Process for dehydrogenation of azaandrostane compounds

Details Process for dehydrogenation of azaandrostane compounds Process for dehydrogenation of azaandrostane compounds

2003-05-06

2004-07-13

Aulakh, Charanjit S. (Department: 1625)

Organic compounds -- part of the class 532-570 series

Organic compounds

Heterocyclic carbon compounds containing a hetero ring...

C546S061000

Reexamination Certificate

active

06762302

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel process for dehydrogenation of azaandrostane compounds.
2. Related Art
The 4-azasteroids are well known specific inhibitors of the testosterone 5&agr;-reductase enzyme. According to previously published data it is apparent that the introduction of the double bond in the C-1,2 position of the molecule is necessary for obtaining the desired effect.
There are both synthetic and biochemical methods for introducing a double bond into the C-1,2 position of 4-azasteroids.
One of the synthetic methods is the introduction of the &Dgr;
1
double bond with benzeneselenic anhydride in boiling chlorobenzene (for example, Patent No. EP 155 096).
Alternatively, dehydrogenation can be carried out with kinones (e.g. 2,3-dichloro-5,6-dicyano-benzokinone) in the presence of silylating agents (e.g., bis-trimethyl-silyl-trifluoro-acetamide) (Patent No. EP 298 652).
According to processes described in EP 428 366 and EP 655 459 the 2-halogen derivative of a steroid can be used as an intermediate to introduce the C-1,2 double bond. Patent No. CA 2 271 974 discloses a process in which the &Dgr;
1
double bond is introduced into an aza-steroid using a 2,2-dibromo-aza-steroid compound as an intermediate.
In another process, 2-halogen derivatives are prepared from the appropriate unsubstituted 4-aza compound via a trialkylsilyl-trifluoro-methanesulfonate intermediate with iodine or trimethylsilyl chloride and iodine (Patent No. EP 473 226). The desired &Dgr;
1
compound is obtained from this 2-halogen-4-aza derivative with potassium tert-butoxide in dimethylformamide solution in about 60% yield.
These synthetic methods require extremely aggressive reaction conditions that have several disadvantages. First, impurities are formed. Second, the impurities, although formed in low concentration (below 0.1%), are not characterized, and some might be toxic. In addition, some of the applied reagents are carcinogenic, flammable, demand completely dry circumstances, or are highly corrosive, and therefore are environmental hazards.
In contrast, the conditions required for biochemical methods for &Dgr;
1
dehydrogenation are gentle and produce fewer and less environmentally hazardous by-products. As a consequence, the formation of toxic compounds is less likely. Moreover, instead of two synthetic steps, the transformation can be carried out in a single step.
Although the &Dgr;
1
-dehydrogenation of steroids is well known in the literature (
Microbial Transformations of Steroids
, Charney, W., Herzog, H. L., Academic Press, New York, London, (1967)), there are very few examples of the microbiological introduction of a double bond into the C-1,2 position of aza-steroids, probably because of the well known antibacterial effect of aza-steroids (
J. Bacteriol
., 93(2), 627-35 (1967);
Steroids
, 27(4), 525-41 (1976);
Chem. Ind
., 52, 1660-1 (1970)).
The microbial introduction of a &Dgr;
1
double bond into 12a-aza compounds has been accomplished with Nocardia sp. and Arthrobacter sp. strains (Mazur et al.,
J. Org. Chem
., 28(9), 2442-3 (1963)). According to Mazur et al., the conversion of 12a-aza-C-homo-1,4-pregnadiene-3,12,20-trione could be realized at 0.29 g/L concentration with a 62% yield; the conversion of 12a-aza- 17&agr;-hydroxy-C-homo-1,4-pregnadiene-3,12,20-trione could be realized at 0.17 g/L concentration with a 17% yield; and the conversion of 12a-aza-C-homo-5&agr;-pregn-1-ene-3,12,20-trione could be realized at 0.29 g/L concentration with a 26% yield.
Similarly, Arthrobacter sp. and Nocardia sp. strains have been used to &Dgr;
1
-dehydrogenate the 17-N-tert-butylcarbamoyl-4-azaandrostan-3-one substrate at 0.1-1.0 g/L concentration with a 20-80% yield (Patent No. EP 786 011). In this method, the &Dgr;
1
