GB2595421A - Chenodeoxycholic acid and preparation method therefor - Google Patents

Abstract

The present application relates to the field of drug synthesis and specifically relates to chenodeoxycholic acid and a preparation method therefor. The preparation method for the chenodeoxycholic acid comprises the following steps: forming an intermediate E after a chemical reaction using 3α, 7α-dihydroxy-5α-cholanic acid as a raw material, and then subjecting the intermediate E to a chemical reaction to form chenodeoxycholic acid; the structural formula of the intermediate E is as below, wherein R1 is an alkyl group, an alkenyl group, or an aromatic group, and R2 is an acyl group. The preparation method can quickly synthesise chenodeoxycholic acid using an extraction by-product as a raw material; the conditions of each step in the process are mild and the yield is high, being suitable for large-scale preparation.

Description

CHENODEOXYCHOLIC ACID AND PREPARATION METHOD

THEREFOR

This application is based on Chinese Patent Application No. 201910167593.3 filed on March 6, 2019, and claims its priority. The entire content of this application is incorporated herein in its entirely.

TECHNICAL FIELD

This application relates to the field of drug synthesis, and in particular, to a chenodeoxycholic acid and a preparation method therefor.

BACKGROUND

Bile acids are important endogenous molecules related to countless biological functions, including absorption and excretion of cholesterol, which play an important role in fat metabolism. Chenodeoxycholic acid is a natural primary bile acid, which is widely present in the bile of human, livestock, and poultry, and is the main component in the bile of poultry such as chickens, ducks, and geese. Since Thistle and Schoenfirld find that chenodeoxycholic acid can be used to treat gallstones, the clinical application of chenodeoxycholic acid has been continuously discovered. Since the 1970s, chenodeoxycholic acid has been mainly used for the treatment of gallstone diseases and other hepatobiliary diseases. Subsequently, it has been discovered that chenodeoxycholic acid can not only be used to treat. various hepatobiliary diseases, but also has medicinal values of anti-bacteria, anti-inflammation, anti-asthma and anti-tussis, promoting digestion in the digestive system, treating cerebrotendinous xanthomatosis, and insulin resistance. In addition, chenodeoxycholic acid is also an important raw material for the synthesis of ursodeoxycholic acid and obeticholic acid. Therefore, with the expansion of medicinal value and the expansion of demand for ursodeoxycholic acid and obeticholic acid, the demand for chenodeoxycholic acid is also increasing.

The current chemically synthetic pathway is mainly to use cholic acid and hyodeoxycholic acid as raw materials for synthesis. There are two pathways for synthesis with cholic acid as a raw material. One is to prepare chenodeoxycholic acid by reducing the carbonyl group at position 12; the other is to prepare chenodeoxycholic acid by hydrogenating the CI I olefin. The yield of chenodeoxycholic acid prepared through these two pathways can reach 40% after process optimization. At present, the yield of chenodeoxycholic acid prepared through the synthetic pathway using hyodeoxycholic acid as a raw material is 26%, but this pathway uses chloranil, which has a great impact on the environment.

SUMMARY

This application provides a method for preparing chenodeoxycholic acid. The preparation method can quickly synthesize chenodeoxycholic acid using an extracted by-product as a raw material, which has mild conditions in each step of the process, has a high yield, and is suitable for large-scale preparation.

This application further provides a chenodeoxycholic acid prepared by the foregoing method.

This application is implemented as follows: A method for preparing chenodeoxycholic acid includes the following steps: allowing 3a,7a-dihydroxy-5a-cholanic acid as a raw material to undergo a chemical reaction to form an intermediate E. and then subjecting the intermediate E to a chemical reaction to form chenodeoxycholic acid, the structural formula of the intermediate E being as R, orr' follows: N2, where RI is an alkyl group, an alkenyl group, or an aryl group, and R2 is an acyl group.

A chenodeoxycholic acid is prepared by the foregoing method for preparing chenodeoxycholic acid.

The beneficial effects of this application are as follows: By using a by-product extracted from duck galls and goose galls as a raw reaction material, this application can achieve waste utilization, reduce synthesis costs, and have a wide source of the raw material. In addition, the chenodeoxycholic acid obtained through the foregoing steps has a high yield up to 32%, which is suitable for large-scale preparation. Moreover, the preparation method has simple operation, strong repeatability, and extremely strong practicability.

BRIEF DESCRIPTION OF THE DRAWINGS

To describe the technical solutions in the specific implementations of this application or in the related art more clearly, the following briefly describes the accompanying drawings required for describing the specific implementations or the related art. Apparently, the accompanying drawings in the following description show some implementations of this application, and a person of ordinary skill in the art may still derive other drawings from these accompanying drawings without creative efforts.

FIG. 1 is a diagram of a synthetic route of a chenodeoxycholic acid according to an embodiment of this application.

DETAILED DESCRIPTION

To make the objectives, technical solutions, and advantages of embodiments of this application more comprehensible, the following clearly and completely describes the technical solutions in the embodiments of this application. Where no specific conditions are given in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. The reagents or instruments for which no manufacturers are noted are all common products commercially available from the market.

The following specifically describes a chenodeoxycholic acid and a preparation method therefor according to the embodiments of this application.

Referring to FIG. 1, a method for preparing chenodeoxycholic acid includes the following steps: Chemical reactions are carried out according to the following: ft ft

OH (A)

OH (B) (C) Ri b HO'

R2 R2 R2 (H) (0) (F) (E) In an embodiment of this application, 3a.,7a-dihydroxy-5a-cholanic acid (allochenodeoxycholic acid) (A) as a raw material is esterified to obtain 3a,7a-clihydroxy-5a-cholanate ester (B), the 3a,7a-dihydroxy-5a-cholanate ester (B) is selectively oxidized at position 3 to obtain 3-keto-7a-hydroxy-5a-cholanate ester (C), the 3-keto-7a-hydroxy-5a-cholanate ester (C) is protected at position 7 to obtain 3-keto-7a-acyloxy-5a-cholanate ester (D), the 3-keto-7a-acyloxy-5a-cholanate ester (D) undergoes an oxidation reaction to obtain A1,4-3-keto-7a-acyloxy-unsaturated cholanate ester (E), the A1,4-3-keto-7a-acyloxy-unsaturated cholanate ester (E) undergoes catalytic hydrogenation to reduce a double bond to obtain 3-keto-7a-acyloxy-50-cholanate ester (F), the 3-keto-7a-acyloxy-5f3-cholanate ester (F) is reduced at position 3 to obtain 3a-hydroxy-7a-acyloxy-5P-cholanate ester (G), and the 3a-hydroxy-7a-acyloxy-5P-cholanate ester (G) is hydrolyzed and deprotected to obtain a chenodeoxycholic acid (H).

