DE1593327C - Process for the production of Delta high 1,4 -androstadien-3,17-dione, Delta high 1,4 -androstadien-3,11,17-trione or Delta high 1,4 -androstadien-1 lalpha-ol-3, 17-dione - Google Patents

Description

1 - 2

The invention relates to a method for the micro- b) oxidation of the 3/9-hydroxy group to 3-keto

biological production of steroid compounds group,

with l ^ -diene-S ^ -dione structure. c) Isomerization of the 5,6 double bond to 4,5 double

The possibility of decomposition of the cheapest steroids pelbinding,

(e.g. cholesterol, sitosterol) was used for the first time 5 _{d) expansion de} ; _u _ _{double bond} .

discussed by Hungarian patent specification 148 093, ■> ^ f p

where it has been proven that with the help of simple after implementation attempts these reactions can

Two-ring connections ensure full oxidation of the station both jointly and separately.

roid molecule can be inhibited. These observa- tions are to be carried out. To support this

tion is utilized, among other things, by the process, io lung were Cholesta-l ^ dien-S-on. Cholest-4-en-

after the Androsta-l, 4-diene-3,17-dione with the help of 3-one, Cholest-5-en-3-one and cholesterol with the

Mycobacterium smegmatis from Cholest-4-en-3-one cultures of the microorganism treated. From all

is provided. We have succeeded in these connections, with good

It has recently become known that it is America-yield androsta-1,4-diene-3,17-dione to be obtained,

Niche authors (C. J. S i h and coworkers, J. Am. 15 It was also found that the active

Chem. Soc, 87, p. 2765 [1965]) has also been successful. Microorganism on the one hand has a significant

has the side chain of the cho-esterase activity with an advantageous yield, e.g. B. he puts out of Chole-

degrade lesterine derivatives. This method is steryl acetate androsta-l, 4-diene-3,17-dione with the same

to manufacture the in the industry extraordinarily. Yield as from cholesterol; other part forms

important steroids with 1.4-diene-3,17-dione-StruktuY 20 he also in the case of a saturated Α-ring the

not suitable. 1,4-diene-3,17-diketo structure. For example educates

During the experiments on the oxidizing agents from 5'-cholestan-3- / 9-ol, 5a-cholestan-3-one or The ability of various mycobacteria was surprisingly 5x-pregnane-3,20-dione with good yield androstadweise found that the Mycobacterium phlei from the 1,4-diene-3,17-dione. The same reactions can Steroids with 1,4-diene-3.17-dione structure from many of 25 also multiply via the corresponding 17-keto group Steroids of various structures can form mating compounds with androstane skeleton. So was z. B. Androsta-1,4-diene-3,17-dione. So it is possible, for. B. from Androstaus Cholesterol in the presence of 8-hydroxy-quinolin 5-en-3 /? - oI-17-one and androst-4-en-3.17-dione as well good yield obtained. if with good yield androsta-1,4-diene-3.17-dione

The invention relates to a method of manufacturing 30.

development of zl ¹ · ⁴ - androstadien - 3,17 - dione / <d ^{I> 4} -An- These basic reactions were also carried out on others

drostadien - 3,11,17 - trione or Δ ¹ · * - androstadien- steroids with a side chain in position 17 /? such as

ll «-ol-3,17-dione through microbiological oxidation on steroids with a modified basic structure.

using mycobacteria from appropriate steroids. Experiments were made for this purpose

Compounds with a 17 /? - side chain or andro- 35 with ergosterol, ergosta-4,22-dien-3-one, ^ -sitosterol,

stan structure in the presence of an overoxidation stigmasterin, Cholesta-4,6-dien-3-one. Pregn-5-en-3 /? - ol-

retardant substance; the process is thereby ge 20-on, pregna - 5.16 - diene - 3/9 - oil - 20 - on - 3-acetate,

indicates that the oxidation with Myco-progesterone, 5 "-Pregnan-3,20-dione and Androsta-

bacterium phlei (deposited under No. 29 in the strain 4,6-dien-3,17-dione and it was

collection of the Institute for Health Care to 40 put that with the breakdown of the side chain the

