We Claims:
1. Rosuvastatin calcium comprising in the range of 1 ppm to 1500
ppm of a compound represented by the following formula (11):
.

2. Rosuvastatin calcium comprising in the range of 1 ppm to 1000
ppm of a compound represented by the following formula (13):
.

DESCRIPTION
Technical Field
[0001]
The present invention relates to a production method of rosuvastatin calcium and an intermediate therefor. Background Art
[0002]
Rosuvastatin is an inhibitor of an enzyme, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), and is useful for, for example, the treatment of hypercholesterolemia and mixed dyslipidemia. Rosuvastatin is a generic name of (E)-7-[4-(4-fluorophenyl)-6-isopropyl-2-
[methyl(methylsulfonyl)amino]pyrimidin-5-yl]-(3R,5S)-3,5-hydroxy-6-heptenoic acid. In treatments, rosuvastatin is administered as a calcium salt thereof. Rosuvastatin calcium is a trade name of CRESTOR (registered trade mark), and is sold as an HMG-CoA reductase inhibitor. Rosuvastatin calcium has the following chemical formula.
[0003]


OH OH e
co2
Ca2+

[0004]
Patent document 1 discloses rosuvastatin, a sodium salt and calcium salt thereof, and production methods of these. According to patent document 1, rosuvastatin and a salt thereof are obtained by condensing methyl (3R)-3-[(tert-butyldimethylsilyl)oxy]-5-oxo-6-triphenylphosphoranylidenehexanoate and 4-(4-fluorophenyl)-6-

isopropyl-2-(N-methyl-N-methanesulfonylamino)-5-pyrimidinecarboxaldehyde to introduce a side chain having one asymmetric center, and performing deprotection of 3-hydroxy group, asymmetric reduction of 5-oxo group, and hydrolysis. Since this method requires extremely low temperature conditions (preferably -85°C - -70°C) during asymmetric reduction, it is not entirely an industrially preferable production method. [0005]
Similar methods for introducing a side chain having two asymmetric centers are also known (patent documents 2, 3 and the like). Since these methods also require extremely low temperature conditions (e.g., about -75°C) during Wittig reaction, they are not entirely industrially preferable production methods. [0006]
Also, methods for introducing an asymmetric center by using an optically active titanium catalyst are known (patent document 4 and the like). Since these methods use an expensive optically active catalyst, and require extremely low temperature conditions (about -80°C - -50°C) during asymmetric reduction, they are not entirely industrially preferable production methods. [0007]
Non-patent documents 1 and 2 describe a method for producing a dihydroxyester derivative by reduction of a diketoester derivative. However, non-patent documents 1 and 2 specifically disclose only the reduction by organic synthesis reaction, and only compounds wherein the diketoester derivative or dihydroxyester derivative is tert-butylester. [0008]
Patent documents 5 and 6 describe a production method using carbonylreductase as a production method of pitavastatin. However, patent documents 5 and 6 do not provide description relating to rosuvastatin. In addition, rosuvastatin has a pyrimidine ring substituted by a sulfonylamino group, whereas

pitavastatin has a quinoline ring, and the chemical structures thereof are vastly different.
[Document List]
[Patent Documents]
[0009]
patent document 1: JP-B-2648897 patent document 2: WO 2010/047296 patent document 3: WO 2005/042522 patent document 4: WO 2008/065410 patent document 5: WO 2002/063028 patent document 6: WO 2003/078634
[non-patent documents]
[0010]
non-patent document 1: IP.com number: IPCOM000144026D, December 14, 2006
non-patent document 2: IP.com number: IPCOM000145623D, January 19, 2007
SUMMARY OF THE INVENTION Problems to be Solved by the Invention
[0011]
Since conventional production methods of rosuvastatin use extremely low temperature reactions and expensive asymmetric catalysts, the development of a more economical production method has been desired. The problem of the present invention is to provide a novel method capable of producing rosuvastatin calcium and intermediates therefor efficiently, inexpensively and with high purity. Means of Solving the Problems
[0012]
The present inventors have conducted intensive studies in an attempt to solve the above-mentioned problems and found that rosuvastatin calcium can be produced efficiently with a high purity under economical reaction conditions by using the following production method and/or intermediate, which resulted in the completion of the present invention.

Therefore, the present invention provides the following. [1] A production method of a compound represented by the following formula (2): [0013]

OH OH
C02R
(2)

[0014]
wherein R is a primary alkyl group having 1-8 carbon atoms or a secondary alkyl group having 3-6 carbon atoms, comprising
(i) a step of reducing a compound represented by the following formula (1):


C02R
(l)

[0016]
wherein R is as defined for R in the aforementioned formula (2), -X1 and -X2 are each independently -OH or =0, and -X1 and/or -X2 are/is =0, comprising reacting the compound with an enzyme having an activity capable of stereoselectively reducing a carbonyl group, a microorganism or cell having an ability to produce the enzyme, a treated product of the microorganism or cell, and/or a culture solution containing the enzyme obtained by culturing the microorganism or cell.
[2] The production method of the above-mentioned [1], wherein the aforementioned enzyme comprises a polypeptide of any of the following (A), (B) or (C) : (A) a polypeptide having carbonylreductase (OCR1) (SEQ ID NO:

2) derived from Ogataea minuta var. nonfermentans NBRC1473,
(B) a polypeptide consisting of an amino acid sequence having a homology of 80% or more to the amino acid sequence shown in SEQ ID NO: 2, and having an activity to convert a compound represented by the aforementioned formula (1) to a compound represented by the aforementioned formula (2),
(C) a polypeptide comprising an amino acid sequence which is the amino acid sequence shown in SEQ ID NO: 2, wherein 1 or several amino acids are substituted, deleted or added, and having an activity to convert a compound represented by the aforementioned formula (1) to a compound represented by the aforementioned formula (2).
[3] The production method of the above-mentioned [1], wherein the gene encoding the aforementioned enzyme is a DNA comprising the base sequence shown in the following (D), (E) or (F):
(D) the base sequence shown in SEQ ID NO: 1,
(E) a base sequence that hybridizes to a DNA consisting of a
sequence complementary to the base sequence shown in SEQ ID NO:
1 under stringent conditions, and encodes a polypeptide having
an activity to act on a compound represented by the
aforementioned formula (1) and convert same to a compound
represented by the aforementioned formula (2),
(F) a base sequence having a base sequence which is the base
sequence shown in SEQ ID NO: 1 wherein 1 or several bases are
substituted, deleted or added, and encodes a polypeptide having
an activity to act on a compound represented by the
aforementioned formula (1) and convert same to a compound
represented by the aforementioned formula (2).
[4] The production method of any of the above-mentioned [1] -[3], wherein the aforementioned step (i) is performed in the presence of polyhydric alcohol.
[5] A production method of a compound represented by the following formula (1): [0017]

X1 X2
OU2K
(1)
[0018]
wherein R is a primary alkyl group having 1-8 carbon atoms or a secondary alkyl group having 3-6 carbon atoms, -X1 and -X2 are each independently -OH or =0, and -X1 and/or -X2 are/is =0, comprising
(ii) a step of condensing a compound represented by the following formula (3): [0019]

N'
. A .
"N N'
o~s=o

,CHO

(3)

[0020]
and a compound represented by the following formula (4]
[0021]


(4)

[0022]
wherein R1 is a linear or branched alkyl group having 1-8 carbon atoms, in the presence of a base.
[6] The production method of the above-mentioned [5], wherein (iia) a step of condensing a compound represented by the following formula (3): [0023]

,CHO
N'
. A ,
(3)
"N N'
o~s=o
[0024]
and a compound represented by the following formula (4a)
[0025]

