Use of an acylpiperazine compound in the preparation of a medicament for preventing or treating metabolic syndrome

Abstract

The present invention relates to the field of biological medicine, and specifically relates to the use of a novel acyl piperazine compound, a urotensin receptor agonist, in the preparation of a medicament for treating and / or preventing metabolic syndrome / type 2 diabetes / obesity / diabetic complications. The compound of the present invention can specifically activate the urotensin receptor, and has the effects of significantly promoting the reduction of the triglyceride content in hepatocytes and reducing the fat accumulation in hepatocytes, and regulating glucose and lipid metabolism.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly relates to a new class of compounds having urotensin-2 receptor (UT) agonist activity, and uses thereof for preventing or treating pathophysiological diseases characterized by low expression or reduced activity of urotensin II or urotensin-like peptides. In particular, it relates to the application in the preparation of drugs for treating and / or preventing metabolic syndrome / type 2 diabetes / obesity / diabetes / liver fat deposition complications. Background Art

[0002] Urotensin II (U-II) is a polypeptide first isolated from the neurosecretory system of the goby fish (Gillichthys mirabilis), and then the polypeptide was also cloned in humans. U-II is a highly active endogenous urotensin-2 receptor (UT receptor) peptide agonist. The UT receptor is a G protein-coupled receptor that can transduce signals through Gαq. Peptides such as U-II have a cyclic structure and contain polypeptides with a length of 11-15 amino acids. Human U-II (hU-II) is an 11-amino acid polypeptide. The UT receptor, also known as GPR14, is involved in the regulation of vasoconstriction. In mammals, the mRNA of the U-II precursor (UTS2) and the UT receptor gene (UTS2R) are widely expressed in the central nervous system and peripheral tissues, including the brain, kidney, liver, lung, pancreas, skeletal muscle and other tissues, as well as vascular and cardiac cells. Consistent with the widespread distribution of U-II and the UT receptor, the urotensinergic system is associated with many pathological conditions, including atherosclerosis, heart failure, hypertension, kidney disease and diabetes.

[0003] With the in-depth study of the UⅡ system mechanism, drug development targeting it has become a key research focus, and some drugs have entered clinical trials. Currently, both agonists and antagonists of UTR have been discovered, with more research on antagonists, which can be classified into peptide and non-peptide types according to their chemical characteristics. The therapeutic indications targeted include cardiovascular diseases, diabetes, tumors, etc. Common UTR peptide antagonists are: urantide, UFP-803, BIM-23127, etc., among which urantide is the most commonly used. Urantide was initially developed for cardiovascular diseases as an indication, but due to unsatisfactory efficacy, it did not enter clinical trials. Currently, some studies have shown that urantide has a certain effect in improving diabetes. It can reverse the ROS production induced by U-II, restore the translocation of insulin-induced glucose transporter 4 (GLUT4) from the cytoplasm to the cell membrane in skeletal muscle, promote glucose uptake in skeletal muscle, and can also reduce the inflammatory response induced by U-II and improve the state of IR. Common UTR non-peptide antagonists are: GSK-1562590, AC-7954, GSK-1440115, palosuran, etc. Among them, palosuran is the most intensively and widely studied UTR inhibitor. It entered clinical trials in 2003 for the indication of diabetic nephropathy, but ultimately terminated in phase II clinical trials in 2007 due to unsatisfactory pharmacodynamics. There are literature reports that palosuran can improve nephropathy, cardiovascular diseases, lung injury, etc. in diabetic patients. Another study has shown that U-II is related to reduced insulin secretion and damage to the insulin signaling pathway, but palosuran did not show the effect of reversing this phenomenon, and the specific mechanism and reasons need further exploration. Generally speaking, drug research targeting UTR is currently mostly in a stagnant state, mostly due to the lack of obvious efficacy. Our research found that U-II itself can improve glucose and lipid metabolism, which explains from another aspect the reason for the poor effect of its antagonists, and also shows that urotensin receptor agonists play an important role in fat regulation and glucose tolerance regulation. Therefore, it is crucial to develop new drugs with urotensin receptor agonist effects to broaden the options for anti-obesity and type 2 diabetes.

