Compound of ursodeoxycholic acid or obeticholic acid and teprenone and its use in the preparation of a drug for preventing and treating liver fibrosis

Abstract

The application discloses a complex of ursodeoxycholic acid or obeticholic acid and teprenone and application of the complex in preparation of a medicine for preventing and treating liver fibrosis. The combination of ursodeoxycholic acid or obeticholic acid and teprenone can significantly reduce the levels and contents of glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST) and hydroxyproline (Hyp) in model mice, thereby the effect of ursodeoxycholic acid or obeticholic acid in treating liver fibrosis can be enhanced. Therefore, the complex of ursodeoxycholic acid or obeticholic acid and teprenone has a potential for preventing and treating liver fibrosis, which is superior to ursodeoxycholic acid or obeticholic acid single component.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the complex of ursodeoxycholic acid or obeticholic acid with teprenone and its application in the preparation of drugs for the prevention and treatment of liver fibrosis. Background Technology

[0002] Currently, there are many types of drugs for treating liver fibrosis. These drugs primarily work by targeting different stages of liver fibrosis to achieve their anti-fibrotic effect. These include drugs that target hepatic stellate cells, drugs that target cytokines, drugs that affect collagen synthesis and metabolism, and drugs that protect against and prevent liver damage. However, some drugs, when used alone, have certain toxicity, poor specificity, low bioavailability, and short half-lives.

[0003] Liver fibrosis is a pathological state, a compensatory repair response to chronic liver damage, characterized by abnormal proliferation of connective tissue within the liver. Liver fibrosis is a major intermediate step in the progression of chronic liver disease to cirrhosis. Therefore, preventing and reversing the development of cirrhosis and promoting the gradual breakdown and absorption of liver fibrosis can not only reduce the degree of liver damage but also prevent the occurrence of cirrhosis. The vast majority of chronic liver diseases caused by various etiologies involve liver fibrosis, with 25%-40% eventually developing into cirrhosis and even liver cancer. Liver fibrosis is a reversible disease, while cirrhosis is irreversible; therefore, anti-fibrotic therapy is an important approach to controlling the progression of liver fibrosis. Liver fibrosis is a dynamic and reversible process and a crucial period for reversing chronic liver disease. Therefore, actively intervening during the liver fibrosis stage to block its progression is of great significance in reducing the risk of chronic liver disease progressing to cirrhosis and even liver cancer. Currently, animal models of liver fibrosis are mainly based on mice, including cholestatic liver fibrosis, irritant-induced liver fibrosis, metabolic liver fibrosis, alcoholic liver fibrosis, and immune-mediated liver fibrosis. Primary biliary cholangitis (PBC) is a chronic cholestasis caused by bile duct dysplasia or bile acid metabolism disorders. This leads to the dissolution of cell membrane lipids, altered permeability, and leakage of cellular contents, eventually progressing to cholestatic liver fibrosis and cirrhosis. Ursodeoxycholic acid and obeticholic acid can be used to treat PBC and related liver fibrosis. Obeticholic acid is specifically for patients who do not respond adequately to or cannot tolerate ursodeoxycholic acid. However, according to the results of a Phase III clinical trial of obeticholic acid against NASH completed by Intercept in February 2019, obeticholic acid improved liver fibrosis symptoms in 23% of patients (compared to 12% improvement with placebo). Although the efficacy showed a statistically significant difference, the improvement was only 11%, and its efficacy did not reach the expected satisfactory level. Summary of the Invention

[0004] The purpose of this invention is to provide a complex of ursodeoxycholic acid or obeticholic acid and teprenone, which has superior potential for the prevention and treatment of liver fibrosis compared to ursodeoxycholic acid or obeticholic acid alone. Specifically, this invention adopts the following technical solution:

[0005] Firstly, this invention provides a complex of ursodeoxycholic acid and teprenone, with the following structural formula (Ⅰ):

[0006]

[0007] Secondly, the present invention also provides a method for preparing the complex of ursodeoxycholic acid and teprenone described above, which involves hydrogenating teprenone with lithium aluminum hydride to obtain intermediate one, then brominating it with phosphorus tribromide to obtain intermediate two, and then reacting it with ursodeoxycholic acid to obtain the final product.

