DE2340162A1 - BICYCLIC CONNECTION - Google Patents

Description

Dr. Ing. A. van der Wirt »- 7. Aua. 1973 Dr. Franz Lecterer PATENT APPLICATION II

RAN 4104/119

F. HoiFmann-La Roche & Co. Aktiengesellschaft, Basel / Switzerland

Bicyclic compound

The invention relates to a bicyclic compound, namely 20β-hydroxy-9-oxo-1,2,3,4,10,19-hexanor-5,9-seco-pregnan-5-acid the formula

HOOC CH ₂
^CH 2

and a method for their production.

Green / 5/28/73

409813/1137

According to the invention, the new compound is obtained by adding 3β, 20β-dihydroxypregn-5-ene or 20β-hydroxypregn-4-en-3-one with Mycobacterium phlei ATCC 19249 adapted to aliphatic hydrocarbons or Fermented mutants of it.

The adaptation of M. phlei ATCC 19249 to aliphatic '' Hydrocarbons can be produced in a manner known per se by removing the microorganism from growing media, which contain glucose, inoculated onto substrates in which the glucose is increased in mass by aliphatic Hydrocarbons, especially C, h-C, o-alkanes, is replaced.

Spontaneous mutants or mutants produced by physical or chemical means can be used as mutants will. Mutants can be caused by irradiation, e.g. with UV light or gamma rays, or by treatment with mutating agents such as N-methyl-nitro-nitrosoguanidine or bromuracil can be generated. Preferably for the In accordance with the invention, spontaneous mutants are used. The selection of the mutants can be done by transferring single colonies to agar plates, which contain the steroid to be broken down as a carbon source. Single colonies obtained from such agar substrates become finally transferred to agar plates as the carbon source the process product, i.e. contain the above-mentioned seco acid. Those cultures based on such Culture media showing no growth are used for the fermentation according to the invention.

Carrying out the method according to the invention can be carried out analogously to the working methods known for the aerobic fermentation of steroids with microorganisms. In particular, liquid media are used as a culture substrate

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Consider having an assimilable nitrogen source and an assimilable carbon source in addition to inorganic salts contain. Come as assimilable sources of nitrogen animal, vegetable, microbial and inorganic nitrogen compounds into consideration, such as meat extracts, peptones, cornsteep, yeast extract, glycine and sodium nitrate. As a carbon source sugars such as glucose and in particular the steroid to be fermented itself can be used. The nutrient medium can also trace elements such as iron, sulfur and phosphorus as well as growth factors such as vitamins such as biotin or Pyridoxine, or auxins, such as indolylacetic acid. To protect against infection, the medium can be sterilized and / or with substances that inhibit the growth of foreign organisms, such as Benzoates, antibiotics, etc. are provided. The vacation is preferably carried out in the neutral or weakly acidic pH range, e.g. at pH 6-7. The fermentation temperature can be between 18 and 40 °, preferably at etv? a 28 ° worked.

The steroid used as the starting material is preferably added to the fermentation culture in solution, e.g. in dimethyl sulfoxide, Dimethylformamide, ethanol or acetone added. It has been shown to be beneficial to the steroid of the fermentation solution to add gradually and to wait for the complete breakdown of the previously added steroid before each new addition.

When the fermentation has ended, the process product can be removed from the culture solution in a manner known per se, for example by Extraction, countercurrent extraction and chromatography, isolated

20β-Hydroxy-9-oxo-1,2,3,4,10,19-hexanor-5,9-seeopregnan-5-acid has not been described so far. She is hiring

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valuable intermediate in the synthesis of steroids, into which it can be converted in analogy to known methods (cf. Belgian patent specification 741826).

example 1 Obtaining the fermentation culture;

Mycobacterium phlei ATCC 19249, adapted to C _{li +} -C, gn-alkanes, was inoculated from slant agar culture doors onto a culture medium with the following composition:

Difco nutrient broth 23 g

C, ₄ -C-, ο-n-alkanes 10 ml

Trace element solution I ml

Distillery. Water ' . 1000 ml

Trace element solution: 11 g ZnSO ₁ , .7 H ₃ O; 6 g of MhSOj ₁ .4 H ₃ O; 1 g PeSO ₄ .7 H ₂ Oj'0.5 g CoSOj ₊ . 7 H ₂ O; 0.04 g CuSO ₄ .5 HgOj 0.06 g 0.01 g KJj 5 g ethylenediaminetetraacetic acid in 1000 ml

H ₇ BO ₇ J

distill. Water.

