HU183154B - Process for preparing 11beta-hydroxy-steroids - Google Patents

Abstract

The present invention relates to a process for the preparation of 11β-hydroxy steroids of formula I, wherein the ring is a single bond or a hatching bond, X is a hydrogen atom or a methyl group, and V is a methylene group or an ethylene group. In the process of the present invention, an 11-deoxy steroid of formula II wherein X and V are as defined above, R1 is hydrogen or C1-C4 alkyl, and R2 is C1-C4 alkyl. It is fermented with a microorganism belonging to the Curvularia lunata species. -1-

Description

The present invention relates to a process for the preparation of 11α-hydroxy steroids of formula I wherein

X is hydrogen or methyl, and V is methylene or ethylene;

which process comprises the preparation of an 11-doxyoxy steroid of formula II, wherein ——, X and V are as defined above,

R 1 is hydrogen or C 1-4 alkyl,

Fermenting a fungus culture belonging R ₂ is C 1-4 alkyl group, a Curvularia lunata species.

As is known, anti-inflammatory 11/3-hydroxy steroid compounds (such as corticoids: hydrocortisone, prednisolone, dexamethasone, betamethasone, prednilidene or flurandrenolone) have so far been prepared from naturally occurring steroids by a very costly, multi-step process.

In the above mentioned multi-step synthesis, the most costly operation and the largest loss due to the formation of by-products is the microbiological introduction of the 11/3-hydroxy group into the steroid skeleton.

In 1966, a process was developed to substantially increase the yield of lβ-hydroxylation of 11-deoxy-17alpha-hydroxy steroids in the pregnane series by first esterifying the 17-alpha-hydroxy group with the help of a fungus of the genus Curvularia the resulting 11β-3-hydroxy-17α-alpha-acyloxy steroid is saponified (German Patent No. 1,618,599).

The 11-deoxy-17-alpha-acyloxy steroid used as starting material in this known process can be prepared by chemical hydrolysis of the 11-deoxy steroid of formula II in a manner known per se.

An advantage of the process of the present invention compared to the above three step process is that it is less expensive than the one step process and the product can be obtained in better yield. Another advantage of the process according to the invention is that it is often carried out with substantially higher substrate concentrations and a shorter reaction time than the known process, and in many cases the difficult hydrolysis of the 11β-hydroxy-17α-alpha-acyloxy steroid eliminates most of the by-products. and in order for a drug to meet purity criteria, the product must in most cases undergo a costly and lossy cleaning.

It is also noteworthy that the process according to the invention provides 11/3-hydroxy-delta steroid in very good yield from the corresponding 11-deoxy-delta ^1,4- steroid of the general formula II, whereas it is not possible to obtain good yield in the prior art.

Finally, it should be noted that the 11-deoxy steroid of the formula II used as starting material in the process of the present invention does not need to be a pure product, for example, the crude product obtained by the following process may be used:

g (4 mmol) of pyridine-tosylate and 10 g (28 mmol) of 17α, 21-dihydroxide steroid in 80 mL of dimethylformamide2 and 700 ml of benzene solution, 300 ml of benzene are distilled off in a water separator in a water separator in order to remove traces of water at a bath temperature of 110-130 ° C (glycerol bath). After cooling rapidly, triacyl ester of orthocarboxylic acid (24 ml, 110-140 mmol) was added, and the remaining benzene was distilled off within 1.5 hours, after which pyridine (5.2 ml) was added and the reaction product was dried under high vacuum (water pump).

The reaction described is quantitative. The 17 alpha, 21- (1-alkoxyalkyldioxy) steroid remaining after evaporation initially has an oily consistency but usually crystallizes in a short time. Its purity is sufficient for microbiological hydroxylation and the small amount of residual reagents used on the crystalline material does not interfere with enzyme catalysis. D-homo steroids of formula II can be prepared in an analogous manner.