[0004] Metabolic syndrome is a complex set of metabolic disorders, a pathological state in which multiple metabolic components are abnormally aggregated, and is a risk factor for diabetes and cardiovascular diseases. It has the following characteristics: ① Multiple metabolic disorders coexist, including obesity, hyperglycemia, hypertension, and dyslipidemia, which are the pathological basis of cardiovascular and cerebrovascular diseases and diabetes; ② There is a common pathological basis. Currently, it is generally believed that their common cause is insulin resistance and hyperinsulinemia caused by obesity, especially central obesity; ③ It can cause the accumulation of multiple diseases, such as hypertension, coronary heart disease, stroke, and even certain cancers, etc.

[0005] Obesity is a chronic metabolic disease caused by excessive accumulation of energy in adipose tissue, and is a major risk factor affecting cardiovascular and cerebrovascular diseases, diabetes, tumors, etc. Strategies such as restricting energy intake, such as dieting and taking weight loss drugs; or increasing energy consumption, such as exercise, make obese patients regain weight due to poor compliance or drug side effects, thus discouraging them from losing weight. Therefore, how to prevent and treat obesity, a global health problem, and find safe and effective treatment methods have become hot issues for medical and nutritional scientists at home and abroad to study.

[0006] Type 2 diabetes is a globally prevalent metabolic disease and the primary risk factor for cardiovascular diseases. Globally, there are approximately 300 million diabetes patients, and the number of patients remains high and is still rising, especially in recent years. In particular, in China, with the improvement of the living standards of the people, great changes have taken place in the diet structure, living habits, environmental conditions, etc. The prevalence of glucose metabolism disorders closely related to genetic, nutritional, metabolic, and environmental factors has increased sharply, and the development trend has not been effectively controlled. Complications such as heart, kidney, brain, eye blood vessels, and nerves caused by metabolic diseases not only seriously affect the quality of life of patients but also bring a heavy family and social medical burden. Therefore, the prevention and treatment of type 2 diabetes are major scientific issues currently faced in China, and exploring effective measures for preventing and treating glucose metabolism diseases has important scientific value and significance.

[0007] Currently, clinically, drugs for treating obesity mainly include appetite suppressants: central appetite-suppressing drugs and gastrointestinal hormones that regulate appetite. Those acting on peripheral fat synthesis and decomposition: those acting on the gastrointestinal tract to reduce fat absorption (lipase inhibitors and sodium-glucose cotransporter 2 inhibitors). Those acting on adipose tissue to reduce fat synthesis and promote release and decomposition. Drugs for treating type 2 diabetes mainly include: insulin and its analogs, sulfonylureas, biguanides, α-glucosidase inhibitors, thiazolidinedione derivatives, insulin secretagogues, traditional Chinese medicines, etc. Currently, there is still no weight loss product that can reduce metabolic syndrome and complications.

[0008] ((5-Nitro-2-furyl)[4-(4-nitrophenyl)-1-piperazinyl]-methanone (Compound 13739) described in the present invention has the molecular formula C 15 H 14 N4O6, a molecular weight of 346.29, and a predicted boiling point of 591.5 °C at 760 Torr. There is currently no research on the pharmacological activity of Compound 13739.

[0009] The compound 13739 described in the present invention is a new discovery obtained through experimental research. The new invention content mainly relates to the preparation of drugs and drug combinations. It provides clinical use for preventing or treating pathological physiological diseases characterized by low expression or reduced activity of urotensin II or urotensin-like peptides and their receptors, especially in the preparation of drugs for treating and / or preventing metabolic syndrome / type 2 diabetes / obesity / diabetes / fatty liver complications. At present, there is no report on the direct or indirect therapeutic effect of the use of compound 13739 in pathological physiological diseases characterized by low expression or reduced activity of urotensin II or urotensin-like peptides and their receptors, especially in the preparation of drugs for treating and / or preventing metabolic syndrome / type 2 diabetes / obesity / diabetes / fatty liver complications. Summary of the invention

[0010] The technical problem to be solved by the present invention is to provide a compound having urotensin receptor agonist activity for use in preparing a drug for treating and / or preventing metabolic syndrome / type 2 diabetes / obesity / diabetes / fatty liver complications.

