US2547726A

US2547726A - Isolation of hyodesoxycholic acid

Info

Publication number: US2547726A
Application number: US83525A
Authority: US
Inventors: Elwood B Trickey
Original assignee: United States Rubber Co
Current assignee: Uniroyal Inc
Priority date: 1949-03-25
Filing date: 1949-03-25
Publication date: 1951-04-03
Anticipated expiration: 1968-04-03

Description

Patented Apr. 3, 1951 2,547,726 ISOLATION OF HYODESOXYCHOLIC ACID Elwood B. Trickey, Claymont, DeL, assignor to United States Rubber Company, New York,

N. Y., a corporation of New Jersey N Drawing. Application March 25,1949,

Serial No. 83,525

8 Claims. (Cl. 260397.1)

. bile by partial salting out of the sodium salt of alpha glycohyodesoxycholic acid with sodium chloride or other soluble inorganic salt, followed by caustic hydrolysis of the amide linkage in the conjugated acid so obtained. The unconjugated acid obtained by acidification of the alkaline hydrolysis mixture may be filtered,

dried and recrystallized from ethyl acetate or other solvent. Hydrolysis of the amide link with NaOH or KOI-I gives the unconjugated acid (hyodesoxycholic acid) asthe alkali metal salt and also the alkali metal salt of glycine. Acidificaticn of the strongly basic hydrolysis mixture with hydrochloric acid releases the free hyodesoxycholic acid. The glycine remains in solution as the hydrochloride.

This process, essentially that of Gumlich as described in Wieland et al., Zeitschrift fiir Physiologische Chemie 215, 18 (1933), requires fresh hog bile and it is often difficult to produce pure hyodesoxycholic acid by this method. The yields of crude crystalline or pure crystalline product are also only 0.84 to 1.09% of the weight of the starting ho bile.

The principal object of the present invention is to provide an improvedprocess of recovering hyodesoxycholic acid from hog bile which gives much higher yields than have been obtainable heretofore. Another object is to provide such a process which is applicable equally to fresh or aged hog bile. Another object is to provide a process which renders unnecessary a salting out procedure. Another object is to provide an improved method of crystallizing the hyodesoXycholic acid by effecting removal of keto acids prior to the final crystallizing step. Numerous other objects Will more fully hereinafter appear.

I have found that if Whole hog bile is subjected to alkaline hydrolysis in the conventional way, which may be that described in the reference cited above, and that if this treatment is followed by a mineral acid hydrolysis, preferably in the presence of a solvent for the crude bile acids and preferably at refluxing temperature,

that a marked improvement in yield of hyode soxycholic acid results. I have found that a further improvement is effected by removing the ketohydroxy acids present in the crude bile acid mixture, prior to final crystallization of the hyodesoxycholic acid, by means of a suitable reagent, for instance that of Anchel and Schoenheimer, J. Biol. Chem. 124, 609-11 (1938) or of Ido and Sakurai, J. Biochem. (Japan) 29, 51-55 (1939). Anchel et al. show the use of ketone reagents, typified by p-carboxyphenylhydrazine, for the separation of ketohydroxy acids. Typically the removal of these ketonic acids is effected by esterifying the crude acid acid mixture, treating with the ketone reagent and distributing the reaction product between ether and dilute aqueous potassium carbonate. The ketonic' acid esters couple with the ketone reagent and form water-soluble potassium salts which are dissolved in the water phase. The hyodesoxycholic acid is recovered by recrystallization of the crude acids derived from the separated ether phase.

I have found the removal of the keto acids in the foregoing manner greatly aids the final crystallization of hyodesoxycholic acid and thus gives a substantial further improvement in yield thereof.

The acid hydrolysis step of my invention is carried out by heating the crude bile acid mixture, resulting from the alkaline hydrolysis of the hog bile, with a mineral acid, preferably in aqueous solution, to a suitable elevated temperature. Any mineral acid, for example, phosphoric acid, sulfuric acid or hydrochloric acid, may be used. The selection of a suitable amount and concentration of acid will be within the skill of workers in this art. The acid should be used in such strength and amount and the temperature and duration of heating should be such as to achieve a substantial degree of hydrolysis. without causing any undesired degradation. Typically, I use an amount of acid (measured as 100% acid) ranging from 30 to by weight of the crude bile acids resulting from the alkaline hydrolysis, and an acid concentration ranging from 10 to 25% by weight of the acid based on water and acid. The temperature almost invariably will be within the range of C. to C. I prefer to carry out the acid hydrolysis by refluxing the mixture. The duration of refluxing may be several hours, say from 2 to 10 hours.

