JP2003026634A - Method for producing pravastatin sodium salt - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing pravastatin sodium salt capable of obtaining a sodium salt thereof in high yield from pravastatin obtained by microbiological conversion from compactin or the like. SOLUTION: Pravastatin amine salt is reacted with an acid in the presence of an organic solvent to form free pravastatin, and sodium alkoxide is added to the obtained organic solvent solution containing free pravastatin to crystallize pravastatin as a sodium salt. A method for producing pravastatin sodium salt, comprising separating crystals.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing pravastatin sodium salt. More specifically, it relates to a method for obtaining pravastatin sodium salt in high yield and high purity from a pravastatin-containing solution obtained by reacting compactin with an enzyme or a microorganism.

[0002]

BACKGROUND OF THE INVENTION Pravastatin is represented by the following formula (I): (+)-(3R, 5R) -3,5-dihydroxy-7
[(1S, 2S, 6S, 8S, 8aR) -6-Hydroxy-2-methyl-8-[(S) -2-methyl-butyryloxy] -1,2,6,7,8,8a-hexahydro-
1-naphthyl] -heptanonic acid.

[0003]

[Chemical 1]

Pravastatin exhibits excellent cholesterol biosynthesis inhibitory activity and organ-selective inhibitory activity, and is useful as an antihyperlipidemic agent for treating or preventing ischemic heart diseases such as arteriosclerosis. From the pharmacological point of view, it is used as pravastatin sodium salt. As for pravastatin, a chemical synthesis method for producing it at low cost is not known.

[0005]

[Chemical 2]

It is produced by a biochemical method using a compactin-producing microorganism and a microorganism (enzyme) that converts compactin into pravastatin. That is, in the first stage, fermentative production of compactin,
In the second step, it is industrially produced by a two-step fermentation in which a hydroxyl group is introduced at the 6-position of compactin by fermentation conversion.
Examples of microorganisms involved in the first stage include Penicillium citrinum , and examples of microorganisms involved in the second stage include Absidia.
dia) genus, Kaningamera (Cunninghamella) genus, synth file last lamb (Syncephalastrum) genus Streptomyces (Streptomyces) specific microorganisms (JP-B-62-54476 JP belonging to the genus), Streptomyces Karubofirasu of (Streptomyces carbophilus) Strain (Patent No. 2603
No. 677 and Japanese Patent No. 2672551) are known. The Applicant also applied to Microtetraspora recticatena ( Microtetra
spora recticatena ) IFO 14525 strain (Japanese Patent Application 2000-104278)
No.), Streptomyces s
p.) TM-6 strain (FERM P-18311) and Streptomyces sp. TM-7 strain (FERM P-18311)
-18312) (Japanese Patent Application No. 2001-166412) is compactin 6β
It has been found to have a hydroxylation activity. In the above-mentioned two-step fermentation method, the reaction liquid finally obtained contains various by-products, so that a step of separating and purifying pravastatin is necessary. Pravastatin has a good octylamine salt crystallinity, and a recrystallization method via an octylamine salt has been carried out (Pharmaceutical Journal, Vol. 111, No. 9, 469).
Pp. 487, 1991).

In order to obtain pravastatin sodium salt from the corresponding amine salt, a method of reacting the amine salt with sodium hydroxide can be considered. WO00 / 46175 describes the following method for obtaining pravastatin sodium salt crystals or powder from pravastatin dibenzylamine salt. 1) A method in which pravastatin dibenzylamine salt is suspended in a mixed solution of isobutyl acetate and water, an aqueous solution of sodium salt is obtained by adding sodium hydroxide, and the solution is freeze-dried after gel filtration treatment. 2) Pravastatin dibenzylamine salt is ethanol. Suspended in water, an ethanol solution of sodium hydroxide is added to obtain an ethanol solution of a sodium salt, which is concentrated, and then acetone is added at a high temperature, followed by cooling to obtain a precipitate. 3) Pravastatin dibenzylamine salt Is dissolved in a warm ethyl acetate-ethanol mixed solution, and an ethanol solution of sodium hydroxide is added, followed by cooling to obtain a sodium salt precipitate.

However, the method of suspending the amine salt of pravastatin in an aqueous solution or a polar solvent and contacting it with sodium hydroxide has a slow conversion rate to a sodium salt,
The method of reacting an amine salt with sodium hydroxide in an ethanol solution has problems such as a large amount of organic solvent used and a low yield.

