JPS60181096A - Purification of bile acid - Google Patents

Abstract

PURPOSE:To remove impurities such as lithocholic acid, etc. selectively from crude bile acid, and to obtain bile acid as precipitate, by adding a water-immiscible organic solvent, an alkaline aqueous solution, and a specific quaternary ammonium salt to a crude bile acid, stirring the mixture, separating the water- layer, and adding an acid to the layer. CONSTITUTION:Crude bile acid containing impurity bile acid such as lithocholic acid, cholanic acid, etc. is mixed with (A) an organic solvent which can be separated from water in layers [preferably 8:2 mixture of cyclohexane and sec- butanol, etc.] and (B) a quaternary ammonium salt of formula I [R1-R4 are 1-30C alkyl or aralkyl (at least one of R1-R4 is >=3C); the group of formula IImay form a heterocyclic group containing N; X is OH or halogen] (preferably trioctyl methyl ammonium hydroxide, etc.), and the mixture is stirred. The aqueous solution layer is separated and added with an acid (e.g. sulfuric acid) to precipitate the bile acid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for purifying ulundeoxycholic acid, chenodeoxycholic acid 1 fc 3α-hydroxy-7 monotocholanic acid. More specifically, monohydroxy bile acids, typified 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. Urundeoxycholic acid, chenodeoxycholic acid, and 3α-trihydroxy-7-ketocholanic acid are produced using, for example, cholic acid (3α, 7α, 12α-trihydroxy-5β-cholanic acid) as a starting material. By-products derived from 1 such as lithol p (3α-hydroxy-5β-furanic acid)
.

colanic acid, 7α (or 7β)-hydroxycholanic acid,
3α,7α-dihydroxy-7-12-ketocholanic acid and unreacted cholic acid remain, making it extremely difficult to separate and purify them. In particular, lithocholic acid has undesirable toxicity, so it is desired to remove it efficiently.

Conventionally known purification methods for ursodeoxycholic acid or chenodeoxycholic acid can be roughly divided into: 1. Methods of esterification or acylation followed by recrystallization or column chromatography (JP-A No. 52-153954. No. 52-1)
53955. 52-'156851), ■Direct recrystallization method from organic solvent (Kagaku Jikken Gakusai Vol. 10, p. 495, 1943, JP-A-52-139053. 53-1)
″37945, 55-79398. 56-3249
8), ■Method using alkali metals (Japanese Unexamined Patent Publication No. 1986-1995)
(J 5s, 5O-126654), etc. However, they were not necessarily satisfactory in terms of ease of operation, yield, purity of N product, etc. In addition, the present inventors had 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 Laid-Open No. 113-202-1982).
), further studies revealed that -monohydroxy bile acids and non-hydroxy bile acids (these are within the ring).

Also includes those with double bonds. ) has been discovered, and the invention has now been completed.

Tocholic acid as a monohydroxy bile acid.

Examples include 7α-hydroxycholanic acid, 7β-hydroxycholanic acid, 3β-hydroxycholanic acid, and the like.

Examples of non-hydroxy bile acids include furanic acid.

It also includes bile acids that have a double bond in the ring produced by the elimination of the 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 the impurity bile acids, lithocholic acid is toxic, and in the normal manufacturing process of chenodeoxycholic acid or ursodeoxycholic acid, it is unavoidable that about 1 liter of lithocholic acid is produced as a by-product. A purification method that efficiently removes it is desired.

The present invention provides a method for selectively and specifically removing the above-mentioned impurity bile acids including tocholic acid.

Next, nine methods of the present invention will be explained.

A crude bile acid containing impurity bile acids such as lithocholic acid and colanic acid is dissolved in an aqueous solution containing an alkali of 10 to 1.5 times the molar amount, preferably an equimolar amount.

In this aqueous solution, a quaternary ammonium salt represented by the following formula is present in an amount of 10 to 150 times the molar amount of the impurity bile acid. Indicates ~30 alkyl groups or aralkyl groups, provided that R□, R,, 'R3, and R4 represent hydroxyl groups or halogen atoms. ] and an organic solvent that can be separated into layers from water, and the resulting mixture is stirred for 5 to 10 minutes. After allowing the liquid to stand until it separates into two layers, separate the aqueous layer. Through this operation, the impurity bile acid, which initially formed an alkaline salt, undergoes a salt exchange with the hydrophobic quaternary ammonium, preferentially forming a hydrophobic ion pair compound with the quaternary ammonium salt. It selectively transfers to the solvent layer and is removed from the water 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 0.8:1.0 to 1.0:9
．． 0 (v/v) is preferred. If a trace amount of organic solvent remains in the separated aqueous solution, it is removed by distillation if necessary. Next, when the aqueous solution is acidified by adding an acid such as dilute hydrochloric acid or dilute sulfuric acid, the target bile acid is precipitated.

