CS220330B2 - Method of producing 11beta-hydroxysteroid - Google Patents

Abstract

A method for producing 11β-hydroxysteroids according to the invention of the invention is that of hydroxylation based on 11-desoxysteroids and these are fermented by the fungal culture of Curvularia. The invention is in the field of steroid chemistry.

Description

The process for the preparation of 11β-hydroxysteroids according to the invention is characterized in that the hydroxylation starts from 11-desoxysteroids and these are fermented with a culture of fungi of the Curvularia species.

The invention is in the field of steroid chemistry.

220

The invention relates to a process for the preparation of 11β-hydroxysteroids of the formula I

CH, OH I ^L

bonds ....... means single bonds or double bonds,

X represents a hydrogen atom or a methyl group,

V represents a methylene group or an ethylene group.

The process for the preparation of the 11? -Hydroxysteroids of the formula I according to the invention is characterized in that the 11-desoxysteroid of formula II

in which the bonds ...... X and V have the meaning given in formula I,

R1 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,

R ₂ represents alkyl having 1 to 6 carbon atoms is fermented with fungal cultures Curvularia species.

It is known to produce anti-inflammatory 11β-hydroxysteroids (for example corticoids: hydrocortisone, prednisolone, dexamethasone, betamethasone, prednylidene or flurandrenolone) from natural steroids (such as diosgenin) by a very expensive multi-stage partial synthesis.

In the multi-stage synthesis of these compounds, the microbiological introduction of the 11? -Hydroxy group into the steroid structure is generally the most costly and, due to the by-products formed, the most lossy synthesis step.

In 1966, a method was developed by which the yield of 11? -Hydroxylation of the 11-desoxy-17? -Hydroxysteroids of the pregnane series can be substantially increased by esterifying the 17? -Hydroxy group, then hydroxylating them with the help of Curvularia fungi. 330

- hydroxy-17 - acyloxysteroids are saponified (German Patent No. 1,618,599).

The 11-desoxy-17α-acyloxysteroids used as starting compounds for this known process can be prepared by a known process by chemical hydrolysis of the 11-desoxysteroids of the general formula ΙΊ.

Compared to this previously known three-step process, the process according to the invention has the advantage that, as a one-step process, it is less expensive and that it results in higher product yields. The process according to the invention also has the advantage that, surprisingly, it can often be carried out at substantially higher substrate concentrations and in a shorter reaction time than the known process.

The process according to the invention also often avoids the truly difficult hydrolysis of 11β-hydroxy-17'-acyloxysteroids, in which intermediates are formed, thus requiring costly and lossy purification of the products obtained to meet the purity criteria required for drug active ingredients.

It should also be noted that the method according to the invention can also produce very good ll | ^3-hydroxy-1 A ^'4 -steroids of general formula I from the appropriate desoxy-A -steroids ^with the general formula II, while the prior art does not possible ^11-deoxy-L4 A -steroids Hydroxylate in a position to 11/3 with a good yield.

Finally, it is noted that pure 11-desoxysteroid products of formula (II) need not be used as starting compounds to carry out the process of the invention when produced as a crude product, for example by the following method:

From a solution of 1 g (4 mmol) of pyridinium tosylate and 10 g (28 mmol) of 17α, 21-dihydroxysteroid in 80 ml of dimethylformamide and 700 ml of benzene, 300 ml are distilled off at a bath temperature of 110-130 ° C (glycerol bath). benzene using a water separator. It is then cooled briefly, 24 ml (110-140 mmol) of orthocarboxylic acid trialkyl ester are added and the remaining benzene is distilled off within 1.5 hours. After addition of 5.2 ml of pyridine, the reaction product is concentrated to dryness under high vacuum.

The reaction proceeds quantitatively. After concentration, the remaining 17α, 21- (1-alkoxyalkylidenedioxy) steroid is initially oily in consistency, but crystallizes after a short time. The degree of purity is fully satisfactory for the subsequent microbiological hydroxylation, the residues of the reagents adhering to the crystallizate do not interfere with the enzymatic catalysis. The D-homosteroids of formula II are prepared analogously.

