Preparation method of ursodeoxycholic acid intermediate and intermediate compound thereof

Abstract

The application relates to the technical field of drug synthesis preparation, in particular to a preparation method of an ursodeoxycholic acid intermediate and an intermediate compound thereof, which takes BA (21-hydroxy-20-methylpregn-4-ene-3-ketone) as a starting material, and prepares the ursodeoxycholic acid intermediate 7K through eight-step reactions of sulfonylation, alkylation, esterification, oxidation, alkaline hydrolysis, hydrogenation, alkaline hydrolysis and decarboxylation. The 3-ketone is protected by adopting the esterification method to generate 3,5-bisalkene esterification products, the esterification reaction product is single, and no other isomer impurities exist, so that the overall yield of the process is improved.

Description

Technical Field

[0001] This invention relates to the field of drug synthesis and preparation technology, and in particular to a method for preparing an ursodeoxycholic acid intermediate and the intermediate compound thereof. Background Technology

[0002] Currently, ursodeoxycholic acid (UDCA) has two main sources: extraction from animal bile and artificial synthesis. However, animal bile extraction resources are limited and cannot meet medical needs, so artificial synthesis is the primary method of production.

[0003] There are two main methods for the artificial synthesis of ursodeoxycholic acid (UDCA): one is semi-synthesis using animal-derived starting materials, such as porcine deoxycholic acid (HDCA), chenodeoxycholic acid (CDCA), and cholic acid (CA), which can be obtained through multiple chemical transformations. However, these animal-derived starting materials may contain residual animal proteins, posing certain safety risks for human consumption. Moreover, UDCA prepared from animal-derived starting materials has complex impurities, is difficult to purify, and is hard to obtain high-purity UDCA. The other method is semi-synthesis using plant-derived starting materials, such as BA (21-hydroxy-20-methylpregn-4-en-3-one), which, through the key intermediate 7K, is further reduced by chemical reduction or enzymatic reduction. This is currently the more commonly used method for preparing UDCA.

[0004] Patents CN116836213 and CN111072744 disclose a method for preparing ursodeoxycholic acid or the key intermediate 7K by using BA as a starting material, introducing a 21-position side chain through oxidation → condensation / Wittig reaction, and then proceeding through multiple steps.

[0005] Patent CN115181150 discloses a method for preparing ursodeoxycholic acid by using BA as a starting material, followed by sulfonation → Grignard reagent alkylation of bromoacetaldehyde derivatives to construct a 21-position side chain, and then through multiple steps:

[0006] Patents CN109415407 and WO2023081657 disclose the preparation of obeticholic acid and other substances by using BA as a raw material, bromination → diethyl malonate alkylation to construct a 21-position side chain, and then through a multi-step reaction.

[0007] In the published patent CN118852307A, we disclosed the following method for preparing 7K:

[0008] This method utilizes ketal protection at the 3-position to prepare a 5-ene ketal, followed by allylic oxidation and hydrolysis to obtain a 3,7-diketone. However, the above technical solution still has the following drawbacks: during the ketal reaction, 10-20% of the isomer impurity 4-ene ketal is inevitably generated. This impurity can be removed during the purification process of the ketal, so the generation of the isomer impurity does not significantly affect the quality of the ketal, but it leads to a decrease in the yield of the ketal reaction, to only 80-85%.

[0009] Summary of the Invention

[0010] In view of this, the present invention proposes a method for preparing the key intermediate 7K of ursodeoxycholic acid, in order to solve the problem that the existing technical solutions for preparing the key intermediate 7K of ursodeoxycholic acid contain isomer impurities, which leads to a decrease in the yield of the key intermediate 7K of ursodeoxycholic acid.

[0011] The technical solution of this invention is achieved as follows: This invention provides a method for preparing ursodeoxycholic acid intermediate 7K, the method comprising the following steps:

[0012] Where R = -CH3, -CH2CH3; R 1 =-COCH3, -COCH2CH3 S1. Compound (II) undergoes a sulfonation reaction to yield sulfonyl compound (III). S2, sulfonyl formula (III) is alkylated to give diester formula (IV); S3, diester (Ⅳ) undergoes esterification to yield trimer (Ⅴ); S4. The esterified compound (V) undergoes an oxidation reaction to yield the esterified oxide compound (VI). S5, the esterified oxide (VI) is hydrolyzed to give the 3,7-diketone (VII); The S6,3,7-diketone formula (VII) is hydrogenated and reduced to give the 7K diester formula (VIII). S7 and 7K diester (VIII) undergo alkaline hydrolysis to yield 7K dicarboxylic acid (IX). The S8 and 7K dicarboxylic acid derivatives (IX) undergo a decarboxylation reaction to yield the ursodeoxycholic acid intermediate 7K (I).

[0013] The technical solution provided by this invention adopts an esterification protection strategy instead of a ketal protection method. By using isopropyl acetate or isopropyl propionate as esterification reagents in the third-step esterification reaction, the carbonyl group at the 3-position of the diester forms an enol ester structure under acid catalysis, generating a structurally simple 3,5-diene triester. This reaction has high regioselectivity and avoids the problem of 4-ene ketal byproduct formation in the ketal protection reaction, thereby eliminating the generation of isomer impurities, significantly improving the reaction yield, improving the economy of the process and product quality, and providing a more efficient and reliable synthetic route for the industrial production of the key intermediate 7K of ursodeoxycholic acid.

[0014] Based on the above technical solution, preferably, in step S1, compound (II) is dissolved in dichloromethane, and 4-dimethylaminopyridine, triethylamine and p-toluenesulfonyl chloride are added in sequence, and the mixture is heated to reflux for 4-5 hours to obtain sulfonate (III).

