Synthesis method of n-chenodeoxycholyl-l-aspartic acid and application thereof in preparation of medicine for improving metabolic related fatty liver disease

Abstract

The application discloses a synthesis method of N-chenodeoxycholyl-L-aspartic acid and application of the N-chenodeoxycholyl-L-aspartic acid in preparation of a medicine for improving metabolic associated fatty liver disease. The method provided by the application uses chenodeoxycholic acid and L-aspartic acid dimethyl ester hydrochloride as raw materials, synthesizes a chenodeoxycholic acid-aspartic acid dimethyl ester intermediate, and efficiently prepares a target compound, N-chenodeoxycholyl-L-aspartic acid, which is a novel structure, the compound exhibits a treatment effect superior to that of obeticholic acid (OCA) in an animal model, and can effectively improve multiple indexes such as liver steatosis, inflammation and fibrosis. The application provides a novel solution for major clinical challenges such as insufficient treatment effect, large side effect and rebound after drug withdrawal of current MAFLD treatment medicines, and provides a candidate compound with broad prospects for developing a new high-efficiency MAFLD treatment medicine.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for synthesizing N-chenodeoxychoyl-L-aspartic acid and its application in the preparation of drugs to improve metabolic-related fatty liver disease. Background Technology

[0002] Metabolic fatty liver disease (MAFLD) affects approximately 30% of the global population. Its progressive form, metabolic steatohepatitis (MASH), can lead to liver fibrosis, cirrhosis, and even liver cancer. It is estimated that the number of MASH patients worldwide will reach 490 million by 2030. Currently, treatment options are limited. Only two drugs have been approved by the FDA (Food and Drug Administration) for the treatment of MASH: First, Resmetirom, approved in March 2024, was the world's first drug approved for treating adult MASH patients with moderate to advanced liver fibrosis. Although its therapeutic effect is statistically and clinically significant, over 70% of patients still fail to achieve the primary goal of MASH remission or fibrosis improvement after 52 weeks of treatment. Second, semaglutide, a GLP-1 receptor agonist approved in August 2025, is used to treat adult MASH patients with moderate to severe liver fibrosis, becoming the first approved GLP-1 drug in this field. However, semaglutide has significant gastrointestinal side effects (such as nausea, vomiting, and diarrhea), which may lead some patients to discontinue use. Its mechanism of action focuses more on metabolic regulation and weight loss, with a relatively indirect and individual-variable effect on direct anti-fibrotic effects. Furthermore, there is a risk of disease rebound after discontinuation, requiring long-term or even lifelong medication, posing challenges to patient adherence and healthcare costs.

[0003] Bile acid drugs, especially farnesoid X receptor (FXR) agonists, are among the most promising and clinically advanced targets in the development of new drugs for MASH. Their core mechanism utilizes bile acids as natural signaling molecules to regulate systemic metabolism and inflammation by activating FXR. Activation of FXR receptors in the liver and intestines can improve insulin sensitivity and reduce hepatic gluconeogenesis; regulate lipid metabolism, inhibit hepatic dendritic lipogenesis (DNL), and promote fatty acid oxidation; simultaneously, it reduces liver inflammation and activation of hepatic stellate cells, thereby slowing the progression of fibrosis. Obeticholic acid (OCA), as the first FXR agonist to be extensively studied, is mainly used clinically for the treatment of primary biliary cholangitis (PBC). The Phase III clinical trial (REGENERATE study) of OCA showed its effect in improving liver fibrosis, but it has not yet been approved by the US FDA for the treatment of MASH. Ursodeoxycholic acid (UDCA) is also a commonly used hepatoprotective and choleretic drug in clinical practice for the treatment of primary biliary cholangitis. Meanwhile, clinical meta-analyses have found that UDCA can reduce serum ALT and AST levels in MAFLD patients, and animal studies have shown that it can slow the progression of MASH. Although both obeticholic acid (OCA) and ursodeoxycholic acid (UDCA) have shown potential in treating MAFLD, they each have significant limitations. For example, OCA has a side effect of itching, with an incidence rate exceeding 50%, and the side effects can be severe. Furthermore, taking OCA leads to elevated low-density lipoprotein cholesterol (LDL-C), potentially increasing the risk of cardiovascular events. Conventional doses of UDCA are ineffective in treating MAFLD; only high doses are effective, but high doses increase gastrointestinal side effects (diarrhea incidence rate 15-20%). Therefore, the development of new drugs for treating MAFLD is imperative. Summary of the Invention

