WO2008127646A2 - Transaminase-based processes for preparation of pregabalin - Google Patents

Transaminase-based processes for preparation of pregabalin Download PDF

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WO2008127646A2
WO2008127646A2 PCT/US2008/004699 US2008004699W WO2008127646A2 WO 2008127646 A2 WO2008127646 A2 WO 2008127646A2 US 2008004699 W US2008004699 W US 2008004699W WO 2008127646 A2 WO2008127646 A2 WO 2008127646A2
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hydrolase
lipase
compound
bacillus
protease
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PCT/US2008/004699
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French (fr)
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WO2008127646A3 (en
WO2008127646A8 (en
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Wolfgang Reinhard Ludwig Notz
Robert William Scott
Lishan Zhao
Junhua Tao
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Dsm Ip Assets B.V.
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Publication of WO2008127646A8 publication Critical patent/WO2008127646A8/en
Publication of WO2008127646A3 publication Critical patent/WO2008127646A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/08—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to hydrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/04—Formation of amino groups in compounds containing carboxyl groups
    • C07C227/06—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid
    • C07C227/08—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid by reaction of ammonia or amines with acids containing functional groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
    • C07C45/41—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by hydrogenolysis or reduction of carboxylic groups or functional derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/09—Preparation of carboxylic acids or their salts, halides or anhydrides from carboxylic acid esters or lactones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/347—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups
    • C07C51/377—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups by splitting-off hydrogen or functional groups; by hydrogenolysis of functional groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/30—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group
    • C07C67/333—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by isomerisation; by change of size of the carbon skeleton
    • C07C67/343—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by isomerisation; by change of size of the carbon skeleton by increase in the number of carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
    • C07C69/67—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of saturated acids
    • C07C69/716—Esters of keto-carboxylic acids or aldehydo-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/26—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D307/30—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/32—Oxygen atoms
    • C07D307/33—Oxygen atoms in position 2, the oxygen atom being in its keto or unsubstituted enol form
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines

Definitions

  • (S)-Pregabalin otherwise known as (3S)-3-(aminomethyl)-5-methylhexanoic acid, is a biologically active compound. This compound is an anticonvulsant drug, a drug for treatment of neuropathic pain, an adjunct therapy for partial seizures, and a drug for anxiety disorders (e.g., see U.S. Pat. Nos. 5,563,175, 6,001,876 and 6,127,418).
  • (S)-Pregabalin activates L-glutamic acid decarboxylase (GAD) and promotes production of gamma-aminobutyric acid (GABA), a major inhibitory neurotrasmitter.
  • GABA gamma-aminobutyric acid
  • S)- Pregabalin binds to voltage-dependent calcium channels in the central nervous system.
  • (S)-pregabalin (Lyrica® (Pfizer, Inc.)) is approved outside the U.S. for treating various peripheral neuropathic pain indications, such as diabetic and post herpetic neuropathic pain, and adjunctive therapy for partial epilepsy in adults.
  • (S)- pregabalin (Lyrica® (Pfizer, Inc.)) is approved for the management of neuropathic pain associated with diabetic peripheral neuropathy and postherpetic neuralgia, and for the adjunctive treatment of partial onset seizures in adults.
  • There are several synthetic methods for preparing pregabalin Hoekstra et al.,
  • (S)-pregabalin ((3S)-3- (aminomethyl)-5-methylhexanoic acid).
  • the method includes chemical or enzymatic amine transfer or amination of an aldehyde precursor.
  • R is selected from among methyl, ethyl, n-butyl, z-butyl, pentyl, hexyl, heptyl, octyl, nonyl, C 1 -C 9 heteroalkyl, cycloalkyl, aryl and heteroaryl.
  • R is C 1 -C 3 alkyl.
  • R is n-butyl or isobutyl.
  • R is C 3 -C 8 aryl.
  • methods for producing pregabalin includes hydrolyzing a compound of formula I:
  • R is an alkyl, alkenyl or aryl, to afford an aldehyde intermediate of formula II
  • n M is hydrogen or a salt
  • the compound of formula I is prepared by reacting 4- methylpentanal with diisobutylamine and an alkyl-3-halo-propanoate.
  • Also provided are methods for producing pregabalin which include a) reacting diisobutylamine and 4-methylpentanal to produce an aldehyde intermediate of the structure:
  • step b) hydrolyzing the reaction product of step a) to yield a compound of formula
  • step c) animating the reaction product of step b) to produce pregabalin.
  • a method for producing pregabalin that includes reacting 4-methylpentanal with diisobutylamine and an alkyl-3-halo- propanoate to produce an aldehyde intermediate of formula I: aminating the aldehyde intermediate to produce a hexanoate intermediate of the structure: cyclizing the hexanoate intermediate to produce a lactam of the structure:
  • a method for producing pregabalin that includes a) reacting 4-methylpentanal with glyoxylic acid hydrate to produce a compound of the following structure:
  • step b) reducing asymmetrically the reaction product of step a) by chemical or enzymatic reduction to produce a compound of the following structure:
  • a method for producing pregabalin that includes a) treating 5-hydroxy-4-isobutylfuran-2(5//)-one: with base to yield a compound of the structure: j where M is hydrogen or a salt; b) enzymatically or chemically reducing the product of step a) to produce a compound of the structure: ,where M is hydrogen or a salt; and c) chemically or enzymatically amidating the product of step c) followed by reduction to provide pregabalin.
  • the compound of formula I is a racemic mixture. In some embodiments, the compound of formula I is the S-enantiomer. In some embodiments, the compound of formula II is a racemic mixture. In some embodiments, the compound of formula II is the S-enantiomer.
  • the amination step is performed chemically under conditions wherein the desired S-stereochemistry is obtained, hi one embodiment, chemical amination includes treating with an ammonia source. In other embodiments, the amination step is performed enzymatically under conditions wherein the desired S-stereochemistry is obtained. In some embodiments, the enzyme is a transaminase.
  • the transaminase is selected from among an Alcaligenes denitrificans transaminase, a Bordetella bronch-iseptica transaminase, a Bordetella parapertussis transaminase, a Brucella melitensis transaminase, a Burkholderia mallei transaminase, a Burkholderia pseudomallei transaminase, a Chromobacterium violaceum transaminase, an Oceanicola granulosus HTCC2516 transaminase, an Oceanobacter sp. RED65 transaminase, an Oceanospirillum sp.
  • the hydrolysis step is performed enzymatically.
  • a hydrolase is used for enzymatic processing of the intermediates.
  • the hydrolase is selected from among an esterase, a lipase and a protease.
  • the hydrolase is a bacterial hydrolase, a fungal hydrolase, an orange peel hydrolase, a porcine liver hydrolase, a rabbit liver hydrolase, a bovine pancreas hydrolase, a porcine pancreas hydrolase, a porcine stomach mucosal hydrolase, a bovine intestine hydrolase, a porcine intestine hydrolase, a porcine kidney hydrolase, a calf stomach hydrolase, a wheat germ hydrolase, a pineapple stem hydrolase and a papaya hydrolase.
  • the hydrolase is selected from among an Achromobacter sp.
  • hydrolase an Alcaligenes sp. hydrolase, and Aspergillus sp. hydrolase, an Apergillus niger hydrolase, an Apergillus oryzae hydrolase, an Aspergillus melleus hydrolase, an Aspergillus mellus hydrolase, an Aspergillus niger hydrolase, an Aspergillus oryzae hydrolase, an Aspergillus saitoi hydrolase, an Aspergillus sojae hydrolase, a Bacillus sp.
  • hydrolase a Bacillus amyloliquefaciens hydrolase, a Bacillus lentus hydrolase, a Bacillus licheniformis hydrolase, a Bacillus polymyxa hydrolase, a Bacillus stearothermophilus hydrolase, a Bacillus subtilis hydrolase, a Bacillus thermoglucosidasius hydrolase, a Bacillus thermoproteolyticus rokko hydrolase, a Burkholderia sp. hydrolase, a Burkholderia cepacia hydrolase, a Candida sp.
  • hydrolase a Candida antarctica hydrolase, a Candida antarctica A hydrolase, a Candida antarctica B hydrolase, a Candida cylindracea hydrolase, a Candida lipolytica hydrolase, a Candida rugosa hydrolase, a Candidia utilis hydrolase, a Carica papaya hydrolase, a Chromobacterium viscosum hydrolase, a Clostridium histolyticum hydrolase, an E. coli hydrolase, a Geotrichum candidum hydrolase, a Mucor javanicus hydrolase, a Mucor miehei hydrolase, a Penicillium sp. hydrolase, a Penicillium sp. I hydrolase, a Penicillium sp. II hydrolase, a Penicillium camembertii hydrolase, a Penicillium roqueforti hydrolase, a
  • Pseudomonas sp. hydrolase a Pseudomonas aeruginosa hydrolase, a Pseudomonas cepacia hydrolase, a Pseudomonas fluorescens hydrolase, a Pseudomonas stutzeri hydrolase, a Pyrococcus furiosis hydrolase, a Rhizomucor miehei hydrolase, a Rhizopus sp.
  • the reducing step is performed enzymatically.
  • the enzyme used for the reducing step is selected from among an alkenal double bond reductase (Pl), an alkenal double bond reductase (P2), an NAD(P)H-dependent alkenal/one oxidoreductase (AOR), an NADP-dependent leukotriene B4 12-hydroxydehydrogenase, an aryl propenal double bond reductase, an NADP-dependent oxidoreductase and a medium-chain dehyrogenase/reductase.
  • Pl alkenal double bond reductase
  • P2 alkenal double bond reductase
  • AOR NAD(P)H-dependent alkenal/one oxidoreductase
  • NADP-dependent leukotriene B4 12-hydroxydehydrogenase an aryl propenal double bond reductase
  • an NADP-dependent oxidoreductase and a medium-chain dehyrogenas
  • the enzymes are free in solution or are immobilized on a solid support.
  • articles of manufacture that include a packaging material, a compound provided herein or an acceptable salt, ester or other pharmaceutically acceptable derivative thereof, within the packaging material, and a label that indicates that the compound is used for the synthesis of pregabalin.
  • kits that include an enzyme and a compound provided herein and acceptable salts, esters and other pharmaceutically acceptable derivative.
  • the enzyme is a hydrolase.
  • the kit includes one or more hydrolases selected from among an esterase, a lipase, and a protease and a combination thereof.
  • the enzyme is a transaminase.
  • Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation and delivery, and treatment of subjects.
  • Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection).
  • Reactions and purification techniques can be performed e.g., using kits according to manufacturer's specifications or as commonly accomplished in the art or as described herein.
  • the foregoing techniques and procedures generally are performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
  • treating encompass either or both responsive and prophylaxis measures, e.g., designed to inhibit, slow or delay the onset of a symptom of a disease or disorder, achieve a full or partial reduction of a symptom or disease state, and/or to alleviate, ameliorate, lessen, or cure a disease or disorder and/or its symptoms.
  • amelioration of the symptoms of a particular disorder by administration of a particular compound or pharmaceutical composition refers to any lessening of severity, delay in onset, slowing of progression, or shortening of duration, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the compound or composition.
  • C 1 -C x includes C 1 -C 2 , Cj-C 3 . . . Ci-C x .
  • alkyl refers to straight or branched chain substituted or unsubstituted hydrocarbon groups, in one embodiment 1 to 40 carbon atoms, in another embodiment, 1 to 20 carbon atoms, in another embodiment, 1 to 10 carbon atoms.
  • the expression “lower alkyl” refers to an alkyl group of 1 to 6 carbon atoms.
  • An alkyl group can be a "saturated alkyl,” which means that it does not contain any alkene or alkyne groups and in certain embodiments, alkyl groups are optionally substituted.
  • An alkyl group can be an "unsaturated alkyl,” which means that it contains at least one alkene or alkyne group.
  • An alkyl group that includes at least one carbon-carbon triple bond (C ⁇ €) also is referred to by the term “alkynyl,” and in certain embodiments, alkynyl groups are optionally substituted.
  • an alkyl contains 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as “1 to 20” refers to each integer in the given range; e.g., "1 to 20 carbon atoms” means that an alkyl group can contain only 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. , up to and including 20 carbon atoms, although the term “alkyl” also includes instances where no numerical range of carbon atoms is designated).
  • An alkyl can be designated as "Ci-C 4 alkyl" or by similar designations.
  • Ci-C 4 alkyl indicates an alkyl having one, two, three, or four carbon atoms, i.e., the alkyl is selected from among methyl, ethyl, propyl, iso-propyl, «-butyl, iso-butyl, sec-butyl and t-butyl.
  • Ci - C 4 includes Ci - C 2 , Ci - C 3 , C 2 - C 3 and C 2 - C 4 alkyl.
  • Alkyls include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, hexenyl, ethynyl, propynyl, butynyl and hexynyl.
  • halogen or halide” refers to F, Cl, Br or I.
  • haloalkyl alone or in combination refers to an alkyl in which at least one hydrogen atom is replaced with a halogen atom. In certain of the embodiments in which two or more hydrogen atom are replaced with halogen atoms, the halogen atoms are all the same as one another. In certain of such embodiments, the halogen atoms are not all the same as one another. Certain haloalkyls are saturated haloalkyls, which do not include any carbon-carbon double bonds or any carbon- carbon triple bonds. Certain haloalkyls are haloalkenes, which include one or more carbon-carbon double bonds. Certain haloalkyls are haloalkynes, which include one or more carbon-carbon triple bonds. In certain embodiments, haloalkyls are optionally substituted.
  • pseudohalides are compounds that behave substantially similar to halides. Such compounds can be used in the same manner and treated in the same manner as halides (X-, in which X is a halogen, such as Cl, F or Br).
  • Pseudohalides include, but are not limited to, cyanide, cyanate, thiocyanate, selenocyanate, trifluoromethoxy, trifluoromethyl and azide.
  • haloalkyl there may be one or more substituents present.
  • haloalkyl may include one or more of the same or different halogens.
  • haloalkyl includes each of the substituents CF 3 , CHF 2 and CH 2 F.
  • heteroatom refers to an atom other than carbon or hydrogen. Heteroatoms are typically independently selected from oxygen, sulfur, nitrogen, and phosphorus, but are not limited to those atoms. In embodiments in which two or more heteroatoms are present, the two or more heteroatoms can all be the same as one another, or some or all of the two or more heteroatoms can each be different from the others.
  • heteroalkyl alone or in combination refers to a group containing an alkyl and one or more heteroatoms.
  • Certain heteroalkyls are saturated heteroalkyls, which do not contain any carbon-carbon double bonds or any carbon-carbon triple bonds.
  • Certain heteroalkyls are heteroalkenes, which include at least one carbon-carbon double bond.
  • Certain heteroalkyls are heteroalkynes, which include at least one carbon-carbon triple bond.
  • Certain heteroalkyls are acylalkyls, in which the one or more heteroatoms are within an alkyl chain.
  • heteroalkyls are optionally substituted.
  • heterohaloalkyl alone or in combination refers to a heteroalkyl in which at least one hydrogen atom is replaced with a halogen atom. In certain embodiments, heteroalkyls are optionally substituted.
  • non-cyclic alkyl refers to an alkyl that is not cyclic (i.e., a straight or branched chain containing at least one carbon atom). Non-cyclic alkyls can be fully saturated or can contain non-cyclic alkenes and/or alkynes. Non- cyclic alkyls can be optionally substituted.
  • ring refers to any covalently closed structure.
  • Rings include, for example, carbocycles (e.g., aryls and cycloalkyls), heterocycles (e.g., heteroaryls and non-aromatic heterocycles), aromatics (e.g., aryls and heteroaryls), and non-aromatics (e.g., cycloalkyls and non-aromatic heterocycles). Rings can be optionally substituted. Rings can form part of a ring system. As used herein, the term “ring system” refers to two or more rings, wherein two or more of the rings are fused. The term “fused" refers to structures in which two or more rings share one or more bonds.
  • carbocycles e.g., aryls and cycloalkyls
  • heterocycles e.g., heteroaryls and non-aromatic heterocycles
  • aromatics e.g., aryls and heteroaryls
  • non-aromatics e.g
  • Carbocycle refers to a ring where each of the atoms forming the ring is a carbon atom.
  • Carbocyclic rings can be formed by three, four, five, six, seven, eight, nine, or more than nine carbon atoms.
  • Carbocycles can be optionally substituted.
  • cycloalkyl refers to a saturated mono- or multicyclic ring system where each of the atoms forming a ring is a carbon atom. Cycloalkyls can be formed by three, four, five, six, seven, eight, nine, or more than nine carbon atoms. In one embodiment, the ring system includes 3 to 12 carbon atoms. In another embodiment, they ring system includes 3 to 6 carbon atoms.
  • the term "cycloalkyl” includes rings that contain one or more unsaturated bonds. As used herein, the terms “cycloalkenyl” and “cycloalkynyl” are unsaturated cycloalkyl ring system. Cycloalkyls can be optionally substituted.
  • a cycloalkyl contains one or more unsaturated bonds.
  • cycloalkyls include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclopentadiene, cyclohexane, cyclohexene, 1,3-cyclohexadiene, 1 ,4-cyclohexadiene, cycloheptane and cycloheptene.
  • cycloalkynyl refers to mono- or multicyclic ring systems that include at least one carbon-carbon triple bond (C ⁇ C).
  • Cycloalkenyl and cycloalkynyl groups include ring systems that include 3 to 12 carbon atoms.
  • the cycloalkenyl groups include 4 to 7 carbon atoms.
  • the cycloalkynyl groups include 8 to 10 carbon atoms.
  • the ring systems of the cycloalkyl, cycloalkenyl and cycloalkynyl groups may be composed of one ring or two or more rings which may be joined together in a fused, bridged or spiro-connected fashion, and may be optionally substituted with one or more alkyl group substituents.
  • heterocycle refers to a ring wherein at least one atom forming the ring is a carbon atom and at least one atom forming the ring is a heteroatom.
  • Heterocyclic rings may be formed by three, four, five, six, seven, eight, nine, or more than nine atoms. Any number of those atoms can be heteroatoms (i.e., a heterocyclic ring can contain one, two, three, four, five, six, seven, eight, nine, or more than nine heteroatoms, provided that at lease one atom in the ring is a carbon atom).
  • a heterocyclic ring can contain one, two, three, four, five, six, seven, eight, nine, or more than nine heteroatoms, provided that at lease one atom in the ring is a carbon atom.
  • the number of carbon atoms in a heterocycle is indicated (e.g., C i-C 6 heterocycle)
  • at least one other atom must be present in the ring.
  • Designations such as "Ci-C 6 heterocycle” refer only to the number of carbon atoms in the ring and do not refer to the total number of atoms in the ring. It is understood that the heterocyclic ring will have additional heteroatoms in the ring. Designations such as “4-6 membered heterocycle” refer to the total number of atoms that comprise the ring (i.e., a four, five, or six membered ring, in which at least one atom is a carbon atom, at least one atom is a heteroatom and the remaining two to four atoms are either carbon atoms or heteroatoms). In heterocycles containing two or more heteroatoms, those two or more heteroatoms can be the same or different from one another.
  • the heterocycle includes 3-12 members. In other embodiments, the heterocycle includes 4, 5, 6, 7 or 8 members.
  • the heterocycle may be optionally substituted with one or more substituents.
  • the substituents of the heterocyclic group are selected from among hydroxy, amino, alkoxy containing 1 to 4 carbon atoms, halo lower alkyl, including trihalomethyl, such as trifluoromethyl, and halogen.
  • the term heterocycle may include reference to heteroaryl. Binding to a heterocycle can be at a heteroatom or via a carbon atom. Examples of heterocycles include, but are not limited to the following: where D, E, F and G independently represent a heteroatom.
  • bicyclic ring refers to two rings, wherein the two rings are fused.
  • Bicyclic rings include, for example, decaline, pentalene, indene, naphthalene, azulene, heptalene, isobenzofuran, chromene, indolizine, isoindole, indole, indoline, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyrididine, quinoxaline, cinnoline, pteridine, isochroman, chroman and various hydrogenated derivatives thereof.
  • Bicyclic rings can be optionally substituted.
  • Each ring is independently aromatic or non-aromatic. In certain embodiments, both rings are aromatic. In certain embodiments, both rings are non-aromatic. In certain embodiments, one ring is aromatic and one ring is non-aromatic.
  • aromatic refers to a planar ring having a delocalized ⁇ -electron system containing 4n+2 ⁇ electrons, where n is an integer.
  • Aromatic rings can be formed by five, six, seven, eight, nine, or more than nine atoms.
  • Aromatics can be optionally substituted. Examples of aromatic groups include, but are not limited to phenyl, naphthalenyl, phenanthrenyl, anthracenyl, tetralinyl, fluorenyl, indenyl and indanyl.
  • aromatic includes, for example, benzenoid groups, connected via one of the ring- forming carbon atoms, and optionally carrying one or more substituents selected from an aryl, a heteroaryl, a cycloalkyl, a non-aromatic heterocycle, a halo, a hydroxy, an amino, a cyano, a nitro, an alkylamido, an acyl, a C )-6 alkoxy, a Ci -6 alkyl, a C 1-6 hydroxyalkyl, a Ci -6 aminoalkyl, a Ci -6 alkylamino, an alkylsulfenyl, an alkylsulfinyl, an alkylsulfonyl, an sulfamoyl, or a trifluoromethyl.
  • an aromatic group is substituted at one or more of the para, meta, and/or ortho positions.
  • aromatic groups containing substitutions include, but are not limited to, phenyl, 3-halophenyl, 4-halophenyl, 3-hydroxyphenyl, 4-hydroxy- phenyl, 3-aminophenyl, 4-aminophenyl, 3-methylphenyl, 4-methylphenyl, 3- methoxyphenyl, 4-methoxyphenyl, 4-trifluoromethoxyphenyl, 3-cyano-phenyl, 4- cyanophenyl, dimethylphenyl, naphthyl, hydroxynaphthyl, hydroxymethyl-phenyl, (trifluoromethyl)phenyl, alkoxyphenyl, 4-mo ⁇ holin-4-ylphenyl, 4-pyrrolidin-l- ylphenyl, 4-pyrazolylphenyl, 4-triazolylphenyl and 4-(2-oxopyrrolidin-l-
  • aryl refers to an aromatic ring wherein each of the atoms forming the ring is a carbon atom.
  • Aryl rings can be formed by three, four, five, six, seven, eight, nine, or more than nine carbon atoms.
  • Aryl groups can be optionally substituted.
  • heteroaryl refers to an aromatic ring in which at least one atom forming the aromatic ring is a heteroatom.
  • Heteroaryl rings can be formed by three, four, five, six, seven, eight, nine and more than nine atoms.
  • Heteroaryl groups can be optionally substituted.
  • heteroaryl groups include, but are not limited to, aromatic C 3-8 heterocyclic groups containing one oxygen or sulfur atom, or two oxygen atoms, or two sulfur atoms or up to four nitrogen atoms, or a combination of one oxygen or sulfur atom and up to two nitrogen atoms, and their substituted as well as benzo- and pyrido-fused derivatives, for example, connected via one of the ring-forming carbon atoms.
  • heteroaryl groups are optionally substituted.