The cultures were in 5OO ml'-Erlenmeyer flasks with each 100 ml nutrient solution for 48 hours at 28 ° on a rotating Shaking machine (160 rpm) cultivated. With the thus obtained Cultures were inoculated into 7 liter small fermenters with a nutrient medium of the following composition:

3β, 20β-dihydroxypregn-5-en

NH..NO, 4 3

KH ₂ PO ₄ K ₂ HPO ₄ Na ₂ HPO ₄ .2 H ₂ O MgSO ₄ .7 H ₂ O KCl

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5 2 G 2 2 G 1 ε 1 G 1 G 0, G 0, G

FeSO ₄ . 7 H ₂ O 0.001 g

Tap water, pH 7.0 1000 ml

The cultures were incubated for 15 days at 28 ° with aeration and stirring. From the cultures thus obtained were Plating made on agar plates as a carbon source only contained 33, 20β-dihydroxypregn ~ 5-ene. the Plating was repeated several times, increasing the steroid content in the agar plates. Finally were Further plate cultures were set up, the agar of which was only 20β-hydroxy-9-oxo-1,2,3,4,10,19-hexanor-5,9 ~ seco-pregnan-5-acid as a carbon source contained. Those cultures that showed no growth on these plates were used for the fermentation according to the invention used. To store the cultures, a suspension in sterile was made of the biomass 1Above skimmed milk solution prepared in 2 ml ampoules was bottled and stored under liquid nitrogen.

ml Example 2 (sterilized separately) 100 % a nutrient solution with 1 % glucose 0.2 % NH ₄ NO 0.1 % KH ₂ PO ₄ ^r 7 TT f \ .
• ι «pt» 0.2 % K ₂ HPO ₄ . H ₂ O 0.1 % Na ₂ HPO ₄ 0.02 % MgSO ₄ 7 0.02 KCl

$ 0.0001 FeSO ₄ . 7 H ₃ O

in tap water, pH 7.0 (adjusted with NaOH) with the contents of 2 ampoules prepared according to Example 1 inoculates. The culture was stirred for 48 hours at 28 ° incubated. 8 liters of nutrient medium were obtained with this preculture

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inoculated in the small fermenter, which was incubated for 2 days at 28 ° with stirring and aeration (640 rpm, 2 liters of air / min), the pH being adjusted to pH 7.0 by adding 28% sodium hydroxide solution. This fermenter content served as inoculum for a 180 liter fermenter. the same nutrient solution which was incubated at 32 °, 1100 rpm and an aeration rate of 100-150 liters / min. After 27 hours the glucose content of the nutrient medium had fallen to 0.2 ^. At this point 180 g of j5β, 20β-dihydroxypregn-5-ene, dissolved in 360 ml of dimethyl sulfoxide, were added. The conversion of the steroid was followed by thin-layer chromatography of samples. The pH was kept between 6.5 and 7.0 by adding sodium hydroxide solution.

After 63 hours, the steroid used as the starting material had completely reacted, whereupon ISO g 3β > 20β-dihydroxypregn-5-ene in diethyl sulfoxide were again added to the fermentation solution. After the

Steroids (90 hours per day) a third addition of 180 g was made Steroid. After 155 hours all steroid had been converted.

The fermentation broth was centrifuged, rinsed with 20 liters of water and adjusted to pH 2.0 with concentrated hydrochloric acid. The solution was extracted twice with the same volume and once with half the volume of methylene chloride. The extracts were concentrated in a rotary evaporator and worked up by means of countercurrent extraction. The yield of 20β-hydroxy-9-oxo-1,2,5,4,10,19-hexanor-5,9-seco-pregnan-5-acid was 82 #. In addition there were 10 $ 9i20-dioxo-1,2,3,4,10,19-hexanor-5-seco-pregnan-5-acid isolated.

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Claims (2)

23A0162 claims 1. Process for the production of 20β ~ hydroxy-9-oxo- j5j ^ jlOil9-hexanor-5 * 9-seco-pregnan-5 ~ acid of the formula CH HOOC CH Wi characterized in that 3ß, 20ß-dihydroxy ~ pregn-5-en or 20β-hydroxy-pregn-4-en-j5-one with an aliphatic hydrocarbons adapted Mycobacterium phlei ATCC 192 ^ 9 or Fermented mutants of it. 2. The method according to claim 1, characterized in that spontaneous mutants are used which contain the 2Oß-hydroxy-9-oxo-l, 2.5, h, 10.19-hexanor-5 * 9-seco-pregnan-5-acid do not use as a carbon source. 13/1137 J5. pregnan-5 acid of the formula HOOC Λ09 813/1137