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WO2006131933A1 - Enzymatic reduction of keto groups in 3-keto-propionic acid derivatives - Google Patents

Enzymatic reduction of keto groups in 3-keto-propionic acid derivatives Download PDF

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Publication number
WO2006131933A1
WO2006131933A1 PCT/IN2005/000187 IN2005000187W WO2006131933A1 WO 2006131933 A1 WO2006131933 A1 WO 2006131933A1 IN 2005000187 W IN2005000187 W IN 2005000187W WO 2006131933 A1 WO2006131933 A1 WO 2006131933A1
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Prior art keywords
formula
compound
keto
mucor
acid derivatives
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PCT/IN2005/000187
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French (fr)
Inventor
Shrivallabh Desai
Surekha K. Prabhu
Ramakrishnan Melarkode
Ganesh Sambasivam
Shrikumar Suryanarayan
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Biocon Limited
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Priority to PCT/IN2005/000187 priority Critical patent/WO2006131933A1/en
Publication of WO2006131933A1 publication Critical patent/WO2006131933A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers

Definitions

  • the instant invention relates to a process for reduction of keto group in 3-keto-propionic acid derivatives to obtain 3-hydroxy propionic acid derivatives using mucor.
  • Compound of formula I may be used as an intermediate for the synthesis of HMG COA reductase inhibitors e.g. Atorvastatin, Rosuvastatin, Fluvastatin and Pitavastatin.
  • the instant invention provides a process for the conversion of keto group to hydroxy group by employing whole cell of fungus belonging to Mucor sp., variants or mutants thereof.
  • the mucor sp. used is preferably Mucor circinelloides MTCC 5187.
  • a compound of formula (I) is produced by stereoselective reduction of a compound of formula (II) using Mucor sp. or a reductase enzyme of mucor sp. in vivo or in vitro.
  • R 1 CH 3 , CH 2 CI, CH 2 CH(OH)CH 2 CN
  • R 2 Me, Et, t-Bu the conformation at CH(OH) in Ri is R
  • R 1 CH 3 , CH 2 CI, CH 2 CH(O)CH 2 CN
  • R 2 Me, Et, t-Bu the conformation at CH(OH) in R x is R FORMULA II
  • the Mucor or the reductase enzyme of the Mucor reduces the keto group to hydroxy group to a preferred stereoisomer which can be used to prepare intermediates for the preparation of pharmaceutical compounds comprising anti-hypercholesterolemic agents i.e. statin e.g. Atorvastatin, Rosuvastatin, Pitavastatin and Fluvastatin.
  • statin e.g. Atorvastatin, Rosuvastatin, Pitavastatin and Fluvastatin.
  • the instant invention relates to a process of reduction of keto group to a hydroxy group by employing whole cells of the fungus belonging to Mucor sp. preferably Mucor circinelloides.
  • keto group in 3-keto- propionic acid derivatives is reduced to obtain 3-hydroxy-propionic acid derivatives (formula I) by employing whole cells of the fungus belonging to Mucor sp. preferably Mucor circinelloides.
  • keto group in 3-keto- propionic acid derivatives (formula II) is reduced to obtain 3-hydroxy- propionic acid derivatives (formula I) by employing the reductase enzyme isolated from whole cells of the fungus belonging to Mucor sp. preferably Mucor circinelloides.
  • the microorganism or the enzyme reduces the keto groups to hydroxy group of preferred stereo chemistry.
  • the hydroxy derivatives can be used to prepare anti-hypercholesterolemic agents such as Atorvastatin, Rosuvastatin, Pitavastatin and Fluvastatin.
  • the process may be carried out by adding a compound of formula II to a culture of the growing organism in a medium capable of supporting growth or to a suspension of the live cells in a medium which preferably contains a carbon source but which lacks one or more nutrients necessary for growth or in isolated enzyme solution with added cofactor.
  • Dead cells may also be used provided the necessary enzymes and co-factors are present; if necessary the cofactors may be added to these cells.
  • a co-factor normally NAD(P)H (nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate) and a system for re-generating the co-factor, for example glucose and glucose dehydrogenase, are required along with the functional enzyme to drive the reaction.
  • suitable co-factors and reduction mechanisms are present in the whole cells, it is preferred to use the whole cells in a nutrient medium which preferably contains a suitable carbon source, which may include one or more of the following: citric acid, a sugar, e.g. maltose, sucrose or preferably glucose, a polyol e.g. glycerol or sorbitol, or a lower alcohol, for example methanol or ethanol.
  • the cells may be immobilized on a support which are contacted with the compound of formula II preferably, in the presence of a suitable carbon source.
  • the process may suitably be carried out within a pH range of 3.5 to 9, preferably 4.5 to 7.0 and more preferably 5.0 to 6.0.
  • the process may suitably be carried out at a temperature of 10 to 50 0 C, preferably 20 to 40 0 C and more preferably 25 to 35 0 C.
  • An aeration rate equivalent to 0.01 to 1.0 volumes of oxygen measured at standard temperature and pressure per volume of the culture medium per minute is suitably employed at the aforesaid conditions of pH and temperature, with considerable variation.
  • the oxygen may be supplied as air.
  • the strain has following characteristics: Identification of the strain
