阅读说明：本技术 生产匹伐他汀钙的方法 (Method for producing pitavastatin calcium ) 是由 渡边尚之 井浦崇敦 大宫秀贵 长滨正树 于 2016-08-05 设计创作，主要内容包括：本发明涉及生产匹伐他汀钙的方法。具体地,根据本发明,在工业规模上以低成本、高产率和高选择性安全地生产匹伐他汀钙。用于生产匹伐他汀钙的该方法的特征在于,该方法包括：通过由通式(1)表示的化合物的缩醛化而获得由通式(3)表示的化合物的步骤(i),通过由通式(3)表示的化合物与酸反应而获得由通式(4)表示的化合物的步骤(ii),以及水解由通式(4)表示的化合物并随后与钙化合物反应的步骤(iii)。(The present invention relates to a method for producing pitavastatin calcium. In particular, according to the present invention, pitavastatin calcium is safely produced on an industrial scale at low cost, high yield and high selectivity. The method for producing pitavastatin calcium is characterized by comprising: a step (i) of obtaining a compound represented by the general formula (3) by acetalization of a compound represented by the general formula (1), a step (4) of obtaining a compound represented by the general formula (4) by reacting a compound represented by the general formula (3) with an acidii), and a step (iii) of hydrolyzing the compound represented by the general formula (4) and then reacting with a calcium compound.)

1. A method for producing a crystal of a compound represented by the following formula (1'):

wherein R is a linear alkyl group having a carbon number of 1 to 8 or a branched alkyl group having a carbon number of 3 to 8, and X¹Is a mixture of methanesulfonic acid and,

the method comprises the following steps:

a compound represented by the following formula (6):

wherein R is as defined above, and wherein,

with methanesulfonic acid at 10 ℃ to 50 ℃, and cooling the reaction mixture to precipitate the compound represented by formula (1') as crystals.

2. The method of claim 1, further comprising:

a compound represented by the following formula (10):

wherein R is a linear alkyl group having a carbon number of 1 to 8 or a branched alkyl group having a carbon number of 3 to 8,

or a compound represented by the following formula (14):

wherein R is as defined above, and wherein,

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 processed 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 formula (6).

3. A crystal of a compound represented by the following formula (1'):

wherein R is ethyl, and X¹Is a mixture of methanesulfonic acid and,

wherein the crystal shows a powder X-ray diffraction pattern comprising peaks with large relative intensities at 2 θ ═ 20.6 ° and 21.8 °.

4. The crystal of claim 3, further exhibiting a powder X-ray diffraction pattern comprising peaks with large relative intensities at 2 θ -6.9 ° and 13.5 °.

Technical Field

The present invention relates to an improved process for the production of pitavastatin calcium.

Background

Pitavastatin calcium has the activity of HMG-CoA reductase, which is a rate-limiting enzyme that specifically and antagonistically inhibits cholesterol synthesis, and is used for the treatment of hypercholesterolemia, familial hypercholesterolemia, and the like.

The chemical name of pitavastatin calcium is bis [ (3R,5S,6E) -7- [ 2-cyclopropyl-4- (4-fluorophenyl) -3-quinolyl ] -3, 5-dihydroxy-6-heptenoic acid ] calcium, which is represented by the following formula:

it is known that pitavastatin calcium can be produced by hydrolysis of its precursor compound, i.e., 2- [ (4R,6S) -6- [ (E) - [ 2-cyclopropyl-4- (4-fluorophenyl) -3-quinolyl ] ethenyl ] -2, 2-dimethyl-1, 3-dioxan-4-yl ] acetate represented by the following formula (16):

wherein R is⁷Is an alkyl group having a carbon number of 1 to 4.

The compound represented by formula (16) can be obtained by a Wittig reaction.

Patent document 1 describes that the above-mentioned compound represented by formula (16) can be obtained by reacting a fused pyridine derivative with an aldehyde compound in the presence of n-butyllithium at-78 ℃.

The method described in patent document 1 requires a special facility for industrial production because the reaction is carried out at an extremely low temperature of-78 ℃ and the yield thereof is low. In addition, there is a safety concern in industrial scale production due to the use of n-butyllithium, which is easily combustible, as a base. In addition, since n-butyllithium and phosphorus tribromide used for the reaction are expensive, the cost becomes high.

Patent document 2 describes the above-mentioned compound represented by formula (16) by reacting a compound represented by the following formula (17):

wherein R is⁷As defined above, the above-mentioned,

and a compound represented by the following formula (18):

wherein R is⁸Is an aryl, aralkyl or alkyl group,

in the presence of an alkali metal salt.

The method described in patent document 2 is insufficient in yield for industrial production. In addition, the compound represented by formula (17) and the compound represented by formula (18) require a multi-step synthesis process, which raises a concern about cost.

[ List of documents ]

[ patent document ]

Patent document 1: JP-A-5-310700

Patent document 2: JP-A-2014-

Disclosure of Invention

[ problem to be solved by the invention ]

There is a need to develop a method for safely producing pitavastatin calcium in a high yield, high selectivity and low cost manner on an industrial scale.

The problem to be solved by the present invention is to provide a method for producing high purity pitavastatin calcium on an industrial scale, which is superior in safety and cost and provides high yield and high selectivity.

[ means for solving the problems ]

The present inventors have made intensive studies and found that the above-mentioned problems can be solved by using a specific intermediate compound, which resulted in the completion of the present invention. That is, the gist of the present invention is as follows.

[1] A method for producing pitavastatin calcium, comprising:

(i) for a compound represented by the following formula (1):

wherein R is a linear alkyl group having a carbon number of 1 to 8 or a branched alkyl group having a carbon number of 3 to 8, and X is an acid,

a step of conducting acetalization to give a compound represented by the following formula (3),

wherein R is³And R⁴Each independently is an alkyl group having a carbon number of 1-4, and R is as defined above;

(ii) a step of reacting the above compound represented by the formula (3) with an acid to give a compound represented by the following formula (4):

wherein R is as defined above; and

(iii) a step of hydrolyzing the above compound represented by the formula (4) and reacting the compound with a calcium compound to give pitavastatin calcium.

[2] [1] A production method further comprising:

(iv) a compound represented by the following formula (6):

wherein R is a linear alkyl group having a carbon number of 1 to 8 or a branched alkyl group having a carbon number of 3 to 8,

a step of reacting with an acid to give a compound represented by the following formula (1),

wherein X is an acid and R is as defined above.

[3] [2] A production method comprising:

(v) a compound represented by the following formula (7):

and a compound represented by the following formula (8):

wherein R is⁵Is a straight chain alkyl group having a carbon number of 1 to 8 or a branched alkyl group having a carbon number of 3 to 8, provided that R⁵Different from R in the above formula (1),

a step of conducting condensation in the presence of a base to give a compound represented by the following formula (9):

wherein R is⁵As defined above;

(vi) a step of reacting the above-mentioned compound represented by the formula (9) obtained in the step (v) with an alcohol represented by R-OH, wherein R is a linear alkyl group having a carbon number of 1 to 8 or a branched alkyl group having a carbon number of 3 to 8, to give a compound represented by the following formula (10),

wherein R is as defined above; and

(vii) (vii) a step of reacting the above-mentioned compound represented by the formula (10) obtained in the step (vi) 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 following formula (6):

wherein R is as defined above.

[4] [2] A production method comprising:

(viii) a compound represented by the following formula (12):

and a compound represented by the following formula (13):

wherein R is a linear alkyl group having a carbon number of 1 to 8 or a branched alkyl group having a carbon number of 3 to 8,

a step of carrying out a reaction in the presence of a titanium catalyst to give a compound represented by the following formula (14):

wherein R is as defined above; and

(ix) (viii) a step of reacting the above-mentioned compound represented by the formula (14) obtained in step (viii) 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 following formula (6):

wherein R is as defined above.

[5] A compound represented by the following formula (1'):

wherein R is a linear alkyl group having a carbon number of 1 to 8 or a branched alkyl group having a carbon number of 3 to 8, and X¹Is methanesulfonic acid.

[6] A method for producing a compound represented by the following formula (1):

wherein X is an acid and R is a linear alkyl group having a carbon number of 1 to 8 or a branched alkyl group having a carbon number of 3 to 8,

the method comprises the following steps

(iv) Reacting a compound represented by the following formula (6) with an acid,

wherein R is as defined above.

The gist of the present invention is as follows.

[1A] A method for producing pitavastatin calcium represented by the following formula (5):

the method comprises the following steps:

(i) for a compound represented by the following formula (1):

wherein R is a linear alkyl group having a carbon number of 1 to 8 or a branched alkyl group having a carbon number of 3 to 8, and X is an acid,

a step of conducting acetalization to give a compound represented by the following formula (3):

wherein R is³And R⁴Each independently is an alkyl group having a carbon number of 1-4, and R is as defined above;

(ii) a compound represented by the following formula (3):

r, R therein³And R⁴As defined above, the above-mentioned,

a step of reacting with an acid catalyst to give a compound represented by the following formula (4),

wherein R is as defined above; and

(iii) for a compound represented by the following formula (4):

wherein R is as defined above, and wherein,

a step of performing hydrolysis and reacting the compound with a calcium compound.

[2A] [1A ] A production method comprising:

(iv) a compound represented by the following formula (6):

wherein R is as defined above, and wherein,

a step of reacting with an acid.

