CN117384113A - Several compounds with FXR agonistic activity - Google Patents

Abstract

The invention belongs to the technical field of medicines, and relates to a compound with FXR (FXR) agonistic activity or pharmaceutically acceptable salts thereof, wherein the compound is 1- (1-adamantanecarbonyl) -4- (5-hydroxy-2-methyl) phenylpiperazine or 1- (1-adamantanecarbonyl) -4- (5-amino-2-methyl) phenylpiperazine. The invention also relates to pharmaceutical preparations of the compounds and application thereof in preparing medicaments for treating and/or preventing fatty liver, liver cirrhosis, hepatitis, liver failure, cholestasis, cholelithiasis, bile acid disorders, hypercholesteremia, atherosclerosis, arteriosclerosis, fibrosis related diseases, thrombus, myocardial infarction, stroke, type 1 or type 2 diabetes and clinical complications thereof, hyperproliferative diseases, neoplastic diseases, inflammatory intestinal diseases and other related diseases mediated by FXR receptors.

Description

Several compounds with FXR agonistic activity

Technical field

The invention belongs to the field of medical technology and relates to 1-(1-adamantanecarbonyl)-4-substituted phenylpiperazine compounds or pharmaceutically acceptable salts thereof and their use in the treatment and/or prevention of FXR receptor-mediated Used in medicines for non-alcoholic fatty liver disease, primary biliary cirrhosis, lipid metabolism disorders, diabetic complications, malignant tumors and other related diseases.

Background technique

Farnesoid FXR has a typical nuclear receptor structure, including a highly conserved DNA-binding domain (DBD) at the N-terminus, a ligand-binding domain (LBD) at the C-terminus that allows receptor dimerization, and a ligand-independent transcriptional activation region at the N-terminus. (AF1), C-terminal ligand-dependent transcription function activation region (AF1) and hinge region. After activation, FXR can form a heterodimer with the retinoid X receptor (RXR) and induce the expression of its target gene SHP, resulting in the transcriptional inhibition 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 major negative regulators of bile acid synthesis and play an important role in regulating bile acid levels in the enterohepatic circulation. In addition, FXR is also directly or indirectly involved in several important metabolic pathways in the body, such as regulating glucose and lipid metabolism. Therefore, activation or inhibition of FXR plays an important role in metabolic homeostasis.

Steroids are the main ligands of FXR, among which chenodeoxycholic acid (CDCA) is the most potent endogenous agonist of FXR. Based on the physiological effects produced by activating FXR, FXR agonists are expected to treat a variety of 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 as a treatment for primary biliary cirrhosis (PBC) and is widely used to treat a variety of chronic cholestasis diseases. Obeticholic acid (OCA) is another bile acid FXR agonist approved for PBC. A phase III clinical trial of OCA has also been conducted for non-alcoholic steatohepatitis. However, many bile acid FXR agonists have been discontinued in preclinical or clinical trials due to serious adverse reactions such as itching, increased total cholesterol and low-density lipoprotein cholesterol levels, and decreased low-density lipoprotein cholesterol levels at effective doses. . Therefore, the development of new FXR receptor agonists is expected to meet clinical needs.

Contents of the invention

This patent provides compounds with novel molecular structures or pharmaceutically acceptable salts thereof. Such compounds can effectively stimulate FXR receptors, inhibit the transcriptional activity of the downstream molecule SREBP, and are FXR receptor agonists for 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 provide possibilities.

In order to solve the technical problems of the present invention, the present invention provides the following technical solutions:

In the first aspect, the compound provided by the invention is:

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

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

In a second aspect, the present invention provides a pharmaceutical composition, which contains the above-mentioned compound or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients.

In a third aspect, the present invention provides the use of the above-mentioned compound or a pharmaceutically acceptable salt thereof in the preparation of farnesoid X receptor agonist.

In a fourth aspect, the present invention provides the use of the above compounds or pharmaceutically acceptable salts thereof for treating and/or preventing FXR-mediated diseases and related diseases, wherein the diseases are selected from fatty liver, cirrhosis, hepatitis, Liver failure, cholestasis, gallstone disease, bile acid disorders, 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.

