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 , 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- 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 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. This is particularly the case for fere With the removal of the product of the reaction (an Water-insoluble organic compounds. In contrast, the appli organic compound) into the organic solvent. It is a signi?cant cant has surprisingly found that these Water-insoluble com advantage of the method of the invention that a biphasic pounds react rapidly and With high yield When simply mixed (aqueous/organic) extraction is avoided. Biphasic extractions With near-dry or damp yeast (i.e. yeast With insu?icient Water are often associated With loW isolated yields. It is also an to provide a visible separate Water layer). This level of Water advantage of the invention that no reagents (in this case, the corresponds to a Water-to-yeast ratio of up to 1.5 ml/ g (ap Water/yeast) are extracted into the organic solvent, so that no proximately 60% W/W). The minimum amount of Water separate puri?cation steps are required. required is approximately 0.2 ml/g of yeast (approximately A broad range of organic compounds can be reduced using 10% W/W). Dry yeast contains at most 1-3% W/W Water, and the method of the present invention. Speci?c classes of com therefore must be Wetted to be activated for use according to pounds that may be reduced by the reaction include ketones, the present invention. Preferably, the Water to yeast ratio is 0.8 alkenes, alkynes, aldehydes, imines (i.e. compounds contain to 1.2 ml/ g of yeast (approximately 44 to 55% W/W). ing the group 4C:Ni) and hydroxamines. Whilst Water is sometimes used as a solvent in organic The reaction is most effective on conjugated or activated reactions (particularly for reactions involving Water-soluble ketones and alkenes. organic reagents), according to the present invention Water is Consequently, particularly suitable classes of organic com not used in a high enough volume to function as a solvent. pounds for subjecting to the method of the present invention Accordingly, it is to be understood that the Water is not a 20 are [3-keto amides, [3-keto esters, enol ethers, activated solvent in the context of the present application. ketones and conjugated (activated) alkenes (i.e. alkenes With Any yeast capable of effecting the reduction reaction may an atom With an electrophilic character, as may be provided, be used. It is economically advantageous to use the cheapest for example by alkenes substituted With N02, iCN, ketone, yeast available, and ordinary baker’s yeast, Saccharomyces ester, amide, aldehyde, thioether, alkene, aromatic groups, cerevisiae, is preferred. Strains of yeast adapted to other 25 halogens, etc). purposes, including breWing yeast and Wine or sherry yeasts Amongst these organic compounds, some classes are could also be employed. For maximum ef?ciency of reaction, industrially very useful precursors in the synthesis of knoWn it is advisable to present the maximum surface area of yeast pharmaceutical agents. Particularly suitable classes of for contact With the reactants. This can be effected by using organic compounds Which may be reacted according to the “active” dried yeast, Which is readily commercially available 30 method of the invention to form useful precursor compounds as “instant dry yeast”, and may be stored at room temperature. include the following: Alternatively, Well-pulverised dry baker’s yeast may be used. Typically “dry yeasts” have 1-3% W/W Water. Other yeasts, Activated ketones (I), (H), and (III): such as those described in Us. Pat. No. 4,734,367, or fungi such as those disclosed in Chenevert et al (1992) (Chenevert, 35 R. Fortier, G. and Rhlid, R. B., Tetrahedron, 1992 48 6769 (I) 6776) may also be used. The person skilled in the art Will O 0 readily be able to test Whether any speci?c organism Will function for the purposes of the invention, using the methods R1 R4 described herein. 