JPH0367080B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for purifying ursodeoxycholic acid, chenodeoxycholic acid or 3α-hydroxy-7-ketocholanic acid. More specifically, the monohydroxy bile acids represented by lithocholic acid, which are contained as impurities in these bile acids (however,
Excluding 3α-hydroxy-7-ketocholanic acid. Same below. ) and bile acids without hydroxyl groups (hereinafter referred to as non-hydroxy bile acids) such as colanic acid.

Ursodeoxycholic acid and chenodeoxycholic acid are useful as gallstone dissolving agents and choleretic agents.
3α-hydroxy-7-ketocholanic acid is useful as an intermediate in their production. Ursodeoxycholic acid, chenodeoxycholic acid and 3α-hydroxy-7-ketocholanic acid are, for example, cholic acid (3α, 7α, 12α-trihydroxy-5α-cholanic acid)
is produced as a starting material, and by-products derived from the respective production steps, such as lithocholic acid (3α-hydroxy-5β-cholanic acid), colanic acid,
7α (or 7β)-hydroxycholanic acid, 3α, 7α-
Dihydroxy-12-ketocholanic acid and unreacted cholic acid remain, making it extremely difficult to separate and purify them. In particular, since lithocholic acid has undesirable toxicity, it is desired to remove it efficiently.

Conventionally known methods for purifying ursodeoxycholic acid or chenodeoxycholic acid can be roughly divided into methods of esterification or acylation followed by recrystallization or column chromatography (Japanese Unexamined Patent Publication No. 1973-
153954, 52-153955, 52-156851), method of direct recrystallization from organic solvent (Chemical Experimental Science No. 10)
Volume, 495 pages, 1943, Japanese Unexamined Patent Publication No. 52-139053, 53-
137945, 55-79398, 56-32498), methods using alkali metals (JP-A-49-95955, 50-
126654) etc. However, these methods were not always satisfactory in terms of ease of operation, yield, purification purity, etc. In addition, the present inventors have previously filed an application for a two-phase extraction method using an organic solvent that is hardly soluble in water and an alkaline aqueous solution as a purification method for crude bile acids (Japanese Patent Application Laid-Open No. 1983-1998-1).
113202), and as a result of further research, we discovered a method for selectively, specifically, and efficiently separating and removing monohydroxy bile acids and non-hydroxy bile acids (including those with double bonds in the ring). He discovered this and completed his invention.

Monohydroxy bile acids include lithocholic acid,
Examples include 7α-hydroxycholanic acid, 7β-hydroxycholanic acid, 3β-hydroxycholanic acid, etc.
Non-hydroxy bile acids include colanic acid and the like, and also include bile acids having a double bond in the ring produced by the elimination of a hydroxyl group (hereinafter, these are collectively referred to as impurity bile acids). These impurity bile acids may be used alone or in combination of two or more. Among impurity bile acids, lithocholic acid is toxic.
Furthermore, in the normal manufacturing process of chenodeoxycholic acid or ursodeoxycholic acid, it is unavoidable that around 1% of lithocholic acid is produced as a by-product.
A purification method that efficiently removes carbon dioxide to 0.1% or less is desired. The present invention provides a method for selectively and specifically removing the above-mentioned impurity bile acids including lithocholic acid.

Next, the method of the present invention will be explained.

A crude bile acid containing impurities such as lithocholic acid and colanic acid is suspended or dissolved in an aqueous solution containing an alkali of 0.85 to 1.1 times the molar amount, and a molar amount of 0.05 to 5.0 times the crude bile acid is added to the aqueous solution using the following formula. Tertiary amine represented by (In the formula, R ₁ , R ₂ and R ₃ are the same or different and represent an alkyl group or an aralkyl group having 1 to 20 carbon atoms. However, at least one of R ₁ , R ₂ and R ₃
One represents an alkyl group or an aralkyl group having 3 or more carbon atoms. ) and an organic solvent that can be separated into layers from water are added and the resulting mixture is stirred for 5-10 minutes. After allowing the liquid to stand until it separates into two layers, separate the aqueous layer. If the tertiary amine used forms a salt, an amount of alkali equivalent to neutralization is further added. By this operation, the impurity bile acid is transferred to the organic solvent layer. This operation is carried out 1 to 3 times depending on the purity of the crude bile acid. The amount of organic solvent and alkaline aqueous solution is 0.8:1 to 1:9.
(v/v) is preferred.