  • the one or more substituents are each independently selected from among halo, hydroxy, amino, cyano, nitro, alkylamido, acyl, Ci -6 -alkoxy, Ci -6 -alkyl, Ci -6 -haloalkyl, Ci -6 -hydroxyalkyl, Ci -6 -aminoalkyl, Ci -6 -alkylamino, alkylsulfenyl, alkylsulfinyl, alkylsulfonyl, sulfamoyl, or trifluoromethyl.
  • heteroaryl groups include, but are not limited to, unsubstituted and mono- or di- substituted derivatives of furan, benzo furan, thiophene, benzothiophene, pyrrole, pyridine, indole, oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole, isothiazole, imidazole, benzimidazole, pyrazole, indazole, tetrazole, quinoline, isoquinoline, pyridazine, pyrimidine, purine and pyrazine, furazan, 1,2,3- oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, triazole, benzotriazole, pteridine, phenoxazole, oxadiazole, benzopyrazole, quinolizine, cinnoline, phthalazine,
  • the substituents are halo, hydroxy, cyano, O-Ci- 6 -alkyl, Ci- 6 -alkyl, hydroxy-Ci -6 -alkyl and amino-Ci -6 -alkyl.
  • non-aromatic ring refers to a ring that does not have a delocalized 4n+2 ⁇ -electron system.
  • non-aromatic heterocycle refers to a non-aromatic ring wherein one or more than one atom forming the ring is a heteroatom.
  • Non- aromatic heterocyclic rings can be formed by three, four, five, six, seven, eight, nine, or more than nine atoms.
  • Non-aromatic heterocycles can be optionally substituted.
  • non-aromatic heterocycles contain one or more carbonyl or thiocarbonyl groups such as, for example, oxo- and thio-containing groups.
  • non-aromatic heterocycles include, but are not limited to, lactams, lactones, cyclic imides, cyclic thioimides, cyclic carbamates, tetrahydrothiopyran, 4H-pyran, tetrahydropyran, piperidine, 1,3-dioxin, 1,3-dioxane, 1,4-dioxin, 1,4-dioxane, piperazine, 1,3-oxathiane, 1 ,4-oxathiin, 1 ,4-oxathiane, tetrahydro-l,4-thiazine, 2H- 1 ,2-oxazine , maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil, morpholine, trioxane, hexahydro-1 ,3,5- triazine, te
  • heteroarylalkyl alone or in combination, refers to an alkyl substituted with a heteroaryl that may be optionally substituted.
  • cyano refers to a group of formula -CN.
  • isocyanato refers to a group of formula -NCO.
  • thiocyanato refers to a group of formula -CNS.
  • isothiocyanato refers to a group of formula -NCS.
  • esters refers to a chemical moiety with formula -(R) n -COOR', where R and R' are independently selected from alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and non-aromatic heterocycle (bonded through a ring carbon), where n is O or 1.
  • amide refers to a chemical moiety with formula
  • an amide can be an amino acid or a peptide.
  • the terms "amine,” “hydroxy,” and “carboxyl” include such groups that have been esterified or amidified.
  • the term "optionally substituted,” refers to a group in which none, one, or more than one of the hydrogen atoms has been replaced with one or more group(s) individually and independently selected from among alkyl, cycloalkyl, heteroalkyl, aryl, heteroaryl, non-aromatic heterocycle, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N carbamyl, O thiocarbamyl, N thiocarbamyl, C amido, N amido, S-sulfonamido, N sulfonamido, C carboxy, O carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, and amino, including mono- and di
  • the compounds provided herein may contain chiral centers. Such chiral centers may be of either the (R) or (S) configuration, or may be a mixture thereof. Thus, the compounds provided herein may be enantiomerically pure, or be stereoisomeric or diastereomeric mixtures. It is to be understood that the chiral centers of the compounds provided herein may undergo epimerization in vivo. As such, one of skill in the art will recognize that administration of a compound in its (R) form is equivalent, for compounds that undergo epimerization in vivo, to administration of the compound in its (S) form.
  • cis and trans are descriptors which show the relationship between two ligands attached to separate atoms which are connected by a double bond or contained in a ring with a double bond.
  • Two ligands are said to be “cis” to each other if they lie on the same side of a plane. If the ligands are on opposite sides, their relative position is described as trans.
  • the appropriate reference plane of a double bond is perpendicular to that of the relevant sigma bond which passes through the double bond.
  • enantiomer refers to one of a pair of molecular entities which are mirror images of each other and non-superimposable.
  • Enantiomeric excess (ee) may be calculated for a mixture of (R) and (S)-enantiomers.
  • the ee is defined as the absolute value of the mole fractions of F (R) minus the mole fraction of F ( s>.
  • the percent ee is the absolute value of the mole fractions of F (R) minus the mole fraction of F ( s> multiplied by 100.
  • optical activity refers to the ability of a sample material to rotate the plane of polarized light. A specific enantiomer causes rotation of light in either a clockwise or counterclockwise direction.
  • optical purity refers to the ratio of observed optical rotation of a sample consisting of a mixture of enantiomers to the optical rotation of one pure enantiomer.
  • substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis, high performance liquid chromatography (HPLC) and mass spectrometry (MS), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance.
  • substantially pure object species e.g., compound
  • is the predominant species present i.e., on a molar basis it is more abundant than any other individual species in the composition.
  • a substantially purified fraction is a composition wherein the object species contains at least about 50 percent (on a molar basis) of all species present.
  • a substantially pure composition will contain more than about 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% of all species present in the composition, hi certain embodiments, a substantially pure composition will contain more than about 80%, 85%, 90%, 95%, or 99% of all species present in the composition.
  • Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art.
  • a substantially chemically pure compound can, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.
  • Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as reverse phase HPLC.
  • the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.
  • all tautomeric forms also are intended to be included.
  • “acid” generally refers to a molecular entity or chemical species capable of donating a hydron (proton), otherwise considered a Br ⁇ nsted acid, or capable of accepting an electron pair, otherwise known as a Lewis acid.
  • base refers to a chemical species or molecular entity having an available pair of electrons capable of forming a bond with a hydron (proton), otherwise known as a Br ⁇ nsted base, or capable of donating an electron pair to form a bond with the vacant orbital of some other species, otherwise known as a Lewis base.
  • activation of a chemical group occurs when some or all of the energy required for a desired transformation is provided by a preceding reaction. For example, in the scheme: A + B ⁇ X ⁇ Y + Z, some or all of the energy required for the reaction of X to form products Y and Z is provided by the first reaction between A and B.
  • chemical resolution occurs when a mixture of stereoisomers is separated into the component diasteriomers and/or enantiomers. Chemical resolution also may be applied to the separation of olefin cis- and trans- isomers.
  • an "aliquot” is a fractional part of known quantity taken from a larger solution or mixture. The properties of the aliquot are usually analyzed and such properties are considered to be representative of the properties of the larger solution or mixture.
  • condensation occurs when two or more reactants, yield a single main product with accompanying formation of water or of some other small molecule such as ammonia, ethanol, acetic acid, or hydrogen sulfide. A condensation reaction also may occur between two or more reactive sites within the same molecular entity.
  • transesterification refers to the reaction which converts one ester into another. Transesterifications are often realized by reacting an ester with an excess of alcohol under acidic or basic conditions.
  • transformation refers to the conversion of a substrate into a particular product irrespective of the reagents or mechanisms involved. Reference to a transformation does not require full description of all reactants or all products necessary to convert the substrate into product.
  • cyclization refers to the formation of a covalently closed ring by formation of a new bond.
  • reduce and “reduction” refers to the transfer of one or more electrons to a molecular entity.
  • a compound can be reduced by the addition of hydrogen.
  • a reduced species also can be formed through the gain of electrons.
  • the reverse process in which one or more electrons is removed from a molecular entity is known as "oxidation.”
  • salting refers to the addition of electrolytes to a solution. Salting is often alters the distribution ratio of a particular solute or changes the miscibility of two liquids.
  • “Baumann conditions” refers to acylation of alcohols with acyl halides in aqueous alkaline solution.
  • a “derivative” is a compound obtained or produced by modification of another compound of similar structure. Derivatives may be produced by one or more modification steps.
  • quenched refers to arresting the course of a chemical or enzymatic reaction by chemical or physical means.
  • protecting groups are often employed. That is, a functional group is temporarily converted into an unreactive form to prevent its interference with transformations to be carried out elsewhere in the molecule. Such temporary functional group modification is known as “protecting" the original group. Subsequent to transformation carried out elsewhere in the molecule, the original unit may be regenerated, i.e., "deprotected," under separate conditions. For example, an alcohol may be protected as a 1,1-dimethylethyl ether by an acid catalyzed reaction of the alcohol with 2-methyl-2-propanol. The resulting ether is inert to some basic, oxidizing, or reducing conditions. The alcohol may be deprotected by removal of the ether group in dilute aqueous acid.
  • Desymmetrization involves the modification of a compound which results in the loss of one or more symmetry elements.
  • Desymmetrization includes the loss of a symmetry element which precludes chirality, such as a mirror plane, center of inversion, or rotational-reflection axis. Desymmetrization may result in the conversion of a prochrial molecular entity into a chiral entity.
  • prochiral refers to a structure which lacks chirality, but which is capable of becoming chiral by addition, removal, or replacement of a substituent.
  • hydrolysis refers to the general rupture of one or more bonds by water molecules.
  • ichael addition refers to the base catalyzed addition methylene compounds to unsaturated systems.
  • EC refers to the Enzyme Commission of the International Union of Biochemistry and Molecular Biology (IUBMB).
  • IUBMB International Union of Biochemistry and Molecular Biology
  • EC numbers such as EC 3.4.21.62, are associated with a recommended name for the respective enzyme. The first number designates the major class, the second number designates the subclass, and the third number designates the sub-subclass. The fourth number indicates the serial number of the enzyme in its sub-subclass.
  • an “esterase” is an enzyme which catalyzes the cleavage of ester bonds. Many esterases show specificity for particular types of esters.
  • proteases are an enzyme which catalyzes the hydrolysis of peptide bonds. Many proteases also are capable of cleaving ester bonds. Proteases often show specificity for particular bond arrangements.
  • hydrolase refers to enzymes that catalyze the cleavage of C- O, C-N, C-C and other bonds by reactions involving the addition or removal of water.
  • product refers to a substance that is formed during a chemical or enzymatic reaction.
  • reaction medium refers to the phase in which a chemical or biological reaction or other such transformation takes place.
  • the reaction medium may include solid, liquid, and gaseous phases and mixtures thereof. Chemical and biological reactants and reagents are commonly dissolved or suspended in various liquid compositions to facilitate a reaction or transformation.
  • a "catalyst” is a substance that increases the rate of a reaction.
  • a catalytic substance is a substance which increases the rate of a reaction.
  • biocatalyst refers to a living organism, enzyme, and/or enzyme complex which catalyses a reaction or otherwise facilitates substrate conversion in various chemical reactions.
  • a “buffer solution” is any substance or mixture of compounds in solution which is capable of neutralizing both acids and bases without appreciably changing the original acidity or alkalinity of the solution. Buffer solutions contain mixture(s) of acid and conjugate base at or near the pK a to minimize pH changes caused by an influx of acid or base. Buffer solutions also may contain additional solutes such as salts and other compounds.
  • enzyme “deactivation” occurs when an enzyme is no longer capable of catalysis.
  • a lipase is one of various enzymes which catalyze the hydrolysis of fats, especially triglycerides and phospholipids, into glycerol and fatty acids. Many lipases selectively cleave ester bonds.
  • substrate refers to the chemical entity involved in a reaction which undergoes conversion to a product or products. Enzymes may catalyze the conversion of substrate(s) to product(s).
  • precipitate refers to the act of separating, e.g., a compound or product, from solution or suspension, usually via a chemical or physical change, often resulting in an insoluble oil or amorphous or crystalline solid, and the term also refers to a substance separated from a solution or suspension by chemical or physical change.
  • supernatant refers to the liquid floating above the surface of a sediment or precipitate.
  • peak area refers to the area between the peak and the baseline of a chromatogram.
  • co-solvent refers to a mixture of liquids.
  • the co-solvent is a material that is not necessarily an acceptable solvent that is added to a generally small amount of an active solvent to form a mixture that has enhanced solvent power.
  • a polar cosolvent can be added into a mixture of an organic liquid and a compound having pendant ionomeric groups to solubilize the pendant ionomeric groups.
  • Co-solvent increase solubility of a compound.
  • the use of cosolvents can increase the solubility by several orders of magnitude.
  • Some commonly used cosolvents in pharmaceutics are propylene glycol, polyethylene glycols, ethanol and sorbitol.
  • the addition of a co-solvent can increase solubility of hydrophobic molecules by reducing the dielectric constant of the solvent.
  • THF tetrahydrofuran
  • article of manufacture is a product that is made and sold and that includes a container and packaging, and optionally instructions for use of the product.
  • articles of manufacture encompass packaged intermediates as disclosed herein.
  • a “kit” refers to a combination of an intermediate provided herein and another item for a purpose including, but not limited to, synthesis of pregabalin or a related compound. Kits also optionally include instructions for use and/or reagents and glassware and other such items for use with the product.
  • substantially identical to a product means sufficiently similar so that the property of interest is sufficiently unchanged so that the substantially identical product can be used in place of the product.
  • the term "monitoring” refers to observing an effect or absence of any effect.
  • effects include, but are not limited to, changes in evolution of gas, the appearance of a reaction product or a disappearance of a substrate or reactant.
  • contacting refers to bringing two or more materials into close enough proximity that they can interact.
  • contacting can be accomplished in a vessel such as, e.g., a test tube, flask, petri dish or mixing tank.
  • contacting can be performed in the presence of additional materials.
  • the term "subject" is an animal, typically a mammal, including human.
  • the term “patient” includes human and animal subjects.
  • carrier refers to a compound that facilitates the incorporation of another compound into cells or tissues.
  • DMSO dimethyl sulfoxide
  • a pharmaceutical composition refers to a chemical compound or composition capable of inducing a desired therapeutic effect in a subject.
  • a pharmaceutical composition contains an active agent, which is the agent that induces the desired therapeutic effect.
  • the pharmaceutical composition can contain a prodrug of the compounds provided herein.
  • a pharmaceutical composition contains inactive ingredients, such as, for example, carriers and excipients.
  • therapeutically effective amount refers to an amount of a pharmaceutical composition sufficient to achieve a desired therapeutic effect.
  • the term "pharmaceutically acceptable” refers to a formulation of a compound that does not significantly abrogate the biological activity, a pharmacological activity and/or other properties of the compound when the formulated compound is administered to a subject. In certain embodiments, a pharmaceutically acceptable formulation does not cause significant irritation to a subject.
  • pharmaceutically acceptable derivatives of a compound include, but are not limited to, salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs thereof.
  • Such derivatives can be readily prepared by those of skill in this art using known methods for such derivatization.
  • the compounds produced can be administered to animals or humans without substantial toxic effects and either are pharmaceutically active or are prodrugs.
  • salts include, but are not limited to, amine salts, such as but not limited to chloroprocaine, choline, N,N'-dibenzyl-ethylenediamine, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N- methylglucamine, procaine, N-benzyl-phenethylamine, l-para-chloro-benzyl-2- pyrrolidin-l'-ylmethyl-benzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)-aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, salts of mineral acids, such as but
  • esters include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids and boronic acids.
  • Pharmaceutically acceptable solvates and hydrates are complexes of a compound with one or more solvent or water molecules, or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solvent or water molecules.
  • salts refers to an assembly of cation and anion.
  • Salts include alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, salts of mineral acids, such as but not limited to hydrochlorides and sulfates; and salts of organic acids, such as but not limited to acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates and fumarates.
  • pregabalin Several methods are known in the art for the preparation of pregabalin. For example, a synthetic scheme using an azide intermediate results in a racemic mixture, which is then subsequently resolved into its R- and S-enantiomers (e.g., see U.S. Pat. No. 5,563,175). Another method uses an intermediate and nitro compounds that can be unstable resulting in an amine, but the reaction can proceed exothermically (e.g. , see Andruszkiewicz et al, Synthesis, 1989:953).
  • Racemic mixtures of pregabalin also have been prepared via a tert-butyl ester route, via an L-leucine conversion, via Stobbe condensation, via malonate synthesis, and by a Hofinann synthesis (e.g., see Hoekstra et al., Organic Process Research & Development 1 : 26-38 (1997); Burk et al, J. Org. Chem. 68: 5731 (2003); U.S. Pat. Nos. 5,840,956; 5,637,767; 5,629,447; and 5,616,793 and U.S. Pat. Pub. 2005283023).
  • a drawback of these methods is that classical methods of resolving a racemate are required to obtain pregabalin.
  • Classical resolution of racemic mixtures involves preparation of a salt with a chiral resolving agent in order to separate and purify the desired S-enantiomer. This involves significant processing, and results in the potential loss of compound as the R-enantiomer, which cannot be efficiently recycled and is often discarded as a waste by-product.
  • the requirement for racemic resolution of these methods also adds additional cost, in processing time and lost product, as well as costs associated with the racemic resolving agent. In many of the reactions, only half of the product is the correct racemate, which results in a maximum theoretical yield of pregabalin, the S-enantiomer, of about 50%. This has a significant negative impact on production cost and capacity.
  • compound 2 is prepared from 4-methylpentanal 1 by known methods (e.g., see Kenda et al, J. Med. Chem. 47: 530 (2004) and Reichelt et al, J. Org. Chem. 67: 4062 (2002)).
  • Compound 2 is then hydrolyzed to 3 by chemical or enzymatic methods.
  • compound 3 is converted to the final product, pregabalin 6, under either chemical or enzymatic amine transfer or amination conditions, and the desired stereoselectivity can be obtained (e.g., Yun et al, Appl. Environ. Microbiol. 70: 2529-2534 (2004); Shin et al, Biosci. Biotechnol. Biochem. 65: 1782-1788 (2001); Mehta et al, Eur. J. Biochem. 214: 549-561 (1993)).
  • the aldehyde 2 is converted to the amino ester 4 under either chemical or enzymatic amine transfer or amination conditions (Yun et al, Appl. Environ. Microbiol. 70: 2529-2534 (2004); Shin et al, Biosci. Biotechnol. Biochem., 65: 1782-1788 (2001); Mehta et al, Eur. J. Biochem. 214: 549-561 (1993)).
  • Compound 4 is cyclized to lactam 5 and hydrolyzed to pregabalin 6 upon hydrolytic ring-opening (e.g., see Hoekstra et al, Org. Proc. Res. Dev. 1 : 26 (1997)). Alternatively, compound 4 is hydrolyzed directly to compound 6.
  • aldehyde precursor 1 to prepare pregabalin is shown in Scheme 2.
  • Compound 1 is condensed with glyoxalic acid to afford lactone 7 (5- hydroxy-4-isobutylfuran-2(5H)-one), the double bond of which is further reduced to afford compound 8 (e.g., see Bourguignon, J. Org Chem. 46: 4889 (1981) and WO 03/093220) or its open-chain equivalent compound 3 or 10.
  • the reduction of the double-bond can be achieved chemically or chemoenzymatically and may proceed via 9, the open-chain equivalent of 7.
  • Compound 8 or 10 is then converted to pregabalin under chemical or enzymatic amine transfer or amination conditions.
  • Compound 3 also may exist in or be converted to its cyclic form 8, (e.g., see Bourguignon, J. Org Chem. 46: 4889 (1981)). As discussed above, compound 8 may be converted to pregabalin under chemical or enzymatic amine transfer or amination conditions.
  • Compound 7 may be converted to its open form, compound 9.
  • hydrolyzing compound 7 affords compound 9.
  • Any method known in the art of hydrolyzing a lactone can be used for the preparation of a compound of formula I from 5-hydroxy-4-isobutylfuran-2(5H)-one.
  • 5-hydroxy-4- isobutylfuran-2(5H)-one can be hydrolyzed under aqueous basic conditions.
  • the base can be selected from among, but is not limited to, sodium hydroxide, potassium hydroxide, sodium t-butoxide, potassium t-butoxide, sodium methoxide, potassium methoxide, lithium methoxide, lithium t-butoxide, potassium carbonate, sodium carbonate, lithium carbonate, cesium carbonate and sodium ethoxide.
  • Saponification of compound 7 also affords the salt of compound 9. Any method of saponification of lactones known in the art can be used. For example, saponification may be accomplished by treating the lactone with a metallic alkali base such as sodium hydroxide or potassium hydroxide. Chemical or enzymatic reduction of compound 9 affords compound 10 (e.g., see Dick et al, J. Bio. Chem.
  • compound 10 is converted to pregabalin by either enzymatic amine transfer or chemical amination conditions.
  • Any solvent can be used in the process.
  • the solvent can be aqueous, nonaqueous, polar or non-polar, hydrophobic or hydrophilic and combinations thereof, such as bi-phasic systems and emulsions.
  • the reaction is carried out at different temperatures and pressures depending upon the substrate.
  • the reaction conditions can be determined by a person skilled in the art on the basis of the substrate, catalysts and rates of conversion, and is appropriately adjusted for the process.
  • the process can be carried out at any pressure known to the skilled artisan to be appropriate for hydrogenation.
  • the pressure can be at atmospheric pressure or above atmospheric pressure.
  • the reaction temperature can be at or above room temperature.
  • the process is conducted in a reaction vessel, for example, in a stirred tank or in membrane reactor, and the process can be operated either as a batch operation, as a semi-continuous process, or as a continuous process.
  • a reaction vessel for example, in a stirred tank or in membrane reactor, and the process can be operated either as a batch operation, as a semi-continuous process, or as a continuous process.
  • continuous or semi -continuous processes can be operated using, e.g., cross-flow filtration mode.
  • a membrane reactor is used.
  • Suitable membrane reactors are described, e.g., in U.S. Pat. No. 6,180,837.
  • the pH of the reaction is varied to optimize the catalysis mediated by the selected hydrolases and/or transaminases and can be determined by a person skilled in the art on the basis of the enzyme stabilities and rates of conversion, and is appropriately adjusted for the process.
  • the pH range for the reaction is selected from about pH 3 to about 11.
  • the enzyme used in the process can be in a free form, such as a homogeneously purified compound.
  • the enzyme is a constituent of an organism, such as a fungus or bacteria, and can be part of an intact organism or a denatured cell mass of the organism, hi other embodiments, the enzyme is immobilized, such as on a solid support (e.g. , see Worsford, Pure and Applied Chem. 67(4): 597-600 (1995); Cao, "Carrier-bound Immobilized Enzymes: Principles, Application and Design” (Wiley, 2006); Okahata et al., Tetrahedron Lett. 38: 1971-1974 (1997); Adlercreutz et al, Biocatalysis 6: 291-305 (1992)).
  • Bayer- Villiger monoxygenases are available as free forms of enzymes and conjugated to a solid support.
  • R is selected from among alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl and heteroaryl, provided that R is not t-butyl.
  • R is alkyl, alkenyl or aryl.
  • R is selected from among methyl, ethyl, n-butyl, i-butyl, pentyl, hexyl, heptyl, octyl, nonyl.
  • R is Cj-C 3 alkyl.
  • R is «-butyl, isobutyl or sec- butyl.
  • R is C 5 -C 8 alkyl.
  • R is C 5 -C 2 O alkyl.