40 R2 R3 The yeast mediated reduction reaction is signi?cantly (11) faster than prior art methods and also provides an improved O result. The applicant has achieved greater than 80% isolated 0 R6 yield as a result of complete reduction of the organic com 45 R1 pound. Little or no side products are produced. No side prod R5 ucts have been detected in the products of the reaction by the R2 present applicant. (111) 0 The proportion of yeast to organic compound may be any thing from 0.1 gram of yeast per mmol of organic compound, 50 R7 up to 50 grams of yeast per mmol of organic compound. R1 HoWever, the preferred range is about 0.8 to 20 g/mmol. R2 R3 While it is possible to speed up the reaction by the use of extra yeast, this is usually unnecessary. The reaction is carried out in non-fermenting conditions at 55 in Which: temperatures betWeen 0 to 50° C. For optimum results, the reaction is carried out at room temperature. Usually the reac R1 is an optionally substituted aryl group; tion is conducted at atmospheric pressure, although it is noted R2, R3, R5 and R6 are H or optionally substituted C 1 -C6 alkyl; that the reaction is not affected by changes in pressure. R4 is an optionally substituted C l-C6 alkoxy, aryloxy, amino, Preferably, the method of the invention involves contacting 60 optionally substituted di-(Cl -C6alkyl) amino, optionally sub the organic compound With the yeast and Water to form a stituted alkarylamino optionally substituted C l-C6 alky mixture, leaving the mixture for su?icient time for the reac tion to take place, adding an organic solvent to the mixture to lamino, optionally substituted cyclic amino, such as pyrroli dissolve the product of the reaction into the organic solvent, dino, piperidino, imidaZolidinyl, piperaZinyl, morpholinyl, Cl_6alkylpyrrolidino or Cl_6alkylpiperidino; and and conducting a solid/liquid separation to separate the prod 65 uct of the reaction from the yeast. Preferably the solvent is R7 is cyano; nitro; halo; OH; NH2; C1_6 alkyl substituted by evaporated to yield the product of the reaction. OH, halo, amine, or C1_6 alkylamino; US 7,723,065 B2 5 6 Conjugated Alkenes: amines and phenylpropylamines such as ProZac. The pre ferred substituents on the phenyl group are hydroxy, methyl, methoxy, hydroxymethyl and tri?uoromethyl. For the synthe (1V) sis of the propionic acid derivatives referred to above from the R9 compound of Formula (IV), the aryl group may be substituted R phenyl (such as p-isobutyl for ibuprofen, 3-phenoxyphenyl R8 \ 10 for fenoprofen, 2-?uoro-4-biphenyl for ?urbiprofen, 4-(l,3 dihydro- l -OXO -2H-isoindol-2-yl)phenyl, 3-benZoylphenyl R1 1 for ketoprofen, p-(2-thenoyl)phenyl for suprofen or p-methy lallylaminophenyl for alminoprofen) or a substituted napthyl (such as 6-methoXy2-napthyl- for naproxen). Consequently, wherein: the substituents on the phenyl and napthyl groups may be R8 is an optionally substituted aromatic group; selected from a Wide variety of substituents. For the preparation of propionic acid derivatives, R10 and R9, R10 and R11 are each independently selected from H, hydroxy, Cl_6alkoxy, mercapto, C1_6 alkylthio, amino, R1 1 are preferably each H, and R9 is carboxy or C l_6alkoxy Cl_6alkylamino, di(Cl_6alkyl)amino, carboxy, Cl_6alkoxy carbonyl. carbonyl, Cl_6aryloxycarbonyl, carbamoyl, Cl_6alkylcar For the preparation of one of the more commonly used bamoyl, di-Cl_6alkylcarbamoyl, Cl_6cycloalkylcarbamoyl, ethylamines containing a substituent on the ot-carbon atom Cl_6alkylsulphonyl, arylsulphonyl, Cl_6alkylaminosulpho (such as amphetamine) from compound (IV), preferably R9 is 20 H or hydroxy. Preferably, one of R10 and R1 1 is selected from nyl, di(Cl_6alkyl)aminosulphonyl, nitro, cyano, cyano-Cl_6 C l_6alkyl, and more preferably methyl or ethyl. Preferably the alkyl, hydroXyC1_6alkyl, amino-Cl_6alkyl, Cl_6alkanoy other of R10 and R11 is selected from Cl_6alkoxycarbonyl, lamino, Cl_6alkoxycarbonylamino, Cl_6alkanoyl, Cl_6al Cl_6aryloxycarbonyl, carbamoyl, Cl_6alkylcarbamoyl, kanoyloxy, Cl_6alkyl, halo, haloCl_6alkyl, or haloCl_6 alkoxy, alkoximino, hydroximino, and alkylimino. di-Cl_6 alkylcarbamoyl, Cl_6cycloalkylcarbamoyl or nitro. 