HK1104024B - Preparation of pregabalin and related compounds - Google Patents

Description

Preparation of pregabalin and related compounds

Background

Technical Field

The present invention relates to methods and materials for the preparation of enantiomerically-enriched gamma-amino acids by enzymatic kinetic resolution, particularly useful for the preparation of peptides exhibiting human alpha₂Gamma-amino acids of delta calcium channel subunit binding affinity, including pregabalin and related compounds.

Discussion of the related Art

Pregabalin, (S) - (+) -3-aminomethyl-5-methyl-hexanoic acid, is associated with the endogenous inhibitory neurotransmitter gamma-aminobutyric acid (GABA), which is involved in the regulation of neuronal activity in the brain. Pregabalin exhibits anti-seizure activity, as discussed in U.S. patent No. 5,563,175 to r.b. silverman et al, and is believed to be useful in the treatment of, among other conditions, pain, physiological conditions associated with psychomotor stimulants, inflammation, gastrointestinal damage, alcoholism, insomnia and a variety of psychiatric disorders, including mania and bipolar disorders. See, respectively, U.S. patent No. 6,242,488 to l.bueno et al, U.S. patent No. 6,326,374 to l.magnus & c.a.segal, and U.S. patent No. 6,001,876 to l.singh; akune et al, U.S. patent No. 6,194,459; U.S. patent No. 6,329,429 to schrier et al; l.bueno et al, U.S. patent No. 6,127,418; l.bueno et al, U.S. patent No. 6,426,368; U.S. Pat. nos. 6,306,910 to l.magnus & c.a.segal; and U.S. patent No. 6,359,005 to a.c. pande, which is incorporated herein by reference in its entirety and for all purposes.

Pregabalin has been prepared in a number of ways. Typically, a racemic mixture of 3-aminomethyl-5-methyl-hexanoic acid is synthesized and subsequently resolved into its R-and S-enantiomers. Such methods may employ azide intermediates, malonate intermediates, or hofmann synthesis. See, respectively, U.S. patent nos. 5,563,175 to r.b. silverman et al; t.m. grote et al, U.S. patent nos. 6,046,353, 5,840,956, and 5,637,767; and U.S. patent nos. 5,629,447 and 5,616,793 to b.k.huckabee & d.m. sobieray, which are incorporated herein by reference in their entirety and for all purposes. In each of these processes, the racemate is reacted with a chiral acid (resolving agent) to form a pair of diastereomeric salts, which are separated by known techniques, such as fractional crystallization and chromatography. These processes thus involve a processing which is significantly more than the preparation of the racemate, which, together with the resolving agent, adds to the production costs. Furthermore, the undesired R-enantiomer is often discarded because it cannot be effectively reused, thus reducing the effective yield of the process by 50%.

Pregabalin has also been synthesized directly using the chiral auxiliary (4R, 5S) -4-methyl-5-phenyl-2-oxazolidinone. See, for example, U.S. patent nos. 6,359,169, 6,028,214, 5,847,151, 5,710,304, 5,684,189, 5,608,090, and 5,599,973 to r.b. silverman et al, which are incorporated herein by reference in their entirety and for all purposes. Although these methods provide pregabalin of high enantiomeric purity, they are less desirable for large scale synthesis because they employ relatively expensive reagents that are difficult to handle (e.g., chiral auxiliaries), and special cryogenic equipment for reaching the required operating temperatures (which can be as low as-78 ℃).

A recently published U.S. patent application discusses a process for preparing pregabalin by asymmetric hydrogenation of cyano-substituted olefins to produce chiral cyano precursors of (S) -3-aminomethyl-5-methylhexanoic acid. See commonly assigned U.S. patent application No. 2003/0212290a1 to Burk et al, published on 11/13/2003, which is incorporated herein by reference in its entirety for all purposes. The cyano precursor is subsequently reduced to form pregabalin. Asymmetric hydrogenation employs chiral catalysts comprising a transition metal, such as (R, R) -Me-DUPHOS, bound to a bisphosphine ligand. The method results in a substantial enrichment of pregabalin relative to (R) -3- (aminomethyl) -5-methylhexanoic acid.

The process discussed in U.S. patent application No. 2003/0212290a1 represents a commercially viable process for the preparation of pregabalin, but for various reasons further improvements may be desirable. For example, bisphosphine ligands, including the proprietary ligand (R, R) -Me-DUPHOS, are often difficult to prepare because they have 2 chiral centers, which increases their cost. Moreover, asymmetric hydrogenation requires the use of special equipment capable of handling H2, which increases capital costs.

Brief description of the invention

The present invention provides materials and methods for preparing enantiomerically enriched γ -amino acids (formula 1), such as pregabalin (formula 9). The process of the present invention comprises the kinetic resolution of a racemic cyanodiester intermediate (formula 4 or formula 12) using an enzyme suitable for enantioselectively hydrolyzing the ester moiety of the intermediate. The resulting substantially enantiomerically pure dicarboxylic acid monoester (formula 3 or formula 11) undergoes further reaction to produce the desired enantiomerically-enriched γ -amino acid (formula 1 or formula 9). The unreacted enantiomer (formula 5 or formula 13) from the kinetic resolution can be reused in the enzymatic resolution after racemization, thereby increasing the overall yield.

The claimed process provides significant advantages over existing methods for preparing enantiomerically-enriched γ -amino acids (formula 1 and formula 9). For example, optically active γ -amino acids can be prepared without the use of chiral auxiliaries or proprietary hydrogenation catalysts, which results in lower unit costs. Since the enzymatic process can be carried out at room temperature and atmospheric pressure, the claimed process helps to minimize scheduling conflicts resulting from the use of specialized equipment capable of handling high and low pressures. As indicated in the examples, the present invention can be used to prepare pregabalin in good yields (26% to 31%) starting from the racemic cyano-substituted diester (formula 12) after a single batch recycle of the unreacted enantiomer (formula 13). This translates into an article cost savings of about 50% compared to the malonate approach described above.

One aspect of the present invention provides a process for preparing a compound of formula 1, or a pharmaceutically acceptable complex, salt, solvate or hydrate thereof,

wherein

R¹And R²Are different from each other and are each independently selected from hydrogen atoms, C_1-12Alkyl radical, C_3-12Cycloalkyl radicalsAnd substituted C_3-12A cycloalkyl group,

the method comprises the following steps:

(a) reacting a compound of formula 2 or a salt thereof with an acid and water,

to produce a compound of formula 1 or a salt thereof; and

(b) optionally converting the compound of formula 1 or a salt thereof, wherein R in formula 2 is to a pharmaceutically acceptable complex, salt, solvate or hydrate¹And R²The same as defined in formula 1.

Another aspect of the present invention provides a process for preparing a compound of formula 1 above, comprising:

(a) reducing the cyano moiety of the compound of formula 6 or a salt thereof,

to produce the compound of formula 7 or a salt thereof,

(b) decarboxylating the compound of formula 7 or a salt thereof to produce a compound of formula 1 or a salt thereof; and

(c) optionally converting the compound of formula 1 or a salt thereof, wherein R in formula 6 and formula 7, into a pharmaceutically acceptable complex, salt, solvate or hydrate¹And R²As defined above in formula 1.

The compound of the above formula 6 can be prepared by hydrolyzing the compound of the formula 3 or a salt thereof,

wherein R in formula 3¹And R²Is as defined above in formula 1, and R³Is C_1-12Alkyl radical, C_3-12Cycloalkyl, or aryl-C_1-6An alkyl group.

Another aspect of the present invention provides a process for preparing a compound of formula 1 above, comprising:

(a) reducing the cyano moiety of the compound of formula 8 or a salt thereof,

to produce a compound of formula 1 or a salt thereof; and

(b) optionally converting the compound of formula 1 or a salt thereof, wherein R in formula 8, into a pharmaceutically acceptable complex, salt, solvate or hydrate¹And R²The same as defined above in formula 1, and R in formula 8⁵Is a hydrogen atom, C_1-12Alkyl radical, C_3-12Cycloalkyl or aryl-C_1-6An alkyl group.

The compound of formula 8 can be prepared by decarboxylating the above compound of formula 3 or a salt thereof, or by hydrolyzing the compound of formula 3 or a salt thereof and decarboxylating it to produce the compound of formula 8 or a salt thereof.

Another aspect of the present invention provides a process for preparing a compound of formula 3 above or a salt thereof, which comprises:

(a) contacting a compound of formula 4 with an enzyme,

to generate the compound shown in the formula 3 and the compound shown in the formula 5,

wherein the enzyme is suitable for enantioselectively hydrolyzing the compound of formula 4 to the compound of formula 3 or a salt thereof;

(b) isolating a compound of formula 3 or a salt thereof; and

(c) optionally racemic formula 5 to produce a compound of formula 4, wherein R in formula 4 and formula 5¹，R²And R³The same as defined above in formula 1 and formula 3; and R in formula 4 and formula 5⁴And R³Are the same or different and are C_1-12Alkyl radical, C_3-12Cycloalkyl or aryl-C_1-6An alkyl group.

Any number of enzymes can be used to enantioselectively hydrolyze a compound of formula 4 to a compound of formula 3 or a salt thereof. Useful enzymes include lipases, such as those derived from Thermomyces lanuginosus.

Another aspect of the present invention provides a compound represented by formula 2 above, including a complex, salt, solvate or hydrate thereof, with the proviso that when R in formula 2 is¹Or R²When one of the substituents is hydrogen, the other substituent is not C_1-3Alkyl or C₅An alkyl group.

Another aspect of the present invention provides a compound of formula 27,

including complexes, salts, solvates or hydrates thereof, wherein

R¹And R²Are different from each other and are each independently selected from hydrogen atoms, C_1-12Alkyl radical, C_3-12Cycloalkyl and substituted C_3-12Cycloalkyl with the proviso that when represented by R¹Or R²When one of the substituents is a hydrogen atom, the other substituent is not a methyl group; and

R⁵and R⁶Independently selected from hydrogen atoms, C_1-12Alkyl radical, C_3-12Cycloalkyl or aryl-C_1-6Alkyl, provided that R is⁵And R⁶If not hydrogen atoms, they are different.

Compounds of formula 27 include those represented by formula 3, formula 4, formula 5, formula 6, and formula 7 above, including complexes, salts, solvates, or hydrates thereof. Useful compounds of formulae 2-7 and 27 include those wherein R¹Is a hydrogen atom, and R²Are those of isobutyl.

Another aspect of the present invention provides a process for preparing a compound of formula 9, or a pharmaceutically acceptable complex, salt, solvate or hydrate thereof,

the method comprises the following steps:

(a) reacting a compound of formula 10 or a salt thereof with an acid and water,

to produce a compound of formula 9 or a salt thereof; and

(b) optionally converting the compound of formula 9 or a salt thereof into a pharmaceutically acceptable complex, salt, solvate or hydrate.