[3A] The method of [2A ], comprising:

(v) a compound represented by the following formula (7):

and a compound represented by the following formula (8):

wherein R is⁵Is a straight chain alkyl group having a carbon number of 1 to 8 or a branched alkyl group having a carbon number of 3 to 8, provided that R⁵In the difference from the R, the ratio of R,

a step of conducting condensation in the presence of a base to give a compound represented by the following formula (9):

wherein R is⁵As defined above;

(vi) (vi) a step of reacting the above-mentioned compound represented by the formula (9) obtained in the step (v) with an alcohol represented by R — OH, wherein R is as defined above, to give a compound represented by the following formula (10):

wherein R is as defined above; and

(vii) (vii) a step of reacting the above-mentioned compound represented by the formula (10) obtained in the step (vi) 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 following formula (6):

wherein R is as defined above.

[4A] [2A ] A production method comprising:

(viii) a compound represented by the following formula (12):

with a compound represented by the following formula (13) in the presence of a Ti catalyst:

a step of carrying out a reaction to give a compound represented by the following formula (14),

wherein R is as defined above; and

(ix) (viii) a step of reacting the above-mentioned compound represented by the formula (14) obtained in step (viii) 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 following formula (6):

wherein R is as defined above.

[5A] A compound represented by the following formula (1):

wherein R is a linear alkyl group having a carbon number of 1 to 8 or a branched alkyl group having a carbon number of 3 to 8, and X¹Is methanesulfonic acid.

[6A] A method for producing a compound represented by the following formula (1):

wherein X is an acid and R is a linear alkyl group having a carbon number of 1 to 8 or a branched alkyl group having a carbon number of 3 to 8,

the method comprises the following steps:

(iv) reacting a compound represented by the following formula (6) with an acid,

wherein R is as defined above.

[ Effect of the invention ]

The production method of the present invention enables the production of pitavastatin calcium safely on an industrial scale with high yield, high selectivity and low cost.

Drawings

FIG. 1 is an X-ray diffraction pattern of DOLE MsOH obtained in example 5.

Detailed Description

The terms used in the present specification are explained in detail below.

R is a linear alkyl group having a carbon number of 1 to 8 or a branched alkyl group having a carbon number of 3 to 8, preferably a linear alkyl group having a carbon number of 1 to 8, more preferably a linear alkyl group having a carbon number of 1 to 4, particularly preferably a methyl group, an ethyl group or an n-propyl group.

R¹、R²、R³And R⁴Each independently is an alkyl group having a carbon number of 1 to 4, preferably a straight-chain alkyl group having a carbon number of 1 to 4, particularly preferably a methyl group, an ethyl group or an n-propyl group. Furthermore, R¹And R²Possibly joined to form a ring.

R⁵Is a straight chain alkyl group having a carbon number of 1 to 8 or a branched alkyl group having a carbon number of 3 to 8, provided that R⁵Different from R. R⁵The branched alkyl group having a carbon number of 3 to 8 is preferable, and isopropyl, sec-butyl, tert-butyl, and tert-pentyl are more preferable, and tert-butyl is particularly preferable.

Ra is an alkyl group having a carbon number of 1 to 10. Ra is preferably a short chain alkyl group having a carbon number of 1 to 4 such as methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, etc., and isopropyl is particularly preferable industrially.

X is an acid, preferably an inorganic acid such as sulfuric acid, hydrochloric acid, or the like, and an organic acid such as formic acid, acetic acid, methanesulfonic acid, p-toluenesulfonic acid, or the like, more preferably sulfuric acid, hydrochloric acid, methanesulfonic acid, or p-toluenesulfonic acid, and particularly preferably methanesulfonic acid.

X¹Is methanesulfonic acid.

In the present specification, "an enzyme having an activity capable of stereoselectively reducing a carbonyl group" means an enzyme having an activity of converting a carbonyl group in a carbonyl group-containing compound into an optically active alcohol by asymmetric reduction.

The presence or absence of "activity capable of stereoselectively reducing a carbonyl group" can be determined by measuring the activity of converting a carbonyl group in a carbonyl group-containing compound into an optically active alcohol by asymmetric reduction using a general assay method. For example, a measurement target enzyme is reacted with a compound represented by the following formula (19):

wherein R is_bIs a straight chain alkyl group having a carbon number of 1 to 8 or a branched alkyl group having a carbon number of 3 to 6, -Q¹and-Q²Each independently is-OH or ═ O, and-Q¹and-Q²At least one of which is ═ O,

and directly measuring the amount of the compound represented by formula (6) converted from the compound represented by formula (19), whereby the enzyme activity can be confirmed.

In the present specification, "enzyme" includes purified enzymes (including partially purified enzymes), enzymes immobilized on a carrier or the like by a conventional immobilization technique, for example, enzymes immobilized on a carrier such as polyacrylamide, carrageenan (carageenan) or the like.

In the present specification, the "microorganism or cell capable of producing an enzyme having an activity of stereoselectively reducing a carbonyl group" (hereinafter sometimes referred to as "microorganism or cell of the present invention") is not particularly limited as long as it has an "activity of stereoselectively reducing a carbonyl group", and it may be a microorganism or cell inherently having the above-mentioned activity, or a microorganism or cell to which the above-mentioned activity has been imparted by breeding (bleeding). As a means for imparting the above-mentioned activity by breeding, known methods such as gene recombination treatment (transformation), mutation treatment and the like can be employed. As the transformation method, methods such as introduction of a target gene, enhancement of expression of an enzyme gene in a biosynthetic pathway of an organic compound, reduction of expression of an enzyme gene in a byproduct biosynthetic pathway, and the like can be used.

As the species of "microorganism or cell of the present invention", there may be mentioned microorganisms or cells described in the following host organisms or host cells. "the microorganism or cell of the present invention" in a frozen state may also be used. In the present specification, "a microorganism or a cell capable of producing an enzyme having the activity" is not limited to a living microorganism or a cell, but also includes a microorganism or a cell which is biologically dead but has an enzyme activity.

In the present invention, the microorganism or cell may be produced, for example, by the method described in WO 2003/078634.

In the present specification, there is no particular limitation on the kind of organism as the "host organism", and there may be mentioned prokaryotes such as Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Corynebacterium (Corynebacterium), Pseudomonas (Pseudomonas) bacteria, Bacillus (Bacillus) bacteria, Rhizobium (Rhizobium) bacteria, Lactobacillus (Lactobacillus) bacteria, Succinobacter (Succobacter) bacteria, Anaerobiospirillum (Anaerobiospirillum) bacteria, Actinobacillus (Actinobacillus) bacteria and the like, fungi such as yeast, filamentous fungi and the like, eukaryotes such as plants, animals and the like. Among them, preferred are Escherichia coli, yeast and coryneform bacteria, and particularly preferred is Escherichia coli.

In the present specification, the kind of the cell as the "host cell" is not particularly limited, and animal cells, plant cells, insect cells, and the like can be used.

In the present specification, an "expression vector" is a genetic element for replicating and expressing a protein having a desired function in a host organism by introducing a polynucleotide encoding the protein having the desired function into the host organism. Examples include, but are not limited to, plasmids, viruses, bacteriophages, cosmids, and the like. Preferred expression vectors are plasmids.

In the present specification, "transformant" means a microorganism or cell into which the above-described expression vector has been introduced and which has obtained the ability to exhibit the desired trait associated with a protein having the desired function.

In the present specification, "a processed product of a microorganism or a cell" means a product obtained by the following process and containing a protein or the like having a desired function: culturing a microorganism or a cell, and 1) treating the microorganism or the cell with an organic solvent or the like, 2) freeze-drying it, 3) immobilizing it on a carrier or the like, 4) physically or enzymatically disrupting it.

In the present specification, "a medium containing an enzyme obtained by culturing a microorganism or a cell" means 1) a medium of a microorganism or a cell, 2) a medium obtained by treating a medium of a microorganism or a cell with an organic solvent or the like, or 3) a medium in which a cell membrane of a microorganism or a cell is physically or enzymatically disrupted.

The production process of pitavastatin calcium of the present invention is explained in detail below. Hereinafter, w/v means weight/volume.

The production method of pitavastatin calcium of the present invention comprises: a step (i) of acetalizing the compound represented by formula (1) to give a compound represented by formula (3), a step (ii) of reacting the compound represented by formula (3) with an acid to give a compound represented by formula (4), and a step (iii) of hydrolyzing the compound represented by formula (4) and reacting it with a calcium compound to give pitavastatin calcium represented by formula (5).

Further, the compound represented by formula (1) may also be obtained by reacting the compound represented by formula (6) with an acid.

Each step of the production method of the present invention is explained in detail below.

Step (i):

in step (i), the compound represented by formula (1) is acetalized to give a compound represented by formula (3).

Specifically, a compound represented by the formula (1) is reacted with an acetalization reagent represented by the following formula (2-1) or (2-2).

The amount of the acetalization reagent used is not particularly limited, and is usually 1mol to 10mol, preferably 1mol to 6mol, with respect to 1mol of the compound represented by formula (1).

The reaction may be carried out in the presence of an acid catalyst.

As the acid catalyst, inorganic acids such as sulfuric acid, hydrochloric acid and the like, organic acids such as formic acid, acetic acid, p-toluenesulfonic acid pyridine and the like, solid acids such as zeolite and the like can be used. One kind thereof may be used, or a mixture of two or more kinds thereof may also be used. Among them, p-toluenesulfonic acid, methanesulfonic acid, or p-toluenesulfonic acid pyridine is industrially preferable.

When an acid catalyst is used, it may be dissolved in a solvent mentioned below and used.

Although there is no particular limitation on the amount of the acid catalyst used, it is usually 0.001mol to 2mol, preferably 0.01mol to 1.5mol, relative to 1mol of the compound represented by formula (1).