25 For the preparation of one of the more commonly used To generate a neW chiral centre, one of R9, R10 and R11 propylamines containing a substituent on the [3-carbon atom must not be H. Accordingly, preferably at least one of R9, R10 (such as ?uoxetine) from compound (IV), preferably R9 is and R11 is not H. hydroxy. More preferably, one of R10 and R1 1 is selected from For the compounds of Formulae (I), (II) and (III), R1 is H and Cl_6alkyl, and more preferably it is H. Preferably the preferably substituted or unsubstituted phenyl or 2-thienyl. 30 other of R10 and R11 is selected from cyano, Cl_6alkoxycar The phenyl group may contain one or more substituents, bonyl, Cl_6aryloxycarbonyl, carbamoyl, Cl_6alkylcarbam preferably selected from hydroxy, methyl, methoxy, oyl, di-Cl_6alkylcarbamoyl and Cl_6cycloalkylcarbamoyl. hydroxymethyl and tri?uoromethyl. According to the present invention, there is also provided a For the compounds of Formulae (I), (II) and (III), R2 is method of synthesising a pharmaceutical compound com preferably H, and R3 is preferably either H, methyl or ethyl. 35 prising the step of subjecting a precursor to a yeast mediated Most preferably R3 is also H. reduction Wherein the reduction is conducted in the absence R4 in the compound of Formula (I) is preferably methoxy, of a solvent; and converting the product of the reduction ethoxy, C1_6 alkylamino, NH2, or di(Cl -C6alkyl)amino. More reaction into the pharmaceutical compound. preferably R4 is NH2 or C 16 alkylamino. Preferably, the pharmaceutical compound is a sympatho R5 and R6 in the compound of Formula (II) are preferably 40 mimetic amine, an ethyl amine, a propylamine or a propionic each H. acid. More preferably, the pharmaceutical compound is an In the situation Where the compound is of Formula (III), arylethylamine, an arylpropylamine, or a propionic acid With preferably R7 is cyano, alkylhalo or C1_6 alkylamino. a 2-aryl substitution. These compounds of Formula (I), (II) and (III) may be Particular pharmaceutical compounds that can be synthe subjected to the method of the present invention to form 45 siZed via the solvent-free yeast mediated reduction step of the precursors for the synthesis of seretonin selective uptake present invention are ?uoxetine (ProZac), tomoxetine, dulox inhibitors and related compounds such as ?uoxetine (Prozac), etine, nisoxetine, epinephrine, , ethylnorepi tomoxetine, duloxetine, nisoxetine, and each of the com nephrine, isoproterenol, isoetharine, metaproterenol, terbyta pounds de?ned in Us. Pat. No. 4,314,081, as Well as epi line, metaproterenol, , , prenalterol, nephrine, norepinephrine, ethylnorepinephrine, isoproter 50 , albuterol and derivatives With N-substitution enol, isoetharine, metaproterenol, terbytaline, such as , derivatives of amphetamine, ephedrine, metaproterenol, phenylephrine, ritodrine, prenalterol, meth , amphetamine and its derivatives such oxamine, albuterol and derivatives With N-substitution such as hydroxyamphetamine, methamphetamine, benZphet as salmeterol, ephedrine, phenylpropanolamine. The routes amine, fen?uramine and propylhexedrine, ibuprofen, to the synthesis of these compounds from the compounds of 55 naproxen, alminoprofen, fenoprofen, ?urbiprofen, indopro Formulae (I), (II) and (III) are described in further detail fen, ketoprofen and suprofen. beloW. The compound of Formula (IV) may be used as the starting DETAILED DESCRIPTION OF THE PREFERRED compound for the synthesis of the pharmaceuticals listed EMBODIMENTS above, together With amphetamine and its derivatives such as 60 hydroxyamphetamine, methamphetamine, benZphetamine, A number of chemical terms used in the above description fen?uramine, propylhexedrine, and propionic acid deriva of the invention are de?ned beloW to avoid any ambiguity. tives, such as ibuprofen, naproxen, alminoprofen, fenopro The term “alkyl” used either alone or in a compound Word fen, ?urbiprofen, indoprofen, ketoprofen and suprofen. such as “optionally substituted alkyl” or “optionally substi For the compounds of Formula (IV), the aromatic group R8 65 tuted alkylamino” denotes straight chain, branched or mono may be substituted or unsubstituted phenyl When the