Ursodesoxycholic acid or obeticholic acid and teprenone compound and application thereof in preparation of medicine for preventing and treating hepatic fibrosis

Abstract

The invention discloses a compound of ursodesoxycholic acid or obeticholic acid and teprenone and application of the compound in preparation of a medicine for preventing and treating hepatic fibrosis. The teprenone is combined with the ursodesoxycholic acid or obeticholic acid for administration, so that the level and content of alanine transaminase (ALT), aspartate transaminase (AST) and hydroxyproline (Hyp) of a model mouse can be remarkably reduced, and the effect of treating hepatic fibrosis by the ursodesoxycholic acid or obeticholic acid can be enhanced. Therefore, the compound of ursodesoxycholic acid or obeticholic acid and teprenone disclosed by the invention has the potential better than that of ursodesoxycholic acid or obeticholic acid single component for preventing and treating hepatic fibrosis.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a complex of ursodeoxycholic acid or obeticholic acid and teprenone and an application thereof in preparing a drug for preventing and treating liver fibrosis. Background Art

[0002] There are many types of drugs for the treatment of liver fibrosis, which mainly achieve the purpose of anti-liver fibrosis by acting on different links of liver fibrosis, including drugs targeting hepatic stellate cells, drugs targeting cytokines, drugs affecting collagen synthesis and metabolism, drugs protecting and preventing liver damage, etc. However, some drugs have certain toxicity when used alone, and have poor specificity, low bioavailability and short half-life.

[0003] Liver fibrosis is a pathological state and a compensatory repair response to chronic liver damage. The connective tissue in the liver shows abnormal proliferation. Liver fibrosis is the main intermediate link for chronic liver disease to further develop into cirrhosis. Therefore, preventing and reversing the development of cirrhosis and promoting the gradual decomposition and absorption of liver fibrosis can not only reduce the degree of liver damage, but also prevent the occurrence of cirrhosis. Most chronic liver diseases caused by various causes have liver fibrosis, of which 25%-40% eventually develop into cirrhosis or even liver cancer. Liver fibrosis is a reversible lesion, and cirrhosis is an irreversible lesion. Therefore, anti-fibrosis treatment is an important way to control the progression of liver fibrosis. Liver fibrosis is a dynamic reversible process and an important period for the reversal of chronic liver disease. Therefore, actively taking intervention measures in the liver fibrosis stage and blocking the process of liver fibrosis are of great significance to reduce the progression of chronic liver disease to cirrhosis or even liver cancer. At present, animal models of liver fibrosis are mainly mice, mainly including cholestatic liver fibrosis, irritant-induced liver fibrosis, metabolic liver fibrosis, alcoholic liver fibrosis and immune liver fibrosis. Primary biliary cholangitis (PBC) is a chronic bile stasis caused by bile duct dysplasia or bile acid metabolism disorder, which causes cell membrane lipid dissolution, permeability changes, and outflow of cell contents, eventually progressing to cholestatic liver fibrosis and cirrhosis. Ursodeoxycholic acid, obeticholic acid, etc. can be used to treat PBC and related liver fibrosis, while obeticholic acid is used for patients who do not respond adequately to or cannot tolerate ursodeoxycholic acid. However, according to the results of the Phase III clinical trial of obeticholic acid against NASH completed by Intercept in February 2019, obeticholic acid can improve the symptoms of liver fibrosis in 23% of patients (placebo can improve 12%). Although the efficacy is statistically significant, it is only increased by 11%, and its efficacy cannot reach the expected satisfactory level. Summary of the invention

[0004] The object of the present invention is to provide a complex of ursodeoxycholic acid or obeticholic acid and teprenone, which has the potential to prevent and treat liver fibrosis better than ursodeoxycholic acid or obeticholic acid alone. Specifically, the present invention adopts the following technical scheme:

[0005] First, the present invention provides a complex of ursodeoxycholic acid and teprenone, the structural formula of which is as follows (I):

[0006]

[0007] Secondly, the present invention also provides a method for preparing the above-mentioned complex of ursodeoxycholic acid and teprenone, which comprises 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 intermediate two.

