Preparation method of ursodeoxycholic acid and intermediate thereof

Abstract

The application provides a preparation method and intermediates of ursodeoxycholic acid. The preparation method uses commercially available compound A as a raw material, and ursodeoxycholic acid is prepared through multiple step reactions. The raw material of compound A is widely available and cheap. The preparation method of the application has mild reaction conditions, does not require special reagents, has simple post-treatment, is simple to operate, and is suitable for industrial production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical chemistry, more specifically, relates to a preparation method of ursodeoxycholic acid and an intermediate.

[0002] BACKGROUND

[0003] Ursodeoxycholic acid (UDCA) has the molecular formula of C 24 H 40 O4. Ursodeoxycholic acid is the main component of the precious traditional Chinese medicine bear bile, and is the first-line treatment drug for primary biliary cirrhosis (PBC) approved by the FDA of the United States. It can also effectively treat gallstone diseases and chronic liver diseases in clinical practice, and has a broad market prospect. The structural formula of ursodeoxycholic acid is as follows:

[0004]

[0005] In the past, UDCA was mainly extracted from natural bear bile, but the live bear resources were very limited, and it violated the animal protection law. With the increase of the clinical dosage of UDCA, it is imperative to develop an efficient and economical synthesis method of UDCA. In recent years, UDCA is mainly prepared from cholic acid (CA), chenodeoxycholic acid (CDCA) or hyodeoxycholic acid (HDCA). However, CA, CDCA and HDCA are all extracted from bile acids, which belong to animal raw materials. The risk brought by animal-derived products always exists in the pharmaceutical industry, mainly because some animal-derived products may carry infectious factors such as viruses, which are difficult to detect. Synthesizing UDCA based on plant-derived raw materials can avoid such risks.

[0006] At present, there are many literatures reporting the synthesis of UDCA using plant-derived raw materials. The method reported in the prior art CN111072744A is shown in route 1, which uses BA (bisnoralcohol) as the raw material (which is obtained by biological fermentation of phytosterol), and uses pyridinium dichromate (PDC), which has the problem of chromium metal pollution. Moreover, the last step of this route generally uses Raney nickel for hydrogenation reduction, which has poor stereoselectivity.

[0007]

[0008] The method reported in the prior art CN113461764A is shown in route 2, which uses the hydroxylation of BA at position 7 as the raw material. However, at present, this technology is not mature, is not easy to scale up production, and has high cost. In addition, in the 5th step of this route, the stereoselectivity of sodium borohydride reduction to generate the hydroxyl group at position 3 is often poor, and the scale-up production of sodium borohydride is dangerous.

[0009]

[0010] The method reported in prior art CN115181150A is shown in Route 3, which is prepared from BA through 7 steps, wherein in step C, the reaction of Grignard reagent is not easy to scale up and is not suitable for industrial scale production, and in step G, the stereoselectivity of hydrogenation reduction by Raney nickel is poor.

[0011]

[0012] Prior art CN115521964A discloses a preparation method of ursodeoxycholic acid as shown in Route 4, and the last step is enzyme-catalyzed 7-position hydroxylation, but this technology is not mature and is not easy to scale up, and the cost is high.

[0013]

[0014] Prior art CN115505622A discloses a preparation method of ursodeoxycholic acid as shown in Route 5, wherein compound I is first reduced to intermediate state I by 3α-steroid dehydrogenase, and then reduced to UDCA by 7β-steroid dehydrogenase.

[0015]

[0016] The raw material compound I used in this preparation method is obtained from compound M, and compound M is obtained from BA through a series of synthesis, so compound I itself is difficult to obtain, and this method needs tedious two-step enzyme-catalyzed reduction and has a long reaction time.

[0017] Prior art WO2023081657 discloses the following preparation method for preparing ursodeoxycholic acid as shown in Route 6

[0018]

[0019] This preparation method has a long step, and ursodeoxycholic acid is prepared from BA through 11 steps, and the yield of several steps is particularly low, the yield of KCEA prepared by decarboxylation reaction in step 4 is only 56%, and the reaction needs to be carried out at a high temperature of 145°C, which is not suitable for industrial production; the yield of compound 4 prepared from KCEA is only 58%; in the reaction of compound 6 to prepare compound 7 by hydrogenation, the hydrogen pressure needs to be 6 bar, which is too high and not suitable for industrial production, and the yield of this step is only 73.2%; 3,7-DKCA needs to be prepared into UDCA through two-step biological reduction, which greatly reduces the production efficiency.

[0020] Prior art CN118638174A discloses the following preparation method for preparing ursodeoxycholic acid as shown in Route 7

[0021]

[0022] In the preparation method, compound C is protected by ethylene glycol to obtain compound D, compound D is oxidized at allyl position to obtain compound E, and compound E is deesterified to obtain compound F. The ethylene glycol protecting group is generally poor in stability, and the protecting group is easy to fall off and return to the raw material during the reaction and post-treatment in the third step. The stable compound D is poor in stability, which leads to difficult purification, low content and relatively difficult long-term storage. In step 4, the ethylene glycol protecting group is unstable and easy to fall off, and compound D returns to compound C, which leads to an impossible oxidation reaction and low yield and many impurities in the fourth step. In step 5, deesterification is carried out in a Lewis acid, which is easy to cause the unstable ethylene glycol protecting group to fall off, leading to many impurities and low yield.

[0023] Therefore, how to obtain a preparation method of ursodeoxycholic acid which is simple in process, mild in conditions, high in product yield, good in product quality, low in impurities and suitable for industrial production is a technical problem to be solved at present. SUMMARY

[0024] In view of the problems in the preparation method of ursodeoxycholic acid in the prior art, the purpose of the present application is to provide a preparation method of ursodeoxycholic acid, which comprises the following steps:

[0025] (1) Compound A is reacted with a halogenating agent or p-toluenesulfonyl chloride (TsCl) to obtain compound B, and the reaction formula is as follows:

[0026] wherein R1 is Cl, Br, I or -OTs,

[0027] (2) Compound B is reacted with a dialkyl malonate to obtain compound C, and the reaction formula is as follows:

[0028] wherein R2 is C1-C6 alkyl, and

[0029] (3) Compound C is reacted with neopentyl glycol to obtain compound D, and the reaction formula is as follows:

[0030]

[0031] In another preferred example, in step (1), the halogenating agent is selected from phosphorus tribromide, phosphorus trichloride, N-bromosuccinimide (NBS), N-chlorosuccinimide or N-iodosuccinimide, more preferably N-bromosuccinimide.

[0032] In another preferred embodiment, in step (1), the molar ratio of compound A to halogenating reagent or TsCl is 1 : 1 to 3, more preferably 1 : 1.0 to 1.5, most preferably 1 : 1.1 to 1.3.

[0033] In another preferred embodiment, R2is methyl or ethyl. In another preferred embodiment, R1is -OTs, and in step (1), the reaction temperature of compound A with p-toluenesulfonyl chloride (TsCl) is 20 °C to 60 °C.

[0034] In another preferred embodiment, in step (1), the solvent used in the reaction is selected from toluene, xylene, dichloromethane, dichloroethane, or a combination thereof.

[0035] In another preferred embodiment, in step (2), the dialkyl malonate is selected from dimethyl malonate or diethyl malonate.

[0036] In another preferred embodiment, in step (2), a base and a phase transfer agent are also present in the reaction system. In another preferred embodiment, the base is selected from sodium carbonate, potassium carbonate, cesium carbonate, potassium tert-butoxide, sodium tert-butoxide, sodium methoxide, sodium ethoxide, or a combination thereof. In another preferred embodiment, the phase transfer agent is selected from tetrabutylammonium iodide, tetrabutylammonium bromide, tetrabutylammonium chloride, or a combination thereof.

[0037] In another preferred embodiment, in step (2), the molar ratio of compound B to dialkyl malonate is 1 : 1 to 5, more preferably 1 : 1.2 to 3. In another preferred embodiment, in step (2), the molar ratio of dialkyl malonate to base is 1 : 1 to 3, more preferably 1 : 1.2 to 3. In another preferred embodiment, in step (2), the molar volume ratio of phase transfer agent to solvent is 0.01 to 0.1 mmol / ml, more preferably 0.02 to 0.005 mmol / ml.

