WO2011106546A1

WO2011106546A1 - A process for the preparation of rosuvastatin intermediate

Info

Publication number: WO2011106546A1
Application number: PCT/US2011/026104
Authority: WO
Inventors: Tihamer Paal; Laszlo Toth; Valerie Niddam-Hildesheim; Brijnath P. Chaurasia
Original assignee: Teva Pharmaceutical Industries Ltd; Teva Pharmaceuticals USA Inc
Current assignee: Teva Pharmaceutical Industries Ltd; Teva Pharmaceuticals USA Inc
Priority date: 2010-02-25
Filing date: 2011-02-24
Publication date: 2011-09-01
Anticipated expiration: 2012-08-25

Abstract

Processes utilizing treatment with Candida Antarctica lipase B ("CAL-B") or Candida Antarctica lipase A ("CAL-A") for desymmetiϊzation of a prochiral di-ester compound of formula (I): wherein R is a C₁-C₄ alkyl group, R₁ is a hydrogen; a C₁-C₄ acyl, preferably acetyl, propanoyl or butyryl; or a C₁-C₆ alkyl group, preferably methyl or ethyl, and n is 1, 2 or 3, to prepare the corresponding optically pure (S)-mono-ester according to formula (II).

Description

A PROCESS FOR THE PREPARATION OF ROSUVASTATIN INTERMEDIATE

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional Applications Nos. 61/308,133, filed February 25, 2010; and 61/317,010, filed March 24, 2010; the disclosures of which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention encompasses a process for desymmetrization of a prochiral di- ester compound to prepare the corresponding optically enriched (S)-mono-ester, one example of which is (S)-methyl 3-hydroxyglutarate, an intermediate of rosuvastatin.

BACKGROUND OF THE INVENTION

Rosuvastatin, (7-[4-(4-fluorophenyl)-6-isopropyl-2-(N-methyl-N-methylsulfonyl- amino) pyrimidin-5-yl]-(3R, 5S)-dihydroxy-(E)-6-heptenoic acid) calcium, having the following chemical formula:

Rosuvastatin calcium is an HMG-CoA reductase inhibitor.

Several processes for the preparation of rosuvastatin, salts and intermediates thereof are known.

Enzyme-catalyzed desymmetrization of prochiral diesters has been described, e.g., in J. Org. Chem. 1996, 61 (17), pp. 6024-6027; Tetrahedron Asymmetry 2004, 15, pp. 1551- 1554; J. Org. Chem. 1984, 49, pp. 3657-3659; Synthetic Communications 1990, 20 (3), pp. 315-319; curr. Org. synth. 2009, 6, pp 362-379; J. Org. Chem. 1999, 64, pp. 1464-1470; Tetrahedron letters 2007, 48, pp 6873-6876 and WO 03/087112, however the optical purity and yield of the obtained chiral products are relatively low.

The present invention provides an enzyme-catalyzed process for desymmetrization of a prochiral di-ester compound to prepare the corresponding optically enriched (S)-mono- ester. The process of the invention can be applied to preparing enantio-enriched (S)-methyl 3-hydroxyglutarate, an intermediate in the synthesis of rosuvastatin, in high optical purity and yield.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a process for desymmetrization of a prochiral di-ester compound of formula I:

I

wherein R is a C1-C4 alkyl group, Ri is a hydrogen; a C1-C4 acyl, preferably acetyl, propanoyl or butyryl; or a C1-C6 alkyl group, preferably methyl or ethyl, and n is 1, 2 or 3, to prepare the corresponding optically pure (S)-mono-ester according to formula II:

II

comprising: (a) combining the prochiral di-ester compound of formula I and Candida Antarctica lipase B ("CAL-B") or Candida Antarctica lipase A ("CAL-A") to obtain a reaction mixture comprising a mono-ester according to formula II A:

