Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
2002-03-27
2004-07-20
Lilling, Hebert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C435S255400
Reexamination Certificate
active
06764842
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a bioreductive process for the efficient preparation of chiral allylic alcohol intermediates which are useful in the asymmetric synthesis of integrin &agr;v&bgr;3 receptor antagonists. The process comprises an enantioselective 1,2-reduction of a prochiral &agr;,&bgr;-unsaturated ketone using a yeast microorganism to afford a chiral allylic alcohol which can be further processed into the desired substituted nonanoic acid derivative, which is useful as an integrin &agr;v&bgr;3 receptor antagonist for the inhibition of bone resorption and treatment of osteoporosis.
BACKGROUND OF THE INVENTION
The present invention provides an efficient process for the preparation of a chiral allylic alcohol of structural formula I,
having the (R)-configuration at the stereogenic center marked with an *; wherein R is hydrogen or methyl.
The preparation of compounds of structural formula I in the racemic form was disclosed in U.S. Pat. No. 6,048,861 (Apr. 11, 2000), which is incorporated by reference herein in its entirety. The racemic allylic alcohols disclosed therein were converted in several steps into the desired 3-(pyrimidin-5-yl)- and 3-(2-methyl-pyrimidin-5-yl)-9-(5,6,7,8-tetrahydro-[1,8]-naphthyridin-2-yl)-nonanoic acids, which are useful as integrin &agr;v&bgr;3 receptor antagonists for the inhibition of bone resorption. The enantiomerically pure forms of the final product were obtained by means of HPLC resolution of the racemic mixture on a chiral solid support. Since only one antipode of the final product is preferred for use as an integrin &agr;v&bgr;3 receptor antagonist, the achiral process disclosed in U.S. Pat. No. 6,048,861 is inefficient in the sense that equal amounts of the less preferred enantiomer are obtained.
The present invention provides a process for the preparation of a chiral allylic alcohol of structural formula I having the (R)-configuration at the indicated stereogenic center in an efficient enantioselective fashion via bioreduction of a prochiral &agr;,&bgr;-unsaturated ketone of structural formula II with a yeast microorganism.
SUMMARY OF THE INVENTION
The present invention is concerned with a process for the preparation of chiral (R)-allylic alcohols of structural formula I. The process utilizes an enantioselective bioreduction with a yeast microorganism under propagation conditions that give rise to enhanced enantioselectivity in the reduction of a prochiral &agr;,&bgr;-unsaturated ketone of structural formula II. The chiral (R)-allylic alcohols obtained in this fashion are key intermediates in the asymmetric synthesis of integrin &agr;v&bgr;3 receptor antagonists, which are useful for inhibiting bone resorption and treating osteoporosis.
DETAILED DESCRIPTION OF THE INVENTION
The process of the present invention involves the preparation of a chiral allylic alcohol of structural formula I having the (R)-configuration at the indicated stereogenic center by an asymmetric bioreduction reaction involving the incubation of an enone substrate of structural formula II with whole cells of a yeast microorganism in a suitable propagation medium.
The chiral allylic alcohols of structural formula I of the present invention can be converted in a 3-step sequence of Claisen rearrangement, hydrogenation, and hydrolysis, as described in U.S. Pat. No. 6,048,861, into the final products of structural formula III, which are useful as integrin &agr;v&bgr;3 receptor antagonists.
Prior to to filing date of the present application, samples of the microorganisms,
Candida chilensis
strain MY1708 and
Candida schatavii
strain MY1831, were deposited at the American Type Culture Collection (ATCC), Manassass, Va. The culture assess designations are ATCC PTA-4078 and 74439, respectively. The deposits will be maintained in the ATCC under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Upon granting of a patent disclosing the deposits, all restrictions imposed by the depositor on the availability to the public of the deposited material will be irrevocably removed.