[0011]

[0012] In order to solve the technical problem of the present invention, the present invention provides the following technical solutions:

[0013] (1) The present invention provides the use of a compound represented by formula (I) (i.e., compound 13739) in the preparation of a drug having urotensin receptor agonist activity.

[0014] (2) The present invention provides the use of a compound as shown in formula (I) in preventing or treating pathological physiological diseases characterized by low expression or reduced activity of urotensin II or urotensin-like peptide and its receptor.

[0015] (3) The present invention provides the use of a compound as shown in formula (I) in the preparation of a drug for treating and / or preventing metabolic syndrome. The metabolic syndrome includes obesity, diabetes, diabetic complications, fatty liver, dyslipidemia, and hypertension. The metabolic syndrome includes a pathological state in which the body's proteins, fats, and carbohydrates undergo metabolic disorders due to various reasons.

[0016] The diabetes described includes type 1 and type 2 diabetes; the obesity described includes obesity caused by congenital, acquired, and drug-induced reasons; the diabetic complications described refer to diabetic large and small vessel lesions; the fatty liver described includes hepatic fatty degeneration caused by various reasons; the dyslipidemia described includes high triglycerides, low high-density lipoproteinemia, and hypercholesterolemia; the hypertension described includes increased systolic and / or diastolic blood pressure, which may or may not be accompanied by functional or organic damage to the heart, brain, and kidneys.

[0017] Furthermore, the diabetes complications include diabetic nephropathy, diabetic peripheral circulatory dysfunction, diabetic peripheral neuropathy, diabetic eye disease, diabetic myopathy, and diabetes mellitus combined with hyperlipidemia.

[0018] (4) The present invention provides an application of a pharmaceutical composition in the preparation of a drug having urotensin receptor agonist activity, in the preparation of a drug for preventing or treating a pathophysiological or psychological disease characterized by low expression or reduced activity of urotensin II or urotensin-like peptide, and in the preparation of a drug for treating and / or preventing metabolic syndrome. The pharmaceutical composition is characterized in that it contains an effective dose of a compound represented by formula (I) and a pharmaceutical excipient; the pharmaceutical composition includes forms such as controlled release, sustained release preparations, and microsomal drug delivery systems.

[0019]

[0020]

[0021] The pharmaceutical composition can be prepared according to methods well known in the art. It can be combined with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants to form any dosage form suitable for human or animal use. The content of the compound of the present invention in its pharmaceutical composition is usually 0.1-95% (by weight).

[0022] The dosage of the pharmaceutical composition of the compound of the present invention can vary within a wide range depending on the nature and severity of the disease to be prevented or treated, the individual conditions of the patient or animal, the route of administration, and the dosage form. Generally speaking, the appropriate daily dosage range of the compound of the present invention is 0.001-150 mg / kg body weight, preferably 0.1-100 mg / kg body weight, more preferably 1-60 mg / kg body weight, and most preferably 2-30 mg / kg body weight. The above dosage can be administered in one dosage unit or divided into several dosage units, which depends on the doctor's clinical experience and the dosing regimen including the use of other therapeutic means. The compound or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs. When there is a synergistic effect between the compound of the present invention and other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0023] The compounds of the present invention or pharmaceutical compositions containing the same can be administered in unit dosage forms, and the administration routes can be enteral or parenteral, such as oral administration, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc. The dosage forms can be liquid dosage forms, solid dosage forms or semi-solid dosage forms. The liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o / w type, w / o type and multiple emulsions), suspensions, injections (including aqueous injections, powder injections and infusions), eye drops, nasal drops, lotions and liniments, etc.; the solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, pellets, dripping pills, suppositories, films, patches, aerosols (powder aerosols), sprays, etc.; the semi-solid dosage forms can be ointments, gels, pastes, etc. The compounds of the present invention can be made into ordinary preparations, as well as sustained-release preparations, controlled-release preparations, targeted preparations and various particulate drug delivery systems.