[0015] Based on the above technical solutions, preferably, the molar ratio of compound formula (II), 4-dimethylaminopyridine, triethylamine and p-toluenesulfonyl chloride is 1:0.13-0.14:2.3-2.4:1.20-1.40; more preferably, the molar ratio of compound formula (II), 4-dimethylaminopyridine, triethylamine and p-toluenesulfonyl chloride is 1:0.135:2.38:1.30.

[0016] Based on the above technical solution, preferably, in step S2, under a nitrogen atmosphere, the sulfonyl compound (III) is dissolved in DMF, potassium carbonate, TBAB and alkylating agent are added, and the reaction is carried out at 50~60℃ for 9-11h to obtain the diester compound (IV), wherein the alkylating agent is diethyl malonate or dimethyl malonate.

[0017] Based on the above technical solutions, preferably, the molar ratio of sulfonyl (III), potassium carbonate, TBAB and alkylating agent is 1:2.8-3.2:0.1:2.8-3.2; more preferably, the molar ratio of sulfonyl (III), potassium carbonate, TBAB and alkylating agent is 1:3.0:0.1:3.0.

[0018] Based on the above technical solution, preferably, in step S3, diester (Ⅳ), dichloromethane, p-toluenesulfonic acid and esterification reagent are mixed and reacted at 35~40℃ for 1-3h. The reaction solution is then cooled to 20-25℃, triethylamine is added, and the mixture is stirred for 0.8-1.2h to obtain diester (Ⅴ). The esterification reagent is isopropyl acetate or isopropyl propionate.

[0019] Based on the above technical solutions, preferably, the molar ratio of diester (Ⅳ), p-toluenesulfonic acid and esterification reagent is 1:0.028-0.036:3.300-3.500; more preferably, the molar ratio of diester (Ⅳ), p-toluenesulfonic acid and esterification reagent is 1:0.034:3.400.

[0020] Based on the above technical solution, preferably, in step S4, the triester formula (V) and acetone are mixed, PDC and NHPI are added, and the mixture is reacted at 40~50℃ for 4-5 hours to obtain the esterified oxide formula (VI).

[0021] Based on the above technical solutions, preferably, the molar ratio of the ester formula (V), PDC and NHPI is 1:1.70-1.75:1.55-1.60; more preferably, the molar ratio of the ester formula (V), PDC and NHPI is 1:1.73:1.58.

[0022] Based on the above technical solution, preferably, in step S5, the esterified oxide (VI) is dissolved in an aqueous solution of tetrahydrofuran, an alkaline catalyst is added, and the reaction is carried out at 40-50°C for 3-5 hours to obtain the 3,7-diketone (VII), wherein the alkaline catalyst is potassium carbonate or sodium carbonate.

[0023] Based on the above technical solutions, preferably, the molar ratio of esterified oxide (VI) and alkaline catalyst is 1:4.8-5.2; more preferably, the molar ratio of esterified oxide (VI) and alkaline catalyst is 1:5.0.

[0024] Based on the above technical solution, preferably, in step S6, 3,7-diketone (Ⅶ) is dissolved in tetrahydrofuran, Raney Ni catalyst is added, and the reaction is carried out in a hydrogen atmosphere at 25-27℃ for 22-26 hours to obtain 7K diester (Ⅷ).

[0025] Based on the above technical solutions, preferably, the molar ratio of 3,7-diketone (VII) to Raney Ni catalyst is 1:15.0-20.0; more preferably, the molar ratio of 3,7-diketone (VII) to Raney Ni catalyst is 1:17.0.

[0026] Based on the above technical solution, preferably, in step S7, 7K diester (VIII) is dissolved in methanol, and an aqueous solution of potassium hydroxide is added. The mixture is reacted at 50~60℃ for 1-3 hours to obtain 7K dicarboxylic acid (IX).

[0027] Based on the above technical solutions, preferably, the molar ratio of 7K diester (VIII) to potassium hydroxide is 1:3.00-3.05; more preferably, the molar ratio of 7K diester (VIII) to potassium hydroxide is 1:3.03.

[0028] Based on the above technical solution, preferably, in step S8, 7K dicarboxylic acid (IX) is dissolved in DMSO solution, sodium chloride and water are added, and the reaction is carried out at 140-150℃ for 4-6 hours to obtain ursodeoxycholic acid intermediate 7K (I).

[0029] Based on the above technical solutions, preferably, the mass ratio of 7K dicarboxylic acid (IX) to sodium chloride is 2:1.

[0030] The present invention also provides an intermediate compound, the structure of which is shown below:

[0031] Where R = -CH3, -CH2CH3; R 1 =-COCH3, -COCH2CH3.

[0032] The method for preparing an ursodeoxycholic acid intermediate and the intermediate compound of the present invention have the following advantages over the prior art: This invention provides a method for preparing ursodeoxycholic acid intermediates, effectively solving the problem of reduced ketal yield caused by the generation of 10-20% isomer impurities in the ketal reaction in existing technologies. By replacing the traditional ketal protection method with an esterification protection strategy, a structurally singular 3,5-dienetriester is generated. This reaction exhibits high regioselectivity and does not generate isomer impurities, thereby increasing the reaction yield from 80-85% in existing technologies to 97.3%, significantly improving the overall process efficiency and product quality. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 The NMR spectrum of the trimer formula (V) in Example 1 of this invention; Figure 2 The NMR spectrum of the esterified oxide formula (VI) in Example 1 of this invention; Figure 3 The NMR spectrum of 3,7-diketone (VII) in Example 1 of the present invention; Figure 4 The NMR spectrum of the 7K dicarboxylic acid compound (IX) in Example 1 of this invention; Figure 5 The NMR spectrum of ursodeoxycholic acid intermediate (I) in Example 1 of this invention. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0036] It should be noted that the HPLC detection method for detecting the reaction process in this invention is as follows: HPLC Method 1: Column: InfinityLab Poroshell 120 EC-C18 (4.6 × 150 mm, 4.6 Micron), Agilent. Mobile phase: water and acetonitrile gradient

