JPS606699A - Purification of bile acid - Google Patents

Abstract

PURPOSE:To purify bile acid useful as a solubilizer for gallstone industrially advantageously, by adding an aqueous solution of an alkalizing agent to a solution of unpurified bile acid in an organic solvent, stirring them, collecting an organic solvent layer, adding an alkali aqueous solution to the organic solvent layer, stirring them, bringing an acid into contact with an aqueous solution layer, precipitating it. CONSTITUTION:Unpurified bile acid dissolved or suspended in an organic solvent (e.g., 2-ethylhexanol, etc.) capable of separating a layer from water, the solution is blended with an aqueous solution of an alkalizing agent (e.g., tetramethylammonium hydroxide, etc.) or a mixed solvent of the organic solvent capable of separating a layer from water and an alkali aqueous solution, they are stirred, and an organic solvent layer is collected. The organic solution is blended with an alkali aqueous solution, stirred, an aqueous solution layer is collected, the collected solution is brought into contact with an acid, and precipitated, to give purified bile acid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides purified bile acids, specifically chenodeoxycholic acid, ursodeoxycholic acid or
The present invention relates to a sheath manufacturing method for obtaining α-hydroxy-7-kedocholanic acid.

Chenodeoxycholic acid and ursodeoxycholic acid are useful as pharmaceuticals such as gallstone dissolving agents and choleretic agents.
α-Hydroxy-7-kedocholanic acid is useful as an intermediate in their production.

Chenodeoxycholic acid, ursodeoxycholic acid, and 3α-hydroxy-7-ketocholanic acid are produced using, for example, cholic acid (3α, 7α, 12α-trihydroxy-5β-cholanic acid) as a starting material, and in this case, the production process of each compound is By-products derived from 2 such as lithocholic acid (
3α-hydroxy-5β-furanic acid), 3α, 7α-
Dihydroxy-12-ketocholanic acid and unreacted cholic acid remain, and it has been considered very difficult to separate and purify them.

Conventionally known purification methods for chenodeoxycholic acid or ursodeoxycholic acid can be roughly divided into: (1) Direct recrystallization method [Journal of Biochemistry (Japan), Off volume, p. 501 (1927), Chemistry Experimental Science, Vol. 1o, p. 495 (1943) and Proceedings of Japan Academy-2, Vol. 30,
(Page 391 (1954)), (2) Method of alkyl esterification and recrystallization or column chromatography (Japanese Patent Publication No. 53-10063 and No. 53-10063)
3-35946) and (3) a method of separating it as an alkali salt (JP-A-49-959'55, JP-A-49-959'55,
0-126654 and 51-110553). However, method (1) did not yield a product of high purity, and method (2) required complicated operations and had a low yield. In addition, in (3) law, Japanese Patent Application Laid-Open No. 49-95955
No. 4 and No. 51-110553 disclose that chenodeoxycholic acid is separated as a calcium salt or strontium salt in methanol, dissolved in water, acidified with acetic acid or propionic acid, extracted with ethyl acetate, and the extract is extracted with water. A method is described in which chenodeoxycholic acid having a melting point of 163 to 5C is obtained by washing, followed by precipitation by adding petroleum ether. However, this method takes time to form the salt (-leaving overnight), and it takes a long time to form the salt, separate it, dissolve it, and extract it to obtain the desired product.

It has disadvantages such as complicated operations such as washing and drying at high temperature for a long time, and there is no description of the purity of the crude chenodeoxycholic acid used as a raw material, so it is unclear how effective it is. JP-A-50-126654 describes a method in which an alkali metal salt of chenodeoxycholic acid is subjected to perforation (continuous liquid/liquid extraction) in an organic solvent, acidified with an acid, and then chenodeoxycholic acid is precipitated with water. has been done. However, this method requires 12 to 24 hours for perforation, and there is no description of the degree of perforation, so it is unclear to what extent the product was purified.

The present inventors have previously filed an application for an industrial purification method for crude bile acids, which is a two-phase extraction method using an organic solvent that is poorly soluble in water and an alkaline aqueous solution, which is easy to operate and can be purified in a short time. (Grant No. 56-209605), further research was conducted to increase the selectivity of impurity removal in the two-phase extraction method,
The present invention has been completed to improve the yield of crude bile acids (chenodeoxycholic acid, ursodeoxycholic acid or 3α-hydroxy-7-ketocholanic acid) with a purity of 60 to 96% using an organic compound that can be separated into layers from water. Solvent 9 Single or mixed solvents such as alcohols having 4 or more carbon atoms, esters such as ethyl acetate, ketones such as methyl ethyl ketone, ethers such as diethyl nitrate, or halogenated hydrocarbons such as chloroform and dichloroethane. Dissolve or suspend in. Ethyl acetate and 5ec-butyl alcohol are used as organic solvents.