(Pyridinium tosyl is prepared as follows: 54 g of p-toluenesulfonic acid crystalline water is evaporated to dryness with sufficient benzene in vacuo and repeated three times to remove any water present. The residue is mixed with 240 ml of pyridine and stirred for 0.5 h. and the product is precipitated with ether, the crystalline material is suctioned off, washed with ether and dried at 50 ° C for one hour in vacuo.

In the 11-deoxy steroids of formula II, the alkyl groups R ₁ and R _{2 have from} 1 to 4 carbon atoms and may be branched or preferably linear. Suitable R 1 and R ₂ alkyl groups include, for example, methyl, ethyl, propyl, isopropyl, butyl. Particularly preferred, when R _is methyl and R ₂ is methyl or ethyl.

For the 11-deoxy steroids of formula I, the X substituent is preferably in the alpha position.

It will be surprising to those skilled in the art that the 11-deoxy steroids of formula II can be hydroxylated and that the corresponding 11β-hydroxysteroids of formula I are formed during the reaction. It was even less predictable that the process of the present invention would yield the desired product in very good yields (about 85-90% of theory).

When other starting materials are used, the process of the invention is carried out under reaction conditions conventionally used for the 11/3-hydroxylation of steroids with fungi of the species Curvularia lunata.

Suitable examples of the hydroxylation reaction include Curvularia lunata NRRL 2380, NRRL 2434, NRRL 2178, ATCC 12017 or IFO (6286).

These microorganisms are cultured under the conditions normally used for fungi inside a suitable aerated medium (subculture). The fungal culture is then added to the substrate (which is dissolved in a suitable solvent or preferably emulsified) and fermented until maximum substrate conversion is achieved. Suitable substrate solvents include methyl alcohol, ethyl alcohol, glycol monomethyl ether, dimethylformamide or dimethylsulfoxide. For example, the emulsification of a substrate may be accomplished by stirring the micro-substrate or a solution thereof in a water-miscible solvent (such as methyl alcohol, ethyl alcohol, acetone, glycol monomethyl ether, dimethylformamide or dimethylsulfoxide) with a conventional emulsifier. containing water. Nonionic emulsifiers such as ethyleneoxy adducts or fatty acid esters of polyglycols are well suited for the reaction. Suitable emulsifiers include commercially available wetting agents called Tegin (R), Tagat (R), Tween ( ^R ) and Span ( ^R ).

Often it is possible to increase the substrate concentration in this way, but of course it is also possible to use an increased substrate concentration for processes other than the process of the invention as known to fermentation experts.

The optimum substrate concentration, substrate dosage and fermentation time depend on the microorganism used and the substrate used. These values, as required for microbiological steroid conversions, are usually determined in some cases by preliminary experiments.

By the process of the present invention, the following 11 β, 17 alpha, 21-trihydroxy-4-pregnen-3,40-dione derivatives can be prepared from the corresponding 11-deoxy steroids of formula II:

?, 3,17alpha, 2'-trihydroxy-4-pregnen-3,20-dione, 11,18,17alpha, 21-trihydroxy-1,4-pregnadiene-3,20-dione, β, 17alpha, 21-trihydroxy-6alpha -methyl-4-pregnen-3,20-dione, β, 17 alpha, 2'-trihydroxy-6-alpha-methyl-1,4-pregnadiene-3,20-dione,

II / 3,17a, 21-trihydroxy-D-homo-4-pregnen-3,20-dione,

II β, 17α, 21 -trihydroxy-D-homo-1,4-pregnadiene-3,20-dione.

The following examples further illustrate the process of the present invention.