[0037] Flow rate: 1 ml / min Detector: Ultraviolet detector (VWD), signal polarity: positive; dual wavelengths 210nm & 254nm Column oven temperature: 29.9℃ Injection volume: 20 μL Data collection time: 30 min Sample preparation solvent: acetonitrile (1 mg / ml); Data processing: No integration for values ​​below 0.05% HPLC Method 2: Column: InfinityLab Poroshell 120 EC-C18 (4.6 × 150 mm, 4.6 Micron), Agilent. Mobile phase: Acetonitrile: Formic acid aqueous solution (0.4%) = 50:50 Flow rate: 0.8 ml / min Detector: Parallax refractometer (RID), signal polarity: positive Detector temperature: 35℃ Column oven temperature: 35℃ Injection volume: 20 μL Data collection time: 70 min

[0038] Example 1 This embodiment provides a method for preparing an intermediate of ursodeoxycholic acid, including the following steps:

[0039] S1. In a 3L reaction flask, add 1L of dichloromethane and 200g of compound (II) (605.12mmol), stir to dissolve and clarify, then add 7.4g of 4-dimethylaminopyridine (81.85mmol) and 200ml of triethylamine (1.44mol) in sequence. Slowly add 150g of p-toluenesulfonyl chloride (786.82mmol) in 400mL of dichloromethane solution. After the addition is complete, heat the reaction solution to reflux for 4.5h until TLC (PE:EA=3:1, UV 254nm color development) shows that the starting material is completely converted.

[0040] The reaction solution was poured into 1L of water, stirred, and allowed to stand for separation. The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure. 200ml of methanol was added, the temperature was lowered to 0~5℃ and stirred for 1.5h, filtered, and dried under reduced pressure at 45℃ to obtain 271g of sulfonyl (III) with a yield of 92.4%. The purity was determined by HPLC method 1 (254nm) to be 98.52%.

[0041] S2. In a 5L reaction flask under nitrogen protection, 270g of sulfonyl (III) (557.05mmol) was dissolved in 2.7L DMF, and 230g of potassium carbonate (1.66mol), 18g of TBAB (55.83mmol), and 270g of diethyl malonate (1.69mol) were added. The mixture was heated to 55℃ and stirred for 10h until TLC (PE:EA=2:1, UV 254nm color development) showed complete conversion of the starting materials.

[0042] The reaction solution was slowly poured into 2700 ml of water and filtered to obtain crude diester. 540 ml of ethanol was added to the crude product, stirred for 30 min, filtered, and dried under reduced pressure at 45 °C to obtain 225 g of white diester (IV), with a yield of 85.4%. The purity was determined by HPLC method 1 (254 nm) to be 92.20%.

[0043] S3. In a 5L reaction flask, add 1500ml of dichloromethane, 1.8g of p-toluenesulfonic acid (10.46mmol), 150g of diester (IV) (371.35mmol), and 127g of isopropyl acetate (1.26mol). Keep the mixture at 38℃ for 2 hours until TLC (developing solvent: PE:EA=3:1, vanillin color development) shows that the starting material is basically completely converted.

[0044] The reaction solution was cooled to 20-25℃, 1.8g of triethylamine was added, and the mixture was stirred for 1 hour, then concentrated to near dryness. 100ml of methanol was added to the concentrate, and the mixture was stirred at 0-5℃ for 1 hour. The mixture was filtered and dried under reduced pressure at 40-50℃ to obtain 159g of the triester (V), with a yield of 82.86%. Purity was determined by HPLC method 1 (254nm) to be 93.12%.

[0045] NMR data for the trimer formula (V) are as follows: Figure 1 As shown: 1 H NMR (600 MHz, DMSO- d6 ) δ 5.66 (s, 1H), 5.38 (s, 1H), 4.18 – 4.07(m, 4H), 3.48 (dd, J = 10.9, 4.1 Hz, 2H), 2.36 (t, J = 15.2 Hz, 1H), 2.14 –2.02 (m, 4H), 1.98 (dd, J = 25.2, 12.2 Hz, 2H), 1.89 – 1.76 (m, 2H), 1.72 –1.46 (m, 4H), 1.43 – 1.33 (m, 2H), 1.31 – 1.22 (m, 2H), 1.21 – 1.13 (m, 8H),1.12 – 1.01 (m, 3H), 0.99 – 0.94 (m, 1H), 0.93 (d, J = 9.4 Hz, 3H), 0.89 (d,J = 6.4 Hz, 3H), 0.65 (s, 3H).

[0046] S4. Under nitrogen protection, add 2L of acetone, 200g of triester (V) (387.06mmol), 200g of PDC (673.17mmol), and 100g of NHPI (613.01mmol) to a 3L reaction flask. Heat to 45℃ and stir for 4.5h until TLC (PE:EA=3:1, vanillin colorimetric) shows complete conversion of the starting materials.

[0047] The reaction solution was concentrated to dryness under reduced pressure, 2L of ethyl acetate was added and stirred for 30 minutes. The mixture was filtered through diatomaceous earth, and the filtrate was washed once with 2L of 1.5M sodium hydroxide aqueous solution and then once with 2L of saturated saline solution. The organic phase was concentrated to dryness under reduced pressure at 45~50℃.

[0048] Add 400 ml of ethanol to the concentrate, heat to reflux until dissolved, cool to room temperature and stir for 1 h, filter, dry the filter cake under reduced pressure at 45 °C to obtain 184 g of white esterified oxide (VI), yield 89.58%, purity 91.41% as determined by HPLC method 1 (254 nm).