(Pyridinium tosylate is produced as follows: 54 g of p-toluenesulfonic acid with water molecule are concentrated to dryness three times under vacuum with sufficient benzene to remove water under vacuum. 240 ml of pyridine are added to the residue, stirred for 1/2 hour and the product precipitated with ether. The crystallizate is filtered off with suction, washed with ether and dried in vacuo at 50 DEG C. for 1 hour.

ill-Desoxysteroidy general formula II · ho mo220330 as alkyl groups, R _t and R ₂ contain a branched chain or preferably a straight chain having 1-6 carbon atoms. Suitable alkyl groups R ₁ and R ₂ are, for example, methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl groups. A particularly preferred R ₁ group is methyl. Particularly preferred R ₂ groups are methyl and ethyl groups.

For 11-desoxysteroids saturated in position

In formula (I) 6,7, X is especially in the alpha position.

It is surprising that one skilled in the art is able to hydroxylate and cleave the 11-desoxysteroids of formula II into the corresponding 11β-hydroxysteroids of formula I. It was even less predictable that in the process of the invention the desired products were obtained in a very high yield (about 85 to 90% of theory).

Apart from the use of other starting compounds, the process according to the invention is carried out under the conditions usually used for 11/3-hydroxylation of steroids by fungi of Curvularia species.

Curvularia fungi suitable for hydroxylation are, for example, Curvularia falcuta QM-102H, Curvularia genticulata IFO (6284), Curvularia lunata NRRL 2380, NRRL 2434, NRRL 2178, ATCC 12 017 or IFO (6286) or Curvularia maculans IFO (6292).

Submerged cultures are cultivated in a suitable culture medium under aeration conditions typically used for these microorganisms. Subsequently, the substrate (dissolved in a suitable solvent or preferably in an emulsified form) is added to the cultures and fermented until maximum transformation of the substrate is achieved.

Suitable substrate solvents are, for example, methanol, ethanol, glycol monomethyl ether, dimethylformamide or dimethylsulfoxide. The emulsification of the substrate can be carried out, for example, by feeding the substrate in micronized form, or dissolved in a water-miscible solvent (such as methanol, ethanol, acetone, glycol monomethyl ether, dimethylformamide or < dimethylsulfoxide), with vigorous stirring into water (preferably Suitable emulsifying aids are nonionic emulsifiers, such as ethylene oxide adducts or fatty acid esters of polyglycols, as well as the conventional commercial wetting agents Tegin ^(R) , Tagat ^(R) , Tween ^(R) and Span ^{(). R)} .

Emulsification of substrates often allows for increased substrate throughput and thus increased substrate concentration. Naturally, it is also possible in the method according to the invention to use other methods which are certainly known to the fermentation expert to increase the substrate throughput.

The optimum substrate concentration, substrate addition time and fermentation time depend on the structure of the substrate used and the type of microorganism used. These quantities must, as is generally necessary in microbiological steroid transformations, be determined in advance on a case-by-case basis, as is customary for a person skilled in the art.

The following 11 / 3,17a, 21-trihydro-4-pregnene-3,20-dione derivatives of the formula I can be prepared, for example, from the corresponding 11-desoxysteroids of the formula II:

11 / 3,17 α, 21-trihydroxy-4-pregnene-3,20-dione, 11 / 3,17α, 21-trihydroxy-1,4-pregnadien-3,20-dione,

11 / 3,17a, 21-trihydroxy-6a-methyl-4-pregnene-3,20-dione, 11 / 3,17a, 21-trihydroxy-6a-methyl-1,4-pregnadien-3,20-dione,

11 / 3,17a, 21-trihydroxy-6-chloro-4,6-pregnadien-3,20-dione, 11 / 3,17a, 21-trihydroxy-6-chloro-1,4,6-pregnatriene-3, 20-dion,

11 / 3,17a, 21-trihydroxy-16a-methyl-4-pregnene-3,20-dione, 11 / 3,17a, 21-trihydroxy-16a-methyl-1,4-pregnadien-3,20-dione,

11 / 3,17a, 21-trihydroxy-16, 3-methyl-4-pregnene-3,20-dione, 11 / 3,17a, 21-trihydroxy-16/3-methyl-1,4-pregnadien-3 , 20-dione,