In addition, crude bile acid is dissolved in advance in an organic solvent together with 1.0 to 5.0 times the molar amount of tertiary amine, and an aqueous solution containing an alkali of 0.85 to 1.1 times the molar amount is added to this organic solvent, and then stirred in the same manner as above. , standing still, fractionation, and acid precipitation.

Note that the organic solvent layer can be washed and reused by the following method.

After separating the aqueous layer, impurity bile acids and third
An alkaline aqueous solution in an amount of 1 to 3 times the molar amount of the impurity bile acid is added to the organic solvent layer containing the class amine in an amount of 0.2 to 3.0 times the amount of the organic solvent (v/v) and stirred. By this operation, the impurity bile acid is transferred to the alkaline aqueous solution layer, and the organic solvent layer becomes a solvent system containing only the tertiary amine from which the impurity bile acid has been removed. It is economical and industrially advantageous because it can be used in the purification process.

If a trace amount of organic solvent remains in the separated aqueous solution containing the target bile acid, it is removed by distillation as necessary. Next, dilute hydrochloric acid was added to the aqueous solution,
When acidified by adding an acid such as dilute sulfuric acid, the target bile acid precipitates.

Examples of the alkali used in the present invention include alkali hydroxides such as sodium hydroxide, potassium hydroxide, and ammonium hydroxide, alkali carbonates such as sodium carbonate, potassium carbonate, and ammonium carbonate, monoethanolamine, piperazine, piperidine, and pyridine. Examples include organic bases.

In addition, examples of tertiary amines include tripropylamine, tributylamine, tripentylamine,
Trihexylamine, triheptylamine, trioctylamine, triisooctylamine, tri-(7-methylheptyl)amine, trilaurylamine, tricetylamine, trieicosylamine, methyldioctylamine, methyldilaurylamine, methyl These include distearylamine, ethyldistearylamine, dimethyloctylamine, dimethyldecylamine, dimethyllaurylamine, dimethylmyristylamine, dimethylpalmitylamine, dimethylstearylamine, dioctylmyristylamine, N,N-dimethylbenzylamine, and the like.

Organic solvents that can be separated into layers from water include n-butanol, sec-butanol, n-amyl alcohol,
Alcohols such as n-hexanol, esters such as ethyl acetate and butyl acetate, ketones such as methyl isobutyl ketone and diisobutyl ketone, ethers such as ethyl ether, isopropyl ether, dibutyl ether, and anisole, chloroform, dichloroethane, and tetrachloride. A single or mixed solvent of halogenated hydrocarbons such as carbon, hydrocarbons such as cyclohexane, benzene, toluene, etc. is used by appropriately selecting a solvent capable of dissolving the tertiary amine used.

The purification method of the present invention will be explained below using Examples.

The content of impurity bile acid in each example was determined by gas chromatography (Hitachi model 163 gas chromatography. Cholanic acid was used as an internal standard) and thin layer chromatography [Silica gel 60 (Merck & Co., Ltd.)].
The developing solvent was ethyl acetate:cyclohexane:glacial acetic acid (50:13:3).

Example 1 (1) 5.0 g of chenodeoxycholic acid containing 0.3% lithocholic acid and 0.1% 7α-hydroxycholanic acid
was dissolved in 42 ml of benzene containing 9.01 g (2 times the molar amount) of trioctylamine, and 50 ml of an aqueous solution containing 0.495 g (0.97 times the molar amount) of sodium hydroxide was added dropwise into this solution over 7 minutes while stirring. . After continuing stirring for an additional 10 minutes, the mixture was allowed to stand for 30 seconds, and the aqueous layer was separated. into the separated aqueous solution
When 2.5% sulfuric acid was added dropwise, an amorphous solid precipitated.
This was filtered, washed with water, and dried to obtain 4.8 g of chenodeoxycholic acid.

