Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy... – Resolution of optical isomers or purification of organic...
Reexamination Certificate
2000-06-02
2002-09-17
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Resolution of optical isomers or purification of organic...
C435S122000, C435S117000, C435S118000
Reexamination Certificate
active
06451587
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel microbial processes. More specifically, the present invention provides microbial asymmetric reduction processes for preparing the alcohol (F)-2-chloro-1-[6-(2,5-dimethyl-pyrrol-1-yl)-pyridin-3-yl]-ethanol from the ketone 2-chloro-1-[6-(2,5-dimethyl-pyrrol-1-yl)-pyridin-3-yl]-ethanone. The alcohol is a known intermediate for the synthesis of certain &bgr;-adrenergic receptor agonists.
BACKGROUND OF THE INVENTION
Microbial asymmetric reductions are known which enable the selection of chirality in synthetic pathways, such as, for example, those processes disclosed in U.S. Pat. Nos. 5,049,497; 5,580,764; and 5,618,707, and in U.S. Provisional Patent Application No. 60/106,233.
U.S. Pat. No. 5,049,497 discloses a process for resolving a racemic derivative of bicyclo[4.2.0]octane by contacting the derivative with Baker's Yeast under conditions sufficient to give a mixture of a ketone and an alcohol of high enantiomeric purity. As described therein, only one enantiomer of the subject racemic ketone is reduced to give an alcohol.
U.S. Pat. No. 5,580,764 discloses an asymmetric reduction process which uses an intact microorganism, or a broken cell preparation thereof, to convert a cyclic ketone to the corresponding chiral alcohol.
U.S. Pat. No. 5,618,707 discloses a process for the stereoselective reduction of ketone substrates by adding the substrates to a culture broth of either
Zygosaccharomyces bailii
ATCC No. 38924 or
Schizosaccharomyces octosporus
ATCC No. 2479, incubating the resultant mixture, and isolating the hydroxy compound through conventional means such as, for example, extraction with organic solvents, adsorption to resins, or chromatography for subsequent use as an intermediate in the preparation of a serum cholesterol lowering agent. The isolated hydroxy compound described therein was analyzed by chiral HPLC or RP-HPLC, or both. Consistent with what would be understood by one of skill in the relevant art, as described therein, many of the large number of microorganisms which were investigated for their ability to reduce the ketone group of the selected substrate failed to reduce the ketone group with the desired specificity or productivity.
U.S. Provisional Patent Application No. 60/106,233 discloses novel microbial stereoselective reductions for preparing a key intermediate in a synthetic pathway for sertraline.
Asymmetric or enantioselective microbial reductions have also been reported in the scientific literature, such as disclosed, for example, by R. N. Patel et al., in “Microbial Synthesis of Chiral Intermediates for &bgr;-3-Receptor Agonists,”
JAOCS
75 (11): 1473-1482 (1998); by M. J. Zmijewski et al., in “Enantioselective reduction of 3,4-methylene-dioxyphenylacetone using
Candida famata
and
Zygosaccharomyces rouxii,” Appl. Microbiol. Biotechnol
. 47(2): 162-166 (1997); by C. Roberge et al., in “Asymmetric Reduction of a Keto Ester to Its Corresponding (S)-Hydroxy Ester by Microbacterium sp. MB 5614
,” J. Ferment. Bioeng
. 81(6): 530-533 (1996); by K. Lorraine et al., in “Asymmetric reduction of a ketosulfone to the corresponding trans-hydroxysulfone by the yeast
Rhodotorula rubra
MY 2169
,” Enzyme Microb. Technol
. 19:250-255 (1996); and by T. Nishida et al., in “Microbial Asymmetric Reduction. Preparation of Optically Active Methyl trans-3-(4-Methoxyphenyl)glycidates,”
Biocatalysis Biotransformation
12: 205-214 (1995).
International PCT Application No. PCT/IB95/00344 published as WO 96/35671, and corresponding to U.S. patent application Ser. No. 08/945,551, discloses certain &bgr;-adrenergic receptor agonists and processes for their preparation. These agonists have utility for, inter alia, the treatment of hyperglycemia, obesity, intestinal motility disorders, depression, prostate disease, dyslipidemia, and airway inflammatory disorders such as asthma and obstructive lung disease. The agonists are also useful for increasing lean meat deposition and/or improving the lean meat to fat ratio in edible animals such as ungulate animals and poultry.
