DE4443377A1 - Process for the preparation of isoursodeoxycholic acid - Google Patents

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

The invention relates to a method for producing Isoursodeoxycholic acid. Isoursodeoxycholic acid is a Main metabolite / metabolite of ursodeoxycholic acid, that arises in the human liver [Beuers, U1., et al. "Formation of iso-ursodeoxycholic acid during administration of ursodeoxycholic acid in man ", Hepatol. 13, 97-103 (1994); Marschall, H.-U., et al .: "Isoursodeoxycholic acid-N- Acetylglucosamide: A major metabolite administered orally Ursodeoxycholic acid ", Ztsch. Gastroenterologie 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 bis 853 (1994)]. To what extent this metabolite is therapeutic Effect of ursodeoxycholic acid in the liver itself Treatment of (cholestatic) liver diseases contributes, is currently unknown [Marschall, H.-U., et al. "Isourso deoxycholic acid-N-acetylglucosamide: a major metabolite orally administered ursodeoxycholic acid ", Ztsch. Gastroenterology 30: 656 (1992); Marschall, H.-U., et al. "N-acetylglu cosaminides 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 production processes known in the prior art of isoursodeoxycholic acid are very poorly realized cash and deliver the desired product in a minimum yields and quantities.

For example, T. Iida, F.C. Chang in J. Org. Chem. 1982, 47, 1966-72, the production of isoursodeoxycholic acid a way that one is different in 3- and 7-position activated derivative of isochenodeoxycholic acid (3β, 7α) by KO₂ crown ether inversion of the OH group in 7ths tion converted into the desired acid.

An educt is also chosen with chenodeoxycholic acid with KO₂ crown ether inversion only after sophisticated To convert protecting group technology into isoursodeoxycholic acid is. The amounts that are obtained are in mg calculated.

A.K. Batta, S.K. Aggar, G.Salen., S.Shefer describe in Journal of Lipid Research 32, 977 (1991) process for isoursodeoxycholic acid, in which chenodeoxy cholic acid (I) is used as the starting material. This first with succinic anhydride to hemisuccinate (II) implemented, oxidized with Jones reagent and then saponified, whereby the protective group is split off and the 7- Oxolithocholic acid (III) is obtained. In another Stage becomes the 3α-OH group of 7-oxolithocholic acid (III) to the 3β-OH group of 7-oxoisolithocholic acid (IV) Mitsunobu reaction inverted. In the last stage it should then 7-oxoisolitho according to this reference cholic acid (IV) with potassium tert-amyl alcohol to form the Isourso Let deoxycholic acid (V) be reduced, with a minor product formation of only 6% is mentioned.  

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

• (a) The formation of hemisuccinate II is involved larger batches (i.e. batches in which more than 50 g Starting material are used) half of the off gear as a no longer usable by-product lost.
• (b) The Jones oxidation and the cleavage the protective group to form 7-oxolithocholic acid (III) is feasible, the hemisuccinate (II) must be very high Purity. There are complex cleaning procedures for this required.
• c) Submitting 7-oxolithocholic acid (III) according to A.K. Batta of Mitsunobu reaction, you get the oxoisolithocholic acid (IV) in with Triphenylphosphine oxide contaminated form. This form has a melting point of 174 ° C, which is significantly different from that by A.K. Batta reported deviations (<240 ° C).
• d) The reduction of oxoisolithocholic acid (IV) with Potassium tert-amyl alcohol causes a conversion of the oxo group in 7-position of 7-oxoisolithocholic acid (IV) in one α-hydroxyl group and not - as stated - in one β-hydroxyl group. The reduction of 7-oxoisolitho cholic acid with NaBH₄ or LiAlH₄ also only leads to 7α- Alcohol.

The invention is therefore based on the object of a method to provide for the production of isoursodeoxycholic acid len, which can be easily carried out in a few steps  and isoursodeoxycholic acid in good yields and amounts and delivers with high purity.

This task is accomplished by providing a procedure solved for the production of isoursodeoxycholic acid, the ge is characterized by the stages:

• (i) Esterification of ursodeoxycholic acid to the ursodeoxycholic acid ester of the following formula (VII): wherein R is C₁-C₄ 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, by Mitsunobu reaction and
• (iii) saponification of the isoursodeoxycholic acid ester (VIII) to isoursodeoxycholic acid (V).

The method according to the invention has compared to the Prior art methods have the advantage that it has only three stages, namely esterification, an inversion and saponification, as described in For melschema is shown below:

Because of the small number of its levels, it delivers Process according to the invention the isoursodeoxycholic acid in we considerably better yields (50%) and with higher purity unit than the methods known in the prior art.  