'-7myl alcohol, n-hexyl alcohol.

2-ethylhexyl alcohol and 4in-4-butyl alcohol are preferred. If necessary, a solvent 9 which is insoluble in water and does not dissolve bile acids, such as n-hegisane, cyclohexane, petroleum ether, benzene, etc., may be added. In this solution, there is an amount slightly in excess of the amount corresponding to the amount of related bile acids that are impurities (0% per mole of crude bile acid).
Add an aqueous solution containing an alkaline agent (an amount equivalent to 0.02 to 0.60 times the mole), stir at room temperature for 5 to 10 minutes, and leave to stand until the liquid separates into two layers, then separate the organic solvent layer. do. This operation is carried out 1 to 3 times depending on the purity of the crude bile acid. This operation mainly produces cholic acid, 3α, 7α-
Dihydroxy-12-ketocholanic acid and the like move to the aqueous solution layer and are removed. The amount of organic solvent and alkaline agent aqueous solution is 1:
1 to 1:2.5 (v/v) is preferred, and the alkali agents used are n-butylamine, 5ec-butylamine, and diethylamine.

Hydroxides of lower alkyl amines such as triethylamine, lower alkyl quaternary ammonium such as tetramethylammonium hydroxide, organic bases such as monoethanolamine, jetanolamine, triethanolamine, aqueous ammonia, and carbonic acid, Examples include tonoammonium salts such as ammonium ammonium and ammonium phosphate, and among them, tetramethylammonium hydroxide-d-=e water rR-formed tetraethylammonium, aqueous ammonia, and ammonium carbonate are preferred. Next, add an alkali agent similar to the above or an alkali such as an alkali metal hydroxide or an alkali metal carbonate, preferably hydroxide, to the organic solvent solution taken in 9 fractions in an amount of α8 to 1.5 times the mole of the crude bile acid. After adding an aqueous solution containing sodium and stirring for 5 to 10 minutes, the solution is left to stand until it separates into two layers, and then the aqueous solution layer is separated.

In this operation, mainly lithocholic acid is transferred into the organic solvent and removed. The amount of organic solvent and alkaline aqueous solution is 1:1 to 1:2.
．． 5 is fine.

If a trace amount of organic solvent remains in the separated aqueous solution, the target bile acid can be obtained with a purity of 99.896 or higher. The purity in the examples was measured by gas chromatography (using Hitachi Model 163 gas chromatography) using colanic acid as an internal standard. Also, thin line chromatography (TLC)
), the thin layer plate was Silica Gel H (manufactured by Merck & Co.), and the developing solvent was ethyl acetate: cyclohexane: glacial acetic acid (50:13).
:, 3) 'Solvent was used.

Example 1 Crude ursodeoxycholic acid 1002 with a purity of 95% was converted into 2-
Ethylhexanol1. OI! and add 1 to this solution.
Normal ammonia water 12.8 ml (0.05 times mole)
Wed 1.51! A solution dissolved in was added and stirred at room temperature for 5 minutes. This solution was allowed to stand and the liquid separated into two layers, and then the upper organic layer was separated. 1N ammonia water in the organic layer
Aqueous solution 1 containing 2.8 ml (0.05 times mole)
．． 5 J was added, stirred, allowed to stand, and the lower layer aqueous solution was removed. The same operation was carried out once more, and one fraction of the organic layer was collected and washed with 1.5 J of water. Next, 2.41 liters of water containing 10.22 (1.0 times mole) of sodium hydroxide was added to the washed organic solution, and the mixture was stirred at room temperature for 10 minutes. After this solution was allowed to stand still and the liquid separated into two layers, the lower layer aqueous solution was collected and the liquid volume was 2.21! It was concentrated until When 0.1N sulfuric acid was added dropwise to this solution, an amorphous solid precipitated.

This was taken off by F and dried to give one spot of ursodeoxycholic acid 87o2 by TLC. Recovery rate 870cm, purity 9
9.8C, melting point 203-204C.

Example 2 Crude chenodeoxycholic acid 200F with a purity of 94% was dissolved in ethyl acetate (hydrated) 1.6%, and to this solution was added 2790F of a 10% aqueous solution of tetramethylammonium hydroxide (0
,06 times mole) in an aqueous solution containing 1.6I! was added and stirred at room temperature for 10 minutes. The liquid was allowed to stand until it separated into two layers, and then the upper organic layer was separated.

