IE73449B1 - Process for the production of chenodeoxycholic - Google Patents

Abstract

Improvements to the stages of the conventional process for the preparation of chenodeoxycholic and ursodeoxycholic acids, which comprise - the preparation of methyl 3 alpha ,7 alpha -diacetoxy-12 alpha -hydroxy- 5ss-cholanates by acetylation of the corresponding alkyl cholates with the stoichiometric quantity of acetic anhydride in an organic solvent in the presence of a catalytic quantity of an N,N-dialkylaniline or a 4-dialkylaminopyridine and a quantity of tertiary amine smaller than one mole per mole of starting material; - the preparation of 12-oxocholanates from alkyl 3,7-diacetate cholates by oxidation with an alkali metal hypochlorite and acetic acid in stoichiometric quantities in the presence of an alkali metal bromide and at a temperature below 40 DEG C; - the reduction of 3 alpha ,7 alpha -dihydroxy-12-oxo-5ss-cholanic acid with hydrazine and an inorganic base, the operation being carried out in an aqueous-organic solvent and by then removing the water by distillation; and - the preparation of 7-oxocholanates, intermediate products in the preparation of ursodeoxycholic acid, by oxidation of chenodeoxycholic acid with an alkali metal hypochlorite and acetic acid in stoichiometric quantities in the presence of an alkali metal bromide and at a temperature below 40 DEG C followed by an optional reduction of 3,7-dioxocholanic acid. b

Description

PROCESS FOR THE PRODUCTION OF CHENODEOXYCHOLIC The present invention relates to an improved process for the preparation of chenodeoxycholic acid (or 3a, 7a-dihydroxy-5jS-cholanic acid) .

Many syntheses of chenodeoxycholic and ursodeoxycholic acids have been described in the literature. Mention may be made, for example, of the syntheses described in American Patents US 3,891,681, US 3,945,562 and US 4,425,273, non-examined Japanese Patent Applications J50088856 (Derwent 11665W9) , J54079260 (Derwent 57301B) and J59039900 (Derwent 84-092393), examined Japanese Patent Applications J75012434 (Derwent 38598W), J81014120 (Derwent 71422A) and J87054439 (Derwent 38484E), Czechoslovakian Patents CS-186067 (Chem. Abst., 100, 175130c) and CS-233418 (Chem. Abst., 106, 102613b), English Patents GB-2,202,850 and GB-2,076,823, French Patent FR-1,372,109, Dutch Patent Application NL-7908972, Spanish Patents ES-489661 (Chem. Abst., 96, 20375m) and ES-499525 (Chem. Abst., 97, 145149t) and European Patents EP-63106 and EP-88637.

In fact, all the processes which are currently used industrially for the preparation of chenodeoxycholic acid are placed in the same reaction scheme which comprises substantially the following stages: stage (a) preparation of an alkyl ester of 3a, 7a-diacetoxy-12a-hydroxy-5£-cholanic acid of formula (I) : in which R is methyl, ethyl or n-propyl, stage (b) conversion of a compound of formula (I) to - 2 a 12-oxo derivative of formula (II) COOR’ (II) in which R' and R represent hydrogen or else R represents an acetyl group and R* has the same meaning as R above, and stage (c) reduction of a compound of formula (II) to chenodeoxycholic acid of formula (III) (III) Each of these changes is well known but, in the embodiments described in the literature, has disadvantages which present serious problems from an industrial viewpoint.

More particularly, as regards stage a) , it is well known that esters of cholic acid can be diacetylated in positions 3 and 7, in order to obtain the corresponding esters of 3α,7a-diacetoxy-12a-hydroxycholanic acid.

According to the conventional methods, this diacetylation is carried out in particular by esterification with acetic anhydride in pyridine, using an excess of both acetic anhydride and pyridine, and, in particular, using the latter two products as reaction solvents.