Another aspect of the present invention provides a process for preparing a compound of formula 9 above, or a pharmaceutically acceptable complex, salt, solvate or hydrate thereof, which comprises:

(a) reducing the cyano moiety of the compound of formula 14 or a salt thereof,

to produce the compound of the formula 15,

or a salt thereof;

(b) decarboxylating the compound of formula 15 or a salt thereof to produce a compound of formula 9 or a salt thereof; and

(c) optionally converting the compound of formula 9 or a salt thereof into a pharmaceutically acceptable complex, salt, solvate or hydrate.

The compound of the above formula 14 can be prepared by hydrolyzing the compound of the formula 11 or a salt thereof,

wherein R in formula 11³As defined above in formula 3.

Another aspect of the present invention provides a process for preparing a compound of formula 9 above, or a pharmaceutically acceptable complex, salt, solvate or hydrate thereof, which comprises:

(a) reducing the cyano moiety of the compound of formula 16 or a salt thereof,

to produce a compound of formula 9 or a salt thereof; and

(b) optionally converting the compound of formula 9 or a salt thereof, wherein R in formula 16 is to a pharmaceutically acceptable complex, salt, solvate or hydrate⁵As defined above in formula 8.

The compound of formula 16 can be prepared by decarboxylating the above compound of formula 11 or a salt thereof (e.g., by heating), or by hydrolyzing the compound of formula 11 or a salt thereof and decarboxylating it.

Another aspect of the present invention provides a process for preparing a compound of formula 11 above or a salt thereof, which comprises:

(a) contacting a compound of formula 12 with an enzyme,

to produce the compound of formula 11 and the compound of formula 13,

wherein the enzyme is suitable for enantioselectively hydrolyzing the compound of formula 12 to a compound of formula 11 or a salt thereof;

(b) isolating a compound of formula 11 or a salt thereof; and

(c) optionally racemic formula 13 to produce a compound of formula 12, wherein

R in formula 12 and formula 13³The same as defined above in formula 3; and is

R in formula 12 and formula 13⁴And R³Are the same or different and are C_1-12Alkyl radical, C_3-12Cycloalkyl or aryl-C_1-6An alkyl group.

In the process for preparing the compound of formula 11, the corresponding salts of the compound of formula 11 include those selected from the group consisting of: alkali metal salts, such as potassium salts; primary amine salts, such as tertiary butylamine; and secondary amine salts. Furthermore, useful enzymes include lipases, such as those derived from Thermomyces lanuginosus.

Another aspect of the invention provides a compound selected from the group consisting of:

3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid,

(3S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid,

(2S, 3S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid,

(2R, 3S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid,

3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid ethyl ester,

(R) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid ethyl ester,

4-isobutyl-2-oxo-pyrrolidine-3-carboxylic acid,

(S) -4-isobutyl-2-oxo-pyrrolidine-3-carboxylic acid,

3-cyano-2-carboxy-5-methyl-hexanoic acid,

(S) -3-cyano-2-carboxy-5-methyl-hexanoic acid,

3-aminomethyl-2-carboxy-5-methyl-hexanoic acid, and

(S) -3-aminomethyl-2-carboxy-5-methyl-hexanoic acid,

including complexes, salts, solvates and hydrates thereof and the opposite enantiomer thereof.

The present invention includes all complexes and salts of the disclosed compounds, whether pharmaceutically acceptable, solvates, hydrates and polymorphs. Certain compounds may contain alkenyl or cyclic groups such that cis/trans (or Z/E) stereoisomers are possible, or may contain keto or oxime groups such that tautomerism may occur. In such cases, the invention generally includes all Z/E isomers and tautomeric forms, whether they be pure, substantially pure, or mixtures.

Brief Description of Drawings

FIG. 1 depicts a scheme for preparing an enantiomerically-enriched γ -amino acid (formula 1).

FIG. 2 depicts a scheme for preparing a cyano-substituted diester (formula 4).

Detailed Description

Definitions and abbreviations

Unless otherwise indicated, the present disclosure uses the definitions provided below. Some definitions and formulas may contain dashes ("-") to indicate bonds between atoms or points of attachment to named or unnamed atoms or groups of atoms. Other definitions and formulas may include an equal sign ("═ or") or a balanced identification ("≡") to indicate a double bond or a triple bond, respectively. Certain formulas may also contain one or more asterisks ("") to indicate stereogenic (asymmetric or chiral) centers, although the absence of an asterisk does not indicate that the compound lacks a stereogenic center. Such formula may refer to a racemate or an individual enantiomer or individual diastereomer, which may or may not be pure or substantially pure.

"substituted" groups are those in which one or more hydrogen atoms are replaced with one or more non-hydrogen groups, provided that the valence requirements are met, and the substitution results in a chemically stable compound.

When used in conjunction with a measurable digital variable, "about" or "approximately" refers to an indicator value for the variable and also refers to all values of the variable that are within experimental error of the indicator value (e.g., within 95% confidence interval of the mean) or within ± 10% of the indicator value (whichever is larger).

"alkyl" refers to straight and branched saturated hydrocarbon groups, typically having the indicated number of carbon atoms (i.e., C)_1-6Alkyl means an alkyl group having 1, 2, 3, 4, 5 or 6 carbon atoms, and C_1-12Alkyl refers to alkyl groups having 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, or 12 carbon atoms). Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pent-1-yl, pent-2-yl, pent-3-yl, 3-methylbutan-1-yl, 3-methylbutan-2-yl, 2, 2, 2-trimethylethan-1-yl, n-hexyl, and the like.

"alkenyl" refers to straight and branched hydrocarbon groups having one or more unsaturated carbon-carbon bonds, and typically having the indicated number of carbon atoms. Examples of alkenyl groups include, but are not limited to, vinyl, 1-propen-1-yl, 1-propen-2-yl, 2-propen-1-yl, 1-buten-2-yl, 3-buten-1-yl, 3-buten-2-yl, 2-buten-1-yl, 2-buten-2-yl, 2-methyl-1-propen-1-yl, 2-methyl-2-propen-1-yl, 1, 3-butan-2-yl, and the like.

"alkynyl" refers to a straight or branched hydrocarbon group having one or more three-carbon bonds and typically having the indicated number of carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propyn-1-yl, 2-propyn-1-yl, 1-butyn-1-yl, 3-butyn-2-yl, 2-butyn-1-yl, and the like.

"alkanoyl" and "alkanoylamino" refer to alkyl-C (O) -and alkyl-C (O) -NH-, respectively, wherein alkyl is as defined above and typically contains the indicated number of carbon atoms, including the carbonyl carbon. Examples of alkanoyl groups include, but are not limited to, formyl, acetyl, propionyl, butyryl, pentanoyl, hexanoyl, and the like.

"alkenoyl" and "alkynoyl" refer to alkenyl-C (O) -and alkynyl-C (O) -respectively, wherein alkenyl and alkynyl are as defined above. References to alkenoyl and alkynoyl groups generally contain the indicated number of carbon atoms, excluding the carbonyl carbon. Examples of alkenoyl include, but are not limited to, acryloyl, 2-methacryloyl, 2-butenoyl, 3-butenoyl, 2-methyl-2-butenoyl, 2-methyl-3-butenoyl, 3-methyl-3-butenoyl, 2-pentenoyl, 3-pentenoyl, 4-pentenoyl, and the like. Examples of alkynoyl include, but are not limited to, propioyl, 2-butynoyl, 3-butynoyl, 2-pentynoyl, 3-pentynoyl, 4-pentynoyl, and the like.

"alkoxy", "alkoxycarbonyl" and "alkoxycarbonylamino" refer to alkyl-O-, alkenyl-O, and alkynyl-O, respectively; alkyl-O-C (O) -, alkenyl-O-C (O) -, alkynyl-O-C (O) -; and alkyl-O-C (O) -NH-, alkenyl-O-C (O) -NH-, and alkynyl-O-C (O) -NH-, wherein alkyl, alkenyl, and alkynyl are as defined above. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, sec-pentoxy, and the like. Examples of alkoxycarbonyl include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, n-butoxycarbonyl, sec-butoxycarbonyl, tert-butoxycarbonyl, n-pentyloxycarbonyl, sec-pentyloxycarbonyl, and the like.

"alkylamino", "alkylaminocarbonyl", "dialkylaminocarbonyl", "alkylsulfonyl", "sulfonylaminoalkyl" and "alkylsulfonylaminocarbonyl" refer to alkyl-NH-, alkyl-NH-C (O) -, respectively₂-N-C (O) -, alkyl-S (O)₂)-，HS(O₂) -NH-alkyl-, and alkyl-S (O) -NH-C (O) -, wherein alkyl is as defined above.

"aminoalkyl" and "cyanoalkyl" refer to NH, respectively₂-alkyl and N ≡ C-alkyl, wherein alkyl is as defined above.

"halo," "halogen," and "halogen" are used interchangeably and refer to fluoro, halo, bromo, and iodo.

"haloalkyl," "haloalkenyl," "haloalkynyl," "haloalkanoyl," "haloalkenoyl," "haloalkynoyl," "haloalkoxyalkoxy," and "haloalkoxycarbonyl" each refer to alkyl, alkenyl, alkynyl, alkanoyl, alkenoyl, alkynoyl, alkoxy, and alkoxycarbonyl groups, respectively, substituted with one or more halogen atoms, wherein alkyl, alkenyl, alkynyl, alkanoyl, alkenoyl, alkynoyl, alkoxy, and alkoxycarbonyl groups are as defined above. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl, and the like.

"hydroxyalkyl" and "oxoalkyl" refer to HO-alkyl and O ═ alkyl, respectively, wherein alkyl is as defined above. Examples of hydroxyalkyl and oxoalkyl include, but are not limited to, hydroxymethyl, hydroxyethyl, 3-hydroxypropyl, oxomethyl, oxoethyl, 3-oxopropyl, and the like.

"cycloalkyl" refers to saturated monocyclic and bicyclic hydrocarbon rings, typically having the indicated number of ring-forming carbon atoms (i.e., C)_3-7Cycloalkyl refers to cycloalkyl having 3, 4, 5,6, or 7 carbon atoms as ring members). The cycloalkyl group may be bonded to the parent group or substrate at any ring atom unless such bonding would violate valence requirements. Likewise, a cycloalkyl group may contain one or more substituents other than hydrogen unless such substitution would violate valence requirements. Useful substituents include, but are not limited to, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, alkoxy, alkoxycarbonyl, alkanoyl, and halo, as defined above, and hydroxy, mercapto, nitro, and amino.

Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. Examples of bicyclic cycloalkyl groups include, but are not limited to, bicyclo [1.1.0] butyl, bicyclo [1.1.1] pentyl, bicyclo [2.1.0] pentyl, bicyclo [2.1.1] hexyl, bicyclo [3.1.0] hexyl, bicyclo [2.2.1] heptyl, bicyclo [3.2.0] heptyl, bicyclo [3.1.1] heptyl, bicyclo [4.1.0] heptyl, bicyclo [2.2.2] octyl, bicyclo [3.2.1] octyl, bicyclo [4.1.1] octyl, bicyclo [3.3.0] octyl, bicyclo [4.2.0] octyl, bicyclo [3.3.1] nonyl, bicyclo [4.2.1] nonyl, bicyclo [4.3.0] nonyl, bicyclo [3.3.2] decyl, bicyclo [4.2.2] decyl, bicyclo [4.3.1] decyl, bicyclo [4.4.0] decyl, bicyclo [3.3.2] undecyl, bicyclo [3.3.0] undecyl, and the like, it may be bonded to the parent group or substrate at any ring atom unless such bonding would violate valence requirements.

"cycloalkenyl" refers to monocyclic and bicyclic hydrocarbon rings having one or more unsaturated carbon-carbon bonds, and typically having the indicated number of ring-forming carbon atoms (i.e., C)_3-7Cycloalkenyl refers to cycloalkenyl having 3, 4, 5,6 or 7 carbon atoms as ring members). The cycloalkenyl group may be bonded to the parent group or substrate at any ring atom unless such bonding would violate valence requirements. Likewise, cycloalkenyl groups can contain one or more substituents other than hydrogen unless such substitution would violate the valence requirement. Useful substituents include, but are not limited to, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, alkoxy, alkoxycarbonyl, alkanoyl, and halo, as defined above, and hydroxy, mercapto, nitro, and amino.

"Cycloalkanoyl" and "cycloalkenoyl" refer to cycloalkyl-C (O) -and cycloalkenyl-C (O) -respectively, wherein cycloalkyl and cycloalkenyl are as defined above. References to cycloalkanoyl and cycloalkenoyl groups generally contain the indicated number of carbon atoms, excluding the carbonyl carbons. Examples of cycloalkanoyl include, but are not limited to, cyclopropanoyl, cyclobutanoyl, cyclopentanoyl, cyclohexanoyl, cycloheptanoyl, 1-cyclobutenoyl, 2-cyclobutenoyl, 1-cyclopentenoyl, 2-cyclopentenoyl, 3-cyclopentenoyl, 1-cyclohexenoyl, 2-cyclohexenoyl, 3-cyclohexenoyl, and the like.

"Cycloalkoxy" and "cycloalkoxycarbonyl" refer to cycloalkyl-O-and cycloalkenyl-O and to cycloalkyl-O-C (O) -and cycloalkenyl-O-C (O) -respectively, wherein cycloalkyl and cycloalkenyl are as defined above. References to cycloalkoxy and cycloalkoxycarbonyl groups generally include the indicated number of carbon atoms, excluding the carbonyl carbon. Examples of cycloalkoxy groups include, but are not limited to, cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexoxy, 1-cyclobutenoxy, 2-cyclobutenoxy, 1-cyclopentenyloxy, 2-cyclopentenyloxy, 3-cyclopentenyloxy, 1-cyclohexenyloxy, 2-cyclohexenyloxy, 3-cyclohexenyloxy, and the like. Examples of the cycloalkoxycarbonyl group include, but are not limited to, cyclopropyloxycarbonyl, cyclobutyloxycarbonyl, cyclopentyloxycarbonyl, cyclohexyloxycarbonyl, 1-cyclobutenyloxycarbonyl, 2-cyclobutenyloxycarbonyl, 1-cyclopentenyloxycarbonyl, 2-cyclopentenyloxycarbonyl, 3-cyclopentenyloxycarbonyl, 1-cyclohexenyloxycarbonyl, 2-cyclohexenyloxycarbonyl, 3-cyclohexenyloxycarbonyl, and the like.

"aryl" and "arylene" refer to monovalent and divalent aromatic radicals, respectively, including 5-and 6-membered monocyclic aromatic radicals containing 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Examples of monocyclic aryl groups include, but are not limited to, phenyl, pyrrolyl, furanyl, thienyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, and the like. Aryl and arylene also include bicyclic groups, tricyclic groups, and the like, including fused 5-and 6-membered rings as described above. Examples of polycyclic aryl groups include, but are not limited to, naphthyl, biphenyl, anthracenyl, pyrenyl, carbazolyl, benzoxazolyl, benzodioxazolyl, benzothiazolyl, benzimidazolyl, benzothienyl, quinolinyl, isoquinolinyl, indolyl, benzofuranyl, purinyl, indolizinyl, and the like. These aryl and arylene groups may be bonded to the parent group or substrate at any of the ring atoms unless such bonding would violate valence requirements. Likewise, aryl and arylene groups may contain one or more substituents other than hydrogen, unless such substitution would violate valence requirements. Useful substituents include, but are not limited to, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, alkanoyl, cycloalkanoyl, cycloalkenoyl, alkoxycarbonyl, cycloalkoxycarbonyl, and halo, as defined above, and hydroxy, mercapto, nitro, amino, and alkylamino.

"heterocycle" and "heterocyclyl" refer to a saturated, partially unsaturated, or unsaturated monocyclic or bicyclic ring having 5-7 or 7-11 ring members, respectively. These groups have ring members consisting of carbon atoms and 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and may comprise any bicyclic group in which any of the monocyclic heterocyclic rings defined above is fused to a benzene ring. The nitrogen and sulfur heteroatoms may optionally be oxidized. The heterocyclic ring may be attached to the parent group or substrate at any heteroatom or carbon atom unless such attachment would violate valence requirements. Likewise, any of the carbon or nitrogen ring members may contain substituents other than hydrogen unless such substitution would violate valence requirements. Useful substituents include, but are not limited to, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, alkanoyl, cycloalkanoyl, cycloalkenoyl, alkoxycarbonyl, cycloalkoxycarbonyl, and halo, as defined above, and hydroxy, mercapto, nitro, amino, and alkylamino.

Examples of heterocycles include, but are not limited to, acridinyl, an azocine gene, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4 aH-carbazolyl, carbolinyl, chromanyl, benzopyranyl, 1, 2-diazanaphthyl, decahydroquinolinyl, 2H, 6H-1, 5, 2-dithiazinyl, dihydrofuro [2, 3-b ] tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, pseudoindolyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isobenzofuranyl, isobenzodihydropyranyl, isoindolyl, isoindolinyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1, 2, 3-oxadiazolyl, 1, 2, 4-oxadiazolyl, 1, 2, 5-oxadiazolyl, 1, 3, 4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, orthophenanthhenanthrenyl, phenazinyl, phenothiazinyl (phenoxathiinyl), phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, quinazolinyl, quinolyl, 4H-quinolizyl, quinoxalinyl, quinuclidinyl, tetrahydrofuryl, tetrahydroisoquinolyl, tetrahydroquinolyl, 6H-1, 2, 5-thiadiazinyl, 1, 2, 3-thiadiazolyl, 1, 2, 4-thiadiazolyl, 1, 2, 5-thiadiazolyl, 1, 3, 4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1, 2, 3-triazolyl, 1, 2, 4-triazolyl, 1, 2, 5-triazolyl, 1, 3, 4-triazolyl, and xanthenyl.

"heteroaryl" and "heteroarylene" refer to monovalent and divalent heterocyclic or heterocyclic groups, respectively, as defined above, which are aromatic. Heteroaryl and heteroarylene represent a subset of aryl and arylene groups, respectively.

"arylalkyl" and "heteroarylalkyl" refer to aryl-alkyl and heteroaryl-alkyl, respectively, wherein aryl, heteroaryl, and alkyl are as defined above. Examples include, but are not limited to, benzyl, fluorenylmethyl, imidazol-2-yl-methyl, and the like.

"arylalkanoyl", "heteroarylalkanoyl", "arylalkenoyl", "heteroarylalkenoyl", "arylalkynylacyl", and "heteroarylalkynoyl" refer to aryl-alkanoyl, heteroaryl-alkanoyl, aryl-alkenoyl, heteroaryl-alkenoyl, aryl-alkynoyl, and heteroaryl-alkynoyl, respectively, wherein aryl, heteroaryl, alkanoyl, alkenoyl, and alkynoyl are as defined above. Examples include, but are not limited to, benzoyl, benzylcarbonyl, fluorenylmethylcarbonyl, imidazol-2-yl-methylcarbonyl, styrenecarbonyl, 1-phenyl-propylenecarbonyl, 2-phenyl-propylenecarbonyl, 3-phenyl-propylenecarbonyl, imidazol-2-yl-ethylenecarbonyl, 1- (imidazol-2-yl) -propylenecarbonyl, 2- (imidazol-2-yl) -propylenecarbonyl, 3- (imidazol-2-yl) -propylenecarbonyl, phenylacetylenecarbonyl, phenylpropylenecarbonyl, (imidazol-2-yl) -acetylenecarbonyl, (imidazol-2-yl) -propynylcarbonyl, and the like.

"arylalkoxy" and "heteroarylalkoxy" refer to aryl-alkoxy and heteroaryl-alkoxy, respectively, wherein aryl, heteroaryl, and alkoxy are as defined above. Examples include, but are not limited to, benzyloxy, fluorenylmethyloxy, imidazol-2-yl-methyloxy, and the like.

"aryloxy" and "heteroaryloxy" refer to aryl-O-and heteroaryl-O-, respectively, wherein aryl and heteroaryl are as defined above. Examples include, but are not limited to, phenoxy, imidazol-2-yloxy, and the like.

"Aryloxycarbonyl," "heteroaryloxycarbonyl," "arylalkoxycarbonyl," and "heteroarylalkoxycarbonyl" refer to aryloxy-C (O) -, heteroaryloxy-C (O) -, arylalkoxy-C (O) -, and heteroarylalkoxy-C (O) -, respectively, wherein aryloxy, heteroaryloxy, arylalkoxy, and heteroarylalkoxy are as defined above. Examples include, but are not limited to, phenoxycarbonyl, imidazol-2-yloxycarbonyl, benzyloxycarbonyl, fluorenylmethyloxycarbonyl, imidazol-2-yl-methyloxycarbonyl, and the like.