The inventors of the present invention suspended the amine salt in a mixed solution of an organic solvent and water, transferred it to the organic layer as an educt under acidic conditions, added sodium hydroxide solution, and added it to the organic solvent. The method of precipitating sodium salt into the solution was investigated. However, in this method, since sodium hydroxide is dissolved in a polar solvent such as water or alcohol and added, the water-soluble sodium salt is dissolved in the aqueous layer, and the yield of the sodium salt that can be precipitated from the organic layer was low. . To improve this problem, it is possible to use another organic solvent as the poor solvent,
Since a large amount of organic solvent is required, there is a problem in terms of impact on the surrounding environment and work environment and safety.

Of the amines used as the starting material for conventionally used amine salts, octylamine is expensive in reagent grade high purity products. In the recrystallization method via octylamine salt,
The removal efficiency of the by-product in which a hydroxyl group is introduced at the 6α position generated in the process of fermentative conversion of compactin is low. Furthermore, since the octylamine salt is water-soluble, a large amount of organic solvent is required for recrystallization. Therefore, in order to obtain high-purity pravastatin that can be used as a drug, recrystallization from an organic solvent must be repeated, and there are problems such as a large problem in terms of environmental load and cost, and a fermentation conversion process from compactin. There has been a demand for a method for efficiently obtaining highly pure pravastatin sodium salt.

[0011]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for efficiently obtaining a sodium salt from pravastatin amine salt. Another object of the present invention is to provide a method for producing bravastatin sodium salt with high purity, high yield and low cost, using pravastatin produced by fermentative conversion of compactin as a raw material.

[0012]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have reacted pravastatin amine salt with an acid in the presence of an organic solvent to give free pravastatin, and obtain the obtained free pravastatin. It was found that the amount of the organic solvent used can be significantly reduced by adding sodium alkoxide to the solution of the organic solvent containing pravastatin to form the sodium salt. Further, by reacting pravastatin with a secondary amine and crystallizing as a secondary amine salt, a highly pure pravastatin amine salt can be obtained in a high yield, to which the above-mentioned conversion method to a sodium salt is applied. Thus, it was confirmed that high-purity pravastatin sodium salt can be efficiently produced, and the present invention was completed.

That is, the present invention provides the following method for producing pravastatin. 1. It is characterized in that pravastatin amine salt is reacted with an acid in the presence of an organic solvent to give free pravastatin, and sodium alkoxide is added to the obtained pravastatin-containing organic solvent solution to crystallize pravastatin sodium salt to separate crystals. And a method for producing pravastatin sodium salt. 2. The method for producing pravastatin sodium salt according to the above 1, wherein the sodium alkoxide is sodium methoxide.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The method for producing pravastatin sodium salt of the present invention comprises a step of adding an acid to pravastatin amine salt in the presence of an organic solvent to give free pravastatin (first step) and the obtained free pravastatin. And a step (second step) of adding sodium alkoxide to an organic solvent solution containing pravastatin to form a sodium salt.

That is, in the method of the present invention, the combination of the first step and the second step is essential, and unless free pravastatin is passed through in the first step, the conversion rate to the sodium salt is remarkably reduced, which is not practical. . Further, if sodium alkoxide is not used in the second step, a sodium salt cannot be formed with a small amount of organic solvent.

In the first step, pravastatin amine salt is suspended in the presence of an organic solvent, preferably in an organic solvent, and then acid is added thereto to obtain free pravastatin. The organic solvent in which the pravastatin amine salt is suspended is for facilitating the conversion to the acid and recovering the free acid, and various organic solvents capable of dissolving the free form can be used. Examples of such organic solvents include esters such as methyl acetate, ethyl acetate, butyl acetate, isobutyl acetate, isopropyl acetate and propyl acetate, aromatic solvents such as toluene, ethers such as ethyl ether, chloroform and dichloromethane. And other halogen-containing organic solvents. Of these, ethyl acetate is preferred. The amount of the organic solvent used should be the minimum limit for smooth conversion into the free form and recovery. Depending on the type of solvent and amine salt, it is usually 5 per 1 g of amine salt.
It is about 50 mL, preferably about 10 to 20 mL.

