Several compounds having fxr agonistic activity

Abstract

The application belongs to the technical field of medicine, and relates to a kind of compounds with FXR agonistic activity or its pharmaceutically acceptable salt, the compound is 1-(1-adamantane carbonyl)-4-(5-hydroxy-2-methyl) phenyl piperazine, 1-(1-adamantane carbonyl)-4-(5-amino-2-methyl) phenyl piperazine.The application further relates to pharmaceutical preparation of these compounds and application in drug for treating and / or preventing related diseases of FXR receptor-mediated fatty liver, liver cirrhosis, hepatitis, liver failure, cholestasis, cholelithiasis, bile acid disorder, hypercholesterolemia, atherosclerosis, arteriosclerosis, fibrosis-related diseases, thrombosis, myocardial infarction, stroke, type 1 or type 2 diabetes and its clinical complications, hyperproliferative diseases, neoplastic diseases and inflammatory bowel diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to 1-(1-adamantanecarbonyl)-4-substituted phenylpiperazine compounds or pharmaceutically acceptable salts thereof and their use in medicaments for the treatment and / or prevention of FXR receptor-mediated non-alcoholic fatty liver disease, primary biliary cirrhosis, lipid metabolism disorders, diabetic complications, and malignant tumors. Background Technology

[0002] The farnesoid X receptor (FXR) is a distinctive member of the metabolic subfamily of nuclear receptor superfamily, a transcription factor expressed in various tissues including the liver, intestine, adipose tissue, and kidney. FXR possesses a typical nuclear receptor structure, including a highly conserved N-terminal DNA-binding domain (DBD), a C-terminal ligand-binding domain (LBD) that allows receptor dimerization, an N-terminal ligand-independent activation region (AF1), a C-terminal ligand-dependent activation region (AF1), and a hinge region. Upon activation, FXR forms a heterodimer with the retinol X receptor (RXR) and induces the expression of its target gene SHP, leading to transcriptional repression of CYP7A1 and LRH-1. FXR can also stimulate the synthesis of FGF-19 and inhibit the expression of CYP7A1 and CYP8B1 through the FGFR4 pathway in hepatocytes. The FXR / SHP and FXR / FGF19 / FGFR4 pathways constitute the main negative regulators of bile acid synthesis, playing a crucial role in regulating bile acid levels in the enterohepatic circulation. Furthermore, FXR is directly or indirectly involved in several important metabolic pathways in vivo, such as regulating glucose and lipid metabolism. Therefore, activation or inhibition of FXR plays a vital role in metabolic homeostasis.

[0003] Steroids are the main ligands for FXRs, with chenodeoxycholic acid (CDCA) being the most potent endogenous FXR agonist. Based on the physiological effects of FXR activation, FXR agonists hold promise for treating various metabolic diseases, such as cholestasis, liver fibrosis, inflammatory bowel disease, type 2 diabetes, atherosclerosis, and erectile dysfunction. Ursodeoxycholic acid (UDCA) is an FXR agonist approved by the FDA for the treatment of primary biliary cirrhosis (PBC) and is widely used to treat various chronic cholestatic diseases. Obeticholic acid (OCA) is another bile acid FXR agonist approved for PBC. A phase III clinical trial of OCA was also conducted for non-alcoholic steatohepatitis (NASH). However, due to serious adverse reactions at effective doses, such as itching, increased total cholesterol and LDL cholesterol levels, and decreased LDL cholesterol levels, many bile acid FXR agonists have been discontinued in preclinical or clinical trials. Therefore, the development of novel FXR receptor agonists holds promise for meeting clinical needs. Summary of the Invention

[0004] This patent provides compounds with novel molecular structures or pharmaceutically acceptable salts thereof, which can effectively activate FXR receptors and inhibit the transcriptional activity of downstream molecule SREBP, thus providing the possibility for the use of FXR receptor agonists in the treatment of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, primary biliary cirrhosis, primary sclerosing cholangitis, lipid metabolism disorders, diabetic complications, and malignant tumors.

[0005] To solve the technical problem of this invention, the present invention provides the following technical solution:

[0006] In a first aspect, the compound provided by the present invention is:

[0007] Q1: 1-(1-adamantanecarbonyl)-4-(5-hydroxy-2-methyl)phenylpiperazine;

[0008] Q2: 1-(1-adamantanecarbonyl)-4-(5-amino-2-methyl)phenylpiperazine.

[0009] In a second aspect, the present invention provides a pharmaceutical composition comprising the above-described compound or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients.

[0010] Thirdly, the present invention provides the use of the above-mentioned compound or a pharmaceutically acceptable salt thereof in the preparation of farnesoid X receptor agonists.

