A process for the preparation of 3beta,7beta-dihydroxy-5beta-cholanic acid

Abstract

The application discloses a preparation method of 3beta, 7beta-dihydroxy-5beta-cholanic acid. The method takes UDCA leftovers containing 3beta, 7beta-dihydroxy-5beta-cholanic acid as a starting raw material, mainly includes three steps of preparation of a taurine cholic acid mixture, separation and purification of 3beta, 7beta-dihydroxy-5beta-cholanic acid-N-taurine, and preparation of 3beta, 7beta-dihydroxy-5beta-cholanic acid. The method has the advantages of simple process route, few side reactions, high conversion rate, high product purity and suitability for mass production.

Description

Technical Field

[0001] This invention belongs to the fields of synthetic chemistry and biomedicine, specifically referring to a method for preparing 3β,7β-dihydroxy-5β-cholanic acid. Background Technology

[0002] Ursodeoxycholic acid (UDCA), chemically known as 3α,7β-dihydroxy-5β-cholanic acid, is a common drug primarily used to dissolve cholesterol gallstones, primary biliary cirrhosis (PBC), and chronic hepatitis C. It is also used for alcoholic liver disease, non-alcoholic fatty liver disease, benign recurrent intrahepatic cholestasis, and congenital cystic dilatation of the internal bile ducts.

[0003]

[0004] UDCA is mainly synthesized by oxidizing chenodeoxycholic acid (3α,7α-dihydroxy-5β-cholanic acid, abbreviated as CDCA) to 7-ketolithocholic acid, followed by reduction to 7β-hydroxy UDCA. Since chenodeoxycholic acid has hydroxyl groups at both positions 3 and 7, the oxidation at position 7 may cause the hydroxyl group at position 3 to be oxidized simultaneously, generating 3,7-diketo-5β-cholanic acid. In the subsequent reduction process, 3β,7β-dihydroxy-5β-cholanic acid may be generated. The synthetic route is shown below:

[0005]

[0006] In fact, most commercially available UDCA contains small amounts of 3β,7β-dihydroxy-5β-cholanic acid impurities. Therefore, the European Pharmacopoeia lists it as a related substance (impurity number H) in its quality standards for ursodeoxycholic acid and controls it accordingly. Although included in the European Pharmacopoeia's list of related substances, the European Pharmacopoeia does not provide a separate, high-purity reference standard for 3β,7β-dihydroxy-5β-cholanic acid impurities. Instead, it only provides a mixture of 3β,7β-dihydroxy-5β-cholanic acid (impurity number H) and chenodeoxycholic acid (impurity number C) as a system suitability control sample. High-purity 3β,7β-dihydroxy-5β-cholanic acid impurities are also rarely available on the market.

[0007] In terms of preparation, when cholanic acid has a carbonyl group at the 3-position, its reduction to a hydroxyl group is still mainly in the α-conformation, so its synthesis is somewhat difficult.

[0008] In the production of UDCA, samples containing 3β,7β-dihydroxy-5β-cholanic acid can be purified and enriched from the by-products through continuous crystallization. However, when the area-normalized purity of 3β,7β-dihydroxy-5β-cholanic acid in the sample is comparable to that of UDCA, the purification effect of crystallization is no longer significant, and further purification and separation to improve purity becomes extremely difficult. When using silica gel column chromatography and C18 column chromatography to separate by-product samples, it is difficult to prepare large quantities of samples with high purity because the retention behavior of 3β,7β-dihydroxy-5β-cholanic acid is too similar to that of UDCA.

[0009] Our company is a manufacturer of UDCA raw materials. We currently have a large amount of by-products rich in 3β,7β-dihydroxy-5β-cholanic acid. Treating these as chemical waste would put pressure on environmental protection efforts, while utilizing them could turn waste into treasure. Furthermore, given that 3β,7β-dihydroxy-5β-cholanic acid is a potential impurity in UDCA products, preparing high-purity samples is crucial for quality control of UDCA drugs and improving the quality of subsequent drug products. It also allows for further pharmacological activity studies, revealing its efficacy in treating diseases such as cholesterol gallstones and primary sclerosing cholangitis, which is of great significance. Summary of the Invention

[0010] The purpose of this invention is to provide a method for preparing 3β,7β-dihydroxy-5β-cholanic acid from UDCA by-products containing 3β,7β-dihydroxy-5β-cholanic acid as starting material. This method has a simple process route, few side reactions, high conversion rate, high product purity, and is suitable for large-scale preparation.