[0024] To prepare the compounds of the present invention into tablets, various excipients well-known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and glidants. The diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agents can be water, ethanol, isopropanol, etc.; the binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, etc.; the lubricants and glidants can be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc. The tablets can be further prepared into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multi-layer tablets. To prepare the dosage unit into capsules, the active ingredient, the compound of the present invention, can be mixed with a diluent and a glidant, and the mixture can be directly placed into hard capsules or soft capsules. Alternatively, the active ingredient, the compound of the present invention, can be first made into granules or pellets with a diluent, a binder, and a disintegrant, and then placed into hard capsules or soft capsules. The varieties of diluents, binders, wetting agents, disintegrants, and glidants used for preparing the tablets of the compounds of the present invention can also be used for preparing the capsules of the compounds of the present invention. To prepare the compounds of the present invention into injections, water, ethanol, isopropanol, propylene glycol, or their mixtures can be used as solvents, and appropriate solubilizers, cosolvents, pH adjusters, and osmotic pressure regulators commonly used in the art can be added. The solubilizers or cosolvents can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjusters can be phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulators can be sodium chloride, mannitol, glucose, phosphates, acetates, etc. When preparing freeze-dried powder injections, mannitol, glucose, etc. can also be added as supporting agents. In addition, if necessary, colorants, preservatives, fragrances, flavoring agents, or other additives can also be added to the pharmaceutical preparations. To achieve the purpose of medication and enhance the therapeutic effect, the drugs or pharmaceutical compositions of the present invention can be administered by any well-known administration methods.

[0025] Beneficial technical effects

[0026] 1. The compound (I) of the present invention can regulate the activity of urotensin receptors. The use of the compound of the present invention for regulating the activity of urotensin receptors is disclosed for the first time.

[0027] 2. Use of the compound (I) of the present invention for preventing or treating pathophysiological diseases characterized by low expression or reduced activity of urotensin II or urotensin-like peptide and their receptors. The report on the use of this compound in this regard is disclosed for the first time.

[0028] 3. Use of the compound (I) of the present invention for preparing drugs for treating and / or preventing metabolic syndrome / type 2 diabetes / obesity / diabetes / liver fatty acid complications. The use of this compound in this regard is disclosed for the first time, broadening the selection of drugs for related diseases.

[0029] 4. Currently internationally, patents on urotensin agonist effects in cardiovascular regulation have been disclosed (US Patent, Patent No. 4533654, Patent Date August 6, 1985). Our previous research first reported the effect of urotensin agonist in improving metabolic syndrome, obesity and type 2 diabetes (Use of a polypeptide for preparing drugs for preventing or treating metabolic syndrome. Application No. CN110038114A). There are no relevant papers or patents published internationally or domestically on the effect of compound 13739 on the urotensin system.

[0030] 5. The compound (I) of the present invention is used for preventing or treating pathophysiological diseases characterized by low expression or reduced activity of urotensin II or urotensin-like peptide and their receptors through a novel target. Use for preparing drugs for treating and / or preventing metabolic syndrome / type 2 diabetes / obesity / diabetes / liver fatty acid complications. It has obvious advantages when developed as a drug. Description of the Drawings

[0031] Figure 1 . Docking results of compound 13739 with the urotensin receptor protein model.

[0032] Figure 2 . ADMET prediction results of compound 13739.

[0033] Figure 3 . Compound 13739 can activate the urotensin receptor in a dose-dependent manner, and its half-maximal effective concentration is 3.70*10 - 8 M.

[0034] Figure 4 . The activation of the urotensin receptor by compound 13739 is specific and does not activate other GPCR receptors.

[0035] Figure 5 . Effect of compound 13739 on glucose uptake in HepG2 hepatocytes.

[0036] Figure 6. Effect of Compound 13739 on Lactate Release in HepG2 Hepatocytes.

[0037] Figure 7 . Effect of Compound 13739 on the Viability of HepG2 Hepatocytes.

[0038] Figure 8 . Compound 13739 Can Improve Lipid Accumulation in an Oleic Acid-Induced HepG2 Non-Alcoholic Fatty Liver Cell Model.

