(12) United States Patent (10) Patent N0.: US 7,723,065 B2 Trewhella Et A]

US007723065B2 (12) United States Patent (10) Patent N0.: US 7,723,065 B2 Trewhella et a]. (45) Date of Patent: May 25, 2010 (54) CONDITIONS FOR REACTIONS MEDIATED Kumar A, Ner DH, Dike SY, A New Chemoenzymatic Enantioselec BY YEAST tive Synthesis of R-(—)-Tomoxetine, R- and S-Fluoxetine, Tetrahe dron Letters, 1991, 32(16): 1901-1904.* (75) Inventors: Maurice Arthur TreWhella, Hoppers Liu X, Zhu T-S, Sun P-D, Xu J-H, Asymmetric Reduction of Aro Crossing (AU); Nick Athanasiou, matic Ketones by the Baker’s Yeast in Organic Solvent Systems, Yarraville (AU); Andrew John Synthetic Communications, 2001, 31(10): 1521-1526.* Smallridge, Hampton East (AU) Chem 231 Lecture No. 10 Sp 1999* Ohta et al. “Asymmeteric Reduction of Nitro Ole?ns by Fermment (73) Assignee: Victoria University, Victoria (AU) ing Baker’sYeast.” J. Org. Chem. vol. 54, pp. 1802-1804. 1989* ( * ) Notice: Subject to any disclaimer, the term of this KaWai et al. “Asymmeteric Reduction of nitroalkenes With baker’s patent is extended or adjusted under 35 yeast.” Tetrahedron: Asymmetry. vol. 12, pp. 309-318. 2001.* U.S.C. 154(b) by 933 days. Y. Gao and K. B. Sharpless-“Asymmetric Synthesis of Both Enantionmers of Tomoxentine and Fluoxentine. Selective Reduction of 2, 3-Epoxycinnamyl Alcohol With Red-A1”, J. Org. Chem. 1988, (21) Appl.No.: 10/814,891 53, 4081-4084. © 1988 American Chemical Society. (22) Filed: Mar. 31, 2004 Badan S. Deol et al., Asymmetric Reduction of Carbonyl Compounds byYeast. II; Aust. J. Chem., 1976, 29, 2459-67, 9 pages. (65) Prior Publication Data * cited by examiner US 2005/0084943 A1 Apr. 21, 2005 Primary ExamineriAnish Gupta (30) Foreign Application Priority Data (74) Attorney, Agent, or FirmiBrooks Kushman RC. Oct. 16, 2003 (AU) ............................ .. 2003905675 (57) ABSTRACT (51) Int. Cl. C12P 1/02 (2006.01) Organic compounds, such as precursors for aryl ethylamines (52) US. Cl. ....................... .. 435/41; 435/128; 435/130; such as ephedrine, aryl propylamines such as ?uoxetine and 435/132; 435/166; 435/171 propionic acid derivatives such as ibuprofen, naproxen and (58) Field of Classi?cation Search ..................... .. None fenoprofen, are subjected to a yeast mediated reduction con See application ?le for complete search history. ducted in the absence of a solvent. The yeast is moistened With Water and contacted With the organic compound. The (56) References Cited yeast may then be contacted With an organic solvent to dis OTHER PUBLICATIONS solve the product of the reaction into the solvent, and a solid/ liquid separation used to separate the product from the yeast. HoWaIth J, James P, Dai J, Immobilized baker’s yeast reduction of ketones in an ionic liquid, [bmim]PF6 and Water mix, Tetrahedron Letters, 2001, 42: 7517-7519* 20 Claims, No Drawings US 7,723,065 B2 1 2 CONDITIONS FOR REACTIONS MEDIATED antihistamine activity; l-ephedrine is Widely used as a bron BY YEAST chodilator, While d-pseudoephedrine is Widely used as a decongestant. Compounds of these groups are present in a BACKGROUND OF THE INVENTION very Wide range of prescription and over-the-counter phar maceutical formulations. 1. Field of the Invention The production of l -phenylacetylcarbinol (PAC), a precur The present invention relates to neW environments in sor of l-ephedrine, by catalysis using Whole baker’s yeast Which to conduct certain classes of chemical reactions. The cells in aqueous medium Was one of the ?rst microbial present invention particularly relates to neW methods and biotransformation processes to be used commercially. This environments for the synthesis of useful pharmaceutical com reaction included the yeast-mediated reduction of a ketone pounds such as aryloxy phenyl propylamines (e.g. Prozac; intermediate to produce the chiral phenylacetylcarbinol, Trade Mark of Eli Lilly, Inc.), 2-aryl ethylamines (eg ephe although today the more common synthetic route involves drine) and propionic acid derivatives (eg. ibuprofen). yeast-mediated condensation betWeen benzaldehyde and 2. Background Art pyruvate to form PAC. Due to the complex molecular structure of many organic The yeast-catalysed systems have utilised aqueous solvent compounds Which have pharmacological activity, it is com systems, Which have been found to be inconvenient for large mon for pharmaceutically-useful agents to include one or scale extraction and puri?cation. Additional problems asso more chiral centres. The complex structure of such com ciated With the aqueous solvent systems are the loW yields and pounds means that their synthesis involves many steps, and loW purity. Whilst the reaction has been improved by utilising consequently Where chiral centres are present, the com 20 immobilised cells, or cells Which have been selected or pounds are usually prepared in the form of racemic mixtures. genetically modi?ed, this adds signi?cantly to the cost of the The pharmacological activity of the compound is often process. The use of puri?ed enzymes is normally prohibi mediated by the binding of the pharmacological agent to a tively expensive, and