Budapest), with the addition of 8-hydroxyquinoline, 1,4-diene-3,17-dione structure in each case

and aerobic conditions. det is, in addition to simultaneous saturation of the <d ^G -Dop-

According to German Auslegeschrift 1135 455, pelbinding in the case of Androsta-4,6-diene. the

are the Pregn-4-en-3.20-dione (progesterone) and Δ ^ double bond in the case of ergosterol and the

the androst-4-en-3,17-dione with the help of a fungus 45 Δ "double bond in the case of pregna-5,16-diene-

(Fusarium caucasium) in the presence of numerous in-3 /? -Ol-20-on-3-acetate.

inhibitors in androsta-1,4-diene-3,17-dione over- those discussed in the present application

out, whereby the end product by degradation the microbiological conversions were with a

Pregnan side chain and formation of a Δ M double bacterial strain carried out, which according to Bergey's

bond arises. The S-hydroxy-quinoline was only 50 "Manual of Determinative Bacteriology" (7th edition,

applied to the latter substrate. 1957) was identified as Mycobacterium phlei.

In contrast, according to the invention, in addition to the bacteria form in the nutrient solution used

according to the above-mentioned German interpretative script, win-

If two substrates turned a large number of tens of grains. For growth, emulsifier

of other steroids in Androsta-1,4-diene-3,17-dione 55 substance (Tween) not absolutely necessary. the

or converted into derivatives of this diene dione. Bacteria take up a lot of nitrogen and

In these reactions, the end product is relatively low in sugar. Reach the cultures

due to very complex microbiological at 37 to 45 ° C the maximum dry matter

Chemical processes (e.g. due to the selective degradation of the content after about 48 hours.

of the sterol side chain in Cholestan, Ergostan and 60. It became the following nutrient solution for breeding

Stigmastane derivatives. the saturation of the zl ⁶ -, / 1 ⁷ -Deri- found to be very suitable:

vate, etc.) - ,, - ,, I. Corn steep water 12 to 20 g

From the chemical point of view, the um- glucose is 10 »

Conversion of cholesterol to androsta-1 4-diene-3,17-dione tap water '".'. '.". ".'.". '.'. '.'. '.'. '.'. '. ad 1000 ml

very complicated and can result in the following reac- 65

The nutrient solution is prepared as follows:

a) Degradation of the 17 /? - side chain. Training is of a 2.4 to 4 ° / ₀ sodium corn steep liquor solution

17-keto group. neutralized and filtered or centrifuged. the

3 4

remaining components of the nutrient solution (glucose, poorly vaporized residue was dissolved in acetone, these Lende amount of water) are then added. The solution on paper impregnated with propylene glycol respective percentage of the corn steep water depends on dripped and then with a mixture of on the quality or the nitrogen content of the carbon tetrachloride and methylcyclohexane in verbs away. 5 ratio of 1: 1 with descending system chromatographic

graphed. The resulting products give im

II corn steep water '12 ° - ultraviolet light strong absorption. Therefore

"Hefeautolysatum 'YYYYYYY 50 ml ^w _u ^urde the dried paper in the contact process

Tap water ad 1000 ml photographist, the spots of the photo were on

ίο referred to the chromatogram. The corresponding Pieces of paper were cut out, with 10 ml abso-

Before the sterilization, the pH was changed to 6.8 lutem alcohol and the extinction was adjusted to 7.2. According to the solution, the yeast autolysate was ηιμ at λ = 244. measured. Thus, the regulations of the technical book »Microorganisms of the amount of the resulting products were established.
Fermentation industry ”(7th edition, Hans Karl, Nürn- 15 The products were obtained as follows: Nach berg, 1956), p. 527, prepared. the microbiological implementation were the Bak

terial cells removed by filtration. The filtrate

III. Corn spring water 5g «) ^vur ^ _x ^d f times ^with ^ ⁰ L ₁ chlorinated hydrocarbon

Glucose 10 ° U ^e 20 percent by volume of the feed) or with a

AsDarasiri 4 s ²⁰ d ^to ren ^e, water-immiscible solvent it ^m

Glutamic acid extracted 3 °. The combined extracts were treated with

Tap water '.YY'. '.'. YYY Y ad 1000 ml of a dilute sodium hydroxide solution of the 8-Hy-