[0038] In another preferred embodiment, in step (2), the solvent used in the reaction is selected from toluene, xylene, or a combination thereof.

[0039] In another preferred embodiment, in step (2), the reaction temperature is 90 °C to 150 °C, more preferably 116 °C to 140 °C.

[0040] In another preferred embodiment, in step (2), the reaction time is 3 to 8 h.

[0041] In another preferred embodiment, in step (3), the reaction of compound C with neopentyl glycol is carried out in the presence of an acid selected from p-toluenesulfonic acid, o-nitrobenzenesulfonic acid, p-chlorobenzenesulfonic acid, boron trifluoride diethyl ether, or a combination thereof, and optionally a water scavenger selected from triethyl orthoformate, trimethyl orthoformate, tributyl orthoformate, or a combination thereof.

[0042] In another preferred embodiment, in step (3), the weight to volume ratio of compound C to neopentyl glycol is 0.1 to 5 g / 10 ml, more preferably 0.5 to 2 g / 10 ml. In another preferred embodiment, in step (3), the molar ratio of compound C to acid is 1 :0.05 to 0.5, more preferably 1 :0.05 to 0.20, most preferably 1 :0.10 to 0.15. In another preferred embodiment, in step (3), the molar ratio of compound C to water absorbing agent is 1 :1.0 to 6.0, more preferably 1 :2.0 to 4.0.

[0043] In another preferred embodiment, in step (3), the solvent is selected from one or a combination of toluene, benzene, ethyl acetate, tetrahydrofuran.

[0044] In another preferred embodiment, in step (3), the reaction temperature is 4°C to 45°C, more preferably 25°C to 35°C.

[0045] In another preferred embodiment, the method for preparing ursodeoxycholic acid further comprises the following steps:

[0046] (4) Compound D is subjected to an oxidation reaction to produce compound E, as shown in the following reaction formula:

[0047]

[0048] In another preferred embodiment, in step (4), the oxidation reaction is carried out in the presence of a cuprous halide catalyst and an oxidizing agent, wherein the oxidizing agent is selected from tert-butyl hydroperoxide, oxygen or air, and the cuprous halide is selected from cuprous chloride, cuprous bromide, cuprous iodide or a combination thereof. More preferably, cuprous iodide.

[0049] In another preferred embodiment, in step (4), the oxidation reaction is carried out in the presence of an imine catalyst, an initiator and an oxygen source, wherein the imine catalyst is selected from N-hydroxyphthalimide, N-hydroxyanthranilic acid imide, N,N'-dihydroxy- pyromellitic acid imide, the initiator is selected from one or a combination of benzoyl peroxide (BPO), azobisisobutyronitrile, azobisisoheptyl nitrile, and the oxygen source is selected from oxygen, air or a combination thereof. In another preferred embodiment, the molar ratio of compound D to imine catalyst is 1 :0.01 to 1.2, more preferably 1 :0.3 to 0.7. In another preferred embodiment, the molar ratio of compound D to initiator is 1 :0.001 to 0.1, more preferably 1 :0.3 to 0.7. In another preferred embodiment, in step (4), the solvent used in the reaction is selected from acetonitrile, dichloromethane, acetone, ethyl acetate, butyl acetate or a combination thereof. In another preferred embodiment, in step (4), the reaction temperature is 4°C to 45°C, more preferably 15°C to 30°C.

[0050] In another preferred embodiment, the method for preparing ursodeoxycholic acid further comprises the following steps:

[0051] (5) Compound E is subjected to de-esterification reaction to form Compound F, and the reaction formula is as follows:

[0052] wherein R2 is C1-C6 alkyl.

[0053] In another preferred embodiment, in step (5), the de-esterification reaction is carried out in the presence of a de-esterification catalyst, and in another preferred embodiment, the de-esterification catalyst is selected from lithium chloride, sodium chloride, or a combination thereof, more preferably lithium chloride.

[0054] In another preferred embodiment, in step (5), the molar ratio of Compound E to the de-esterification catalyst is 1:1-3, more preferably 1:1.0-1.5, and most preferably 1:1.1-1.3.

[0055] In another preferred embodiment, in step (5), the solvent used in the reaction is selected from N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, or a combination thereof.

[0056] In another preferred embodiment, in step (5), the reaction temperature is 100°C-220°C, more preferably 120°C-150°C.

[0057] In another preferred embodiment, in step (5), the reaction time is 10-20 h.

[0058] In another preferred embodiment, the method for preparing ursodeoxycholic acid further comprises the following steps:

[0059] (6) Compound F is subjected to de-ketalization reaction under the catalysis of an acid to form Compound G, and the reaction formula is as follows:

[0060]

[0061] In another preferred embodiment, in step (6), the acid is hydrochloric acid, oxalic acid, acetic acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, trifluoroacetic acid, triflic acid, toluenesulfonic acid, or a combination thereof, more preferably hydrochloric acid.

[0062] In another preferred embodiment, in step (6), the solvent used in the reaction is selected from dichloromethane, dichloroethane, chloroform, acetone, butanone, tetrahydrofuran, methanol, ethyl acetate, or a combination thereof.

[0063] In another preferred embodiment, in step (6), the reaction temperature is 5°C-40°C, and the reaction time is 2-10 h.

[0064] In another preferred embodiment, the method for preparing ursodeoxycholic acid further comprises the following steps:

[0065] (7) Compound G is subjected to hydrogenation reduction reaction to form Compound H, and the reaction formula is as follows:

[0066]

[0067] In another preferred embodiment, in step (7), the hydrogenation reduction is carried out in the presence of a hydrogen source and a catalyst. In another preferred embodiment, the hydrogen source is selected from hydrogen gas or ammonium formate. In another preferred embodiment, the catalyst is selected from palladium on carbon, palladium hydroxide, Raney nickel, or a combination thereof.

[0068] In another preferred embodiment, the hydrogen source is hydrogen gas, and the hydrogen gas pressure during the hydrogenation reduction is 0.01-2.0 MPa. In another preferred embodiment, the hydrogen source is ammonium formate, and the molar ratio of compound G:ammonium formate is 1:5.0-10.0.

[0069] In another preferred embodiment, the catalyst is selected from palladium on carbon, and the hydrogen source is hydrogen gas, and the mass ratio of compound G:palladium on carbon is 10-60:1.

[0070] In another preferred embodiment, in step (7), the hydrogenation reduction temperature is 5°C-40°C, and the reaction time is 12-20 h.

[0071] In another preferred embodiment, the method for preparing ursodeoxycholic acid further comprises the following step:

[0072] (8) Compound H is subjected to an ester hydrolysis reaction to generate compound I, and the reaction formula is as follows:

[0073]

[0074] In another preferred embodiment, in step (8), the ester hydrolysis reaction is carried out in the presence of a base, and the base is selected from sodium hydroxide, potassium hydroxide, lithium hydroxide, or a combination thereof.

[0075] In another preferred embodiment, in step (8), the molar ratio of compound H:base is 1:1-10, more preferably 1:2-5.

[0076] In another preferred embodiment, in step (8), the solvent used for the reaction is selected from methanol, ethanol, isopropanol, water, or a combination thereof.

[0077] In another preferred embodiment, in step (8), the reaction temperature is 40°C-70°C, more preferably 50°C-60°C.

[0078] In another preferred embodiment, in step (8), the reaction time is 1 h-3 h.

[0079] In another preferred embodiment, the method for preparing ursodeoxycholic acid further comprises the following step:

[0080] (9) Compound I is subjected to a carbonyl reduction to generate compound H, and the reaction formula is as follows:

[0081]

[0082] The carbonyl reduction is carried out in one step in the presence of 3α-steroid dehydrogenase with amino acid sequence as shown in SEQ ID NO. 1 and 7β-steroid dehydrogenase with amino acid sequence as shown in SEQ ID NO. 2.

[0083] In another preferred embodiment, in step (9), the carbonyl reduction is carried out in the presence of 3α-steroid dehydrogenase with amino acid sequence as shown in SEQ ID NO. 1, 7β-steroid dehydrogenase with amino acid sequence as shown in SEQ ID NO. 2 and glucose dehydrogenase with amino acid sequence as shown in SEQ ID NO. 3, wherein the 3α-steroid dehydrogenase with amino acid sequence as shown in SEQ ID NO. 1, 7β-steroid dehydrogenase with amino acid sequence as shown in SEQ ID NO. 2 and glucose dehydrogenase with amino acid sequence as shown in SEQ ID NO. 3 are provided by the same engineered bacteria.