[0004] To address the shortcomings and deficiencies of existing technologies, this invention aims to provide a novel method for synthesizing bile acid derivatives, and the application of these derivatives in the preparation of drugs for the prevention and / or treatment of metabolic-associated fatty liver disease (MAFLD), specifically involving the synthesis method and application of N-chenodeoxychoyl-L-aspartic acid.

[0005] The first objective of this invention is to provide a bile acid derivative, N-chenodeoxychoyl-L-aspartic acid, for improving metabolic-associated fatty liver disease, with the structural formula shown in Formula I: Formula I.

[0006] A second objective of this invention is to provide a medicament for improving metabolic-related fatty liver disease, comprising the bile acid derivative N-chenodeoxychoyl-L-aspartic acid as the main active ingredient.

[0007] A third object of the present invention is to provide the use of the bile acid derivative N-chenodeoxychoyl-L-aspartic acid in the preparation of a medicament for improving metabolic-related fatty liver disease.

[0008] A fourth object of the present invention is to provide a method for preparing the cholic acid derivative N-chenodeoxychoyl-L-aspartic acid, comprising the following steps: S1. Add L-aspartic acid dimethyl ester hydrochloride and triethylamine to methanol and stir for 20-45 min, then cool to -5℃ to 5℃. Add and dissolve chenodeoxycholic acid, 1-hydroxybenzotriazole, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and react at 20℃-30℃ for 12-24 h. Remove methanol, dissolve the residue in dichloromethane, wash, dry, and filter the residue. Remove dichloromethane from the filtrate to obtain chenodeoxycholic acid-aspartic acid dimethyl ester. The addition ratio of methanol, L-aspartic acid dimethyl ester hydrochloride, triethylamine, chenodeoxycholic acid, 1-hydroxybenzotriazole, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 100 mL: 10-13.33 mmol: 60-66.67 mmol: 5-6.67 mmol: 12-20 mmol: 12-16.67 mmol; S2. Dissolve the chenodeoxycholic acid-aspartic acid dimethyl ester obtained in step S1 in an organic solvent, and add an aqueous solution containing an alkaline reagent dropwise while stirring; the ratio of organic solvent, chenodeoxycholic acid-aspartic acid dimethyl ester and alkaline reagent is 10 mL: 0.93~1 mmol: 1.86 mmol~4 mmol; stir at 0℃~30℃ in the dark for 6~18 h; pour into water to cool, adjust the pH to 1.5~2.5 with an acidic solution; extract with an extraction reagent, dry and filter the extraction reagent after extraction, remove the extraction reagent from the filtrate to obtain N-chenodeoxycholic acid-L-aspartic acid.

[0009] Preferably, the method includes the following steps: S1. Add L-aspartic acid dimethyl ester hydrochloride and triethylamine to methanol and stir for 20-45 min, then cool to -5℃ to 5℃; add and dissolve chenodeoxycholic acid, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, heat to 20℃-30℃ and react for 12-36 h, remove methanol, dissolve the residue with dichloromethane, wash, dry and filter the residue, remove dichloromethane from the filtrate to obtain chenodeoxycholic acid-aspartic acid dimethyl ester; the addition ratio of methanol, L-aspartic acid dimethyl ester hydrochloride, triethylamine, chenodeoxycholic acid, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 100 mL: 10 mmol: 60 mmol: 5 mmol: 15 mmol: 15 mmol; S2. Dissolve the chenodeoxycholic acid-aspartic acid dimethyl ester obtained in step S1 in an organic solvent, and add an aqueous solution containing an alkaline reagent dropwise while stirring; the ratio of organic solvent, chenodeoxycholic acid-aspartic acid dimethyl ester and alkaline reagent is 10 mL: 1 mmol: 4 mmol; stir at 25 °C in the dark for 12 h; pour into water to cool, and adjust the pH to 2.0 with an acidic solution; extract with an extraction reagent, dry the extraction reagent after extraction, filter, remove the extraction reagent from the filtrate, and obtain N-chenodeoxycholic acid-L-aspartic acid.