[0008] As a preferred technical solution, the preparation method of the above-described complex of ursodeoxycholic acid and teprenone may include the following steps:

[0009] (1) Dissolve 20g of teprenone in tetrahydrofuran, cool to below 0℃ under nitrogen protection, add 2-3g of lithium aluminum hydride in batches, add 2-3g of water dropwise under ice-water bath, solidify after the addition is complete, add 50wt% sodium hydroxide, add anhydrous sodium sulfate and stir to remove water, filter with diatomaceous earth, wash with EA (ethyl acetate), evaporate the mother liquor to dryness to obtain intermediate one;

[0010] (2) Add 18-19g of intermediate one, 50-1500mL of methyl tert-butyl ether and 1-2g of pyridine to a reaction flask, stir to dissolve, cool to below 0℃ under nitrogen protection, add dropwise a methyl tert-butyl ether solution containing 9-10g of phosphorus tribromide, complete the addition within 1h, and react for 1-2h; add water to dissolve the solid in the reaction solution, separate the liquid, wash with potassium carbonate solution, wash with water, wash with saturated sodium chloride, dry with anhydrous sodium sulfate, filter, and evaporate the solvent at room temperature to obtain intermediate two;

[0011] (3) Dissolve 20-22g of ursodeoxycholic acid in DMF, then add 20-22g of intermediate di and potassium carbonate, react at room temperature for 10-20h, then react at 40-60℃ for 10-15h; add water to the reaction solution, separate the ethyl acetate, extract the aqueous layer with ethyl acetate, combine the ethyl acetate, wash with water, wash with saturated sodium chloride, dry with anhydrous sodium sulfate, filter, and purify the mother liquor by column chromatography with silica gel.

[0012] Thirdly, the present invention provides a complex of obeticholic acid and teprenone, with the following structural formula (II):

[0013]

[0014] Fourth, the present invention also provides a method for preparing the above-mentioned complex of obeticholic acid and teprenone, which involves hydrogenating teprenone with lithium aluminum hydride to obtain intermediate one, and then reacting it with obeticholic acid to obtain the final product.

[0015] As a preferred technical solution, the preparation method of the above-mentioned obeticholic acid and teprenone complex is characterized by comprising the following steps:

[0016] (1) Dissolve 40g of teprenone in tetrahydrofuran, cool to below 0℃ under nitrogen protection, add 4-6g of lithium aluminum hydride in batches, and wait for the reaction to be complete; add 4-6g of water dropwise under an ice-water bath, solidify after the dropwise addition, add 50wt% sodium hydroxide, add anhydrous sodium sulfate and stir to remove water, filter with diatomaceous earth, wash with EA, and evaporate the mother liquor to dryness to obtain intermediate one.

[0017] (2) Add 8-9g of obeticholic acid, 8-9g of intermediate one, 5-6g of DCC (N,N'-dicyclohexylcarboimide), 0.5-1g of DMAP (4-dimethylaminopyridine), and DCM to a reaction flask, stir, and react at room temperature for 20-30h; filter, and purify the mother liquor by column chromatography to obtain the final product.

[0018] Fifth, the complexes of ursodeoxycholic acid and teprenone, as well as the complexes of obeticholic acid and teprenone of the present invention, can be further used as drugs for the prevention and treatment of liver fibrosis.

[0019] In this invention, teprenone is a terpene compound with tissue repair effects, particularly enhancing anti-ulcer effects. It is a gastric mucosa protectant and is mainly used clinically to treat gastric ulcers and acute and chronic gastritis. When combined with ursodeoxycholic acid or obeticholic acid, it can significantly reduce the levels and contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and hydroxyproline (Hyp) in model mice, thereby enhancing the therapeutic effect of ursodeoxycholic acid or obeticholic acid on liver fibrosis. Attached Figure Description

[0020] Figure 1 To analyze liver function in mice using alanine aminotransferase (ALT) levels.

[0021] Figure 2 Aspartate aminotransferase (AST) analysis of liver function in mice

[0022] Figure 3 The determination of hydroxyproline (Hyp) content indicates collagen deposition in the liver of mice.

[0023] Compared with the control group, ###P<0.001; compared with the model group, *P<0.05,**P<0.01,***P<0.001. Detailed Implementation

[0024] The following embodiments are further illustrations of the present invention and serve as explanations of the technical content of the present invention. However, the essence of the present invention is not limited to the embodiments described below. Those skilled in the art can and should know that any simple changes or substitutions based on the spirit of the present invention should fall within the protection scope claimed by the present invention.

