PURIFICATION PROCESS FOR CHENODEOXYCHOLIC ACID

TECHNICAL FIELD

The present invention relates to a process for purifying chenodeoxycholic acid

(SαJα-dihydroxy-Sβ-cholic acid). In particular, the present invention relates to a process

for purifying chenodeoxycholic acid from low grade chenodeoxycholic acid mixture

contained in swine bile solid, with high yield and purity.

BACKGROUND ART

Chenodeoxycholic acid is generally contained in bile of cow, swine, bear, or

poultry such as chicken or goose, as well as in bile of human. Chenodeoxycholic acid is

used as starting material for the preparation of ursodeoxycholic acid which is effective to

alleviate biliary system diseases, hyperlipidemia, cholelithiasis, and chronic liver diseases,

and a typical process for preparing ursodeoxycholic acid known in the art is as follows.

A typical process for preparing chenodeoxycholic acid comprises the steps of:

esterifying cholic acid (3α,7α,12θ!-trihydroxy cholic acid) with methyl; protecting the

hydroxyl group of 3α and Ia position by acetylating them with anhydrous acetic acid;

oxidizing the hydroxyl group of 12α position to carbonyl group by using chromic acid,

and then removing the carbonyl group by Wolff-kichner reduction reaction; hydrolyzing

and deprotecting the obtained product to yield chenodeoxycholic acid. The above

process requires the reaction to be maintained at a high temperature of more than 200 ^° C ,

and the supply of raw material may be interrupted by bovine spongiform encephalopathy,

etc.

Bile ,of poultry contains chenodeoxycholic acid, lithocholic acid, and a small

amount of cholic acid. Thus, the process for separating chenodeoxycholic acid from

poultry is well known in the art, but is not economically reasonable due to the supply

decrease of raw material and low yield [see, Windhaus et al, I Physiol. Chem., 140,

177-185 (1924)].

US Patent No. 4,186,143 disclosed a process for purely separating and purifying

chenodeoxycholic acid from chenodeoxycholic acid mixture derived from natural swine

bile. This process comprises the major steps of: pre-treatment to remove 3ohydroxy-6-

oxo-5/3-cholic acid by saponification of bile; esterification of bile acid; acetylation of bile

acid ester; removal of intermediate product by using non-polar organic solvent;

crystallization of acetylated ester of formula I; deprotection; and production of the

compound of formula I by using crystallization in organic solvent. However, this patent

does not describe HPLC content for acetylated ester of formula I, and the purity of the

final product is very low since the specific rotatory power is [ofo ²⁵ +13.8° (c=l, CHCl ₃ ),

and the melting point is 119-121 ^° C [STD: [α] _D ²⁵ +15.2°(c=l, CHCl ₃ ), melting point

127- 129 "C]. Also, the crystallization for purifying the final product requires a very

long time (i.e., 16-48 hours), and the entire process is complex as eight (8) steps. Thus,

when purifying the compound of formula I by using the above process, the yield of the

final product becomes low, and the reaction time is as long as 12 days. Therefore, the

process is not economically reasonable.

In particular, when purifying the acetylated ester of formula I from the swine bile

solid having 5-35 wt% of chenodeoxycholic acid content used in the present invention by

using the above process, despite two times of recrystallization in ethanol solvent, the

content of the final product is as low as 80%.

To overcome the above problems, the object of the present invention is to provide

a new process for purifying the compound of formula I in high purity and yield, with

reducing the time required for the entire process.

DISCLOSURE OF THE INVENTION

The present invention provides a process for purifying the compound of formula I,

comprising the steps of pre-treatment of swine bile; esterification of bile acid; acetylation

of bile acid ester; removal of intermediate product by using non-polar organic solvent;

crystallization of acetylated bile acid ester; and deprotection; wherein the process is

characterized in

1) dissolving swine bile solid having 5—35 wt% of chenodeoxycholic acid content

in organic solvent containing salt, as pre-treatment step;

2) crystallizing the product obtained from the pre-treatment step in methanol or

isopropanol, within the temperature range of 0 ~ 15 ^° C , as crystallization step; and

3) deprotecting the product obtained from the crystallization step by adding base,

and crystallizing the deprotected product in the presence of water by adding acid, as

deprotection step.