[0011] The starting material of this invention is a UDCA by-product containing 3β,7β-dihydroxy-5β-cholanic acid, mainly composed of cholic acid components, primarily consisting of 3β,7β-dihydroxy-5β-cholanic acid, UDCA (ursodeoxycholic acid), CDCA (chenodeoxycholic acid), and other impurities. Since the hydroxyl groups at positions 3 and 7 of 3β,7β-dihydroxy-5β-cholanic acid, UDCA, and CDCA exhibit different α- and β-hydroxyl forms, their structural formula can be represented by general formula (V):

[0012]

[0013] in:

[0014] UDCA 3α-OH, 7β-OH

[0015] CDCA 3α-OH, 7α-OH

[0016] 3β,7β-Dihydroxy-5β-cholanoic acid 3β-OH,7β-OH.

[0017] This invention discloses a method for preparing 3β,7β-dihydroxy-5β-cholanic acid, comprising the following steps:

[0018] (1) Preparation of taurocholic acid mixture

[0019] The UDCA by-product containing 3β,7β-dihydroxy-5β-cholanic acid was first activated with acyl chloride to generate mixed acid anhydrides, which were then condensed with taurine to prepare a taurcholic acid mixture.

[0020] (2) Isolation and purification of 3β,7β-dihydroxy-5β-cholanyl-N-taurine

[0021] The mixture of taurocholic acid compounds was separated into 3β,7β-dihydroxy-5β-cholanyl-N-tauroic acid by C18 column chromatography;

[0022] (3) Preparation of 3β,7β-dihydroxy-5β-cholanic acid

[0023] Hydrolyzing 3β,7β-dihydroxy-5β-cholanoyl-N-taurine yields 3β,7β-dihydroxy-5β-cholanoic acid.

[0024] The structural formula of 3β,7β-dihydroxy-5β-cholanyl-N-taurine is as follows:

[0025]

[0026] In this invention, the UDCA by-product containing 3β,7β-dihydroxy-5β-cholanic acid is composed of the following components by mass percentage, wherein:

[0027]

[0028] This invention discloses a method for preparing 3β,7β-dihydroxy-5β-cholanic acid, which specifically includes the following steps:

[0029] (1) Preparation of taurocholic acid mixture

[0030] a. Suspend UDCA by-products (Formula I) containing 3β,7β-dihydroxy-5β-cholanic acid in a nonpolar solvent, add pentanoyl chloride, then add triethylamine dropwise, and react at -5°C to generate a mixed anhydride (Formula II) solution.

[0031] The ratio of UDCA by-products containing 3β,7β-dihydroxy-5β-cholanic acid, nonpolar solvent, pentanoyl chloride, and triethylamine is 392g:2-8L:1.0-1.5mol:1.0-1.5mol.

[0032] The nonpolar solvent is an aprotic solvent that is immiscible with water. Further, the nonpolar solvent is one of dichloromethane, toluene, and C5-C8 alkanes. More preferably, dichloromethane is used.

[0033] b. Extract the mixed anhydride solution obtained in step a with purified water at 0-10℃, take the organic phase and add taurine solution, stir and react to obtain a taurine sodium salt (Formula III) solution;

[0034] The taurine solution is prepared from taurine, sodium hydroxide, and water. The ratio of UDCA by-products of 3β,7β-dihydroxy-5β-cholanic acid to taurine, sodium hydroxide, and water is 392g:1-1.5mol:1-1.5mol:300-500g.

[0035] c. Extract the sodium taurocholic acid solution obtained in step b with water, take the aqueous phase, add hydrochloric acid to adjust the pH to ≤3, precipitate the solid, filter, and dry to obtain a taurocholic acid mixture (Formula IV).

[0036] The reaction process for preparing the taurocholic acid mixture is shown in route (A):

[0037]

[0038] (2) Isolation and purification of 3β,7β-dihydroxy-5β-cholanyl-N-taurine

[0039] C18 bonded silica gel was soaked in acetonitrile, packed into a chromatography column, and equilibrated with acetonitrile-water containing trifluoroacetic acid as the eluent. The taurine mixture was dissolved in acetonitrile-water, loaded onto the column, and then eluted with the eluent. The eluent was collected and evaporated to dryness. The solid was dissolved in ethanol and precipitated with acetone to obtain 3β,7β-dihydroxy-5β-cholanyl-N-taurine.

[0040] The eluent contains 0.01% to 1% trifluoroacetic acid, more preferably 0.1%; and 10% to 45% acetonitrile, more preferably 20% to 35%.