[0025] Example 1

[0026] Preparation of ursodeoxycholic acid / teprenone complex (XHH007)

[0027] 1.1 Process Route

[0028]

[0029] 1.2 Experimental Procedure

[0030] 1.2.1 Step 1: Preparation of XHH007-I

[0031] (1) Feeding ratio

[0032] name relative molecular weight Feeding amount equivalent source Teprenone 330.56 20g 1.0 Sigma-Aldrich Lithium aluminum hydride 38 2.8g 0.56eq Aladdin THF / 200ml / Cologne

[0033] (2) Operation process

[0034] Reaction: Teprenone and tetrahydrofuran were added to a reaction flask, stirred and dissolved, and cooled to below 0°C under nitrogen protection. Lithium aluminum hydride was added in batches, and the reaction was carried out by TLC (PE / EA = 10:1) until complete. 2.8 g of water was added dropwise under an ice-water bath. After solidification, 50 ml of THF was added, followed by 8.4 g of 50% sodium hydroxide. Anhydrous sodium sulfate was added and stirred to remove water. The mixture was filtered through diatomaceous earth, washed with EA, and the mother liquor was evaporated to dryness to obtain 18.5 g of XHH007-I. MS (m / z): 332.57, yield 92.7%.

[0035] 1.2.2 Step Two: Preparation of XHH007-II

[0036] (1) Experimental Consumables

[0037]

[0038]

[0039] (2) Operation process

[0040] Reaction: XHH007-I, methyl tert-butyl ether, and pyridine were added to a reaction flask and stirred to dissolve. Under nitrogen protection, the mixture was cooled to below 0°C, and phosphorus tribromide / methyl tert-butyl ether was added dropwise over 1 hour. The reaction was allowed to proceed for 1 hour. The solid was dissolved in 50 ml of water, separated, and washed twice with 50 ml of 0.5% potassium carbonate, once with 50 ml of water, and once with 50 ml of saturated sodium chloride. The mixture was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated at room temperature to obtain 28 g of liquid, yielding 90%. MS (m / z): 392.58.

[0041] 1.2.3 Step 3: Preparation of XHH007

[0042] (1) Experimental Consumables

[0043] name relative molecular weight Feeding amount equivalent source Ursodeoxycholic acid 392.58 20.6g 1.0 Nightingale XHH007-II 395.74 20.8g 1eq self made Potassium carbonate 138.21 8.7g 1.2eq Shanghai Qiangsheng DMF / 360mL / Xilong

[0044] (2) Operation process

[0045] Reaction: Ursodeoxycholic acid and DMF were added to a reaction flask and stirred to dissolve. Then XHH007-II and potassium carbonate were added. The reaction was carried out at room temperature (25℃) for 15 hours and at 50℃ for 12 hours.

[0046] The reaction solution was dissolved in 2000 ml of water and 1 L of ethyl acetate. The mixture was separated, and the aqueous layer was extracted twice with ethyl acetate (500 ml × 2). The ethyl acetate was combined, washed once with water (300 ml), washed once with saturated sodium chloride (30 ml), dried over anhydrous sodium sulfate, filtered, and the mother liquor was purified by column chromatography with 60 g of silica gel (eluent PE / EA = 2:1 petroleum ether: ethyl acetate) to obtain 2.8 g of pure oily product (batch number: 1015620).

[0047] 1 H NMR (600MHz, DMSO) δ5.06 (s, 4H), 4.77 (s, 1H), 4.45 (d, J = 3.5Hz, 1H), 3.86 (d, J = 6.5Hz, 1H) ,2.32-2.10(m,2H),1.97(ddd,J=25.8,15.0,7.8Hz,17H),1.84(s,1H),1.66(dd,J=30.9,1 7.1Hz,8H),1.59-1.51(m,12H),1.46(d,J=7.9Hz,4H),1.40-1.30(m,6H),1.15(dt,J=21.9 ,11.5Hz,10H),1.01(d,J=9.6Hz,1H),0.87(d,J=7.7Hz,7H),0.60(s,3H).MS(m / z):707.14.