ΠΑ

wherein R is a C1 -C4 alkyl group, Ri is a hydrogen; a C1 -C4 acyl, preferably acetyl, propanoyl or butyryl; or a Ci-C₆ alkyl group, preferably, methyl or ethyl, and n is 1, 2 or 3; and wherein greater than 90% of the mono-ester is the (S)-mono-ester compound according to formula II, and less than 10% of the mono-ester is present as the corresponding (R)-mono-ester as assessed by HPLC; and (b) further reacting the reaction mixture formed in step (a) with CAL- B at a pH from about 5 to about 8, preferably from about 5 to about 6.5, to obtain the mono- ester compound according to formula IIA, wherein greater than 99% of the mono-ester compound is the (S)-mono-ester according to formula II; and less than 1% of the mono-ester is present as the corresponding (R)-mono-ester by HPLC.

According to some embodiments, the product obtained by the above described processes contains optically pure mono-ester according to formula IIA, which is greater than 99.5% (S)-mono-ester and less than 0.5% (R)-mono-ester by HPLC. According to other embodiments, the product obtained contains optically pure mono-ester according to formula IIA, which is greater than 99.85% (S)-mono-ester and less than 0.15% (R)-mono-ester by HPLC.

According to another embodiment, the invention provides a process for preparing optically pure (S)-methyl-hydroxyglutarate comprising: (a) combining dimethyl 3-hydroxy- glutarate and Candida Antarctica lipase B ("CAL-B") or Candida Antarctica lipase A

("CAL-A") to obtain a reaction mixture containing methyl 3-hydroxyglutarate wherein greater than 90% by HPLC of the methyl 3-hydroxyglutarate is (S)-methyl 3-hydroxyglutarate, and less than 10% by HPLC of the mono-ester is present as the corresponding (R)- methyl 3-hydroxyglutarate; and (b) further reacting the reaction mixture formed in step (a) with CAL-B at a pH from about 5 to about 8, or about 5 to about 6.5, to obtain a product that contains optically pure (S)-methyl 3-hydroxyglutarate which is greater than 99% (S)-methyl 3-hydroxyglutarate and less than 1% (R)-methyl 3-hydroxyglutarate by HPLC. According to some embodiments, the product obtained contains optically pure (S)-methyl 3- hydroxyglutarate which is greater than 99.5% (S)-methyl 3-hydroxyglutarate and less than 0.5% (R)-methyl 3-hydroxyglutarate acid by HPLC. According to other embodiments, the product obtained contains optically pure (S)-methyl 3-hydroxyglutarate which is greater than 99.85% (S)-methyl 3-hydroxyglutarate and less than 0.15% (R)-methyl 3-hydroxyglutarate by HPLC. In yet another embodiment, the present invention provides a process for preparing rosuvastatin or a pharmaceutically acceptable salt thereof comprising prepaiing (S)-methyl 3- hydroxyglutarate by the process of the present invention; and converting it to Rosuvastatin or a pharmaceutically acceptable slat thereof, preferably Rosuvastatin Calcium.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : HPLC pattern of a sample obtained after the desymmetrization process (stereoselective hydrolysis followed by a ldnetic resolution).

Figure 2: HPLC pattern of a sample obtained after stereo-selective hydrolysis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses a process for desymmetrization of a prochiral di- ester compound to prepare the corresponding optically enriched (S)-mono-ester, one example of which is (S)-methyl 3 -hydroxyglutarate, an intermediate of Rosuvastatin.

As used herein, the term "optically pure" refers to compounds having an optical purity of above at least about 99%, as measured by HPLC.

As used herein, the HPLC purity is determined by the area under the peaks observed after separation on an analytical chiral column, followed by detection using a UN detector at a wavelength of 220 nm or mass spectrometric (MS) detection. Preferably the chiral column contains a silica support coated with a polysaccharide chiral stationary phase. More preferably, the column is a Chiralpack AD-H analytical column, preferably of dimensions 250 mm (length) x 4.6 mm (internal diameter), 5 microns (particle size) (Daicel Chemical Industries Ltd). Preferably, the eluent is methanol: water rformic acid, preferably in a ratio of 80:20:0.1. Typically the flow rate is about 0.20 ml/min. Preferred HPLC and MS detection parameters conditions are set out in the examples.