[0049] NMR data for esterified oxide (VI) are as follows: Figure 2 As shown: 1 H NMR (600 MHz, DMSO- d6 ) δ 7.85 (d, J = 8.7 Hz, 4H), 5.89 (s, 1H), 4.75 (s, 1H), 4.21 – 4.06 (m, 4H), 3.49 (dd, J = 10.8, 4.1 Hz, 1H), 2.62 –2.52 (m, 1H), 2.22 (dd, J = 48.5, 16.0 Hz, 2H), 2.07 – 1.99 (m, 3H), 1.88 –1.79 (m, 1H), 1.67 (td, J = 14.4, 4.0 Hz, 1H), 1.58 – 1.43 (m, 3H), 1.41 –1.35 (m, 4H), 1.28 (ddd, J = 27.4, 19.0, 9.8 Hz, 3H), 1.18 (dt, J = 13.9, 7.1Hz, 7H), 1.06 – 0.99 (m, 1H), 0.96 (td, J = 11.5, 4.2 Hz, 1H), 0.90 (d, J =6.3 Hz, 3H), 0.72 (s, 3H).

[0050] S5. In a 3L reaction flask, add 900ml tetrahydrofuran, 100ml water and 100g esterified oxide (VI) (188.43mmol), stir to dissolve, add 131g potassium carbonate (946.12mmol), heat to 45℃ and stir for 4h until TLC (PE:EA=2:1, UV 254nm color development) shows complete conversion of the starting material.

[0051] The reaction solution was neutralized with 1000 ml of 1N hydrochloric acid aqueous solution, and the layers were separated. The aqueous layer was extracted with 500 ml of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and 300 ml of petroleum ether was added. The mixture was stirred at room temperature for 1 h, filtered, and the filter cake was dried under reduced pressure at 45 °C to obtain 82.20 g of white 3,7-diketone (VII) solid, with a yield of 89.64%. The purity was determined by HPLC method 1 (254 nm) to be 95.13%.

[0052] NMR data for 3,7-diketone (VII) are as follows: Figure 3 As shown: 1 H NMR (600 MHz, CDCl3) δ 5.69 (d, J = 19.5 Hz, 1H), 4.32 – 4.09 (m,4H), 3.54 – 3.35 (m, 2H), 3.06 (t, J = 15.3 Hz, 1H), 2.62 – 2.37 (m, 3H),2.26 – 2.12 (m, 2H), 2.04 (tdd, J = 23.2, 12.9, 6.7 Hz, 3H), 1.74 (td, J =14.1, 4.6 Hz, 1H), 1.68 – 1.56 (m, 2H), 1.52 – 1.43 (m, 2H), 1.43 – 1.34 (m, 5H), 1.28 (dq, J = 14.5, 7.2 Hz, 7H), 1.20 – 1.10 (m, 2H), 1.04 (ddd, J =24.4, 12.2, 6.5 Hz, 1H), 0.95 (t, J = 5.8 Hz, 3H), 0.70 (d, J = 5.2 Hz, 3H).

[0053] S6. Add 1.6 L of tetrahydrofuran and 80 g of 3,7-diketone (Ⅶ) (164.61 mmol) to a 3 L reaction flask, stir to dissolve, add 240 g of Raney Ni (2.80 mol), replace the air in the reaction flask with nitrogen three times, then replace it with hydrogen three times, and then maintain a slightly positive pressure hydrogen atmosphere and stir at room temperature for 24 h until TLC (PE:EA=2:1, phosphomolybdic acid color development) shows complete conversion of the starting material, filter the catalyst, concentrate the filtrate to dryness under reduced pressure to obtain 7K diester (Ⅷ).

[0054] S7. Dissolve the 7K diester (VIII) obtained in step S6 directly in 560 ml of methanol, slowly add 48 ml of aqueous solution of 28 g potassium hydroxide (499.02 mmol), heat to 55 °C and stir for 2 h until TLC (dichloromethane:methanol = 20:1, phosphomolybdic acid colorimetric) shows that the hydrolysis reaction is complete.

[0055] The reaction solution was cooled to room temperature, 1.2 L of water was added, and the pH was adjusted to 4-5 with 3 M hydrochloric acid. A large amount of solid precipitated out. The mixture was stirred at room temperature for 2 hours, filtered, and the filter cake was dried under reduced pressure at 45 °C to obtain 67.54 g of white 7K dicarboxylic acid compound (IX). The yield of the two-step hydrogenation hydrolysis was 94.55%, and the purity was determined by HPLC method 2 to be 90.99%.

[0056] NMR data for 7K dicarboxylic acid (IX) are as follows: Figure 4 As shown: 1 H NMR (600 MHz, DMSO- d6 ) δ 12.63 (s, 2H), 4.48 (s, 1H), 3.29 – 3.21(m, 1H), 2.90 (dd, J = 12.3, 6.0 Hz, 1H), 2.44 (t, J = 11.3 Hz, 1H), 2.11 – 2.01(m, 2H), 2.00 – 1.89 (m, 2H), 1.89 – 1.75 (m, 2H), 1.75 – 1.64 (m, 3H), 1.54– 1.43 (m, 2H), 1.39 (t, J = 10.1 Hz, 2H), 1.31 (dd, J = 19.4, 10.6 Hz, 3H), 1.21(dd, J = 21.8, 10.2 Hz, 1H), 1.17 – 0.95 (m, 8H), 0.95 – 0.84 (m, 4H), 0.60 (s, 3H).

[0057] S8. In a 3L reaction flask, add 600ml DMSO and 60g 7K dicarboxylic acid (IX) (142.67mmol), stir to dissolve, add 30g sodium chloride and 36ml water, heat to 145℃ and reflux for 5h until TLC (dichloromethane:methanol = 20:1, phosphomolybdic acid colorimetric) shows complete conversion of the starting material.

