Preparation method of cholic acid derivative

Abstract

The invention belongs to the technical field of biomedicine synthesis, and particularly relates to the technical field of chemical synthesis of natural drugs / prodrugs for controlling metabolic abnormalities such as blood sugar and blood fat. The invention provides a preparation method of a cholic acid derivative. The preparation method comprises the following steps: adding benzyl bromide into an alkaline solution containing cholic acid to obtain benzyl cholate; the method comprises the following steps: adding a dichloromethane solution containing acyl chloride into a dichloromethane solution containing benzyl cholate and 4-dimethylaminopyridine under protective gas to obtain a colorless oily product; and introducing hydrogen into the absolute ethyl alcohol in which the colorless oily product is dissolved to obtain the cholic acid derivative. The chemical synthesis of the cholic acid derivative is realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical synthesis technology, specifically relating to the chemical synthesis technology of natural drugs / produce for controlling metabolic abnormalities such as blood sugar and blood lipids. Background Technology

[0002] Cholic acid is an organic compound with the molecular formula C6H2O. 24 H 40 O5, found in the bile of mammals such as cattle, sheep, and pigs, is a colorless flaky substance or a white crystalline powder. It is used in biochemical research and as a pharmaceutical intermediate. Sodium cholate is a choleretic drug used to treat cholecystitis, bile deficiency, and indigestion.

[0003] There is an important interaction between bile acids and gut microbiota. Gut microbiota is the microbial community living in the human gut, including bacteria, fungi, viruses, etc. These microorganisms play a key role in the metabolism and circulation of bile acids. The following are some key points about the relationship between bile acids and gut microbiota: 1) Bile acid metabolism: Gut microbiota participate in the metabolism of bile acids. The main mechanisms of bile acid metabolism are as follows: 1) Through the action of a series of enzymes, microorganisms can convert bile acids in bile into derivatives such as hypocholic acid. This metabolic activity occurs in the colon and is called bile acid redox reaction; 2) Formation of hypocholic acid: Microorganisms can convert bile acids, which are mainly present in bile, into hypocholic acid. This conversion changes the biological activity of bile acids and affects their absorption and recycling in the intestine; 3) Recycling of bile salts: Hypocholic acid produced by microbial-mediated bile acid metabolism is absorbed into the blood, returns to the liver, and is secreted back into bile, forming bile salt recycling. This process is crucial for the homeostatic cycling of bile salts and the absorption of intestinal lipids; 4) Effects on energy balance: The interaction between bile acid metabolism and microorganisms may affect the host's energy balance. Studies have shown that bile acid metabolism mediated by microorganisms may be related to energy metabolism and weight regulation; 5) Influence on metabolic diseases: The interaction between gut microbiota and bile acids may be related to the occurrence and development of metabolic diseases. For example, in the study of non-alcoholic fatty liver disease, the interaction between bile acids and microorganisms has become a field of great interest.

[0004] Overall, the interaction between bile acids and gut microbiota has significant biological and medical implications for bile acid metabolism, bile salt cycling, energy balance, and the understanding of some metabolic diseases. Research in this area will contribute to a deeper understanding of the regulatory role of gut microbiota in host physiological processes.

[0005] Obeticholic acid, developed by Intercept Pharmaceuticals in the United States, is the first drug developed in twenty years for the treatment of cholestatic liver disease. Obeticholic acid is a farnesoid X receptor agonist, indirectly inhibiting the gene expression of cytochrome 7A1 (CYP7A1) by activating the farnesoid X receptor. Since CYP7A1 is the rate-limiting enzyme in bile acid biosynthesis, obeticholic acid can inhibit bile acid synthesis and is used to treat primary biliary cirrhosis and non-alcoholic fatty liver disease.