The Candida antarctica lipase A (CAL-A) amino acid sequence: D4PHA8, specified at http://www.imiprot.org/uniprot/D4PHA8. The CAL-A used in the examples herein was purchased from Codexis.

The Candida antarctica lipase B (CAL-B) amino acid sequence: P41365, specified at http://www.uniprot.org/uniprot/P41365. The CAL-A used in the examples herein was purchased from Codexis or from Novozymes. The present invention provides a process, wherein prochiral diesters of dicarboxylic acids are first hydrolyzed by an enzyme-catalyzed enantio-selective process, and then are enantiomerically emiched by a kinetic resolution enzymatic process. The hydrolysis and the kinetic resolution can be done by the same enzyme, and the process may be a one-pot process.

The process comprises: combining substrate prochiral diester of a dicarboxylic acid and Candida Antarctica lipase B ("CAL-B") or Candida Antarctica lipase A ("CAL-A") to obtain a reaction mixture, maintaining the reaction mixture for a time sufficient to produce a mono-ester according to formula IIA:

IIA

wherein R is a C C alkyl group and ¾ is a hydrogen; a C_rC₄ acyl, preferably acetyl, propanoyl, or butyryl; or a Ci-C₆ alkyl group, preferably methyl or ethyl; and n is 1, 2 or 3; and wherein greater than 90% of the product mono-ester in the reaction mixture is the (S)-mono-ester according to formula II:

II

wherein R is a C1-C4 alkyl group; ¾ is a hydrogen, a C1-C4 acyl, preferably acetyl, propanoyl, or butyryl; or a Ci-C₆ alkyl group, preferably methyl or ethyl; and n is 1, 2 or 3; and less than 10% of the mono-ester is present as the corresponding (R)-mono-ester, as assessed by HPLC; and

(b) further reacting the reaction mixture formed in step (a) with CAL-B at a pH from about 5 to about 8, preferably from about 5 to about 6.5; to obtain the mono-ester according to formula IIA, wherein greater than 99% of the mono-ester is the (S)-mono-ester according to formula II, and less than 1% of the mono-ester is present as the corresponding (R)-mono- ester, as assessed by HPLC. According to a sub-embodiment, the process of the invention can be used to prepare optically pure (S)-methyl 3 -hydroxyglutarate which is greater than 99% (S)-methyl 3- hydroxyglutarate and less than 1% (R)-methyl 3 -hydroxyglutarate by HPLC. According to some embodiments, the (S)-methyl 3 -hydroxyglutarate product according to the claimed process contains optically pure (S)-methyl 3 -hydroxyglutarate which is greater than 99.5% or greater than 99.85% (S)-methyl 3 -hydroxyglutarate and less than 0.5%, or less than 0.15%, respectively of the corresponding (R)-methyl 3 -hydroxyglutarate by HPLC.

The process includes two steps: a) stereo-selective hydrolysis of a substrate prochiral diester of a dicarboxylic acid to obtain an enriched mono-ester according to formula IIA, which contains greater than 90% of the (S) enantiomer by HPLC and less than 10% of the corresponding (R)-enantiomer by HPLC; and b) kinetic resolution of (R)-enantiomer

produced in step a) to obtain the mono-ester according to formula IIA, wherein greater than 99% of the mono-ester is the (S)-mono-ester according to formula II, and less than 1% of the mono-ester is present as the corresponding (R)-mono-ester by HPLC. The process may be illustrated by the following scheme:

er)