The reaction with the acetalizing agent is preferably carried out using a solvent. Although the solvent is not particularly limited as long as the reaction can proceed, it is preferable to use a hydrocarbon solvent such as cyclohexane, n-hexane, n-heptane, toluene, etc.; ether solvents such as t-butyl methyl ether, Tetrahydrofuran (THF), cyclopentyl methyl ether (CPME), and the like; ester solvents such as ethyl acetate, propyl acetate, butyl acetate, and the like; alcohol solvents such as methanol, ethanol, n-propanol, etc.; ketone solvents such as acetone, methyl ethyl ketone, and the like; acetonitrile, and the like. One of these solvents may be used, or a mixture of two or more thereof may also be used.

The amount of the solvent used is not particularly limited, and it is usually 1mL-100mL, preferably 1mL-20mL, relative to 1g of the compound represented by formula (1).

The reaction temperature below the boiling point of the solvent used and above the melting point of the solvent used hardly affects the reaction or the yield of the objective product. Therefore, the reaction temperature can be appropriately selected from this temperature range. Industrially, it is preferably from 0 ℃ to 80 ℃, more preferably from 10 ℃ to 70 ℃.

The reaction time is usually 0.5hr to 24hr, preferably 1hr to 12 hr.

Further, it is also possible to produce the compound represented by the formula (3) having a low epimer content percentage by further reacting the reaction product containing the compound represented by the formula (3) obtained in the step (i) with an acid to partially decompose the acetal constituent portion of the compound represented by the formula (3).

As the acid, inorganic acids such as sulfuric acid, hydrochloric acid, and the like; organic acids such as formic acid, acetic acid, p-toluenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid pyridine, and the like. One kind thereof may be used, or a mixture of two or more kinds thereof may also be used. Among them, sulfuric acid, hydrochloric acid or methanesulfonic acid is industrially preferable. The acid may be used as an aqueous solution, or may be dissolved in a solvent mentioned below and used.

The amount of the acid used is not particularly limited, and it is usually 0.01mol to 1mol, preferably 0.05mol to 0.5mol, relative to 1mol of the compound represented by the formula (3).

The reaction with the acid is preferably carried out using a solvent. Although the solvent is not particularly limited as long as the reaction can proceed, it is preferable to use a hydrocarbon solvent such as cyclohexane, n-hexane, n-heptane, toluene, etc.; ether solvents such as t-butyl methyl ether, Tetrahydrofuran (THF), cyclopentyl methyl ether (CPME), and the like; ester solvents such as ethyl acetate, propyl acetate, butyl acetate, and the like; alcohol solvents such as methanol, ethanol, n-propanol, etc.; ketone solvents such as acetone, methyl ethyl ketone, and the like; acetonitrile, and the like. One of these solvents may be used, or a mixture of two or more thereof may also be used.

The amount of the solvent used is not particularly limited, and it is usually 1mL-100mL, preferably 1mL-20mL, relative to 1g of the compound represented by formula (1).

The reaction temperature below the boiling point of the solvent used and above the melting point of the solvent used hardly affects the reaction or the yield of the objective product. Therefore, the reaction temperature can be appropriately selected from this temperature range. Industrially, it is preferably from 0 ℃ to 60 ℃, more preferably from 10 ℃ to 50 ℃.

The percentage of the epimer content can be controlled by the degree of progress of the reaction, for example, by carrying out the reaction so that the conversion is 5% to 30%, the percentage of the epimer content can be set to 0.3% or less.

The compound represented by formula (3) obtained in step (i) may be used directly in step (ii), or may be used in step (ii) after reducing the epimer content percentage as described above, or may be used in step (ii) after purification.

As a purification method, purification by column chromatography, crystallization, or the like can be suitably combined. In the case of column chromatography, for example, a nonpolar solvent such as n-heptane, n-hexane, toluene, or the like is combined with a polar solvent such as ethyl acetate, methyl ethyl ketone, THF, ethanol, or the like to perform purification. In the case of crystallization, for example, a nonpolar solvent such as n-heptane, n-hexane, toluene, or the like or water is combined with a polar solvent such as ethyl acetate, methyl ethyl ketone, THF, methanol, ethanol, n-propanol, or the like to perform purification.

Further, the compound of formula (1) wherein X is methanesulfonic acid (methanesulfonate) is preferable because the compound represented by formula (3) can be crystallized by merely adding water to the reaction system after completion of step (i) or after reduction of the epimer content percentage, and the compound represented by formula (3) can be easily obtained. When water is added, the compound represented by formula (3) obtained separately may be used as seed crystals to promote crystallization. During crystallization, a base may be added to neutralize the system. As the base, inorganic salts such as sodium hydrogencarbonate, potassium hydrogencarbonate and the like, organic bases such as triethylamine and the like can be mentioned, and sodium hydrogencarbonate and potassium hydrogencarbonate are preferable.

Step (ii):

in step (ii), the compound represented by formula (3) is reacted with an acid for deacetalization to give a compound represented by formula (4).

As the acid, inorganic acids such as sulfuric acid, hydrochloric acid, and the like; organic acids such as formic acid, acetic acid, p-toluenesulfonic acid pyridine, and the like. One kind thereof may be used, or a mixture of two or more kinds thereof may also be used. Among them, sulfuric acid or hydrochloric acid is industrially preferable.

The acid may be used as an aqueous solution, or may be dissolved in a solvent mentioned below and used.

The amount of the acid used is not particularly limited, but it is usually 1mol to 5mol, preferably 1mol to 2mol, relative to 1mol of the compound represented by the formula (3).

The reaction with the acid is preferably carried out using a solvent. Although the solvent is not particularly limited as long as the reaction can proceed, it is preferable to use a hydrocarbon solvent such as cyclohexane, n-hexane, n-heptane, toluene, etc.; ether solvents such as t-butyl methyl ether, Tetrahydrofuran (THF), cyclopentyl methyl ether (CPME), and the like; ester solvents such as ethyl acetate, propyl acetate, butyl acetate, and the like; alcohol solvents such as methanol, ethanol, etc.; ketone solvents such as acetone, methyl ethyl ketone and the like. One of these solvents may be used, or a mixture of two or more thereof may also be used.

The amount of the solvent used is not particularly limited, and it is usually 1mL-100mL, preferably 1mL-20mL, relative to 1g of the compound represented by formula (3).

The reaction temperature below the boiling point of the solvent used and above the melting point of the solvent used hardly affects the reaction or the yield of the objective product. Therefore, the reaction temperature can be appropriately selected from this temperature range. Industrially, it is preferably from 0 ℃ to 70 ℃, more preferably from 10 ℃ to 50 ℃.

The reaction time is usually 1hr to 10hr, preferably 1hr to 5 hr.

Step (iii):

in step (iii), the compound represented by formula (4) is hydrolyzed and reacted with a calcium compound to give pitavastatin calcium represented by formula (5). Preferably, the compound represented by the formula (4) is hydrolyzed by reacting with a basic catalyst, and the obtained compound is reacted with a calcium compound to give pitavastatin calcium. Generally, pitavastatin calcium precipitated as crystals, and thus the precipitated pitavastatin calcium was filtered and dried. As the base catalyst, an inorganic base may be used, and preferably, an alkali metal hydroxide such as lithium hydroxide, sodium hydroxide, potassium hydroxide, or the like may be used. Sodium hydroxide is particularly preferred industrially.

The amount of the base catalyst used is not particularly limited, and it is usually 1mol to 2mol, preferably 1mol to 1.5mol, relative to 1mol of the compound represented by the formula (4).

The hydrolysis is preferably carried out using a solvent. As the solvent, water is preferable. If necessary, an ethereal solvent such as THF, 1, 4-dioxane, etc.; alcohol solvents such as methanol, ethanol, etc.; ketone solvents such as acetone and the like. One of these solvents may be used, or a mixture of two or more thereof may also be used.

The amount of the solvent used is not particularly limited, and it is usually 1mL-100mL, preferably 1mL-50mL, relative to 1g of the compound represented by formula (4).

The hydrolysis temperature below the boiling point of the solvent used and above the melting point of the solvent used hardly affects the hydrolysis or the yield of the target product. Therefore, the hydrolysis temperature can be appropriately selected from this temperature range. Industrially, it is preferably from 0 ℃ to 70 ℃, more preferably from 10 ℃ to 50 ℃.

The hydrolysis time is usually 1hr-24hr, preferably 1hr-12 hr.

The compound obtained by hydrolysis is reacted with a calcium compound to give pitavastatin calcium. The compounds obtained by hydrolysis do not have to be isolated. Industrially, it is preferably hydrolyzed and continuously reacted with a calcium compound.

As the calcium compound, an inorganic salt or an organic salt of calcium can be used, and calcium chloride, calcium acetate, or the like can be preferably used.

The reaction temperature with the calcium compound, which is lower than the boiling point of the solvent used and higher than the melting point of the solvent used, hardly affects the reaction or the yield of the target product. Therefore, the reaction temperature can be appropriately selected from this temperature range. Industrially, it is preferably from 0 ℃ to 70 ℃, more preferably from 10 ℃ to 60 ℃.

The reaction time is usually 0.1hr to 10hr, preferably 0.5hr to 5 hr.

The method for filtering and drying pitavastatin calcium is not particularly limited, and a known method can be used.

The water content of pitavastatin calcium is usually 5% to 15%, preferably 7% to 13%, particularly preferably 8% to 12% by weight. The obtained pitavastatin calcium can be adjusted to the desired water content by drying or can be adjusted to the desired water content by wetting after drying.

Step (iv):

in step (iv), the compound represented by formula (1) used in step (i) is obtained. Specifically, the compound represented by formula (6) is reacted with an acid to give a compound represented by formula (1).

