A method for rapid synthesis of aramchol

Abstract

A rapid synthesis method for Aramchol belongs to the field of organic synthesis technology. The rapid synthesis method of Aramchol of this invention first synthesizes 3-ketocholic acid from cholic acid under the action of an enzyme, then synthesizes 3-oximecholic acid from 3-ketocholic acid under the action of hydroxylamine hydrochloride; 3-oximecholic acid is reduced to synthesize 3-aminocholic acid with corresponding chirality under a chiral ruthenium catalyst, and then 3-aminocholic acid and arachidic acid are condensed to prepare Aramchol. The synthesis of 3-ketocholic acid of this invention uses 3α,7α,12α-trihydroxy-5β-cholanic acid as a starting material, and prepares 3-keto-7α,12α-dihydroxy-5β-cholanic acid under the catalysis of oxidized coenzyme I, 3α-hydroxysteroid dehydrogenase, and NADH oxidase. The use of specific enzyme catalysis makes the reaction more efficient and cleaner, with a high yield, and the oxidation reaction proceeds in a directed manner without producing byproducts. The entire reaction utilizes the method of converting carbonyl groups to amino groups. The reaction is simple and has a high yield. During the reduction, a metal catalyst is used, which can selectively reduce the carbonyl group to a chiral amino group, making it suitable for industrial application.

Description

Technical Field

[0001] This invention relates to a method for synthesizing a known compound. Specifically, it relates to a method for the rapid synthesis of Aramchol, belonging to the field of organic synthesis technology. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic liver disease closely associated with insulin resistance and genetic susceptibility. With the global prevalence of obesity, NAFLD has become the most common cause of chronic liver disease. A 2021 review published in *The Lancet* indicated that NAFLD has multiple stages. Simple hepatic steatosis is characterized by intrahepatic lipid deposition, while non-alcoholic steatohepatitis (NASH) can further develop into cirrhosis and hepatocellular carcinoma, and may lead to cardiovascular disease and complications in other organs. Poorly controlled NAFLD can progress to non-alcoholic steatohepatitis (NASH). According to the IPD database, the global prevalence of NAFLD is approximately 25% across all age groups, and the prevalence of NASH is approximately 4%. In China, the prevalence of NAFLD is approximately 18%, and the prevalence of NASH is approximately 2.4%. Currently, there are no specific drugs for NASH. Therefore, researching and developing an oral specific drug for NAFLD is of great significance for the treatment of both NAFLD and NASH.

[0003] Aramchol (Arachidyl amido cholanoic acid) is a novel oral drug for non-alcoholic fatty liver disease (NAFLD) developed by Galmed Pharmaceuticas in Israel. As a novel fatty acid-bile acid conjugate, it targets and inhibits stearoyl-CoA desaturase 1 (SCD1). SCD1 is a key enzyme in hepatic lipogenesis, converting saturated fatty acids into monounsaturated fatty acids. It is currently in Phase III clinical trials. Results from the Aramchol IIb clinical trial showed that at week 52, patients taking 600 mg of Aramchol had a significantly improved liver fat percentage compared to the placebo group (p=0.0045), and the number of patients with an improvement rate exceeding 5% was significantly higher in the Aramchol group than in the placebo group (p=0.0279). Furthermore, 600 mg of Aramchol also showed significant efficacy in alleviating NASH symptoms (without worsening fibrosis), improving liver fibrosis, and reducing the progression of cirrhosis.

[0004] Existing methods for preparing aramchol mainly include the sodium azide-to-hydroxyl conversion method, the DPPA-to-hydroxyl conversion method, and the photo-extending reaction, i.e., the Mitsunobu amination-to-hydroxyl conversion method. However, each method has several drawbacks: the sodium azide-to-hydroxyl conversion method was an early approach to the synthesis of aramchol and was significant in pioneering its chemical synthesis; however, its low yield and the highly toxic and explosive nature of sodium azide limit its industrial application. The DPPA-to-hydroxyl conversion method, while using DPPA as a safer alternative to sodium azide, suffers from high costs and does not improve overall yield. The Mitsunobu amination-to-hydroxyl conversion method, although using the commonly used reagent phthalimide, involves too many reaction excipients, such as triphenylphosphine and diethyl azodicarbonate, resulting in numerous post-processing impurities and limiting its industrial production value.

[0005] Therefore, it is essential to design a new method for the rapid synthesis of Aramchol that is simple to operate, has short steps, convenient post-processing, and high yield, in order to broaden its application. Summary of the Invention

[0006] To address the problems existing in the background art, the purpose of this invention is to provide a novel method for the rapid synthesis of Aramchol that is simple to operate, has short steps, convenient post-processing, and high yield. To achieve the above objective, the technical solution of this invention is as follows: A rapid method for synthesizing Aramchol includes the following steps: first, 3-ketocholic acid is synthesized from cholic acid under enzymatic action; then, 3-oximecholic acid is synthesized from 3-ketocholic acid under the action of hydroxylamine hydrochloride; 3-oximecholic acid is reduced to 3-aminocholic acid with corresponding chirality under a chiral ruthenium catalyst, and then condensed with arachidic acid to prepare 3β-arachidoamino-7α,12α-dihydroxy-5β-cholan-24-acid (Aramchol). The synthetic route is shown in the appendix. Figure 1 .

