JPH0120880B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the general formula: [Bond in the formula] represents a single bond or double bond,
represents a hydrogen atom, a fluorine atom, a chlorine atom, or a methyl group, and V represents a methylene group, an ethylene group, an ethylidene group, or a vinylidene group], which has the general formula: [In the formula], X and V represent the above, and R ₁
represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and R ₂ represents an alkyl group having 1 to 6 carbon atoms] is fermented in a bacterial culture of the genus Curvularia. It is characterized by causing

As is well known, 11β-hydroxysteroids (e.g. corticoids; hydrocortisone, prednisolone, dexamethasone, betamethasone, prednylidene or flurandrenolone) with anti-inflammatory effects can be produced naturally by a very expensive multi-step partial synthesis. Manufactured from a steroid (Diosgenin).

In the multi-step synthesis of these compounds, the microbiological introduction of the 11β-hydroxy group into the steroid skeleton is generally a very expensive and very lossy synthesis due to the by-products formed. It is a process.

In 1966, the pregnane 11-desoxy-17α
-When 11β-hydroxylating a hydroxysteroid, the 17α-hydroxy group is esterified,
A method has been developed which allows the yield to be significantly increased by hydroxylation using bacteria of the genus Curvularia and saponification of the resulting 11β-hydroxy-17α-acyloxysteroids (West German Patent No. 1618599).

11 used as starting compound in this known method
-Desoxy-17α-acyloxysteroids can be produced by chemical hydrolysis of 11-desoxysteroids of the general formula, as is well known.

Compared to this known three-step process, the process according to the invention has the advantage that it is a single-step process, resulting in lower outlays and higher yields of the process product. Furthermore, the process according to the invention surprisingly often has the advantage that it can be carried out with significantly higher substrate concentrations and shorter reaction times than the known processes. The process of the invention also eliminates the need for hydrolysis of the 11β-hydroxy-17α-acyloxysteroids, which is often quite difficult; this hydrolysis almost always produces by-products and is therefore often In order for the product obtained to correspond to the purity standards required for pharmaceutical active substances, a complex and lossy purification of the product is necessary.

Furthermore, although the process according to the invention allows 11β-hydroxy-Δ ^1,4 -steroids of the general formula to be produced very well from the corresponding 11-desoxy-Δ ^1,4 -steroids of the general formula, the known techniques At the level
It is also noteworthy that 11-desoxy- ^Δ1,4 -steroids cannot be hydroxylated at the 11β-position with good yields.

Finally, in carrying out the process of the present invention, it is not necessary to use the purified product as a starting compound if the 11-desoxysteroid of the general formula is produced as a purified product, for example by the following method. : Dimethylformamide 80ml and benzene 700ml
1 g (40 mmol) of pyridinium tosylate in
and 10g of 17α,21-dihydroxy-steroid
300 ml of benzene are distilled off using a water separator at a bath temperature of 110-130 °C (glycerin bath) to remove traces of water from the solution of (28 mmol). This is followed by brief cooling, 24 ml (110-140 mmol) of orthocarboxylic acid trialkyl ester are added, and the remaining benzene is distilled off over 1.5 hours. After addition of 5.2 ml of pyridine, the reaction product is concentrated to dryness in a high vacuum.

The reaction proceeds quantitatively. Remains after concentration
17α,21-(1-Alkoxy-alkylidenedioxy)-steroids initially have an oily consistency but in most cases crystallize after a short time. The purity is completely sufficient for the subsequent microbial hydroxylation, and any residues of the reaction components used, even if attached to the crystals, do not interfere with the enzyme catalysis. D-homo-steroids of the general formula are prepared in a similar manner.

Pyridinium tosylate is prepared as follows: 54 g of P-toluenesulfonic acid.H ₂ O are concentrated to dryness in vacuo three times with sufficient benzene to remove water. 240 ml of pyridine are added to the residue, stirred after 1/2 hour and the product is precipitated with ether. The crystals were collected by suction filtration, washed with ether and incubated in vacuo at 50°C.
Let dry for 1 hour.