The organic solution was then diluted with 10% tetramethylammonium hydroxide.
Extracted twice with 1.6 t of an aqueous solution containing 13.95 F (0.03 times mole) of water, and 1.61 t of water. Washed with. In this organic solution, sodium hydroxide 1837f (0
, 90 times the strength) was added to the mixture, and the mixture was stirred at room temperature for 10 minutes. This solution was allowed to stand to separate into two layers, and the lower layer aqueous solution was separated. After distilling off the organic solvent present in the aqueous solution, α1N sulfuric acid was added dropwise to precipitate an amorphous solid. This was taken off and dried, and one spot of chenodeoxycholic acid 1742 was obtained by TLC. Recovery rate 870%
, purity 99.8% Example 3 10v/v to crude chenodeoxycholic acid 52 with purity 94%
%n-butanol-containing diisopropylene ether 50m
A! and triethylamine α179mA! An aqueous solution 5ON containing (0.1 times the mole) was added, stirred for 10 minutes, left to stand, and then the upper organic layer was separated. Triethylamine αQ9mJ? It was extracted again with 5Qml of an aqueous solution containing (Q, 05 times the mole). (hydroxide in this organic solution)
50 ml of an aqueous solution containing IJum α51 F (1.0 times the mole) was added and stirred for 10 minutes.

After standing still, the water bath liquid in the lower layer was separated, the remaining child solvent was distilled off, and αl normal sulfuric acid was added dropwise to precipitate an amorphous solid. This was dried several times, and one spot of chenodeoxycholic acid 432 was obtained by TLC. Recovery rate: 86 cm, purity: 99.8
Related Example 4 Crude 3α-hydroxy-7-kedocholanic acid 2 with a purity of 96%
2 was added to a mixed solvent of triethanolamine 7 (3.4 mf (0.1 times mole), 40 ml of methyl ethyl ketone, and 50 ml of water and stirred at room temperature for 1 α minute. The liquid was allowed to stand until it separated into two layers, and then the upper layer The organic layer was separated. To the organic solution was added 45.9! (0.06 times mole) of triethanolamine and 40 ml of water for extraction, and the organic solution layer was separated. Then, 185 mjl of sodium hydroxide (185 mjl) was added to the organic solution.
Add 40 ml of water containing 0.90 times mole) at room temperature.
The mixture was stirred for α minutes. After the liquid was separated into two layers, the lower aqueous solution was separated and the remaining organic solvent was distilled off. When 01N sulfuric acid was added dropwise to this aqueous solution, an amorphous solid precipitated. This was dried by F and 1 spot of 3α-hydroxy-7-kedocolane @1.79 was obtained by TLC. Recovery rate: 870%, purity: 99.8%, melting point: 203-204C.

Patent Applicant: Tokyo Tanabe Pharmaceutical Co., Ltd. Agent Masashi Hisataka Shingai - Famous Procedural Amendment (Method) % Formula % 1. Patent Application No. 114,331/1982 2. Name of the Invention: Purification of Bile Acid Method 3, Relationship with the person making the amendment Patent applicant Tokyo Tanabe Pharmaceutical Co., Ltd. 4, Agent 6, Subject of amendment Full text of the application and specification 7, Contents of the amendment As shown in the attached document, the engraving of the application and specification (
(No changes to the contents).

Claims (1)

[Scope of Claims] 1. Crude bile acid is dissolved or suspended in an organic solvent that can be separated into layers from water, and an aqueous alkaline solution is added to this solution, or a mixed solvent of an organic solvent and an aqueous alkaline solution that can be separated into layers from water. After stirring, the organic solvent layer is separated. Next, an alkaline aqueous solution is added to the separated organic solution and stirred, the aqueous solution layer is separated, and the separated water-bath liquid is brought into contact with an acid to extract bile. M-split blade method for bile acids, which is characterized by precipitating the acid. 2. The purification method according to claim 1, wherein the purified bile acid is chenodeoxycholic acid, ursodeoxycholic acid, or 3α-hydroxy-7-kedocholanic acid. 3. Claim 1, wherein the organic solvent capable of phase separation from water is a single or mixed solvent of alcohols, esters, ketones, 1-fl, or halogenated hydrocarbons having 4 or more carbon atoms. Purification method described in section. 4. Lower alkyl amines that are easily soluble in water,
Hydroxide of lower alkyl quaternary ammonium. The purification method according to claim 1, which is a lower alkyl alcohol amine, ammonia water or ammonium salt alone or in a mixture.