[0011] Fourthly, the present invention provides the use of the above-mentioned compounds or pharmaceutically acceptable salts thereof for the treatment and / or prevention of FXR-mediated diseases and related diseases selected from fatty liver, cirrhosis, hepatitis, liver failure, cholestasis, gallstones, bile acid disorders, hypercholesterolemia, atherosclerosis, arteriosclerosis, fibrosis-related diseases, thrombosis, myocardial infarction, stroke, type 1 or type 2 diabetes and its clinical complications, proliferative diseases, neoplastic diseases and inflammatory bowel diseases.

[0012] Preferably, the fatty liver is selected from alcoholic fatty liver and non-alcoholic fatty liver; the cirrhosis is selected from primary biliary cirrhosis and primary bile duct cirrhosis; the hepatitis is selected from hyperlipidemic chronic hepatitis, chronic hepatitis, non-viral hepatitis, alcoholic fatty liver, and non-alcoholic fatty liver; the cholestasis is selected from benign intrahepatic cholestasis, progressive familial intrahepatic cholestasis, extrahepatic cholestasis, drug-induced cholestasis, pregnancy-related cholestasis, cholestasis related to gastrointestinal nutrition, and extrahepatic cholestasis; the diabetic complication is selected from diabetic nephropathy, diabetic neuropathy, and diabetic retinopathy; and the neoplastic disease is selected from hepatocellular carcinoma, colonic adenoma, polyposis, colonic adenocarcinoma, breast cancer, pancreatic cancer, esophageal cancer, and other forms of gastrointestinal and liver neoplastic diseases.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] This experiment marks the first discovery of a novel structural type of FXR agonist that inhibits the transcriptional activity of the downstream molecule SREBP, offering potential applications for the treatment of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, primary biliary cirrhosis, primary sclerosing cholangitis, lipid metabolism disorders, type 1 or type 2 diabetes and its clinical complications, and malignant tumors. Because this structural type differs from previously reported FXR inhibitors, it warrants further investigation and has significant potential for clinical application. Attached Figure Description

[0015] Figure 1 Dose-response curves and EC50 of compounds Q1 and Q2 activating FXR 50

[0016] The results show that the EC in Q1 and Q2 50 The values ​​were 2.90 μM and 0.79 μM, respectively.

[0017] Figure 2 Effects of compound Q2 on serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in high-fat fed obese C57 mice

[0018] The results showed that Q2 significantly reduced the levels of TC and LDL-C in the serum of obese C57BL / 6J mice fed a high-fat diet.

[0019] Figure 3 Effects of compound Q2 on total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and alanine aminotransferase (ALT) in the liver and blood of obese C57 mice fed a high-fat diet.

[0020] The results showed that Q2 (100 mg / kg) significantly reduced the levels of TC, LDL-C in the liver and ALT in the blood.

[0021] Figure 4 Effects of compound Q2 on fasting blood glucose and area under the glucose curve (AUC) in insulin tolerance test (ITT) in high-fat fed obese C57 mice

[0022] The results showed that Q2 (100 mg / kg) significantly reduced fasting blood glucose, and Q2 (100 mg / kg) reduced blood glucose and area under the blood glucose curve (AUC) at 40 min and 90 min after insulin administration in the ITT experiment. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments. However, the embodiments are only for illustrating the present invention and are not intended to limit the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0024] I. Preparation and Detection of New Compounds

[0025] Example 1: 1-(1-adamantanecarbonyl)-4-(5-hydroxy-2-methyl)phenylpiperazine (Q1)

[0026]

[0027] 2-Amino-4-chlorophenol (1 g, 1 eq), di(2-chloroethyl)amine hydrochloride (1.48 g, 1.5 eq), potassium iodide (1.28 g, 1.1 eq), potassium carbonate (1.06 g, 1.1 eq) and 20 mL xylene were added to a round-bottom flask. The reaction mixture was heated under reflux at 150 °C for 10 h. The solvent was removed by rotary evaporation. Ethyl acetate and water were added for extraction. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography. The solvent was removed by rotary evaporation to give yellow-brown intermediate 1 (1.16 g, yield: 78%).

[0028] Add 1-adamantanecarboxylic acid (500 mg, 1 eq), HATU (1.16 g, 1.1 eq), and 30 mL of dichloromethane to a round-bottom flask, then add triethylamine (962 μL, 2.5 eq). Stir the reaction mixture at room temperature for 10 min, then add intermediate 1 (706 mg, 1.2 eq). Continue stirring the reaction mixture at room temperature for 2 h. Wash the reaction mixture with 1 M HCl aqueous solution, saturated sodium bicarbonate solution, and saturated brine, respectively. Dry the mixture with anhydrous sodium sulfate and separate the product by silica gel column chromatography to obtain 1-(1-adamantanecarbonyl)-4-(5-hydroxy-2-methyl)phenylpiperazine (Q1) (758 mg, yield: 73%).

