WO2001004342A1

WO2001004342A1 - Process to prepare exemestane

Info

Publication number: WO2001004342A1
Application number: PCT/US2000/016298
Authority: WO
Inventors: Mark A. Krook; Bradley D. Hewitt
Original assignee: Pharmacia and Upjohn Co; Upjohn Co
Current assignee: Pharmacia and Upjohn Co
Priority date: 1999-07-07
Filing date: 2000-06-27
Publication date: 2001-01-18
Anticipated expiration: 2002-01-07
Also published as: AU5873300A

Abstract

The present invention is an enzymatic Δ1-dehydrogenation of 6-methyleneandrost-4-ene-3,17-dione (II) to produce 6-methyleneandrosta-1,4-diene-3,17-dione (III) which is a useful pharmaceutical agent.

Description

PROCESS TO PREPARE EXEMESTANE BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is an enzymatic Δ'-dehydrogenation of a 6-methylene steroid. 2. Description of the Related Art

Biotechnology and Bioengineering, 37, 97-102 (1991) discloses the Δ'- dehydrogenation of 6α-methylhydrocortisone 21 -acetate by A. simplex in an organic solvent. The steroid of the present invention is not a 6α-methyl steroid.

US Patent 4,684,610 claims a process for converting 1,2-saturated steroids to 1,2- dehydro steroids by contacting the 1,2-saturated steroid with A. simplex or B. cyclooxydans in the presence of exogenous electron carrier and a water-immiscible aromatic hydrocarbon solvent. No 6-methylene steroids were exemplified.

US Patent 4,808,616 (EXAMPLE 1) discloses the transformation of 6- methyleneandrost-4-ene-3,17-dione to 6-methyleneandrosta-l,4-diene-3,17-dione by nonmicrobial chemical means, using DDQ. The present invention uses biotransformation to accomplish the Δ'-dehydrogenation.

US Patent 4,990,635 (EXAMPLES 2-4) discloses the transformation of 6- methyleneandrost-4-ene-3,17-dione to 6-methyleneandrosta-l,4-diene-3,17-dione by nonmicrobial chemical means. The 2-bromo intermediate is formed followed by debromination. The present invention does not form any 2-bromo intermediates but rather uses biotransformation to accomplish the Δ'-dehydrogenation.

US Patent 4,876,045 (EXAMPLE 2) discloses the transformation of 6- methyleneandrost-4-ene-3,17-dione to 6-methyleneandrosta-l,4-diene-3,17-dione by use of Jones reagent. The present invention does not use a chemical process but rather uses a biotransformation to accomplish the Δ'-dehydrogenation.

US Patent 4,749,649 discloses the use to scavengers of toxic oxygen species in the microbial Δ'-dehydrogenation of steroids.

SUMMARY OF INVENTION Disclosed is a process for the preparation of 6-methyleneandrosta-l,4-diene-3,17- dione (UT) which comprises contacting 6-methyleneandrost-4-ene-3,17-dione (H) with Δ¹- dehydrogenating enzymes of A. simplex in the presence of a water-immisible organic solvent and an exogenous electron carrier.

l - DETAILED DESCRIPTION OF THE INVENTION Androst-4-ene-3,l 7-dione (I) is known, see US Patent 3,112,305. It can be transformed to 6-methyleneandrost-4-ene-3,l 7-dione (II) by methods well known to those skilled in the art. 6-Methyleneandrost-4-ene-3,l 7-dione (II) is known, see US Patents 3,112,305

(EXAMPLES 4 and 39) and 4,990,635 (Example 1). It is dehydrogenated to form 6- methyleneandrosta-l,4-diene-3,l 7-dione (in) as generally set forth below and more specifically as set forth in Examples A and 1-20.