R

symmetric di-acid OR-i O

wherein R is a Cj-C₄ all yl group, R] is a hydrogen, a C1-C4 acyl, preferably acetyl, propanoyl, or butyryl; or a C i-C₆ alkyl group, preferably methyl or ethyl; and n is 1, 2 or 3. The stereo-selective hydrolysis in step (a) is typically done in the presence of water or an aqueous solvent. Typically, the aqueous solvent comprises water and may further comprise one or more suitable co-solvents. Suitable co-solvents include for example Cj-C₄ alcohols, such as methanol, ethanol, propanol, isopropanol and butanol; C₃-C₇ ketones, such as acetone and methylethylketone (MEK); C₄-C₆ ethers, such as diethyl ether, methyl-t-butyl ether, tetraliydrofuran and dioxane; C₅-C]₀ hydrocarbons, such as pentane, hexane, and cyclohexane; Ci-C₆ halogenated hydrocarbons, such as methylene chloride, chloroform, and 1,1,1-trichloroethane; C₂-C₆ nitriles, such as acetonitrile and propionitrile; C₂-C₇ amides, such as dimethylformamide and dimethylacetamide, and dimethyl sulfoxide. Preferred solvents are n-hexane, toluene, diisopropyl ether and tert-butyl methyl ether.

If an alcoholic solvent is used, it is preferably used in amount of up to about 5% (v/v).

The aqueous solvent may further contain a suitable buffer. Examples of suitable buffers include, for example, triethanolamine-HCl buffer, triethanolamine-H₂S0₄ buffer and KH2PO4 E 2HPO4 buffer.

Typically, enzymes are used in a combination with a buffer. The buffer provides a pH suitable for the enzyme activity. A suitable pH for CAL-B and CAL-A activity is from about 3.5 to about 10, or from about 5 to about 8.5.

Preferably, the process of step (a) is done a t pH of about 6 to about 8.5, preferably about 7.5.

Typically, a suitable base is added to the reaction mixture during the reaction to control the pH of the reaction mixture. Suitable bases include alkali metal hydroxides, such as sodium hydroxide and potassium hydroxide; alkaline earth metal hydroxides, such as calcium hydroxide and magnesium hydroxide; ammonium hydroxides, and alkali metal carbonates, such as such as sodium or potassium carbonate; hydrogen carbonates, such as sodium or potassium bicarbonate alkaline earth metal carbonates, such as calcium carbonate; and ammonium carbonates; or organic bases such as C]-C₆ primary amines, e.g., methyl amine or ethyl amine, C2-C₈ secondary amines, e.g., dimethyl amine, diethyl amine, piperidine, C₃-C₁₀ tertiary amines, e.g., trimethyl amine, triethylamine, and diisopropylethylamine, guanidine, heterocyclic amines, e.g., pyridine; ethanolamine; diethanolamine; triethanolamine, and polyethyleneimines. Examples of suitable bases thus include KOH and K₂C0₃.

The substrate prochiral diester of a dicarboxylic acid (e.g., dimethyl 3- hydroxyglutarate) and the solvent are combined to form a reaction mixture. The enzyme (CAL-B or CAL-A) is added to the thus-formed reaction mixture, at a suitable temperature. Suitable temperatures for this enzymatic reaction are, for example from about -5°C to about 20°C, or from about 0°C to about 15°C, or about 5°C.

The reaction mixture containing the substrate prochiral diester of a dicarboxylic acid (e.g., dimethyl 3 -hydroxy glutarate), the CAL-B or CAL-A and optionally the co-solvent, is maintained, for example, at a temperature of about -5°C to about 20°C, or from about 0°C to about 15°C, or about 5°C, for a time from about 90 minutes to about 10 hours, or from about 4 hours to about 5 hours to obtain an enriched mono-ester according to formula IIA, which contains greater than 90% of the (S) enantiomer by HPLC and less than 10% of the corresponding (R)-enantiomer by HPLC, e.g., a mixture that is greater than 90% of (S)- methyl-hydroxyglutarate and less than 10% of (R)-methyl-hydroxyglutarate.

The enriched (S)-mono-ester, e.g., (S)-methyl-hydroxyglutarate can be obtained in an optical purity of about 95% to about 99% by HPLC, while the corresponding (R)-enantiomer of the product mono-ester, e.g., (R)-methyl-hydroxyglutarate can be obtained in an optical purity of about 5% to about 1% by HPLC.