[0084] In another preferred embodiment, the 3α-steroid dehydrogenase, 7β-steroid dehydrogenase and glucose dehydrogenase in step (9) are in the form of bacteria, and are provided by wet cells of the engineered bacteria. In another preferred embodiment, the wet cells of the engineered bacteria are obtained by centrifuging the fermentation broth containing the engineered bacteria at 4°C, 6000 rpm for 5 min.

[0085] In another preferred embodiment, in step (9), the mass ratio of compound I to the wet cells of the engineered bacteria is 1:0.5-1.5, more preferably 1:1.

[0086] In another preferred embodiment, in step (9), a coenzyme is further present in the reaction system.

[0087] In another preferred embodiment, in step (9), the coenzyme is selected from NADH, NADPH, NAD, NADP or a salt thereof. In another preferred embodiment, the coenzyme is selected from NADP disodium salt.

[0088] In another preferred embodiment, in step (9), the final concentration of the coenzyme is 0.1 mM-1 mM, more preferably the final concentration is 0.5 mM.

[0089] In another preferred embodiment, in step (9), a co-substrate for coenzyme regeneration is further present in the reaction system, and the co-substrate is glucose, and the mass ratio of compound I to glucose is 1:0.5-2.

[0090] In another preferred embodiment, in step (9), the reaction temperature is 15-40°C, and the time is 0.5-3 h, more preferably the reaction temperature is 30°C, and the time is 2 h.

[0091] In another preferred embodiment, in step (9), the pH value of the reaction system is 6.0-7.0, more preferably, the pH value of the reaction system is 6.2-6.5.

[0092] In another preferred embodiment, the method for preparing ursodeoxycholic acid provided by the present application comprises the following steps:

[0093] Compound I is subjected to carbonyl reduction to obtain ursodeoxycholic acid, and the reaction formula is as follows:

[0094] wherein R is selected from C1-C6 alkyl,

[0095] wherein, the carbonyl reduction is carried out in one step in the presence of 3α-steroid dehydrogenase with the amino acid sequence shown in SEQ ID NO. 1, 7β-steroid dehydrogenase with the amino acid sequence shown in SEQ ID NO. 2 and glucose dehydrogenase with the amino acid sequence shown in SEQ ID NO. 3.

[0096] In another more preferred embodiment, the carbonyl reduction is carried out in the presence of 3α-steroid dehydrogenase with the amino acid sequence shown in SEQ ID NO. 1, 7β-steroid dehydrogenase with the amino acid sequence shown in SEQ ID NO. 2 and glucose dehydrogenase with the amino acid sequence shown in SEQ ID NO. 3, and glucose, wherein the 3α-steroid dehydrogenase with the amino acid sequence shown in SEQ ID NO. 1, the 7β-steroid dehydrogenase with the amino acid sequence shown in SEQ ID NO. 2 and the glucose dehydrogenase with the amino acid sequence shown in SEQ ID NO. 3 are provided by the same engineered bacterium. In another preferred embodiment, the 3α-steroid dehydrogenase, the 7β-steroid dehydrogenase and the glucose dehydrogenase are in the form of bacterial cells, and are provided by the wet cells of the same engineered bacterium. In another preferred embodiment, the wet cells of the engineered bacterium are obtained by centrifuging the fermentation broth containing the engineered bacterium at 4°C and 6000 rpm for 5 min. In another preferred embodiment, the mass ratio of Compound I to the wet cells of the engineered bacterium is 1:0.5-1.5, more preferably 1:1. In another preferred embodiment, a coenzyme is further present in the reaction system. In another preferred embodiment, the coenzyme is selected from NADH, NADPH, NAD, NADP or a salt thereof. In another preferred embodiment, the final concentration of the coenzyme is 0.1 mM-1 mM, more preferably, the final concentration is 0.5 mM. In another preferred embodiment, a co-substrate for coenzyme regeneration is further present in the reaction system, and the co-substrate is glucose, and the mass ratio of Compound I to glucose is 1:0.5-2. In another preferred embodiment, the reaction temperature is 15-40°C, and the time is 0.5-3 h, more preferably, the reaction temperature is 30°C, and the time is 2 h. In another preferred embodiment, the pH value of the reaction system is 6.0-7.0, more preferably, the pH value of the reaction system is 6.2-6.5.

[0097] In another preferred embodiment, the present application provides a method for preparing ursodeoxycholic acid comprising the steps of:

[0098] Compound F is subjected to a de-ketalization reaction to form compound G, as shown in the following reaction scheme:

[0099]

[0100] Compound G is subjected to a hydrogenation reduction reaction to form compound H, as shown in the following reaction scheme:

[0101]

[0102] Preferably, the hydrogenation reduction reaction is carried out in the presence of a hydrogen source and a catalyst,

[0103] Preferably, the hydrogen source is selected from hydrogen gas or ammonium formate; and the catalyst is selected from palladium on carbon, palladium hydroxide or Raney nickel.

[0104] Compound H is subjected to an ester hydrolysis reaction to form compound I, as shown in the following reaction scheme:

[0105]

[0106] Compound I is subjected to a carbonyl reduction reaction to form compound H, as shown in the following reaction scheme:

[0107] wherein the carbonyl reduction reaction is carried out in the presence of 3a-steroidal dehydrogenase having an amino acid sequence as shown in SEQ ID NO. 1 and 7b-steroidal dehydrogenase having an amino acid sequence as shown in SEQ ID NO. 2.

[0108] In another preferred embodiment, the present application provides a method for preparing ursodeoxycholic acid comprising the steps of:

[0109] Compound E is subjected to a de-esterification reaction to form compound F, as shown in the following reaction scheme:

[0110] wherein R2 is C1-C6 alkyl,

[0111] Preferably, the de-esterification reaction is carried out in the presence of a de-esterification catalyst, preferably selected from lithium chloride, sodium chloride or a combination thereof.

[0112] Compound F is subjected to a de-ketalization reaction to form compound G, as shown in the following reaction scheme:

[0113]

[0114] Compound G is subjected to a hydrogenation reduction reaction to form compound H, as shown in the following reaction scheme:

[0115]

[0116] Preferably, the hydrogenation reduction reaction is carried out in the presence of a hydrogen source and a catalyst,

[0117] Preferably, the hydrogen source is selected from hydrogen gas or ammonium formate; and the catalyst is selected from palladium on carbon, palladium hydroxide or Raney nickel.

[0118] Compound H is subjected to an ester hydrolysis reaction to generate compound I, as shown in the following reaction formula:

[0119]

[0120] Compound I is subjected to a carbonyl reduction to generate compound H, as shown in the following reaction formula:

[0121] wherein the carbonyl reduction is carried out in the presence of 3a-steroidal dehydrogenase having an amino acid sequence as shown in SEQ ID NO. 1 and 7b-steroidal dehydrogenase having an amino acid sequence as shown in SEQ ID NO. 2.

[0122] In another preferred embodiment, the present application provides a method for preparing ursodeoxycholic acid, comprising the following steps:

[0123] Compound D is subjected to an oxidation reaction to generate compound E, as shown in the following reaction formula:

[0124]

[0125] Compound E is subjected to a de-esterification reaction to generate compound F, as shown in the following reaction formula:

[0126] wherein R2 is C1-C6 alkyl,

[0127] Preferably, the de-esterification reaction is carried out in the presence of a de-esterification catalyst, and preferably the de-esterification catalyst is selected from lithium chloride, sodium chloride or a combination thereof.

[0128] Compound F is subjected to a de-ketal protecting group reaction to generate compound G, as shown in the following reaction formula:

[0129]

[0130] Compound G is subjected to a hydrogenation reduction reaction to generate compound H, as shown in the following reaction formula:

[0131]

[0132] Preferably, the hydrogenation reduction reaction is carried out in the presence of a hydrogen source and a catalyst,

[0133] Preferably, the hydrogen source is selected from hydrogen gas or ammonium formate; and the catalyst is selected from palladium on carbon, palladium hydroxide or Raney nickel.

