US3285830A - 16-oxygenation of steroids by stagonospora species - Google Patents

Description

United States Patent 3,285,830 16-0XYGENATIUN 0F STEROIDS BY STAGONOSPORA SPECIES Jan de Flines and Willem Frederik van der Waard, Delft, Netherlands, assignors, by mesne assignments, to Koninklijke Nederlandse Gist & Spiritusfabrieken, a corporation of the Netherlands No Drawing. Filed Oct. 15, 1963, Ser. No. 316,433 Claims priority, application Netherlands, Oct. 16, 1962, 284,411 9 Claims. (Cl. 19551) The invention relates to the oxygenation of steroids at position 16 with the aid of micro-organisms.

In the present patent application the term steroid compounds is used to denote compounds having a cyclopentanoperhydrophenanthrene skeleton, particularly those having a methyl group at positions and 13 of said skeleton. Suitable starting substances are therefore not only steroids of the normal series (10,8, 9a-C0nfiguf3- tion) but also steroids of the retro-series (IOa-methyl, 9/3-configuration) and the pyro-series (IOa-methyl, 9nc-COI1fi guration) The starting substance may contain different substituents, for example one or more etherifier, non-etherifier, esterified or non-esterified hydroxy-groups, one or more keto-oxygen atoms, one or more alkyl-groups with 1 to 6 carbon atoms, which may be unsaturated, one or more halogen atoms, for example Cl, Br or F. There may be hydroxy-groups at the positions 1, 2, 3, 6, 11, 17 or 21 (however, not in l7a-position), keto oxygen atoms at the positions 3, 11 and/or 20 and alkyl-groups such as methylor ethyl-groups at the positions 1, 2, 4, 6' or 17, however in the latter case not at the Uri-position, in conjunction with a 17a-hydroxy-group. The starting substances must furthermore contain one or more double bonds e.g. starting from the carbon atoms 1, 2, 3, 4, 5, 6, 14 or 17 (20). Preferably the starting steroid contains a 3-keto-A a 3-keto-A or a 3-hydroxy-A -group or groups functionally derived therefrom e.g. ethers or esters.

Suitable starting materials are 6-dehydro-retro-progesterone, retro-progesterone, pyro-progesterone, retro-testosterone or l7B-alkyl-retro-testosterone.

The steroids oxygenated at position 16 have an interesting pharmacological activity either themselves or subsequent to the conversion into other steroids. The l6-hydroxy-group can be converted in known manner into other groups. The 16-hydroxy-steroids are therefore important intermediate products in the production of pharmacologically interesting steroids. It should be noted that with the introduction of a l6-hydroxy group in accordance with the method of this invention it is probably orientated in the Ot-POSlllOIl.

It is known that a hydroxy-group at the position 16 of a normal (lop-methyl, 9a)Sl8IOld can be introduced by contacting such a steroid, having two hydrogen atoms at position 16, with the enzyme system of certain microorganisms. Mention may be made of Pestalotia funerea of the class of the Fungi Imperfecti.

Among the genus of Streptomyces the species of S. roseochromogenus (Waksman Collection 3689), S. sp. ATCC 11.009, S. roseoclzromogenus (ATCC 3347) are also suitable for the introduction of a 16-hydroxyl-group into steroids. Other species of Streptomyces, which could also be suitable for the introduction of hydroxy-group at position 16 are: S. viridus and S. olivaceus.

In accordance with the invention it has been found that a hydroxyl-group at osition 16 of steroids can be introduced with the aid of the enzyme system of fungi of the family of Sphaerioidaceae, of the order of the Sphaeropsidales, forming part of the class of the Fungi Imperfecti.

3,285,830 Patented Nov. 15, 1966 ICC bon atom 1 of normal steroids and that fungi of said order, i.e. those of the family of Sphaerioidaceae can be employed for the selective introduction of a ZI-hydroxygroup into normal steroids.

The invention relates to a method of oxygenating steroids with the aid of enzyme systems or the spores of micro-organisms of the family of Sphaerioidaceae and is characterized in that a steroid having two hydrogen atoms at position 16 is converted into a steroid having a ketooxygen atom or a hydroxy-group at position 16.

It has been found that particularly micro-organisms of the species of Stagonospora curtisii (Berk. et Cke.) Sacc. are suitable for the method according to the invention.

It has furthermore been found that the invention is particularly advantageous with steroids of the IDOL-methyl, 9,3-series (retro-steroids). With conversions according to the invention high yields of more than were found in this series, whilst substantially no disturbing byproducts were found in the ultimate processing.

A further advantage consists in that the culture medium can be made from cheap raw material and requires only a short incubation period with normal aeration.