I prefer to conduct the acid hydrolysis under such conditions that the crude bile acids remain in solution. This is achieved by having present 'such an amount of a solvent capable of dissolving the crude bile acids that they remain in solution throughout this step. Examples of suitable solvents are: water, aliphatic saturated alcohols, especially the lower alcohols especially those ranging from ethyl to amyl, ketones such as acetone and methyl ethyl ketone, etc. I find it especially desirable to use the lower alkanols such as those ranging from ethyl to amyl since these are very good solvents for the bile acids. When such lower alkanols are used, they esterify a part of the bile acids during the hydrolysis step. To avoid loss of bile acids as a result of this partial esterification, I prefer to subject the mixture resulting from acid hydrolysis in the presence of such lower alkanols to a further alkaline hydrolysis, by heating the mixture with alkali metal hydroxide to saponify the esters thus formed. Thereafter the saponified mixture is cooled and acidified to precipitate the crude mixture of bile acids which is then treated in any suitable way to recover its hyodesoxycholic acid content in purified condition. The final purification of the hyodesoxycholic acid is preferably effected by crystallization from any suitable organic solvent for hyodesoxycholic acid, ethyl acetate and acetone being especially suitable.

If an inert solvent such as water or a ketone is present during the acid hydrolysis, the mixture resulting from the acid hydrolysis may be treated in any suitable way to recover the hyodesoxycholic acid content, for example, by concentration by evaporation of solvent followed by recovery of pure hyodesoxycholic acid from the precipitated crude bile acid mixture.

It is unnecessary to give details of the alkaline hydrolysis which precedes the acid hydrolysis, since it is merely the conventional alkaline hydrolysis of hog bile which is well known, being described in detail in Gumlich, in Anchel et al., and in numerous other references. Typically, whole bile is heated, for example, boiled or autoclaved, with 100% NaOH in an amount ranging from 5 to 20% by weight based on the weight of the whole bile for a period of time of from to 30 hours, cooled, diluted with water and neutralized with dilute I-ICl to precipitate the crude bile acids which are then subjected to acid hydrolysis as detailed above.

Th reason for the improvement in yield due to the acid hydrolysis of the process of my invention is obscure but may be due to hydrolysis of glycoside or other linkages amenable to attack with hot mineral acid and whose presence would interfere with crystallization of the hyodesoxycholic acid. Using these improvements I have found that fresh hog bile is not required, nor is a salting out procedure necessary, and that the yields of hyodesoxycholic acid are 1.76% of th weight of the starting bile, which is double the yield or nearly so, of that of the usually used procedure of Gumlich before referred to.

This discovery is new and surprising and makes readily available an abundant supply of a compound useful as a therapeutic agent or as an intermediate in the preparation of therapeutic agents.

The following examples serve to illustrate my invention.

EXAMPLE 1 Acid hydrolysis 183 grs. of the crude bile acids prepared by alkaline hydrolysis of whole hog bile according to the method described by Anchel at al., cited above, are mixed with 1500 cc. of n-amyl alcohol, 300 cc. of ethyl alcohol and 500 cc. of 18% 1-101 and refluxed 3 hours. I believe that by this process any glycosidic or other linkages amenable to attack with hot mineral acid are severed. At the same time a portion of the bile acids is esterified as a result of being heated in an alcohol in the presence of the mineral acid. Therefore to regain the free acids the esters formed are saponified by another treatment with sodium hydroxide. After heating the acid mixture for 3 hours, as much as possible of the uncombined alcohol is steam distilled off and enough sodium hydroxide added to hydrolyze the esters formed and the solution again steam distilled to remove alcohol freed by hydrolysis. The strongly caustic solution is then cooled to 20 C. and acidified with dilute hydrochloric acid with stirring when a finely divided, nongummy mixture of acids separates and is filtered, washed and dried at C. The product resinifies upon drying at this temperature but can be crystallized from ethyl acetate or other solvent to give pure hyodesoxycholic acid.

EXAMPLE 2 Removal of keto acids If the precipitated crude non-crystalline mixture of bile acids obtained above before crystallizing from a solvent, is treated with a reagent to remove the keto hydroxy acids present as described for example by Anchel et al., above cited, or by Ido et al., above cited, then a further improvement in yield can be effected.