The acid used to release the pravastatin amine salt as a free form is not particularly limited, but an inorganic strong acid is preferable from the viewpoint that the conversion proceeds rapidly and it is not mixed in the organic layer in the second step. Examples of such inorganic acids include sulfuric acid, hydrochloric acid and nitric acid. The concentration and amount of the acid to be added and the pH of the solution may be such that the pravastatin amine salt is converted into the free acid, but for example, 0.1 to 1
The M acid may be used in an amount of about 0.1 to 10 times (equivalent to about 1 to 3 times) the amount of the organic solvent. If it is less than 0.1M, conversion to acid does not proceed sufficiently.

After the acid is added to the suspension of pravastatin amine salt in the organic solvent, the reaction is allowed to proceed with stirring. Additional solvent may be added after conversion to the acid to increase recovery efficiency. The amount of the organic solvent added here may be equal to or less than the initial amount of the organic solvent. After completion of the reaction, the organic layer is collected, washed with water and dehydrated.

In the second step, the solution of pravastatin in the organic solvent obtained in the first step is reacted with sodium alkoxide to convert it into a sodium salt. In order to avoid side reactions, the reaction is performed at room temperature or lower, preferably -20 to 10 ° C,
More preferably, it is carried out in the range of -5 to 5 ° C. The amount of sodium alkoxide used may be equivalent to or more than pravastatin, but may be used in excess in consideration of hydrolysis by water in the air or water in the solvent. Sodium alkoxide may be added as a corresponding alcohol solution. In the present invention, it is necessary that the sodium alkoxide is not easily affected by water in the reaction system, and sodium methoxide is suitable in this respect. The reaction is usually completed in several hours or less, for example, in about 30 minutes to 2 hours. After completion of the reaction, stirring is further continued, and if necessary, an organic solvent is added to promote crystallization. The organic solvent is selected from the above range. The same solvent as that used for the addition reaction to the acid may be used, or a different solvent may be used. The amount of the solvent for promoting crystallization is not more than twice the amount used for the addition reaction to the acid.

The method for producing pravastatin sodium salt of the present invention is preferably applied to secondary amine salts. Examples of the secondary amine that forms a salt with pravastatin include:

Embedded image R ¹ R ² NH (wherein R ¹ and R ² are the same or different and have 1 to 8 carbon atoms)
Is an alkyl group, a cyclic alkyl group, an allyl group, an aralkyl group, or an optionally substituted phenyl group. )
Those represented by are preferred. Examples of such secondary amines include dimethylamine, methylethylamine, diethylamine, ethylpropylamine, di (n-propyl) amine, di (isopropyl) amine, di (n-butyl) amine, di (isobutyl). Aliphatic dialkylamines such as amines, di (t-butyl) amine, dipentylamine, dihexylamine, diheptylamine, di-2-ethylhexylamine, dioctylamine (the carbon chains below dipentylamine are branched at any position. A), a cycloaliphatic dialkylamine such as dicyclohexylamine, diallylamine, dibenzylamine, diphenylamine and the like. Among these, dicyclohexylamine and diethylamine are preferable from the viewpoint of recovery rate and purity of pravastatin.

As described above, according to the method of crystallizing an appropriate amine salt (dicyclohexylamine salt and diethylamine salt) from a pravastatin-containing solution, pravastatin having a higher purity than that in the case of using monoalkylamine or ammonia is used. Can be obtained in high yield. When a trialkylamine was used, the salt did not crystallize and could not be purified.

Since the dicyclohexylamine salt is difficult to dissolve in an aqueous solvent, it can be recrystallized from an aqueous isopropanol solution, has a small environmental load, and is an economically advantageous purification method. Further, in the recrystallization, when an aqueous solvent is used, 6
It is possible to significantly reduce α epimers. Furthermore, when recrystallized from an organic solvent, there was a problem that the formed crystals aggregated or adhered to the wall surface, except for the ammonium salt, but recrystallization from an aqueous solvent caused both aggregation and adhesion to the wall surface. Not observed.