com or poly-cyclic alkyl, preferably C1_6 alkyl or cycloalkyl. pound is to be used for the synthesis of the sympathomimetic Examples of straight chain and branched C1_6 alkyl include US 7,723,065 B2 7 8 methyl, ethyl, propyl, isopropyl, butyl, isbutyl, sec-butyl, tert 1 . Preparation of ProZac (as One Example) from [3-keto Esters butyl, amyl, isoamyl, sec-amyl, 1,2-dimethylpropyl, 1,1 or Amides. dimethylpropyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 1,1-dimethylbutyl, 2,2 dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3 O O dimethylbutyl, 1 ,2,2-trimethylpropyl and 1, 1 ,2-trimethylpro Yeast R —> Pyl 1 minimal H2O R2 R3 The term “aryl” used either alone or in compound Words (1) such as “optionally substituted aryl”, “optionally substituted aryloxy” or “optionally substituted heteroaryl” denotes For Prozac: R1 is phenyl; single, polynuclear, conjugated and fused residues of aro R2 and R3 are H matic hydrocarbons or aromatic heterocyclic ring systems. Examples of aryl include phenyl, biphenyl, terphenyl, quater phenyl, phenoxyphenyl, naphtyl, tetrahydronaphthyl, anthra cenyl, dihydroanthracenyl, benZanthracenyl, dibenZanthra cenyl, phenanthrenyl, ?uorenyl, pyrenyl, indenyl, aZulenyl, chrysenyl, pyridyl, 4-phenylpyridyl, 3-phenylpyridyl, thie 20 nyl, furyl, pyrryl, pyrrolyl, furanyl, imadaZolyl, pyrrolydinyl, pyridinyl, piperidinyl, indolyl, pyridaZinyl, pyraZolyl, pyraZi 1. Substitute OH for ether OR13 nyl, thiaZolyl, pyrimidinyl, quinolinyl, isoquinolinyl, benZo 2. Reduce carbonyl group and substitute R4 if necessary for furanyl, benZothienyl, purinyl, quinaZolinyl, phenaZinyl, suitable amine acridinyl, benZoXaZolyl, benZothiaZolyl and the like. Prefer 25 ably, the aromatic heterocyclic ring system contains 1 to 4 QR13 heteratoms independently selected from N, O and S and con : NHMe taining up to 9 carbon atoms in the ring. 30 Rl/YV R R In the description provided above, reference is made to For Prozac: 2 3 R1 is phenyl; optional substituents. In this speci?cation “optionally substi R2 and R3 are H tuted” means that a group may or may not be further substi tuted With one or more groups selected from alkyl, alkenyl, alkynyl, aryl, halo, haloalkyl, haloalkenyl, haloalkynyl, 35 haloaryl, hydroxy, alkoxy, alkenyloxy, aryloxy, benZyloxy, The above reaction scheme illustrates the synthesis of haloalkoxy, haloalkenyloxy, haloaryloxy, nitro, cyano, ProZac from a [3-keto ester or amide, in accordance With the nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl, nitrohetero present invention. Suitable reagents and reaction conditions cyclyl, amino, alkylamino, dialkylamino, alkenylamino, 40 for conducting the steps folloWing the yeast mediated reduc alkynylamino, arylamino, diarylamino, benZylamino, diben tion are outlined in]. Org. Chem 53 (17) 4081, particularly for Zylamino, acyl, alkenylacyl, alkynylacyl, arylacyl, acy the situation Where R4 is iCHZOH. lamino, diacylamino, acyloxy, alkylsulphonyloxy, arylsul Other propylamines can be synthesiZed using this tech phenyloxy, heterocyclyl, heterocycloxy, heterocyclamino, nique by using the appropriate reagents. Table 1 details suit haloheterocyclyl, alkylsulphenyl, arylsulphenyl, car 45 able target propylamines. boalkoxy, carboaryloxy, mercapto, alkylthio, benZylthio, acylthio, phosphorus-containing groups, aZo, imino, nitrile, TABLE 1 carboxylate and the like. Preferably the substituents are nd R1 R2 R3 selected from C1_6 alkyl, halo, tri?uoromethyl, hydroxy, and 50 Cl_6alkoxy. U013 Ph H H EXAMPLES 0 55 Ph)\/\ NHMe Fluoxetine The folloWing reaction schemes are provided to illustrate hoW the method of the present invention can be incorporated Ph H H into a reaction scheme for the stereoselective synthesis of a 60 target pharmaceutical compound. The speci?c compounds Me : : referred to above have similar structures With different sub 0 stituents, and methods for their synthesis are Well knoWn. The knoWn synthetic methods can me modi?ed to incorporate the Ph)\/\ NHMe 65 Tomoxetine neW solvent-free yeast mediated stereoselective reduction step of the present invention in one of the folloWing Ways. US 7,723,065 B2