  • Colony habit Colony on malt extract -agar fast growing, colourless to greyish ; reverse yellowish. Growth and sporulation at 35 deg C; growth but no sporulation at 37 deg C. No growth at 40 deg C. sexual reproduction (zygospore formation) not tested. Nitrate utilization positive.
  • Morphology Sporangiophores (15-20 ⁇ m ⁇ erect , either long (- 12mm), seldom branched , or short (1-2 mm) frequently branched .
  • sporangia on long sporangiophores at first yellowish , becoming light brown , upto 80 ⁇ m ⁇ ; walls deliquescing .
  • Purified enzymes may be isolated by prior art techniques well documented in literature the techniques selected from cell lysis, centrifugation, filtration, extraction, precipitation, chromatography, lyophilization or drying.
  • the enzyme is isolated by centrifuging of a suspension of disintegrated cells and separating a supernatant from debris, separating the desired enzyme from the solution for example by ion exchange chromatography suitably with elution from the column with liquid of increasing ionic strength and/or by selective precipitation by the addition of an ionic material, for example ammonium sulphate.
  • an ionic material for example ammonium sulphate.
  • Mucor cirdnelloides MTCC 5187 was maintained on Malt Extract Agar (MEA) plates prepared in sterilized MEA (5%) in distilled water.
  • a loopful of microbial cells was aseptically transferred from an agar plate to a 250 mL baffled conical flask containing 50 ml of medium having the following composition (per liter) - K 2 HPO 4 (1.9 g), NaH 2 PO 4 .2H 2 O (2.02 g) (NH 4 ) 2 SO 4 (1.8 g), MgSO 4 7H 2 O (0.2 g), FeCI 3 (0.97 mg) and trace elements solution (1 ml).
  • Trace elements solution consisted of (per liter) CuSO 4 5H 2 O (0.02 g), MnSO 4 4H 2 O (0.1 g), ZnSO 4 .7H 2 O (0.1 g) and CaCO 3 (1.8 g).
  • the above minimal medium was supplemented with 0.2% (w/v) yeast extract and 2.25% (w/v) glucose.
  • the microorganism was grown at 28 0 C on an orbital shaker at 220 rpm for 24-48 hours. Microbial cells were harvested by centrifuging at 7000 rpm for 20 minutes at 10° C. The cell pellet was resuspended in 10 ml of 50 mM sodium phosphate buffer, pH 5.4 and the cells were washed by centrifuging as above. The cell pellet was finally resuspended in 10 ml of the above buffer. To the 10 ml of cell suspension in a 50 ml baffled flask glucose (10 g per liter) was added and the compound of formula IV (2 g per liter). The cells were incubated at 28 degree C on a rotary shaker at 150 rpm for 24-48 hours.
  • the entire reaction broth was extracted with ethyl acetate.
  • the separated ethyl acetate extracts were pooled together and dried over anhydrous sodium sulphate and the solvent removed by vacuum distillation to afford a golden oil.
  • the extent of conversion of the compound of formula IV to the compound of formula III and the enantiomeric / diastereomeric composition of the compound of formula IV was determined by HPLC (high pressure liquid chromatography).
  • Mucor circinelloides MTCC 5187 was grown in fed-batch culture in a Braun Biostat ED/ER10 L fermenter.
  • Fermentations were performed under the following conditions of pH, temperature, aeration and agitation until a dry cell weight in the range 10 to 15 g/L was obtained:
  • the preparation of the compound of formula III was initiated by the addition of the compound of formula IV as a crude oil of approximately 80% w/w concentration, the balance being substantially solvent.
  • the compound of formula IV was added as a continuous feed at a rate sufficient to maintain its concentration at typically 2 g/L.
  • Solid glucose was added to the fermentation broth as co-substrate to maintain a concentration of 2 to 10 g/L.
  • the preparation of the compound of formula (I) was performed under the same conditions of temperature, pH, aeration and agitation as for the growth stage of the microorganism.
  • the concentration of the compounds of formula III and IV in the fermentation broth were measured by reverse phase HPLC.
  • a loopful of microbial cells was aseptically transferred from an agar plate to a 250 mL baffled conical flask containing 50 ml of medium having the following composition (per liter) - K 2 HPO 4 (1.9 g), NaH 2 PO 4 .2H 2 O (2.02 g) (NH 4 ) 2 SO 4 (1.8 g), MgSO 4 7H 2 O (0.2 g), FeCb (0.97 mg) and trace elements solution (1 ml).
  • Trace elements solution consisted of (per liter) CuSO 4 5H 2 O (0.02 g), MnSO 4 4H 2 O (0.1 g), ZnSO 4 JH 2 O (0.1 g) and CaCO 3 (1.8 g).
  • the above minimal medium was supplemented with 0.2% (w/v) yeast extract and 2.25% (w/v) glucose.
  • the microorganism was grown at 28 0 C on an orbital shaker at 220 rpm for 24-48 hours. Microbial cells were harvested by centrifuging at 7000 rpm for 20 minutes at 10° C. The cell pellet was resuspended in 10 ml of 50 mM sodium phosphate buffer, pH 5.4 and the cells were washed by centrifuging as above. The cell pellet was finally resuspended in 10 ml of the above buffer. To the 10 ml of cell suspension in a 50 ml baffled flask glucose (10 g per liter) was added and the compound of formula VI (2 g per liter). The cells were incubated at 28 degree C on a rotary shaker at 150 rpm for 24-48 hours.
  • the entire reaction broth was extracted with ethyl acetate.
  • the separated ethyl acetate extracts were pooled together and dried over anhydrous sodium sulphate and the solvent removed by vacuum distillation to afford a golden oil.
  • the extent of conversion of the compound of formula VI to the compound of formula V and the enantiomeric / diastereomeric composition of the compound of formula VI was determined by HPLC (high pressure liquid chromatography).

