IE47691B1

IE47691B1 - Process for the manufacture of 21-hydroxy-20-methylpregnane derivatives

Info

Publication number: IE47691B1
Application number: IE2486/78A
Authority: IE
Original assignee: Schering Ag
Priority date: 1977-12-19
Filing date: 1978-12-18
Publication date: 1984-05-30
Also published as: GB2010276B; DK144708C; DE2861249D1; IE782486L; EP0002535A2; YU285578A; AT363625B; EP0002535A3; CS202517B2; ATA904778A; EP0002535B1; DD139859A5; DK570578A; JPS6350000B2; SU862830A3; GB2010276A; JPS5581A; HU181505B; DE2757156A1; DK144708B

Abstract

A steroid of the formula in which @ is a single or double bond is manufactured by fermenting at pH 6 to 8 a sterol containing a free hydroxyl group in the 3-position or a corresponding compound containing another oxygen- containing function in the 3- position with a culture of a microorganism selected from Mycobacterium spec. NRRL B-3683 and B-3805 and their variants and mutants, at least a part of the fermentation being carried out in the presence of a boron- containing agent selected from borate ions and organic boron compounds.

Description

The present invention is concerned with a process for the manufacture of 21-hydroxy-20-methyl-pregnane derivatives.

It has been known that cultures of Mycobacterium spec.

NRRL B -3683 are able to form 2l-hydroxy-20-methyl-pregnane derivatives of the general formula I'given below from zooor phytosterols (Applied Microbiology 23, 1972, 72 et seq. and Applied and Environmental Microbiology 32, 1976, 310 et seq.). However, these compounds are formed only in trace amounts or in very small yields, so that this known XO process can hardly be used on an industrial scale.

The present invention provides a process for the manufacture of a 21-hydroxy-20-methyl-pregnane derivative of the general formula I in which the bond in the 1,2-position is a carbon-tocarbon single bond or a carbon-to-carbon double bond, wherein a sterol, as hereinafter defined, is fermented at a pH-value within the range of from 6.0 to 8.0 with a culture of a microorganism selected form Mycobacterium spec. NRRL B-3683 and Mycobacterium spec. NRRL B-3805 and their variants and mutants, at least a part of the fermentation being carried out in the presence of a boroncontaining agent selected from borate ions and organic boron compounds.

The term sterol is understood herein to include not only a sterol containing a free hydroxyl group in the 3-position but also a corresponding compound containing another oxygen-containing function in the 3-position.

By means of the process of the present invention there can be obtained significantly higher yields of 21-hydroxy-20methyl-pregnane derivatives of the general formula I than by the aforesaid known process.

As the sterol starting material there may be used a zoo20 or phyto-sterol of the general formula II 4789 1 in which each of the .... bonds in the 4,5- and 5,6-positions is a carbon-to-carbon single bond or one of the bonds is a carbon-to-carbon single bond and the other is a carbon-to5 carbon double bond, the . ., bond in the 22, 23-position is a carbon-to-carbon single bond or a carbon-to-carbon double bond, X represents an oxo group or a hydrogen atom together with a free or functionally modified hydroxyl group and R^ represents a hydrogen atom, a methyl group or an ethyl group.

Suitable zoo- or phyto-sterols for the fermentation are, for example, chloesterol, stigmasterol, campesterol, brassicasterol or the sitosterols, the 3-acyl-derivatives, for example the acetates, of these sterols, or the 3-keto15 Δ -steroids obtainable from these sterols by, for example, the Oppenauer oxidation, for example A4-chiesten-3-one, A4 -stigmasten-3-one, a Δ4 -sitosten-3-one or Δ4 -campesten3-one. - 5 In accordance with the process of the present invention the fermentation is carried out in the presence of borate ions and/or organic boron compounds. The fermentation maybe carried out in the presence of borate ions by adding an agent yielding borate ions, for example orthoboric acid, metaboric acid or poly-horic acid or an alkali or alkaline earth metal salt thereof, for example sodium metaborate, disodium tetraborate or calcium metaborate. The agent yielding borate ions is preferably used in an amount of 1 gram to 5 grams per litre of the culture.

As the organic boron compound that may be used for the fermentation there may be mentioned, for example, triphenyl borate. The organic borate compound is likewise preferably used in an amount of 1 gram to 5 grams per litre of the culture.

The boron-containing agent used for the fermentation according to the process of the present invention is preferably added 10 to 30 hours after the beginning of the fermentation.