The 11-desoxysteroids of the general formula can have branched or preferably straight-chain radicals having 1 to 6 carbon atoms in the alkyl radicals R ₁ and R ₂ . For example, suitable alkyl groups R ₁ and R ₂ are methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl. A particularly good alkyl group R ₁ is the methyl group. Particularly preferred alkyl groups R ₂ are the methyl group and the ethylene group.

In 11-desoxysteroids of the general formula which are saturated in the 6,7-position, the substituent X is in particular in the .alpha.-position.

11-desoxysteroids of the general formula can be hydroxylated and the corresponding 11β of the general formula
- cleavage to hydroxysteroids is surprising to the person skilled in the art. The method according to the invention provides very high yields of the desired process product (approx. 85-90% of theory)
I couldn't have predicted what I would get.

Except for the use of different starting compounds, the process of the invention is carried out under conditions generally applicable to the 11β-hydroxylation of steroids by fungi of the genus Curvularia.

For example, a fungus of the genus Curvularia suitable for hydroxylation is Curvularia fulcta.
falcuta) QM-102H, Curvularia genticulata IFO (6284), Curvularia lunata NRRL2380, NRRL2434,
NRRL2178, ATCC12017 or IFO (6286) or Curvularia maculans
maculans) IFO (6292).

These microorganisms are cultured submerged under aeration in a suitable medium under the culture conditions generally applicable. To this medium is added the substrate (dissolved in a suitable solvent or preferably in emulsified form) and fermented until the maximum substrate conversion is achieved.

For example, suitable substrate solvents are methanol, ethanol, glycol monomethyl ether, dimethylformamide or dimethyl sulfoxide.
For example, emulsification of the substrate can be carried out in finely divided form or in water-miscible solvents such as methanol, ethanol, acetone, glycol monomethyl ether,
This can be carried out by injection into water (preferably demineralized) containing the customary emulsification aids under strong turbulence. Suitable emulsifying agents are nonionic emulsifiers, such as ethylene oxide adducts of polyglycols or fatty acid esters. Suitable emulsifiers include, ^for example, the commercially available wetting agents Tegin®, ^Tagat® , ^Tween® and ^Span® .

Emulsification of the substrate often allows for high substrate throughputs and thus increases in substrate concentration.
However, it is of course possible to apply other methods for increasing the substrate throughput which are well known to fermenters in the method of the invention.

The optimum substrate concentration, substrate feeding time and fermentation time depend on the structure of the substrate used and the type of microorganism used. These degrees must be determined in each individual case by preliminary experiments, which are well known to those skilled in the art, as is generally necessary in microbiological steroid conversions.

By the method of the present invention, the following 11β having the general formula is obtained from the corresponding 11-desoxysteroid of the general formula:
17α,21-trihydroxy-4-pregnene-
3,20-dione derivatives can be prepared: 11β,17α,21-trihydroxy-4-pregnene-3,20-dione, 11β,17α,21-trihydroxy-1,4-pregnadiene-3, 20-dione, 11β,17α,21-trihydroxy-6α-methyl-4-pregnene-3,20-dione, 11β,17α,21-trihydroxy-6α-methyl-1,4-pregnadiene-3,20- dione, 11β,17α,21-trihydroxy-6-chloro-4,6-pregnadiene-3,20-dione, 11β,17α,21-trihydroxy-6-chloro-1,4,6-pregnatriene-3, 20-dione, 11β,17α,21-trihydroxy-16α-methyl-4-pregnene-3,20-dione, 11β,17α,21-trihydroxy-16α-methyl-1,4-pregnadiene-3,20- dione, 11β,17α,21-trihydroxy-16β-methyl-4-pregnene-3,20-dione, 11β,17α,21-trihydroxy-16β-methyl-1,4-pregnadiene-3,20-dione, 11β,17α,21-trihydroxy-16-methyl-4-pregnene-3,20-dione, 11β,17α,21-trihydroxy-16-methylene-1,4-pregnadiene-3,20-dione, 11β, 17α,21-trihydroxy-D-homo-
4-pregnene-3,20-dione, 11β,17α,21-trihydroxy-D-homo-
1,4-pregnadiene-3,20-dione, 6α-fluoro-11β,17α,21-trihydroxy-4-pregnene-3,20-dione, 6α-fluoro-11β,17α,21-trihydroxy-1, 4-Pregnadiene-3,20-dione.