[0007] The enzyme is a mixture of oxidized coenzyme I, 3α-hydroxysteroid dehydrogenase (3α-HSDH), and NADH oxidase; the cholic acid is 3α,7α,12α-trihydroxy-5β-cholanoic acid; the 3-ketocholic acid is 3-keto-7α,12α-dihydroxy-5β-cholanoic acid; the 3-oximecholic acid is 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid; and the 3-aminocholic acid is 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid.

[0008] Preferably, the ruthenium catalyst is any one of dichloro[(S)-(-)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl]ruthenium(II) or (S,S)-N-(p-toluenesulfonyl)-1,2-diphenylethanediamine (p-isopropyltoluene) ruthenium chloride. The ruthenium catalyst is any one of dichloro[(S)-(-)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl]ruthenium(II) or (S,S)-N-(p-toluenesulfonyl)-1,2-diphenylethanediamine (p-isopropyltoluene) ruthenium chloride. The molecular formula of dichloro[(S)-(-)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl]ruthenium(II) is C 44 H 32 Cl2P2Ru, molecular weight 794.66, CAS number: 134524-84-8; the molecular formula of (S,S)-N-(p-toluenesulfonyl)-1,2-diphenylethanediamine (p-isopropyltoluene)ruthenium chloride is C 31 H 35 ClN2O2RuS, molecular weight 637.2, CAS number: 192139-90-5.

[0009] The method for rapid synthesis of Aramchol includes the following specific steps: Preparation of S1: 3-Keto-7α,12α-Dihydroxy-5β-cholanic acid Add the specified amount of 3α,7α,12α-trihydroxy-5β-cholanic acid to water, and while stirring, add sodium hydroxide solution dropwise until all 3α,7α,12α-trihydroxy-5β-cholanic acid is dissolved. Adjust the pH with hydrochloric acid; then add oxidized coenzyme I (NAD) sequentially. + ), 3α-hydroxysteroid dehydrogenase (3α-HSDH) and NADH oxidase, stirred at 25-35℃ for 4-6 h, the reaction solution was heated to 80℃ and kept at that temperature for 30 min, and then filtered to remove the enzymes; the filtrate was cooled, filtered and dried to obtain white 3-keto-7α,12α-dihydroxy-5β-cholanic acid powder for later use.

[0010] Preparation of S2: 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid At room temperature, 3-keto-7α,12α-dihydroxy-5β-cholanic acid, hydroxylamine hydrochloride, and potassium hydroxide prepared in step S1 were added to ethanol, and the mixture was heated to reflux. After reacting for 5 hours, thin-layer chromatography (TLC) was used to detect that 3-keto-7α,12α-dihydroxy-5β-cholanic acid had completely reacted. The mixture was then cooled to room temperature, filtered, and the filter cake was washed with ethanol. The filtrates were combined, concentrated, and dried to obtain a yellow 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid powder for later use.

[0011] Preparation of S3: 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid The 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid prepared in step S2 was added to methanol, followed by the addition of a ruthenium catalyst. After three hydrogen purgings, the reaction proceeded overnight at room temperature under hydrogen protection. TLC was used to confirm the complete reaction of the 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid. The reaction solution was directly filtered, and the filtrate was concentrated and dried to obtain a yellow powder of 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid, which was then set aside.

[0012] S4: Preparation of Aramchol The 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid prepared in step S3 was added to dichloromethane solvent (DCM), followed by the addition of arachidic acid (CH3(CH2)). 18 COOH, AA), N,N'-dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine (DMAP) were reacted overnight at room temperature. The reaction of 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid was detected by TLC until the reaction was complete. The reaction solution was flushed into water and then extracted twice by DCM. The organic phases were combined, dried and concentrated, and purified by silica gel plate chromatography to obtain white 3β-arachidoamino-7α,12α-dihydroxy-5β-cholan-24-acid (Aramchol) powder.

[0013] Preferably, the 3α,7α,12α-trihydroxy-5β-cholanoic acid described in step S1 and oxidized coenzyme I (NAD) + The mass ratio of the two components is 1:0.006~0.012; the amount of 3α-hydroxysteroid dehydrogenase (3α-HSDH) added is 0.01~0.02 g per gram of substrate; the amount of NADH oxidase added is 0.005~0.01 g per gram of substrate.