11 / 3,17 α, 21-trihydroxy-16-methyl-4-pregnene-3,20-dione, 11,13,17a, 21-trihydroxy-16-methylene-1,4-pregnadien-3,20-dione, 11 (3,17a, α, 21-trihydroxy-D-homo-4-pregnene-3,20-dione, II) / 3,17a, α, 21-trihydroxy-D-homo-1,4-pregnadien-3,20 -dion,

6'-Fluoro-III / 3,17a, 21-trihydroxy-4-pregnene-3,20-dione,

6a-fluoro-11,13a, 21-trihydroxy-1,4-pregnadien-3,20-dione.

The following examples illustrate the process of the invention.

He did.

(a) Dissolve 8 g of pyrodinium tosylate and 80 g of 17α, 21-dihydroxy-4-pregnene-3,20-dione in 560 ml of dimethylformamide and dilute

3,5 l benzene. Then, at a bath temperature of 120 ° C, 1.5 liters of benzene are distilled off through a water separator. 192 ml of triethyl orthoacetate are slowly added to the hot reaction solution, and then benzene and other slightly volatile reaction components are distilled off for 1 hour. 96 ml of pyridine are now added and concentrated to dryness under high vacuum at 60 DEG C. over 2 hours. The oily residue initially crystallizes rapidly. 113 g of 17α, 21- (1-ethoxy-ethylidenedioxy) -4-pregnene-3,20-dione are obtained in the form of yellowish crystals, m.p. 89-90 ° C.

(b) 2 liter Erlenmeyer flask containing 500 ml of nutrient solution sterilized for 30 minutes at 120 ° C in an autoclave consisting of 3% glucose, 1% corn extracts, 0,2% NaNO ₃ , 0,1% KH ₂ PO ₄ 0.2% K ₂ HPO ₄ , 0.05% MgSO ₄ .7H ₂ O, 0.002% FeSO ₄ . . 7H ₂ O and 0.05% KC1 was seeded with a culture of Curvularia lunata lyophilic (NRRL 2380), and 60 hours is moved at 30 ° C on a rotary shaker.

This seed culture (250 ml) is used to inoculate a 201 liter seed fermenter which is filled with 15 L of culture medium sterilized at 121 ° C and 0.11 MPa and consisting of 2% glucose and 2% corn extract, adjusted to pH 6.5.

With the addition of Silicone SH as antifoam, it is now allowed to grow for 24 hours at 29 ° C and 0.07 MPa, aeration (10 L / min) and stirring (220 rpm). Then 1.5 liters of this culture are removed under sterile conditions and inoculated with a 201 liter main fermenter packed with 14 liters sterilized as a nutrient medium from 3% corn extract and 0.7% glucose, adjusted to pH 6.5.

After a growth phase of 12 hours under seed fermentor conditions, 7.5 g of crude 17α, 21- (1-ethoxyethylidendloxy) -4-pregnene-3,20-dione containing 6.43 g of pure substance dissolved in 100 ml of dimethylformamide are added. , under sterile conditions, and further mixed and aerated.

The fermentation process is controlled by taking samples which are extracted with methyl isobutyl ketone and analyzed by thin layer chromatography. After 38 hours of treatment, the substrate reaction is complete.

The fermentor contents were filtered, the culture filtrate and the filtered mycels were extracted with methyl isobutyl ketone, the extracts were combined and first concentrated in a circulating evaporator, then evaporated to dryness in a rotary evaporator at 50 ° C under vacuum. The residue was dissolved in warm methanol, filtered off from the silicone oil which had left undissolved and again evaporated to dryness. The residue (9.4 g) was dissolved in heat in 40 ml of ethyl acetate and initially at room temperature, then crystallized in a deep-freeze box. The crystallized product is filtered off with suction, washed with a small amount of ethyl acetate and dried in a vacuum oven at 60 [deg.] C. for 5 hours. There was obtained 4.18 g of hydrocortisone (11 (3,17 a, 21-trihydroxy-4-pregnene-3,20-dione), m.p. 212 DEG-214 DEG C. The mother liquor after crystallization was heated with activated carbon, filtered. After cooling and standing overnight at room temperature, an additional 0.7 g of hydrocortisone, m.p. 208 DEG-210 DEG C., crystallizes, giving an overall yield of 87.1% of theory.

