MXPA99009970A - Microbial reduction stereoselectiva of a tetralone racém - Google Patents

Abstract

The present invention relates to processes for carrying out the following steroselective microbial reduction of a racemic tetralone, comprising: contacting a compound of formula (1) with a microorganism, or an enzyme reduction system capable of carrying out the present reduction, comprising an enzyme derived from said microorganism and a cofactor for said enzyme, and incubating the resulting mixture under conditions sufficient to produce the (4R) tetralol of formula (II) and leaving substantially unreacted the (4S) tetralone of formula (V) or "chiral tetralone." The chiral tetralone can be used in the synthesis of sertraline. The present process optionally further comprises separating the (4S) tetralone of formula (V) from (4R) tetralol of formula (II). The (4R) tetralol can be recycled to produce the compound of formula (I) and the present process is repeated to produce more desired (4S) tetralone of formula (

Description

STEREOSELECTIVE MICROBIAL REDUCTION OF A RACEMICA TETRALONE FIELD OF THE INVENTION The present invention relates to novel processes for preparing the (4S) enantiomer of 4- (3,4-dichlorophenyl) -3,4-dihydro-1 (2H) -naphthalenone (hereinafter referred to as "tetralone chiral" or "tetralone") (4S) ") and, more especially, refers to the stereoselective microbial reduction of racemic 4- (3,4-dichlorophenyl) -3,4-dihydro-1 (2H) -naphthalenone (hereinafter also referred to as" racemic tetralone ") ") to the chiral tetralone.

BACKGROUND OF THE INVENTION The chiral tetralone prepared by the methods of the present invention can be further reacted to prepare cis- (1S) (4S) -N-methyl-4- (3,4-dichlorophenyl) -1, 2,3,4-tetrahydro -1-naphthalenamine pure, usually called sertraline. Sertraline is well known for its usefulness, for example, as an antidepressant and anorectic agent and in the treatment of chemical dependencies, disorders related to anxiety, premature ejaculation, cancer and after a myocardial infarction. For the preparation of sertraline, methods are known in the art, such as, for example, those described in U.S. Patent Nos. 4,536,518, 4,777,288, 4,839,104, 4,855,500, 4,940,731, 4. 962,128, 5,082,970, 5,130,338, 5,196,607, 5,248,699, 5,442,116, . 463.126, 5.466.880, 5.597.826 and 5.750.794 and, in the W: M: Welch article, Jr. et al., Which was published in the Journal of Medicinal Chemistry, vol. 27, No. 11, p. 1508 (1984). Some of the aforementioned patents refer to the synthesis of cis and trans isomer mixtures of racemic N-methyl-4- (3,4-dichlorophenyl) -1, 2,3,4-tetrahydro-1 -naphthalenamine. As described therein, the cis and trans isomers, as well as the (S) and (R) enantiomers, can be separated by methods known to those skilled in the art including, for example, fractional crystallization and chromatography. It is also known to select the chirality previously desired in the synthesis of sertraline. For example, the aforementioned U.S. Patent No. 5,750,794 describes a process for preparing chiral tetralone by reacting the racemic tetralone with a reducing agent based on an asymmetric ketone to provide the corresponding cis and trans alcohols, depending on the the chirality of the asymmetric reactant employed and, subsequently, separate the alcohols and oxidize the alcohols (1S, 4S) and / or (1 r, 4S) to the (4S) -tetralone. It is also known in the art that chiral compounds can be synthesized using microorganisms, such as fungi, for example, yeasts. For example, the use of yeasts to reduce ketones to chiral alcohols is well known. However, as is known to those skilled in the art, the chemical and optical yields, for example of the particular enantiomers and their amounts, of such microbial reductions, generally vary substantially depending on, for example, the particular microorganism. chosen, as well as the substituents of the starting material. U.S. Patent No. 5,049,497 describes 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 enatomeric purity. As described in said document, only one enantiomer of the subject racemic ketone is reduced to an alcohol. U.S. Patent 5,576,764 discloses an asymmetric reduction process using an intact microorganism, or a preparation of used cells thereof, to convert a cyclic ketone into the corresponding chiral alcohol. U.S. Patent No. 5,618,707 describes a process for the stereoselective reduction of ketonic substrates by adding the substrates to a culture medium of Zygosaccharomyces bailii ATCC (American Type Culture Collection) No. 38924 or Schizosaccharomyces octosporus ATCC No. 2479, incubating the resulting mixture and isolating the hydroxy compound by conventional means such as, for example, extraction with organic solvents, adsorption on resins, or chromatography for later use as an intermediate in the preparation of an agent that lowers serum cholesterol. The isolated hydroxylated compound described in said document was analyzed by chiral high performance liquid chromatography (HPLC), reverse phase HPLC or both. Consistent with what would be appreciated by one skilled in the corresponding art, as described therein, many of the large number of microorganisms that were investigated for their ability to reduce the ketone group of the selected substrate failed to reduce the ketone group with specificity or desired productivity. It has now been unexpectedly discovered that a series of microorganisms, including fungi, for example yeasts and actinomycetes, substantially substantially reduces a racemic tetralone. More especially, the present stereoselective microbial reduction object selectively reduces the (4R) tetralone of the racemic mixture leaving the (4S) tetralone substantially unreacted. On the other hand, the unwanted tetralol (4R) produced by the present process can be oxidized and converted to the racemic tetralone and the present process repeated to provide even more (4S) tetralone. The (4S) tetralone produced by the present process can be used in the synthesis of sertraline. All documents cited in the present, including the foregoing, are incorporated herein by reference in their entirety.