O
O
o
'OR*
(4a)
[0026]
wherein R2 is a branched alkyl group having 3-8 carbon atoms, which is different from the above-mentioned R, in the presence of a base; and
(iib) a step of reacting a compound represented by the following formula (5):


C02R2
(5)
[0028]
wherein R2 is as defined for R2 in the aforementioned formula (4a), which is obtained in the aforementioned step (iia) and an alcohol represented by R-OH wherein R is a primary alkyl group having 1-8 carbon atoms or a secondary alkyl group having 3-6 carbon atoms.
[7] The production method of the above-mentioned [5], comprising
(iia) a step of condensing a compound represented by the following formula (3): [0029]

XHO
1ST ^V'

[0030]
and a compound represented by the following formula (4a):
[0031]
o o o .
(4a)
[0032]
wherein R2 is a branched alkyl group having 3-8 carbon atoms, which is different from the aforementioned R, in the presence of a base;
(iib) a step of reacting a compound represented by the following formula (5): [0033]


CQ2R2
(5)
[0034] wherein R2 is as defined for R2 in the aforementioned formula
(4a), which is obtained in the aforementioned step (iia), and an alcohol represented by R-OH wherein R is a primary alkyl group having 1-8 carbon atoms or a secondary alkyl group having 3-6 carbon atoms; and
(ia) a step of obtaining compound(s) represented by the following formula (lb) and/or (lc):
[0035]


C02R
(lb)

[0036]
wherein R is as defined for R in the aforementioned formula (la),


C02R
(lc)

[0038] wherein R is as defined for R in the aforementioned formula
(la), comprising acting an enzyme having an activity capable of stereoselectively reducing a carbonyl group, a microorganism or cell having an ability to produce the enzyme, a treated product of the microorganism or cell, and/or a culture solution containing the enzyme obtained by culturing the microorganism or cell on a compound represented by the following formula
(la) :


C02R
(la)
[0040]
wherein R is a primary alkyl group having 1-8 carbon atoms or a secondary alkyl group having 3-6 carbon atoms, to reduce the compound.

[8] The production method of the above-mentioned [7], wherein the aforementioned enzyme comprises any of the polypeptide shown in the following (A), (B) or (C):
(A) a polypeptide having carbonylreductase (0CR1) (SEQ ID NO: 2) derived from Ogataea minuta var. nonfermentans NBRC1473,
(B) a polypeptide consisting of an amino acid sequence having a homology of 80% or more to the amino acid sequence shown in SEQ ID NO: 2, and having an activity to convert a compound represented by the aforementioned formula (1) to a compound represented by the aforementioned formula (2),
(C) a polypeptide comprising an amino acid sequence which is the amino acid sequence shown in SEQ ID NO: 2, wherein 1 or several amino acids are substituted, deleted or added, and having an activity to convert a compound represented by the aforementioned formula (1) to a compound represented by the aforementioned formula (2).
[9] The production method of the above-mentioned [7], wherein the gene encoding the aforementioned enzyme is a DNA comprising the base sequence shown in the following (D), (E) or (F):
(D) the base sequence shown in SEQ ID NO: 1,
(E) a base sequence that hybridizes to a DNA consisting of a
sequence complementary to the base sequence shown in SEQ ID NO:
1 under stringent conditions, and encodes a polypeptide having
an activity to act on a compound represented by the
aforementioned formula (1) and convert same to a compound
represented by the aforementioned formula (2),
(F) a base sequence having a base sequence which is the base
sequence shown in SEQ ID NO: 1 wherein 1 or several bases are
substituted, deleted or added, and encodes a polypeptide having
an activity to act on a compound represented by the
aforementioned formula (1) and convert same to a compound
represented by the aforementioned formula (2).
[10] The production method of any of the above-mentioned [7] -[9], wherein the aforementioned step (ia) is performed in the presence of polyhydric alcohol.

[11] A compound represented by the following formula [0041]

:ia) :

O 0
C02R

(la)

[0042]
'lb) or
wherein R is a primary alkyl group having 1-8 carbon atoms o secondary alkyl group having 3-6 carbon atoms. [12] A compound represented by the following formula (lc) : [0043]


C02R
(lb)
[0044]
wherein R is a primary alkyl group having 1-8 carbon atoms o secondary alkyl group having 3-6 carbon atoms, [0045]

O OH
C02R
(lc)

[0046]
wherein R is a primary alkyl group having 1-8 carbon atoms o secondary alkyl group having 3-6 carbon atoms. [13] A crystal of a compound represented by the following formula:


C02n-Pr

[0048]
which shows a powder X-ray diffraction pattern having characteristic peaks at 26=8.7°, 16.3°, 19.7°, 21.2°, 21.3° (±0.2°) .
[14] A production method of rosuvastatin calcium represented by the following formula (6): [0049]


OH OH 0 C02
Ca"

(6)

[0050]
comprising (iiia) a step of hydrolyzing a compound represented by the aforementioned formula (2) : [0051]


C02R
(2)

[0052]
wherein R is a primary alkyl group having 1-8 carbon atoms or a secondary alkyl group having 3-6 carbon atoms, which is obtained by the production method of the above-mentioned [1],

with a base, and reacting same with a calcium compound. [15] The production method of the above-mentioned [14], wherein the aforementioned hydrolysis in the aforementioned step (iiia) is performed in the presence of mixed solvent of a polar solvent, and at least one solvent selected from the group consisting of ether solvent, hydrocarbon solvent, and halogenated solvent.
[16] The production method of the above-mentioned [14] or [15], wherein the reaction with the calcium compound in step (iiia) is initiated at pH 5-10.
[17] A production method of rosuvastatin calcium represented by the following formula (6): [0053]


OH OH e
co2
Ca"

(6)

[0054]
comprising (iiib) a step of hydrolyzing a compound represented by the aforementioned formula (2) : [0055]

OH OH
C02R

(2)

[0056]
wherein R is a primary alkyl group having 1-8 carbon atoms or a secondary alkyl group having 3-6 carbon atoms, which is obtained by the production method of the above-mentioned [1], with a base, treating same with an acid, reacting the obtained

compound represented by the following formula (8): [0057]


CO,H
(8)

[0058]
with an amine compound, subjecting the obtained compound represented by the following formula (9): [0059]
I
OH OH

N
o=s=o

HNR3R4

(9)

[0060]
wherein R3 and R4 are each independently an alkyl group having 1-8 carbon atoms, to salt-exchange with a base, and reacting same with a calcium compound.
[18] A production method of rosuvastatin calcium represented by the following formula (6): [0061]


OH OH e
co2
Ca2+

(6)

[0062]
comprising (iiic) a step of hydrolyzing a compound represented
by the aforementioned formula (2) :

OH OH
C02R
[0063] F

(2)

[0064]
wherein R is a primary alkyl group having 1-8 carbon atoms or a secondary alkyl group having 3-6 carbon atoms, which is obtained by the production method of the above-mentioned [1], with a base, subjecting same to intramolecular dehydration condensation in the presence or absence of an acid catalyst, and reacting the obtained compound represented by the following formula (10): [0065]
F O

(10)
[0066]
with a calcium compound.
[19] A crystal of (E)-7-[4-(4-fluorophenyl)-6-isopropyl-2-[methyl(methylsulfonyl)amino]pyrimidin-5-yl]-(3R,5S)-3,5-hydroxy-6-heptenoic acid n-propylamine salt, which shows a powder X-ray diffraction pattern having characteristic peaks at 29=19.8°, 22.9° (±0.2°) .
[20] A crystal of (E)-7-[4-(4-fluorophenyl)-6-isopropyl-2-[methyl(methylsulfonyl)amino]pyrimidin-5-yl]-(3R,5S)-3,5-hydroxy-6-heptenoic acid dimethylamine salt, which shows a powder X-ray diffraction pattern having characteristic peaks at 29=6.6°, 17.0° (±0.2°) . [21] Rosuvastatin calcium comprising not less than 1 ppm and

not more than 1500 ppm of a compound represented by the
following formula (11):
[0067]