Next, the method of the present invention will be explained in detail with reference to examples.

Example 1 (a) 8 g of pyridinium tosylate and 17α,21−
80 g of dihydroxy-4-pregnene-3,20-dione are dissolved in 560 ml of dimethylformamide and diluted with 3.5 g of benzene. Thereafter, 1.5 benzene is distilled off via a water separator at a bath temperature of 120°C. 192 ml of orthoacetic acid triethyl ester are gradually introduced into the hot reaction solution, followed by distillation of benzene and other easily volatile reaction components for 1 hour. Add 96 ml of pyridine and concentrate completely to dryness in a high vacuum for 2 hours at a bath temperature of 60°C. The initially oily residue quickly crystallizes out. 17α, 21−
113 g of (1-ethoxy-ethylidenedioxy)-4-pregnene-3,20-dione has a melting point of 89-90.
Obtained in yellow crystalline form at °C.

(b) sterilized in an autoclave at 120°C for 30 min;
Glucose 3%, Corn Steep 1%,
_NaNO3 0.2%, _{KH2PO4 0.1} %, _{K2HPO4 0.2} %,
_MgSO4・_7H2O0.05 %, _FeSO4・_7H2O0.002 %
A 2-erlenmeyer flask containing 500 ml of a culture medium consisting of C. and 0.05% KCl is inoculated with a freeze-dried culture of Curvularia lunata (NRRL) and moved on a rotary shaker at 30° C. for 60 hours. This culture (250
ml) consisting of 2% glucose and 2% corn steep, adjusted to pH 6.5, heated at 121°C and
1.1Atu¨ sterilized medium 15 is charged 20
- Used to inoculate prefermenters. 29℃ and pressure with addition of silicone SH as foam suppressant
Aeration (10/min) and stirring (220 r.p. at 0.7 atu¨)
m.) Allow to germinate under 24 hours. 1.5 of this medium was then taken under sterile conditions and charged with medium 14, sterilized as above, consisting of 3% corn steep and 0.7% glucose, and adjusted to a pH of 6.5. Inoculate the fermenter. Crude 17α,21-(1-ethoxy-ethylidenedioxy)-4-pregnene-3,20-dione dissolved in 100 ml of dimethylformamide after a growth period of 12 hours under prefermenter conditions.
7.5 g (containing 6.43 g of pure material) are added under sterile conditions and further stirred and vented. The progress of the fermentation is checked by taking samples, extracting them with methyl isobutyl ketone and analyzing them by thin layer chromatography. Conversion of the substrate is complete after a contact time of 38 hours.

The fermenter contents were filtered, the culture filtrate and also the filtered micelles were extracted with methyl isobutyl ketone, the extracts were combined and concentrated first in a reflux evaporator and then in a rotary evaporator under vacuum. Medium bath temperature
Concentrate to dryness at 50℃. The residue is dissolved in warm methanol, the undissolved residual silicone oil is filtered off and evaporated again to dryness. The residue (9.4 g) is dissolved hot in 40 ml of ethyl acetate and crystallized first at room temperature and then on standing at low temperature. The crystalline product is filtered off with suction, washed with a small amount of cold acetate and dried for 5 hours at 60° C. in a vacuum drying box. 4.18 g of hydrocortisone (11β,17α,21-trihydroxy-4-pregnene-3,20-dione) with a melting point of 212-214°C are obtained. The crystal mother liquor is heated with activated charcoal, filtered and concentrated to a volume of 15 ml. Upon cooling and standing overnight at room temperature, a further 0.7 g of hydrocortisone with a melting point of 208 DEG-210 DEG C. crystallizes. Therefore, the total yield is the theoretical amount
It is 87.1%.