Droxy-chinohn freed and the residue after drying out Crystallized from a suitable solvent. The pH was reduced to 6.8 25 prior to sterilization.

set to 7.2. The inventive method is on hand

The nutrient solution was sterilized. explained in more detail in the following examples:

The flask broths were washed with cell suspensions washed with agar slants, the. Laboratory remote

mentor, on the other hand, with a daily shake flask

culture vaccinated. The inoculated flasks were at
37 ° C on a circular shaker with a speed of 300 revolutions per minute and a 5 liter nutrient solution No. I were shaken in a glass rash of 2 cm for 2 days. It sterilized fermentator of 10 liter content and was worked in the fermentor at 37 to 45 ° C with a rotary 35 100 ml one-day shaken culture of the same number of 250 revolutions per minute, nutrient solution inoculated. The fermentor was stirred at 37 ° C. and a volume of air per liter of nutrient solution and at 250 revolutions per minute and 5 liters per minute were blown onto the surface of the culture on the surface of the culture. After 2 days, appropriate blown sterile air was incubated for 2 days. Afterwards culture gained. There was no foam inhibition 4 °, the culture was sedimented and the nutrient solution used medium. The full-grown culture was sedimented verdanach with a 0.4 ° / ₀ sodium chloride solution. After the sedimentation was exchanged. Then 3 g of cholesterol and 360 mg of the nutrient solution were poured off the cells and 8-hydroxy-quinoline was dissolved in 60 ml of ethanol, which was added through sterile water or preferably through a culture.

0.4 ° / ₀ by weight sodium chloride solution equal in amount 45 The fermentation was already below the anersetzt. Experience has shown that the given growth conditions continue to decrease, and the remaining sugar content and possibly other grains. For 6 hours per 100 ml components of the nutrient solution, the steroid conversion samples were taken for 6 hours. The samples were made with dichloro-ability of the bacteria. Then the umzuethane were extracted. The combined extracts were dissolved releasing substances and 8-hydroxy-quinoline in 5 ° with a l ° / _o sodium hydroxide solution from alcohol, acetone or dioxane and culture of 8-hydroxy-quinoline free, then neuzugefügt with water. The steroid reaction was neutralized, dried with anhydrous sodium sulphate and dried with the growth conditions of the Bak- and evaporated to dryness. The residue was terminated after 2 days. The ferment solution was taken up in 5 ml of acetone. This solution was was then filtered or centrifuged because the 55 resulting in at 40 ° / ₀ ethanol containing propylene glycol l, 4-diene-3,17-dione in the fermentate or impregnated paper Macherey-Nagel no. 214 is to find gein the supernatant . The cells that drip off and are saturated with propylene glycol are capable of remixing carbon tetrachloride and methyl substitution in a new salt solution after adding steroid and inhibiting cyclohexane in a ratio of 1: 1 in a decreasing medium Avieder. 60 system chromatographed. The papers were after

The amount of steroids that were dehydrated in the course of the fermentation with contact methods in the ultraviolet, a 1,4-diene-3,17-dione structure photographed and thus the spots of the steroids was determined as follows: The determined. The resulting products were measured with the amount of fermentate was dissolved with 10 ml of absolute alcohol. The extinction chloroethane was thoroughly extracted and afterwards through 65 of the alcohol solution was determined at λ = 244 πιμ repeated rinsing with water from the 8-hydroxy- and the extinction values obtained were immediately freed from quinoline. The extract was dried with an anhydrous bar to Androsta-1,4-diene-3,17-dione content, reverse sodium sulfate and evaporated. That takes into account.

