saturated sodium-chloride successively
The aqueous solution is washed, dried with anhydrous magnesium sulfate, and vacuum rotary steam removes organic solvent, obtains protecting product 2a and 2b mixing
Thing, reaction equation are
（2）Methylenation：Sodium acetate, dichloromethane and dimethoxym ethane are added in reaction bulb, is slowly added dropwise under stirring
POCl3, certain temperature is heated under nitrogen protection, is reacted 1 hour at this temperature；Upper protection product 2a and 2b mixture is dissolved in
Dichloromethane, on this dichloromethane solution of product will be protected to be slowly added dropwise to reaction solution, then POCl is slowly added dropwise3, reaction one
Fix time, TLC detects no raw material point, is cooled to room temperature, adds ammoniacal liquor and adjusts pH value to alkalescence, separates organic phase, and aqueous phase is with two
Chloromethanes extracts, and merges organic phase；Organic phase washed with water is washed, washed with saturated sodium-chloride water solution, done with anhydrous sodium sulfate
Dry, vacuum rotary steam removes organic solvent, obtains six upper methylene product 3a and 3b mixture, and reaction equation is
（3）Hydrogenation：Six upper a of methylene product 3 and 3b mixture is dissolved in the mixed liquor of ethanol and acetic acid, added
Cyclohexene, palladium carbon, it is heated to flowing back, reacts certain time in certain temperature, stop reaction, palladium carbon, hot second are recovered by filtration while hot
Alcohol washs insoluble matter, merges organic solvent, and most of organic solvent is removed under reduced pressure, and adds sodium hydrate aqueous solution and adjusts pH value into
Property, dichloromethane extraction is removed under reduced pressure organic solvent, obtains hydrogenated products 4, reaction equation is
（4）Hydrolysis：Hydrogenated products 4 are added into ethanol, are cooled to 0 DEG C, add concentrated hydrochloric acid in the timing of certain temperature reaction one
Between, TLC detects no raw material point, stops reaction, pours into elutriation in frozen water, filters, and 50 DEG C of dryings, ethanol refines, and drying obtains
6 Alpha-Methyl hydrocortisone products 5, reaction equation are
（5）Dehydrogenation reaction：It is that substrate carries out oxidative dehydrogenation with 6 Alpha-Methyl hydrocortisone products 5, produces methylprednisolone,
Comprise the following steps：
S51：Seed culture：Add in the fermentation tank of jacketed and trained with 6 Alpha-Methyl hydrocortisone products 5 for the fermentation of substrate
Base is supported, is fed, sterilizing, certain temperature is cooled to, accesses a certain amount of Arthrobacter simplex and fermented；Fermentation is in two stages：
First stage is the thalli growth stage：Inoculation, certain temperature and air mass flow are adjusted, cultivate certain time, the dense OD of bacterium620Reach
During certain value, fed intake；Second stage is the microorganism conversion stage：Feed intake, adjust certain temperature and air mass flow, conversion one
Fix time, fermentation is completed；
S52：Hydrolysis kinetics：Zymotic fluid is carried out to be filtrated to get fermentation filter cake, fermentation filter cake is entered using acetone-water mixed solvent
Row Hydrolysis kinetics, are concentrated under reduced pressure to extract solution, obtain methylprednisolone 6, and reaction equation is
Further, step（1）Described in the ratio of hydrocortisone 1, formalin and concentrated hydrochloric acid be 1W:5W:5W~
1W:20W:20W, the concentration of hydrocortisone 1 is 0.04~0.15mol/L.
Further, step（1）Described in reaction temperature be 20 DEG C ~ 30 DEG C, the reaction time be 6 ~ 10 hours.
Further, step（2）Described in upper protection product 2, sodium acetate, dichloromethane, dimethoxym ethane and POCl3Ratio
For 1W:1W:10W:13W:5W~1W:4W:20 W:26W:10W.
Further, step（2）Described in reaction temperature be 40 DEG C ~ 60 DEG C, the reaction time be 4 ~ 8 hours.