The raw material used is 3a,7a-dihydroxy-Su-cholanic acid, which is a by-product extracted from duck galls and goose galls. By using die foregoing discarded by-product as the raw material, the chenodeoxycholic acid can be quickly prepared. Waste is used as the raw material, which has a wide source and sufficient supply, and can also reduce synthesis costs. In addition, the chenodeoxycholic acid obtained through the foregoing steps has a high yield up to 32%, which is suitable for large-scale preparation.

In an intermediate E in the foregoing steps, R1 is an alkyl group, an alkenyl group, or an aryl group, and R2 is an acyl group. The intermediate E is an intermediate obtained by allowing 3a,7a-dihydroxy-5a-cholanic acid to undergo first csterification, oxidation, second esterification, and dehydrogenation.

S 1. Prepare an intermediate B. A first esterification is an esterification between 3a,7a-dihydroxy-5a-cholanic acid and alcohol, specifically, is to allow 3a,7a-dihydroxy-5a-cholanic acid to react with alcohol at 50-90°C under the action of a catalyst to obtain the inteimediate B. The temperature is preferably 60-70°C, and the reaction temperature may be within the foregoing range and may be adjusted according to actual needs. For example, the reaction temperature may be 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, and the like.

The reaction time is 2-6 h. The reaction time may be adjusted according to the progress of the esterification to complete the esterification. For example, the reaction time may be 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, 6 h, and the like.

The catalyst is an acidic substance, more preferably any one of concentrated hydrochloric acid, concentrated sulfuric acid, benzenesulfonic acid, methanesulfonic acid, and p-toluenesulfonic acid, and further preferably concentrated hydrochloric acid or concentrated sulfuric acid. The use of the foregoing catalyst can ensure its catalytic effect and the formation of the intermediate B. The molar ratio of the 3a,7a-dihydroxy-5a-cholanic acid, the alcohol, and the catalyst is 1:2-30:0.1-1, which can ensure that the 3a,7a-dihydroxy-5a-cholanic acid is smoothly esterified within this range. For example, in different implementations, the molar ratio of the 3a,7a-dihydroxy-5a-cholanic acid to the alcohol may be 1:2, 1:4, 1:6, 1:8, 1:10, 1:12, 1:14, 1:16, 1:18, 1:20, 1:25, 1:30, and the like, and the molar ratio of the 3a,7a-dihydroxy-5a-cholanic acid to the catalyst may be 1:0.1, 1:0.3, 1:0.5, 1:0.7, 1:0.9, 1:1, and the like.

Further, the alcohol is monoalcohol, more preferably Cl-C10 monoalcohol, further preferably methanol, ethanol, isopropanol, ally] alcohol, or benzyl alcohol, and most preferably methanol or isopropanol. The use of the foregoing alcohol can ensure the yield of the prepared chenodeoxycholic acid.

After the reaction, the reaction solution is purified. The purification is to reduce the content of impurities in the intermediate B, thereby ensuring the purity of the prepared chenodeoxycholic acid and ensuring the yield thereof.

The purification method is as follows: After the reaction, the reaction solution obtained after the first esterification is cooled to room temperature, methanol is removed, ethyl acetate is added, the mixture is washed with a saturated sodium bicarbonate solution and a saturated sodium chloride solution sequentially and is then dried, and a solvent is removed, to obtain the intermediate B. The mass of the added ethyl acetate is preferably 10-20 times that of the raw material 3a,7a-clihydroxy-5a-cholanic acid, so as to ensure the purification effect.

S2. Prepare an intermediate C. The prepared intermediate B reacts with a first oxidizing agent for 2-36 h to form the intermediate C, which is oxidation. The oxidation is selective oxidation at position 3.

Specifically, a compound B, the first oxidizing agent, diatomaceous earth, and a first organic solvent are mixed and reacted under reflux, to obtain a compound C. The foregoing method ensures a high yield of the prepared intermediate C. Further, the first oxidizing agent is any one of peroxide, metal compound, or bromoimide; more preferably, the peroxide is peroxybenzoic acid or hydrogen peroxide; the metal compound is any one of sodium hypochlorite, silver carbonate, manganese dioxide, or chromium trioxide; the bromoimide is N-bromosuccinimide; and most preferably, the first oxidizing agent is sodium hypochlorite, silver carbonate, or manganese dioxide. The use of the foregoing oxidizing agent can ensure the oxidation effect of the oxidation, thereby ensuring the synthesis rate and yield of the intermediate C. Further, the first organic solvent includes toluene, dichloromethane, tetrahydrofuran, chloroform, and acetonitrile, preferably toluene. The use of the foregoing first organic solvent can provide a good reaction atmosphere for the oxidation to ensure the smooth progress of the reaction.

The molar ratio of the intermediate B to the first oxidizing agent is 1:0.5-3, preferably 1:0.8-2. For example, in different implementations, the molar ratio may be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, and the like. The molar ratio of the intermediate B to the diatomaceous earth is 1:5-15. For example, in different implementations, the molar ratio may be 1:6, 1:8, 1:10, 1:12, 1:15, and the like. Preferably, the mass ratio of the intermediate B to the first organic solvent is 1:5-20. The mass ratio may be adjusted within this range. Using the foregoing ratio can ensure the smooth progress of the reaction process and ensure the yield of the intermediate C. Further, after the reaction, the reaction solution obtained from the oxidation is purified to form the intermediate C. The purification method is as follows: After the reaction, the reaction solution from the oxidation is filtered, the filtrate is collected and concentrated to obtain a residual sample, and the residual sample is recrystallized with methanol and water in a ratio of 5:1 and then filtered to obtain the intermediate C. S3. Prepare an intermediate D. The intermediate C reacts with 4-dimethylaminopyridine, triethylamine, and organic acid anhydride for 1-15 h, and then the pH of the reaction solution is adjusted to 6-7, to form the intermediate D, which is second esterification. The second esterification is esterification at position 7. Specifically, a compound C is dissolved in a second organic solvent, 4-dimethylaminopyridine, triethylaminc, and organic acid anhydride are added to react at room temperature for 1-15 h, then the reaction solution is added to water to adjust the pH to 6-7, and an organic phase is collected through fractionation, to obtain the intermediate D. The second organic solvent is ethyl acetate, and the organic acid anhydride is acetic anhydride.