-dehydrogenase enzymes of the above strains were induced by the addition of hydrocortisone, the separated biomass was suspended in a buffer (pH=6-8) saturated with organic solvent and the bioconversion was performed in the presence of menadione or phenazine methosulfate. The bioconversion was carried out in a small volume. In the only disclosed example the conversion was carried out for 3 days in a volume of 20 ml at 0.26 g/L substrate concentration with a 60% yield. However, for industrial realization, one must determine the appropriate concentration level and scale up the procedure. According to our experiments, when this procedure is scaled up, the conversion drops drastically. Therefore, a fundamental modification of this process is essential for industrial realization.
SUMMARY OF THE INVENTION
The aim of the present invention is to provide a bioconversion process that avoids the disadvantages of wholly synthetic methods while achieving industrial realization. More specifically, the invention relates to a process for the production of compounds of general formula (I), wherein R
1
is a —NH-tert-butyl group or a 4-methyl-piperidino group, by bioconversion of compounds of general formula (II), wherein R
1
is as described above, using a biocatalyst having steroid-&Dgr;
1
-dehydrogenase enzyme activity, wherein the activity of the enzyme needed for the bioconversion is produced by induction.
In one preferred embodiment, the present invention encompasses a method for producing a compound of formula I, comprising the steps of:
a) preparing a culture of a biocatalyst or an extract of a biocatalyst;
b) adding to the culture or extract an inducer of steroid-&Dgr;
1
-dehydrogenase activity;
c) adding to the culture or extract an electron carrier, a stabilizer, and a compound of formula II; and
d) incubating the culture or extract for a time sufficient for bioconversion of the compound of formula II to the compound of formula I to occur.
In another preferred embodiment, the present invention encompasses a method for producing a compound of formula I, comprising the steps of:
a) preparing a culture of a biocatalyst or an extract of a biocatalyst, wherein said culture or extract has a volume of at least about 1 L;
b) adding to the culture or extract an inducer of steroid-&Dgr;
1
-dehydrogenase;
c) adding to the culture or extract an electron carrier, a stabilizer, a complexing agent or emulsifier, and a compound of formula II; and
d) incubating the culture or extract for a time sufficient for bioconversion of the compound of formula II to the compound of formula I to occur.
In another preferred embodiment, the present invention encompasses a method for producing a compound of formula I, comprising the steps of:
a) preparing a culture of a biocatalyst or an extract of a biocatalyst;
b) adding to the culture or extract an inducer of steroid-&Dgr;
1
-dehydrogenase;
c) adding to the culture or extract an electron carrier at a concentration of about 0.05 to about 3.5 g/L and a stabilizer at a concentration of about 0.01 to about 0.1 g/L and a compound of formula II; and
d) incubating the culture or extract for a time sufficient for bioconversion of the compound of formula II to the compound of formula I to occur; provided that an oxygen scavenger is not added to the culture or extract.
DETAILED DESCRIPTION OF THE INVENTION
The invention relates to a process for the production of compounds of general formula (I), wherein R
1
is a —NH-tert-butyl group or a 4-methyl-piperidino group, by bioconversion of compounds of general formula (II), wherein R
1
is as described above, using a biocatalyst having steroid-&Dgr;1-dehydrogenase enzyme activity, by inducing the activity of the enzyme, maintaining the activity of the enzyme at the necessary level by adding a stabilizer, promoting the continuous dissolution of the substrate by adding a complexing agent, and completing the conversion by adding an electron carrier.
The term bioconversion is defined as the conversion of compound (II) to compound (I) through the use of a biocatalyst such that at least a measurable amount of compound (I) is produced.
The term biocatalyst is defined as any microorganism that contains or can be induced to contain steroid-&Dgr;
1
-dehydrogenase enz

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