SUMMARY OF THE INVENTION The present invention relates to the microbiological reduction of carbonyl groups comprising, contacting a ketone compound, tetralone recémica of formula (I), with a microorganism, or an enzyme reduction system, capable of carrying out the present reduction , comprising an enzyme derived from said microorganism and a cofactor for said enzyme, and incubating the resulting mixture under suitable conditions so that a compound having a hydroxy group, specifically, (4R) tetralol can be formed and accumulated in the medium. of formula (II) and, a compound having the desired stereochemistry, the (4S) tetralone of formula (V) below, remains substantially unreacted. The tetralone (4S) of formula (V) below, that is, the chiral tetralone, can then be isolated by any suitable method, for example, chromatography or crystallization. In addition, the (4R) tetralol of formula (II) can be separated from the compounds of formulas (III) to (V), oxidized and form the racemic tetralone and the present stereoselective microbial reduction repeated to produce even more desired chiral ketone. Accordingly, the present invention provides procedures for carrying out the following stereospecific microbial reduction: comprising: contacting a compound of formula (I) with a microorganism, an enzymatic reduction system capable of carrying out the present reduction, comprising an enzyme derived from said microorganism and a cofactor for said enzyme, and incubating the mixture resulting under conditions sufficient to produce more compound of formula (II) than compound of formula (III), thus leaving more compound of formula (V) unreacted than unreacted compound of formula (IV). The present stereospecific reduction can also be represented by: (0 (II) (V) comprising: contacting a compound of formula (I) with a microorganism, or an enzyme reduction system capable of carrying out the present reduction, comprising an enzyme derived from said microorganism and a cofactor for said enzyme, and incubating the resulting mixture under conditions sufficient to produce the (4R) tetralol of formula (II) and leave substantially unreacted the (4S) tetralone of formula (IV). The stereoselective reduction further comprises, optionally, the separation of the (4S) tetralone of formula (V) from the (4R) tetralol of formula (II). The (4R) tetralol can then be oxidized to produce the (4R) tetralone, which is then reacted, for example, with a base, to produce the racemic tetralone of formula (I) and the present stereoselective microbial reduction can be repeated for producing even more (4S) tetralone of formula (V), ie, the (4S) enantiomer of the racemic tetralone of formula (I). The present invention provides methods comprising the stereoselective microbial reduction of a compound of formula (I) to a compound of formula (II): by contacting a compound of formula (I) with a microorganism, or an enzyme reduction system capable of carrying out the present reduction, comprising an enzyme derived from said microorganism and a cofactor for said enzyme, and incubating the resulting mixture under conditions sufficient to produce a compound of formula (II), whereby substantially no further compound of formula (V) which is composed of formula (IV) and substantially more compound of formula (II) than compound of formula (III) is produced. In a preferred embodiment, the contacting of the compound of formula (I) is carried out with an enzymatic reduction system. In another preferred embodiment, the contacting of the compound of formula (I) is carried out with an enzymatic reduction system in which the enzyme is immobilized. In a particularly preferred embodiment, the contacting of the compound of formula (I) is carried out with an enzyme reduction system derived from Hansenula polymorpha ATCC No. 26012. In another preferred embodiment, the microorganism is a preparation of used cells thereof. In yet another preferred embodiment, the microorganism is an enzyme powder preparation in acetone thereof. In an especially preferred embodiment of the present invention intact microorganism is used. In a preferred embodiment in which the microorganism is an intact microorganism, the compound of formula (I) is contacted with a fermentation medium, culture broth or solvent, comprising the microorganism. In another preferred embodiment in which the microorganism is an intact microorganism, the compound of formula (I) is contacted with an intact washed microorganism. In another preferred embodiment in which the microorganism is intact, the compound of formula (I) is contacted with an immobilized intact microorganism.

In an especially preferred embodiment of the present invention, the microorganism is an intact microorganism that develops in a fermentation medium and the contact is produced by the addition of the compound of formula (I) thereto. In another especially preferred embodiment of the present invention, the microorganism is an intact microorganism that grows in a growth medium for about forty-eight hours, and contact occurs in said growth medium by the addition of the compound of formula (I). ) to the same one and the incubation lasts approximately five days. In another preferred embodiment of the present invention, the microorganism in a fungus, for example, a yeast, an actinomycete, or one of its mutants that can carry out the stereoselective reduction. In another preferred embodiment of the present invention, the microorganism is a fungus. In another still more preferred embodiment in which the microorganism is a fungus, the fungus is Absidia coerulea ATCC No. 20137. In a particularly preferred embodiment of the present invention, the microorganism is a yeast. In an especially preferred embodiment of the present invention wherein the microorganism is a yeast, the yeast is Hansenula polymorpha ATCC No. 26012, also deposited as ATCC No. 74449.

When Hansenula polymorpha ATCC No. 26012, also deposited as ATCC No. 74449, is used as microorganism, the present stereoselective microbial reduction appreciably reduces only one enantiomer of the compound of formula (I), giving the corresponding alcohol, ie, the compound of formula (II), while the other enantiomer of the compound of formula (I), ie, the compound of formula (V) remains substantially unreacted. As described above, the methods of the present invention optionally further include separation, for example carried out using crystallization or chromatography, from the compound of formula (V) of the compounds of formula (II) to (IV) and the use of said separate compound of formula (V) in the synthesis of sertraline using any of the methods known for this. As also described above, it is preferred to oxidize the isolated (4R) tetralol of formula (II) to the (4R) tetralone of formula (IV). Next, it is further preferred to form the racemic, preferably by reacting the (4R) tetralone with a base, by transforming the (4R) tetralone of formula (IV) into the racemic tetralone of formula (I). Oxidation and racemic formation recycles the unwanted (4R) tetralol to another cycle of stereoselective microbial reduction according to the methods of the present invention. Recycling the desired (4S) tetralone and decreasing the amount of unwanted (4R) tetralol discarded. Oxidation and racemic formation of the oxidized product can be carried out using any of the methods known for this.

DETAILED DESCRIPTION OF THE INVENTION Those skilled in the art will fully understand the terms used herein to describe the present invention; however, the following terms are used herein as described below. "Cofactor" means any suitable cofactor comprising the enzyme reduction system such as, for example, NADH, NADPH, FADH, FMNH and / or PQQ or any of the cofactors that occur with the enzyme in the microorganism. "Enzymatic reduction system" means any suitable microbial oxido-reductase enzyme and the reduced form of a cofactor for the enzyme oxido-reductase, the cofactor of the selected microorganism being derivable or being derived from any suitable source. The enzyme comprising the enzyme reduction system can be in free form or immobilized, for example, in a column or linked to spheres. "Microbial reduction" means the stereoselective reduction of the present invention, as carried out by the enzyme reduction system, the enzyme reduction system comprising the intact microorganism or any of its preparations and the like.