The pravastatin-containing solution to which the method for producing pravastatin sodium salt according to the present invention is applied is not particularly limited, but the enzymatic or microbial conversion reaction of compactin showing the typical processes shown in the following (1) to (4) It is particularly useful for the reaction solution containing pravastatin obtained by (1) The reaction solution (culture solution) obtained by the enzymatic or microbial conversion reaction of compactin is filtered according to a conventional method (such as Celite filtration) to remove insoluble matter. (2) The filtrate is subjected to an appropriate pretreatment, if desired, and then pravastatin is extracted under acidic conditions with ethyl acetate or butyl acetate. (3) After concentrating the obtained extract,
An amine, preferably a secondary amine, is added to this, and the precipitated crystals are separated by filtration and dried to give crystals. (Four)
After converting the pravastatin amine salt into the free form by the above-mentioned method, sodium alkoxide is allowed to act to obtain pravastatin sodium salt.

Each step will be described below. (1) Enzymatic or microbial conversion reaction solution of compactin The conversion reaction of compactin is performed, for example, by adding an enzyme such as cytochrome P-450 to compactin, a ring-opened body thereof or a salt thereof, or reacting the above-mentioned microorganism. It can be done by acting. For example, Streptomyces sp. TM-6 strain (FERM P-18311)
And Streptomyces sp
p.) TM-7 strain (FERM P-18312) (all strains have been deposited with the National Institute of Advanced Industrial Science and Technology on April 25, 2001).

(2) Pretreatment and Extraction Procedure In the above reaction (culture) solution, pravastatin is usually contained as a salt (particularly sodium salt) and extracted under acidic conditions with ethyl acetate or butyl acetate. It can be extracted with a solvent. Prior to the extraction, (a) the filtrate is washed with butyl acetate or the like under alkaline conditions, or (b) under acidic conditions pravastatin is extracted as a free form with ethyl acetate or the like, and an alkaline aqueous solution is added to this. The pravastatin salt may be collected in an aqueous layer, and the pravastatin salt may be subjected to a treatment with an ion-exchange resin for preliminary purification. Here, the ion exchange resin treatment is performed by neutralizing the aqueous layer containing pravastatin, immersing it in the ion exchange resin to adsorb pravastatin to the resin, and then eluting pravastatin with, for example, an acetone-containing aqueous solution.

(3) Concentration and separation procedure Next, the pravastatin extract is concentrated. Concentration is carried out to the extent that 1 mg or more of pravastatin is contained per 1 ml, depending on the concentration of impurities. An amine is added to the concentrated extract thus obtained to precipitate the amine salt of pravastatin. Examples of the amine that forms a salt with pravastatin include

Embedded image ^Second represented by R ¹ R ² NH (R ¹ and R ² are the same as above)
Primary amines are preferred. Add 1 equivalent or more, preferably 1 to 3 equivalents of amine to pravastatin, and add 1 at room temperature.
After stirring for several hours, the amine salt of pravastatin is precipitated.

When a primary amine (monoalkylamine) is used, the yield and purity are low and the salt produced is also water-soluble. Therefore, in order to obtain a high-purity product, many times from an organic solvent is used. Recrystallization is not preferable. In addition, tertiary amines cannot be used because pravastatin salts do not crystallize. Ammonia gas may be blown into the pravastatin extract to form an ammonium salt, but the salt obtained is water-soluble and must be crystallized from an organic solvent. Further, ammonia gas is not preferable in terms of work environment and safety because it has a bad odor and toxicity.

According to the above method, highly pure pravastatin (absolute purity 99 to 100%) can be obtained in a high yield simply by subjecting the culture solution containing pravastatin to simple pretreatment and crystallization. However, the amine salt may be further purified by recrystallization. The specific operation of recrystallization depends on the type of amine used.

The dicyclohexylamine salt of pravastatin is poorly soluble in an aqueous solvent and can be purified by recrystallization from an aqueous solvent such as an aqueous isopropanol solution (isopropanol concentration of about 1 to 30%). For example, as a recrystallization operation, a dicyclohexylamine salt is dissolved in an isopropanol aqueous solution while heating and then left to cool to precipitate crystals. After the precipitation, it is preferably cooled to about 0 to 10 ° C and stirred for about 15 minutes to 1 hour.

Water-soluble amine salts other than the dicyclohexylamine salt are purified by recrystallization from an organic solvent.
For example, the amine salt is dissolved in methanol, a non-polar solvent such as isopropyl acetate or ethyl acetate is added to precipitate crystals, and the mixture is stirred at room temperature for about 1 to 2 hours.