OH OH
F

[0068]
[22] A purification method of a compound represented by the
following formula (2):
[0069]

OH OH
C02R
(2)

[0070]
wherein R is a primary alkyl group having 1-8 carbon atoms or a secondary alkyl group having 3-6 carbon atoms, comprising dissolving the compound in an organic solvent, or a mixed solvent of an organic solvent and water, and cooling same at a cooling rate of 15°C/hr or below to precipitate a crystal of the compound represented by the aforementioned formula (2). [23] A production method of rosuvastatin calcium, comprising (B) a step of converting a compound represented by the following formula (12): [0071]


OH O
C02M
(1 2)

[0072]
wherein M is an alkali metal element, an alkaline earth metal element or hydrogen, to a compound represented by the following formula (13): [0073]


(1 3)

[0074]
[24] The production method of the above-mentioned [23],
comprising, prior to the aforementioned step (B), (Aa) a step
of converting a mixture of a compound represented by the
following formula (14):
[0075]


OH O
COoR
(14)

[0076]
wherein R is a primary alkyl group having 1-8 carbon atoms or a secondary alkyl group having 3-6 carbon atoms, and a compound

represented by the following formula (15): [0077]
F
OH OH
C02R
(15)

[0078]
wherein R is as defined above, to a mixture of a compound represented by the following formula (16): [0079]

OH OH
0O2M
F

(1 6)

[0080]
wherein M is an alkali metal element, an alkaline earth metal element or hydrogen, and a compound represented by the aforementioned formula (12), by hydrolyzing same in the presence of a base.
[25] The production method of the above-mentioned [23] or [24], comprising, after the aforementioned step (B), (C) a step of removing the compound represented by the aforementioned formula (13) .
[26] The production method of the above-mentioned [25], comprising, after the aforementioned step (C), (D) a step of reacting the compound obtained by the aforementioned step (C) and a calcium compound.
[27] A purification method of rosuvastatin calcium comprising a compound represented by the following formula (12):

OH O
C02M
[0081] F

(1 2

[0082]
wherein M is an alkali metal element, an alkaline earth metal element or hydrogen, comprising (B) a step of converting the compound represented by the formula (12) to a compound represented by the following formula (13): [0083]

O
F

(13)

[0084]
[28] The purification method of the above-mentioned [27], comprising, prior to the aforementioned step (B), (Ab) a step of dissolving rosuvastatin calcium comprising a compound represented by the aforementioned formula (12) in a solvent. [29] The purification method of the above-mentioned [27] or [28], comprising, after the aforementioned step (B) , (C) a step of removing the compound represented by the aforementioned formula (13).
[30] The purification method of the above-mentioned [29], comprising, after the aforementioned step (C), (D) a step of reacting the compound obtained by the aforementioned step (C) and a calcium compound.
[31] Rosuvastatin calcium comprising not less than 1 ppm and not more than 1000 ppm of a compound represented by the

following formula (13): [0085]

O
F

(1 3)

[0086]
[Effect of the Invention]
[0087]
According to the production method of the present invention, rosuvastatin calcium and intermediates therefor having a high purity can be efficiently produced under economical conditions and at an industrial scale, without using an extremely low temperature reaction or an expensive asymmetric catalyst. [Brief Description of the Drawings] [0088]
Fig. 1 shows the powder X-ray diffraction pattern of the compound (DOXP(n-propyl (E)-7-[4-(4-fluorophenyl)-6-isopropyl-2-[methyl(methylsulfonyl)amino]pyrimidin-5-yl]-3,5-dioxo-6-heptenoate)) obtained in Example 2, wherein the vertical axis shows intensity and the horizontal axis shows 26 (°) .
Fig. 2 shows the powder X-ray diffraction pattern of the compound obtained in Example 2', wherein the vertical axis shows intensity and the horizontal axis shows 26 (°) .
Fig. 3 shows the powder X-ray diffraction pattern of the compound (DOLP(n-propyl ((3R),(5S),(6E))-7-[4-(4-fluorophenyl)-6-isopropyl-2-[methyl(methylsulfonyl)amino]pyrimidin-5-yl]-3,5-dihydroxy-6-heptenoate)) obtained in Example 5, wherein the vertical axis shows intensity and the horizontal axis shows 26(°) .
Fig. 4 shows the powder X-ray diffraction pattern of the

compound (DOLP) obtained in Example 11, wherein the vertical axis shows intensity and the horizontal axis shows 26 (°) .
Fig. 5 shows the powder X-ray diffraction pattern of the propylamine salt obtained in Example 7, wherein the vertical axis shows intensity and the horizontal axis shows 26 (°) .
Fig. 6 shows the powder X-ray diffraction pattern of the dimethylamine salt obtained in Example 9, wherein the vertical axis shows intensity and the horizontal axis shows 26 (°) . [Description of Embodiments] [0089]
The terms used in the present specification are explained in detail the following.
In the present specification, the "primary alkyl group having 1-8 carbon atoms" means methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, n-hexyl group, n-octyl group.
In the present specification, the "primary alkyl group having 1-4 carbon atoms" means methyl group, ethyl group, n-propyl group, n-butyl group.
In the present specification, the "secondary alkyl group having 3-6 carbon atoms" means isopropyl group, cyclopropyl group, sec-butyl group, 1-methylbutyl group, 1-methylheptyl group, 1-ethylpropyl group, 1-ethylbutyl group.
In the present specification, the "secondary alkyl group having 3-4 carbon atoms" means isopropyl group, cyclopropyl group, sec-butyl group.
In the present specification, the "linear or branched alkyl group having 1-8 carbon atoms" means methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, n-hexyl group, n-octyl group, isopropyl group, cyclopropyl group, sec-butyl group, 1-methylbutyl group, 1-methylheptyl group, tert-butyl group, tert-amyl group.
In the present specification, the "branched alkyl group having 3-8 carbon atoms" means isopropyl group, cyclopropyl group, sec-butyl group, 1-methylbutyl group, 1-methylheptyl

group, tert-butyl group, tert-amyl group.
In the present specification, the "calcium compound" means a compound such as calcium chloride, calcium acetate and the like, which can convert a carboxylic acid to a calcium salt thereof. Preferably, the calcium compound is calcium chloride.
In the present specification, the "amine compound" means a compound such as n-propylamine, isopropylamine, dimethylamine and the like, which can convert a carboxylic acid to an amine salt thereof. Preferably, the amine compound is n-propylamine or dimethylamine.
The compound of the present invention also encompasses salts, anhydrides, hydrates, solvates and the like of the compound. [0090]
In the present specification, the "enzyme having an activity capable of reducing a carbonyl group stereoselectively" means an enzyme having an activity to convert a carbonyl group in a carbonyl group-containing compound to optically active alcohol by asymmetric reduction.
Whether the "activity capable of reducing a carbonyl group stereoselectively" is present can be determined by measuring an activity to convert a carbonyl group in a carbonyl group-containing compound to an optically active alcohol by asymmetric reduction by a general assay method. For example, a measurement target enzyme is reacted with a compound represented by the formula (1), the amount of a compound represented by the formula (2) converted from the compound represented by the formula (1) is directly measured, whereby the enzyme activity can be confirmed.
The "enzyme" in the present specification includes purified enzyme (including partially purified enzyme), an enzyme immobilized by a conventional immobilization technique, for example, one immobilized on a carrier such as polyacrylamide, carageenan gel and the like.
In the present specification, the "microorganism or cell