[0058] The reaction solution was cooled to room temperature and poured into 3L of water. The mixture was stirred for 1 hour, and a crude solid product precipitated. The product was then filtered. 60ml of methanol was added to the wet filter cake, and the mixture was stirred at room temperature for 1 hour. The mixture was then filtered, and the filter cake was dried under reduced pressure at 45°C to obtain 51.59g of a white ursodeoxycholic acid intermediate of formula (Ⅰ), with a yield of 92.59%. The purity was determined by HPLC method 2 to be 93.46%.

[0059] NMR data for ursodeoxycholic acid intermediate formula (I) are as follows: Figure 5 As shown: 1 H NMR (600 MHz, DMSO) δ 4.57 (s, 1H), 3.35 (s, 1H), 2.90 (dd, J =12.3, 6.0 Hz, 1H), 2.44 (t, J = 11.3 Hz, 1H), 2.22 (ddd, J = 15.0, 9.5, 5.1Hz, 1H), 2.14 – 2.02 (m, 2H), 1.93 (d, J = 12.7 Hz, 1H), 1.87 – 1.74 (m, 2H), 1.75 – 1.62 (m, 4H), 1.49 (dd, J = 21.9, 10.7 Hz, 2H), 1.43 – 1.27 (m, 4H), 1.26 – 1.15 (m, 3H), 1.14 (s, 3H), 1.12 – 0.97 (m, 5H), 0.96 – 0.84 (m, 4H), 0.61 (s, 3H).

[0060] Example 2 This embodiment provides a method for preparing an intermediate of ursodeoxycholic acid, including the following steps: S1. In a 3L reaction flask, add 1L of dichloromethane and 200g of compound (II) (605.12mmol), stir to dissolve and clarify, then add 7.11g of 4-dimethylaminopyridine (78.67mmol) and 84.72ml of triethylamine (0.61mol) in sequence. Slowly add 138.43g of p-toluenesulfonyl chloride (726.14mmol) in 400mL of dichloromethane solution. After the addition is complete, heat the reaction solution to reflux for 4h until TLC (PE:EA=3:1, UV 254nm color development) shows that the starting material is completely converted.

[0061] The reaction solution was poured into 1L of water, stirred, and allowed to stand for separation. The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and 200ml of methanol was added. The mixture was cooled to 0-5℃ and stirred for 1-2 hours. After filtration, the mixture was dried under reduced pressure at 40℃ to obtain 270.11g of sulfonyl (III) with a yield of 92.09%. The purity was determined to be 98.46% by HPLC method 1 (254nm).

[0062] S2. In a 5L reaction flask under nitrogen protection, 270g of sulfonyl (III) (557.05mmol) was dissolved in 2.7L DMF, and 230g of potassium carbonate (1.66mol), 18g of TBAB (55.83mmol), and 177.34g of diethyl malonate (1.11mol) were added. The mixture was heated to 50℃ and stirred for 11h until TLC (PE:EA=2:1, UV 254nm color development) showed complete conversion of the starting material.

[0063] The reaction solution was slowly poured into 2700 ml of water and filtered to obtain crude diester. 540 ml of ethanol was added to the crude product, stirred for 30 min, filtered, and dried under reduced pressure at 40 °C to obtain 224.74 g of white diester (IV), with a yield of 85.35%. The purity was determined by HPLC method 1 (254 nm) to be 92.17%.

[0064] S3. In a 5L reaction flask, add 1500ml of dichloromethane, 2.05g of p-toluenesulfonic acid (11.88mmol), 150g of diester (IV) (371.35mmol), and 111.13g of isopropyl acetate (1.11mol). Keep the mixture at 35℃ for 3 hours until TLC (developing solvent: PE:EA=3:1, vanillin color development) shows that the starting material is basically completely converted.

[0065] The reaction solution was cooled to 20°C, 1.8 g of triethylamine was added, and the mixture was stirred for 1.2 h and concentrated to near dryness. 100 ml of methanol was added to the concentrate, and the mixture was stirred at 0–5°C for 1 h. The mixture was filtered and dried under reduced pressure at 40°C to obtain 158.51 g of the triester (V), with a yield of 82.61%. Purity was determined to be 93.08% by HPLC method 1 (254 nm).

[0066] S4. Under nitrogen protection, add 2L of acetone, 200g of triester (V) (387.06mmol), 70.02g of PDC (658.00mmol), and 97.88g of NHPI (599.94mmol) to a 3L reaction flask. Heat to 40℃ and stir for 5 hours until TLC (PE:EA=3:1, vanillin colorimetric) shows complete conversion of the starting materials.

[0067] The reaction solution was concentrated to dryness under reduced pressure, 2L of ethyl acetate was added and stirred for 30 minutes. The mixture was filtered through diatomaceous earth, and the filtrate was washed once with 2L of 1.5M sodium hydroxide aqueous solution and then once with 2L of saturated saline solution. The organic phase was concentrated to dryness under reduced pressure at 45°C.

[0068] Add 400 ml of ethanol to the concentrate, heat to reflux until dissolved, cool to room temperature and stir for 1 h, filter, and dry the filter cake under reduced pressure at 40 °C to obtain 183.19 g of white esterified oxide (VI), with a yield of 89.18%. The purity was determined by HPLC method 1 (254 nm) to be 91.38%.

[0069] S5. In a 3L reaction flask, add 900ml tetrahydrofuran, 100ml water and 100g esterified oxide (VI) (188.43mmol), stir to dissolve, add 125.00g potassium carbonate (904.46mmol), heat to 40℃ and stir for 5h until TLC (PE:EA=2:1, UV 254nm color development) shows complete conversion of the starting material.