[0041] This invention employs reversed-phase column chromatography to separate and purify 3β,7β-dihydroxy-5β-cholanyl-N-taurine. Since 3β,7β-dihydroxy-5β-cholanic acid exhibits similar retention behavior to UDCA and CDCA in silica gel and C18 chromatography, and their solubility in commonly used solvents is also extremely similar, conventional methods are difficult to use for separation. However, after 3β,7β-dihydroxy-5β-cholanic acid combines with taurine to form a bound cholic acid, its retention behavior on C18 is significantly different from that of tauroursodeoxycholic acid and taurourchedeoxycholic acid. This allows for a simple and effective separation of 3β,7β-dihydroxy-5β-cholanyl-N-taurine. Furthermore, 3β,7β-dihydroxy-5β-cholanic acid can be obtained by a single hydrolysis step.

[0042] (3) Preparation of 3β,7β-dihydroxy-5β-cholanic acid

[0043] Sodium hydroxide and water were added to 3β,7β-dihydroxy-5β-cholanoyl-N-taurine, and the mixture was subjected to reflux hydrolysis. After the reaction was completed, the pH of the solution was slowly adjusted to 3-4 with hydrochloric acid, and a white solid precipitated. The solid was filtered, the filter cake was washed with water, and dried to obtain 3β,7β-dihydroxy-5β-cholanoic acid.

[0044] The weight ratio of the 3β,7β-dihydroxy-5β-cholanoyl-N-taurine to the added sodium hydroxide and water is 1:0.5 to 2:5 to 20.

[0045] The reflux hydrolysis temperature is 90–110°C, and the hydrolysis time is 4–10 h.

[0046] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0047] (1) The starting material used in this invention is UDCA by-product, which mainly contains cholic acid components such as 3β,7β-dihydroxy-5β-cholanic acid, UDCA, and CDCA. The raw materials are abundant and inexpensive. After separating 3β,7β-dihydroxy-5β-cholanic acid, the tailings are a mixture of UDCA and CDCA, which can be further separated and recycled. This method can effectively improve the comprehensive utilization rate of materials and reduce environmental pressure.

[0048] (2) The synthesis conditions of this invention are mild and simple, environmentally friendly, and have a high conversion rate, making it suitable for large-scale preparation.

[0049] (3) The present invention employs a method for separating and purifying 3β,7β-dihydroxy-5β-cholanyl-N-taurine by reversed-phase column chromatography, which is suitable for the preparation of large samples and the purity of the separated samples reaches more than 98.5%.

[0050] Instruction manual illustrations

[0051] Figure 1The image shows the proton NMR spectrum of the 3β,7β-dihydroxy-5β-cholanyl-N-taurine sample. Detailed Implementation

[0052] The present invention will be further described below with reference to specific embodiments, but is not limited thereto.

[0053] Example 1

[0054] 4.70 g of UDCA by-products containing 3β,7β-dihydroxy-5β-cholanic acid (3β,7β-dihydroxy-5β-cholanic acid area normalized percentage 38%) was suspended in 50 ml of dichloromethane. The mixture was cooled to below -5°C with stirring. 1.8 ml of pentanoyl chloride was added, followed by the slow addition of 2.0 ml of triethylamine. The reaction was maintained below -5°C for 1 h. After the reaction was complete, 100 ml of purified water pre-cooled to 0–5°C was added, and the mixture was slowly stirred and extracted. The organic phase was separated, and then extracted twice more with 100 ml of purified water. After extraction, the organic phase was added to an aqueous solution containing 1.8 g of taurine, 4.8 g of water, and 0.58 g of sodium taurate. The mixture was stirred at 30°C for 3 h. After the reaction was complete, 2.5 g of purified water was added, the mixture was stirred, and the layers separated. The aqueous phase was collected, and 1.4 ml of concentrated hydrochloric acid was added. The mixture was stirred to precipitate a solid, which was then filtered and dried to obtain 5.2 g of taurine-like substances.