[0134] Compound H is subjected to an ester hydrolysis reaction to produce Compound I, as shown in the following reaction scheme:

[0135]

[0136] Compound I is subjected to a carbonyl reduction to produce Compound H, as shown in the following reaction scheme:

[0137] wherein the carbonyl reduction is performed in one step in the presence of 3a-steroidal dehydrogenase having an amino acid sequence as shown in SEQ ID NO. 1 and 7β-steroidal dehydrogenase having an amino acid sequence as shown in SEQ ID NO. 2.

[0138] In another preferred embodiment, the present application provides a method for preparing ursodeoxycholic acid, comprising the following steps:

[0139] Compound C is reacted with neopentyl glycol to produce Compound D, as shown in the following reaction scheme:

[0140]

[0141] Compound D is subjected to an oxidation reaction to produce Compound E, as shown in the following reaction scheme:

[0142]

[0143] Compound E is subjected to a de-esterification reaction to produce Compound F, as shown in the following reaction scheme:

[0144] wherein R2 is C1-C6 alkyl,

[0145] Preferably, the de-esterification reaction is performed in the presence of a de-esterification catalyst, preferably, the de-esterification catalyst is selected from lithium chloride, sodium chloride or a combination thereof.

[0146] Compound F is subjected to a de-ketal protecting group reaction to produce Compound G, as shown in the following reaction scheme:

[0147]

[0148] Compound G is subjected to a hydrogenation reduction reaction to produce Compound H, as shown in the following reaction scheme:

[0149]

[0150] Preferably, the hydrogenation reduction reaction is performed in the presence of a hydrogen source and a catalyst,

[0151] Preferably, the hydrogen source is selected from hydrogen gas or ammonium formate; and the catalyst is selected from palladium on carbon, palladium hydroxide or Raney nickel.

[0152] Compound H is subjected to an ester hydrolysis reaction to produce Compound I, as shown in the following reaction scheme:

[0153]

[0154] Compound I is subjected to a carbonyl reduction to produce Compound H, as shown in the following reaction scheme:

[0155] wherein the carbonyl reduction is performed in one step in the presence of 3a- steroidal dehydrogenase having an amino acid sequence as shown in SEQ ID NO. 1 and 7β- steroidal dehydrogenase having an amino acid sequence as shown in SEQ ID NO. 2.

[0156] In another preferred embodiment, the present application provides a method for preparing ursodeoxycholic acid, comprising the following steps:

[0157] Compound B is reacted with a dialkyl malonate to produce Compound C, as shown in the following reaction scheme:

[0158] wherein R1 is Cl, Br, I or

[0159] -OTs, R2 is C1-C6 alkyl;

[0160] Compound C is reacted with neopentyl glycol to produce Compound D, as shown in the following reaction scheme:

[0161]

[0162] Compound D is subjected to an oxidation reaction to produce Compound E, as shown in the following reaction scheme:

[0163]

[0164] Compound E is subjected to a de-esterification reaction to produce Compound F, as shown in the following reaction scheme:

[0165] wherein R2 is C1-C6 alkyl,

[0166] Preferably, the de-esterification reaction is performed in the presence of a de-esterification catalyst, preferably, the de-esterification catalyst is selected from lithium chloride, sodium chloride or a combination thereof.

[0167] Compound F is subjected to a de-ketal protecting group reaction to produce Compound G, as shown in the following reaction scheme:

[0168]

[0169] Compound G is subjected to a hydrogenation reduction reaction to form Compound H, as shown in the following reaction formula:

[0170]

[0171] Preferably, the hydrogenation reduction reaction is carried out in the presence of a hydrogen source and a catalyst,

[0172] Preferably, the hydrogen source is selected from hydrogen gas or ammonium formate; and the catalyst is selected from palladium on carbon, palladium hydroxide or Raney nickel.

[0173] Compound H is subjected to an ester hydrolysis reaction to form Compound I, as shown in the following reaction formula:

[0174]

[0175] Compound I is subjected to a carbonyl reduction to form Compound H, as shown in the following reaction formula:

[0176] The carbonyl reduction is carried out in the presence of 3α-steroidal dehydrogenase having an amino acid sequence as shown in SEQ ID NO. 1 and 7β-steroidal dehydrogenase having an amino acid sequence as shown in SEQ ID NO. 2.

[0177] The present application also provides a compound having the following structural formula:

[0178] wherein R2 is C1-C6 alkyl, or

[0179] wherein R2 is C1-C6 alkyl. BRIEF DESCRIPTION OF DRAWINGS

[0180] Figure 1 is the nuclear magnetic hydrogen spectrum of Compound B-1;

[0181] Figure 2 is the nuclear magnetic hydrogen spectrum of Compound C-1;

[0182] Figure 3 is the nuclear magnetic hydrogen spectrum of Compound E-1;

[0183] Figure 4 is the nuclear magnetic hydrogen spectrum of Compound F-1;

[0184] Figure 5 is the nuclear magnetic hydrogen spectrum of Compound G-1;

[0185] Figure 6 is the nuclear magnetic hydrogen spectrum of Compound I;

[0186] Figure 7 is the nuclear magnetic hydrogen spectrum of Compound UDCA;

[0187] Figure 8 Figure 1 is a SDS-PAGE chart of recombinant E. coli co-expressing 3α-steroid dehydrogenase (3α-HSDH), 7β-steroid dehydrogenase (7β-HSDH) and glucose dehydrogenase (GDH). DETAILED DESCRIPTION

[0188] The present inventors, in view of the defects in the prior art method for preparing ursodeoxycholic acid, have conducted in-depth research and obtained the technical solution of the present application. The method of the present application has readily available raw materials, mild reaction conditions, no need for column chromatography purification in post-treatment, and simple operation, which is suitable for industrial production.

[0189] In the description of the present application, "room temperature" refers to 0℃ to 40℃, for example, 15℃ to 35℃, 20℃ to 30℃, 22℃, 25℃, 28℃, etc.

[0190] In the description of the present application, "C1-C6 alkyl" refers to an alkyl group containing 1 to 6 carbons. For example: methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, etc.

[0191] In one specific embodiment of the present application, the method for preparing ursodeoxycholic acid of the present application comprises the following steps:

[0192] (1) Compound A is reacted with a halogenating agent or TsCl to generate compound B;

[0193] (2) Compound B is reacted with a dialkyl malonate to generate compound C;

[0194] (3) Compound C is reacted with neopentyl glycol to generate compound D;

[0195] (4) Compound D is subjected to an oxidation reaction to generate compound E;

[0196] (5) Compound E is subjected to a de-esterification reaction to generate compound F;

[0197] (6) Compound F is subjected to a de-ketal protecting group reaction to generate compound G;

[0198] (7) Compound G is subjected to a hydrogenation reduction reaction to generate compound H;

[0199] (8) Compound H is subjected to an ester hydrolysis reaction to generate compound I;

[0200] (9) Compound I is subjected to a carbonyl reduction to generate compound H,

[0201] The reaction formula is as follows:

[0202]

[0203] The reaction formula is as follows: wherein R1is CI, Br, I, or -OTs, and R2is C1-C6 alkyl.

[0204] In step (1), compound A is reacted with a halogenating reagent or TsCl, so that the hydroxyl group is substituted by halogen or p-toluenesulfonyl group. The reaction is carried out according to the conventional method in the art, and the halogenating reagent includes but is not limited to phosphorus tribromide, phosphorus trichloride, N-bromosuccinimide, N-chlorosuccinimide, N-iodosuccinimide. In some embodiments, compound A is reacted with p-toluenesulfonyl chloride (TsCl) in the presence of a base (e.g., triethylamine) and a catalyst (e.g., 4-dimethylaminopyridine), and after the reaction is completed as detected by TLC, the organic phase is concentrated, slurried with ethanol, filtered, and the residue is dried to obtain the target compound.

[0205] In step (2), the reaction of compound B with dialkyl malonate (preferably dimethyl malonate or diethyl malonate) is carried out in the presence of a base and a phase transfer agent. The base includes but is not limited to sodium carbonate, potassium carbonate, cesium carbonate, potassium tert-butoxide, sodium tert-butoxide, sodium methoxide, and sodium ethoxide. The phase transfer agent includes but is not limited to tetrabutylammonium iodide, tetrabutylammonium bromide, and tetrabutylammonium chloride. In some embodiments of the present application, compound B is reacted with dimethyl malonate, and after the reaction is completed as detected by TLC, the reaction solution is filtered after cooling, the filtrate is concentrated to obtain a residue, and the residue is dried to obtain the target compound.