capable of producing an enzyme having an activity capable of reducing a carbonyl group stereoselectively" (hereinafter sometimes referred to as "the microorganism or cell of the present invention") is not particularly limited as long as it has an "activity capable of reducing a carbonyl group stereoselectively", and it may be a microorganism or cell inherently having the aforementioned activity, or a microorganism or cell imparted with the aforementioned activity by bleeding. As a means for imparting the aforementioned activity by bleeding, known methods such as a gene recombinant treatment (transformation), a mutation treatment and the like can be adopted. As a method of transformation, methods such as introduction of the object gene, enhanced expression of an enzyme gene in the biosynthetic pathway of organic compounds, reduction of expression of an enzyme gene in the by-product biosynthetic pathway and the like can be used.
As the kind of the "microorganism or cell", those described in the below-mentioned host organism or host cell can be mentioned. A "microorganism or cell" in a state of being frozen can also be used. In the present specification, the "microbial or cell capable of producing an enzyme having the activity" is not limited to a living microorganism or cell, but also includes one which is biologically dead but has an enzymatic activity.
The microorganism or cell in the present invention can be produced by the method described in WO 2003/078634.
In the present specification, the kind of the organism to be a "host organism" is not particularly limited, and prokaryotes such as Escherichia coli, Bacillus subtilis, corynebacterium, Pseudomonas bacterium, Bacillus bacterium, Rhizobium bacterium, Lactobacillus bacterium, Succinobacillus bacterium, Anaerobiospirillum bacterium, Actinobacillus bacterium and the like, fungi such as yeast, filamentous fungi and the like, eucaryotes such as plant, animal and the like can be mentioned. Of these, preferred are Escherichia coli, yeast

and corynebacterium, and particularly preferred is Escherichia coli.
In the present specification, the kind of the cell to be a "host cell" is not particularly limited, and animal cell, plant cell, insect cell and the like can be used.
In the present specification, an "expression vector" is a genetic factor used for replicating and expressing a protein having a desired function in the aforementioned host organism, by introducing a polynucleotide encoding a protein having a desired function into a host organism. Examples thereof include, but are not limited to, plasmid, virus, phage, cosmid and the like. Preferable expression vector is a plasmid.
In the present specification, a "transformant" means a microorganism or cell into which the aforementioned expression vector has been introduced, and which has acquired an ability to show a desired trait associated with a protein having a desired function.
In the present specification, a "treated product of microorganism or cell" means a product obtained by culturing a microorganism or cell, and 1) treating the microorganism or cell with an organic solvent and the like, 2) freeze-drying same, 3) immobilizing same on a carrier and the like, 4) physical or enzymatical destruction and containing a protein having a desired function and the like.
In the present specification, a "culture solution containing enzyme obtained by culturing microorganism or cell" means 1) a culture solution of microorganism or cell, 2) a culture solution obtained by treating a culture solution of microorganism or cell with an organic solvent and the like, or 3) a culture solution wherein cellular membrane of microorganism or cell is physically or enzymatically destroyed. [0091] [The production method of the present invention]
The production method of the present invention is explained in detail below. In the following, w/v means

weight/volume.
The production method of the present invention includes, as shown below, step (i) for converting a compound represented by the formula (1) to a compound represented by the formula (2), and step (iiia), (iiib) ((iiib-1) - (iiib-3)) or (iiic) ((iiic-1) - (iiic-2)) for converting a compound represented by the formula (2) or rosuvastatin calcium represented by the formula (6) .

Step (iiib-1) T i
N^V^
/S0
Step
(iiii.-2)
OH OH
COnR
,C02H
OH OH e
co2
Step Ca2+ (iiii,-3)

(6)
[0093]
wherein, R is a primary alkyl group having 1-8 carbon atoms or a secondary alkyl group having 3-6 carbon atoms, preferably a primary alkyl group having 1-4 carbon atoms or a secondary alkyl group having 3-4 carbon atoms. As R, methyl group, ethyl group, n-propyl group, isopropyl group or n-butyl is preferable. Of these, R is more preferably n-propyl group or isopropyl group, particularly preferably n-propyl group to efficiently perform step (i).
-X1 and -X2 are each independently -OH or =0, and -X1 and/or -X2 are/is =0.
R3 and R4 are each independently a hydrogen atom or an alkyl group having 1-8 carbon atoms, preferably a hydrogen atom or an alkyl group having 1-4 carbon atoms.

[0094]
The production method of the present invention includes, as shown below, as a production method of a compound represented by the formula (1) used in step (i), step (ii) for converting a compound represented by the formula (3) and a compound represented by the formula (4) to a compound represented by the formula (1).
Furthermore, as a preferable embodiment of step (ii), step (iia) for converting a compound represented by the formula (3) and a compound represented by the formula (4a) to a compound represented by the formula (5), and step (iib) for converting a compound represented by the formula (5) to a compound represented by the formula (1) are included. [0095]
As another embodiment of step (i), moreover, step (ia) for converting a compound represented by the formula (la) to a compound represented by the formula (lb) and/or a compound represented by the formula (lc), and step (ib) for converting a compound represented by the formula (lb) and/or a compound represented by the formula (lc) to a compound represented by the formula (2) are also included in the production method of the present invention. [0096]

C09R
CHO

o o
A~A^CO2RI
CHO
(4)
CO-R
COnR
o o
Step (ia)
[0097] wherein R, -X1 and -X2 are as defined above.
R1 is a linear or branched alkyl group having 1-8 carbon atoms, preferably a primary alkyl group having 1-8 carbon atoms or a secondary alkyl group having 3-6 carbon atoms, more preferably a primary alkyl group having 1-4 carbon atoms or a secondary alkyl group having 3 or 4 carbon atoms. As R1, methyl group, ethyl group, n-propyl group, isopropyl group or n-butyl is preferable. Of these, R1 is more preferably n-propyl group or isopropyl group, particularly preferably n-propyl group, since step (i) can be performed efficiently.
R2 is a branched alkyl group having 3-8 carbon atoms, and is different from the above-mentioned R. R2 is preferably isopropyl group, s-butyl group, tert-butyl group, tert-amyl group, particularly preferably tert-butyl group.
[0098]

In the following, each step of the production method of the present invention is explained in detail.
[0099] Step (i):
In step (i), a compound represented by the formula (1) is reduced by reaction with an enzyme having an activity capable of stereoselectively reducing a carbonyl group, a microorganism or cell having an ability to produce the enzyme (the microorganism or cell of the present invention), a treated product of the microorganism or cell, and/or a culture solution containing the enzyme obtained by culturing the microorganism or cell (hereinafter these are sometimes collectively referred to as "the enzyme etc. of the present invention") to give a compound represented by the formula (2).
[0100]



OH OH
C02R
C02R

[0101]
wherein, R, -X1 and -X2 are as defined above. [0102]
As the enzyme used in step (i), one having the amino acid sequence shown in SEQ ID NO: 2 (hereinafter sometimes to be referred to as "OCR1") or a homologue of the amino acid sequence can be used. Specifically, an enzyme containing a polypeptide of the following (A), (B) or (C) or a homologue of these can be mentioned.
(A) a polypeptide having carbonylreductase (OCR1) (SEQ ID NO: 2) derived from Ogataea minuta var. nonfermentans NBRC1473 described in JP-B-4270918,
(B) a polypeptide consisting of an amino acid sequence having a homology of 80% or more to the amino acid sequence shown in SEQ