These methods include the use of very large amounts of reactants and the formation of a not insignificant amount of triacetoxylated byproduct. In order to avoid the formation of excessively large amounts of this byproduct, it is necessary to watch over the progress of the reaction, controlling either the temperature or the duration of the reaction, in order to halt it at the appropriate time.

The oxidation of hydroxycholanates provided for in stage (h) , generally carried out using an alkali metal hypochlorite and acetic acid, is also well known in the literature, this reaction being carried out using an excess of acetic acid. The main disadvantage of this reaction is that of the release of chlorine including, inter alia, problems of monitoring in order to avoid ecological damage.

The reduction of the compound of formula (II) to chenodeoxycholic acid (III) , which is well known as the Huang-Minlon modification of the Wolff-Kishner reduction, has been described, in the context of the preparation of chenodeoxycholic acid, by L.F. Fieser et al., J. Am. Chem. Soc., 1950, 72, 5530-5536 and by E. Hauser et al., Helv. Chim. Acta, 1960, 43., 1595-1600 and is the method commonly used in the context of the manufacture of chenodeoxycholic acid.

In any case, this reaction does not give good yields of final product and the main reason is due to the fact that, according to the conventional methods, the reaction must be carried out or completed at a very high temperature, which causes the formation of not insignificant amounts of byproducts.

It has now been found that it is possible to improve each of these stages, the problems posed by the known methods being overcome.

As regards the diacetylation at positions 3 and 7 of esters of cholic acid, provided for in stage (a) , it has now been found that, generally, the diacetylation at positions 3 and 7 is better if the starting ester does not contain traces of the alcohol which esterifies the alkyl cholate and that the removal of the alcohol can be carried out by washing an alkyl cholate solution with water and azeotropic distillation.

It has also been found that a selective diacetylation of the esters of cholic acid can be carried out by using a stoichiometric amount of acetic anhydride, if the preparation is carried out in the presence of catalytic amounts of an Ν,Ν-dialkylaniline or of a 4-dialkylaminopyridine and of an amount of tertiary amine of less than one mol of amine per mole of starting material.

Thus, the preparation of alkyl 3a,7a-diacetoxy12a-hydroxy-50-cholanates of formula (I) in which R represents methyl, ethyl or n-propyl, is suitably carried out by acetylation of the corresponding esters of cholic acid of formula (VI) in which R is as defined above, with the stoichiometric amount of acetic anhydride in an organic solvent in the presence of a catalytic amount of a compound of formula (VII) in which Y' and Y independently represent an alkyl containing 1 to 4 carbon atoms or else, together, form, with the nitrogen atom to which they are bonded, a pyrrolidino, piperidino or morpholino group and Z represents a nitrogen atom or a group, and of a tertiary amine in an amount of less than one mole of amine per mole of starting material.

The preferred starting material is methyl cholate (formula VI, R = methyl). 4-Dimethylaminopyridine, hereinafter designated n4-DMAPn (formula VII, Y' = Y = methyl, Z = 'n) , is used Use may be made, as organic solvent, of an aromatic hydrocarbon, such as benzene or toluene, a halogenated solvent, such as methylene chloride or 1,2-dichloroethane, an ether, such as methyl isobutyl ether or 1,2-dimethoxyethane, a ketone, such as acetone or methyl isobutyl ketone, an ester, such as ethyl acetate, or else a mixture of two of these solvents. In particular, a second solvent is added in order to complete the solubilization of the reactants, without using excessively large volumes.

The acetylation reaction is preferably carried out in a toluene/acetone mixture or in ethyl acetate.

The tertiary amine is used in amounts which can vary between 2 0 and 50 % by weight with respect to the compound (VI). The preferred amines are aliphatic amines, such as trimethylamine or triethylamine, but heterocyclic amines, such as 1-methylpiperidine or 1-methylmorpholine, or aromatic amines, such as pyridine, can also be used.

This acetylation is carried out at a temperature of 0-5°C, as in all the cases of this type of acetylation.