[Formula I]

DETAILED DESCRIPTION OF THE INVENTION

In the present specification, the phrase "swine bile solid" represents solid derived

from swine bile, and contains the mixture of chenodeoxycholic acids of Formulae I~IV.

[Formula I]

[Formula II]

[Formula III]

[Formula IV]

wherein, the compound of formula I represents chenodeoxycholic acid (3α,7α!-

dihydroxy-5/3-cholic acid, CDCA); the compound of formula II represents hyodeoxycholic

acid (3θ!,6θ!-dihydroxy-5j8-cholic acid, HDCA); the compound of formula I represents

hyocholic acid (3α;6α;7α-trihydroxy-5/3-cholic acid, HCA); and the compound of formula

IV represents 3α-hydroxy-6-oxo-5/?-cholic acid(keto).

Hereinafter, each step for purifying chenodeoxycholic acid according to the

present invention will be exemplified in detail.

Step 1 : Pre-treatment of swine bile solid

To use swine bile solid having 5-35 wt% of chenodeoxycholic acid content in the

purification step, all of the swine bile solid is stirred and dissolved in organic solvent with

reflux. Then, the mixture is cooled to room temperature, and more stirred for 1-2 hours.

Then, insoluble materials are removed from the mixture with using filter paper, preferably

filter paper and diatomaceous earth. The organic solvent is removed under reduced

pressure to obtain residues (CDCA, HDCA, HCA and keto), which are used in the next

step. Salt used in the present step can be optionally selected as long as it does not affect

the compounds in the reactant. Preferably, the salt is at least one selected from the group

consisting of sodium chloride, anhydrous magnesium sulfate (MgSO ₄ ), and anhydrous

sodium sulfate, more preferably sodium chloride. The amount of salt used in the present

step is preferably 5~10 wt%, based on the amount of organic solvent. If the amount of

salt is less than 5 wt%, water and insoluble materials (such as fatty acids, etc.) in the swine

bile solid are not sufficiently removed, which makes the filtration difficult and reduces the

yield and velocity of esterification reaction. If the amount of salt is more than 10 wt%,

superfluous salt remains as impurity, which makes the purification difficult. Preferably,

the organic solvent can be optionally selected from ones which can dissolve

chenodeoxycholic acid of the swine bile solid and have no adverse effects thereto. More

preferably, the solvent is ethyl acetate or acetone.

Step 2: Esterification of chenodeoxycholic acid

Alcohol is added to the chenodeoxycholic acid mixture residue obtained from the

prior step, and then the solution is stirred with reflux before the residue is completely

dissolved. Then, the solution is cooled to 0-5 ^° C . Acid catalyst is added to the solution,

which is stirred with reflux at room temperature until the esterificaton reaction of

chenodeoxycholic acid mixture is completed. When the reaction is completed, the

solution is neutralized by adding base, and then filtered. The filtered material is washed

with alcohol and concentrated under reduced pressure to obtain chenodeoxycholic acid

ester mixture (CDCA-Me, HDCA-Me, HCA-Me and keto-Me) as residue. Alcohol used

in the present step is not specifically limited, but preferably lower alcohol having 1-4 of

carbon atoms, more preferably methanol, for easy esterification reaction. The acid

catalyst used in the present step is preferably sulfuric acid or para-toluenesulfonic acid

(PTSA), and the base is sodium bicarbonate, sodium carbonate or potassium carbonate.

Step 3: Acetylation of chenodeoxycholic acid ester

All the hydroxy groups in chenodeoxycholic acid ester mixture are acetylated by

adding anhydrous acetic and weak base to the residue obtained from the prior step with

reflux. When the reaction is completed, toluene is added to the reaction solution with

stirring at reflux. Then, the anhydrous acetic acid, acetic acid, and base remaining after

the reaction are removed by concentrating the reaction solution under reduced pressure, to

obtain a mixture of acetylated chenodeoxycholic acid ester (CDCA-diAc-Me, HDCA-

diAc-Me, HCA-triAc-Me and keto-Ac-Me) as residue. The weak base used in the

present step is preferably anhydrous sodium acetate or pyridine, more preferably

anhydrous sodium acetate.