ID NO: 2, and having an activity to convert a compound represented by the formula (1) to a compound represented by the formula (2),
(C) a polypeptide comprising an amino acid sequence which is
the amino acid sequence shown in SEQ ID NO: 2, wherein 1 or
several amino acids are substituted, deleted or added, and
having an activity to convert a compound represented by the
formula (1) to a compound represented by the formula (2).
[0103]
A homologue of the above-mentioned (B) is a protein having at least 80%, preferably 85% or more, more preferably 90% or more, further preferably 95% or more, homology with the full-length amino acid sequence shown in SEQ ID NO: 2.
A homologue of the above-mentioned (C) has an amino acid sequence which is the amino acid sequence shown in SEQ ID NO: 2, wherein 1 or several amino acids are deleted, added or substituted, as long as the activity capable of stereoselectively reducing the carbonyl group is not inhibited. As used herein, "1 or several amino acids" is specifically 20 or less, preferably 10 or less, more preferably 5 or less, amino acids.
The gene encoding the above-mentioned enzyme is a DNA comprising the base sequence shown in the following (D), (E) or (F) or a homologue thereof:
(D) the base sequence shown in SEQ ID NO: 1,
(E) a base sequence that hybridizes to a DNA consisting of a
sequence complementary to the base sequence shown in SEQ ID NO:
1 under stringent conditions, and encodes a polypeptide having
an activity to act on a compound represented by the formula (1)
and convert same to a compound represented by the formula (2),
(F) a base sequence having a base sequence which is the base
sequence shown in SEQ ID NO: 1 wherein 1 or several bases are
substituted, deleted or added, and encodes a polypeptide having
an activity to act on a compound represented by the formula (1)
and convert same to a compound represented by the formula (2).

Here, the "base sequence that hybridizes under stringent conditions" in the above-mentioned (E) means a base sequence of a DNA obtained by colony hybridization method, plaque hybridization method, or Southern blot hybridization method and the like under stringent conditions by using DNA as a probe. Examples of the stringent conditions in colony hybridization method and plaque hybridization method include conditions of hybridization using a filter immobilizing a colony- or plaque-derived DNA or a fragment of the DNA in the presence of a 0.7 mol/L - 1.0 mol/L aqueous sodium chloride solution at 65°C, and washing the filter with 0.1 - 2*SSC solution (composition of lxSSC, 150 mmol/L aqueous sodium chloride solution, 15 mmol/L aqueous sodium citrate solution) at 65°C.
Each hybridization can be performed according to the method described in Molecular Cloning: A Laboratory Mannual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989 and the like.
A homologue of the above-mentioned (F) has a base sequence which is the base sequence shown in SEQ ID NO: 1, wherein 1 or several bases are deleted, added or substituted, as long as the activity capable of stereoselectively reducing the carbonyl group is not inhibited. As used herein, "1 or several bases" is specifically 60 or less, preferably 30 or less, more preferably 15 or less, bases.
In step (i), since the enzyme etc. of the present invention is superior in the handling property, and easily added to a reaction system, it can also be used in a frozen state. When frozen enzyme etc. of the present invention are used, the shape thereof is not particularly limited and, for example, prismatic, cylindrical, bulk, spherical shape and the like can be employed. [0104]
In step (i), a compound represented by the formula (1) to be a reaction substrate is generally used at a substrate concentration of 0.01%w/v - 20%w/v, preferably 0.1%w/v - 10%w/v.

A reaction substrate may be added at once at the start of the reaction. When substrate is inhibited by the enzyme, the enzyme can also be added continuously or intermittently to reduce the influence thereof or improve accumulation concentration of the resultant product.
Step (i) is preferably performed in the presence of coenzyme NAD(P)+ or NAD(P)H. In this case, the above-mentioned coenzyme is preferably added at a concentration of generally 0.001 mmol/L - 100 mmol/L, preferably 0.01 mmol/L - 10 mmol/L.
When the above-mentioned coenzyme is added, regeneration of NAD(P)+ produced from NAD(P)H into NAD(P)H in the reaction system is preferable, since production efficiency can be improved. Examples of the regeneration method include
1) a method utilizing an ability to generate NAD(P)H from NAD(P)+ of the microorganism or cell itself in the present invention, i.e., NAD(P)+ reduction ability,
2) a method comprising addition of one or more kinds from a microorganism or a treated product thereof having an ability to generate NAD(P)H from NAD(P)+, or an enzyme utilizable for regeneration of NAD(P)H such as glucose dehydrogenase, formate dehydrogenase, alcohol dehydrogenase, amino acid dehydrogenase, organic acid dehydrogenase (malic acid dehydrogenase and the like) and the like (hereinafter to be referred to as "regenerating enzyme") to a reaction system,
3) a method comprising concurrent introduction of one or more kinds of the above-mentioned regenerating enzyme gene into a host organism or host cell when producing the microorganism or cell in the present invention, and the like.
In the above-mentioned method of 1), glucose, ethanol, 2-propanol or formic acid and the like are preferably added to the reaction system.
In the above-mentioned method of 2), a microorganism having an ability to produce the above-mentioned regenerating enzyme, a treated product of microorganism such as the microorganism treated with acetone or freeze-dry treated,

physically or enzymatically disrupted and the like, the enzyme fraction obtained as a crude product or purified product, and further, these after immobilization on a carrier such as polyacrylamide gel, carageenan gel and the like, and the like may be used, or a commercially available enzyme may also be used.
In this case, the amount of the above-mentioned regenerating enzyme to be used is such amount that renders the enzyme activity generally 0.01-fold to 100-fold, preferably about 0.5-fold to 20-fold, as compared to the carbonyl reduction activity of the enzyme of the present invention having an ability to stereoselectively reduce a carbonyl group.
While addition of a compound to be the substrate of the above-mentioned regenerating enzyme, for example, glucose when glucose dehydrogenase is utilized, formic acid when formate dehydrogenase is utilized, ethanol or isopropanol when alcohol dehydrogenase is utilized and the like, is also necessary, the amount thereof to be added is generally 0.1 equivalent - 20 equivalents, preferably 1 equivalent - 10 equivalents, relative to a compound represented by the formula (1) to be the reaction starting material.
In the method of the above-mentioned 3), a method for incorporating a DNA of the above-mentioned regenerating enzyme into chromosome along with a DNA encoding the enzyme used in step (i), a method for introducing both DNAs into a single expression vector and transforming a host organism or cell, or a method for introducing both DNAs into separate expression vectors, and transforming a host organism or cell can be used. In the method for introducing both DNAs into separate expression vectors, and transforming a host organism or cell, an expression vector needs to be selected in consideration of the incompatibility between both expression vectors.
When plural genes are introduced into a single expression vector, a method of connecting regions involved in the control of expression such as a promoter and a terminator and the like

to each gene, as well as expression as an operon containing multiple cistrons such as lactose operon are also possible.
[0105]
Step (i) is performed in an aqueous medium or a mixture of the aqueous medium and an organic solvent, which contains a compound represented by the formula (1) and the above-mentioned enzyme, a microorganism or cell having an ability to produce the enzyme, a treated product of the microorganism or cell, and/or a culture solution containing the enzyme obtained by culturing the microorganism or cell and, where necessary, various coenzymes (a regenerating system thereof, i.e., more preferably the coenzyme can be regenerated). A compound represented by the formula (1) can be produced by the below-mentioned method.
As the aqueous medium, water and buffers such as potassium phosphate buffer, sodium citrate buffer, Tris-HCl buffer and the like can be mentioned.
As the organic solvent, a solvent in which a compound represented by the formula (1) shows high solubility, such as ethyl acetate, isopropyl acetate, butyl acetate, toluene, chloroform, n-hexane, n-heptane, dimethyl sulfoxide, methanol, ethanol, n-propanol, 2-propanol and the like can be used. Of these, dimethyl sulfoxide, methanol, ethanol are preferable as an organic solvent, since a compound represented by the formula
(1) shows high solubility. Furthermore, dimethyl sulfoxide is more preferable since conversion ratio is high.
Step (i) can also be performed in the presence of polyhydric alcohol such as glycerol, ethylene glycol, propylene glycol, erythritol, inositol, sorbitol, xylitol and the like. The aforementioned polyhydric alcohol may be a polymer or derivative, and one kind thereof may be used or a mixture of two or more kinds thereof can also be used. When step (i) is performed in the presence of polyhydric alcohol, the conversion ratio tends to be improved. Among those, glycerol is preferable since it is assumed to maintain enzymatic activity