[0029] White solid. ESI-MS (m / z): 375.19 [M+H] + . 1 H NMR (500MHz, Chloroform-d) δ7.10–7.04(m,2H),6.90(d,J=8.6Hz,1H),3.87(s,4H),2.86(t,J=5.0Hz,4H),2.08–2.00(m,9H),1.74(s,6H).

[0030] Example 2: 1-(1-adamantanecarbonyl)-4-(5-amino-2-methyl)phenylpiperazine (Q2)

[0031]

[0032] 4-Chloro-2-fluoronitrobenzene (1 g, 1 eq), piperazine (1.48 g, 3 eq) and 30 mL isopropanol were added to a round-bottom flask. The reaction mixture was stirred at room temperature for 2 h. The solvent was removed by rotary evaporation. Ethyl acetate and water were added for extraction. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation to give yellow intermediate 2 (1.17 g, yield: 85%).

[0033] Add 1-adamantanecarboxylic acid (500 mg, 1 eq), HATU (1.16 g, 1.1 eq), and 30 mL of dichloromethane to a round-bottom flask, then add triethylamine (962 μL, 2.5 eq). Stir the reaction mixture at room temperature for 10 min, then add intermediate 2 (803 mg, 1.2 eq). Continue stirring the reaction mixture at room temperature for 2 h. Wash the reaction mixture with 1 M HCl aqueous solution, saturated sodium bicarbonate solution, and saturated brine, respectively. Dry the mixture with anhydrous sodium sulfate and separate it by silica gel column chromatography to obtain yellow intermediate 3 (840 mg, yield: 75%).

[0034] Yellow intermediate 3 (840 mg, 1 eq), zinc powder (1.33 g, 10 eq), ammonium chloride (1.10 g, 10 eq) and 20 mL of dichloromethane were added to a round-bottom flask. The reaction mixture was stirred at room temperature for 2 h. The solvent was removed by rotary evaporation. The mixture was extracted with ethyl acetate and saturated sodium carbonate solution. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography to obtain 1-(1-adamantanecarbonyl)-4-(5-amino-2-methyl)phenylpiperazine (Q2) (450 mg, yield: 58%).

[0035] White solid. ESI-MS (m / z): 374.25 [M+H] + .1H NMR(500MHz,Chloroform-d)δ

[0036] 7.20(s,1H),6.91(q,J=4.5,3.8Hz,1H),6.87–6.83(m,1H),3.89(d,J=72.3Hz,

[0037] 4H), 2.90–2.78 (m, 4H), 2.07–1.96 (m, 9H), 1.72 (dd, J = 21.8, 9.7 Hz, 6H). II. Bioactivity Assay

[0038] Bioactivity evaluation of compounds

[0039] Experimental Example 1: Preliminary Screening of Farnesol X Receptor (FXR) Activity

[0040] 1. Methods: Reporter gene plasmid systems PCMX-Gal4-FXRLBD and Peak12-Gal4UAS-Luci were constructed using genetic engineering techniques. 293T cells were transiently transfected with the PCMX-Gal4-FXRLBD / Peak12-Gal4UAS-luci plasmid system, and different concentrations of FXR receptor agonist drugs were added to verify the reactivity and specificity of the FXR receptor agonist drugs in the cell model. Specifically, the expression level and activity of reporter gene luciferase reflected the activation activity of the FXR receptor, with DMSO as a blank control. The agonistic effect of the compound on FXR was measured by the ratio of the fluorescence intensity of the reporter gene luciferase after compound treatment to that after DMSO treatment. The initial screening concentration was 10 μM.

[0041] 2. The agonistic activity of the compound at a concentration of 10 μM on FXR is shown in Table 1.

[0042] Experimental Example 2: Compounds activate EC transcriptional activity of FXR 50 Measurement

[0043] 1. Methods: 293T cells were transfected using a reporter gene plasmid system. Different concentrations of the FXR test compound were added. The activation activity of FXR was reflected by the enzyme activity of the reporter gene luciferase. The EC50 of the compound was calculated using GraphPadPrism 6 software. 50 .

[0044] 2. Dose-response curves and EC50 of the compound FXR agonist 50 See Figure 1 The results show that the ECB in Q1 and Q2... 50 The values ​​were 2.90 μM ( Figure 1 A), 0.79μM ( Figure 1 B).

[0045] Experimental Example 3: Effects of Compound Q2 on Serum Total Cholesterol (TC) and Low-Density Lipoprotein Cholesterol (LDL-C) in High-Fat-Fed Obese C57 Mice

[0046] 1. Methods: Male 11-week-old C57BL / 6J mice, weighing 28-32g, were fed a high-fat diet for 12 weeks. Ten mice of the same age were fed a normal diet as a normal control group. After 12 weeks, the high-fat diet mice reached a weight of about 45g. Based on body weight, random blood glucose, fasting blood glucose, 40-minute blood glucose decrease rate, serum triglyceride content, and total cholesterol content, they were randomly divided into two groups: the model group (Control) and the Q2 group (100mg / kg). The Q2 was thoroughly ground, dissolved in 0.5% CMC-Na, and brought to a final volume. The mice were administered the drug by gavage once a day. Blood was collected from the tail tip at the third week of administration to measure the serum TC and LDL-C content.