"Microorganism" includes any intact microorganism or its preparations, including for example, a preparation of used cells of the microorganism; a dehydrated preparation of the microorganism, for example, an enzyme preparation powder in acetone; microorganism washing event of, for example, fermentation medium, culture medium and the like; immobilized microorganism, for example, in a column, attached to spheres and the like. The methods provided by the present invention comprise the stereoselective microbial reduction of a compound of formula (I) to a compound of formula (II): contacting a compound of formula (I) with a microorganism, or an enzymatic reduction system capable of carrying out the present reduction, comprising an enzyme derived from said microorganism and a cofactor for said enzyme, and incubating the resulting mixture under sufficient conditions to produce a compound of formula (II), whereby, more compound of formula (V) than compound of formula (IV) remains unreacted and substantially more compound of formula (II) than compound of formula (III) is produced ). As will be understood by those skilled in the art, the compound of formula (I), the racemic tetralone, is a mixture of the (4S) tetralone and (4R) tetralone as shown below: (trans) (1 S. 4R) (cis) (1 R, 4R).

The compounds, or more especially, the tetraloles of formula (II) are: (trans) (1 S.4R) (cis) (1 R, 4R) The compounds, or more especially, the tetraloles of formula (III) are: The compounds of formula (II) and (III) are described and claimed in the aforementioned U.S. Patent No. 5,750,794. The desired compound of formula (V) can be isolated as described below from the undesired compounds of formula (II) and any of the compounds of formula (II) or (IV) which may have been produced or will be left without react, respectively, depending on, for example, the microorganism selected and the incubation conditions. The compounds of formula (II) can be converted into a compound of formula (I), for example, by oxidizing or forming the racemic, and the present stereoselective microbial reduction carried out to produce additional amounts of the (4S) tetralone of formula ( V). The process of the present invention is carried out easily. Thus, the microorganism is fermented (intanus microorganism) or incubated (preparation of used cells, dehydrated preparation or any other suitable preparation of the microorganism) in the presence of the racemic tetralone, represented by the formula (I), to modify the racemic tetralone and , more especially, to reduce the undesired enantiomer (4R) of the racemic ketone to its corresponding alcohol, represented by the formula (II), leaving the desired (4S) enantiomer, represented by the formula (V), substantially unreacted, of this mode in one step, giving rise to the optically enriched enantiomer (4S). The (4S) enantiomer can be further reacted by methods well known to those skilled in the corresponding art as described, for example, in U.S. Patent Nos. 4,536,518, 4,777,288, 4,839,104, 4,855. 500, 4,940,731, 4,962,128, 5,082,970, 5,130,338, 5,196,607, 5,248,699, 5,442,116, 5,463,126, 5,466,880, 5,597,826, and 5,750,794 and, in the cited WM article Welch, Jr. and others, to finally produce sertraline. The activity, methods for assaying activities, dosages, dosage forms, administration procedures and prior information relating to sertraline are described, for example, in U.S. Patent Nos. 4,536,517, 4,777,288 and 4,839,104 previously. cited and in the WM article Welch, Jr. and others, previously cited. Any suitable microorganism can be used in the process of the present invention. As described before, the microorganism used in the present method may be intact, or in the form of any of its preparations, for example, a preparation of used cells thereof, a dehydrated preparation thereof and be free or immobilized. However, when a non-intact microorganism is employed in the present invention as, for example, a preparation of used cells, for example a cell extract, enzyme preparation powder in acetone or in enzyme derived therefrom, those skilled in the art They will understand that a cofactor for the enzyme will also be included. Those skilled in the art will understand from the description set forth herein and their related knowledge how to prepare a cell preparation used as described, for example, by R.N. Patel et al. In the article "Oxidation of Secondary Alcohols to Methyl Ketones by Yeast" published in Apllied and Enviromental Microbiology, 38 (2): 219-223 (197). Those skilled in the art will understand from the description set forth herein and their related knowledge how to prepare an enzyme preparation powder in acetone as described, for example, by K. Nakamura et al. In the article "Asymmetric Reduction of Ketones by the Acetone Powder of Geotrichum candidum "published in 'Tetrahedron Letters, 37 (10): 1629-1632 (1996). In addition, an enzyme (for example an oxide reductase) of any suitable microorganism can also be used in the present methods, and this enzyme can be isolated from the microorganism by any suitable method known to those skilled in the art and, as in the case of the intact microorganism can be used in the present process in free or immobilized form. Those skilled in the art will understand from the description set forth herein and their related knowledge as to isolate and purify the enzyme from the suitable microorganism as generally described, for example, in the articles of: M. Wada et al. " Purification and Characterization of NADPH-Dependent Carbonyl Reduced, Involved in Stereoselective Reduction of Etyl 4-Chloro-3-oxobutanoate, from Candida magnoliae "published in Biosci, Biotechnol.

