DE1568932B - Process for the production of 1,4 an drostadien 3,17 dione and 4 androsten 3,17 dione by microbiological degradation - Google Patents

Description

For the manufacture of steroid hormones, naturally occurring steroids are used as the starting material used. However, there was no method to find steroids that had an aliphatic side chain in the 17-position contain at least 8 carbon atoms, easily convertible into 17-ketosteroids, which are used as starting compounds for the production of other steroids with androgenic, estrogenic, can serve anabolic and conception-preventing effect.

It was known that microorganisms of the genus Mycobacterium are able to use steroids an aliphatic side chain with at least 8 carbon atoms in the 17-position, such as cholesterol, completely dismantle (see Zentralblatt Bakteriologie, II, Abt. 37 [1913], p. 595).

In 1942 T a k devoted a research paper to the same phenomenon, in the course of which he isolated a bacterium which he named Mycobacterium cholesterolicum (cf.Ant. van Leeuwenhoek, Vol. 8 [1942], p. 32). After that, a number of other publications appeared on exposure of microorganisms of the genera Proactinomyces, Azotobacter, Flavobacterium, Aerobacter, Pseudomonas, Nocardia and Streptomyces for Cholesterol.

In these processes, cholesterol completely turns into low molecular weight compounds degraded, and there are no significant amounts of compounds with an intact steroid structure on. Occasionally, 4-cholesterol and 7-dehydrocholesterol have been found in small amounts. T a k found small amounts of 4-cholestene-3,6-dione in his investigations.

When breaking down cholesterol with a Nocardia species in the presence of 8-hydroxyquinoline, it was possible to 3-keto-bisnor-4-cholenic acid, 3-ketone-bisnor-1,4-choladienoic acid as well as small amounts of 1,4-androstadiene-3,17-dione and 4-androstene-3,17-dione to be detected (see Biochemical Journal, Vol. 90 [1964], p. 23P).

The action of microorganisms of the genera Nocardia and Mycobacterium on 1,3,5 (10) -cholestatrien brings no degradation (see Abstracts of Papers of the 150th Meeting of the American Chemical Society [1965], 13Q). On the other hand, there were 19-hydroxy-4-cholesten-3-one and 6,19-oxido-4-cholesten-3-one easily converted by these microorganisms into oestrone or 6,19-oxido-4-androstene-3,17-dione.

The invention is based on the surprising finding that the side chain in the 17-position of Steroids with formation of a 17-keto group and maintenance of the steroid structure with microorganisms of the genus Mycobacterium can break down if you use certain parent steroids and the carries out microbiological conversion in the presence of an inhibitor.

The invention thus provides a process for the preparation of 1,4-androstadiene-3,17-dione and 4-Androstene-3,17-dione by microbiological breakdown of steroids with a saturated or unsaturated aliphatic side chain with at least 8 carbon atoms in the 17-position and an oxygen function in the 3-position using a microorganism of the genus Mycobacterium which is characterized in that the microbiological conversion in the presence of nickel, Carries out cobalt, lead, cadmium or selenite ions as an inhibitor.

The starting steroids are z. B. esters of sterols, especially cholesteryl betaine chloride, cholesteryl acetate, Cholesteryl hemisuccinate, sa-chlorocholestanol, or sterols in which the Α ring contains one or more additional substituents, in Ask.

The amount of the inhibitor to be added depends on the circumstances. When the concentration is too low, the steroids will be completely broken down into low molecular weight compounds.

If the concentration is too high, the conversion of the steroid is significantly inhibited and the bacteria are killed. When using NiSO ₄ · 7 H ₂ O, a concentration of 10 to 1000 mg per liter is generally sufficient. When using cobalt chloride, lead acetate, cadmium sulfate or sodium selenite, the concentration is preferably also in the aforementioned range.

Mycobacterium phlei and Mycobacterium are preferably used in the method according to the invention butyricum used.

The method of the invention is preferably carried out as follows:

The microorganism is grown in a nutrient medium that has inorganic or organic nitrogen sources contains, such as amino acids, ammonium salts, alkali nitrates or alkaline earth nitrates. Peptones, Corn steep liquor, yeast extracts, cottonseed meal, soybean meal or soluble residues from alcohol production can also be used to advantage. The culture media do not need an assimilable one Contain carbon source such as sugar, starch or polyhydric alcohols, their presence in the otherwise has no adverse effect on the course of the proceedings.

A nutrient medium suitable for the method of the invention preferably also contains the usual amounts of salts such as phosphates, sulfates, alkali chlorides and alkaline earth chlorides. The necessary Trace elements are then present in sufficient quantities when tap water is used will.

If a complex mixture such as corn steep water is used as the natural nitrogen source,

3 4

so the addition of mineral salts can usually under- The culture is 48 hours at 30 ⁰ C on a

remain. Rotary shaker at a speed of

The nutrient media are sterilized in the usual way - 250 revolutions per minute and a stroke of siert and shaken to a pH value of 4.0 to 8.5 in 2 ¹ I ₂ cm, after which the bacteria stand up. A pH of around 7 is sufficient to have increased in most 5.