The compound represented by formula (1) is a salt of the compound represented by formula (6). Preferred are salts with inorganic acids such as sulfate, hydrochloride, and the like; and salts with organic acids such as formate, acetate, methanesulfonate, p-toluenesulfonate and the like, more preferably sulfate, hydrochloride, methanesulfonate or p-toluenesulfonate. From the aspect of crystallinity, the compound represented by the formula (1) is industrially preferably a methanesulfonate, i.e., a compound represented by the following formula (1'):

wherein X¹Is methanesulfonic acid and R is as defined above.

As the acid, inorganic acids such as sulfuric acid, hydrochloric acid, and the like; organic acids such as formic acid, acetic acid, methanesulfonic acid, p-toluenesulfonic acid and the like. One kind thereof may be used, or a mixture of two or more kinds thereof may also be used. Among them, sulfuric acid, hydrochloric acid, methanesulfonic acid, or p-toluenesulfonic acid is industrially preferable.

The amount of the acid used is not particularly limited, and it is usually 1mol to 2mol, preferably 1mol to 1.5mol, relative to 1mol of the compound represented by the formula (6).

The reaction is preferably carried out using a solvent. Although the solvent is not particularly limited as long as the reaction can proceed, it is preferable to use a hydrocarbon solvent such as cyclohexane, n-hexane, n-heptane, toluene, etc.; ether solvents such as t-butyl methyl ether, Tetrahydrofuran (THF), cyclopentyl methyl ether (CPME), and the like; ester solvents such as ethyl acetate, propyl acetate, butyl acetate, and the like; alcohol solvents such as methanol, ethanol, etc.; ketone solvents such as acetone, methyl ethyl ketone, and the like; acetonitrile, and the like. One of these solvents may be used, or a mixture of two or more thereof may also be used.

The amount of the solvent used is not particularly limited, and it is usually 1mL-100mL, preferably 1mL-50mL, relative to 1g of the compound represented by formula (6).

The reaction temperature below the boiling point of the solvent used and above the melting point of the solvent used hardly affects the reaction or the yield of the objective product. Therefore, the reaction temperature can be appropriately selected from this temperature range. Industrially, it is preferably from 0 ℃ to 70 ℃, more preferably from 10 ℃ to 50 ℃.

The reaction time is usually 0.1hr to 10hr, preferably 0.5hr to 5 hr.

The compound represented by formula (6) sometimes contains impurities derived from the step of obtaining the compound. For example, when the compound represented by formula (6) is obtained by a biological reaction step such as the method of (a) or (b) mentioned below, the resulting reaction product contains impurities in addition to the compound represented by formula (6). Therefore, it is preferable to remove the impurities by purification or the like and use the compound in step (i).

The compound represented by the formula (1), particularly the methanesulfonate salt, obtained by the step (iv) of the present invention is superior in crystallinity. Therefore, it is easily precipitated as crystals. Therefore, the compound represented by formula (1) can be easily isolated.

The present invention having step (iv) can efficiently remove impurities from the previous step. Therefore, in the production on an industrial scale, the purification step can be omitted or simplified.

The compound represented by formula (6) may be obtained by an organic synthesis step or a biological reaction step. In the present invention, the compound represented by formula (6) is preferably produced by the method of the following (a) or (b).

Method (a):

method (a) characteristically comprises step (v), step (vi) and step (vii).

Step (v):

in step (v), the compound represented by formula (7) and the compound represented by formula (8) are condensed in the presence of a base to give a compound represented by formula (9).

As the base, there can be used a metal hydride such as sodium hydride, potassium hydride, calcium hydride or the like, an amino metal such as sodium amide or the like, an organolithium such as butyllithium, lithium diisopropylamide or the like, a Grignard reagent such as t-butylmagnesium chloride or the like, an alkoxide such as sodium ethoxide, sodium t-butoxide, potassium t-butoxide or the like. In particular, sodium amide, sodium tert-butoxide and sodium hydride are preferred.

The amount of the base used is not particularly limited, and it is usually 1mol to 5mol, preferably 1mol to 4mol, relative to 1mol of the compound represented by the formula (7).

The condensation reaction may be carried out using a solvent. Although the solvent is not particularly limited as long as the reaction can proceed, 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 t-butyl methyl ether, Tetrahydrofuran (THF), cyclopentyl methyl ether (CPME) and the like, polar solvents such as N-methyl-2-pyrrolidone, N-dimethylformamide, N-dimethylacetamide, dimethylsulfoxide and the like can be used. One kind thereof may be used, or a mixture of two or more kinds thereof may also be used.

Although there is no particular limitation on the amount of the solvent used, it is usually 5mL-100mL, preferably 5mL-30mL, relative to 1g of the compound represented by formula (7).

The reaction temperature below the boiling point of the solvent used and above the melting point of the solvent used hardly affects the condensation reaction or the yield of the objective product. Therefore, the reaction temperature can be appropriately selected from this temperature range. Industrially, it is usually from 0 ℃ to 100 ℃ and preferably from 0 ℃ to 50 ℃.

The reaction temperature is usually 0.1hr to 200hr, preferably 1hr to 24 hr.

The compound represented by formula (7) can be produced by, for example, the method described in JP-B-2569746, and a commercially available product can also be used.

The compound represented by formula (8) can be obtained according to a known method. It can be obtained, for example, by the methods described in SYNTHETIC COMMUNICATIONS,18(7),735-739 (1988). Commercially available compounds may also be used.

The compound represented by formula (8) has a pH of preferably not more than 4, more preferably not more than 3. By setting the pH of the compound represented by formula (8) to fall within this range, the storage stability of the compound represented by formula (8) is improved, and the formation of impurities during the reaction can be reduced. The pH of the compound represented by formula (8) is a value obtained by mixing the compound represented by formula (8) with water at a volume ratio of 1:1 and measuring the pH of the aqueous layer. When the pH is too high (e.g., pH above 4), it may be lowered using an acid such as acetic acid, hydrochloric acid, sulfuric acid, or the like, if necessary.

In terms of crystallinity, R in the formula (9)⁵The branched alkyl group having a carbon number of 3 to 8 is preferable, and isopropyl, sec-butyl, tert-butyl, and tert-pentyl are more preferable, and tert-butyl is particularly preferable. The compound represented by the formula (9) having high crystallinity is industrially preferable because it can be obtained in high purity without complicated purification such as chromatography and the like.

Step (vi):

in step (vi), the compound represented by formula (9) is reacted with an alcohol represented by R — OH to give a compound represented by formula (10).

The amount of the alcohol represented by R-OH used is not particularly limited, and it is usually 1mL-100mL, preferably 1mL-10mL, relative to 1g of the compound represented by the formula (9).

The reaction may be carried out using a solvent. Although the solvent is not particularly limited as long as the reaction can proceed, ester solvents such as ethyl acetate, methyl acetate, isopropyl acetate, etc.; nonpolar solvents such as cyclohexane, n-hexane, n-heptane, toluene, etc.; halogenated solvents such as dichloromethane, chloroform, carbon tetrachloride and the like; ether solvents such as t-butyl methyl ether (MTBE), Tetrahydrofuran (THF), cyclopentyl methyl ether (CPME), and the like; polar solvents such as N-methyl-2-pyrrolidone, N-dimethylformamide, N-dimethylacetamide, dimethylsulfoxide and the like. One of these solvents may be used, or a mixture of two or more thereof may also be used. Further, an alcohol represented by R-OH itself can be used as the solvent.

Although there is no particular limitation on the amount of the solvent used, it is usually 1mL-100mL, preferably 1mL-10mL, relative to 1g of the compound represented by formula (9).

The reaction temperature below the boiling point of the solvent used and above the melting point of the solvent used hardly affects the reaction or the yield of the objective product. Therefore, the reaction temperature can be appropriately selected from this temperature range. In industry, it is generally from 30 ℃ to 150 ℃ and preferably from 40 ℃ to 110 ℃.

The reaction time is usually 1hr to 48hr, preferably 2hr to 24 hr.

In terms of reaction efficiency in the biological reaction step (vii), R — OH and R in the formula (10) are preferably a straight-chain alkyl group having a carbon number of 1 to 8, more preferably a straight-chain alkyl group having a carbon number of 1 to 4, and particularly preferably a methyl group, an ethyl group, or an n-propyl group. Such a compound represented by formula (10) is preferable because the stereoselective reduction of a carbonyl group is efficiently performed in the biological reaction step (vii).

Step (vii): biological reaction step

In step (vii), the compound represented by formula (10) is reduced by reaction with an enzyme having an activity capable of stereoselectively reducing a carbonyl group, a microorganism or cell (the microorganism or cell of the present invention) having an ability to produce the enzyme, a treated product of the microorganism or cell, and/or a medium containing the enzyme obtained by culturing the microorganism or cell (hereinafter, they are sometimes collectively referred to as "the enzyme of the present invention, etc.) to give a compound represented by formula (6).

As the enzyme used in step (vii), an enzyme having the amino acid sequence shown in SEQ ID NO:2 (hereinafter sometimes referred to as "OCR 1") or a homologue of the amino acid sequence may be used. Specifically, there may be mentioned an enzyme containing the polypeptide of the following (A), (B) or (C) or a homologue thereof.

(A) A polypeptide having carbonyl reductase (OCR1) (SEQ ID NO:2) derived from non-fermentative variant of methanol-inducible yeast (Ogataea minuta var. nonfermentants) NBRC1473 described in JP-B-4270918,

(B) a polypeptide consisting of an amino acid sequence having 80% or more homology with the amino acid sequence shown in SEQ ID NO. 2 and having an activity of converting a compound represented by formula (10) into a compound represented by formula (6),

(C) a polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO. 2, and having an activity of converting a compound represented by formula (10) into a compound represented by formula (6).

The homologue of the above (B) is a protein having at least 80%, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more homology with the full-length amino acid sequence shown in SEQ ID NO. 2, as long as the stereoselective carbonyl group reducing activity is not impaired.