Further, step（3）Described in six upper methylene products 3, ethanol, acetic acid, cyclohexene, the ratios of palladium carbon be
1W:4W:5W:1W:0.1W~1W:15W:20 W:4W:0.5 W.
Further, step（3）Described in reaction temperature be 60 DEG C ~ 90 DEG C, the reaction time be 3 ~ 10 hours.
Further, step（4）Described in hydrogenated products 4, ethanol, the ratio of concentrated hydrochloric acid be 1W:0.5W:2W~1W:4W:
10W。
Further, step（4）Described in reaction temperature be -5 DEG C ~ 10 DEG C, the reaction time be 0.5 ~ 2 hour.
Further, step（5）Described in fermentation medium components include：6~8g/L of glucose, the g/ of yeast extract 6~8
L, the g/L of corn steep liquor 10~12, the g/L of potassium dihydrogen phosphate 1, pH=7.2~7.5 of fermentation medium；Thalli growth stage, thalline
Inoculum concentration is 8~10%, and cultivation temperature is 30~32 DEG C, and incubation time is 8~10h, the dense OD of bacterium620Fed intake during=30-40%；
The feed concentrations in microorganism conversion stage are 0.5~10g/L, and conversion temperature is 33~34 DEG C, and transformation time is 24~40h.
Compared with prior art, the beneficial effects of the invention are as follows：
Traditional methylprednisolone preparation technology is the complex process using mould dehydrogen substance as raw material or key intermediate, into
This height；And the present invention designs brand-new process route first using hydrocortisone as raw material, by ketal protection reaction, methylene
Glycosylation reaction, hydrogenation, hydrolysis and the step of dehydrogenation reaction five obtain methylprednisolone, cost with 60% weight yield
It is low, there is very strong cost competitiveness, simultaneous reactions are gentle, and reaction is simple, and selectivity is good, easily amplification, are adapted to industrialized production.
Embodiment
Presently in connection with embodiment, the present invention is further detailed explanation, and application of the invention is not limited to following
Embodiment, any formal accommodation done to the present invention fall within protection scope of the present invention.
In following embodiments, the reaction equation of the ketal protection reaction of the first step is：
The reaction equation of the methylenation of second step is：
The reaction equation of the hydrogenation of 3rd step is：
The reaction equation of the hydrolysis of 4th step is：
The reaction equation of the dehydrogenation reaction of 5th step is：
Embodiment 1
The first step, ketal protection reaction：Hydrocortisone 1（10g, 1W）Dichloromethane (200mL, 20V) is dissolved in, under stirring
Add formalin（100mL, 10V）, concentrated hydrochloric acid is slowly added dropwise（100ml,10V）, it is stirred at room temperature 8 hours, TLC detections do not have
There is raw material point, stop reaction, separate organic phase, aqueous phase is extracted with dichloromethane, merges organic phase.Organic phase uses saturated carbon successively
Sour hydrogen sodium water solution washing, saturated sodium-chloride water solution washing, anhydrous magnesium sulfate are dried.Vacuum rotary steam removes organic solvent, obtains
To upper protection product 2a and 2b mixture (11.02g, 110%), product 2a HPLC (240nm, 55%), product 2b HPLC
(240nm, 37%)。
Second step, methylenation：Sodium acetate is added in reaction bulb（20g,2W）, dichloromethane（100mL,10V）With
Dimethoxym ethane (200mL, 20V), POCl is slowly added dropwise under stirring3（20mL,2V）, it is heated to 50 degree under nitrogen protection and reacts 1 hour.