  • R is Cj-C 8 alkenyl.
  • R is C 1 -C 8 heteroalkyl.
  • R is C 3 -C 8 cycloalkyl or C 3 -C 8 cycloheteroalkyl having one to eight heteroatoms selected from among O, N, S and P.
  • R is C 3 -C 8 aryl or C 3 -C 8 heteroaryl having one to eight heteroatoms selected from among O, N, S and P.
  • Compounds of formula I can be a racemic mixture, or can be enantiomerically enriched as the R-enantiomer or the S-enantiomer:
  • the (R)- and (S)-enantiomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as reverse phase HPLC.
  • hydrolase as noted. Any hydrolase can be can be used. If needed, hydrolases, libraries thereof and/or libraries of modified hydrolases can be screened to identify more suitable or suitable hydrolases for the methods herein.
  • Hydrolases are enzymes that catalyze the hydrolysis of a chemical bond. These enzymes are classified as EC 3 in the EC number classification of enzymes, and are further classified into several subclasses, based on the bonds upon which they act. Bonds that can be hydrolyzed by these enzymes include, but are not limited to, ester bonds, bonds to sugars, ether bonds, peptide bonds, carbon-nitrogen bonds, acid anhydrides, carbon-carbon bonds, halide bonds, phosphorus-nitrogen bonds, sulfur- nitrogen bonds, carbon-phosphorus bonds, sulfur-sulfur bonds and carbon-sulfur bonds.
  • hydrolases can hydrolyze esters, including, but not limited to, various proteases, and those enzymes classified as esterases, including lipases. Any enzyme that can hydrolyze the ester bond of compound 2, described above, to yield compound 3, can be used in the methods described herein.
  • the hydrolase is an esterase, a lipase, or a protease.
  • the hydrolase can be of mammalian, including human, origin, or can be of non-mammalian origin, including but not limited to, plant, bacterial, viral, yeast and fungal origin.
  • the hydrolase used in the methods provided herein to hydrolyze the ester bond of compound 2 can be a wild-type protein or a variant thereof.
  • a hydrolase variant is used that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations, such as amino acid substitutions, deletions, additions, or combinations thereof.
  • hydrolase variants can be generated by directed evolution such that the substrate specificity and enzymatic activity of the variants are optimized for the purposes herein (see, e.g., Bornscheuer et ah, Curr. Opin. Biotech. 7:2169-2173 (1999)).
  • the properties of a candidate hydrolase, such as substrate specificity and enzymatic activity can be assessed using any method known in the art, as described below.
  • high-throughput screening of multiple hydrolases is performed (e.g., see Yazbeck et ah, Adv.
  • Esterases display broad substrate specificity and also can exhibit enantioselectivity. Many esterases have been described in the art to hydrolyze different substrates. For example, esterase enzymes cleaving carboxyl esters have been identified in leukocytes; phosphatases, such as alkaline and acidic phosphatases, hydrolyse phosphoric acid esters; glycosidases, such as galactosidases, glucosidases, mannosidases and amylases can cleave glycosidic bonds.
  • esterase enzymes cleaving carboxyl esters have been identified in leukocytes; phosphatases, such as alkaline and acidic phosphatases, hydrolyse phosphoric acid esters; glycosidases, such as galactosidases, glucosidases, mannosidases and amylases can cleave glycosidic bonds.
  • esterases include, but are not limited to, lipases, acetylesterases, thiolester hydrolases, phosphoric monoester hydrolases, phosphoric diester hydrolases, triphosphoric monoester hydrolases, sulfuric ester hydrolases, diphosphoric monoester hydrolases, phosphoric triester hydrolases, exonucleases and endonucleases.
  • lipases acetylesterases, thiolester hydrolases, phosphoric monoester hydrolases, phosphoric diester hydrolases, triphosphoric monoester hydrolases, sulfuric ester hydrolases, diphosphoric monoester hydrolases, phosphoric triester hydrolases, exonucleases and endonucleases.
  • lipases acetylesterases, thiolester hydrolases, phosphoric monoester hydrolases, phosphoric diester hydrolases, triphosphoric monoester hydrolases, sulfuric ester
  • Lipases also known as triacylglycerol ester hydrolases, (E.C.3.1.1.3) are a subclass of esterases which belongs to the a/ ⁇ hydrolase super family. They are ubiquitous enzymes that can be generally divided into the following four groups according to their specificity in hydrolysis reaction: substrate specific lipases, regio- selective lipases, fatty acid specific Upases, and stereo-specific lipases.
  • substrate specific lipases can be obtained from a variety of sources including, but not limited to, plants, animals, yeast and bacteria.
  • Exemplary lipases include, but are not limited to, Upases from Acinetobacter calcoaceticus, Acinetobacter sp. , Alcaligenes sp.
  • proteases can be hydrolyzed by proteases, including, but not limited to, serine proteases and cysteine proteases.
  • proteases including, but not limited to, serine proteases and cysteine proteases.
  • the activity of proteases in the serine protease family is dependent on a set of amino acid residues that form the active site. One of the residues is always a serine, hence their designation as serine proteases.
  • serine proteases also can hydrolyze ester bonds. They can be obtained from a variety of sources including, but not limited to, animals (including humans), plants, yeast and bacteria.
  • Exemplary eukaryotic serine proteases include, but are not limited to, acrosin; blood coagulation factors VII, IX, X, XI and XII, thrombin, plasminogen, and protein C; cathepsin G; chymotrypsins; complement components CIr, CIs, C2, and complement factors B, D and I; complement-activating component of RA-reactive factor; cytotoxic cell proteases (granzymes A to H); duodenase I; elastases 1 , 2, 3 A, 3B (protease E), leukocyte (medullasin); enterokinase (enteropeptidase); hepatocyte growth factor activator; hepsin; glandular (tissue) kallikreins (including EGF-binding protein types A, B, and C, NGF- ⁇ chain, 7-renin, prostate specific antigen (PSA) and tonin); plasma kallikrein; mast
  • Exemplary prokaryotic serine protease include, but are not limited to, subtilisins from Bacillus sp.; alkaline elastase YaB from Bacillus sp.; alkaline serine exoprotease A from Vibrio alginolyticus; aqualysin I from Thermus aquaticus; AspA from Aeromonas salmonicida; bacillopeptidase F (esterase) from Bacillus subtilis; C5A peptidase from Streptococcus pyogenes; cell envelope-located proteases PI, PII, and PIII from Lactococcus lactis; extracellular serine protease from Serratia marcescens; extracellular protease from Xanthomonas campestris; intracellular serine protease (ISP) from various Bacillus; minor extracellular serine protease epr from Bacillus subtilis; minor extracellular serine protease
  • alkaline extracellular protease (AEP) from Yarrowia lipolytica; alkaline proteinase from Cephalosporium acremonium; cerevisin (vacuolar protease B) from yeast; cuticle-degrading protease (prl) from Metarhizium anisopliae.; KEX-I protease from Kluyveromyces lactis.
  • AEP alkaline extracellular protease
  • Cephalosporium acremonium alkaline proteinase from Cephalosporium acremonium
  • cerevisin (vacuolar protease B) from yeast
  • cuticle-degrading protease (prl) from Metarhizium anisopliae.
  • KEX-I protease from Kluyveromyces lactis.
  • kexin from yeast
  • oryzin alkaline proteinase
  • Aspergillus proteinase K from Tritirachium album
  • proteinase R from Tritirachium album
  • proteinase T from Tritirachium album
  • subtilisin-like protease III from yeast
  • thermomycolin from Malbranchea sulfur ea.
  • cysteine proteases have a common catalytic mechanism that involves a cysteine amino acid residue in the active site of the protease.
  • Exemplary cysteine proteases that could be used in the methods herein include, but are not limited to, vertebrate lysosomal cathepsins B, H, L, and S; vertebrate lysosomal dipeptidyl peptidase I (also known as cathepsin C); vertebrate calpains; mammalian cathepsin K, which seems involved in osteoclastic bone resorption; human cathepsin O; bleomycin hydrolase; barley aleurain; EP-B 1/B4; kidney bean EP-Cl; rice bean SH-EP; kiwi fruit actinidin; papaya latex papain; chymopapain; caricain; proteinase IV; pea turgor-responsive protein 15 A; pineapple stem bromelain; rape COT44;
  • hydrolases useful for the methods herein Any method known in the art to screen enzymes for substrate specificity and enzymatic activity can be used to identify hydrolases useful for the methods provided herein ⁇ see, e.g., Gupta et al, (2003) Biotechnol. Appli. Biochem. 37:63-71, Yazbeck et al., (2003) Adv. Synth. Catal. 345:524-532, Kim et al., (2006) Prot. Exp. Purif. 45:315-323).
  • high throughput methods are employed to screen multiple enzymes for their ability to hydrolyze the ester bond of compound 2. Such methods are typically performed in, for example, 96-well assay plates, such that multiple hydrolases (e.g. , a hydrolase library) can be simultaneously screened for hydrolysis of a chosen substrate.
  • the hydrolases are obtained from commercial sources, such as those provided in Table 1.
  • the hydrolases are specifically engineered for this purpose, such as through directed evolution methods (Bornscheuer et al., (1999) Curr. Opin. Biotech. 7: 2169-2173). Table 1. Commercially available hydrolases.
  • the reactions are typically carried out in volumes of about 100 ⁇ l of a suitable buffer, such as 0.1 M potassium phosphate buffer, containing 1 mg/ml of substrate, 10 mg/ml of enzyme and 10% organic solvent.
  • a suitable buffer such as 0.1 M potassium phosphate buffer
  • the pH of the reaction is generally maintained at between 7.0 and 7.4.
  • water immiscible solvents such as methyl tert-butyl ether (MTBE), ethyl acetate, dichloro- methane, toluene or DIPE can be used for lipase screening, resulting in a biphasic system.
  • Proteases and esterases can be screened in water miscible solvents, such as acetonitrile, methanol, acetone or ethanol. Following incubation at an appropriate temperature, typically ranging from O 0 C to 6O 0 C, the samples are analyzed to assess hydrolysis.
  • a variety of methods can be used in the analysis of the sample, including, but not limited to, high performance liquid chromatography (HPLC), capillary electrophoresis (CE), gas chromatography (GC), UV spectrophotometry, thin layer chromatography (TLC) and liquid chromatography coupled with mass spectrometry (LC-MC). e.
  • hydrolases Conditions for use of hydrolases in the methods herein
  • One of skill in the art can determine suitable conditions for the enzymatic hydrolysis of compound 2 to compound 3, taking into account the type of hydrolase being employed in the reaction.
  • the reaction is carried out in an organic-aqueous biphasic system.
  • Suitable organic solvents that can be used for this purpose include, but are not limited to, hexane, cyclohexane, ethyl acetate, 1 - hexanol, chloroform, dichloroethane, dichloromethane, toluene, tert-pentyl alcohol, methyl isobutyl ketone (MIBK), methyl tert-butyl ether (MTBE) and di-isopropyl ether (DIPE).
  • the reaction is carried out in an aqueous buffer.
  • Suitable aqueous phases include, but are not limited to, buffers such as glutamic acid- glutamate, phosphoric acid-phosphate, acetic acid-acetate and citric acid-citrate buffers.
  • buffers such as glutamic acid- glutamate, phosphoric acid-phosphate, acetic acid-acetate and citric acid-citrate buffers.
  • 0.1 M potassium phosphate buffer can be used in the methods herein.
  • a suitable pH for the reaction can be determined by one of skill in the art, taking into account the activity and stability of the hydrolase. A pH range from 3 to 11 is contemplated for the methods herein.
  • a neutral environment is maintained, such that the pH of the reaction is at or about 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 or 7.7.
  • the temperature during the reaction can be between O 0 C to 6O 0 C. In some embodiments, an ambient temperature is maintained throughout the reaction. In other embodiments,
  • the amount of hydrolase used to hydrolyze compound 2 is determined by the activity of the enzyme.
  • An IU International Unit designates that amount of an enzyme preparation which catalyzes the formation of one micromole of product per minute. Such determinations can be made using methods well known in the art. For the methods herein, typically 10 to 10,000 IU of hydrolase is added to the reaction for every gram of substrate. The mixture is then typically agitated throughout the reaction. The reaction can be monitored by, for example, gas chromatography (GC), high performance liquid chromatography (HPLC) or thin layer chromatography (TLC) to determine the point of completion. The resulting compound can be isolated by extraction, evaporation, or other suitable separation methods. In one embodiment, the reaction is monitored by GC and the product is purified by extraction. The reaction mixture is saturated with sodium chloride and repeatedly extracted with ethyl acetate until complete recovery of the product. The organic layers are then dried over anhydrous sodium sulfate and filtered and concentrated. 2. Enzymatic reduction of carbon-carbon double bonds
  • pregabalin involves an enzymatic reduction of carbon-carbon double bonds such that compound 10 is produced from compound 9. Chirality also is introduced in this step to facilitate the final production of the (S)-enantiomeric form of pregabalin.
  • Enzymes that effect reduction of carbon-carbon bonds of a variety of substrates, including a broad range of ⁇ ,0-unsaturated aldehydes and ketones, have been described in the art (Eur. Pat. No 1236796, Wanner et al, Eur. J. Biochem. 255:271-278 (1998), Ensor et al, Biochem. J. 330:103-108 (1998), Mano et al., Eur. J. Biochem.
  • Enzymes that can effect the reduction of carbon-carbon double bonds have been identified in a variety of species, including, but not limited to, mammalian, plant, yeast and bacterial species. Exemplary of these are set forth in Table 2. Table 2. Enzymes that can effect the reduction of carbon-carbon double bonds
  • the enzyme used in the reduction of compound 9 to compound 10 can be of mammalian, including human, origin, or can be of non-mammalian origin, including but not limited to, plant, bacterial, viral, yeast and fungal origin.
  • the enzyme can be a wild-type protein or variant thereof.
  • an enzyme variant is used that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations, such as amino acid substitutions, deletions, additions, or combinations thereof.
  • enzyme variants can be generated by directed evolution such that the substrate specificity and enantioselectivity of the variants are optimized for the purposes herein.
  • substrate specificity of one or more enzymes can be determined spectrophotometrically by monitoring the rate of NADPH oxidation at 340 ran (Dick et al, J. Biol. Chem. 276:40803-40810 (2001)).
  • HPLC methods can be used (Dick et al, J. Biol. Chem. 276:40803-40810 (2001), and Youn et al, J. Biol. Chem.
  • the enantiomeric composition of the reduction products can be determined using one of a variety of chromatographic methods well known in art, including, but not limited to, HPLC, GC, TLC and LC-MS.
  • GC is performed to determine the enantiomeric selectivity of an enzyme using compound 9 as a substrate (Wanner et al, Eur. J. Biochem. 255:271-278 (1998)).
  • a reaction mixture containing the enzyme, compound 9 and NADPH in an appropriate buffer such as, 0.1 M potassium phosphate buffer, can be incubated for between 2 and 48 hours before being saturated with NaCl and extracted with ether.
  • reaction product can then be dried over sodium sulphate and concentrated, before being analyzed by chiral GC using an appropriate stationary phase, such as for example, heptakis-(3-O-acetyl-2,6-di-O-pentyl)-j8-cyclodextrin.
  • stationary phase such as for example, heptakis-(3-O-acetyl-2,6-di-O-pentyl)-j8-cyclodextrin.
  • Suitable conditions for the enzymatic reduction of carbon-carbon bonds are well known in the art, and can be employed in the methods provided herein (see e.g., Wanner et al, Eur. J. Biochem. 255:271-278 (1998), Ensor et al, Biochem. J. 330:103-108 (1998), Mano et al, Eur. J. Biochem. 267:3661-3671 (2000), Dick et al, J. Biol. Chem. 276:40803-40810 (2001), Dick et al, J. Biol. Chem. 279:17269-17277 (2004), and Youn et al., J. Biol. Chem. 281 :40076-40088 (2006)).
  • aqueous buffers include, but are not limited to, phosphate buffers, sodium acetate buffers, citric acid buffers, Tris buffers and MES (2-(N-morpholino)ethanesulfonic acid) buffers, hi some embodiments, additional reagents such as Triton X-100 or bovine serum albumin (BSA) also are included in the reaction.
  • a suitable pH for the reaction can be determined by one of skill in the art, taking into account the activity and stability of the enzyme. A pH range from 3 to 11 is contemplated for the methods herein.
  • a neutral environment is maintained, such that the pH of the reaction is at or about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 or 7.7.
  • the temperature during the reaction can be between O 0 C to 6O 0 C. hi some embodiments, an ambient temperature is maintained throughout the reaction.
  • NADPH or NADP cofactor also is included in the reaction.
  • Concentrations of NADP or NADPH used in the reaction can range from 0.0001 to 10 molar equivalents to the substrate or more.
  • NADPH or NADH also can be recycled using a cofactor regenerating system well known to the skill of the art.
  • the amount of purified enzyme used to catalyze the reduction of compound 9 is determined by the activity of the enzyme.
  • An IU International Unit designates that amount of an enzyme preparation which catalyzes the formation of one micromole of product per minute. Such determinations can be made using methods well known in the art.
  • GC gas chromatography
  • HPLC high performance liquid chromatography
  • TLC thin layer chromatography
  • the resulting compound 10 can be isolated by extraction, evaporation, or other suitable separation methods.
  • the reaction is monitored by GC and the product is purified by extraction.
  • the reaction mixture is saturated with sodium chloride and repeatedly extracted with ether until complete recovery of the product.
  • the organic layers are then dried over anhydrous sodium sulfate and filtered and concentrated.
  • the processes described herein for the synthesis of pregabalin includes an enzymatic amination of one or more substrates to introduce an amine group into the compound.
  • enzymatic amination can be used to convert compound 2 to compound 4 and/or compound 3 to compound 6.
  • enzymatic amination can be used to convert compound 8 to compound 6 and/or compound 10 to compound 6.
  • chirality also is introduced into the compounds to facilitate production of the (S) enantiomer of pregabalin.
  • transaminases also known as aminotransferases (Mehta et al, Eur. J. Biochem. 214:549-561 (1993)).
  • Transaminases catalyze the transfer of an amino group, a pair of electrons, and a proton from a primary amine to the carbonyl group of an acceptor molecule (e.g., compound 2, 3, 8, or 10) in a process called transamination. They can be divided into four subgroups (I, II, III and IV) based on their amino acid sequence similarity (Mehta et al, Eur. J. Biochem. 214:549-561 (1993)).
  • the ⁇ - transaminases also known as beta-alanine-pyruvate transaminase (EC 2.6.1.18 ), can utilize substrates where the amino group is not adjacent to a carboxylate group.
  • subgroup II transaminases are classified as subgroup II transaminases, and are, therefore, structurally related to other subgroup II transaminases, including, but not limited to, N-acetyl-L- ornithine transaminase, L-ornithine transaminase, 4-amino-butyrate transaminase, and 7,8-diaminopelargonate transaminase, ⁇ - Transaminases are pyridoxal phosphate- dependent enzymes which, upon transamination, can yield optically pure amines (Shin et al, Biosci. Biotechnol. Biochem. 65:1782-1788 (2001), Iwasaki et al, Biotech. Lett.
  • transaminases result in 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 94%, 95%, 96%, 98%, 99%, or more, ee with a particular set of amino donors and acceptors.
  • ⁇ - Transaminases have been isolated from various microorganisms, including, but not limited to, Alcaligenes denitrificans (SEQ ID NO: 12), Bordetella bronch- iseptica (SEQ ID NO:13), Bordetella parapertussis (SEQ ID NO:14), Brucella melitensis (SEQ ID NOS: 15 and 16), Burkholderia mallei (SEQ ID NO: 17), Burkholderia pseudomallei (SEQ ID NO: 18), Chromobacterium violaceum (SEQ ID NO: 19), Oceanicola granulosus HTCC2516 (SEQ ID NO:20), Oceanobacter sp.
  • Alcaligenes denitrificans SEQ ID NO: 12
  • Bordetella bronch- iseptica SEQ ID NO:13
  • Bordetella parapertussis SEQ ID NO:14
  • Brucella melitensis SEQ ID NOS: 15 and 16
  • RED65 SEQ ID NO:22
  • Oceanospirillum sp. MED92 SEQ ID NO:21
  • Pseudomonas putida SEQ ID NO:23
  • Ralstonia solanacearum SEQ ID NO:26
  • Rhizobium meliloti SEQ ID NO:25
  • Rhizobium sp. strain NGR234
  • Vibrio fluvialis Bacillus thuringienis, and Klebsiella pneuminiae (Shin et al., (2001) Biosci. Biotechnol. Biochem. 65:1782-1788).
  • transaminases including other subgroup II transaminases, for amino donor and amino acceptor (e.g., compounds 2, 3, 8, or 10) specificity and enantioselectivity to determine their suitability for use in the methods provided herein for the production of pregabalin.
  • the one or more transaminases used in the methods herein can be wild-type proteins or variants thereof, such as those described in U.S. Pat. No. 7,172,885.
  • an enzyme variant is used that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations, such as amino acid substitutions, deletions, additions, or combinations thereof.
  • enzyme variants can be generated by mutagenesis such that the substrate specificity and enantioselectivity of the variants are optimized for the purposes herein (Reetz et al, (2003) in Directed Evol. Prot., Ch 6, pp 245-279).
  • Identification of transaminases for the methods herein If necessary, any transaminase can be tested for use in the methods herein. Any method known in the art to screen transaminases for amino acceptor specificity and enantioselectivity can be used to identify enzymes useful for the transamination of compounds 2, 3, 8, and 10. Libraries of transaminases or modified forms thereof or other collections or individual transaminases can be screened. Screening can employ assays.
  • Such assay typically can include incubation of the transaminase with the amino acceptor compound of interest, pyridoxal phosphate (the cofactor), and an appropriate amino donor.
  • the products of the reaction are then analyzed using one of a variety of methods than can separate and/or detect enantiomers, including, but not limited to, high performance liquid chromatography (HPLC), capillary electrophoresis (CE), gas chromatography (GC), UV spectrophotometry, thin layer chromatography (TLC) and liquid chromatography coupled with mass spectrometry (LC-MC) (Yazbeck et al, Adv. Synth. Catal. 345:524-532 (2003), Reetz et al, Catal. Today 67:389-96 .,(2001),
  • Suitable conditions for the transamination of compounds are well known in the art, and can be employed in the methods provided herein ⁇ see e.g. Yun et al., (2004) Appl. Environ. Microbiol. 70:2529-2534, Shin et al, Biosci. Biotechnol. Biochem. 65:1782-1788 (2001), Hwang et al, Enzyme Microbiol. Technol. 34: 429-426 (2004)). If necessary such conditions can be identified and/or optimized further empirically.
  • the reactions can be carried out in an aqueous buffer or an organic-aqueous biphasic medium.
  • Suitable aqueous buffers include, but are not limited to, phosphate buffers, sodium acetate buffers, citric acid buffers and Tris buffers. In one embodiment, 0.1 M potassium phosphate buffer is used as an aqueous buffer.
  • the cofactor, pyridoxal phosphate, and an appropriate amino donor also are added to the reaction. Suitable concentrations of pyridoxal phosphate can range from 2 ⁇ M to 200 ⁇ M, but can be more or less.