The stereoselective hydrolysis can lead to the formation of an alcohol, e.g., methanol, ethanol, propanol or isopropanol corresponding to the C]-C₄ alkyl group on the substrate diester. Prior to the kinetic resolution portion of the process, that alcohol can be removed. Preferably, the alcohol by-product R-OH formed in step (a) is removed after step (a) in any of the above embodiments of the process of the invention. Preferably, in any of the above embodiments of the process, step (b) involves the addition of CAL-B after the removal of the alcohol by-product

The reaction in step b) is typically done while adjusting the pH to about 5 to about 8, preferably to about 5 to about 6.5, more preferably to about 6.5; and adding additional amount of CAL-B, or adding by CAL-A.

Typically, the amount of CAL-A or CAL-B added in step (b) is about 1.5 to about 2 times of the amount of CAL-B used in step (a).

The addition of the enzyme (CAL-B or CAL-A) and the reaction in step (b) is carried out at a suitable pH to form the reaction mixture for the second step of the process.

According to some embodiments, the suitable pH is from about 5 to about 8, preferably to about 5 to about 6.5, more preferably, about 6.5. This reaction mixture can be maintained, for example, at a temperature of about -5°C to about 20°C, or about 5°C, for about 1 day to about

3 days to obtain the optically pure (S)-mono-ester, e.g., (S)-methyl-hydroxyglutarate, i.e., the (S)-mono-ester having an optical purity greater than about 99%, or greater than about 99.5%, or greater than about 99.85% by HPLC.

The obtained optically pure (S)-mono-ester, e.g., (S)-methyl-hydroxyglutarate may be recovered from the reaction mixture that also contains the corresponding symmetric diacid, e.g., 3-hydroxy glutaric acid. The recovery may be done for example by extraction from ethyl acetate/ethanol at suitable pH, for example, a pH of about 2.0 to about 3.5, or a pH of about

2.5 to about 3.0 or apH of about 2.5.

Optically pure (S)-methyl-hydroxyglutarate obtained as a product of the claimed process may be used to prepare rosuvastatin.

Having thus described the invention with reference to particular preferred

embodiments and illustrative examples, those in the art can appreciate modifications to the invention as described and illustrated that do not depart from the spirit and scope of the invention as disclosed in the specification. The Examples are set forth to aid in

understanding the invention but are not intended to, and should not be construed to limit its scope in any way.

HPLC method:

Column and packing: Chiralpak AD-H 250,0 x 4,6 mm, 5 μπι, (Daicel Chemical Industries

Ltd. ADHOCE-FD037)

Eluent: Methanol: water :formic acid = 80:20:0.1

Flow rate: 0.20 ml/min.

Stop time: 70 min.

Column temperature: 4.0°C

Sample temperature: 10.0°C

Detection wavelength: 220nm

Sample volume: ΙΟμΙ

Diluent: Methanol: water = 40:60

Needle wash: Diluent

HPLC-MS detection method: Chromatographic conditions:

Column and packing: Chiralpalc AD-H 250.0 x 4.6 mm, 5 μπι, (Daicel Chemical Industries Ltd. ADHOCE-FD037), polar phase mode usage.

Eluent: Methanol: water: formic acid = 80:20:0.1

Flow rate: 0.20 ml/min.

Stop time: 70 min.

Column temperature: 5.0°C

Sample temperature: 10.0°C

Detection: mass spectrometric (MS)

Sample volume: ΙΟμΙ

Diluent: Methanol: water = 40:60

• Needle wash: Diluent

MS detection: Principle: MRM (multiple reaction monitoring)

Monitored fragments 163→ 127

163→ 99

163→ 95

Ion source TurboSpray (electrospray)

Polarity positive

Curtain gas 15

Collision gas 10

Spray voltage 4500

Temperature 100

Ion source gas 1 45

Ion source gas 2 30

Declustering potential 20

Focusing potential 400

Entrance potential 9

Collision energy 22

Collision cell exit point 6

Diverter valve To waste between -23.5 and 35.0 min Flow rate and temperature in the HPLC conditions may be slightly varied in order to achieve the required system suitability. The mass spectrometric parameters may be varied in order to obtain the desired sensitivity.

