JP3463875B2 - How to purify pravastatin - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a compound of formula CH ₃ CO ₂ R (), which is used as an organic solvent in a step of extracting pravastatin from a culture concentrate containing pravastatin produced by a bacterium using an organic solvent. In the above formula, R represents an alkyl group having 3 or more carbon atoms.), And relates to the step of isolating and purifying pravastatin or a pharmacologically acceptable salt thereof.

[0002] The present invention also relates to a method for isolating and purifying pravastatin or a pharmacologically acceptable salt thereof, which comprises the step of decomposing impurities with an inorganic acid.

Further, the present invention relates to a method for isolating and purifying pravastatin or a pharmacologically acceptable salt thereof, which comprises the step of decomposing impurities using an inorganic base.

The present invention also relates to a method for isolating and purifying pravastatin or a pharmacologically acceptable salt thereof, which comprises two or more steps selected from the above-mentioned three steps.

Further, the present invention provides the formula (I)

[0006]

[Chemical 4]

It relates to the invention relating to the removal of the compounds having the amount up to 0.1% by weight, based on industrially produced pravastatin sodium.

[0008]

BACKGROUND ART Pravastatin is disclosed in JP-A-57-224.
No. 0 (USP4346227), which is a compound disclosed as an HMG-CoA reductase inhibitor,
Formula (II) below

[0009]

[Chemical 5]

Currently, pravastatin sodium is on sale as a therapeutic agent for hyperlipidemia.

[0011]

Many compounds other than pravastatin are known as HMG-CoA reductase inhibitors. For example, atorvastatin, fluvastatin, itavastatin, etc. are all synthetically produced. It is a compound.

On the other hand, lovastatin and simvastatin are produced by fermentation similarly to pravastatin, but are produced by one-step fermentation, which is different from the two-step fermentation of pravastatin. That is, pravastatin sodium is produced by conversion culture with a bacterium in two steps as shown in the following formula.

[0013]

[Chemical 6]

In the first place, when a bacterium is fermented, many unexpected impurities are produced as compared with the case where the synthesis is carried out by a chemical reaction. Even if purification is performed in each step, impurities cannot be removed completely, and the products of each step in mass culturing particularly for industrial production are all the more important.

On the other hand, "one of the important factors for producing a safe and effective medicine is to obtain a highly pure product. Chemical substances are chemically synthesized from raw materials and produced by living organisms. It is obtained by a method such as isolating and purifying a substance, or isolating and purifying the substance produced by production using a genetically modified cell, etc. In general, any method including a gene recombination method can be used. Although adopted, it is often difficult to obtain a 100% pure chemical substance produced due to the purity of raw materials, incomplete reaction, decomposition in the isolation / purification process, etc. In the field of
It is clear that the therapeutic agent cannot be denied the possibility of adversely affecting the diagnosis and treatment when it contains impurities, and it is clear that it belongs to the common general technical knowledge in the technical field to which the present invention belongs. Obtaining the product is important. (Tokyo High Court, 1997 (Goke) No. 30
Case 2 (judgment on February 17, 2012).

The HMG-CoA reductase inhibitor is effective in lowering blood cholesterol.
Since the drug has a long administration period, it is required to have a particularly high purity.

Therefore, as described above, the pravastatin produced by the two-step fermentation contains more impurities than the simvastatin and lovastatin produced by the one-step fermentation, and thus the purification step is particularly important, Studies have continued to find ways to remove impurities and to isolate and purify highly pure pravastatin.

The inventors of the present invention, in particular, when the pravastatin is produced by two-stage fermentation, the following general formula (I)

[0019]

[Chemical 7]

It was found that the compound having the formula (1) is always produced, and the production amount of the above compound (I) is the largest among the impurities produced during the conversion culture into pravastatin. However, as described above, in order to obtain highly pure pravastatin, it was found that it is an important issue to reliably remove the compound (I) among the compounds that are regarded as impurities in the production of a drug. However, since the compound (I) is an optical isomer of a hydroxyl group on one asymmetric carbon of pravastatin, it was very difficult to remove it as compared with other impurities.

Therefore, pravastatin or a pharmacologically acceptable salt thereof is isolated and purified without being as complicated as chromatography, without impairing the process productivity and with such a high purity that it can be purified by chromatography. There has been a demand for a simple isolation / purification method that can remove the compound (I) to obtain highly pure pravastatin.

On the other hand, the following methods are known as methods for purifying the HMG-CoA reductase inhibitor. (1) WO92 / 16276 (Patent Document 6-506
No. 210) This publication describes “a method for purifying an HMG-CoA reductase inhibitor characterized by using high performance liquid chromatography, and HMG-C obtained by the purification method”.
oA reductase inhibitor ".

The purification method of the present publication is characterized by using high performance liquid chromatography in the purification step of the HMG-CoA reductase inhibitor. On the other hand, the isolation / purification method of the present invention has a formula CH ₃ CO ₂ R such as n-propyl acetate or n-butyl acetate (wherein R represents an alkyl group having 3 or more carbon atoms). The solvent is used to remove impurities of pravastatin and / or the impurities are removed by decomposing with an inorganic acid and / or an inorganic base to obtain high purity without using high performance liquid chromatography. Is characterized in that pravastatin can be obtained, which is different from the purification method of this publication.