On the addition of the agents yielding borate ions care must be taken that the pH-value of the fermentation culture is adjusted in the usual manner to a value between 5.0 and 8.0.

In other respects the fermentation is carried out under the usual conditions. The microorganisms are grown in. submerged cultures in a suitable nitrient medium with aeration. The substrate (dissolved in a suitable solvent or preferably in an emulsified form) is then added to the cultures and fermentation is carried out until maximum conversion of the substrate has been achieved (about 96 to 160 hours).

Suitable substrate solvents are, for example, methanol, ethanol, glycol monojmethyl ether, dimethyl-formamide and dimethyl sulphoxide. The emulsification of the substrate may be effected, for example, by injecting it in micronized form or dissolved in a water-miscible solvent (for example methanol, ethanol, acetone, glycol monomethyl ether, dimethylformamide or diemthyl sulphoxide) with strong turbulence into water (preferably decalcified) which contains the usual emulsification assistants. Suitable emulsification assistants are non-ionic emulsifiers, for example ethylene oxide adducts or fatty acid esters of polyglycols. As suitable emulsifiers there may be mentioned by way of example V the usual commercial wetting agents Tegin ( Trade Mark), Tagat, Tween ( 'Trade Mark) and Span (Registered Trade Mark).

In the fermentation the emulsification of the substrate often enables the throughput of substrate to be increased and consequently the concentration of substrate to be increased. However, it Is of course also possible In the process of the present invention to use other methods of increasing the throughput of substrate, for example those 47681 that are well known to the fermentation expert.

The optimum concentration of substrate, the time at which to add the substrate and the duration of the fermentation depend on the structure of the substrate used. These factors must be determined, as is generally necessary in microbiological conversions of steriods, in each particular case by preliminary tests of the type that are familiar to the expert.

A further increase in the yield of the products of the process of the present invention can be obtained by the selection or mutation of the Mycobacteria species in the usual manner.

Thus, for example, Mycobacterium spec. NRRL B-3805 advantageously after treatment with mutagens - can be spread out on blood-agar plates, whereby separate colonies of different morphological appearance are obtained. These separate colonies are isolated and tested for their ability to form 21-hydroxy-20-methyl-pregnane derivatives of the general formula I , and in this way it is found that, especially among the selection strains that form round colonies, there are strains that form a yield of 21hydroxy-20-methyl-pregnane derivatives 1.5 to 3 times higher than does a non-selected strain.

The 21-hydroxy-20-methyl-pregnane derivatives of the general formula I are valuable intermediates for the synthesis of pharmacologically active steriods. Thus, 4769 1 for example it is possible to oxidise these compounds to form the corresponding pregnane-20-carboxylic acids of the general formula III which in accordance with the process described by H.Ruschig et al. (Chem. Ber. 88, 1955, 883 et seq.) can be converted into the corresponding pregnane - 3,20 - dione derivatives of the general formula IV.

(Y = hydrogen or 17a -hydroxy) The compounds so obtained are distinguished, as is known, by their gestagenic activity and are also valuable intermediates for the synthesis of numerous pharmacologically active steriods.

The following examples illustrate the invention: Example 1 (a) An Erlenmeyer flask having a capacity of 2 litres and charged with 500 ml of a sterile nutrient medium containing 1% of yeast extract 0.45% of disodium hydrogen phosphate 0.35% of potassium dihydrogen phosphate 0.2% of Tagat 02 - adjusted to a pH-value of 6.7 15 was inoculated with a washing of a dry culture of Mycobacterium spec. NRRL B-3805 and agitated for 3 days at o C and at 190 revolutions per minute . (b) 22 gm of sitosterol were emulisifed for 25 minutes with 4.4 gms of Tegin and 430 ml of water at 95°C by means of an Ultra Turrax ( Trade Mark - firm Jahnke and Kunkel) and then made up to 513 gms with water. The emulsion was sterilized for 20 minutes at 120° C. (c) An Erlenmeyer flask having a capacity of 500 ml and charged with 65 ml of a sterile nutrient medium containing gms of cornsteep liquor 0.3 gm of diammonium hydrogen phosphate 0.25 gm of Tagat 02 - adjusted to a pH-value of 6.5 was inoculated with 5 ml of the grown Mycobacterium spec, culture prepared as described under 1 (a). There were then added 28 ml of the emulsion (corresponding to 1.2 gms of sitosterol) prepared as described under 1(b) and after 24 hrs 4 ml of an aqueous solution of 4% strength of disodium tetraborate, and the whole was further fermented for 120 hours at 30°C while agitating.· After the fermentation, the culture liquor was extracted twice with 100 ml of ethylene chloride each time. To the combined ethylene chloride extracts were then added 11 gms of active carbon and the mixture was filtered through a folded filter. The filtrate was then concentrated at after 40°C in a rotary evaporator and/chromatography there were obtained 135 mg of 21-hydroxy-20-methyl- Δ4 -pregnen-3one melting at 140 - 141°C (from ethyl acetate).