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Abstract

This invention claims a process of reduction of keto groups to hydroxy groups, e.g. a compound of formula (I) is produced by stereo/diastereoselectively reducing a compound of formula (II) using a reductase of Mucor, in vivo or in vitro. Formula (I): the conformation at CH(OH) in R1 is R. Formula (II): the conformation at CH(OH) in R1 is R. Mucor circinelloides produces the preferred stereoisomers which can be used to prepare intermediates for the preparation of pharmaceutical compounds comprising anti-hypercholesterolemic agents i.e. statin e.g. Atorvastatin, Rosuvastatin, Pitavastatin and Fluvastatin.

Description

TITLE OF THE INVENTIOM
ENZYMATIC REDUCTION OF KETO GROUPS IN 3-KETO- PROPIONIC ACID DERIVATIVES FIELD OF THE INVENTION
The instant invention relates to a process for reduction of keto group in 3-keto-propionic acid derivatives to obtain 3-hydroxy propionic acid derivatives using mucor. BACKGROUND INFORMATION:
Conversion of keto group into a hydroxy group is an important step in the synthesis of pharmaceutical compounds e.g. statins, Compound of formula I may be used as an intermediate for the synthesis of HMG COA reductase inhibitors e.g. Atorvastatin, Rosuvastatin, Fluvastatin and Pitavastatin.
It is known from Brower et al, Tetrahedron Lett. 1992, 2279-2282 to synthesize a compound of formula (I) by diastereoselective reduction of a compound of formula (II) using the method of Chen et al, Tetrahedron Lett. 1987, 155 and Chem. Lett. 1987, 1923,
A cross Claisen approach to compound (I) was also reported; this route involved the use of temperatures of -90° C, which renders it expensive and inconvenient. Stereoselective reductions in chemistry require special reactants and reaction conditions, which make the process often expensive, inconvenient and at times hazardous.
The instant invention provides a solution to the drawbacks of the prior art processes. SUMMARY OF THE INVENTION
The instant invention provides a process for the conversion of keto group to hydroxy group by employing whole cell of fungus belonging to Mucor sp., variants or mutants thereof. The mucor sp. used is preferably Mucor circinelloides MTCC 5187. A compound of formula (I) is produced by stereoselective reduction of a compound of formula (II) using Mucor sp. or a reductase enzyme of mucor sp. in vivo or in vitro.
Figure imgf000003_0001
R1 = CH3, CH2CI, CH2CH(OH)CH2CN R2 = Me, Et, t-Bu the conformation at CH(OH) in Ri is R
FORMULA I
Figure imgf000003_0002
R1 = CH3, CH2CI, CH2CH(O)CH2CN R2 = Me, Et, t-Bu the conformation at CH(OH) in Rx is R FORMULA II
The Mucor or the reductase enzyme of the Mucor reduces the keto group to hydroxy group to a preferred stereoisomer which can be used to prepare intermediates for the preparation of pharmaceutical compounds comprising anti-hypercholesterolemic agents i.e. statin e.g. Atorvastatin, Rosuvastatin, Pitavastatin and Fluvastatin.
The instant invention has following advantages over the prior art processes:
1. Economic
2. Ease of operation 3. Industrially feasible
4. Use of biological methods of conversion (green chemistry) over chemical conversion at ambient temperature.
5. Use of whole cells of the organism provides co-factors necessary for the reaction. 6. Use of whole cells of the organism avoids the need to separate the desired enzyme. DESCRIPTION OF THE INVENTION
The instant invention relates to a process of reduction of keto group to a hydroxy group by employing whole cells of the fungus belonging to Mucor sp. preferably Mucor circinelloides.
In the first embodiment of the invention, keto group in 3-keto- propionic acid derivatives (formula II) is reduced to obtain 3-hydroxy-propionic acid derivatives (formula I) by employing whole cells of the fungus belonging to Mucor sp. preferably Mucor circinelloides. In the second embodiment of the invention, keto group in 3-keto- propionic acid derivatives (formula II) is reduced to obtain 3-hydroxy- propionic acid derivatives (formula I) by employing the reductase enzyme isolated from whole cells of the fungus belonging to Mucor sp. preferably Mucor circinelloides. The microorganism or the enzyme reduces the keto groups to hydroxy group of preferred stereo chemistry. The hydroxy derivatives can be used to prepare anti-hypercholesterolemic agents such as Atorvastatin, Rosuvastatin, Pitavastatin and Fluvastatin.