[0010] Preferably, the washing involves washing 2 to 4 times each with a saturated aqueous solution of NaCl and a sodium bicarbonate solution with a volume fraction of 3% to 7%.

[0011] Preferably, the drying is performed using anhydrous sodium sulfate, and the removal of dichloromethane is achieved by rotary evaporation concentration.

[0012] Preferably, the organic solvent is an organic solvent containing one or more of tetrahydrofuran, methanol, ethanol and 1,4-dioxane; the alkaline reagent is an alkaline reagent containing one or more of lithium hydroxide, sodium hydroxide, potassium hydroxide, sodium carbonate and potassium carbonate; and the acidic solution is an aqueous solution of citric acid, hydrochloric acid or sulfuric acid.

[0013] Preferably, the extraction with the extraction reagent is performed 3 to 5 times, and the removal of the extraction reagent is performed by vacuum concentration. The extraction reagent is dichloromethane, ethyl acetate, or diethyl ether.

[0014] The beneficial effects of this invention are: 1. Synthetic Method: This invention provides a method for synthesizing the key intermediate "chenodeoxycholic acid-aspartic acid dimethyl ester" through a chemical reaction using chenodeoxycholic acid and L-aspartic acid dimethyl ester hydrochloride as starting materials, and further preparing the target compound "N-chenodeoxychoyl-L-aspartic acid". This synthetic route is simple and efficient.

[0015] 2. Novel Compound and Application: The N-chenodeoxychoyl-L-aspartic acid prepared in this invention is a novel bile acid derivative. Experiments have shown that this compound can effectively improve liver function indicators related to MAFLD and reduce the levels of hepatic steatosis, inflammation, and fibrosis. Compared with obeticholic acid, the N-chenodeoxychoyl-L-aspartic acid compound provided by this invention shows better therapeutic effects in animal models, indicating its higher clinical application value and development potential. Attached Figure Description

[0016] Figure 1 It is a 1H NMR spectrum analysis of the structure of chenodeoxycholic acid-aspartic acid dimethyl ester.

[0017] Figure 2 This is a 1H NMR spectrum analysis of the structure of N-chenodeoxychoyl-L-aspartic acid.

[0018] Figure 3 This is a graph showing the results of determining the molecular weight of chenodeoxycholic acid using liquid chromatography-mass spectrometry.

[0019] Figure 4 This is a chromatogram showing the molecular weight determination of chenodeoxycholic acid-aspartic acid dimethyl ester using liquid chromatography-mass spectrometry (LC-MS). The mass spectrometry result indicates that the product requires the binding of a chloride ion to determine its molecular weight; therefore, the chemical reagent used contains a hydrochloride salt. Thus, the molecular formula in the graph needs to include the "Cl" ion. - "Remove Cl" - The relative atomic mass of chenodeoxycholic acid-aspartic acid dimethyl ester is approximately 535.26 g / mol.

[0020] Figure 5 This is a graph showing the results of determining the molecular weight of N-chenodeoxychoyl-L-aspartic acid using liquid chromatography-mass spectrometry.

[0021] Figure 6 The graph shows the changes in body weight (A) and liver weight (B) of mice in each group.

[0022] Figure 7 This is a graph showing the results of the levels of aspartate aminotransferase (A) and alanine aminotransferase (B) in the serum of mice in each group.

[0023] Figure 8 The expression of lipid metabolism, inflammation, and fibrosis-related genes in the liver tissue of mice in each group. Detailed Implementation

[0024] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0025] L-Aspartic acid dimethyl ester hydrochloride: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number: A109002.

[0026] Triethylamine: Purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number: T140677.