Step 4: Removal of intermediate products of formulae III and IV

Non-polar solvent is added to the residue. The mixture is stirred with reflux

until all the residue is dissolved and cooled to room temperature. With maintaining the

temperature of solvent within 20~25 ^° C , intermediate products of formulae III and IV; and

a part of intermediate product of formula II (HCA-triAc-Me, keto-Ac-Me and part of

HDCA-diAc-Me) are crystallized and removed by filtration. Thus filtered material is

additionally washed with non-polar solvent, and then the filtered and washed solution is

concentrated under reduced pressure, and dried in vacuum. The non-polar solvent used

in the present step is preferably hexane, heptane, octane, isooctane and the like, more

preferably hexane or heptane.

Step 5 : Production of chenodeoxycholic acid-diacetate-methyl-ester

To produce chenodeoxycholic acid-diacetate-methyl-ester(CDCA-diAc-Me) of

formula V which is an intermediate product for preparing the compound of formula I,

alcohol solvent is added to the product obtained from the prior step, and then the

compound of formula V is crystallized by standing the mixture for 2~3 hours at 0 ^° C ~15 ^° C ,

preferably 0 ^° C~5 ^° C .

[Formula V]

If the temperature is less than O ^° C , the content for the compound of formula V

decreases, and if the temperature is more than 15 ^° C , the crystallization is not done

sufficiently. When the compound of formula V is crystallized, the intermediate product

of formula II is removed from the solvent by using filtration. Thus filtered material is

washed with alcohol solvent, and dried in vacuum to obtain chenodeoxycholic acid-

diacetate-methyl-ester of formula V as crude product. To purify the compound of

formula V in high purity, recrystallization is performed until the content for the compound

of formula V becomes 98.5% or more, preferably 99% or more, under the same conditions.

To obtain the content of 99% or more, it is preferable to perform the recrystallizaton three

(3) times or more. The alcohol used in the crystallization is preferably lower alcohol,

more preferably methanol or isopropanol, most preferably methanol, considering the

content for the compound of formula V. The amount of alcohol used in the

crystallization is 0.5-3 times, preferably 1.5-3 times, to the amount of residue. If the

amount is less than 0.5 times, the filtration is difficult since crystals coagulate each other.

If the amount becomes more than 3 times, the content for the compound of formula V is

not affected by the amount.

Step 6: Deprotection and crystallization of chenodeoxycholic acid

The compound of formula V obtained from the prior step is deprotected in the

presence of base, and the pH of the reaction solution is adjusted to 4 or lower, preferably

2-3 in acid condition, to form chenodeoxycholic acid of formula I. Simultaneously, the

reaction solution stands at 35-45 ^° C , preferably 35-40 ^° C, to crystallize the compound of

formula I in the presence of water. The reaction solution is filtered, washed with water,

and dried in vacuum to refine chenodeoxycholic acid purely. The base used for the

deprotection is not specifically limited, but sodium hydroxide or potassium hydroxide is

preferred for the post-treatment step. If the pH is more than 4, crystals are not formed,

and if the pH is less than 2, the purity of the final product is reduced due to superfluous

acid. If the crystallization temperature in water is less than 35 ^° C , the purity of the

compound of formula I decreases, and if the temperature is more than 45 °C , the filtration

is difficult since crystals derived from the compound of formula I coagulate each other.

The acid used in neutralizing the reaction solution is also not specifically limited, but

hydrochloric acid or sulfuric acid is preferred for the post-treatment step. Since the

residue obtained from the prior step contains 98.5% or more of the compound of formula

V, preferably 99% or more, the compound of formula I can be purely refined without

additional crystallization using organic solvent since the content of impurities is low.

The product containing the compound of formula I crystallized in water according to the

present invention is suitable for industrial manufacturing process since its melting point is

about 20 ^° C higher, and has lower volume, than crystallized compound of formula I in

organic solvent.

The present invention will be more specifically explained in the following

examples. However, it should be understood that the following examples are intended to

illustrate the present invention, and cannot limit the scope of the present invention in any

manner.

Analytical method

HPLC was used to confirm the intermediate products separated form each step,

and the test conditions are as follows:

Column: Capcell pak UGl 20 Cl 8 (4.6 X 250mm, Shiseido)

Mobile phase: acetonitrile/water (85:15)

Detector: ultraviolet spectrometer (210nm)

Flow rate: l.Oml/min

Insertion: 20 μJi

Example

Step 1 : Pre-treatment of swine bile solid

15Og of swine bile solid having 30-35 wt% of chenodeoxycholic acid content, and 6Og of

sodium chloride in 600 ml of ethyl acetate were stirred with reflux for 1 hour, to dissolve

all the swine bile solid. Then, the mixture was cooled to 20-25 ^° C , stirred for 1 hour, and

filtered through diatomaceous earth, and thus filtered material was washed with 60 ml of

ethyl acetate. Organic solvent was removed by concentrating the filtered material under

reduced pressure to obtain chenodeoxycholic acid mixture (CDCA, HDCA, HCA and

keto) as residue.