The method according to the invention is carried out in a manner similar to the known microbiological conversions, for example, by contacting the substrate under suitable conditions with a culture of a fungus as mentioned above and/or enzyme systems thereof. To this end, for example, first a culture of the fungus is developed under aerobic conditions in a nutrient solution, after which a fermentation medium containing the steroid to be oxygenated, which may be added in a solution or a suspension, is subjected to the oxybiontic dissimilation activity of the mycelium formed. The nutrient solution consists mainly of a carbon source and a nitrogen source, for example a carbohydrate e.g. glucose, maltose or starch and an organic nitrogen source e.g. corn steep liquor, or yeast extract, protein hydrolysates, amino acids or an inorganic nitrogen source e.g. ammonium salts or alkaline metal nitrates. To the medium containing the steroid to be oxygenated and one or more of the aforesaid nutrient sources may, if desired, be added an antifoam agent, for example glycerylmonostearate.

The most suitable fermentation temperature usually lies between 20 C. and 28 C., although higher or lower temperatures between 15 C. and 35 C. are, in general, also suitable.

The pH value of the medium can be adjusted in a conventional manner and is preferably brought to a value between 6 and 7.

The time required for the oxidation of the steroid may vary between wide limits, but usually the oxygenation period is at an optimum between 10 and 48 hours for full conversion.

The l6a-hydroxy-steroid compounds obtained after the termination of the oxygenation process may be separated out of the medium and/or the mycelium in any of the known manners, preferably by extraction with organic solvents not miscible with water, e.g. diethylether, ethylacetate, amylacetate, methylisobutylketone or other esters and ketones. Particularly methylisobutylketone is a suitable extraction agent.

The oxygenated steroid may also be isolated and purified by chromatographic methods in conjunction or not in conjunction with extraction from the fermentation me dium.

In accordance with the invention the l6-hydroxysteroids may also be formed by reacting spores of the said microorganisms with solutions or dispersions of the starting steroids mentioned above.

The invention will be described more fully with reference to the following examples.

Example I.Cnversi0n 0f ren -progesterone into 1 6 ot-hydroxy-retro-progesterone A nutrient solution containing gms. of inspissated corn steep liquor, gms. of glucose and 5 grns. of caseine hydrolysate per 1000 mls. of tap water, subsequent to the adjustment of a pH value of 6.4 by 2 N sodium hydroxide and to sterilisation with 120 C. for 20 minutes, was inoculated with a culture of Stagonospora curtisii grown on oats agar per portion of 250 mls. in shaking flasks of 1000 mls. These inoculation cultures were shaken for 24 hours at 27 C. on a rotating shaking machine (250 rev./min.), after which the mycelium formed of four shaking flasks was transferred to a fermentation tank of 100 litres of stainless steel, provided with an agitator and an aerator and containing the following fermentation medium, sterilised under the aforesaid conditions:

Corn steel liquor (calculated on the dry substance) gms 200 Glucose grns 200 Glycerylmonostearate gms 25 Tap water to liters 40 pH=6.4 (sodium hydroxide solution) To said fermentation medium was added under aseptic conditions 13.7 grns. of retro-progesterone, suspended in 300 mls. of sterile water, after which for 48 hours the fermentation took place at 26 C. (speed of 140 rev./ min.); supply of air: 0.7 m. /m. of bottom surface per minute.

The filtered culture (39.2 litres) was then extracted three times with 8 litres of methylisobutylketone. The collected extracts were evaporated to 1000 mls. and then washed with aqueous sodium hydroxide solution and Water and finally treated with activated carbon. The filtered extract was then evaporated in vacuo to 100'm1s., from which subsequent to cooling 12.3 gms. of crystalline product was obtained. The crystallisate was recrystallised from 60 mls. of methanol-water (2:1) so that 12.05 gms. of white crystalline final product was obtained, which was identified to be 16ot-hydroxy-retroprogesterone having a melting point of 173l74.5 C.

(a) =92.3 (c.=1%, chloroform).

U.V. absorption spectrum in methanol: A 244 m (e=16,900).

Elementary analysis: C, 76.20%; H, 9.19%. Calculated for C H O C, 76.32%; H, 9.15%.

By evaporation to dryness and recrystallisation the mother liquors yielded a further 0.85 gm. of pure product.

Example lI,-Conversi0n of 6-dehydro-retra-progesterone into. 6-dehydr0-J 6a-hydr0xy-retr0-pr0gester0ne A 24-hours inoculation culture as described in Example I of 2.5 litres was transferred to a fermentation tank of 400 litres, provided with an agitator and an aerator and containing 250 litres of nutrient medium consisting of a sterilised solution of 3 kgs. of inspissated corn steep liquor (calculated on the dry substance) and 2 kgs. of glucose in tap water, to which 250 gms. of glycerylmonostearate was added. While stirring (speed 200 rev./ min.) and aeration (0.7 m. /m. of bottom surface per minute), 120 gms. of 6-dehydro-retro-progesterone was blown in under aseptic conditions in a finely divided state after 12 hours cultivation, after which the fermentation was continued for 36 hour and then terminated. The culture filtrate was processed in the manner described in Example I and in total 101.5 gms. of crystalline final product was obtained, which was identified to be 6-dehydro- 16a-hydroxy-retro-progesterone. The product melted at 202.5 to 204 C.

(00 520 (c.=1%, chloroform).

U.V. absorption spectrum in methanol: A 286 m (e=26,600).

Elementary analysis: C, 77.15%; H, 8.40%. lated for C H O C, 76.79%; H, 8.59%.