[0047] 2. Experimental results are as follows Figure 2 As shown, Q2 can reduce the levels of TC and LDL-C in the serum of obese C57BL / 6J mice fed a high-fat diet.

[0048] Experiment Example 4: Effects of compound Q2 on total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and alanine aminotransferase (ALT) in the liver and blood of high-fat fed obese C57 mice

[0049] 1. Methods: Male 11-week-old C57BL / 6J mice, weighing 28-32g, were fed a high-fat diet for 12 weeks. Ten mice of the same age were fed a normal diet as a normal control group. After 12 weeks, the high-fat diet-fed mice reached a weight of approximately 45g and were randomly divided into two groups based on body weight, random blood glucose, fasting blood glucose, 40-minute blood glucose decrease rate, serum triglyceride content, and total cholesterol content: a model group (Control) and a Q2 group (100mg / kg). The Q2 group was thoroughly ground, dissolved in 0.5% CMC-Na, and brought to a final volume. It was administered by gavage once daily for 6 weeks. After gavage administration, the animals were sacrificed, and their livers and blood were collected to measure the TC and LDL-C content in the liver and the ALT level in the blood.

[0050] 2. Experimental results are as follows Figure 3 As shown, Q2 (100 mg / kg) can significantly reduce the levels of TC, LDL-C in the liver and ALT in the blood.

[0051] Experimental Example 5: Effects of Compound Q2 on Fasting Blood Glucose and Insulin Tolerance in High-Fat-Fed Obese C57 Mice

[0052] 1. Methods: Male C57BL / 6J mice, aged 11 weeks and weighing 28-32g, were fed a high-fat diet for 12 weeks. Ten mice of the same age were fed a normal diet as a control group. Mice fed the high-fat diet reached approximately 45g in weight after 12 weeks and were then divided into groups. Q2 was thoroughly ground, dissolved completely in 0.5% CMC-Na, and brought to a final volume. The drug was administered by gavage once daily. After 5 weeks of gavage administration, blood was collected from the tail tip to measure fasting blood glucose and blood glucose levels 40 and 90 minutes after subcutaneous insulin injection (insulin tolerance test, ITT).

[0053] 2. Experimental results are as follows Figure 4 As shown, Q2 (100 mg / kg) significantly reduced fasting blood glucose. Q2 (100 mg / kg) also reduced blood glucose and the area under the blood glucose curve (AUC) at 40 min and 90 min after insulin administration in the ITT experiment.

[0054] Table 1: Agonistaltic activity of representative compounds against farnesol X receptor at a concentration of 10 μM (percentage of activity relative to the positive control drug OCA)

[0055]

[0056]

[0057]

Claims

1. A class of compounds or pharmaceutically acceptable salts thereof, characterized in that, The compounds are selected from the following group: Q1： ； Q2： 。 2. A pharmaceutical composition, characterized in that, It includes the compound of claim 1 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

3. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutical composition is selected from injections, tablets, pills, capsules, suspensions, or emulsions.

4. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of farnesoid X receptor agonists.

5. The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment and / or prevention of FXR-mediated diseases and related diseases, wherein the diseases are selected from fatty liver, cirrhosis, hepatitis, liver failure, cholestasis, gallstones, bile acid disorders, hypercholesterolemia, arteriosclerosis, fibrosis-related diseases, thrombosis, myocardial infarction, stroke, type 1 or type 2 diabetes and its clinical complications, proliferative disorders and inflammatory bowel disease.

6. The use according to claim 5, characterized in that, The fatty liver is selected from alcoholic fatty liver and non-alcoholic fatty liver; the cirrhosis is selected from primary biliary cirrhosis and primary bile duct cirrhosis; the hepatitis is selected from chronic hepatitis, non-viral hepatitis, alcoholic steatohepatitis, and non-alcoholic steatohepatitis; the cholestasis is selected from benign intrahepatic cholestasis, progressive familial intrahepatic cholestasis, drug-induced cholestasis, pregnancy-related cholestasis, cholestasis related to gastrointestinal nutrition, and extrahepatic cholestasis; the diabetic complications are selected from diabetic nephropathy, diabetic neuropathy, and diabetic retinopathy; the proliferative diseases are selected from neoplastic diseases, and the neoplastic diseases are selected from hepatocellular carcinoma, colonic adenoma, polyposis, colonic adenocarcinoma, breast cancer, pancreatic cancer, and esophageal cancer.

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