  • Amino donors that can be used include, but are not limited to, L-alanine, L-aspartate or L-glutamate. In one embodiment, L-aspartate is included in the reaction as the amino donor.
  • additional reagents such as oxaloacetate decarboxylase also are added to the assay to remove the side product such as oxaloacetate from deamination of the amino donor L-aspartate, thus shifting the reaction equilibrium to completion.
  • a suitable pH for the reaction can be determined by one of skill in the art, taking into account the activity and stability of the enzyme. A pH range from 3 to 11 is contemplated for the methods herein. Typically, however, a neutral environment is maintained, such the pH of the reaction is at or about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 or 7.7.
  • the temperature during the reaction can be between O 0 C to 6O 0 C. In some embodiments, an ambient temperature is maintained throughout the reaction. In other embodiments, a temperature of approximately 37 0 C is maintained throughout the reaction.
  • the amount of purified enzyme used to transaminate the amino acceptor ⁇ i.e., compound 2, 3, 8, or 10) is determined by the activity of the enzyme.
  • An IU International Unit designates that amount of an enzyme preparation which catalyzes the formation of one micromole of product per minute. Such determinations can be made using methods well known in the art. For the methods herein, typically 10 to 10,000 IU of enzyme is added to the reaction for every gram of amino acceptor. The mixture is then typically agitated throughout the reaction. The reaction can be monitored by, for example, gas chromatography (GC), high performance liquid chromatography (HPLC) or thin layer chromatography (TLC) to determine the point of completion. The resulting compound can be isolated by extraction, evaporation, or other suitable separation methods, as described above.
  • GC gas chromatography
  • HPLC high performance liquid chromatography
  • TLC thin layer chromatography
  • n-propyl 4-methylpentanoate was prepared by refluxing 4-methylpentanoic acid (0.1 mol) in n-propyl alcohol (80 ml) containing concentrated sulfuric acid (8 ml) for 2 h. The reaction product was extracted into ethyl acetate, and the extract was evaporated to dryness under reduced pressure. The resulting residual oil was purified by distillation to give n-propyl 4- methylpentanoate.
  • This compound is prepared in a manner analogous to the one described in Kenda et al., J. Med. Chem 47: 530-549 (2004).
  • a solution of diisobutylamine and 4-methylpentanal in toluene is heated at reflux until formation of water has ceased.
  • the solution is cooled to room temperature and methyl bromoacetate is added.
  • the solution is stirred at room temperature for 18 hours and then at 90 0 C for 1 hour. Water is added to the solution at 90 0 C, and then the solution is cooled to room temperature.
  • the organic layer is washed with 1 N HCl and saturated aqueous sodium bicarbonate, dried over magnesium sulfate, filtered, and evaporated to afford the title compound.
  • the product can be purified by distillation or other methods known in the art to provide the title compound.
  • the amination of 3-formyl-5-methylhexanoate is catalyzed by a transaminase enzyme.
  • the reactions can be carried out in an aqueous buffer or an organic-aqueous biphasic medium.
  • Suitable aqueous buffers include, but are not limited to, phosphate buffers, sodium acetate buffers, citric acid buffers and Tris buffers.
  • 0.1 M potassium phosphate buffer is used as an aqueous buffer.
  • the cofactor, pyridoxal phosphate and an appropriate amino donor also are added to the reaction. Suitable concentrations of pyridoxal phosphate can range from 2 ⁇ M to 200 ⁇ M, but can be more or less.
  • Amino donors that can be used include, but are not limited to, L-alanine, L- valine or L-glutamate.
  • L-glutamate is included in the reaction as the amino donor.
  • additional reagents such as ⁇ -ketoglutate also are added to the assay to shift the reaction equilibrium to completion.
  • a suitable pH for the reaction can be determined by one of skill in the art, taking into account the activity and stability of the enzyme. A pH range from 3 to 11 is contemplated for the methods herein. Typically, however, a neutral environment is maintained, such the pH of the reaction is at or about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 or 7.7.
  • the temperature during the reaction can be between O 0 C to 6O 0 C. In some embodiments, an ambient temperature is maintained throughout the reaction. In other embodiments, a temperature of approximately 37 0 C is maintained throughout the reaction.
  • Pregabalin Compound 6, Scheme D (S)-4-isobutylpyrrolidin-2-one is treated with hydrochloric acid to produce pregabalin hydrochloride.
  • the pregabalin hydrochloride salt is broken with triethylamine. Filtration affords pregabalin.
  • the solvent is evaporated to dryness and the resulting residue is extracted with ethyl ether.
  • the organic layer is dried over anhydrous magnesium sulfate and the solvent is evaporated.
  • the crude product is purified by distillation under reduced pressure. Alternatively, the crude oil can be recrystallized from isopropyl ether, or an isopropyl ether/hexane solution, or acetone/chloroform solution.
  • 5-hvdroxy-4-isobutyldihvdrofuran-2(3H)-one (Compound 8, Scheme H) 5-Hydroxy-4-isobutylfuran-2(5H)-one is hydrogenated with a hydrogen source such as H 2 , HCO 2 NH 4 , and HCO 2 H, in the presence of a metal catalyst, such as Pt, Pd, Ni, Ru, Rh, or Ir) to provide 5-hydroxy-4-isobutyldihydrofuran-2(3H)-one.
  • a hydrogen source such as H 2 , HCO 2 NH 4 , and HCO 2 H
  • a metal catalyst such as Pt, Pd, Ni, Ru, Rh, or Ir
  • 5-Hydroxy-4-isobutyldihydrofuran-2(3H)-one was treated with an ammonia source and hydrogenated with a hydrogen source such as H 2 , HCO 2 NH 4 , and HCO 2 H, in the presence of a metal catalyst, e.g., Pt, Pd, Ni, Ru, Rh, or Ir) to provide pregabalin.
  • a metal catalyst e.g., Pt, Pd, Ni, Ru, Rh, or Ir
  • 5-hydroxy-4-isobutylfuran-2(5H)-one can be treated with base (e.g., aqueous sodium hydroxide) to afford (Z)-3-formyl-5-methylhex-2-enoic acid as the salt.
  • base e.g., aqueous sodium hydroxide
  • Compound 10 is made chiral (as shown) by an enzymatic transformation, not a chemical reduction.
  • the reduction of compound 9 to afford compound 10 is catalyzed by an enzyme that selectively reduces a carbon-carbon double bond.

Abstract

Provided herein are compounds and processes to prepare intermediates and precursors of pregabalin and related compositions, and processes for the preparation of pregabalin.

Description

TRANSAMINASE-BASED PROCESSES FOR PREPARATION OF
PREGABALIN
Related Applications Benefit of priority is claimed herein to U.S. Provisional Patent Application
Serial No. 60/922,994, filed April 1 1, 2007, to Wolfgang Reinhard Ludwig Note, Robert William Scott, Lishan Zhao and Junhua Tao, entitled "TRANSAMINASE- BASED PROCESSES FOR PREPARATION OF PREGABALIN ANDROGEN RECEPTOR MODULATOR COMPOUNDS AND METHODS." Where permitted, the disclosure of the above-referenced U.S. provisional application is incorporated herein by reference in its entirety.
Field
Provided herein are compounds and processes to prepare intermediates and precursors of pregabalin and related compositions, and processes for the preparation of pregabalin.
Background
(S)-Pregabalin, otherwise known as (3S)-3-(aminomethyl)-5-methylhexanoic acid, is a biologically active compound. This compound is an anticonvulsant drug, a drug for treatment of neuropathic pain, an adjunct therapy for partial seizures, and a drug for anxiety disorders (e.g., see U.S. Pat. Nos. 5,563,175, 6,001,876 and 6,127,418). (S)-Pregabalin activates L-glutamic acid decarboxylase (GAD) and promotes production of gamma-aminobutyric acid (GABA), a major inhibitory neurotrasmitter. (S)- Pregabalin binds to voltage-dependent calcium channels in the central nervous system. (S)-pregabalin (Lyrica® (Pfizer, Inc.)) is approved outside the U.S. for treating various peripheral neuropathic pain indications, such as diabetic and post herpetic neuropathic pain, and adjunctive therapy for partial epilepsy in adults. In the United States, (S)- pregabalin (Lyrica® (Pfizer, Inc.)) is approved for the management of neuropathic pain associated with diabetic peripheral neuropathy and postherpetic neuralgia, and for the adjunctive treatment of partial onset seizures in adults. There are several synthetic methods for preparing pregabalin (Hoekstra et al.,
Org. Proc. Res. Dev. 1 :26 (1997); Burk et al., J. Org. Chem. 68: 5731 (2003); Sammis and Jacobsen, JACS 125:4442-4443 (2003); PCT Pat. App. Publication WO2006/110783; U.S. Pat. No. 6,891,059; and U.S. Pat. Pub. 2005283023). For example, U.S. Patent No. 6,891,059 describes the asymmetric hydrogenation of a cyano-substituted olefin. This produces an optically enriched cyano compound, which is reduced to obtain (S)-pregabalin. U.S. Pat. Pub. 2005283023 describes an enzymatic resolution process with the possibility of recycling of the wrong enantiomer. U.S. Pat. Pub. Sammis and Jacobsen report a method that uses an aluminum salen catalyst to add hydrogen cyanide to an alpha-beta unsaturated imide (JACS, 125:4442-4443, 2003). An alternative method that proceeds through a substituted lactam is described in WO2006/110783. Each of these syntheses is either inefficient, expensive or poses difficulty for the large scale production of (S)- pregabalin.
Hence, a need exists for an efficient, cost effective, and safe method for the large-scale synthesis of (S)-pregabalin. Accordingly, among the objects herein, it is an object to provide alternative pathways for synthesis of (S)-pregabalin. Summary
Provided are processes for the production of (S)-pregabalin ((3S)-3- (aminomethyl)-5-methylhexanoic acid). Provided are methods to synthesize racemic and enantiomerically enriched intermediates toward pregabalin. In one embodiment, the method includes chemical or enzymatic amine transfer or amination of an aldehyde precursor.
Also provided are methods for synthesis of racemic and enantiomerically enriched intermediates in the synthesis of pregabalin. Also provided are methods for separation of such racemic intermediates into their respective enantiomers.
Provided herein are compounds of formula I:
Figure imgf000003_0001
where R is selected from among methyl, ethyl, n-butyl, z-butyl, pentyl, hexyl, heptyl, octyl, nonyl, C1-C9 heteroalkyl, cycloalkyl, aryl and heteroaryl. In some embodiments, R is C1-C3 alkyl. In some embodiments, R is n-butyl or isobutyl. In some embodiments, R is C3-C8 aryl. Also provided herein are methods for producing pregabalin. In one embodiment, the method includes hydrolyzing a compound of formula I:
Figure imgf000004_0001
in R is an alkyl, alkenyl or aryl, to afford an aldehyde intermediate of formula II
Figure imgf000004_0002
n M is hydrogen or a salt; and subjecting the aldehyde intermediate of formula II to chemical or enzymatic amine transfer or amination conditions to yield pregabalin:
Figure imgf000004_0003
In some embodiments, the compound of formula I is prepared by reacting 4- methylpentanal with diisobutylamine and an alkyl-3-halo-propanoate.
Also provided are methods for producing pregabalin, which include a) reacting diisobutylamine and 4-methylpentanal to produce an aldehyde intermediate of the structure:
Figure imgf000004_0004
b) hydrolyzing the reaction product of step a) to yield a compound of formula
II: ^
Figure imgf000004_0005
where M is hydrogen or a salt, which cyclizes to form a compound of the structure:
Figure imgf000004_0006
c) animating the reaction product of step b) to produce pregabalin.
In another embodiment, provided herein is a method for producing pregabalin that includes reacting 4-methylpentanal with diisobutylamine and an alkyl-3-halo- propanoate to produce an aldehyde intermediate of formula I:
Figure imgf000005_0001
aminating the aldehyde intermediate to produce a hexanoate intermediate of the structure:
Figure imgf000005_0002
cyclizing the hexanoate intermediate to produce a lactam of the structure:
Figure imgf000005_0003
treating the lactam with hydrochloric acid followed by addition of triethylamine to yield pregabalin.
In another embodiment, provided herein is a method for producing pregabalin that includes a) reacting 4-methylpentanal with glyoxylic acid hydrate to produce a compound of the following structure:
Figure imgf000005_0004
b) reducing asymmetrically the reaction product of step a) by chemical or enzymatic reduction to produce a compound of the following structure:
Figure imgf000005_0005
c) aminating the reaction product of step b) to produce pregabalin. In another embodiment, provided herein is a method for producing pregabalin that includes a) treating 5-hydroxy-4-isobutylfuran-2(5//)-one:
Figure imgf000005_0006
with base to yield a compound of the structure:
Figure imgf000005_0007
j where M is hydrogen or a salt; b) enzymatically or chemically reducing the product of step a) to produce a compound of the structure:
Figure imgf000006_0001
,where M is hydrogen or a salt; and c) chemically or enzymatically amidating the product of step c) followed by reduction to provide pregabalin.
In some embodiments, the compound of formula I is a racemic mixture. In some embodiments, the compound of formula I is the S-enantiomer. In some embodiments, the compound of formula II is a racemic mixture. In some embodiments, the compound of formula II is the S-enantiomer. In one embodiment, the amination step is performed chemically under conditions wherein the desired S-stereochemistry is obtained, hi one embodiment, chemical amination includes treating with an ammonia source. In other embodiments, the amination step is performed enzymatically under conditions wherein the desired S-stereochemistry is obtained. In some embodiments, the enzyme is a transaminase. In some embodiments, the transaminase is selected from among an Alcaligenes denitrificans transaminase, a Bordetella bronch-iseptica transaminase, a Bordetella parapertussis transaminase, a Brucella melitensis transaminase, a Burkholderia mallei transaminase, a Burkholderia pseudomallei transaminase, a Chromobacterium violaceum transaminase, an Oceanicola granulosus HTCC2516 transaminase, an Oceanobacter sp. RED65 transaminase, an Oceanospirillum sp. MED92 transaminase, a Pseudomonas putida transaminase, a Ralstonia solanacearum transaminase, a Rhizobium meliloti transaminase, a Rhizobium sp. transaminase, a Vibrio fluvialis transaminase, a Bacillus thuringiensis transaminase and a Klebsiella pneuminiae transaminase. hi some embodiments, the hydrolysis step is performed enzymatically. For enzymatic processing of the intermediates, a hydrolase is used. In one embodiment, the hydrolase is selected from among an esterase, a lipase and a protease. In some embodiments, the hydrolase is a bacterial hydrolase, a fungal hydrolase, an orange peel hydrolase, a porcine liver hydrolase, a rabbit liver hydrolase, a bovine pancreas hydrolase, a porcine pancreas hydrolase, a porcine stomach mucosal hydrolase, a bovine intestine hydrolase, a porcine intestine hydrolase, a porcine kidney hydrolase, a calf stomach hydrolase, a wheat germ hydrolase, a pineapple stem hydrolase and a papaya hydrolase. In some embodiments, the hydrolase is selected from among an Achromobacter sp. hydrolase, an Alcaligenes sp. hydrolase, and Aspergillus sp. hydrolase, an Apergillus niger hydrolase, an Apergillus oryzae hydrolase, an Aspergillus melleus hydrolase, an Aspergillus mellus hydrolase, an Aspergillus niger hydrolase, an Aspergillus oryzae hydrolase, an Aspergillus saitoi hydrolase, an Aspergillus sojae hydrolase, a Bacillus sp. hydrolase, a Bacillus amyloliquefaciens hydrolase, a Bacillus lentus hydrolase, a Bacillus licheniformis hydrolase, a Bacillus polymyxa hydrolase, a Bacillus stearothermophilus hydrolase, a Bacillus subtilis hydrolase, a Bacillus thermoglucosidasius hydrolase, a Bacillus thermoproteolyticus rokko hydrolase, a Burkholderia sp. hydrolase, a Burkholderia cepacia hydrolase, a Candida sp. hydrolase, a Candida antarctica hydrolase, a Candida antarctica A hydrolase, a Candida antarctica B hydrolase, a Candida cylindracea hydrolase, a Candida lipolytica hydrolase, a Candida rugosa hydrolase, a Candidia utilis hydrolase, a Carica papaya hydrolase, a Chromobacterium viscosum hydrolase, a Clostridium histolyticum hydrolase, an E. coli hydrolase, a Geotrichum candidum hydrolase, a Mucor javanicus hydrolase, a Mucor miehei hydrolase, a Penicillium sp. hydrolase, a Penicillium sp. I hydrolase, a Penicillium sp. II hydrolase, a Penicillium camembertii hydrolase, a Penicillium roqueforti hydrolase, a
Pseudomonas sp. hydrolase, a Pseudomonas aeruginosa hydrolase, a Pseudomonas cepacia hydrolase, a Pseudomonas fluorescens hydrolase, a Pseudomonas stutzeri hydrolase, a Pyrococcus furiosis hydrolase, a Rhizomucor miehei hydrolase, a Rhizopus sp. hydrolase, a Rhizopus arrhizus hydrolase, a Rhizopus delemar hydrolase, a Rhizopus niveus hydrolase, a Rhizopus oryzae hydrolase, a Saccharomyces cerevisiae hydrolase, a Schizophyllum commune hydrolase, a Streptomyces sp. hydrolase, a Streptomyces diastatochromogenes hydrolase, a Streptomyces griseus hydrolase, a Thermoanaerobium brockii hydrolase, a Thermomyces lanuginosus hydrolase and a Tritirachium album hydrolase. In some embodiments, the reducing step is performed enzymatically. In some embodiments, the enzyme used for the reducing step is selected from among an alkenal double bond reductase (Pl), an alkenal double bond reductase (P2), an NAD(P)H-dependent alkenal/one oxidoreductase (AOR), an NADP-dependent leukotriene B4 12-hydroxydehydrogenase, an aryl propenal double bond reductase, an NADP-dependent oxidoreductase and a medium-chain dehyrogenase/reductase.
The enzymes are free in solution or are immobilized on a solid support. Also provided are articles of manufacture that include a packaging material, a compound provided herein or an acceptable salt, ester or other pharmaceutically acceptable derivative thereof, within the packaging material, and a label that indicates that the compound is used for the synthesis of pregabalin.
Also provided are combinations and kits that include an enzyme and a compound provided herein and acceptable salts, esters and other pharmaceutically acceptable derivative. In one embodiment, the enzyme is a hydrolase. In one embodiment, the kit includes one or more hydrolases selected from among an esterase, a lipase, and a protease and a combination thereof. In another embodiment, the enzyme is a transaminase. Detailed Description A. Definitions
Unless specific definitions are provided, the nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, biochemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those known in the art. All patents, patent applications, and published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety for any purpose. In the event that there are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.
It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the subject matter claimed. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation and delivery, and treatment of subjects. Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Reactions and purification techniques can be performed e.g., using kits according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures generally are performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
As used herein, the abbreviations for any protective groups, amino acids and other compounds are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (1972) Biochem., 11 : 942-944.
As used herein, "or" means "and/or" unless stated otherwise. Furthermore, use of the term "including" as well as other forms, such as "includes," and "included," is not limiting.
As used herein, use of the singular includes the plural unless specifically stated otherwise.
As used herein, the terms "treating" or "treatment" encompass either or both responsive and prophylaxis measures, e.g., designed to inhibit, slow or delay the onset of a symptom of a disease or disorder, achieve a full or partial reduction of a symptom or disease state, and/or to alleviate, ameliorate, lessen, or cure a disease or disorder and/or its symptoms.
As used herein, amelioration of the symptoms of a particular disorder by administration of a particular compound or pharmaceutical composition refers to any lessening of severity, delay in onset, slowing of progression, or shortening of duration, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the compound or composition.
As used herein, C1-Cx includes C1-C2, Cj-C3 . . . Ci-Cx. The term "alkyl" refers to straight or branched chain substituted or unsubstituted hydrocarbon groups, in one embodiment 1 to 40 carbon atoms, in another embodiment, 1 to 20 carbon atoms, in another embodiment, 1 to 10 carbon atoms. The expression "lower alkyl" refers to an alkyl group of 1 to 6 carbon atoms. An alkyl group can be a "saturated alkyl," which means that it does not contain any alkene or alkyne groups and in certain embodiments, alkyl groups are optionally substituted. An alkyl group can be an "unsaturated alkyl," which means that it contains at least one alkene or alkyne group. An alkyl group that includes at least one carbon-carbon double bond (C=C) also is referred to by the term "alkenyl," and in certain embodiments, alkenyl groups are optionally substituted. An alkyl group that includes at least one carbon-carbon triple bond (C ≡€) also is referred to by the term "alkynyl," and in certain embodiments, alkynyl groups are optionally substituted.
In certain embodiments, an alkyl contains 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as "1 to 20" refers to each integer in the given range; e.g., "1 to 20 carbon atoms" means that an alkyl group can contain only 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. , up to and including 20 carbon atoms, although the term "alkyl" also includes instances where no numerical range of carbon atoms is designated). An alkyl can be designated as "Ci-C4 alkyl" or by similar designations. By way of example only, "Ci-C4 alkyl" indicates an alkyl having one, two, three, or four carbon atoms, i.e., the alkyl is selected from among methyl, ethyl, propyl, iso-propyl, «-butyl, iso-butyl, sec-butyl and t-butyl. Thus "Ci - C 4" includes Ci - C2 , Ci - C 3, C2 - C 3 and C2 - C 4 alkyl. Alkyls include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, hexenyl, ethynyl, propynyl, butynyl and hexynyl. As used herein, "halogen" or "halide" refers to F, Cl, Br or I.
As used herein, the term "haloalkyl" alone or in combination refers to an alkyl in which at least one hydrogen atom is replaced with a halogen atom. In certain of the embodiments in which two or more hydrogen atom are replaced with halogen atoms, the halogen atoms are all the same as one another. In certain of such embodiments, the halogen atoms are not all the same as one another. Certain haloalkyls are saturated haloalkyls, which do not include any carbon-carbon double bonds or any carbon- carbon triple bonds. Certain haloalkyls are haloalkenes, which include one or more carbon-carbon double bonds. Certain haloalkyls are haloalkynes, which include one or more carbon-carbon triple bonds. In certain embodiments, haloalkyls are optionally substituted.
As used herein, pseudohalides are compounds that behave substantially similar to halides. Such compounds can be used in the same manner and treated in the same manner as halides (X-, in which X is a halogen, such as Cl, F or Br). Pseudohalides include, but are not limited to, cyanide, cyanate, thiocyanate, selenocyanate, trifluoromethoxy, trifluoromethyl and azide.
Where the number of any given substituent is not specified (e.g., "haloalkyl"), there may be one or more substituents present. For example, "haloalkyl" may include one or more of the same or different halogens. For example, "haloalkyl" includes each of the substituents CF3, CHF2 and CH2F.
As used herein, the term "heteroatom" refers to an atom other than carbon or hydrogen. Heteroatoms are typically independently selected from oxygen, sulfur, nitrogen, and phosphorus, but are not limited to those atoms. In embodiments in which two or more heteroatoms are present, the two or more heteroatoms can all be the same as one another, or some or all of the two or more heteroatoms can each be different from the others.