Upper protection product 2a and 2b mixture（10g,1W）It is dissolved in dichloromethane（40mL,4V）.Protect the dichloromethane of product molten on this
Liquid is slowly added dropwise to reaction solution, then POCl is slowly added dropwise3（20mL,2V）, back flow reaction 6 hours.TLC detects no raw material point,
Room temperature is cooled to, ammoniacal liquor is added and adjusts pH value to separate organic phase, aqueous phase is extracted with dichloromethane, merges organic phase to alkalescence.It is organic
Mutually it is washed with water successively, saturated sodium-chloride water solution washing, anhydrous sodium sulfate drying.Vacuum rotary steam removes organic solvent, obtains six
Methylene product 3a and 3b mixture (10.2g, 102%), product 3a HPLC (240nm, 60%), product 3b HPLC on position
(240nm,31%)。
3rd step, hydrogenation：Six upper methylene products 3 are added in reaction bulb（10g, 1W）, ethanol（125mL,
12.5V）And acetic acid（125mL, 12.5V）, dissolved clarification is stirred, adds cyclohexene（25mL, 2.5V）, palladium carbon（2.0g, 0.2W）,
Reacted 5 hours in 80 degree under nitrogen protection, stop reaction, palladium carbon is recovered by filtration while hot, hot ethanol washing insoluble matter, merges organic
Solvent, most of organic solvent is removed under reduced pressure, adds 10% sodium hydrate aqueous solution and adjust pH value to neutrality, dichloromethane extraction
（200mLx2）, organic solvent is removed under reduced pressure, obtains clear yellow viscous thing (9.0g, 90%), crude product HPLC (240nm):Product 4a
(73%), product 4b (15%).
4th step, hydrolysis：Hydrogenated products 4（9g, 1W）, add ethanol（9mL, 1V）, 0 DEG C is cooled to, is added dense
Hydrochloric acid（45mL, 5V）, 0 DEG C is reacted 1 hour, and TLC detects no raw material point, is stopped reaction, is poured into elutriation in frozen water, is filtered,
50 DEG C of dryings, add ethanol（4.5mL, 0.5V）It is heated to reflux 1 hour, is cooled to 0 DEG C of discharging, 50 DEG C of dryings, obtains hydrolysis production
Thing 5 (6.3g, 70%), product HPLC (240nm, 96%).
5th step, dehydrogenation reaction：Fermentation medium components：Glucose 7g/L, yeast extract 7 g/L, corn steep liquor 11g/L, phosphorus
The g/L of acid dihydride potassium 1, PH=7.3, substrate are 6 Alpha-Methyl hydrocortisones 5, and during the fermentation, Arthrobacter simplex is by methyl hydrogen
Change cortisone and carry out oxidative dehydrogenation, obtain methylprednisolone 6.
Fermentation process is as follows：Secondary seed culture, culture medium, coefficient are added in the 10L fermentation tanks of jacketed
0.6, sterilizing, 32 DEG C are cooled to, access Arthrobacter simplex, inoculum concentration 9%, fermentation is in two stages：1. the thalli growth stage：
After inoculation, air mass flow 1.0m3/h is adjusted, 32 DEG C of temperature, cultivates 9h, the dense OD of monitoring bacterium620=36%, fed intake.2. microorganism
Transformation stage：Feed concentrations 1g/L, hydrogenated methyl cortisone 6g is thrown, adjust air mass flow 0.5m3/h, temperature is 34 DEG C, conversion
Time is 36h, conversion ratio 95%.
Hydrolysis kinetics：Zymotic fluid is filtered, and fermentation filter cake carries out Hydrolysis kinetics using 85% acetone-water mixed solvent, carries
Take liquid to be concentrated under reduced pressure, obtain methylprednisolone 6 (5.1g, 85%), product HPLC (240nm, 99%).
Embodiment 2
The first step, ketal protection reaction：Hydrocortisone 1（10g, 1W）Dichloromethane (100mL, 10V) is dissolved in, under stirring
Add formalin（46mL, 4.6V）, concentrated hydrochloric acid is slowly added dropwise（42ml,4.2V）, 30 DEG C are stirred 10 hours, stop reaction,
Organic phase is separated, aqueous phase is extracted with dichloromethane, merges organic phase.Organic phase is washed with saturated sodium bicarbonate aqueous solution successively,
Saturated sodium-chloride water solution is washed, and anhydrous magnesium sulfate is dried.Vacuum rotary steam removes organic solvent, obtains protecting product 2a and 2b
Mixture (10.4g, 104%), product 2a HPLC (240nm, 30%), product 2b HPLC (240nm, 40%).