"leaving group" refers to any group that leaves the molecule during the cleavage process, including substitution, elimination, and addition-elimination reactions. Leaving groupThe group may be nucleofugic, in which the group leaves with a pair of electrons that previously acted as a bond between the leaving group and the molecule, or may be ionogenic, in which the group leaves without an electron pair. The leaving ability of a nucleofugic leaving group depends on its base strength, with the strongest base being the worst leaving group. Common nucleofugal leaving groups include nitrogen (e.g., from diazonium salts); sulfonates, including alkylsulfonates (e.g., methanesulfonate), fluoroalkylsulfonates (e.g., trifluoromethylsulfonate, hexafluoromethylsulfonate, nonafluoromethylsulfonate, and trifluoroethylsulfonate), and arylsulfonates (e.g., tosylate, brosylate, chlorobenzenesulfonate, and p-nitrobenzenesulfonate). Others include carbonate, halide ions, carboxylate anions, phenolate ions, and alkoxides. By treatment with acids, some stronger bases such as NH can be made₂ ^-And OH^-Becomes a better leaving group. Common electron leaving groups include protons, CO₂And a metal.

"enantiomeric excess" or "ee" is a measure, for a given sample, of the amount of an enantiomer in excess of that of a racemic sample of a chiral compound, and is expressed as a percentage. Enantiomeric excess is defined as 100 × (er-1)/(er +1), where "er" is the ratio of the richer enantiomer to the less abundant enantiomer.

"diastereomeric excess" or "de" is a measure, for a given sample, of the amount of one diastereomer over a sample having an equivalent amount of diastereomer, and is expressed as a percentage. Diastereomeric excess is defined as 100 × (dr-1)/(dr +1), where "dr" is the ratio of the more abundant diastereomer to the less abundant diastereomer.

"stereoselective," "enantioselective," "diastereoselective," and variations thereof, refer to a given process (e.g., ester hydrolysis, hydrogenation, hydroformylation, pi-allylpalladium coupling, hydrosilation, hydrocyanation, olefin transfer, hydroacylation, allylamine isomerization, etc.) that produces one stereoisomer, enantiomer, or diastereomer, respectively, over another.

"high level of enantioselectivity," "high level of diastereoselectivity," and variants thereof, refer to a given process that produces a product with an excess of one stereoisomer, enantiomer, or diastereomer, which comprises at least about 90% of the product. For enantiomeric or diastereomeric pairs, high levels of enantioselectivity or diastereoselectivity correspond to at least about 80% ee or de.

"stereoisomerically enriched," enantiomerically enriched, "diastereomerically enriched," and variations thereof refer to a sample of a compound having one stereoisomer, enantiomer or diastereomer, respectively, more than the other. The degree of enrichment can be measured by% of total product, or for enantiomeric or diastereomeric pairs, by ee or de.

"substantially pure stereoisomers," "substantially pure enantiomers," "substantially pure diastereomers," and variations thereof, respectively, refer to samples that contain stereoisomers, enantiomers, or diastereomers, which comprise at least about 95% of the sample. For enantiomeric and diastereomeric pairs, a substantially pure enantiomer or diastereomer corresponds to a sample having an ee or de of about 90% or greater.

"pure stereoisomers," "pure enantiomers," "pure diastereomers," and variations thereof, respectively, refer to samples that contain stereoisomers, enantiomers, or diastereomers, which comprise at least about 99.5% of the sample. For enantiomeric and diastereomeric pairs, a pure enantiomer or pure diastereomer "corresponds to a sample having an ee or de of about 99% or greater.

"opposite enantiomer" refers to a molecule that is a non-superimposable mirror image of a reference molecule, which can be obtained by inverting all stereogenic centers of the reference molecule. For example, if the reference molecule has S absolute stereochemical configuration, the opposite enantiomer has R absolute stereochemical configuration. Similarly, if the reference molecule has S, S absolute stereochemical configuration, the opposite enantiomer has R, R stereochemical configuration, and so on.

The "stereoisomers" of a given compound refer to the opposite enantiomer of that compound, and any diastereomer or geometric isomer (Z/E) of that compound. For example, if a given compound has S, R, Z stereochemical configuration, its stereoisomers include its opposite enantiomer having R, S, Z configuration, its diastereomer having S, Z configuration and R, Z configuration, and its geometric isomers having S, R, E configuration, R, S, E configuration, S, E configuration, and R, E configuration.

"enantioselectivity value" or "E" refers to the ratio of the specificity constants for each enantiomer of a compound undergoing chemical reaction or conversion, and can be calculated from the following expression (for the S-enantiomer),

wherein K_SAnd K_R1 order velocity constants for the conversion of the S-and R-enantiomers, respectively; k_SMAnd K_RMThe Michaelis constants of the S-and R-enantiomers, respectively; x is the conversion of the substrate; ee_pAnd ee_sThe enantiomeric excess of the product and substrate (reactant), respectively.

"Lipase Unit" or "LU" refers to the amount of enzyme (in grams) that releases 1. mu. mol of titratable butyric acid per minute when contacted with tributyrin and emulsifier (gum Arabic) at 30 ℃ and pH7.

"solvate" refers to a molecular complex comprising a disclosed or claimed compound, and a stoichiometric or non-stoichiometric amount of one or more solvent molecules (e.g., EtOH).

"hydrate" refers to a solvate comprising a disclosed or claimed compound, and a stoichiometric or non-stoichiometric amount of water.

"pharmaceutically acceptable complex, salt, solvate, or hydrate" refers to a complex, acid or base addition salt, solvate, or hydrate of the claimed and disclosed compounds which is, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use.

"procatalyst" or "catalyst precursor" refers to a compound or group of compounds that is converted to a catalyst prior to use.

"treating" or "treatment" refers to reversing, alleviating, inhibiting the progression of, or preventing the disorder or condition to which the term applies, or preventing one or more symptoms of the disorder or condition.

"treatment" refers to the act of "treating", as defined above.

Table 1 lists the abbreviations used in this specification.

TABLE 1 list of abbreviations

In some of the reaction schemes and examples below, certain compounds may be prepared using protecting groups that prevent undesired chemical reactions at other reaction sites. Protecting groups may also be used to enhance solubility or otherwise alter the physical properties of the compound. For discussion of Protecting group strategies, descriptions of materials and methods for installing and removing Protecting Groups, a compilation of Protecting Groups useful for common functional Groups (including amines, carboxylic acids, alcohols, ketones, aldehydes, etc.) are found in t.w.greene and p.g.wuts, Protective Groups in organic chemistry (1999) and p.kocienski, Protective Groups (2000), which are incorporated herein by reference in their entirety for all purposes.

Additionally, some of the schemes and examples below may omit details of common reactions, including oxidation, reduction, etc., which are known to those of ordinary skill in the art of organic chemistry. Details of such reactions can be found in a number of papers, including Richard Larock, Comprehensive Organic transformations (1999), and the multivolume series edited by Michael B.Smith et al, Complex of Organic Synthetic Methods (1974-2003). In general, starting materials and reagents may be obtained from commercial sources or may be prepared from literature sources.

Generally, the chemical transformations described herein may be achieved using substantially stoichiometric amounts of the reactants, although certain reactions may benefit from the use of an excess of one or more of the reactants. In addition, many of the reactions disclosed in this specification, including the enantioselective hydrolysis of the racemic diester (formula 4) described in detail below, can be carried out at about RT, but a particular reaction may require the use of higher or lower temperatures, depending on reaction kinetics, yield, etc. Moreover, many chemical transformations may employ one or more compatible solvents, which may affect reaction rates and yields. Depending on the nature of the reactants, the one or more solvents may be polar protic solvents, polar aprotic solvents, non-polar solvents, or some combination. Any reference in this disclosure to concentration ranges, temperature ranges, pH ranges, catalyst loading ranges, etc., whether or not the word "range" is used explicitly, includes the indicated endpoints.

The present invention provides materials and methods for preparing optically active gamma-amino acids (formula 1), including pharmaceutically acceptable salts, esters, amides, or prodrugs thereof. The compounds of formula 1 include substituents R as defined above¹And R². Useful compounds of formula 1 thus include those wherein R is¹Is a hydrogen atom, and R²Is C_1-12Alkyl radical, C_3-12Cycloalkyl, or substituted C_3-12Cycloalkyl, or a compound wherein R is²Is a hydrogen atom, and R¹Is C_1-12Alkyl radical, C_3-12Cycloalkyl, or substituted C_3-12A cycloalkyl group. Particularly useful compounds of formula 1 include those wherein R is¹Is a hydrogen atom, and R²Is C_1-6Alkyl or C_3-7Cycloalkyl, or a compound wherein R is²Is a hydrogen atom, and R¹Is C_1-6Alkyl or C_3-7A cycloalkyl group. Particularly useful compounds of formula 1 include those wherein R is¹Is a hydrogen atom, and R²Is C_1-4Alkyl groups such as pregabalin (formula 9).

FIG. 1 shows a process for preparing an optically active gamma-amino acid (formula 1). TheThe method comprises the following steps: contacting or combining a reaction mixture consisting of a cyano-substituted diester (formula 4) and water with an enzyme to produce a product mixture comprising an optically active dicarboxylic acid monoester (formula 3) and an optically active diester (formula 5). The cyano-substituted diester (formula 4) has a stereogenic center, which is marked with an asterisk ("") and, as described below, can be prepared according to the reaction scheme shown in FIG. 2. Prior to contacting the enzyme, the cyano-substituted diester (formula 4) typically comprises a racemic (equimolar) mixture of the diester of formula 5 and its opposite enantiomer. The substituent R in the formula 3, formula 4, and formula 5¹，R²And R³And a substituent R in the formulae 4 and 5⁴As defined above with respect to formula 1. In general, except where stated differently, when a formula is first defined a particular substituent identifier (R)¹，R²，R³Etc.), the same substituent designations used in the subsequent formulae will have the same meaning as in the preceding formulae.

The enzyme (or biocatalyst) may be any protein that, although having little or no effect on the compound of formula 5, will catalyze the hydrolysis of its opposite enantiomer to produce the dicarboxylic acid monoester (formula 3). Useful enzymes for enantioselectively hydrolyzing the compound of formula 4 to the compound of formula 3 may thus include hydrolases, including lipases, certain proteases, and other enantioselective esterases. Such enzymes can be obtained from a number of natural sources, including animal organs and microorganisms. See, for example, Table 2 for non-limiting examples of commercially available hydrolases.

TABLE 2 commercially available hydrolases

As shown in the examples section, enzymes useful for enantioselective conversion of cyano-substituted diesters (formula 4 and formula 12) to desired optically active dicarboxylic monoesters (formula 3 and formula 11) include lipases. Particularly useful lipases include those derived from the microorganism Thermomyces lanuginosus, for example under the trade name Thermomyces lanuginosus(CAS number 9001-62-1) those obtained from Novo-Nordisk A/S. By submerged fermentation of an Aspergillus oryzae microorganism genetically modified with DNA encoding the amino acid sequence of a lipase from Thermomyces lanuginosus DSM4109An enzyme.100L and100T can be obtained as a liquid solution and a granular solid, respectively, each having a nominal activity of 100 kLU/g. Other formsComprises that50L of a compound havingHalf of the activity of 100L, and100L of a compound having a structure of100L of the same activity, but food grade.