TABLE l-continued TABLE 2

nd R1 R2 R3 Compound R1 R2 R5 R6

/{ H H C133 Ph H H H

S 10 O O 0 Ph)\/\ NHMe / \ Fluoxetine S NHMe 15

Me Ph H H H Duloxetine Nisoxetine H H

O

2. Preparation of Prozac from [3-keto Epoxide: 20 Ph)\/\ NHM6 Tomoxetine

O 0 R6 Yeast 25 H H H minimal H2O / \ R1 R5 S R2

(1) 30 O For Prozac: / \ R1 is phenyl; NHM6 R2 and R3 and H S

3 5 Duloxetine Nisoxetine H H H

40 3. Preparation of Prozac from Enol Ether of [3-keto Ester

R2 R9 0 (VI) 45 Yeast —> 1. Substitute OH for R8 R12 mm‘mal H2O ether OR13 R 2. Reduce epoxide and, if 11 necessary, replace R5 (1V) and/or R6 With suitable 50 _ _ amine- R12 is O-C1,6all

' NHMe Rl/XV R8 ; R12 R2 R3 in For Prozac: R1 is phenyl; 60 (VII) R2 and R3 are H R13 15 p'cFrphenyl 1. Substitute OH for ether group 2. Reduce carbonyl group _ _ _ _ and substitute R12 ifnecessary Similarly to method 1 outlined above, this method can be 65 for Suitable 3mm; applied to the synthesis of the compounds outlined in Table 2. See J. Org. Chem. 53(17) 4081. US 7,723,065 B2

-continued TABLE 3-continued c1:3 Compound R8 R2 R11

5 Tomoxetine 9 / \ H H M NHMe R8 5 S 5 10 R11

0 Again, aside from the yeast-mediated reduction step, / \ appropriate reaction reagents and conditions are set out in J. 15 NHM6 Org. Chem. 53(17) 4081. The yeast-mediated reduction is S conducted in accordance With the procedure outlined in the Dvloxetme Experimental_ section._ Nisoxetme H H This procedure can also be used for the synthesis of the _ _ _ Compounds Outlined in Table 3' 20 4. Preparation of Amphetamine from Conjugated Alkene.

TABLE 3 R9 R9 R Yeast R Compound R8 R2 R11 25 R8 10 minimal H2O R8

C133 Ph H H R11 R11 (1V) (VIII) R3 is phenyl O 30 R9 is H R10 is No2

P hM NHM6 for R11amphetamine is cH3 1. Convert R10 (N02) into amine, NHMe for amphetamine 35 R9

Fluoxetine NHM6 Rs 40 Me Ph H H R11 NHM6 R8 is phenyl R9 is H R11 is CH3 0 for amphetamine

PhM NHMe 45 The procedure outlined above can likeWise be utilised for the synthesis of the compounds outlined in the Table 4.

