EP0600946A1 - METHOD OF PREPARING 14$g(a)-HYDROXY-4-ANDROSTENE-3,17-DIONE - Google Patents

METHOD OF PREPARING 14$g(a)-HYDROXY-4-ANDROSTENE-3,17-DIONE

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Publication number
EP0600946A1
EP0600946A1 EP92917129A EP92917129A EP0600946A1 EP 0600946 A1 EP0600946 A1 EP 0600946A1 EP 92917129 A EP92917129 A EP 92917129A EP 92917129 A EP92917129 A EP 92917129A EP 0600946 A1 EP0600946 A1 EP 0600946A1
Authority
EP
European Patent Office
Prior art keywords
dione
androstene
hydroxy
culture
fermentation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP92917129A
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German (de)
French (fr)
Inventor
Alfred Weber
Mario Kennecke
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer Pharma AG
Original Assignee
Schering AG
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Publication date
Application filed by Schering AG filed Critical Schering AG
Publication of EP0600946A1 publication Critical patent/EP0600946A1/en
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
    • C12P33/06—Hydroxylating
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
    • C07J1/0003—Androstane derivatives
    • C07J1/0011—Androstane derivatives substituted in position 17 by a keto group

Definitions

  • the invention relates to a process for the preparation of 14 ⁇ -hydroxy-4-androstene-3,17-dione by fermentation of 4-androstene-3,17-dione with a culture of Curvularia lunata, which is characterized in that the 4- Androsten-3,17-dione suspended in water and the suspension obtained fermented.
  • SU patent 801 517 (ref. CA. 110, 133697u) describes a process for the preparation of 14 ⁇ -hydroxy-4-androstene-3,17-dione by fermentation of 4-androstene-3,17-dione a culture of Curvularia lunata.
  • this patent does not contain any reference from which public stock collection and under which number this microorganism can be obtained, so that it is not possible to rework the described method.
  • the 4-androstene-3,17-dione is dissolved in ethanol or dimethylformamide, 0.5 to 1.5 g / l of substrate is fermented for 40 to 66 hours and 35 to 70% is obviously obtained very strongly contaminated crude 14 ⁇ -hydroxy-4-androsten-3,17-dione product.
  • the fermentation batch is extracted with chloroform, the chloroform phase is concentrated and the residue is boiled with hexane.
  • the product obtained must therefore contain all extractable substances insoluble in hexane, not all of them Steroids contained in the fermentation batch but also the corresponding Curvularia ingredients and media components.
  • the process according to the invention is carried out under the fermentation conditions which are also used in the known microbiological conversions of substrates with Curvularia lunata cultures.
  • Under the culture conditions usually used for this microorganism submerged cultures are grown in a suitable nutrient medium with aeration. Then an aqueous suspension of 4-androstene-3,17-dione is added to the culture and cemented until a maximum substrate conversion is reached.
  • the aqueous suspension of 4-androsten-3,17-dione can be prepared in a simple manner by, for example, grinding this substance with twice at five times the amount of water with the addition of a non-ionic surfactant in a ball mill until it has a particle size of approximately 1 to 5 ⁇ .
  • Suitable surfactants are, for example, ethylene oxide adducts or fatty acid esters of polyglycols.
  • Suitable surfactants include, for example, commercially available wetting agents such as Tegin ®, Tween ®, or Span ®.
  • the culture is prepared in the customary manner, for example by extracting it with an alcohol, ketone or ester which is poorly soluble in water, concentrating the extract and purifying the residue obtained by chromatography and / or crystallization.
  • a 501 fermenter with 301 sterile nutrient solution as indicated under a) is inoculated with 1 l of the Curvularia lunata culture and incubated for 18 hours at 30 ° C. with aeration of 2 m 2 / hour and stirring at 220 revolutions per minute.
  • the culture broth is extracted three times with 201 methyl isobutyl ketone, the extract in a rotary evaporator at max. Concentrated 50 ° C under vacuum. This is followed by purification by chromatography on a silica gel column using a hexane-acetone gradient.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Steroid Compounds (AREA)

Abstract

Il est décrit un procédé de production de 14alpha-hydroxy-4-androstène-3,17-dione par fermentation de 4-androstène-3,17-dione avec une culture de Curvularia lunata, caractérisé en ce qu'on met en suspension dans l'eau la 4-androstène-3,17-dione et qu'on effectue la fermentation de la suspension obtenue.There is described a process for the production of 14alpha-hydroxy-4-androstene-3,17-dione by fermentation of 4-androstene-3,17-dione with a culture of Curvularia lunata, characterized in that one suspends in water 4-androstene-3,17-dione and that the fermentation of the suspension obtained is carried out.