A variety of screening techniques can be used to identify suitable enzymes. For example, a large number of commercially available enzymes can be screened using the high throughput screening techniques described in the examples section below. Other enzymes (or microbial sources of enzymes) can be screened using enrichment separation techniques. Such techniques typically involve the use of carbon-limited or nitrogen-limited media supplemented with an enrichment substrate, which may be a racemic substrate (formula 4) or a structurally similar compound. Potentially useful microorganisms are selected for further study based on their ability to grow in media containing enriched substrate. Subsequently, the ability of these microorganisms to enantioselectively catalyze ester hydrolysis is evaluated by contacting a suspension of microbial cells with a racemic substrate and testing for the presence of the desired optically active dicarboxylic acid monoester (formula 3) using analytical methods such as chiral HPLC, gas liquid chromatography, LC/MS, and the like.

Once a microorganism having the desired hydrolytic activity has been isolated, enzyme engineering can be employed to modify the properties of the enzyme it produces. For example, and without limitation, enzymatic engineering can be used to increase the yield and enantioselectivity of ester hydrolysis, broaden the temperature and pH operating range of the enzyme, and improve the tolerance of the enzyme to organic solvents. Useful enzyme engineering techniques include rational design approaches such as site-directed mutagenesis, and in vitro-directed evolution techniques using successive rounds of random mutagenesis, gene expression, and high throughput screening to optimize desired properties. See, e.g., k.m.koeller & c. -h.wong, "Enzymes for chemical synthesis," Nature 409: 232-240(11 Jan.2001), and the references cited therein, the entire contents of which are incorporated herein by reference.

The enzyme may be in the form of an intact microbial cell, a permeabilized microbial cell, an extract of a microbial cell, a partially purified enzyme, a purified enzyme, or the like. The enzyme may comprise a dispersion of particles having an average particle size of less than about 0.1 mm (fine dispersion) or about 0.1 mm or greater (coarse dispersion) on a volume basis. The crude enzyme dispersion offers potential processing advantages over the fine dispersion. For example, the crude enzyme particles may be reused in a batch process or a semi-continuous or continuous process, and may generally be more easily separated from other components of the bioconversion (e.g., by filtration) than a fine dispersion of the enzyme.

Useful crude enzyme dispersions include cross-linked enzyme crystals (CLECs) and cross-linked enzyme aggregates (CLEAs), which consist essentially of the enzyme. Other raw dispersions may include enzymes immobilized onto or into an insoluble support. Useful solid supports include polymer matrices comprising calcium alginate, polyacrylamide,and other polymeric materials, and inorganic matrices, e.g.For a general description of CLEC and other enzyme immobilization techniques, see m.a. navia&St.st.clair, U.S. patent No. 5,618,710. General discussion on CLEAs, including their preparation and use, see l.cao&Us patent application No. 2003/0149172 to j. See also a.m. anderson, biocat.biotransform, 16: 181(1998) and P.L pez-Serrano et al, Biotechnol. Lett.24: 1379-83(2002). The entire contents of the above documents are incorporated herein by reference for all purposes.

The reaction mixture may comprise a single phase, or may comprise multiple phases (e.g., a two-or three-phase system). Thus, for example, enantioselective hydrolysis as shown in FIG. 1 can take place in a single aqueous phase which contains the enzyme, the initial racemic substrate (formula 4), the undesired optically active diester (formula 5) and the desired optically active dicarboxylic acid monoester (formula 3). Alternatively, the reaction mixture may comprise a multiphase system comprising an aqueous phase in contact with a solid phase (e.g., the enzyme or product), an aqueous phase in contact with an organic phase, or an aqueous phase in contact with an organic phase and a solid phase. For example, enantioselective hydrolysis can be carried out in a two-phase system consisting of a solid phase containing the enzyme and an aqueous phase containing the initial racemic substrate, the undesired optically active diester, and the desired optically active dicarboxylic acid monoester.

Alternatively, enantioselective hydrolysis can be carried out in a three-phase system consisting of a solid phase containing the enzyme, an organic phase initially containing the racemic substrate (formula 4), and an aqueous phase initially containing a small fraction of the racemic substrate. Since the desired optically active dicarboxylic acid monoester (formula 3) has a lower pKa than the unreacted optically active diester (formula 5), and thus exhibits greater water solubility, the organic phase is enriched in the unreacted diester as the reaction proceeds, while the aqueous phase is enriched in the desired dicarboxylic acid monoester.

The amounts of racemic substrate (formula 4) and biocatalyst used in the enantioselective hydrolysis depend on, among other things, the particular cyano-substituted diester and the nature of the enzyme. In general, however, the reaction may employ a substrate having an initial concentration of about 0.1M to about 3.0M, and in many cases, about 1.5M to about 3.0M. In addition, the reaction may generally employ from about 1% to about 10% enzyme loading, and in many cases, from about 3% to about 4% (v/v) enzyme loading.

Enantioselective hydrolysis can be carried out over a wide range of temperatures and pH. For example, the reaction may be carried out at a temperature of from about 100 ℃ to about 500 ℃, but is typically carried out at about RT. Such temperatures generally allow for substantially complete conversion (e.g., about 42% to about 50%) of the racemate (formula 4) for a reasonable amount of time (about 2h to about 24h) without inactivation of the enzyme. In addition, enantioselective hydrolysis may be carried out at a pH of about 5 to a pH of about 10, more typically at a pH of about 6 to a pH of about 9, and often at a pH of about 6.5 to a pH of about 7.5.

Without pH control, the reaction mixture pH would decrease as the hydrolysis of the substrate (formula 4) proceeds due to the formation of the dicarboxylic acid monoester (formula 3). To compensate for this change, the hydrolysis reaction may be carried out under internal pH control (i.e., in the presence of a suitable buffer) or may be carried out under external pH control (by addition of a base). Suitable buffering agents include potassium phosphate, sodium acetate, ammonium acetate, calcium acetate, BES, BICINE, HEPES, MES, MOPS, PIPES, TAPS, TES, TRICINE, tris,or other buffers having a pKa of about 6 to about 9. The buffer concentration is generally from about 5mM to about 1mM, typically from about 50mM to about 200 mM. Suitable bases include those containing KOH, NaOH, NH₄0H, etc., having a concentration of about 0.5M to about 15M, or more typically, about 5M to about 10M. Other inorganic additives, such as calcium acetate, may also be used.

Following or during the enzymatic conversion of the racemate (formula 4), the desired optically active dicarboxylic acid monoester (formula 3) is isolated from the product mixture using standard techniques. For example, in the case of a single (aqueous) phase batch reaction, the product mixture may be extracted one or more times with a non-polar organic solvent, such as hexane or heptane, which separates the desired dicarboxylic acid monoester (formula 2) and the unreacted diester (formula 5) in the aqueous and organic phases, respectively. Alternatively, in the case of a heterogeneous reaction using an aqueous phase and an organic phase enriched with the desired monoester (formula 3) and unreacted diester (formula 5), respectively, the monoester and diester may be separated batch by batch after the reaction, or may be separated semi-continuously or continuously during the enantioselective hydrolysis.

As shown in FIG. 1, the unreacted diester (formula 5) can be separated from the organic phase and racemized to produce a racemic substrate (formula 4). The resulting racemate (formula 4) may be recycled or combined with unconverted racemic substrate, which is then subjected to enzymatic conversion to formula 3 as described above. Recycling of the unreacted diester (formula 5) increases the overall yield of enantioselective hydrolysis by over 50%, thereby improving the atom economy of the process and reducing the costs associated with disposing of the undesired enantiomer.

Treatment of the diester (formula 5) with a strong base sufficient to extract the acidic alpha-proton of the malonate moiety typically results in the inversion of the stereogenic center and the production of the racemic substrate (formula 4). Useful bases include organic bases such as alkoxides (e.g., sodium ethoxide), linear aliphatic amines, and cyclic amines, and inorganic bases such as KOH, NaOH, NH₄OH, and the like. The reaction is carried out in a compatible solvent, including polar protic solvents, such as EtOH, or aprotic polar solvents, such as MTBE. Reaction temperatures in excess of room temperature typically increase the yield of the racemization process.

As shown in FIG. 1, a substantially enantiomerically pure dicarboxylic acid monoester (formula 3) can be converted into an optically active γ -amino acid (formula 1) using at least 3 different methods. In one method, the monoester (formula 3) is hydrolyzed in the presence of an acid catalyst or a base catalyst to form an optically active cyano-substituted dicarboxylic acid (formula 6) or a corresponding salt. The cyano moiety of the resulting dicarboxylic acid (or its salt) is reduced to produce an optically active γ -aminodicarboxylic acid (formula 7) or the corresponding salt, which is subsequently decarboxylated by treatment with an acid, by heating, or both, to produce the desired optically active γ -amino acid (formula 1). By reacting with H in the presence of catalytic amounts of Raney nickel, palladium, platinum, etc₂By reaction with reducing agents, e.g. LiAlH₄，BH₃-Me₂S, etc., and the cyano moiety may be reduced. Acids useful for hydrolysis and decarboxylation reactions include inorganic acids, such as HClO₄，HI，H₂SO₄HBr, HCl, etc. Useful base catalysts for the hydrolysis reaction include a variety of alkali and alkaline earth metal hydroxides and oxides, including LiOH, NaOH, KOH, and the like.

In another method, the dicarboxylic acid monoester (formula 3) undergoes reductive cyclization to form optically active cyclic 3-carboxy-pyrrolidin-2-one (formula 2), which is subsequently treated with an acidThus, the desired enantiomerically-enriched γ -amino acid (formula 1) is produced. By reacting a monoester of formula 3 with H in the presence of a catalytic amount of Raney nickel, palladium, platinum, or the like₂The reaction may be a reductive cyclization. The lactam acids (formula 2) obtained from hydrolysis and decarboxylation may be used with one or more acids, including inorganic acids such as HClO₄，HI，H₂SO₄HBr, and HCl, and organic acids such as HOAc, TFA, p-TSA, and the like. The concentration of the acid may range from about 1N to about 12N, and the amount of the acid may range from about 1eq to about 7 eq. The hydrolysis and decarboxylation reactions may be carried out at a temperature of about RT or greater, or at a temperature of about 60 ℃ or greater, or in a temperature range of about 60 ℃ to about 130 ℃.