In the conversion reaction of compactin by the microorganism, pravastatin (6β form) and 6α form which is an inactive epimer in vivo are by-produced, but the 6α form can be removed by recrystallization treatment. Particularly, when recrystallized from an aqueous solvent, 6α-form can be largely removed.

(4) Conversion of amine salt to sodium The amine salt obtained as described above can be converted to a sodium salt by the above-mentioned method.

[0033]

The present invention will be described in more detail with reference to Examples, Reference Examples and Comparative Examples, but the present invention is not limited to the following description. In the following example,
The "content" is obtained by measuring the concentration of pravastatin in the analyte sample solution using the analytical value (calibration curve) of a standard product of known content.

In all the examples, the pravastatin purity was measured by HPLC under the following conditions. Device: Shimadzu Corporation CLASS-VP Column: YMC-Pack ODS-A307 (YMS Co., Ltd.) Column size: 75 x 4.6 mm (inner diameter) Mobile phase: 50% methanol (containing 0.1% each of triethylamine and acetic acid) ) Flow rate: Adjusted so that the elution time of pravastatin is 4.6 minutes (about 1.3 mL / min) Column temperature: 40 ° C Detection wavelength: 238 nm Analysis time: 15 minutes

Example 1: Preparation of pravastatin amine salt (dicyclohexylamine salt) 250 m containing 50 mL of TSB medium having the following composition (Table 1)
An L-volume Erlenmeyer flask was inoculated with 1 mL of a frozen seed matrix of Streptomyces sp. TM-7 strain (FERM P-18312), and it was inoculated at 28 ° C and 220 rpm for 24 hours.
After culturing for a time, seed culture was performed. Subsequently, 2% of seed seed was added to 50 mL of TSB medium containing compactin at a final concentration of 500 mg / L and FeSO ₄ .7H ₂ O at a final concentration of 100 mg / L, and the mixture was cultured at 28 ° C. and 220 rpm. 48
After 500 hours and 72 hours compactin at a final concentration of 500
mg / L was added and the culture was continued for 96 hours to obtain a converted culture medium.

[0036]

[Table 1] Table 1: Trypsin soybean (TSB) medium (manufactured by Difco) Casein digested product 1.7% Soybean powder papain digested product 0.3% Glucose 0.25% NaCl 0.5% K ₂ HPO ₄ 0. 25%

The compactin-pravastatin conversion reaction culture solution (3 L) was filtered through Celite, the pH was adjusted to 9.0 with a 6 mol / L sodium hydroxide solution, and then washed with butyl acetate (1.27 L). The aqueous layer was adjusted to pH 4.3 with 6 mol / L sulfuric acid and extracted with ethyl acetate (3.54 L). A part (1500 mL) of this extract was concentrated to 75 mL. To this, 1.3 equivalents of dicyclohexylamine was added, and the mixture was stirred at room temperature for 1.5 hours. The precipitated crystal was filtered and dried to obtain 2.32 g (content of plastabatin dicyclohexylamine salt: 1.66 g) of crude crystal.

Reference Example 1 200 mg of the crude crystal obtained in Example 1 (content: 147 mg) was suspended in 4 mL of butyl acetate, and 0.01 mol / M sulfuric acid was added in the order of 4 mL, 2 mL and 2 mL.
Even at the addition stage, the crude crystals remained in a suspended state and the conversion reaction to acid did not proceed. Similarly, crude crystal 20 obtained in Example 1
0 mg (content 147 mg) was suspended in 4 mL of butyl acetate, and 0.05 mol / M sulfuric acid was added to 4 mL, 2 mL, 2 mL.
However, the crude crystals remained in a suspended state and the conversion reaction to the acid did not proceed even when a total of 8 mL was added. As a result of repeating the same experiment, it was confirmed that when 4 mL of 0.1 mol / M sulfuric acid was added, the crude crystals were completely dissolved and the conversion reaction to the acid proceeded. Based on this result, the experiments of Examples 2 and 3 were performed.