Other steps are the same as embodiment 1.
Embodiment 3
The first step, ketal protection reaction：Hydrocortisone 1（10g, 1W）Dichloromethane (400mL, 40V) is dissolved in, under stirring
Add formalin（185mL, 18.5V）, concentrated hydrochloric acid is slowly added dropwise（170ml,17V）, 20 DEG C are stirred 6 hours, are stopped anti-
Should, organic phase is separated, aqueous phase is extracted with dichloromethane, merges organic phase.Organic phase is washed with saturated sodium bicarbonate aqueous solution successively
Wash, saturated sodium-chloride water solution washing, anhydrous magnesium sulfate is dried.Vacuum rotary steam removes organic solvent, obtains protecting product 2a
With 2b mixture (111g, 111%), product 2a HPLC (240nm, 50%), product 2b HPLC (240nm, 43%).
Other steps are the same as embodiment 2.
Embodiment 4
Second step, methylenation：Sodium acetate is added in reaction bulb（10g,1W）, dichloromethane（50mL,5V）And dimethoxym ethane
(150mL, 15V), POCl is slowly added dropwise under stirring3（15mL,1.5V）, it is heated to 60 degree under nitrogen protection and reacts 1 hour.Upper guarantor
Protect product 2a and 2b mixture（10g,1W）It is dissolved in dichloromethane（25mL,2.5V）.The dichloromethane solution of product is protected on this
It is slowly added dropwise to reaction solution, then POCl is slowly added dropwise3（15mL,1.5V）, back flow reaction 8 hours.Room temperature is cooled to, adds ammoniacal liquor
PH value is adjusted to separate organic phase, aqueous phase is extracted with dichloromethane, merges organic phase to alkalescence.Organic phase washed with water is washed, saturation chlorine
Change sodium water solution washing, anhydrous sodium sulfate drying.Vacuum rotary steam removes organic solvent, obtains six upper methylene product 3a and 3b
Mixture (90g, 90%), product 3a HPLC (240nm, 67%), product 3b HPLC (240nm, 15%).
Other steps are the same as embodiment 2.
Embodiment 5
Second step, methylenation：Sodium acetate is added in reaction bulb（40g,4W）, dichloromethane（100mL,10V）Contracted with first
Aldehyde (300mL, 30V), POCl is slowly added dropwise under stirring3（30mL,3V）, it is heated to flowing back under nitrogen protection, 40 degree of reactions 1 are small
When.Upper protection product 2a and 2b mixture（10g,1W）It is dissolved in dichloromethane（50mL,5V）.The dichloromethane of product is protected on this
Solution is slowly added dropwise to reaction solution, then POCl is slowly added dropwise3（30mL,3V）, back flow reaction 4 hours.Room temperature is cooled to, adds ammonia
Water adjusts pH value to separate organic phase, aqueous phase is extracted with dichloromethane, merges organic phase to alkalescence.Organic phase washed with water is washed, saturation
Sodium-chloride water solution washs, anhydrous sodium sulfate drying.Vacuum rotary steam removes organic solvent, obtain six upper methylene product 3a and
3b mixture (9.3g, 93%), product 3a HPLC (240nm, 72%), product 3b HPLC (240nm, 20%).
Other steps are the same as embodiment 3.
Embodiment 6
3rd step, hydrogenation：Six upper methylene products 3 are added in reaction bulb（10g, 1W）, ethanol（50mL, 5V）And vinegar
Acid（50mL, 5V）, dissolved clarification is stirred, adds cyclohexene（12.5mL,1.25V）, palladium carbon（1.0g, 0.1W）, nitrogen protection under in
90 degree are reacted 10 hours, stop reaction, and palladium carbon is recovered by filtration while hot, hot ethanol washing insoluble matter, merges organic solvent, decompression removes
Most of organic solvent is removed, 10% sodium hydrate aqueous solution is added and adjusts pH value to neutrality, dichloromethane extraction（200mLx2）, decompression
Organic solvent is removed, obtains clear yellow viscous thing (9.4g, 94%), crude product HPLC (240nm):Product 4a (53%), product 4b
(13%)。
Other steps are the same as embodiment 3.