The starting material in the process according to the invention Ursodeoxycholic acid (VI) used is a commercially available Chemical and can be obtained, for example, from Aldrich, Steinheim, as well as the manufacturers of ursodeoxychol acid PCA-Prodotti Chimici Alimentari S.P.A., Basaluzzo / Italy and Tokyo Tanabe Co., Ltd., Tokyo / Japan will. The production of ursodeoxycholic acid is also in Japanese Patent Application JP-A-92/165867.

Step (i) of the process according to the invention, i. H. the Esterification of ursodeoxycholic acid to ursodeoxycholic acid ester (VII), according to methods familiar to the expert carried out. As alcohols used in the esterification of the original Sodeoxycholic acid can be used, for example wise lower aliphatic alcohols, such as methanol, ethanol, Propanol, isopropanol or butanol are suitable. According to one preferred embodiment of the method according to the invention stage (i) is carried out in methanolic HCl.

According to stage (ii) of the process according to the invention, Urso deoxycholic acid ester (VII) to isoursodeoxycholic acid ester (VIII) implemented. This implementation is in the literature as "Mitsunobu reaction" known and causes the hydroxy group in the 3-position of the ursodeoxycholic acid ester (VII) is inverted, causing the isoursodeoxycholic acid ester (VIII) is obtained.

The Mitsunobu reaction has been litera for more than 10 years turbekennt (see Mitsunobu, O., Synthesis, 1981, 1). In the classic Mitsunobu reaction becomes a combination of Triphenylphosphine and dialkyl azodicarboxylic acid inserted sets to alcohols for nucleophilic substitution reactions to activate. Recent investigations by D. Camp and I.D. 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 Tri butylphosphine, successfully used in the Mitsunobu reaction can be set.

According to a preferred embodiment of the fiction According to the procedure, the ursodeoxycholic acid ester (VII) is included Triphenylphosphine and Azodicarbonsäurediethylester solution in Presence of an organic carboxylic acid such as formic acid or benzoic acid.

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

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

After the reaction has ended, the reaction mixture is opened Cooled to room temperature, whereupon the reaction in the ge formed phosphine oxide precipitates, which is then filtered off becomes. A further purification of the isoursodeoxy obtained cholic acid ester (VII) is not required.

The stage of saponification [stage (iii)] of the invention The method can be carried out by methods familiar to the person skilled in the art be performed. Corresponding saponification processes are in every internship book of organic chemical laboratory technology, for example in Hünig, Märkl, Sauer, Integrated Organi internship, Verlag Chemie Weinheim. According to  a preferred embodiment of the Ver The isoursodeoxycholic acid ester (VIII) is first used implemented with methanolic KOH and then the Iso ursodeoxycholic acid (V) from its potassium salt by acidification released with dilute HCl.

Isoursodeoxycholic acid (V) is preferably replaced by Um crystallization or chromatographically cleaned. For this the isoursodeoxycholic acid in an inert, little po laren solvent, such as chloroform, slurried and on a chromatography column is applied. Then eluted the column with the same solvent, making the Verunreini still contained in the isoursodeoxycholic acid conditions are removed. As soon as there are no more impurities can be eluted, which can be recognized from the fact that in a last eluate, no residue when distilling off the chloroform If more remains, you can then elute with a pola ren solvent, such as acetone, around the purified Isoursodes to recover oxycholic acid. After filtering the solution the solvent is distilled off and the reaction pro product of solvent residues by steam distillation exempted. The reaction product, isoursodeoxycholic acid, is obtained in a yield of 50%.

Another, even more preferred form of workup exists in that the isoursodeoxychol obtained as a crude product acid is recrystallized. As a solvent for the order Examples of crystallization are ethyl acetate and mixtures genes of ethyl acetate with other solvents, such as in for example methanol, suitable. Preferably ethyl acetate used for recrystallization. Residual solvents can also in this cleaning process by expulsion with Water vapor (water vapor distillation) can be removed. This form of processing offers the advantage that the Isoursodeoxycholic acid with higher yield (58%) and with higher purity can be obtained by the higher  Melting point of the reaction product (161 to 163 ° C in Ver equal to 158 to 160 ° C in the by chromatography isoursodeoxycholic acid) is displayed.