[0039] Figure 9 . Compound 13739 Can Reduce the Synthesis of Intracellular Triglycerides in Oleic Acid-Induced HepG2 Non-Alcoholic Fatty Liver Cells. Detailed Implementation Modes

[0040] The following further describes the use of Compound (I) in regulating the activity of urotensin receptors and preventing or treating pathophysiological diseases characterized by low expression or reduced activity of urotensin II or urotensin-like peptides and their receptors. Its use in the preparation of drugs for treating and / or preventing metabolic syndrome / type 2 diabetes / obesity / diabetes / fatty liver complications.

[0041] The following examples illustrate the present invention in more detail and are not any limitation to the present invention. Therefore, those skilled in the art who make non-essential improvements and adjustments to the implementation modes based on the above-mentioned invention content still fall within the protection scope of the present invention.

[0042] Example 1. Dissolution and Use of Compound 13739

[0043] Compound 13739 was purchased from Anjie Kai Biopharmaceutical Co., Ltd. (Wuhan, China), product number: ajcz0112 - 300mg, obtained by chemical synthesis method, and the LCMS purity > 97%. The compound powder was dissolved in DMSO to prepare a 10 mM solution. After aliquoting, it was stored at -80 °C for later use. When used, it was diluted to the corresponding concentration with physiological saline.

[0044] Example 2. Docking of Compound 13739 with the Urotensin Receptor Protein Model

[0045] Experimental method: Import the pdb file of the urotensin receptor protein structure and the sdf file of compound 13739 into Discovery Studio, and use the CDOCKER flexible docking method in Discovery Studio for protein-ligand docking. First, click "Prepare Ligands" and "Prepare Protein" to process the compound and the protein, define the protein as the receptor molecule, and predict the active site of the receptor structure; use the CDOCKER module in "Receptor-Ligand Interactions" to perform the docking operation of the protein and compound 13739, and set the corresponding parameters. After the program runs to completion, output the docking results and analyze the docking results.

[0046] Experimental results: Molecular docking is a theoretical simulation method for studying the interactions between molecules (such as ligands and receptors) and predicting their binding modes and affinities. Generally, the lower the energy when the conformation of the ligand-receptor binding is stable, the greater the possibility of the occurrence of the interaction. The binding energy of compound 13739 with the receptor protein is -18.18 kcal / mol, indicating that compound 13739 has a relatively high binding activity with the receptor. The two-dimensional planar diagram of the ligand-protein interaction generated by compound 13739 and the receptor is shown in Figure 1 , compound 13739 forms hydrogen bond interactions with the amino acid residues Arg168 and Lys163, forms hydrophobic binding Pi-Alkyl interactions with the amino acid residues Ala151 and Ala84, and forms van der Waals forces with multiple residues, increasing the interaction between the molecule and the protein. The results are shown in Figure 1 .

[0047] Example 3. Druggability evaluation of compound 13739

[0048] Experimental method: Use the ADMET module in Discovery Studio software to import the sdf file of compound 13739 into the DS software. In the "Small Molecules" module, select "Calculate Molecular Properties", click ADMET descriptors for parameter settings, click to run, and predict the absorption, distribution, metabolism, excretion, and toxicity of the compound to obtain data on the passive intestinal absorption, water solubility at 25°C, blood-brain barrier permeability, binding to human cytochrome CYP4502D6 enzyme, hepatotoxicity, plasma protein binding, etc. of compound 13739.

[0049] Experimental results: The ADMET prediction results are shown in Figure 2。The water solubility value logSW corresponding to Compound 13739 is -3.60, indicating that Compound 13739 has good water solubility. The inhibition number of Compound 13739 on CYP450 2D6 enzyme is 0.0054, and within the 0 level range, there is no inhibitory effect. The hepatotoxicity measurement value of Compound 13739 is 0.27, showing no hepatotoxicity. The plasma protein binding value of Compound 13739 is 2.18, indicating that it is not bound to plasma proteins, is in the free form, and has drug activity. The results are shown in Table 1 and Figure 2 。

[0050] Table 1 ADMET Prediction Results of Compound 13739

[0051]