[0014] Preferably, the oxidized coenzyme I (NAD) described in step S1 + The mass ratio of 3α-hydroxysteroid dehydrogenase (3α-HSDH) and NADH oxidase is 1:1:1. The oxidized coenzyme I (NAD...) + 3α-hydroxysteroid dehydrogenase (3α-HSDH) and NADH oxidase are both commercially available reagents, such as NAD... +The enzymes are available from Sigma-Aldrich (product number N7004, purity ≥98%); 3α-HSDH is available from Sigma-Aldrich (product number H7009, enzyme activity ≥10 U / mg); NADH oxidase is available from Sigma-Aldrich (product number N9785, enzyme activity ≥5 U / mg). Those skilled in the art can choose similar products from other suppliers as needed. In this invention, the enzyme mass ratio is based on its purified form; in actual use, it can be equivalently converted and adjusted according to enzyme activity units (U).

[0015] Preferably, the pH is adjusted to 7.7-8.0 using hydrochloric acid as described in step S1.

[0016] Preferably, the molar ratio of 3-keto-7α,12α-dihydroxy-5β-cholanic acid, hydroxylamine hydrochloride, and potassium hydroxide in step S2 is 3-ketocholanic acid: hydroxylamine hydrochloride: potassium hydroxide = 1.0: 1.0~1.5: 1.0~1.5.

[0017] Preferably, the molar ratio of 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid and ruthenium catalyst in step S3 is 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid:ruthenium catalyst = 1.0: 0.04~0.1.

[0018] Preferably, in step S4, the molar ratio of 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid, dichloromethane, arachidic acid, N,N'-dicyclohexylcarbodiimide, and dimethylaminopyridine is 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid: arachidic acid: N,N'-dicyclohexylcarbodiimide: dimethylaminopyridine = 1.0:1.0~1.5:1.0~1.5:0.1~0.3; and the volume ratio of arachidic acid to dichloromethane is 1:20.

[0019] 1. The rapid synthesis method of Aramchol of the present invention uses 3α,7α,12α-trihydroxy-5β-cholanic acid as a starting material to prepare intermediates under the catalysis of oxidized coenzyme I, 3α-hydroxysteroid dehydrogenase (3α-HSDH), and NADH oxidase. The use of specific enzyme catalysis makes the reaction more efficient and cleaner, with high yields, and the oxidation reaction proceeds in a directed manner without producing byproducts. The reaction process does not use hazardous reagents, thus reducing reaction risks and facilitating industrialization.

[0020] 2. The rapid synthesis method of Aramchol of the present invention first synthesizes 3-ketocholic acid from cholic acid under the action of an enzyme, and then synthesizes 3-oximecholic acid from 3-ketocholic acid under the action of hydroxylamine hydrochloride. 3-oximecholic acid is then reduced to 3-aminocholic acid with corresponding chirality under a chiral ruthenium catalyst, and subsequently condensed with arachidic acid to prepare Aramchol. This reaction ingeniously utilizes the carbonyl group to amino group conversion method, resulting in a simple reaction with high yield. The use of a metal catalyst during reduction allows for selective reduction to chiral amino groups. Furthermore, steps S2, S3, and S4 are all carried out at room temperature, and step S1 is also close to room temperature. The overall reaction conditions are mild and controllable, and the product yield is high, making it very suitable for industrial application. Attached Figure Description

[0021] Figure 1 This is a synthetic route diagram for the rapid synthesis of Aramchol according to the present invention; Figure 2 The 1H NMR spectrum of 3-keto-7α,12α-dihydroxy-5β-cholanic acid prepared in Example 1; Figure 3 The 1H NMR spectrum of 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid prepared in Example 1 of this invention; Figure 4 The 1H NMR spectrum of Aramchol prepared in Example 1 of this invention; Figure 5 This is the mass spectrum of Aramchol prepared in Example 1 of the present invention. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] In the following examples, unless otherwise specified, the reagents used are in the following forms: oxidized coenzyme I (NAD) + For solid powder, weigh directly; 3α-hydroxysteroid dehydrogenase (3α-HSDH) should be prepared into an aqueous solution with a concentration of 20 mg / mL before use; NADH oxidase should be prepared into an aqueous solution with a concentration of 40 mg / mL before use.

[0024] Example 1: Rapid Synthesis of Aramchol Preparation of S1: 3-Keto-7α,12α-Dihydroxy-5β-cholanic acid Weigh 30.00 g (73.5 mmol, M=408.3) of 3α,7α,12α-trihydroxy-5β-cholanic acid and add it to 700 mL of water. While stirring, add 1 M sodium hydroxide solution dropwise until all 3α,7α,12α-trihydroxy-5β-cholanic acid (CA) is dissolved. Adjust the pH to 7.7–8.0 with hydrochloric acid. Then add 0.20 g of oxidized coenzyme I (NAD⁺), 15 mL of 3α-hydroxysteroid dehydrogenase (3α-HSDH) solution (0.30 g as solid enzyme), and 5 mL of NADH oxidase solution (0.20 g as solid enzyme). Make up the volume with water to 1 L. Start the reaction in a 30°C water bath with stirring for 4–6 h.