Example 2

(a) 7 g of pyridinium tosylate and 70 g of 17α, 21-dihydroxy-4-pregnene-3,20-dione are dissolved in 560 ml of dimethylformamide and diluted with 4,9 l of benzene. At a bath temperature of 130 ° C, 2.1 L of benzene was distilled off through a water separator and after brief cooling, 168 ml of triethyl orthobutyrate were added. The remaining benzene was distilled off at 130 ° C over 1.5 hours. 84 ml of pyridine are added to the solution and then concentrated to dryness on a rotary evaporator under high vacuum. 103.8 g of 17α, 21- (1-ethoxy-butylidenedioxy) -4-pregnene-3,20-dione are obtained as an oil.

bj Under the conditions of Example 1b, 7.5 g of crude 17α, 21- (1-ethoxybutylidenedioxy) -4-pregnene-3,20-dione containing 6.3 grams of pure substance was fermented with a Curvularia lunata culture for 47 hours. After treatment of the batch and crystallization of the silicone-free ethyl acetate residue, a total of 4.3 g of hydrocortisone of melting point 210 DEG-213 DEG C. is obtained.

Example 1 a d 3

(a) 5.3 g of pyrodinium tosylate and 53 g of 17α, 21-dihydroxy-6α-methyl-4-pregnene-3,20-dione are dissolved in 370 ml of dimethylformamide and diluted with 2.5 l of benzene. Then, at a bath temperature of 130 DEG C., 1 l of benzene is distilled off through a water separator. 127 ml of ethyl orthoacetic acid are slowly added to the hot reaction solution and then benzene and other volatile reaction components are distilled off for one hour. Pyridine (63 ml) was then added and completely evaporated to dryness under high vacuum at 60 ° C over 2 hours. 66.8 g of oily 17α, 21- (1-ethoxy-ethylidenedioxy) -6α-methyl-4-pregnene-3,20-dione remained.

A sample of the oily residue is vigorously stirred with distilled water to crystallize the oil. Mp 75/92 to 95 ° C.

b) Under the conditions of Example 1b, 6 g of crude 17α, 21- (1-ethoxy-ethylidenedioxy) -6α-methyl-4-pregnene-3,20-dione containing 4.8 grams of pure substance were fermented for 51 hours with a culture of Curvularia lunata . Working up of the batch and crystallization of the residue after extraction with diisopropyl ether gave 3.4 g of 6α-methylhydrocortisone, m.p. 217-219 ° C.

Example 4

(a) 4 g of pyrodinium tosylate and 40 g of 17α, 21-dihydroxy-1,4-pregnadien-3,20-dione are dissolved in 280 ml of dimethylformamide and diluted with 2 l of benzene. 600 ml of benzene are then distilled off at a bath temperature of 120 DEG C. through a water separator. 96 ml of triethyl ortho-acetic acid is slowly introduced into the hot reaction solution and then further benzene and other slightly volatile reaction components are distilled off. 48 ml of pyridine are now added and evaporated to dryness in a high vacuum over a period of 2 hours at a bath temperature of 60 ° C. 58 g of 17a, 21- (1-ethoxy220330) are obtained

ethyl-ethylenedioxy] -1,4-pregnadien-3,20-dione as a yellowish crystallizate.

After treatment with activated carbon, the sample crystallizes from ether with 1% acetone to give pure product, m.p. 153-154 ° C.

bj A 2 liter Erlenmeyer flask containing 500 ml of the nutrient solution sterilized for 30 minutes at 120 ° C in an autoclave consisting of 3% glucose, 1% corn extract, 0,2% NaNO ₃ , 0,1% KH ₂ PO ₄ , 0.2% K ₂ HPO ₄ , 0.05% MgSO ₄ .7H ₂ O, 0.002 why. FeSO ₄ .7H ₂ O and 0.05% KCl were inoculated with a lyophilic culture of Curvularia lunata (NRRL 2380) and shaken at 30 ° C on a rotary shaker for 60 hours. This seed culture (250 ml) was used to inoculate a 201 liter seed fermenter containing 15 liters of culture medium sterilized at 121 degrees Celsius and 0.11 MPa, consisting of 2% glucose and 2% corn extract, adjusted to pH 6.5.