TABLE 4 Compound R8 R9 R10 R11

UYNH; Ph H NO2 CH3 Amphetamine

QH HO OR NO2 H

Ho : NHMe

HO

HO Epinephrine US 7,723,065 B2

TABLE 4-c0ntinued Compound R8 R9 R10 R11 QH H0 OR N02 H HO : NHZ :@ HO

HO Nor epinephrine

OH HO OR NO2 H HO : NHEt 1Q HO

HO Ethylnorepinephrine

QH H0 OR N02 H E H HO N Y HO HO Isoproterenol

QH H0 OR N02 CH2CH3 E H HO N

HO Isoetharine

QH H0 OR N02 H E H HO N

OH

OH Metaproterenol

OH HO OR NO2 H i H HO Nj<

OH

OH

OH HO OR NO2 H

HO NHMe

PhenylephrineQ?

OH OR No2 cH3

HO HO Q1; £36 Ritodrine US 7,723,065 B2 15 16

TABLE 4-c0ntinued

Compound R8 R9 R10 R11

OH O OR NO H \ 2 H O N Y HO HO Prenalterol OMe OH NHZ iOMe OR N02 CH3

OMe OMe Methoxalnine

OH HO OR N02 H H N HO HO

HO Albuterol

OR NO H OH HO 2 H

HO N\/\/\/\ O HO

HO Sahneterol

OH Ph OR N02 CH3

NHMe

Ephedrine ©)YNH2OH Ph OR N02 CH3 Phenylpropanolamine

UYNHNR H N02 CH3 HO HO Hydroxyalnphetamine mNHMe Ph H NO2 CH3 Methamphetamine US 7,723,065 B2 17 18

TABLE 4-continued Compound R8 R9 R10 R11 Ph H No2 cH3 <31 Benzphetamine

NHMe H No2 cH3

%CF3 CF3 Fen?uramine 3 NHMe H No2 CH3 Propylhexedrine

5. Preparation of Ibuprofen -continued

R9 R 30 9 COZH Yeast (IX) : R H—> R10 R8 minimal H2O R8

R11 R11 35 iei (IV) (1X) (CH3)ZCHCHZ R8 is p-isobutylphenyl R9 is COZH R8 is p-isobutyphenyl R10 is H R9 is COZH R11 is H R10 is H for ibuprofen R1 1 is H 40

for ibuprofen _ _ _ The procedure outlined above can likewise be used for the synthesis of the compounds outlined in Table 5.

TABLE 5

Compound R8 R9 R 10 R1 1

COOH H H

co2H

Ibuprofen

COOH H H

co2H MeO

MeO Naproxen US 7,723,065 B2 19 20

TABLE 5-continued Compound /@ COOH H H cozn Yb]