[0052] Example 4. Activation of Compound 13739 on Vasopressin Receptor

[0053] Experimental method: HTLA cells were cultured in 4.5 g / L DMEM high-glucose culture medium containing 10% inactivated fetal bovine serum and placed in a cell culture incubator at 37 °C and 5% CO2. When the cell density reached 80%, subculture was carried out at a ratio of 1:3. When the cell confluence reached 70%, the original culture medium was discarded and 8 ml of DMEM was used for continued culture for 4 h. After 4 h, 4 ml of DMEM was aspirated from the culture medium and 4 ml of DMEM culture medium containing 10% fetal bovine serum was added. The transfection solution was prepared at a ratio of DMEM:UTR:PEI = 800 μl:10 μg:40 μl per dish, and the amount of transfection reagent was adjusted according to different plasmid concentrations. The transfection solution was mixed evenly, allowed to stand for 15 - 20 min, and then dropped into the cells. 24 h after cell transfection, the original culture medium was discarded, the cells were washed twice with 4 mL of normal saline, 1 mL of trypsin was added, and the cells were placed in the incubator for digestion for 1 - 2 min. The cells were transferred into a centrifuge tube, centrifuged at 800 r for 3 min, the supernatant was discarded, and DMEM culture medium containing 10% fetal bovine serum was added. The cells were repeatedly pipetted until they were mixed evenly, and then seeded at 1.5*10 5 / well into a 96-well plate and cultured in an incubator at 37 °C and 5% CO2 for 24 h. 24 h after seeding the cells, the stock solution of Compound 13739 was serially diluted with normal saline to make the final concentrations 10 -10 M, 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, 10 -5 M, and 10 μl / well of Compound 13739 at different concentrations was added. 24 h after drug administration to the cells, the culture medium supernatant was discarded, 50 μl / well of Bright-Glo reagent was added, and the chemiluminescence intensity was detected using an M5 microplate reader.

[0054] Experimental results: After stimulating HTLA cells with different concentrations of compound 13739, within the range of 10 -10 M - 10 -5 M, the agonistic effect of compound 13739 showed a good concentration - dependent relationship, that is, with the increase in the concentration of compound 13739, the agonistic effect became stronger. After calculation, the half - maximal effective concentration of compound 13739 was 3.70*10 -8 M. The results are shown in Table 2 and Figure 3 .

[0055] Table 2 Activation effect of compound 13739 on urotensin receptor

[0056]

[0057] Example 5. Activation effect of compound 13739 on other GPCR receptors

[0058] Experimental method: HTLA cells were cultured in 4.5 g / L DMEM high - glucose medium containing 10% inactivated fetal bovine serum and placed in a 37°C, 5% CO2 cell culture incubator. When the cell density reached 80%, they were passaged at a ratio of 1:3. When the cell confluence reached 70%, the original medium was discarded and 8 ml of DMEM was used to continue culturing for 4 h. After 4 h, 4 ml of DMEM was aspirated from the medium and 4 ml of DMEM medium containing 10% fetal bovine serum was added. GPCR plasmids FFA1, FFA2, and FFA3 were transfected respectively. The transfection solution was prepared at a ratio of DMEM: plasmid: PEI = 800 μl: 10 μg: 40 μl, and the amount of transfection reagent was adjusted according to different plasmid concentrations. The transfection solution was mixed evenly, allowed to stand for 15 - 20 min, and then dropped into the cells. The remaining operations were the same as in Example 4.

[0059] Experimental results: After stimulating HTLA cells with different concentrations of compound 13739, only the activation effect of HTLA cells transfected with the UTS2R plasmid showed a concentration - dependent relationship, while HTLA cells transfected with FFA1, FFA2, and FFA3 plasmids had no obvious activation effect. It is suggested that compound 13739 has specificity in activating the urotensin receptor and does not activate other GPCR receptors. The results are shown in Table 3 and Figure 4 .