by retaining conformation of the enzyme, and is easily available. The amount of glycerol to be used is preferably not less than 40 g/L, more preferably not less than 170 g/L, and preferably not more than 600 g/L, more preferably not more than 400 g/L.
The below-mentioned step (ia) and/or step (ib) can also be performed in the presence of the aforementioned polyhydric alcohol.
Step (i) is generally performed at a reaction temperature of 4°C - 70°C, preferably 20°C - 60°C, generally at pH 3 - 11, preferably pH 4-8. The reaction time is generally 0.5 hr -48 hr, preferably 0.5 hr - 24 hr. It can also be performed utilizing a membrane reactor and the like.
A compound represented by the formula (2) obtained in step (i) can be purified by separating cells, polypeptide and the like by centrifugation, filtration and the like, adjusting to a suitable pH, extraction with an organic solvent such as hexane, ethyl acetate, toluene and the like, and applying an appropriate combination of purification by column chromatography, crystallization and the like. [0106]
When a compound represented by the formula (2) is purified by crystallization, as an organic solvent that can be used, a solvent in which a compound represented by the formula (2) shows high solubility, for example, hydrocarbon solvents such as cyclohexane, n-hexane, n-heptane, toluene and the like, halogenated solvents such as chlorobenzene, dichlorobenzene and the like, ether solvents such as tert-butyl methyl ether, tetrahydrofuran (THF), cyclopentyl methyl ether (CPME) and the like, alcoholic solvents such as methanol, ethanol, n-propanol, isopropanol and the like, polar solvents such as N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide and the like, and the like can be used. These organic solvents can be used singly, and a mixed solvent of these organic solvents and water can also be used.

When a compound represented by the formula (2) is purified by crystallization, a crystal of a compound represented by the aforementioned formula (2) is preferably precipitated by dissolving a compound represented by the aforementioned formula (2) in an organic solvent, or a mixed solvent of an organic solvent and water, and then cooling same at a cooling rate of 15°C/hr or below (this step of crystal precipitation by cooling is hereinafter to be referred to as a "cooling step").
In the cooling step, a temperature at which cooling is started is preferably 15°C - 60°C, more preferably 20°C - 55°C.
The cooling rate in the cooling step is preferably not more than 15°C/hr, more preferably not more than 9°C/hr, further preferably not more than 6°C/hr, particularly preferably not more than 5°C/hr. In this way, the purity of the obtained compound represented by the formula (2) can be increased.
The cooling rate can be changed during the cooling step. Particularly, slow cooling is preferable within a temperature range of preferably not more than 45°C, more preferably not more than 40°C. To be specific, the cooling rate is more preferably 9°C/hr, further preferably not more than 6°C/hr, particularly preferably not more than 5°C/hr. [0107]
Also, it is preferable to include a step of aging (hereinafter to be referred to as "aging step") after dissolving a compound represented by the aforementioned formula (2) in the aforementioned solvent and before the aforementioned cooling step. The aging step preferably has a aging step at a high temperature and a low temperature aging step wherein aging is performed at a temperature lower than that in the high temperature aging step. In the aging step, the order of the high temperature aging step and the low temperature aging step is not particularly limited, and a high temperature aging step is preferably performed after a low temperature aging step. In

addition, a low temperature aging step and a high temperature aging step may be repeated several times as necessary. [0108]
The low temperature aging step in the aging step is a step wherein aging is performed after dissolving a compound represented by the aforementioned formula (2) in the aforementioned organic solvent or a mixed solvent of an organic solvent and water, and at a temperature lower than the temperature of dissolution in the aforementioned organic solvent and the like, and lower than the aging temperature of the below-mentioned high temperature aging step.
The aging temperature in the low temperature aging step is preferably lower than the temperature of dissolution in the aforementioned organic solvent and the like by not less than 1°C, more preferably not less than 5°C, particularly preferable not less than 10°C. A specific aging temperature is preferably 0°C - 59°C, more preferably 5°C - 50°C.
In the low temperature aging step, the temperature can be changed on the way. When the temperature is changed, for example, aging can be performed first at a comparatively high temperature (e.g., 35°C - 45°C) for 5 min - 12 hr, after which at a comparatively low temperature (e.g., 30°C - 40°C) for 10 min - 5 hr.
The low temperature aging step is preferably performed for 10 min - 24 hr, more preferably 20 min - 10 hr.
In the low temperature aging step, the temperature is not merely retained but, where necessary, the solution may be stirred or a seed crystal may be added. [0109]
The high temperature aging step in the aging step is a step wherein aging is performed at a temperature higher than the aging temperature of the aforementioned low temperature aging step.
The aging temperature in the high temperature aging step is preferably higher than the aging temperature in the

aforementioned low temperature aging step by not less than 1°C, more preferably not less than 3°C, particularly preferably not less than 5°C. A specific aging temperature is preferably 20°C - 60°C, more preferably 25°C - 55°C. Generally, the aforementioned cooling step is started from the aging temperature of the high temperature aging step (final temperature of the high temperature aging step when the high temperature aging step is performed plural times). The temperature can be changed on the way also in the high temperature aging step.
The high temperature aging step is preferably performed for 10 min - 24 hr, more preferably 20 min - 10 hr.
In the high temperature aging step, the temperature is not merely retained but, where necessary, the solution may be stirred.
By forming such aging step, effects of improved filtration efficiency and improved purity of the object product can be obtained.
When a compound represented by the formula (2) is purified by crystallization, purification is preferably achieved by performing the aforementioned aging step (the aforementioned high temperature aging step, and the aforementioned low temperature aging step) and the aforementioned cooling step after dissolving a compound represented by the aforementioned formula (2) in an organic solvent, or a mixed solvent of an organic solvent and water.
The purity of the obtained crystal can be further improved by crystallization by such method. [0110]
Step (i) can also be performed in two steps of step (ia) and step (ib) as shown below. [0111] Step (ia):
In step (ia), a compound represented by the formula (la) which is the formula (1) wherein -X1 and -X2 are =0, is reduced

by reaction with an enzyme having an activity capable of stereoselectively reducing a carbonyl group, a microorganism or cell having an ability to produce the enzyme, a treated product of the microorganism or cell, and/or a culture solution containing the enzyme obtained by culturing the microorganism or cell to give a compound represented by the formula (lb) which is the formula (1) wherein -X1 is -OH and -X2 is =0, and/or a compound represented by the formula (lc) which is the formula (1) wherein -X1 is =0 and -X2 is -OH.
A compound represented by the formula (la) can be reduced by a method similar to that in step (i). [0112]




OH O
C02R
C02R
O OH
C02R

[0113]
wherein R is as defined above. [0114]
The compound(s) represented by the formula (lb) and/or (lc) obtained in step (ia) may be purified by, for example, crystallization before subjecting to step (ib). [0115] Step (ib):
In step (ib), a compound represented by the formula (lb) and/or a compound represented by the formula (lc) obtained in

step (ia) is reduced by applying the above-mentioned enzyme having an activity capable of stereoselectively reducing a carbonyl group, a microorganism or cell having an ability to produce the enzyme, a treated product of the microorganism or cell, and/or a culture solution containing the enzyme obtained by culturing the microorganism or cell to give a compound represented by the formula (2).
A compound represented by the formula (lb) and/or a compound represented by the formula (lc) can be reduced by a method similar to that in step (i).