Further, the pH is adjusted by hydrochloric acid with a concentration of 1-3 M. The molar ratio of the intermediate C, the 4-dimethylaminopyridine, the triethylamine, and the organic acid anhydride is 1:0.01-0.5:1.5-3:0.8-4. The mass ratio of the intermediate C to the second organic solvent is 1:10-15. Using the foregoing ratio can ensure the smooth progress of the second esterification and ensure the yield of the intermediate D. Further, the organic phase obtained from the second esterification is purified to form the intermediate D. The purification method is as follows: The organic phase is washed with a saturated sodium bicarbonate solution and a saturated sodium chloride solution and is then dried, and a solvent is removed, to obtain the intermediate D. S4. Prepare an intermediate E. The intermediate D reacts with a second oxidizing agent at 20-90°C for 3-72 h, which is dehydrogenation. The dehydrogenation is a reaction of simultaneous dehydrogenation and oxidation at positions 1, 2, and 4, 5 to form olefin. Specifically, the intermediate D is dissolved in a third organic solvent, the second oxidizing agent and trifluoroacetic acid are added, and the mixture is heated to react under the protection of nitrogen, to obtain the intermediate E. The third organic solvent is preferably dimethyl sulfoxide. The second oxidizing agent is an iodine reagent, preferably a hypervalent iodine reagent, and more preferably 2-iocloxybenzoic acid. The use of the foregoing second oxidizing agent and third organic solvent can ensure the production efficiency of the intermediate E. The molar ratio of the intermediate D to the second oxidizing agent is 1:2-5. The molar ratio of the intermediate D to the trilluoroacetic acid is 1:0.1-0.5, and the mass ratio of the intermediate D to the third organic solvent is 1:10-35.

Further, after the reaction, the reaction solution from the dehydrogenation is purified. The purification method is as follows: The reaction solution is added into water and extracted with ethyl acetate, an organic phase is collected through fractionation, the organic phase is washed with water and a saturated sodium chloride solution and is then dried, a solvent is removed, and separation is carried out by column chromatography, to obtain the intermediate E. Further preferably, an eluant of the column chromatography separation is ethyl acetate and petroleum ether, and the volume ratio of die ethyl acetate to the petroleum ether is 1:10-20. The use of the foregoing purification method can further ensure the purification effect, and then ensure the purity and yield of chenodeoxycholic acid prepared subsequently.

After the foregoing operations, the intermediate E is obtained, and then the intermediate E sequentially undergoes catalytic hydrogenation and reduction, reduction at position 3, and hydrolysis to obtain the chenodeoxycholic acid.

55. Prepare an intermediate F. The catalytic hydrogenation and reduction is a reaction of simultaneous hydrogenation and reduction at positions 1, 2, and 4, 5. Specifically, the intermediate E is mixed with a catalyst and triethylaminc to undergo hydrogenation at 20-60°C for 0.5-4 h to form the intermediate F. Specifically, the intermediate E, the catalyst, an alcohol solvent, and the triethylamine react under a reaction pressure of 0.8-1.2 MPa. The catalyst is palladium on carbon, preferably 5% of palladium on carbon. The alcohol solvent is methanol.

Preferably, the molar ratio of the intermediate E. the catalyst, and the triethylamine is 1:2-10:0.001-0.5, and the mass ratio of the intermediate E to the alcohol solvent is 1:10-20. Using the foregoing ratio can ensure the smooth progress of the catalytic hydrogenation and reduction and ensure the yield of the intermediate F. Then, the reaction solution obtained from the catalytic hydrogenation and reduction is purified. The purification method is as follows: After the reaction, the alcohol solvent undergoes rotary drying, the resulting solution is dissolved with ethyl acetate and then is washed with hydrochloric acid and a saturated sodium chloride solution, and a solvent is removed, to obtain the intermediate F. The mass of the added ethyl acetate is preferably 20-30 limes that of the intermediate F. During washing, similar volumes of hydrochloric acid with a concentration of 1-3 M and the saturated sodium chloride solution are used for washing, preferably in the same volume. The foregoing purification method can ensure the yield and purity of the prepared intermediate F. S6. Prepare an intermediate G The reduction at position 3 is to allow the intermediate F to react with a reducing agent. The reduction at position 3 refers to that a carbonyl group at position 3 undergoes catalytic hydrogenation to obtain a hydroxyl group.

Specifically, the intermediate F is dissolved in a fourth organic solvent, and the reducing agent is added at 0-10°C to react at room temperature, to obtain the intermediate G. The reducing agent is a hydride, more preferably sodium borohydride. The fourth organic solvent is preferably anhydrous methanol. The reaction time is monitored by thin-layer chromatography (TLC). A developing agent is ethyl acetate and petroleum ether with a volume ratio of 1:6-1:2. The reaction by using the foregoing method can ensure the smooth progress of the reaction and ensure the yield of the prepared intermediate G. The molar ratio of the intermediate F to the reducing agent is 1:0.5-10. The mass ratio of the intermediate F to the fourth organic solvent is 1:30-50 Using the foregoing ratio can further ensure the preparation effect of the intermediate G. Further, after the reaction, the reaction solution obtained from the reduction at position 3 is purified. The purification method is as follows: Water and ethyl acetate are added into the reaction solution, the mixture is fully stirred and left for separation, an organic phase is collected, the organic phase is washed with a saturated sodium bicarbonate solution and a saturated sodium chloride solution sequentially and is then dried, and a solvent is removed, to obtain the intermediate F. During purification, the masses of the added water and ethyl acetate are preferably 5-10 times and 10-30 times the mass of the reaction solution, respectively.

S7. Prepare a chenodeoxycholic acid.