[0055] Weigh 50g of C18 bonded silica gel for chromatography, soak it in acetonitrile for 1 hour, and then pack it into a chromatography column. Equilibrate with 5 column volumes of eluent (acetonitrile:water:trifluoroacetic acid = 25:75:0.1). Dissolve 5.0g of taurine derivatives in 10ml of 25% acetonitrile, load the sample, and elute with eluent (acetonitrile:water:trifluoroacetic acid = 25:75:0.1), collecting one 10ml vial. Monitor the eluent by HPLC, combine the eluents mainly containing 3β,7β-dihydroxy-5β-cholanyl-N-taurine, and evaporate to dryness. Dissolve the solid in ethanol, precipitate with acetone, and obtain 1.5g of 3β,7β-dihydroxy-5β-cholanyl-N-taurine with a purity of 98.8%. 1H NMR (400MHz, DMSO) δ7.67(s,1H,25-NH-),3.98–3.68(m,6H,3-CH,7-OH,3-OH,H2O,28-SO3H),3.28(dd,J=13.3,6.1Hz,3H,7-CH,26-CH2),2.09(s ,2H,27-CH2),2.07–2.00(m,1H,23-CH2),1.97–1.86(m,2H,23-CH2,12-CH2),1.85–1.78(m,1H,-15-CH2),1.69(td,J=13.4,4.0Hz,5H,16-CH2,2 2-CH2,1-CH2,6-CH2,2-CH2),1.34(dt,J=16.7,10.7Hz,11H,4-CH2,6-CH2,9-CH,11-CH2,5CH,15-CH2,20-CH,8-CH,22-CH2,16-CH2),1.15(dt,J =20.8,10.0Hz,5H,11-CH2,14-CH,12-CH2,2-CH2,17-CH),0.99(q,J=9.4Hz,1H,1-CH2),0.92–0.82(m,6H,21-CH3,19-CH3),0.61(s,3H,18-CH3). See the hydrogen spectrum Figure 1 .

[0056] Add 1.5g of 3β,7β-dihydroxy-5β-cholanoyl-N-taurine from the previous step to 1.5g of sodium hydroxide and 15ml of purified water, and saponify at 100℃ for 5h. Adjust the pH of the saponified solution to 3-4 with hydrochloric acid, and a white solid precipitates. Filter, rinse the filter cake with purified water, and dry to obtain 1.0g of 3β,7β-dihydroxy-5β-cholanoic acid.

[0057] Example 2

[0058] 10.8 g of UDCA by-products containing 3β,7β-dihydroxy-5β-cholanic acid (3β,7β-dihydroxy-5β-cholanic acid area normalized percentage 25%) was suspended in 100 ml of dichloromethane. The mixture was cooled to below -5°C with stirring. 4.0 ml of pentanoyl chloride was added, followed by the slow addition of 4.6 ml of triethylamine. The reaction was maintained below -5°C for 1 h. After the reaction was complete, 200 ml of purified water pre-cooled to 0–5°C was added, and the mixture was slowly stirred and extracted. The organic phase was separated, and then extracted twice more with 200 ml of purified water. After extraction, the organic phase was added to an aqueous solution of sodium taurate containing 4.2 g of taurine, 10.8 g of water, and 1.32 g of sodium hydroxide. The mixture was stirred at 25°C for 5 h. After the reaction was complete, 9.2 g of purified water was added, the mixture was stirred, and the layers separated. The aqueous phase was collected, and 3.4 ml of concentrated hydrochloric acid was added. The mixture was stirred to precipitate a solid, which was then filtered and dried to obtain 11.2 g of taurine-like substances.

[0059] Weigh 100g of C18 bonded silica gel for chromatography, soak it in acetonitrile for 1 hour, then pack it into a chromatography column and equilibrate it with 5 column volumes of eluent (acetonitrile:water:trifluoroacetic acid = 30:70:0.1). Dissolve 11.2g of taurocholic acid in 20ml of 30% acetonitrile, load the sample, and elute with eluent. Collect one 10ml vial per flask, monitor the eluent by HPLC, combine the eluents containing the main product, and evaporate to dryness. Dissolve the solid in ethanol, precipitate with acetone, and obtain 2.2g of 3β,7β-dihydroxy-5β-cholanyl-N-taurine with a purity of 99.2%.

[0060] In the previous step, 2.2 g of 3β,7β-dihydroxy-5β-cholanoyl-N-taurine was added to 2.2 g of sodium hydroxide and 22 ml of purified water, and saponified at 100 °C for 5 h. The saponification solution was adjusted to pH 3-4 with hydrochloric acid, and a white solid precipitated. The solid was filtered, the filter cake was washed with purified water, and dried to obtain 1.5 g of 3β,7β-dihydroxy-5β-cholanoic acid.