The alkali used in the present invention includes alkali hydroxides such as sodium hydroxide, potassium hydroxide, and ammonium hydroxide, and alkali carbonates such as sodium carbonate, potassium carbonate, and ammonium carbonate.

Monoethanolamine, piperazine, piperidine.

Examples include organic bases such as pyridine. Further, the 4R ammonium salt represented by the above formula is specifically:

Tetrapropylammonium hydroxide, Tetrabutylammonium hydroxide, Tetrapentylammonium hydroxide,
Tetrahexylammonium hydroxide.

Tetraheptylammonium hydroxide, hytrioctylmethylammonium hydroxide, cetyltrimethylammonium hydroxide, benzyltrimethylammonium hydroxide,
Benzyltriethylammonium hydroxide, 7enethyltrimethylammonium hydroxide, styryltrimethylammonium hydroxide, l-laurylpyridinium hydroxide, llauryl-4-picolinium hydroxide, trieicosylmethylammonium hydroxide, tetraacontylammonium hydroxide , tetraheptyl ammonium chloride, 1-chloride
These include 2uryl-4-picolium, benzyltrimethylammonium chloride, benzyltributylammonium chloride, and the like. Examples of organic solvents that can be separated into layers from water include n-butanol and 5ec-butanonyl.

Alcohols such as n-amyl alcohol and n-hexane, and esters such as ethyl acetate and butyl acetate.

Ketones such as methyl isobutyl ketone, ethers such as ethyl ether, inglovir ether, n-butyl ether, anisole, and ethylene glycol cyphthyl ether, and halogenated hydrocarbons such as chloroform, dichloroethane, and carbon tetrachloride.

It is a single or mixed solvent of hydrocarbons such as benzene, cyclohexane, n-hexane, etc., and is preferably used as a mixed solvent.

The purification method of the present invention will be explained below with reference to Examples. However, the content of impurity bile acid in each example was determined by gas chromatography (Hitachi Model 163 gas chromatography. Furanic acid or cholic acid was used as an internal standard) and thin layer chromatography [Silica gel 60 (Merck M), The developing solvent was ethyl acetate: cyclohexane; glacial acetic acid (50:
13:3)].

Example 1 Lithocholic acid 0.5%, 7β-hydroxycholanic acid 03
%, 100 t of ursodeoxycholic acid containing 10% of chenodeoxycholic acid was added to 10.2'r of sodium hydroxide.
(equimolar amount), and in this aqueous solution, 347 g of trioctylmethylammonium hydroxide (
A mixed decomposition solvent 11 of cyclohexane-5ec-butanol (8:2) and cyclohexane-5ec-butanol (8:2) was added and stirred for 5 minutes. The solution was allowed to stand until it separated into two layers, and then the lower aqueous layer was separated. A mixed solvent 1e of trioctylmethylammonium hydroxide 2311 (2.39 times the molar amount relative to the impurity bile acid) and cyclohexane-5ec-butanol (8:2) was added to the aqueous layer again, and the same operation as above was performed. The aqueous layer was separated. Then,
The solution was concentrated to a liquid volume of 2.71 to remove a trace amount of organic solvent present in the aqueous solution, and 09l normal sulfuric acid was dropped into the aqueous solution to precipitate an amorphous solid. This was collected by filtration, washed with water, and dried to give 94.5 It of ursodeoxycholic acid.
' (recovery rate of 94.5%) was obtained. Lithocholic acid is 0.
0.04%, 7β-hydroxycholanic acid decreased to less than 0.001%, and chenodeoxycholic acid decreased to 10%.

Example 2 Lithocholic acid 0.4%, 7α-hydroxycholanic acid 02
%, 3α, 7α-dihydroxy-12-ketocholanic acid 2
Chenodeoxycholic acid 3007 containing 5% and 0.5% cholic acid was dissolved in an aqueous solution 91 containing 45°3f (equimolar amount) of potassium hydroxide.