C02R
OH OH
C02R
O OH
C02R

[0117]
wherein R is as defined above. [0118]
Among the compounds represented by the formula (2) obtained in step (i), or step (ia) and (ib), since a compound of the formula (2) wherein R is an n-propyl group or isopropyl group has high crystallinity, it can be obtained with a high purity.
A crystal of a compound of the formula (2) wherein R is an n-propyl group has, for example, a powder X-ray diffraction pattern shown below (powder X-ray diffraction patterns shown

below were obtained in the below-mentioned Example 5).
[0119] Table 1

26 relative intensity 26 relative intensity 26 relative intensity 26 relative intensity
7.7 16 15.0 12 21.1 100 25.1 5
8.4 65 16.1 26 22.1 8 26.4 6
11.2 8 16.8 36 22.5 13 28.5 8
12.6 32 18.1 7 24.2 6 29.5 6
12.9 9 18.4 11 24.4 9 - -
14.6 11 19.5 35 24.7 10 - -
[0120]
That is, a crystal of a compound of the formula (2) wherein R is an n-propyl group has a powder X-ray diffraction pattern showing characteristic peaks at 26=8.4°, 16.1°, 21.1° (±0 . 2°) . Furthermore, a powder X-ray diffraction pattern showing peaks at 29=7.7°, 8.4°, 16.1°, 19.5°, 21.1°(±0.2°) is preferable, and a powder X-ray diffraction pattern showing peaks at 29=7.7°, 8.4°, 15.0°, 16.1°, 19.5°, 21.1°, 22.5°, (±0.2°) is more preferable. In addition, a powder X-ray diffraction pattern showing peaks at 29=16.3°, 19.7°, 21.3° (±0.2°) is also preferable, and further, a powder X-ray diffraction pattern showing characteristic peaks at 29=7.9°, 16.3°, 19.7°, 21.3°, 22.7°, 24.9°(±0.2°) is more preferable. [0121] Step (ii):
Step (ii) is a step for preparing a compound represented by the formula (1) to be used in step (i). Specifically, a compound represented by the formula (3) and a compound represented by the formula (4) are condensed in the presence of a base. [0122]



CO,R
OR1
(4)
[0123] wherein, R1, R, -X1 and -X2 are as defined above.
As the base, metal hydrides such as sodium hydride, potassium hydride, calcium hydride and the like, metal amides such as sodium amide and the like, organic lithiums such as butyllithium, lithium diisopropylamide and the like, Grignard reagents such as tert-butylmagnesium chloride and the like, alkoxides such as sodium ethoxide, sodium tert-butoxide, potassium tert-butoxide and the like, and the like can be used, particularly, sodium hydride, sodium amide and sodium tert-butoxide are preferable. The amount of the base to be used is generally 1 equivalent - 6 equivalents, preferably 1.5 equivalents - 6 equivalents, relative to a compound represented by the formula (3).
The reaction can be performed using a solvent. While the solvent is not particularly limited as long as the reaction proceeds, hydrocarbon solvents such as cyclohexane, n-hexane, n-heptane, toluene and the like, halogenated solvents such as chlorobenzene, dichlorobenzene and the like, ether solvents such as tert-butyl methyl ether, tetrahydrofuran (THF), cyclopentyl methyl ether (CPME) and the like, polar solvents such as N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide and the like, and the like can be used. One kind thereof may be used or a mixture of two or more kinds thereof can also be used, and a mixture of a polar solvent and a nonpolar solvent can also be used.
The amount of the solvent to be used is, generally 5 mL -100 mL, preferably 5 mL - 30 mL, relative to 1 g of a compound represented by the formula (3).

The reaction temperature is generally -10°C - 200°C, preferably -5°C - 40°C.
The reaction time is generally 0.1 hr - 200 hr, preferably 1 hr - 24 hr.
[0124]
A compound represented by the formula (3) can be produced by the method described in, for example, JP-B-2648897, and a commercially available can also be used.
A compound represented by the formula (4) can be produced according to a known method, for example, the method described in SYNTHETIC COMMUNICATIONS, 18(7), 735-739(1988), and the method described in Reference Example 1 in the present specification, and a commercially available compound can also be used.
A compound represented by the formula (4) has a pH of preferably not more than 4, more preferably not more than 3. The pH of a compound represented by the formula (4) is a value obtained by mixing a compound represented by the formula (4) and water at 1:1 (volume ratio), and measuring the pH of the aqueous layer. When the pH value is too high (e.g., pH higher than 4), it can be lowered as necessary with an acid such as acetic acid, hydrochloric acid, sulfuric acid and the like. As a result, the preservation stability of a compound represented by the formula (4) is improved, and impurity formation during the reaction can be reduced.
[0125]
When R1 in the formula (4a) is a group different from R in the formula (1), a compound represented by the formula (1) is obtained by reacting a compound obtained by the above-mentioned condensation and alcohol represented by R-OH. In this step, a method similar to that in the below-mentioned step
(iib) can be employed.
[0126]
Step (ii) particularly preferably includes the following steps (iia) and (iib).

[0127] Step (iia):
In step (iia), a compound represented by the formula (5) is obtained by condensing a compound represented by the formula (3) and a compound represented by the formula (4a) which is the formula (4) wherein R1 is R2, in the presence of a base. [0128]



CO,R2
O
(4a)
[0129] wherein R2 is as defined above.
As the base, metal hydrides such as sodium hydride, potassium hydride, calcium hydride and the like, metal amides such as sodium amide and the like, organic lithiums such as butyllithium, lithium diisopropylamide and the like, Grignard reagents such as tert-butylmagnesium chloride and the like, alkoxides such as sodium ethoxide, sodium tert-butoxide, potassium tert-butoxide and the like, and the like can be used, particularly, sodium amide, sodium tert-butoxide and sodium hydride are preferable. The amount of the base to be used is generally 1 equivalent - 6 equivalents, preferably 1.5 equivalents - 6 equivalents, relative to a compound represented by the formula (3).
The reaction can be performed using a solvent. While the solvent is not particularly limited as long as the reaction proceeds, hydrocarbon solvents such as cyclohexane, n-hexane, n-heptane, toluene and the like, halogenated solvents such as chlorobenzene, dichlorobenzene and the like, ether solvents such as tert-butyl methyl ether, THF, CPME and the like, polar solvents such as N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide and the like, and the

like can be used. One kind thereof may be used or a mixture of two or more kinds thereof can also be used, and a mixture of a polar solvent and a nonpolar solvent can also be used.
The amount of the solvent to be used is, generally 5 mL -100 mL, preferably 5 mL - 30 mL, relative to 1 g of a compound represented by the formula (3).
The reaction temperature is generally 0°C - 200°C, preferably 0°C - 40°C.
The reaction time is generally 0.1 hr - 200 hr, preferably 1 hr - 24 hr. [0130]
Since a compound represented by the formula (5) has high crystallinity, it can be obtained with a high purity without performing a complicated purification such as chromatography and the like. [0131] Step (iib):
A compound represented by the formula (5) is reacted with alcohol represented by R-OH to give a compound represented by the formula (la).
Here, R is a primary alkyl group having 1-8 carbon atoms or a secondary alkyl group having 3-6 carbon atoms, preferably a primary alkyl group having 1-4 carbon atoms or a secondary alkyl group having 3-4 carbon atoms. As R, methyl group, ethyl group, n-propyl group, isopropyl group or n-butyl group is preferable, n-propyl group or isopropyl group is more preferable, and n-propyl group is particularly preferable.
The amount of alcohol represented by R-OH to be used is generally 1 mL - 100 mL, preferably 1 mL - 10 mL, relative to 1 g of a compound represented by the formula (5). [0132]



