JPH0364508B2 - - 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-5β-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.
Examples of non-hydroxy bile acids include colanic acid, and other bile acids that have a double bond in the ring formed when a hydroxyl group is removed (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 impurity bile acids such as lithocholic acid and colanic acid is dissolved in an aqueous solution containing an alkali of 1.0 to 1.5 times the molar amount, preferably an equimolar amount. Quaternary ammonium salt represented by the following formula with 15.0 times the molar amount [In the formula, R ₁ , R ₂ , R ₃ and R ₄ are the same or different and represent an alkyl group or an aralkyl group having 1 to 30 carbon atoms (provided that among R ₁ , R ₂ , R ₃ , R ₄ At least one is an alkyl group or an aralkyl group having 3 or more carbon atoms, and

[Formula] may form a nitrogen-containing heterocycle. ), X represents a hydroxyl group or a halogen atom. ] 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 that initially formed an alkaline salt undergoes a salt exchange with a hydrophobic quaternary ammonium, preferentially forming a quaternary ammonium salt and a hydrophobic ion pair compound. Therefore, it is selectively transferred to the organic solvent layer and removed from the aqueous 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 ~ 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 as 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. 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. or,
Specifically, the quaternary ammonium salt represented by the above formula includes tetrapropylammonium hydroxide, tetrabutylammonium hydroxide, tetrapentylammonium hydroxide, tetrahexylammonium hydroxide, tetraheptylammonium hydroxide,
trioctylmethylammonium hydroxide, cetyltrimethylammonium hydroxide, benzyltrimethylammonium hydroxide, benzyltriethylammonium hydroxide, phenethyltrimethylammonium hydroxide, styryltrimethylammonium hydroxide, 1-laurylpyridinium hydroxide, 1-lauryl hydroxide -4-picolinium, trieicosylmethylammonium hydroxide, tetraacontylammonium hydroxide, tetraheptylammonium chloride, 1-lauryl-4-picolium chloride, benzyltrimethylammonium chloride, benzyltributylammonium chloride, and the like. Examples of organic solvents that can be phase-separated from water include n-butanol, sec
-Alcohols such as butanol, n-amyl alcohol, and n-hexane, esters such as ethyl acetate and butyl acetate, ketones such as methyl isobutyl ketone, ethyl ether, isopropyl ether, n-butyl ether, anisole, ethylene glycol dibutyl ether It is a single or mixed solvent of ethers such as chloroform, dichloroethane, halogenated hydrocarbons such as carbon tetrachloride, and hydrocarbons such as benzene, cyclohexane, n-hexane, and is preferably used as a mixed solvent. The purification method of the present invention will be explained below using Examples. However, the content of impurity bile acid in each example is determined by gas chromatography (Hitachi 163 model gas chromatography. Cholanic acid or cholic acid is used as an internal standard) and thin layer chromatography [Silica gel 60 (manufactured by Merck & Co., Ltd.)] , the developing solvent was ethyl acetate:cyclohexane:glacial acetic acid (50:13:3)]. Example 1 Lithocholic acid 0.5%, 7β-hydroxycholanic acid
100 g of ursodeoxycholic acid containing 0.3% and 1.0% of chenodeoxycholic acid was added to sodium hydroxide.
In this aqueous solution, 3.47 g of trioctylmethylammonium hydroxide (purity 85%, 3.59 times the molar amount relative to the impurity bile acid) and cyclohexane-sec-butanol ( 8:2) mixed solvent 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. 2.31 g of trioctylmethylammonium hydroxide (2.39 times the molar amount relative to the impurity bile acid) and cyclohexane were added to the aqueous layer again.
One portion of a mixed solvent of sec-butanol (8:2) was added and the same operation as above was performed to separate the aqueous layer.