[0027] Chenodeoxycholic acid: purchased from MCE, product number: 474-25-9.

[0028] 1-Hydroxybenzotriazole: Purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number: H684271.

[0029] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number: E106172.

[0030] Example 1 This embodiment illustrates operating conditions near the midpoint of the parameter range that achieves the purpose of the invention.

[0031] 1. Reaction preparation: In a 250 mL round-bottom flask, L-aspartic acid dimethyl ester hydrochloride (1.9726 g, 10.0 mmol) was dissolved in 100 mL of anhydrous methanol, and triethylamine (5.0 mL, 60.0 mmol) was added. The mixture was stirred at room temperature for 30 min. After stirring, the mixture was cooled to 0 °C using an ice-water bath to obtain the mixture.

[0032] 2. Condensation reaction: Chenodeoxycholic acid (1.9629 g, 5.0 mmol, molecular weight 392.58 g / mol, molecular structure formula as shown) was added sequentially to the mixture obtained in step 1. Figure 3 The mixture of 1-hydroxybenzotriazole (2.0268 g, 15.0 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.8755 g, 15.0 mmol) was completely dissolved to give reaction mixture 1. The ice-water bath was then removed, and the flask was transferred to a constant temperature water bath at 25 °C with continuous stirring for 24 h.

[0033] 3. Post-treatment and purification: Methanol in reaction mixture 1 was removed under reduced pressure using a rotary evaporator. The residue was dissolved in 150 mL of dichloromethane and transferred to a separatory funnel. The mixture was washed three times with 100 mL of saturated NaCl solution, followed by three times with 100 mL of 5% sodium bicarbonate solution to remove excess reactants (such as triethylamine, L-aspartic acid dimethyl ester hydrochloride, 1-hydroxybenzotriazole, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride). The washed organic phase (containing the dissolved intermediate target product in dichloromethane) was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by rotary evaporation to remove the dichloromethane, yielding a white powdery solid.

[0034] 4. Product identification: such as Figure 1 and Figure 4 As shown, the proton nuclear magnetic resonance spectrum (NMR) 1 Analysis by ¹H NMR and determination by liquid chromatography-mass spectrometry confirmed that the obtained white powdery solid was the intermediate target product chenodeoxycholic acid-aspartic acid dimethyl ester, with a molecular weight of 535.26 g / mol and a yield of 89%.

[0035] 5. Dissolve chenodeoxycholic acid-aspartic acid dimethyl ester (1.0705 g, 2.0 mmol) in 20 mL of tetrahydrofuran to obtain reaction mixture 2.

[0036] 6. Dissolve lithium hydroxide (0.0958 g, 4.0 mmol) in 6 mL of water to obtain an aqueous solution of lithium hydroxide. While stirring, slowly add the aqueous solution of lithium hydroxide dropwise to reaction mixture 2 to obtain reaction mixture 3.

[0037] 7. After the addition was complete, the reaction mixture 3 was magnetically stirred for 12 h at room temperature (25°C) in the dark. The reaction was confirmed to be complete by thin-layer chromatography (TLC).

[0038] 8. Pour the fully reacted reaction mixture 3 into 15 mL of ice water. While cooling in an ice water bath, slowly add a 10% (w / w) citric acid aqueous solution to precisely adjust the pH to 2.0.

[0039] 9. Extract three times with dichloromethane, 100 mL each time. Combine the organic layers (the liquids obtained from the three dichloromethane extractions) and dry with anhydrous sodium sulfate. Filter and concentrate under reduced pressure to remove dichloromethane, giving a white solid product in 92% yield. Figure 2 and Figure 5 As shown, via 1H NMR and liquid chromatography-mass spectrometry confirmed that the white solid product was N-chenodeoxychoyl-L-aspartic acid with a molecular weight of 507.67 g / mol. The molecular structure of N-chenodeoxychoyl-L-aspartic acid is shown in Formula I. Formula I.

[0040] Example 2 This embodiment demonstrates the use of milder, shorter reaction time operating conditions within a parameter range that enables the invention's objectives to be achieved.

