WO1996017859A1

WO1996017859A1 - Process for preparing isoursodesoxycholic acid

Info

Publication number: WO1996017859A1
Application number: PCT/EP1995/004533
Authority: WO
Inventors: Hans Günter THOMAS
Original assignee: Dr. Falk Pharma Gmbh
Priority date: 1994-12-06
Filing date: 1995-11-17
Publication date: 1996-06-13
Also published as: DE4443377A1

Abstract

The invention concerns a process for preparing isoursodesoxycholic acid, wherein the ursodesoxycholic acid is first esterified and the resultant ursodesoxycholic acid ester is reacted by the Mitsunobu reaction to form isoursodesoxycholic acid ester. The isoursodesoxycholic acid ester is then saponified to form isoursodesoxycholic acid. The process is distinguished by the simplicity with which it is carried out and the high yields and amounts in which the isorusodesoxycholic acid is obtained.

Description

DESCRIPTION

Process for the preparation of isoursodeoxycholic acid

The invention relates to a process for the production of isoursodeoxycholic acid. Isoursodeoxycholic acid is a

Main metabolite / metabolite of ursodeoxycholic acid, which is produced in the human liver [Beuerε, Ul., Et al.

"Formation of iso-ursodeoxycholic acid during administration of ursodeoxycholic acid in man", Hepatol. 13, 91-103 (1994); Marschall, H.-U., et al .: "Isoursodeoxycholic acid-N-acetylglucosamide: a major metabolite of orally administered ursodeoxycholic acid", Ztsch. Gastroenterology 30, 656

(1992); Marschall, H.-U., et al. "N-Acetylglucosaminides are major urinary metabolites of orally given ursodeoxycholic acid in healthy humans", Hepatology 16, No. 4, Part 2, p. 257 A, Abstr. No. 850 (1992); Marschall, H.-U., et al. "The major metabolites of ursodeoxycholic acid in human urine are conjugated with N-acetylglucosamine", Hepatology 20, 845 to 853 (1994)]. To what extent this metabolite contributes to the therapeutic effect of ursodeoxycholic acid in the liver itself for the treatment of (cholestatic) liver diseases is currently unknown [Marschall, H.-U., et al. "Isoursodeoxycholic acid-N-acetylglucosamide: a major metabolite of orally administered ursodeoxycholic acid", Ztsch. Gastroenterology 30, 656 (1992); Marschall, H.-U., et al. "N-Acetylglucosaminides are major urinary metabolites of orally given ursodeoxycholic acid in healthy humans", Hepatology 16, No. 4, Part 2, p. 257 A, Abstr. No. 850 (1992)]. The processes known in the prior art for the production of isoursodeoxycholic acid are very difficult to implement and at most provide the desired product in low yields and amounts.

For example, T. Iida, FC Chang in J. Org. Chem. 1982, 47, 1966-72, describe the preparation of isoursodeoxycholic acid in a way that a derivative of isochenodeoxycholic acid (3β, 7α) activated differently in the 3- and 7-position converted into the desired acid by KO ₂ crown ether inversion of the OH group in the 7-position.

An educt is also selected with chenodeoxycholic acid, which can only be converted into isoursodeoxycholic acid using KO ₂ crown ether inversion using sophisticated protective group technology. The amounts that are obtained are calculated according to mg.

A.K. Batta, S.K. Aggar, G. Sälen., S. Shefer describe in Journal of Lipid Research 32, 977 (1991) a production process for isoursodeoxycholic acid, in which chenodeoxycholic acid (I) is used as the starting material. This is first reacted with succinic anhydride to form the hemisuccinate (II), oxidized with Jones' reagent and then saponified, whereby the protective group is split off and the 7-oxolithocholic acid (III) is obtained. In a further stage, the 3α-OH group of 7-oxolithocholic acid (III) becomes the 3β-OH group of 7-oxoisolithocholic acid (IV)

Mitsunobu reaction inverted. In the last stage, 7-oxoisolithocholic acid (IV) should then be reduced with potassium tert-amyl alcohol to isoursodeoxycholic acid (V) according to this reference, with a by-product formation of only 6% being mentioned.