The values obtained are as follows:

Time of Content of sampling Androsta-1,4-diene-3,17-dione 0 hours 0 µg / ml 6 hours 42 µg / ml 12 hours 90 μ§ / ΐη1 18 hours 149 µg / ml 24 hours 162 µg / ml 30 hours 190 µg / ml 36 hours 191 μ ^ ΏΛ 42 hours 202 µg / ml 48 hours 238 µg / ml 60 hours 229 μ ^ ΐηΐ

After the end of the reaction, the residue of the fermentation solution was filtered and the filtrate was mixed three times with 20 percent by volume of dichloroethane extra ¹ each time. The extracts were combined and, after dehydration, evaporated to dryness. A solution of 1 g / 5-naphthol. in 20 ml of dichloroethane was added to the residue and kept for 24 hours at 0 ⁰ C. The crystalline molecular compound androsta-l, 4-diene-3,17-dione-y? Naphthol was filtered (mp. 168-170 ⁰ C) dissolved in 200 ml of dichloroethane, with l ° / o'g ^he aqueous sodium hydroxide solution of freed from the / ^ - naphthol, then evaporated to dryness after rinsing with water and dehydration and crystallized from a little ether. The crystals were filtered and dried. 850 mg of androsta-1,4-diene-3,17-dione were obtained (melting point 141 to 142 ° C.); [oc] _D = 115 ° (c = 1, chloroform); λ "%? = 244 ΐημ. A further 60 mg androsta-1,4-diene-3,17-dione could be obtained from the mother liquor. Yield: 38 percent by weight.

Example 2

The procedure was as in Example 1, with the difference that instead of cholesterol, 5 g of cholest-4-en-3-one were added to the culture. The control of the implementation and the processing took place also in the manner described in Example 1. Yield 40 weight percent.

Example 3

The procedure was as in Example 1, with the difference that instead of cholesterol 3 g of 5'-cholesterol-3 /? - ol were added to the culture. The control of the implementation and the processing also took place in the manner described in Example 1. Yield: 34 percent by weight.

Example 4

The procedure was as in Example 1, with the difference that instead of cholesterol 3 g /? - sitosterol were added to the culture. The control of the implementation and the processing also took place in the manner described in Example 1. Yield: 18 percent by weight.

Example 5

The procedure was as in Example 1, with the difference that instead of cholesterol Ig Pregn-5-en-3 /? - oI-20-on-3-acetate was added to the culture. The control of the reaction and the work-up were also carried out in the manner described in Example 1 Wise. Yield: 63.5 percent by weight.

Example 6

The procedure was as in Example 1, with the difference that instead of cholesterol 1 g of androst-5-en-3 /? - ol-17-one acetate was added to the culture. The control of the reaction and the work-up were also carried out in the manner described in Example 1 Wise. Yield: 70 percent by weight.

Example 7

100 ml of sterile nutrient solution III were mixed with 1 ml of a 5-day agar slant culture 10 ml of cell suspension prepared with broth inoculated. The waxing lasted 2 days at 28 ° C on the Shaking table. The culture was sedimented and the nutrient solution with a 0.4% sterile sodium chloride solution exchanged by the same amount. The culture was given 20 mg of Cholesta-1,4-dien-3-one in 1 ml Ethanol and 7.2 mg of 8-hydroxy-quinoline in 0.5 ml of acetone were added. The flask was at 2 days Shaken 37 ° C. The work-up and the determination of the steroid content were carried out in accordance with the procedure described in Example 1 described way. In a series of tests of ten flasks, an average of 41 percent by weight Androsta-1,4-diene-3,17-dione detected.

Example 8

The procedure was as in Example 7, but with the difference that instead of Cholesta-1,4-dien-3-one 5a-cholestan-3-one was added to the culture.

There were 38 percent by weight androsta-1,4-diene-3,17-dione proven as a product.

Example 9

The procedure was as in Example 7, but with the difference that instead of Cholesta-1,4-dien-3-one Cholest-5-en-3-one was added to the culture.

There were 38 percent by weight androsta-1,4-diene-3,17-dione proven as a product.

Example 10

The procedure was as in Example 7, however

with the difference that instead of Cholesta-1,4-dien-3-one Cholest-4,6-dien-3-one was added to the culture. There were 36 percent by weight androsta-1,4-diene-3,17-dione proven as a product.

Example 11

The procedure was as in Example 7, but with the difference that instead of Cholesta-1,4-dien-3-one Ergosterol was added to the culture. There were 28 percent by weight androsta-1,4-diene-3,17-dione proven as a product.