Next, the aqueous solution was concentrated to a liquid volume of 2.7 to remove a trace amount of organic solvent present in the aqueous solution, and when 0.1N sulfuric acid was dropped into this aqueous solution, an amorphous solid was precipitated. This was collected by filtration, washed with water, and dried to obtain 94.5 g of ursodeoxycholic acid (recovery rate 94.5%). Lithocholic acid was reduced to 0.04%, 7β-hydroxycholanic acid was reduced to 0.01% or less, and chenodeoxycholic acid was 1.0%. Example 2 Lithocholic acid 0.4%, 7α-hydroxycholanic acid
300 g of kenooxycholic acid containing 0.2%, 2.5% of 3α,7α-dihydroxy-12-ketocholanic acid, and 0.5% of cholic acid was mixed with 45.3 g of potassium hydroxide (equimolar amount).
To this aqueous solution was added 4.83 g of tetrapentylammonium hydroxide (3.20 times the molar amount relative to the impurity bile acid) and a mixed solvent of chloroform and carbon tetrachloride (1:1) 5.
Stir for a minute. The liquid was allowed to stand until it separated into two layers, and then the upper aqueous layer was separated. Further, the same operation as above was carried out twice using 2.4 g of tetrapentylammonium hydroxide (1.59 times the molar amount relative to the impurity bile acid). The separated aqueous solution was concentrated under reduced pressure until the liquid volume reached 8 to remove a trace amount of organic solvent present in the aqueous solution, and when 0.1N sulfuric acid was added dropwise to this aqueous solution, an amorphous solid precipitated. Filter this out,
Washed with water and dried to give 286.5g of chenodeoxycholic acid.
(recovery rate 95.5%). Lithocholic acid is 0.04%,
7α-hydroxycholanic acid decreased to less than 0.01%,
3α,7α-dihydroxy-12-ketocholanic acid is 2.5
%, and cholic acid was 0.5%. Example 3 Contains 4.0% lithocholic acid and 2.0% colanic acid
Dissolve 2 g of 3α-hydroxy-7-ketocholanic acid in 50 ml of an aqueous solution containing 0.286 g (equimolar amount) of sodium carbonate, and add 114.2 mg of tetraheptylammonium hydroxide (0.824 times the molar amount to the impurity bile acid) to this aqueous solution. and n-butyl ether-n
25 ml of a mixed solvent of -amyl alcohol (9:1) 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. 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, which was collected by filtration, washed with water, and dried to yield 1.70 g of 3α-hydroxy-7-ketocholanic acid.
(recovery rate 85.0%). Lithocholic acid is 0.1%,
Colanic acid decreased to below 0.05%. Example 4 Lithocholic acid 0.5%, 7β-hydroxycholanic acid
Add 50 g of ursodeoxycholic acid containing 0.3% and 10.0% of chenodeoxycholic acid to sodium hydroxide.
1.74 g of trioctylmethylammonium hydroxide (purity 85%, 3.60 times the molar amount relative to the impurity bile acid) and n-hexane-n were dissolved in an aqueous solution 1.3 containing 7.66 g (1.5 times the molar amount). 0.5 of a mixed solvent of -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. To the aqueous layer again were added 1.16 g of trioctylmethylammonium hydroxide (2.41 times the molar amount relative to the impurity bile acid) and 0.5 g of the same mixed solvent as above, and the same operation as above was carried out to separate the aqueous layer. The fractionated aqueous solution was concentrated under reduced pressure until the liquid volume became 1.1 to remove a trace amount of organic solvent, and when 0.1N hydrochloric acid was added dropwise to this aqueous solution, an amorphous solid precipitated. This was collected by filtration, washed with water, and dried to yield 47.25 g of ursodeoxycholic acid (recovery rate
94.5%). Lithocholic acid was 0.04%, 7β-hydroxycholanic acid was reduced to 0.01% or less, and chenodeoxycholic acid was 9.9%. Example 5 Lithocholic acid 0.3%, 7α-hydroxycholanic acid
200 g of chenodeoxycholic acid containing 0.3% was dissolved in aqueous solution 6 containing 32.84 g (equimolar amount) of monoethanolamine, and 4.36 g of tetraheptylammonium chloride (3.07 times the molar amount relative to the impurity bile acid) and Mixed solvent 1 of chloroform-carbon tetrachloride (1:1) was 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, 2.18 g of tetraheptyl ammonium chloride (based on the impurity bile acid) was added to the aqueous layer.