[0006] FXR agonists can alleviate cholestasis by stimulating the bile salt output pump (BSEP) to increase bile acid-dependent bile flow and by stimulating MRP2 to increase non-bile acid-dependent bile flow. FXR holds promise as a novel drug target for screening treatments of cholestatic diseases and other metabolic disorders, including non-alcoholic steatohepatitis. Furthermore, current research suggests that FXR agonists may have therapeutic and research value in diseases such as atherosclerosis, cholestatic diseases caused by bile acid disorders, liver fibrosis, cirrhosis, and cancer. Summary of the Invention

[0007] This invention provides 3-acylated bile acid derivatives isolated from human intestinal microorganisms. These novel bile acid derivatives play a crucial role in regulating intestinal flora and controlling overall health. Therefore, there is an urgent need to obtain 3-acylated products with different substitutions through chemical synthesis.

[0008] This invention discloses a synthetic route for cholic acid acylated derivatives, which have the following chemical structural formula:

[0009]

[0010] R=C n H 2n-1 , where n is from 1 to 17.

[0011] The synthetic route is shown below. It is a general synthetic route for the derivative modified by the 3-position acylation of cholic acid, which involves three chemical transformations.

[0012] First, the carboxyl group of cholic acid is subjected to an affinity substitution reaction under alkaline conditions of potassium carbonate to synthesize the corresponding benzyl cholic acid intermediate. Then, it is acylated by different acyl chlorides. Finally, the benzyl protecting group is removed under the action of palladium carbon hydrogen to obtain the target product of 3-position cholic acid acylation.

[0013]

[0014] This invention also provides a method for the chemical synthesis of bile acid derivatives, comprising the following steps:

[0015] 1) At low temperature, a certain amount of cholic acid and potassium carbonate were added sequentially to a round-bottom flask equipped with a magnetic stir bar, dissolved in anhydrous dimethylformamide, and stirred on a magnetic stirrer. Benzyl bromide was slowly added dropwise to the above solution, and the reaction mixture was stirred at room temperature. The reaction progress was monitored by thin-plate chromatography. After the reactants had completely reacted, a certain amount of dilute hydrochloric acid was added to quench the reaction. The reaction was then diluted and extracted with dichloromethane. The organic phase was washed sequentially with saturated sodium bicarbonate solution and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, and the mixture was concentrated under reduced pressure. The crude mixture was purified by silica gel combined rapid column chromatography to obtain the benzyl cholate intermediate.

[0016] 2) Under argon protection, a certain amount of benzyl cholate and 4-dimethylaminopyridine were added sequentially to a round-bottom flask equipped with a magnetic stirrer and dissolved in anhydrous dichloromethane. The mixture was stirred on a magnetic stirrer. The reaction solution was cooled to a low temperature, and the corresponding acyl chloride was slowly added dropwise to the solution, followed by the slow addition of triethylamine. The reaction mixture was stirred at room temperature, and the reaction progress was monitored by thin-plate chromatography. After the reactants had reacted completely, an aqueous acetic acid solution was added to quench the reaction. The reaction was then diluted and extracted with dichloromethane. The organic phase was washed sequentially with saturated sodium bicarbonate solution and saturated brine. The combined organic layers were dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude product was purified by rapid column chromatography to obtain a colorless oily product.

[0017] 3) Under argon protection, the target product was dissolved in anhydrous ethanol, and 10% by weight of palladium on carbon was added. The atmosphere was then replaced with hydrogen, and the reaction was allowed to proceed for approximately 1 hour. After the reaction was complete, the palladium on carbon was removed by filtration through a Buchner funnel, the mixture was washed with anhydrous ethanol, concentrated under reduced pressure, and evaporated to dryness. The crude mixture was then purified by silica gel combined rapid column chromatography to obtain the cholic acid acylated derivative. Attached Figure Description

[0018] Figure 1 This is the hydrogen spectrum of the compound in Example 1.

[0019] Figure 2 This is the carbon spectrum of the compound in Example 1.

[0020] Figure 3 This is the hydrogen spectrum of the compound in Example 2.

[0021] Figure 4 This is the carbon spectrum of the compound in Example 2.

[0022] Figure 5 This is the hydrogen spectrum of the compound in Example 3.

[0023] Figure 6 This is the carbon spectrum of the compound in Example 3.

[0024] Figure 7 This is the hydrogen spectrum of the compound in Example 4.

[0025] Figure 8 This is the carbon spectrum of the compound in Example 4.