As used herein, the term "heteroalkyl" alone or in combination refers to a group containing an alkyl and one or more heteroatoms. Certain heteroalkyls are saturated heteroalkyls, which do not contain any carbon-carbon double bonds or any carbon-carbon triple bonds. Certain heteroalkyls are heteroalkenes, which include at least one carbon-carbon double bond. Certain heteroalkyls are heteroalkynes, which include at least one carbon-carbon triple bond. Certain heteroalkyls are acylalkyls, in which the one or more heteroatoms are within an alkyl chain. Examples of heteroalkyls include, but are not limited to, CH3CC=O)CH2-, CH3CC=O)CH2CH2-, CH3CH2CC=O)CH2CH2-, CH3CC=O)CH2CH2CH2-, CH3OCH2CH2-, CH3OCC=O)CH2- and CH3NHCH2-. hi certain embodiments, heteroalkyls are optionally substituted.
As used herein, the term "heterohaloalkyl" alone or in combination refers to a heteroalkyl in which at least one hydrogen atom is replaced with a halogen atom. In certain embodiments, heteroalkyls are optionally substituted. As used herein, the term "non-cyclic alkyl" refers to an alkyl that is not cyclic (i.e., a straight or branched chain containing at least one carbon atom). Non-cyclic alkyls can be fully saturated or can contain non-cyclic alkenes and/or alkynes. Non- cyclic alkyls can be optionally substituted. As used herein, the term "ring" refers to any covalently closed structure.
Rings include, for example, carbocycles (e.g., aryls and cycloalkyls), heterocycles (e.g., heteroaryls and non-aromatic heterocycles), aromatics (e.g., aryls and heteroaryls), and non-aromatics (e.g., cycloalkyls and non-aromatic heterocycles). Rings can be optionally substituted. Rings can form part of a ring system. As used herein, the term "ring system" refers to two or more rings, wherein two or more of the rings are fused. The term "fused" refers to structures in which two or more rings share one or more bonds.
As used herein, the term "carbocycle" refers to a ring where each of the atoms forming the ring is a carbon atom. Carbocyclic rings can be formed by three, four, five, six, seven, eight, nine, or more than nine carbon atoms. Carbocycles can be optionally substituted.
As used herein, "cycloalkyl" refers to a saturated mono- or multicyclic ring system where each of the atoms forming a ring is a carbon atom. Cycloalkyls can be formed by three, four, five, six, seven, eight, nine, or more than nine carbon atoms. In one embodiment, the ring system includes 3 to 12 carbon atoms. In another embodiment, they ring system includes 3 to 6 carbon atoms. The term "cycloalkyl" includes rings that contain one or more unsaturated bonds. As used herein, the terms "cycloalkenyl" and "cycloalkynyl" are unsaturated cycloalkyl ring system. Cycloalkyls can be optionally substituted. In certain embodiments, a cycloalkyl contains one or more unsaturated bonds. Examples of cycloalkyls include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclopentadiene, cyclohexane, cyclohexene, 1,3-cyclohexadiene, 1 ,4-cyclohexadiene, cycloheptane and cycloheptene.
As used herein, the term "cycloalkenyl" refers to mono- or multicyclic ring systems that includes at least one carbon-carbon double bond (C=C).
As used herein, the term "cycloalkynyl" refers to mono- or multicyclic ring systems that include at least one carbon-carbon triple bond (C ≡C). Cycloalkenyl and cycloalkynyl groups include ring systems that include 3 to 12 carbon atoms. In some embodiments, the cycloalkenyl groups include 4 to 7 carbon atoms. In some embodiment, the cycloalkynyl groups include 8 to 10 carbon atoms. The ring systems of the cycloalkyl, cycloalkenyl and cycloalkynyl groups may be composed of one ring or two or more rings which may be joined together in a fused, bridged or spiro-connected fashion, and may be optionally substituted with one or more alkyl group substituents.
As used herein, the term "heterocycle" refers to a ring wherein at least one atom forming the ring is a carbon atom and at least one atom forming the ring is a heteroatom. Heterocyclic rings may be formed by three, four, five, six, seven, eight, nine, or more than nine atoms. Any number of those atoms can be heteroatoms (i.e., a heterocyclic ring can contain one, two, three, four, five, six, seven, eight, nine, or more than nine heteroatoms, provided that at lease one atom in the ring is a carbon atom). Herein, whenever the number of carbon atoms in a heterocycle is indicated (e.g., C i-C6 heterocycle), at least one other atom (the heteroatom) must be present in the ring.
Designations such as "Ci-C6 heterocycle" refer only to the number of carbon atoms in the ring and do not refer to the total number of atoms in the ring. It is understood that the heterocyclic ring will have additional heteroatoms in the ring. Designations such as "4-6 membered heterocycle" refer to the total number of atoms that comprise the ring (i.e., a four, five, or six membered ring, in which at least one atom is a carbon atom, at least one atom is a heteroatom and the remaining two to four atoms are either carbon atoms or heteroatoms). In heterocycles containing two or more heteroatoms, those two or more heteroatoms can be the same or different from one another. In one embodiment, the heterocycle includes 3-12 members. In other embodiments, the heterocycle includes 4, 5, 6, 7 or 8 members. The heterocycle may be optionally substituted with one or more substituents. In some embodiments, the substituents of the heterocyclic group are selected from among hydroxy, amino, alkoxy containing 1 to 4 carbon atoms, halo lower alkyl, including trihalomethyl, such as trifluoromethyl, and halogen. As used herein, the term heterocycle may include reference to heteroaryl. Binding to a heterocycle can be at a heteroatom or via a carbon atom. Examples of heterocycles include, but are not limited to the following:
Figure imgf000014_0001
where D, E, F and G independently represent a heteroatom. Each of D, E, F and G can be the same or different from one another. As used herein, the term "bicyclic ring" refers to two rings, wherein the two rings are fused. Bicyclic rings include, for example, decaline, pentalene, indene, naphthalene, azulene, heptalene, isobenzofuran, chromene, indolizine, isoindole, indole, indoline, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyrididine, quinoxaline, cinnoline, pteridine, isochroman, chroman and various hydrogenated derivatives thereof. Bicyclic rings can be optionally substituted. Each ring is independently aromatic or non-aromatic. In certain embodiments, both rings are aromatic. In certain embodiments, both rings are non-aromatic. In certain embodiments, one ring is aromatic and one ring is non-aromatic.
As used herein, the term "aromatic" refers to a planar ring having a delocalized π-electron system containing 4n+2 π electrons, where n is an integer. Aromatic rings can be formed by five, six, seven, eight, nine, or more than nine atoms. Aromatics can be optionally substituted. Examples of aromatic groups include, but are not limited to phenyl, naphthalenyl, phenanthrenyl, anthracenyl, tetralinyl, fluorenyl, indenyl and indanyl. The term aromatic includes, for example, benzenoid groups, connected via one of the ring- forming carbon atoms, and optionally carrying one or more substituents selected from an aryl, a heteroaryl, a cycloalkyl, a non-aromatic heterocycle, a halo, a hydroxy, an amino, a cyano, a nitro, an alkylamido, an acyl, a C)-6 alkoxy, a Ci-6 alkyl, a C1-6 hydroxyalkyl, a Ci-6 aminoalkyl, a Ci-6 alkylamino, an alkylsulfenyl, an alkylsulfinyl, an alkylsulfonyl, an sulfamoyl, or a trifluoromethyl. In certain embodiments, an aromatic group is substituted at one or more of the para, meta, and/or ortho positions. Examples of aromatic groups containing substitutions include, but are not limited to, phenyl, 3-halophenyl, 4-halophenyl, 3-hydroxyphenyl, 4-hydroxy- phenyl, 3-aminophenyl, 4-aminophenyl, 3-methylphenyl, 4-methylphenyl, 3- methoxyphenyl, 4-methoxyphenyl, 4-trifluoromethoxyphenyl, 3-cyano-phenyl, 4- cyanophenyl, dimethylphenyl, naphthyl, hydroxynaphthyl, hydroxymethyl-phenyl, (trifluoromethyl)phenyl, alkoxyphenyl, 4-moφholin-4-ylphenyl, 4-pyrrolidin-l- ylphenyl, 4-pyrazolylphenyl, 4-triazolylphenyl and 4-(2-oxopyrrolidin-l-yl)phenyl.
As used herein, the term "aryl" refers to an aromatic ring wherein each of the atoms forming the ring is a carbon atom. Aryl rings can be formed by three, four, five, six, seven, eight, nine, or more than nine carbon atoms. Aryl groups can be optionally substituted.
As used herein, the term "heteroaryl" refers to an aromatic ring in which at least one atom forming the aromatic ring is a heteroatom. Heteroaryl rings can be formed by three, four, five, six, seven, eight, nine and more than nine atoms. Heteroaryl groups can be optionally substituted. Examples of heteroaryl groups include, but are not limited to, aromatic C3-8 heterocyclic groups containing one oxygen or sulfur atom, or two oxygen atoms, or two sulfur atoms or up to four nitrogen atoms, or a combination of one oxygen or sulfur atom and up to two nitrogen atoms, and their substituted as well as benzo- and pyrido-fused derivatives, for example, connected via one of the ring-forming carbon atoms. In certain embodiments, heteroaryl groups are optionally substituted. In one embodiment, the one or more substituents are each independently selected from among halo, hydroxy, amino, cyano, nitro, alkylamido, acyl, Ci-6-alkoxy, Ci-6-alkyl, Ci-6-haloalkyl, Ci-6-hydroxyalkyl, Ci-6-aminoalkyl, Ci-6-alkylamino, alkylsulfenyl, alkylsulfinyl, alkylsulfonyl, sulfamoyl, or trifluoromethyl. Examples of heteroaryl groups include, but are not limited to, unsubstituted and mono- or di- substituted derivatives of furan, benzo furan, thiophene, benzothiophene, pyrrole, pyridine, indole, oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole, isothiazole, imidazole, benzimidazole, pyrazole, indazole, tetrazole, quinoline, isoquinoline, pyridazine, pyrimidine, purine and pyrazine, furazan, 1,2,3- oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, triazole, benzotriazole, pteridine, phenoxazole, oxadiazole, benzopyrazole, quinolizine, cinnoline, phthalazine, quinazoline and quinoxaline. In some embodiments, the substituents are halo, hydroxy, cyano, O-Ci-6-alkyl, Ci-6-alkyl, hydroxy-Ci-6-alkyl and amino-Ci-6-alkyl.
As used herein, the term "non-aromatic ring" refers to a ring that does not have a delocalized 4n+2 π-electron system.
As used herein, the term "non-aromatic heterocycle" refers to a non-aromatic ring wherein one or more than one atom forming the ring is a heteroatom. Non- aromatic heterocyclic rings can be formed by three, four, five, six, seven, eight, nine, or more than nine atoms. Non-aromatic heterocycles can be optionally substituted. In certain embodiments, non-aromatic heterocycles contain one or more carbonyl or thiocarbonyl groups such as, for example, oxo- and thio-containing groups. Examples of non-aromatic heterocycles include, but are not limited to, lactams, lactones, cyclic imides, cyclic thioimides, cyclic carbamates, tetrahydrothiopyran, 4H-pyran, tetrahydropyran, piperidine, 1,3-dioxin, 1,3-dioxane, 1,4-dioxin, 1,4-dioxane, piperazine, 1,3-oxathiane, 1 ,4-oxathiin, 1 ,4-oxathiane, tetrahydro-l,4-thiazine, 2H- 1 ,2-oxazine , maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil, morpholine, trioxane, hexahydro-1 ,3,5- triazine, tetrahydrothiophene, tetrahydrofuran, pyrroline, pyrrolidine, pyrrolidone, pyrrolidione, pyrazoline, pyrazolidine, imidazoline, imidazolidine, 1,3-dioxole, 1,3- dioxolane, 1,3-dithiole, 1,3-dithiolane, isoxazoline, isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazoline, thiazolidine and 1,3-oxathiolane. As used herein, the term "arylalkyl" alone or in combination, refers to an alkyl substituted with an aryl that can be optionally substituted.
As used herein, the term "heteroarylalkyl" alone or in combination, refers to an alkyl substituted with a heteroaryl that may be optionally substituted.
As used herein, the term "O-carboxy" refers to a group of formula RC(=O)O-. As used herein, the term "C-carboxy" refers to a group of formula -C(=O)OR.
As used herein, the term "acetyl" refers to a group of formula -CC=O)CH3. As used herein, the term "cyano" refers to a group of formula -CN. As used herein, the term "isocyanato" refers to a group of formula -NCO.
As used herein, the term "thiocyanato" refers to a group of formula -CNS.
As used herein, the term "isothiocyanato" refers to a group of formula -NCS.
As used herein, the term "C-amido" refers to a group of formula -C(=O)-NR2. As used herein, the term "N-amido" refers to a group of formula RC(=O)NH-.
As used herein, the term "ester" refers to a chemical moiety with formula -(R)n-COOR', where R and R' are independently selected from alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and non-aromatic heterocycle (bonded through a ring carbon), where n is O or 1. As used herein, the term "amide" refers to a chemical moiety with formula
-(R)n-C(O)NHR' or -(R)n-NHC(O)R', where R and R' are independently selected from alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon), where n is O or 1. In certain embodiments, an amide can be an amino acid or a peptide. As used herein, the terms "amine," "hydroxy," and "carboxyl" include such groups that have been esterified or amidified. Procedures and specific groups used to achieve esterification and amidification are known to those of skill in the art and can readily be found in reference sources such as Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, which is incorporated herein in its entirety.
As used herein, the term "together form a bond" refers to the instance in which two substituents to neighboring atoms are null the bond between the neighboring atoms becomes a double bond. For example, if A and B below "together form a bond"
Figure imgf000017_0001
^ the resulting structure is: Unless otherwise indicated, the term "optionally substituted," refers to a group in which none, one, or more than one of the hydrogen atoms has been replaced with one or more group(s) individually and independently selected from among alkyl, cycloalkyl, heteroalkyl, aryl, heteroaryl, non-aromatic heterocycle, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N carbamyl, O thiocarbamyl, N thiocarbamyl, C amido, N amido, S-sulfonamido, N sulfonamido, C carboxy, O carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, and amino, including mono- and di- substituted amino groups, and the protected derivatives of amino groups. Such protective derivatives (and protecting groups that can form such protective derivatives) are known to those of skill in the art and can be found in references such as Greene and Wuts, above. In embodiments in which two or more hydrogen atoms have been substituted, the substituent groups can together form a ring.
Throughout the specification, groups and substituents thereof can be chosen by one skilled in the field to provide stable moieties and compounds. It is to be understood that the compounds provided herein may contain chiral centers. Such chiral centers may be of either the (R) or (S) configuration, or may be a mixture thereof. Thus, the compounds provided herein may be enantiomerically pure, or be stereoisomeric or diastereomeric mixtures. It is to be understood that the chiral centers of the compounds provided herein may undergo epimerization in vivo. As such, one of skill in the art will recognize that administration of a compound in its (R) form is equivalent, for compounds that undergo epimerization in vivo, to administration of the compound in its (S) form.
As used herein, "cis" and "trans" are descriptors which show the relationship between two ligands attached to separate atoms which are connected by a double bond or contained in a ring with a double bond. Two ligands are said to be "cis" to each other if they lie on the same side of a plane. If the ligands are on opposite sides, their relative position is described as trans. The appropriate reference plane of a double bond is perpendicular to that of the relevant sigma bond which passes through the double bond. As used herein, "enantiomer" refers to one of a pair of molecular entities which are mirror images of each other and non-superimposable. Enantiomeric excess (ee) may be calculated for a mixture of (R) and (S)-enantiomers. The ee is defined as the absolute value of the mole fractions of F(R) minus the mole fraction of F(s>. The percent ee is the absolute value of the mole fractions of F(R) minus the mole fraction of F(s> multiplied by 100.
As used herein, "optical activity" refers to the ability of a sample material to rotate the plane of polarized light. A specific enantiomer causes rotation of light in either a clockwise or counterclockwise direction. As used herein, "optical purity" refers to the ratio of observed optical rotation of a sample consisting of a mixture of enantiomers to the optical rotation of one pure enantiomer.
As used herein, the term "substantially pure" means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis, high performance liquid chromatography (HPLC) and mass spectrometry (MS), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Thus, substantially pure object species (e.g., compound) is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition). In certain embodiments, a substantially purified fraction is a composition wherein the object species contains at least about 50 percent (on a molar basis) of all species present. In certain embodiments, a substantially pure composition will contain more than about 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% of all species present in the composition, hi certain embodiments, a substantially pure composition will contain more than about 80%, 85%, 90%, 95%, or 99% of all species present in the composition. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound can, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound. The instant disclosure is meant to include all such possible isomers, as well as, their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as reverse phase HPLC. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms also are intended to be included. As used herein, "acid" generally refers to a molecular entity or chemical species capable of donating a hydron (proton), otherwise considered a Brønsted acid, or capable of accepting an electron pair, otherwise known as a Lewis acid.
As used herein, "base" refers to a chemical species or molecular entity having an available pair of electrons capable of forming a bond with a hydron (proton), otherwise known as a Brønsted base, or capable of donating an electron pair to form a bond with the vacant orbital of some other species, otherwise known as a Lewis base.
As used herein, "activation" of a chemical group occurs when some or all of the energy required for a desired transformation is provided by a preceding reaction. For example, in the scheme: A + B → X → Y + Z, some or all of the energy required for the reaction of X to form products Y and Z is provided by the first reaction between A and B.
As used herein, "chemical resolution" occurs when a mixture of stereoisomers is separated into the component diasteriomers and/or enantiomers. Chemical resolution also may be applied to the separation of olefin cis- and trans- isomers.
As used herein, an "aliquot" is a fractional part of known quantity taken from a larger solution or mixture. The properties of the aliquot are usually analyzed and such properties are considered to be representative of the properties of the larger solution or mixture. As used herein, "condensation" reaction occurs when two or more reactants, yield a single main product with accompanying formation of water or of some other small molecule such as ammonia, ethanol, acetic acid, or hydrogen sulfide. A condensation reaction also may occur between two or more reactive sites within the same molecular entity. As used herein, "transesterification" refers to the reaction which converts one ester into another. Transesterifications are often realized by reacting an ester with an excess of alcohol under acidic or basic conditions.
As used herein, "transformation" refers to the conversion of a substrate into a particular product irrespective of the reagents or mechanisms involved. Reference to a transformation does not require full description of all reactants or all products necessary to convert the substrate into product. As used herein, "cyclization" refers to the formation of a covalently closed ring by formation of a new bond.
As used herein, "reduce" and "reduction" refers to the transfer of one or more electrons to a molecular entity. For example, a compound can be reduced by the addition of hydrogen. A reduced species also can be formed through the gain of electrons. The reverse process in which one or more electrons is removed from a molecular entity is known as "oxidation."
As used herein, "salting" refers to the addition of electrolytes to a solution. Salting is often alters the distribution ratio of a particular solute or changes the miscibility of two liquids.
As used herein, "Baumann conditions" refers to acylation of alcohols with acyl halides in aqueous alkaline solution.
As used herein, a "derivative" is a compound obtained or produced by modification of another compound of similar structure. Derivatives may be produced by one or more modification steps.
As used herein, "quenched" refers to arresting the course of a chemical or enzymatic reaction by chemical or physical means.
To remove potentially complicating reactive functionality of chemical moieties, the usage of "protecting groups" is often employed. That is, a functional group is temporarily converted into an unreactive form to prevent its interference with transformations to be carried out elsewhere in the molecule. Such temporary functional group modification is known as "protecting" the original group. Subsequent to transformation carried out elsewhere in the molecule, the original unit may be regenerated, i.e., "deprotected," under separate conditions. For example, an alcohol may be protected as a 1,1-dimethylethyl ether by an acid catalyzed reaction of the alcohol with 2-methyl-2-propanol. The resulting ether is inert to some basic, oxidizing, or reducing conditions. The alcohol may be deprotected by removal of the ether group in dilute aqueous acid.
As used herein, "desymmetrization" involves the modification of a compound which results in the loss of one or more symmetry elements. Desymmetrization includes the loss of a symmetry element which precludes chirality, such as a mirror plane, center of inversion, or rotational-reflection axis. Desymmetrization may result in the conversion of a prochrial molecular entity into a chiral entity. The term, "prochiral" refers to a structure which lacks chirality, but which is capable of becoming chiral by addition, removal, or replacement of a substituent.
As used herein, "hydrolysis" refers to the general rupture of one or more bonds by water molecules.
As used herein, "Michael addition" refers to the base catalyzed addition methylene compounds to unsaturated systems.
As used herein, "EC" refers to the Enzyme Commission of the International Union of Biochemistry and Molecular Biology (IUBMB). As used herein, EC numbers, such as EC 3.4.21.62, are associated with a recommended name for the respective enzyme. The first number designates the major class, the second number designates the subclass, and the third number designates the sub-subclass. The fourth number indicates the serial number of the enzyme in its sub-subclass.
As used herein, an "esterase" is an enzyme which catalyzes the cleavage of ester bonds. Many esterases show specificity for particular types of esters.
As used herein, a "protease" is an enzyme which catalyzes the hydrolysis of peptide bonds. Many proteases also are capable of cleaving ester bonds. Proteases often show specificity for particular bond arrangements.
As used herein, "hydrolase" refers to enzymes that catalyze the cleavage of C- O, C-N, C-C and other bonds by reactions involving the addition or removal of water.
As used herein, "product" refers to a substance that is formed during a chemical or enzymatic reaction.
As used herein, "reaction medium" refers to the phase in which a chemical or biological reaction or other such transformation takes place. The reaction medium may include solid, liquid, and gaseous phases and mixtures thereof. Chemical and biological reactants and reagents are commonly dissolved or suspended in various liquid compositions to facilitate a reaction or transformation.
As used herein, a "catalyst" is a substance that increases the rate of a reaction. A catalytic substance is a substance which increases the rate of a reaction. As used herein, "biocatalyst" refers to a living organism, enzyme, and/or enzyme complex which catalyses a reaction or otherwise facilitates substrate conversion in various chemical reactions. As used herein, a "buffer solution" is any substance or mixture of compounds in solution which is capable of neutralizing both acids and bases without appreciably changing the original acidity or alkalinity of the solution. Buffer solutions contain mixture(s) of acid and conjugate base at or near the pKa to minimize pH changes caused by an influx of acid or base. Buffer solutions also may contain additional solutes such as salts and other compounds.
As used herein, enzyme "deactivation" occurs when an enzyme is no longer capable of catalysis.
As used herein, a "lipase" is one of various enzymes which catalyze the hydrolysis of fats, especially triglycerides and phospholipids, into glycerol and fatty acids. Many lipases selectively cleave ester bonds.
As used herein, "substrate" refers to the chemical entity involved in a reaction which undergoes conversion to a product or products. Enzymes may catalyze the conversion of substrate(s) to product(s). As used herein, the term "precipitate" refers to the act of separating, e.g., a compound or product, from solution or suspension, usually via a chemical or physical change, often resulting in an insoluble oil or amorphous or crystalline solid, and the term also refers to a substance separated from a solution or suspension by chemical or physical change. As used herein, "supernatant" refers to the liquid floating above the surface of a sediment or precipitate.
As used herein, "peak area" refers to the area between the peak and the baseline of a chromatogram.