Example 1

(a) A solution of 8 g of pyridinium tosylate and 80 g of 17a, 21-dihydroxy-4-pregnen-3,20-dione in 560 ml of dimethylformamide is diluted with 3.5 l of benzene and

At a bath temperature of 120 ° C, 1.5 L of benzene are distilled off on a water separator. To the hot reaction mixture was slowly added triethyl ester of orthoacetic acid (192 mL), and benzene and other volatile reaction components were distilled off from the reaction mixture for another hour. Pyridine (96 mL) was then added and completely dried in a water jet vacuum for 2 hours at 60 ° C. The residual oily material crystallized rapidly, yielding 113 g of 17α, 21- (1-ethoxyethylenedioxy-4-pregnen-3,20-dione) as a yellow crystalline solid, m.p. 89-90 ° C.

(b) In a 2 L Erlenmeyer flask, autoclave sterilized 500 ml of medium containing 3% glucose, 1% corn jam, 2% NaNO ₃ , 0.1% KH ₂ PO ₄ , 0.2 ml. Contains% K ₂ HPO ₄ , 0.05% MgSO ₄ .7H ₂ O, 0.002% FeSO ₄ .7H ₂ O, and 0.05% KCl, inoculated with lyophilized culture of Curvularia lunata (NRRL 2380) and the flask. Shake at 30 ° C on a rotary shaker for 60 hours. This culture (250 ml) was inoculated in a 20 L pre-fermenter,

15 L of medium sterilized at 121 ° C and 2.1 atm, containing 2% glucose and 2% corn jam and adjusted to pH 6.5. Subsequently, while adding Silicone SH as an antifoam agent, it was aerated (10 L / min) and stirred (220 rpm) at 29 ^D C and 0.7 atm _; After 24 hours growth, 1.5 liters of this culture was inoculated under sterile conditions with a 20 L master fermenter containing 14 L of sterilized medium as above. This medium contains 3% corn jam and 0.7% glucose and is adjusted to pH 6.5.

After 12 hours of growth under the same conditions as the pre-fermentation, 7.5 g of crude 17a, 21- (1-ethoxyethylenedioxy) -4-pregnen-3,20-dione, 6.43, dissolved in 100 ml of dimethylformamide, were obtained. g of pure material, added under sterile conditions, and further stirred and aerated. The evolution of the fermentation process is monitored by periodically taking a sample, extracting it with methyl isobutyl ketone and analyzing it by thin layer chromatography. Within 38 hours, substrate conversion is complete.

The fermentation broth is filtered, the filtrate and the filtered mycelium are extracted with methyl isobutyl ketone, the extracts are combined, first concentrated in a rotary evaporator and finally evaporated to dryness in a rotary evaporator under vacuum at 50 ° C. The residue was dissolved in warm methanol, the remaining insoluble silicone oil was removed by filtration and the solution was again evaporated to dryness. The residue (9.4 g) was dissolved in ethyl acetate (40 mL) and crystallized first at room temperature and then frozen. The crystallized product is filtered off with suction, washed with a little cold ethyl acetate and dried at 60 ° C in a vacuum oven for 5 hours. 4.18 g of hydrocortisone (11 / 3.17a, 21-trihydroxy-4-pregnen-3,20-dione) are obtained, m.p. 212-214 ° C. The mother liquor was heated with activated carbon, filtered and concentrated to a volume of 15 ml. After cooling and standing at room temperature overnight, an additional 0.7 g of hydrocortisone crystallizes, m.p. 208210 ° C.

The total yield is 87.1% of the theoretical value.

Example 2

(a) A solution of 7 g of pyridinium tosylate and 70 g of 17a, 21-dihydroxy-4-pregnene-3,20-dione in 560 ml of dimethylformamide is diluted with 4.9 l of benzene and

At a bath temperature of 130 ° C, 2.1 L of benzene are distilled off on a water separator. After cooling briefly, trimethyl ester of orthobutyric acid (168 ml) was added and the remaining benzene was distilled off at 130 ° C within 1.5 hours. To the solution was added pyridine (84 mL) and evaporated to dryness under high vacuum in a rotary evaporator. 103.8 g of oily 17a, 21- (1-ethoxybutylidenedioxy) -4-pregnen-3,20-dione are obtained.

b) Under the reaction conditions described in Example 1b, 7.5 g of crude 17a, 21- (1-ethoxybutylidenedioxy) -4-pregnen-3,20-dione containing 6.3 g of pure material were cultured with Curvularia lunata. It is fermented for 47 hours. Workup and crystallization of the residue from the silicone-free extract with ethyl acetate: After 33,154, a total of 4.3 g of hydrocortisone is obtained, m.p. 210-213 ° C.

