WO2018227940A1 - Method for preparing ursodeoxycholic acid via chemical-enzymatic process - Google Patents

Abstract

A method for preparing ursodeoxycholic acid via a chemical-enzymatic process, comprising: adding hyodeoxycholic acid to a first organic solvent, and oxidising under the effect of an oxidising agent to obtain 6-oxo-lithocholic acid; adding the 6-oxo-lithocholic acid and a sulphonyl hydrazide derivative to a second organic solvent, so that the 6-oxo-lithocholic acid and the sulphonyl hydrazide derivative undergo a nucleophilic addition-elimination reaction to obtain a hydrazone compound; in an inert gas environment, using a reducing agent to reduce the hydrazone compound to obtain lithocholic acid; performing a hydroxylation reaction on the lithocholic acid under the catalytic effect of a hydroxylase and a coenzyme to obtain ursodeoxycholic acid. The method uses hyodeoxycholic acid as an initial raw material, and obtains ursodeoxycholic acid via a three-step chemical process and a one-step biological enzymatic process. The overall synthesis process has few steps, simple operations, high yields and low costs, and may be widely applied to industrial scale production.

Description

Method for preparing ursodeoxycholic acid by chemical-enzymatic method Technical field

The invention relates to the technical field of biomedicine, in particular to a method for preparing ursodeoxycholic acid by a chemical-enzymatic method.

Background technique

Ursodeoxycholic acid (UDCA), the molecular formula is C ₂₄ H ₄₀ O ₄ , which is the main component of the traditional Chinese medicine bear bile. Its chemical name is (3α,7β)-dihydroxy-5β-cholanoic acid, which is a goose. 7β-hydroxy epimer of denodeoxycholic acid (CDCA). UDCA was primarily used to treat cholelithiasis. In recent years, the application of UDCA in the treatment of various acute and chronic liver diseases has been reported abroad. New research shows that UDCA not only has a good effect on the treatment of primary biliary cirrhosis, primary sclerosing cholangitis, chronic active hepatitis, but also can be used to treat chronic hepatitis and rejection after liver transplantation. Therefore, with the deepening of research, the use value of UDCA is increasingly recognized and valued by people, and the demand for UDCA is also increasing year by year.

Because the Chinese medicine bear bile is made by cutting the bear's gallbladder, the source is limited and it is contrary to animal protection. China now adopts artificial breeding, and live bears extract UDCA, but the steps are many, the cycle is long, the yield is low, and the medical requirements cannot be met. Therefore, artificial synthesis of UDCA is of great significance. At present, UDCA's production process mainly uses chenodeoxycholic acid (CDCA) as raw material, which is prepared by esterification, oxidation and reduction. The domestic and international market demand for this product is large, while CDCA resources are limited, and its market price is increasing year by year. The cost of synthetic routes using chenodeoxycholic acid is high. Relatively speaking, hyodeoxycholic acid (HDCA) has a wide range of sources and low cost. Therefore, it is of great significance to synthesize UDCA from HDCA. However, the current synthesis of UDCA from HDCA is a chemical method, and the synthesis step requires about 7 steps, and the yield is low; in addition, chemical methods often use highly contaminated reagents such as pyridinium dichromate.

Therefore, it is becoming more and more urgent to develop a synthetic method that produces fewer UDCA steps, has a higher conversion rate, and is more environmentally friendly.

Summary of the invention

In order to solve the above technical problems, the present invention provides a chemical-enzymatic method for preparing ursodeoxycholic acid, which involves a three-step chemical method and a one-step enzymatic method, which greatly reduces the number of synthetic steps in the preparation process of the conventional method, and simultaneously The method for preparing ursodeoxycholic acid by the chemical-enzymatic method of the invention has the characteristics of low cost, high yield and low pollution.

The invention provides a chemical-enzymatic method for preparing ursodeoxycholic acid, comprising:

(1) adding hyodeoxycholic acid to the first organic solvent, and oxidizing the hyodeoxycholic acid under the action of an oxidizing agent to obtain a 6-oxo-lithocholic acid having a chemical structural formula of the formula (I).

(2) adding the 6-oxo-lithocholic acid and a sulfonylhydrazine derivative to a second organic solvent to cause nucleophilicity of the 6-oxo-lithocholic acid and the sulfonylhydrazide derivative Addition-elimination reaction to obtain an anthracene compound of the formula (II)

(II), in the formula (II), R is hydrogen, methyl, ethyl, propyl and butyl;

(3) reducing the quinone compound to obtain lithocholic acid (LCH) using a reducing agent under an inert gas atmosphere;

(4) The choline acid is subjected to hydroxylation reaction under the catalytic action of hydroxylase and coenzyme to obtain ursodeoxycholic acid.