The hydrolysis is to hydrolyze the intermediate G under an alkaline condition to obtain the chenodeoxycholic acid, that is, 3a,7a-dihydroxy-50-cholanic acid. Specifically, the intermediate G, an alcohol solvent, water, and an alkali metal hydroxide are mixed and reacted under reflux, the mixture is concentrated to remove the alcohol solvent, water and hydrochloric acid are added to adjust the p1-1 to 3-4 to precipitate a precipitate, and the precipitate is filtered and collected, to obtain the chenodeoxycholic acid. Further preferably, the reaction is carried out at 60-80°C for 4-8 it The alcohol solvent is methanol. The alkali metal hydroxide includes sodium hydroxide and potassium hydroxide, preferably sodium hydroxide. The foregoing preparation process ensures the production efficiency and purity.

Further, the mass ratio of the intermediate G, the alcohol solvent, and water is 1:10-20:5-10. The molar ratio of the intermediate G to the alkali metal hydroxide is 1:2-6. Using the foregoing ratio can ensure the full progress of the reaction and reduce the production of by-products.

The amount of the added water is preferably the same volume as the reaction solution. The concentration of the added hydrochloric acid is 1-3 M. This application further provides a chenodeoxycholic acid prepared by the foregoing method for preparing chenodeoxycholic acid.

In summary, in this application, the reaction conditions of each step are relatively mild, and the process is simple. The product is obtained through the 7-step reaction, which is easy to be controlled, and the target compound can be obtained without complex equipment.

The features and performance of this application are further described in detail below with reference to embodiments.

Embodiment 1 This embodiment provides a method for preparing chenodeoxycholic acid, including the following steps: Sl. Prepare an intermediate B. Allochenodeoxycholic acid (1.0 g, 2.6 mmol), 15 mL of anhydrous methanol, and 100 pL, of concentrated sulfuric acid were added into a reaction vessel equipped with a dry reflux condenser. After the addition, the reaction temperature was increased to 67°C. The mixture was allowed to react with stirring for 4 h. After the reaction, the solvent methanol was removed by rotary evaporation, 20 nth of ethyl acetate was added to dissolve die residue, and the dissolved residue was washed with 10 mL of saturated NaHCO3 solution and water sequentially. An organic phase was dried with anhydrous MgSO4 to remove water and then filtered to remove the anhydrous MgSO4, the resulting organic phase was collected, and a solvent was removed by distillation under reduced pressure, to obtain 1.0 g of intermediate B as a white solid with a yield of 99%.

The structural characterization data of the intermediate B is as follows: 11-1 NMR (600 MHz, CD30D) 8=403-3.90 (m, 1H), 3.77 (d, J=2.1 Hz, 1H), 3.64 (s, 1H), 0.93 (dd, J=13.1, 8.9 Hz, 1H), 0.80 (s, 1H), 0.68 (s, 1H).

13C NMR (151 MHz, CD30D) 6 =175.06, 67.36, 65.81, 55.85, 50.61, 50.34, 45.63, 42.30, 39.52, 39.51, 36.35, 35.81, 35.34, 35.10, 31.90, 31.18, 30.85, 30.43, 28.13, 27.74, 23.11, 20.31, 17.36, 10.86, 9.24.

HRMS: Cab-xi for C25H4204 [M+Nar 429.2983, Found 429.2971.

S2. Prepare an intermediate C. The intemiediate B (0.5 g, 1 23 mmol) was dissolved in toluene (10 nth), and diatomaceous earth (0.74 g, 12.23 mmol) was added to stir uniformly. Silver carbonate (0.68 g, 2.46 mmol) was added in the dark. The temperature was increased to reflux. The mixture was allowed to react with stirring for 24 h. A precipitate was removed by filtration. The toluene was concentrated. 3 mL of methanol and water (methanol: water = 5:1) were added for recrystallization. Suction filtration was carried out to obtain the intermediate C with a yield of 83%.

The structural characterization data of the intermediate C is as follows: 11-1 NMR (600 MHz, CD30D) 8=3.79 (d, J=2.6 Hz, 1H), 3.64 (s, 3H), 1.05 (s, 3H), 0.94 (d. J=6.6 Hz, 3H). 0.72 (s, 3H).

13C NMR (151 MHz, COIOD) 6 =213.32, 175.02. 66.81, 55.88. 50.62. 50.24, 45.08, 43.69, 42.30, 39.46, 39.30, 38.23), 37.56, 36.53, 35.46, 35.33, 30.82, 30.43, 27.76, 23.13, 20.84, 17.35, 10.86, 9.30.

HRMS: Calcd for C25H4004 [M+Na] 427.2827, Found 427.2822.

S3. Prepare an intermediate D. The intermediate C (0.2 g, 0.49 mmol) and ethyl acetate (3 mL) were added into the reaction vessel and stirred to dissolve, and then DMAP (6 mg, 0.049 minol), triethylamine (170!IL, 1.23 mmol), and acetic anhydride (151 pL, 1.61 mmol) were added sequentially and allowed to react with stining at room temperature for 10.5 h. 0.5 N hydrochloric acid was added to adjust the pH to 6-7. An organic phase was collected through fractionation. The organic phase was washed with water (3x5 mL) and a saturated sodium chloride solution (3x5 mL) sequentially and dried with anhydrous MgSO4 to remove water. The anhydrous MgSO4 was removed by suction filtration. A solvent was removed by concentration, to obtain 0.22 g of the intermediate D as a white solid with a yield of 99.5%.

The structural characterization data of the intermediate D is as follows: NMR (600 MHz, CD30D) 6=4.94-4.90 (m, 11-1). 3.64 (s, 111), 2.03 (s, 1H), 1.07 (d, J=7.5 Hz, 1H), 0.94 (t, J=4.9 Hz, 1H), 0.73 (d, J=6.8 Hz, 1H).

NMR (151 MHz, CD30D) 6=212.70, 174.96, 171.06, 70.99 (s), 55.79, 50.61, 50.31, 46.53, 43.32, 42.46, 40.09, 39.34, 38.01, 38.00, 37.46, 35.32, 35.26, 33.01, 30.76, 30.40, 27.58, 23.22, 20.96, 19.73, 17.31, 10.76, 9.33.

HRMS: Calcd for C27H4205 [M+Na] 469.2932, Found 469.2920.