(2) In order to wash and regenerate the benzene solution separated above, add 0.051 ml of sodium hydroxide into the solution.
40 ml of an aqueous solution containing g was added under stirring, stirring was continued for 10 minutes, the mixture was allowed to stand, and the benzene solution layer was separated. By this operation, lithocholic acid and 7α-byroxycholanic acid were transferred to the aqueous layer.

(3) Dissolve 4.8 g of chenodeoxycholic acid obtained in (1) in the benzene solution obtained in (2), add 50 ml of an aqueous solution containing 0.475 g (0.97 times the molar amount) of sodium hydroxide to the solution, and stir for 10 minutes. After that, let it stand,
The aqueous layer was separated. When 2.5% sulfuric acid was added dropwise into the separated aqueous solution, an amorphous solid precipitated, which was collected by filtration, washed with water, and dried to produce chenodeoxycholic acid.
4.65g (recovery rate 93%) was obtained. Lithocholic acid is
The concentration decreased to 0.04%, and 7α-hydroxycholanic acid was not detected.

Example 2 (1) Ursodeoxycholic acid containing 4.0% lithocholic acid and 1.0% 7β-hydroxycholanic acid
39.2g to 4.0g (equimolar amount) of sodium hydroxide
was dissolved in 500 ml of an aqueous solution containing trilaurylamine, 600 ml of a toluene solution containing 156.6 g (3 times the molar amount) of trilaurylamine was added to this solution, and the mixture was stirred for 10 minutes.
After standing still, the aqueous layer was separated.

(2) Add 300 ml of an aqueous solution containing 0.8 g of sodium hydroxide to the toluene solution layer separated above, stir for 10 minutes, let stand, separate the toluene layer, and wash with 300 ml of water to separate the toluene solution. Washed and regenerated.

(3) After adding 50 ml of 0.1N sulfuric acid to the aqueous layer separated in (1) to lower the pH of the liquid, further adding the toluene solution washed and regenerated in (2) and performing the extraction operation again. The layers were separated.

(4) The toluene solution separated in (3) was washed and regenerated in the same manner as in (2).

(5) After adding 50 ml of 0.1N sulfuric acid to the aqueous layer separated in (3) to lower the pH of the liquid, further adding the toluene solution washed and regenerated in (4) and stirring for 10 minutes.
The mixture was allowed to stand still, and the aqueous layer was separated. into the separated aqueous solution
2.5% sulfuric acid was added dropwise, and the precipitated solid was collected by filtration, washed with water, and dried to obtain 34.3 g of urosodeoxycholic acid (recovery rate: 87.5%).

Lithocholic acid decreased to 0.05% and 7β-hydroxycholanic acid decreased to 0.01%.

Example 3 (1) 3.92 g of 3α-hydroxy-7-ketocholanic acid containing 0.2% lithocholic acid was added to potassium hydroxide.
Trihexylamine is dissolved in 80 ml of an aqueous solution containing 0.56 g (equimolar amount), and 13.48 g of trihexylamine is added to this aqueous solution.
diisobutyl ketone containing g (5.0 times the molar amount)
50 ml was added and stirred for 10 minutes, and after standing still, the aqueous layer was separated.

(2) Add potassium hydroxide to the organic solution separated above.
Add 40 ml of an aqueous solution containing 0.168 g, stir for 10 minutes, let stand, separate the organic solvent layer, and add 40 ml of water.
Washed with ml.

(3) After adding 3 ml of 0.1N sulfuric acid to the aqueous layer separated in (1) to lower the pH of the liquid, the organic solution washed and regenerated in (2) was further added and mixed and stirred for 10 minutes.
The mixture was allowed to stand still, and the aqueous layer was separated. 2.5% into the aqueous solution
Add hydrochloric acid dropwise, filter out the precipitated amorphous fixation,
After washing with water and drying, 3.67 g of 3α-hydroxy-7-ketocholanic acid (recovery rate 94.1%) was obtained. Lithocholic acid decreased to 0.02%.

Example 4 (1) 50 g of chenodeoxycholic acid containing 0.3% lithocholic acid and 0.2% 7α-hydroxycholanic acid
was dissolved in aqueous solution 1 containing 5.10 g (equimolar amount) of sodium hydroxide, and toluene solution 1 containing 187.3 g (4 times the molar amount) of methyldilaurylamine was added to this solution, stirred for 10 minutes, and allowed to stand still. Then, the aqueous layer was separated.