Description

Verfahren zur Herstellung von 14α- Hydroxy-4-Androsten-3,17-dion Process for the preparation of 14α-hydroxy-4-androstene-3,17-dione
Die Erfindung betrifft ein Verfahren zur Herstellung von 14α-Hydroxy-4-androsten- 3,17-dion durch Fermentation von 4-Androsten-3,17-dion mit einer Kultur von Curvularia lunata, welches dadurch gekennzeichnet ist, daß man das 4-Androsten-3,17- dion in Wasser suspendiert und die erhaltene Suspension fermentiert.The invention relates to a process for the preparation of 14α-hydroxy-4-androstene-3,17-dione by fermentation of 4-androstene-3,17-dione with a culture of Curvularia lunata, which is characterized in that the 4- Androsten-3,17-dione suspended in water and the suspension obtained fermented.
In der SU-Patentschrift 801 517 (ref. CA. 110, 133697u) wird ein Verfahren zur Her¬ stellung von 14α-Hydroxy-4-androsten-3,17-dion durch Fermentation von 4- Androsten-3,17-dion mit einer Kultur von Curvularia lunata beschrieben. In diesem Patent ist aber kein Hinweis enthalten, aus welcher öffentlichen Stammsammlung und unter welcher Nummer dieser Mikroorgnismus bezogen werden kann, so daß eine Nacharbeitung des beschriebenen Verfahrens nicht möglich ist. Nach der Lehre dieser Patentschrift löst man das 4-Androsten-3,17-dion in Ethanol oder Dimethyl.formamid, fermentiert 0,5 bis 1,5 g/1 Substrat 40 bis 66 Stunden lang und erhält 35 bis 70 % offensichtlich sehr stark verunreinigtes 14α-Hydroxy-4-androsten-3,17-dion- Rohprodukt. (Das dem so ist, erkennt man an der Art der Aufarbeitung: Der Fermentationsansatz wird mit Chloroform extrahiert, die Chloroformphase eingeengt und der Rückstand mit Hexan ausgekocht. Das erhaltene Produkt muß also alle extrahierbaren, in Hexan unlöslichen Stoffe enthalten, dies sind nicht nur alle im Fermentationsansatz enthaltenen Steroide sondern auch die entsprechenden Curvularia- Inhaltsstoffe und Medienbestandteile.)SU patent 801 517 (ref. CA. 110, 133697u) describes a process for the preparation of 14α-hydroxy-4-androstene-3,17-dione by fermentation of 4-androstene-3,17-dione a culture of Curvularia lunata. However, this patent does not contain any reference from which public stock collection and under which number this microorganism can be obtained, so that it is not possible to rework the described method. According to the teaching of this patent, the 4-androstene-3,17-dione is dissolved in ethanol or dimethylformamide, 0.5 to 1.5 g / l of substrate is fermented for 40 to 66 hours and 35 to 70% is obviously obtained very strongly contaminated crude 14α-hydroxy-4-androsten-3,17-dione product. (You can tell that from the way it is worked up: the fermentation batch is extracted with chloroform, the chloroform phase is concentrated and the residue is boiled with hexane. The product obtained must therefore contain all extractable substances insoluble in hexane, not all of them Steroids contained in the fermentation batch but also the corresponding Curvularia ingredients and media components.)
Demgegenüber kann man mit Hilfe des erfindungsgemäßen Verfahrens mehr als 5 g 4-Androsten-3,17-dion pro Liter innerhalb von etwa 12 Stunden reproduzierbar in 14α-Hydroxy-4-androsten-3,17-dion umzuwandeln. Die Ausbeute an Verfahrensprodukt beträgt etwa 60 % Reinprodukt.In contrast, more than 5 g of 4-androstene-3,17-dione per liter can be reproducibly converted into 14α-hydroxy-4-androstene-3,17-dione within about 12 hours using the process according to the invention. The yield of process product is about 60% pure product.
Das erfindungsgemäße Verfahren wird unter den Fermentationsbedingungen durchgeführt, welche man auch bei den bekannten mikrobiologischen Umwandlungen von Substraten mit Curvularia lunata Kulturen anwendet. Unter den für diesen Mikroorganismus üblicherweise verwendeten Kulturbedingungen werden in einem geeigneten Nährmedium unter Belüften Submerskulturen angezüchtet. Dann setzt man der Kultur eine wässrige Suspension des 4-Androsten-3,17-dion zu und fementiert bis eine maximale Substratumwandlung erreicht ist. Die wässrige Suspension des 4-Androsten-3,17-dions läßt sich in einfacher Weise herstellen, indem man beispielsweise dieser Substanz mit der zweifachen bei fünffachen Menge Wasser unter Zusatz eines nichtiomschen Tensids in einer Kugelmühle mahlt bis sie eine Korngröße von ca. 1 bis 5 μ besitzt. Geeignete Tenside sind beispielsweise Ethylenoxidaddukte oder Fettsäureester von Polyglykolen. Als geeignete Tenside seien beispielsweise die handelsüblichen Netzmittel wie Tegin ® , Tween® , oder Span® genannt.The process according to the invention is carried out under the fermentation conditions which are also used in the known microbiological conversions of substrates with Curvularia lunata cultures. Under the culture conditions usually used for this microorganism, submerged cultures are grown in a suitable nutrient medium with aeration. Then an aqueous suspension of 4-androstene-3,17-dione is added to the culture and cemented until a maximum substrate conversion is reached. The aqueous suspension of 4-androsten-3,17-dione can be prepared in a simple manner by, for example, grinding this substance with twice at five times the amount of water with the addition of a non-ionic surfactant in a ball mill until it has a particle size of approximately 1 to 5 μ. Suitable surfactants are, for example, ethylene oxide adducts or fatty acid esters of polyglycols. Suitable surfactants include, for example, commercially available wetting agents such as Tegin ®, Tween ®, or Span ®.