Further, there is no description or suggestion of the isolation / purification method of the present invention "characterized in that the above compound (I) is removed up to 0.1% by weight or less based on pravastatin sodium". .

By the way, high performance liquid chromatography becomes more serious in the case of large-scale production because it requires a large amount of solvent and may contain a small amount of non-volatile impurities. Upon removing the solvent, these impurities are mixed in with the sample. In addition, the use of such a large amount of solvent causes serious environmental pollution and large distillation costs.

Therefore, high performance liquid chromatography is complicated and is not considered to be an operation used for industrial isolation and purification of pravastatin.

In the industrial production of pravastatin, it is difficult to obtain the desired purity of pravastatin by removing the compound (I), which is an optical isomer of pravastatin, even if high performance liquid chromatography is used. (2) WO99 / 42601 gazette This gazette says, "After adjusting the concentrated culture solution of HMG-CoA reductase inhibitor to pH 4.5 to 7.5 with acid, extract HMG-CoA reductase inhibitor with ethyl acetate. Then, it is subjected to lactonization if desired, and then crystallized to obtain an HMG-CoA reductase inhibitor having a purity of 99.6% or more ”.

In the purification method of this publication, ethyl acetate is used in the step of extracting pravastatins from a culture concentrate containing pravastatins produced by a bacterium using an organic solvent. The purification method of the present invention comprises the steps of formula CH ₃ such as n-purupyl acetate and n-butyl acetate.
This is different from the isolation / purification method of the present invention in that a solvent having CO ₂ R (in the above formula, R represents an alkyl group having 3 or more carbon atoms) is used.

Further, the isolation / purification method of the present invention is characterized in that highly pure pravastatin is obtained by performing a step of decomposing pravastatin impurities with an inorganic acid and / or an inorganic base. This publication does not describe or suggest the step of decomposing the impurities of the HMG-CoA reductase inhibitor using an inorganic acid and / or an inorganic base.

The isolation / purification method of the present invention "characterized in that the compound (I) is removed up to 0.1% by weight or less of pravastatin sodium" is described and suggested. Absent.

Although it is true that Example 3 of the present publication describes a method for isolating and purifying pravastatin, in this Example, the purity of pravastatin extracted with ethyl acetate was less than 70.3%. Although there is a description that the purification is performed using a chromatography different from the isolation / purification method of the present invention thereafter, the purity of pravastatin finally obtained is not described at all. Therefore,
Whether or not highly pure pravastatin is finally obtained is unknown in the first place, and in addition, it is described whether the above compound (I) is purified to 0.1 wt% or less with respect to pravastatin. Neither suggested nor suggested.

In the embodiment of this publication, 99.6 is used.
Even with lovastatin, which is said to be obtained with a high purity of more than%, it is unlikely that lovastatin with a purity of more than 99.6% was obtained, as seen from the HPLC chart of the final product (Fig.4). , Not credible. (3) WO00 / 17182 gazette This gazette "the purification method of the HMG-CoA reductase inhibitor characterized by using displacement chromatography, and the HMG-CoA reductase inhibitor obtained by this purification method". Regarding

The purification method of the present publication is characterized by using displacement chromatography in the purification step of the HMG-CoA reductase inhibitor. On the other hand, the isolation / purification method of the present invention has a formula CH ₃ CO ₂ R such as n-propyl acetate or n-butyl acetate (wherein R represents an alkyl group having 3 or more carbon atoms). Solvent is used to remove pravastatin impurities and / or inorganic acid and / or
Or, by removing impurities by decomposing using an inorganic base, without using any chromatography including substitution chromatography, high-purity pravastatin is characterized in that the purification method of the present disclosure and Is different.

The isolation / purification method of the present invention "characterized in that the compound (I) is removed to 0.1% by weight or less of pravastatin sodium" is described and suggested. Absent.

In the industrial production of pravastatin, even if substitution chromatography is used, it is complicated and it is difficult to remove the compound (I) which is an optical isomer of pravastatin.

In addition to the above-mentioned purification method, the isolation / purification method usually adopted by those skilled in the art includes, for example, analytical high pressure liquid chromatography (HPLC). Isolation of pravastatin
It is not a practical method as a purification method. Analytical HPLC
Is a very thin straight packed column (diameter 4.6 mm x length 2
5 cm) and about 1 is applied to the column at high pressure for each analysis.
A sample of 0 μg (1 × 10 ^-2 mg) is added, and one analysis requires about 30 minutes. Although the sample eventually flows out of the column, it is in the form of an extremely diluted solution, and although the components can be detected by the analytical system, collection is not practical.
Furthermore, analytical HPLC does not come with a collection system, and even if it is installed, removal of the solvent is required when it is desired to obtain a solid sample. Analytical H for isolation and purification of pravastatin
Using PLC, 500 analyzes and 2 was used to isolate and purify the pravastatin needed to make just one 5 mg tablet.
It takes 50 hours and it is clear that analytical HPLC is an unrealistic operation for the purpose of isolating and purifying pravastatin.

Further, preparative HPLC cannot be said to be a method suitable for commercial-scale isolation / purification of pravastatin for the above-mentioned reason.

Silica gel chromatography can be roughly classified into column chromatography and flash chromatography based on the size of silica gel particles, but was not expected to be a method suitable for commercial-scale isolation / purification. .