[0008] As a preferred technical solution, the method for preparing the complex of ursodeoxycholic acid and teprenone may include the following steps:

[0009] (1) 20 g of teprenone was dissolved in tetrahydrofuran, and the temperature was lowered to below 0° C. under nitrogen protection, and 2-3 g of lithium aluminum hydride was added in batches. 2-3 g of water was added dropwise under an ice-water bath. After the solidification was completed, 50 wt % of sodium hydroxide was added, and anhydrous sodium sulfate was added and stirred to remove water. The mixture was filtered with diatomaceous earth, and washed with EA (ethyl acetate). The mother liquor was evaporated to dryness to obtain intermediate 1;

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

[0011] (3) Dissolve 20-22 g of ursodeoxycholic acid in DMF, add 20-22 g of intermediate II and potassium carbonate, react at room temperature for 10-20 h, and then react at 40-60° C. for 10-15 h; add water to the reaction solution, separate the layers with 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 mixing with silica gel column.

[0012] Thirdly, the present invention provides a complex of obeticholic acid and teprenone, the structural formula of which is as follows (II):

[0013]

[0014] Fourthly, the present invention also provides a method for preparing the above-mentioned complex of obeticholic acid and teprenone, which is obtained by hydrogenating teprenone with lithium aluminum hydride to obtain intermediate 1, and then reacting it with obeticholic acid.

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

[0016] (1) 40 g of teprenone was dissolved in tetrahydrofuran, and the temperature was lowered to below 0° C. under nitrogen protection, and 4-6 g of lithium aluminum hydride was added in batches until the reaction was complete; 4-6 g of water was added dropwise under an ice-water bath, and after solidification, 50 wt % of sodium hydroxide was added, and anhydrous sodium sulfate was added and stirred to remove water, and diatomaceous earth was used for filtration, EA was eluted, and the mother liquor was evaporated to dryness to obtain intermediate 1;

[0017] (2) Add 8-9 g of obeticholic acid, 8-9 g of intermediate 1, 5-6 g of DCC (N,N'-dicyclohexylcarbodiimide), 0.5-1 g of DMAP (4-dimethylaminopyridine), and DCM into a reaction bottle, stir, and react at room temperature for 20-30 h; filter with suction, and purify the mother liquor through a column.

[0018] Fifthly, the complex of ursodeoxycholic acid and teprenone and the complex of obeticholic acid and teprenone of the present invention can be further used as a drug for preventing and treating liver fibrosis.

[0019] In the present invention, teprenone is a terpene compound, which has a tissue repair effect, and can particularly enhance the anti-ulcer effect. It is a gastric mucosal protective drug, and is clinically mainly used to treat gastric ulcers and acute and chronic gastritis. Its administration in combination with ursodeoxycholic acid or obeticholic acid can significantly reduce the levels and contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and hydroxyproline (Hyp) in model mice, thereby enhancing the effect of ursodeoxycholic acid or obeticholic acid in treating liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Alanine aminotransferase (ALT) was used to analyze the liver function of mice.

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

[0022] Figure 3 To determine the hydroxyproline (Hyp) content in the liver of mice collagen deposition.

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

[0024] The following embodiments are further descriptions of the content of the present invention as an explanation of the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required 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 process

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

[0031] (1) Feed 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: Add teprenone and tetrahydrofuran to a reaction flask, stir to dissolve, cool to below 0°C under nitrogen protection, add lithium aluminum hydride in batches, and the reaction is complete according to TLC (PE / EA=10:1); add 2.8 g of water dropwise under an ice-water bath, solidify after adding, add 50 ml of THF, add 8.4 g of 50% sodium hydroxide, add anhydrous sodium sulfate, stir to remove water, filter with diatomaceous earth, rinse with EA, and evaporate the mother liquor to dryness to obtain 18.5 g of XHH007-I, MS (m / z): 332.57, yield 92.7%.