[0206] In step (3), the reaction of compound C with neopentyl glycol is carried out in the presence of a catalyst (including but not limited to p-toluenesulfonic acid, o-nitrobenzenesulfonic acid, p-chlorobenzenesulfonic acid, boron trifluoride etherate) and a water absorbent (including but not limited to triethyl orthoformate, trimethyl orthoformate, tributyl orthoformate). After the reaction is completed, the reaction solution is quenched by dropwise addition of saturated aqueous sodium bicarbonate solution, and the product is precipitated by dropwise addition of water, filtered, and dried to obtain the target compound.

[0207] In step (4), in some embodiments, the oxidation reaction is carried out in the presence of an oxidizing agent (including but not limited to oxygen, air), an imine catalyst (including but not limited to N-hydroxyphthalimide, N-hydroxyanthranilic acid imide, N,N'-dihydroxyisophthalic acid imine), and an initiator (including but not limited to dibenzoyl peroxide (BPO), azobisisobutyronitrile, azobisisoheptyl nitrile). After the reaction is completed, the solvent is removed by concentration, dissolved in dichloromethane, filtered to remove insoluble matter, the filtrate is washed with an aqueous base, the organic phase is concentrated, the obtained residue is slurried with an alcohol to obtain compound C which can be directly used in the next step of the reaction. In some embodiments of the present application, the oxidizing agent is oxygen.

[0208] In step (5), compound E is de-esterified to form compound F in the presence of a de-esterification catalyst. The de-esterification catalyst includes, but is not limited to, lithium chloride, sodium chloride. In some embodiments of the present application, the de-esterification catalyst is lithium chloride. After the reaction is completed, water is added to the reaction solution, which is stirred thoroughly, filtered, and the filter cake is slurried with ethanol, filtered, and dried to obtain the target compound.

[0209] In step (6), compound F is de-protected to form compound G, which can be carried out according to conventional methods in the art for such reactions. For example, in the presence of an acid, which includes, but is not limited to, hydrochloric acid, oxalic acid, acetic acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, trifluoroacetic acid, triflic acid, toluenesulfonic acid. In some embodiments of the present application, the acid is hydrochloric acid, and the solvent is dichloromethane. After the reaction is completed, the reaction solution is adjusted to neutral with a base, stirred thoroughly, and extracted with dichloromethane. The organic phase is retained, concentrated, and then slurried with methanol, filtered, and dried to obtain the target compound.

[0210] In step (7), the hydrogen source for the hydrogenation reaction includes, but is not limited to, hydrogen gas, ammonium formate. The catalyst includes, but is not limited to, palladium on carbon, palladium hydroxide, Raney nickel. In some embodiments of the present application, the catalyst is Pd / C, and the hydrogen source is hydrogen gas. After the reaction is completed, the reaction solution is filtered, and the filtrate is concentrated, and the residue is dissolved in ethanol and concentrated to obtain compound F, which can be directly used in the next step.

[0211] In step (8), compound H is hydrolyzed to form compound I in the presence of a base. The base includes, but is not limited to, sodium hydroxide, potassium hydroxide, and lithium hydroxide. In some embodiments of the present application, the base used for hydrolysis is sodium hydroxide. After the reaction is completed, the reaction solution is concentrated, and then water and dichloromethane are added. The aqueous phase obtained is adjusted to a pH of 2-3 with hydrochloric acid, and a solid is precipitated, which is filtered and dried to obtain the target compound.

[0212] In step (9), the compound I is subjected to carbonyl reduction to form compound H. In some embodiments of the present application, the carbonyl reduction is carried out in one step in the presence of 3α-steroid dehydrogenase having the amino acid sequence shown in SEQ ID NO. 1, 7β-steroid dehydrogenase having the amino acid sequence shown in SEQ ID NO. 2, and glucose dehydrogenase having the amino acid sequence shown in SEQ ID NO. 3, coenzyme, and glucose, wherein the 3α-steroid dehydrogenase, 7β-steroid dehydrogenase, and glucose dehydrogenase are provided by the same engineered bacterium. The reaction solvent is n-octanol and phosphate buffer (pH 6.3), and during the reaction, the pH of the system is maintained at 6.2-6.5. After the reaction is completed, the pH of the system is adjusted to 13 with aqueous NaOH, and then the pH of the system is adjusted to about 2 with hydrochloric acid, and the solid is precipitated. The product is dissolved in methanol, filtered, the filtrate is concentrated to dryness, and the obtained filter cake is dried to obtain the target compound.

[0213] Compared with the prior art, the preparation method of ursodeoxycholic acid has the following beneficial effects:

[0214] 1. The raw materials are easy to obtain, and the reaction conditions of each step are relatively mild. No special reagents are required, and no column chromatography or multiple recrystallization purification operations are required for post-treatment. The operation is simple and suitable for industrial production.

[0215] 2. The carbonyl is protected by neopentyl glycol. After the carbonyl is protected by neopentyl glycol, the rigidity of the substrate is increased, the substrate has good stability, and the purification is easy with less impurities. The substrate is not easy to fall off in subsequent oxidation reaction and deesterification reaction, reducing the generation of impurities, and high-purity intermediates can be easily purified. The neopentyl glycol protecting group removal reaction is also very simple and fast. Therefore, after the carbonyl is protected by neopentyl glycol, the intermediates are relatively stable, have less impurities, are easy to purify, and have high yield.

[0216] 3. The combination of chemical synthesis and biosynthesis method has high product purity, which can reach 99.91%.

[0217] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following examples are not specified, and are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0218] Unless otherwise specified, the raw materials or reagents used in the examples can be obtained commercially or prepared according to conventional methods.

[0219] Example 1 Preparation of compound B-1

[0220]

[0221] To the reaction flask was added compound A (CAS: 40736-33-2) (200.0 g, 605.1 mmol), triethylamine (122.4 g, 1209.6 mmol), 4-dimethylaminopyridine (10.4 g, 84.8 mmol), dichloromethane (2000 ml), cooled to 0 °C, added p-toluenesulfonyl chloride (150.0 g, 786.6 mmol) in 3-5 batches, then warmed to 40 °C, stirred for 3 h, TLC showed no starting material remained. Concentrated the organic phase, added ethanol (800 ml), slurried for 2 h, filtered to give white solid, dried to give 283.0 g, yield 96.0%, HPLC purity 99.5%. MS: m / z = 485.7 [M+1] + .

[0222] Preparation of Example 2 compound C-1

[0223]

[0224] To the reaction flask was added compound B-1 (29.1 g, 59.74 mmol, purity about 99.5%), potassium carbonate (12.4 g, 89.61 mmol), tetrabutylammonium iodide (2.2 g, 5.97 mmol), dimethyl malonate (11.84 g, 89.61 mmol), toluene (60 ml), replaced with nitrogen, warmed to 110 °C, reacted for 5 h, TLC showed no starting material remained, cooled to 25 °C, filtered, concentrated the filtrate to give light yellow solid, added methanol (60 ml), slurried for 2 h at room temperature, filtered, dried the filter cake to give 23.7 g, yield 88.6%, HPLC purity 99.2%. MS: m / z = 445.6 [M+1] + .

[0225] Preparation of Example 3 compound D-1

[0226]

[0227] At room temperature, compound C-1 (20.0 g, 44.62 mmol, purity approximately 99.2%), neopentyl glycol (100.00 g, 960.15 mmol), toluene (60 mL), and p-toluenesulfonic acid (0.77 g, 4.50 mmol) were added to a reaction flask. Stirring was started, and triethyl orthoformate (20.0 g, 44.98 mmol) was added dropwise over 3 hours. After the addition was complete, the mixture was stirred at 25–35 °C for 10 hours. TLC analysis showed no residual starting material. The mixture was then cooled to 0–10 °C in an ice-water bath, and the reaction was quenched by adding 30 mL of saturated sodium bicarbonate solution. The mixture was then concentrated to remove toluene until no more droplets were added. Water (200 mL) was slowly added and stirred for 2 hours. The mixture was filtered, and the filter cake was dried to give 22.5 g of a pale yellow solid, yield 95.0%, HPLC purity 99.6%. MS: m / z = 531.8 [M+1] + .