In a third method, the ester moiety of the dicarboxylic acid monoester (formula 3) is first hydrolyzed to produce the cyano-substituted dicarboxylic acid (formula 6 or its salt) as described above. The resulting dicarboxylic acid (or salt thereof) is then decarboxylated to produce an optically active cyano-substituted carboxylic acid or salt thereof (formula 8, wherein R is⁵Is a hydrogen atom, although R⁵Or may be C as indicated below_1-12Alkyl radical, C_3-12Cycloalkyl, or aryl-C_1-6Alkyl groups). The same conditions for decarboxylation of the lactam acid (formula 2) or the γ -aminodicarboxylic acid (formula 7) can be used. Instead of first hydrolyzing the ester moiety, the dicarboxylic acid monoester (formula 3) can be first decarboxylated directly by heating an aqueous solution of the dicarboxylic acid monoester (as a salt) from a temperature of about 50 ℃ to reflux to produce the cyano-substituted monoester (formula 8). Krapcho conditions (DMSO/NaCl/water) can also be used. In either case, subsequent reduction of the cyano moiety of the compound of formula 8 produces an optically active γ -amino acid (formula 1). In addition to raney nickel, a number of other catalysts may be used to reduce the cyano moiety of the compounds of formulae 3, 6 and 8. They include, but are not limited to, heterogeneous catalysts containing from about 0.1% to about 20%, more typically from about 1% to about 5% (by weight) of a transition metal such as Ni, Pd, Pt, Rh, Re, Ru, and Ir, including oxides and combinations thereof, typically supported on a variety of materials, including Al₂O₃，C，CaCO₃，SrCO₃，BaSO₄，MgO，SiO₂，TiO₂，ZrO₂And so on. Many such metals, including Pd, can be doped with an amine, sulfide, or a second metal, such as Pb, Cu, or Zn. Useful catalysts thus include palladium catalysts such as Pd/C, Pd/SrCO₃，Pd/Al₂O₃，Pd/MgO，Pd/CaCO₃，Pd/BaSO₄PdO, Pd black, PdCl₂Etc., containing from about 1% to about 5% Pd by weight. Other useful catalysts include Rh/C, Ru/C, Re/C, PtO₂，Rh/C，RuO₂And so on.

Catalytic reduction of the cyano moiety is typically carried out in the presence of one or more polar solvents, including, but not limited to, water, alcohols, ethers, esters and acids, such as MeOH, EtOH, IPA, THF, EtOAc, and HOAc. The reaction may be carried out at a temperature ranging from about 5 ℃ to about 100 ℃, although reactions at room temperature are common. Generally, the ratio of substrate to catalyst can range from about 1: 1 to about 1000: 1 by weight, and H₂The pressure range may be about atmospheric, 0psig, to about 1500 psig. More typically, the ratio of substrate to catalyst ranges from about 4: 1 to about 20: 1, and H₂The pressure range is from about 25psig to about 150 psig.

All of the foregoing methods can be used to convert the substantially enantiomerically pure monoester (formula 3) to the optically active γ -amino acid (formula 1), but each may provide certain advantages over the other methods. For example, the lactam acid (formula 2) may be isolated and purified by extracting it into an organic solvent after acid work-up using a reductive cyclization process, while the cyano-substituted dicarboxylic acid (formula 6) may be more difficult to isolate because of its relatively higher water solubility. The isolation of the lactam acid (formula 2) reduces the transfer of water soluble impurities to the final product mixture and allows for higher reactant concentrations (e.g., about 1M to about 2M) during hydrolysis and decarboxylation, thereby increasing process throughput. In addition, direct decarboxylation by heating an aqueous solution of the dicarboxylic acid monoester (formula 3) provides a cyano monoester of high enantiomeric purity (formula 8). The compound can be separated from the reaction medium by extraction in an organic solvent, or by direct phase separation, ensuring efficient removal of inorganic impurities from the aqueous phase. The avoidance of high reaction flux and strong acidic conditions is two advantages of this process.

FIG. 2 illustrates a method for preparing cyano-substituted diesters (formula 4) that can be used as substrates for the enzymatic enantioselective hydrolysis shown in FIG. 1. The method comprises a cross aldol condensation comprising reacting an asymmetric ketone or aldehyde (formula 17) with a malonic diester (formula 18) in the presence of a catalytic amount of a base to form an α, β -unsaturated malonic diester (formula 19) wherein R is¹，R²，R³And R⁴As defined above with respect to formula 1. This type of cross aldol reaction is known as Knoevenagel condensation, which is described in many literature reviews. See, e.g., b.k.wilk, Tetrahedron 53: 7097-7100(1997), and the references cited therein, the entire contents of which are incorporated herein by reference for all purposes.

In general, any base that can generate an enol type ion from a malonic acid diester (formula 18) can be used, including secondary amines such as di-n-propylamine, di-isopropylamine, pyrrolidine, and the like, and salts thereof. The reaction may include a carboxylic acid, such as HOAc, to neutralize the product and minimize enolization of the asymmetric ketone or aldehyde (formula 17). Reactions involving unsymmetrical ketones, Lewis acids such as titanium tetrachloride, zinc chloride, zinc acetate, and the like, may also be employed to facilitate the reaction. The reaction is typically carried out under reflux conditions in a hydrocarbon solvent. Useful solvents include hexane, heptane, cyclohexane, toluene, methyl tert-butyl ether, and the like, and water is removed azeotropically.

In a subsequent step, a cyanide source, such as HCN, acetone cyanohydrin, alkali metal cyanide (NaCN, KCN, etc.), or alkaline earth metal cyanide (magnesium cyanide, etc.), undergoes conjugate addition to the β -carbon of the α, β -unsaturated malonic diester (formula 19). The reaction is typically carried out in one or more polar protic solvents, including EtOH, MeOH, n-propanol, isopropanol, or polar aprotic solvents, such as DMSO, and the like. Subsequent acid work-up produces the cyano-substituted diester (formula 4). For the use of the process shown in FIG. 2 in the preparation of a pregabalin precursor (formula 12), see U.S. Pat. No. 5,637,767 to Grote et al, which is incorporated herein by reference in its entirety for all purposes.

The desired (S) -or (R) -enantiomer of any of the compounds disclosed herein can be further enriched by classical resolution, chiral chromatography or recrystallization. For example, optically active γ -amino acids (formula 1 or formula 9) can be reacted with enantiomerically-pure compounds (e.g., acids or bases) to form diastereomeric pairs, each of which consists of a single enantiomer, which are separated, for example, by fractional recrystallization or chromatography. The desired enantiomer is then regenerated from the appropriate diastereomer. In addition, when it is available in sufficient amounts (e.g., typically not more than about 85% ee, and in some cases not more than about 90% ee), the desired enantiomer can often be further enriched by recrystallization from a suitable solvent.

As described in the specification, many of the disclosed compounds have stereoisomers. Some of these compounds may exist as single enantiomers (enantiomerically pure compounds) or mixtures of enantiomers (enriched and racemic samples), which may be optically active depending on the relative excess of one enantiomer over the other in the sample. Such stereoisomers, which are non-superimposable mirror images, have a stereogenic axis or one or more stereogenic centers (i.e., chirality). Other disclosed compounds may be non-mirror image stereoisomers. Such stereoisomers are referred to as diastereomers and may be chiral or achiral (contain no stereogenic centers). They include molecules containing alkenyl or cyclic groups such that cis/trans (or Z/E) stereoisomers are possible, or molecules containing 2 or more stereogenic centers, where inversion of a single stereogenic center results in the corresponding diastereomer. Unless stated or otherwise clear (e.g., by use of a stereo bond, stereocenter descriptor, etc.), the scope of the present invention generally includes reference to a compound and its stereoisomers, whether each is pure (e.g., enantiomerically pure) or a mixture (e.g., enantiomerically enriched or racemic).

Some compounds may also contain ketone or oxime groups, such that tautomerism may occur. In such cases, the invention generally includes tautomeric forms, whether each is pure or a mixture.

Many of the compounds described herein, including those represented by formula 1 and formula 9, are capable of forming pharmaceutically acceptable salts. Such salts include, but are not limited to, acid addition salts (including diacids) and base salts. Pharmaceutically acceptable acid addition salts include non-toxic salts derived from inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, hydrofluoric, phosphorous, and the like, as well as non-toxic salts derived from organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Such salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, hydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, trifluoroacetate, propionate, octanoate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, malate, tartrate, methanesulfonate, and the like.

Pharmaceutically acceptable base salts include non-toxic salts derived from bases, including metal cations, such as alkali or alkaline earth metal cations, and amines. Examples of suitable metal cations include, but are not limited to, sodium cation (Na)⁺) Potassium cation (K)⁺) Magnesium cation (Mg)²⁺) Calcium cation (Ca)²⁺) And so on. Examples of suitable amines include, but are not limited to, N, N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamineEthylenediamine, N-methylglucamine, procaine, and tert-butylamine. For a discussion of useful acid addition and base Salts, see s.m. berge et al, "Pharmaceutical Salts," 66 j.of pharm.sci., 1-19 (1977); see also Stahl and Wermuth, Handbook of Pharmaceutical Salts: properties, Selection, and Use (2002).

Pharmaceutically acceptable acid addition salts (or base salts) can be prepared by contacting the free base (or free acid) or zwitterion of the compound with a sufficient amount of the desired acid (or base) to form a non-toxic salt. If the salt precipitates from solution, it can be isolated by filtration; otherwise, the salt may be recovered by evaporation of the solvent. The free base (or free acid) may also be regenerated by contacting the acid addition salt with a base (or the base salt with an acid). Although certain physical properties of the free base (or free acid) and its respective acid addition salt (or base salt) may differ (e.g., solubility, crystal structure, hygroscopicity, etc.), the free base and acid addition salt of a compound (or its free acid and base salt) are the same for purposes of the present invention.

The disclosed and claimed compounds may exist in unsolvated and solvated forms and as other types of complexes besides salts. Useful complexes include clathrates or compound-host inclusion complexes in which the compound and host are present in stoichiometric or non-stoichiometric amounts. Useful composites may also contain 2 or more stoichiometric or non-stoichiometric amounts of organic, inorganic, or organic and inorganic components. The resulting complex may be ionized, partially ionized or unionized. For reviews on such complexes, see j.k.haleblian, j.pharm.sci.64 (8): 1269-88(1975). Pharmaceutically acceptable solvates also include hydrates and solvates, where the crystallization solvent may be isotopically substituted, e.g. D₂O，d₆-acetone, d₆-DMSO, etc. In general, for the purposes of this disclosure, reference to a compound in unsolvated form also includes the corresponding compound in solvated or hydrated form.