In the present invention, the specific process route of the chemical-enzymatic method for preparing ursodeoxycholic acid is as follows:

Wherein, the first three steps in the process are chemical methods, the fourth step is a biological enzymatic method; the chemical structure of the hyodeoxycholic acid is as shown in formula (III), the lithocholic acid (or referred to as 3α) a chemical structural formula of -hydroxy-5β-cholestane-24-acid, or 3α-hydroxy-5β-cholanoic acid, as shown in formula (IV), wherein the chemical structure formula of ursodeoxycholic acid is as defined V) is shown.

Optionally, in the step (1), the oxidizing agent comprises a high-valent iodine oxidizing agent or a chromium-based oxidizing agent. Optionally, the high valent iodine oxidant comprises 2-Iodoxybenzoic acid (IBX), and the chromic oxidant comprises pyridinium dichromate (PDC). For example, the oxidizing agent is 2-iodobenzoic acid or a dichromate pyridinium salt.

Further, in the step (1) of the present invention, when the oxidizing agent is 2-iodobenzoic acid, contamination can be avoided, and green safety and environmental protection can be achieved. The IBX is an inexpensive and mild oxidant. It is also an environmentally friendly oxidant. It is stable in the air and can be stored for a long time. It does not require inert gas protection during the reaction, and can even react under water conditions. The operation is simple, the yield is high, the selectivity is good, and many functional groups are not affected during the reaction; compared with the commonly used chromium-based oxidants, such as dichromate pyridinium salt (PDC), the cost can be greatly reduced, and the Reduce pollution.

Optionally, the molar ratio of the oxidizing agent to the hyodeoxycholic acid is 1: (0.5-5). Further optionally, the molar ratio of the oxidizing agent to the hyodeoxycholic acid is 1: (0.5-3). Preferably, the molar ratio of the oxidizing agent to the hyodeoxycholic acid is 1: (1-3). For example, the molar ratio of the oxidizing agent to the hyodeoxycholic acid is 1:1, or 1:1.5, or 1:2. In the present invention, a preferred molar content of the oxidizing agent is effective to oxidize the hydroxyl group at the 6 position of the HDCA to a carbonyl group.

Optionally, in the step (1), the first organic solvent is a non-alcoholic organic reagent. Further optionally, the first organic solvent comprises one or more of dichloromethane (CH ₂ Cl ₂ ), tetrahydrofuran (THF), acetone (CH ₃ COCH ₃ ), and dimethyl sulfoxide (DMSO). . For example, the first organic solvent is dichloromethane, or a mixed solution of dichloromethane and dimethyl sulfoxide, or a mixed solution of dichloromethane, tetrahydrofuran and dimethyl sulfoxide. The first organic solvent of the present invention has good solubility to both the hyodeoxycholic acid and the oxidizing agent.

Further optionally, in the step (1), when the oxidizing agent is 2-iodobenzoic acid, the first organic solvent comprises dimethyl sulfoxide. Optionally, the first organic solvent is a mixed solution of one or more of dichloromethane, tetrahydrofuran and acetone and dimethyl sulfoxide. The dimethyl sulfoxide of the invention has good solubility to 2-iodobenzoic acid and can improve the oxidation effect of the oxidizing agent.

Optionally, the step (1) further comprises recrystallizing the 6-oxo-lithocholic acid (I). The recrystallization process is a further purification of the 6-oxo-lithocholic acid (I), which facilitates the subsequent reaction and indirectly increases the yield of UDCA.

Optionally, in the step (2), the sulfonyl hydrazide derivative comprises benzenesulfonyl hydrazide, p-toluenesulfonyl hydrazide, p-ethylbenzenesulfonyl hydrazide, p-propyl benzene sulfonyl hydrazide and p-butyl One or more of benzenesulfonylhydrazide, and the molar ratio of the sulfonylhydrazine derivative to the 6-oxo-lithocholic acid (I) is (1-5):1. Further, the molar ratio of the sulfonyl hydrazide derivative to the 6-oxo-lithocholic acid is (1 - 3.5): 1. Preferably, the molar ratio of the sulfonyl hydrazide derivative to the 6-oxo-lithocholic acid is (1 - 2.5):1. For example, the molar ratio of the sulfonyl hydrazide derivative to the 6-oxo-lithocholic acid is 1:1, or 2:1. The chemical structural formula of the sulfonyl hydrazide derivative is as shown in the formula (VI):

(VI) wherein R is hydrogen, methyl, ethyl, propyl and butyl.

