Preparation method and intermediate of ursodesoxycholic acid

Abstract

The invention provides a preparation method and an intermediate of ursodesoxycholic acid, and the preparation method is characterized in that a commercially available compound A is used as a raw material, and ursodesoxycholic acid is prepared through a plurality of steps of reactions. The raw materials of the compound A are wide in source and low in price, and the preparation method is mild in reaction condition, free of special reagents, simple in post-treatment, easy and convenient to operate and suitable for industrial production. # imgabs0 #

Description

Technical Field

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

[0002] Background Field

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

[0004]

[0005] In the past, UDCA was mainly extracted from natural bear bile, but the resources of live bears are very limited and it violates the Animal Protection Act. With the increase in the clinical use of UDCA, it is imperative to develop an efficient and economical method for synthesizing UDCA. In recent years, UDCA has been mainly used as raw materials to prepare ursodeoxycholic acid using cholic acid (CA), chenodeoxycholic acid (CDCA) or hyodeoxycholic acid (HDCA). However, CA, CDCA and HDCA are all extracted from bile acid and are animal raw materials. The risks brought by animal-derived products always exist for the pharmaceutical industry, mainly because some animal-derived products may carry infectious factors such as viruses and are difficult to detect. The synthesis of UDCA based on plant-derived raw materials can avoid such risks.

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

[0007]

[0008] The method reported in CN113461764A is shown in route 2. The raw material of this method is obtained by hydroxylating the 7-position of BA. However, at present, this technology is immature, difficult to scale up production, and the cost is high. In addition, in step 5 of this route, the stereoselectivity of sodium borohydride reduction to generate the 3-position hydroxyl group is often poor, and the scaled production of sodium borohydride is dangerous.

[0009]

[0010] The method reported in the prior art CN115181150A is shown in route 3. This method uses BA as a raw material and prepares UDCA through 7 steps of reaction. In step C, the reaction scale-up operation using a Grignard reagent is not easy and is not suitable for industrial scale-up production. In addition, in step G, the stereoselectivity of the hydrogenation reduction using Raney nickel is poor.

[0011]

[0012] Prior art CN115521964A discloses a method for preparing ursodeoxycholic acid as shown in route 4, wherein the last step is enzyme-catalyzed 7-hydroxylation. However, the technology is immature, difficult to scale up, and has high cost.

[0013]

[0014] Prior art CN115505622A discloses a method for preparing 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 the preparation method is obtained from compound M, and compound M is obtained from BA through a series of syntheses. Therefore, compound I itself is difficult to obtain, and the method requires cumbersome two-step enzyme-catalyzed reduction and a long reaction time.

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

[0018]

[0019] The preparation method of this route has a long process. It takes 11 steps of reaction to prepare ursodeoxycholic acid using BA as a raw material, and the yields of several steps are particularly low. The yield of preparing KCEA through 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 preparing compound 4 from KCEA is only 58%; in the reaction of preparing compound 7 from compound 6 through hydrogenation reaction, 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%; UDCA can only be prepared from 3,7-DKCA through two steps of bioreduction, 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 for carbonyl group to obtain compound D, compound D is subjected to allylic oxidation reaction to obtain compound E, and compound E is deesterified to obtain compound F. However, the stability of the carbonyl group protected by ethylene glycol is generally poor. During the reaction and post-treatment in step 3, the protecting group is easily detached and returned to the raw material, and the stability of the product compound D is poor, resulting in difficulty in purification, low content, and relatively difficult long-term storage; in step 4, the ethylene glycol carbonyl protecting group is unstable and easily detached, and compound D will be converted back to compound C, resulting in the inability to carry out the oxidation reaction, thereby resulting in a low yield and many impurities in the reaction in step 4; in step 5, the deesterification is carried out in Lewis acid, which easily causes the unstable ethylene glycol protecting group to fall off, resulting in many impurities and low yield.

[0023] Therefore, how to obtain a preparation method of ursodeoxycholic acid with simple process, mild conditions, high product yield, good product quality and few impurities and suitable for industrial production is a technical problem that needs to be solved at present. Summary of the invention

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

[0025] (1) Compound A reacts with a halogenating agent or p-toluenesulfonyl chloride (TsCl) to generate compound B. The reaction formula is as follows:

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

[0027] (2) Compound B reacts with dialkyl malonate to generate compound C. The reaction formula is as follows:

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

[0029] (3) Compound C reacts with neopentyl glycol to generate compound D. The reaction formula is as follows:

[0030]

[0031] In another preferred embodiment, 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 the halogenating agent or TsCl is 1:1-3, more preferably 1:1.0-1.5, and most preferably 1:1.1-1.3.

[0033] In another preferred embodiment, R2 is methyl or ethyl. In another preferred embodiment, R1 is -OTs, and in step (1), the reaction temperature of compound A and 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 1:1-5, more preferably 1:1.2-3. In another preferred embodiment, in step (2), the molar ratio of dialkyl malonate to base is 1:1-3, more preferably 1:1.2-3. In another preferred embodiment, in step (2), the molar volume ratio of phase transfer agent to solvent is 0.01-0.1 mmol / ml, more preferably 0.02-0.005 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 hours.

[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 and optionally a water absorbent, wherein the acid is selected from p-toluenesulfonic acid, o-nitrobenzenesulfonic acid, p-chlorobenzenesulfonic acid, boron trifluoride etherate or a combination thereof, and the water absorbent is selected from triethyl orthoformate, trimethyl orthoformate, tributyl orthoformate or a combination thereof.