[0025] The solution after the above reaction was heated to 80℃ and kept at that temperature for 30 min, followed by filtration to remove the enzyme. The resulting filtrate was cooled to 0℃ and then filtered under vacuum. The filter cake was dried to obtain 29.53 g of 3-keto-7α,12α-dihydroxy-5β-cholanic acid white powder, with a yield of 98.4%.

[0026] H-NMR (400MHz, DMSO-d6): 11.94 (s, 1H), 4.34 (d, J=3.2Hz, 1H), 4.20 (d, J=2.8Hz, 1H), 3.84 (s, 1H), 3.70 (s, 1H), 3.45 (t, J=13.6Hz, 1H), 2.32-0.96 (m, 23H), 0.64 (s, 3H).

[0027] Preparation of S2: 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid At room temperature, 10.00 g (24.6 mmol, M=406.3) of 3-keto-7α,12α-dihydroxy-5β-cholanic acid prepared in step S1, 2.05 g (29.5 mmol, M=69.49) of hydroxylamine hydrochloride, and 1.65 g (29.4 mmol, M=56.1) of potassium hydroxide were added to 100 mL of ethanol. The mixture was heated to reflux and reacted for 5 h. After TLC detection showed that the 3-ketocholanic acid reaction was complete, the mixture was cooled to room temperature, filtered, and the filter cake was washed with 30 mL of ethanol. The filtrates were combined, concentrated, and dried to obtain 5.73 g of 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid as a yellow powder.

[0028] H-NMR (400MHz, DMSO-d6): 11.86 (δ, 1H), 9.90 (s, 1H), 4.23 (d, J=3.2 Hz, 1H), 4.14 (d, J=2.8 Hz, 1H), 3.80 (s, 1H), 3.66 (s, 1H), 2.87-2.70 (m, 2H), 2.25-0.86 (m, 28H), 0.61 (s, 3H).

[0029] Preparation of S3: 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid 3.00 g (7.1 mmol, M=421.3) of 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid prepared in step S2 was added to 50 mL of methanol, followed by 0.45 g of dichloro[(S)-(-)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl]ruthenium(II) (M=782.65 g / mol, 0.575 mmol). After three hydrogen purgings, the reaction was carried out overnight at room temperature under hydrogen protection. TLC was used to determine the complete reaction of 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid. The reaction solution was directly filtered, and the filtrate was concentrated and dried to obtain 2.5 g of 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid as a yellow powder, with a yield of 86.2%.

[0030] H-NMR (400MHz, DMSO-d6): 12.06 (δ, 1H), 7.56 (s, 1H), 4.53 (d, J=4.2 Hz, 2H), 4.25 (d, J=3.6 Hz, 1H), 4.20 (d, J=3.2 Hz, 1H), 3.88 (s, 1H), 3.76 (s, 1H), 2.87-2.68 (m, 2H), 2.25-0.86 (m, 28H), 0.63 (s, 3H).

[0031] S4: Preparation of Aramchol 2.50 g (6.1 mmol, M=407.3) of the 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid prepared in step S3 was added to 50 ml of DCM, followed by 2.30 g (7.36 mmol, M=312.54) of arachidic acid, 1.52 g (7.37 mmol, M=206.33) of DCC and 0.08 g (0.65 mmol, M=122.17) of DMAP. The reaction was carried out overnight at room temperature. After the reaction of 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid was complete, the reaction solution was flushed into 100 ml of water, and then extracted twice with DCM. The organic phases were combined, dried, concentrated, and purified by silica gel slab chromatography through a 200-mesh silica gel column to obtain 3.85 g of 3β-arachidoamino-7α,12α-dihydroxy-5β-cholan-24-acid white powder, with a yield of 89.5%.

[0032] H-NMR (400MHz, DMSO-d6): 12.06 (s, 1H), 7.54 (s, 1H), 4.11 (d, J=29.2 Hz, 2H), 3.83 (d, J=21.6Hz, 2H), 3.65 (s, 1H), 2.24-0.89 (m, 69H), 0.62 (s, 3H).

[0033] Example 2: Rapid Synthesis of Aramchol Preparation of S1: 3-Keto-7α,12α-Dihydroxy-5β-cholanic acid Weigh 30.00 g (73.5 mmol, M=408.3) of 3α,7α,12α-trihydroxy-5β-cholanic acid and add it to 700 mL of water. While stirring, add 1 M sodium hydroxide solution dropwise until CA is completely dissolved. Adjust the pH to 7.7–8.0 with hydrochloric acid. Then add 0.30 g of oxidized coenzyme I (NAD⁺), 15 mL of 3α-hydroxysteroid dehydrogenase (3α-HSDH) solution (0.30 g as solid enzyme), and 5 mL of NADH oxidase solution (0.20 g as solid enzyme). Make up the volume with water to 1 L. Start the reaction in a 30°C water bath with stirring for 4–6 h.