[0060] Table 3 Specific activation effect of compound 13739 on urotensin receptor

[0061]

[0062] Example 6. Effect of compound 13739 on glucose uptake in HepG2 hepatocytes

[0063] Experimental method: HepG2 hepatocytes were cultured in high-glucose DMEM medium containing 10% inactivated fetal bovine serum and placed in a cell culture incubator at 37°C and 5% CO2. When the cell density reached 90%, they were digested with trypsin and subcultured at a ratio of 1:3. Cells in the logarithmic growth phase were inoculated into 96-well plates at 100 μl / well (1*10 5 cells / ml) and cultured for 24 h. After 24 h, the original medium was discarded, and 100 μl / well of phenol red-free 1640 medium containing 10% fetal bovine serum was added. At the same time, the stock solution of compound 13739 was serially diluted with physiological saline to final concentrations of 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, 10 -5 M, and 10 μl / well of compound 13739 at different concentrations was added. After continued culture for 48 h, 10 μl of the culture medium supernatant was aspirated from each well and added to a new 96-well plate, along with 10 μl of the standard control and 10 μl of pure water; 100 μl / well of glucose test working solution R1 was added, mixed well, incubated at 37°C for 4 min, and the absorbance was measured at 340 nm; then 25 μl / well of glucose test working solution R2 was added, mixed well, incubated at 37°C for 5 min, and the absorbance was measured at 340 nm.

[0064] Experimental results: After compound 13739 acted for 48 h, compared with the control group, glucose uptake by hepatocytes HepG2 was significantly promoted at 10 -5 M. The results are shown in Table 4 and Figure 5 .

[0065] Table 4 Effects of compound 13739 on glucose uptake by HepG2 hepatocytes

[0066]

[0067] Example 7. Effects of compound 13739 on lactate release by HepG2 hepatocytes

[0068] Experimental method: For cell culture, seeding, and drug administration, refer to Example 6. The method for detecting lactate content is as follows: After the cells were cultured for 48 h, 10 μl of the culture medium supernatant was aspirated from each well, diluted 10-fold, and 20 μl was added to a new 96-well plate, along with 20 μl of the standard control and 20 μl of pure water; 50 μl / well of the enzyme working solution and 10 μl / well of the chromogenic solution were added, mixed well, and accurately reacted in a 37°C air bath for 10 min; then 100 μl / well of the termination solution was added, and after thorough mixing, the absorbance was measured at 530 nm.

[0069] Experimental results: After compound 13739 acted for 48 h, compared with the control group, at 10-9 M-10 -5 There is a tendency for the lactic acid production of HepG2 hepatocytes to decrease within the range of M, and a significant difference appears at a concentration of 10 -9 M. The results are shown in Table 5 and Figure 6 .

[0070] Table 5 Effects of Compound 13739 on Lactic Acid Release of HepG2 Hepatocytes

[0071]

[0072] Example 8. Effects of Compound 13739 on the Viability of HepG2 Hepatocytes

[0073] Experimental method: For cell culture, seeding the plates, and drug administration, refer to Example 6. The method for detecting cell viability is as follows: After culturing the cells for 48 h, discard the original culture medium, add 100 μl / well of CCK8 dilution solution to the 96-well plates, and incubate them in an incubator at 37 °C for about 30 min, then read the absorbance value at 450 nm.

[0074] Experimental results: After the action of Compound 13739 for 48 h, compared with the control group, there is no significant effect on the viability of HepG2 hepatocytes within the range of 10 -9 M-10 -5 M. The results are shown in Table 6 and Figure 7 .

[0075] Table 6 Effects of Compound 13739 on the Viability of HepG2 Hepatocytes

[0076]

[0077] Example 9. Effects of Compound 13739 on Lipid Accumulation in an Oleic Acid-Induced HepG2 Non-Alcoholic Fatty Liver Cell Model

[0078] Experimental method: HepG2 hepatocytes are cultured in 4.5 g / L DMEM high-glucose culture medium containing 10% inactivated fetal bovine serum and placed in a cell culture incubator at 37 °C and 5% CO2. When the cell density reaches 90%, digest with trypsin and passage the cells at a ratio of 1:3. Take cells in the logarithmic growth phase at 100 μl / well (6*10 4Inoculate into a 96-well plate at a density of (e.g., number of cells per milliliter) and culture for 24 h. After 24 h, discard the original culture medium. In the normal group, add 100 μl / well of 4.5 g / L high-glucose DMEM culture medium containing 10% inactivated fetal bovine serum. In the model group, add 100 μl / well of 4.5 g / L high-glucose DMEM culture medium containing 0.25 mM oleic acid and 10% inactivated fetal bovine serum. In the drug administration group, add 90 μl / well of 4.5 g / L high-glucose DMEM culture medium containing 0.25 mM oleic acid and 10% inactivated fetal bovine serum. At the same time, dilute the stock solution of compound 13739 with physiological saline and add 10 μl / well of 10 μM compound 13739. After co-incubating compound 13739 with oleic acid for 24 h, discard the culture medium, wash 1 - 2 times with PBS, fix with 4% paraformaldehyde for 20 - 30 min, stain with Oil Red O working solution at room temperature for 1 h, wash 3 - 5 times with PBS, and then observe and take pictures under a microscope.