Actually, various chromatographic methods were used to separate the compound (I) and other impurities. However, since the compound (I) is a stereoisomer of pravastatin, the compound was separated from pravastatin. could not.

Recrystallization is an isolation / purification operation widely used in the pharmaceutical field. However, the use of recrystallization is also effective in selectively separating the above compound (I) structurally similar to pravastatin from pravastatin and improving the purity of pravastatin in the composition to the desired level. There wasn't.

Gas chromatography, distillation,
Other isolation and purification methods were also considered, but both are based on separation based on the difference in sample molecule size, and both require sample heating. However, since pravastatin tends to decompose at its melting point, these cannot be isolated / purified.

Further, even if any of the above-mentioned isolation / purification methods is repeated to remove the compound (I), the yield of pravastatin is also decreased.

Further, even if any conventionally known isolation / purification method as described above is combined, the above-mentioned compound (I) is removed to an amount of 0.1% or less to produce pravastatin for industrial production. Could not be applied to.

Therefore, without impairing the process productivity,
An isolation / purification method by which highly pure pravastatin, particularly pravastatin of high purity, can be obtained by removing a compound having the above-mentioned general formula (I), which is an analogue of pravastatin, has been completely known. Absent.

The present inventors have conducted diligent studies on a pharmaceutical composition containing pravastatin, and as a result, have isolated and purified a method for obtaining highly pure pravastatin, and further include the compound (I), which is a related substance of pravastatin. The isolation / purification method for removing the impurities from the pharmaceutical composition, particularly the isolation / purification method for removing the compound (I) to an amount of 0.1% or less based on the total amount of the pharmaceutical composition containing pravastatin. Heading, completed the present invention.

[0046]

MEANS FOR SOLVING THE PROBLEMS The present invention provides (1) a compound of formula CH 2 as an organic solvent in the step of extracting pravastatins from a culture concentrate containing pravastatins produced by a bacterium using an organic solvent. _A method for isolating / purifying pravastatin or a pharmacologically acceptable salt thereof, which comprises using a solvent having ₃ CO ₂ R (wherein R represents an alkyl group having 3 or more carbon atoms). (2)
In the step of isolating / purifying pravastatin or a pharmacologically acceptable salt thereof, a step of decomposing impurities by using an inorganic acid is performed, and isolation / purification of pravastatin or a pharmacologically acceptable salt thereof. Purification method, (3) pravastatin or a pharmacologically acceptable salt thereof, characterized in that in the step of isolating and purifying pravastatin or a pharmacologically acceptable salt thereof, a step of decomposing impurities by using an inorganic base is carried out. And a method for isolating and purifying a salt.

Preferably, (4) R is an n-propyl group or
(1) A method for isolating / purifying pravastatin or a pharmacologically acceptable salt thereof which is an n-butyl group, (5) An inorganic acid is phosphoric acid, (2) pravastatin or a pharmacological method thereof. An acceptable salt isolation / purification method, (6) the pH of the step of decomposing using an inorganic acid is 2 to 5,
Examples include the method for isolating and purifying pravastatin or a pharmacologically acceptable salt thereof described in (2) or (5).

Further, the present invention (7) uses a compound of the formula CH ₃ CO ₂ R () as an organic solvent in the step of extracting pravastatins from a culture concentrate containing pravastatins produced by the bacterium using an organic solvent. In the above formula, R represents an alkyl group having 3 or more carbon atoms) and a step of decomposing impurities with an inorganic acid is carried out, pravastatin or a pharmacologically acceptable salt thereof. (8) In the step of isolating and purifying pravastatin or a pharmacologically acceptable salt thereof, the step of decomposing impurities with an inorganic acid, and the step of using impurities with an inorganic base And a method for isolating and purifying pravastatin or a pharmacologically acceptable salt thereof, (9) A culture concentrate containing pravastatin produced by a bacterium. From the above, in the step of extracting pravastatins using an organic solvent, a solvent having the formula CH ₃ CO ₂ R (wherein R represents an alkyl group having 3 or more carbon atoms) is used as the organic solvent. , And
A method of isolating and purifying impurities such as pravastatin or a pharmacologically acceptable salt thereof, which comprises performing a step of decomposing impurities using an inorganic acid, and a step of decomposing impurities using an inorganic base, preferably (10) The method for isolating / purifying pravastatin or a pharmaceutically acceptable salt thereof according to (7) or (9), wherein R is an n-propyl group or an n-butyl group, (11) the inorganic acid is , Phosphoric acid, a method for isolating / purifying pravastatin or a pharmacologically acceptable salt thereof according to any one of (7) to (10), (12) decomposing using an inorganic acid The pH of the process
The method for isolating and purifying pravastatin or a pharmacologically acceptable salt thereof according to any one of (7) to (11), which is 2 to 5, can be mentioned. The present invention also provides a compound of formula CH ₃ CO ₂ R (the above formula) as an organic solvent in the step of extracting pravastatins from a culture concentrate containing pravastatins produced by (13) bacteria using an organic solvent. Wherein R represents an alkyl group having 3 or more carbon atoms) and a step of decomposing impurities with an inorganic base is carried out, and pravastatin or a pharmacologically acceptable thereof. Regarding a method for isolating / purifying a salt, (14) a method for isolating / purifying pravastatin or a pharmaceutically acceptable salt thereof according to (13), wherein R is an n-propyl group or an n-butyl group. Can be mentioned.