[0034] The solution after the above reaction was heated to 80℃ and kept at that temperature for 30 min, followed by filtration to remove the enzyme. The resulting filtrate was cooled to 0℃ and then filtered under vacuum. The filter cake was dried to obtain 29.53 g of 3-keto-7α,12α-dihydroxy-5β-cholanic acid white powder, with a yield of 98.4%.

[0035] H-NMR (400MHz, DMSO-d6): 11.94 (s, 1H), 4.34 (d, J=3.2Hz, 1H), 4.20 (d, J=2.8Hz, 1H), 3.84 (s, 1H), 3.70 (s, 1H), 3.45 (t, J=13.6Hz, 1H), 2.32-0.96 (m, 23H), 0.64 (s, 3H).

[0036] Preparation of S2: 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid At room temperature, 19.50 g (48 mmol, M=406.3) of 3-keto-7α,12α-dihydroxy-5β-cholanic acid prepared in step S1, 3.34 g (48 mmol, M=69.49) of hydroxylamine hydrochloride, and 2.69 g (48 mmol, M=56.1) of potassium hydroxide were added to 200 mL of ethanol. The mixture was heated to reflux and reacted for 4-5 h. After TLC detection showed that the reaction of 3-keto-7α,12α-dihydroxy-5β-cholanic acid was complete, the mixture was cooled to room temperature and filtered. The filter cake was washed with 60 mL of ethanol, and the filtrates were combined, concentrated, and dried to obtain 12.30 g of 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid yellow powder.

[0037] H-NMR (400MHz, DMSO-d6): 11.86 (δ, 1H), 9.90 (s, 1H), 4.23 (d, J=3.2 Hz, 1H), 4.14 (d, J=2.8 Hz, 1H), 3.80 (s, 1H), 3.66 (s, 1H), 2.87-2.70 (m, 2H), 2.25-0.86 (m, 28H), 0.61 (s, 3H).

[0038] S3: 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid 12.30 g (29.2 mmol, M=421.3) of 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid prepared in step S2 was added to 200 mL of methanol, followed by 1.15 g (1.4 mmol) of dichloro[(S)-(-)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl]ruthenium(II) ruthenium catalyst. After three hydrogen purgings, the reaction was carried out overnight at room temperature under hydrogen protection. TLC analysis showed that compound 2 was completely reacted. The reaction solution was directly filtered, and the filtrate was concentrated and dried to give 10.46 g of 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid yellow powder, with a yield of 88.4%.

[0039] H-NMR (400MHz, DMSO-d6): 12.06 (δ, 1H), 7.56 (s, 1H), 4.53 (d, J=4.2 Hz, 2H), 4.25 (d, J=3.6 Hz, 1H), 4.20 (d, J=3.2 Hz, 1H), 3.88 (s, 1H), 3.76 (s, 1H), 2.87-2.68 (m, 2H), 2.25-0.86 (m, 28H), 0.63 (s, 3H).

[0040] S4: Preparation of Aramchol 10.40 g (25.5 mmol, M=407.3) of the 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid prepared in step S3 was added to 200 ml of DCM, followed by 7.98 g (25.5 mmol, M=312.54) of arachidic acid, 5.27 g (25.5 mmol, M=206.33) of DCC and 0.66 g (5.4 mmol, M=122.17) of DMAP. The mixture was reacted overnight at room temperature. TLC was used to detect 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid. After the reaction was complete, the reaction solution was flushed into 400 ml of water, and then extracted twice with DCM. The organic phases were combined, dried, concentrated, and purified by silica gel slab chromatography on a 200 μm silica gel column to obtain 16.32 g of 3β-arachidoamino-7α,12α-dihydroxy-5β-cholan-24-acid white powder, with a yield of 91.2%.

[0041] H-NMR (400MHz, DMSO-d6): 12.06 (s, 1H), 7.54 (s, 1H), 4.11 (d, J=29.2 Hz, 2H), 3.83 (d, J=21.6Hz, 2H), 3.65 (s, 1H), 2.24-0.89 (m, 69H), 0.62 (s, 3H).

[0042] Example 3: Rapid Synthesis of Aramchol Preparation of S1: 3-Keto-7α,12α-Dihydroxy-5β-cholanic acid Weigh 30.00 g (73.5 mmol, M=408.3) of 3α,7α,12α-trihydroxy-5β-cholanic acid and add it to 700 mL of water. While stirring, add 1 M sodium hydroxide solution dropwise until CA is completely dissolved. Adjust the pH to 7.7–8.0 with hydrochloric acid. Then add 0.40 g of oxidized coenzyme I (NAD⁺), 15 mL of 3α-hydroxysteroid dehydrogenase (3α-HSDH) solution (0.30 g as solid enzyme), and 5 mL of NADH oxidase solution (0.20 g as solid enzyme). Make up the volume with water to 1 L. Start the reaction in a 30°C water bath with stirring for 4–6 h.