The disclosed compounds also include all pharmaceutically acceptable isotopic variations, in which at least one atom is replaced by an atom having the same atomic sequence, but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes suitable for inclusion in the disclosed compounds include, but are not limited to, isotopes of hydrogen, such as²H and³h; isotopes of carbon, e.g.¹³C and¹⁴c; isotopes of nitrogen, e.g.¹⁵N; isotopes of oxygen, e.g.¹⁷O and¹⁸o; isotopes of phosphorus, e.g.³¹P and³²p; isotopes of sulfur, e.g.³⁵S; isotopes of fluorine, e.g.¹⁸F; and isotopes of chlorine, e.g.³⁶And (4) Cl. The use of isotopic variations (e.g., deuterium,²H) certain therapeutic advantages resulting from greater metabolic stability may be provided, for example, increased in vivo half-life or reduced dosage requirements. In addition, certain isotopic variations of the disclosed compounds can incorporate a radioactive isotope (e.g., tritium,³h, or¹⁴C) It can be used in drug and/or substrate tissue distribution studies.

Examples

The following examples are intended to illustrate, not to limit, and represent specific embodiments of the present invention.

General materials and methods

Enzyme screening was performed using 96-well plates, which are described in d.yazbeck et al, synth.cata.345: 524-32(2003), the entire contents of which are incorporated herein by reference for all purposes. All enzymes used in the screening plates (see Table 2) were obtained from commercial enzyme suppliers including Amano (Nagoya, Japan), Roche (Basel, Switzerland), Novo Nordisk (Bagsvaerd, Denmark), Altus Biologics Inc. (Cambridge, MA), Biocatalytics (Pasadena, CA), Toyobo (Osaka, Japan), Sigma-Aldrich (St. Louis, MO) and Fluka (Buchs, Switzerland). In Eppendorf Thermomixer-R (VWR), a screening reaction was performed. Subsequent larger scale enzymaticThe splitting is carried out100L and100T, available from Novo-Nordisk A/S (CAS number 9001-62-1).

Nuclear magnetic resonance

BRUKER300UltraShield equipped with 5mm PHQNP probe capable of automatic switching^TMTo obtain 300MHz¹H NMR and 75MHz¹³C NMR spectrum. Spectra are typically collected near room temperature and standard auto-lock, auto-compensation and auto-gain approaches are employed. The sample was rotated, typically at 20Hz, and the 1D experiment was performed. Acquisition using a 16 scan with 30-degree apex angle pulse, 1.0 second recirculation delay, and 0.25 Hz/point resolution¹H NMR spectrum. The acquisition window is typically 8000Hz, from +18 to-2 ppm (at 0ppm with reference to TMS), and is treated with 0.3Hz line broadening. Typical acquisition times are 5-10 seconds. Conventional 2048 scans were acquired using 30-degree corner angle pulses, 2.0 second recirculation delay, and 1 Hz/point resolution¹³C NMR spectrum. The spectral width is typically 25KHz, from +235 to-15 ppm (at 0ppm with reference to TMS). Proton decoupling was applied continuously and 1Hz line broadening was applied during treatment. A typical acquisition time is 102 minutes.

Mass spectrometry

Mass spectrometry was performed on HEWLETT PACKARD1100MSD using HP Chemstation Plus software. The LC was equipped with an Agilent 1100 quaternary LC system and Agilent liquid handler as an autosampler. Data were acquired under electrospray ionization with ACN/water (containing 0.1% formic acid) as solvent (10% ACN to 90%, 7 min). Temperature: the probe was 350 ℃ and the source was 150 ℃. The corona discharge of the cations was 3000V and the anions were 3000V.

High performance liquid chromatography

High Performance Liquid Chromatography (HPLC) was performed on a 1100 Agilent tecchologies instrument set equipped with an Agilent 220 HPLC autosampler, quaternary pump and UV detector. LC was PC controlled using HP Chemstation Plus software. Normal phase Chiral HPLC was performed using Chiral HPLC columns from Chiral Technologies (Exton, PA) and Phenomenex (Torrance, CA).

Gas chromatography

Gas Chromatography (GC) was performed on a 110 volt Agilent 6890N network GC system equipped with FID detector (with electrometer), 7683 component flow/no flow capillary injector, relay board to monitor 4 external events, and in-cabin printer/plotter. The diester (formula 13, R) was carried out using a CHIRALDEX G-TA column (30 m.times.0.25 mm) at 135 ℃ with a helium carrier gas³＝R⁴Et) and monoesters (formula 11, R)³Et) in enantiomeric excess. Under such conditions, the monoester decomposed to give (S) -3-cyano-5-methyl-ethyl hexanoate, and ee was determined based on the decomposition product. The chiral GC column used in the analysis was obtained from Astec, Inc (whitepany, NJ).

EXAMPLE 1 enzymatic screening by enzymatic hydrolysis of (R/S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid ethyl ester (formula 20) to produce (3S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid (formula 21)

Enzyme screening was performed using a screening kit containing individual enzymes placed in separate wells of a 96-well plate previously prepared according to d.yazbeck et al, synth.cata.345: 524-32 (2003). Each well had a void volume of 0.3mL (shallow wells plate) 96-well plate one well contained only phosphate buffer (10. mu.L, 0.1M, pH7.2), the other well contained only ACN (10. mu.L), and the remaining wells contained one of the 94 enzymes listed in Table 2 (10. mu.L, 100 mg/mL). Prior to use, the screening kit was removed from storage at-80 ℃ and the enzyme was allowed to thaw for about 5 minutes at room temperature. Potassium phosphate buffer (85. mu.L, 0.1M, pH7.2) was dispensed into each well using a multichannel pipette. Subsequently, concentrated substrate (formula 20, 5 μ L) was added to each well by multichannel pipette and 96 reaction mixtures were incubated at 30 ℃ and 750 rpm. After 24h, the reaction was quenched by transferring each reaction mixture to a separate well of a second 96-well plate and sampling was performed. Each well had a 2mL void volume (deep well plate) and contained EtOAc (1mL) and HCl (1N, 100 μ L). The components of each well were mixed by pipetting the contents of the well. The second plate was centrifuged and 100 μ L of organic supernatant was transferred from each well to a separate well of a third 96-well plate (shallow plate). The wells of the third plate were then sealed using a penetrable gasket cover. Once the wells are sealed, the third plate is transferred to a GC system and the optical purity (ee) is determined.

Table 3 lists some of the enzymes screened, the trade names, the suppliers, and the E values. For a given enzyme, the E value can be interpreted as the relative reactivity of a pair of enantiomers (substrates). The E values listed in table 3 were calculated from HPLC data (conversion, x, and Ee) using a computer program called Ee2, available from Graz university. In general, enzymes exhibiting S-selectivity and an E value of about 35 or greater are suitable for scale-up.

TABLE 3 results of the screening reaction of example 1

Example 2 enzymatic resolution of (R/S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid ethyl ester (formula 20) to give (3S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid potassium salt (formula 23) and (R) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid ethyl ester (formula 22)

A reactor (392L) equipped with overhead stirring was charged with potassium phosphate buffer (292.2L, 10mM, pH8.0) and100L, EX type (3.9L). The mixture was stirred at 800RPM for 1 minute and KOH (2M) was added to adjust the pH to 8.0. (R/S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid ethyl ester (formula 20, 100kg) was added, and the resulting mixture was titrated with aqueous NaOH (50%) during hydrolysis, maintaining pH 8.0. By HPLC (C)₁₈Column, 4.6mm × 150mm, detection at 200 nm), the extent of the reaction was monitored. After about 40-45% conversion is achieved (e.g., after about 24 hours), the reaction mixture is transferred to a separatory funnel. The aqueous mixture was extracted with heptane (205L). EtOH (anhydrous) was added (up to about 5% v/v), breaking the light emulsion formed and separating the aqueous and organic layers. The extraction step is repeated 2 times and the aqueous layer containing (3S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid potassium salt (formula 23) (e.g., 25-50% of its original volume) can be further concentrated under vacuum. The organic layers containing (R) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid ethyl ester (formula 22) were combined, dried, and concentrated. Subsequently, the diethyl ester obtained was racemized according to example 6. MS M/z [ M + H ]]⁺227。¹H NMR(300MHz，D₂O)：δ2.35(dd，6H)，2.70(t，3H)，2.85(m，1H)，2.99(m，1H)，3.25(m，1H)，4.75(m，1H)，5.60(q，2H)。¹³C NMR(75ppm，D₂O)δ 172.19，171.48，122.85，62.70，59.49，40.59，31.83，27.91，23.94，21.74，14.77。

Example 3 enzymatic resolution of (R/S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid ethyl ester (formula 20) to give (3S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid potassium salt (formula 23) and (R) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid ethyl ester (formula 22)

Supply withA stirred reactor (3.92L) was set up and charged with calcium acetate buffer (1.47L, 100mM, pH7.0) and (R/S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid ethyl ester (formula 20, 1 kg). The mixture was stirred at 1100RPM for 5 minutes and KOH (5M) was added to adjust the pH to 7.0. Adding into100L, EX type (75mL), and during hydrolysis, the resulting mixture was titrated with KOH (5M) to maintain pH 7.0. By HPLC (C)₁₈Column, 4.6mm × 150mm, detection at 200 nm), the extent of the reaction was monitored. After about 42% to 45% conversion is achieved (e.g., after about 20-25 h), the reaction mixture is transferred to a separatory funnel. The aqueous mixture was extracted with hexane (100% v/v). EtOH (anhydrous) was added (up to about 5% v/v), breaking the light emulsion formed and separating the aqueous and organic layers. The extraction step was repeated 2 times to obtain an aqueous layer containing (3S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid potassium salt (formula 23), which can be used without separation in the subsequent conversion. The organic layers containing (R) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid ethyl ester (formula 22) were combined, dried, and concentrated. Subsequently, the diethyl ester obtained was racemized according to example 6.

Example 4 preparation of (S) -4-isobutyl-2-oxo-pyrrolidine-3-carboxylic acid (formula 10) from (3S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid potassium salt (formula 23)

The pot was loaded with an aqueous solution containing (3S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid potassium salt (formula 23, 411L, from example 2). Raney nickel (50% aqueous, Sigma-Aldrich) was added to the mixture and hydrogen was introduced into the tank over 20h, maintaining a pressure of 50psig in the headspace of the tank during the reaction. H through the can contents₂Absorption and HPLC analysis (C)₁₈Column, 4.6 mm. times.150 mm, detection at 200 nm), the hydrogenation reaction was monitored. After the reaction, the aqueous mixture is filtered to remove the Raney nickelA catalyst. The pH of the concentrated solution was adjusted to 3.0 using 37% HCl (about 14L). The resulting solution was extracted 3 times with EtOAc (50% v/v). The combined organic layers were concentrated in vacuo to give (S) -4-isobutyl-2-oxo-pyrrolidine-3-carboxylic acid (formula 10). MS M/z [ M + H ]]⁺186.1130。¹³C NMR(75ppm，CDCl₃) δ 175.67, 172.23, 54.09, 47.62, 43.69, 37.22, 26.31, 23.34, 22.54. The yield is 40-42%; 97% ee.