Cases preferred. The main fermentation medium is increased by using tap water under constant aeration in thin 5 g corn steep water liquid (calculated from the nutrient medium, as dry matter) with tap water. B. cholesterol or adjusting the liquid to a pH value of 7 / S-sitosterol, are added, either dissolved in io prepared with dilute sodium hydroxide solution. A suitable solvent such as Ν, Ν-dimethyl medium is sterilized in three formamide bottles for 30 minutes, or in the form of a fine aqueous solution at HO ^{0 C.} Each of the three 21 capacity suspensions. Bottle contains 11 culture medium. The bottles

The starting steroid will now be 0 to 72 hours after each with 50 ml of the aforementioned

added to inoculation; inoculated 15 seed cultures even if added later. Then the bottles

quite good results are achieved. The addition shaken for 48 hours at 30 ^{0 C,}

of the steroid is preferably given for 24 to 48 hours.

given after inoculation of the nutrient medium pre-taken sterol in the form of an aqueous suspension,

men, if the microorganisms are sufficiently reduced by grinding the steroid with the addition of

have increased. A very early addition, e.g. B. was obtained immediately 20 water that 0.1% of a nonionic

directly after inoculation, is also possible and contains wetting agent. Furthermore, each bottle came with

gives good results. a sterile aqueous solution of 750 mg NiSO ₄ ■

The conversion of the steroid takes place in general 7H ₂ O offset. This amount corresponds to 157 mg mine with constant aeration at temperatures nickel ions. The bottles are now held further from 20 to 45 ° C; The procedure continues however 25 shakes. Just 12 hours after adding, even at higher or lower temperatures. Cholesterol can be 1,4-androstadiene-3,17-dione. The conversion is preferably allowed to proceed in thin-layer chromatography on silica gel G according to the temperature that is shown to be the optimum. The solvent used is a temperature for the microorganism in question. Mixing of chloroform and ethyl acetate in volume-This temperature is generally around 3 ° ratio 10: 1 used.

3O ⁰ C. It is not necessary that growth and after 3 days of shaking becomes the contents of the three

Conversion at the same temperature in front of bottles and combined three times with the mixture

walk. So z. B. the microorganism first a fifth of its volume methyl isobutyl ketone

grown for 24 hours at 26 ° C, extracted. The extract is reduced under

and after adding the steroid and the inhibitor 35 pressure evaporated to dryness; the residue

the temperature is increased to 30 ⁰ C. is extracted with 20 ml of boiling methanol. Out

The conversion of the steroid takes 1 to 7 days the residue can regain 1.6 g of cholesterol.

in claim; in general, the amount of recovered decreases. The methanol extract is used for

End product does not noticeably increase after three days. Evaporated to dryness and the residue in 8 ml

If compounds are used as starting steroids, 4 ° benzene is added. The benzene solution is columnar

are used that do not contain any further sub-chromatography on 30 g of aluminum oxide (Al ₂ O ₃

contain substituents, the main product is neutral, activity III) purified. As a solvent

l, 4-androstadiene-3,17-dione together with low is a mixture of benzene and acetone (10: 1)

Amounts of 4-androstene-3,17-dione. used. By thin layer chromatography

The products formed can be determined from the culture, in which fractions the 1,4-andro liquid is obtained and in which stage 3,17-dione is present according to customary methods. These fractions are further purified. When the threads are finished, they are caught and evaporated. You can mentation the residue from the culture liquid using an organic solvent such as methyliso- (1: 3) is recrystallized from a mixture of acetone and heptane. Yield 160 mg. After still butyl ketone, ethyl acetate, methylene chloride or chloro 5 ° recrystallization, 138 mg of pure proform are extracted. These extracts are obtained for the domestic product, melting at 137.5 to 139 ⁰ C. Yield evaporated to dryness. The residue is recrystallized from a 13.4%, based on converted consumed chole suitable solvent or if sterol.

necessary z. B. by chromatography and subsequent B e i s d i e 1 2

Crystallization separated into its components. 55

The examples illustrate the invention. According to Example 1, a seed culture of

_,. . . "Mycobacterium phlei, strain KNGSF 75-3 / 53 produced

^Beis P ^IE11 asked.

A seed culture of Mycobacterium phlei. The main fermentation is carried out in three 500 ml fas strain KNGSF 70 is produced by sending 60 bottles, each of which contains 100 ml inoculating a culture of this bacterium on broth nutrient medium. Here, too, the agar and incubation for three days at 3O ⁰ C culture culture in the same concentration, ie of in a nutrient medium that is used from a solution of 10 g 5%, based on the volume of the main yeast extract in 11 tap water of pH 6.8 fermentation medium. The bottles are withstood for 48 hours, previously ^{shaken for 30 minutes at HO 0} C 65 den. After that, each bottle has been sterilized with 200 mg. The nutrient medium contains cholesterol and 50 mg NiSO ₄ · 7H ₂ O added to it in 2 liter bottles, and each bottle is shaken further. After 3 days, the three holds 500 ml of nutrient medium. Bottles total 1,4-androstadiene-3,17-dione in

5 6

an amount from 188.9 to 236 mg. Usually 5 mg of cadmium sulfate is added to each of the bottles,

Even small amounts of 4-androstene-3,17-dione before- After shaking for days, the presence of

act. ... "" "1,4-androstadiene-3,17-dione and 4-androstene-3,17-dione

The yield is between 42.8 and 53.6% as demonstrated by thin layer chromatography

based on the cholesterol used. 5 become. There are also two other unknown spots

visible showing a positive Zimmerman reaction

Example 3 show.