[0026] Figure 9 This is the hydrogen spectrum of the compound in Example 5.

[0027] Figure 10 This is the carbon spectrum of the compound in Example 5. Detailed Implementation

[0028] The following examples are helpful in understanding the present invention, but are not limited to the content of the present invention.

[0029] Example 1

[0030]

[0031] At 0 °C, benzyl bromide (41.8 mg, 0.245 mmol) was added dropwise to a dimethylformamide (1 mL) solution containing a suspension of cholic acid (50.0 mg, 0.122 mmol) and potassium carbonate (33.8 mg, 0.245 mmol). The reaction mixture was stirred at room temperature for 12 hours. The mixture was diluted with dichloromethane (10 mL), washed with saturated sodium bicarbonate solution (2 × 5 mL) and 2N HCl (2.5 mL), and the organic layer was dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure. The crude mixture was purified by rapid column chromatography to obtain the benzyl cholate intermediate.

[0032] Under argon protection and at 0°C, acetyl chloride (0.122 mmol) was dissolved in dichloromethane (2.0 mL) and slowly added dropwise to a dichloromethane (40 mL) solution of benzyl cholate and 4-dimethylaminopyridine (17.7 mg, 0.15 mmol). The mixture was then stirred and heated to ambient temperature. After 3 hours, 250 mL of AcOH aqueous solution (1.2%) was added to wash the aqueous layer and dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude product was purified by rapid column chromatography to obtain a colorless oily product.

[0033] Under argon protection, the above colorless oily product was dissolved in anhydrous ethanol (5 mL), and approximately 20 mg of 10 wt% Pd / C was added. The atmosphere was then replaced with hydrogen, and the reaction was allowed to proceed for approximately 1 hour. After the reaction was complete, the palladium on carbon was removed by filtration, and the mixture was dried under vacuum. The crude mixture was purified by rapid column chromatography to obtain 13.7 mg of the 3-acetylated bile acid derivative, with an overall yield of approximately 25%.

[0034] The characterization data of the obtained products are as follows:

[0035] 1H NMR (500MHz, CDCl3) δ4.57(ddt,J=11.3,6.7,4.3Hz,1H),4.01(t,J=3.0Hz,1H),3.87(q,J=3.0Hz,1H),2.41(dd,J=10.6,5.2Hz,1H),2.38–2.1 7(m,3H),2.10–1.87(m,6H),1.87–1.37(m,14H),1.37–1.23(m,1H),1.1 4(dd,J=12.2,6.0Hz,1H),1.09–0.96(m,4H),0.91(s,3H),0.70(s,3H).

[0036] 13 C NMR (126MHz, CDCl3) δ179.0,170.9,74.3,73.0,68.4,47.0,46.5,41.9,41.1,39.5,35 .2,35.1,34.9,34.7,34.3,30.9,30.7,28.2,27.5,26.6,23.1,22.5,21.5,17.3,12.5.

[0037] Example 2

[0038]

[0039] At 0 °C, benzyl bromide (41.8 mg, 0.245 mmol) was added dropwise to a dimethylformamide (1 mL) solution containing a suspension of cholic acid (50.0 mg, 0.122 mmol) and potassium carbonate (33.8 mg, 0.245 mmol). The reaction mixture was stirred at room temperature for 12 hours. The mixture was diluted with dichloromethane (10 mL), washed with saturated sodium bicarbonate solution (2 × 5 mL) and 2N HCl (2.5 mL), and the organic layer was dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure. The crude mixture was purified by rapid column chromatography to obtain the benzyl cholate intermediate.

[0040] Under argon protection and at 0°C, n-propionyl chloride (0.12 mmol) dissolved in dichloromethane (2.0 mL) was slowly added dropwise to a dichloromethane (40 mL) solution of benzyl cholate and 4-dimethylaminopyridine (17.7 mg, 0.15 mmol). The mixture was then stirred and heated to ambient temperature. After 3 hours, 250 mL of AcOH aqueous solution (1.2%) was added to wash the aqueous layer and dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude product was purified by rapid column chromatography to obtain a colorless oily product.