Example 2 (a) 7 g of pyridinium tosylate and 17α,21−
70 g of dihydroxy-4-pregnene-3,20-dione are dissolved in 560 ml of dimethylformamide and diluted with 4.9 g of benzene. Orthobutyric acid triethyl ester after distilling off benzene 2.1 through a water separator at a melt temperature of 130°C and after short cooling.
Add 168ml. The remaining benzene is distilled off at 130°C for 1.5 hours. 84 ml of pyridine are added to this solution and it is subsequently concentrated to dryness in a high vacuum using a rotary evaporator. 17α,21-(1-ethoxy-butylidenedioxy)-4-pregnene-3,
103.8 g of 20-dione are obtained as an oil.

(b) 7.5 g of crude 17α,21-(1-ethoxy-butylidenedioxy)-4-pregnene-3,20-dione containing 6.3 g of pure material under the conditions of Example 1(b).
g was fermented for 47 hours in Curvularia lunata medium. Hydrocortisone (melting point
210-213°C) 4.3g is obtained.

Example 3 (a) 5.3 g of pyridinium tosylate and 17α,21−
53 g of dihydroxy-6α-methyl-4-pregnene-3,20-dione was dissolved in dimethylformamide.
Dissolve in 370 ml and dilute with benzene 2.5. Thereafter, benzene 1 is distilled off via a water separator at a bath temperature of 130°C. Gradually pour 127 ml of orthoacetic acid ethyl ester into the hot reaction solution.
Benzene and other easily volatile reactants are then added to 1
Allow time to evaporate. 63 ml of pyridine are added and the mixture is completely concentrated to dryness in a high vacuum at a bath temperature of 60° C. for 2 hours. 66.8 g of oily 17α,21-(1-ethoxy-ethylidenedioxy)-6α-methyl-4-pregnene-3,20-dione are obtained.

When a sample of the oily residue is vigorously stirred with distilled water, the oil gradually turns crystalline. melting point
75/92~95℃.

(b) Crude 17α,21-(1-ethoxy-ethylidenedioxy)-6α-methyl-4-pregnene-3, containing 4.8 g of pure material under the conditions of Example 1(b).
20-6 g of dione in Curvularia lunata medium
Ferment for 51 hours. After working up the batch and crystallizing the extraction residue from diisopropyl ether, 3.4 g of 6α-methyl-hydroxycortisone are obtained, melting point 217-219°C.

Example 4 (a) 4 g of pyridinium tosylate and 17α,21−
dihydroxy-1,4-pregnadiene-3,
20-40g of dione and 280ml of dimethylformamide
and diluted with 2 parts of benzene. Then benzene 600 ml via water separator with bath temperature 120℃
ml is distilled off. 96 ml of orthoacetic acid triethyl ester was gradually introduced into the hot reaction solution.
Benzene and other easily volatile reaction components are then further distilled off. Add 48 ml of pyridine and completely concentrate to dryness in a high vacuum at a bath temperature of 60°C for 2 hours. 17α,21-(1-ethoxy-ethylidenedioxy)-1,4-pregnadiene-3,20-
58 g of dione are obtained as yellowish crystals. The sample is recrystallized from ether containing 1% acetone after treatment with activated charcoal and a pure product with a melting point of 153 DEG-154 DEG C. is obtained.

(b) 3% glycose, 1% corn steep;
_NaNO3 0.2%, _{KH2PO4 0.1} %, _{K2HPO4 0.2} %,
_MgSO4・_7H2O0.05 %, _FeSO4・_7H2O0.002 %
and KCl 0.05%, in autoclave
A 2-erlenmeyer flask containing 500 ml of culture sterilized for 30 minutes at 120°C is inoculated with freeze-dried medium of Curvularia lunata (NRRL 2380) and run for 60 hours at 30°C on a rotary shaker. This culture was mixed with 2% glucose and 2% corn steep.
121℃ and 1.1atu¨ and adjusted to PH6.5
20 - Used to inoculate the pre-fermenter, which is charged with medium 15 sterilized by . Germinate for 24 hours under aeration (10/min) and stirring (220 r.pm) at 29° C. and a pressure of 0.7 atu¨ with the addition of silicone SH as a foam suppressant. Thereafter, take 1.5 of this culture under sterile conditions and transfer it to a cone steeper.
% and glucose 0.7%, sterilized as above and adjusted to pH 5.5.
20- Inoculate the main fermenter.

