FR2510120A1 - PROCESS FOR THE PRODUCTION OF 6-FLUORO-9A-HYDROXY-ANDROST-4-ENE-3,17-DIONE BY FERMENTATION - Google Patents

Abstract

THE INVENTION RELATES TO A PROCESS FOR THE PRODUCTION OF THE INTERMEDIATE COMPOUND CALLED 6-FLUORO-9A-HYDROXY-ANDROST-4-ENE-3,17-DIONE (6-FLUORO-HAD). THE PROCESS OF THE INVENTION CONSISTS OF CONVERTING 6-FLUORO-D-3-KETONIC DERIVATIVES OF STEROLS SUCH AS CHOLESTEROL, SITOSTEROL, STIGMASTEROL, ETC., BY FERMENTATION UNDER THE ACTION OF THE MICRO-ORGANISM MYCOBACTERIUM FORTUITUM NRRL B-8. 6-FLUORO-HAD IS USEFUL AS AN INTERMEDIATE COMPOUND IN THE SYNTHESIS OF INTERESTING CORTICOIDS.

Description

U.S. Patent No. 4,029,549 describes and claims

  the use of Mycobacterium fortuitum

  NRRL B-8119 for the preparation of 9-hydroxy-3-oxoacid

  4-pregnene-20-carboxylic acid-9-hydroxybisnoracid 7 The same microorganism is used for the preparation of the

  9-hydroxy-4-androstene-3,17-dione / -9-hydroxyandrostene

  dione_ 7 (HAD) in the United States patent

  No. 4,035,236; and for the preparation of the methyl ester

  9-hydroxy-3-oxo-4-pregnene-20-carboxylic acid / 9-hydroxybisnoracidyl methyl ester in the patent

  United States of America N4 214 051.

  The present invention provides a method of

  fermentation preparation of the intermediate compound used

  called 6-fluoro-9α-hydroxy-androst-4-ene-3,17-dione (6-

  fluoro-HAD) by conversion of 6-fluoro-A 4-3-keto-

  sterols, for example cholesterol, sitosterol, stigmasterol, etc., according to the following scheme I:

SCHEME I

O I Ferrrentation>

F F

  This process is carried out using M. fortuitum NRRL B-8119. The process of the present invention

  also encompasses the use of mutants obtained from

  from the genera of microorganisms described in U.S. Patent No. 4,029,549, that is Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia and Streptomyces. microorganisms of these genera are all well known for their ability to degrade

  sterols The wild-type strains of these genera degrade

  Non-selectively sterols are obtained from low molecular weight compounds, for example CO 2 + H. Mutants can be created from these wild types according to the procedures described in Example 1 of the US Pat. America N 4,029,549 cited above This example describes the

  preparation of M fortuitum NRRL B-8119.

  The process of the invention is the only process

  known for the preparation of 6-fluoro-HAD.

  Micro-organisms Mutants that are characterized by their

  ability to selectively convert 6-fluoro-A 4- derivatives

  3-ketone sterols having 17-alkyl side chains

  and to accumulate 6-fluoro-HAD in a broth of

  fermentation can be obtained by mutation of micro-

  organisms of the following genera: Arthrobacter, Bacillus,

  Brevibacterium, Corynebacterium, Microbacterium, Mycobac-

  Terium, Nocardia, Protaminobacter, Serratia and Streptomyces.

  Mycobacterium fortuitum NRRL B-8119 is described in U.S. Patent No. 4,029,549 M.

  fortuitum NRRL B-8119 has been deposited in the collection

  Managed by the Northern Regional Research Laboratory, United States Department of Agriculture, Peoria, Illinois, USA. It has been made available to the public at least since the issuance of United States patents.

  mentioned above describing this micro-organism It should be noted that

  that being able to dispose of the culture does not mean that the present invention can be

  derogation from the laws of Industrial Property.

  6-fluoro-HAD is useful as an international compound

  For example, by application of current technology as described in the steroid literature, fluoro-HAD derivatives can be converted to pharmacologically important steroids such as fluocinolone-acetonide, fluocinonide, flurandrenolide,

paramethasone, fluprednisolone.

  The following are examples illustrating the fermentation process of the present invention. These examples are only illustrations and therefore must

  not be interpreted as limiting All percentages

  are expressed by weight and all the proportions of the solvent mixtures are expressed in volume, except

contrary specification.