[0048] Example 2

[0049] Preparation of obeticholic acid / teprenone complex (XHH009)

[0050] 2.1 Process Route

[0051]

[0052] 2.2 Experimental Procedure

[0053] 2.2.1 Step 1: Preparation of XHH009-I

[0054] (1) Experimental Consumables

[0055] name relative molecular weight Feeding amount Mole ratio source Teprenone 330.56 40g 1.0 Sigma-Aldrich Lithium aluminum hydride 38 5.6g 0.56eq Aladdin THF / 300ml / Cologne

[0056] (2) Operation process

[0057] Reaction: Teprenone and tetrahydrofuran were added to a reaction flask and stirred to dissolve. Under nitrogen protection, the mixture was cooled to below 0°C. Lithium aluminum hydride was added in portions, and the reaction was allowed to proceed until complete. TLC (PE / EA = 10:1, iodine colorimetric analysis) was performed. 4.9 g of water was added dropwise under an ice-water bath. After the mixture solidified, 15 g of 50% sodium hydroxide was added, followed by anhydrous sodium sulfate and stirring to remove water. The mixture was filtered through diatomaceous earth, washed with EA, and the mother liquor was evaporated to dryness to obtain 36.8 g of XHH009-I.

[0058] 2.2.2 Step Two: Preparation of XHH009

[0059] (1) Experimental Consumables

[0060] name relative molecular weight Feeding amount Mole ratio source obeticholic acid 420.63 8.83g 1.0 Nightingale XHH009-I 332.57 8.5g 1.2eq self made DCC 206.33 5.29g 1.2eq McLean DMAP 122.17 0.78g 0.3eq / DCM / 150mL / Cologne

[0061] (2) Experimental procedure

[0062] Reaction: Obeticholic acid, XHH009-I, DCC (N,N'-dicyclohexylcarboimide), DMAP (4-dimethylaminopyridine), and DCM (dichloromethane) were added to a reaction flask, stirred, and reacted at room temperature (15℃) for 24 h. After the reaction was completed, the mixture was filtered, and the mother liquor was purified by column chromatography (eluent PE / EA = 2:1) to obtain 3.7 g of pure product as an oily substance (batch number: 1015623).

[0063] 1H NMR (600MHz, DMSO) δ5.06 (s, 4H), 4.76 (s, 1H), 4.30 (d, J = 4.5Hz, 1H), 3.49 (s, 1H), 3 .12(s,1H),2.26(s,1H),2.16(s,1H),2.06-1.88(m,18H),1.77(dd,J=41.2,29.1Hz ,6H),1.63(d,J=6.9Hz,4H),1.59-1.51(m,13H),1.43(d,J=13.0Hz,5H),1.21-1.05 (m,12H),0.98(s,1H),0.85(dd,J=37.4,6.9Hz,10H),0.59(s,3H).MS(m / z):735.19.

[0064] Example 3

[0065] Serum ALT and AST are key indicators for assessing liver function impairment, and hydroxyproline is one of the main components of collagen. Therefore, the status of collagen in liver tissue can be assessed by measuring the hydroxyproline content, and the liver function of mice can be analyzed by measuring alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0066] Preparation of a CCl4-induced liver fibrosis mouse model: Male 6-8 week old C57BL / 6 mice were intraperitoneally injected with 10% CCl4-sterile corn oil solution, 10 mL / kg, twice a week for 6 weeks to establish a mouse fibrosis model. The control group received an equal volume of sterile corn oil solution intraperitoneally. At week 5, the model group mice were randomly divided into four groups: model group (10% CCl4), CCl4 + ursodeoxycholic acid group, CCl4 + obeticholic acid group, CCl4 + XHH007 group, and CCl4 + XHH009 group. The control group and the solvent group received an equal volume of sterile corn oil solution orally once daily for 4 weeks. After treatment, blood was collected from the mice using the ocular sampling method. After standing, the blood was centrifuged at 3000 rpm for 10 min, and the supernatant was collected as serum. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured according to the experimental operation method of the kit; liver samples were taken, and hydroxyproline (Hyp) levels were measured using the kit to evaluate the treatment effects of each group.

[0067] like Figure 1-3Compared with the blank control group, the serum ALT and AST levels of mice in the model group were increased. Compared with the model group, the serum AST and ALT levels of the treatment groups were decreased, but the XHH007 and XHH009 groups had a more significant effect on reducing AST and ALT than the ursodeoxycholic acid and obeticholic acid groups. In addition, by detecting Hyp content, it was found that the Hyp content in the liver of mice in the model group was increased compared with the blank control group. Compared with the model group, the ursodeoxycholic acid and obeticholic acid treatment groups had the effect of reducing Hyp content, but the XHH007 and XHH009 groups could reduce Hyp content more significantly, thereby significantly inhibiting collagen deposition in the liver of CCl4 model mice. Therefore, compared with ursodeoxycholic acid alone or obeticholic acid alone, the XHH007 group and the XHH009 group significantly reduced the levels and contents of ALT, AST and Hyp, indicating that the complex of the present invention can enhance the therapeutic effect of ursodeoxycholic acid or obeticholic acid on liver fibrosis.