(2) Add 800 ml of an aqueous solution containing 1.02 g of sodium hydroxide to the toluene solution layer separated above, stir for 5 minutes, let stand, separate the toluene layer, and wash with 800 ml of water to wash the toluene solution. Replayed.

(3) Add 38.3ml of 0.1N sulfuric acid to the aqueous layer separated in (1) to lower the pH of the liquid, then add the toluene solution washed and regenerated in (2) and perform the extraction operation again to separate the aqueous layer. I took it.

(4) The toluene solution separated in (3) was washed and regenerated in the same manner as in (2).

(5) After adding 38.3 ml of 0.1N sulfuric acid to the aqueous layer separated in (3) to lower the pH of the liquid, further adding the toluene solution washed and regenerated in (4) and stirring for 10 minutes.
The mixture was allowed to stand still, and the aqueous layer was separated. into the separated aqueous solution
Add 2.5% sulfuric acid dropwise, filter the precipitated solid, wash with water, and dry to obtain chenodeoxycholic acid.
46.9g (93.8%) was obtained.

Lithocholic acid decreased to 0.04%, and 7α-hydroxycholanic acid decreased to less than 0.01%.

Example 5 (1) Urosodeoxycholic acid 100 containing 0.3% lithocholic acid and 0.3% 7β-hydroxycholanic acid
g was dissolved in aqueous solution 4 containing 14.3 g (equimolar amount) of potassium hydroxide, and N, N
- Benzene 3 containing 103.3 ml (3 times the molar amount) of dimethylbenzylamine was added, stirred for 10 minutes, allowed to stand, and the aqueous layer was separated.

(2) Aqueous solution 2 containing 2.86 g of potassium hydroxide was added to the benzene solution separated above, stirred for 10 minutes, allowed to stand, and the benzene solution layer was separated and further washed with water 2.

(3) Add 76.5 ml of 0.1N sulfuric acid to the aqueous layer separated in (1) to lower the pH of the liquid, then add the benzene solution washed and regenerated in (2) and mix and stir for 10 minutes. The mixture was allowed to stand still, and the aqueous layer was separated. to the aqueous solution
2.5% sulfuric acid was added dropwise, and the precipitated amorphous solid was collected by filtration, washed with water, and dried to obtain 91.2 g of ursodeoxycholic acid (recovery rate 91.2%). Lithocholic acid is 0.06% and 7β-hydroxycholanic acid is 0.02%.
decreased to

Claims (1)

[Scope of Claims] 1. Crude bile acid, an organic solvent capable of phase separation from water, an alkaline aqueous solution, and a tertiary amine represented by the following formula. (In the formula, R ₁ , R ₂ and R ₃ are the same or different and represent an alkyl group or an aralkyl group having 1 to 20 carbon atoms. However, at least one of R ₁ , R ₂ and R ₃
One represents an alkyl group or an aralkyl group having 3 or more carbon atoms. ) is added and stirred, the aqueous solution layer is separated, and an acid is added to the separated aqueous solution to precipitate the bile acids. 2 The bile acid to be purified is ursodeoxycholic acid,
Chenodeoxycholic acid or 3α-hydroxy-
The purification method according to claim 1, wherein the purification method is 7-ketocholanic acid. 3. The purification method according to claim 1, wherein the impurity bile acid contained in the crude bile acid is lithocholic acid, colanic acid, 7α-hydroxycholanic acid or 7β-hydroxycholanic acid. 4. The purification method according to claim 1, wherein the tertiary amine is trihexylamine, trioctylamine, trilaurylamine, methyldilaurylamine or N,N-dimethylbenzylamine. 5. Add and mix an organic solvent that can be separated into layers from water, an alkaline aqueous solution, and a tertiary amine (same meaning as above) to the crude bile acid, and then add the aqueous solution layer to the remaining organic solvent layer again. The purification method according to claim 1, wherein the organic solvent that has been washed and regenerated by adding and mixing an aqueous alkaline solution and separating and removing the aqueous solution layer is reused.