S4. Prepare an intermediate E. The intermediate D (1.0 g, 2.24 mmol) and 2-iodoxybenzoic acid (2.5 g, 8.96 mmol) were added into the reaction vessel. and DMSO (30 mL) was added under a nitrogen atmosphere to stir and dissolve. Trifluoroacetic acid (0.1 mL, 0.67 mmol) was added. The temperature was increased to 65°C. TLC (Vali)" acetateNpetroleum ether= 1:3) was used for monitoring. After the reaction, water was added for dilution, and extraction with ethyl acetate (3x20 mL) was carried out for three times. The obtained organic phase was washed with water (3x20 mL) and a saturated sodium chloride solution (3x20 mL) sequentially and dried with anhydrous MgSO4 to remove water. The anhydrous MgSO4 was removed by filtration. The resulting organic phase was concentrated to obtain a crude product. The crude product was separated by column chromatography, and an elmmt is ethyl acetate and petroleum ether with a volume ratio of 1:10, to obtain 0.5 2 of intermediate E as a yellow solid with a yield of 50%.

The structural characterization data of the intermediate E is as follows: 1H NMR (600 MHz, DMSO-d6) 6=721 (d, 1=10.1 Hz, 1H), 613 (dd, J101, 1.5 Hz, 1H), 5.94 (s, 1H), 4.96 (d, J=2.3 Hz, 1H), 3.57 (s, 1H), 1.94 (s, 1H), 1.22 (s, 1H), 0.87 (d, 1=6.5 Hz, 1H), 0.71 (s, 1H).

13C NMR (151 MHz, DMSO-d6) 8=185.00, 174.15, 170.25, 165.36, 156.47, 127.35, 126.09, 72.31, 55.52, 51.63, 50.23, 45.49, 43.46, 42.78, 39.03, 37.69, 37.15, 35.13, 30.96, 30.79, 27.83, 23.61, 22.46, 21.24, 18.70, 18.50, 12.09.

HRMS: Calcd for C271-14005 [M+Nar 465.2619, Found 465.2624.

55. Prepare an intermediate F. The intermediate E (0.1 g, 0.22 mmol) was dissolved in methanol (2 mL), palladium on carbon (0.2 g, 1.88 mmol) was added, and two drops of triethylamine were added dropwise. The reaction system was reacted at room temperature under 1 MPa for 1 h. The methanol underwent rotary drying. Ethyl acetate (2 mL) was added. The mixture was washed with 1 M hydrochloric acid (3x2 mL) and a saturated sodium chloride solution (3x2 mL) sequentially and dried with anhydrous MgSO4 to remove water. The anhydrous MgSO4 was removed by filtration. The resulting reaction system was concentrated to obtain the intermediate F with a yield of 99%.

The structural characterization data of the intermediate F is as follows: NMR (600 MHz, CD30D) 8=4.98-4.91 (m, 111), 3.64 (s, 1H), 2.02 (s, 114), 1.07 (s, 1H), 0.96 (d, J=6.6 Hz, 1H), 0.74 (s, 1H).

13C NMR (151 MHz, CD30D) 6=213.86, 174.97, 170.74, 71.33, 55.77, 50.62, 50.25, 44.32, 42.76, 42.52, 39.36, 37.78, 36.33, 36.29, 35.26, 34.75, 34.70, 30.77, 30.62, 30.40, 27.64, 23.19, 20.78, 20.75, 19.91, 17.34, 10.73.

HRMS: Calcd for C271-14405 [M+NaL 469.2932, Found 469.2934.

S6. Prepare an intermediate G. The intermediate F (34 m2, 0.076 mmol) was dissolved in methanol (2 mL). The temperature was reduced to 0°C. Sodium borohydride (14 mg, 0.368 mmol) was added in batches. After the addition, the reaction system was slowly raised to room temperature and allowed to react with stirring. TLC (Vethyi acerate:Vp,oteun, ed"r=1:2) was used for monitoring. After the reaction, distilled water (5 mL) and ethyl acetate (20 mL) were added to be fully stirred and left for separation, and an organic phase was separated. An aqueous phase was extracted with ethyl acetate (2x5 mL) and the organic phases were combined. The combined phases were washed with a saturated sodium bicarbonate solution (3x15 mL) and a saturated sodium chloride solution (3x15 mL) sequentially and then dried with anhydrous MgSO4. The anhydrous MgSO4 was removed by filtration. The resulting solution was concentrated to obtain 30 mg of intermediate G as a white solid with a yield of 88%.

The structural characterization data of the intermediate G is as follows: 11-1 NMR (600 MHz, CD30D) 6=4.85 (d, 1=2.8 Hz, 1H), 3.64 (s, 1H), 3.41 (tt, J=11.1, 4.3 Hz, 1H), 2.04 (d, J=3.7 Hz, 1H), 0.95 (s, 1H), 0.94 (d, J=6.6 Hz, 1H), 0.70 (s, 1H).

13C NMR (151 MHz, CD30D) 6=174.99, 171.13, 71.64, 70.96, 55.80, 50.62, 50.39, 42.45, 41.19, 39.54, 38.44, 37.83, 35.27, 34.90, 34.47, 34.13, 31.08, 30.78, 30.41, 29.97, 27.66, 23.19, 21.87, 20.39, 20.06, 17.34, 10.72.

HRMS: Calcd for C27H4405 [M+Na] 471.3089, Found 471.3076.

S7. Prepare a chenodeoxycholic acid.

At room temperature, the intermediate G (20 mg, 0.044 mmol) was added into a mixed solution of methanol (0.5 mL) and water (0.1 mL). Sodium hydroxide (12 nig, 03 mmol) was added to stir under reflux for 6 h. The methanol was removed by concentration 1 mL of water was added for dilution. Then, 1 N hydrochloric acid was added to adjust the pH to 3-4 to precipitate a while precipitate. The precipitate was filtered and dried to obtain 16 mg of chenodeoxycholic acid as a white solid with a yield of 90.9% and a purity of 98.7%.

The structural characterization data of the chenodeoxycholic acid is as follows: 11-1 NMR (600 MHz, CD30D) 6=3.79 (d, J=2.4 Hz, 1H), 3.41-3.32 (m, 1H), 0.96 (d, J=6.5 Hz, 111), 0.92 (d, J=10.1 Hz, 111), 0.69 (s, 1H).