[0041] 1. Reaction preparation: In a 100 mL round-bottom flask, L-aspartic acid dimethyl ester hydrochloride (1.5809 g, 8.0 mmol) was dissolved in 60 mL of anhydrous methanol. Triethylamine (3.3 mL, 40.0 mmol) was added, and the mixture was stirred at room temperature for 20 min. Then it was cooled to 5 °C to obtain the mixture.

[0042] 2. Condensation reaction: Chenodeoxycholic acid (1.5703 g, 4.0 mmol), 1-hydroxybenzotriazole (1.6214 g, 12.0 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.0 g, 10.0 mmol) were added sequentially to the mixture obtained in step 1. After complete dissolution, reaction mixture 1 was obtained. Subsequently, the ice-water bath was removed, and the flask was transferred to a constant temperature water bath at 25°C with continuous stirring for 16 h.

[0043] 3. Post-treatment and purification: Methanol was removed from the reaction mixture 1 under reduced pressure using a rotary evaporator. The residue was dissolved in 100 mL of dichloromethane and transferred to a separatory funnel. The solution was then dissolved in 100 mL of dichloromethane and washed twice each with 80 mL of saturated NaCl aqueous solution and 5% sodium bicarbonate solution to remove excess reactants. The washed organic phase (containing the dissolved dichloromethane intermediate) was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by rotary evaporation to remove the dichloromethane, yielding a white powdery solid.

[0044] 4. Product identification: Identification was performed using 1H NMR spectroscopy (NMR spectroscopy). 1 ¹H NMR analysis confirmed that the obtained white powdery solid was the intermediate target product chenodeoxycholic acid-aspartic acid dimethyl ester, with a yield of 85%.

[0045] 5. Dissolve chenodeoxycholic acid-aspartic acid dimethyl ester (1.4933 g, 2.79 mmol) in 30 mL of a mixed solvent of methanol and 1,4-dioxane (v / v = 1:1) to obtain reaction mixture 2.

[0046] 6. Dissolve sodium hydroxide (0.2232 g, 5.58 mmol) in 8 mL of water to obtain an aqueous sodium hydroxide solution, and slowly add it dropwise to the reaction mixture 2 obtained in step 5 while stirring to obtain reaction mixture 3.

[0047] 7. Place reaction mixture 3 in a 30°C constant temperature water bath and stir for 6 h in the dark. The reaction was confirmed to be complete by HPLC monitoring.

[0048] 8. Pour the reaction mixture 3, which has been used to complete the reaction, into 15 mL of ice water. While cooling in an ice water bath, adjust the pH to 1.5 with a 10% (w / w) citric acid aqueous solution.

[0049] 9. Extract three times with ethyl acetate, 120 mL each time. Combine the organic layers (the liquids obtained from the three ethyl acetate extractions) and dry with anhydrous sodium sulfate. Filter and concentrate under reduced pressure to remove ethyl acetate, giving an off-white solid product. 1 HNMR and liquid chromatography-mass spectrometry confirmed that the off-white solid product was N-chenodeoxychoyl-L-aspartic acid (structural formula as shown in Example 1, Formula I), with a yield of 90%.

[0050] Example 3 This embodiment demonstrates enhanced conditions using higher reactant concentrations and longer reaction times within the parameter range that enables the invention to achieve its objective.

[0051] 1. Reaction preparation: In a 500 mL round-bottom flask, L-aspartic acid dimethyl ester hydrochloride (2.7667 g, 14.0 mmol) was dissolved in 140 mL of anhydrous methanol, and triethylamine (7.5 mL, 90.0 mmol) was added. The mixture was stirred for 45 min and then cooled to -5 °C (using an ice-salt bath) to obtain the final mixture.

[0052] 2. Condensation reaction: Chenodeoxycholic acid (2.7481 g, 7.0 mmol), 1-hydroxybenzotriazole (2.8375 g, 21.0 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.8340 g, 20.0 mmol) were added sequentially to the mixture obtained in step 1. After complete dissolution, reaction mixture 1 was obtained. Subsequently, the ice-water bath was removed, and the flask was transferred to a constant temperature water bath at 30°C with continuous stirring for 24 h.