[0035] 1.2.2 Step 2: Preparation of XHH007-II

[0036] (1) Experimental consumables

[0037]

[0038]

[0039] (2) Operation process

[0040] Reaction: Add XHH007-I, methyl tert-butyl ether and pyridine to a reaction flask, stir and dissolve, cool to below 0°C under nitrogen protection, add phosphorus tribromide / methyl tert-butyl ether dropwise, drip completely within 1 hour, and react for 1 hour; add 50 ml of water to the reaction solution to dissolve the solid, separate the liquids, wash twice with 50 ml of 0.5% potassium carbonate, once with 50 ml of water, and once with 50 ml of saturated sodium chloride, dry with anhydrous sodium sulfate, filter with suction, and evaporate the solvent at room temperature to obtain 28 g of liquid, with a yield of 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 Lark XHH007-II 395.74 20.8g 1eq self made Potassium carbonate 138.21 8.7g 1.2eq Shanghai Strong DMF / 360mL / Xilong

[0044] (2) Operation process

[0045] Reaction: Add ursodeoxycholic acid and DMF into the reaction bottle, stir to dissolve, then add XHH007-II and potassium carbonate, react at room temperature (25°C) for 15h, and at 50°C for 12h;

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

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

[0054] (1) Experimental consumables

[0055] name Relative molecular weight Feeding amount Molar 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: Add teprenone and tetrahydrofuran to the reaction bottle, stir and dissolve, cool to below 0°C under nitrogen protection, add lithium aluminum hydride in batches, and wait for the reaction to be complete by TLC (PE / EA=10:1, iodine color development). Add 4.9g of water dropwise under an ice-water bath, solidify after the dropwise addition, add 15g of 50% sodium hydroxide, add anhydrous sodium sulfate, stir to remove water, filter with diatomaceous earth, rinse with EA, and evaporate the mother liquor to dryness to obtain 36.8g of XHH009-I.

[0058] 2.2.2 Step 2: Preparation of XHH009

[0059] (1) Experimental consumables

[0060] name Relative molecular weight Feeding amount Molar Ratio source Obeticholic acid 420.63 8.83g 1.0 Lark XHH009-I 332.57 8.5g 1.2eq self made DCC 206.33 5.29g 1.2eq Maclean DMAP 122.17 0.78g 0.3eq / DCM / 150mL / Cologne

[0061] (2) Experimental process

[0062] Reaction: Add obeticholic acid, XHH009-I, DCC (N,N'-dicyclohexylcarboximide), DMAP (4-dimethylaminopyridine), and DCM (dichloromethane) into a reaction bottle, stir, and react at room temperature (15°C) for 24 hours. After the reaction is completed, filter and purify the mother liquor by column (eluent PE / EA = 2:1) to obtain 3.7g of pure product, oil (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] ALT and AST in serum are key indicators for evaluating liver function damage. Hydroxyproline is one of the main components of collagen. Therefore, the content of hydroxyproline can be used to evaluate the collagen in liver tissue, and the liver function of mice can be analyzed by measuring alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0066] Preparation of CCl4 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 blank group was intraperitoneally injected with an equal amount of sterile corn oil solution. In the 5th week, the model group mice were randomly divided into model group (10% CCl4), CCl4+ursodeoxycholic acid group, CCl4+obeticholic acid group, CCl4+XHH007 group, and CCl4+XHH009 group, and the mice were gavaged at 10 mL / kg. The blank group and the solvent group were gavaged with an equal amount of sterile corn oil once a day for 4 consecutive weeks. After the treatment, the blood of the mice was collected by eyeball bleeding method, and the blood was centrifuged at 3000 rpm / min for 10 minutes after standing, and the supernatant was taken to obtain serum. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were detected according to the experimental operation method of the kit; the liver was taken and the hydroxyproline (Hyp) content was detected using a kit to evaluate the treatment effect of each group.