[0228] Example 4 Preparation of compound E-1

[0229]

[0230] At room temperature, compound D-1 (10.0 g, 18.77 mmol, purity approximately 99.6%), N-hydroxyphthalimide (3.07 g, 18.84 mmol), benzoyl peroxide (0.05 g, 0.19 mmol), methanol (160 mL), and dichloromethane (40 mL) were added to a reaction flask. The mixture was purged three times with nitrogen and then three times with oxygen under oxygen bulb protection. The mixture was stirred at room temperature for 6 h. The organic phase was concentrated, and the resulting residue was slurried with dichloromethane (300 mL) for 1 h and filtered. The filtrate was washed once with sodium bicarbonate aqueous solution and once with saturated sodium chloride. The organic phase was concentrated, slurried with methanol, filtered, and dried to give 9.3 g of a white solid, yield 90.9%, HPLC purity 99.8%. MS: m / z = 545.7 [M+1] + . 1 H NMR (400MHZ, CDCl3), δ5.74(s,1H),3.78-3.75(m,4H),3.58-3.41(m,5H),2.89-2.85(m,1H),2.47-0.96(m,34H),0.70(s,3H).

[0231] Example 5 Preparation of compound F-1

[0232]

[0233] To the reaction flask was added compound E-1 (10.0 g, 18.32 mmol, purity about 99.8%), lithium chloride (1.81 g, 42.78 mmol), water (0.5 ml), N,N-dimethylacetamide (100 ml) at room temperature, warmed to 125 °C for 12 h, TLC detection no raw material left, ice water bath cooling to 0-5 °C, drop water (30 ml) into the reaction liquid, stirring at 0-5 °C for 1-2 h, filtration, the filter cake was added ethanol (45 ml) at 0-5 °C for 1-2 h, filtration to get white solid, dried to get 8.7 g, yield 97.1%, HPLC purity 99.5%. MS: m / z = 487.7 [M+1] + . 1 H NMR (400 MHZ, CDC13), δ 5.71 (s, 1H), 3.68 (s, 3H), 3.58 (s, 2H), 3.48-3.41 (m, 2H), 2.87-2.84 (m, 1H), 2.47-0.97 (m, 34H), 0.70 (s, 3H).

[0234] Preparation of compound G-1 in Example 6

[0235]

[0236] To the reaction flask was added compound F-1 (20.0 g, 40.89 mmol, HPLC purity about 99.5%), dichloromethane (100 ml) at room temperature, ice water bath cooling to 0-10 °C, drop 37% concentrated hydrochloric acid (20 ml), warmed to 25 °C for 5 h, TLC detection no raw material left, liquid-liquid separation, dichloromethane phase was retained, ice water bath cooling to 0-5 °C, drop sodium bicarbonate aqueous solution to adjust pH to neutral, liquid-liquid separation, water phase was extracted with dichloromethane (100 ml), combined dichloromethane phase, concentrated, the residue was added methanol (40 ml) at 0-5 °C for 1-2 h, filtration to get white solid, dried to get 15.6 g, yield 95.1%, HPLC purity 99.8%. MS: m / z = 401.6 [M+1] + .

[0237] Preparation of compound H-1 in Example 7

[0238]

[0239] To a reaction flask was added compound G-1 (1.0 g, 2.49 mmol, purity about 99.8%), methanol (25 ml), 10% Pd / C (0.05 g), hydrogen balloon protection after hydrogen replacement, stirring at room temperature for 16 h. Subsequently, filter, concentrated to no obvious liquid outflow, the residue was added to ethanol (25 ml), continue to concentrate, 45 °C vacuum drying to obtain white solid 0.98 g, yield 97.6%, HPLC purity 99.8%. MS: m / z = 403.6 [M+1] + .

[0240] Preparation of compound I of example 8

[0241]

[0242] To a reaction flask was added compound H-1 (0.9 g, 2.23 mmol, purity about 99.8%), ethanol (10 ml), nitrogen replacement three times, then add sodium hydroxide aqueous solution (0.24 g sodium hydroxide dissolved in 5 ml water), then heated to internal temperature 55 °C, stirring for 2 h. Subsequently, concentrated to no obvious liquid outflow, the obtained residue was added to a mixture of water (30 ml) and dichloromethane (10 ml), stirred for 30 min, then static layering, the water phase was retained, the water phase was adjusted to pH 2-3 with 1M hydrochloric acid aqueous solution, a large amount of light yellow solid precipitated, stirred for 2 h, then filtered to obtain light yellow solid, dried to obtain 0.80 g, yield 92.0%, purity 99.6%. MS: m / z = 389.6 [M+1] + . 1 H NMR (400 MHZ, CDC13), δ 2.88 (dd, 1H), 2.50 (t, 1H), 2.38-2.43 (m, 1H), 2.18-2.30 (m, 7H), 2.04-2.11 (m, 2H), 1.82-1.98 (m, 4H), 1.43-1.67 (m, 5H), 1.33-1.39 (m, 1H), 1.31 (s, 3H), 1.12-1.29 (m, 3H), 0.96-1.00 (m, 1H), 0.98 (d, 3H), 0.73 (s, 3H).

[0243] Preparation of compound UDCA of example 9

[0244]

[0245] Into a 250 mL three-necked round bottom flask, add compound I (15.00 g, 38.45 mmol, purity about 99.6%), coenzyme substrate monohydrate glucose (22.92 g, 115.65 mmol), add n-octanol (45 ml), pH 7.0 0.1 M phosphate buffer (45 ml), final concentration of 1 mM NADP disodium salt (CAS No: 24292-60-2), mix the materials at 35 °C, 400-600 rpm mechanical stirring. After the material is mixed, add E. coli wet cell suspension (500 g / L, 30 ml), continue to stir, measure the initial pH of the reaction as 6.3, monitor the pH during the reaction, adjust the pH of the reaction to 6.2-6.5 using 4% NaOH. After 2 h of reaction, add 40% NaOH to the above reaction bottle to adjust the pH to about 13, after the product is completely dissolved, add concentrated hydrochloric acid to adjust the pH of the system to about 2, so that the product is completely precipitated, suction filtration to obtain the filter cake, and wash the filter cake with water to further remove water-soluble impurities. Add methanol (300 ml) to the filter cake, stir to completely dissolve the product in methanol, suction filter, and wash the filter cake with methanol, collect the methanol solution to remove bacteria and proteins. Concentrate the methanol solution to dryness by rotary evaporation, add 150 mL petroleum ether to the residue, beat, suction filter to obtain a filter cake that removes impurities and n-octanol, wash the filter cake with petroleum ether, dry and weigh after drying to obtain 13.90 g of white solid, yield 92.0%, purity 99.91%, optical rotation +59.2° (pharmacopoeia standard +59.0-62.0°). MS: m / z = 393.6 [M+1] + . 1 H NMR (400 MHz, CD3OD), δ 3.48-3.56 (m, 2H), 2.30-2.38 (m, 1H), 2.18-2.25 (m, 1H), 2.03 (dt, 1H), 1.83-1.91 (m, 5H), 0.99-1.66 (m, 24H), 0.75 (s, 3H).