Example 2 506 mg of the crude crystal obtained in Example 1 (content: 362 mg)
Was suspended in 9 mL of butyl acetate, 9 mL of 0.1 mol / L sulfuric acid was added thereto, and the mixture was stirred for 2 hours. Collect the organic layer and water 2.
After washing with mL, the mixture was dehydrated with sodium sulfate, filtered, and washed. 10.5 mL of this solution was cooled to 0 ° C., and 1.1 mL of methanol in which 44.5 mg of sodium methoxide was dissolved was added thereto over 2 hours. After returning to room temperature, butyl acetate 3
mL was added and the mixture was stirred for 1 hour. The precipitated crystals are filtered,
After drying, 332 mg (content 329 mg, yield 90.9%) of white crystalline powder was obtained. The amount of solvent used in the precipitation step is
14.6 mL, 40 times the volume of pravastatin (mL /
g).

Example 3 518 mg of crude crystals obtained in Example 1 (content: 371 mg)
Was suspended in 9 mL of ethyl acetate, 9 mL of 0.1 mol / L sulfuric acid was added thereto, and the mixture was stirred for 2 hours. Collect the organic layer and water 2.
After washing with mL, the mixture was dehydrated with sodium sulfate, filtered, and washed. 10.5 mL of this solution was cooled to 0 ° C., and 1.1 mL of methanol in which 45.5 mg of sodium methoxide was dissolved was added thereto over 2 hours. After returning to room temperature, ethyl acetate 3
mL was added and the mixture was stirred for 1 hour. The precipitated crystals are filtered,
After drying, 330 mg (content 326 mg, yield 87.9%) of white crystalline powder was obtained. The amount of solvent used in the precipitation step is
14.6 mL, 39 times the volume of pravastatin (mL /
g).

Example 4: Conversion to the sodium salt 582 mg of the dicyclohexylamine salt of pravastatin obtained in Example 1 (content: 417 mg) was added to 10 parts of ethyl acetate.
The mixture was suspended in mL, 10 mL of 0.1 mol / L sulfuric acid was added thereto, and the mixture was stirred for 2 hours. The organic layer was collected, washed with 2 mL of water, and then dehydrated with sodium sulfate. Add 11 mL of this solution to 0
After cooling to 0 ° C, 1.2 mL of methanol in which 51.2 mg of sodium methoxide was dissolved was added over 30 minutes. After returning to room temperature, 10 mL of ethyl acetate was added and the mixture was stirred for 1 hour. The precipitated crystals were filtered and dried to obtain 385 mg (content 376 mg, yield 90.2%) of white crystalline powder. The amount of the solvent used in the precipitation step was 22.2 mL, which was 53 times the amount (mL / g) of pravastatin.

Comparative Example 1 Dicyclohexylamine salt 10 obtained in the same manner as in Example 1
00 mg (content: 737 mg) was suspended in 20 mL of ethyl acetate, and 20 mL of 0.1 mol / L sulfuric acid was added thereto, followed by stirring for 30 minutes. The organic layer was collected, washed with 5 mL of water, and then dehydrated with sodium sulfate. To 20 mL of this solution methanol 4.1
mL and 268 μL of 6 mol / L sodium hydroxide solution
Was added and stirred for 30 minutes. 45 mL of ethyl acetate was added and the mixture was further stirred for 2 hours, and the precipitated crystals were filtered and dried to obtain 614 mg (content 593 mg, yield 80.5%) of white crystalline powder. The amount of solvent used in the precipitation step was 69.4.
94 times the amount of pravastatin in mL (mL / g)
Met.

Comparative Example 2 100 mg of the dicyclohexylamine salt obtained in Example 1
(Content of 72 mg) was suspended in 2 mL of butyl acetate and 2 mL of water, and the aqueous layer was adjusted to pH 4.3 with 1 mol / L sulfuric acid and stirred for 1 hour. To this, 2 mL of ethyl acetate was added, and after stirring for 1 hour, 2 mL of 0.2 mol / L sulfuric acid was added and further stirred for 1 hour. The organic layer was collected, washed with 5 mL of water, dehydrated with sodium sulfate, filtered, and washed. 0.3 mL of methanol and 24 μL of 6 mol / L sodium hydroxide solution were added to 7.3 mL of this solution, and the mixture was stirred for 1 hour. Butyl acetate 6
mL was added and the mixture was stirred for additional 16 hours, and the precipitated crystals were filtered and dried to 55 mg (content 55 mg, yield 76.4%).
A white crystalline powder of was obtained. The amount of solvent used in the precipitation step was 13.6 mL, which was 189 times the amount (m) of pravastatin.
L / g).