The invention is illustrated by the example below purifies.

example 1. Preparation of the ursodeoxycholic acid methyl ester

10 g of ursodeoxycholic acid (VI) are placed in 50 ml for 3 hours 3% methanolic HCl heated under reflux. The reak tion mixture is left overnight. Subsequently become the volatile components on the rotary evaporator deducted to dryness of the reaction product. The back Stand is taken up in 100 ml of chloroform and with 30 ml Water, 30 ml saturated sodium carbonate solution and again washed with 30 ml of water. The solution is about magnesium dried sulfate and after filtering off the desiccant evaporated on a rotary evaporator. There will be 9.8 g Ursodes get oxycholic acid methyl ester (yield = 95% of theory), the a melting point of 153-155 ° C (not corrected) points.

2. Preparation of Isoursodeoxycholic Acid (V)

10 g of ursodeoxycholic acid methyl ester are absorbed in 120 ml lutem toluene and dissolved with 20 g triphenylphosphine and 4 ml 98% formic acid added. Become this mixture 40 ml diethyl azodicarboxylic acid solution (38-40% in To luol) added dropwise with stirring. The reaction is easy exothermic. After the reaction mixture for 48 hours is kept at 80 ° C, is cooled to room temperature. The triphenylphos formed in the reaction falls phinoxide, which is filtered off and discarded. The fil  is rotated in and the residue with 200 ml of 5% me ethanolic KOH refluxed for 3 hours. To cooling, 200 ml of water are added and the reacti The mixture is concentrated to half in vacuo at 40 ° C. The reaction mixture is filtered and the filtrate under Ice cooling acidified with dilute HCl. There will be 8.3 g isoursodeoxychol contaminated with triphenylphosphine oxide Acid (V) obtained.

The raw product is then air dried and then taken up in about 50 ml of chloroform, the most of the contamination goes into solution. After the Ab suck 6.8 g of isoursodesoycholic acid (V) are obtained.

A chromatography column (length 40 cm, diameter 3 cm) is ge with 30 g of silica gel slurried in chloroform fills and the isoursodeoxychol slurried in 150 ml acid applied. Then wash with 500 ml of Chlo roform or more until all impurities are removed which can be seen from the fact that in a last eluate at Ab Distilling the chloroform leaves no residue. The Acid is returned by rinsing the column with 500 ml acetone won. The acetone solution thus obtained is cloudy and must be filtered. After removal of the acetone on the rotation the residue is evaporated by steam distillation freed from solvent residues. There will be 4.8 g of Isoursodes oxycholic acid (yield = 50% of theory) with a melting point of 158-160 ° C (not corrected).

Another form of processing is that one Crude product recrystallized from ethyl acetate (for 10 g raw acid 100 ml ethyl acetate). This gives 5.6 g of acid (V) a melting point of 161 to 163 ° C. Solvent residues can only be expelled here with steam (What steam distillation).  

Analytical data:
3β, 7β-dihydroxy-5β-cholic acid (isoursodeoxycholic acid)

Melting point: 158 to 160 ° C
1 H-NMR ([D₆] 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 ([D₆] 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 (10)

1. A process for producing isoursodeoxycholic acid represented by the following formula (V) characterized by the levels:

• (i) Esterification of ursodeoxycholic acid to the ursodeoxycholic acid ester of the following formula (VII): wherein R is C₁-C₄ 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).

2. The method according to claim 1, characterized records that the ursodeoxycholic acid ester (VII) with triphenylphosphine and azodicarboxylic acid diethyl ester in the presence of an organic carboxylic acid such as formic acid or benzoic acid, to the isoursodeoxycholic acid ester (VIII) implements. 3. The method according to claim 1 or 2, characterized ge indicates that stage (i) in methano hCl. 4. The method according to claims 1 to 3, characterized ge indicates that stage (ii) in toluene or benzene at 70 to 110 ° C. 5. The method according to claims 1 to 4, characterized ge indicates that the isoursodeoxychol acid ester (VIII) first reacted with methanolic KOH and acidify the reaction product with dilute HCl.   6. The method according to claims 1 to 5, characterized characterized by a further level of chromatogra phical cleaning. 7. The method according to claim 6, characterized records that the chromatographic purification first with chloroform and then with acetone as the elu agent. 8. The method according to claims 1 to 5, characterized characterized by a further stage of cleaning by recrystallization. 9. The method according to claim 8, characterized records that one recrystallizes the Isour Sodeoxycholic acid (V) in ethyl acetate or in mixtures of Performs ethyl acetate and methanol. 10. The method of claims 6 to 9, characterized ge indicates that solvent residues Steam distillation removed.