Nach erfolgter Fermentation wird die Kultur in üblicher Weise aufbereitet, beispielsweise indem man sie mit einem in Wasser schwer löslichen Alkohol, Keton oder Ester extrahiert, den Extrakt einengt und den erhaltenen Rückstand durch Chromatographie und/oder Kristallisation auf reinigt.After the fermentation has taken place, the culture is prepared in the customary manner, for example by extracting it with an alcohol, ketone or ester which is poorly soluble in water, concentrating the extract and purifying the residue obtained by chromatography and / or crystallization.
Die Weiterverarbeitung des erhaltenen 14α-Hydroxy-4-androsten-3,17-dions zu pharmakologisch wirksamen Steroiden ist bekannt oder erfolgt nach an sich bekannten Verfahren (J. Amer. Chem. Soc, 91, 1969, 1228 ff; EP-A 0300062 und weitere).The further processing of the 14α-hydroxy-4-androsten-3,17-dione obtained is known to pharmacologically active steroids or is carried out by processes known per se (J. Amer. Chem. Soc. 91, 1969, 1228 ff; EP-A 0300062 and more).
Das nachfolgende Ausführungsbeispiel dient zur näheren Erläuterung des erfindungs¬ gemäßen Verfahrens. The following exemplary embodiment serves to explain the method according to the invention in more detail.
Beispiel:Example:
a) Ein 21 Erlenmeyer mit 11 sterilem Nährmedium enthaltenda) Containing a 21 Erlenmeyer with 11 sterile nutrient medium
1 9/6 Cornsteep liqour1 9/6 Cornsteep liqour
1,25 % Sojapuder1.25% soy powder
-eingestellt auf pH 6,2- wird mit 5 ml einer Suspension einer Curvularia lunata NRRL 2380 - Kultur beimpft und 60 Stunden bei 30 °C mit 180 Umdrehungen pro Minute geschüttelt.- adjusted to pH 6.2 - inoculated with 5 ml of a suspension of a Curvularia lunata NRRL 2380 - culture and shaken for 60 hours at 30 ° C at 180 revolutions per minute.
b) Ein 501-Fermenter mit 301 steriler Nährlösung wie unter a) angegeben wird mit 1 1 der Curvularia lunata-Anzuchtkultur beimpft und 18 Stunden bei 30 °C unter Belüftung von 2 m^ pro Stunde und Rühren mit 220 Umdrehungen pro Minute inkubiert.b) A 501 fermenter with 301 sterile nutrient solution as indicated under a) is inoculated with 1 l of the Curvularia lunata culture and incubated for 18 hours at 30 ° C. with aeration of 2 m 2 / hour and stirring at 220 revolutions per minute.
c) Ein 501-Fermenter mit 401 steriler Nährlösung enthaltendc) Containing a 501 fermenter with 401 sterile nutrient solution
1 % Cornsteep liqour1% Cornsteep liqour
1,25 % Sojapuder1.25% soy powder
0,3 % ß-Cyclodextrin0.3% β-cyclodextrin
-eingestellt auf pH 6,2- wird mit 41 der Curvularia lunata- Vorkultur beimpft und unter Belüftung von 2,4 m3 pro Stunde und Rühren mit 150 Umdrehungen pro Minute bei 30 °C 7 Stunden inkubiert.- adjusted to pH 6.2 - inoculated with 41 of the Curvularia lunata preculture and incubated with aeration of 2.4 m3 per hour and stirring at 150 revolutions per minute at 30 ° C for 7 hours.
Dann setzt man der Kultur 250 g 4-Androstaen-3,17-dion als Mahlung in 1000 ml Wasser mit 25 g Tween®80 zu und fermentiert weitere 12 Stunden lang unter Rühren mit 220 Umdrehungen pro Minute und einer Belüftung von 2,4 m^ pro Stunde.Then 250 g of 4-androstaen-3,17-dione are added to the culture as a grinding in 1000 ml of water with 25 g of Tween®80 and fermented for a further 12 hours with stirring at 220 revolutions per minute and aeration of 2.4 m ^ per hour.
d) Nach erfolgter Fermentation wird die Kulturbrühe dreimal mit je 201 Methylisobutylketon extrahiert, der Extrakt im Rotationsverdampfer bei max. 50 °C unter Vakuum eingeengt. Anschließend erfolgt eine Reinigung durch Chromatographie über eine Kieselgelsäule mittels Hexan-Aceton-Gradienten.d) After fermentation, the culture broth is extracted three times with 201 methyl isobutyl ketone, the extract in a rotary evaporator at max. Concentrated 50 ° C under vacuum. This is followed by purification by chromatography on a silica gel column using a hexane-acetone gradient.
Man erhält 150 g 14α-Hydroxy-androsta-4-en-3,17-dion vom Schmelzpunkt 257- 259 °C. 150 g of 14α-hydroxy-androsta-4-en-3,17-dione with a melting point of 257-259 ° C. are obtained.