In general, after 10-12 hours at this temperature, the reaction is complete but its duration beyond 12 hours is not critical because no subsequent formation of byproducts is observed, even leaving the reaction mixture at 0-5°C for 24 hours. This constitutes the main advantage with respect to the conventional processes.

The final product is isolated according to conventional techniques and can optionally be crystallized from toluene or from methanol.

To obtain good yields of product (I), it is desirable that the starting material which has to be subjected to the acetylation should be dry, in particular that it should not contain the alcohol used for the prior esterification.

However, according to another aspect of the present invention, it is possible to use a starting methyl, ethyl or n-propyl cholate which contains 5 to 30 % of the esterifying alcohol. In this case, the said alcohol can he removed by dissolving the starting material in a mixture formed by an organic solvent, which has a boiling point at ambient pressure greater than 100°C, preferably the same solvent which will be used for the acetylation reaction, and water and by removal first of water and then, azeotropically, of the solvent/water mixture.

It is preferable to dissolve the starting material in the solvent which will be used for the acetylation reaction and to use an amount of water of 10 to 25 % by volume with respect to the amount of organic solvent used.

The removal can be carried out first of water and then of the mixture of water and of the alcohol possibly still present by azeotropic distillation at a temperature greater than 100°C.

According to this aspect of the present invention, it is therefore advantageous to use toluene as solvent having a boiling point at ambient pressure greater than 100°C.

It was thus found that, as regards stage (b) , the oxidation reaction of the alkyl esters of 3a,7a-diacetoxy-12a-hydroxy-50-cholanic acid (I) can be carried out efficiently and without control problems by slowly adding an essentially stoichiometric amount of an alkali metal hypochlorite to a solution of the 12a-hydroxy derivative of formula (I) and of acetic acid in a stoichiometric amount, in the presence of an alkali metal bromide, at a temperature of less than 40°C and preferably of less than 10°C.

Thus, the preparation of the 12-oxocholanate of formula (II) in which the two R and R' represent hydrogen or else the two R represent an acetyl group and R' has the same meaning as R above, is more suitably carried out by slowly adding an alkali metal hypochlorite to a solution of a 12a-hydroxycholanate of formula (I) COOR (I) in which R is as defined above, containing acetic acid in a stoichiometric amount and an alkali metal bromide, at a temperature of less than 40°C, and by optionally saponifying the product thus obtained, in order to prepare the compound of formula (II) in which R' and R represent hydrogen.

The methyl ester of 3a,7a-diacetoxy-12a-hydroxy5/3-cholanic acid is used as the preferred starting hydroxycholanate.

This reaction can be carried out in an organic or aqueous/organic solvent, the organic solvent being a halogenated hydrocarbon, for example methylene chloride or 1,2-dichloroethane, ethyl acetate or an ether, for example dimethoxyethane or tetrahydrofuran.

The alkali metal bromide is preferably used in a proportion of 0.30-1.15 mol per mole of starting hydroxycholanate, the cation preferably being that of the hypochlorite used. Sodium hypochlorite and sodium bromide are preferably used.

The reaction temperature must be less than 40°C in order to avoid release of chlorine, but it is advantageous to carry out the oxidation below 10°C and preferably below 0°C.

In general, according to a preferential procedure, the reaction is completed after 16-24 hours at a temperature between -10 and 0°C and the product of formula (II) is isolated according to conventional techniques.

For example, the product thus obtained can be isolated in the crystalline form by pouring the reaction mixture into water, preferably in the presence of an alkali metal sulphite which serves to destroy the excess oxidizing agent still present, by removing the aqueous phase and by evaporating the solvent. The 12-oxocholanate thus obtained can also be purified by crystallizing it from an appropriate solvent or solvent system, such as acetone or acetone/water mixtures.

An ester of 3a,7a-diacetoxy-12-oxo-50-cholanic acid is thus obtained which can be saponified by using an aqueous sodium hydroxide solution in order to isolate 3a,7a-dihydroxy-12-oxo-5/3-cholanic acid.