[0068] It should be noted that the above-described technical content of this invention is merely an explanation and clarification to enable those skilled in the art to understand the technical essence of this invention, and therefore is not intended to limit the scope of protection of this invention. The scope of protection of this invention should be determined by the claims. Those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made based on the essential spirit of this invention should be within the scope of protection of this invention.

Claims

1. The complex of ursodeoxycholic acid and teprenone has the following structural formula (Ⅰ): Equation (Ⅰ).

2. The method for preparing the ursodeoxycholic acid and teprenone complex according to claim 1, wherein teprenone is hydrogenated with lithium aluminum hydride to obtain intermediate one. The intermediate di was obtained by bromination with phosphorus tribromide. It is then reacted with ursodeoxycholic acid to obtain the final product.

3. The preparation method according to claim 2, characterized in that, Includes the following steps: (1) Dissolve 20g of teprenone in tetrahydrofuran, cool to below 0℃ under nitrogen protection, add 2-3g of lithium aluminum hydride in batches, add 2-3g of water dropwise under ice-water bath, solidify after the addition is complete, add 50wt% sodium hydroxide, add anhydrous sodium sulfate and stir to remove water, filter with diatomaceous earth, wash with ethyl acetate, evaporate the mother liquor to dryness to obtain intermediate one; (2) Add 18-19g of intermediate one, 50-150mL of methyl tert-butyl ether and 1-2g of pyridine to a reaction flask, stir to dissolve, cool to below 0℃ under nitrogen protection, add dropwise a methyl tert-butyl ether solution containing 9-10g of phosphorus tribromide, complete the addition within 1h, and react for 1-2h; add water to dissolve the solid in the reaction solution, separate the liquid, wash with potassium carbonate solution, wash with water, wash with saturated sodium chloride, dry with anhydrous sodium sulfate, filter, and evaporate the solvent at room temperature to obtain intermediate two; (3) Dissolve 20-22g of ursodeoxycholic acid in DMF, then add 20-22g of intermediate di and potassium carbonate, react at room temperature for 10-20h, then react at 40-60℃ for 10-15h; add water to the reaction solution, separate the ethyl acetate, extract the aqueous layer with ethyl acetate, combine the ethyl acetate, wash with water, wash with saturated sodium chloride, dry with anhydrous sodium sulfate, filter, and purify the mother liquor by column chromatography with silica gel.

4. The complex of obeticholic acid and teprenone has the following structural formula (II): Formula (II).

5. The method for preparing the obeticholic acid and teprenone complex according to claim 4, wherein teprenone is hydrogenated with lithium aluminum hydride to obtain intermediate one. Then it reacts with obeticholic acid to obtain...

6. The preparation method according to claim 5, characterized in that, Includes the following steps: (1) Take 40g of teprenone and dissolve it in tetrahydrofuran. Cool it to below 0°C under nitrogen protection. Add 4-6g of lithium aluminum hydride in batches. Add 4-6g of water dropwise under an ice-water bath. After the water is added, solidify it. Add 50wt% sodium hydroxide and anhydrous sodium sulfate and stir to remove water. Filter with diatomaceous earth and wash with EA. Evaporate the mother liquor to obtain intermediate one. (2) Add 8-9g of obeticholic acid, 8-9g of intermediate one, 5-6g of DCC (N,N'-dicyclohexylcarboimide), 0.5-1g of DMAP (4-dimethylaminopyridine), and dichloromethane to a reaction flask, stir, and react at room temperature for 20-30h; filter, and purify the mother liquor by column chromatography to obtain the final product.

7. The use of the complex of ursodeoxycholic acid and teprenone obtained by the preparation method according to claim 1 or any one of claims 2-3 in the preparation of drugs for the prevention and treatment of liver fibrosis.

8. The use of the complex of obeticholic acid and teprenone obtained by the preparation method according to claim 4 or any one of claims 5-6 in the preparation of drugs for the prevention and treatment of liver fibrosis.

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