With the addition of Silicone SH as antifoam, at 29 ° C and 0.07 MPa pressure, with aeration (10 l / min) and with stirring 220 rpm. allow 24 hours to grow. Then, 1.5 L of this culture is removed under sterile conditions and inoculated with a 201 liter main fermenter, which is filled with 14 liters as above sterilized broth from 3% corn extract and 0.7% glucose, adjusted to pH 5.5 .

After a growth phase of 12 hours under seed fermentor conditions, 7.5 g of 17 ', 21- (1-ethoxy-ethylidenedioxy) -1,4-pregnadien-3,20-dione dissolved in 100 ml of dimethylformamide are added under sterile conditions and stirred and airs on. The fermentation process is controlled by taking samples which are extracted with methyl isobutyl ketone and analyzed by thin layer chromatography. After 36 hours contact time, the substrate reaction is complete. The fermentor contents were filtered, the culture filtrate and the filtered mycels were extracted with methyl isobutyl ketone, the extracts were combined, concentrated in a circulating evaporator, then concentrated to 100 ml in a rotary evaporator at 50 ° C under vacuum and allowed to crystallize at room temperature. The product is filtered off with suction, dried in vacuo to give 5.7 g of prednisolone, m.p. 229-231 ° C. Example 5

(a) Dissolve 0,62 g of pyridinium tosylate and 6,2 g of D-homo Reichstein compound S (17 α, 21-dihydroxy-D-homo-4-pregnene-3,20-dione) in 43,4 ml of dimethylformamide and dilute 270 ml benzene.

Then, at a bath temperature of 120 ° C, 116 ml of benzene are distilled off through a water separator. 14.9 ml of trimethyl ortho-acetic acid is slowly added to the hot reaction solution, and then benzene and other slightly volatile reaction components are distilled off for 1 hour. Pyridine (7.5 ml) was added and evaporated to dryness at 60 ° C bath temperature over 2 hours. The oily residue crystallized from ether. 7.3 g of 17,21- (1-methoxy-ethylidenedioxy) -D-homo-4-pregnene-3,20-dione are obtained in the form of yellow crystals, m.p. 167-169 ° C.

(b) Two liter Erlenmeyer flask containing 500 ml of nutrient solution sterilized for 30 min at 120 ° C in an autoclave consisting of 3% glucose, 1% corn extract, 0,2% NaNO ₃ , 0,1% KH ₂ PO ₄ 0.2% K ₂ HPO ₄ , 0.05% MgSO ₄ .7H ₂ O, 0.002 why. FeSO ₄ .7H ₂ O and 0.05% KCl were inoculated with a lyophilic culture of Curvularia lunata (NRRL 2380) and moved at 30 ° C on a rotary shaker for 60 hours.

This seed culture (250 ml) is used to inoculate a 201 liter seed fermenter which is charged with 15 L of culture medium sterilized at 121 ° C and 0.11 MPa, consisting of 2% glucose and 2% corn extract, adjusted to pH 6.5. After addition of Silicone SH as antifoam, it is now allowed to grow for 24 hours at 29 ° C and 0.07 MPa under aeration (10 L / min and stirring (220 rpm). Then 1.5 L is removed under sterile conditions. of this culture and inoculated with a 201-liter main fermenter, which is filled as above-sterilized nutrient medium of 3% corn extract and 0.7% glucose, adjusted to pH 6.5. 7.3 g of 17,21- (1-methoxy-ethyl-idendloxy) -D-homo-4-pregnene-3,20-dione dissolved in 100 ml of dimethylformamide was added under stirring conditions and further stirred and airborne. vzo After 48 hours of contact time, the substrate reaction is complete.