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

[0043] In another preferred embodiment, in step (3), the solvent is selected from one or a combination of toluene, benzene, ethyl acetate and 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 undergoes oxidation reaction to generate compound E. The reaction formula is as follows:

[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 oxidant, the oxidant 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-hydroxy-o-sulfonylbenzoimide, N,N'-dihydroxypyromellitic acid imide, and the initiator is selected from one or a combination of dibenzoyl peroxide (BPO), azobisisobutyronitrile, and azobisisoheptanenitrile. The oxygen source is selected from oxygen, air or a combination thereof. In another preferred embodiment, the molar ratio of compound D to the 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 the 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 undergoes a deesterification reaction to generate compound F. The reaction formula is as follows:

[0052] Wherein R2 is C1~C6 alkyl.

[0053] In another preferred embodiment, in step (5), the deesterification reaction is carried out in the presence of a deesterification catalyst. In another preferred embodiment, the deesterification 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 deesterification 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 to 220°C, more preferably 120°C to 150°C.

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

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

[0059] (6) Compound F undergoes a deketalization reaction under acid catalysis to generate compound G. 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, trifluoromethanesulfonic 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 to 40°C, and the reaction time is 2 to 10 hours.

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

[0065] (7) Compound G undergoes a hydrogenation reduction reaction to generate compound H. The reaction formula is as follows:

[0066]

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

[0068] In another preferred embodiment, the hydrogen source is hydrogen, and during the hydrogenation reduction reaction, the hydrogen pressure 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 carbon, the hydrogen source is hydrogen gas, and the mass ratio of compound G: palladium carbon is 10 to 60:1.

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

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

[0072] (8) Compound H undergoes 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 to the base is 1:1-10, more preferably 1:2-5.

[0076] In another preferred embodiment, in step (8), the solvent used in 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 to 70°C, more preferably, 50°C to 60°C.

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

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

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

[0081]

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

[0083] In another preferred embodiment, in step (9), carbonyl reduction is carried out in the presence of 3α-steroid dehydrogenase as shown in SEQ ID NO.1, 7β-steroid dehydrogenase as shown in SEQ ID NO.2, glucose dehydrogenase as shown in SEQ ID NO.3 and glucose, wherein the 3α-steroid dehydrogenase as shown in SEQ ID NO.1, the 7β-steroid dehydrogenase as shown in SEQ ID NO.2 and glucose dehydrogenase 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 bacterial form and are provided by wet cells of engineered bacteria. In another preferred embodiment, the wet cells of engineered bacteria are obtained by centrifuging a fermentation broth containing the engineered bacteria at 4°C and 6000 rpm for 5 minutes.

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

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

[0087] In another preferred embodiment, in step (9), the coenzyme is selected from NADH, NADPH, NAD, NADP or their salts. 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 to 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 also 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 reaction time is 0.5-3h. More preferably, the reaction temperature is 30°C and the reaction time is 2h.

[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 preparation method of ursodeoxycholic acid provided by the present invention 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] The carbonyl reduction is carried out in a one-step reaction in the presence of 3α-steroid dehydrogenase as shown in SEQ ID NO.1, 7β-steroid dehydrogenase as shown in SEQ ID NO.2 and glucose dehydrogenase as shown in SEQ ID NO.3.

[0096] In another more preferred embodiment, carbonyl reduction is carried out in the presence of 3α-steroid dehydrogenase as shown in SEQ ID NO.1, 7β-steroid dehydrogenase as shown in SEQ ID NO.2, and glucose dehydrogenase as shown in SEQ ID NO.3, and glucose, wherein the 3α-steroid dehydrogenase as shown in SEQ ID NO.1, the 7β-steroid dehydrogenase as shown in SEQ ID NO.2, and the glucose dehydrogenase as shown in SEQ ID NO.3 are provided by the same engineered bacteria. In another preferred embodiment, the 3α-steroid dehydrogenase, 7β-steroid dehydrogenase and glucose dehydrogenase are in bacterial form and are provided by wet cells of the same engineered bacteria. In another preferred embodiment, the engineered bacteria wet cells are obtained by centrifuging the fermentation broth containing the engineered bacteria at 4°C and 6000rpm for 5min. In another preferred embodiment, the mass ratio of compound I to the engineered bacteria wet cells is 1:0.5-1.5, preferably 1:1. In another preferred embodiment, a coenzyme is also 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 0.1mM to 1mM, and more preferably, the final concentration is 0.5mM. In another preferred embodiment, a cosubstrate for coenzyme regeneration is also present in the reaction system, and the cosubstrate is glucose, and the mass ratio of compound I to glucose is 1:0.5 to 2. In another preferred embodiment, the reaction temperature is 15 to 40°C, and the time is 0.5 to 3h, and more preferably, the reaction temperature is 30°C and the time is 2h. In another preferred embodiment, the pH value of the reaction system is 6.0 to 7.0, and more preferably, the pH of the reaction system is 6.2 to 6.5.

[0097] In another preferred embodiment, the preparation method of ursodeoxycholic acid provided by the present invention comprises the following steps:

[0098] Compound F undergoes a deketalization reaction under acid catalysis to generate compound G. The reaction formula is as follows:

[0099]

[0100] Compound G undergoes a hydrogenation reduction reaction to generate compound H. The reaction formula is as follows:

[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 or ammonium formate; the catalyst is selected from palladium carbon, palladium hydroxide or Raney nickel;

[0104] Compound H undergoes ester hydrolysis reaction to generate compound I, and the reaction formula is as follows:

[0105]

[0106] Compound I undergoes carbonyl reduction to generate compound H. The reaction formula is as follows:

[0107] The carbonyl reduction is carried out in a one-step reaction in the presence of 3α-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.1 and 7β-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.2.