13C NMR (151 MHz, CD30D) 6 =176.80, 71.46, 67.66, 55.93, 50.13, 42.28, 41.78, 39.65, 39.37, 39.07, 35.37, 35.17, 34.83, 34.49, 32.65, 30.95, 30.59, 29.96, 27.84, 23.23, 22.01, 20.39, 17.41, 10.78.

HRMS: Cal cd for C24H4004 1M+1-11+ 393.2927, Found 393.2097.

This embodiment further provides a chenodeoxycholic acid prepared by the foregoing preparation method. The total yield was 32, and the purity was 98.7%.

Embodiments 2-10 The methods for preparing chenodeoxycholic acid provided in Embodiments 2 to 10 are basically similar to the method for preparing chenodeoxycholic acid provided in Embodiment 1, except those raw materials used and specific conditions of each step are different.

Embodiment 2: Si. Prepare an intermediate B. The alcohol was isopropanol, the reaction temperature was 85°C, the reaction time was 6 h, the catalyst was concentrated sulfuric acid, the molar ratio of 3a,7a-dihydroxy-5a-cholanic acid, the isopropanol, and the concentrated sulfuric acid was 1:5:0.5. and the amount of ethyl acetate used for purification was 10 times that of the 3a,7a-dihydroxy-5a-cholanic acid. The yield was 99%.

The characterization data is as follows: 1HNMR (600 MHz, CDC13) 5503-4.96 (m, 1H), 4.06-4.03 (m, 1H), 3.82 (d, J=2.4 Hz, 1H), 1.23 (s, 1H), 1.22 (s, 1H), 0.91 (t. J=5.9 Hz, 1H), 0.77 (s, 1H), 0.65 (s, 1H).

13C NMR (151 MHz, CDC13) 5=173.92, 68.05, 67.37, 66.44, 55.80, 50.64, 45.87, 42.65, 39.52, 39.49, 36.24, 36.16, 35.51, 35.35, 31.98, 31.63, 31.51, 31.00, 28.83, 28.09, 23.57, 21.88, 20.55, 18.27, 11.86, 10.15.

FIRMS: Calcd for C271-14604 [M+Nar 434.3396, Found 434.3300.

52. Prepare an intermediate C. The reaction time was 36 h, the first oxidizing agent was peroxybenzoic acid, the first organic solvent was tetrahydrofuran, the molar ratio of the intermediate B to the first oxidizing agent was 1:0.5, and the molar ratio of the intermediate B to diatomaceous earth was 1:5.

53. Prepare an intermediate D. The reaction time was 15 h, the pH was 6.2-6.5, the concentration of the hydrochloric acid used to adjust the pH was 1 M, and the molar ratio of the intermediate C, the 4-dimethylaminopyricline, the triethylamine, and the acetic anhydride was 1:0.1:1.5:2.

S4. Prepare an intermediate E. The reaction temperature was 90°C, the reaction time was 48 h, the molar ratio of the intermediate D to 2-iodoxybenzoic acid was 1:2, the molar ratio of the intermediate D to tritluoroacetic acid was 1:0.5, and the mass ratio of the intermediate D to the third organic solvent was 1:10. The volume ratio of ethyl acetate to petroleum ether during purification was 1:20.

S5. Prepare an intermediate F. The reaction temperature was 60°C, the reaction time was 0.5 h, the reaction pressure was 0.8 MPa, the molar ratio of the intermediate E, 5% of palladium on carbon, and triethylamine was 1:2:0.001, and the mass ratio of the intermediate E to methanol was 1:10.

S6. Prepare an intermediate G. The temperature of adding sodium borohydride was 10°C, the developing agent was ethyl acetate and petroleum ether with a volume ratio of 1:5, the molar ratio of the intermediate F to the sodium borohydride was 1:10, and the mass ratio of the intermediate F to anhydrous methanol was 1:30.

S7. Prepare a chenodeoxycholic acid. The pH was adjusted to 3, the reaction temperature was 80°C, the reaction time was 4 h, and the mass ratio of the intermediate G, methanol, and water was 1:10:5. The molar ratio of the intermediate G to potassium hydroxide was 1:2, and the concentration of the added hydrochloric acid was 3 M. The total yield was 30%, and the purity was 97%.

Embodiment 3 Si. Prepare an intermediate B. The alcohol was ethanol, the reaction temperature was 50°C, the reaction time was 3 h, the catalyst was concentrated hydrochloric acid, the molar ratio of 3a,7a-dihydroxy-5a-cholanic acid, the ethanol, and the concentrated hydrochloric acid was 1:15:1, and the amount of ethyl acetate used for purification was 20 times that of the 3a,7a-dihydroxy-5a-cholanic acid.

S2. Prepare an intermediate C. The reaction time was 15 h, the first oxidizing agent was chromium trioxide, the first organic solvent was dichloromethane, the molar ratio of the intermediate B to the first oxidizing agent was 1:1.5, and the molar ratio of the intermediate B to diatomaceous earth was 1:9.

S3. Prepare an intermediate D. The reaction time was 14 h, the pH was 6.2-6.6, the concentration of the hydrochloric acid used to adjust the pH was 3 M. and the molar ratio of the intermediate C, the 4-dimethylaminopyridine, the thethylamine, and the acetic anhydride was 1:0.4:2.5:2.5.

S4. Prepare an intermediate E. The reaction temperature was 75°C, the reaction time was 34 h, the molar ratio of the intermediate D to 2-iodoxybenzoic acid was 1:4.5, the molar ratio of the intermediate D to trifluoroacetic acid was 1:0.4, and the mass ratio of the intermediate D to the third organic solvent was 1:13. The volume ratio of ethyl acetate to petroleum ether during purification was 1:12.

55. Prepare an intermediate F. The reaction temperature was 40°C, the reaction time was 2 h, the reaction pressure was 0.9 MPa, the molar ratio of the intermediate E, 5% of palladium on carbon, and triethylanaine was 1:5:0.005, and the mass ratio of the intermediate E to methanol was 1:15.

56. Prepare an intermediate G. The temperature of adding sodium borohydride was 7°C, the developing agent was ethyl acetate and petroleum ether with a volume ratio of 1:3, the molar ratio of the intermediate F to the sodium borohydride was 1:9, and the mass ratio of the intermediate F to anhydrous methanol was 1:35.