When the fermentation was carried out under the same conditions, but without the addition of the disodium tetraboarate solution, there were obtained 45 mg of 21-hydroxy-20-methyl-A^-pregnen-3-one. - 11 Example 2 an (a, From 22 gms of Δ -cholesten-3-one 513 gms of/emulsion were prepared as described in Example 1(b). (b) To 70 ml of a culture prepared as described in Example 1(a) by inoculating a nutrient medium with a grown Mycobac5 terium spec. NRRL B-3805 culture was added 28 ml of the Δ4 -cholesten-3-one emulsion prepared as described under 2(a) and after 24 hours 4 ml of an aqueous solution of 4% strength of disodium tetraborate, and the whole was fermented for a further 120 hours at 30°C while agitating.

The fermentation batch was worked up as described in Example 1(c) and 95 mg of 21-hydroxy-20-methylpregnen-3-one melting at 142 - 144°C were obtained.

When the reaction was carried out under the same conditions, but without the addition of the disodium tetraborate solution Λ 4 there were obtained 40 mg of 21-hydroxy-20-methyl-Δ pregnen-3-one.

Example 3 ml of the sitosterol emulsion prepared as described in Example 1(b) were fermented as described in Example 1(c) with 70 ml of a Mycobacterium spec. NRRL B-3805 culture, and instead of the aqueous solution of disodium tetraborate of 6 ml of a suspension of 4% strength in water of calcium metaborate was used. - 12 After working up the fermentation batch as described in Example 1(c) there were obtained 120 mg of 21-hydroxy20-methyl- Δ4 -pregnen-3-one melting at 141 - 143°C.

Example 4 28 ml of the sitosterol emulsion prepared as described in Example 1(b) were fermented as described in Example 1(c) with 70 ml of a Mycobacterium spec- NRRL B-3805 culture, and instead of the aqueous solution of disodium tetraborate an addition of 5 ml of a solution of 4% strength of triphenyl borate was used.

After working up the fermentation batch as described in Example 1(c), there were obtained 125 mg of 21-hydroxy20-methyl- Δ4- pregnen-3-one melting at 142-143°C.

Example 5 (a) A Mycobacterium spec. NRRL B-3683 culture was grown under the conditions described in Example 1(a). (b) To 65 ml of the nutrient medium described in example 1(c) were added 5 ml of the grown Mycobacterium spec.

NRRL B-3683 culture prepared as described under 5(a).

There were then added to the culture 28 ml of the sitosterol emulsion prepared as described in Example 1(b) and after 24 hours 4 ml of a solution of 4% strength of disodium tetraborate, and the whole was further fermented for 120 hours. The fermentation batch was worked up as described in Example 1(c) and there were obtained 90 mg of 2147691 hydroxy -20- methyl- A4-pregnen-3-one melting at 142 - 143° C.

Example 5 (a) A Mycobacterium spec. NRRL B-3683 culture was grown under the conditions described in Example 1(a). (c) To 65 ml of the nutrient medium described in Example 1(c) were added 5 ml of the grown Mycobacterium spec.

NRRL B-3683 culture prepared as described under 1(a).

There were then added to the culture 28 ml of the sitosterol emulsion prepared as described in Example 1(b) and after 24 hours 4 ml of a solution of 4% strength of disodium tetraborate, and the whole was further fermented for 120 hours. The fermentation batch was worked up as described in Example 1(c) and there were obtained 90 mg of 21-hydroxy20-methyl- A^'4-pregnadien-3-one melting at 180 - 182°C (from ethyl acetate-acetone).

Example 6 ml of a grown culture of Mycobacterium spec. NRRL B3805 prepared as described in Example 1(a) - were centrifuged at 4000 revolutions per minute.

The bacterial mass so obtained was then washed twice with a salt solution buffered to pH 6 containing 0.5% of sodium chloride, 0.012% of magnesium sulphate (Hepta-hydrate) and 1.36% of potassium dihydrogen phosphate, and then suspended in 40 ml of such a salt solution and 10 ml of a 0.5% - 14 l-methyl-3-nitro-l-nitrosoguanidine were added.