The bottles contain a mixture of 1,4-andro-

According to Example 2, a seed culture of stadien-3,17-dione and 4-androstene-3,17-dione in one

Mycobacterium phlei, strain KNGSF 70 was used. io amount of 29.4 mg. Yield of 10%> based

400 mg of cholesterol are added to each bottle. on used cholesterol.
After 4 days, 93 mg of 1,4-androstadiene

3,17-dione in the manner described in Example 1 Example 9
to be isolated. The amount of unconverted

Cholesterol is 230 mg per bottle. The procedure of Example 7 is repeated, that is to say at least 170 mg of cholesterol in each bottle, however, instead of cobalt chloride, 50 mg are converted. The theoretical yield would be added lead acetate to each of the bottles. After 124 mg are 1,4-androstadiene-3,17-dione. These 2 days can already show an amount of 1,4-androstadiene numbers that the conversion in an ex-3,17-dione can be shown by thin-layer chromatography to yield 75%> based on the converted chole-20,
sterol, and of 31.6%> based on used Bei s Di el 10
Cholesterol.

_R. . . The procedure of Example 7 is repeated,

Example 4, however, instead of cobalt chloride, 50 mg. According to Example 2, the process with Myco-25 sodium selenite is added to each of the bottles. Carried out by bacteriumbutyricum ATCC 11 314. Thin-layer chromatography can isolate and purify the presence as described in Example 1. of 1,4-androstadiene-3,17-dione and 4-androstene-30 mg 1,4-androstadiene-3,17-dione are each 3.17 -dion can be detected.
Bottle received after 3 days of shaking. The yield is 20.4 ° / ₀ > based on the cholesterol used 30 B is ρ ie 11
sterol. .

The procedure of Example 3 is repeated ^{as follows}

Instead of cholesterol, however, 200 mg of 4-cholesterol

According to example 2, the procedure with mycosten-3-one is added to each bottle. After three days

bacterium phlei, strain KNGSF 70. 35 Shaking can be done using the UV absorption spec-

Instead of cholesterol, however, 400 mg trums the presence of 25 to 30 mg 1,4-andro-

/ S-sitosterol added to each bottle. After 4 days stadien-3,17-dione can be found. Yield 16.9

37 mg 1,4-androstadiene-3,17-dione are obtained from each up to 20.2%, based on the 4-cholesten-3-one used. of the bottles according to Example 1 isolated. yield

13.5%> based on the ^ -Sitosterol used. 4.0 B e i s ρ i e 1 12

B eis ρ ie 1 6 _The procedure of Example 3 is repeated at

It is carried out according to the procedure of Example 3 instead of cholesterol, however, 200 mg des

It works, however, 400 mg of stigmasterol in each water-soluble cholesterylbetaine chloride in each of the

of the bottles. given. After 3 days it can be given by 45 bottles. After 4 days of shaking

Thin-layer chromatography, as in Example 1, results in the product in the manner described in Example 1

wrote, 1,4-androstadiene-3,17-dione detected isolated and purified. 20 to 25 mg of 1,4-an

will. . drostadien-3,17-dione and small amounts of 4-andro-

Example 7 sten-3,17-dione was obtained. Yield 18.3 to 22.9% "

50 based on the cholesteryl betaine chloride used.
According to Example 3, a seed culture of

Mycobacterium phlei, strain KNGSF 70 produced, Example 13
instead of 50 mg of nickel sulfate, the same

Amount of cobalt chloride added to each of the bottles. The procedure of Example 4 is repeated on

will. After 2 days of shaking at 30 ° C, 55% of cholesterol can, however, be 200 mg of 5 «-chlorine-

1,4-androstadiene cholestanol was added to each of the bottles by thin layer chromatography. The iso-

3,17-dione and 4-androstene-3,17-dione detected lation and cleaning is carried out according to Example 1. It

will. are 16.7 mg 1,4-androstadien-3,17-dione and

Example 8 3.9 mg of 4-androstene-3,17-dione obtained; the total

60 yield is 15.3%. based on used

It is following the procedure of Example 7 gear-Sa-chlorocholestanol. The products formed are

Working, however, instead of cobalt chloride, 18.9% 4-androstene-3,17-dione are used.

Claims (1)

Claim: Process for the preparation of 1,4-androstadiene-3,17-dione and 4-androstene-3,17-dione by microbiological degradation of steroids with a saturated or unsaturated aliphatic side chain with at least 8 carbon atoms in the 17-position and an oxygen function in the 3-position using a microorganism of the genus Mycobacterium, thereby characterized in that the microbiological conversion in the presence of Performs nickel, cobalt, lead, cadmium or selenite ions as an inhibitor.