Preferably, the fatty liver is selected from alcoholic fatty liver and non-alcoholic fatty liver; the cirrhosis is selected from primary biliary cirrhosis, primary bile duct cirrhosis, and the hepatitis is selected from hyperlipidemia chronic Hepatitis disease, chronic hepatitis, non-viral hepatitis, alcoholic steatohepatitis, non-alcoholic steatohepatitis; the cholestasis is selected from benign intrahepatic cholestasis, progressive familial intrahepatic cholestasis, extrahepatic cholestasis, drugs Induced cholestasis, cholestasis of pregnancy, cholestasis associated with gastrointestinal nutrition, extrahepatic cholestasis; the diabetic complications are selected from diabetic nephropathy, diabetic neuropathy, and diabetic retinopathy; the neoplastic disease Selected from hepatocellular carcinoma, colon adenoma, polyposis, colon adenocarcinoma, breast cancer, pancreatic cancer, esophageal cancer and other forms of neoplastic diseases of the gastrointestinal tract and liver.

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

For the first time, the experiment discovered a new structural type of FXR agonist, which can inhibit the transcriptional activity of the downstream molecule SREBP and is used to treat 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 provide possibilities. Since this structural type is different from reported FXR inhibitors, it has the value of in-depth research and potential for clinical application.

Description of the drawings

Figure 1: Dose-response curves and EC ₅₀ of compounds Q1 and Q2 stimulating FXR

The results showed that the EC ₅₀ values of Q1 and Q2 were 2.90 μM and 0.79 μM respectively.

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

The results showed that Q2 could significantly reduce the levels of TC and LDL-C in the serum of high-fat fed C57BL/6J obese mice.

Figure 3: Effect of compound Q2 on total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and alanine aminotransferase (ALT) in the blood of high-fat fed C57 obese mice

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

Figure 4: Effect of compound Q2 on fasting blood glucose and area under the blood glucose curve (AUC) of insulin tolerance test (ITT) in C57 obese mice fed with high fat

The results showed that Q2 (100mg/kg) significantly reduced fasting blood glucose, and Q2 (100mg/kg) had the effect of reducing blood glucose 40min, 90min after insulin administration and the area under the blood glucose curve (AUC) in the ITT experiment.

Detailed ways

The present invention will be further described below with reference to specific examples, but the examples are only used to illustrate the present invention, rather than limit the present invention. Unless otherwise stated, the experimental methods used in the examples are conventional methods; unless otherwise stated, the materials and reagents used are commercially available reagents and materials.

1. Preparation and detection of new compounds

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

Add 2-amino-4-chlorophenol (1g, 1eq), bis(2-chloroethyl)amine hydrochloride (1.48g, 1.5eq), potassium iodide (1.28g, 1.1eq), and carbonic acid to the round-bottomed flask. Potassium (1.06g, 1.1eq) and 20mL xylene, the reaction solution was heated to reflux at 150°C for 10h, 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. Separate by silica gel column chromatography, and remove the solvent by rotary evaporation to obtain yellow-brown intermediate 1 (1.16g, yield: 78%).

Add 1-adamantanecarboxylic acid (500mg, 1eq), HATU (1.16g, 1.1eq) and 30mL dichloromethane to the round-bottomed flask, then add triethylamine (962μL, 2.5eq), and stir the reaction solution at room temperature for 10 minutes. , then intermediate 1 (706 mg, 1.2 eq) was added, and the reaction solution was continued to stir at room temperature for 2 h. The reaction solution was washed with 1M HCl aqueous solution, saturated sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, and layered on a silica gel column. 1-(1-adamantanecarbonyl)-4-(5-hydroxy-2-methyl)phenylpiperazine (Q1) (758 mg, yield: 73%) was obtained by analysis.

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).

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

Add 4-chloro-2-fluoronitrobenzene (1g, 1eq), piperazine (1.48g, 3eq) and 30mL isopropyl alcohol to the round-bottomed flask. Stir the reaction solution at room temperature for 2h. Remove the solvent by rotary evaporation and add Extract with ethyl acetate and water, wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, and remove the solvent by rotary evaporation to obtain yellow intermediate 2 (1.17g, yield: 85%).