Further, according to the present invention, in the step of extracting pravastatins from a culture concentrate containing pravastatins produced by (15) fungus using an organic solvent, the organic solvent of the formula CH ₃ CO ₂ R (In the above formula, R
Represents an alkyl group having 3 or more carbon atoms. ) Is used and the step of decomposing the impurities with an inorganic acid is carried out to give a compound of general formula (I)

[0050]

[Chemical 8]

A method for isolating and purifying pravastatin sodium, characterized in that the compound having the above is removed up to an amount of 0.1% by weight or less based on pravastatin sodium, (16) Isolating and purifying pravastatin sodium The step of decomposing the impurities with an inorganic acid and the step of decomposing the impurities with an inorganic base in the step of

[0052]

[Chemical 9]

A method for isolating and purifying pravastatin sodium, characterized in that the compound having is removed to an amount of 0.1% by weight or less with respect to pravastatin sodium, (17) pravastatins produced by a bacterium In the step of extracting pravastatin using an organic solvent from the culture concentrate containing
A solvent having CH ₃ CO ₂ R (in the above formula, R represents an alkyl group having 3 or more carbon atoms) is used, and a step of decomposing impurities with an inorganic acid, and an impurity of inorganic base By carrying out the step of decomposing by using the general formula (I)

[0054]

[Chemical 10]

A method for isolating and purifying pravastatin sodium, characterized in that the compound having the formula (1) is removed to an amount of 0.1% by weight or less with respect to pravastatin sodium. The method for isolating / purifying pravastatin sodium according to (15) or (17), which is an n-propyl group or an n-butyl group, (19) From (15) to (18), wherein the inorganic acid is phosphoric acid (20) The method for isolating / purifying pravastatin sodium according to any one of (1) to (15) to (19), wherein the pH of the step of decomposing with pravastatin sodium is 2 to 5
The method for isolating / purifying pravastatin sodium according to any one of the above can be mentioned.

The present invention further provides (21) in the step of extracting pravastatins from a culture concentrate containing pravastatins produced by a bacterium using an organic solvent,
Using a solvent having the formula CH ₃ CO ₂ R (wherein R represents an alkyl group having 3 or more carbon atoms) as the organic solvent, and performing a step of decomposing impurities with an inorganic base. By the general formula (I)

[0057]

[Chemical 11]

A method for isolating and purifying pravastatin sodium, characterized in that the compound having the formula (1) is removed to an amount of 0.1% by weight or less with respect to pravastatin sodium, preferably (22) (I)

[0059]

[Chemical 12]

An industrially produced composition containing pravastatin sodium, characterized in that the compound having the formula (1) is contained in an amount of 0.1% by weight or less based on pravastatin sodium, (23) R The method for isolating / purifying pravastatin sodium described in (21), which is an n-propyl group or an n-butyl group, can be mentioned.

The present invention also provides (24) a compound of the general formula (I), which is obtained by the isolation / purification method according to any one of (1) to (23).

[0062]

[Chemical 13]

The present invention relates to a composition containing pravastatin sodium, which is characterized in that the compound having the formula (1) is contained in an amount of 0.1% by weight or less based on pravastatin sodium.

In the present invention, "pravastatins" means the following formula (II)

[0065]

[Chemical 14]

Pravastatin or a pharmacologically acceptable salt thereof and a compound having a structure similar to that of the above formula (II), that is, an analogue of pravastatin (for example, the above compound (I) can be mentioned). Say.

Isolation / purification of pravastatin or a pharmaceutically acceptable salt thereof is usually accomplished by the isolation / purification method described below.

First, the culture medium containing pravastatin, which has been subjected to the conversion culture, is separated into bacterial cells by a conventional method by filtration and / or centrifugation, and then concentrated to obtain a culture concentrated solution.

The culture concentrate obtained is, if desired, adjusted in pH with an acid, and then pravastatin is extracted with an organic solvent such as ethyl acetate, which is immiscible with water.
For example, in WO99 / 42601, HMG-C
The culture concentrate containing the oA reductase inhibitor is adjusted to pH 4.5 to 7.5 (preferably 5.5 to 7.5) with an acid and then extracted with ethyl acetate. .

The pravastatin thus obtained is collected from the above extract according to a conventional method. For example,
After washing the above extract with water, saturated saline, etc., sodium hydroxide is added, and the solution is extracted and separated to obtain an aqueous solution of pravastatin salt as a back-extracted aqueous layer. The pravastatin salt obtained can also be crystallized if desired.

Crystallization, if desired, is generally carried out by methods well known in the art of synthetic organic chemistry, eg Ullman.
n's, Encyclopedia of Industrial Chemistry "Vol. A2
It can be carried out as follows by the method described in 4, 5th edition (1993), pp. 437-505.

As a crystallization method, for example, pravastatin as a crystalline substance is obtained by adding an organic solvent and water to the obtained composition of pravastatin or a pharmacologically acceptable salt thereof, heating and dissolving the mixture, and inoculating the composition. Can be obtained.