Example 12

The procedure was as in Example 7, however

with the difference that instead of Cholesta-1,4-dien-3-one Ergosta-4,22-dien-3-one was added to the culture.

There were 30 percent by weight androsta-1,4-diene-3,17-dione proven as a product.

Example 13

The procedure was as in Example 7, but with the difference that instead of Cholesta-1,4-dien-3-one Stigmaster was inflicted on the culture. There were 27 percent by weight androsta-1,4-diene-3,17-dione proven as a product.

Example 14

The procedure was as in Example 7, but with the difference that instead of Cholesta-1,4-dien-3-one Pregna-5,16-dien-3 /? - ol-20-on-3-acetate was added to the culture. It became 64 percent by weight Androsta-1,4-diene-3,17-dione detected as a product.

Example 15

The procedure was as in Example 7, but with the difference that instead of Cholesta-1,4-dien-3-one Progesterone was added to the culture. There were 66 percent by weight androsta-1,4-diene-3,17-dione proven as a product.

Example 16

The procedure was as in Example 7, but with the difference that instead of Cholesta-1,4-dien-3-one Androst-4-en-3,17-dione was added to the culture. There were 85 percent by weight androstal, 4-diene-3,17-dione proven as a product.

Example 17

The procedure was as in Example 7, but with the difference that instead of Cholesta-1,4-dien-3-one Androsta-4,6-diene-3,17-dione was added to the culture. There were 61 percent by weight androstal, 4-diene-3,17-dione proven.

Example 18

The residue was crystallized from diethyl ether (0.550 g), furthermore, after treatment with activated charcoal, recrystallized from a mixture of acetone and petroleum ether. 0.37 g of Androsta-1,4-diene-3,11,17-trione (37 percent by weight) were obtained; Mp 195 to 196 ° C; [ec] _D = 235 ° (c = 1, acetone); λ * £ ° ^Η = 238 ΐημ.

3 °

Under the fermentation conditions of Example 1, 1 g of 11-keto-progesterone was added to the culture instead of cholesterol. The amount of androsta-l, 4-diene-3, ll, 17-trione formed was determined essentially in the manner described in Example 1, by measuring the extinction at λ = 238 ηιμ. After the reaction had ended, the fermentation solution was filtered and the filtrate was extracted three times with 20 percent by volume of dichloroethane each time. The extracts were combined, washed with l ° / _o sodium hydroxide solution to 8-oxy-quinolin-free further with water until neutral and evaporated to dryness after dewatering. The example 19

Under the fermentation conditions of Example 1, 1 g of loc-hydroxyprogesterone was added to the culture instead of cholesterol. The amount of Androsta -1,4 - diene -11 "- oil - 3,17-dione formed was determined essentially in the manner described in Example 1 by means of absorbance measured at λ = 247 ΐημ. After the reaction had ended, the fermentation solution was filtered and the filtrate was extracted three times with 20 percent by volume of ethyl acetate each time. The combined extracts were washed to 8-oxyquinoline-free with a 1% sodium hydroxide solution and then washed with water until the reaction was neutral and evaporated to dryness. The residue (0.400 g) was crystallized from a mixture of acetone-ether. So 0.180 g of androsta-1,4-dien-lla-ol-3,17-dione was obtained (18 percent by weight); Mp 209-212 ° C; [x] _D = 87.5 ° (c = 1, chloroform); λ ^Ε ™ = 247 ητμ.

Claims (1)

Claim: Process for the production of zJ ^{1> 4} -androstadiene - 3.17 - dione, Δ ^1Λ - androstadiene - 3.11-17 - trione or Δ ^1Λ - androstadiene - 11 «- ol-3,17-dione by means of microbiological oxidation Mycobacteria from corresponding steroid compounds with a 17 / S side chain or androstane structure in the presence of an over-oxidation inhibiting substance, characterized in that the oxidation with Mycobacterium phlei (deposited under No. 29 in the collection of the Institute for Health in Budapest) under Addition of 8-hydroxy-quinoline and aerobic conditions. 109520/381