The homologue of the above (C) has an amino acid sequence in which one or several amino acids are deleted, added or substituted in the amino acid sequence shown in SEQ ID NO. 2, as long as the stereoselective carbonyl group reducing activity is not impaired. As used herein, "one or several amino acids" is in particular 20 or less, preferably 10 or less, more preferably 5 or less amino acids.

The gene encoding the above enzyme is a DNA comprising the base sequence shown in the following (D), (E) or (F) or a homologue thereof:

(D) 1 in the nucleotide sequence shown in SEQ ID NO,

(E) a base sequence that hybridizes under stringent conditions to a DNA consisting of a sequence complementary to the base sequence shown in SEQ ID NO. 1 and encodes a polypeptide having an activity that acts on and converts a compound represented by formula (10) into a compound represented by formula (6),

(F) a base sequence having a base sequence in which one or several bases are substituted, deleted or added in the base sequence shown in SEQ ID NO. 1, and encodes a polypeptide having an activity to act on the compound represented by formula (10) and convert it into the compound represented by formula (6).

Here, the "base sequence which hybridizes under stringent conditions" in the above-mentioned (E) means a base sequence of DNA obtained by a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method or the like under stringent conditions by using the DNA as a probe. In the colony hybridization method and plaque hybridization method, examples of stringent conditions include conditions in which a filter on which DNA or DNA fragments derived from colonies or plaques are immobilized is used, hybridization is carried out at 65 ℃ in the presence of 0.7mol/L to 1.0mol/L aqueous sodium chloride solution, and the filter is washed with 0.1 to 2 XSSC solution (composition of 1 XSSC: 150mmol/L aqueous sodium chloride solution, 15mmol/L aqueous sodium citrate solution) at 65 ℃.

Each hybridization can be carried out according to the methods described in the following documents: molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989, and others.

The homologue of the above (F) has a base sequence in which one or several bases are deleted, added or substituted in the base sequence shown in SEQ ID NO. 1 as long as the stereoselective carbonyl group reducing activity is not impaired. As used herein, "one or several bases" is specifically 60 or less, preferably 30 or less, more preferably 15 or less, more preferably 10 or less, particularly preferably 5 or less bases.

In step (vii), since the enzyme or the like of the present invention is superior in handling property and is easily added to the reaction system, it can also be used in a frozen state. When the frozen enzyme of the present invention or the like is used, the shape thereof is not particularly limited, and, for example, a prism, a cylinder, a lump, a spherical shape, or the like can be used.

In step (vii), the compound represented by formula (10) as a reactant is generally used at a substrate concentration of 0.01% w/v-20% w/v, preferably 0.1% w/v-10% w/v. The reactants may be present in the reaction system beforehand, or may be added immediately at the start of the reaction. When the substrate is inhibited by the enzyme, the enzyme may also be added continuously or intermittently from the start of the reaction to reduce its influence or to increase the cumulative concentration of the product produced.

Step (vii) is preferably performed in coenzyme NAD (P)⁺Or NAD (P) H. In this case, the coenzyme is preferably added at a concentration of usually 0.001mmol/L to 100mmol/L, preferably 0.01mmol/L to 10 mmol/L.

When the above-mentioned coenzyme is added, NAD (P) produced from NAD (P) H in the reaction system is added from the viewpoint of reaction efficiency⁺Regeneration to NAD (P) H is preferred. Examples of regeneration methods include:

1) derived from NAD (P) by the microorganism or cell of the present invention⁺Ability to produce NAD (P) H, i.e. NAD (P)⁺A method for the reduction of a protein in a protein,

2) a method comprising adding to the reaction system one or more selected from the group consisting of: having a structure derived from NAD (P)⁺Microorganisms having the ability to produce NAD (P) H or treated products thereof, or enzymes useful for NAD (P) H regeneration such as glucose dehydrogenase, formate dehydrogenase, alcohol dehydrogenase, amino acid dehydrogenase, organic acid dehydrogenase (malate dehydrogenase, etc.) and the like (hereinafter referred to as "regenerative enzymes"),

3) including a method of simultaneously introducing one or more of the above-mentioned regenerant genes into a host organism or a host cell upon production of the microorganism or cell of the present invention, and the like.

In the method of 1) above, it is preferable to add glucose, ethanol, 2-propanol, formic acid or the like to the reaction system from the viewpoint of production efficiency.

In the method of the above 2), a microorganism having an ability to produce the above regenerated enzyme, a treated product of the microorganism such as a microorganism treated with acetone, glutaraldehyde or freeze-drying treatment, physical or enzyme disruption or the like, an enzyme fraction obtained as a crude product or a purified product, and these substances after being immobilized on a carrier such as polyacrylamide gel, carrageenan or the like, or the like may be used, or a commercially available enzyme may also be used.

The amount of the above-mentioned regenerated enzyme to be used is preferably an amount which provides an enzyme activity of usually 0.01 to 100 times, preferably 0.5 to 20 times as much as the carbonyl group-reducing activity of the enzyme of the present invention having the ability to stereoselectively reduce a carbonyl group.

Since it is also necessary to add a compound serving as a substrate of the above-mentioned regenerative enzyme, for example, glucose in the case of using glucose dehydrogenase, formic acid in the case of using formate dehydrogenase, ethanol or isopropanol in the case of using alcohol dehydrogenase, etc., the amount added is usually 0.1mol to 20mol, preferably 1 equivalent to 10mol, based on 1mol of the compound represented by formula (10).

In the method of 3) above, a method of incorporating the above-mentioned DNA for regenerating an enzyme into a chromosome together with the DNA encoding the enzyme used in step (i), a method of introducing both DNAs into a single expression vector and transforming a host organism or cell, a method of introducing both DNAs into separate expression vectors and transforming a host organism or cell, or the like can be used. In the method of introducing the two DNAs into separate expression vectors and transforming host organisms or cells, the expression vectors need to be selected in consideration of incompatibility between the two expression vectors.

When a plurality of genes are introduced into a single expression vector, a method of ligating a region involved in expression control, such as a promoter and a terminator, to each gene and expressing as an operon containing a plurality of cistrons, such as the lactose operon, is also possible.

Step (vii) is carried out in an aqueous medium or a mixture of an aqueous medium and an organic solvent. The aqueous medium or the mixture of the aqueous medium and the organic solvent contains the compound represented by formula (10) 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 medium containing the enzyme obtained by culturing the microorganism or cell. In addition, various coenzymes may be contained, if necessary. When a coenzyme is contained, a regeneration system thereof is more preferable, that is, more preferably, the coenzyme can be regenerated.

The compound represented by formula (10) can also be produced by the method mentioned below.

As the aqueous medium, water and pH buffer such as potassium phosphate buffer, sodium citrate buffer, Tris-HCl buffer, etc. may be mentioned.

As the organic solvent, a solvent in which the compound represented by formula (10) exhibits high solubility, for example, ethyl acetate, isopropyl acetate, butyl acetate, toluene, chloroform, n-hexane, n-heptane, dimethyl sulfoxide, methanol, ethanol, n-propanol, 2-propanol, etc., may be used. Among them, dimethyl sulfoxide, methanol, ethanol are preferable as the organic solvent because the compound represented by formula (10) shows high solubility therein. Further, dimethyl sulfoxide is more preferable because of high conversion.

Step (vii) is generally carried out at a reaction temperature of from 4 ℃ to 70 ℃, preferably from 30 ℃ to 60 ℃, generally at a pH of from 3 to 11, preferably from 4 to 8. The reaction time is usually 0.5hr to 48hr, preferably 0.5hr to 24 hr.

The compound represented by formula (6) obtained in step (vii) may be purified by the following method: bacterial cells, polypeptides, and the like are isolated by centrifugation, filtration, or the like, adjusted to a suitable pH, subjected to a suitable combination of extractions using organic solvents such as hexane, ethyl acetate, toluene, and the like, and purified by column chromatography, crystallization, and the like.

Method (b):

method (b) characteristically comprises step (viii) and step (ix).

Step (viii):

in step (viii), the compound represented by formula (12) is reacted with the compound represented by formula (13) in the presence of a titanium catalyst (Ti catalyst) represented by the following formula (15) to give a compound represented by formula (14).

Wherein Ra is an alkyl group having a carbon number of 1 to 10.

The amount of the compound represented by formula (13) used is not particularly limited, and it is usually 1mol to 10mol, preferably 1mol to 5mol, relative to 1mol of the compound represented by formula (12).

The binaphthyl structure of the Ti catalyst represented by formula (15) is preferably in the S conformation. As Ra, short chain alkyl groups having a carbon number of 1 to 4 such as methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, and the like are preferable, and industrially, isopropyl is particularly preferable.

The amount of the Ti catalyst to be used is not particularly limited, and it is usually 0.001mol to 1mol, preferably 0.01mol to 0.5mol, relative to 1mol of the compound represented by the formula (12).

The reaction is preferably carried out using a solvent. Although the solvent is not particularly limited as long as the reaction can proceed, it is preferable to use a hydrocarbon solvent such as cyclohexane, n-hexane, n-heptane, toluene, etc.; ether solvents such as t-butyl methyl ether, Tetrahydrofuran (THF), cyclopentyl methyl ether (CPME), and the like; ester solvents such as ethyl acetate, propyl acetate, butyl acetate, and the like; acetonitrile, and the like. One of these solvents may be used, or a mixture of two or more thereof may also be used.

The amount of the solvent used is not particularly limited, and it is usually 1mL-100mL, preferably 1mL-50mL, relative to 1g of the compound represented by formula (12).

It is also possible to add lithium chloride and/or synthetic zeolite to the reaction system. As a result, a desired product having high optical purity can be obtained.