[0108] In another preferred embodiment, the preparation method of ursodeoxycholic acid provided by the present invention comprises the following steps:

[0109] Compound E undergoes a deesterification reaction to generate compound F. The reaction formula is as follows:

[0110] Wherein R2 is C1~C6 alkyl,

[0111] Preferably, the deesterification reaction is carried out in the presence of a deesterification catalyst, and preferably the deesterification catalyst is selected from lithium chloride, sodium chloride or a combination thereof;

[0112] Compound F undergoes a ketal protection group removal reaction to generate compound G. The reaction formula is as follows:

[0113]

[0114] Compound G undergoes a hydrogenation reduction reaction to generate compound H. The reaction formula is as follows:

[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 or ammonium formate; the catalyst is selected from palladium carbon, palladium hydroxide or Raney nickel;

[0118] Compound H undergoes ester hydrolysis reaction to generate compound I, and the reaction formula is as follows:

[0119]

[0120] Compound I undergoes carbonyl reduction to generate compound H. The reaction formula is as follows:

[0121] The carbonyl reduction is carried out in a one-step reaction in the presence of 3α-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.1 and 7β-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.2.

[0122] In another preferred embodiment, the preparation method of ursodeoxycholic acid provided by the present invention comprises the following steps:

[0123] Compound D undergoes an oxidation reaction to generate compound E. The reaction formula is as follows:

[0124]

[0125] Compound E undergoes a deesterification reaction to generate compound F. The reaction formula is as follows:

[0126] Wherein R2 is C1~C6 alkyl,

[0127] Preferably, the deesterification reaction is carried out in the presence of a deesterification catalyst, preferably, the deesterification catalyst is selected from lithium chloride, sodium chloride or a combination thereof;

[0128] Compound F undergoes a ketal protection group removal reaction to generate compound G. The reaction formula is as follows:

[0129]

[0130] Compound G undergoes a hydrogenation reduction reaction to generate compound H. The reaction formula is as follows:

[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 or ammonium formate; the catalyst is selected from palladium carbon, palladium hydroxide or Raney nickel;

[0134] Compound H undergoes ester hydrolysis reaction to generate compound I, and the reaction formula is as follows:

[0135]

[0136] Compound I undergoes carbonyl reduction to generate compound H. The reaction formula is as follows:

[0137] The carbonyl reduction is carried out in a one-step reaction in the presence of 3α-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.1 and 7β-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.2.

[0138] In another preferred embodiment, the preparation method of ursodeoxycholic acid provided by the present invention comprises the following steps:

[0139] Compound C reacts with neopentyl glycol to generate compound D. The reaction formula is as follows:

[0140]

[0141] Compound D undergoes an oxidation reaction to generate compound E. The reaction formula is as follows:

[0142]

[0143] Compound E undergoes a deesterification reaction to generate compound F. The reaction formula is as follows:

[0144] Wherein R2 is C1~C6 alkyl,

[0145] Preferably, the deesterification reaction is carried out in the presence of a deesterification catalyst, preferably, the deesterification catalyst is selected from lithium chloride, sodium chloride or a combination thereof;

[0146] Compound F undergoes a ketal protection group removal reaction to generate compound G. The reaction formula is as follows:

[0147]

[0148] Compound G undergoes a hydrogenation reduction reaction to generate compound H. The reaction formula is as follows:

[0149]

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

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

[0152] Compound H undergoes ester hydrolysis reaction to generate compound I, and the reaction formula is as follows:

[0153]

[0154] Compound I undergoes carbonyl reduction to generate compound H. The reaction formula is as follows:

[0155] The carbonyl reduction is carried out in a one-step reaction in the presence of 3α-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.1 and 7β-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.2.

[0156] In another preferred embodiment, the preparation method of ursodeoxycholic acid provided by the present invention comprises the following steps:

[0157] Compound B reacts with dialkyl malonate to generate compound C. The reaction formula is as follows:

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

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

[0160] Compound C reacts with neopentyl glycol to generate compound D. The reaction formula is as follows:

[0161]

[0162] Compound D undergoes an oxidation reaction to generate compound E. The reaction formula is as follows:

[0163]

[0164] Compound E undergoes a deesterification reaction to generate compound F. The reaction formula is as follows:

[0165] Wherein R2 is C1~C6 alkyl,

[0166] Preferably, the deesterification reaction is carried out in the presence of a deesterification catalyst, preferably, the deesterification catalyst is selected from lithium chloride, sodium chloride or a combination thereof;

[0167] Compound F undergoes a ketal protection group removal reaction to generate compound G. The reaction formula is as follows:

[0168]

[0169] Compound G undergoes a hydrogenation reduction reaction to generate compound H. The reaction formula is as follows:

[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 or ammonium formate; the catalyst is selected from palladium carbon, palladium hydroxide or Raney nickel;

[0173] Compound H undergoes ester hydrolysis reaction to generate compound I, and the reaction formula is as follows:

[0174]

[0175] Compound I undergoes carbonyl reduction to generate compound H. The reaction formula is as follows:

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

[0177] The present invention also provides a compound, the structural formula of which is shown below:

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

[0179] Wherein R2 is C1~C6 alkyl. BRIEF DESCRIPTION OF THE DRAWINGS

[0180] Figure 1 is the H NMR spectrum of compound B-1;

[0181] Figure 2 is the H NMR spectrum of compound C-1;

[0182] Figure 3 is the H NMR spectrum of compound E-1;

[0183] Figure 4 is the H NMR spectrum of compound F-1;

[0184] Figure 5 is the H NMR spectrum of compound G-1;

[0185] Figure 6 is the H NMR spectrum of compound I;

[0186] Figure 7 This is the H NMR spectrum of the compound UDCA;

[0187] Figure 8 This is an SDS-PAGE image of recombinant Escherichia coli induced to co-express three proteins: 3α-steroid dehydrogenase (3α-HSDH), 7β-steroid dehydrogenase (7β-HSDH) and glucose dehydrogenase (GDH). DETAILED DESCRIPTION

[0188] The inventors of the present application have obtained the technical solution of the present invention after in-depth research on the defects in the preparation method of ursodeoxycholic acid in the prior art. The method of the present invention has easy-to-obtain raw materials, mild reaction conditions, no need for column chromatography purification in post-treatment, simple operation and is suitable for industrial production.