Optionally, in the step (2), the second organic solvent is an organic solvent without a carbonyl group. Further optionally, the second organic solvent is an alcoholic organic reagent without a carbonyl group. Further optionally, the second organic solvent comprises one or more of methanol and ethanol. The second organic solvent of the present invention has good solubility to both the 6-oxo-lithocholic acid (I) and the sulfonyl hydrazide derivative.

Optionally, in the step (2), the second organic solvent further comprises an acidic organic reagent having a volume fraction of 0.1-5%. Further optionally, the acidic organic reagent comprises one or more of acetic acid, oxalic acid and propionic acid. For example, the acidic organic reagent is acetic acid, or oxalic acid, or propionic acid, or a mixed solution of acetic acid and propionic acid, or a mixed solution of acetic acid and oxalic acid. When the nucleophilic addition-elimination reaction of the step (2) of the present invention is carried out under weakly acidic conditions, the protons in the reaction system are combined with the carbonyl oxygen atom, thereby enhancing the activity of the carbonyl group, facilitating the forward progress of the reaction and improving Product conversion rate.

Optionally, in the step (2), after the nucleophilic addition-elimination reaction, the method further comprises: adding an inorganic salt solution to the reaction system, filtering, and drying the filter cake; the inorganic salt solution comprises The mass fraction is from 50% to 80% of a carbonate solution, a bicarbonate solution or a hydrogen sulfate. Optionally, the inorganic salt comprises one or more of a sodium salt and a potassium salt. For example, the inorganic salt solution is a sodium hydrogencarbonate solution, or a potassium hydrogencarbonate solution, or a sodium hydrogen sulfate solution, or a sodium carbonate solution, or a potassium carbonate solution. In the present invention, the volume of the inorganic salt solution depends on the actual reaction conditions, and the inorganic salt solution can cause the hydrazine compound (II) to precipitate from the reaction system. In the present invention, the inorganic salt solution is capable of effectively terminating the nucleophilic addition-elimination reaction, and the quinone compound (II) produced by the nucleophilic addition-elimination reaction is in the inorganic salt solution system. The solubility is very low, so the inorganic salt solution can function to promote product precipitation.

Optionally, in the step (3), the reducing agent comprises catechol borane or sodium borohydride; and the molar ratio of the reducing agent to the quinone compound is (0.5-5):1. Optionally, the molar ratio of the reducing agent to the terpenoid is (0.5-3):1. Further optionally, the molar ratio of the reducing agent to the quinone compound is (0.5-2.5):1. For example, the molar ratio of the reducing agent to the terpenoid is 2:1, or 1.5:1, or 1:1. Preferably, the reducing agent is catechol borane. The catechol borane has the molecular formula C ₆ H ₅ BO ₂ , also known as o-phthalodioxane, which is commonly used in the hydrogenation reaction in organic synthesis, and is a highly active reducing reagent, in the step (3) of the present invention. In the above, the quinone compound (II) can be efficiently reduced to obtain lithocholic acid (IV) under the reduction of the catechol borane to increase the yield of the lithocholic acid (IV).

Optionally, in the step (3), the process of reducing the quinone compound (II) to obtain lithocholic acid (IV) by using a reducing agent comprises: dissolving the quinone compound and the reducing agent The mixture is stirred in a third organic solvent for 0.5-2 hours. After the completion of the stirring, an alkaline solution is added, and the mixture is stirred at normal temperature for 1-5 hours, filtered, and recrystallized to obtain the choline acid (IV).

Alternatively, the quinone compound and the reducing agent are dissolved in the third organic solvent in a molar ratio of 1: (1-5).

Optionally, the third organic solvent is a non-oxidizing organic solvent. Further optionally, the third organic solvent comprises one or more of dichloromethane and tetrahydrofuran. For example, the third organic solvent is dichloromethane, or tetrahydrofuran, or a mixed solution of dichloromethane and tetrahydrofuran.

Optionally, the alkaline solution comprises one or more of a sodium hydroxide solution and a potassium hydroxide solution, the alkaline solution having a concentration of 0.5-4 mol/L. For example, the alkaline solution is a sodium hydroxide solution, or a potassium hydroxide solution, or a mixed solution of sodium hydroxide and potassium hydroxide. Further optionally, the concentration of the alkaline solution is 1-3 mol/L. For example, the concentration of the alkaline solution is 2 mol/L, or the concentration of the alkaline solution is 4 mol/L. Optionally, the volume of the alkaline solution is from 20% to 60% by volume of the total reactant system in step (3). In the present invention, the quinone compound and the reducing agent are first reacted in an anhydrous third organic solvent to form an intermediate complex, and when added, the volume fraction of the reactant system is 20% to 60%. After the alkaline solution, the intermediate complex is further reacted to obtain the lithocholic acid.