As regards the reduction of a compound (II) in order to obtain chenodeoxycholic acid (III) (stage (c)), it has now been found that, if the Wolff-Kishner reaction is carried out in an aqueous/organic solvent, water and part of the solvent being slowly removed by azeotropic distillation, the progress of the reduction is easily controlled and chenodeoxycholic acid is obtained in the pure state with a virtually quantitative yield.

More particularly, it has been found that chenodeoxycholic acid can be obtained with a good yield and substantially free of impurities, in particular of lithocholic acid, by treating 3a,7a-dihydroxy-12-oxo5/S-cholanic acid or one of its esters of formula (II) in which R' and R represent hydrogen or R represents an acetyl group and R* has the same meaning as R above, with hydrazine and an inorganic base in a mixture of water and of a hydroxylated organic solvent, which has a boiling point at ambient pressure greater than 100°C and forms an azeotrope with water, at a temperature of 85-110°C, the water and part of the solvent are then distilled off by increasing the temperature of the reaction mixture to 115-145°C and the reaction is left to come to completion at this temperature.

The term hydroxylated organic solvent is understood to mean an organic solvent chosen from alkanols and the lower alkyl ethers of ethylene or propylene glycol.

Appropriate organic solvents comprise 2-methoxyethanol (tradename: Methyl Cellosolve), 2-ethoxyethanol, 2-butoxyethanol, 2-methoxypropanol, 1-butanol and 2-pentanol, 2-methoxyethanol and 1-butanol being preferred.

The amount of water present in the reaction mixture with the hydroxylated organic solvent can vary between 10 % and 30 % by volume and is preferably present in a percentage of approximately 20 % by volume with respect to the organic solvent.

The use of this solvent system makes it possible, under the conditions provided for this reaction, to remove water slowly and at the same time completely by azeotropic distillation, thus making it possible to continue the reaction to the end.

The preferred starting compound is that of formula (II) above, where R' = R = H, but the methyl ester of the diacetate (formula (II), R' = methyl, R = acetyl) is also suitable.

As, in any case, the desired final product is chenodeoxycholic acid (III), the amount of inorganic base must be calculated so as to cause the saponification of all the esters optionally present.

Use is preferably made, as inorganic base, of potassium hydroxide (80-85 %) and hydrazine is also used, in the base form, as an 80-85 % aqueous solution.

The reaction is slowly initiated by heating to the temperature of 85-110 °C over approximately 45-75 minutes and the reaction mixture is then left at the same temperature for a variable period of time between 1 and hours.

In order to bring the reaction to completion, the reaction mixture is distilled, the temperature of the mixture being slowly increased to 115-145°C, and the mixture is then left to react at the same temperature.

Normally, after heating for 5-10 hours, the reaction is completed and chenodeoxycholic acid (III) is isolated by simple acidification and filtration.