In this aqueous solution, 4.8% tetrapentylammonium hydroxide was added.
3F (3.20 times the molar amount relative to the impurity bile acid) and a mixed solvent 11 of chloroform-carbon tetrachloride (1:1') were added and stirred for 5 minutes. The liquid was allowed to stand until it separated into two layers, and then the upper aqueous layer was separated.

Furthermore, the same operation as above was carried out twice using 2.41 times the molar amount of tetrapentylammonium hydroxide (159 times the molar amount relative to the impurity bile acid). The volume of the separated aqueous solution is 84
The aqueous solution was concentrated under reduced pressure to remove a trace amount of organic solvent present in the aqueous solution, and 0.1N sulfuric acid was dropped into the aqueous solution to precipitate an amorphous solid. This was collected by filtration, washed with water, and dried to yield 286.52 (recovery rate of 9).
5.5%). Lithocholic acid is 0.04%, 7
α-hydroxycholanic acid decreased to less than 0.01%, 3
α,7α-dihydroxy-12-ketocholanic acid is 2.5
%, cholic acid was 0.5%.

Example 3 3 containing 4.0% lithocholic acid and 2.0% furanic acid
α-hydroxy-7-ketocholanic acid 2f in an aqueous solution containing 0.286 P (equimolar amount) of sodium carbonate 50
Tetraheptylammonium hydroxide 11'4.2 rn:i (impurity 11
25 rrtl of a mixed solvent of 0824 times molar amount relative to Jl acid) and +1-butyl ether-〇-amyl alcohol (9:1) were added and stirred for 5 minutes. The solution was allowed to stand until it separated into two layers, and then the lower aqueous layer was separated.

Furthermore, the same operation as above was performed twice on the aqueous layer, and the aqueous layer was separated. When 0.1N sulfuric acid was added dropwise to this aqueous solution, an amorphous solid precipitated and was collected by filtration.

It was washed with water and dried to obtain 1.70 j of 3α-hydroxy-7-kedocholanic acid (recovery rate: 850%). -Lithocholic acid decreased to 0.1% and furanic acid decreased to 0.05% or less.

Example 4 Lithocholic acid 05%, 7β-hydroxycholanic acid 0.3
50!7' of ursodeoxycholic acid containing 10.0% of chenodeoxycholic acid and 7.6% of sodium hydroxide.
Aqueous solution 1.3'A containing 6f (1.5 times the molar amount)
1.74 t of trioctylmethylammonium hydroxide (purity 85%) was dissolved in this aqueous solution.

A mixed solvent of 3.60 times the molar amount of impurity bile acid) and n-hexane-〇-butanol (1:1) was added and stirred for 5 minutes. The solution was allowed to stand until it separated into two layers, and then the lower aqueous layer was separated. Add 1° of trioctylmethylammonium hydroxide and 0% of the same mixed solvent as above to the aqueous layer again.
．． 57 was added and the same operation as above was performed to separate the aqueous layer. The fractionated aqueous solution is concentrated under reduced pressure until the liquid volume becomes 1.11 to remove a trace amount of organic solvent.
When 1N hydrochloric acid was added dropwise, an amorphous solid precipitated. This was collected by filtration, washed with water, and dried to produce ursodeoxycholic acid 47.
．． 251 (recovery rate 94.5%) was obtained. Lithocholic acid is 0.04%, 7β-hydroxycholanic acid is 0.01%
%, and cutudeoxycholic acid was 9.9%.

Example 5 Lithocholic acid 03%, 7α-hydroxycholanic acid 03%
An aqueous solution 6 containing chenodeoxycholic acid 2007 containing monoethanolamine 32842 (equimolar amount)
A mixed solvent 11 of 4.369 tetraheptylammonium chloride (307 times the molar amount relative to the impurity bile acid) and chloroform-carbon tetrachloride (1:1) was added to this aqueous solution, and the mixture was stirred for 5 minutes. The liquid was allowed to stand until it separated into two layers, and then the upper aqueous layer was separated. Furthermore, in the aqueous layer, 2.187 tetraheptylammonium chloride (153 times the molar amount relative to the impurity bile acid) and the same mixed solvent 11 as above were added.
was added and the same operation as above was performed twice. After concentrating the separated aqueous solution to a liquid volume of 51, 0.1N sulfuric acid was added dropwise to precipitate an amorphous solid. This was collected by filtration, washed with water for 70 minutes, and dried to give chenodeoxycholic acid 1'90. '
O' y (recovery rate 95.0%) was obtained. Lithocholic acid decreased to 0.003%, and 7α-hydroxycholanic acid decreased to 0.01% or less.