The reworking of the AK Batta et al. However, the method described showed that this method has the following disadvantages:

(a) In the formation of hemisuccinate II, larger batches (i.e. batches in which more than 50 g of starting material are used) lose half of the starting material as a no longer usable by-product.

(b) The hemisuccinate (II) must have a very high purity so that the Jones oxidation and the deprotection can be carried out with the formation of 7-oxolithocholic acid (III). This requires complex cleaning procedures. c) If the 7-oxolithocholic acid (III) is subjected to the Mitsunobu reaction according to A.K.Batta, the oxoisolithocholic acid (IV) in is obtained with

Triphenylphosphine oxide contaminated form. This form has a melting point of 174 ° C, which differs considerably from that reported by AK Batta (> 240 ° C). d) The reduction of the oxoisolithocholic acid (IV) with potassium tert-amyl alcohol causes the oxo group in the 7-position of the 7-oxoisolithocholic acid (IV) to be converted into an α-position hydroxyl group and not - as stated - into a β-position hydroxyl group . The reduction of 7-oxoisolithocholic acid with NaBH ₄ or LiAlH ₄ also only leads to 7α-alcohol.

The invention is therefore based on the object of providing a process for the preparation of isoursodeoxycholic acid which can be carried out simply in a few steps and provides isoursodeoxycholic acid in good yields and quantities and with high purity.

This object is achieved by the provision of a process for the production of isoursodeoxycholic acid, which is characterized by the steps:

(i) Esterification of ursodeoxycholic acid to

Ursodeoxycholic acid esters of the following formula (VII):

where R is C ₁ -C ₄ alkyl,

(ii) conversion of the ursodeoxycholic acid ester (VII) to the isoursodeoxycholic acid ester of the following formula (VIII):

where R is as defined above,

through Mitsunobu reaction and

(iii) saponification of the isoursodeoxycholic acid ester (VIII) to the isoursodeoxycholic acid (V). The process according to the invention has the advantage over the processes known in the prior art that it has only three stages, namely esterification, inversion and saponification, as shown in the formula scheme below:

Because of the small number of its stages, the process according to the invention provides isoursodeoxycholic acid in significantly better yields (≥ 50%) and with a higher purity than the processes known in the prior art. The ursodeoxycholic acid (VI) used as starting material in the process according to the invention is a commercially available chemical and can be obtained, for example, from the companies Aldrich, Steinheim and the manufacturers of ursodeoxycholic acid PCA-Prodotti Chimici Alimentari SPA, Basaluzzo / Italy and Tokyo Tanabe Co., Ltd. , Tokyo / Japan. The preparation of ursodeoxycholic acid is also described in Japanese patent application JP-A-92/165867.

Step (i) of the process according to the invention, i.e. the esterification of ursodeoxycholic acid to the ursodeoxycholic acid ester (VII) is carried out according to methods familiar to the person skilled in the art. Alcohols which can be used in the esterification of ursodeoxycholic acid are, for example, lower aliphatic alcohols, such as methanol, ethanol, propanol, isopropanol or butanol. According to a preferred embodiment of the process according to the invention, stage (i) is carried out in methanolic CCl.

In step (ii) of the process according to the invention, ursodeoxycholic acid ester (VII) is converted to isoursodeoxycholic acid ester (VIII). This reaction is known in the literature as the "Mitsunobu reaction" and causes the hydroxyl group in the 3-position of the ursodeoxycholic acid ester (VII) to be inverted, which results in the isoursodeoxycholic acid ester

(VIII) is obtained.

The Mitsunobu reaction has been known in the literature for more than 10 years (see Mitsunobu, O., Synthesis, 1981, 1). The classic Mitsunobu reaction uses a combination of triphenylphosphine and dialkyl azodicarboxylate to activate alcohols for nucleophilic substitution reactions. Recent studies by D. Camp and ID Jenkins (see "The Mechanism of the Mitsunobu Reaction. The Use of Tributylphosphine", Aust. J. Chem., 1992, 45, 47-55) show, however, that other phosphines, such as tributylphosphine, can also be used successfully in the Mitsunobu reaction.

According to a preferred embodiment of the process according to the invention, the ursodeoxycholic acid ester (VII) is reacted with triphenylphosphine and diethyl azodicarboxylate in the presence of an organic carboxylic acid, such as formic acid or benzoic acid.