S7. Prepare a chenodeoxycholic acid. The pH was adjusted to 3.5, the reaction temperature was 75°C, the reaction time was 6 h, and the mass ratio of the intermediate G, methanol, and water was 1:18:6. The molar ratio of the intermediate 0 to potassium hydroxide was 1:5, and the concentration of the added hydrochloric acid was 1.5 M. The total yield was 28%, and the purity was 98%.

Embodiment 4 S I. Prepare an intermediate B. The alcohol was allyl alcohol, the reaction temperature was 90°C, the reaction time was 4 h. the catalyst was methanesulfonic acid, the molar ratio of 3a,7a-dihydroxy-5a-cholanic acid, the allyl alcohol, and the methanesulfonic acid was 1:10:0.7, and the amount of ethyl acetate used for purification was 15 times that of the 3a,7a-clihyclroxy-5a-cholanic acid.

S2. Prepare an intermediate C. The reaction time was 30 h, the first oxidizing agent was N-bromosuccinimide the first organic solvent was acetonitrile, the molar ratio of the intermediate B to the first oxidizing agent was 1:0.8, and the molar ratio of the intermediate B to diatomaceous earth was 1:12.

S3. Prepare an intermediate D. The reaction time was 13 h, the pH was 6.1-6.8, the concentration of the hydrochloric acid used to adjust the pH was 2 M, and the molar ratio of the intermediate C. the 4-dimethylaminopyridine, the triethylamine, and the acetic anhydride was 1:0.2:2:3.

S4. Prepare an intermediate E. The reaction temperature was 50°C, the reaction time was 36 h, the molar ratio of the intermediate D to 2-iodoxybenzoic acid was 1:3, the molar ratio of the intermediate D to trifluoroacetic acid was 1:0.2, and the mass ratio of the intermediate D to the third organic solvent was 1:11. The volume ratio of ethyl acetate to petroleum ether during purification was 1:15.

S5. Prepare an intermediate F. The reaction temperature was 20°C, the reaction time was 4 h, the reaction pressure was 1.2 MPa, the molar ratio of the intermediate E, 5% of palladium on carbon, and triethylamine was 1:10:0.01, and the mass ratio of the intermediate E to methanol was 1:20.

S6. Prepare an intermediate G. The temperature of adding sodium borohydride was 2°C, the developing agent was ethyl acetate and petroleum ether with a volume ratio of 1:6, the molar ratio of the intermediate F to the sodium borohydride was 1:5, and the mass ratio of the intermediate F to anhydrous methanol was 1:40.

S7. Prepare a chenodeoxycholic acid. The pH was adjusted to 3.2, the reaction temperature was 70°C, the reaction time was 5 h, and the mass ratio of the intermediate G, methanol, and water was 1:15:8. The molar ratio of the intermediate G to potassium hydroxide was 1:4, and the concentration of the added hydrochloric acid was 2 M. The total yield was 28%, and the purity was 99%.

Embodiment 5 Si. Prepare an intermediate B. The alcohol was benzyl alcohol, the reaction temperature was 70°C, the reaction time was 5 h, the catalyst was benzenesulfonic acid, the molar ratio of 3a,7a-dihydroxy-5a-cholanic acid, the benzyl alcohol, and the benzenesulfonic acid was 1:7:0.55. and the amount of ethyl acetate used for purification was 17 times that of the 3a,7a-dihydroxy-5a-cholanic acid.

S2. Prepare an intermediate C. The reaction time was 2 h, the first oxidizing agent was manganese dioxide, the first organic solvent was chloroform, the molar ratio of the intermediate B to the first oxidizing agent was 1:3, and the molar ratio of the intermediate B to diatomaceous earth was 1:15.

S3. Prepare an intermediate D. The reaction time was 10 h, the pH was 6.7-6.9, the concentration of the hydrochloric acid used to adjust the pH was 1.5 M, and the molar ratio of the intermediate C. the 4-dimethylaminopyridine, the niethylamine, and the acetic anhydride was 1:0.5:3:3.5.

S4. Prepare an intermediate E. The reaction temperature was 20°C, the reaction time was 12 h, the molar ratio of the intermediate D to 2-iodoxybenzoic acid was 1:5, the molar ratio of the intermediate D to trifluoroacetic acid was 1:0.1, and the mass ratio of the intermediate D to the third organic solvent was 1:15. The volume ratio of ethyl acetate to petroleum ether during purification was 1:10.

S5. Prepare an intermediate F. The reaction temperature was 50°C, the reaction time was 3 h, the reaction pressure was 1.1 MPa, the molar ratio of the intermediate E. 5% of palladium on carbon, and triethylamine was 1:7:0.008, and the mass ratio of the intermediate E to methanol was 1:17.

S6. Prepare an intermediate G. The temperature of adding sodium borohydride was 0°C, the developing agent was ethyl acetate and petroleum ether with a volume ratio of 1:2, the molar ratio of the intermediate F to the sodium borohydride was 1:2, and the mass ratio of the intermediate F to anhydrous methanol was 1:50.

S7. Prepare a chenodeoxycholic acid. The pH was adjusted to 4, the reaction temperature was 60°C, the reaction time was 8 h, and the mass ratio of the intermediate G, methanol, and water was 1:20:10. The molar ratio of the intermediate G to potassium hydroxide was 1:6, and the concentration of the added hydrochloric acid was 1 M. The total yield was 26%, and the purity was 98%.

Embodiment 6 The difference between the preparation method of this embodiment and Embodiment 1 lies in the preparation of the intermediate C: The intermediate B (0.5 g, 1.23 mmol) was dissolved in dichloromethane (10 mL), and activated manganese dioxide (1 6 g 12 3 mmol) was added to react at room temperature. TLC (Vethvl acetate:Vpetroleum ether-1:1) was used for monitoring. The reaction was stopped until the raw materials were no longer reduced. Suction filtration was carried out. A crude product was obtained by concentration. The crude product was separated by column chromatography, and an eluant is ethyl acetate and petroleum ether with a volume ratio of 1:1, to obtain 0.3 2 of intermediate C as a while solid with a yield of 60%. The total yield was 24%, and the purity was 98%.

Embodiment 7 The difference between the preparation method of this embodiment and Embodiment 1 lies in that the amount of acetic anhydride for preparing the intermediate D was 0.98 mmol. The total yield was 30%, and the purity was 99%.