[0067] like Figure 1-3Compared with the blank control group, the ALT and AST levels in the serum of the model group mice were increased. Compared with the model group, the AST and ALT levels in the serum of the drug-treated group were decreased, but the XHH007 group and the XHH009 group had a more significant reduction effect on AST and ALT than the ursodeoxycholic acid group and the obeticholic acid group. In addition, through the detection of Hyp content, it can be found that compared with the blank control group, the Hyp content in the liver of the model group mice was increased. Compared with the model group, the ursodeoxycholic acid and obeticholic acid treatment groups had the effect of reducing the Hyp content, but the XHH007 group and the XHH009 group were able to more significantly reduce the Hyp content, thereby significantly inhibiting the collagen deposition in the liver of the CCl4 model mice. Therefore, compared with the administration of ursodeoxycholic acid alone or obeticholic acid alone, the XHH007 group and the XHH009 group had a significant reduction effect on 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-mentioned technical contents of the present invention are only for explanation and clarification to enable those skilled in the art to understand the technical essence of the present invention, so the technical contents are not used to limit the substantial protection scope of the present invention. The substantial protection scope of the present invention shall be subject to the claims. Those skilled in the art should know that any modification, equivalent substitution and improvement based on the substantial spirit of the present invention shall be within the substantial protection scope of the present invention.

Claims

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

2. A method for preparing the complex of ursodeoxycholic acid and teprenone according to claim 1, which comprises hydrogenating teprenone with lithium aluminum hydride to obtain an intermediate 1, brominating it with phosphorus tribromide to obtain an intermediate 2, and then reacting it with ursodeoxycholic acid to obtain the intermediate 2.

3. The preparation method according to claim 2, characterized in that: The steps include: (1) 20 g of teprenone was dissolved in tetrahydrofuran, and the temperature was lowered to below 0° C. under nitrogen protection, and 2-3 g of lithium aluminum hydride was added in batches. 2-3 g of water was added dropwise under an ice-water bath. After the addition was completed and solidified, 50 wt % of sodium hydroxide was added, and anhydrous sodium sulfate was added and stirred to remove water. The mixture was filtered with diatomaceous earth, washed with ethyl acetate, and the mother liquor was evaporated to dryness to obtain intermediate 1; (2) Add 18-19 g of intermediate 1, 50-150 mL of methyl tert-butyl ether and 1-2 g of pyridine to a reaction flask, stir to dissolve, cool to below 0° C. under nitrogen protection, dropwise add 9-10 g of methyl tert-butyl ether solution containing phosphorus tribromide within 1 hour, and react for 1-2 hours; add water to the reaction solution to dissolve the solid, separate the liquids, wash with potassium carbonate solution, wash with water, wash with saturated sodium chloride, dry with anhydrous sodium sulfate, filter with suction, and evaporate the solvent at room temperature to obtain intermediate 2; (3) Dissolve 20-22 g of ursodeoxycholic acid in DMF, add 20-22 g of intermediate II and potassium carbonate, react at room temperature for 10-20 h, and then react at 40-60° C. for 10-15 h; add water to the reaction solution, separate the layers with 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 mixing with silica gel column.

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

5. A method for preparing the complex of obeticholic acid and teprenone according to claim 4, which comprises hydrogenating teprenone with lithium aluminum hydride to obtain an intermediate 1, which is then reacted with obeticholic acid to obtain the complex.

6. The preparation method according to claim 5, characterized in that: The steps include: (1) 40 g of teprenone was dissolved in tetrahydrofuran, and the temperature was lowered to below 0° C. under nitrogen protection, and 4-6 g of lithium aluminum hydride was added in batches. 4-6 g of water was added dropwise under an ice-water bath. After the addition was completed and solidified, 50 wt % of sodium hydroxide was added, and anhydrous sodium sulfate was added and stirred to remove water. The mixture was filtered with diatomaceous earth, washed with EA, and the mother liquor was evaporated to dryness to obtain intermediate 1; (2) Add 8-9 g of obeticholic acid, 8-9 g of intermediate 1, 5-6 g of DCC (N,N'-dicyclohexylcarbocyanine), 0.5-1 g of DMAP (4-dimethylaminopyridine), and dichloromethane into a reaction flask, stir, and react at room temperature for 20-30 h; filter with suction, and purify the mother liquor through a column to obtain the product.

7. Use of the complex of ursodeoxycholic acid and teprenone as claimed in claim 1 or obtained by the preparation method according to any one of claims 2 to 3 in the preparation of drugs for preventing and treating liver fibrosis.

8. Use of the complex of obeticholic acid and teprenone according to claim 4 or obtained by the preparation method according to any one of claims 5 to 6 in the preparation of drugs for preventing and treating liver fibrosis.

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