Biochem, 62 (2): 280-285 (1998), P. Trots et al., "Purification and Properties of NAP (P) H: (quinone-acceptor) oxidoreductase of sugarbeet cells" published in Eur. J. Biochem, 234 : 452-458 (1995), KM Madyastha and TL Gururaja, "Purification and Some of the Properties of a Novel Secondary Alcohol Dehydrogenase from Alcaligenes eutrophus", published in Biochemical and Biophysical Research Communications, 211 (2): 540-546 (1995 ), O. Bortolini et al., "Kinetic resolution of victools by Bacillus stearothermophilus diacetyl reducíase" published in Tetrahedron: Asymmetry, 9: 647-651 (1998), RN Patel et al., "Stereospecific microbial reduction of 4,5-dihydro- 4- (4-methoxyphenyl) -6- (trifluoromethyl-1 H-1) -benzazepin-2-one "published in Enzyme Microb. Technol., 3: 906-912 (1991) and R.N. Patel et al., "Stereoselecive microbial / enzymatic oxidation of (exo, exo) -7-oxabicyclo [2.2.2] hepyane-2,3-dimethanol to the corresponding chiral lacíol and lacfone" published in Enzyme Microb. Technol., 14: 778-784 (1992); and by U.S. Patent Nos. 5,523,223 and the aforementioned 5,580,764. Suitable microorganisms include Hansenula polymorpha ATCC No. 26012, Hansenula polymorpha ATCC No. 74449, Absidia coerulea ATCC No. 20137, Geotrichum candidum ATCC No. 34614, Geotrichum candidum ATCC No. 62401, Mortierella isabelina ATCC No. 42613, Mortierella isabellina ATCC No. 38063, Mortierella vinacea ATCC No. 09515, Pencillium notatum ATCC No. 36740, Blastoschizomyces capitatus ATCC No. 28575, Monosporium olivacerum v. ATCC No. 36300, Aureobasidium pullulans ATCC No. 16623, Debaryomyces polymorphus ATCC No. 20280, Saccharomyces cerevisiae ATCC No. 15248, Candida schatavii ATCC No. 24409, Pichia fabianii ATCC 16755 and Streptomyces rimmosus ss. RIMOSUS ATCC No. 10970; and their mutants which are known or can be obtained in any other way by those skilled in the corresponding art and can, in spite of said mutation, carry out the stereoselective microbial reduction described herein. Preferred intact microorganisms will be those that substantially reduce the (4R) tetralone by leaving the (4S) tetralone leaving the (4S) tetralone substantially unreacted, including the reduction reaction or any other intrinsic activity that may degrade or cause a negative impact on the (4R) tetralone. 4S) tetralone desired at any stage of the present process. As will be appreciated by those skilled in the art of the present disclosure, said undesired reaction of (4S) tetralone can be substantially prevented, for example, by using the enzyme derived from the microorganism selected against the intact microorganism. Suitable microorganisms for use in the present stereospecific microbial reduction can be prepared by any method known to those skilled in the corresponding art. The following is an example of a method suitable for the preparation of a microorganism from a commercially purchased stock solution. The method set out below can be used for any microorganism suitable for use in the present process and those skilled in the art will understand the description set forth herein to modify any part of the process, for example, a method for preparing the microorganism, intact or preparation, for example of used or dehydrated, free or immobilized cells; a process for preparing a suitable enzyme derived from said microorganisms; a method for contacting the racemic tetralone with the microorganism or the enzyme comprising the enzyme reduction system derived therefrom; the components of the growth medium and the conditions, for example, temperature, pH and the like; or the incubation conditions; to achieve the desired result in any particular procedure. Those skilled in the art will understand from the description set forth herein and their related knowledge how to prepare immobilized intact microorganisms as described, for example, by A. Bauer et al., in the article "Polyvinyl alcohol-immobilized whole-cell preparations for biotransformation of nitriles" published in Biotechnology Letters, 18 (3): 343-348 (March 1996). Any suitable method for contacting the compound of formula (I) with the microorganism or enzyme reduction system can be used in the present invention. The compound of formula (I) can be contacted with the microorganism or enzyme reduction system in any suitable order. For example, the compound of formula (I) may be added to a medium, such as a culture medium, comprising the microorganism, free or immobilized, or any combination thereof; or the medium can comprise the compound of formula (I) and the microorganism can then be added to said medium; or the compound of formula (I) and the microorganism can be added together to said medium; or the compound of formula (I) can be added to a preparation of used cells thereof; or the compound of formula (I) can be added to a dehydrated preparation of the microorganism; or the compound of formula (I) or the microorganism or the enzyme reduction system can be added to a suitable solvent comprising the other; and similar. Those skilled in the art will understand from the description set forth herein to modify any of the parts of the present process according to their wish. It is especially preferred in the present invention that the microorganism, or the enzyme reduction system, is derived from Hansenula polymorpha ATCC No. 26012. A lyophilized sample of Hansenula polymorpha ATCC No. 26012 (originally provided by DW Levine) was deposited with the ATCC, located at 10801 University Boulevard, Manassas, Virginia, 20110-2209, United States of America, under the conditions of Budapest Treaty on June 26, 1998. This newly deposited crop was assigned the ATCC deposit number 74449. Therefore, it is also especially preferred in the present invention that the microorganism be Hansenula polymorpha ATCC No. 74449. All limitations on the availability to the public of the culture of microorganisms thus deposited will be irrevocably removed after the granting of a patent from the specification of the present invention. The cultures of Hansenula polymorpha ATCC No. 26012 can be obtained from the ATCC and an example of a suitable process for its preparation from said commercially purchased stock solution is given below. A culture thus obtained is added to a suitable growth medium and incubated with agitation until growth occurs, both being as considered by those skilled in the art. The culture thus prepared can be used to inoculate tilted cultures, with portions of these frozen tilted cultures as stock solutions. Alternatively, cultures of the liquid stock can be prepared by adding about 10% to about 20% glycerol, then frozen at about -80 ° C, preferably in small cryogenic tubes.