[0043] The solution after the above reaction was heated to 80℃ and kept at that temperature for 30 min, followed by filtration to remove the enzyme. The resulting filtrate was cooled to 0℃ and then filtered under vacuum. The filter cake was dried to obtain 28.53 g of 3-keto-7α,12α-dihydroxy-5β-cholanic acid white powder, with a yield of 95.1%.

[0044] H-NMR (400MHz, DMSO-d6): 11.94 (s, 1H), 4.34 (d, J=3.2Hz, 1H), 4.20 (d, J=2.8Hz, 1H), 3.84 (s, 1H), 3.70 (s, 1H), 3.45 (t, J=13.6Hz, 1H), 2.32-0.96 (m, 23H), 0.64 (s, 3H).

[0045] Preparation of S2: 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid At room temperature, 20.00 g (49.2 mmol, M=406.3) of 3-keto-7α,12α-dihydroxy-5β-cholanic acid prepared in step S1, 5.12 g (73.5 mmol, M=69.49) of hydroxylamine hydrochloride, and 4.12 g (73.5 mmol, M=56.1) of potassium hydroxide were added to 200 mL of ethanol. The mixture was heated to reflux and reacted for 4-5 h. After TLC detection showed that the reaction of 3-keto-7α,12α-dihydroxy-5β-cholanic acid was complete, the mixture was cooled to room temperature and filtered. The filter cake was washed with 60 mL of ethanol, and the filtrates were combined, concentrated, and dried to obtain 12.50 g of 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid yellow powder.

[0046] H-NMR (400MHz, DMSO-d6): 11.86 (δ, 1H), 9.90 (s, 1H), 4.23 (d, J=3.2 Hz, 1H), 4.14 (d, J=2.8 Hz, 1H), 3.80 (s, 1H), 3.66 (s, 1H), 2.87-2.70 (m, 2H), 2.25-0.86 (m, 28H), 0.61 (s, 3H).

[0047] S3: 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid 12.50 g (29.7 mmol, M=421.3) of 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid prepared in step S2 was added to 200 mL of methanol, followed by 2.43 g (2.96 mmol) of dichloro[(S)-(-)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl]ruthenium(II) ruthenium catalyst. After three hydrogen purgings, the reaction was carried out overnight at room temperature under hydrogen protection. TLC analysis showed that compound 2 was completely reacted. The reaction solution was directly filtered, and the filtrate was concentrated and dried to give 10.95 g of 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid yellow powder, with a yield of 92.5%.

[0048] H-NMR (400MHz, DMSO-d6): 12.06 (δ, 1H), 7.56 (s, 1H), 4.53 (d, J=4.2 Hz, 2H), 4.25 (d, J=3.6 Hz, 1H), 4.20 (d, J=3.2 Hz, 1H), 3.88 (s, 1H), 3.76 (s, 1H), 2.87-2.68 (m, 2H), 2.25-0.86 (m, 28H), 0.63 (s, 3H).

[0049] S4: Preparation of Aramchol 10.95 g (26.9 mmol, M=407.3) of the 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid prepared in step S3 was added to 200 ml of DCM, followed by 12.58 g (40.2 mmol, M=312.54) of arachidic acid, 8.27 g (40.2 mmol, M=206.33) of DCC and 0.99 g (8.0 mmol, M=122.17) of DMAP. The mixture was reacted overnight at room temperature. TLC was used to detect 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid. After the reaction was complete, the reaction solution was flushed into 400 ml of water, and then extracted twice with DCM. The organic phases were combined, dried, concentrated, and purified by silica gel slab chromatography through a 200-mesh silica gel column to obtain 16.32 g of 3β-arachidoamino-7α,12α-dihydroxy-5β-cholan-24-acid white powder, with a yield of 91.2%.

[0050] H-NMR (400MHz, DMSO-d6): 12.06 (s, 1H), 7.54 (s, 1H), 4.11 (d, J=29.2 Hz, 2H), 3.83 (d, J=21.6Hz, 2H), 3.65 (s, 1H), 2.24-0.89 (m, 69H), 0.62 (s, 3H).

[0051] Example 4: Rapid Synthesis of Aramchol Preparation of S1: 3-Keto-7α,12α-Dihydroxy-5β-cholanic acid Weigh 30.00 g (73.5 mmol, M=408.3) of 3α,7α,12α-trihydroxy-5β-cholanic acid and add it to 700 mL of water. While stirring, add 1 M sodium hydroxide solution dropwise until CA is completely dissolved. Adjust the pH to 7.7–8.0 with hydrochloric acid. Then add 0.40 g of oxidized coenzyme I (NAD⁺), 15 mL of 3α-hydroxysteroid dehydrogenase (3α-HSDH) solution (0.30 g as solid enzyme), and 5 mL of NADH oxidase solution (0.20 g as solid enzyme). Make up the volume with water to 1 L. Start the reaction in a 30°C water bath with stirring for 4–6 h.