Example 1

  Fermentation of 6α-F-cholestenone The biotransformation medium contains (per liter) 8.0 g of "Ucon"; 5.0 g of "Cerelose"; 3.0 g of NH 4 Cl; 3.0 g of CaCO 3; 3.0 g of Na 3 -citrate 7H 2 O; 2.0 g of "Tween 80"; 1.0 g of soy flour; 0.5 g of

  KH 2 PO 4; 0.5 g of urea and 2.0 g of 68-F-cholestenone in.

  city water with p H adjusted to 7.0 vials

  containing 100 ml portions of this medium are inoculated

  with 10 ml of M fortuitum NRRL B-8119 seed cultures, grown at 28 in medium containing (per liter), 8.0 g of nutrient broth; 5.0 g of glycerol; 1.0 g of yeast extract and 1.0 g of "Tween 80" in distilled water, p H being adjusted to 7.0. The cultures are then incubated at 28 for 336 hours on a rotary shaker. After the incubation, the

  mixture with an equal volume of CH 2 Cl 2 and isolate the

  duit, namely 6-fluoro-HAD, of this extract using

  conventional chromatographic methods.

Example 2

  Substituting 6-fluorocholestenone for 63-fluorocholestenone in Example 1 gives the

6α-fluoro-9α-hydroxy-AD.

Example 3

  Substituting a fluoroketone derivative of another sterol, for example sitosterol, stigmasterol, etc.,

  6-fluoro-HAD is obtained: Sterols can be used

  individually or in mixtures and in the form of

pure or in the raw form.

Example 4

  Using a sterol-degrading microorganism selected from the genera Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Nocardia, Protaminobacter, Serratia and Streptomyces, in place of Mycobacterium fortuitum NRRL B-8119 in the process described in the US Pat. No. 4,029,549 for the preparation of M fortuitum NRRL B-8119, mutant microorganisms are obtained which are characterized by their

  ability to selectively convert 6-fluoro-A 4- derivatives

  3-ketone sterols carrying a C-17 side chain

  and accumulating 6-fluoro-HAD product.

Example 5

  Substituting 6-fluorocholestenone in Example 1 for a 6-fluoro-4-3-ketone derivative of another sterol such as sitosterol, stigmasterol, etc.,

  6-fluoro-HAD is obtained. Sterols can be used

  individually or in mixtures and in the form of

pure or in the raw form.

Example 6

  Replacing M fortuitum NRRL B-8119 in Examples 1 and 3 with the mutants obtained in the example

4, 6 g-fluoro-HAD is obtained.

Example 7

  Using the mutants obtained in Example 4, in place of M fortuitum NRRL B-8119 in the examples

  2 and 5, 6-fluoro-HAD is obtained.

Example 8

  The 6-fluoro-4-4-ketone derivatives of sterols can be prepared following the procedures described in the United States patent.

N 4 100 027.

Claims (6)

  Fermentation process for the preparation of a compound of formula O O   F characterized in that it consists in cultivating Mycobacterium fortuitum NRRL B-8119 in an aqueous nutrient medium in   aerobic conditions in the presence of a 6-fluoro-A 4- derivative   3-ketone of a sterol having a lateral chalne in   C-17 and collect the desired product.   2 fermentation process for the preparation of a compound according to claim 1, characterized in that it consists in culturing a mutant microorganism selected from the genera Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Nocardia, Protaminobacter, Serratia and Streptomyces, said mutant being characterized   by its ability to selectively convert the 6- derivative   sterol fluoro-A 4-3 -ketone having a C-17 side chain and accumulating 6-fluoro-HAD in the fermentation broth in an aqueous nutrient medium   under aerobic conditions in the presence of a 6- derivative   4-3-keto fluoro-A of a sterol with or without a chain lateral C-17.   3 Fermentation process for the preparation   of a compound as defined in claim 1, which is   tered by cultivating a mutant of Myco-   bacterium which is characterized by its ability to selectively convert the sterol 6-fluoro-A 4-3-ketone derivative.   wearing a C-17 side chain and accumulating 6-   fluoro-HAD in the fermentation broth, in an aqueous nutrient medium under aerobic conditions in the presence   a steroid carrying a C-17 cha Xne.   4 Process according to any one of the   1 to 3, wherein said sterol is selected from the group consisting of sitosterol, cholesterol   terol, stigmasterol and campesterol or a mixture of so-called sterols.   As a novel compound, 6α-fluoro-9α-hydroxy-androst-4-ene-3,17dione.   Process according to claim 1, characterized   characterized in that said 6-fluoro-A 4-3-ketone derivative of a   Sterol is 6-fluorocholestenone.   The method of claim 1, wherein   characterized in that said 6-fluoro-A 4-3-ketone derivative of a   sterol is 6a-fluorocholestenone.