As will be appreciated by those skilled in the art for any selected microorganism and as specifically set forth hereinbelow in the examples for Absidia coerulea ATCC No. 20137 and the especially preferred Hansenula polymorpha ATCC No. 26012 or ATCC No. 74449, a suitable method for preparing the microorganism is as follows: the microorganism is inoculated from a culture of the frozen stock solution as described above (approximately a 17% stock solution in glycerol) in flasks or in a glass tube with a metal closure containing a growth medium (containing an aliquot of a sterile solution including Tween® 80, glycerol and distilled water) whose composition is described in more detail later. The fermentation is carried out at temperatures ranging from about 22 ° C to about 32 ° C and, preferably at about 29 ° C, with suitable agitation, preferably from about 200 rpm to about 220 rpm, and, most preferably, at approximately 210 rpm. When desired, the pH of the growth medium can be maintained using suitable buffers incorporated in the fermentation medium and / or adjusted periodically by the addition of base or acid., as needed. In the present invention any suitable growth time of the microorganism can be used, by contacting the microorganism with the compound of formula (I) and incubating the compound of formula (I) with the microorganism. Proper growth of the microorganism can be achieved, for example, in about 24 hours, at which time a suitable aliquot of a racemic tetralone solution in a suitable solvent, preferably ethanol, can be added to the culture. The fermentation may then continue for, for example, from two to about six days and, preferably, for about five days, at which time the fermentation medium can be extracted using any suitable extraction procedure in which a suitable solvent, For example, ethyl acetate, methyl butyl ketone, metii etii ketone, methylene chloride and the like, preferably ethyl acetate, extract the organic components from the fermentation medium. After extraction of the fermentation medium and separation of the organic and aqueous phases, the compounds comprising the organic residue can be determined using any suitable method, such as, for example, chromatography, preferably chiral HPLC. Any suitable growth medium can be used in the process of the present invention and the suitable growth medium will contain a source or sources of assimilable carbon, assimilable nitrogen and inorganic salts containing the essential minerals. In general, many carbohydrates, such as glucose, maltose, sugar, sucrose, starch, glycerin, millet jelly, molasses, soybean seeds and the like, can be used as assimilable sources of carbon. Sources of assimilable nitrogen include, for example, materials such as yeast and casein hydrolysates, primary yeast, yeast extracts, cottonseed meal, soybean solids, wheat germ, meat extracts, peptone, maceration liquor of corn and ammonium salts. Nutrients of inorganic salts suitable for use in the culture medium of the present invention include, for example, the usual salts containing sodium, iron, magnesium, potassium, cobalt, phosphate and the like. More especially, growth media suitable for use in the present invention include, for example: (a) dextrose (approximately 20 g), yeast extract (approximately 5 g), soybean meal (approximately 5 g), NaCl (approximately 5 g), K2OP4 (approximately 5 g) and distilled water (approximately 1 liter (I)), pH adjusted to approximately pH 7.0 with aqueous H2SO4.; (b) dextrin (approximately 10 g), meat extract (approximately 3 g), ardamine pH (approximately 5 g), NZ type E amine (approximately 5 g), MgSO4 7H2O (approximately 0.5 g), KH2PO4 (approximately 0.37 g) ), CaCO3 (approximately 0.5 g) and distilled H2O (approximately 1 I), pH adjusted to approximately 7.1 with aqueous HCl, followed by a second glucose stage (approximately 10 g), Hy-Case SF® (approximately 2 g), meat extract (approximately 1 g), corn steep liquor (approximately 3 g) and distilled H2O (approximately 1 I), pH adjusted to approximately pH 7.0; (c) glucose (approximately 10 g), corn steep liquor (approximately 6 g), KH2PO4 (approximately 3 g), CaCO3 (approximately 3.5 g), soybean oil (crude, approximately 2.2 ml), yeast extract ( approximately 2.5 g) and distilled H2O (about 1 I), adjusted pH from about pH 7.0 to about pH 7.3 with aqueous HCl; (d) malt syrup (approximately 20 g), soy flour (approximately 5 g), casein (approximately 1 g), dried yeast (approximately 1 g), NaCl (approximately 5 g) and distilled H2O (approximately 1 I); (e) lactose (approximately 75 g), Pharmamedia® (substitute yeast extract, approximately 40 g), CaCO3 (approximately g), Na 2 SO 4 (approximately 4 g) and distilled H 2 O (approximately 1; (f) ISP No. 2 (see for example page 460 of Handbook of Microbial Media by RM Atlas, published by LC Parks, CRC Press, Inc 1993 ("Handbook"); (g) ISP n ° 3 (see page 460 of Handbook); (h) ISP n ° 4 (see page 461 of Handbook); (i) ISP n ° 5 (see pages 461-462 of Handbook), and the like A particularly preferred culture medium is 2X from (a) discussed above With reference to buffers, media, reagents, contacting or culture conditions and the like, it is not intended be limiting, but will be construed to include all related materials that those of ordinary skill in the art may know as of interest or value in the particular context in which the present disclosure is presented.

For example, it is often possible to substitute a buffer system or culture medium for another, so that a different but known form is used to achieve the same purpose for which the suggested method, material or composition is directed. In addition, it will be understood that the present invention includes scaling up the present process for commercial purposes. Therefore, as will be understood by those skilled in the art, variations in the growth medium, fermentation conditions and / or the amount of racemic tetralone can be altered to control the performance of the resulting compounds and their relative production rates. . In general, the techniques employed in the present invention will be chosen with respect to industrial efficiency. The growth media, the fermentation conditions and the relative amounts of microorganisms or the enzyme reduction system and the racemic tetralone described herein are merely illustrative of the wide range of media, fermentation conditions and amounts of starting materials that they may be employed suitably in the present invention, as will be appreciated by those skilled in the art, and are not intended to be limiting in any way.

Any suitable process for isolating and / or purifying any of the products of the present process can be used in the present invention, including filtration, extraction, crystallization, column chromatography, thin layer chromatography, low pressure liquid chromatography or preparative HPLC or any suitable combination of such procedures. In addition, one skilled in the art will appreciate that the corresponding undesired alcohol of the (4R) tetralone, the compound of formula (II), produced by the methods described herein can be recycled, for example, oxidized and form the racemic as above. described herein, by any known method, forming the racemic tetralone of formula (I) and the methods of the present invention repeated to produce, again, the desired tetralone (4S) of formula (V). Oxidation of (4R) tetralol to (4R) ketone can be accomplished by methods known to those skilled in the art. The racemization reaction can be carried out in any suitable manner although it is generally carried out at a temperature from about 0 ° C to about 100 ° C, preferably from about 25 ° C to about 65 ° C. The (4R) tetralone is reacted with a base at a temperature of about 25 ° C to about 85 ° C, preferably about 50 ° C to about 65 ° C. Suitable bases for this racemization reaction include potassium t-butoxide, sodium hydroxide, sodium methoxide and potassium hydroxide. A preferred base is potassium t-butoxide. The detailed examples set forth below show that a series of microorganisms, including fungi, for example, yeasts and actinomycetes, stereoselectively reduce the racemic tetralone, providing the desired (4S) tetralone of formula (V), i.e., the chiral tetralone , which can then be separated from the undesired compounds and subsequently reacted according to procedures known in the art to provide sertraline. The present invention is illustrated by the following examples. The above and following description of the present invention and the different embodiments are not intended to limit the invention, but are merely illustrative thereof. Therefore, it will be understood that the invention is not limited to the specific details of these examples.