As used herein, "co-solvent" refers to a mixture of liquids. In some embodiments, the co-solvent is a material that is not necessarily an acceptable solvent that is added to a generally small amount of an active solvent to form a mixture that has enhanced solvent power. For example, a polar cosolvent can be added into a mixture of an organic liquid and a compound having pendant ionomeric groups to solubilize the pendant ionomeric groups. Co-solvent increase solubility of a compound. The use of cosolvents can increase the solubility by several orders of magnitude. Some commonly used cosolvents in pharmaceutics are propylene glycol, polyethylene glycols, ethanol and sorbitol. The addition of a co-solvent can increase solubility of hydrophobic molecules by reducing the dielectric constant of the solvent.
As used herein, "THF" refers to tetrahydrofuran.
As used herein, the term "combination" refers to any association between two or more items or elements.
As used herein, the term "article of manufacture" is a product that is made and sold and that includes a container and packaging, and optionally instructions for use of the product. For purposes herein, articles of manufacture encompass packaged intermediates as disclosed herein. As used herein, a "kit" refers to a combination of an intermediate provided herein and another item for a purpose including, but not limited to, synthesis of pregabalin or a related compound. Kits also optionally include instructions for use and/or reagents and glassware and other such items for use with the product.
As used herein, "substantially identical to a product" means sufficiently similar so that the property of interest is sufficiently unchanged so that the substantially identical product can be used in place of the product.
As used herein, the term "monitoring" refers to observing an effect or absence of any effect. In certain embodiments, one monitors a reaction after addition of a reactant or change in reaction conditions, such as temperature or pressure. Examples of effects that can be monitored include, but are not limited to, changes in evolution of gas, the appearance of a reaction product or a disappearance of a substrate or reactant.
As used herein, the term "contacting" refers to bringing two or more materials into close enough proximity that they can interact. In certain embodiments, contacting can be accomplished in a vessel such as, e.g., a test tube, flask, petri dish or mixing tank. In certain embodiments, contacting can be performed in the presence of additional materials.
As used herein, the term "subject" is an animal, typically a mammal, including human.
As used herein, the term "patient" includes human and animal subjects. As used herein, the term "carrier" refers to a compound that facilitates the incorporation of another compound into cells or tissues. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier for improving incorporation of certain organic compounds into cells or tissues.
As used herein, the term "pharmaceutical composition" refers to a chemical compound or composition capable of inducing a desired therapeutic effect in a subject. In certain embodiments, a pharmaceutical composition contains an active agent, which is the agent that induces the desired therapeutic effect. The pharmaceutical composition can contain a prodrug of the compounds provided herein. In certain embodiments, a pharmaceutical composition contains inactive ingredients, such as, for example, carriers and excipients. As used herein, the term "therapeutically effective amount" refers to an amount of a pharmaceutical composition sufficient to achieve a desired therapeutic effect.
As used herein, the term "pharmaceutically acceptable" refers to a formulation of a compound that does not significantly abrogate the biological activity, a pharmacological activity and/or other properties of the compound when the formulated compound is administered to a subject. In certain embodiments, a pharmaceutically acceptable formulation does not cause significant irritation to a subject.
As used herein, pharmaceutically acceptable derivatives of a compound include, but are not limited to, salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs thereof. Such derivatives can be readily prepared by those of skill in this art using known methods for such derivatization. The compounds produced can be administered to animals or humans without substantial toxic effects and either are pharmaceutically active or are prodrugs. Pharmaceutically acceptable salts include, but are not limited to, amine salts, such as but not limited to chloroprocaine, choline, N,N'-dibenzyl-ethylenediamine, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N- methylglucamine, procaine, N-benzyl-phenethylamine, l-para-chloro-benzyl-2- pyrrolidin-l'-ylmethyl-benzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)-aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, salts of mineral acids, such as but not limited to hydrochlorides and sulfates; and salts of organic acids, such as but not limited to acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates and fumarates. Pharmaceutically acceptable esters include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids and boronic acids. Pharmaceutically acceptable enol ethers include, but are not limited to, derivatives of formula C=C(OR) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl. Pharmaceutically acceptable enol esters include, but are not limited to, derivatives of formula C=C(OC(O)R) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl. Pharmaceutically acceptable solvates and hydrates are complexes of a compound with one or more solvent or water molecules, or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solvent or water molecules.
As used herein, "salt" refers to an assembly of cation and anion. Salts include alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, salts of mineral acids, such as but not limited to hydrochlorides and sulfates; and salts of organic acids, such as but not limited to acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates and fumarates. B. Processes for the Production of Pregabalin 1. Description of processes
Several methods are known in the art for the preparation of pregabalin. For example, a synthetic scheme using an azide intermediate results in a racemic mixture, which is then subsequently resolved into its R- and S-enantiomers (e.g., see U.S. Pat. No. 5,563,175). Another method uses an intermediate and nitro compounds that can be unstable resulting in an amine, but the reaction can proceed exothermically (e.g. , see Andruszkiewicz et al, Synthesis, 1989:953). Racemic mixtures of pregabalin also have been prepared via a tert-butyl ester route, via an L-leucine conversion, via Stobbe condensation, via malonate synthesis, and by a Hofinann synthesis (e.g., see Hoekstra et al., Organic Process Research & Development 1 : 26-38 (1997); Burk et al, J. Org. Chem. 68: 5731 (2003); U.S. Pat. Nos. 5,840,956; 5,637,767; 5,629,447; and 5,616,793 and U.S. Pat. Pub. 2005283023).
A drawback of these methods is that classical methods of resolving a racemate are required to obtain pregabalin. Classical resolution of racemic mixtures involves preparation of a salt with a chiral resolving agent in order to separate and purify the desired S-enantiomer. This involves significant processing, and results in the potential loss of compound as the R-enantiomer, which cannot be efficiently recycled and is often discarded as a waste by-product. The requirement for racemic resolution of these methods also adds additional cost, in processing time and lost product, as well as costs associated with the racemic resolving agent. In many of the reactions, only half of the product is the correct racemate, which results in a maximum theoretical yield of pregabalin, the S-enantiomer, of about 50%. This has a significant negative impact on production cost and capacity.
Methods have been described that uses a chiral auxiliary to introduce the stereochemical configuration desired in the final product. For example, one such method includes use of H-butyllithium at low temperatures (-35°C) and the use of (4R,5S)-4-methyl-5-phenyl-2-oxazolidinone, a costly reagent, as the chiral auxiliary (U.S. Pat. No. 5,563,175). Thus, although such a synthesis scheme might provide pregabalin, its high cost for chiral materials makes the synthesis scheme impractical for large-scale or commercial processing.
Provided herein are intermediates and methods that address the deficiencies in the prior art synthetic schemes. Exemplary processes for the production of pregabalin are depicted below. Provided herein is a process for synthesizing pregabalin via amination of an aldehyde precursor, as shown in Scheme 1. Scheme 1
Figure imgf000028_0001
4 5
In this route, compound 2 is prepared from 4-methylpentanal 1 by known methods (e.g., see Kenda et al, J. Med. Chem. 47: 530 (2004) and Reichelt et al, J. Org. Chem. 67: 4062 (2002)). Compound 2 is then hydrolyzed to 3 by chemical or enzymatic methods. Subsequently compound 3 is converted to the final product, pregabalin 6, under either chemical or enzymatic amine transfer or amination conditions, and the desired stereoselectivity can be obtained (e.g., Yun et al, Appl. Environ. Microbiol. 70: 2529-2534 (2004); Shin et al, Biosci. Biotechnol. Biochem. 65: 1782-1788 (2001); Mehta et al, Eur. J. Biochem. 214: 549-561 (1993)).
Alternatively, the aldehyde 2 is converted to the amino ester 4 under either chemical or enzymatic amine transfer or amination conditions (Yun et al, Appl. Environ. Microbiol. 70: 2529-2534 (2004); Shin et al, Biosci. Biotechnol. Biochem., 65: 1782-1788 (2001); Mehta et al, Eur. J. Biochem. 214: 549-561 (1993)).
Compound 4 is cyclized to lactam 5 and hydrolyzed to pregabalin 6 upon hydrolytic ring-opening (e.g., see Hoekstra et al, Org. Proc. Res. Dev. 1 : 26 (1997)). Alternatively, compound 4 is hydrolyzed directly to compound 6.
A further use of aldehyde precursor 1 to prepare pregabalin is shown in Scheme 2. Compound 1 is condensed with glyoxalic acid to afford lactone 7 (5- hydroxy-4-isobutylfuran-2(5H)-one), the double bond of which is further reduced to afford compound 8 (e.g., see Bourguignon, J. Org Chem. 46: 4889 (1981) and WO 03/093220) or its open-chain equivalent compound 3 or 10. The reduction of the double-bond can be achieved chemically or chemoenzymatically and may proceed via 9, the open-chain equivalent of 7. Compound 8 or 10 is then converted to pregabalin under chemical or enzymatic amine transfer or amination conditions. This amine transfer or animation reaction may proceed through open-chain equivalent compound 3. (e.g., see Yun et al., Appl. Environ. Microbiol. 70: 2529-2534 (2004); Shin et al., Biosci. Biotechnol. Biochem. 65: 1782-1788 (2001); Mehta et al., Eur. J. Biochem. 214: 549-561 (1993)). Scheme 2
Figure imgf000029_0001
lin)
Figure imgf000029_0002
M = H, salt 10
Compound 3 also may exist in or be converted to its cyclic form 8, (e.g., see Bourguignon, J. Org Chem. 46: 4889 (1981)). As discussed above, compound 8 may be converted to pregabalin under chemical or enzymatic amine transfer or amination conditions.
Compound 7 may be converted to its open form, compound 9. For example, hydrolyzing compound 7 affords compound 9. Any method known in the art of hydrolyzing a lactone can be used for the preparation of a compound of formula I from 5-hydroxy-4-isobutylfuran-2(5H)-one. For example, 5-hydroxy-4- isobutylfuran-2(5H)-one can be hydrolyzed under aqueous basic conditions. The base can be selected from among, but is not limited to, sodium hydroxide, potassium hydroxide, sodium t-butoxide, potassium t-butoxide, sodium methoxide, potassium methoxide, lithium methoxide, lithium t-butoxide, potassium carbonate, sodium carbonate, lithium carbonate, cesium carbonate and sodium ethoxide. Saponification of compound 7 also affords the salt of compound 9. Any method of saponification of lactones known in the art can be used. For example, saponification may be accomplished by treating the lactone with a metallic alkali base such as sodium hydroxide or potassium hydroxide. Chemical or enzymatic reduction of compound 9 affords compound 10 (e.g., see Dick et al, J. Bio. Chem. 279: 17269 (2004) and Youn et al., J. Bio. Chem. 281 : 4007 (2006)). Finally, compound 10 is converted to pregabalin by either enzymatic amine transfer or chemical amination conditions. Any solvent can be used in the process. The solvent can be aqueous, nonaqueous, polar or non-polar, hydrophobic or hydrophilic and combinations thereof, such as bi-phasic systems and emulsions. The reaction is carried out at different temperatures and pressures depending upon the substrate. The reaction conditions can be determined by a person skilled in the art on the basis of the substrate, catalysts and rates of conversion, and is appropriately adjusted for the process. The process can be carried out at any pressure known to the skilled artisan to be appropriate for hydrogenation. For example, the pressure can be at atmospheric pressure or above atmospheric pressure. The reaction temperature can be at or above room temperature. The process is conducted in a reaction vessel, for example, in a stirred tank or in membrane reactor, and the process can be operated either as a batch operation, as a semi-continuous process, or as a continuous process. These processes are well-known in the art (e.g., see "Engineering Processes for Bioseparations," (L. R. Weatherley, ed.), Butterworth-Heinemann Publishers (1994), pages 135-165). For example, continuous or semi -continuous processes can be operated using, e.g., cross-flow filtration mode. In one embodiment, a membrane reactor is used. Suitable membrane reactors are described, e.g., in U.S. Pat. No. 6,180,837. The pH of the reaction is varied to optimize the catalysis mediated by the selected hydrolases and/or transaminases and can be determined by a person skilled in the art on the basis of the enzyme stabilities and rates of conversion, and is appropriately adjusted for the process. In general, the pH range for the reaction is selected from about pH 3 to about 11. The enzyme used in the process can be in a free form, such as a homogeneously purified compound. In other embodiments, the enzyme is a constituent of an organism, such as a fungus or bacteria, and can be part of an intact organism or a denatured cell mass of the organism, hi other embodiments, the enzyme is immobilized, such as on a solid support (e.g. , see Worsford, Pure and Applied Chem. 67(4): 597-600 (1995); Cao, "Carrier-bound Immobilized Enzymes: Principles, Application and Design" (Wiley, 2006); Okahata et al., Tetrahedron Lett. 38: 1971-1974 (1997); Adlercreutz et al, Biocatalysis 6: 291-305 (1992)). Commercially available Bayer- Villiger monoxygenases are available as free forms of enzymes and conjugated to a solid support.
2. Description of compounds of formula I
Provided herein are compounds for the synthesis of pregabalin, related compounds, and intermediates. Among these are compounds of formula I:
Figure imgf000031_0001
where R is selected from among alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl and heteroaryl, provided that R is not t-butyl. In some embodiments, R is alkyl, alkenyl or aryl. In some embodiments, R is selected from among methyl, ethyl, n-butyl, i-butyl, pentyl, hexyl, heptyl, octyl, nonyl. In some embodiments, R is Cj-C3 alkyl. In some embodiments, R is «-butyl, isobutyl or sec- butyl. In some embodiment, R is C5-C8 alkyl. In some embodiments, R is C5-C2O alkyl. In some embodiments, R is Cj-C8 alkenyl. In some embodiments, R is C1-C8 heteroalkyl. In some embodiments, R is C3-C8 cycloalkyl or C3-C8 cycloheteroalkyl having one to eight heteroatoms selected from among O, N, S and P. In some embodiments, R is C3-C8 aryl or C3-C8 heteroaryl having one to eight heteroatoms selected from among O, N, S and P.
Compounds of formula I can be a racemic mixture, or can be enantiomerically enriched as the R-enantiomer or the S-enantiomer:
Figure imgf000031_0002
R-enantiomer S-enantiomer
Compounds of formula I may be prepared, e.g., by reacting 4-methylpentanal with diisobutylamine and an alkyl-3-halo-propanoate to afford an aldehyde compound of formula I:
Figure imgf000031_0003
The (R)- and (S)-enantiomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as reverse phase HPLC. C. Enzymatic processing
1. Enzymatic hydrolysis of esters
The methods provided herein can employ a hydrolase as noted. Any hydrolase can be can be used. If needed, hydrolases, libraries thereof and/or libraries of modified hydrolases can be screened to identify more suitable or suitable hydrolases for the methods herein.
Hydrolases are enzymes that catalyze the hydrolysis of a chemical bond. These enzymes are classified as EC 3 in the EC number classification of enzymes, and are further classified into several subclasses, based on the bonds upon which they act. Bonds that can be hydrolyzed by these enzymes include, but are not limited to, ester bonds, bonds to sugars, ether bonds, peptide bonds, carbon-nitrogen bonds, acid anhydrides, carbon-carbon bonds, halide bonds, phosphorus-nitrogen bonds, sulfur- nitrogen bonds, carbon-phosphorus bonds, sulfur-sulfur bonds and carbon-sulfur bonds. Several classes of hydrolases can hydrolyze esters, including, but not limited to, various proteases, and those enzymes classified as esterases, including lipases. Any enzyme that can hydrolyze the ester bond of compound 2, described above, to yield compound 3, can be used in the methods described herein. In some embodiments, the hydrolase is an esterase, a lipase, or a protease. The hydrolase can be of mammalian, including human, origin, or can be of non-mammalian origin, including but not limited to, plant, bacterial, viral, yeast and fungal origin. The hydrolase used in the methods provided herein to hydrolyze the ester bond of compound 2 can be a wild-type protein or a variant thereof. In some embodiments, a hydrolase variant is used that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations, such as amino acid substitutions, deletions, additions, or combinations thereof. For example, hydrolase variants can be generated by directed evolution such that the substrate specificity and enzymatic activity of the variants are optimized for the purposes herein (see, e.g., Bornscheuer et ah, Curr. Opin. Biotech. 7:2169-2173 (1999)). The properties of a candidate hydrolase, such as substrate specificity and enzymatic activity, can be assessed using any method known in the art, as described below. In one embodiment, high-throughput screening of multiple hydrolases is performed (e.g., see Yazbeck et ah, Adv. Synth. Catal. 345:524-532 (2003)). Thus, one of skill in the art can readily identify one or more hydrolases that can catalyze the hydrolysis of compound 2 to compound 3. Exemplary classes of hydrolases that can be screened for such activities are described in the following sections. a. Esterases Esterases (classified as EC 3.1 in the EC number classification of enzymes) are enzymes that hydrolyze esters into an acid and an alcohol in a chemical reaction with water. This is mediated through nucleophilic attack of the active serine in the esterase on the carbonyl of the substrate in a charge-relay system with two other amino acid residues in the esterase. Together, the three amino acid residues are called the catalytic triad. Esterases display broad substrate specificity and also can exhibit enantioselectivity. Many esterases have been described in the art to hydrolyze different substrates. For example, esterase enzymes cleaving carboxyl esters have been identified in leukocytes; phosphatases, such as alkaline and acidic phosphatases, hydrolyse phosphoric acid esters; glycosidases, such as galactosidases, glucosidases, mannosidases and amylases can cleave glycosidic bonds. Other esterases include, but are not limited to, lipases, acetylesterases, thiolester hydrolases, phosphoric monoester hydrolases, phosphoric diester hydrolases, triphosphoric monoester hydrolases, sulfuric ester hydrolases, diphosphoric monoester hydrolases, phosphoric triester hydrolases, exonucleases and endonucleases. One of skill in the art can screen any esterase for substrate specificity and enzymatic activity to determine its suitability for use in the methods provided herein for the production of pregabalin. b. Lipases
Lipases, also known as triacylglycerol ester hydrolases, (E.C.3.1.1.3) are a subclass of esterases which belongs to the a/β hydrolase super family. They are ubiquitous enzymes that can be generally divided into the following four groups according to their specificity in hydrolysis reaction: substrate specific lipases, regio- selective lipases, fatty acid specific Upases, and stereo-specific lipases. Lipases can be obtained from a variety of sources including, but not limited to, plants, animals, yeast and bacteria. Exemplary lipases include, but are not limited to, Upases from Acinetobacter calcoaceticus, Acinetobacter sp. , Alcaligenes sp. , Aspergillus carneus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bacillus sp., Bacillus stearothermophilus, Bacillus subtilis, Bacillus thermocatenulatus, Burkholderia sp., Candida antarctica, Candida parapsilosis, Candida rugosa, Cephaloleia presignis, Chromobacterium viscosum, Homo sapiens, Mus musculus, Oryza sativa, Penicillium candidum, Penicillium roquefortii, Penicillium wortmanii, Pichia burtonii, Proteus vulgaris, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas cepacia, Pseudomonas βuorescens, Pseudomonas pseudoalcaligenes, Pseudomonas sp., Rattus norvegicus, Rhizomucor miehei, Rhizopus delemar, Rhizopus japonicus, Rhizopus oryzae, Rhodotorula glutinis, Saccharomyces cerevisiae, Staphylococcus epidermidis, Staphylococcus simulans, Streptomyces rimosus, Sus scrofa, Thermomyces lanuginosa, Triticum aestivum, Staphylococcus warneri, Bacillus pumilus and Candida rugosa. One of skill in the art can screen any lipase for substrate specificity and enzymatic activity to determine its suitability for use in the methods provided herein for the production of pregabalin. c. Proteases Esters also can be hydrolyzed by proteases, including, but not limited to, serine proteases and cysteine proteases. The activity of proteases in the serine protease family is dependent on a set of amino acid residues that form the active site. One of the residues is always a serine, hence their designation as serine proteases. In addition to their natural proteolytic activity, serine proteases also can hydrolyze ester bonds. They can be obtained from a variety of sources including, but not limited to, animals (including humans), plants, yeast and bacteria. Exemplary eukaryotic serine proteases include, but are not limited to, acrosin; blood coagulation factors VII, IX, X, XI and XII, thrombin, plasminogen, and protein C; cathepsin G; chymotrypsins; complement components CIr, CIs, C2, and complement factors B, D and I; complement-activating component of RA-reactive factor; cytotoxic cell proteases (granzymes A to H); duodenase I; elastases 1 , 2, 3 A, 3B (protease E), leukocyte (medullasin); enterokinase (enteropeptidase); hepatocyte growth factor activator; hepsin; glandular (tissue) kallikreins (including EGF-binding protein types A, B, and C, NGF-γ chain, 7-renin, prostate specific antigen (PSA) and tonin); plasma kallikrein; mast cell proteases (MCP) 1 (chymase) to 8; myeloblastin (proteinase 3) (Wegener's autoantigen); plasminogen activators (urokinase-type, and tissue-type); trypsins I, II, III, and IV; tryptases; snake venom proteases such as ancrod, batroxobin, cerastobin, flavoxobin, and protein C activator; collagenase from common cattle grub and collagenolytic protease from Atlantic sand fiddler crab; apolipoprotein(a); Blood fluke cercarial protease; Drosophila trypsin like proteases: a, easter, snake-locus; Drosophila protease stubble (gene sb); and major mite fecal allergen Der p III. Exemplary prokaryotic serine protease include, but are not limited to, subtilisins from Bacillus sp.; alkaline elastase YaB from Bacillus sp.; alkaline serine exoprotease A from Vibrio alginolyticus; aqualysin I from Thermus aquaticus; AspA from Aeromonas salmonicida; bacillopeptidase F (esterase) from Bacillus subtilis; C5A peptidase from Streptococcus pyogenes; cell envelope-located proteases PI, PII, and PIII from Lactococcus lactis; extracellular serine protease from Serratia marcescens; extracellular protease from Xanthomonas campestris; intracellular serine protease (ISP) from various Bacillus; minor extracellular serine protease epr from Bacillus subtilis; minor extracellular serine protease vpr from Bacillus subtilis; nisin leader peptide processing protease nisP from Lactococcus lactis; serotype-specific antigene 1 from Pasteurella haemolytica; thermitase from Thermoactinomyces vulgaris; calcium-dependent protease from Anabaena variabilis; halolysin from halophilic bacteria sp. 172pl ; alkaline extracellular protease (AEP) from Yarrowia lipolytica; alkaline proteinase from Cephalosporium acremonium; cerevisin (vacuolar protease B) from yeast; cuticle-degrading protease (prl) from Metarhizium anisopliae.; KEX-I protease from Kluyveromyces lactis. ; kexin from yeast; oryzin (alkaline proteinase) from Aspergillus; proteinase K from Tritirachium album; proteinase R from Tritirachium album; proteinase T from Tritirachium album; subtilisin-like protease III from yeast; and thermomycolin from Malbranchea sulfur ea.