The process may be carried out by adding a compound of formula II to a culture of the growing organism in a medium capable of supporting growth or to a suspension of the live cells in a medium which preferably contains a carbon source but which lacks one or more nutrients necessary for growth or in isolated enzyme solution with added cofactor. Dead cells may also be used provided the necessary enzymes and co-factors are present; if necessary the cofactors may be added to these cells.
In general a co-factor, normally NAD(P)H (nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate) and a system for re-generating the co-factor, for example glucose and glucose dehydrogenase, are required along with the functional enzyme to drive the reaction. As suitable co-factors and reduction mechanisms are present in the whole cells, it is preferred to use the whole cells in a nutrient medium which preferably contains a suitable carbon source, which may include one or more of the following: citric acid, a sugar, e.g. maltose, sucrose or preferably glucose, a polyol e.g. glycerol or sorbitol, or a lower alcohol, for example methanol or ethanol.
If desired the cells may be immobilized on a support which are contacted with the compound of formula II preferably, in the presence of a suitable carbon source.
The process may suitably be carried out within a pH range of 3.5 to 9, preferably 4.5 to 7.0 and more preferably 5.0 to 6.0.
The process may suitably be carried out at a temperature of 10 to 50 0C, preferably 20 to 40 0C and more preferably 25 to 35 0C.
It is preferred to operate under aerobic conditions if live whole cells of the aforesaid organisms are employed. An aeration rate equivalent to 0.01 to 1.0 volumes of oxygen measured at standard temperature and pressure per volume of the culture medium per minute is suitably employed at the aforesaid conditions of pH and temperature, with considerable variation. The oxygen may be supplied as air.
The microorganism i.e. Mucor circinelloides, Biocon culture collection number BICC 5389 was deposited in MTCC- IDA under Budapest Treaty, Chandigarh with accession number MTCC 5187. The strain has following characteristics: Identification of the strain
Colony habit: Colony on malt extract -agar fast growing, colourless to greyish ; reverse yellowish. Growth and sporulation at 35 deg C; growth but no sporulation at 37 deg C. No growth at 40 deg C. sexual reproduction (zygospore formation) not tested. Nitrate utilization positive.
Morphology : Sporangiophores (15-20 μm φ erect , either long (- 12mm), seldom branched , or short (1-2 mm) frequently branched . sporangia on long sporangiophores at first yellowish , becoming light brown , upto 80 μm φ ; walls deliquescing . sporangiospores ellipdoidal , 7*5 μm , chlamydospores present.
Purified enzymes may be isolated by prior art techniques well documented in literature the techniques selected from cell lysis, centrifugation, filtration, extraction, precipitation, chromatography, lyophilization or drying. Preferably the enzyme is isolated by centrifuging of a suspension of disintegrated cells and separating a supernatant from debris, separating the desired enzyme from the solution for example by ion exchange chromatography suitably with elution from the column with liquid of increasing ionic strength and/or by selective precipitation by the addition of an ionic material, for example ammonium sulphate. Such operations may be repeated if desired to enhance purity.
The following examples further illustrate the invention, it being understood that the invention is not intended to be limited by the details disclosed therein. EXAMPLES EXAMPLE 1
Preparation of 6-cyano-3, 5- dihydroxy-hexanoic add derivative
Mucor cirdnelloides MTCC 5187 was maintained on Malt Extract Agar (MEA) plates prepared in sterilized MEA (5%) in distilled water.
For growth in liquid medium a loopful of microbial cells was aseptically transferred from an agar plate to a 250 mL baffled conical flask containing 50 ml of medium having the following composition (per liter) - K2 HPO4 (1.9 g), NaH2 PO4.2H2 O (2.02 g) (NH4)2 SO4 (1.8 g), MgSO4 7H2 O (0.2 g), FeCI3 (0.97 mg) and trace elements solution (1 ml). Trace elements solution consisted of (per liter) CuSO45H2 O (0.02 g), MnSO44H2 O (0.1 g), ZnSO4.7H2 O (0.1 g) and CaCO3 (1.8 g). The above minimal medium was supplemented with 0.2% (w/v) yeast extract and 2.25% (w/v) glucose.
The microorganism was grown at 280C on an orbital shaker at 220 rpm for 24-48 hours. Microbial cells were harvested by centrifuging at 7000 rpm for 20 minutes at 10° C. The cell pellet was resuspended in 10 ml of 50 mM sodium phosphate buffer, pH 5.4 and the cells were washed by centrifuging as above. The cell pellet was finally resuspended in 10 ml of the above buffer. To the 10 ml of cell suspension in a 50 ml baffled flask glucose (10 g per liter) was added and the compound of formula IV (2 g per liter). The cells were incubated at 28 degree C on a rotary shaker at 150 rpm for 24-48 hours.