The fermentor contents are filtered, the culture filtrate and the filtered mycels are extracted with methyl isobutyl ketone, the extracts are combined and first concentrated in a circulating evaporator, then concentrated to dryness in a rotary evaporator at 50 ° C in a rotary evaporator. The residue was dissolved in warm methanol, filtered from silicone oil which remained undissolved and evaporated again to dryness. The residue is chromatographed over a silica gel column using a gradient elution of 4.5 L methylene chloride / 0.5 L acetone - 4 L methylene chloride / 1 L acetone. It is then crystallized from acetone. 5.7 g of ocortisone D-homohydrate (11d, 17a, 21-trihydroxy-D-homo-4-pregnene-3,20-dione), m.p. 212 DEG-215 DEG C. (decomposition), are obtained.

Example 6

(a) Dissolve 1,5 g of pyridinium tosylate and 15 g of 17α, 21-dihydroxy-D-homo-1,4-pregnedien-3,20-dione in 120 ml of dimethylformamide and dilute with 800 ml of benzene. Then, at a bath temperature of 120 ° C, 250 ml of benzene are distilled off through a water separator. Ml of trimethyl orthoacetic acid is slowly introduced into the hot reaction solution and then further benzene and other volatile reaction components are distilled off. Pyridine (20 ml) was then added and concentrated to dryness over 2 hours at a bath temperature of 60 ° C under high vacuum. 18.8 g of an oily crystalline product remain. After treatment with activated carbon, a sample of isopropyl ether crystallizes to give pure 17α, 21- (1-methoxy-ethylidenedioxy) -D-homo-1,4-pregnadien-3,20-dione, m.p. 142-144 ° C.

b'j Two liter Erlenmeyer flask containing 500 ml of autoclaved sterilized solution at 120 ° C for 30 minutes, composed of 3% glucose, 1% corn extract, 0,2% NaNO ₃ , 0,1% KH ₂ PO ₄ 0.2% K ₂ HPO ₄ , 0.05 O / o MgSO 2. 7H ₂ O, 0.002% FeSO ₄ . . 7H ₂ O, 0.05% KC1, is inoculated with a culture of Curvularia lunata lyophilic (NRRL 2380) and 60 hours at 30 ° C on a rotary shaker moves. This seed culture (250 ml) was used to inoculate a 201 liter seed fermenter filled with 15 L of culture medium sterilized at 121 ° C and 0.11 MPa consisting of 2% glucose and 2% corn extract adjusted to pH 6.5. After addition of Silicone SH as antifoam, it is allowed to grow for 24 hours at 29 ° C and 0.07 MPa, under aeration (10 L / min and stirring (220 rpm)).

Then, 1.5 L of this culture is harvested under sterile conditions and inoculated with a 201 L main fermenter which is filled with 14 L of sterilized medium as. above, which consists of 3% corn extract and 0.7% glucose and is adjusted to a pH of 5.5.

After a growth phase of 12 hours under seed fermentor conditions, 7.5 g of 17 ', 21- (1-methoxy-ethylidenedioxy) -D-homo-1,4-pregnadien-3,20-dione dissolved in 100 ml are added under sterile conditions. dimethylformamide and further stirred and airborne.

The fermentation process is controlled by taking samples which are extracted with methyl isobutyl ketone and analyzed by thin layer chromatography. After 52 hours of contact time, the substrate reaction is complete. The fermentor content is filtered, the culture filtrate and the filtered mycels are extracted with methyl isobutyl ketone, the extracts are combined and concentrated first in a circulating evaporator, then at 50 ° C bath temperature, concentrated to 100 ml in a rotary evaporator under vacuum and allowed to crystallize at room temperature. . The product is filtered off with suction and dried under vacuum. 6.1 g of D-homoprednisolone (11j, 17a, 21-trihydroxy-D-homo-1,4-pregnadien-3,20-dione) are obtained, m.p. 226-228 ° C.

Claims (2)

A process for the preparation of 11β-hydroxysteroids of the general formula I CH, OH t ^L in which the bonds ....... denote single bonds or double bonds, X represents a hydrogen atom or a methyl group, V represents a methylene group or an ethylene group, characterized in that the 11-desoxysteroid of the formula II is ........ X and V are as previously defined, R _x represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, R ₂ represents alkyl having 1 to 6 carbon atoms is fermented with fungal cultures Curvularia species. 2. Process according to claim 1, characterized in that the fermentation is carried out with a culture of fungi of the species Curvularia lunata.