Comparative Example 3 200 mg of the crude crystal obtained in Example 1 (content 72 mg) was suspended in 4 mL of butyl acetate, and sodium methoxide 18 was added.
0.4 mL of methanol in which mg was dissolved was added. The mixture was stirred for 1 hour as it was, but the crude crystals remained in a suspended state, the conversion reaction to the sodium salt did not proceed, and almost the entire amount remained as a dicyclohexylamine salt.

Example 5 1.5 g of crude crystals (pravastatin octylamine salt) obtained in the same manner as in Example 1 except that octylamine was used instead of dicyclohexylamine was added to 45 m of methanol.
After dissolving in L, the mixture was stirred for 1 hour while adding 1000 mL of isopropyl acetate at room temperature. The precipitated crystals were filtered and dried to obtain 1.365 g of crystals as a white solid. The same operation was repeated again, and 1.2 g of octylamine salt crystals (content
0.96 g) was obtained. 310 mg of this octylamine salt
(Content 248 mg) is dissolved in 6 mL of water, and the aqueous layer is separated with 6 mo
After adjusting pH to 4.3 with 1 / L sulfuric acid, ethyl acetate 6m
L was added and stirred for 1 hour. The organic layer was collected, dehydrated with sodium sulfate, filtered, and washed. 6.9 mL of this solution was cooled to 0 ° C, to which 25.0 mg of sodium methoxide was added.
0.6 mL of dissolved methanol was added over 1 hour. After returning to room temperature, 2 mL of ethyl acetate was added and stirred for 1 hour. The precipitated crystals were filtered and dried, 213 mg
A white crystalline powder having a content of 208 mg and a yield of 83.9% was obtained. The amount of the solvent used in the precipitation step was 9.5 mL, which was 38 times the amount (mL / g) of pravastatin.

Comparative Example 4 310 mg of crystals of pravastatin octylamine salt (content 248 mg) obtained in the same manner as in Example 5 was dissolved in 6 mL of water, and the aqueous layer was adjusted to pH 4.3 with 6 mol / L sulfuric acid. 6 mL of ethyl acetate was added and stirred for 1 hour. The organic layer was collected, dehydrated with sodium sulfate, filtered, and washed. To 6.9 mL of this solution, 1 mL of methanol and 6 mol /
80 μL of L sodium hydroxide solution was added and stirred for 1 hour. 17 mL of ethyl acetate was added and the mixture was further stirred for 16 hours, but a small amount of precipitate could not be collected. The amount of the solvent used in the precipitation step was 25 mL, which was 101 times the amount (mL / g) of pravastatin.

Example 6 320 mg of crystals of pravastatin octylamine salt (content 256 mg) obtained in the same manner as in Example 5 were dissolved in 6 mL of water, and the aqueous layer was adjusted to pH 4.3 with 6 mol / L sulfuric acid, 6 mL of isobutyl acetate was added and stirred for 1 hour. The organic layer was collected, dehydrated with sodium sulfate, filtered, and washed. This solution (8.2 mL) was cooled to 0 ° C., and sodium methoxide (25.0 mg) dissolved in methanol (0.6 mL) was added to the solution.
mL was added over 1 hour. After returning to room temperature, 4 mL of isobutyl acetate was added and stirred for 16 hours. The precipitated crystals were filtered and dried to obtain 239 mg (content 232 mg, yield 90.6%) of white crystalline powder. The amount of solvent used in the precipitation step was 12.8 mL, which was 50% relative to pravastatin.
It was a double amount (mL / g).

Comparative Example 5 Pravastatin octylamine salt crystals 326 mg (content 261 mg) obtained in the same manner as in Comparative Example 4 were dissolved in 6 mL of water, the aqueous layer was adjusted to pH 4.3 with 6 mol / L sulfuric acid, and then acetic acid was added. 6 mL of isobutyl was added and stirred for 1 hour.
The organic layer was collected, dehydrated with sodium sulfate, filtered, and washed. To 8.2 mL of this solution, 1 mL of methanol and 6 mol /
82 μL of L sodium hydroxide solution was added and stirred for 1 hour. Isobutyl acetate (9 mL) was added and the mixture was stirred for additional 16 hours, and the precipitated crystals were filtered and dried to give 191 mg.
A white crystalline powder having a content of 190 mg and a yield of 72.8% was obtained. The amount of the solvent used in the precipitation step was 18.3 mL, which was 70 times the amount (mL / g) of pravastatin.