[0052] The solution after the above reaction was heated to 80℃ and kept at that temperature for 30 min, followed by filtration to remove the enzyme. The resulting filtrate was cooled to 0℃ and then filtered under vacuum. The filter cake was dried to obtain 29.0 g of 3-keto-7α,12α-dihydroxy-5β-cholanic acid white powder, with a yield of 95.6%.

[0053] H-NMR (400MHz, DMSO-d6): 11.94 (s, 1H), 4.34 (d, J=3.2Hz, 1H), 4.20 (d, J=2.8Hz, 1H), 3.84 (s, 1H), 3.70 (s, 1H), 3.45 (t, J=13.6Hz, 1H), 2.32-0.96 (m, 23H), 0.64 (s, 3H).

[0054] Preparation of S2: 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid At room temperature, 28.85 g (71.0 mmol, M=406.3) of 3-keto-7α,12α-dihydroxy-5β-cholanic acid prepared in step S1, 4.93 g (71.0 mmol, M=69.49) of hydroxylamine hydrochloride, and 3.98 g (71.0 mmol, M=56.1) of potassium hydroxide were added to 200 mL of ethanol. The mixture was heated to reflux and reacted for 4-5 h. After TLC detection showed that the reaction of 3-keto-7α,12α-dihydroxy-5β-cholanic acid was complete, the mixture was cooled to room temperature and filtered. The filter cake was washed with 60 mL of ethanol, and the filtrates were combined, concentrated, and dried to obtain 19.23 g of 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid yellow powder.

[0055] H-NMR (400MHz, DMSO-d6): 11.86 (δ, 1H), 9.90 (s, 1H), 4.23 (d, J=3.2 Hz, 1H), 4.14 (d, J=2.8 Hz, 1H), 3.80 (s, 1H), 3.66 (s, 1H), 2.87-2.70 (m, 2H), 2.25-0.86 (m, 28H), 0.61 (s, 3H).

[0056] S3: 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid 19.23 g (45.6 mmol, M=421.3) of 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid prepared in step S2 was added to 300 mL of methanol, followed by 1.43 g (2.27 mmol, M=631.2) of (S,S)-N-(p-toluenesulfonyl)-1,2-diphenylethanediamine (p-isopropyltoluene)ruthenium chloride. After three purgings with hydrogen, the reaction was carried out overnight at room temperature under hydrogen protection. TLC analysis showed that compound 2 was completely reacted. The reaction solution was directly filtered, and the filtrate was concentrated and dried to give 17.20 g of 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid yellow powder, with a yield of 94.4%.

[0057] S4: Preparation of Aramchol 17.20 g (42.2 mmol, M=407.3) of the 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid prepared in step S3 was added to 200 ml of DCM, followed by 15.81 g (50.6 mmol, M=312.54) of arachidic acid, 10.39 g (50.4 mmol, M=206.33) of DCC and 1.04 g (8.5 mmol, M=122.17) of DMAP. The mixture was reacted overnight at room temperature. TLC was used to detect 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid. After the reaction was complete, the reaction solution was flushed into 400 ml of water, and then extracted twice with DCM. The organic phases were combined, dried, concentrated, and purified by silica gel slab chromatography through a 200-mesh silica gel column to obtain 25.50 g of 3β-arachidoamino-7α,12α-dihydroxy-5β-cholan-24-acid white powder, with a yield of 90.7%.

[0058] H-NMR (400MHz, DMSO-d6): 12.06 (s, 1H), 7.54 (s, 1H), 4.11 (d, J=29.2 Hz, 2H), 3.83 (d, J=21.6Hz, 2H), 3.65 (s, 1H), 2.24-0.89 (m, 69H), 0.62 (s, 3H).

[0059] In summary, the rapid synthesis method for Aramchol of this invention utilizes the conversion of a carbonyl group to an amino group throughout the reaction. A metal catalyst is used during reduction, allowing for selective reduction to a chiral amino group. The yield of 3β-arachidoamino-7α,12α-dihydroxy-5β-cholan-24-acid is over 89%, and the reaction is simple, highly efficient, and reproducible.

[0060] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for rapid synthesis of Aramchol, characterized in that... The process includes the following steps: First, 3-ketocholic acid is synthesized from cholic acid under the action of an enzyme. Then, 3-oximecholic acid is synthesized from 3-ketocholic acid under the action of hydroxylamine hydrochloride. 3-oximecholic acid is reduced to 3-aminocholic acid with corresponding chirality under a chiral ruthenium catalyst. After that, it is condensed with arachidic acid to prepare 3β-arachidoamino-7α,12α-dihydroxy-5β-cholan-24-acid, namely Aramchol.