The full taxonomic profiles of
Candida chilensis
and
Candida schatavii
are detailed in: The Yeasts, A taxonomic study 4th edition (Editors Kurtzman and Fell 1998 Elsevier). The following is a brief description of growth, morphological and taxonomic characteristics of
Candida chilensis
and
Candida schatavii.
(a)
Candida chilensis
strain MY 1708 could be propagated on complex growth media including Saboraud Dextrose Broth (SDB, Difco), Yeast Extract/Peptone, and Potato Dextrose Broth. Galactose, glycerol, and glucose were demonstrated to be assimilated carbon sources and either glutamate or ammonium could satisfy nitrogen source requirement when defined yeast basal media were employed. Fermentative growth was possible only with glucose as a carbon source. Growth on SDB agar plates at 25 C. was apparent within 48 hours. Growth at 29 C. was sluggish and no growth at 35 C. could be observed. Colonies were smooth, white to beige, and irregular at edges. Microscopic evaluation indicated ovoidal to longer elliptical forms (average approximately 4 by 4 uM) typically as single units but with some tendency to cluster.
(b)
Candida schatavii
strain MY1831 could also be propagated on the complex growth media described above for
Candida chilensis
. Galactose, glycerol, and glucose also served as carbon sources and glutamate or ammonium could be utilized as carbon source. Either glucose or galactose could be used as carbon source to support fermentative growth. Colony formation was evident on SDB medium within 48 hours at 25 C., 29 C., and in contrast to
Candida chilensis
, 35 C. Colonies were white to cream and shiny. Ovoid to elliptical morphology (average 4 by 4 uM, similar to chilensis) was observed microscopically typically as single cells or in small clusters (<3).
In one embodiment of the enantioselective bioreduction of the present invention, the yeast microorganism is
Candida chilensis
strain MY1708 or
Candida schatavii
strain MY1831. In a class of this embodiment, the yeast microorganism is
Candida chilensis
strain MY1708.
The bioreduction requires growth of the microorganism, such as
Candida chilensis
or
Candida schatavii
, in the presence of a suitable propagation medium, such as Yeast media (YM), Sabouraud dextrose broth (SDB), and Yeast nitrogen base (YNB), or a yeast medium as defined in Table 1, for 24-96 hours followed by a period of contact with the ketone substrate (I) under appropriate incubation conditions. The presence of the allylic alcohol product is observed postcharge when solvent-extracted samples are analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC). In a second embodiment, the enone substrate of formula II is supplied at a final concentration of 0.5 to 15 grams per liter. The incubation conditions consist of aeration at a temperature of about 22° C. to about 30° C. at a pH of about 6.5 to about 8.5, and about 5-50 g/L of glucose to regenerate the required cofactor in whole cells.
In another embodiment, the bioreduction of the present invention is carried out in the presence of a carboxylic acid of structural formula IV:
wherein
“a” represents a single bond or a double bond;
n is 0, 1, or 2;
R
1
is hydrogen, phenyl, or methyl; and
R
2
is C
1-4
alkyl or aryl wherein aryl is selected from the group consisting of phenyl, naphthyl, biphenyl, pyridyl, furanyl, thienyl, pyrrolyl, benzofuranyl, benzothiophenyl, and indolyl, wherein the aryl group is unsubstituted or substituted with one to five substituents independently selected from halogen, amino, C
1-4
alkylamino, di-(C
1-4
alkyl)amino, hydroxy, C
1-4
alkoxy, and C
1-4
alkyl. When “a” represents a double bond, both the “cis” and “trans” geometric isomers are intended to be encompassed within the carboxylic acids of formula IV.
In one class of this embodiment, “a” represents a double bond, n is 0, R
1
is hydrogen, and R
2
is aryl. In a subclass of this class
Boyd Russell Fieldon
Gbewonyo Kodzo
McWilliams James Christopher
Nti-Gyabaah Joseph
Pollard David J.
Lilling Hebert J.
Merck & Co. , Inc.
Shatynski Patricia A.
Winokur Melvin
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