Examples of the organic solvent used for crystallization include aliphatic hydrocarbons such as hexane and heptane;
Aromatic hydrocarbons such as toluene and xylene; Esters such as methyl acetate, ethyl acetate, n-propyl acetate and n-butyl acetate; Organic acids such as acetic acid; methanol;
Ethanol, n-propanol, isopropanol, n
-Alcohols such as butanol, isobutanol, t-butanol, isoamyl alcohol, diethylene glycol, glycerin, octanol; ketones such as acetone and methyl ethyl ketone; diethyl ether, diisopropyl ether, tetrahydrofuran, Ethers such as dioxane, dimethoxyethane, diethylene glycol dimethyl ether; amides such as formamide, dimethylformamide, dimethylacetamide, hexamethylphosphoric triamide; sulfoxides such as dimethyl sulfoxide; water and one or more of the above organics A mixed solvent with a solvent, preferably a mixed solvent of water and one or more kinds of the above organic solvents, and more preferably selected from water and alcohols, esters and ketones. More than one type A mixed solvent of an organic solvent,
Most preferably, it is a mixed solvent of water, alcohols and esters.

In the isolation / purification method of the present invention, after the culture concentrate is obtained according to the conventional method as described above, not the ethyl acetate but the formula CH ₃ CO ₂ R (in the above formula, R has 3 carbon atoms). Extraction is performed using an organic solvent having the above alkyl group).

In the above formula, the “alkyl group having 3 or more carbon atoms” in the definition of R is, for example, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl,
Pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl,
4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1, 3-dimethylbutyl, 2,3-dimethylbutyl, 1-ethylbutyl, 2-
A group such as an ethylbutyl group, preferably having 3 carbon atoms
To 6 straight or branched chain alkyl groups, preferably C ₃ -C ₄ alkyl groups, most preferably n-propyl or n-butyl groups.

The isolation / purification method of the present invention comprises the step of decomposing pravastatin impurities with an inorganic acid in the above-mentioned method of isolating / purifying pravastatin, and / or impurities It is characterized in that a step of decomposing it is performed.

When the step of decomposing the impurities of pravastatin with an inorganic acid and the step of decomposing the impurities with an inorganic base are carried out, the desired steps in any order can be appropriately carried out in the isolation / purification method. . Specific examples of such an isolation / purification method include, for example, (a) in the step of extracting pravastatins from a culture concentrate containing pravastatins produced by a bacterium using an organic solvent, A solvent having the formula CH ₃ CO ₂ R (wherein R represents an alkyl group having 3 or more carbon atoms) is used, and a step of decomposing impurities with an inorganic acid is performed. A method for isolating / purifying pravastatin or a pharmacologically acceptable salt thereof, (b) in the step of extracting pravastatin using an organic solvent from a culture concentrate containing pravastatin produced by a bacterium, A step of using a solvent having the formula CH ₃ CO ₂ R (wherein R represents an alkyl group having 3 or more carbon atoms) as a solvent, and decomposing impurities with an inorganic base. A method of isolating / purifying pravastatin or a pharmacologically acceptable salt thereof, characterized in that (c) using an inorganic acid as an impurity in the step of isolating / purifying pravastatin or a pharmacologically acceptable salt thereof. (D) a method of isolating and purifying pravastatin or a pharmacologically acceptable salt thereof, characterized in that the step of decomposing by means of and the step of decomposing impurities by using an inorganic base are carried out in no particular order. From the culture concentrate containing pravastatin
In the step of extracting pravastatins using an organic solvent, a solvent having the formula CH ₃ CO ₂ R (wherein R represents an alkyl group having 3 or more carbon atoms) is used as the organic solvent, and And a method for isolating and purifying pravastatin or a pharmacologically acceptable salt thereof, which comprises performing a step of decomposing impurities with an inorganic acid and a step of decomposing impurities with an inorganic base. You can

The step of decomposing the impurities of pravastatin or a pharmaceutically acceptable salt thereof with an inorganic acid is carried out in the presence or absence (preferably, in the presence) of an inert solvent, pravastatin or a pharmaceutically acceptable salt thereof. It is carried out under the condition that the salt is added to pH 2 to 5 (preferably pH 3 to 4).

The "inorganic acid" used in the decomposition of impurities with inorganic acids is not particularly limited as long as it is an inorganic substance usually used as an acid, and examples thereof include hydrobromic acid, hydrochloric acid, sulfuric acid,
Mention may be made of inorganic acids such as perchloric acid, phosphoric acid, nitric acid, preferably phosphoric acid or sulfuric acid.

The reaction temperature and the reaction time depend on the pH value, but basically, the reaction time needs to be long when the reaction temperature is low, and the reaction time needs to be short when the reaction temperature is high. However, for example, the treatment is performed at 20 ° C. to 80 ° C. (preferably 40 ° C. to 60 ° C.) for 1 minute to 6 hours (preferably 5 minutes to 20 minutes).

The "inert solvent" used in the inorganic acid decomposition of impurities is not particularly limited as long as it is usually used as a solvent, and examples thereof include methanol, ethanol and n.
-Propanol, isopropanol, n-butanol,
Isobutanol, t-butanol, isoamyl alcohol
Alcohol, diethylene glycol, glycerin, octanol, cyclohexanol, methyl cellosolve; water; a mixed solvent of water and the above organic solvent; preferably water or a mixture of water and the above organic solvent. A solvent, most preferably water or a mixed solvent of water and ethanol.