17α, dissolved in 100 ml of dimethylformamide after a growth period of 12 hours under prefermenter conditions.
21-(1-ethoxy-ethylidenedioxy)-
1,4-pregnadiene-3,20-dione 7.5
g under aseptic conditions and further stirred and aerated. The progress of the fermentation is checked by taking samples, which are extracted with methyl isobutyl ketone and analyzed by thin layer chromatography. Conversion of the substrate is complete after a contact time of 36 hours. The contents of the fermenter were filtered, the culture filtrate and the filtered micelles were extracted with methyl isobutyl ketone, the extracts were combined and concentrated first in a reflux evaporator and then in a rotary evaporator under vacuum. Concentrate to 100 ml at a medium bath temperature of 50°C and crystallize at room temperature.
The product was filtered off with suction and dried in vacuo, melting point
Obtain 5.7 g of prednisolone at 229-231°C.

Example 5 (a) 0.62 g of pyridinium tosylate and 6.2 g of D-homo-Reichscheutain S are dissolved in 43.4 ml of dimethylformamide and diluted with 270 ml of benzene. Thereafter, 116 ml of benzene is distilled off via a water separator at a bath temperature of 120°C. 14.9 ml of orthoacetic acid trimethyl ester are slowly introduced into the hot reaction solution and the benzene and other easily volatile reaction components are then distilled off for 1 hour. pyridine
Add 7.5 ml and completely concentrate to dryness in a high vacuum at a bath temperature of 60°C for 2 hours. 17,21-(1-methoxy-ethylidenedioxy)-D-homo-4-
7.3g of pregnene-3,20-dione has a melting point of 167~
Obtained in yellow crystalline form at 169°C.

(b) 3% glucose, 1% corn steep;
_NaNO3 0.02%, _{KH2PO4 0.1} %, _{K2HPO4 0.2}
%, _MgSO4・_7H2O0.05 %, _FeSO4・
500 ml of culture solution consisting of 0.002% 7H ₂ O and 0.05% KCl, sterilized in an autoclave at 120°C for 30 minutes.
A 2-erlenmeyer flask containing a 2-erlenmeyer flask was inoculated with a lyophilized culture of Curvularia lunata (NRRL 2380) and run for 60 hours at 30°C on a rotary shaker. This culture (250 ml) was added to 2% glucose
and corn steep 2%, sterilized at 120°C and 1.1 atu¨ and adjusted to pH 6.515
20 - Used to inoculate the pre-fermenter. With the addition of silicone SH as a foam suppressor
Germinate for 24 hours at 29° C. and pressure 0.7 atu¨ under aeration (10/min) and stirring (220 r.pm). This culture 1.5 was then harvested under sterile conditions and placed in a medium 14 sterilized as described above consisting of 3% corn steep and 0.7% glucose and adjusted to a pH of 6.5. Inoculate the fermenter. Growth time 12 under conditions of pre-fermentation
17,21-(1-methoxy-ethylidenedioxy)-D-homo-4-pregnene-3,20- dissolved in 100 ml of dimethylformamide after hours.
7.3 g of dione are added under aseptic conditions, followed by stirring and aeration. The progress of the fermentation is checked by taking samples, which are extracted with methyl isobutyl ketone and analyzed by thin layer chromatography. Conversion of the substrate is complete after a contact time of 48 hours.

Filter the fermentation contents, extract the culture filtrate and filtered micelles with methyl isobutyl ketone,
The extracts were combined and concentrated first in a reflux evaporator and then in a rotary evaporator in vacuo at a bath temperature of 50°C.
Concentrate to dryness at °C. The residue is dissolved in hot methanol, the undissolved residual silicone oil is filtered off and concentrated again to dryness. The residue is chromatographed on a silicic acid gel column (gradient 4.5 methylene chloride/0.5 acetone to 4 methylene chloride/1 acetone) and crystallized from acetone. D-homo-hydrocortisone (11β, 17α, 21-trihydroxy-D
-homo-4-pregnene-3,20-dione)
5.7g is obtained.