The entire reaction broth was extracted with ethyl acetate. The separated ethyl acetate extracts were pooled together and dried over anhydrous sodium sulphate and the solvent removed by vacuum distillation to afford a golden oil. The extent of conversion of the compound of formula IV to the compound of formula III and the enantiomeric / diastereomeric composition of the compound of formula IV was determined by HPLC (high pressure liquid chromatography).
Conversion of compound of formula III to compound of formula I was >90%.
EXAMPLE 2
Preparation of 6-cyano-3,5- dihydroxy-hexanoic acid derivatives
Growth of Mucor circinelloides MTCC 5187 in fermenter and In Situ Preparation of the Compound of Formula III.
Mucor circinelloides MTCC 5187 was grown in fed-batch culture in a Braun Biostat ED/ER10 L fermenter.
Growth in batch culture was performed in 9 L of medium (media composition used is given in Example 1). Fermentation using fed-batch culture was initiated by the addition of an inoculum of Mucor circinelloides MTC 5187. The inoculum was prepared in 2*500 ml_ of the medium described in Example 1 and was grown at 28 0C on an orbital shaker at 220 rpm for 48 hours.
Fermentations were performed under the following conditions of pH, temperature, aeration and agitation until a dry cell weight in the range 10 to 15 g/L was obtained:
Temperature: 28 0C, 34 0C, 40 0C. pH: 4.5, 5.5, 6.5
Aeration: 0.1 to 1.0 wm (volume air per volume of medium per minute)
Agitation: 400- 800 rpm The preparation of the compound of formula III was initiated by the addition of the compound of formula IV as a crude oil of approximately 80% w/w concentration, the balance being substantially solvent. The compound of formula IV was added as a continuous feed at a rate sufficient to maintain its concentration at typically 2 g/L. Solid glucose was added to the fermentation broth as co-substrate to maintain a concentration of 2 to 10 g/L. In each experiment the preparation of the compound of formula (I) was performed under the same conditions of temperature, pH, aeration and agitation as for the growth stage of the microorganism.
The concentration of the compounds of formula III and IV in the fermentation broth were measured by reverse phase HPLC.
On completion of the biotransformation stage, the fermentation broth was centrifuged at 5000 rpm for 20 minutes at 20-22° C. The compound of formula III was isolated from the supernatant by extraction with ethyl acetate. The solvent extracts were pooled and dried over anhydrous sodium sulphate and the solvent was removed by vacuum distillation to afford the product of formula III. EXAMPLE 3
Preparation of 4-choro-3-hγdroxγ-butanoϊc acid derivative Mucor circinelloides MTCC 5187 was maintained on Malt Extract Agar (MEA) plates prepared in sterilized MEA (5%) in distilled water.
For growth in liquid medium a loopful of microbial cells was aseptically transferred from an agar plate to a 250 mL baffled conical flask containing 50 ml of medium having the following composition (per liter) - K2 HPO4 (1.9 g), NaH2 PO4.2H2 O (2.02 g) (NH4)2 SO4 (1.8 g), MgSO4 7H2 O (0.2 g), FeCb (0.97 mg) and trace elements solution (1 ml). Trace elements solution consisted of (per liter) CuSO45H2 O (0.02 g), MnSO44H2 O (0.1 g), ZnSO4JH2 O (0.1 g) and CaCO3 (1.8 g). The above minimal medium was supplemented with 0.2% (w/v) yeast extract and 2.25% (w/v) glucose.
The microorganism was grown at 280C on an orbital shaker at 220 rpm for 24-48 hours. Microbial cells were harvested by centrifuging at 7000 rpm for 20 minutes at 10° C. The cell pellet was resuspended in 10 ml of 50 mM sodium phosphate buffer, pH 5.4 and the cells were washed by centrifuging as above. The cell pellet was finally resuspended in 10 ml of the above buffer. To the 10 ml of cell suspension in a 50 ml baffled flask glucose (10 g per liter) was added and the compound of formula VI (2 g per liter). The cells were incubated at 28 degree C on a rotary shaker at 150 rpm for 24-48 hours.
The entire reaction broth was extracted with ethyl acetate. The separated ethyl acetate extracts were pooled together and dried over anhydrous sodium sulphate and the solvent removed by vacuum distillation to afford a golden oil. The extent of conversion of the compound of formula VI to the compound of formula V and the enantiomeric / diastereomeric composition of the compound of formula VI was determined by HPLC (high pressure liquid chromatography).