Example 3

(a) A solution of 5.3 g of pyridinium tosylate and 53 g of 17a, 21-dihydroxy-6-methyl-4-propyl-3,2-dionionyl in 370 ml of dimethylformamide is diluted with 2.5 l of benzene. Then, at a bath temperature of 130 ° C, 1 liter of benzene was distilled off on a water separator. 127 ml of orthoacetic triethyl ester are slowly added to the hot reaction solution, and benzene and the other volatile reaction components are distilled off for an additional 1 hour. Pyridine (63 mL) was then added and evaporated to dryness in a water pump vacuum bath at 60 ° C for 2 hours. The residual oily substance was 66.8 g of 17a, 21- (1-ethoxyethylenedioxy) -6a-methyl-4-pregnen-3,20-dione.

If a smaller sample of the residue is stirred vigorously with distilled water, the oil will slowly crystallize. 92-95 ° C.

b) Under the reaction conditions described in Example 1b, 6 g of crude 17a, 21- (1-ethoxyethylidenedioxy) -6a-methyl-4-pregnen-3,20-dione containing 4.8 g of pure material, Curvularia lunata was cultured for 51 hours. After working-up and recrystallization of the residue from diisopropyl ether, 3.4 g of 6a-methylhydrocortisone (6a-methyl-11,17a, 21-trihydroxy-4-pregnen-3,20-dione) are obtained. mp 217-219 ° C.

Example 4

a) A solution of 4 g of pyridinium tosylate and 40 g of 17a, 21-dihydroxy-1,4-pregnadiene-3,20-dione in 280 ml of dimethylformamide is diluted with 2 l of benzene. thereafter

At a bath temperature of 120 ° C, 600 ml of benzene are distilled off on a water separator. 96 ml of triethyl ester of orthoacetic acid are slowly added to the hot reaction mixture, followed by distillation of benzene and other volatile reaction components, followed by 48 ml of pyridine. At 2 ° C bath temperature, evaporate to dryness completely within 2 hours. This gave 58 g of 17a, 21- (1-ethoxyethylenedioxy) -1,4-pregnadiene-3,20-dione as a yellowish crystalline solid.

Recrystallization of a sample from ether containing 1% acetic acid after treatment with activated charcoal gives a pure product, m.p. 153-154 ° C.

(b) 500 ml of medium containing 3% glucose, 1% corn jam, 0.2% NaNO ₃ , 0.1% KH ₂ PO ₄ sterilized in a 2 L Erlenmeyer flask for 30 minutes in an autoclave at 120 ° C. -ot, 0.2% K ₂ HPO ₄ , 0.05% MgSO ₄ . Containing 7H ₂ O, 0.002% FeSO ₄ .7H ₂ O and 0.05% KCl, inoculated with a lyophilized culture of Curvularia lunata (NRRL 2380) and shaking the flask at 30 ° C on a rotary shaker for 60 hours. This culture (250 ml) was inoculated with 15 L of medium sterilized at 121 ° C and 2.1 atm in a 20 L pre-fermenter containing 2% glucose and 2% corn jam and pH

Set to 6.5. With the addition of Silicon SH as an antifoam agent, it is incubated at 29 ° C and 0.7 atm at aeration (10 L / min) and agitated (220 rpm) for 24 hours. Subsequently, 1.5 L of the culture was removed under sterile conditions and an inoculum of 20 L containing 14 L of sterilized medium as above was inoculated. This medium contains 3% corn jam and 0.7% glucose and its pH is adjusted to 5.5.