[0246] The preparation process of the above E. coli wet cell suspension is as follows:

[0247] (1) Construction of recombinant E. coli

[0248] The 3a-steroid dehydrogenase (3a-HSDH) derived from Comamonas testosteroni (NCBI accession number: WP_003078312.1) shown in SEQ ID NO. 1, the 7b-steroid dehydrogenase (7b-HSDH) derived from Clostridium nigeriense (NCBI accession number: WP_066892209.1) shown in SEQ ID NO. 2, and the glucose dehydrogenase (GDH) derived from Bacillus subtilis (NCBI accession number: WEZ47479.1) shown in SEQ ID NO. 3 were codon-optimized by Shengwo Biotechnology (Shanghai) Co., Ltd., and then synthesized and cloned into the EcoRI / HindIII enzyme sites of the pET28a plasmid (the codon-optimized nucleotide sequence of 3a-HSDH is shown in SEQ ID NO. 4, the codon-optimized nucleotide sequence of 7b-HSDH is shown in SEQ ID NO. 5, and the codon-optimized nucleotide sequence of GDH is shown in SEQ ID NO. 6). A pET28a-3aHSDH-7bHSDH-GDH three-enzyme co-expression recombinant plasmid was constructed and transformed into E. coli BL21 (DE3) to obtain a recombinant E. coli (engineered bacteria) E. coli BL21 (DE3)-pET28a-3aHSDH-7bHSDH-GDH with a kanamycin resistance gene, which was subsequently named AU060021. SDS-PAGE protein expression verification (see Figure 8 ) showed that the three enzymes were successfully expressed, with a 3a-HSDH protein molecular weight of 23.2 kDa, a 7b-HSDH protein molecular weight of 25.7 kDa, and a GDH protein molecular weight of 24.8 kDa.

[0249] (2) Culturing the AU060021 recombinant E. coli

[0250] The AU060021 recombinant E. coli was inoculated into LB liquid medium containing 50 mg / L kanamycin and cultured at 37°C and 100-250 rpm until the mid-logarithmic growth phase to obtain a seed solution. The composition of the LB medium was: yeast powder 5 g / L, peptone 10 g / L, NaCl 10 g / L, solvent: deionized water, pH 6.8-7.2. The seed solution was inoculated into TB medium containing 50 mg / L kanamycin at a volume of 1%-5%, and cultured at 37°C for 3-6 h until the bacterial OD 600nm~0.6-0.8, IPTG is added to a final concentration of 0.5-1 mM, and the culture is incubated at 25℃, 100-250 rpm for 12-16 h. The TB culture medium consists of: 20 g / L of tryptone, 40 g / L of yeast extract, 12.5 g / L of K2HPO4, 2.3 g / L of KH2PO4, 0.4% of glycerol, and the solvent is deionized water.

[0251] (3) Obtain a bacterial suspension

[0252] The AU060021 recombinant E. coli bacterial solution obtained by culturing the above TB culture medium is centrifuged at 4℃, 6000 rpm for 5 min, the supernatant is poured off, and an equal amount of pH 7.5 100 mM phosphate buffer is added to resuspend the bacterial suspension, which is ready for use.

[0253] The sequence involved in the present application

[0254] SEQ ID NO. 1 (3α-steroid dehydrogenase amino acid sequence)

[0255] Source: Comamonas testosteroni

[0256] NCBI number: WP_003078312.1

[0257] IRDAEVIADLSTAEGRKQAIADVLAKCSKGMDGLVLCAGLGPQTKVLGNVVSVNYFGATELMDAFLPALKKGHQPAAVVISSVASAHLAFDKNPLALALEAGEEAKARAIVEHAGEQGGNLAYAGSKNALTVAVRKRAAAWGEAGVRLNTIAPGATETPLLQAGLQDPRYGESIAKFVPPMGRRAEPSEMASVIAFLMSPAASYVHGAQIVIDGGIDAVMRPTQF

[0258] SEQ ID NO. 2 (7β-steroid dehydrogenase amino acid sequence)

[0259] Source: Clostridium nigeriense, NCBI number: WP_066892209.1

[0260] MNVVMVGRREEMLKALGEDISSKYGVKHLVIKADFSDPNSTDEIFEKTKDLDMGFMSYVACFHTFGKLQDTPWEKHEQMLNVNVITFLKCFYHYMKIFSKQDRGAIINVSSLTGISASPYNAQYGAGKSYILKLTEAVAYEASKTNVDVEVITLGTTITPSLLKNLPGGPAGEAVMKAALTPEACVEEAFENLGKKFSIIAGEHNKASIHDWKANHTEDEFISYMGSFYER

[0261] SEQ ID NO. 3 (glucose dehydrogenase)

[0262] Source: Bacillus subtilis, NCBI No. WEZ47479.1

[0263] KVVINYYSNKQDPNEVKEEVIKAGGEAVVVQGDVTKEEDVKNIVQTAIKEFGTLDIMINNAGLENPVPSHEMPLKDWDKVIGTNLTGAFLGSREAIKYFVENDIKGNVINMSSVHEVIPWPLFVHYAASKGGIKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPKQKADVESMIPMGYIGEPEEIAAVAAWLASKEASYVTGITLFADGGMTQYPSFQAGRG

[0264] SEQ ID NO. 4 (nucleotide sequence of 3a-steroid dehydrogenase)

[0265] ATCGGTGCGGCGACCCGTAAAGTTCTGGAAGCGGCGGGTCACCAGATCGTTGGTATCGATATCCGTGATGCGGAAGTTATCGCGGATCTGAGCACCGCGGAAGGTCGTAAACAGGCGATCGCGGATGTTCTGGCGAAATGCAGTAAAGGCATGGATGGCCTGGTTCTGTGCGCTGGTTTGGGCCCGCAGACCAAAGTTCTGGGCAACGTTGTTAGCGTTAACTACTTCGGCGCGACCGAACTGATGGATGCGTTCCTGCCGGCGCTGAAAAAAGGCCACCAGCCGGCGGCGGTTGTTATCAGCTCCGTTGCGAGCGCGCACCTGGCGTTCGATAAAAACCCGCTCGCGCTGGCGCTGGAAGCGGGCGAAGAAGCGAAAGCGCGTGCGATCGTGGAACACGCAGGTGAACAGGGTGGTAACCTGGCGTACGCGGGTTCTAAAAACGCGCTGACCGTTGCGGTTCGTAAACGTGCGGCGGCATGGGGTGAAGCGGGTGTTCGTCTGAACACCATCGCCCCGGGTGCGACCGAAACCCCGCTGCTGCAGGCGGGTCTGCAGGATCCGCGTTACGGTGAAAGCATCGCAAAATTTGTGCCGCCGATGGGTCGTCGTGCGGAACCGAGCGAAATGGCGAGCGTCATCGCATTCCTGATGTCCCCGGCGGCGAGCTACGTCCACGGCGCGCAGATCGTTATCGATGGTGGCATTGATGCAGTGATGCGTCCGACCCAGTTCTAAAAGCTT

[0266] SEQ ID NO. 5 (nucleotide sequence of 7β-steroid dehydrogenase)

[0267] ATGGGGCATCATCCTGGGCGCGACCGAAGGCGTTGGTAAAGCGTTCTGCGAAAAAATCGCGAGCGAAGGCATGAACGTTGTTATGGTTGGTCGTCGTGAAGAAATGCTGAAAGCGCTGGGTGAAGATATCAGCTCTAAATACGGCGTTAAACACCTGGTTATCAAAGCGGATTTCAGCGATCCGAACAGCACCGATGAAATCTTCGAAAAAACCAAAGATCTGGATATGGGCTTCATGTCTTATGTTGCGTGCTTCCACACCTTCGGTAAACTGCAGGATACCCCGTGGGAAAAACACGAACAGATGCTGAACGTGAACGTTATCACCTTCCTGAAATGCTTCTACCACTACATGAAAATCTTCTCCAAACAGGATCGTGGCGCGATCATCAACGTTTCTAGCCTGACCGGAATCTCCGCGAGCCCGTACAACGCGCAGTACGGTGCTGGCAAAAGCTACATCCTGAAACTGACCGAAGCGGTTGCGTACGAAGCGAGCAAAACCAACGTTGATGTTGAAGTTATCACCCTGGGTACCACCATCACCCCGAGCCTGCTGAAAAACCTGCCGGGCGGTCCGGCGGGTGAAGCGGTTATGAAAGCGGCGCTGACCCCGGAAGCGTGCGTTGAAGAAGCGTTCGAAAACCTGGGCAAGAAGTTCTCTATCATCGCAGGTGAGCATAATAAAGCTAGCATTCATGACTGGAAAGCTAACCACACCGAAGATGAGTTCATTAGCTACATGGGTAGCTTCTATGAACGCTAAAAGCTT

[0268] SEQ ID NO. 6 (nucleotide sequence of glucose dehydrogenase)

[0269]

Claims

1. A method for preparing ursodeoxycholic acid, characterized by, The preparation method comprises the following steps: (1) Compound A reacts with a halogenating reagent or p-toluenesulfonyl chloride to generate Compound B, and the reaction formula is as follows: wherein R1is Cl, Br, I or -OTs, (2) Compound B reacts with a malonic acid dialkyl ester to generate Compound C, and the reaction formula is as follows: wherein R2is C1-C6alkyl, and (3) Compound C reacts with neopentyl glycol to generate Compound D, and the reaction formula is as follows: ， (4) Compound D is subjected to an oxidation reaction to generate Compound E, and the reaction formula is as follows: 。 2. The method of preparing ursodeoxycholic acid according to claim 1, characterized in that, The halogenating reagent is selected from phosphorus tribromide, phosphorus trichloride, N-bromosuccinimide, N-chlorosuccinimide or N-iodosuccinimide.