EXAMPLES Example 1 : Preparation of CSVMethyl 3-hvdroxyRlutarate.

Ice-cooled dimethyl 3-hydroxyglutarate (200 g) was added to 660 ml of

triethanolamine-HCl buffer (pH = 7.5, made from 16.9 g of triethanolamine dissolved in distilled water, pH was adjusted using HCl), in a glass reactor cooled to 5°C. Lipozyme CAL-B L (10.0 g, Novozymes, > 5000 U/g specific activity) was added to the mixture with vigorous stirring. The stirring was maintained with cooling to 5°C. The pH was kept constant at 7.5 with KOH (4.0 M aqueous), with the aid of a pH-stat (during 4 h of desymmetrization 285 ml of KOH 4.0 M was consumed).

The methanol formed during the stereo-selective hydrolysis, and the water, was partially evaporated from the reaction mixture (evaporation to 50% of the initial volume) under vacuum, at 20 °C. Then the mixture was diluted back to the initial volume (1130 ml) with distilled water. The pH of the thus-formed mixture was adjusted to 6.5 with a few ml of HCl (36%) and Lipozyme CAL-B L (17.43 g) was added. The resulting reaction mixture was maintained at pH = 6.5 (controlled by pH-stat) at 20°C for 36 h. The volume of the reaction mixture was reduced twice (after 36 h and 60 h of enantioenrichment) during the reaction by applying vacuum (7-20mbar) at 20°C, the evaporated volume was replaced with an equivalent volume of ice-cooled distilled water. The reaction mixture was stirred at 20°C between the evaporations and at 5°C after the last evaporation. The product, (S^-methyl 3- hydroxyglutarate was isolated after 132 h by extraction of the reaction mixture at pH = 2.5, (using HCl) with ethyl acetate/ethanol (9:1 vol/vol; 6 x 700 ml). The extracts were combined and dried overnight over anhydrous Na₂S0₄, then filtered and concentrated under vacuum to provide 153.21 g of crude (5)-methyl 3-hydroxyglutarate. Assay = 93.1% (according to HPLC). Optical purity: 99.85% (determined by HPLC with UV detection). Yield of pure (5)- methyl 3-hydroxyglutarate: 77.5%.

Example 2: Preparation of GS Methyl 3-hvdroxyglutarate (comparative example)

Ice-cooled dimethyl 3-hydroxyglutarate (200 g) was added to KH₂P0₄/K₂HP0₄ buffer

(660 ml, 0.1 M, pH = 7.5. potassium dihydrogen phosphate was dissolved in distilled water. The pH was adjusted with KOH) in a glass reactor cooled to 5°C. Lipozyme CAL-B L (10.0 g, Novozymes, > 5000 U/g specific activity) was added to the mixture with vigorous stirring. The stining was maintained under cooling to 5°C. The pH was maintained, with the aid of a pH-stat by adding K₂C0₃ (2.0M, 425 ml over 4 hours).

The product, (S)-methyl 3-hydroxyglutarate was isolated after 4 h by extraction. In the extraction, the unreacted dimethyl 3-hydroxyglutarate was first extracted at pH = 8.5 with ethyl acetate (3 x 800 ml). Then the pH was adjusted to 2.5 and the product was extracted with ethyl acetate/ethanol (9/1 ( V); 6 x 800 ml). The product extracts were combined and dried overnight over anhydrous Na₂S0₄, then filtered and concentrated under vacuum to yield 143.3 g of crude (5)-methyl 3-hydroxyglutarate. Assay = 94.6% (HPLC). Optical purity: 99.0% (determined by HPLC with UV detection). Yield of pure (5)-methyl 3- hydroxyglutarate: 73.6%.

Example 3: Preparation of (^-Methyl 3-hydroxyglutarate.