Add 1-adamantanecarboxylic acid (500mg, 1eq), HATU (1.16g, 1.1eq) and 30mL dichloromethane to the round-bottomed flask, then add triethylamine (962μL, 2.5eq), and stir the reaction solution at room temperature for 10 minutes. , then intermediate 2 (803 mg, 1.2 eq) was added, and the reaction solution was continued to stir at room temperature for 2 h. The reaction solution was washed with 1M HCl aqueous solution, saturated sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, and layered on a silica gel column. The yellow intermediate 3 (840 mg, yield: 75%) was isolated by analysis.

Add yellow intermediate 3 (840 mg, 1 eq), zinc powder (1.33 g, 10 eq), ammonium chloride (1.10 g, 10 eq) and 20 mL dichloromethane to the round bottom flask. The reaction solution is stirred at room temperature for 2 hours and rotary evaporated. The solvent was removed, and ethyl acetate and saturated sodium carbonate solution were added for extraction. 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%).

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

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,

4H), 2.90–2.78 (m, 4H), 2.07–1.96 (m, 9H), 1.72 (dd, J = 21.8, 9.7Hz, 6H). 2. Determination of biological activity

Evaluation of biological activity of compounds

Experimental Example 1: Preliminary screening of farnesoid X receptor (FXR) activity

1. Method: Genetic engineering technology was used to construct the reporter gene plasmid systems of PCMX-Gal4-FXRLBD and Peak12-Gal4UAS-Luci. 293T cells were transiently transfected with PCMX-Gal4-FXRLBD/Peak12-Gal4UAS-luci gene plasmid system, and different concentrations of FXR receptor agonist drugs were added to verify the responsiveness and specificity of FXR receptor agonist drugs in the cell model. That is, the expression level and enzyme activity of the reporter gene luciferase reflect the activation activity of the FXR receptor, DMSO is used as a blank control, and the ratio of the fluorescence intensity of the reporter gene luciferase after compound treatment to the fluorescence intensity after DMSO treatment is measured. Compound's agonistic intensity on FXR. The initial screening concentration is 10 μM.

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

Experimental Example 2: EC ₅₀ determination of compound agonistically activated FXR transcriptional activity

1. Method: Use the reporter gene plasmid system to transfect 293T cells, add FXR test compounds at different concentrations, and use the enzyme activity of the reporter gene luciferase to reflect the activation activity of FXR. According to the value, use GraphPadPrism 6 software to analyze the data. _EC50 of the compound.

2. The dose-effect curve and EC ₅₀ of the compound stimulating FXR are shown in Figure 1. The results showed that the EC ₅₀ values of Q1 and Q2 were 2.90 μM (Figure 1A) and 0.79 μM (Figure 1B) respectively.

Experimental Example 3: Effect of compound Q2 on serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in high-fat fed C57 obese mice

1. Method: Male 11-week-old C57BL/6J mice with a size of 28-32g were fed with high-fat diet for 12 weeks, and 10 mice of the same age were fed with normal diet as the normal control group (Normal) and high-fat diet. The fed mice reached a weight of about 45g after 12 weeks. They were randomly divided into 2 groups based on body weight, random blood sugar, fasting blood sugar, 40-minute blood sugar drop rate, serum triglyceride content, and total cholesterol content, namely the model group (Control) and Q2. group (100mg/kg), Q2 was fully ground, 0.5% CMC-Na was fully dissolved and then diluted to volume, administered once a day by gavage, and blood was collected from the tail tip in the third week of administration to measure the TC and LDL-C contents in the serum. .

2. The experimental results are shown in Figure 2. Q2 can reduce the levels of TC and LDL-C in the serum of high-fat fed C57BL/6J obese mice.