After a 12-hour growth phase under the same conditions as the pre-fermenter, 7.5 g of 17a, 21- (1-ethoxyethylidenedioxy) -1,4-pregnadiene-3,20-dione in sterile conditions were added in 100 ml of dimethylformamide. and continue stirring and aeration. The progress of the fermentation process is monitored by periodically taking a sample, extracting it with methyl isobutyl ketone and analyzing it by thin layer chromatography. Within 36 hours, substrate conversion is complete. The fermentation broth is filtered, the filtrate and the filtered mycelium are extracted with methyl isobutyl ketone, the extracts are combined and concentrated first in a circulating evaporator, then concentrated to 100 ml in a rotary evaporator at 50 [deg.] C. and allowed to stand at room temperature. . The product was filtered off with suction, dried in vacuo to give 5.7 g of prcdnisolone (η 7,17a, 21-trihydroxy-1,4-prgnadiene-3,20-dione), m.p. 229231 ° C.

Example 5

a) Preparation of 0.62 g of pyridinium tosylate and 6.2 g of D-homo-Reichstein S substance (a-homo-17a, 21-dihydroxy-4-pregnene-3,20-diene) in 43.4 ml of dimethylformamide The solution is diluted with 270 ml of benzene and 116 ml of benzene are distilled off at 120 [deg.] C. in a water separator. 14.9 ml of trimethyl ester of orthoacetic acid are added slowly to the hot reaction solution and benzene and other volatile reaction components are distilled off for an additional 1 hour. 7.5 ml of pyridine are then added and the mixture is completely dried at 60 [deg.] C. under a water jet vacuum for 2 hours. The resulting oily residue was crystallized from ether to give 17.21- (1-methoxyethylenedioxy) -D-homo-4-pregnen-3,20-dione (7.3 g) as a yellow crystalline solid, m.p. -169 ° C.

(b) 500 ml of medium containing 3% glucose, 1% corn jam, 0.02% NaNO ₃ , 0.1% KH _z PO _{4 in} a 2 L Erlenmeyer flask autoclaved for 30 minutes at 120 ° C. containing 0.2% K ₂ HPO ₄ , 0.05% MgSO ₄ .7H ₂ O, 0.002% FeSO ₄ .7H ₂ O, and 0.05% KCl, lyophilized from Curvularia lunata (NRRL 2380). culture and shake the flask on a rotary shaker for 60 hours at 30 ° C. This culture (250 ml) was inoculated with 15 L of medium sterilized at 121 ° C and 2.1 atm in a 20 L pre-fermenter containing 2% glucose and 2% corn jam and pH 6. , Set to 5. Aeration (10 L / min) at 29 ° C and 0.7 atm while adding Silicon SH as an antifoam agent and stirring

-4183 154 (220 rpm) was grown for 24 hours. Subsequently, 1.5 L of the culture was removed under sterile conditions, and a 20 L main fermentor containing 14 L of sterilized medium was inoculated.

This medium contains 3% corn jam and 0.7% glucose and is adjusted to pH 6.5. After 12 hours of growth under the same conditions as the pre-fermentation, 7.3 g of 17,21- (1-methoxyethylidenedioxy) -D-homo-4-pregnen-3,20-dione dissolved in 100 ml of dimethylformamide were added. under sterile conditions and further agitated and aerated. The progress of the fermentation process is monitored by periodically taking a sample, extracting it with methyl isobutyl ketone and analyzing it by thin layer chromatography. Within 48 hours, substrate conversion is complete.

The fermentation broth is filtered, the filtrate and the filtered mycelium are extracted with methyl isobutyl ketone, the extracts are combined and first concentrated in a rotary evaporator and then evaporated to dryness in a rotary evaporator at 50 ° C in a vacuum. The residue is dissolved in warm methanol, the insoluble silicone oil is filtered off and the silicone free solution is again evaporated to dryness. The residue was chromatographed on a silica gel column (gradient: 4.5 L methylene chloride / 0.5 L acetone to 4 L methylene chloride / 1 L acetone) and crystallized from acetone. This gives 5.7 g of D-homobihydrocortisone (110.17 µL, 2L-trihydroxy-D-homo-4-pregnen-3,20-dione), m.p. 212-30 215 ° C (dec). .