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

[0190] In the description of the present invention, "C1-C6 alkyl" refers to an alkyl group containing 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, etc.

[0191] In a specific embodiment of the present invention, the preparation method of ursodeoxycholic acid of the present invention comprises the following steps:

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

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

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

[0195] (4) Compound D undergoes oxidation reaction to generate compound E;

[0196] (5) Compound E undergoes a deesterification reaction to generate compound F;

[0197] (6) Compound F undergoes a ketal protecting group removal reaction to generate compound G;

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

[0199] (8) Compound H undergoes ester hydrolysis to generate Compound I;

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

[0201] The reaction formula is as follows:

[0202]

[0203] Wherein R1 is Cl, Br, I or -OTs, and R2 is C1~C6 alkyl.

[0204] In step (1), compound A reacts with a halogenating agent or TsCl so that the hydroxyl group is replaced by a halogen or p-toluenesulfonyl group. The conventional method for carrying out such a reaction in the art is carried out, and the halogenating agent includes but is not limited to phosphorus tribromide, phosphorus trichloride, N-bromosuccinimide, N-chlorosuccinimide, and N-iodosuccinimide. In some specific embodiments, a base (e.g., triethylamine) and a catalyst (e.g., 4-dimethylaminopyridine) are also present in the system in which compound A reacts with p-toluenesulfonyl chloride (TsCl). After the reaction is completed by TLC detection, the organic phase is concentrated, pulped 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 specific embodiments of the present invention, compound B reacts with dimethyl malonate, and after the TLC detection reaction is completed, the reaction solution is cooled and filtered, and the filtrate is concentrated to obtain a residue, and the residue is dried to obtain the target compound.

[0206] In step (3), 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) are also present in the reaction system of compound C and neopentyl glycol. After the reaction is completed, a saturated sodium bicarbonate aqueous solution is added dropwise to the reaction solution to quench the reaction, and water is added dropwise to precipitate the product, which is then filtered and dried to obtain the target compound.

[0207] In step (4), in some specific embodiments, the oxidation reaction is carried out in the presence of an oxidant (including but not limited to oxygen, air), an imide catalyst (including but not limited to N-hydroxyphthalimide, N-hydroxybenzoylsulfonyl imide, N,N'-dihydroxypyromellitic acid imide) and an initiator (including but not limited to dibenzoyl peroxide (BPO), azobisisobutyronitrile, azobisisoheptanenitrile). After the reaction is completed, the solvent is removed by concentration, dissolved with dichloromethane, the insoluble matter is filtered, the filtrate is washed with an alkaline aqueous solution, the organic phase is concentrated, and the obtained residue is slurried with alcohol to obtain a compound C that can be directly used in the next step. In some specific embodiments of the present invention, the oxidant is oxygen.

[0208] In step (5), compound E is deesterified in the presence of a deesterification catalyst to generate compound F. Deesterification catalysts include but are not limited to lithium chloride and sodium chloride. In some specific embodiments of the present invention, the deesterification catalyst is lithium chloride. After the reaction is completed by TLC detection, water is added to the reaction solution, and after sufficient stirring, the reaction solution is filtered, and the filter cake is slurried with ethanol, filtered and dried to obtain the target compound.

[0209] In step (6), the ketal protecting group of compound F is removed to generate compound G, which can be carried out according to conventional methods for such reactions in the art. For example, it is carried out in the presence of an acid, and the acid includes but is not limited to hydrochloric acid, oxalic acid, acetic acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, trifluoroacetic acid, trifluoromethanesulfonic acid, and toluenesulfonic acid. In some specific embodiments of the present invention, the acid is hydrochloric acid, and the solvent is dichloromethane. After the reaction is completed by TLC detection, the reaction solution is adjusted to neutral with a base, and after sufficient stirring, the dichloromethane extraction is performed, and the organic phase is retained. After concentration, it is pulped with methanol, filtered and dried to obtain the target compound.

[0210] In step (7), the hydrogen source used in the hydrogenation reduction reaction includes but is not limited to hydrogen and ammonium formate. The catalyst includes but is not limited to palladium carbon, palladium hydroxide, and Raney nickel. In some specific embodiments of the present invention, the catalyst is Pd / C, the hydrogen source is hydrogen, and after the reaction is completed, the reaction solution is filtered, 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 reaction.

[0211] In step (8), compound H is hydrolyzed in the presence of a base to generate compound I, which can be carried out according to conventional methods for such reactions in the art. The base includes but is not limited to sodium hydroxide, potassium hydroxide and lithium hydroxide. In some specific embodiments of the present invention, the base used for hydrolysis is sodium hydroxide. After the reaction is completed, the reaction solution is concentrated, and water and dichloromethane are added. The obtained aqueous phase is adjusted to pH 2-3 with hydrochloric acid, solids are precipitated, filtered, and the obtained filter cake is dried to obtain the target compound.