The amount of lithium chloride used is not particularly limited, and it is usually 0.001mol to 2mol, preferably 0.01mol to 1mol, relative to 1mol of the compound represented by formula (12).

Examples of the synthetic zeolite include molecular sieves 3A, 4A, 5A, 13X and the like, and preferably molecular sieves 3A, 4A.

The amount of the synthetic zeolite used is not particularly limited, and it is usually 0.01 to 1g, preferably 0.1 to 0.5g, relative to 1g of the compound represented by the formula (12).

The reaction temperature below the boiling point of the solvent used and above the melting point of the solvent used hardly affects the reaction or the yield of the objective product. Therefore, the reaction temperature can be appropriately selected from this temperature range. Industrially, it is preferably from 0 ℃ to 100 ℃, more preferably from 10 ℃ to 50 ℃.

The reaction time is usually 1hr to 24hr, preferably 1hr to 12 hr.

The compound represented by formula (12) can be obtained according to a known method. It can be obtained, for example, by the method described in WO 2000/42016. In addition, commercially available products may also be used.

The compound represented by formula (13) can also be obtained according to a known method as shown below.

It can be obtained, for example, by the method described in WO 2003/420180.

Step (ix):

in step (ix), the compound represented by formula (14) is reduced by reaction with an enzyme having an activity capable of stereoselectively reducing a carbonyl group, a microorganism or cell (microorganism or cell of the present invention) having an ability to produce the enzyme, a treated product of the microorganism or cell, and/or a medium containing the enzyme obtained by culturing the microorganism or cell, to give a compound represented by formula (6).

Step (ix) may be performed in the same manner as in step (vii) above.

[ examples ]

The present invention is explained in further detail below with reference to examples; however, the present invention is not limited by the examples.

Abbreviations in the examples are shown as the following compounds.

PT-DOXE: (6E) -7- [ 2-cyclopropyl-4- (4-fluorophenyl) -3-quinolinyl ] -3, 5-dioxo-6-heptenoic acid ethyl ester

PT-ALD: [ 2-cyclopropyl-4- (4-fluorophenyl) -3-quinolyl ] carbaldehyde

DHAB: 3, 5-Dioxoheptenoic acid tert-butyl ester

PT-DOXB: (6E) -7- [ 2-cyclopropyl-4- (4-fluorophenyl) -3-quinolinyl ] -3, 5-dioxo-6-heptenoic acid tert-butyl ester

PT-DOLE: (3R,5S,6E) -7- [ 2-cyclopropyl-4- (4-fluorophenyl) -3-quinolyl ] -3, 5-dihydroxy-6-heptenoic acid ethyl ester

DOLE MsOH: (3R,5S,6E) -7- [ 2-cyclopropyl-4- (4-fluorophenyl) -3-quinolyl ] -3, 5-dihydroxy-6-heptenoic acid ethyl ester methanesulfonate

5S-MOLE: (E) - (5S) -7- [ 2-cyclopropyl-4- (4-fluorophenyl) -quinolin-3-yl ] -5-hydroxy-3-oxo-hept-6-enoic acid ethyl ester

ACPT: (4R,6S,1E) -2- [ 2-cyclopropyl-4- (4-fluorophenyl) -3-quinolyl ] -2, 2-dimethyl-1, 3-dioxolane-4-acetic acid ethyl ester

Me: methyl radical

Et: ethyl radical

t-Bu: tert-butyl radical

EtOH: ethanol

THF: tetrahydrofuran (THF)

DMF: n, N-dimethylformamide

The assay analysis in the examples includes measurement using HPLC (high performance liquid chromatography) under the following conditions.

< chemical purity of PT-DOXE >

Column: capcell Pak C18 MG (4.6 mm. times.75 mm,3 μm), manufactured by Shiseido Co., Ltd

Mobile phase:

a: water/acetic acid/ammonium acetate 1000/100/7.7 (mL/g)

B：THF

Gradient program (concentration of B): 41 vol% (0min) → 41 vol% (17min) → 90 vol% (27min)

Flow rate: 1mL/min

Column temperature: 40 deg.C

Detection wavelength: UV 254nm

< chemical purity of PT-DOLE >

Column: L-Column ODS (4.6 mm. times.250 mm,5 μm) manufactured by CERI

Mobile phase:

a: 10mmol/L ammonium acetate

B: ethanol/THF (vol/vol) 15/1

Gradient program (concentration of B): 48 vol% (0min) → 48 vol% (35min) → 64 vol% (55min)

Flow rate: 1.0mL/min

Column temperature: 40 deg.C

Detection wavelength: UV 245nm

< chemical purity of ACPT >

Column: unison UK-C18(4.6 mm. times.250 mm,3 μm) manufactured by Imtact

Mobile phase:

a: 30mmol/L ammonium acetate in water/acetonitrile 20/80 (vol.)

B: 30mmol/L ammonium acetate in water/acetonitrile 5/95 (vol.)

Gradient program (concentration of B): 0 vol% (0min) → 0 vol% (10min) → 90 vol% (30min)

Flow rate: 1mL/min

Column temperature: 40 deg.C

Detection wavelength: UV 245nm

< chemical purity of pitavastatin calcium >

Column: unison UK-C18(4.6 mm. times.250 mm,3 μm) manufactured by Imtact

Mobile phase:

a: 0.1 vol% formic acid

B: acetonitrile

Gradient program (concentration of B): 25 vol% (0min) → 35 vol% (20min) → 90 vol% (30min) → 90 vol% (35min)

Flow rate: 1mL/min

Column temperature: 40 deg.C

Detection wavelength: UV 331nm

< measurement conditions for powder X-ray diffraction >

A measuring device: XRD6000 type, manufactured by SHIMADZU Corporation

Radiation source: cu

Wavelength:

a monochromator: use of

Tube voltage: 40.0Kv

Tube current: 40.0mA

Divergence: 1.00deg

Scattering: 1.00deg

Receiving: 0.15mm

Mode (2): continuous scanning

A transmission shaft: theta-2 theta

Data range: 2-40 deg

Step level: 0.02deg

Scanning speed: 2.0000deg/min

Rotation speed: 60rpm

Example 1 (production of PT-DOXE)

Sodium hydride (20.8g, purity 59.5%, 515mmol) and THF (200mL) were charged to the flask and cooled to 17 ℃ under a nitrogen atmosphere. To this mixture was added dropwise a solution of DHAB (53.9g, purity 91.9%, 247.1mmol) in THF (200mL) over 2hr, and after completion of the dropwise addition the solution was stirred at 25 ℃ for 13 hr.

To the stirred solution was added dropwise a solution of PT-ALD (40.0g, 137.3mmol) in THF (400mL) over 4hr, and after the dropwise addition was complete, the solution was stirred at 25 ℃ for 1 hr. The stirred solution was analyzed by HPLC and found to have a conversion to PT-DOXB of 99.2%.

To the stirred solution, n-heptane (200mL) and water (400mL) were added dropwise at 25 ℃ and the solution was separated. Subsequently, the organic layer was washed with a 4 wt% aqueous sodium chloride solution, a 10 wt% aqueous citric acid solution and a 10 wt% aqueous sodium chloride solution. The obtained organic layer was analyzed by HPLC, and the yield of PT-DOXB was found to be 88.2%.

The resulting organic layer was concentrated under reduced pressure and ethanol (200mL) was added. The resulting solution was heated in an autoclave (sealed vessel) to an internal temperature of 100 ℃ to 105 ℃ and maintained. After 10hr had elapsed, the resulting solution was analyzed by HPLC, and the conversion to PT-DOXE was found to be 99.0%. The resulting solution was cooled to 64 ℃ and stirred for 1 hr. The stirred solution was gradually cooled to 0 ℃ to-5 ℃ and crystals were recovered by filtration.

The resulting crystals and ethanol (200mL) were charged into a flask and heated to a temperature at which ethanol refluxed. Subsequently, the mixture was cooled to an internal temperature of 70 ℃ and stirred for 1 hr. The stirred solution was gradually cooled to 0 ℃ to-5 ℃, and the crystals were recovered by filtration and dried under reduced pressure. The obtained crystals were analyzed by HPLC, and found to have a purity of PT-DOXE of 99.1 area%, and an amount of 39.7g (yield 64.9%).

Example 2 (production of PT-DOXB)

Sodium hydride (15.6g, purity 59.5%, 386mmol), THF (150mL) and N, N-dimethylformamide (75mL) were charged to the flask and cooled to 5 ℃ under a nitrogen atmosphere. To this mixture was added dropwise a solution of DHAB (40.4g, purity 91.9%, 185.4mmol) in THF (150mL) over 2hr, and after completion of the dropwise addition, the mixed solution was stirred at 8 to 10 ℃ for 14 hr.

To the stirred mixed solution was added dropwise a solution of PT-ALD (30.0g, 103.0mmol) in THF (225mL) over 3hr, and the solution after completion of the dropwise addition was stirred at 8 to 10 ℃ for 6 hr. The stirred solution was analyzed by HPLC and found to have a conversion to PT-DOXB of 98.5%.

To the stirred solution, n-heptane (150mL) and water (150mL) were added dropwise at 10 ℃ and the solution was separated. Subsequently, the organic layer was washed with a 4 wt% aqueous sodium chloride solution, a 10 wt% aqueous citric acid solution and a 10 wt% aqueous sodium chloride solution. The obtained organic layer was analyzed by HPLC, and the yield of PT-DOXB was found to be 88.9%.