Comparison of yields by old and present processes 150 grs. of the hog bile acids obtained by the procedure of Example 2, when dried and ground to a powder and boiled with 200 cc. of ethyl acetate, on cooling deposits 41 grs. of crystalline hyodesoxycholic acid which melts at One recrystallization from acetone gives material melting at 196 C. Pure hyodesoxycholic acid melts at 197 C. Gumlich gives as his yield 680 to 880 grs. of hyodesoxycholic acid from 81 litres of hog bile. From an equivalent amount of bile by my process I can isolate 1425 grs.

Having thus described my invention, what I claim and desire to protect by Letters Patent is:

1. The process of obtaining hyodesoxycholic acid from hog bile which comprises subjecting the hog bile to an alkaline hydrolysis, then subjecting the resulting bile materials to an acid hydrolysis by heating with a mineral acid, and recovering hyodesoxycholic acid from the resulting bile material.

2. The process of claim 1 wherein said acid hydrolysis is effected by heating with aqueous hydrochloric acid under refluxing conditions.

3. The process of claim 1 wherein said acid hydrolysis is conducted in the presence of such an amount of a solvent for the bile acids that they remain in solution throughout the acid hydrolysis.

4. The process of claim 1 wherein the crude bile acid mixture resulting from the acid hydrolysis is treated to remove the ketohydroxy acids present therein after which hyodesoxycholic acid is recovered from the residual bile acid mixture by crystallization .from an organic solvent for hyodesoxycholic acid.

5. The process of claim 1 wherein said acid hydrolysis is effected by refluxing a mixture of the crude bile acids resulting from the alkaline hydrolysis, aqueous hydrochloric acid and a lower saturated monohydric alcohol, and wherein the esters thus formed are thereafter saponified by heating with alkali metal hydroxide, the mixture is then acidified with precipitation of a crude mixture of bile acids, and pure hyodesoxycholic acid is recovered from said mixture by crystallization from an organic solvent for hyodesoxycholic acid.

6. The process of claim 5 wherein the crude mixture of bile acids obtained upon acidification is treated to remove the ketohydroxy acids present before said crystallization step whereby a still further improvement in yield of hyodesoxycholic acid is effected.

7. The process of obtaining hyodesoxycholic acid from hog bile which comprises heating the hog bile with alkali metal hydroxide to effect alkaline hydrolysis thereof, neutralizing the resulting mixture to precipitate the crude bile acids, subjecting said crude bile acids to an acid hydrolysis by heating for from 2 to hours at from 90 C. to 125 C. in the presence of a 10 to aqueous solution of a mineral acid, the amount of said mineral acid, measured as 100% acid, ranging from to by weight of said crude bile acids, and recovering hyodesoxycholic acid from the resulting mixture.

8. The process of obtaining hyodesoxycholic acid from hog bile which comprises heating the hog bile with alkali metal hydroxide to effect alkaline hydrolysis thereof, neutralizing the resulting mixture to precipitate the crude bile acids, subjecting said crude bile acids to an acid hydrolysis by heating for from 2 to 10 hours at from C. to 125 C. in the presence of a 10 to 25% aqueous solution of a mineral acid, the amount of said mineral acid, measured as% acid, ranging from 30 to 80% by weight of said crude bile acids and in the presence of a lower saturated monohydric alcohol in such amount that the crude bile acids remain in solution throughout the acid hydrolysis step, subjecting the resulting mixture to a further alkaline hydrolysis by heating with alkali metal hydroxide and thereby effecting saponification of esters of the bi1e acids and said alcohol formed during the acid hydrolysis, acidifying the resulting mixture to precipitate the crude mixture of bile acids, and recovering hyodesoxycholic acid from said crude mixture.

ELWOOD B. TRICKEY.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Name Date 1,861,319 Peyer May 31, 1932 2,438,232 Sifierd Mar. 23, 1948

Claims

1. THE PROCESS OF OBTAINING HYODESOXYCHOLIC ACID FROM THE HOG BILE WHICH COMPRISES SUBJECTING THE HOG BILE TO AN ALKALINE HYDROLYSIS, THEN SUBJECTING THE RESULTING BILE MATERIALS TO AN ACID HYDROLYSIS BY HEATING WITH A MINERAL ACID, AND RECOVERING HYODESOXYCHOLIC ACID FROM THE RESULTING BILE MATERIAL.