Claims

1. A method for preparing 3β,7β-dihydroxy-5β-cholanic acid, characterized in that... Includes the following steps: (1) Preparation of taurocholic acid mixture The UDCA by-product containing 3β,7β-dihydroxy-5β-cholanic acid was first activated with acyl chloride to generate mixed acid anhydrides, and then condensed with taurine solution to prepare a taurochlic acid mixture. (2) Isolation and purification of 3β,7β-dihydroxy-5β-cholanyl-N-taurine The mixture of taurocholic acid compounds was separated into 3β,7β-dihydroxy-5β-cholanyl-N-tauroic acid by C18 column chromatography; (3) Preparation of 3β,7β-dihydroxy-5β-cholanic acid Hydrolyzing 3β,7β-dihydroxy-5β-cholanoyl-N-taurine yields 3β,7β-dihydroxy-5β-cholanoic acid. In step (1), the UDCA by-product containing 3β,7β-dihydroxy-5β-cholanic acid is composed of the following components by mass percentage, wherein: 。 2. The method for preparing 3β,7β-dihydroxy-5β-cholanic acid according to claim 1, characterized in that, In step (1), the preparation of the taurocholic acid mixture includes the following steps: a. Suspend UDCA by-products (Formula I) containing 3β,7β-dihydroxy-5β-cholanic acid in a nonpolar solvent, add pentanoyl chloride, then add triethylamine dropwise, and react at -5°C to generate a mixed anhydride (Formula II) solution. b. Extract the mixed anhydride solution obtained in step a with purified water at 0-10℃, take the organic phase and add taurine solution, stir and react to obtain a taurine sodium salt (Formula III) solution; c. Extract the sodium taurocholic acid solution obtained in step b with water, take the aqueous phase, add hydrochloric acid to adjust the pH to ≤3, precipitate the solid, filter, and dry to obtain a taurocholic acid mixture (Formula IV). The reaction process for preparing the taurocholic acid mixture is shown in route (A): 。 3. The method for preparing 3β,7β-dihydroxy-5β-cholanic acid according to claim 2, characterized in that: In step a, the nonpolar solvent is one of dichloromethane, toluene, or C5-C8 alkanes.

4. The method for preparing 3β,7β-dihydroxy-5β-cholanic acid according to claim 3, characterized in that: In step a, the ratio of the UDCA by-product containing 3β,7β-dihydroxy-5β-cholanic acid, the nonpolar solvent, the pentanoyl chloride, and the triethylamine is 392g:2~8L:1.0~1.5mol:1.0~1.5mol.

5. The method for preparing 3β,7β-dihydroxy-5β-cholanic acid according to claim 2, characterized in that: In step b, the taurine solution is prepared from taurine, sodium hydroxide, and water. The ratio of the UDCA by-product of 3β,7β-dihydroxy-5β-cholanic acid to taurine, sodium hydroxide, and water is 392g:1-1.5mol:1-1.5mol:300-500g.

6. The method for preparing 3β,7β-dihydroxy-5β-cholanic acid according to claim 1, characterized in that, The specific operation method for step (2) is as follows: C18 bonded silica gel was soaked in acetonitrile and packed into a chromatography column. The column was equilibrated with acetonitrile-water containing trifluoroacetic acid as the eluent. The taurine mixture was dissolved in acetonitrile-water, loaded onto the column, and then eluted with the eluent. The eluent was collected and evaporated to dryness. The solid was dissolved in ethanol and precipitated with acetone to obtain 3β,7β-dihydroxy-5β-cholanyl-N-taurine.

7. The method for preparing 3β,7β-dihydroxy-5β-cholanic acid according to claim 6, characterized in that: The eluent contains 0.01% to 1% trifluoroacetic acid and 10% to 45% acetonitrile.

8. The method for preparing 3β,7β-dihydroxy-5β-cholanic acid according to claim 1, characterized in that, The specific operation method of step (3) is as follows: Sodium hydroxide and water were added to 3β,7β-dihydroxy-5β-cholanoyl-N-taurine, and the mixture was subjected to reflux hydrolysis. After the reaction was completed, the pH of the solution was slowly adjusted to 3-4 with hydrochloric acid, and a white solid was precipitated. The solid was filtered, the filter cake was washed with water, and dried to obtain 3β,7β-dihydroxy-5β-cholanoic acid.

9. The method for preparing 3β,7β-dihydroxy-5β-cholanic acid according to claim 8, characterized in that: The weight ratio of the 3β,7β-dihydroxy-5β-cholanoyl-N-taurine to the added sodium hydroxide and water is 1:0.5 to 2:5 to 20.

Images