1.53 times the molar amount) and the same mixed solvent 1 as above were added and the same operation as above was performed twice. After concentrating the separated aqueous solution to a liquid volume of 5, 0.1N sulfuric acid was added dropwise to precipitate an amorphous solid. This was collected by filtration, washed with water, and dried to obtain 190.0 g (recovery rate 95.0%) of chenodeoxycholic acid. Lithocholic acid decreased to 0.03% and 7α-hydroxycholanic acid decreased to less than 0.01%. Example 6 Lithocholic acid 0.5%, 7β-hydroxycholanic acid
Add 10 g of urosodeoxycholic acid containing 0.3% and 1.2% chenodeoxycholic acid to sodium hydroxide.
Dissolve it in 300 ml of an aqueous solution containing 1.02 g (equimolar amount), and add 760 mg of a 30% aqueous solution of 1-lauryl-4-picolinium chloride (3.59 times the molar amount to the impurity bile acid) and 100 ml of chloroform to this aqueous solution. Stir for a minute. The solution was allowed to stand until it separated into two layers, and then the lower aqueous layer was separated. Add 30% aqueous solution of 1-lauryl-4-picolinium chloride to the aqueous layer again.
507 mg (2.40 times the molar amount relative to the impurity bile acid) and 100 ml of chloroform were added, and the same operation as above was performed to separate the aqueous layer. Next, the solution was concentrated under reduced pressure until the liquid volume became 270 ml, and then 0.1N hydrochloric acid was added dropwise into the aqueous solution to precipitate an amorphous solid. This was collected by filtration, washed with water, and dried to obtain 9.47 g of ursodeoxycholic acid (recovery rate 94.7%). Lithocholic acid was reduced to 0.05%, 7β-hydroxycholanic acid was reduced to less than 0.01%, and chenodeoxycholic acid was 1.1%. Example 7 20 g of chenodeoxycholic acid containing 0.3% lithocholic acid and 0.3% colanic acid was mixed with 2.86 g of potassium hydroxide.
Dissolved in 600 ml of an aqueous solution containing g (equimolar amount),
Add to this aqueous solution 636 mg of a 50% aqueous solution of benzyltributylammonium chloride (relative to the impurity bile acid).
3.13 times the molar amount) and 100 ml of a mixed solvent of chloroform-n-butanol (8:2) were 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. Then, the liquid volume is
After concentrating to 550 ml to remove a trace amount of organic solvent in the aqueous solution, 0.1N sulfuric acid was added dropwise to precipitate an amorphous solid. Filter this out,
Washed with water and dried to give 18.7g of chenodeoxycholic acid.
(recovery rate 93.5%). Lithocholic acid is 0.06%,
Colanic acid decreased to 0.03%.

Claims (1)

[Scope of Claims] 1. Crude bile acid, an organic solvent capable of phase separation from water, an alkaline aqueous solution, and a quaternary ammonium salt represented by the following formula. [In the formula, R ₁ , R ₂ , R ₃ and R ₄ are the same or different and represent an alkyl group or an aralkyl group having 1 to 30 carbon atoms (provided that among R ₁ , R ₂ , R ₃ , R ₄ At least one is an alkyl group or an aralkyl group having 3 or more carbon atoms, and [Formula] may form a nitrogen-containing heterocycle), and X represents 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 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 quaternary ammonium salt is trioctylmethylammonium hydroxide, tetrapentylammonium hydroxide, tetraheptylammonium hydroxide, or tetraheptylammonium chloride.