The conversion of 6-methyleneandrost-4-ene-3,l 7-dione (H) to 6- methyleneandrosta- 1 ,4-diene-3 , 17-dione (in) with A. simplex can be performed using whole cells or cell free preparations. It is preferred to use whole cells using a two phase system. The non-aqueous phase contains the 6-methyleneandrost-4-en-3,l 7-dione (H), an exogenous electron carrier and a water-immiscible organic solvent. It is preferred that the 6-methyleneandrost-4-en-3,l 7-dione (H) be present in a concentration of from about 10 g/L to about 125 g/L; it is more preferred that the 6-methyleneandrost-4-ene-3,l 7-dione (H) is present in a concentration from about 50 g/L to about 125 g/L. Operable exogenous electron carriers are selected from the group consisting of menadione, menadione bisulfite, 1,4-naphthoquinone, phenazine methosulfate, phenazine ethosulfate and vitamin K-type compounds. It is preferred that the exogenous electron carrier is selected from the group consisting of menadione and 1,4-naphthoquinone; it is more preferred that the exogenous electron carrier is menadione. It is preferred to add the exogenous electron carrier in catalytic amounts, for example, in an amount of from about 3 to about 5% (wt exogenous electron carrier/wt of 6-methyleneandrost-4-ene-3,l 7-dione (IT)). It is more preferred that the exogenous electron carrier is present in an amount of about 4%. Operable water-immiscible organic solvents are selected from the group consisting of toluene, xylene, benzene, heptane, methylene chloride, n-octanol or mixtures thereof and the alike. It is preferred that the water-immiscible organic solvent be toluene. It is preferred that the water-immiscible organic solvent be present in a range of from about 1 to about 99% it is more preferred that the water-immiscible organic solvent is present in a range of from about 60 to about 95%.

It is preferred that the aqueous phase contain both catalase and a buffer. Virtually any buffer which will maintain the pH in the range of from about a low end of pH range 8 to about high end of pH range 9 is operable and these buffers are well known to those skilled in the art. It is preferred that the buffer be phosphate. The aqueous phase is prepared by mixing catalase, the buffer, and a cell concentrate of A. simplex and water.

The aqueous phase is preferably added to the briskly agitated non-aqueous phase to begin the bioconversion. The temperature is controlled at about 30° and the pH is controlled between 8.7 and 8.9 by the addition of base, preferably hydroxide, and more preferably sodium hydroxide. The reaction mixture is preferably sparged with both air (about 0.10 SCHF) flow and nitrogen (0.30 SCHF) flow and permitted to react until less than about 0.1% residual 6-methyleneandrost-4-ene-3,l 7-dione (H) remains. If necessary, additional source of enzyme is added to assure completion. When the reaction is complete, the pH control, the air and nitrogen feeds and agitation are turned off and the reaction mixture permitted to settle. The aqueous phase is drawn off from the bottom of the reactor and then the non-aqueous phase is removed. Additional water-immiscible organic solvent is added to the bioconversion vessel, the agitator is again started and the aqueous phase is added. Again the agitation is stopped and the aqueous phase is permitted to settle. The spent aqueous cell layer is discarded and the water-immiscible organic solvent phase is drained from the reactor. The water-immiscible organic solvent phase is filtered to assure cell removal. The clarified filtrate is then concentrated and the desired 6-methyleneandrosta-l,4-diene-3,l 7-dione (in) obtained by means well known to those skilled in the art. 6-Methyleneandrosta-l,4-diene-3,l 7-dione (lTf) is known, see US Patent 4,808,616, claim 1 (Example 1) and US Patent 4,990,635 (Example 4). The compound is generically claimed in US Patent 4,904,650, which discloses the utility of 6-methyleneandrosta-l,4- dien-3,17-dione (IH).

DEFINITIONS AND CONVENTIONS The definitions and explanations below are for the terms as used throughout this entire document including both the specification and the claims.

DEFINITIONS All temperatures are in degrees Centigrade. DDQ refers to 2,3-dichloro-5,6-dicyano-l,4-benzoquinone. Chromatography (column and flash chromatography) refers to purification separation of compounds expressed as (support, eluent). It is understood that the appropriate fractions are pooled and concentrated to give the desired compound(s).

Pharmaceutically acceptable refers to those properties and/or substances which are acceptable to the patient from a pharmacological/toxicological point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding composition, formulation, stability, patient acceptance and bioavailability.

When solvent pairs are used, the ratios of solvents used are volume/volume (v/v). When the solubility of a solid in a solvent is used the ratio of the solid to the solvent is weight volume (wt/v).

SCFH refers to standard cubic feet hr.

EXAMPLES Without further elaboration, it is believed that one skilled in the art can, using the preceding description, practice the present invention to its fullest extent. The following detailed examples describe how to prepare the various compounds and/or perform the various processes of the invention and are to be construed as merely illustrative, and not limitations of the preceding disclosure in any way whatsoever. Those skilled in the art will promptly recognize appropriate variations from the procedures both as to reactants and as to reaction conditions and techniques. EXAMPLE A Bioconversion Of 6-Methyleneandrost-4-ene-3 , 17-dione (H) To 6- Methyleneandrosta- 1 ,4-diene-3 , 17-dione (HI) 6-Methyleneandrost-4-ene-3, 17-dione (II, 50 g) and menadione (2.0 g) are mixed with toluene (800 mL).