[0053] 3. Post-treatment and purification: After the reaction was completed, methanol in reaction mixture 1 was removed under reduced pressure using a rotary evaporator. The resulting residue was dissolved in 200 mL of dichloromethane and transferred to a separatory funnel. The mixture was washed four times each with 120 mL of saturated NaCl aqueous solution and 5% sodium bicarbonate solution to remove excess reactants. The washed organic phase (containing the dissolved intermediate target product in dichloromethane) was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by rotary evaporation to remove the dichloromethane, yielding a white powdery solid.

[0054] 4. Product identification: Identification was performed using 1H NMR spectroscopy (NMR spectroscopy). 1 ¹H NMR analysis confirmed that the obtained white powdery solid was the intermediate target product chenodeoxycholic acid-aspartic acid dimethyl ester, with a yield of 91%.

[0055] 5. Dissolve chenodeoxycholic acid-aspartic acid dimethyl ester (0.5352 g, 1.0 mmol) in 10 mL of tetrahydrofuran to obtain reaction mixture 2.

[0056] 6. Dissolve potassium carbonate (0.2764 g, 2.0 mmol) in 3 mL of water to obtain an aqueous solution of potassium carbonate, and add it dropwise to reaction mixture 2 in the reaction flask to obtain reaction mixture 3.

[0057] 7. Place reaction mixture 3 in a 0°C ice-water bath and stir for 18 h in the dark.

[0058] 8. After the reaction is complete, pour reaction mixture 3 into 10 mL of water and adjust the pH to 2.5 with 0.1 mol / L dilute hydrochloric acid.

[0059] 9. Extract four times with dichloromethane, 60 mL each time. Combine the organic phases (liquids obtained from the four dichloromethane extractions), dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to remove dichloromethane to obtain the product. 1 H NMR and liquid chromatography-mass spectrometry confirmed that it was N-chenodeoxychoyl-L-aspartic acid (structural formula as shown in Example 1, Formula I), with a yield of 87%.

[0060] In Examples 1-3, the reagent used to dissolve chenodeoxycholic acid-aspartic acid dimethyl ester in step 5 can be one or more organic solvents such as tetrahydrofuran, methanol, 1,4-dioxane, or ethanol; the lithium hydroxide, sodium hydroxide, or potassium carbonate in step 6 can be replaced with one or more alkaline reagents containing lithium hydroxide, sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate, and the amount added is 2 to 4 times that of chenodeoxycholic acid-aspartic acid dimethyl ester. Specifically, the purpose of using alkaline reagents is to remove chenodeoxycholic acid-aspartic acid dimethyl ester. The methyl ester on the dimethyl ester of chenodeoxycholic acid-aspartic acid contains two methyl ester groups. Therefore, the molar ratio of the alkaline reagent to dimethyl chenodeoxycholic acid-aspartic acid must be greater than 2. A ratio lower than 2 may cause incomplete hydrolysis, while a ratio greater than 4 may cause degradation of dimethyl chenodeoxycholic acid-aspartic acid. The solution used to adjust the pH in step 8 can be an acidic solution such as citric acid aqueous solution, hydrochloric acid aqueous solution, or sulfuric acid aqueous solution. The reagent used for extraction in step 9 can be an organic reagent such as dichloromethane, ethyl acetate, or diethyl ether.

[0061] Example 4 In this embodiment, a mouse model was constructed, and N-chenodeoxychoyl-L-aspartic acid (Asp-CDCA) prepared in Example 3 was used as the experimental sample to detect the effect of Asp-CDCA on improving the progression of MAFLD.

[0062] Asp-CDCA and obeticholic acid (OCA) were dissolved in a 0.5% (w / w) aqueous solution of sodium carboxymethyl cellulose (CMC-Na) to prepare Asp-CDCA and OCA solutions with a concentration of 10 mg / mL.