A first subject of the present invention is 10 therefore a process for the preparation of chenodeoxycholic acid which takes place essentially according to the following stages: stage (a) - 3,7-diacetylation of methyl, ethyl or n-propyl cholate in order to obtain the corres15 ponding ester of formula (I) in which R is methyl, ethyl or n-propyl, stage (b) - oxidation of a compound of formula (I) to the 12-oxo derivative of formula (II) in which R' and R represent hydrogen or else R represents an acetyl group and Rz has the same meaning as R above, and stage (c) - reduction of a compound of formula (II) to chenodeoxycholic acid of formula (III) characterized in that : (a) : a compound of formula (I) is prepared by treating an ester of cholic acid of formula (VI) in which R is methyl, ethyl or n-propyl, with the stoichiometric amount of acetic anhydride in an organic solvent in the presence of a catalytic amount of a compound of formula (VII) Y’ \ N Z Y in which Y' and Y independently represent a (Cj-C^) alkyl group or else, together, form, with the nitrogen atom to which they are bonded, a pyrrolidino, piperidino or morpholino group and Z represents a (b) nitrogen atom or a ^£H group, and of a tertiary amine in an amount of less than one mole of amine per mole of starting material; a compound of formula (I) is oxidized to the 12-oxo by slowly adding an in an essentially a solution of a derivative of formula (II) alkali metal hypochlorite stoichiometric amount to 12a-hydroxycholanate of formula (I), in which R is as defined above, and of acetic acid in a stoichiometric amount, in the presence of an alkali metal bromide, at a temperature of less than 40°C, and by optionally saponifying the product thus obtained in order to obtain the compound of formula (II) in which R' and R are hydrogen; and (c) : a compound of formula (II) is reduced to chenodeoxycholic acid (III) by treating the said compound of formula (II) , in which R' and R are as defined above, with hydrazine and an inorganic base in a mixture formed by water and a hydroxylated organic solvent, which has a boiling point at ambient pressure greater than 100°C and which forms an azeotrope with water, at a temperature of 85-110°C, the water and part of the solvent are then distilled off by increasing the temperature of the reaction mixture to 115-145°C and the reaction is left to come to completion at this temperature.

It is understood that all the particulars relating to the still more advantageous reaction conditions which have been mentioned above for each stage also apply to the overall process for the preparation of chenodeoxycholic acid and represent preferred embodiments of the said process.

The following examples illustrate the present invention without, however, limiting it.

Stage (a) - Preparation of the alkyl esters of 3a.7a-diacetoxv-12a-hvdroxv-5g-cholanic acid (I) Example 1 A mixture of 105 kg of methyl cholate, arising directly from the esterification of cholic acid with methanol and containing 15 % of methanol, 580 1 of toluene and 90 1 of water is heated to 40°C; the aqueous phase is then separated, the organic phase is heated to reflux and the water is removed azeotropically to 110°C. The reaction mixture is cooled, 135 1 of acetone are added and, at a temperature of less than 20°C, 0.78 kg of 4-DMAP and 35 kg of triethylamine are first added and then, slowly, 44 kg of acetic anhydride are added. The temperature is brought to 0-5°C and the reaction mixture is left at this temperature until the reaction is complete. The reaction mixture is then washed with 180 1 of water and the organic phase is concentrated to a concentration of 1:2. Crystallization is initiated at 60°C and the product is isolated at 0-5°C by centrifuging and washed with toluene and water. After crystallizing from toluene, methyl 3a,7a-diacetoxy-12a-hydroxy-5/3-cholanate is obtained which is identical to an authentic sample and which has an assay of greater than 99 %, measured by gas phase chromatography. Yield of crystallized product: 76 % on the basis of an estimated 90 kg of dry starting material.

Example 2 By carrying out the preparation as described in Example 1, but replacing the 35 kg of triethylamine with 35 kg of pyridine, methyl 3a, 7a-diacetoxy-12a-hydroxy5/3-cholanate is obtained which has the same degree of purity and with the same yield.

Stage (b) - Oxidation of a compound (I) to a 12-oxo derivative (II) Example 3 18.48 1 of a 14.9 % weight/volume solution of sodium hypochlorite are added over 5 hours to a solution, cooled to -10°C-0°C, of 17 kg of methyl 3a,7a-diacetoxy12a-hydroxy-5/?-cholanate and 1.04 kg of sodium bromide in kg of ethyl acetate, 3.6 kg of acetic acid and 3.4 kg of water. The reaction mixture is left for 16 hours at the same temperature, 17 kg of a 10 % sodium sulphite solution are then added and the reaction mixture is heated to 40°C. The aqueous phase is removed, washing is carried out with a sodium bicarbonate solution to neutrality and 1.1 volumes of water are added per volume of methyl 3a,7a-diacetoxy-12a-hydroxy-50-cholanate. Distillation is carried out at 100°C and the suspension is cooled to a temperature of less than 50°C. 3.1 volumes of acetone are added thereto and heating is carried out at reflux for one hour until a solution is obtained. The solution is cooled and allowed to crystallize. The temperature is lowered to 0-5 °C for 4 hours and the crystallized product is centrifuged and washed with a 1/1 ice-cold water/acetone mixture. 15.57 kg of methyl 3a, 7a-diacetoxy-12-oxo-5/J-cholanate are obtained, which product has an assay of greater than 99 % with respect to an authentic sample. It is also possible, after neutralization of the solution of the product in ethyl acetate, to evaporate the solvent on a water bath, to cool and to filter, methyl 3a,7a-diacetoxy-12-oxoδβ-cholanate being obtained with a quantitative yield.