Embodiment 7
3rd step, hydrogenation：Six upper methylene products 3 are added in reaction bulb（10g, 1W）, ethanol（190mL, 19V）With
Acetic acid（190mL, 19V）, dissolved clarification is stirred, adds cyclohexene（50mL,5V）, palladium carbon（5.0g, 0.5W）, nitrogen protection under in
60 degree are reacted 3 hours, stop reaction, and palladium carbon is recovered by filtration while hot, hot ethanol washing insoluble matter, merges organic solvent, decompression removes
Most of organic solvent is removed, 10% sodium hydrate aqueous solution is added and adjusts pH value to neutrality, dichloromethane extraction（200mLx2）, decompression
Organic solvent is removed, obtains clear yellow viscous thing (8.5g, 85%), crude product HPLC (240nm):Product 4a (83%), product 4b
(10%)。
Other steps are the same as embodiment 4.
Embodiment 8
4th step, hydrolysis：Hydrogenated products 4（9g, 1W）, add ethanol（6mL, 0.6V）, -5 DEG C are cooled to, is added dense
Hydrochloric acid（18mL, 2V）, -5 DEG C are reacted 2 hours, and TLC detects no raw material point, are stopped reaction, are poured into elutriation in frozen water, take out
Filter, 50 DEG C of dryings, add ethanol（4.5mL, 0.5V）It is heated to reflux 1 hour, is cooled to 0 DEG C of discharging, 50 DEG C of dryings, obtains water
Solve product 5 (6.0g, 66%), product HPLC (240nm, 93%).
Other steps are the same as embodiment 5.
Embodiment 9
4th step, hydrolysis：Hydrogenated products 4（9g, 1W）, add ethanol（45mL, 5V）, 10 DEG C are cooled to, adds dense salt
Acid（76.5mL, 8.5V）, 10 DEG C are reacted 0.5 hour, and TLC detects no raw material point, are stopped reaction, are poured into elutriation in frozen water,
Filter, 50 DEG C of dryings, add ethanol（4.5mL, 0.5V）It is heated to reflux 1 hour, is cooled to 0 DEG C of discharging, 50 DEG C of dryings, obtains
Hydrolysate 5 (5.5g, 61%), product HPLC (240nm, 97%).
Other steps are the same as embodiment 6.
Embodiment 10
In 5th step, fermentation medium components：Glucose 6g/L, the g/L of yeast extract 6, the g/L of corn steep liquor 10, potassium dihydrogen phosphate 1
G/L, PH=7.2, substrate are 6 hydrogenated methyl cortisones, and during the fermentation, Arthrobacter simplex carries out 6 hydrogenated methyl cortisones
Oxidative dehydrogenation, obtain methylprednisolone.
Fermentation process is as follows：First order seed culture, culture medium, coefficient are added in the 10L fermentation tanks of jacketed
0.6, sterilizing, 30 DEG C are cooled to, access Arthrobacter simplex, inoculum concentration 8%, fermentation is in two stages：1. the thalli growth stage：
After inoculation, air mass flow 0.6m3/h is adjusted, 30 DEG C of temperature, cultivates 10h, the dense OD of monitoring bacterium620=40%, fed intake.2. micro- life
The thing transformation stage：Feed concentrations 0.5g/L, hydrogenated methyl cortisone 3g to be thrown, adjust the m3/h of air mass flow 0.3, temperature is 33 DEG C,
Transformation time is 40h, conversion ratio 90%.
Hydrolysis kinetics：Zymotic fluid is filtered, and fermentation filter cake carries out Hydrolysis kinetics using 85% acetone-water mixed solvent, carries
Take liquid to be concentrated under reduced pressure, obtain methylprednisolone 6 (2.4g, 80%), product HPLC (240nm, 98%).
Other steps are the same as embodiment 7.