C02R2
C02R
+ R-OH

[0133]
wherein R2, R, -X1 and -X2 are as defined above. [0134]
Of compounds represented by the formula (1), a compound represented by the following formula (la) is particularly preferable. [0135]

O 0
C02R
(la)
[0136] wherein R is as defined above.
The reaction can also be performed in a solvent. While the solvent is not particularly limited as long as the reaction proceeds, ester solvents such as ethyl acetate, methyl acetate, isopropyl acetate and the like, nonpolar solvents such as cyclohexane, n-hexane, n-heptane, toluene and the like, halogenated solvents such as methylene chloride, chloroform, carbon tetrachloride and the like, ether solvents such as tert-butyl methyl ether (MTBE), THF, CPME and the like, polar solvents such as N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide and the like, and the like can be used. One kind thereof may be used or a mixture of two or more kinds thereof can also be used, and a mixture of a polar solvent and a nonpolar solvent can also be used. In addition, alcohol itself represented by R-OH may also be used

as a solvent.
The amount of the solvent to be used is generally 1 mL -100 mL, preferably 1 mL - 10 mL, relative to 1 g of a compound represented by the formula (5).
The reaction temperature is generally 30°C - 150°C, preferably 40°C - 110°C.
The reaction time is generally 1 hr - 48 hr, preferably 2 hr - 2 4 hr. [0137]
A compound represented by the formula (1) obtained as mentioned above, particularly a compound represented by the formula (la), among them, a compound of the formula (la) wherein R is an n-propyl group or isopropyl group has high crystallinity, it can be obtained with a high purity.
A crystal of a compound of the formula (la) wherein R is an n-propyl group preferably has, for example, a powder X-ray diffraction pattern shown below (powder X-ray diffraction patterns shown below were obtained in the below-mentioned Example 2).
[0138] Table 2

26 relative intensity 26 relative intensity 26 relative intensity 26 relative intensity
8.3 32.3 17.4 20.5 23.9 18.7 30.3 4
11.1 21.5 17.7 29.1 24.9 32.5 31.5 11.5
12.1 6.4 18.9 19.2 26.2 9.7 31.8 8.9
13.0 25.3 19.2 9.9 26.6 8.4 33.5 7.7
13.9 23.1 20.6 33.7 27.5 7 37.8 6
15.2 11.7 21.0 100 28.1 7 - -
16.5 41.5 22.0 33.1 28.6 13.5 - -
[0139]
That is, it has a powder X-ray diffraction pattern showing characteristic peaks at 26=8.3°, 16.5°, 21.0° (±0 . 2°) . Furthermore, a powder X-ray diffraction pattern showing

characteristic peaks at 29=8.3°, 16.5°, 21.0°, 22.0° (±0.2°) is preferable, and a powder X-ray diffraction pattern showing characteristic peaks at 29=8.3°, 13.0°, 13.9°, 16.5°, 17.7°, 21.0°, 22.0°, 24.9° (±0.2°) is more preferable. In addition, a powder X-ray diffraction pattern showing peaks at 29=16.7°, 17.6°, 20.8°, 22.1°(±0.2°) is also preferable.
Moreover, a crystal of a compound of the formula (la) wherein R is an n-propyl group also preferably has a powder X-ray diffraction pattern shown below (powder X-ray diffraction patterns shown below were obtained in the below-mentioned Example 2'). [0140] Table 3

26 relative intensity 26 relative intensity 26 relative intensity 26 relative intensity
5.2 6 15.5 5 20.6 17 27.1 5
6.5 6 16.5 30 21.5 63 29.7 6
7.9 7 17.5 8 21.9 10 30.7 5
9.8 15 17.9 5 23.7 19 31.0 5
10.3 100 18.4 46 24.1 5 31.5 5
11.8 57 19.0 19 25.3 10 35.7 8
14.1 35 19.5 21 26.1 12 38.2 5
[0141]
That is, it has a powder X-ray diffraction pattern showing characteristic peaks at 29=10.3°, 11.8°, 21.5° (±0.2°) . Furthermore, a powder X-ray diffraction pattern showing characteristic peaks at 29=10.3°, 11.8°, 14.1°, 18.4°, 21.5° (±0.2°) is preferable, and a powder X-ray diffraction pattern showing characteristic peaks at 29=10.3°, 11.8°, 14.1°, 16.5°, 18.4°, 19.0°, 19.5°, 20.6°, 21.5°, 23.7°(±0.2°) is more preferable. In addition, a powder X-ray diffraction pattern showing peaks at 29=16.7°, 19.2°, 20.8°, 21.3° (±0.2°) is also preferable. [0142]

Step (iiia):
In step (iiia), a compound represented by the formula (2) is hydrolyzed with a base, reacted with a calcium compound, and the obtained resultant product is isolated, whereby rosuvastatin calcium shown by the formula (6) is obtained. [0143]



OH OH
OH OH e
co2
C02R
Ca2+
(6)

[0144] wherein R is as defined above.
In step (iiia), a compound represented by the formula (2) is first hydrolyzed with a base.
As the base, sodium hydroxide, potassium hydroxide and the like can be used, and particularly, sodium hydroxide is preferable. The amount of the base to be used is generally 0.9 equivalents - 2 equivalents, preferably 1 equivalent - 1.5 equivalents, relative to a compound represented by the formula
(2) .
The reaction can be performed in a solvent. While the solvent is not particularly limited as long as the reaction proceeds, hydrocarbon solvents such as cyclohexane, n-hexane, n-heptane, toluene and the like, halogenated solvents such as methylene chloride, chloroform, carbon tetrachloride and the like, ether solvents such as MTBE, THF, CPME and the like, polar solvents such as N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, water and the like can be used. Furthermore, a mixed solvent of such polar solvent and at least one kind selected from the group consisting of ether solvent, hydrocarbon solvent, and halogenated solvent is preferable, and a mixed solvent of a

polar solvent and an ether solvent is preferable. Using such mixed solvent, a resultant product obtained by hydrolysis (e.g., sodium salt) is transferred to an aqueous layer, and impurity is transferred to an organic solvent layer, whereby the resultant product and impurity can be preferably separated easily.
When a mixed solvent of a polar solvent and at least one kind selected from the group consisting of ether solvent, hydrocarbon solvent, and halogenated solvent is used as a solvent, of those mentioned above, water or a mixed solvent of water and other polar solvent (e.g., THF, N-methyl-2-pyrrolidone, dimethyl sulfoxide etc.) is preferable as a polar solvent. MTBE and CPME are preferable as ether solvent, cyclohexane and toluene are preferable as hydrocarbon solvent, and methylene chloride is preferable as halogenated solvent. Of these, since toxicity of the solvent is low, a mixed solvent of water and MTBE or a mixed solvent of water and CPME is particularly preferably used.
The amount of the solvent to be used is generally 1 mL -100 mL, preferably 2 mL - 50 mL, more preferably 5 mL - 30 mL, relative to 1 g of a compound represented by the formula (2).
The reaction temperature is generally -10°C - 50°C, preferably 0°C - 40°C.
The reaction time is generally 1 hr - 48 hr, preferably 2 hr - 2 4 hr.
The pH during reaction is preferably pH 8 or more, more preferably pH 9 or more. In this range, the reaction efficiency can be improved. The upper limit thereof is preferably not more than pH 13. [0145]
After hydrolysis of a compound represented by the formula (2), it can be subjected to a reaction with the below-mentioned calcium compound. Where necessary, washing, extraction, concentration, drying and the like may be performed and, for example, it can be isolated as a sodium salt.