Example 7 323 mg of crystals of pravastatin octylamine salt (content: 259 mg) obtained in the same manner as in Example 5 were dissolved in 6 mL of water, and the aqueous layer was adjusted to pH 4.3 with 6 mol / L sulfuric acid. 6 mL of butyl acetate was added and stirred for 1 hour. The organic layer was collected, dehydrated with sodium sulfate, filtered, and washed. 8.5 mL of this solution was cooled to 0 ° C., and 0.6 mL of methanol in which 25.0 mg of sodium methoxide was dissolved
Was added over 1 hour. After returning to room temperature, 4 mL of butyl acetate was added and stirred for 16 hours. The precipitated crystals were filtered and dried to give 240 mg (content 236 mg, yield 91.1
%) White crystalline powder was obtained. The amount of the solvent used in the precipitation step was 13.1 mL, which was 51 times the amount (mL / g) of pravastatin.

Comparative Example 6 327 mg of pravastatin octylamine salt crystals (content 262 mg) obtained in the same manner as in Comparative Example 4 was dissolved in 6 mL of water, the aqueous layer was adjusted to pH 4.3 with 6 mol / L sulfuric acid, and then acetic acid was added. 6 mL of butyl was added and stirred for 1 hour. The organic layer was collected, dehydrated with sodium sulfate, filtered, and washed.
To 8.5 mL of this solution, 1 mL of methanol and 85 μL of a 6 mol / L sodium hydroxide solution were added, and the mixture was stirred for 1 hour. 9 mL of butyl acetate was added and the mixture was further stirred for 16 hours, and the precipitated crystals were filtered and dried to give 205 mg (content 2
(04 mg, 77.9% yield) was obtained as a white crystalline powder. The amount of the solvent used in the precipitation step was 18.6 mL, which was 71 times the amount (mL / g) of pravastatin.

As described above, according to the method of the present invention, in which an amine salt such as pravastatin dicyclohexylamine is crystallized as a sodium salt using sodium alkoxide and purified, the amount of the solvent used is kept to a minimum and pravastatin sodium salt is obtained. It can be obtained in high yield.

[0052]

EFFECTS OF THE INVENTION According to the method for producing a sodium salt from pravastatin amine salt of the present invention, the sodium salt can be obtained by minimizing the amount of the organic solvent used. Further, when producing parastabatine sodium salt from a fermentation broth containing plastavatin obtained by microbiological conversion of compactin, high purity pravastatin can be obtained by passing through a specific amine salt such as dicyclohexylamine salt. Since it can be obtained in a high yield, a high-purity sodium salt can be efficiently obtained by the method of converting into the above-mentioned sodium salt via an amine salt. further,
Economical water-based solvent with low environmental load in the amine salt formation process High-purity pravastatin can be obtained in high yield, and the amount of organic solvent used in the sodium salt formation process can be reduced. Can be greatly reduced. Further, in the amine salt forming step, recrystallization from an aqueous solvent is possible, so that the crystals do not aggregate or adhere to the wall surface, which is advantageous in terms of management such as maintenance of production facilities.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ identification code FI theme code (reference) (C12P 7/62 C12R 1: 465 C12R 1: 465) (72) Inventor Osamu Shirodo Kamei, Fujisawa City, Kanagawa Prefecture No. 3-18-12 (72) Inventor Kunio Isshiki 2-2-17 Minami Kurihara, Zama City, Kanagawa Prefecture F-term (reference) 4B064 AD62 CA04 CE07 CE17 DA01 4H006 AA02 AC46 BB14 BB17 BC10 BC19 BC31 BJ30 BN10 BN20 BS10 KC12

Claims (2)

[Claims] 1. A pravastatin amine salt is reacted with an acid in the presence of an organic solvent to give free pravastatin, and sodium alkoxide is added to the obtained pravastatin-containing organic solvent solution to crystallize pravastatin sodium salt to form crystals. A method for producing pravastatin sodium salt, which comprises separating. 2. The method for producing pravastatin sodium salt according to claim 1, wherein the sodium alkoxide is sodium methoxide.