Embodiment 8 The difference between the preparation method of this embodiment and Embodiment 1 lies in that the amount of acetic anhydride for preparing the intermediate D was 1.72 mmol. The total yield was 31%, and the purity was 98%.

Embodiment 9 The difference between the preparation method of this embodiment and Embodiment 1 lies in that the amount of 2-iodoxybenzoic acid for preparing the intermediate E was 4.48 mmol. The total yield was 28%, and the purity was 98%.

Embodiment 10 The difference between the preparation method of this embodiment and Embodiment 1 lies in that the amount of sodium borohydride for preparing the intermediate 0 was 0.148 mmol. The total yield was 29%, and the purity was 98%.

Embodiment 11 The difference between the preparation method of this embodiment and Embodiment 1 lies in that the amount of sodium borohydride for preparing the intermediate G was 0.74 mmol. The total yield was 31%, and the purity was 98%.

In summary, by using a discarded by-product extracted from duck galls and goose galls as a raw reaction material, this application can achieve waste utilization, reduce synthesis costs, and have a wide source of the raw material and sufficient supply.

In addition, the chenodeoxycholic acid obtained through the foregoing steps has a high yield up to 32%, which is suitable for large-scale preparation. Moreover, the preparation method has simple operation, strong repeatability, and extremely strong practicability The foregoing descriptions are merely preferred implementations of this application, but are not intended to limit this application. A person skilled in the art may make various modifications and changes to this application. Any modification, equivalent replacement, or improvement made without departing from the spirit and principle of this application shall fall within the scope of protection of this application.

Claims (10)

1. CLAIMS1. A method for preparing chenodeoxycholic acid, comprising the following steps: allowing 3a,7a-dihydroxy-5a-cholanic acid as a raw material to undergo a chemical reaction to form an intermediate E. and then subjecting the intermediate E to a chemical reaction to form chenodeoxycholic acid, the structural formula of the intermediate E being as follows: * wherein RI is an alkyl group, an alkenyl group, or an aryl group, and R2 is an acyl group.
2. 2. The method for preparing chenodeoxycholic acid according to claim 1, wherein the intermediate E is an intermediate obtained by allowing 3a,7a-dihydroxy-5a-cholanic acid to undergo first esterification, oxidation, second esterification, and dehydrogenation; preferably, the first esterification is an esterification between 3a,7a-dihydroxy-5a-cholanic acid and alcohol; preferably, the oxidation is a selective oxidation at position 3; preferably, the second esterification is an esterification at position 7; and preferably, the dehydrogenation is a reaction of simultaneous dehydrogenation at positions 1, 2, and 4, 5 to form olefin.
3. 3. The method for preparing chenodeoxycholic acid according to claim 2, wherein the first esterification is to allow 3a,7a-dihydroxy-5a-cholanic acid to react with alcohol at 50-90°C under the action of a catalyst to obtain an intermediate B; preferably, the molar ratio of the 3a,7a-dihydroxy-5a-cholanic acid, the alcohol, and the catalyst is 1:2-30:0.1-1; preferably, the alcohol is monoalcohol, more preferably Cl-C10 monoalcohol, further preferably methanol. ethanol. isopropanol, ally] alcohol. or benzyl alcohol, and most preferably methanol or isopropanol; and preferably, the catalyst is an acidic substance, more preferably any one of concentrated hydrochloric acid, concentrated sulfuric acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid, and further preferably concentrated hydrochloric acid or concentrated sulfuric acid.
4. 4. The method for preparing chenodeoxycholic acid according to claim 3, wherein the oxidation is to allow the intermediate B to react with a first oxidizing agent for 2-36 h to form an intermediate C; preferably, the molar ratio of the intermediate B to the first oxidizing agent is 1:0.5-3, preferably 1:0.8-2; and preferably, the first oxidizing agent is any one of peroxide, metal compound, or bromoimide; more preferably, the peroxide is peroxybenzoic acid or hydrogen peroxide; the metal compound is any one of sodium hypochlorite, silver carbonate, manganese dioxide, or chromium trioxide; the bromoimide is N-bromosuccinimide; and most preferably, the first oxidizing agent is silver carbonate, sodium hypochloritc, or manganese dioxide.
5. 5. The method for preparing chenodeoxycholic acid according to claim 4, wherein the second esterification is to allow the intermediate C to react with 4-dirnethylaminopyridine, triethylamine, and organic acid anhydride for 1-15 h and then adjust the pH of a reaction solution to 6-7 to form an intermediate D; preferably, the molar ratio of the intermediate C, the 4-dimethylaminopyridine, the triethylamine, and the organic acid anhydride is 1:0.01-0.5:1.5-3:0.8-4.
6. 6. The method for preparing chenodeoxycholic acid according to claim 5, wherein the dehydrogenation is to allow the intermediate D to react with a second oxidizing agent at 20-90°C for 3-72 h; preferably, the second oxidizing agent is an iodine reagent, preferably a hypervalent iodine reagent, and more preferably 2-iodoxybenzoic acid; and preferably, the molar ratio of the intermediate D to the second oxidizing agent is 1:2-5.
7. 7. The method for preparing chenodeoxycholic acid according to claim 1, wherein the intermediate E sequentially undergoes catalytic hydrogenation and reduction, reduction at position 3, and hydrolysis to obtain the chenodeoxycholic acid; preferably, the catalytic hydrogenation and reduction is a reaction of simultaneous hydrogenation and reduction at positions 1, 2, and 4, 5.
8. 8. The method for preparing chenodeoxycholic acid according to claim 7, wherein the catalytic hydrogenation and reduction is to mix the intermediate E with a catalyst and triethylamine to undergo hydrogenation at 20-60°C for 0.5-4 h to form an intermediate F; preferably, the molar ratio of the intermediate E, the catalyst and the triethylamine is 1:0.02-10:0.001-0.5; and preferably, the catalyst is palladium on carbon, preferably 5-10% of palladium on carbon.
9. 9. The method for preparing chenodeoxycholic acid according to claim 8, wherein the reduction at position 3 is to allow the intermediate F to react with a reducing agent; preferably, the molar ratio of the intermediate F to the reducing agent is 1:0.5-10; and preferably, the reducing agent is a hydride, more preferably sodium borohydride.
10. 10. A chenodeoxycholic acid, prepared by the method for preparing chenodeoxycholic acid according to claim 1.