[0079] Experimental results: Lipid accumulation was observed in oleic acid-induced HepG2 hepatocytes. After 24 h of intervention with compound 13739, compared with the model group, the red lipid droplets in HepG2 hepatocytes were significantly reduced, indicating that compound 13739 can improve lipid accumulation in HepG2 hepatocytes. The results are shown in Table 7 and Figure 8 .

[0080] Table 7 Effects of compound 13739 on lipid accumulation in an oleic acid-induced HepG2 non-alcoholic fatty liver cell model

[0081]

[0082] Example 10. Effects of compound 13739 on triglyceride synthesis in oleic acid-induced HepG2 non-alcoholic fatty liver cells

[0083] Experimental method: HepG2 hepatocytes were cultured in 4.5 g / L high-glucose DMEM culture medium containing 10% inactivated fetal bovine serum and placed in a 37 °C, 5% CO2 cell culture incubator. When the cell density reached 90%, they were digested with trypsin and subcultured at a ratio of 1:3. Take cells in the logarithmic growth phase at 2 ml / well (30 * 10 4Inoculate at a density of 1×10⁶ cells / ml into a 6-well plate and culture for 24 h. After 24 h, discard the original medium. Add 2 ml / well of 4.5 g / L high-glucose DMEM culture medium containing 10% inactivated fetal bovine serum to the normal group, add 1.8 ml / well of 4.5 g / L high-glucose DMEM culture medium containing 10% inactivated fetal bovine serum and 0.25 mM oleic acid to the model group, and add 1.8 ml / well of 4.5 g / L high-glucose DMEM culture medium containing 10% inactivated fetal bovine serum and 0.25 mM oleic acid to the drug administration group. At the same time, dilute the stock solution of compound 13739 with physiological saline and add 200 μl / well of 10 μM compound 13739. After co-incubating compound 13739 with oleic acid for 24 h, discard the supernatant. Add 100 μl of lysis buffer to each well, collect the cells, mix well, and let stand at room temperature for 10 min; transfer an appropriate amount of supernatant to a 1.5 ml EP tube, heat at 70 °C for 10 min, and then centrifuge at 2000 rpm at room temperature for 5 min; take 10 μl of the supernatant sample and add 190 μl of working solution, and measure the absorbance value at 550 nm.

[0084] Experimental results: The triglyceride content in HepG2 hepatocytes induced by oleic acid increased significantly. After 24 h of intervention with compound 13739, compared with the model group, the triglyceride content in HepG2 hepatocytes decreased significantly, suggesting that compound 13739 can reduce the synthesis of triglycerides in hepatocytes. The results are shown in Table 8 and Figure 9 .

[0085] Table 8 Effects of compound 13739 on the synthesis of triglycerides in oleic acid-induced HepG2 non-alcoholic fatty liver cells

[0086]

Claims

1. Use of a compound represented by formula (I) in the preparation of a medicament for treating and / or preventing type 2 diabetes and / or non-alcoholic fatty liver; the structure of the compound is as follows:

2. Use of a pharmaceutical composition in the preparation of a medicament for treating and / or preventing type 2 diabetes and / or non-alcoholic fatty liver, characterized in that, The pharmaceutical composition contains an effective dose of the compound represented by formula (I) and a pharmaceutical excipient; 3. The application according to claim 2, characterized in that, The pharmaceutical composition includes forms of controlled release, sustained release preparations and microsomal drug delivery systems.

Images