[0041] Under argon protection, the target product was dissolved in anhydrous ethanol (5 mL), and approximately 20 mg of 10 wt% Pd / C was added. The atmosphere was then replaced with hydrogen, and the reaction was allowed to proceed for approximately 1 hour. After the reaction was complete, palladium on carbon was removed by filtration, and the mixture was dried under vacuum. The crude mixture was purified by rapid column chromatography to obtain 11.9 mg of the 3-propionylcholine derivative, with an overall yield of approximately 21%.

[0042] The characterization data of the obtained products are as follows:

[0043] 1 H NMR (500MHz, CDCl3) δ4.59 (td, J=11.3, 5.6Hz, 1H), 4.00 (t, J=3.0Hz, 1H), 3.86 (d, J=3.2Hz,1H),2.43(ddd,J=15.2,9.8,4.9Hz,2H),2.32–2.24(m,6H),1.99–1.88(m, 3H),1.85–1.72(m,4H),1.68(t,J=8.9Hz,2H),1.65–1.41(m,8H),1.19(dd,J=12.2, 5.9Hz, 1H), 1.12 (t, J = 7.6Hz, 3H), 1.00 (d, J = 6.3Hz, 3H), 0.91 (s, 3H), 0.71 (s, 3H).

[0044] 13 C NMR (126MHz, CDCl3) δ178.4,174.4,74.2,73.1,68.5,47.3,46.7,42.2,41.3,39.7,35.4,35.3 ,35.3,35.0,34.8,34.5,30.9,30.8,28.5,28.1,27.6,26.9,26.8,23.3,22.7,17.4,12.7,9.3.

[0045] Example 3

[0046]

[0047] At 0 °C, benzyl bromide (41.8 mg, 0.245 mmol) was added dropwise to a solution of anhydrous dimethylformamide (1 mL) containing a suspension of cholic acid (50.0 mg, 0.122 mmol) and potassium carbonate (33.8 mg, 0.245 mmol). The reaction mixture was stirred at room temperature for 12 hours. The mixture was diluted with dichloromethane (2 mL), washed with saturated sodium bicarbonate solution (2 × 5 mL) and 2N HCl (2.5 mL), and the organic layer was dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure. The crude mixture was purified by rapid column chromatography to obtain the benzyl cholate intermediate.

[0048] Under argon protection, n-butyryl chloride (0.12 mmol) dissolved in dichloromethane (2.0 mL) was slowly added dropwise to a dichloromethane (40 mL) solution of benzyl cholate (60 mg, 0.12 mmol) and 4-dimethylaminopyridine (17.7 mg, 0.15 mmol). The mixture was then stirred and heated to ambient temperature. After 3 hours, the aqueous layer and dichloromethane were washed with 250 mL of AcOH aqueous solution (1.2%). The combined organic layers were dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude product was purified by rapid column chromatography to obtain a colorless oily product.

[0049] Under argon protection, the target product was dissolved in anhydrous ethanol (5 mL), and approximately 20 mg of 10% Pd / C was added. The atmosphere was then replaced with hydrogen, and the reaction was allowed to proceed for approximately 1 hour. After the reaction was complete, palladium on carbon was removed by filtration, and the mixture was dried under vacuum. The crude mixture was purified by rapid column chromatography to obtain 16.3 mg of the 3-butyrylated cholic acid derivative, with an overall yield of approximately 28%.

[0050] The characterization data of the obtained products are as follows:

[0051] 1 H NMR (500MHz, CDCl3) δ4.69–4.53(m,1H),4.00(t,J=3.0Hz,1H),3.87(q,J=3.0Hz,1H),2.40(dd,J=10.1,4.8Hz,1H),2.37–2.16(m,5H) ,2.03–1.88(m,3H),1.88–1.26(m,18H),1.22–1.11(m,1H),1.11–1.03(m,1H),1.00(d,J=6.1Hz,3H),0.97–0.88(m,6H),0.70(s,3H).