After completion of the reaction, pravastatin, which is the target compound, is collected from the reaction solution according to a conventional method. For example,
A water-immiscible organic solvent such as ethyl acetate is added, the organic layer containing the target compound is separated, washed with water or the like, and the solvent is distilled off. If necessary, add activated charcoal to decolorize, filter to remove activated charcoal, then add reagents that form salts such as sodium hydroxide, sodium methoxide, and sodium ethoxide, and concentrate under reduced pressure using a rotary evaporator. Thus, the salt of pravastatin can be obtained.

The step of decomposing the impurities of pravastatin or a pharmacologically acceptable salt thereof with an inorganic base can be carried out in the presence or absence (preferably, in the presence) of an inert solvent for pravastatin or a pharmacologically acceptable salt thereof. PH of 10 to 14
Is applied (preferably pH 11 to 13).

The "inorganic base" used in the decomposition of impurities with an inorganic base is not particularly limited as long as it is an inorganic substance used as a base in a normal reaction, and examples thereof include lithium carbonate, sodium carbonate and potassium carbonate. Alkali metal carbonates such as; alkali metal bicarbonates such as lithium hydrogen carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate; alkali metal hydrides such as lithium hydride, sodium hydride, potassium hydride; lithium hydroxide ,
Alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkali metal alkoxides such as lithium methoxide, sodium methoxide, sodium ethoxide and potassium t-butoxide can be mentioned, and preferably, Alkali metal hydroxides, most preferably sodium hydroxide.

The reaction temperature and the reaction time depend on the pH value, but basically, the reaction time needs to be long when the reaction temperature is low, and the reaction time needs to be short when the reaction temperature is high. Is, for example, at -10 ° C to 110 ° C (preferably 0 ° C to 100 ° C) for 15 minutes to 60 hours (preferably 30 minutes to 72 hours), and more preferable. The temperature is around 0 ° C. for about 30 hours.

The "inert solvent" used for the decomposition of impurities with an inorganic base is not particularly limited as long as it is inert to the reaction, but is similar to the inert solvent used for the decomposition of impurities with an inorganic acid. I can list things.

After completion of the reaction, pravastatin which is the target compound is collected from the above reaction solution according to a conventional method. For example, an acid such as an aqueous solution of sulfuric acid is added, an immiscible organic solvent such as ethyl acetate is added, the organic layer containing the target compound is separated, washed with water or the like, and the solvent is obtained by distillation. To be If necessary, add activated carbon to decolorize, remove the activated carbon by filtration, add reagents that form salts such as sodium methoxide, sodium ethoxide, and sodium hydroxide, and concentrate under reduced pressure with a rotary evaporator. Thus, the salt of pravastatin can be obtained.

Further, the isolation / purification method of the present invention may include a step of crystallizing the obtained pravastatin salt, and the crystallizing method can be carried out by a conventional method as described above.

The analysis of the degree of purification of the present invention can be carried out by high performance liquid chromatography (HPLC). The HPLC measurement conditions are: A: mobile phase: 20% acetonitrile, 30% methanol, 50% T
EAP buffer _{(0.3% Triethylamine-H 3 PO} 4 (pH3.2)); Detection wavelength: UV238nm; Column: Waters Co. reverse phase column Symmetry C18 3.5μm, φ
4.6mm × 15cm; Flow rate: 1ml / min B: Mobile phase: Methanol: Water: Acetic acid (100): Triethylamine mixed solution (600: 400: 1: 1); Detection wavelength: UV238nm; Column: Elma Optical column ERC-ODS -1262 φ6mm x 1
0 cm; Column temperature: 30 ° C .; Flow rate: 1 ml / min C: Mobile phase: Methanol: Water: Acetic acid (100): Triethylamine mixed solution (450: 550: 1: 1); Detection wavelength: UV238 nm; Column: BECKMAN column ULTRASPHE
RE ODS φ 4.6 mm × 15 cm 5 μm; Column temperature: 25 ° C .; Flow rate: 1.3 ml / min.

The present invention will be described in more detail below with reference to Examples, but the scope of the present invention is not limited thereto.

[0091]

Example 1 Example 1 Using n-butyl acetate from the culture concentrate
10 L of the pravastatin culture solution which had been subjected to the concentration conversion culture of the extracted (1a) culture solution was adjusted to pH 12 with sodium hydroxide, then heated to 50 ° C. and stirred for 30 minutes. After cooling the culture solution to room temperature, 500 g of Celite 545 (trademark) (manufactured by Celite Corporation) as a filter aid was added and filtered. Add 3 L of water to the separated cells,
It was suspended again and then filtered. The obtained two concentrates were combined to obtain 10 L of culture concentrate. (1b) Extraction with n-butyl acetate The obtained culture concentrate was adjusted to pH 5.7 with 25% sulfuric acid, and then 5 L of n-butyl acetate was added and stirred to extract pravastatin. The separated aqueous layer was adjusted to pH 5.7 with 75% sulfuric acid, and then 5 L of acetic acid was added.
n-Butyl was added, and the mixture was stirred and extracted. 2 acetic acid obtained
The n-butyl layer was combined, 2 L of saturated saline was added thereto, and the mixture was stirred and washed. 1 to 8 L of extract obtained by separating the upper layer
After adding L of water and adding 25% sodium hydroxide with stirring to adjust the pH to 9.5, the aqueous layer was separated to obtain 1 L of an aqueous solution of pravastatin sodium salt. The purity of pravastatin sodium by HPLC (condition A) was 90% or more. From the results of this example, it is clear that highly pure pravastatin sodium was obtained by using n-butyl acetate.