Reference example 1 (preparation of bacterial cells)

[ preparation of recombinant Escherichia coli JM109/pKV32OCR1-GDH co-expressing carbonyl reductase (hereinafter referred to as OCR1) and glucose-1-dehydrogenase (hereinafter referred to as GDH) ]

(1) Cloning of genes

Primers OCR1_ F (SEQ ID NO:3) and OCR1_ R (SEQ ID NO:4) for amplifying the full-length OCR1 gene were designed and synthesized on the basis of the gene sequence (OCR1) encoding OCR 1(SEQ ID NO:2 described in JP-B-4270918) derived from a non-fermentative variant (Ogataea minor var. nonfermenters) NBRC (formerly IFO)1473 of methanol-inducible yeast. Then, PCR was carried out according to a conventional method using chromosomal DNA of a methanol-inducible yeast non-fermentative variant (octaea minor var. nonfermentans) as a template, giving a DNA fragment of about 0.8 kbp.

Then, primers GDH _ F1(SEQ ID NO:7) and GDH _ R1(SEQ ID NO:8) for amplifying the full-length GDH gene, which is glucose-1-dehydrogenase encoded by a gene derived from Bacillus subtilis (GeneBank accession AL009126.3) in which the 96 th amino acid residue glutamic acid was substituted with alanine, were designed and synthesized on the basis of the gene sequence (GDH (SEQ ID NO:5) hereinafter) encoding GDH (SEQ ID NO: 6). Then, PCR was carried out according to the conventional method to give a DNA fragment of about 0.8 kbp.

(2) Preparation of expression plasmid

The DNA fragment of OCR1 obtained in the above (1) was digested with restriction enzymes EcoRI and HindIII, and introduced into the downstream of trc promoter in ｃA plasmid pKV32 (described in JP-A-2005-34025) digested with MunI and HindIII using the Ligation-Convenience kit (manufactured by Nippon Gene Co., Ltd.), giving pKV32OCR 1.

Then, the DNA fragment of GDH obtained in the above (1) was digested with restriction enzymes EcoRI and XbaI, and introduced into the downstream of trc promoter in plasmid pKV32 digested with MunI and XbaI using Ligation-convergence kit (manufactured by Nippon Gene co., ltd.) to give pKV32 GDH.

PCR was carried out using pKV32GDH as a template and using the primers GDH _ F2(SEQ ID NO:9) and GDH _ R2(SEQ ID NO:10) to which the restriction enzyme site HindIII was added, and the resulting fragment was digested with the restriction enzyme HindIII and inserted into the downstream of the plasmid pKV32OCR1 which had been digested with the restriction enzyme HindIII in advance, giving pKV32OCR 1-GDH. The orientation of the gdh gene in the resulting plasmid was confirmed by PCR.

(3) Preparation of expression Strain

Using the pKV32OCR1-GDH obtained in the above (2), Escherichia coli JM109 (manufactured by TAKARA BIO INC.) was transformed according to a conventional method to give recombinant Escherichia coli JM109/pKV32OCR 1-GDH.

Example 3 (production of PT-DOLE)

Ion-exchanged water (57mL), glucose monohydrate (30g, 151.5mmol), and NADP⁺(30mg, 0.04mmol), potassium dihydrogenphosphate (2.76g, 20.3mmol) and dipotassium hydrogenphosphate (0.39g, 2.3mmol) were charged into and dissolved in a 250mL fermentor. Frozen bacterial cells (45g) of recombinant Escherichia coli JM109/pKV32OCR1-GDH prepared by the method of reference example 1 and the entire amount of a substrate solution prepared by dissolving PT-DOXE (1.2g, 2.69mmol) obtained in a similar manner to example 1 in dimethyl sulfoxide (DMSO) (36.3g) were added thereto, and the mixture was reacted by stirring at an internal temperature of 50 ℃ for 4 hr. During the reaction, 25 wt% aqueous sodium hydroxide solution was added dropwise to maintain pH 6. Diluting the obtained reaction mixture with acetonitrileReleasing 36 times, filtering through a filter membrane with the pore size of 0.2nm, and obtaining filtrate. The filtrate was analyzed by HPLC and the conversion from PT-DOXE to PT-DOLE was found to be 85.7%.

Example 4 (production of PT-DOLE)

Ion-exchanged water (12L), glucose monohydrate (750g), NADP⁺(2.9g), dipotassium hydrogenphosphate (20.85g, 0.12mol) and potassium dihydrogenphosphate (147.0g, 1.08mol) were charged into a 30L fermenter and dissolved therein. Frozen bacterial cells (2.1kg) of recombinant Escherichia coli JM109/pKV32OCR1-GDH prepared by the method of reference example 1 and the entire amount of substrate solution prepared by dissolving 5S-MOLE (produced as described in the national publication of International patent application No. 2005-516064) (0.3kg, 0.67mol) in dimethyl sulfoxide (DMSO) (3.3kg) were added thereto, and the mixture was reacted by stirring at an internal temperature of 50 ℃ for 3 hr. During the reaction, 25 wt% aqueous sodium hydroxide solution was added dropwise to maintain pH 6.5. The resulting reaction mixture was centrifuged at 10,000rpm for 10min to give a precipitate consisting of bacterial cells and the resulting reaction product. The precipitate was suspended in a 5 wt% aqueous sodium sulfate solution and extracted with ethyl acetate. The extraction with ethyl acetate was repeated three times. The obtained extracts were combined to give a solution. The resulting solution was analyzed by HPLC, and found to have a purity of 97.6 area% for PT-DOLE and a yield of 236g (78.7% yield).

Example 5 (production of DOLE MsOH)

PT-DOLE obtained by a method similar to example 4 was concentrated under reduced pressure, and a PT-DOLE solution prepared to a concentration of 11.6 wt% was obtained. To the resulting PT-DOLE solution (377.6g, 97.4mmol) was added methanesulfonic acid (0.5g, 5.2mmol) at 30 deg.C and the mixture was stirred at the same temperature for 1.5 hr. A solution of methanesulfonic acid (8.9g, 92.6mmol) dissolved in ethyl acetate (39.3g) was added dropwise. After the dropwise addition was complete, the reaction mixture was cooled to-3 ℃.

After cooling, the resulting suspension was filtered and the residue was dried at an external temperature of 40 ℃ under reduced pressure. The resulting solid was analyzed by HPLC and found to have a DOLE MsOH yield of 52.4g (98% yield). Melting point of the obtained solid,¹H-NMR and powder X-ray diffraction (XRD) measurements were as follows.

Melting point: 132 deg.C

¹H-NMR(400MHz,DMSO)δ1.22-1.24(6H,m),1.46(2H,s),1.49(1H,m),2.22-2.33(2H,m),2.37(3H,s),2.61(1H,m),3.76(1H,m),4.08(2H,t,J＝8Hz),4.14(1H,m),5.67(1H,dd,J＝4Hz,16 Hz),6.53(1H,d,J＝16 Hz),7.35-7.40(5H,m),7.56(1H,m),7.82(1H,m),8.09(1H,m)

XRD measurement results:

TABLE 1

2θ	Relative strength	2θ	Relative strength
				6.9	20	26.2	8
13.5	28	26.9	6
				15.3	9	27.8	9
15.7	5	28.2	9
				16.5	6	29.6	7
17.0	14	29.8	6
				18.8	13	30.1	5
19.8	9	30.6	5
				20.6	100	30.9	8
21.1	16	33.0	5
				21.8	42	33.6	7
22.2	16	34.2	5
				23.9	11	34.6	5
24.5	7	34.8	5
				24.7	8	35.6	5
25.0	10	39.0	5

Example 6 (production of ACPT)

A flask was charged with DOLE MsOH (15.0g, 27.5mmol) obtained by a method similar to example 5 and acetonitrile (58.5 g). Acetone dimethyl acetal (11.3g, 109mmol) was added to the mixture at an internal temperature of 20 ℃. After stirring for 3hr, sodium bicarbonate (2.9g) and water (19.5g) were added. Water (13.0g) was further added and the mixture was stirred for 12 hr. Subsequently, water (42.2g) was added, the internal temperature was cooled to-4 ℃, and the resulting crystals were collected by filtration and dried under reduced pressure to give ACPT (13.3g, yield 99%) as crude crystals.

The obtained crude crystals of ACPT (10g), ethanol (58.8g) and water (14.7g) were charged into a flask, and dissolution was carried out by raising the internal temperature to about 60 ℃. The resulting solution was cooled to an internal temperature of about 37 ℃ and stirred for a while to precipitate crystals. Subsequently, the internal temperature was cooled to-3 ℃ over 6 hr. The resulting crystals were collected by filtration and dried under reduced pressure. The obtained crystals were analyzed by HPLC, and found to have an ACPT purity of 99.8 area% and a yield of 8.9g (yield 89%).

Example 7 (production of pitavastatin calcium)

ACPT crystals (8g, 16.3mmol) obtained by a method similar to example 6 and ethanol (40mL) were charged into a flask. To the mixture was added a mixed solution of 35 wt% hydrochloric acid (2.4g) and water (21.6g) at an internal temperature of about 30 ℃. After stirring for 3.5hr, ethyl acetate (90mL) and 7 wt% aqueous sodium bicarbonate solution (45g) were added. The resulting solution was subjected to liquid separation, ethyl acetate (45mL) was added to the aqueous layer, and the mixture was subjected to liquid separation. The resulting organic layers were combined, washed with 20 wt% aqueous sodium chloride (45g) and concentrated.