In the step (3) of the present invention, the recalcification process of the choline acid produced by the reaction may not be carried out, and the filter cake obtained by the filtration may be directly dried and then subjected to the next reaction; the lithocholic acid obtained after the recrystallization The purity and yield of the final product, ursodeoxycholic acid (V), can be effectively enhanced by carrying out the reaction of step (4).

Optionally, in the step (4), the coenzyme includes one or more of an oxidizing coenzyme and a reducing coenzyme, and when the coenzyme includes the oxidizing coenzyme, the reaction of the hydroxylation reaction One or more of alcohol dehydrogenase and Glucose dehydrogenase (GDH) are also included in the system.

Optionally, the alcohol dehydrogenase comprises one or more of methanol dehydrogenase (MDH) and alcohol dehydrogenase (ADH).

Optionally, the oxidative coenzyme comprises one or more of NAD ⁺ and NADP ⁺ , and the reducing coenzyme comprises one or more of NADH and NADPH. Optionally, the molar ratio of the alcohol dehydrogenase, glucose dehydrogenase, or a mixture of alcohol dehydrogenase and glucose dehydrogenase to the oxidative coenzyme is 1: (5-15).

Optionally, in the step (4), the hydroxylation reaction is carried out in a buffer solution having a temperature of 30-45 ° C and a pH of 6-8, and the concentration of the buffer solution is 50-150 mmol/L. Optionally, the buffer solution comprises a phosphate buffer, a Tris-HCl buffer or other buffering reagent. Optionally, the buffer solution has a concentration of 50-120 mmol/L. Further optionally, the buffer solution has a concentration of 60-100 mmol/L. Preferably, the buffer solution has a concentration of 80-120 mmol/L. For example, the concentration of the buffer solution is 60 mmol/L, or 80 mmol/L, or 90 mmol/L, or 100 mmol/L, or 110 mmol/L.

Optionally, in the step (4), when the alcohol dehydrogenase is included in the reaction system of the hydroxylation reaction, the reaction system of the hydroxylation reaction further includes isopropyl alcohol. Optionally, the isopropanol is dissolved in the buffer solution, and the concentration of the isopropanol in the buffer solution is 0.5-5 mol/L. Optionally, the concentration of the isopropanol in the buffer solution is from 0.5 to 3 mol/L. Further optionally, the concentration of the isopropanol in the buffer solution is from 0.8 to 2.5 mol/L. For example, the concentration of the isopropanol in the buffer solution is 1 mol/L, or 1.5 mol/L, or 2.5 mol/L.

Optionally, in the step (4), when the glucose dehydrogenase is included in the reaction system of the hydroxylation reaction, glucose (Glucose) is further included in the reaction system of the hydroxylation reaction. Optionally, the glucose is dissolved in the buffer solution, and the concentration of the glucose in the buffer solution is 0.5-5 mol/L. Alternatively, the concentration of the glucose in the buffer solution is from 0.5 to 3 mol/L. Further optionally, the concentration of the glucose in the buffer solution is from 0.8 to 2.5 mol/L. For example, the concentration of the glucose in the buffer solution is 1 mol/L, or 1.5 mol/L, or 2.5 mol/L.

Alternatively, in the step (4), the hydroxylase may promote the formation of a β-configuration hydroxyl group at the 7 position of the lithocholic acid. Alternatively, the hydroxylase is derived from Fusarium oxysporum or Zea mays; the hydroxylase comprises 7β-hydroxylase (7β-LAH).

Optionally, in the step (4), the molar ratio of the alcohol dehydrogenase to the hydroxylase is 1: (0.5-1.5), and the concentration of the alcohol dehydrogenase is (0.3-1) g/ L. Further optionally, the molar ratio of the alcohol dehydrogenase to the hydroxylase is 1: (0.5-1.0). For example, the molar ratio of the alcohol dehydrogenase to the hydroxylase is 1:1, or 1:1.2, or 1:1.5. Further optionally, the concentration of the alcohol dehydrogenase is (0.5-1) g/L. For example, the alcohol dehydrogenase concentration is 0.5 g/L, or 0.8 g/L, or 1 g/L.