Ice-cooled dimethyl 3-hydroxyglutarate (48.3 g) was added to triethanolamine-sulfate buffer (made from 2.98 g of triethanolamine, corresponding to 0.1 M triethanolamine in the final reaction mixture. Triethanolamine was dissolved in distilled water, the pH was adjusted using H₂S0₄, the volume was adjusted to 200 ml with H₂0, the pH to 7.5. Lipozyme CAL-B L (2.41 g, Novozymes, > 5000 U/g specific activity) was added to the mixture cooled to 5°C, in a glass reactor. The stirring was maintained under cooling, the pH was kept constant with K₂C0₃ (3.0 M, aqueous), with the aid of a pH-stat, for 5 h. During the last hour the pH was let to drop until 6.5.

The methanol formed during the stereo-selective hydrolysis was partially evaporated from the reaction mixture (evaporation to 50% of the initial volume; methanol- ¾0 mixture has been evaporated), under vacuum, at 20 °C. Then the mixture was diluted back to the initial volume, the pH of the thus-formed mixture was adjusted to 6.5 with a few ml of HC1 (36%) and Lipozyme CAL-B L was added up to totally 24.16 mg/ml enzyme preparation. The resulting reaction mixture was maintained at pH = 6.5 (controlled by pH-stat with K₂C0₃ 3.0 M) at 5°C for 72 h. Meanwhile the methanol formed during the reaction was evaporated twice (as methanol-H₂0 mixture after 24 and 48 h, under vacuum at 20 °C), by reducing the initial reaction volume to 70%, and replacing the evaporated amount of solvent with ¾0.

The product, (S)-methyl 3-hydroxyglutarate was isolated from a sample obtained after 72 h by repeated extraction of the reaction mixture at pH = 2.5, with ethyl acetate (3*), the extracts were dried on anhydrous Na₂S0₄ and evaporated. Optical purity: 99.96% (detemiined by HPLC with MS detection).

Claims

We Claim:

1. A process for desymmetrization of a prochiral di-ester compound of formula I:

I

wherein:

R is a C]-C₄ alkyl group;

R] is selected from hydrogen; CrC₄acyl, and Cj-C₆ alkyl; and

n is 1, 2 or 3,

to prepare the corresponding optically pure (S)-mono-ester compound according to formula II:

II

said process comprising:

(a) combining the prochiral di-ester compound of formula I and an enzyme selected from Candida Antarctica lipase B ("CAL-B") and Candida Antarctica lipase A ("CAL- A") to obtain a reaction mixture comprising a mono-ester compound according to formula IIA:

IIA

wherein R, R_\ and n are as defined, and wherein greater than 90% of the mono-ester compound is the (S)-mono-ester compound according to formula II, and less than 10% of the mono-ester compound is present as the corresponding (R)-mono-ester compound as assessed by HPLC; and

(b) further reacting the reaction mixture formed in step (a) with CAL-B at a pH from about 5 to about 8; to obtain the mono-ester compound according to formula IIA, wherein greater than 99% of the mono-ester compound is the (S)-mono-ester compound according to formula II, and less than 1% of the mono-ester compound is present as the

corresponding (R)-mono-ester compound by HPLC.

2. The process of claim 1, wherein the enzyme used in steps (a) and (b) is CAL-B.

3. The process of claim 1, wherein the amount of enzyme added in step (b) is about 1.5 to about 2 times of the amount of enzyme used in step (a).

4. The process of claim 1 or claim 2, wherein step a) is done at a pH of about 6 to about 8.5.

5. The process according to any preceding claim, wherein the said process is done in the presence of a solvent, wherein said solvent is water or an aqueous solvent.

6. The process of claim 5, wherein the solvent contains a buffer.

7. The process of claim 6, wherein the buffer is selected from a triethanolamine-HCl buffer, a triethanolamine-H₂S0₄ buffer and a KH₂P04/K₂HP0₄ buffer.

8. The process according to any of of claims 5 to 7, wherein the solvent further comprises a co-solvent.

9. The process of claim 8, wherein the co-solvent is selected from at least one of a Cj-C₄ alcohol, a C₃-C₇ ketone, a C₄-C₆ ether, a C₅-C₁₀ hydrocarbon, a Q-Q halogenated hydrocarbon, a C₂-C₆ nitrile, a C₂-C₇ amide, and dimethyl sulfoxide.