3. The method of preparing ursodeoxycholic acid according to claim 1, characterized by, The oxidation reaction is performed in the presence of an imine catalyst, an initiator and an oxygen source, the imine catalyst is selected from N-hydroxyphthalimide, N-hydroxyanthranilic acid imide, N,N'-dihydroxy pyromellitic imide, the initiator is selected from dibenzoyl peroxide, azobisisobutyronitrile, azobisisoheptyl nitrile or a combination, and the oxygen source is selected from air or oxygen, or The oxidation reaction is performed in the presence of a cuprous halide catalyst and an oxidant, the cuprous halide is selected from cuprous chloride, cuprous bromide, cuprous iodide or a combination thereof, and the oxidant is selected from tert-butyl hydroperoxide, oxygen or air.

4. The method of preparing ursodeoxycholic acid according to claim 1, characterized by, The preparation method further comprises the following steps: (5) Compound E is subjected to a de-esterification reaction to generate Compound F, and the reaction formula is as follows: wherein R2is C1-C6alkyl.

5. The method of preparing ursodeoxycholic acid according to claim 4, characterized in that, The de-esterification reaction is performed in the presence of a de-esterification catalyst, and the de-esterification catalyst is selected from lithium chloride, sodium chloride or a combination thereof.

6. The method of preparing ursodeoxycholic acid according to claim 4, characterized by, The preparation method further comprises the following steps: (6) Compound F is subjected to a de-ketal protecting group reaction to generate Compound G, and the reaction formula is as follows: 。 7. The method of preparing ursodeoxycholic acid according to claim 6, characterized by, The preparation method further comprises the following steps: (7) Compound G is subjected to a hydrogenation reduction reaction to generate Compound H, and the reaction formula is as follows: 。 8. The method of preparing ursodeoxycholic acid according to claim 7, characterized by, The hydrogenation reduction reaction is performed in the presence of a hydrogen source and a catalyst, the hydrogen source is selected from hydrogen gas or ammonium formate, and the catalyst is selected from palladium on carbon, palladium hydroxide or Raney nickel.

9. The method of preparing ursodeoxycholic acid according to claim 7, characterized by, The preparation method further comprises the following steps: (8) Compound H is subjected to an ester hydrolysis reaction to generate Compound I, and the reaction formula is as follows: 。 10. The method of preparing ursodeoxycholic acid according to claim 9, characterized in that, The preparation method further comprises the following steps: (9) Compound I is subjected to a carbonyl reduction to generate ursodeoxycholic acid, and the reaction formula is as follows: ， The carbonyl reduction is performed in the presence of 3α-steroidal dehydrogenase with an amino acid sequence as shown in SEQ ID NO. 1 and 7β-steroidal dehydrogenase with an amino acid sequence as shown in SEQ ID NO.

2.

11. A method for preparing ursodeoxycholic acid, characterized by, The preparation method comprises the following steps: Compound D is subjected to an oxidation reaction to generate Compound E, and the reaction formula is as follows: wherein R2is C1-C6alkyl, Compound E is subjected to a de-esterification reaction to generate Compound F, and the reaction formula is as follows: ， Compound F is subjected to a de-ketal protecting group reaction to generate Compound G, and the reaction formula is as follows: ， Compound G is subjected to a hydrogenation reduction reaction to generate Compound H, and the reaction formula is as follows: ， Compound H is subjected to an ester hydrolysis reaction to generate Compound I, and the reaction formula is as follows: ， Compound I is subjected to a carbonyl reduction to generate Compound ursodeoxycholic acid, and the reaction formula is as follows: wherein the carbonyl reduction is performed in one step in the presence of a 3a- steroidal dehydrogenase having the amino acid sequence of SEQ ID NO. 1 and a 7b- steroidal dehydrogenase having the amino acid sequence of SEQ ID NO.

2.

12. The method of preparing ursodeoxycholic acid according to claim 11, characterized in that, The de-esterification reaction is performed in the presence of a de-esterification catalyst, and the de-esterification catalyst is selected from lithium chloride, sodium chloride or a combination thereof, and / or The hydrogenation reduction reaction is carried out in the presence of a hydrogen source selected from hydrogen gas or ammonium formate and a catalyst selected from palladium on carbon, palladium hydroxide or Raney nickel.

13. A method for preparing ursodeoxycholic acid, characterized by, The preparation method comprises the following steps: Compound C is reacted with neopentyl glycol to generate compound D, and the reaction formula is as follows: wherein R2is C1-C6alkyl, Compound D is subjected to an oxidation reaction to generate compound E, and the reaction formula is as follows: ， Compound E is subjected to a de-esterification reaction to generate compound F, and the reaction formula is as follows: ， Compound F is subjected to a de-ketal protecting group reaction to generate compound G, and the reaction formula is as follows: ， Compound G is subjected to a hydrogenation reduction reaction to generate compound H, and the reaction formula is as follows: ， Compound H is subjected to an ester hydrolysis reaction to generate compound I, and the reaction formula is as follows: ， Compound I is subjected to a carbonyl reduction to generate compound ursodeoxycholic acid, and the reaction formula is as follows: wherein the carbonyl reduction is performed in one step in the presence of a 3a- steroidal dehydrogenase having the amino acid sequence of SEQ ID NO. 1 and a 7b- steroidal dehydrogenase having the amino acid sequence of SEQ ID NO.

2.

14. The method of preparing ursodeoxycholic acid according to claim 13, characterized in that, The de-esterification reaction is carried out in the presence of a de-esterification catalyst selected from lithium chloride, sodium chloride or a combination thereof, and / or The hydrogenation reduction reaction is carried out in the presence of a hydrogen source and a catalyst.

15. The method of preparing ursodeoxycholic acid according to claim 14, characterized in that, The hydrogen source is selected from hydrogen gas or ammonium formate, and the catalyst is selected from palladium on carbon, palladium hydroxide or Raney nickel.

16. A method for preparing ursodeoxycholic acid, characterized by, The preparation method comprises the following steps: Compound B is reacted with dialkyl malonate to generate compound C, and the reaction formula is as follows: wherein R1is CI, Br, I or -OTs, R2is C1-C6 alkyl, Compound C is reacted with neopentyl glycol to generate compound D, and the reaction formula is as follows: ， Compound D is subjected to an oxidation reaction to generate compound E, and the reaction formula is as follows: ， Compound E is subjected to a de-esterification reaction to generate compound F, and the reaction formula is as follows: wherein R2is C1-C6alkyl, Compound F is subjected to a de-ketal protecting group reaction to generate compound G, and the reaction formula is as follows: ， Compound G is subjected to a hydrogenation reduction reaction to generate compound H, and the reaction formula is as follows: ， Compound H is subjected to an ester hydrolysis reaction to generate compound I, and the reaction formula is as follows: ， Compound I is subjected to a carbonyl reduction to generate compound ursodeoxycholic acid, and the reaction formula is as follows: wherein the carbonyl reduction is performed in one step in the presence of a 3a- steroidal dehydrogenase having the amino acid sequence of SEQ ID NO. 1 and a 7b- steroidal dehydrogenase having the amino acid sequence of SEQ ID NO.

2.

17. The method of preparing ursodeoxycholic acid according to claim 16, characterized in that, The de-esterification reaction is carried out in the presence of a de-esterification catalyst selected from lithium chloride, sodium chloride or a combination thereof, and / or The hydrogenation reduction reaction is carried out in the presence of a hydrogen source and a catalyst.

18. A compound, characterized in that, The structural formula is as follows: ， R2 is C1-C6 alkyl.

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