Example 4 The preparation is carried out as described above as far as the crystallization of the methyl 3a, 7a-diacetoxy-12-oxo-5jS-cholanate, 4 equivalents of 40 % sodium hydroxide are then added and the reaction mixture is heated at reflux until saponification is complete. On acidification with hydrochloric acid, 3a, 7a-dihydroxy-12-oxo-50-cholanic acid is obtained which has an assay of greater than 99 % with respect to an authentic sample.

Stage (c) - Reduction of a compound (II) to chenodeoxycholic acid Example 5 g of 85 % potassium hydroxide and then 13 ml of 80 % hydrazine are added to a mixture of 40 g of 3a,7a-dihydroxy-12-oxo-50-cholanic acid, 80 ml of Methyl Cellosolve and 17 ml of water. The temperature is brought to 110°C and heating is continued at the same temperature for 4 hours. The reaction mixture is then distilled to the temperature of 135-138°C and is left at the same temperature for 8 hours. The reaction mixture is cooled, 120 ml of water are added and the solution is then poured into 160 ml of water containing 40 ml of 50 Be sulphuric acid at a temperature of less than 20°C, the pH being adjusted to approximately 2. The precipitate is filtered off and washed to neutrality. 40 g of pure chenodeoxycholic acid are thus obtained, which product has an assay of 86.5 %, the major impurities being water and potassium sulphate. The amount of lithocholic acid, assessed by thin layer chromatography, is < 0.05 %.

Example 6 A mixture of 50 g of methyl ester of 3a, 7a-diacetoxy-12-oxo-5jS-cholanic acid, 250 ml of n-butyl alcohol and 25 g of 80 % hydrazine hydrate is heated to 103°C and heating is continued at the same temperature for 2 hours. The reaction mixture is then distilled to the temperature of 117-122°C, 50 g of 88 % potassium hydroxide are added and the reaction mixture is distilled to the temperature of 138-142°C. Heating is continued at the same temperature for 24 hours. The reaction mixture is cooled, 200 ml of water are added, the mixture is distilled until all the n-butyl alcohol has been removed, the mixture is cooled, 130 ml of water are added, 60% formic acid is added to pH 7-8, 100 ml of ethyl acetate are added and formic acid is added to pH 4-5. The organic phase is separated, which phase is washed with an aqueous sodium bicarbonate solution to neutrality, and the organic phase is separated; the product is extracted with an aqueous sodium bicarbonate solution.

The aqueous phase is separated and distillation is carried out until the ethyl acetate has been completely removed.

The solution is cooled and is poured into an amount of 2 0 % sulphuric acid in order to obtain a final pH of approximately 2. The precipitate is filtered off and washed to neutrality. 36.8 g of pure chenodeoxycholic acid are thus obtained, which product has an assay of 97.2 %, the major impurity being water.

Example 7 The preparation is carried out substantially as described in Example 6, methyl cellosolve being used in place of n-butyl alcohol. Heating is carried out for 10 hours in place of 24 and the chenodeoxycholic acid is isolated according to the method described in Example 5. 38.1 g of pure chenodeoxycholic acid are obtained, which product has an assay of 98.5 %, the major impurity being water.