Alminoprofen

0 COOH H H COZH O \@ Q?Fenoprofen QQ COOH H H

cozn

Ph5; Flurbiprofen 0e COOH H H cozn N

PLIndoprofen

COOH H H

cont

0

0%Ketopro fen COOH H H

/ I cozn S

Experimental Procedure: 55 . The present invention Will noW be described in further fomlnued detail With reference to the following Examples. :mmoI O Ph OEt 1. Preparation of Ethyl (R)-3-hydroxy-3-phenylpropanoate 60 (Method 1). Ethyl benZoyl acetate (192 mg, 1 mmol) Was added to Water (10 mL, 1 mL/ g yeast) in a 70 mL Pyrex test tube and O O vortexed until an even dispersion of substrate throughout the M Yeast 65 Water Was achieved (opaque mixture persists). Yeast (10 Ph OEt g/mmol) Was then added quickly and vortex Was maintained for a further 5 minutes. This procedure produced a moist US 7,723,065 B2 21 22 pliable yeast that ?rmed up a feW minutes after Water had been 6. Preparation of Ethyl (S)-3 -methoxybutanoate incorporated into the yeast. The reaction Was left at room temperature for 24 hours. The product Was extracted from the yeast using ethyl acetate (2x30 mL). Evaporation under OMe O Yeast reduced pressure produced an essentially pure ethyl (R)-3 —> hydroxy-3-phenylbutanoate as an oil Which can be further RMOH puri?ed by distillation if necessary (isolated yield 86%) This reaction Was repeated several times and achieved similar yields. 2. Preparation of Ethyl (R)-3-hydroxy-3-phenylpropanoate R I Me: 70% yield (Method 11) Ethyl benZoyl acetate (192 mg, 1 mmol) Was added to Ethyl 3-methoxy-2-butenoate (144 mg, 1 mmol) Was reacted according to Method 1. The product Was obtained in diethylether (10 mL) and the solution applied to a sheet of 70% yield. Reaction according to method ll gave the product ?lter paper. The solvent Was left to evaporate. Yeast (10 in 70% yield. g/mmol) Was mixed With Water (10 ml) and the resultant paste spread onto the ?lter paper and left for 24 h. The product Was 7. Preparation of 2-phenylethanol extracted from the yeast using ethyl acetate (2x30 mL). Acetophenone (120 mg, 1 mmol) Was reacted With yeast (5 20 g) and Water (5 ml) according to method I. The product Was Evaporation under reduced pressure produced an essentially obtained in 50% yield. Reaction according to Method ll gave pure ethyl (R)-3 -hydroxy-3 -phenylbutanoate as an oil. the product in 52% yield. 3. Preparation of Ethyl (S)-3 -hydroxybutanoate Unlike a solvent based yeast mediated reduction reaction, there is no interference in the isolation process from extracted Ethyl acetoacetate (130 mg, 1 mmol) Was reacted With biomass material product and therefore chromatographic yeast (2 g) and Water (2 ml) according to method I. Extraction puri?cation is not needed to obtain pure product; and unlike of the yeast gave the pure product in 80% yield. The reaction an aqueous based yeast mediated reduction reaction system, Was repeated folloWing the general procedure of Method 11 biphasic extractions, often associated With loW isolated outlined above to yield a pure product in 78% yield. yields, are avoided. 30 The steps required to synthesise the range of pharmaceu 4. Preparation of 2-nitro-3 -phenylpropane tical compounds from the precursors described in this appli cation are Will Within the skill and knoWledge of the person in the art of the invention. N02 Yeast The foregoing Examples are provided for illustration of the 35 concept of the invention only. Modi?cations may be made to the preferred embodiments Without departing from the spirit and scope of the invention. No2 The invention claimed is: 40 1. A method of reducing an organic compound of Formula IV:

(1V) Z-2-nitro-3 -phenyl-2-propene (151 mg 1 mmol) Was reacted With yeast (5 g) and Water (5 ml) according to Method 1. The product Was isolated as a racemic mixture of products in 41% yield. Reaction according to method 11 also resulted in a racemic mixture of products in 38% yield.