Members of the class of cysteine proteases have a common catalytic mechanism that involves a cysteine amino acid residue in the active site of the protease. Exemplary cysteine proteases that could be used in the methods herein include, but are not limited to, vertebrate lysosomal cathepsins B, H, L, and S; vertebrate lysosomal dipeptidyl peptidase I (also known as cathepsin C); vertebrate calpains; mammalian cathepsin K, which seems involved in osteoclastic bone resorption; human cathepsin O; bleomycin hydrolase; barley aleurain; EP-B 1/B4; kidney bean EP-Cl; rice bean SH-EP; kiwi fruit actinidin; papaya latex papain; chymopapain; caricain; proteinase IV; pea turgor-responsive protein 15 A; pineapple stem bromelain; rape COT44; rice oryzain a, β, and γ, cathepsin B-like proteinases from the worms Caenorhabditis elegans, Schistosoma mansoni, Haemonchus contortus, and Ostertagia ostertagi; slime mold cysteine proteinases CPl and CP2; Cruzipain from Trypanosoma cruzi and bruce; Throphozoite cysteine proteinase (TCP) from various Plasmodium sp.; Baculoviruses cathepsin-like enzyme (v-cath); Drosophila small optic lobes protein (gene sol); Yeast thiol protease BLHl /Y CPl /LAP3; Aminopeptidase C from Lactococcus lactis: Thiol protease tpr from Porphyromonas gingivalis; and mammalian caspases, caspase-1, -2, -3, -4, -5, - 6, -7, -8, -9, -10, -11, -12, 13, and -14. One of skill in the art can screen any protease for substrate specificity and enzymatic activity to determine its suitability for use in the methods provided herein for the production of pregabalin. d. Identification of hydrolases useful for the methods herein Any method known in the art to screen enzymes for substrate specificity and enzymatic activity can be used to identify hydrolases useful for the methods provided herein {see, e.g., Gupta et al, (2003) Biotechnol. Appli. Biochem. 37:63-71, Yazbeck et al., (2003) Adv. Synth. Catal. 345:524-532, Kim et al., (2006) Prot. Exp. Purif. 45:315-323). In one embodiment, high throughput methods are employed to screen multiple enzymes for their ability to hydrolyze the ester bond of compound 2. Such methods are typically performed in, for example, 96-well assay plates, such that multiple hydrolases (e.g. , a hydrolase library) can be simultaneously screened for hydrolysis of a chosen substrate. In some embodiments, the hydrolases are obtained from commercial sources, such as those provided in Table 1. In other embodiments, the hydrolases are specifically engineered for this purpose, such as through directed evolution methods (Bornscheuer et al., (1999) Curr. Opin. Biotech. 7: 2169-2173). Table 1. Commercially available hydrolases.
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
The reactions are typically carried out in volumes of about 100 μl of a suitable buffer, such as 0.1 M potassium phosphate buffer, containing 1 mg/ml of substrate, 10 mg/ml of enzyme and 10% organic solvent. The pH of the reaction is generally maintained at between 7.0 and 7.4. One of skill in the art can modify the reaction conditions depending on the type of hydrolase being screened. For example, water immiscible solvents such as methyl tert-butyl ether (MTBE), ethyl acetate, dichloro- methane, toluene or DIPE can be used for lipase screening, resulting in a biphasic system. Proteases and esterases can be screened in water miscible solvents, such as acetonitrile, methanol, acetone or ethanol. Following incubation at an appropriate temperature, typically ranging from O0C to 6O0C, the samples are analyzed to assess hydrolysis. A variety of methods can be used in the analysis of the sample, including, but not limited to, high performance liquid chromatography (HPLC), capillary electrophoresis (CE), gas chromatography (GC), UV spectrophotometry, thin layer chromatography (TLC) and liquid chromatography coupled with mass spectrometry (LC-MC). e. Conditions for use of hydrolases in the methods herein One of skill in the art can determine suitable conditions for the enzymatic hydrolysis of compound 2 to compound 3, taking into account the type of hydrolase being employed in the reaction. In some embodiments, the reaction is carried out in an organic-aqueous biphasic system. Suitable organic solvents that can be used for this purpose include, but are not limited to, hexane, cyclohexane, ethyl acetate, 1 - hexanol, chloroform, dichloroethane, dichloromethane, toluene, tert-pentyl alcohol, methyl isobutyl ketone (MIBK), methyl tert-butyl ether (MTBE) and di-isopropyl ether (DIPE). In other embodiments, the reaction is carried out in an aqueous buffer. Suitable aqueous phases include, but are not limited to, buffers such as glutamic acid- glutamate, phosphoric acid-phosphate, acetic acid-acetate and citric acid-citrate buffers. For example, 0.1 M potassium phosphate buffer can be used in the methods herein. A suitable pH for the reaction can be determined by one of skill in the art, taking into account the activity and stability of the hydrolase. A pH range from 3 to 11 is contemplated for the methods herein. Typically, however, a neutral environment is maintained, such that the pH of the reaction is at or about 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 or 7.7. The temperature during the reaction can be between O0C to 6O0C. In some embodiments, an ambient temperature is maintained throughout the reaction. In other embodiments, a temperature of 3O0C or 370C is maintained.
The amount of hydrolase used to hydrolyze compound 2 is determined by the activity of the enzyme. An IU (International Unit) designates that amount of an enzyme preparation which catalyzes the formation of one micromole of product per minute. Such determinations can be made using methods well known in the art. For the methods herein, typically 10 to 10,000 IU of hydrolase is added to the reaction for every gram of substrate. The mixture is then typically agitated throughout the reaction. The reaction can be monitored by, for example, gas chromatography (GC), high performance liquid chromatography (HPLC) or thin layer chromatography (TLC) to determine the point of completion. The resulting compound can be isolated by extraction, evaporation, or other suitable separation methods. In one embodiment, the reaction is monitored by GC and the product is purified by extraction. The reaction mixture is saturated with sodium chloride and repeatedly extracted with ethyl acetate until complete recovery of the product. The organic layers are then dried over anhydrous sodium sulfate and filtered and concentrated. 2. Enzymatic reduction of carbon-carbon double bonds
The process described herein for the synthesis of pregabalin involves an enzymatic reduction of carbon-carbon double bonds such that compound 10 is produced from compound 9. Chirality also is introduced in this step to facilitate the final production of the (S)-enantiomeric form of pregabalin. Enzymes that effect reduction of carbon-carbon bonds of a variety of substrates, including a broad range of α,0-unsaturated aldehydes and ketones, have been described in the art (Eur. Pat. No 1236796, Wanner et al, Eur. J. Biochem. 255:271-278 (1998), Ensor et al, Biochem. J. 330:103-108 (1998), Mano et al., Eur. J. Biochem. 267:3661-3671 (2000), Dick et al, J. Biol. Chem. 276:40803-40810 (2001), Dick et al, J. Biol. Chem. 279:17269- 17277 (2004), and Youn et al, J. Biol. Chem. 281 :40076-40088 (2006)). Furthermore, such enzymes also can exhibit enantioselectivity when a prochiral substrate is being reduced (Wanner et al, Eur. J. Biochem. 255:271-278 (1998)). Enzymes that can effect the reduction of carbon-carbon double bonds have been identified in a variety of species, including, but not limited to, mammalian, plant, yeast and bacterial species. Exemplary of these are set forth in Table 2. Table 2. Enzymes that can effect the reduction of carbon-carbon double bonds
Figure imgf000044_0001
These enzymes effect carbon-carbon double bond reduction in an NAD(P)H-dependent manner, and, although quite diverse, typically can be grouped within the MDR (medium-chain dehyrogenases/reductases) superfamily (Nordling et al, Eur. J. Biochem. 269:4267-4276 (2002)).
One of skill in the art can screen these and other enzymes for substrate specificity and enantioselectivity to determine their suitability for use in the methods provided herein for the production of pregabalin. The enzyme used in the reduction of compound 9 to compound 10 can be of mammalian, including human, origin, or can be of non-mammalian origin, including but not limited to, plant, bacterial, viral, yeast and fungal origin. The enzyme can be a wild-type protein or variant thereof. In some embodiments, an enzyme variant is used that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations, such as amino acid substitutions, deletions, additions, or combinations thereof. For example, enzyme variants can be generated by directed evolution such that the substrate specificity and enantioselectivity of the variants are optimized for the purposes herein. a. Identification of carbon-carbon double bond reductases useful for the methods herein
Any method known in the art to screen enzymes for substrate specificity and enantioselectivity can be used to identify those enzymes useful for the reduction of compound 9 to compound 10. For example, substrate specificity of one or more enzymes can be determined spectrophotometrically by monitoring the rate of NADPH oxidation at 340 ran (Dick et al, J. Biol. Chem. 276:40803-40810 (2001)). In instances where the substrate also has significant absorbance at 340 ran, thereby interfering with the detection of NADPH oxidation, HPLC methods can be used (Dick et al, J. Biol. Chem. 276:40803-40810 (2001), and Youn et al, J. Biol. Chem.
281 :40076-40088 (2006)). The enantiomeric composition of the reduction products can be determined using one of a variety of chromatographic methods well known in art, including, but not limited to, HPLC, GC, TLC and LC-MS. In one embodiment, GC is performed to determine the enantiomeric selectivity of an enzyme using compound 9 as a substrate (Wanner et al, Eur. J. Biochem. 255:271-278 (1998)). For example, a reaction mixture containing the enzyme, compound 9 and NADPH in an appropriate buffer, such as, 0.1 M potassium phosphate buffer, can be incubated for between 2 and 48 hours before being saturated with NaCl and extracted with ether. The reaction product can then be dried over sodium sulphate and concentrated, before being analyzed by chiral GC using an appropriate stationary phase, such as for example, heptakis-(3-O-acetyl-2,6-di-O-pentyl)-j8-cyclodextrin. b. Conditions for the use of carbon-carbon double bond reductases in the methods herein
Suitable conditions for the enzymatic reduction of carbon-carbon bonds are well known in the art, and can be employed in the methods provided herein (see e.g., Wanner et al, Eur. J. Biochem. 255:271-278 (1998), Ensor et al, Biochem. J. 330:103-108 (1998), Mano et al, Eur. J. Biochem. 267:3661-3671 (2000), Dick et al, J. Biol. Chem. 276:40803-40810 (2001), Dick et al, J. Biol. Chem. 279:17269-17277 (2004), and Youn et al., J. Biol. Chem. 281 :40076-40088 (2006)). The reactions are typically carried out using aqueous buffers. Suitable aqueous buffers include, but are not limited to, phosphate buffers, sodium acetate buffers, citric acid buffers, Tris buffers and MES (2-(N-morpholino)ethanesulfonic acid) buffers, hi some embodiments, additional reagents such as Triton X-100 or bovine serum albumin (BSA) also are included in the reaction. A suitable pH for the reaction can be determined by one of skill in the art, taking into account the activity and stability of the enzyme. A pH range from 3 to 11 is contemplated for the methods herein. Typically, however, a neutral environment is maintained, such that the pH of the reaction is at or about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 or 7.7. The temperature during the reaction can be between O0C to 6O0C. hi some embodiments, an ambient temperature is maintained throughout the reaction.
In reactions using NAD(P)H-dependent enzymes as the catalysts, the appropriate NADPH or NADP cofactor also is included in the reaction. Concentrations of NADP or NADPH used in the reaction can range from 0.0001 to 10 molar equivalents to the substrate or more. NADPH or NADH also can be recycled using a cofactor regenerating system well known to the skill of the art. The amount of purified enzyme used to catalyze the reduction of compound 9 is determined by the activity of the enzyme. An IU (International Unit) designates that amount of an enzyme preparation which catalyzes the formation of one micromole of product per minute. Such determinations can be made using methods well known in the art. For the methods herein, typically 10 to 10,000 IU of enzyme is added to the reaction for every gram of substrate. The mixture is then typically agitated throughout the reaction. The reaction can be monitored by, for example, gas chromatography (GC), high performance liquid chromatography (HPLC) or thin layer chromatography (TLC) to determine the point of completion. The resulting compound 10 can be isolated by extraction, evaporation, or other suitable separation methods. In one embodiment, the reaction is monitored by GC and the product is purified by extraction. The reaction mixture is saturated with sodium chloride and repeatedly extracted with ether until complete recovery of the product. The organic layers are then dried over anhydrous sodium sulfate and filtered and concentrated. 3. Enzymatic animation
The processes described herein for the synthesis of pregabalin includes an enzymatic amination of one or more substrates to introduce an amine group into the compound. For example, as shown in Scheme 1 above, enzymatic amination can be used to convert compound 2 to compound 4 and/or compound 3 to compound 6. Similarly, as shown in Scheme 2 above, enzymatic amination can be used to convert compound 8 to compound 6 and/or compound 10 to compound 6. In the amination of compound 2 to compound 4 in Scheme 1 , chirality also is introduced into the compounds to facilitate production of the (S) enantiomer of pregabalin. These enzymatic reactions can be effected by a class of enzymes called transaminases, also known as aminotransferases (Mehta et al, Eur. J. Biochem. 214:549-561 (1993)). Transaminases catalyze the transfer of an amino group, a pair of electrons, and a proton from a primary amine to the carbonyl group of an acceptor molecule (e.g., compound 2, 3, 8, or 10) in a process called transamination. They can be divided into four subgroups (I, II, III and IV) based on their amino acid sequence similarity (Mehta et al, Eur. J. Biochem. 214:549-561 (1993)).
Whereas most transaminases accept only substrates with at least one carboxyl group, the ω- transaminases, also known as beta-alanine-pyruvate transaminase (EC 2.6.1.18 ), can utilize substrates where the amino group is not adjacent to a carboxylate group. They are classified as subgroup II transaminases, and are, therefore, structurally related to other subgroup II transaminases, including, but not limited to, N-acetyl-L- ornithine transaminase, L-ornithine transaminase, 4-amino-butyrate transaminase, and 7,8-diaminopelargonate transaminase, ω- Transaminases are pyridoxal phosphate- dependent enzymes which, upon transamination, can yield optically pure amines (Shin et al, Biosci. Biotechnol. Biochem. 65:1782-1788 (2001), Iwasaki et al, Biotech. Lett. 25:1843-1846 (2003), Iwasaki et al, Appl. Microb. Biotech. 69:499-505 (2004), Yun et al, Appl. Environ. Microbiol. 70:2529-2534 (2004), and Hwang et al, Enzyme Microbiol. Technol. 34: 429-426 (2004)). The optical purity of the resulting compound is measured as enantiomer excess (ee), which is often expressed as a percentage to describe the percentage of the selective enantiomer in the mixture. Highly selective transaminases result in 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 94%, 95%, 96%, 98%, 99%, or more, ee with a particular set of amino donors and acceptors. The more selective the transaminase is, the higher the optical purity of the resulting chiral amine. ω- Transaminases have been isolated from various microorganisms, including, but not limited to, Alcaligenes denitrificans (SEQ ID NO: 12), Bordetella bronch- iseptica (SEQ ID NO:13), Bordetella parapertussis (SEQ ID NO:14), Brucella melitensis (SEQ ID NOS: 15 and 16), Burkholderia mallei (SEQ ID NO: 17), Burkholderia pseudomallei (SEQ ID NO: 18), Chromobacterium violaceum (SEQ ID NO: 19), Oceanicola granulosus HTCC2516 (SEQ ID NO:20), Oceanobacter sp. RED65 (SEQ ID NO:22), Oceanospirillum sp. MED92 (SEQ ID NO:21), Pseudomonas putida (SEQ ID NO:23), Ralstonia solanacearum (SEQ ID NO:26), Rhizobium meliloti (SEQ ID NO:25), Rhizobium sp. (strain NGR234) (SEQ ID NO:24), and Vibrio fluvialis, Bacillus thuringienis, and Klebsiella pneuminiae (Shin et al., (2001) Biosci. Biotechnol. Biochem. 65:1782-1788). One of skill in the art can screen these and other transaminases, including other subgroup II transaminases, for amino donor and amino acceptor (e.g., compounds 2, 3, 8, or 10) specificity and enantioselectivity to determine their suitability for use in the methods provided herein for the production of pregabalin. The one or more transaminases used in the methods herein can be wild-type proteins or variants thereof, such as those described in U.S. Pat. No. 7,172,885. In some embodiments, an enzyme variant is used that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations, such as amino acid substitutions, deletions, additions, or combinations thereof. For example, enzyme variants can be generated by mutagenesis such that the substrate specificity and enantioselectivity of the variants are optimized for the purposes herein (Reetz et al, (2003) in Directed Evol. Prot., Ch 6, pp 245-279). a. Identification of transaminases for the methods herein If necessary, any transaminase can be tested for use in the methods herein. Any method known in the art to screen transaminases for amino acceptor specificity and enantioselectivity can be used to identify enzymes useful for the transamination of compounds 2, 3, 8, and 10. Libraries of transaminases or modified forms thereof or other collections or individual transaminases can be screened. Screening can employ assays. Such assay typically can include incubation of the transaminase with the amino acceptor compound of interest, pyridoxal phosphate (the cofactor), and an appropriate amino donor. The products of the reaction are then analyzed using one of a variety of methods than can separate and/or detect enantiomers, including, but not limited to, high performance liquid chromatography (HPLC), capillary electrophoresis (CE), gas chromatography (GC), UV spectrophotometry, thin layer chromatography (TLC) and liquid chromatography coupled with mass spectrometry (LC-MC) (Yazbeck et al, Adv. Synth. Catal. 345:524-532 (2003), Reetz et al, Catal. Today 67:389-96 .,(2001),
Ramseier et al, Electrophoresis 20:2726-38 (1999) , Reetz et al, Angew Chem Int Ed. 39:3891-93 (2000)). High throughput screening of multiple transaminases against multiple amino donors and acceptor also can be performed using spectrophotometric methods (Hwang et al, Enz. Microbiol. Technol. 34: 429-426 (2004)). b. Conditions for the use of transaminases in the methods herein
Suitable conditions for the transamination of compounds are well known in the art, and can be employed in the methods provided herein {see e.g. Yun et al., (2004) Appl. Environ. Microbiol. 70:2529-2534, Shin et al, Biosci. Biotechnol. Biochem. 65:1782-1788 (2001), Hwang et al, Enzyme Microbiol. Technol. 34: 429-426 (2004)). If necessary such conditions can be identified and/or optimized further empirically. The reactions can be carried out in an aqueous buffer or an organic-aqueous biphasic medium. Suitable aqueous buffers include, but are not limited to, phosphate buffers, sodium acetate buffers, citric acid buffers and Tris buffers. In one embodiment, 0.1 M potassium phosphate buffer is used as an aqueous buffer. The cofactor, pyridoxal phosphate, and an appropriate amino donor also are added to the reaction. Suitable concentrations of pyridoxal phosphate can range from 2 μM to 200 μM, but can be more or less. Amino donors that can be used include, but are not limited to, L-alanine, L-aspartate or L-glutamate. In one embodiment, L-aspartate is included in the reaction as the amino donor. In some embodiments, additional reagents such as oxaloacetate decarboxylase also are added to the assay to remove the side product such as oxaloacetate from deamination of the amino donor L-aspartate, thus shifting the reaction equilibrium to completion. A suitable pH for the reaction can be determined by one of skill in the art, taking into account the activity and stability of the enzyme. A pH range from 3 to 11 is contemplated for the methods herein. Typically, however, a neutral environment is maintained, such the pH of the reaction is at or about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 or 7.7. The temperature during the reaction can be between O0C to 6O0C. In some embodiments, an ambient temperature is maintained throughout the reaction. In other embodiments, a temperature of approximately 370C is maintained throughout the reaction.
The amount of purified enzyme used to transaminate the amino acceptor {i.e., compound 2, 3, 8, or 10) is determined by the activity of the enzyme. An IU (International Unit) designates that amount of an enzyme preparation which catalyzes the formation of one micromole of product per minute. Such determinations can be made using methods well known in the art. For the methods herein, typically 10 to 10,000 IU of enzyme is added to the reaction for every gram of amino acceptor. The mixture is then typically agitated throughout the reaction. The reaction can be monitored by, for example, gas chromatography (GC), high performance liquid chromatography (HPLC) or thin layer chromatography (TLC) to determine the point of completion. The resulting compound can be isolated by extraction, evaporation, or other suitable separation methods, as described above.
D. Examples Example 1
^CHO 4-Methylpentanal (Compound 1 , Scheme 1 )
4-methylpentanal was prepared according to the procedure in Matsuura et al., Arch Biochem Biophys. 328(2):265-71 (1996). First, n-propyl 4-methylpentanoate was prepared by refluxing 4-methylpentanoic acid (0.1 mol) in n-propyl alcohol (80 ml) containing concentrated sulfuric acid (8 ml) for 2 h. The reaction product was extracted into ethyl acetate, and the extract was evaporated to dryness under reduced pressure. The resulting residual oil was purified by distillation to give n-propyl 4- methylpentanoate. To a stirred solution of n-propyl 4-methylpentanoate (37.9 mmol) in dry hexane (300 ml) was added dropwise diisobutylaluminium hydride (53 ml of 0.93 M solution in hexane) at -78°C. After stirring for 30 minutes, the reaction was quenched by the addition of methanol (7 ml). The mixture was poured into water (100 ml), acidified with 1 N sulfuric acid, and stirred vigorously for 30 min. The organic layer was washed with water, dried over anhydrous sodium sulfate, and evaporated keeping the temperature below 100C to dryness. The resulting residual oil was purified by distillation to afford 4-methylpentanal.
Example 2
Figure imgf000051_0001
Methyl 3-formyl-5-methylhexanoate (Compound 2, Scheme I, R = methyl)
This compound is prepared in a manner analogous to the one described in Kenda et al., J. Med. Chem 47: 530-549 (2004). In a flask fitted with a Dean-Stark apparatus under argon, a solution of diisobutylamine and 4-methylpentanal in toluene is heated at reflux until formation of water has ceased. The solution is cooled to room temperature and methyl bromoacetate is added. The solution is stirred at room temperature for 18 hours and then at 90 0C for 1 hour. Water is added to the solution at 90 0C, and then the solution is cooled to room temperature. The organic layer is washed with 1 N HCl and saturated aqueous sodium bicarbonate, dried over magnesium sulfate, filtered, and evaporated to afford the title compound. The product can be purified by distillation or other methods known in the art to provide the title compound.
Example 3
Figure imgf000051_0002
Sodium 3-formyl-5-methylhexanoate (Compound 3. Scheme L M = sodium) 3-Formyl-5-methylhexanoate is hydrolyzed with aqueous sodium hydroxide to afford sodium 3-formyl-5-methylhexanoate.
Example 4
Figure imgf000051_0003
Pregabalin (Compound 6, Scheme I) Method A: Chemical Animation.
Sodium 3-formyl-5-methylhexanoate is treated with an ammonia source in the presence of a reducing agent to afford methyl 3-(aminomethyl)-5-methylhexanoate. Method B: Enzymatic amination.