This inversion reaction is preferably carried out in toluene, but other solvents, such as benzene, are also suitable as a medium for carrying out the Mitsunobu reaction. The reaction temperature is generally 70 to 110 ° C, preferably 80 to 90 ° C and most preferably 80 ° C. The reaction time is not critical and is generally 20 hours to 60 hours, preferably 30 hours to 55 hours and most preferably 48 hours.

The molar ratios are not critical in the Mitsunobu reaction and are generally 1: 2: 2: 2 to 1: 4: 4: 4.5 in relation to the ratio of alcohol / phosphine / dialkyl azodicarboxylate / organic carboxylic acid used.

After the reaction has ended, the reaction mixture is cooled to room temperature, whereupon the phosphine oxide formed in the reaction precipitates, which is then filtered off. A further purification of the isoursodeoxycholic acid ester (VII) obtained is not necessary.

The step of saponification [step (iii)] of the process according to the invention can be carried out by methods familiar to the person skilled in the art. Corresponding saponification processes are described in every internship book of organic chemical laboratory technology, for example in Hünig, Märkl, Sauer, Integrated Organic Internship, Verlag Chemie Weinheim. According to In a preferred embodiment of the process according to the invention, the isoursodeoxycholic acid ester (VIII) is first reacted with methanolic KOH and then the isoursodeoxycholic acid (V) is released from its potassium salt by acidification with dilute HCl.

The isoursodeoxycholic acid (V) is preferably purified by recrystallization or by chromatography. For this purpose, the isoursodeoxycholic acid is slurried in an inert, slightly polar solvent, such as chloroform, and applied to a chromatography column. The column is then eluted with the same solvent, which removes the impurities still present in the isoursodeoxycholic acid. As soon as impurities can no longer be eluted, which can be seen from the fact that in a last eluate no residue remains when the chloroform is distilled off, the mixture is then eluted with a polar solvent such as acetone in order to recover the purified isoursodeoxycholic acid. After filtering the solution, the solvent is distilled off and the reaction product is freed from solvent residues by steam distillation. The reaction product, isoursodeoxycholic acid, is obtained in a yield of 50%.

Another, even more preferred form of working up is that the isoursodeoxycholic acid obtained as the crude product is recrystallized. Suitable solvents for the recrystallization are, for example, ethyl acetate and mixtures of ethyl acetate with other solvents, such as, for example, methanol. Preferably ethyl acetate is used for recrystallization. In this cleaning process, solvent residues can also be removed by expelling them with steam (steam distillation).

This form of processing offers the advantage that the

Isoursodeoxycholic acid can be obtained with higher yield (58%) and with higher purity, which is due to the higher Melting point of the reaction product (161 to 163 ° C compared to 158 to 160 ° C for the chromatography-purified isoursodeoxycholic acid) is displayed.

The invention is illustrated by the example below.

Example

1. Preparation of the ursodeoxycholic acid methyl ester

10 g of ursodeoxycholic acid (VI) are refluxed in 50 ml of 3% methanolic HCl for 3 hours. The reaction mixture is left to stand overnight. The volatile constituents are then stripped off on a rotary evaporator until the reaction product is dry. The residue is taken up in 100 ml of chloroform and washed with 30 ml of water, 30 ml of saturated sodium carbonate solution and again with 30 ml of water. The solution is dried over magnesium sulfate and, after the desiccant has been filtered off, evaporated on a rotary evaporator. 9.8 g of methyl ursodeoxycholic acid (yield = 95% of theory) are obtained, which has a melting point of 153-155 ° C. (not corrected).

2. Preparation of Isoursodeoxycholic Acid (V)

10 g of ursodeoxycholic acid methyl ester are dissolved in 120 ml of absolute toluene and 20 g of triphenylphosphine and 4 ml of 98% formic acid are added. 40 ml of diethyl azodicarboxylic acid solution (38-40% in toluene) are added dropwise to this mixture with stirring. The reaction is slightly exothermic. After the reaction mixture is kept at 80 ° C. for a further 48 hours, the mixture is cooled to room temperature. The triphenylphosphine oxide formed in the reaction precipitates, which is filtered off and discarded. The fil is rotated in and the residue is heated with 200 ml of 5% methanolic KOH for 3 hours under reflux. After cooling, 200 ml of water are added, and the reaction mixture is concentrated in half at 40 ° C. in vacuo. The reaction mixture is filtered and the filtrate is acidified with dilute HCl while cooling with ice. 8.3 g of isoursodeoxycholic acid (V) contaminated with triphenylphosphine oxide are obtained.