Wherein the step (4) of the present invention is a biological enzymatic method, wherein the hydroxylase, alcohol dehydrogenase or glucose dehydrogenase may be a commercial protease powder, or may be capable of expressing the hydroxyl group by fragmentation. Obtained by cells or cells of the enzyme or alcohol dehydrogenase. Wherein, the NAD ⁺ is Nicotinamide adenine dinucleotide (NAD ⁺ ), also known as oxidized coenzyme I; the NADH is a reduced state of nicotinamide adenine dinucleotide, also called Is a reduced coenzyme I; the NADP ⁺ is nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), also known as oxidized coenzyme II; the NADPH is nicotinamide adenine dinucleotide The reduced state of phosphoric acid, also known as reduced coenzyme II. In the presence of oxygen (O ₂ ), an electron circulation system is formed between the coenzyme, alcohol dehydrogenase and isopropanol, and hydroxylation of lithocholic acid (IV) can be effectively achieved under the action of hydroxylase. The reaction forms a hydroxyl group in the β configuration at the 7 position of the choline acid (IV) to form ursodeoxycholic acid (V). In the presence of oxygen (O ₂ ), an electron circulation system is formed between the coenzyme, glucose dehydrogenase and glucose, and the hydroxylation reaction of lithic acid (IV) can be effectively realized under the action of hydroxylase. A hydroxyl group in the β configuration is formed at the 7 position of the choline acid (IV) to form ursodeoxycholic acid (V).

Optionally, in the step (4), after the hydroxylation reaction is finished, heat treatment is performed to inactivate the enzyme, then filtered, the filtrate is collected, the pH of the filtrate is adjusted to be between 2-3, and the mixture is dried. After distillation, the ursodeoxycholic acid is obtained. Optionally, in the step (4), the step of extracting comprises extracting with an extraction solution, the extraction times are 2-5 times, and the extraction solution comprises ethyl acetate.

The beneficial effects of the present invention include the following aspects:

1. The method for preparing ursodeoxycholic acid by the chemical-enzymatic method according to the present invention, which comprises a three-step chemical method and a one-step biological enzymatic method, using hyodeoxycholic acid as a starting material, has low production cost, less process steps, and reaction Mild conditions, greatly improving the yield of the product;

2. The invention adopts the chemical-enzymatic method for preparing ursodeoxycholic acid, and can not use or not produce compounds which cause harm to the environment and the human body during the reaction process, avoiding environmental pollution problems from the root source, further reducing the cost, and can be widely used. Suitable for industrial scale production;

3. The ursodeoxycholic acid prepared by the method of the invention has high purity and good activity and can be widely used in the field of biomedicine.

DRAWINGS

1 is a nuclear magnetic resonance spectrum of ursodeoxycholic acid provided by an embodiment of the present invention;

2 is a nuclear magnetic resonance carbon spectrum of ursodeoxycholic acid provided by an embodiment of the present invention.

detailed description

The following are the preferred embodiments of the embodiments of the present invention, and it should be noted that those skilled in the art can make some improvements and refinements without departing from the principles of the embodiments of the present invention. And retouching is also considered to be the scope of protection of the embodiments of the present invention. Unless otherwise stated, the raw materials and other chemical reagents used in the examples of the present invention are all commercially available.

Embodiment 1

A chemical-enzymatic method for preparing ursodeoxycholic acid, comprising:

(1) 10 g of hyodeoxycholic acid was dissolved in 200 mL of dichloromethane, 60 mL of DMSO was further added, and the mixture was stirred until dissolved. 7.50 g of 2-iodobenzoic acid was added and stirred at room temperature for 4 hours, followed by filtration, and the filtrate was concentrated under reduced pressure to about 40 mL. 200 mL of deionized water was slowly dropped, and 6-oxo-lithocholic acid was precipitated and dried. The product was further recrystallized, including dissolving the 6-oxo-lithocholic acid obtained in the above process in 50 mL of methanol and filtering, and slowly dropping 100 mL of deionized water into the filtrate, filtering and drying to obtain a product of 9.18 g. Recrystallized 6-oxo-lithocholic acid.

(2) The above 9.18 g of 6-oxo-lithocholic acid and 8.50 g of p-toluenesulfonylhydrazine were dissolved in a mixed solution of 200 mL of methanol and 2 mL of acetic acid, stirred at room temperature, and then 100 mL of 70% sodium hydrogencarbonate was added thereto, and filtered. The filter cake was dried to give 13.68 g of 3?-hydroxy-6-p-toluenesulfonylhydrazide-5?-cholane acid as a white solid.

(3) Weigh 13.68 g of 3α-hydroxy-6-p-toluenesulfonyl hydrazide-5β-cholanoic acid dissolved in 200 mL of anhydrous dichloromethane, pass N ₂ , dissolve at room temperature, and slowly add 75 mL of 1 mol/L. The catechol borane was stirred for 1 hour, and then 200 mL of a 2 mol/L NaOH solution was added. After stirring for 4 hours, the filter cake was collected and collected to obtain lithocholic acid. Further recrystallizing the lithic acid, comprising dissolving the lithocholic acid in 100 mL of methanol, and then slowly adding 150 mL of deionized water, filtering, and drying to obtain 7.85 g of recrystallized stone urchin in the form of a white solid. acid.