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

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

[0214] 1. The reaction raw materials are easily available, 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. Neopentyl glycol is used to protect the carbonyl group. It is relatively easy to add the protective group. After protecting the carbonyl group with neopentyl glycol, the rigidity of the substrate is increased, the substrate has good stability, and the purification is easy with less impurities. It is not easy to fall off in the subsequent oxidation reaction and deesterification reaction, which reduces the generation of impurities and is easy to purify to obtain a high-purity intermediate. The removal reaction of the neopentyl glycol protective group is also simple and quick. Therefore, after the carbonyl group is protected by neopentyl glycol, the intermediate is relatively stable, with less impurities, easy to purify and high yield.

[0216] 3. Combining chemical synthesis and biosynthesis methods, the product purity is high, up to 99.91%.

[0217] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

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

[0219] Example 1 Preparation of Compound B-1

[0220]

[0221] At room temperature, 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) were added to the reaction bottle, cooled to 0°C, p-toluenesulfonyl chloride (150.0 g, 786.6 mmol) was added in 3 to 5 batches, then heated to 40°C, stirred for 3 h, and no raw material was left after TLC detection. The organic phase was concentrated, ethanol (800 ml) was added, slurried for 2 h, and filtered to obtain a white solid, which was 283.0 g after drying, with a yield of 96.0% and a purity of 99.5% by HPLC. MS: m / z = 485.7 [M+1] + .

[0222] Example 2 Preparation of Compound C-1

[0223]

[0224] At room temperature, 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) were added to the reaction bottle, nitrogen was replaced and nitrogen was protected, the temperature was raised to 110°C, the reaction was continued for 5 hours, TLC detected that there was no residual raw material, the temperature was lowered to 25°C, filtered, and the filtrate was concentrated to obtain a light yellow solid, methanol (60 ml) was added and the mixture was slurried at room temperature for 2 hours, filtered, and the filter cake was dried to obtain 23.7 g, with a yield of 88.6% and a purity of 99.2% by HPLC. MS: m / z=445.6[M+1] + .

[0225] Example 3 Preparation of Compound D-1

[0226]

[0227] At room temperature, compound C-1 (20.0 g, 44.62 mmol, purity about 99.2%), neopentyl glycol (100.00 g, 960.15 mmol), toluene (60 mL), p-toluenesulfonic acid (0.77 g, 4.50 mmol) were added to the reaction bottle, stirring was started, and triethyl orthoformate (20.0 g, 44.98 mmol) was added dropwise to the reaction bottle. It took 3 hours. After the addition was completed, it was stirred at 25-35 ° C for 10 hours. TLC detected that there was no raw material remaining. The temperature was cooled to 0-10 ° C in an ice-water bath, and a saturated sodium bicarbonate aqueous solution (30 ml) was added dropwise to quench the reaction. Then, toluene was removed by concentration until no liquid droplets were left. Water (200 mL) was slowly added and stirred for 2 hours. After filtering, the filter cake was dried to obtain 22.5 g of a light yellow solid with a yield of 95.0% and a purity of 99.6% by HPLC. 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 about 99.6%), N-hydroxyphthalimide (3.07 g, 18.84 mmol), dibenzoyl peroxide (0.05 g, 0.19 mmol), methanol (160 mL), dichloromethane (40 mL) were added to the reaction bottle, nitrogen was replaced three times and then oxygen was replaced three times, oxygen ball protection was continued, stirring was continued at room temperature for 6 hours, the organic phase was concentrated, the obtained residue was slurried with dichloromethane (300 ml) for 1 hour and then filtered, the filtrate was washed once with sodium bicarbonate aqueous solution and once with saturated sodium chloride, the organic phase was concentrated and methanol was added for slurrying, and 9.3 g of white solid was obtained after filtration and drying, with a yield of 90.9% and a purity of 99.8% by HPLC. 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] At room temperature, 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) were added to the reaction bottle, the temperature was raised to 125 ° C for 12 hours, TLC detected that there was no residual raw material, the temperature was cooled to 0-5 ° C in an ice-water bath, water (30 ml) was added dropwise to the reaction solution, stirred at 0-5 ° C for 1-2 hours, filtered, the filter cake was added to ethanol (45 ml), stirred at 0-5 ° C for 1-2 hours, filtered to obtain an off-white solid, which was 8.7 g after drying, with a yield of 97.1% and a purity of 99.5% by HPLC. MS: m / z=487.7[M+1] + . 1 H NMR (400MHZ, CDCl3), δ5.71(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] Example 6 Preparation of Compound G-1

[0235]

[0236] At room temperature, compound F-1 (20.0 g, 40.89 mmol, HPLC purity of about 99.5%) and dichloromethane (100 ml) were added to the reaction bottle, cooled to 0-10°C in an ice-water bath, 37% concentrated hydrochloric acid (20 ml) was added dropwise, and the temperature was raised to 25°C for reaction for 5 h. TLC detected that there was no residual raw material, and the liquids were separated, and the dichloromethane phase was retained. The temperature was cooled to 0-5°C in an ice-water bath, and sodium bicarbonate aqueous solution was added dropwise to adjust the pH to neutral. The liquids were separated, and the aqueous phase was extracted with dichloromethane (100 ml). The dichloromethane phases were combined and concentrated. The residue was added to methanol (40 ml), stirred at 0-5°C for 1-2 h, and filtered to obtain an off-white solid, which was 15.6 g after drying, with a yield of 95.1% and a HPLC purity of 99.8%. MS: m / z=401.6[M+1] + .