Embodiment 11
In 5th step, fermentation medium components：Glucose 8g/L, yeast extract 8g/L, the g/L of corn steep liquor 12, the g/ of potassium dihydrogen phosphate 1
L, PH=7.5, substrate are 6 hydrogenated methyl cortisones, and during the fermentation, 6 hydrogenated methyl cortisones are carried out oxygen by Arthrobacter simplex
Fluidized dehydrogenation, obtain methylprednisolone.
Fermentation process is as follows：Three-level seed culture, culture medium, coefficient are added in the 10L fermentation tanks of jacketed
0.6, sterilizing, 33 DEG C are cooled to, access Arthrobacter simplex, inoculum concentration 10%, fermentation is in two stages：1. the thalli growth stage：
After inoculation, air mass flow 1.6m3/h is adjusted, 33 DEG C of temperature, cultivates 8h, the dense OD of monitoring bacterium620=30%, fed intake.2. microorganism
Transformation stage：Feed concentrations 10g/L, hydrogenated methyl cortisone 60g is thrown, adjust the m3/h of air mass flow 0.8, temperature is 35 DEG C, is turned
The change time is 40h, conversion ratio 93%.
Hydrolysis kinetics：Zymotic fluid is filtered, and fermentation filter cake carries out Hydrolysis kinetics using 85% acetone-water mixed solvent, carries
Take liquid to be concentrated under reduced pressure, obtain methylprednisolone 6 (50.4g, 84%), product HPLC (240nm, 98%).
Other steps are the same as embodiment 8.
Obtained methylprednisolone in various embodiments above is detected, mass spectrum：m/z 375 (M+H+), fusing point：
228-237oC, compareed with methylprednisolone reference material, comply fully with the characteristic of methylprednisolone.
It is complete by above-mentioned description, relevant staff using the above-mentioned desirable embodiment according to the present invention as enlightenment
Various changes and amendments can be carried out without departing from the scope of the technological thought of the present invention' entirely.The technology of this invention
Property scope is not limited to the content on specification, it is necessary to determines its technical scope according to right.
Claims (10)
1. prepare the method for methylprednisolone, it is characterised in that comprise the following steps：
（1）Ketal protection reaction：Hydrocortisone 1 is dissolved in dichloromethane, stirs lower addition formalin, slowly drop
Enriching hydrochloric acid, the stirring reaction certain time under certain temperature, TLC detect no raw material point, stop reaction, separate organic phase,
Aqueous phase is extracted with dichloromethane, merges organic phase；Organic phase is washed with saturated sodium bicarbonate aqueous solution, uses saturated sodium-chloride successively
The aqueous solution is washed, dried with anhydrous magnesium sulfate, and vacuum rotary steam removes organic solvent, obtains protecting product 2a and 2b mixing
Thing, reaction equation are
（2）Methylenation：Sodium acetate, dichloromethane and dimethoxym ethane are added in reaction bulb, POCl is slowly added dropwise under stirring3,
Certain temperature is heated under nitrogen protection, is reacted 1 hour at this temperature；Upper protection product 2a and 2b mixture is dissolved in dichloromethane
Alkane, on this dichloromethane solution of product will be protected to be slowly added dropwise to reaction solution, then POCl is slowly added dropwise3, certain time is reacted,
TLC detects no raw material point, is cooled to room temperature, adds ammoniacal liquor and adjusts pH value to separate organic phase, aqueous phase is extracted with dichloromethane to alkalescence
Take, merge organic phase；Organic phase washed with water is washed, washed with saturated sodium-chloride water solution, being revolved with anhydrous sodium sulfate drying, decompression
Organic solvent is evaporated off, obtains six upper methylene product 3a and 3b mixture, reaction equation is
（3）Hydrogenation：Six upper a of methylene product 3 and 3b mixture is dissolved in the mixed liquor of ethanol and acetic acid, added
Cyclohexene, palladium carbon, it is heated to flowing back, reacts certain time in certain temperature, stop reaction, palladium carbon, hot second are recovered by filtration while hot