5. Preparation of Ethyl (R)-3 -methoxy-3 -phenylpropanoate Wherein: R8 is an optionally substituted aromatic group; R9, R10 and R11 are each independently selected from H, hydroxy, Cl_6alkoxy, mercapto, Cl_6 alkylthio, amino, 55 Cl_6alkylamino, di(Cl_6alkyl)amino, carboxy, Cl_6alkoxycarbonyl, Cl_6aryloxycarbonyl, carbamoyl, Cl_6alkylcarbamoyl, di-Cl_6alkylcarbamoyl, Cl_6cy cloalkylcarbamoyl, Cl_6alkylsulphonyl, arylsulphonyl, R OEt Cl_6alkylaminosulphonyl, di(Cl_6alkyl)aminosulpho 60 nyl, nitro, cyano, cyano-Cl_6alkyl, hydroxyCl_6alkyl, R I Ph: 54% yield amino-Cl_6alkyl, C l_6alkanoylamino, C l_6alkoxycarbo nylamino, Cl_6alkanoyl, Cl_6alkanoyloxy, Cl_6alkyl, halo, haloCl_6alkyl, or haloCl_6alkoxy, alkoximino, Ethyl 3-methoxy-3-phenylpropenoate (206 mg, 1 mmol) hydroximino, and alkylimino; Was reacted With yeast (10 g) and Water (10 ml) according to 65 the method comprising subjecting the organic compound method I. The product Was obtained in 54% yield. Reaction to a yeast-Water paste of a yeast mediated reduction in according to method 11 gave the product in 60% yield. the presence of an amount of Water that is su?icient for US 7,723,065 B2 23 24 enzymes to be hydrated and but insu?icient to provide a 14. The method ofclaim 1, Wherein R10 and R1 1 are each H, visibly separate Water layer Wherein the reduction is and R9 is carboxy or C l_6alkoxycarbonyl. conducted in the absence of any additional solvents, and 15. The method of claim 1, Wherein R9 is H or hydroxy, one Wherein a Water-to-yeast ratio is up to 1.5 ml/ g. ofR1O and R1 1 is selected from Cl_6alkyl, and the other ofR1O 2. The method of claim 1, Wherein the Water-to-yeast ratio and R1 1 is selected from the group consisting of C l_6alkoxy is betWeen 0.2 ml/g and 1.5 ml/g. carbonyl, C l_6aryloxycarbonyl, carbamoyl, C l_6alkylcar 3. The method of claim 2, Wherein the Water-to-yeast ratio is betWeen 0.8 and 1.2 ml/g of yeast. bamoyl, di-Cl_6alkylcarbamoyl, Cl_6cycloalkylcarbamoyl 4. The method of claim 1, Wherein the reduction is con and nitro. ducted in the presence of Water in an amount of 44 to 55% 16. The method of claim 1, Wherein R9 is hydroxy, one of W/W based on the Weight of yeast. R10 and R1 1 is selected from H and Cl_6alkyl, and the other of 5. The method of claim 1, Wherein the proportion of yeast R10 and R11 is selected from the group consisting of cyano, to organic compound is from 0.1 gram of yeast per mmol of Cl_6alkoxycarbonyl, Cl_6aryloxycarbonyl, carbamoyl, organic compound, up to 50 grams of yeast per mmol of Cl_6alkylcarbamoyl, di-Cl_6alkylcarbamoyl, and Cl_6cy organic compound. cloalkylcarbamoyl. 6. The method of claim 5, Wherein the proportion of yeast 17. The method of claim 1, Wherein the compound of to organic compound is 0.8 to 20 g/mmol. Formula (IV) is a precursor for the synthesis of a pharmaceu 7. The method of claim 1, Wherein the reaction is carried tical selected from the group consisting of ?uoxetine, tomox out in non-fermenting conditions at temperatures betWeen 0 etine, duloxetine, nisoxetine, epinephrine, norepinephrine, to 50° C. 20 ethylnorepinephrine, isoproterenol, isoetharine, metaproter 8. The method of claims 1, Wherein the reaction is carried enol, terbytaline, metaproterenol, phenylephrine, ritodrine, out at room temperature. prenalterol, methoxamine, albuterol or a derivative thereof, 9. The method of claim 1, Wherein the reaction is con salmeterol, ephedrine and phenylpropanolamine, amphet ducted at atmospheric pressure. amine or a derivative thereof, hydroxyamphetamine, meth 10. The method of claim 1, Wherein the method comprises 25 amphetamine, benZphetamine, fen?uramine, propyl the steps of contacting the organic compound With the yeast hexedrine, ibuprofen, naproxen, alminoprofen, fenoprofen, and Water in the absence of any additional solvents to form a ?urbiprofen, indoprofen, ketoprofen and suprofen, the mixture, leaving the mixture for su?icient time for the reac method further comprising the step of converting the precur tion to take place, adding an organic solvent to the mixture to sor into the pharmaceutical. dissolve a product of the reaction into the organic solvent, and 30 18. The method of claim 1, Wherein the organic compound conducting a solid/liquid separation to separate the product of and the yeast-Water paste forms a moist pliable yeast. the reaction from the yeast. 11. The method of claim 10, further comprising evaporat 19. The method of claim 1, Wherein the method avoids ing the solvent to isolate the product of the reaction. biphasic extractions before the step of adding the organic solvent to the mixture. 12. The method of claim 1, Wherein one of R9, R10 and R1 1 35 is not H. 20. The method of claim 1, Wherein the yeast mediated 13. The method of claim 1, the group R8 is selected from reduction is carried out in non-fermenting conditions. the group consisting of phenyl, substituted phenyl, napthyl and substituted napthyl.