(4) In 100 mM potassium phosphate buffer (100 mL, pH=7.0), add 7.85 g of lithocholic acid, 5 g of 7β-hydroxylase, 5 g of glucose alcohol dehydrogenase, 50 mg of NAD ⁺ , and 18 g of glucose at 35 ° C. The reaction was stirred for 24 hours at an external temperature. After the completion of the reaction, the protein was denatured by heating at 70 ° C, and the protein was removed by filtration, adjusted to pH = 2 with HCl, and extracted three times with ethyl acetate. The organic phase was combined, dried and evaporated to give 7.21 g of a solid product. The prepared solid product was subjected to nuclear magnetic resonance detection to obtain a nuclear magnetic spectrum as shown in Figs. 1 and 2, and it was confirmed that the final product was ursodeoxycholic acid. In the present example, the yield of ursodeoxycholic acid was 72.1%, and the purity of ursodeoxycholic acid was 99.2% by HPLC. Among them, 7β-hydroxylase is derived from Fusarium oxysporum, which is obtained by ultrasonication and centrifugal pretreatment. The reaction process of this embodiment is specifically as follows:

Embodiment 2

A chemical-enzymatic method for preparing ursodeoxycholic acid, comprising:

(1) 10 g of hyodeoxycholic acid was dissolved in 200 mL of dichloromethane, 40 mL of DMSO was further added, stirred until dissolved, and 8.2 g of 2-iodobenzoic acid was added thereto at room temperature for 3 hours, followed by filtration, and the filtrate was concentrated under reduced pressure to about 40 mL. 200 mL of deionized water was slowly dropped, and 6-oxo-lithocholic acid was precipitated and dried. Further recrystallizing the above product, comprising dissolving the 6-oxo-lithocholic acid obtained in the above process in 50 mL of methanol and filtering, slowly dropping 100 mL of deionized water into the filtrate, filtering and drying to obtain a product of 9.05 g. Recrystallized 6-oxo-lithocholic acid.

(2) The above 9.05 g of 6-oxo-lithocholic acid and 8.2 g of p-toluenesulfonylhydrazine were dissolved in 200 mL of ethanol and 2 mL of acetic acid solution, stirred at room temperature, then 100 mL of 80% sodium carbonate was added, and the filter cake was filtered. Drying gave 13.35 g of 3α-hydroxy-6-p-toluenesulfonyl hydrazide-5β-cholanoic acid as a white solid.

(3) Weigh 13.35 g of 3α-hydroxy-6-p-toluenesulfonyl hydrazide-5β-cholanoic acid dissolved in 200 mL of anhydrous dichloromethane, pass N ₂ , stir to dissolve at room temperature, and slowly drip into 100 mL of 1 mol/L. After the catechol borane was stirred for 2 hours, 200 mL of a 3 mol/L NaOH solution was added, and stirring was continued for 5 hours, followed by filtration and collection of the filter cake to obtain lithocholic acid. Further recrystallizing the lithocholic acid, comprising dissolving the lithocholic acid in 100 mL of methanol, then slowly adding 150 mL of deionized water, filtering, and drying to obtain 7.54 g of recrystallized stone bile in the form of a white solid. acid.

(4) In a 100 mM potassium phosphate buffer (100 mL, pH=7.0), add 7.54 g of lithocholic acid, 7 g of 7β-hydroxylase, 7 g of alcohol dehydrogenase, 50 mg of NADP ⁺ , 15 mL of isopropanol, at 40 The reaction was stirred for 24 hours with an open temperature at °C. After the reaction, the protein was denatured by heating at 70 ° C, the protein was removed by filtration, adjusted to pH = 3 with HCl, and extracted three times with ethyl acetate. The organic phases were combined, dried, and distilled under reduced pressure to obtain 6.75 g of ursodeoxycholic acid. . In the present example, the yield of ursodeoxycholic acid was 67.5%, and the purity of the product ursodeoxycholic acid was 99.3% by HPLC. Among them, 7β-hydroxylase is derived from Fusarium oxysporum by ultrasonication and centrifugal pretreatment. The reaction process of this embodiment is specifically as follows:

Embodiment 3

A chemical-enzymatic method for preparing ursodeoxycholic acid, comprising:

(1) 10 g of hyodeoxycholic acid was dissolved in 150 mL of acetone, 50 mL of DMSO was stirred until dissolved, and 7.50 g of 2-iodobenzoic acid was added and stirred at room temperature for 3 hours, followed by filtration, and the filtrate was concentrated under reduced pressure to about 40 mL, and 150 mL of deionized slowly. Water, precipitated to give 6-oxo-lithocholic acid, and dried. Further recrystallizing the above product, comprising dissolving the 6-oxo-lithocholic acid obtained in the above process in 50 mL of methanol and filtering, slowly dropping 100 mL of deionized water into the filtrate, filtering and drying to obtain a product of 9.25 g. Recrystallized 6-oxo-lithocholic acid.