[0237] Example 7 Preparation of Compound H-1

[0238]

[0239] At room temperature, compound G-1 (1.0 g, 2.49 mmol, purity about 99.8%), methanol (25 ml), 10% Pd / C (0.05 g) were added to the reaction bottle, and hydrogen was replaced with a hydrogen balloon for protection, and stirred at room temperature for 16 h. Subsequently, the mixture was filtered and concentrated until no obvious liquid flowed out. The residue was added to ethanol (25 ml), and the mixture was further concentrated and dried in a vacuum at 45 ° C to obtain 0.98 g of a white solid, with a yield of 97.6% and a HPLC purity of 99.8%. MS: m / z = 403.6 [M+1] + .

[0240] Example 8 Preparation of Compound I

[0241]

[0242] At room temperature, compound H-1 (0.9 g, 2.23 mmol, purity about 99.8%) and ethanol (10 ml) were added to the reaction bottle. After nitrogen replacement three times, nitrogen protection was applied, and sodium hydroxide aqueous solution (0.24 g sodium hydroxide was dissolved in 5 ml water) was added dropwise, and then the internal temperature was heated to 55°C and stirred for 2 h. Subsequently, the mixture was concentrated until no obvious liquid flowed out, and the obtained residue was added to a mixture of water (30 ml) and dichloromethane (10 ml). After stirring for 30 min, the mixture was allowed to stand for stratification, and the aqueous phase was retained. The aqueous phase was adjusted to pH 2-3 with 1 M hydrochloric acid aqueous solution. A large amount of light yellow solid precipitated. After stirring for 2 h, it was filtered to obtain a light yellow solid, which was 0.80 g after drying, with a yield of 92.0% and a purity of 99.6%. MS: m / z=389.6[M+1] + . 1 H NMR(400MHZ, CDCl3), δ2.88(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] Example 9 Preparation of Compound UDCA

[0244]

[0245] At room temperature, compound I (15.00 g, 38.45 mmol, purity about 99.6%), coenzyme substrate monohydrate glucose (22.92 g, 115.65 mmol), n-octanol (45 ml), pH 7.0 0.1 M phosphate buffer (45 ml), NADP disodium salt (CAS No.: 24292-60-2) with a final concentration of 1 mM were added to a 250 mL three-necked round-bottom flask, and the materials were mixed at 35° C. and 400-600 rpm mechanical stirring conditions. After the materials were mixed, a wet suspension of Escherichia coli (500 g / L, 30 ml) was added, and stirring was continued. The initial pH of the reaction was measured to be 6.3, the pH of the reaction process was monitored, and 4% NaOH was used to adjust the reaction pH to 6.2-6.5. After 2 hours of reaction, add 40% NaOH to the above reaction flask 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, filter to obtain a filter cake, and wash the filter cake with water to further remove water-soluble impurities. The filter cake is added to methanol (300 ml), stirred to completely dissolve the product in methanol, filtered, and the filter cake is washed with methanol to collect the methanol solution from which the bacteria and protein are removed. The methanol solution is concentrated to dryness by rotary evaporation, and the obtained residue is added to 150 mL of petroleum ether, slurried, and filtered to obtain a filter cake from which impurities and n-octanol are removed. The filter cake is washed with petroleum ether, dried and weighed to obtain 13.90 g of a white solid, with a yield of 92.0%, a purity of 99.91%, and an optical rotation of +59.2° (Pharmacopoeia standard +59.0~62.0°). MS: m / z=393.6[M+1] + . 1 H NMR (400MHz, CD3OD), δ3.48-3.56(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-mentioned Escherichia coli wet bacterial suspension is as follows:

[0247] (1) Construction of recombinant Escherichia coli

[0248] The amino acid sequences of 3α-steroid dehydrogenase from Comamonas testosteroni (referred to as 3α-HSDH, NCBI accession number: WP_003078312.1) as shown in SEQ ID NO.1, 7β-steroid dehydrogenase from Clostridium nigeriense (referred to as 7β-HSDH, NCBI accession number: WP_066892209.1) as shown in SEQ ID NO.2, and glucose dehydrogenase from Bacillus subtilis (referred to as GDH, NCBI accession number: WEZ47479.1) as shown in SEQ ID NO.3 were fully synthesized after codon optimization by Sangon Biotech (Shanghai) Co., Ltd. and cloned into the EcoRI / HindIII restriction sites of pET28a plasmid (the nucleotide sequence of 3α-HSDH after codon optimization is shown in SEQ ID NO.4, the nucleotide sequence of 7β-HSDH after codon optimization is shown in SEQ ID NO.5, and the nucleotide sequence of GDH after codon optimization is shown in SEQ ID NO.6). NO.6), construct pET28a-3αHSDH-7βHSDH-GDH three enzymes co-expression recombinant plasmid, and transform into E. coli BL21 (DE3), obtain recombinant E. coli BL21 (DE3)-pET28a-3αHSDH-7βHSDH-GDH with kanamycin resistance gene, and subsequently name the recombinant E. coli AU060021. After SDS-PAGE protein expression verification (see Figure 8 ), the results showed that the three enzymes were successfully expressed, corresponding to the molecular weight of 3α-HSDH protein of 23.2kDa, 7β-HSDH protein of 25.7kDa, and GDH protein of 24.8kDa.

[0249] (2) Cultivation of AU060021 recombinant Escherichia coli

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

[0251] (3) Obtaining bacterial suspension

[0252] The AU060021 recombinant Escherichia coli cultured in the above TB medium was centrifuged at 4°C, 6000 rpm for 5 min, the supernatant was discarded, and 100 mM phosphate buffer with a pH of 7.5 and an equal mass to the wet bacteria was added, and the suspension was resuspended by shaking to obtain a bacterial suspension for use.