Claims

Patentansprüche: Claims:
1. Verfahren zur Herstellung von 14α-Hydroxy-4-androsten-3,17-dion durch Fermentation von 4-Androsten- 3,17-dion mit einer Kultur von Curvularia lunata, dadurch gekennzeichnet, daß man das 4-Androsten-3,17-dion in Wasser suspendiert und die erhaltenen Suspensionen fermentiert.1. Process for the preparation of 14α-hydroxy-4-androstene-3,17-dione by fermentation of 4-androstene-3,17-dione with a culture of Curvularia lunata, characterized in that the 4-androstene-3, 17-dione suspended in water and the suspensions obtained fermented.
2. Verfahren zur Herstellung von 14α-Hydroxy-4-androsten-3,17-dion gemäß Patentanspruch 1, dadurch gekennzeichnet, daß man zur Fermentation eine Kultur von Curvularia lunata NRRL 2380 verwendet. 2. Process for the preparation of 14α-hydroxy-4-androsten-3,17-dione according to claim 1, characterized in that a culture of Curvularia lunata NRRL 2380 is used for the fermentation.
EP92917129A 1991-08-28 1992-08-07 METHOD OF PREPARING 14$g(a)-HYDROXY-4-ANDROSTENE-3,17-DIONE Withdrawn EP0600946A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE4129005 1991-08-28
DE19914129005 DE4129005A1 (en) 1991-08-28 1991-08-28 METHOD FOR PRODUCING 14 (ALPHA) -HYDROXY-4-ANDROSTEN-3,17-DION
PCT/EP1992/001797 WO1993005170A1 (en) 1991-08-28 1992-08-07 METHOD OF PREPARING 14α-HYDROXY-4-ANDROSTENE-3,17-DIONE

Publications (1)

Publication Number Publication Date
EP0600946A1 true EP0600946A1 (en) 1994-06-15

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EP92917129A Withdrawn EP0600946A1 (en) 1991-08-28 1992-08-07 METHOD OF PREPARING 14$g(a)-HYDROXY-4-ANDROSTENE-3,17-DIONE

Country Status (4)

Country Link
EP (1) EP0600946A1 (en)
JP (1) JPH07501689A (en)
DE (1) DE4129005A1 (en)
WO (1) WO1993005170A1 (en)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR1267758A (en) * 1956-12-19 1961-07-28 Merck & Co Inc Preparation process
WO1988005781A1 (en) * 1987-02-06 1988-08-11 Snow Brand Milk Products Co., Ltd. Novel androst-4-ene-3,17-dione derivatives and process for their preparation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9305170A1 *

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DE4129005A1 (en) 1993-03-04
JPH07501689A (en) 1995-02-23
WO1993005170A1 (en) 1993-03-18

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