The amination of 3-formyl-5-methylhexanoate is catalyzed by a transaminase enzyme. The reactions can be carried out in an aqueous buffer or an organic-aqueous biphasic medium. Suitable aqueous buffers include, but are not limited to, phosphate buffers, sodium acetate buffers, citric acid buffers and Tris buffers. In one embodiment, 0.1 M potassium phosphate buffer is used as an aqueous buffer. The cofactor, pyridoxal phosphate and an appropriate amino donor also are added to the reaction. Suitable concentrations of pyridoxal phosphate can range from 2 μM to 200 μM, but can be more or less. Amino donors that can be used include, but are not limited to, L-alanine, L- valine or L-glutamate. In one embodiment, L-glutamate is included in the reaction as the amino donor. In some embodiments, additional reagents such as α-ketoglutate also are added to the assay to shift the reaction equilibrium to completion. A suitable pH for the reaction can be determined by one of skill in the art, taking into account the activity and stability of the enzyme. A pH range from 3 to 11 is contemplated for the methods herein. Typically, however, a neutral environment is maintained, such the pH of the reaction is at or about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 or 7.7. The temperature during the reaction can be between O0C to 6O0C. In some embodiments, an ambient temperature is maintained throughout the reaction. In other embodiments, a temperature of approximately 370C is maintained throughout the reaction.
Example 5
Figure imgf000052_0001
(S)-4-isobutylpyrrolidin-2-one (Compound 5, Scheme L R = methyl) Compound 5 is produced by the cyclization of intermediate 4. The nitrogen of compound 4 can react with the carbonyl carbon of compound 4 via an intramolecular cyliczation reaction to afford the lactam 5.
Example 6
Figure imgf000052_0002
Pregabalin (Compound 6, Scheme D (S)-4-isobutylpyrrolidin-2-one is treated with hydrochloric acid to produce pregabalin hydrochloride. The pregabalin hydrochloride salt is broken with triethylamine. Filtration affords pregabalin.
Example 7
Figure imgf000053_0001
5-hvdroxy-4-isobutylfuran-2(5H)-one (Compound 7, Scheme ID Glyoxylic acid hydrate in the presence of a base is reacted with 4- methylpentanal to provide compound 7. For a related procedure, see Bourguignon, J. Org Chem. 46:4889-4894 (1981). Briefly, glyoxylic acid hydrate and powdered morpholinium hydrochloride are dispersed in dioxane. Water is added dropwise until the medium becomes homogeneous. Freshly distilled 4-methylpentanal is added to the solution. The reaction is maintained at room temperature for 1 hour followed by refluxing for 24 hours. Once there reaction is complete, the solvent is evaporated to dryness and the resulting residue is extracted with ethyl ether. The organic layer is dried over anhydrous magnesium sulfate and the solvent is evaporated. The crude product is purified by distillation under reduced pressure. Alternatively, the crude oil can be recrystallized from isopropyl ether, or an isopropyl ether/hexane solution, or acetone/chloroform solution.
Example 8
Figure imgf000053_0002
5-hvdroxy-4-isobutyldihvdrofuran-2(3H)-one (Compound 8, Scheme H) 5-Hydroxy-4-isobutylfuran-2(5H)-one is hydrogenated with a hydrogen source such as H2, HCO2NH4, and HCO2H, in the presence of a metal catalyst, such as Pt, Pd, Ni, Ru, Rh, or Ir) to provide 5-hydroxy-4-isobutyldihydrofuran-2(3H)-one. Example 9
Figure imgf000053_0003
Pregabalin (Compound 6, Scheme II)
5-Hydroxy-4-isobutyldihydrofuran-2(3H)-one was treated with an ammonia source and hydrogenated with a hydrogen source such as H2, HCO2NH4, and HCO2H, in the presence of a metal catalyst, e.g., Pt, Pd, Ni, Ru, Rh, or Ir) to provide pregabalin. Example 10
Figure imgf000054_0001
5-hvdroxy-4-isobutyldihvdrofuran-2(3H)-one (Compound 8, Scheme ID Sodium 3-formyl-5-methylhexanoate is converted to its cyclic form under acidic conditions. For example, dissolve sodium 3-formyl-5-methylhexanoate in water with stirring. The pH is adjusted with hydrochloric acid solution to provide the desired product.
Example 11
Figure imgf000054_0002
(Z)-3-formyl-5-methylhex-2-enoic acid (Compound 9, Scheme H)
5-hydroxy-4-isobutylfuran-2(5H)-one can be treated with base (e.g., aqueous sodium hydroxide) to afford (Z)-3-formyl-5-methylhex-2-enoic acid as the salt.
Example 12
Figure imgf000054_0003
(S)-3-formyl-5-methylhexanoic acid (Compound 10, Scheme II)
Compound 10 is made chiral (as shown) by an enzymatic transformation, not a chemical reduction. The reduction of compound 9 to afford compound 10 is catalyzed by an enzyme that selectively reduces a carbon-carbon double bond.
Example 13
Figure imgf000054_0004
Pregabalin (Compound 6, Scheme H)
Chemical Amination. (S)-3-Formyl-5-methylhexanoic acid is treated with an ammonia source followed by reduction to provide pregabalin.
Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.

Claims

CLAIMS:
1. A method for producing pregabalin, comprising: hydrolyzing a compound of formula I:
Figure imgf000055_0001
^ wnerein R is an alkyl, alkenyl or aryl, to afford an aldehyde intermediate of formula II
Figure imgf000055_0002
} wherein M is hydrogen or a salt; and subjecting the aldehyde intermediate of formula II to chemical or enzymatic amine transfer or animation conditions to yield pregabalin:
Figure imgf000055_0003
2. The method of claim 1, wherein the compound of formula I is prepared by reacting 4-methylpentanal with diisobutylamine and an alkyl-3-halo-propanoate.
3. A method for producing pregabalin, comprising: a) reacting diisobutylamine and 4-methylpentanal to produce an aldehyde intermediate of the structure
Figure imgf000055_0004
b) hydrolyzing the reaction product of step a) to yield a compound of formula II:
Figure imgf000055_0005
wherein M is hydrogen or a salt, which cyclizes to form a compound of the structure:
Figure imgf000055_0006
c) animating the reaction product of step b) to produce pregabalin.
4. A method for producing pregabalin, comprising: reacting 4-methylpentanal with diisobutylamine and an alkyl-3-halo- propanoate to produce an aldehyde intermediate of formula I:
Figure imgf000056_0001
aminating the aldehyde intermediate to produce a hexanoate intermediate of the structure:
Figure imgf000056_0002
cyclizing the hexanoate intermediate to produce a lactam of the structure:
Figure imgf000056_0003
treating the lactam with hydrochloric acid followed by addition of triethylamine to yield pregabalin.
5. A method for producing pregabalin, comprising: a) reacting 4-methylpentanal with glyoxylic acid hydrate to produce a compound of the following structure:
Figure imgf000056_0004
b) reducing asymmetrically the reaction product of step a) by chemical or enzymatic reduction to produce a compound of the following structure:
Figure imgf000056_0005
c) aminating the reaction product of step b) to produce pregabalin.
6. A method for producing pregabalin, comprising: a) treating 5-hydroxy-4-isobutylfuran-2(5H)-one with base to yield a compound of the structure:
Figure imgf000057_0001
t wherein M is hydrogen or a salt; b) enzymatically or chemically reducing the product of step a) to produce a compound of the structure:
Figure imgf000057_0002
, wherein M is hydrogen or a salt; and c) chemically or enzymatically amidating the product of step b) followed by reduction to provide pregabalin.
7. The method of any of claims 1-3, wherein the compound of formula II is a racemic mixture.
8. The method of any of claims 1 -3, wherein the compound of formula II is the S-enantiomer.
9. The method of any of claims 1, 2 and 4, wherein the compound of formula I is a racemic mixture.
10. The method of any of claims 1, 2 and 4, wherein the compound of formula I is the S-enantiomer.
11. The method of any of claims 1 -6, wherein the amination step is performed enzymatically to produce the S-enantiomer.
12. The method of claim 11, wherein the enzyme is a transaminase.
13. The method of claim 12, wherein the transaminase is selected from among an Alcaligenes denitrificans transaminase, a Bordetella bronch-iseptica transaminase, a Bordetella parapertussis transaminase, a Brucella melitensis transaminase, a Burkholderia mallei transaminase, a Burkholderia pseudomallei transaminase, a Chromobacterium violaceum transaminase, an Oceanicola granulosus HTCC2516 transaminase, an Oceanobacter sp. RED65 transaminase, an Oceanospirillum sp. MED92 transaminase, a Pseudomonas putida transaminase, a Ralstonia solanacearum transaminase, a Rhizobium meliloti transaminase, a Rhizobium sp. transaminase, a Vibrio fluvialis transaminase, a Bacillus thuringiensis transaminase and a Klebsiella pneuminiae transaminase.
14. The method of claim 11, wherein the transaminase comprises a sequence of amino acid residues as set forth in any of SEQ ID NOS: 12-26.
15. The method of any of claims 1-6, wherein the amination step is performed chemically to produce the S-enantiomer.
16. The method of claim 15, wherein the chemically amidating step comprises treating with an ammonia source.
17. The method of any of claims 1-3, wherein an enzyme hydrolyzes the compound of formula I.
18. The method of claim 17, wherein the enzyme is a hydrolase.
19. The method of claim 18, wherein the hydrolase is selected from among an esterase, a lipase and a protease.
20. The method of claim 18, wherein the enzyme is selected from among a bacterial hydrolase, a fungal hydrolase, an orange peel hydrolase, a porcine liver hydrolase, a rabbit liver hydrolase, a bovine pancreas hydrolase, a porcine pancreas hydrolase, a porcine stomach mucosal hydrolase, a bovine intestine hydrolase, a porcine intestine hydrolase, a porcine kidney hydrolase, a calf stomach hydrolase, a wheat germ hydrolase, a pineapple stem hydrolase and a papaya hydrolase.
21. The method of claim 18, wherein the hydrolase is selected from among an Achromobacter sp hydrolase, an Alcaligenes sp. hydrolase, an Aspergillus sp. hydrolase, an Apergillus niger hydrolase, an Apergillus oryzae hydrolase, an
Aspergillus melleus hydrolase, an Aspergillus mellus hydrolase, an Aspergillus niger hydrolase, an Aspergillus oryzae hydrolase, an Aspergillus saitoi hydrolase, an Aspergillus sojae hydrolase, a Bacillus sp. hydrolase, a Bacillus amyloliquefaciens hydrolase, a Bacillus lentus hydrolase, a Bacillus licheniformis hydrolase, a Bacillus polymyxa hydrolase, a Bacillus stearothermophilus hydrolase, a Bacillus subtilis hydrolase, a Bacillus thermoglucosidasius hydrolase, a Bacillus thermoproteolyticus rokko hydrolase, a Burkholderia sp. hydrolase, a Burkholderia cepacia hydrolase, a Candida sp. hydrolase, a Candida antarctica hydrolase, a Candida antarctica A hydrolase, a Candida antarctica B hydrolase, a Candida cylindracea hydrolase, a Candida lipolytica hydrolase, a Candida rugosa hydrolase, a Candidia utilis hydrolase, a Carica papaya hydrolase, a Chromobacterium viscosum hydrolase, a Clostridium histolyticum hydrolase, an E. coli hydrolase, a Geotrichum candidum hydrolase, a Mucor javanicus hydrolase, a Mucor miehei hydrolase, a Penicillium sp. hydrolase, a Penicillium sp. I hydrolase, a Penicillium sp. II hydrolase, a Penicillium camembertii hydrolase, a Penicillium roqueforti hydrolase, a Pseudomonas sp. hydrolase, a Pseudomonas aeruginosa hydrolase, a Pseudomonas cepacia hydrolase, a Pseudomonas fluorescens hydrolase, a Pseudomonas stutzeri hydrolase, a Pyrococcus furiosis hydrolase, a Rhizomucor miehei hydrolase, a Rhizopus sp. hydrolase, a Rhizopus arrhizus hydrolase, a Rhizopus delemar hydrolase, a Rhizopus niveus hydrolase, a Rhizopus oryzae hydrolase, a Saccharomyces cerevisiae hydrolase, a Schizophyllum commune hydrolase, a Streptomyces sp. hydrolase, a Streptomyces diastatochromogenes hydrolase, a Streptomyces griseus hydrolase, a Thermoanaerobium brockii hydrolase, a Thermomyces lanuginosus hydrolase and a Tritirachium album hydrolase.
22. The method of claim 19, wherein the esterase is selected from among an acetylesterase, a thiolester hydrolase, a phosphoric monoester hydrolase, a phosphoric diester hydrolase, a triphosphoric monoester hydrolase, a sulfuric ester hydrolase, a diphosphoric monoester hydrolase, a phosphoric triester hydrolase, an exonuclease and an endonuclease.
23. The method of claim 19, wherein the lipase is selected from among an Acinetobacter calcoaceticus lipase, an Acinetobacter sp. lipase, an Alcaligenes sp. lipase, an Aspergillus carneus lipase, an Aspergillus nidulans lipase, an Aspergillus niger lipase, an Aspergillus oryzae lipase, a Bacillus sp. lipase, a Bacillus stearothermophilus lipase, a Bacillus subtilis lipase, a Bacillus thermocatenulatus lipase, a Burkholderia sp. lipase, a Candida antarctica lipase, a Candida parapsilosis lipase, a Candida rugosa lipase, a Cephaloleia presignis lipase, a Chromobacterium viscosum lipase, a Homo sapiens lipase, a Mus musculus lipase, an Oryza sativa lipase, a Penicillium candidum lipase, a Penicillium roquefortii lipase, a Penicillium wortmanii lipase, a Pichia burtonii lipase, a Proteus vulgaris lipase, a Pseudomonas aeruginosa lipase, a Pseudomonas alcaligenes lipase, a Pseudomonas cepacia lipase, a Pseudomonas fluorescens lipase, a Pseudomonas pseudoalcaligenes lipase, a Pseudomonas sp. lipase, a Rattus norvegicus lipase, a Rhizomucor miehei lipase, a Rhizopus delemar lipase, a Rhizopus japonicus lipase, a Rhizopus oryzae lipase, a Rhodotorula glutinis lipase, a Saccharomyces cerevisiae lipase, a Staphylococcus epidermidis lipase, a Staphylococcus simulans lipase, a Streptomyces rimosus lipase, a Sus scrofa lipase, a Thermomyces lanuginose lipase, a Triticum aestivum lipase, a Staphylococcus warneri lipase, a Bacillus pumilus lipase and a Candida rugosa lipase.
24. The method of claim 19, wherein the protease is selected from among acrosin; blood coagulation factors VII, IX, X, XI and XII; thrombin; plasminogen; protein C; cathepsin G; a chymotrypsin; complement components CIr, CIs, C2, and complement factors B, D and I; complement-activating component of RA-reactive factor; cytotoxic cell proteases (granzymes A to H); duodenase I; elastases 1, 2, 3 A, 3B (protease E), leukocyte (medullasin); enterokinase (enteropeptidase); hepatocyte growth factor activator; hepsin; a glandular (tissue) kallikrein; plasma kallikrein; mast cell proteases (MCP) 1 (chymase) to 8; myeloblastin (proteinase 3); trypsins I, II, III, and IV; tryptases; a snake venom protease; ancrod; batroxobin; cerastobin; flavoxobin; collagenase from common cattle grub; collagenolytic protease from Atlantic sand fiddler crab; apolipoprotein(a); Blood fluke cercarial protease; Drosophila trypsin-like proteases: a, easter, snake-locus; Drosophila protease stubble (gene sb); a subtilisin from Bacillus sp.; alkaline elastase YaB from Bacillus sp.; alkaline serine exoprotease A from Vibrio alginolyticus; aqualysin I from Thermus aquaticus; AspA from Aeromonas salmonicida; bacillopeptidase F (esterase) from Bacillus subtilis; C5A peptidase from Streptococcus pyogenes; cell envelope-located proteases PI, PII, and PIII from Lactococcus lactis; extracellular serine protease from Serratia marcescens; extracellular protease from Xanthomonas campestris; intracellular serine protease (ISP) from various Bacillus; minor extracellular serine protease epr from Bacillus subtilis; minor extracellular serine protease vpr from Bacillus subtilis; nisin leader peptide processing protease nisP from Lactococcus lactis; serotype-specific antigene 1 from Pasteurella haemolytica; thermitase from Thermoactinomyces vulgaris; calcium- dependent protease from Anabaena variabilis; halolysin from halophilic bacteria sp. 172pl; alkaline extracellular protease (AEP) from Yarrowia lipolytica; alkaline proteinase from Cephalosporium acremonium; cerevisin (vacuolar protease B) from yeast; cuticle-degrading protease (prl) from Metarhizium anisopliae.; KEX-I protease from Kluyveromyces lactis. ; kexin from yeast; oryzin (alkaline proteinase) from Aspergillus; proteinase K from Tritirachium album; proteinase R from Tritirachium album; proteinase T from Tritirachium album; subtilisin-like protease III from yeast; and thermomycolin from Malbranchea sulfurea.
25. The method of claim 19, wherein the protease is a cysteine protease selected from among vertebrate lysosomal cathepsins B, H, L, and S; vertebrate lysosomal dipeptidyl peptidase I (also known as cathepsin C); vertebrate calpains; mammalian cathepsin K; human cathepsin O; bleomycin hydrolase; barley aleurain; EP-B1/B4; kidney bean EP-Cl; rice bean SH-EP; kiwi fruit actinidin; papaya latex papain; chymopapain; caricain; proteinase IV; pea turgor-responsive protein 15 A; pineapple stem bromelain; rape COT44; rice oryzain a, β, and γ, cathepsin B-like proteinases from the worms Caenorhabditis elegans, Schistosoma mansoni,
Haemonchus contortus, and Ostertagia ostertagi; slime mold cysteine proteinases CPl and CP2; cruzipain from Trypanosoma cruzi and bruce; throphozoite cysteine proteinase (TCP) from a Plasmodium sp.; Baculoviruses cathepsin-like enzyme (v- cath); Drosophila small optic lobes protein (gene sol); yeast thiol protease BLHl /YCP1/LAP3; aminopeptidase C from Lactococcus lactis; thiol protease tpr from Porphyromonas gingivalis; and mammalian caspases, caspase-1, -2, -3, -4, -5, - 6, -7, -8, -9, -10, -11, -12, 13, and -14.
26. The method of claim 18, wherein chirality is introduced in the compound by the hydrolase.
27. The method of claim 6, wherein the product of step a) is reduced by an enzyme selected from among an alkenal double bond reductase (Pl), an alkenal double bond reductase (P2), an NAD(P)H-dependent alkenal/one oxidoreductase (AOR), an NADP-dependent leukotriene B4 12-hydroxydehydrogenase, an aryl propenal double bond reductase, an NADP-dependent oxidoreductase and a medium- chain dehydrogenase/reductase.
28. The method of claim 6, wherein the product of step a) is reduced by an enzyme comprising a sequence of amino acid residues as set forth in any of SEQ ID NOS:1-11.
29. The method of any of claims 11-14 and 17-28, wherein the enzyme is in solution or is immobilized on a solid support.
30. The method of claim 29, wherein the solid support is selected from among glass, plastic, a film, nitrocellulose or a sol-gel polymer.
31. The method of any of claims 1 -30, wherein the reaction is performed in an organic or aqueous solvent.
32. The method of any of claims 1-30, wherein the reaction is performed in a 2-phase system or in an emulsion.
33. The method of claim 31 , wherein the organic solvent is selected from among diisopropyl ether, methyl tert-butyl ether (MTBE), dibutyl ether, and ethyl acetate.
34. The method of claim 31 , wherein the aqueous solvent comprises a buffer.
35. The method of claim 34, wherein the buffer is selected from among a glutamic acid-glutamate buffer, a phosphoric acid-phosphate buffer, an acetic acid- acetate buffer and a citric acid-citrate buffer.
36. A compound of formula I:
Figure imgf000062_0001
wherein: R is selected from among methyl, ethyl, n-butyl, i-butyl, pentyl, hexyl, heptyl, octyl, nonyl, Ci-C9 heteroalkyl, cycloalkyl, aryl and heteroaryl.
37. The compound of claim 36, wherein R is selected from among methyl, ethyl, H-butyl, i-butyl, pentyl, hexyl, heptyl, octyl and nonyl.
38. An article of manufacture, comprising: packaging material; a compound of claim 36 or an acceptable salt thereof, within the packaging material; and a label that indicates that the compound is used for the synthesis of pregabalin.
39. A kit, comprising: a hydrolase; and a compound of claim 36 or an acceptable salt thereof.
40. The kit of claim 39, wherein the hydrolase is selected from among an esterase, a lipase, and a protease.
41. The kit of claim 39, wherein the hydrolase is selected from among a bacterial hydrolase, a fungal hydrolase, an orange peel hydrolase, a porcine liver hydrolase, a rabbit liver hydrolase, a bovine pancreas hydrolase, a porcine pancreas hydrolase, a porcine stomach mucosal hydrolase, a bovine intestine hydrolase, a porcine intestine hydrolase, a porcine kidney hydrolase, a calf stomach hydrolase, a wheat germ hydrolase, a pineapple stem hydrolase and a papaya hydrolase.
42. The kit of claim 39, wherein the hydrolase is selected from among an
Achromobacter sp. hydrolase, an Alcaligenes sp. hydrolase, an Aspergillus sp. hydrolase, an Apergillus niger hydrolase, an Apergillus oryzae hydrolase, an Aspergillus melleus hydrolase, an Aspergillus mellus hydrolase, an Aspergillus niger hydrolase, an Aspergillus oryzae hydrolase, an Aspergillus saitoi hydrolase, an Aspergillus sojae hydrolase, a Bacillus sp. hydrolase, a Bacillus amyloliquefaciens hydrolase, a Bacillus lentus hydrolase, a Bacillus licheniformis hydrolase, a Bacillus polymyxa hydrolase, a Bacillus stearothermophilus hydrolase, a Bacillus subtilis hydrolase, a Bacillus thermoglucosidasius hydrolase, a Bacillus thermoproteolyticus rokko hydrolase, a Burkholderia sp. hydrolase, a Burkholderia cepacia hydrolase, a Candida sp. hydrolase, a Candida antarctica hydrolase, a Candida antarctica A hydrolase, a Candida antarctica B hydrolase, a Candida cylindracea hydrolase, a Candida lipolytica hydrolase, a Candida rugosa hydrolase, a Candidia utilis hydrolase, a Carica papaya hydrolase, a Chromobacterium viscosum hydrolase, a Clostridium histolyticum hydrolase, an E. coli hydrolase, a Geotrichum candidum hydrolase, a Mucor javanicus hydrolase, a Mucor miehei hydrolase, a Penicillium sp. hydrolase, a Penicillium sp. I hydrolase, a Penicillium sp. II hydrolase, a Penicillium camembertii hydrolase, a Penicillium roqueforti hydrolase, a Pseudomonas sp. hydrolase, a Pseudomonas aeruginosa hydrolase, a Pseudomonas cepacia hydrolase, a Pseudomonas fluorescens hydrolase, a Pseudomonas stutzeri hydrolase, a Pyrococcus furiosis hydrolase, a Rhizomucor miehei hydrolase, a Rhizopus sp. hydrolase, a Rhizopus arrhizus hydrolase, a Rhizopus delemar hydrolase, a Rhizopus niveus hydrolase, a Rhizopus oryzae hydrolase, a Saccharomyces cerevisiae hydrolase, a Schizophyllum commune hydrolase, a Streptomyces sp. hydrolase, a Streptomyces diastatochromogenes hydrolase, a Streptomyces griseus hydrolase, a Thermoanaerobium brockii hydrolase, a Thermomyces lanuginosus hydrolase and a Tritirachium album hydrolase.
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