[0063] 1. Asp-CDCA can reduce body weight and liver weight in MAFLD mice. To explore the effect of Asp-CDCA in improving MAFLD, 6-week-old BSK strain spontaneously diabetic mice (db / db) were selected and fed a Western diet to establish an MAFLD model. Simultaneously, Asp-CDCA was administered via gavage (Western diet + Asp-CDCA group) at a dose of 50 mg / kg body weight, once daily. Body weight was measured every 3 days, and the experiment ended after 42 days (6 weeks), at which point liver weight was measured. Age-matched BSK strain spontaneously diabetic mice not fed a Western diet but administered 0.5% CMC-Na aqueous solution via gavage served as the control group. Meanwhile, age-matched BSK strain spontaneously diabetic mice fed a Western diet were either not administered gavage (Western diet group) or administered the same dose of OCA solution via gavage (Western diet + OCA group).

[0064] Figure 6The results showed that, compared with the Western diet group, the mice treated with Asp-CDCA had significantly lower body weight (P<0.05) and significantly lower liver weight (P<0.01) after day 30. Compared with oral administration of obeticholic acid (OCA), Asp-CDCA showed a more significant trend in improving body weight and liver weight in mice.

[0065] 2. Asp-CDCA can improve liver function and lipid metabolism in MAFLD mice. To investigate the effects of Asp-CDCA on liver function in MAFLD mice, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. Figure 7 As can be seen, compared with the control group mice, the ALT and AST levels of the Western diet group mice were significantly increased, indicating that the Western diet significantly aggravated liver damage in db / db mice; after administration of Asp-CDCA, the ALT and AST levels of the Asp-CDCA treatment group mice were significantly decreased compared with the Western diet model group mice (P<0.05).

[0066] 3. Asp-CDCA can improve lipid metabolism, reduce inflammatory response and fibrosis in MASLD mice. To further demonstrate the role of Asp-CDCA in improving MAFLD, RNA was extracted from the liver tissues of mice in each group and real-time quantitative PCR was performed. The GAPDH gene was used as an internal reference gene to detect lipid metabolism, inflammation and fibrosis indicators in the liver. The primers used are shown in Table 1. Figure 8 The results showed that, compared with the control group, the Western diet significantly upregulated the expression of genes in the liver of mice, including lipid metabolism-related genes: fatty acid synthase (FASN), human leukocyte differentiation antigen 36 (CD36), inflammation-related chemokines (CCL5, CXCL9), and fibrosis-related genes (Col4a1, Col5a2). Administration of Asp-CDCA significantly inhibited the expression of lipid metabolism, inflammation, and fibrosis-related genes, with a greater degree of inhibition than OCA.

[0067] Table 1. Quantitative primer sequence information

Claims

1. A drug for improving metabolic-related fatty liver disease, characterized in that, The active ingredient is N-chenodeoxychoyl-L-aspartic acid, a cholic acid derivative; the structural formula of the N-chenodeoxychoyl-L-aspartic acid derivative is shown in Formula I: Formula I.

2. The application of the bile acid derivative N-chenodeoxychoyl-L-aspartic acid in the preparation of drugs for improving metabolic-related fatty liver disease, characterized in that, The structural formula of the bile acid derivative N-chenodeoxychoyl-L-aspartic acid is shown in Formula I: Formula I.

3. The application according to claim 2, characterized in that, The preparation method of the cholic acid derivative N-chenodeoxychoyl-L-aspartic acid includes the following steps: S1. Add L-aspartic acid dimethyl ester hydrochloride and triethylamine to methanol and stir for 20-45 min, then cool to -5℃ to 5℃. Add and dissolve chenodeoxycholic acid, 1-hydroxybenzotriazole, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and react at 20℃-30℃ for 12-36 h. Remove methanol, dissolve the residue in dichloromethane, wash, dry, and filter the residue. Remove dichloromethane from the filtrate to obtain chenodeoxycholic acid-aspartic acid dimethyl ester. The addition ratio of methanol, L-aspartic acid dimethyl ester hydrochloride, triethylamine, chenodeoxycholic acid, 1-hydroxybenzotriazole, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 100 mL: 10-13.33 mmol: 60-66.67 mmol: 5-6.67 mmol: 12-20 mmol: 12-16.67 mmol; S2. Dissolve the chenodeoxycholic acid-aspartic acid dimethyl ester obtained in step S1 in an organic solvent, and add an aqueous solution containing an alkaline reagent dropwise while stirring; the ratio of organic solvent, chenodeoxycholic acid-aspartic acid dimethyl ester and alkaline reagent is 10 mL: 0.93~1 mmol: 1.86 mmol~4 mmol; stir at 0℃~30℃ in the dark for 6~18 h; cool in water, adjust the pH to 1.5~2.5 with an acidic solution; extract with an extraction reagent, dry and filter the extraction reagent after extraction, remove the extraction reagent from the filtrate, and obtain N-chenodeoxycholic acid-L-aspartic acid.