Example 2 n-propyl acetate from the culture concentrate
Using n-propyl acetate instead of n-butyl acetate, the same treatment as in Example 1 was carried out to obtain an aqueous sodium salt solution of pravastatin. The purity of pravastatin sodium by HPLC (condition A) was 85% or more. From the results of this example, it is clear that highly pure pravastatin sodium was obtained by using n-propyl acetate.

Example 3 Decomposition of Impurities with Phosphoric Acid The aqueous solution obtained in Example 1 [compound (I) / pravastatin sodium 9.3% by HPLC (condition A)]
After adding 50 ml of ethanol and adjusting to pH 3.0 with phosphoric acid
The mixture was stirred at 50 ° C for 10 minutes. The compound (I) / pravastatin sodium content by HPLC (condition A) was 0.9%. From the results of this example, by using phosphoric acid,
It is clear that the compound (I) is significantly removed.

Example 4 Decomposition of Impurities with Sulfuric Acid The same treatment as in Example 3 was carried out using sulfuric acid instead of phosphoric acid. Compound (I) / pravastatin sodium was 3% by HPLC (condition A). From the results of this example, it is clear that the compound (I) was remarkably removed by using sulfuric acid.

Example 5 Impurities due to sodium hydroxide
Decomposition, extraction and crystallization of (5a) Decomposition of impurities with sodium hydroxide 500 ml of culture concentrate of pravastatin [Compound (I) / pravastatin sodium by HPLC (condition B)]
12.1%] was heated to 100 ° C. and then 2 equivalents of sodium hydroxide were added as an aqueous solution (pH 11.3). 3 at 100 ° C
After stirring for an hour, the mixture was cooled and adjusted to pH 8.5 with a 20% aqueous sulfuric acid solution at room temperature to obtain an alkali-treated solution with sodium hydroxide
(Pravastatin sodium content: 56.6 g). HPL
Compound (I) / pravastatin sodium according to C (condition B) was 0.41%. From the results of this example, it is clear that the compound (I) is remarkably removed by using sodium hydroxide. (5b) Extraction after decomposition of impurities with sodium hydroxide and crystallization Using n-butyl acetate in 260 g of alkaline treatment liquid with sodium hydroxide obtained in (5a) (content of pravastatin sodium: 19 g) The aqueous layer was separated in the same manner as in Example (1b) to obtain an aqueous sodium salt solution of pravastatin.

The obtained aqueous solution of pravastatin sodium salt was concentrated under reduced pressure to about 1/2 volume, 77 ml of water was added, and the mixture was concentrated under reduced pressure again. Adjust the volume of the concentrated solution with water so that it will be 6 times the amount of pravastatin free form, cool to 0-5 ° C, add 20% sulfuric acid aqueous solution dropwise, and obtain pravastatin free form crystals at pH 4.6. It was precipitated, stirred overnight and then filtered. The crystals were washed with cold dilute sulfuric acid (pH 4) to obtain pravastatin free body wet crystals.

63 ml of ethyl acetate was added to pravastatin free body hygroscopic product, dissolved at room temperature, and then stirred at room temperature for 20 minutes.
The pH was adjusted to 4.5 with a% sulfuric acid aqueous solution, and after stirring and separation, 21 ml of water was added to the ethyl acetate layer for extraction, washing and separation. After 25 ml of ethanol was added to the extracted and washed ethyl acetate layer, the pH was adjusted to 8 with a 6% sodium hydroxide-ethanol solution while stirring at room temperature.
It was adjusted to 7 to obtain a sodium chloride solution. After that, it was crystallized by a conventional method, and crystals of pravastatin sodium salt 8.95.
got g. The purity of pravastatin sodium salt obtained by this method according to HPLC (condition C) was 99.67%, and compound (I) / pravastatin sodium = 0.1%.

From the results of this example, it is clear that the compound (I) was remarkably removed by the step of decomposing impurities with sodium hydroxide and the step of extracting with n-butyl acetate.

Example 6 Extraction with n-butyl acetate
Decomposition of Impurities by Acid and Crystallization The acid-treated solution obtained in Example 3 was adjusted to pH 12 with 25% sodium hydroxide and further stirred at 50 ° C. for 30 minutes. Concentrate the solution under reduced pressure on a rotary evaporator to 1 L,
A concentrated solution containing pravastatin sodium salt was obtained.
The obtained concentrated solution was adjusted to pH 4.0 with sulfuric acid, and pravastatin was extracted with 0.5 L of ethyl acetate.
Washed with. After adding 5 g of activated carbon to the extract and letting it stand for 10 minutes, it was filtered with filter paper and the filtrate was adjusted to pH with 25% sodium hydroxide.
After adjusting to 8.7, 50 g of pravastatin sodium salt was obtained by drying on a rotary evaporator under reduced pressure.
Crystallization was performed according to a conventional method to obtain 34 g of crude crystals of pravastatin sodium salt. The crude crystals were recrystallized using a solvent of the same composition to obtain pure crystals of pravastatin sodium salt 3
I got 2 g.