(2) The above 9.25 g of 6-oxo-lithocholic acid and 10.0 g of p-toluenesulfonylhydrazine were dissolved in a mixed solution of 200 mL of ethanol and 4 mL of acetic acid, stirred at room temperature, then 100 mL of 80% sodium carbonate was added, and filtered. The cake was dried to give 13.93 g of 3?-hydroxy-6-p-toluenesulfonyl hydrazide-5?-cholanoic acid as a white solid.

(3) Weigh 13.93g of 6-p-toluenesulfonyl hydrazide-lithocholic acid dissolved in 200mL of anhydrous tetrahydrofuran, pass N ₂ , stir and dissolve at room temperature, slowly drip 100mL 1mol / L catechol borane, stir for 2h Then, 200 mL of a 1 mol/L NaOH solution was added, and stirring was continued for 3 hours, followed by filtration and collection of the filter cake to obtain 7.98 g of choline acid.

(4) In a 100 mM Tris-HCl buffer (100 mL, pH=7.0), 7.98 g of lithocholic acid, 6 g of 7β-hydroxylase, and 100 mg of NADPH were added, and the reaction was stirred at 20 ° C for 20 hours with open exposure. After the reaction, the protein was denatured by heating at 70 ° C, and the protein was removed by filtration, adjusted to pH between 2.5 with HCl, and extracted three times with ethyl acetate. The organic phase was combined, dried, and distilled under reduced pressure to obtain 7.32 g of ursodeoxycholic acid. . In the present example, the yield of ursodeoxycholic acid was 73.2%, and the purity of the product ursodeoxycholic acid was 99.2% by HPLC. Among them, 7β-hydroxylase is derived from Gibberella zeii, which is obtained by ultrasonication and centrifugation pretreatment. The reaction process of this embodiment is specifically as follows:

Embodiment 4

A chemical-enzymatic method for preparing ursodeoxycholic acid, comprising:

(1) 10 g of hyodeoxycholic acid was dissolved in 200 mL of tetrahydrofuran, and 30 mL of DMSO was added thereto, and the mixture was stirred until dissolved. 8 g of 2-iodobenzoic acid was added thereto at room temperature for 3.5 hours, followed by filtration, and the filtrate was concentrated under reduced pressure to about 50 mL. 200 mL of deionized water was added dropwise, precipitated, and dried to obtain 9.35 g of 6-oxo-lithocholic acid.

(2) The above 9.35 g of 6-oxo-lithocholic acid and 7.5 g of benzenesulfonyl hydrazide were dissolved in 200 mL of ethanol and 8 mL of acetic acid solution, stirred at room temperature, then 100 mL of 80% sodium hydrogencarbonate was added, and the filter cake was filtered. Drying gave 13.15 g of 3?-hydroxy-6-benzenesulfonylhydrazide-5?-cholanoic acid as a white solid.

(3) Weigh 13.19g of 3α-hydroxy-6-benzenesulfonyl hydrazide-5β-cholanoic acid dissolved in 200mL of anhydrous dichloromethane, pass N ₂ , stir and dissolve at room temperature, slowly drip into 130mL 1mol / L The tea phenol borane was stirred for 2.5 h, and then 200 mL of a 3 mol/L NaOH solution was added. After stirring for 4 hours, the filter cake was filtered and collected, and dried to obtain 7.23 g of choline acid as a white solid.

(4) In 100 mM potassium phosphate buffer (100 mL, pH=7.0), add 7.23 g of lithocholic acid, 5 g of 7β-hydroxylase, 6 g of alcohol dehydrogenase, 20 mg of NADP ⁺ , 15 mg of NAD ⁺ , 20 mL of isopropyl The alcohol was stirred at room temperature for 24 hours at 40 ° C. After the reaction, the protein was denatured by heating at 70 ° C, and the protein was removed by filtration, adjusted to pH between 2.5 and HCl, and extracted three times with ethyl acetate. The organic phase was combined, dried, and distilled under reduced pressure to obtain 6.55 g of ursodeoxycholic acid. . In the present example, the yield of ursodeoxycholic acid was 65.5%, and the purity of the product ursodeoxycholic acid was 99.0% by HPLC. Among them, 7β-hydroxylase is derived from Gibberella zeii, which is obtained by ultrasonication and centrifugation pretreatment. The reaction process of this embodiment is specifically as follows.