[0070] The reaction solution was neutralized with 1000 ml of 1N hydrochloric acid aqueous solution, and the layers were separated. The aqueous layer was extracted with 500 ml of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and 300 ml of petroleum ether was added. The mixture was stirred at room temperature for 1 h, filtered, and the filter cake was dried under reduced pressure at 40 °C to obtain 82.05 g of white 3,7-diketone (VII) solid, with a yield of 89.48%. The purity was determined by HPLC method 1 (254 nm) to be 95.11%.

[0071] S6. Add 1.6L of tetrahydrofuran and 80g of 3,7-diketone (Ⅶ) (164.61mmol) to a 3L reaction flask, stir to dissolve, add 211.60g of Raney Ni (2.47mol), replace the air in the reaction flask with nitrogen three times, then replace it with hydrogen three times, and then maintain a slightly positive pressure hydrogen atmosphere and stir at room temperature for 22h until TLC (PE:EA=2:1, phosphomolybdic acid color development) shows complete conversion of the starting material, filter the catalyst, concentrate the filtrate to dryness under reduced pressure to obtain 7K diester (Ⅷ).

[0072] S7. Dissolve the 7K diester (VIII) obtained in step S6 directly in 560 ml of methanol, slowly add 50 ml of aqueous solution of 27.71 g potassium hydroxide (493.83 mmol), heat to 50 °C and stir for 3 h until TLC (dichloromethane:methanol = 20:1, phosphomolybdic acid colorimetric) shows that the hydrolysis reaction is complete.

[0073] The reaction solution was cooled to room temperature, 1.2 L of water was added, and the pH was adjusted to 4-5 with 3 M hydrochloric acid. A large amount of solid precipitated out. The mixture was stirred at room temperature for 2 hours, filtered, and the filter cake was dried under reduced pressure at 40 °C to obtain 67.28 g of white 7K dicarboxylic acid compound (IX). The yield of the two-step hydrogenation hydrolysis was 97.19%, and the purity was 90.84% ​​as determined by HPLC method 2.

[0074] S8. In a 3L reaction flask, add 600ml DMSO and 60g 7K dicarboxylic acid (IX) (142.67mmol), stir to dissolve, add 30g sodium chloride and 37.5ml water, heat to 140℃ and reflux for 6h until TLC (dichloromethane:methanol = 20:1, phosphomolybdic acid colorimetric) shows complete conversion of the starting material.

[0075] The reaction solution was cooled to room temperature and poured into 3L of water. The mixture was stirred for 1 hour, and a crude solid product precipitated. The product was then filtered. 60ml of methanol was added to the wet filter cake, and the mixture was stirred at room temperature for 1 hour. The mixture was then filtered, and the filter cake was dried under reduced pressure at 40℃ to obtain 51.36g of a white ursodeoxycholic acid intermediate of formula (Ⅰ), with a yield of 92.18%. The purity was determined by HPLC method 2 to be 93.27%.

[0076] Example 3 This embodiment provides a method for preparing an intermediate of ursodeoxycholic acid, including the following steps:

[0077] S1. In a 3L reaction flask, add 1L of dichloromethane and 200g of compound (II) (605.12mmol), stir to dissolve and clarify, then add 7.66g of 4-dimethylaminopyridine (84.72mmol) and 252.78ml of triethylamine (1.82mol) in sequence, and slowly add 161.51g of p-toluenesulfonyl chloride (847.17mmol) in 400mL of dichloromethane solution. After the addition is complete, heat the reaction solution to reflux for 5h until TLC (PE:EA=3:1, UV 254nm color development) shows that the starting material is completely converted.

[0078] The reaction solution was poured into 1L of water, stirred, and allowed to stand for separation. The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and 200ml of methanol was added. The mixture was cooled to 0-5℃ and stirred for 2h. After filtration, it was dried under reduced pressure at 50℃ to obtain 271.89g of sulfonyl (III) with a yield of 92.70%. The purity was determined to be 98.50% by HPLC method 1 (254nm).

[0079] S2. In a 5L reaction flask under nitrogen protection, 270g of sulfonyl (III) (557.05mmol) was dissolved in 2.7L DMF, and 230g of potassium carbonate (1.66mol), 18g of TBAB (55.83mmol), and 294.61g of dimethyl malonate (2.23mol) were added. The mixture was heated to 60℃ and stirred for 9 hours until TLC (PE:EA=2:1, UV 254nm color development) showed complete conversion of the starting material.

[0080] The reaction solution was slowly poured into 2700 ml of water and filtered to obtain crude diester. 540 ml of ethanol was added to the crude product, stirred for 30 min, filtered, and dried under reduced pressure at 50 °C to obtain 226.32 g of white diester (IV), with a yield of 91.38%. The purity was determined to be 92.25% by HPLC method 1 (254 nm).

[0081] S3. In a 5L reaction flask, add 1500ml of dichloromethane, 1.91g of p-toluenesulfonic acid (11.10mmol), 137.03g of diester (IV) (308.20mmol), and 175.76g of isopropyl propionate (1.54mol). Keep the mixture at 40℃ for 1h until TLC (developing solvent: PE:EA=3:1, vanillin color development) shows that the starting material is basically completely converted.

[0082] The reaction solution was cooled to 25°C, 1.8 g of triethylamine was added, and the mixture was stirred for 0.8 h and concentrated to near dryness. 100 ml of methanol was added to the concentrate, and the mixture was stirred at 0–5°C for 1 h. The mixture was filtered and dried under reduced pressure at 50°C to obtain 150.30 g of the triester (V), with a yield of 97.4%. Purity was determined to be 93.12% by HPLC method 1 (254 nm).

[0083] S4. Under nitrogen protection, add 2L of acetone, 194.55g of triester (V) (388.58mmol), 202.04g of PDC (680.02mmol), and 101.42g of NHPI (621.73mmol) to a 3L reaction flask. Heat to 50℃ and stir for 4 hours until TLC (PE:EA=3:1, vanillin colorimetric) shows complete conversion of the starting materials.