[0253] The sequence of the present invention

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

[0255] Source: Comamonastestosteroni

[0256] NCBI ID: WP_003078312.1

[0257] IRDAEVIADLSTAEGRKQAIADVLAKCSKGMDGLVLCAGLGPQTKVLGNVVSVNYFGATELMDAFLPALKKGHQPAAVVISSVASAHLAFDKNPLALALEAGEEAKARAIVE HAGEQGGNLAYAGSKNALTVAVRKRAAAWGEAGVRLNTIAPGATETPLLQAGLQDPRYGESIAKFVPPMGRRAEPSEMASVIAFLMSPAASYVHGAQIVIDGGIDAVMRPTQF

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

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

[0260] MNVVMVGRREEMLKALGEDISSKYGVKHLVIKADFSDPNSTDEIFEKTKDLDMGFMSYVACFHTFGKLQDTPWEKHEQMLNVNVITFLKCFYHYMKIFSKQDRGAIINVSSLTGISASPYNAQYGAGKSYILKLTEAVAYEASKTNVDVEVITLGTTITPSLLKNLPGGPAGEAVMKAALTPEACVEEAFENLGKKFSIIAGEHNKASIHDWKANHTEDEFISYMGSFYER

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

[0262] Source: Bacillus subtilis, NCBI accession number: WEZ47479.1

[0263] KVVINYYSNKQDPNEVKEEVIKAGGEAVVVQGDVTKEEDVKNIVQTAIKEFGTLDIMINNAGLENPVPSHEMPLKDWDKVIGTNLTGAFLGSREAIKYFVENDIKGNVINMSSVHEVIPWPLFVHYAASKGGIKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPKQKADVESMIPMGYIGEPEEIAAVAAWLASKEASYVTGITLFADGGMTQYPSFQAGRG

[0264] SEQ ID NO.4 (Nucleotide sequence of 3α - 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 in that: The preparation method comprises the following steps: (1) Compound A reacts with a halogenating agent or p-toluenesulfonyl chloride to generate compound B. The reaction formula is as follows: Wherein R1 is Cl, Br, I or -OTs, Preferably, the halogenating agent is selected from phosphorus tribromide, phosphorus trichloride, N-bromosuccinimide, N-chlorosuccinimide or N-iodosuccinimide. (2) Compound B reacts with dialkyl malonate to generate compound C. The reaction formula is as follows: wherein R2 is a C1-C6 alkyl group, and (3) Compound C reacts with neopentyl glycol to generate compound D. The reaction formula is as follows:

2. The method for preparing ursodeoxycholic acid according to claim 1, characterized in that: The preparation method further comprises the following steps: (4) Compound D undergoes oxidation reaction to generate compound E. The reaction formula is as follows:

3. The method for preparing ursodeoxycholic acid according to claim 2, characterized in that: 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-hydroxy-o-sulfonylbenzoimide, N,N'-dihydroxypyromellitic acid imide, the initiator is selected from dibenzoyl oxide, azobisisobutyronitrile, azobisisoheptanenitrile or a combination thereof, and the oxidant is selected from air or oxygen; or The oxidation reaction is carried out in the presence of a cuprous halide catalyst and an oxidant, wherein 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 for preparing ursodeoxycholic acid according to claim 2, characterized in that: The preparation method further comprises the following steps: (5) Compound E undergoes a deesterification reaction to generate compound F. The reaction formula is as follows: Wherein R2 is C1~C6 alkyl, Preferably, the deesterification reaction is carried out in the presence of a deesterification catalyst. Preferably, the deesterification catalyst is selected from lithium chloride, sodium chloride or a combination thereof.

5. The method for preparing ursodeoxycholic acid according to claim 4, characterized in that: The preparation method further comprises the following steps: (6) Compound F is subjected to a ketal protection group removal reaction to generate compound G. The reaction formula is as follows:

6. The method for preparing ursodeoxycholic acid according to claim 5, characterized in that: The preparation method further comprises the following steps: (7) Compound G undergoes a hydrogenation reduction reaction to generate compound H. The reaction formula is as follows: Preferably, the hydrogenation reduction reaction is carried out in the presence of a hydrogen source and a catalyst. Preferably, the hydrogen source is selected from hydrogen or ammonium formate; and the catalyst is selected from palladium carbon, palladium hydroxide or Raney nickel.

7. The method for preparing ursodeoxycholic acid according to claim 6, characterized in that: The preparation method further comprises the following steps: (8) Compound H undergoes ester hydrolysis reaction to generate compound I, and the reaction formula is as follows:

8. The method for preparing ursodeoxycholic acid according to claim 7, characterized in that: The preparation method further comprises the following steps: (9) Compound I is subjected to carbonyl reduction to generate compound H, and the reaction formula is as follows: The carbonyl reduction is carried out in a one-step reaction in the presence of 3α-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.1 and 7β-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.

2.

9. A method for preparing ursodeoxycholic acid, characterized in that: The preparation method comprises the following steps: Compound F undergoes a ketalization protection group removal reaction under acid catalysis to generate compound G. The reaction formula is as follows: Compound G undergoes a hydrogenation reduction reaction to generate compound H. The reaction formula is as follows: Preferably, the hydrogenation reduction reaction is carried out in the presence of a hydrogen source and a catalyst. Preferably, the hydrogen source is selected from hydrogen or ammonium formate; the catalyst is selected from palladium carbon, palladium hydroxide or Raney nickel; Compound H undergoes ester hydrolysis reaction to generate compound I, and the reaction formula is as follows: Compound I undergoes carbonyl reduction to generate compound H. The reaction formula is as follows: The carbonyl reduction is carried out in a one-step reaction in the presence of 3α-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.1 and 7β-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.