The crude product is then air-dried and then taken up in about 50 ml of chloroform, with most of the impurities going into solution. After suctioning off, 6.8 g of isoursödesoycholic acid (V) are obtained.

A chromatography column (length 40 cm, diameter 3 cm) is filled with 30 g of silica gel slurried in chloroform and the isoursodeoxycholic acid slurried in 150 ml is applied. It is then washed with 500 ml of chloroform or more until all impurities have been removed, which can be seen from the fact that no residue remains in a last eluate when the chloroform is distilled off. The acid is recovered by rinsing the column with 500 ml acetone. The acetone solution thus obtained is cloudy and must be filtered. After removal of the acetone on a rotary evaporator, the residue is freed from solvent residues by steam distillation. 4.8 g of isoursodeoxycholic acid (yield = 50% of theory) with a melting point of 158-160 ° C. (not corrected) are obtained.

Another form of working up consists in recrystallizing the crude product from ethyl acetate (100 ml of ethyl acetate for every 10 g of crude acid). This gives 5.6 g of acid (V) with a melting point of 161 to 163 ° C. Here too, solvent residues can only be removed by expelling them with steam (steam distillation). Analytical data:

3ß, 7ß-dihydroxy-5ß-cholic acid (isoursodeoxycholic acid)

Melting point: 158 to 160 ° C

1H-NMR ([D ₆ ] DMSO, 300 MHz):

δ = 0.62 (s, 3H); 0.85-1.5 (m, 23H);

1.6-2.3 (m, 9H); 3.35 (s, 1H);

3.82 (s, 2H); 4.17 (s, 1H);

12.0 (s, 1H, COOH).

¹³ C-NMR ([D ₆ ] DMSO, 75 MHz):

δ = 11.95; 18.21; 21.11; 23.77; 26.60; 27.35;

28.08; 29.42; 30.65; 34.11; 34.22; 34.75; 36.71; 37.28; 37.96; 42.76; 43.00; 54.62; 55.74; 64.33 (C-3); 69.37 (C-7); 174.79 (COOH)

Claims

PATENT CLAIMS

1. Process for the preparation of isoursodeoxycholic acid of the following formula (V):

g e k e n n e i c h n e t through the stages:

(i) Esterification of ursodeoxycholic acid to

Ursodeoxycholic acid esters of the following formula (VII):

where R is C ₁ -C ₄ alkyl,

where R is as defined above,

through Mitsunobu reaction and

(iii) saponification of the isoursodeoxycholic acid ester (VIII) to the isoursodeoxycholic acid (V).

2. The method according to claim 1, characterized in that the ursodeoxycholic acid ester (VII) is reacted with triphenylphosphine and diethyl azodicarboxylate in the presence of an organic carboxylic acid, such as formic acid or benzoic acid, to give the isoursodeoxycholic acid ester (VIII).

3. The method according to claim 1 or 2, characterized in that step (i) is carried out in methanolic HCl.

4. Process according to claims 1 to 3, characterized in that step (ii) is carried out in toluene or benzene at 70 to 110 ° C.

5. Process according to claims 1 to 4, characterized in that the isoursodeoxycholic acid ester (VIII) is first reacted with methanolic KOH and the reaction product is acidified with dilute HCl.

6. The method according to claims 1 to 5, g e k e n n z e i c h n e t by a further stage of the chromatographic purification.

7. The method according to claim 6, characterized in that the chromatographic purification is carried out first with chloroform and then with acetone as the eluent.

8. The method according to claims 1 to 5, g e k e n e z e i c h n e t by a further step of purification by recrystallization.

9. The method according to claim 8, characterized in that the recrystallization of isoursodeoxycholic acid (V) in ethyl acetate or in mixtures of ethyl acetate and methanol is carried out.

10. The method according to claims 6 to 9, characterized in that solvent residues are removed by steam distillation.