[0084] The reaction solution was concentrated to dryness under reduced pressure, 2L of ethyl acetate was added and stirred for 30 minutes. The mixture was filtered through diatomaceous earth, and the filtrate was washed once with 2L of 1.5M sodium hydroxide aqueous solution and then once with 2L of saturated saline solution. The organic phase was concentrated to dryness under reduced pressure at 50°C.

[0085] Add 400 ml of ethanol to the concentrate, heat to reflux until dissolved, cool to room temperature and stir for 1 h, filter, dry the filter cake under reduced pressure at 50 °C to obtain 183.19 g of white esterified oxide (VI), yield 91.6%, purity 91.48% as determined by HPLC method 1 (254 nm).

[0086] S5. In a 3L reaction flask, add 900ml tetrahydrofuran, 100ml water and 97.34g esterified oxide (VI) (189.14mmol), stir to dissolve, add 136.18g potassium carbonate (983.53mmol), heat to 50℃ and stir for 3h until TLC (PE:EA=2:1, UV 254nm color development) shows complete conversion of the starting material.

[0087] The reaction solution was neutralized with 1000 ml of 1N hydrochloric acid aqueous solution, and the layers were separated. The aqueous layer was extracted with 500 ml of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and 300 ml of petroleum ether was added. The mixture was stirred at room temperature for 1 h, filtered, and the filter cake was dried under reduced pressure at 50 °C to obtain 77.94 g of white 3,7-diketone (VII) solid, with a yield of 89.85%. The purity was determined by HPLC method 1 (254 nm) to be 95.10%.

[0088] S6. Add 1.6 L of tetrahydrofuran and 75.49 g of 3,7-diketone (Ⅶ) (164.61 mmol) to a 3 L reaction flask, stir to dissolve, add 281.85 g of Raney Ni (3.29 mol), replace the air in the reaction flask with nitrogen three times, then replace it with hydrogen three times, and then maintain a slightly positive pressure hydrogen atmosphere and stir at room temperature for 26 h until TLC (PE:EA=2:1, phosphomolybdic acid color development) shows complete conversion of the starting material, filter the catalyst, concentrate the filtrate to dryness under reduced pressure to obtain 7K diester (Ⅷ).

[0089] S7. Dissolve the 7K diester (VIII) obtained in step S6 directly in 560 ml of methanol, slowly add 47 ml of aqueous solution of 28.17 g potassium hydroxide (502.06 mmol), heat to 60 °C and stir for 1 h until TLC (dichloromethane:methanol = 20:1, phosphomolybdic acid colorimetric) shows that the hydrolysis reaction is complete.

[0090] The reaction solution was cooled to room temperature, 1.2 L of water was added, and the pH was adjusted to 4-5 with 3 M hydrochloric acid. A large amount of solid precipitated out. The mixture was stirred at room temperature for 2 hours, filtered, and the filter cake was dried under reduced pressure at 50 °C to obtain 67.69 g of white 7K dicarboxylic acid compound (IX). The yield of the two-step hydrogenation hydrolysis was 94.62%, and the purity was 91.03% as determined by HPLC method 2.

[0091] S8. In a 3L reaction flask, add 600ml DMSO and 60g 7K dicarboxylic acid (IX) (138.07mmol), stir to dissolve, add 30g sodium chloride and 36ml water, heat to 150℃ and reflux for 4h until TLC (dichloromethane:methanol = 20:1, phosphomolybdic acid colorimetric) shows complete conversion of the starting material.

[0092] The reaction solution was cooled to room temperature and poured into 3L of water. The mixture was stirred for 1 hour, and a crude solid product precipitated. The product was then filtered. 60ml of methanol was added to the wet filter cake, and the mixture was stirred at room temperature for 1 hour. The mixture was then filtered, and the filter cake was dried under reduced pressure at 50°C to obtain 50.21g of a white ursodeoxycholic acid intermediate of formula (Ⅰ), with a yield of 93.12%. The purity was determined to be 93.45% by HPLC method 2.

[0093] Comparative Example 1 This comparative example provides a method for preparing an ursodeoxycholic acid intermediate, comprising the following steps:

[0094] Steps S1-S2 are the same as in Example 1; S3. In a 3L reaction flask, add 800ml ethylene glycol, 420mL triethyl orthoformate, 7.28g p-toluenesulfonic acid and 400g diester (IV). Keep the mixture at 20~25℃ for 10h until TLC (developing solvent: PE:EA=3:1, vanillin color development) shows that the raw materials are basically completely converted.

[0095] The reaction solution was poured into 800 ml of water, stirred for 1 h, and filtered to obtain the crude ketal. 800 ml of ethanol was added to the crude product, and the mixture was stirred at 50-60 °C for 1 h, then cooled to 0-10 °C and stirred for another 1 h. The mixture was filtered and dried under reduced pressure at 40-50 °C to obtain 372 g of the ketal (V'), with a yield of 85%. Purity was determined to be 92.67% by HPLC method 1 (210 nm).

[0096] S4. Under nitrogen protection, add 2L of acetone, 200g of ketal (V'), 200g of PDC, and 100g of NHPI to a 3L reaction flask. Heat to 40~50℃ and stir for 4~5 hours until TLC (PE:EA=3:1, vanillin color development) shows complete conversion of the raw materials.

[0097] The reaction solution was concentrated to dryness under reduced pressure, 2L of ethyl acetate was added and stirred for 30 minutes. The mixture was filtered through diatomaceous earth, and the filtrate was washed once with 2L of 1.5M sodium hydroxide aqueous solution and then once with 2L of saturated saline solution. The organic phase was concentrated to dryness under reduced pressure at 45~50℃.