2.

10. A method for preparing ursodeoxycholic acid, characterized in that: The preparation method comprises the following steps: Compound E undergoes a deesterification reaction to generate compound F. The reaction formula is as follows: Wherein R2 is C1~C6 alkyl, Preferably, the deesterification reaction is carried out in the presence of a deesterification catalyst, wherein the deesterification catalyst is selected from lithium chloride, sodium chloride or a combination thereof; Compound F undergoes a ketal protection group removal reaction to generate compound G. The reaction formula is as follows: Compound G undergoes a hydrogenation reduction reaction to generate compound H. The reaction formula is as follows: Preferably, the hydrogenation reduction reaction is carried out in the presence of a hydrogen source and a catalyst. Preferably, the hydrogen source is selected from hydrogen or ammonium formate; the catalyst is selected from palladium carbon, palladium hydroxide or Raney nickel; Compound H undergoes ester hydrolysis reaction to generate compound I, and the reaction formula is as follows: Compound I undergoes carbonyl reduction to generate compound H. The reaction formula is as follows: The carbonyl reduction is carried out in a one-step reaction in the presence of 3α-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.1 and 7β-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.

2.

11. A method for preparing ursodeoxycholic acid, characterized in that: The preparation method comprises the following steps: Compound D undergoes an oxidation reaction to generate compound E. The reaction formula is as follows: Compound E undergoes a deesterification reaction to generate compound F. The reaction formula is as follows: Wherein R2 is C1~C6 alkyl, Preferably, the deesterification reaction is carried out in the presence of a deesterification catalyst, wherein the deesterification catalyst is selected from lithium chloride, sodium chloride or a combination thereof. Compound F undergoes a ketal protection group removal reaction to generate compound G. The reaction formula is as follows: Compound G undergoes a hydrogenation reduction reaction to generate compound H. The reaction formula is as follows: Preferably, the hydrogenation reduction reaction is carried out in the presence of a hydrogen source and a catalyst. Preferably, the hydrogen source is selected from hydrogen or ammonium formate; the catalyst is selected from palladium carbon, palladium hydroxide or Raney nickel; Compound H undergoes ester hydrolysis reaction to generate compound I, and the reaction formula is as follows: Compound I undergoes carbonyl reduction to generate compound H. The reaction formula is as follows: The carbonyl reduction is carried out in a one-step reaction in the presence of 3α-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.1 and 7β-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.

2.

12. A method for preparing ursodeoxycholic acid, characterized in that: The preparation method comprises the following steps: Compound C reacts with neopentyl glycol to generate compound D. The reaction formula is as follows: Compound D undergoes an oxidation reaction to generate compound E. The reaction formula is as follows: Compound E undergoes a deesterification reaction to generate compound F. The reaction formula is as follows: Wherein R2 is C1~C6 alkyl, Preferably, the deesterification reaction is carried out in the presence of a deesterification catalyst, wherein the deesterification catalyst is selected from lithium chloride, sodium chloride or a combination thereof; Compound F undergoes a ketal protection group removal reaction to generate compound G. The reaction formula is as follows: Compound G undergoes a hydrogenation reduction reaction to generate compound H. The reaction formula is as follows: Preferably, the hydrogenation reduction reaction is carried out in the presence of a hydrogen source and a catalyst. Preferably, the hydrogen source is selected from hydrogen or ammonium formate; the catalyst is selected from palladium carbon, palladium hydroxide or Raney nickel; Compound H undergoes ester hydrolysis reaction to generate compound I, and the reaction formula is as follows: Compound I undergoes carbonyl reduction to generate compound H. The reaction formula is as follows: The carbonyl reduction is carried out in a one-step reaction in the presence of 3α-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.1 and 7β-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.

2.

13. A method for preparing ursodeoxycholic acid, characterized in that: The preparation method comprises the following steps: Compound B reacts with dialkyl malonate to generate compound C. The reaction formula is as follows: Wherein R1 is Cl, Br, I or -OTs, and R2 is C1-C6 alkyl; Compound C reacts with neopentyl glycol to generate compound D. The reaction formula is as follows: Compound D undergoes an oxidation reaction to generate compound E. The reaction formula is as follows: Compound E undergoes a deesterification reaction to generate compound F. The reaction formula is as follows: Wherein R2 is C1~C6 alkyl, Preferably, the deesterification reaction is carried out in the presence of a deesterification catalyst, wherein the deesterification catalyst is selected from lithium chloride, sodium chloride or a combination thereof; Compound F undergoes a ketal protection group removal reaction to generate compound G. The reaction formula is as follows: Compound G undergoes a hydrogenation reduction reaction to generate compound H. The reaction formula is as follows: Preferably, the hydrogenation reduction reaction is carried out in the presence of a hydrogen source and a catalyst. Preferably, the hydrogen source is selected from hydrogen or ammonium formate; the catalyst is selected from palladium carbon, palladium hydroxide or Raney nickel; Compound H undergoes ester hydrolysis reaction to generate compound I, and the reaction formula is as follows: Compound I undergoes carbonyl reduction to generate compound H. The reaction formula is as follows: The carbonyl reduction is carried out in a one-step reaction in the presence of 3α-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.1 and 7β-steroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.

2.

14. A compound, characterized in that Its structural formula is as follows: Wherein R2 is C1-C6 alkyl, or Wherein R2 is C1~C6 alkyl.

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