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

1. Method: Male 11-week-old C57BL/6J mice with a size of 28-32g were fed with high-fat diet for 12 weeks, and 10 mice of the same age were fed with normal diet as the normal control group (Normal) and high-fat diet. The fed mice reached a weight of about 45g after 12 weeks, and were divided into two groups according to body weight, random blood sugar, fasting blood sugar, 40-min blood sugar drop rate, serum triglyceride content, and total cholesterol content, which were the model group (Control). ), Q2 group (100mg/kg). Q2 was fully ground, 0.5% CMC-Na was fully dissolved and the volume was adjusted, and it was administered by gavage once a day. The animals were killed after 6 weeks of gavage administration, and the liver and blood were collected to measure the TC and LDL-C contents in the liver and blood. Moderate ALT levels.

2. The experimental results are shown in Figure 3. Q2 (100mg/kg) can significantly reduce the levels of TC, LDL-C in the liver and ALT in the blood.

Experimental Example 5: Effect of compound Q2 on fasting blood glucose and insulin tolerance in C57 obese mice fed with high fat

1. Method: Male 11-week-old C57BL/6J mice with a size of 28-32g were fed with high-fat diet for 12 weeks, and 10 mice of the same age were fed with normal diet as the normal control group (Normal) and high-fat diet. After 12 weeks, the mice were fed and their weight reached about 45g, and they were divided into groups. Q2 was fully ground, 0.5% CMC-Na was fully dissolved and the volume was adjusted, and it was administered once a day by intragastric administration. After 5 weeks of intragastric administration, blood was collected from the tip of the tail to measure fasting blood glucose and blood glucose levels at 40 and 90 minutes after subcutaneous injection of insulin (insulin tolerance test ,ITT).

2. The experimental results are shown in Figure 4. Q2 (100mg/kg) significantly reduces fasting blood glucose. Q2 (100mg/kg) has the effect of reducing blood glucose 40min and 90min after insulin administration and the area under the blood glucose curve (AUC) in the ITT experiment. .

Table 1: Agonistic activity of representative compounds at the farnesoid X receptor at a concentration of 10 μM (% activity relative to the positive drug OCA)

Claims (6)

1. 1- (1-adamantanecarbonyl) -4-substituted phenylpiperazine compounds or pharmaceutically acceptable salts thereof, wherein the compounds are selected from the group consisting of:

q1:1- (1-adamantanecarbonyl) -4- (5-hydroxy-2-methyl) phenylpiperazine;

q2:1- (1-adamantanecarbonyl) -4- (5-amino-2-methyl) phenylpiperazine.

2. A pharmaceutical composition comprising a 1- (1-adamantanecarbonyl) -4-substituted phenylpiperazine compound of claim 1 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

3. Pharmaceutical composition according to claim 2, characterized in that it is selected from the group consisting of injections, tablets, pills, capsules, suspensions or emulsions.

4. Use of a compound of claim 1, or a pharmaceutically acceptable salt thereof, for the preparation of a farnesoid X receptor agonist.

5. The use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment and/or prophylaxis of FXR mediated diseases selected from fatty liver, liver cirrhosis, hepatitis, liver failure, cholestasis, cholelithiasis, bile acid disorders, 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.

6. Use according to claim 5, characterized in that the fatty liver is selected from alcoholic fatty liver, non-alcoholic fatty liver; the liver cirrhosis is selected from primary biliary cirrhosis, and the hepatitis is selected from hyperlipidemia chronic hepatitis, non-viral hepatitis, alcoholic steatohepatitis, and non-alcoholic steatohepatitis; the cholestasis is selected from benign intrahepatic cholestasis, progressive familial intrahepatic cholestasis, extrahepatic cholestasis conditions, drug-induced cholestasis, gestational cholestasis, cholestasis associated with gastrointestinal nutrition, extrahepatic cholestasis conditions; the diabetic complications are selected from diabetic nephropathy, diabetic neuropathy, and diabetic retinopathy; the neoplastic disease is selected from the group consisting of hepatocellular carcinoma, colon adenoma, polyposis, colon adenocarcinoma, breast cancer, pancreatic cancer, esophageal cancer, and other forms of gastrointestinal and liver neoplastic disease.