Example 6 (a) 1.5 g of pyridinium tosylate and 17α,21−
15 g of dihydroxy-D-homo-1,4-pregnadiene-3,20-dione are dissolved in 120 ml of dimethylformamide and diluted with 800 ml of benzene. Thereafter, 250 ml of benzene is distilled off via a water separator at a bath temperature of 120°C. 40 ml of orthoacetic acid trimethyl ester are slowly introduced into the hot reaction solution and the benzene and other easily volatile reaction components are then further distilled off. pyridine 20
ml and completely concentrated to dryness in a high vacuum at a bath temperature of 60°C for 2 hours. 18.8 g of oily crystalline crude remain. The sample is treated with activated carbon and then recrystallized from isopropyl ether. Pure 17α,21-(1-methoxy-ethylidenedioxy)-D-homo-1,4-pregnadiene-3,20-dione is obtained with a melting point of 142 DEG -144 DEG C.

(b) 3% glucose, 1% corn steep;
_NaNO3 0.2%, _{KH2PO4 0.1} %, _{K2HPO4 0.2} %,
_MgSO4・_7H2O0.05 %, _FeSO4・_7H2O0.002 %
and KCl 0.05%, in autoclave
A 2-erlenmeyer flask containing 500 ml of culture sterilized for 30 minutes at 120°C is inoculated with a lyophilized culture of Curvularia lunata (NRRL 2380) and run on a rotary shaker for 60 hours at 30°C. This culture (250 ml) was incubated at 121° C. with 2% glucose and 2% corn steep and adjusted to pH 6.5.
1.1Atu¨20 is charged with sterilized medium 15
- Used for inoculating prefermenters. 29℃ and pressure 0.7atu¨ with addition of silicone SH as foam suppressant
Germinate for 24 hours under aeration (10/min) and stirring (220 rpm). Then, this culture
1.5 was collected under sterile conditions and made up of 3% corn soup and 0.7% glucose;
Medium 14 sterilized as above and adjusted to pH 5.5
Inoculate the 20-main fermentor that is charged with.

17α, dissolved in 100 ml of dimethylformamide after a growth period of 12 hours under pre-fermentation conditions.
21-(1-methoxy-ethylidenedioxy)-D
7.5 g of homo-1,4-pregnadiene-3,20-dione are added under sterile conditions, followed by stirring and aeration. The progress of the fermentation is checked by taking samples, which are extracted with methyl isobutyl ketone and analyzed by thin layer chromatography. After a contact time of 52 hours, the conversion of the substrate is complete. The fermenter contents were filtered, the culture filtrate and the filtered micelles were extracted with methyl isobutyl ketone, the extracts were combined and concentrated first in a reflux evaporator and then in a vacuum bath in a rotary evaporator. Concentrate to 100 ml at 50°C and crystallize at room temperature. The product is filtered off with suction and dried in vacuo. D-homo-prednisolone (11β,17α,21-trihydroxy-D-homo-1,4-pregnadiene-
6.1 g of 3,20-dione) are obtained.

Claims (1)

[Claims] 1. General formula: [Bond in the formula] represents a single bond or double bond,
represents a hydrogen atom, a fluorine atom, a chlorine atom, or a methyl group, and V represents a methylene group, an ethylene group, an ethylidene group, or a vinylidene group], the general formula: [In the formula], X and V represent the above, and R ₁
represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and R ₂ represents an alkyl group having 1 to 6 carbon atoms] is fermented in a culture medium of Curvularia spp. Characteristic method for producing 11β-hydroxysteroids. 2. The production method according to claim 1, wherein the fermentation is carried out in a culture medium of Curvularia lunata species. 3. The method according to claim 1 or 2 for producing a 11β-hydroxy-D-homo-steroid having the general formula.