More specifically, WO 96/35671 discloses a synthetic process for the preparation of 4-(2-(2-(6-aminopyridin-3-yl)-2(R)-hydroxyethyl-amino)ethoxy)-phenylacetic acid. The majority of the &bgr;-adrenergic receptor agonist activity of this compound resides in its (R)-enantiomer. Accordingly, since the process generates a chiral diol comprising both (S)- and (R)-enantiomers, the enantiomers are preferably separated (e.g., by fractional crystallization or chromatography).
It has now been unexpectedly found that a range of microorganisms, including bacteria and fungi such as yeasts, stereoselectively reduce a ketone to its corresponding alcohol of the desired stereochemistry.
More specifically, the microbial stereoselective reductions of 2-chloro-1-[6-(2,5-dimethyl-pyrrol-1-yl)-pyridin-3-yl]-ethanone provided by the present invention result in the formation of (R)-2-chloro-1-[6-(2,5-dimethyl-pyrrol-1-yl)-pyridin-3-yl]-ethanol which can be used, for example, in the synthesis of certain of the &bgr;-adrenergic receptor agonists disclosed in WO 96/35671. The use of an intermediate having the desired stereochemistry enables an earlier committment to the desired stereochemistry of the final product and fewer steps to achieve that ultimate product.
All of the documents cited herein, including the foregoing, and any and all of the incorporated documents of the foregoing, are incorporated by reference herein in their entireties.
SUMMARY OF THE INVENTION
The present invention relates to microbiological reduction of carbonyl groups which comprises contacting an amount of a ketone compound, the compound of Formula A below, with a microorganism, or an enzyme reduction system capable of accomplishing the subject reduction comprising an enzyme derived from said microorganism and a co-factor for said enzyme, and incubating the resultant mixture under suitable conditions such that more of the compound of Formula B below than the compound of Formula C below can be formed and accumulated in the medium. The compound of Formula B is the desired corresponding alcohol of the compound of Formula A due to its having a hydroxy group of the desired stereochemistry.
The compound of Formula A (“substrate”) has the following structure:
The desired corresponding alcohol product, the compound of Formula B, has the following structure:
Where produced, the other alcohol product of the reduction, the compound of Formula C, has the following structure:
Accordingly, the present invention provides processes for carrying out a microbial asymmetric reduction
which comprises: contacting an amount of the compound of Formula A with a microorganism, or an enzyme reduction system capable of accomplishing the subject reduction comprising an enzyme derived from said microorganism and a co-factor for said enzyme, and incubating the resulting mixture under conditions sufficient to yield more of the compound of Formula B than of the compound of Formula C.
In a preferred embodiment, the microorganism is a bacterium. In a preferred embodiment wherein the microorganism is a bacterium, the bacterium is selected from the group consisting of
Rhodococcus globerulus
ATCC No. 21505, Rhodococcus sp. ATCC No. 15592, and any suitable mutants thereof.
In another preferred embodiment, the microorganism is a fungus. In a preferred embodiment wherein the microorganism is a fungus, the fungus is selected from
Mucor ambiguus
IFO 06742
, Geotrichum candidum
ATCC No. 07341, and any suitable mutants thereof.
In another preferred embodiment, the microorganism is a yeast. In a preferred embodiment wherein the microorganism is a yeast, the yeast is selected from the group consisting of
Pachysolen tannophilus
ATCC No. 32691
, Rhodotorula mucilaginosa
v.
mucilaginosa
ATCC No. 90687
, Rhodotorula mucilaginosa
v.
mucilaginosa
ATCC No. 04558
, Zygosaccharomyces bailii
ATCC No. 38924 (also deposited as PTA-660),
Rhodotorula mucilaginosa
v.
mucilaginosa
ATC
Burns Michael P.
Wong John W.
Benson Gregg C.
Kispert Jennifer A.
Marx Irene
Pfizer Inc.
Richardson Peter C.
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