[0052] 13 C NMR (126MHz, CDCl3) δ179.0,173.4,74.0,73.0,68.4,47.1,46.511,42.0,41.2,39.5,36.6,35.2 ,35.2,34.9,34.7,34.4,30.9,30.7,28.3,27.5,26.7,26.6,23.1,22.5,18.5,17.3,13.7,12.5.

[0053] Example 4

[0054]

[0055] At 0 °C, benzyl bromide (41.8 mg, 0.245 mmol) was added dropwise to a solution of anhydrous dimethylformamide (1 mL) containing a suspension of cholic acid (50.0 mg, 0.122 mmol) and potassium carbonate (33.8 mg, 0.245 mmol). The reaction mixture was stirred at room temperature for 12 hours. The mixture was diluted with dichloromethane (2 mL), washed with saturated sodium bicarbonate solution (2 × 5 mL) and 2N HCl (2.5 mL), and the organic layer was dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure. The crude mixture was purified by rapid column chromatography to obtain the benzyl cholate intermediate.

[0056] Under argon protection, valeryl chloride (0.12 mmol) dissolved in dichloromethane (2.0 mL) was slowly added dropwise to a dichloromethane solution of benzyl cholate (60 mg, 0.12 mmol) and 4-dimethylaminopyridine (17.7 mg, 0.15 mmol). The mixture was then stirred and heated to ambient temperature. After 3 hours, the aqueous layer and dichloromethane were washed with 250 mL of AcOH aqueous solution (1.2%). The combined organic layers were dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude product was purified by rapid column chromatography to obtain a colorless oily product.

[0057] Under argon protection, the target product was dissolved in anhydrous ethanol (5 mL), and approximately 20 mg of 10 wt% Pd / C was added. The atmosphere was then replaced with hydrogen, and the reaction was allowed to proceed for approximately 1 hour. After the reaction was complete, palladium on carbon was removed by filtration, and the mixture was dried under vacuum. The crude mixture was purified by rapid column chromatography to obtain 10.8 mg of the 3-pentanoylcholine derivative, with an overall yield of approximately 18%.

[0058] The characterization data of the obtained products are as follows:

[0059] 1 H NMR (500MHz, CDCl3) δ4.58(ddd,J=11.3,6.9,4.5Hz,1H),4.01(d,J=3.1Hz,1H),3.87(t,J=3.1Hz,1H),2.47–2.16(m,5H),2.01–1.87(m,2H),1.87– 1.38(m,15H),1.38–1.23(m,5H),1.15(qd,J=11.9,5.8Hz,1H),1.06(td,J =14.2,3.2Hz,1H),1.00(d,J=6.2Hz,3H),0.96–0.84(m,6H),0.70(s,3H).

[0060] 13C NMR (126MHz, CDCl3) δ179.0,173.7,74.0,73.1,68.4,47.1,46.5,42.0,41.1,39.5,35.2,35.7, 34.9,34.7,34.5,34.4,30.8,30.7,28.3,27.4,27.1,26.7,23.1,22.5,22.3,17.3,13.7,12.5.

[0061] Example 5

[0062]

[0063] At 0 °C, benzyl bromide (41.8 mg, 0.245 mmol) was slowly added dropwise to a suspension of cholic acid (50.0 mg, 0.122 mmol) and potassium carbonate (33.8 mg, 0.245 mmol) in anhydrous dimethylformamide (1 mL). The reaction mixture was stirred at room temperature for 12 hours. The mixture was then diluted with dichloromethane (2 mL) and washed with saturated sodium bicarbonate solution (2 × 5 mL) and 2N hydrochloric acid (2.5 mL). The washed organic layer was dried over anhydrous sodium sulfate, and the mixture was concentrated under reduced pressure. The crude benzyl cholate intermediate was purified by silica gel rapid column chromatography.

[0064] Under argon protection, 0.12 mmol of n-octadecyl chloride was dissolved in 2.0 mL of dichloromethane and slowly added dropwise to a 40 mL solution of benzyl cholate and 4-dimethylaminopyridine (17.7 mg, 0.15 mmol) in dichloromethane. The mixture was stirred and gradually heated to room temperature for 3 hours. The reaction mixture was then poured into 250 mL of 1.2% aqueous acetic acid solution, and the aqueous layer was washed with dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate, and the organic solvent was evaporated under reduced pressure. The crude product was purified by rapid column chromatography to obtain a colorless oil.