EXAMPLE 1 Reduction of a racemic tetralone using Hansenula polymorph ATCC No. 26012 A. Fermentation of yeast Hansenula polymorph ATCC No. 26012 A control culture (Cl) and a test culture (T1) were prepared as follows: about 2.5 ml of sterile growth medium (approximately 40 g / l of approximately 10 g / l of nutritious soy flour, approximately 10 g / l of yeast extract, approximately g / l of NaCl and dextrose, approximately 10 g / l of K HPO 4, with the pH adjusted to approximately 7.0 with H2SO4) to each of two 16 x 125 mm glass tubes each having a metal plug (C1 , T1), followed by the addition of approximately 0.2 ml of a solution A (approximately 25 g of Tween® 80, approximately 100 g of glycerol and approximately 250 ml of distilled water, sterilized by filtering), to each of the two cultures. T1 was inoculated in approximately 25 μl of a stock solution frozen in glycerol approximately 17% of Hansenula polymorpha ATCC no. 26012. The two tube cultures were incubated at approximately 29 ° C with shaking at approximately 210 rpm. After approximately 24 hours, approximately 50 μl of a stock solution (approximately 5 mg / ml in approximately 100% ethanol, final concentration approximately 100 μg / ml) of a racemic tetralone ( Formula (I) comprising the compounds of formulas (IV) and (V), at about 5 mg / ml in ethanol). After about five days, one ml of NaCl (saturated) was added to each of the two tube cultures. The fermentation medium from each tube culture (approximately 3.6 ml) was extracted with an equal volume of ethyl acetate (neat): ethyl acetate was added, the tube culture was vortexed and then centrifuged at approximately 2,000. rpm (IEC® centrifuge '300 Second Avenue, Needham Heights, Massachusetts 02194). The ethyl acetate layer was removed and the aqueous layer was extracted for a second period. The combined organic extracts were dried under nitrogen in a water bath at about 50 ° C.

B. Configuration of the residual ketone: Compounds of formulas (IV) and Each extract, prepared as described above, was resuspended in approximately one ml of ethanol, and approximately 20 μl of each extract resuspended by injection was analyzed on an HPLC column: Chiralcel OK protection column (4.6 x 50 mm, Diacel Chemical Industries, LTD, 730 Springdale Drive, PO Box 564, Exton Pennsylvania 19341) coupled to a Chiralcel OK column (4.6 x 250 mm, Diacel). The compounds contained in each of the injected resuspended extracts were separated in a Socratic manner at approximately 0.8 ml per minute in a mobile phase (ethanol: ethyl acetate, 85:15) and the compounds included in the extracts were detected using a detector. 996 PDA (Waters®, 34 Maple Street, Milford, Massachusetts 01757) adjusted to 254 nm. As illustrated by the data of C1 and T1 in the following Table I, the chiral HPLC analysis showed that the inclusion of the microorganism, ie, Hansenula polymorpha ATCC No. 26012 gave a ratio of 16: 1 ((4S) tetralone of unreacted formula (V) against (4R), tetralone of formula (IV) without reacting), which further illustrates the stereospecific character of the present microbial reduction process. The results expressed below are based on the known amount of each added enantiomer (approximately 50 μg / ml of each of the compounds of formulas (IV) and (V)). As mentioned above, the starting racemic tetralone of formula (I) had a concentration of approximately 100 // lg / ml.

TABLE 1 The results of the chiral analysis show that the cultivation of Hansenula polymorpha ATCC No. 26012 (T1) substantially reduces the (4R) tetralone, the (4S) tetralone remaining almost unreacted (approximately 4.7% of (4R) tetralone remains against 76% of (4S) tetralone). It was determined that the (4S) tetralone was present in an ee of about 88% ("percentage amount of enantiomeric excess") by chiral HPLC. As also shown by the data in Table I, especially by the ratio (4S) :( 4R), that is, 16, remains substantially more (4S) unreacted tetralone than (4R) tetralone. Accordingly, the inclusion of the intact microorganism, ie Hansenula polymorpha ATCC No. 26012, produced a substantial stereospecific reduction of more starting (4R) tetralone of formula (IV) than starting (4S) tetralone of formula (V) ( (4S): (4R)) and provided mostly (4R) tetralol of formula (II) against (4S) tetralol of formula (lll) (data not shown). The majority of the (4R) tetralol produced was (1S, 4R) tetralol and the majority of the minority amount of (4S) tetralol produced was (1S.4S) tetralol.

EXAMPLE II REDUCTION OF A RACEMICA TETRALONE USING Absidia coerulea ATCC no. 20137 A. Fermentation of the fungus Absidia coerulea ATCC no. 20137 A control culture (C2) and a test culture were prepared (T2) as follows: approximately 2.5 ml of sterile growth medium (approximately 20 g / l of dextrose, approximately 5 g / l of nutritious soybean meal, approximately 5 g / l of yeast extract, approximately 5 g / l) were added. l of NaCl and about 5 g / l of K2HPO4, with the pH adjusted to about 7.0 with H2SO4) to each of two 16 x 125 mm glass tubes each having a metal plug (C2, T2). Approximately 25 μl of a stock solution in frozen 17% giicerol of Absidia coerulea ATCC no. 20137. The two tube cultures were incubated at approximately 29 ° C with shaking at approximately 210 rpm. After about 48 hours, approximately 50 μl (approximately 5 mg / ml in ethanol, final concentration of approximately 100 // l / ml) of a racemic tetralone (as described in Example I, a) was added to C2 and T2. about 5 mg / ml in about 100% ethanol). After about five days, one ml of NaCl (saturated) was added to each of the two tube cultures. The fermentation medium of each tube culture (approximately 3.6 ml) was extracted with approximately 3 ml of ethyl acetate (neat): ethyl acetate was added, the tube culture was vortexed and then centrifuged at approximately 2,000. rpm (IEC centrifuge). The ethyl acetate layer was removed and the aqueous layer was extracted for a second period. The combined organic extracts were dried under nitrogen in a water bath at about 50 ° C.