2. The enzyme mentioned is oxidized coenzyme I (NAD). + The mixture comprises 3α-hydroxysteroid dehydrogenase (3α-HSDH) and NADH oxidase; wherein the cholic acid is 3α,7α,12α-trihydroxy-5β-cholanic acid, the 3-ketocholic acid is 3-keto-7α,12α-dihydroxy-5β-cholanic acid, the 3-oximecholic acid is 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid, and the 3-aminocholic acid is 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid.

3. The method for rapid synthesis of Aramchol according to claim 1, characterized in that... Includes the following steps: Preparation of S1: 3-Keto-7α,12α-Dihydroxy-5β-cholanic acid Add the specified amount of 3α,7α,12α-trihydroxy-5β-cholanic acid to water, and add sodium hydroxide solution dropwise while stirring until all 3α,7α,12α-trihydroxy-5β-cholanic acid is dissolved. Adjust the pH with hydrochloric acid. Then, add oxidized coenzyme I, 3α-hydroxysteroid dehydrogenase (3α-HSDH), and NADH oxidase in sequence. Stir the reaction at 25-35℃ for 4-6 hours. Heat the reaction solution to 80℃ and keep it at that temperature for 30 minutes. Then filter to remove the enzymes. Cool the filtrate, filter and dry it to obtain white 3-keto-7α,12α-dihydroxy-5β-cholanic acid powder for later use. Preparation of S2: 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid At room temperature, the 3-keto-7α,12α-dihydroxy-5β-cholanic acid, hydroxylamine hydrochloride, and potassium hydroxide prepared in step S1 were added to ethanol. The mixture was heated to reflux and reacted for 5 hours. After the reaction of 3-keto-7α,12α-dihydroxy-5β-cholanic acid was detected by thin-layer chromatography and the mixture was cooled to room temperature, filtered, and the filter cake was washed with ethanol. The filtrates were combined, concentrated, and dried to obtain a yellow 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid powder for later use. Preparation of S3: 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid The 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid prepared in step S2 was added to methanol in the specified proportions. Then, a ruthenium catalyst was added, and the mixture was purged with hydrogen three times. The reaction was carried out overnight at room temperature under hydrogen protection. Thin-layer chromatography was used to detect the complete reaction of 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid. The reaction solution was directly filtered, and the filtrate was concentrated and dried to obtain a yellow powder of 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid for later use. S4: Preparation of Aramchol The 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid prepared in step S3 was added to dichloromethane solvent, followed by the addition of arachidic acid, N,N'-dicyclohexylcarbodiimide, and dimethylaminopyridine. The reaction was carried out overnight at room temperature. After the complete reaction of 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid was detected by thin-layer chromatography, the reaction solution was flushed into water, and then extracted twice with dichloromethane. The organic phases were combined, dried, concentrated, and purified by silica gel plate chromatography to obtain a white 3β-arachidoamino-7α,12α-dihydroxy-5β-cholan-24-acid powder.

4. A method for rapid synthesis of Aramchol according to any one of claims 1 or 2, characterized in that: The ruthenium catalyst is either dichloro[(S)-(-)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl]ruthenium(II) or (S,S)-N-(p-toluenesulfonyl)-1,2-diphenylethanediamine(p-isopropyltoluene)ruthenium chloride.

5. A method for rapid synthesis of Aramchol according to claim 2, characterized in that: The 3α,7α,12α-trihydroxy-5β-cholanoic acid and oxidized coenzyme I (NAD) described in step S1 + The mass ratio of the two components is 1:0.006~0.012; the amount of 3α-hydroxysteroid dehydrogenase (3α-HSDH) added is 0.01~0.02 g per gram of substrate; the amount of NADH oxidase added is 0.005~0.01 g per gram of substrate.

6. A method for rapid synthesis of Aramchol according to any one of claims 2 or 4, characterized in that: The mass ratio of oxidized coenzyme I, 3α-hydroxysteroid dehydrogenase (3α-HSDH), and NADH oxidase in step S1 is 1:1:

1.

7. The method for rapid synthesis of Aramchol according to claim 2, characterized in that: The pH is adjusted to 7.7-8.0 using hydrochloric acid as described in step S1.

8. The method for rapid synthesis of Aramchol according to claim 2, characterized in that: The molar ratio of 3-keto-7α,12α-dihydroxy-5β-cholanic acid, hydroxylamine hydrochloride and potassium hydroxide in step S2 is 1.0:1.0~1.5:1.0~1.

5.

9. A method for rapid synthesis of Aramchol according to claim 2, characterized in that: The molar ratio of 7α,12α-dihydroxy-3-oxime-5β-cholan-24-acid and ruthenium catalyst in step S3 is 1.0: 0.04~0.

1.

10. The method for rapid synthesis of Aramchol according to claim 2, characterized in that: The molar ratio of 3β-amino-7α,12α-dihydroxy-5β-cholan-24-acid, arachidic acid, N,N'-dicyclohexylcarbodiimide and dimethylaminopyridine in step S4 is 1.0:1.0~1.5:1.0~1.5:0.1~0.3.

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