After concentration, ethanol (74mL) and water (74mL) were added to the resulting residue. To the mixture was added 8 wt% aqueous sodium hydroxide solution (9.9 g). After stirring for 3hr, the resulting solution was concentrated under reduced pressure. After concentration under reduced pressure, tert-butyl methyl ether (37mL) was added to the obtained residue, and the mixture was subjected to liquid separation. This operation was performed twice. The aqueous layer was concentrated under reduced pressure, and a solution of calcium chloride dihydrate (2.6g) dissolved in water (104g) was added dropwise at an internal temperature of about 32 ℃. Subsequently, the resulting reaction mixture was cooled to about 2 ℃, the resulting crystals were collected by filtration, and the wet crystals were dried under reduced pressure. When the water content of the crystals reached 9.6 wt%, drying was discontinued. The obtained crystals were analyzed by HPLC to find that the purity of pitavastatin calcium was 99.93 area%, and the yield was 9.7g (yield 94%).

Example 8 (production of pitavastatin calcium)

ACPT crystals (70g, 143mmol) obtained by a method similar to example 6 and ethanol (350mL) were charged in a flask. To the mixture was added 35 wt% hydrochloric acid (208g) at an internal temperature of around 30 ℃. After stirring for 2hr, ethanol (210mL) was added and 8 wt% aqueous sodium hydroxide (100g) was added. The mixture was concentrated to a volume of 630 mL. Subsequently, 3.5 wt% hydrochloric acid (178g) was added, and the mixture was stirred at room temperature for 2 hr. Subsequently, 8 wt% aqueous sodium hydroxide solution (178g) was added, and the mixture was stirred at room temperature for 2 hr. 210mL of water were added to the mixture and the mixture was concentrated to a volume of 700 mL. To the concentrate was added water (350mL) and tert-butyl methyl ether (350mL), the mixture was partitioned, and the aqueous layer was concentrated to a volume of 980 mL. To the resulting concentrate was added dropwise a mixed solution of calcium chloride dihydrate (22.9g) and water (173g) at a temperature of about 30 ℃. The resulting suspension was cooled to room temperature, the resulting crystals were collected by filtration, and the wet crystals were dried under reduced pressure. The water content of the obtained crystals was 7.8 wt%. Humidified nitrogen was flowed through the resulting crystals to adjust the water content to 10.9 wt%. The obtained crystals were analyzed by HPLC to find that the purity of pitavastatin calcium was 99.96 area%, and the yield was 66.0g (yield 93%).

[ Industrial Applicability ]

According to the process of the present invention, pitavastatin calcium can be produced safely on an industrial scale with high yield, high selectivity and low cost.

The present application is based on patent application No. 2015-154864 filed in japan, the entire contents of which are incorporated herein.

Sequence listing

<110> API from Kabushiki Kaisha

<120> method for producing pitavastatin calcium

<130> SPI213396-51

<150> JP2015-154864

<151> 2015-08-05

<160> 10

<170> PatentIn 3.5 edition

<210> 1

<211> 753

<212> DNA

<213> non-fermentative variant of methanol-inducible yeast (Ogataea minuta var. nonfermentans)

<220>

<221> CDS

<222> (1)..(753)

<400> 1

atg gct aaa act gtt tac ttc atc gca ggt gct tcc aga ggt atc ggt 48

Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly

1 5 10 15

ctc gag gtt gct tcc cag ctg agt gca aac cca gac aat tat gtt att 96

Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile

20 25 30

gca tcc tat aga tct gaa aag tct gct tca gga ctt ttg gag ctg gca 144

Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala

35 40 45

aag aag gat aat gtc gac aca att gtg ttg gat att gca agc cag gaa 192

Lys Lys Asp Asn Val Asp Thr Ile Val Leu Asp Ile Ala Ser Gln Glu

50 55 60

tcg att gat gct gtt cca gca cag att tcc aag ctg act gat gga atc 240

Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile

65 70 75 80

gat gtt gcc ttg atc aac gct gga att gcc aac gct atg tgt ccg att 288

Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile

85 90 95

ctc gaa tgt tct aga gag tcc tac act gat cac tgg aca acc aat gcc 336

Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala

100 105 110

ttg ggt cca atc atg ctc tac caa gct att cat aag ttc atg ctc cag 384

Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln

115 120 125

aga gag acc aga aaa gtg ttc ttt acc acg agt gct ggt ggt tcc att 432

Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile

130 135 140

cag gct aag ata ccc gtg cct gtg agt ggt tac ggt atg tcc aag gct 480

Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Met Ser Lys Ala

145 150 155 160

gcg ctt aat tat gct gtg aga aaa ctt gct gac gag tgc tac aag gac 528

Ala Leu Asn Tyr Ala Val Arg Lys Leu Ala Asp Glu Cys Tyr Lys Asp

165 170 175

aac ttc act att gtg ttg ctg cat cct ggt ttt gtt aag acg gac atg 576

Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met

180 185 190

ggt caa agc gcc att cag aag atg tca aat gga aat gct gag ctt ctt 624

Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu

195 200 205

gct tac att gac tca atg act att gat gtt cct acc agt gct ggc caa 672

Ala Tyr Ile Asp Ser Met Thr Ile Asp Val Pro Thr Ser Ala Gly Gln

210 215 220

atc gtc ggt gcc att atg acc ttg gac aag cag agc agc ggt aga ttt 720

Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe

225 230 235 240

atc aac gct gct gac cag ttt gac atg cca ttt 753

Ile Asn Ala Ala Asp Gln Phe Asp Met Pro Phe

245 250

<210> 2

<211> 251

<212> PRT

<213> non-fermentative variant of methanol-inducible yeast (Ogataea minuta var. nonfermentans)

<400> 2

Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly

1 5 10 15

Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile

20 25 30

Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala

35 40 45

Lys Lys Asp Asn Val Asp Thr Ile Val Leu Asp Ile Ala Ser Gln Glu

50 55 60

Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile

65 70 75 80

Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile

85 90 95

Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala

100 105 110

Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln

115 120 125

Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile

130 135 140

Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Met Ser Lys Ala

145 150 155 160

Ala Leu Asn Tyr Ala Val Arg Lys Leu Ala Asp Glu Cys Tyr Lys Asp

165 170 175

Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met

180 185 190

Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu

195 200 205

Ala Tyr Ile Asp Ser Met Thr Ile Asp Val Pro Thr Ser Ala Gly Gln

210 215 220

Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe

225 230 235 240

Ile Asn Ala Ala Asp Gln Phe Asp Met Pro Phe

245 250

<210> 3

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 3

cggaattcat ggctaaaact gtttacttc 29

<210> 4

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 4

gggaagctta ttactaaaat ggcatgtcaa actgg 35

<210> 5

<211> 783

<212> DNA

<213> Bacillus subtilis

<220>

<221> CDS

<222> (1)..(783)

<400> 5

atg tat ccg gat tta aaa gga aaa gtc gtc gct att aca gga gct gct 48

Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala

1 5 10 15

tca ggg ctc gga aag gcg atg gcc att cgc ttc ggc aag gag cag gca 96

Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala

20 25 30

aaa gtg gtt atc aac tat tat agt aat aaa caa gat ccg aac gag gta 144

Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val

35 40 45

aaa gaa gag gtc atc aag gcg ggc ggt gaa gct gtt gtc gtc caa gga 192

Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly

50 55 60

gat gtc acg aaa gag gaa gat gta aaa aat atc gtg caa acg gca att 240

Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile

65 70 75 80

aag gag ttc ggc aca ctc gat att atg att aat aat gcc ggt ctt gca 288

Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Ala

85 90 95

aat cct gtg cca tct cac gaa atg ccg ctc aag gat tgg gat aaa gtc 336

Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val

100 105 110

atc ggc acg aac tta acg ggt gcc ttt tta gga agc cgt gaa gcg att 384

Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile

115 120 125

aaa tat ttc gta gaa aac gat atc aag gga aat gtc att aac atg tcc 432

Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser

130 135 140

agt gtg cac gaa gtg att cct tgg ccg tta ttt gtc cac tat gcg gca 480

Ser Val His Glu Val Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala

145 150 155 160

agt aaa ggc ggg ata aag ctg atg aca gaa aca tta gcg ttg gaa tac 528

Ser Lys Gly Gly Ile Lys Leu Met Thr Glu Thr Leu Ala Leu Glu Tyr

165 170 175

gcg ccg aag ggc att cgc gtc aat aat att ggg cca ggt gcg atc aac 576

Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn

180 185 190

acg cca atc aat gct gaa aaa ttc gct gac cct aaa cag aaa gct gat 624

Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp

195 200 205

gta gaa agc atg att cca atg gga tat atc ggc gaa ccg gag gag atc 672

Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile

210 215 220

gcc gca gta gca gcc tgg ctt gct tcg aag gaa gcc agc tac gtc aca 720

Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr

225 230 235 240

ggc atc acg tta ttc gcg gac ggc ggt atg aca caa tat cct tca ttc 768

Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Gln Tyr Pro Ser Phe

245 250 255

cag gca ggc cgc ggt 783

Gln Ala Gly Arg Gly

260

<210> 6

<211> 261

<212> PRT

<213> Bacillus subtilis

<400> 6

Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala

1 5 10 15

Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala

20 25 30

Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val

35 40 45

Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly

50 55 60

Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile

65 70 75 80

Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Ala

85 90 95

Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val

100 105 110

Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile

115 120 125

Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser

130 135 140

Ser Val His Glu Val Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala

145 150 155 160

Ser Lys Gly Gly Ile Lys Leu Met Thr Glu Thr Leu Ala Leu Glu Tyr

165 170 175

Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn

180 185 190

Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp

195 200 205

Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile

210 215 220

Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr

225 230 235 240

Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Gln Tyr Pro Ser Phe

245 250 255

Gln Ala Gly Arg Gly

260

<210> 7

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 7

ggggaattca tgtatccgga tttaaaagga aaagtcg 37

<210> 8

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 8

gggtctagat taaccgcggc ctgcctggaa tg 32

<210> 9

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 9

cccaagctta gttaacttta gaaggagaca attc 34

<210> 10

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 10

ctgccgcccg actatcac 18