again Without the use of immobilised target site. The more accurate the 3-dimensional ?t betWeen enzymes the yields tend to be loW and puri?cation dif?cult. In the pharmacological agent and the target site, the more potent 25 vieW of the dif?culty of large- scale extraction and puri?cation the pharmacological activity, and the loWer the likelihood of With the aqueous solvent systems, organic systems, super unWanted side-effects. critical ?uid systems and lique?ed gas systems have been As a consequence of this, it is not unexpected that indi investigated. vidual enantiomeric forms of a chiral compound shoW differ In our earlier International Application PCT/AUOO/Ol 543, ent pharmacological activity, differences in metabolic behav 30 We shoWed that yeast-mediated acyloin condensation of ben iour and different spectra of undesirable side-effects. zaldehyde could be achieved in supercritical or lique?ed car It is therefore desirable to ensure Where possible that the bon dioxide or in lique?ed petroleum gas. The use of super end-products of synthesis of pharmaceutical compounds are critical ?uids as the reaction medium in large scale reactions enantiomerically pure. is advantageous as compared With conventional organic sol Physicochemical methods for production of enantiomeri 35 vents since the puri?cation and processing of the products is cally pure compounds usually involve multi-step synthesis simpler. HoWever, the use of such reagents requires special incorporating one or more steps Which are asymmetric, and ised equipment design and control that add to expense. laborious puri?cation procedures. Such methods are not only There is accordingly still room for the current systems for tedious, but frequently provide relatively poor yields. Alter synthesising pharmaceutical compounds to be improved natively enantiomerically-pure starting materials canbe used, 40 upon. together With enantioselective reaction steps; hoWever, such It has noW been surprisingly found by the present applicant pure starting materials are available only for a very limited that yeast mediated reduction reactions of organic com number of desired compounds. pounds can be conducted in the absence of a solvent. The In recent years, efforts have been directed toWards devel present applicant has established that a broad range of impor opment of methods Which are highly selective, provide a good 45 tant pharmaceutical compounds containing chiral centres can rate of transformation, and enable easy, non-chromato graphic be synthesized using a route in Which a starting compound is separation and puri?cation of the product. It has also been subjected to a yeast-mediated reduction reaction to provide a considered particularly desirable for the reactions to be car product, Which may be enantiomerically pure, and Which can ried out in non-aqueous solvents, since these are particularly then be converted into one isomer of the target pharmaceuti convenient for large-scale reactions and puri?cations. In 50 cal compound. In cases Where the product is a racemic mix addition, Where enantiomerically-pure reaction products can ture, the process provides improvements in process ef?cien not be obtained, changes in the physical environment in cies, such as the simple isolation of a product Without a Which the reactions are conducted can lead to improvements liquid-liquid separation step. in the overall ef?ciency of the reaction system. Some principle candidate classes of pharmaceutical com 55 SUMMARY OF THE INVENTION pounds containing chiral centres Which may be advanta geously stereospeci?cally synthesized include aryl ethy According to one aspect of the present invention there is lamines such as ephedrine and the other sympathomimetic provided a method of reducing an organic compound, com amines, aryl propylamines such as ?uoxetine (Prozac) and the prising subjecting the organic compound to a yeast mediated other serotonin selective uptake inhibitors, and propionic acid 60 reduction Wherein the reduction is conducted in the absence derivatives such as ibuprofen, naproxen and fenoprofen. of a solvent. Ephedrine ((X-[ l -(methylamino)ethyl]benzene-methanol), It Will be understood to a person skilled in the art that the originally isolated from plants of the genus Ephedra, occurs yeast mediated reaction requires some Water for the reaction as the naturally occurring isomers l-ephedrine and d-pseu to take place. Su?icient Water is required for enzymes to be doephedrine, and other pharmacologically active isomers 65 hydrated and take the appropriate con?guration. A “mono include d-ephedrine and l-pseudoephedrine. These com layer” of Water around the enzymes is required. For many pounds are adrenergic sympathomimetic agents and have compounds, the presence of larger volumes of Water (i.e. US 7,723,065 B2 3 4 suf?cient Water to provide a separate Water layer) prevents or The Water that is present in the mixture is present in such a substantially prevents the yeast-mediated reduction of that small amount that it “sticks” to the yeast, and does not inter compound from taking place.

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