Example 5 preparation of pregabalin (formula 9) from (S) -4-isobutyl-2-oxo-pyrrolidine-3-carboxylic acid (formula 10)

The reactor tank (60L) was loaded with (S) -4-isobutyl-2-oxo-pyrrolidine-3-carboxylic acid (formula 10), HCl (36-38%, 30L), and water (29L). HOAc (1L) was added to the solution and the resulting slurry was heated at 80 ℃ for 36-38h and at 110 ℃ for an additional 6 h. By HPLC (C)₁₈Column, 4.6mm × 150mm, detection at 200 nm), the extent of the reaction was monitored. Water and excess HCl were evaporated to give an oil which was washed with MTBE (2X 15L). Water was added to the oil and the mixture was stirred until the solution was clear. The pH of the solution was adjusted to 5.2-5.5 using KOH (about 6kg), which resulted in the precipitation of pregabalin. The mixture was heated to 80 ℃ and subsequently cooled to 4 ℃. After 10h, the crystalline pregabalin was filtered and washed with IPA (12L). The filtrate was concentrated under vacuum to give the remaining oil. Water (7.5L) and EtOH (5.0L) were added to the remaining oil and the resulting mixture was heated to 80 ℃ and then cooled to 4 ℃. After 10h, the second batch of pregabalin crystals was filtered and washed with IPA (1L). The combined pregabalin crystals were dried in a vacuum oven at 45 ℃ for 24 h. MS M/z [ M + H ]]⁺160.1340。¹H NMR(300MHz，D₂O): δ 2.97(dd, J ═ 5.4, 12.9Hz, 1H), 2.89(dd, J ═ 6.6, 12.9Hz, 1H), 2.05-2.34(m, 2H), 1.50-1.70 (heptad, J ═ 6.9Hz, 1H), 1.17(t, J ═ 7.0Hz, 2H), 0.85(dd, J ═ 2.2, 6.6Hz, 6H).¹³C NMR(75ppm，D₂O) δ 181.54, 44.32, 41.28, 32.20, 24.94, 22.55, 22.09. The yield is 80-85%; ee is more than 99.5%.

Example 6 preparation of (R/S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid Ethyl ester (formula 20) by racemization of (R) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid Ethyl ester (formula 22)

The reactor was charged with (R) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid ethyl ester (formula 22, 49.5kg) and EtOH (250L). Sodium ethoxide (21% w/w in EtOH, 79.0L, 1.1eq) was added to the mixture and heated at 80 ℃ for 20 h. After completion of the reaction, the mixture was cooled to room temperature and neutralized by addition of HOAc (12.2L). After evaporation of EtOH, MTBE (150L) was added to the mixture, the resulting solution was filtered and evaporated to give (R/S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid ethyl ester (formula 20) in quantitative yield.

EXAMPLE 7 preparation of (S) -3-cyano-5-methyl-hexanoic acid ethyl ester (formula 24) from (3S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid (formula 21)

A50 mL round bottom flask was charged with (3S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid (formula 21, 3.138g, 13.79mmol), NaCl (927mg, 1.15eq), deionized water (477 μ L, 1.92eq), and DMSO (9.5 mL). The resulting mixture was heated to 88 ℃ and maintained at this temperature for 17 h. Samples were taken for LC and LC/MS analysis, which showed the presence of starting material (formula 21) and product (formula 24 and formula 25). Subsequently, the temperature of the mixture was raised to 135 ℃ and reacted for an additional 3.5 h. A second sample was taken for LC and LC/MS analysis, which showed the absence of starting material (formula 21) and the presence of unidentified by-products in addition to the desired product (formula 24 and formula 25). (S) -3-cyano-5-methyl-hexanoic acid ethyl ester (formula 24): 97.4% ee after 88 ℃; 97.5% ee after 135 ℃.

Example 8 determination of optical purity (ee) of (S) -4-isobutyl-2-oxo-pyrrolidine-3-carboxylic acid (formula 10)

The optical purity of (S) -4-isobutyl-2-oxo-pyrrolidine-3-carboxylic acid (formula 10) was determined by the derivatization method. A sample of (S) -4-isobutyl-2-oxo-pyrrolidine-3-carboxylic acid was esterified with EtOH at 70 ℃ in the presence of catalytic amounts of anhydrous HCl in dioxane. The resulting lactam ester was analyzed by HPLC (CHIRALPAK AD-H, 4.6 mm. times.250 mm), using a mobile phase of hexane and EtOH (95: 5), a flow rate of 1.0mL/min, an injection volume of 10. mu.L, a column temperature of 35 ℃ and detection at 200 nm.

Example 9 determination of optical purity (ee) of Pregabalin (formula 9)

The optical purity of pregabalin was analyzed by the derivatization method. Derivatization of a sample of pregabalin with Marfey reagent (1-fluoro-2-4-dinitrophenyl-5-L-alaninamide) followed by HPLC (LUNA C)₁₈(2) Column, 0.46 mm. times.150 mm, 3 μm), using aqueous NaPO₄(20nM, pH2.0) and ACN (90: 10 for 10 min, 10: 90 for 3 min, 90: 10 for 5 min), flow rate of 1.2 mL/min, injection volume of 10. mu.L, column temperature of 35 ℃ for analysis at 200 nM.

EXAMPLE 10 enzymatic resolution of (R/S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid ethyl ester (formula 20) to give (3S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid sodium salt (formula 23) and (R) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid ethyl ester (formula 22)

A reactor (16000L) equipped with overhead stirrer was charged with calcium acetate (254kg), deionized water (1892.7kg) andTL100L (food grade)983.7 kg). After thorough mixing, (R/S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid ethyl ester (formula 20, 9000kg, 85% purity determination) was loaded and the mixture was stirred for 24 h. During the reaction, NaOH (2068kg of a 30% solution) was added to maintain the pH at 7.0. By HPLC (C)₁₈Column, 4.6mm × 150mm, detection at 200 nm), the extent of the reaction was monitored. After about 42% to 45% conversion is achieved (e.g., after about 20-25 h), the titration and stirring are stopped. The organic phase was immediately separated and the aqueous phase was washed 2 times with toluene (780 kg). The aqueous layer containing (3S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid sodium salt (formula 23) was used without isolation in the subsequent conversion (example 11). The organic layers containing (R) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid ethyl ester (formula 22) were combined and concentrated. Subsequently, the diethyl ester obtained was racemized according to example 6.

EXAMPLE 11 preparation of (S) -3-cyano-5-methyl-hexanoic acid ethyl ester (formula 24) from (3S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid sodium salt (formula 23)

A reactor (16000L) equipped with overhead stirring was charged with the final aqueous solution (9698.6L, containing (3S) -3-cyano-2-ethoxycarbonyl-5-methyl-hexanoic acid sodium salt, formula 23), NaCl (630kg) and toluene (900L) from example 10. The mixture was stirred under reflux conditions (75-85 ℃) for 2 h. Stopping stirring; the organic phase was immediately separated and the aqueous phase was washed 2 times with toluene (900L). The organic layers containing (S) -3-cyano-5-methyl-hexanoic acid ethyl ester (formula 24) were combined and concentrated. The ethyl ester (formula 24) was then hydrolyzed according to example 12.

EXAMPLE 12 preparation of (S) -3-cyano-5-methyl-hexanoic acid potassium salt (formula 26) from (S) -3-cyano-5-methyl-hexanoic acid ethyl ester (formula 24)

A reactor (12000L) equipped with overhead stirring was charged with (S) -3-cyano-5-methyl-hexanoic acid ethyl ester (formula 24, 2196L from example 11). KOH (1795.2kg, 45% solution, w/w) and H were added with vigorous stirring₂O (693.9kg) was added to the reaction mixture. The temperature was maintained at 25 ℃. After 4h, the reaction mixture was charged to the hydrogenation tank (example 13) without further work-up.

Example 13 preparation of pregabalin (formula 9) from (S) -3-cyano-5-methyl-hexanoic acid potassium salt (formula 26)

The hydrogenator (12000L) was charged with water (942.1L) and the reaction mixture from example 12 containing the potassium salt of (S) -3-cyano-5-methyl-hexanoic acid (formula 26, 4122.9L). Raney nickel suspension (219.6kg, 50% w/w in H) was added₂In O). Hydrogenation was carried out at 50psig, 35 ℃. After 6h, the Raney nickel is filtered and the filtrate obtained is transferred into a reactor (16000L) and crystallized. Addition of H₂After O (1098L), the pH of the solution was adjusted to 7.0-7.5 using HOAc (864.7 kg). The resulting precipitate was filtered and washed with H₂O (549L) was washed once and 2 times with IPA (2,586L each). With IPA (12296L) and H₂O (6148L) recrystallizes the solid. The mixture was heated to 70 ℃ and subsequently cooled to 4 ℃. After 5-10h, the crystalline solid was filtered, washed with IPA (5724L), and dried in a vacuum oven at 45 deg.C for 24h to yield pregabalin as a white crystalline solid (1431kg, 30.0% overall yield, 99.5% purity and 99.75% ee).

It should be noted that, as used in this specification and the appended claims, singular articles such as "a," "1," and "the" may refer to a single object or a plurality of objects unless the context clearly dictates otherwise. Thus, for example, a composition containing "a compound" may comprise a single compound or two or more compounds. It is to be understood that the above description is intended to be illustrative, and not restrictive. Many embodiments will be apparent to those of skill in the art upon reading the above description. The scope of the invention should, therefore, be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. The contents of all papers and references, including patents, patent applications, and publications, are incorporated herein by reference in their entirety and for all purposes.

Claims (1)

1. A process for preparing a compound of formula 11,

wherein R is³Is C_1-12Alkyl radical, C_3-12Cycloalkyl or aryl-C_1-6An alkyl group, the method comprising:

(a) contacting a compound of formula 12 with an enzyme,

to produce the compound of formula 11 and the compound of formula 13,

wherein the enzyme is suitable for enantioselectively hydrolyzing the compound of formula 12 to a compound of formula 11 or a salt thereof;

(b) isolating a compound of formula 11 or a salt thereof; and

(c) optionally racemic formula 13 to produce a compound of formula 12, wherein

R in formula 12 and formula 13³The same as defined in formula 11; and is

R in formula 12 and formula 13⁴And R³Are the same or different and are C_1-12Alkyl radical, C_3-12Cycloalkyl or aryl-C_1-6An alkyl group, a carboxyl group,

wherein the enzyme is selected from the group consisting of Thermomyces lanuginosus lipase, Rhizopus delemar lipase, Rhizoctonia nikoensis lipase, Rhizomucor miehei esterase, Pseudomonas species lipase, Rhizomucor miehei lipase, Rhizopus oryzae lipase, Candida antarctica lipase-A and Candida antarctica lipase-B.