The pravastatin sodium salt obtained by this method had a purity by HPLC (condition A) of 99.7% or higher, and the compound (I) / pravastatin sodium was 0.1%.

From this example, it is apparent that the compound (I) was removed to 0.1% or less by the steps of extraction with n-butyl acetate and decomposition of impurities with phosphoric acid to obtain highly pure pravastatin sodium. .

Further, by adding the step of decomposing impurities with sodium hydroxide to the step of decomposing impurities with n-butyl acetate and the step of decomposing impurities with phosphoric acid, compound (I) was further removed, and highly pure pravastatin sodium was obtained. It is clear that it was done.

Example 7 Impurities due to sodium hydroxide
Of the alkali-treated solution with sodium hydroxide obtained in Example (5a), using ethyl acetate to give Example (5b).
Extraction was carried out in the same manner as in 1. to obtain an aqueous solution of pravastatin sodium salt. To the resulting aqueous solution was added 350 ml of ethanol, the pH was adjusted to 1.5 with sulfuric acid, and the mixture was stirred at 20 ° C for 1 hr. Crystallization according to a conventional method gave pure crystals of pravastatin sodium salt. The compound (I) / pravastatin sodium was 0.1% or less by HPLC (condition C).

According to this example, the steps of decomposing impurities with sulfuric acid and impurities with sodium hydroxide
The compound (I) was removed to 0.1% or less to obtain highly pure pravastatin sodium.

Comparative Example 1 Extraction of Pravastatin with Ethyl Acetate Using ethyl acetate instead of n-butyl acetate and treating in the same manner as in Example (1a), an aqueous sodium salt solution of pravastatin was obtained. The purity of pravastatin sodium by HPLC (condition A) was about 70%.

Compound (I) / pravastatin sodium (%) when the organic solvent for extracting pravastatin from the culture concentrate is ethyl acetate, n-propyl acetate and n-butyl acetate is as follows: Is. From the results below, it is clear that highly pure pravastatin was obtained by extracting with n-propyl acetate and n-butyl acetate rather than with ethyl acetate.

[0107]

[Table 1] Example (1a) Example 2 Comparative Example 1 Extraction solvent n-butyl acetate n-propyl acetate ethyl acetate Puravastatin purity (%)>90> 85 About 70

[0108]

INDUSTRIAL APPLICABILITY The purification method of the present invention does not use a complicated and low productivity method such as column chromatography, which is not suitable for industrial production, and therefore impairs the process productivity and the purity of pravastatin. , High-purity pravastatin or a pharmacologically acceptable salt thereof could be obtained.

By the isolation / purification method of the present invention, the above compound (I) was removed to an amount of 0.1% or less with respect to pravastatin.

─────────────────────────────────────────────────── ─── Continuation of front page (72) Kiyoshi Hamano Kiyoshi Hamano 1-2-58 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. (72) Inventor Takayuki Yasuyuki 389-4 Sankyo, Izumi-cho, Iwaki, Fukushima Prefecture Incorporated (72) Inventor Minoru Hagisawa 1-12-1, Shinomiya, Hiratsuka, Kanagawa Sankyo Co., Ltd. (56) References International Publication 00/017182 (WO, A1) International Publication 00/046175 (WO, A1 ) International Publication 99/042601 (WO, A1) (58) Fields investigated (Int.Cl. ⁷ , DB name) C07C 67/58 C07C 69/732 CA (STN) REGISTRY (STN)

Claims (9)

(57) [Claims] 1. A method comprising pravastatins produced by a bacterium.
Pravastatin from the culture concentrate using an organic solvent
In the step of extracting the compounds, an organic solvent of the formula CH ₃ CO ₂ R (wherein R is an alkyl group having 3 or 4 carbon atoms) is used.
Indicates a rukyl group. ) Is used, and
Process of decomposing pure substances with inorganic acid, impurities as inorganic base
Combines the process of decomposing by using and the process of performing crystallization
The compound of general formula (I) : To the compound pravastatin sodium
A content of 0.1% by weight or less,
Contains commercially produced pravastatin sodium
Composition. 2. The inorganic base is an alkali metal hydroxide
Containing pravastatin sodium according to claim 1.
Composition. 3. Reaction in the process of decomposing with an inorganic base
The responsive temperature is 50 ° C., according to claim 1 or claim 2.
A composition containing pravastatin sodium. 4. Reaction in the process of decomposing with an inorganic base
The temperature according to claim 1 or claim 2, wherein the reaction temperature is 100 ° C.
A composition containing the listed pravastatin sodium. 5. R is an n-propyl group or an n-butyl group,
The program according to any one of claims 1 to 4.
A composition containing lavastatin sodium. 6. The inorganic acid is phosphoric acid or sulfuric acid,
The pravas according to any one of 1 to 5
A composition containing tatin sodium. 7. The pH of the step of decomposing using an inorganic acid is 2
Any one selected from Claim 1 thru | or 6 which is
A composition containing pravastatin sodium according to the item 1.
object. 8. Reaction in the process of decomposing using inorganic acid
The temperature is 20 ° C to 80 ° C, and the temperature is selected from 1 to 7.
Pravastatin Natliu according to any one of the following:
A composition containing a mu. 9. Reaction temperature of decomposition process using inorganic acid
Is 40 ° C. to 60 ° C., selected from claims 1 to 7.
Including pravastatin sodium according to any one of
A composition having.