Claims

CLAIMS We claim
1. A process for stereoselective reduction of keto group to hydroxy group employing a fungus, Mucor species.
2. A process for stereoselective reduction of keto group to hydroxy group employing a reductase enzyme isolated from a fungus, Mucor species.
3. The process of claim 1 or 2, wherein the fungus is specifically Mucor circinelloides.
4. A process of any preceding claim wherein the fungus is Mucor circinelloides MlCC 5187.
5. A process of any preceding claim, wherein a compound of formula II is reduced to afford a compound of formula I.
OH O
R1 = CH3, CH2CI, CH2CH(OH)CH2CN R2 = Me, Et, t-Bu the conformation at CH(OH) in Ri is R FORMULA I
Figure imgf000010_0001
R1 = CH3, CH2CI, CH2CH(O)CH2CN R2 = Me, Et, t-Bu the conformation at CH(OH) in Ri is R FORMULA II
6. A process of claim 5, wherein the hydroxy derivative thus obtained is used for preparation of pharmaceutical compounds comprising anti- hypercholesterolemic agents.
7. A process of claim 6, wherein the antihypercholesterolemic compound is selected from Atorvastatin, Rosuvastatin Fluvastatin and Pitavastatin.
8. A process of any preceding claim, wherein compound of formula IV is converted to compound of formula III.
Figure imgf000011_0001
Figure imgf000011_0002
9. A process of any preceding claim, wherein compound of formula VI is converted to formula V.
Figure imgf000011_0003
FORMULA Vl
Figure imgf000011_0004
FORMULA V
PCT/IN2005/000187 2005-06-08 2005-06-08 Enzymatic reduction of keto groups in 3-keto-propionic acid derivatives WO2006131933A1 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104388488A (en) * 2014-10-28 2015-03-04 尚科生物医药（上海）有限公司 Preparation of (3R,5R)-6-cyan-3,5-dihydroxy ter-butyl caproate employing immobilized whole-cell catalysis
US10676441B2 (en) 2015-08-05 2020-06-09 Api Corporation Method for producing pitavastatin calcium

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US10815201B2 (en) 2015-08-05 2020-10-27 Api Corporation Method for producing pitavastatin calcium
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