B. Configuration of the residual ketone: Compounds of formula (IV) and (V).

Each extract, prepared as described above, was resuspended in approximately one ml of ethanol, and approximately 20 μl of each extract resuspended was analyzed by injection on an HPLC column: Chiralcel OK protection column (4.6 x 50 mm) coupled to a Chiracel OK column (4.6 x 250 mm). The compounds contained in each of the injected resuspended extracts were separated in a Socratic manner at approximately 0.8 ml per minute in a mobile phase (ethanol: ethyl acetate, 85:15) and the compounds included in the extracts were detected using a detector. 996 PDA (Waters®) adjusted to 254 nm. As illustrated by the HPLC data for Cl and TI, inclusion of the microorganism, ie, Absidia coerulea ATCC No. 20137 provided an estero-specific reduction of more (4R) tetralone starting formula (IV) that of the starting tetralone (4S) of formula (V). More specifically, the results of the chiral analysis show that the culture of Absidia coerule ATCC No. 20137 reduces the (4R) tetralone while leaving substantially unreacted the (4S) tetralone (approximately 13.6% of (4R) tetralone remains against approximately 40.5% of the (4S) tetralone). It was determined that the (4S) tetralone was present in an ee of approximately 50% by this chiral HPLC. As illustrated by the data for C2 and T2 in Table II below, the chiral HPLC analysis showed that the inclusion of the microorganism, ie, Absidia coerulea ATCC No. 20137 (T2) produced a ratio of at least two times as much of (4S) tetralone that remained unreduced against the unreduced (4R) tetralone, further demonstrating the stereospecificity of the present microbial reduction process. The results expressed below are based on the known amount of each added enantiomer (approximately 50 μg / ml of each, as described in example 1 above). As mentioned above, the starting racemic tetralone had a concentration of about 10 μg / ml.

TABLE ll EXAMPLE III Reduction of a racemic tetralone using fungi, yeasts and an actinomycete As will be understood by those skilled in the corresponding art, the microorganisms listed in Table Ill, which were used in the present reduction, Geotrichum candidum ATCC No. 62401, Mortierella isabellin ATCC No. 38063, Mortierella vinacea ATCC No. 09515, Penicillium nonatum ATCC No. 36740, Blastoschizomyces capitatus ATCC No. 28757, Monosporium olivaceum v. major ATCC No. 36300, Aureobasidium pullulans ATCC No. 16623, Pichia fabianii ATCC No. 16755, and Streptomyces rimosis ss. rimosmos ATCC No. 10970 were separated as described in example II; Geotrichum candidum ATCC No. 34614, Mortierella isabellin ATCC No. 42613, Debaryomyces polymorphus ATCC No. 20280 and Saccharomyces cerevisiae ATCC No. 15248 were prepared as described in Example II, except that extraction was repeated; and Candida schatavii ATCC No. 24409 was prepared as indicated below. Candida schatavii ATCC No. 24409 was prepared and used according to the present invention as follows: about 2.5 ml of sterile growth medium (approximately 20 g / l of dextrose, approximately 5 g / l of nutritious soy flour, approximately 5 g / l of yeast extract, approximately 5 g / l of NaCl and approximately 5 g / l of K2PO4, with the pH adjusted to approximately 7.0 with H2SO4) to a glass tube of 16 x 125 mm with a metal stopper, followed by the addition of approximately 0.1 ml of a sterilized solution by filtration of approximately 25 g of Tween® 80, approximately 100 g of glycerol and approximately 250 ml of distilled water to the culture. Next, approximately 25 μl of a stock solution in glycerol to about 17% frozen of Candida schatavii ATCC No. 24409 was inoculated into the culture. The culture was developed at about 29 ° C, with stirring at about 210 rpm. After 48 hours, approximately 50 μl of a stock solution (approximately 5 mg / ml in approximately 100% ethanoi, final concentration of approximately 100 μl / ml of a racemic tetralone (compound of formula (I) comprising the compounds of formulas (IV) and (V), at about 5 mg / ml in about 100% ethanol).

After four more days, the culture fermentation medium (approximately 2.6 ml) was extracted with an equal volume of ethyl acetate (neat), the culture was vortexed and then centrifuged at approximately 2000 rpm (IEC® centrifuge) . The extraction was repeated. The extracts were dried under nitrogen in a water bath at about 50 ° C. The extract was resuspended in approximately one ml of ethanol, and approximately 5 μl of the resuspended extract was analyzed by injection on an HPLC column: Chiralcel OD protection column (4.6 x 50 mm, Diacel Chemical Industries LTD.) Coupled to a Chiralcel column OD (4.6 x 250 mm, Diacel). The compounds contained in the injected resuspended extract were separated in a Socratic manner at approximately 0.9 ml per minute in a mobile phase (hexane: isopropanol, 95: 5) and the compounds in the extract were detected using a 996 PDA detector (Waters®) set to 210 nm. As shown by the chiral HPLC (carried out as in Example I and II), the data set forth in Table III, each of the microorganisms listed in the following Table ll stereospecifically reduced more (4R) tetralone than ( 4S) tetralone and there was generally a ratio of at least about twice as much (4S) tetralone that remained unreacted with respect to the unreacted (4R) tetralone.

PICTURE It will be appreciated that, while Monosporium olivaceum v. ATTC No. 36300, as illustrated by the data in Table III, reduces substantially more (4R) tetralone than (4S) tetralone and, as such would be a preferred microorganism for use in the present process, however, it is also disclosed for this culture undesirable degradation of (4R) tetralone and (4S) tetralone. The undesirable degradation may be due, for example, to other enzymes and the like comprising the intact microorganism. Therefore, as will be understood by those skilled in the art from the description set forth herein, the use of the enzyme isolated from Monosporium olivaceum v. Is preferred. ATCC major n ° 36300 against Monosporium olivaceum v. ATCC major No. 36300 intact.