The bacterial suspension was incubated for one hour at 30°C, and the bacteria were centrifuged off, washed twice with a salt solution as defined above and spread out on blood-agar plates (manufacturing firm Oxoid Ltd,, London) From among the individual colonies formed there were chosen those forming round colonies, and from the latter grown cultures were produced as described in Example 1(a), The grown colonies were used to carry out the fermentation described in Example 1(c) and the following results were obtained.

No. of the tested selection strains. - The yield of 21-hydroxy-20-methyl-Δ^-pregnen- 3-one Without addition of disodium tetraborate With addition of disodium tetraborate 5 19 0 - 200 mg — 8 201 - 400 mg — 94 401 - 600 mg -- 35 601 - 800 mg — 7 801 - 1000 mg 1550 - 2100 mg 10 0 1001 - 1020 mg — 6 1021 - 1040 mg 1900 - 3000 mg 1 1041 - 1060 mg (1050 mg) 2900 mg 0 1061 - 1080 mg __ 15 1 1081 - 2000 mg 3450 mg (1095 mg)

Claims

1. A process for the manufacture of a 21-hydroxy-20-methyl -pregnane derivative of the general formula I 5 in which the ... bond ’ in the 1,

2. -position is a carbonto-carbon single bond or a carbon-to-carbon double bond, wherein a sterol, as hereinbefore defined, is fermented at a pH-value within the range of from 6.0 to 8.0 with a culture of a microorganism selected from Mycobacterium 10 spec. NREL B-3683 and Mycobacterium spec. NRRL B-3805 and their variants and mutants, at least a part of the fermentation being carried out in the presence of a boroncontaining agent selected from borate ions and organic boron compounds. 15 2. A process as claimed in claim 1, wherein the sterol is a zoo- or phyto-sterol of the general formula II in which each of the ____bonds in the 4,5- and 5,6-positions is a carbon-to-carbon single bond or one of the bonds is a carbon-to-carbon single bond and the other, is a carbon5 to-carbon double bond, the .. bond in the 22,23-position is a carbon-to-carbon single bond or a carbon-to-carbon double bond, X represents an oxo group or a hydrogen atom together with a free or functionally modified hydroxyl group and represents a hydrogen atom, a methyl group or 10 an ethyl group.

3. A process as claimed in claim 1, wherein the sterol is cholesterol, stigmasterol, campesterol, brassicasterol or a sitosterol.

4. A process as claimed in claim 1, wherein the sterol 15 is a 3-acyl derivative of cholesterol, stigmasterol, campesterol, brassicasterol or a sitosterol. 478 91 - 18

5. A process as claimed in claim 4, wherein the 3-acyl derivative is the 3-acetate.

6. A process as claimed in claim 1, wherein the sterol is Δ 4 -cholest-3-one, Δ 4 -stigmasten-3-one, a Δ 4 -sitosten5 3-one or A 4 -campesten-3-one.

7. A process as claimed in any one of claims 1 to 6, wherein the fermentation is carried out in the persence of borate ions yielded by orthoboric acid, metaboric acid or polyboric acid or an alkali or alkaline earth metal 10 salt thereof.

8. A process as claimed in claim 7, wherein the borate Ions are yielded by sodium metaborate, disodium tetraborate or calcium metaborate.

9. A process as claimed in claim 7 or 8, wherein the 15 acid or salt is used in an amount of 1 gram to 5 grams per litre of the culture.

10. A process as claimed in any one of claims 1 to 6, wherein the fermentation is carried out in the presence of an organic boron compound, the compound being triphenyl 20 borate.

11. A process as claimed in any one of claims 1 to 6, and 10, wherein the organic boron compound is used in an amount of 1 gram to 5 grams per litre of the culture. 47631

12. A process as claimed in any one of claims 1 to 11, wherein the boron-containing agent is added 10 to 30 hours after the beginning of the fermentation.

13. A process as claimed in claim 1, conducted substantially 5 as described herein.

14. A process as claimed in claim 1, conducted substantially as described in any one of Examples 1 to 6 herein.

15. A 21-hydroxy-20-methyl-pregnane derivative of the general fonnula I given in claim 1 in which the .... bond 10 in the 1, 2-position has the meaning given in claim 1, whenever made by the process claimed in any one of claims 1 to 14.