The aqueous phase is prepared as a mixture of catalase (0.015 g), potassium dibasic phosphate (2.2 g), A. simplex cell concentrate (obtained by fermentation, concentrated to contain about 20% dried solids, 50 mL) and water (50 mL). The aqueous phase is added to the briskly agitated toluene mixture (500 RPM) to begin the bioconversion. The temperature is controlled at 30° using a water bath and the pH is controlled between 8.7 and 8.9 using sodium hydroxide (2N) additions. The reaction mixture is sparged with a regulated flow air (0.10 SCFH) and nitrogen (0.30 SCFH) flow, respectively.

The mixture is allowed to react until less than 0.1 % residual 6-methyleneandrost-4- ene-3, 17-dione remains. If necessary, more A. simplex cells are added during the reaction to assure completion.

When the reaction is complete, the pH control, air feed, nitrogen feed, and agitation are turned off and the aqueous phase is permitted to settle. The aqueous layer is drawn from the bottom of the reactor and then the toluene mixture is removed.

Additional toluene (600 mL) is added to the bioconversion vessel. The agitator is started and the aqueous cell layer is added. The agitation is stopped and the aqueous phase is allowed to settle. The spent aqueous cell layer is discarded and the toluene extract is drained from the reactor.

The two toluene extracts are filtered. The clarified filtrate is then concentrated to about 100 mL. Octane (250 mL) is then added over a 30 - 45 minute period at 20-25°. The crystal slurry is cooled to -5 to -10 ° and held for 1 hr.

The crystals are collected by filtration, washed with octane (100 mL) and dried in a vacuum oven at 50° to give the desired crystalline 6-methyleneandrosta-l,4-diene-3, 17- dione (TJD. EXAMPLES 1 thru 20 Production of 6-Methyleneandrosta- 1 ,4-diene-3, 17-dione (III)

Following the general procedure of EXAMPLE A, and making non-critical variations (including amount of substrate, amount of cells, and reaction time as set forth below) the title compound is obtained.

CHART A PROCESS

Claims

CLAIMS 1. A process for the preparation of 6-methyleneandrosta-l,4-diene-3, 17-dione (HI) which comprises:

(1) contacting 6-methyleneandrost-4-ene-3, 17-dione (II) with Δ'-dehydrogenating enzymes of A. simplex in the presence of a water-immiscible organic solvent and an exogenous electron carrier.

2. A process according to claim 1 where the Δ'-dehydrogenating enzymes of A. simplex are present as whole cells of A. simplex.

3. A process according to claim 1 where the Δ'-dehydrogenating enzymes of A. simplex are present as a cell free preparation of A. simplex.

4. A process according to claim 1 where the water-immiscible organic solvent is selected from the group consisting of toluene, xylene, benzene, heptane, methylene chloride, n- octanol or mixtures thereof.

5. A process according to claim 5 where the water-immiscible organic solvent is toluene.

6. A process according to claim 1 where the water-immiscible organic solvent is present in a range of from about 1 to about 99%.

7. A process according to claim 7 where the water-immiscible organic solvent is present in a range of from about 60 to about 95%.

8. A process according to claim 1 where the exogenous electron carrier is selected from the group consisting of menadione, menadione bisulfite, 1,4-naphthoquinone, phenazine methosulfate, phenazine ethosulfate and vitamin K-type compounds.

9. A process according to claim 8 where the exogenous electron carrier is selected from the group consisting of menadione and 1 ,4-naphthoquinone.

10. A process according to claim 9 where the exogenous electron carrier is selected from the group consisting of menadione.

11. A process according to claim 1 where the exogenous electron carrier is present in an amount of from about 3 to about 5% (weight exogenous electron carrier/weight of 6- methyleneandrost-4-ene-3, 17-dione (H)).

12. A process according to claim 11 where the exogenous electron carrier is present in an amount of about 4%.

13. A process according to claim 1 where the contacting is performed in the presence of a buffer.

14. A process according to claim 13 where the buffer is selected from the group consisting of phosphate.

15. A process according to claim 1 where the reaction is performed in the presence of a regulated oxygen supply.

16. A process according to claim 1 where the reaction is performed in the presence of a regulated nitrogen supply.

17. A process according to claim 1 where the reaction is performed in the presence of catalase.

18. A process according to claim 1 where the 6-methyleneandrost-4-ene-3, 17-dione (H) is present in a concentration from about 10 g/L to about 125 g/L.

19. A process according to claim 18 where the 6-methyleneandrost-4-ene-3, 17-dione (IT) is present in a concentration from about 50 g/L to about 125 g/L.