Example 6 Lithocholic acid 05%, 7β-hydroxycholanic acid 0.3
%, ursodeoxycholic acid 107 containing chenodeoxycholic acid 12% is dissolved in 300 g of an aqueous solution containing 1.029 (equimolar amount) of sodium hydroxide, and 30% of 1-lauryl-4-picolinium chloride is dissolved in this aqueous solution. 76 oq of an aqueous solution (3.59 times the molar amount relative to the impurity bile acid) and 10'OmJ of chloroform were added and stirred for 5 minutes. The solution was allowed to stand until it separated into two layers, and then the lower aqueous layer was separated.

Add 30% of 1-lauryl-4-picolinium chloride to the aqueous layer again.
% aqueous solution so7my (2.40 times the molar amount relative to the impurity bile acid) and chloroform 100me were added, and the same operation as above was performed to separate the aqueous layer. Then, the liquid volume is 270
After concentrating under reduced pressure until it becomes rrtl, 0 is added to the aqueous solution.
．． When 1N hydrochloric acid was added dropwise, an amorphous solid precipitated. This was collected by filtration, washed with water, and dried to produce ursodeoxycholic acid 9.
47 g (recovery rate 947%) was obtained. Lithocholic acid is 00
5%, 7β-hydroxycholanic acid decreased to less than 001%, and chenodeoxycholic acid decreased to 11%.

Example 7 Chenodeoxycholic acid 202 containing 0.3% lithocholic acid and 0.3% furanic acid was mixed with potassium hydroxide 286 (
equimolar amount) in an aqueous solution containing 600 rug of
Add 5% of benzyltributylammonium chloride to this aqueous solution.
0% aqueous solution 636 μm (3.13 times the molar amount relative to the impurity bile acid) and chloroform-I+-but/-ol (8:
2)+17) 100ml of mixed solvent was added and stirred for 5 minutes. After the liquid was allowed to stand until it separated into two layers, the upper aqueous layer was separated. The same operation as above was performed twice on the aqueous layer, and the aqueous layer was separated.

Next, the solution was concentrated to a volume of 550 ml to remove trace amounts of organic solvent present in the aqueous solution.

When 0.1N sulfuric acid was added dropwise, an amorphous solid precipitated.

This was collected by filtration, washed with water, and dried to obtain chenodeoxycholic acid 18.7r (recovery rate 935%). Lithocholic acid decreased to 0.06% and furanic acid decreased to 0.03%.

Applicant: Tokyo Tanabe Pharmaceutical Co., Ltd. Agent: Masanobu Hisataka (foreign name) Procedure correction ii (method) June 19, 1981 Mr. Kazuo Wakasugi, Commissioner of the Patent Office ], Incident display Special request No. 59-35,360 2. Name of the invention Method for purifying bile acids 3. Person who makes corrections Relationship to the incident: Patent applicant Tokyo Tanabe Pharmaceutical Co., Ltd. 4 agents

Claims (4)

[Claims] (1) An organic solvent capable of phase separation from water in the crude bile acid, an alkaline aqueous solution, and a quaternary ammonium 3 represented by the following formula [wherein the formula □, ■2. ■. and R2 are the same or different and represent an alkyl group or an aralkyl group having 1 to 30 carbon atoms, provided that R1, to R8, generally represent a hydroxyl group or a halogen atom. ] A method for purifying bile acids, which comprises adding and stirring, separating an aqueous solution layer, and adding an acid to nine aliquots of the aqueous solution to precipitate the bile acids. (2) The bile acid to be purified is ursodeoxycholic acid. The purification method according to claim E(1), which is chenodeoxycholic acid or 3α-hydroxy-7-kedocholanic acid. (3) Impurity bile acid contained in crude bile acid is lithocholic acid, colanic acid, 7α-hydroxycholanic acid or 7β
-Hydroxycholanic acid. (4) The quaternary ammonium salt is trioctylmethylammonium hydroxide, tetrapentylammonium hydroxide,
The purification method according to claim E(1), which is tetraheptylammonium hydroxide or tetraheptylammonium chloride.