10. The process according to any preceding claim, wherein the reaction in step a) is done in the presence of a base.

11. The process of claim 10, wherein the base is selected from an alkali metal, alkalme earth metal or ammonium hydroxide; an alkali metal, alkaline earth metal or ammonium carbonates; a hydrogen carbonate, and an organic base, such as a C_\-Ce primary amine, a C₂-C₈ secondary amine, a C₃-C₁₀ tertiary amine, guanidine, a heterocyclic amine, ethanolamine, diethanolamine, triethanolamine, or a polyethyleneimine.

12. The process according to any preceding claim, wherein an alcohol by product corresponding to the CrC alkyl group on the substrate diester formed in step a) is removed prior to step (b).

13. The process according to any preceding claim, wherein step (b) is done at a pH of about 5 to about 6.5.

14. The process of claim 13, wherein step b) is done at a pH of about 6.5.

15. The process according to any preceding claim, wherein the obtained mono-ester compound according to formula II A has an optical purity of greater than 99.5% of the (S)-mono-ester compound and less than 0.5% of the corresponding (R)-mono-ester compound by HPLC.

16. The process of claim 15, wherein the obtained mono-ester compound according to formula IIA has an optical purity of greater than 99.85% of the (S)-mono-ester compound and less than 0.15% of the (R)-mono-ester compound by HPLC.

17. The process according to any preceding claim, further comprising converting the obtained mono-ester compound according to formula II to Rosuvastatin or a

pharmaceutically acceptable salt thereof.

18. The process of claim 16, wherein the obtained Rosuvastatin or pharmaceutically acceptable salt thereof has an optical purity of greater than 99.85%) by HPLC.

19. The process according to any preceding claim, wherein R is methyl, Rj is hydrogen and n is 1, said process comprising:

(a) combining dimethyl 3 -hydroxy glutarate and Candida Antarctica lipase B ("CAL- B") or Candida Antarctica lipase A ("CAL-A") to obtain a reaction mixture containing methyl 3-hydroxyglutarate wherein greater than 90% by HPLC of the methyl 3-hydroxy- glutarate is (S)-methyl 3-hydroxyglutarate, and less than 10% by HPLC of the mono-ester is present as the corresponding (R)-methyl 3-hydroxyglutarate; and

(b) further reacting the reaction mixture formed in step (a) with CAL-B at a pH from about 5 to about 8; to obtain a product that contains optically pure (S)-methyl 3- hydroxyglutarate which is greater than 99% (S)-methyl 3-hydroxyglutarate and less than 1%) (R)-methyl 3-hydroxyglutarate by HPLC.

20. The process of claim 19, wherein step (a) is done at a pH of about 6 to about 8.5.

21. The process of claim 19, wherein step (b) is done at a pH of about 6.5.

22. A process for enantiomeric purification of a mixture of the (S)-monoester and (R)- monoester of a compound according to formula IIA:

wherein the (S)-monoester compound is present in an amount of at least 90%, and the (R)-monoester compound is present in an amount of about 1-10%; said process comprising combining the mixture of said (S)-monoester and (R)-monoester with CAL-B at a pH of about 5 to about 8.

23. The process of claim 22, wherein the said process is done in the presence of a solvent, wherein said solvent is water or an aqueous solvent.

24. The process of claim 23, wherein the solvent contains a buffer.

25. The process of claim 24, wherein the solvent buffer is selected from a

triethanolamine-HCl buffer, a triethanolamine-H₂S0₄ buffer and a KH₂P0 /K₂HP0₄ buffer.

26. The process according to any one of claims 23-25, wherein the solvent further comprises a co-solvent.

27. The process of claim 26, wherein the co-solvent is selected from at least one of: a C_\- C₄ alcohol, a C₃-C₇ ketone, a C₄-C₆ ether, a C₅-C₁₀ hydrocarbon, a C_!-C₆ halogenated hydrocarbon, a C₂-C₆ nitrile, a C₂-C₇ amide, and dimethyl sulfoxide.