Step 2: Esterification of chenodeoxycholic acid

To the residue obtained from the prior step was added 375ml of methanol, and the

mixed solution was stirred with reflux for 30 minutes until the residue was completely

dissolved. This solution was cooled to 0~10 ^° C, 4.88ml of sulfuric acid was added to the

solution with stirring, and then the esterification reaction of chenodeoxycholic acid

mixture was completed by stirring at 20-25 ^° C for 2 hours. When the esterification

reaction is completed, the solution was neutralized with 53.9g of sodium bicarbonate, and

then filtered. Thus filtered material was washed with 150ml of methanol, and

concentrated under reduced pressure to obtain 134g of chenodeoxycholic acid ester

mixture (CDCA-Me, HDCA-Me, HCA-Me and keto-Me) as residue.

Step 3: Acetylation of chenodeoxycholic acid ester

To 134g of chenodeoxycholic acid ester mixture obtained from the prior step were

added 2Og of anhydrous sodium acetate and 200ml of anhydrous acetic acid. The mixed

solution was reflux ed at 120-140 ^° C for 5 hours, and then immediately concentrated under

reduced pressure. Anhydrous acetic acid and acetic acid were completely removed by

adding 25ml of toluene to the reaction solution, stirring with reflux for 15 minutes, and

concentrating under reduced pressure, to obtain acetylated chenodeoxycholic acid ester

mixture (CDCA-diAc-Me, HDCA-diAc-Me, HCA-triAc-Me and keto-Ac-Me) as residue.

HPLC result for the residue (RT): HCA-triAc-Me (8.76min), keto-Ac-Me (9.05min),

CDCA-diAc-Me (12.21min), and HDCA-diAc-Me (12.81min)

Step 4: Removal of intermediate products of formulae III and IV

To the residue obtained from the prior step was added non-polar solvent (400ml

of hexane), and then the mixed solution was stirred with reflux for 30 minutes. Then,

hexane solvent was cooled to 25-35 ^° C , stirred for 3 hours, and then filtered. Thus

filtered material (HCA-triAc-Me, keto-Ac-Me and part of HDCA-diAc-Me) was

additionally washed with 65ml of hexane, and the filtered and washed solution was

concentrated under reduced pressure to obtain the intermediate products of formulae I and

II (CDC A-di Ac-Me, HDCA-diAc-Me) as residue. HPLC result for the residue (RT):

CDCA-diAc-Me (12.21min) and HDCA-diAc-Me (12.81min).

Step 5: Production of chenodeoxycholic acid-diacetate-methyl-ester

To the residue obtained from the prior step was added 270ml of methanol, and the

mixed solution is stirred with reflux for 30 minutes, cooled to 0~10 ^° C , more stirred for 2

hours, and then filtered. The filtered material (CDCA-diAc-Me) was washed with

methanol 70ml, and dried in vacuum at 60 "C to obtain 85% content of crude product.

Then, to the crude product was added 72ml of methanol, and recrystallization was

performed to the mixture at 0~5 ^° C for 2 hours. Recrystallization is additionally

performed to the mixture one more time to obtain 99% content of chenodeoxycholic acid-

diacetate-ester. The yield is 24.5g (19.5g+mother liquor 5g). m.p.: 128-129 ^° C .

HPLC result for the residue (RT): CDCA-diAc-Me (12.21 min).

Step 6: Deprotection and crystallization of chenodeoxycholic acid

To 220ml of water were added 24.5g of chenodeoxycholic acid-diacetate-ester

and 29.5g of sodium hydroxide, and then the solution was stirred with reflux for 4 hours.

To the solution was added 370ml of water. The solution's pH is adjusted to 2.0-3.0 by

using 59ml of hydrochloric acid. Then, the solution was stirred at 35-45 ^° C for 1 hour,

and then filtered. The filtered material was washed with 24.5ml of water and dried in

vacuum at 70 ^° C to obtain 19.5g of pure chenodeoxycholic acid, m.p.: 160-161 ^° C, [α]o ²⁵

+13.0°(c=l, CHCl ₃ ).