The above-mentioned embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims (15)

1. A chemical-enzymatic method for preparing ursodeoxycholic acid, which comprises:

   (1) adding hyodeoxycholic acid to the first organic solvent, and oxidizing the hyodeoxycholic acid under the action of an oxidizing agent to obtain a 6-oxo-lithocholic acid having a chemical structural formula of the formula (I).

   

   (2) adding the 6-oxo-lithocholic acid and a sulfonylhydrazine derivative to a second organic solvent to cause nucleophilicity of the 6-oxo-lithocholic acid and the sulfonylhydrazide derivative Addition-elimination reaction to obtain an anthracene compound of the formula (II)

   

   In the formula (II), R is hydrogen, methyl, ethyl, propyl and butyl;

   (3) reducing the quinone compound to obtain lithocholic acid using a reducing agent under an inert gas atmosphere;

   (4) The choline acid is subjected to hydroxylation reaction under the catalytic action of hydroxylase and coenzyme to obtain ursodeoxycholic acid.
2. The method according to claim 1, wherein in the step (1), the oxidizing agent comprises a high-valent iodine oxidizing agent or a chromium-based oxidizing agent.
3. The method according to claim 1, wherein in the step (2), the sulfonyl hydrazide derivative comprises benzenesulfonyl hydrazide, p-toluenesulfonyl hydrazide, p-ethylbenzenesulfonyl hydrazide, p-propylbenzene. One or more of a sulfonyl hydrazide and a p-butylbenzene sulfonyl hydrazide.
4. The method according to claim 1, wherein in the step (3), the reducing agent comprises catechol borane or sodium borohydride.
5. The method according to claim 1, wherein in the step (3), the reducing the quinone compound to obtain lithocholic acid using a reducing agent comprises:

   Dissolving the hydrazine compound and the reducing agent in a third organic solvent, then stirring for 0.5-2 hours, adding an alkaline solution after completion of stirring, stirring at room temperature for 1-5 hours, filtering, and recrystallizing and collecting. Said cholesteric acid.
6. The method according to claim 5, wherein the alkaline solution comprises one or more of a sodium hydroxide solution and a potassium hydroxide, the alkaline solution having a concentration of 0.5 to 4 mol/L; The triorganic solvent includes one or more of dichloromethane and tetrahydrofuran.
7. The method according to claim 1, wherein in the step (4), the coenzyme includes one or more of an oxidative coenzyme and a reducing coenzyme, and when the coenzyme includes the oxidative coenzyme, The reaction system of the hydroxylation reaction further includes one or more of an alcohol dehydrogenase and a glucose dehydrogenase.
8. The method according to claim 7, wherein in the step (4), the oxidizing coenzyme comprises one or more of NAD ⁺ and NADP ⁺ , and the reducing coenzyme includes one of NADH and NADPH Kind or more.
9. The method according to claim 7, wherein when the reaction system of the hydroxylation reaction includes an alcohol dehydrogenase, the reaction system of the hydroxylation reaction further comprises isopropyl alcohol; when the hydroxylation reaction When glucose dehydrogenase is included in the reaction system, glucose is also included in the reaction system of the hydroxylation reaction.
10. The method according to claim 1, wherein in the step (4), the hydroxylation reaction is carried out in a buffer solution having a temperature of 30 to 45 ° C and a pH of 6 to 8. The concentration of the buffer solution is 50-150 mmol/L.
11. The method according to claim 1, wherein in the step (4), after the completion of the hydroxylation reaction, heat treatment is performed to inactivate the enzyme, followed by filtration, and the filtrate is collected to adjust the pH of the filtrate to 2-3. The ursodeoxycholic acid is obtained after extraction, drying, and distillation.
12. The method according to claim 1, wherein in the step (1), the first organic solvent comprises one or more of dichloromethane, tetrahydrofuran, acetone, and dimethyl sulfoxide.
13. The method according to claim 1, wherein in the step (2), the second organic solvent comprises one or more of methanol and ethanol.
14. The method according to claim 1, wherein in the step (2), the second organic solvent further comprises an acidic organic reagent having a volume fraction of 0.1 to 5%.
15. The method according to claim 1, wherein in the step (2), after the nucleophilic addition-elimination reaction, the inorganic salt solution is added to the reaction system, and the filter cake is dried. The inorganic salt solution includes a carbonate solution, a hydrogencarbonate solution or a hydrogen sulfate salt having a mass fraction of 50% to 80%.

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