Example III.ConversiOm 0] retro-testosterone into 1 6ot-hydr0vty-retr -testosteron'e To ten, 24 hours aged shaked cultures of Stagonospora curtisii of 1000 mls. each (nutrient medium: inspissated corn steep liquor (calculated on the dry substance) 1%, glucose 1%, yeast extract 0.1%, primary ammonium phosphate 0.1%) was added per culture 200 mgs. of retro-testosterone in 15 mls. of acetone. After an incubation of 28 hours (speed 250 rev./min.) at 26 C. the mixture of the cultures was separated by filtration from the mycelium and then extracted four times with 1.5 litres of methylisobutylketone. The extract was concentrated in vacuo to 350 mls., then washed with 4 N sodium hydroxide in water and treated with activated carbon. The extract thus treated was evaporated in vacuo to 60 mls. 1.65 grns. of crystallineproduct was separated out. The mother liquor was then evaporated to dryness and the residue was recrystallised from a mixture of 4 mls. of acetone and 6 mls. of heptane after a treatment with activated carbon. A further 0.27 gms. of crystallisate was obtained. The total product was then recrystallised from 35 mls. of methanol-water (2:1), whereupon 1.84 gins. of the product was obtained, which was identified to be 16a-hydroxy-retro-testosterone, having a melting point of 210212 C.

Elementary analysis: C, 74.84%; H, 9.21%. Calcu- Iated for C19H28O3: C, H, 9.27%.

U.V. absorption spectrum in methanol: )t 242 mu (e=16,600).

What is claimed is:

1. A method of converting a steroid into a 16cc hydroxy derivative thereof which comprises subjecting a steroid selected from the group consisting of steroids of the pregnane and androstane series containing at least one carbon to carbon double bond at the position 1, 2, 3, 4, 5, 6, 14 and 17(20) and free of hydroxy at the 17m position, to the action of enzymes of a microorganism of the genus Stagonospora under oxidizing conditions and recovering the resultant 16a hydroxy steroid.

2. The method of claim 1 in which the steroid contains methyl in the 1011 position.

3. The method of claim wherein the microorganism is Stagonospora curtisii.

4. The method of claim 1 wherein the steroid contains a moiety selected from the group consisting of 3-keto-A 3-keto-A and 3-hydroxy-A 5. A method of converting a steroid into a 16a hydroxy derivative thereof which comprises subjecting a steroid selected from the group consisting of steroids of the pregnane and androstane series containing at least one carbon to carbon double bond at the positions 1, 2, 3, 4, 5, 6, 14 and 17(20) and free of hydroxy at the 170: position, to the action of spores of a microorganism of the genus Stagonospora under oxidizing conditions and recovering the resultant 16ot hydroxy steroid.

6. A method of preparing 16a hydroxy retro-progesterone which comprise subjecting retro-progesterone to the action of enzymes of Stagonospora curtisii under oxidizing conditions and recovering the resultant thydroxy-retro-progesterone.

7. A method of preparing 6-dehydro-16a hydroxy retro-progesterone which comprises subjecting 6-dehydroretro-progesterone to the action of enzymes of Stag0n0spara curtisii under oxidizing conditions and recovering the resultant 6-dehydro-16a-hydroxy retro-progesterone. 8. A method of preparing 16a-hydroxy retro-testosterone which comprises subjecting retro-testosterone to the action of enzymes of Stagonospora curlisl'i under oxidizing conditions and recovering the resultant 16a-hydroxyretro-testosterone.

Calcu- 9. A method of converting a steroid into a 16a hy- References Cited by the Examiner droxy derivative thereof which comprises subjecting a steroid selected from the group consisting of steroids of UNITED STATES PATENTS the pregnane and androstane series containing at least 2,777,843 1/1957 Chemerda et a1. 19551 one carbon to carbon double bond at the postions 1, 2, 5 3,054,725 9/1962 Ilavsky et a1. 19551 3, 4, 5, 6, 14 and 17(20) and free of hydroxy at the 1701. position, to the action of enzymes of a microorganism A LOUIS MQNACELL, Pri Exa in of the genus Stagonospora under oxidizing conditions and recovering the resultant 16a hydroxy steroid by extrac- STEPHENS, Assistant Exammertion with methylisobutyl ketone. 10

Claims (1)

1. A METHOD OF CONVERTING A STERIOD INTO THE 16A HYDROXY DERIVATIVE THEREOF WHICH COMPRISES SUBJECTING A STERIOD SELECTED FROM THE GROUP CONSISTING OF STEROIUS OF THE PREGNANE AND ANDROSTANE SERIES CONTAINING AT LEAST ONE CARBON TO CARBON DOUBLE BOND AT THE POSITION 1, 2, 3, 4, 5, 6, 14 AND 17(20) AND FREE OF HYDROXY AT THE 17A POSITION, TO THE ACTION OF ENZYMES OF A MICROORGANISM OF THE GENUS STAGONOSPORA UNDER OXIDIZING CONDITIONS AND RECOVERING THE RESULTANT 16A HYDROXY STEROID.