4. The application according to claim 3, characterized in that, The preparation method of the cholic acid derivative N-chenodeoxychoyl-L-aspartic acid includes the following steps: S1. Add L-aspartic acid dimethyl ester hydrochloride and triethylamine to methanol and stir for 20-45 min, then cool to -5℃ to 5℃; add and dissolve chenodeoxycholic acid, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, heat to 20℃ to 30℃ and react for 12-24 h, remove methanol, dissolve the residue with dichloromethane, wash, dry and filter the residue, remove dichloromethane from the filtrate to obtain chenodeoxycholic acid-aspartic acid dimethyl ester; The addition ratio of methanol, L-aspartic acid dimethyl ester hydrochloride, triethylamine, chenodeoxycholic acid, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was 100 mL: 10 mmol: 60 mmol: 5 mmol: 15 mmol: 15 mmol; S2. Dissolve the chenodeoxycholic acid-aspartic acid dimethyl ester obtained in step S1 in an organic solvent, and add an aqueous solution containing an alkaline reagent dropwise while stirring; the ratio of organic solvent, chenodeoxycholic acid-aspartic acid dimethyl ester and alkaline reagent is 10 mL: 1 mmol: 4 mmol; stir at 25°C in the dark for 12 h; pour into water to cool, and adjust the pH to 2.0 with an acidic solution; extract with an extraction reagent, dry the extraction reagent after extraction, filter, remove the extraction reagent from the filtrate, and obtain N-chenodeoxycholic acid-L-aspartic acid.

5. The application according to claim 3, characterized in that, The washing process in the preparation method of the cholic acid derivative N-chenodeoxychoyl-L-aspartic acid involves washing 2 to 4 times each with a saturated aqueous solution of NaCl and a sodium bicarbonate solution with a volume fraction of 3% to 7%.

6. The application according to claim 3, characterized in that, The drying process in the preparation method of the cholic acid derivative N-chenodeoxychoyl-L-aspartic acid is performed using anhydrous sodium sulfate, and the removal of dichloromethane in the preparation method of the cholic acid derivative N-chenodeoxychoyl-L-aspartic acid is performed by rotary evaporation concentration to remove dichloromethane.

7. The application according to claim 3, characterized in that, The organic solvent used in the preparation method of the cholic acid derivative N-chenodeoxychoyl-L-aspartic acid is one or more organic solvents selected from tetrahydrofuran, methanol, ethanol and 1,4-dioxane; the alkaline reagent used in the preparation method of the cholic acid derivative N-chenodeoxychoyl-L-aspartic acid is one or more alkaline reagents selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, sodium carbonate and potassium carbonate; the acidic solution used in the preparation method of the cholic acid derivative N-chenodeoxychoyl-L-aspartic acid is an aqueous solution of citric acid, hydrochloric acid or sulfuric acid.

8. The application according to claim 3, characterized in that, In the preparation method of the cholic acid derivative N-chenodeoxychoyl-L-aspartic acid, the extraction with the extraction reagent is performed 3 to 5 times. In the preparation method of the cholic acid derivative N-chenodeoxychoyl-L-aspartic acid, the extraction reagent is removed by vacuum concentration. The extraction reagent is dichloromethane, ethyl acetate, or diethyl ether.

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