Alcohol washs insoluble matter, merges organic solvent, and most of organic solvent is removed under reduced pressure, and adds sodium hydrate aqueous solution and adjusts pH value into
Property, dichloromethane extraction is removed under reduced pressure organic solvent, obtains hydrogenated products 4, reaction equation is
（4）Hydrolysis：Hydrogenated products 4 are added into ethanol, are cooled to 0 DEG C, add concentrated hydrochloric acid in the timing of certain temperature reaction one
Between, TLC detects no raw material point, stops reaction, pours into elutriation in frozen water, filters, and 50 DEG C of dryings, ethanol refines, and drying obtains
6 Alpha-Methyl hydrocortisone products 5, reaction equation are
（5）Dehydrogenation reaction：It is that substrate carries out oxidative dehydrogenation with 6 Alpha-Methyl hydrocortisone products 5, produces methylprednisolone,
Comprise the following steps：
S51：Seed culture：Add in the fermentation tank of jacketed and trained with 6 Alpha-Methyl hydrocortisone products 5 for the fermentation of substrate
Base is supported, is fed, sterilizing, certain temperature is cooled to, accesses a certain amount of Arthrobacter simplex and fermented；Fermentation is in two stages：
First stage is the thalli growth stage：Inoculation, certain temperature and air mass flow are adjusted, cultivate certain time, the dense OD of bacterium620Reach
During certain value, fed intake；Second stage is the microorganism conversion stage：Feed intake, adjust certain temperature and air mass flow, conversion one
Fix time, fermentation is completed；
S52：Hydrolysis kinetics：Zymotic fluid is carried out to be filtrated to get fermentation filter cake, fermentation filter cake is entered using acetone-water mixed solvent
Row Hydrolysis kinetics, are concentrated under reduced pressure to extract solution, obtain methylprednisolone 6, and reaction equation is
。
2. according to the method for claim 1, it is characterised in that step（1）Described in hydrocortisone 1, formalin
Ratio with concentrated hydrochloric acid is 1W:5W:5W~1W:20W:20W, the concentration of hydrocortisone 1 is 0.04~0.15mol/L.
3. according to the method for claim 1, it is characterised in that step（1）Described in reaction temperature be 20 DEG C ~ 30 DEG C, instead
It is 6 ~ 10 hours between seasonable.
4. according to the method for claim 1, it is characterised in that step（2）Described in upper protection product 2, sodium acetate, dichloro
Methane, dimethoxym ethane and POCl3Ratio be 1W:1W:10W:13W:5W~1W:4W:20 W:26W:10W.
5. according to the method for claim 1, it is characterised in that step（2）Described in reaction temperature be 40 DEG C ~ 60 DEG C, instead
It is 4 ~ 8 hours between seasonable.
6. according to the method for claim 1, it is characterised in that step（3）Described in six upper methylene products 3, ethanol,
Acetic acid, cyclohexene, the ratio of palladium carbon are 1W:4W:5W:1W:0.1W~1W:15W:20 W:4W:0.5 W.
7. according to the method for claim 1, it is characterised in that step（3）Described in reaction temperature be 60 DEG C ~ 90 DEG C, instead
It is 3 ~ 10 hours between seasonable.
8. according to the method for claim 1, it is characterised in that step（4）Described in hydrogenated products 4, ethanol, concentrated hydrochloric acid
Ratio is 1W:0.5W:2W~1W:4W:10W.
9. according to the method for claim 1, it is characterised in that step（4）Described in reaction temperature be -5 DEG C ~ 10 DEG C, instead
It is 0.5 ~ 2 hour between seasonable.
10. according to the method for claim 1, it is characterised in that step（5）Described in fermentation medium components include：Portugal
Grape 6~8g/L of sugar, the g/L of yeast extract 6~8, the g/L of corn steep liquor 10~12, the g/L of potassium dihydrogen phosphate 1, the pH of fermentation medium=
7.2~7.5；Thalli growth stage, thalline inoculum concentration are 8~10%, and cultivation temperature is 30~32 DEG C, and incubation time is 8~10h,
The dense OD of bacterium620Fed intake during=30-40%；The feed concentrations in microorganism conversion stage are 0.5~10g/L, conversion temperature is 33~
34 DEG C, transformation time is 24~40h.
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