Comparative Example

Step 1 : Pre-treatment of bile

150g of concentrated swine bile was dissolved in 1000ml of hot water. Then,

lOOg of sodium hydroxide was added, and the solution was stirred with reflux for 20 hours.

This solution was cooled to 25 ^° C . 1500ml of water was added to the solution, which was

kept cool for one day. 1Og of diatomaceous earth was added to the reaction solution,

which was then stirred and filtered to remove precipitated sodium 3α-hydroxy-6-

ketocholate of formula IV. The filtrate was adjusted to pH 8 by using cone, sulfuric acid,

and then stirred for 15 minutes after adding 5g of sodium hydrosulfite. Then, 400ml of

ethyl acetate was added to the solution, which was then adjusted to pH 5 by using diluted

sulfuric acid. The solution was stirred for 30 minutes, and aqueous layer was removed

therefrom by layer separation. To the organic layer were added 7g of diatomaceous earth

and 7g of active carbon, which was then stirred for 30 minutes and filtered. Thus filtered

material was washed with 50ml of ethyl acetate, and concentrated under reduced pressure.

Step 2: Esterfication of bile acid

The residue obtained from the prior step was dissolved in 300ml of methanol.

Then, the solution was neutralized with sodium bicarbonate (pH 7), filtered, and then

concentrated under reduced pressure.

Step 3: Removal of methyl ester of formula II

The residue obtained from the prior step was dissolved in 320ml of hot benzene,

and the mixed solution was concentrated to 225ml, and kept cold for one day. Then, the

solution was filtered; thus filtered material (methyl ester benzene adduct of formula II)

was washed with benzene, and the benzene-filtered and washed solution was concentrated

under reduced pressure.

Step 4: Acetylation of bile acid ester

To the residue obtained from the prior step were added 75ml of anhydrous acetic

acid and 7.5g of anhydrous sodium acetate, and then the mixed solution was stirred with

reflux for 5 hours. The remaining anhydrous acetic acid was removed by distilling

anhydrous acetic acid, stirring with reflux for 15 minutes after adding 35ml of methanol,

and then distilling under reduced pressure.

Step 5: Removal of acetylated ester of formula IH

The residue obtained form the prior step was reflux ed in 200ml of hexane solvent,

and the solution was stored at 20 ^° C for one day, and filtered. Thus filtered material

(crude crystal of HDC A-tri Ac-Me) was washed with hexane, and the filtered and washed

solution was distilled under reduced pressure.

Step 6: Separation of the compound of formula V

The residue obtained form the prior step was dissolved in 46ml of hot ethanol, and

then kept cool for one day. This solution was filtered, and thus filtered material was

washed with 27ml of cold ethanol, and dried in vacuum at 60 ^° C. 21.5g of the compound

of formula V was recrystallized 3 times by using ethanol to obtain 18.5g of product, m.p.

119-121 ⁰ C; [α] _D ²⁵ +10.4°(c=l, Dioxane); [α] _D ²⁵ +13.8°(c=l, CHCl ₃ ).

Step 7: Saponification and neutralization

To 185ml of water were added 18.5g of chenodeoxycholic acid-diacetate-methyl-

ester and 18.5g of sodium hydroxide, and then the mixed solution was stirred with reflux

for 14 hours. Then, the solution's pH was adjusted to 4.5 by using cone, sulfuric acid.

Step 8: Production of the compound of formula I

The reaction solution was extracted by using ethyl acetate, and aqueous layer was

discarded therefrom. Ethyl acetate layer in the solution was washed with 6% saline, and

the solution was distilled to about 90ml. This solution was cooled, kept cool for one day

after adding 90ml of hexane, and filtered. Thus filtered material was washed with 20ml

of hexane, and dried in vacuum at 60 ^° C to produce 12.7g of chenodeoxycholic acid. m.p.

142-145 ⁰ C; [α] _D ²⁵ +13.0°(c=l, CHCl ₃ ).

INDUSTRIAL APPLICABILITY

The present invention can purify chenodeoxycholic acid of formula I from swine

bile solid in high yield and purity. Also, the present invention is suitable for industrial

purification by reducing the purification time.