[0065] Under argon protection, the above product was dissolved in anhydrous ethanol (5 mL), and 10% palladium on carbon catalyst (approximately 20 mg) was added. The argon gas was replaced with hydrogen, and the reaction was allowed to proceed for approximately 1 hour. After the reaction was complete, the palladium on carbon was removed by filtration, and the product was dried under vacuum. The crude product was finally purified by silica gel rapid column chromatography to obtain 12.4 mg of the 3-cholic acid n-octadecylyl derivative, with an overall yield of approximately 15%.

[0066] The characterization data of the obtained products are as follows: 1H NMR(400MHz,CDCl3)δ4.58(ddd,J=11.5,6.9,4.5Hz,1H),4.06–3.95(m,1H),3.86(d,J=3.0Hz,1H),2.43–2.17(m,6H),1.98–1.42(m,22H),1.25(s,26H),1.20–1.07(m,2H),1.00(d,J=6.2Hz,3H),0.93–0.84(m,6H),0.70(s,3H). 13 C NMR(101 MHz,CDCl3)δ179.2,173.5,74.1,73.2,68.4,47.0,46.4,41.8,41.1,39.3,35.3,35.1,34.9,34.8,34.7,34.4,31.9,31.0,30.6,29.6,29.6,29.6,29.4,29.3,29.3,29.2,28.0,27.5,26.7,26.4,25.0,23.1,22.6,22.3,17.2,14.1,12.4。

Claims

1. A method for preparing bile acid derivatives, characterized in that, Includes the following steps: Benzyl bromide is added to an alkaline solution containing cholic acid to obtain benzyl cholate; Under a protective atmosphere, a dichloromethane solution containing acyl chloride is added to a dichloromethane solution containing benzyl cholate and 4-dimethylaminopyridine to obtain a colorless oily product. Hydrogen gas is passed through anhydrous ethanol containing a colorless oily product to obtain a cholic acid derivative.

2. The method for preparing the bile acid derivative according to claim 1, characterized in that, The bile acid derivative It has the following chemical structural formula: R=C n H 2n-1 , where n is from 1 to 17.

3. The method for preparing the cholic acid derivative according to claim 2, characterized in that, The alkaline solution refers to a dimethylformamide solution containing alkalis such as potassium carbonate.

4. The method for preparing the cholic acid derivative according to claim 2, characterized in that, The acyl chloride is acetyl chloride, propionyl chloride, butyryl chloride, valeryl chloride, hexanoyl chloride, dodecyl chloride, or octadecyl chloride.

5. The method for preparing the bile acid derivative according to claim 2, characterized in that, The benzyl bromide is added to an alkaline solution containing cholic acid by dropwise addition.

6. The method for preparing the bile acid derivative according to claim 2, characterized in that, The benzyl bromide was added to the alkaline solution containing cholic acid at a temperature of 0°C.

7. The method for preparing the cholic acid derivative according to claim 2, characterized in that, The dichloromethane solution containing acyl chloride is added to the dichloromethane solution containing benzyl cholate and 4-dimethylaminopyridine, wherein the addition is made by slow dropwise addition.

8. The method for preparing the cholic acid derivative according to claim 3, characterized in that, The addition of benzyl bromide to an alkaline solution containing cholic acid includes adding benzyl bromide dropwise to an anhydrous dimethylformamide solution containing a cholic acid suspension and potassium carbonate, and stirring at room temperature for 12 hours.

9. The method for preparing the cholic acid derivative according to claim 2, characterized in that, Under a protective atmosphere, a dichloromethane solution containing acyl chloride is added to a dichloromethane solution containing benzyl cholate and 4-dimethylaminopyridine. This includes dissolving the acyl chloride in dichloromethane and slowly adding it dropwise to the dichloromethane solution containing benzyl cholate and 4-dimethylaminopyridine, then stirring the mixture and heating it to ambient temperature for 3 hours.

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