[0098] Add 400 ml of ethanol to the concentrate, heat to reflux until dissolved, cool to room temperature and stir for 1 h, filter, dry the filter cake under reduced pressure at 40-50 °C to obtain 144 g of white ketal oxide (VI'), yield 70%, purity 97.47% as determined by HPLC method 1 (254 nm).

[0099] S5. In a 3L reaction flask, add 900ml tetrahydrofuran, 100ml water and 100g ketal oxide (VI'), stir to dissolve, add 100ml hydrochloric acid, heat to 40~50℃ and stir to react for 4h until TLC (PE:EA=2:1, UV 254nm color development) shows that the starting material is completely converted.

[0100] The reaction solution was neutralized with 1000 ml of saturated sodium bicarbonate aqueous solution, and the layers were separated. The aqueous layer was extracted with 500 ml of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and 300 ml of petroleum ether was added. The mixture was stirred at room temperature for 1 h, filtered, and the filter cake was dried under reduced pressure at 40-50 °C to obtain 82.5 g of white 3,7-diketone (VII) solid, with a yield of 92%. The purity was determined by HPLC method 1 (254 nm) to be 81.35%.

[0101] The preparation methods for S6-S8 are the same as in Example 1. In S7, 68.8 g of white 7K dicarboxylic acid compound (IX) was prepared with a two-step hydrogenation and hydrolysis yield of 92.4% and a purity of 90.12% as determined by HPLC method 2. In S8, 48 g of white ursodeoxycholic acid intermediate (I) was prepared with a yield of 89.13% and a purity of 96.40% as determined by HPLC method 2.

[0102] As can be seen from the comparison between Example 1 and Comparative Example 1, the carbonyl group is protected by ketal reaction in Comparative Example 1, which inevitably produces the isomer impurity 4-ene ketal, resulting in a significant decrease in product purity and yield. Moreover, the isomer impurity is not easy to remove, which in turn leads to a decrease in the yield of the final product. In contrast, the technical solution of Example 1 of the present invention replaces the traditional ketal protection method with an esterification protection strategy, which can avoid the generation of isomer impurities and thus significantly improve the yield of the final product.

[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an intermediate of ursodeoxycholic acid, characterized in that: The preparation method of the ursodeoxycholic acid intermediate includes the following steps: ； Where R = -CH3, -CH2CH3; R 1 =-COCH3, -COCH2CH3 S1. Compound (II) undergoes sulfonation to yield sulfonyl compound (III). S2, sulfonyl formula (III) is alkylated to give diester formula (IV); S3, diester (Ⅳ) undergoes esterification to yield trimer (Ⅴ); S4. The esterified compound (V) undergoes an oxidation reaction to yield the esterified oxide compound (VI). S5, the esterified oxide (VI) is hydrolyzed to give the 3,7-diketone (VII); The S6,3,7-diketone formula (VII) is hydrogenated and reduced to give the 7K diester formula (VIII). S7 and 7K diester (VIII) undergo alkaline hydrolysis to yield 7K dicarboxylic acid (IX). The S8 and 7K dicarboxylic acid derivatives (IX) undergo a decarboxylation reaction to yield the ursodeoxycholic acid intermediate 7K (I). In step S3, diester (Ⅳ), dichloromethane, p-toluenesulfonic acid and esterification reagent are mixed and reacted at 35~40℃ for 1-3h. When the reaction solution is cooled to 20-25℃, triethylamine is added and stirred for 0.8-1.2h to obtain diester (Ⅴ). The esterification reagent is isopropyl acetate or isopropyl propionate. In step S4, the trimeric formula (V) and acetone are mixed, PDC and NHPI are added, and the mixture is reacted at 40~50℃ for 4-5 hours to obtain the esterified oxide formula (VI). In step S5, the esterified oxide (VI) is dissolved in an aqueous solution of tetrahydrofuran, an alkaline catalyst is added, and the reaction is carried out at 40-50°C for 3-5 hours to obtain the 3,7-diketone (VII). The alkaline catalyst is potassium carbonate or sodium carbonate.

2. The method for preparing an ursodeoxycholic acid intermediate as described in claim 1, characterized in that: In step S1, compound (II) is dissolved in dichloromethane, and 4-dimethylaminopyridine, triethylamine and p-toluenesulfonyl chloride are added in sequence. The mixture is heated to reflux for 4-5 hours to obtain sulfonyl compound (III).

3. The method for preparing an ursodeoxycholic acid intermediate as described in claim 1, characterized in that: In step S2, under a nitrogen atmosphere, sulfonyl compound (III) is dissolved in DMF, potassium carbonate, TBAB and alkylating agent are added, and the mixture is reacted at 50~60℃ for 9-11 h to obtain diester compound (IV), wherein the alkylating agent is diethyl malonate or dimethyl malonate.

4. The method for preparing an ursodeoxycholic acid intermediate as described in claim 1, characterized in that: In step S6, the 3,7-diketone (VII) is dissolved in tetrahydrofuran, Raney Ni catalyst is added, and the reaction is carried out at 25-27°C for 22-26 h in a hydrogen atmosphere to obtain the 7K diester (VIII).

5. The method for preparing an ursodeoxycholic acid intermediate as described in claim 1, characterized in that: In step S7, 7K diester (VIII) is dissolved in methanol, and potassium hydroxide aqueous solution is added. The mixture is reacted at 50-60℃ for 1-3 hours to obtain 7K dicarboxylic acid (IX).

6. The method for preparing an ursodeoxycholic acid intermediate as described in claim 1, characterized in that: In step S8, 7K dicarboxylic acid (IX) is dissolved in DMSO solution, sodium chloride solution is added, and the reaction is carried out at 140-150℃ for 4-6 hours to obtain ursodeoxycholic acid intermediate 7K (I).

7. An intermediate compound, characterized in that: The structure of the intermediate compound is shown below: ； Where R = -CH3, -CH2CH3; R 1 =-COCH3, -COCH2CH3.

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