Claims (35)

NOVELTY OF THE INVENTION CLAIMS

1. - A process for the steroselective microbial reduction of a compound of formula (I) to compounds of formulas (II) and comprising: contacting a compound of formula (I) with a microorganism, or an enzyme reduction system capable of carrying out said reduction, comprising an enzyme derived from said microorganism and a cofactor for said enzyme, and incubating the mixture resulting under conditions sufficient to produce more compound of formula (II) than compound of formula (III), thus leaving more compound of formula (V) unreacted than unreacted compound of formula (IV), said microorganism being selected from the group consisting of: Hansenula polymorpha ATCC No. 26012, Hansenula polymorpha ATCC No. 74449, Absidia coerulea ATCC No. 20137, Geotrichum candidum ATCC No. 34614, Geotrichum candidum ATCC No. 62401, Mortierella isabellina ATCC No. 42613, Mortierella isabellina ATCC No. 38063, Mortierella vinacea ATCC No. 09515, Penicillium notatum ATCC No. 36740, Blastoschizomyces capitatus ATCC No. 28575,

Monosporium olivaceum v. ATCC No. 36300, Aureobasidium pullulans ATCC No. 16623, Debaryomyces polymorphus ATCC No. 20280,

Saccharomyces cerevisiae ATCC No. 15248, Candida schatavii ATCC nc 24409, Pichia fabianii ATCC 16755 and Streptomyces rimosus ss. RIMOSUS ATCC No. 10970; and its mutants capable of carrying out said reduction. 2. The process according to claim 1, wherein the compound of formula (V) is separated from the compounds of formulas (II), (III) and (IV). 3. Process according to claim 2, wherein said separation is carried out by chromatography.

4. Process according to claim 2, wherein said separation is carried out by crystallization.

5. Process according to claim 2, wherein said compound of formula (II) is separated from said compounds of formulas (III) and (IV).

6. The process according to claim 5, wherein said separate compound of formula (II) is recycled to a compound of formula (I) by oxidizing said separate compound of formula (II) and subjecting said oxidized compound to racemization said compound of formula (I).

7. The process according to claim 6, wherein said racemization comprises reacting said oxidized compound with a base.

8. The method according to claim 1, wherein said compound of formula (I) is prepared as defined in claim 6.

9. The method according to claim 1, wherein said contacting occurs with a microorganism. .

10. The method according to claim 1, wherein said contacting takes place with said enzyme reduction system.

11. The method according to claim 9, wherein said microorganism is an intact microorganism.

12. Method according to claim 9, wherein said microorganism is a preparation of used cells thereof.

13. Process according to claim 9, wherein said microorganism is a dehydrated preparation thereof.

14. The method according to claim 11, wherein said intact microorganism comprises washed cells of said intact microorganism.

15. The method according to claim 14, wherein said washed cells are immobilized.

16. The method according to claim 10, wherein said enzyme of said enzyme reduction system is immobilized.

17. - Process according to claim 13, wherein said dehydrated preparation is an enzyme preparation powder in acetone.

18. The method according to claim 9, wherein said microorganism is in a culture medium.

19. Process according to claim 18, wherein said contacting is carried out by adding said compound of formula (I) to said culture medium.

20. Process according to claim 10, wherein said enzyme reduction system is in a solvent.

21. Process according to claim 20, wherein said solvent is an appreciably organic solvent.

22. Process according to claim 10, wherein said contacting is carried out by adding said compound of formula (I) to said solvent.

23. The method according to claim 9, wherein said microorganism is said Hansenula polymorpha ATCC No. 26012 or said Hansenula polymorpha ATCC No. 74449 or its aforementioned mutants.

24. The method according to claim 10, wherein said enzyme comprising said enzyme reduction system is derived from said Hansenula polymorpha ATCC No. 26012 or Hansenula polymorpha ATCC No. 74449 or from said mutants.

25. The method according to claim 18, wherein said microorganism is said Hansenula polymorpha ATCC No. 26012 or said Hansenula polymorpha No. 74449 or its aforementioned mutants.

26. The method according to claim 19, wherein said microorganism is said Hansenula polymorpha ATCC No. 26012 or said Hansenula polymorpha No. 74449 or its aforementioned mutants.

27. The method according to claim 9, wherein said microorganism and Absidia coerule ATCC No. 20137 or its aforementioned mutants.

28. The method according to claim 10, wherein said enzyme comprising said enzyme reduction system is derived from said Absidia coerulea ATCC No. 20137 or its aforementioned mutants.

29. The method according to claim 10, wherein said enzyme comprising said enzyme reduction system is derived from said Monosporium olivaceum v. ATCC No. 36300 or its aforementioned mutants.

30. A process for the stereoselective microbial reduction of a compound of formula (I) to compounds of formulas (II) and (III) comprising: contacting a compound of formula (I) with a microorganism, and incubating the resulting mixture under conditions sufficient to produce more compound of formula (II) than compound of formula (III), thereby leaving more compound of formula (V) ) which is composed of unreacted formula (IV), said microorganism being selected from the group consisting of: Hansenula polymorpha ATCC No. 26012, Hansenula polymorpha ATCC No. 74449 and its mutants capable of carrying out said reduction.

31. The method according to claim 30, wherein said microorganism is in a culture medium.

32. The method according to claim 31, wherein said contacting is carried out by adding said compound of formula (I) to said culture medium. 33.- A procedure for stereoselective microbial reduction of a compound of formula (I) to compound of formulas (II) and (III) comprising: contacting a compound of formula (I) with an enzyme reduction system capable of carrying out said reduction comprising an enzyme derived from a microorganism and a cofactor for said enzyme, and incubating the resulting mixture under conditions sufficient to producing more compound of formula (II) than compound of formula (III), thus leaving more compound of formula (V) unreacted than unreacted compound of formula (IV), said microorganism being selected from the group consisting of: Hansenula polymorpha ATCC no. 26012, Hansenula polymorpha ATCC No. 74449 and its mutants capable of carrying out said reduction. 34. The method according to claim 33, wherein said enzyme reduction system is in a solvent. 35. The method according to claim 34, wherein said contacting is carried out by adding said compound of formula (I) to said solvent. SUMMARY OF THE INVENTION The present invention relates to methods for carrying out the following steroselective microbial reduction of a racemic tetralone: comprising: contacting a compound of formula (I) with a microorganism, or an enzyme reduction system capable of carrying out the present reduction, comprising an enzyme derived from said microorganism and a cofactor for said enzyme, and incubating the resulting mixture under conditions sufficient to produce the (4R) tetralol of formula (II) and leave substantially unreacted the (4S) tetralone of formula (V) or "chiral tetralone". The chiral tetralone can be used in the synthesis of sertraline. The present process optionally further comprises separating the (4S) tetralone of formula (V) from (4R) tetralol of formula (II). The (4R) tetralol can be recycled to produce the compound of formula (I) and the present procedure is repeated to produce more desired (4S) tetralone of formula (V). PF / all * P99 / 1350