Claims (12)

1. Process for the preparation of chenodeoxycholic acid, which takes place according to the following stages : stage (a): 3,7-diacetylation of methyl, ethyl or n-propyl cholate in order to obtain the corresponding ester of formula (I) in which R is methyl, ethyl or n-propyl, stage (b) : oxidation of a compound of formula (I) to the 12-oxo derivative of formula (II) in which R' and R represent hydrogen or else R represents an acetyl group and R' has the same meaning as R above, and stage (c) : reduction of a compound of formula (II) to chenodeoxycholic acid of formula (III) characterized in that : (a) : a compound of formula (I) as defined above is prepared by treating an ester of cholic acid of formula (VI) in which R is as defined above, with a stoichiometric amount of acetic anhydride in an organic solvent in the presence of a catalytic amount of a compound of formula (VII) (VII) in which Y' and Y independently represent a (C^-C*) alkyl group or else, together, form, with the nitrogen atom to which they are bonded, a pyrrolidino, piperidino or morpholino group and Z represents a nitrogen atom or a CH group, and of a tertiary amine in an amount of less than one mole of amine per mole of starting material; (b) : a compound of formula (I) is oxidized to the 12-oxo derivative of formula (II) by slowly adding an alkali metal hypochlorite to a solution of 12a-hydroxycholanate of formula (I), in which R is as defined above, and of acetic acid in a stoichiometric amount, in the presence of an alkali metal bromide, at a temperature of less than 40°C, and by optionally saponifying the product thus obtained in order to obtain the compound of formula (II) in which R' and R are hydrogen; (c) : a compound of formula (II) is reduced to chenodeoxycholic acid (III) by treating the said compound of formula (II) , in which R' and R are as defined above, with hydrazine and an inorganic base in a mixture formed by water and a hydroxylated organic solvent, which has a boiling point at ambient pressure greater than 100°C and which forms an azeotrope with water, at a temperature of 85-110°C, the water and part of the solvent are then distilled off by increasing the temperature of the reaction mixture to 115-145°C and the reaction is left to come to completion at this temperature.

2. Process according to Claim 1, characterized in that a methyl, ethyl or n-propyl cholate which contains 5 to 30 % of the esterifying alcohol is used as starting compound in stage (a) and in that the said alcohol is removed by dissolving in a mixture formed by an organic solvent, which has a boiling point at amHi pnt- pressure greater than 100°C, and water and by removal first of water and then, azeotropically, of the solvent/water mixture.

3. Process according to either of Claims 1 and 2, characterized in that methyl cholate is used as starting material in stage (a) and toluene is used as organic solvent.

4. Process according to any one of Claims 1 to 3, characterized in that 4-dimethylaminopyridine is used as catalyst in stage (a).

5. Process according to any one of Claims 1, 2 and 4, characterized in that stage (a) is carried out in a toluene/acetone mixture or in ethyl acetate.

6. Process according to Claim 1, characterized in that, in stage (b), methyl 3a, 7a-diacetoxy-12a-hydroxy5/5-c hoi ana te is used as starting material.

7. Process according to Claim 6, characterized in that, in stage (b), the alkali metal bromide is used in a proportion of 0.30 - 1.15 mol per mole of starting hydroxycholanate.

8. Process according to either of Claims 6 and 7, characterized in that, in stage (b), sodium hypochlorite and sodium bromide are used.

9. Process according to Claim 1, characterized in that, in stage (c), the water is present in the reaction mixture in an amount of 10 to 30 % by volume with respect to the hydroxylated organic solvent.

10. Process according to Claim 9, characterized in that, in stage (c) , 2-methoxyethanol or 1-butanol is used as hydroxylated organic solvent.

11. Process according to either of Claims 9 and 10, characterized in that, in stage (c), free 3a,7adihydroxy-12-oxo-5jS-cholanic acid is used as starting material.

12. Process for the preparation of chenodeoxycholic acid according to Claim 1, substantially as herein described with reference to the Examples.