Example 6

(a) A solution of 1.5 g of pyridinium tosylate and 15 g of 17a, 21-dihydroxy-35-D-homo-1,4-pregnadiene-3,20-dione in 120 ml of dimethylformamide is diluted with 800 ml of benzene and then 120 ° C. At bath temperature C, 250 ml of benzene are distilled off on a water separator. To the hot reaction solution is slowly added 40 ml of orthoacetic acid 40 trimethyl ester and the benzene and other volatile reaction components are further distilled. Pyridine (20 ml) was added and the mixture was completely dried in a water jet vacuum for 2 hours at 60 ° C to leave 18.8 g of a crude crystalline crude oil (18.8 g). A sample is crystallized from isopropyl ether after treatment with activated carbon to give pure 17β, 21- (1-methoxyethylidenedioxy) -D-homo-1,4-pregnadiene-3,20-dione, m.p. 142-144 ° C. 50

(b) 500 ml of medium containing 3% glucose, 1% corn jam, 0.2% NaNO ₃ in a 2 L Erlenmeyer flask autoclaved for 30 minutes at 120 ° C,

0.1% KH ₂ PO ₄ , 0.2% K ₂ HPO ₄ -0t, 0.05% MgSO ₄ . 55 .7H ₂ O, 0.002% FeSO ₄ .7H ₂ O was contains 0.05% KCl, Curvularia lunata (NRRL 2380) was inoculated with a lyophilized culture and the flask was shaken for 60 hours at 30 ° C on a rotary shaker set Nai . This culture (250 ml) was inoculated with 15 l of medium sterilized at 121 ° C and 2.1 atm in a 20 L pre-fermenter containing 2% glucose and 2% corn jam and pH 6, Set to 5. Silicon SHnak as antifoaming agent with addition of aeration (101 / min) and stirring (220 rev / min) were grown for 24 hours at 29 ^c C and an overpressure of 0.7 atm. Subsequently, 1.5 l of the culture is removed under sterile conditions and an inoculum of 20 l containing 14 l of sterilized medium as above is inoculated. This medium contains 3% corn jam and 0.7% glucose and its pH is adjusted to 5.5.

After 12 hours of growth under the same conditions as the pre-fermentation, 7.5 g of 17?, 21- (1-methoxyethylenedioxy) -D-homo-1,4-pregnadiene-3,20- was dissolved in 100 ml of dimethylformamide. dione was added under sterile conditions and further stirred and aerated. The progress of the fermentation process is monitored by occasionally taking a sample, extracting it with methyl isobutyl ketone and analyzing it by thin layer chromatography. Within 52 hours the substrate transformation is complete. The fermentation broth is filtered, the filtrate and the filtered mycelium are extracted with methyl isobutyl ketone, the extracts are combined and first concentrated to 100 ml in a rotary evaporator and then at 50 [deg.] C. in a vacuum rotary evaporator and allowed to crystallize at room temperature. The product is aspirated and dried in vacuo. 6.1 g of D-homo-prednisolone (110.17a, 21-trihydroxy-D-homo-1,4-pregnadiene-3,20-dione) are obtained, m.p. 226-228 ° C.

Claims (1)

A patent claim A process for the preparation of 110-hydroxy steroids of formula I wherein: is a bond or a double bond, X is hydrogen or methyl, and V is a methylene group or an ethylene group characterized in that an 11-deoxy steroid of formula II wherein X and V are as defined above, R 1 is a hydrogen atom or a C 1-4 alkyl group, and R ₂ is C 1-4 alkyl, It is fermented with a microorganism belonging to the species Cu vularia lunata.