A GLP-1R agonist

Abstract

The present invention relates to a GLP-1R agonist, comprising anisomycin, compound 1, compound 2, or a pharmaceutically acceptable salt thereof as an effective ingredient. The present invention finds that anisomycin and its derivatives have the effect of promoting insulin secretion under high blood sugar concentration, can treat diabetes and prevent the occurrence of hypoglycemia. The mechanism of action thereof is studied, and it is found that it is highly correlated with GLP-1R, indicating that it is a small molecule GLP-1R agonist, which can effectively treat a variety of GLP-1R-mediated diseases including obesity and diabetes, and has good commercial application prospects and scientific research value.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a GLP-1R agonist. Background Art

[0002] The present invention is a divisional application of CN202110103556.3.

[0003] Glucagon-like peptide-1 (GLP-1) is secreted by gastrointestinal L cells and exerts a hypoglycemic effect after the intake of glucose and other nutrients. Studies on the physiological effects of GLP-1 have shown that it can not only enhance insulin secretion from β cells, but also reduce glucagon secretion by acting on α cells, thereby reducing liver glucose output, and act on the central nervous system and stomach to suppress appetite and slow gastric emptying, thereby reducing β cell load. However, since GLP-1 is rapidly degraded by dipeptidyl peptidase 4 (DPP-4) in the body, its duration of action is limited. Therefore, the current methods of improving blood sugar control based on GLP-1 include exogenous drugs that simulate the effects of GLP-1 and drugs that prolong the activity of endogenous GLP-1. GLP-1 receptor agonists exert their hypoglycemic effects based on this.

[0004] So far, traditional drugs used in the clinic to treat type 2 diabetes (T2DM), such as insulin and sulfonylureas, can effectively reduce hyperglycemia, but they can continue to play a hypoglycemic role after controlling blood sugar within the normal range, which will lead to the risk of hypoglycemia in T2DM patients. Studies have reported that GLP-1 receptor agonists stimulate insulin secretion in a glucose concentration-dependent manner through the GLP-1 receptor (GLP-1R), which is the biggest advantage of GLP-1 receptor agonists over other hypoglycemic drugs and can avoid the risk of hypoglycemia. In addition, GLP-1 receptor agonists have some effects other than hypoglycemia, including weight loss, cardiovascular benefits, and improvement of Alzheimer's disease and fatty liver.

[0005] As hormones secreted by intestinal cells under the stimulation of food intake, incretin can regulate the response of insulin to food intake, and its insulin secretion effect accounts for about 60% of the total insulin secretion after a meal, playing an important role in blood sugar regulation. GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) are two major incretin hormones, of which the former has a significantly reduced secretion in patients with type 2 diabetes (even those with impaired glucose tolerance), making it an important therapeutic target. Studies on the physiological effects of GLP-1 have shown that it can not only enhance the response of β cells, but also reduce glucagon secretion by acting on α cells, thereby reducing liver glucose output, and act on the feeding center and stomach to suppress appetite and slow gastric emptying, thereby reducing β cell load. However, since GLP-1 is rapidly degraded by dipeptidyl peptidase 4 (DPP-4) in the body, its action time is limited. Therefore, the current methods to improve blood sugar control mainly based on GLP-1 include exogenous drugs that simulate the action of GLP-1 and drugs that prolong the activity of endogenous GLP-1. GLP-1 receptor agonists exert their hypoglycemic effects based on this.

[0006] GLP-1(7-36)amide is the main active form of GLP-1. After GLP-1(7-36)amide enters the blood circulation, dipeptidyl peptidase IV (DPP-IV) in the blood and cell membranes quickly cleaves it into inactive GLP-1(9-36), which has a half-life of only 1-2 minutes. Therefore, it is not suitable for the long-term treatment of type 2 diabetes mellitus (T2DM). Furthermore, scientific research has made corresponding changes and modifications to the structure of GLP-1 to increase its half-life and thus prolong its biological effects in vivo. Diabetes is a chronic disease characterized by hyperglycemia caused by insufficient (relative or absolute) insulin secretion or impaired insulin action in the human body. Type 2 diabetes is a progressive disease. The patient's condition progressively worsens over time, and it is difficult to control blood sugar well in the long term even with conventional drug treatment. Whether using metformin, sulfonylureas or insulin monotherapy, it is difficult to continuously reach the target for glycated hemoglobin (HbA1c). The reason for this is related to the progressive decline of β-cell function. GLP-1 can also increase insulin synthesis in the body, promote β-cell proliferation and differentiation, and inhibit β-cell apoptosis. These properties may help reverse or even prevent diabetes.

[0007] The GLP-1R agonists known in the prior art for the treatment or prevention of diabetes are generally large-molecule polypeptides, such as lixisenatide, liraglutide, and exenatide, which are difficult to synthesize and have high costs. They need to be injected, resulting in poor patient compliance. The development of small-molecule GLP-1R agonists will improve patient compliance, ease of use, and reduce drug costs, and has broad clinical market prospects. Therefore, the development of a class of GLP-1R agonists based on small-molecule structures has very important research significance and commercial value.

[0008] The chemical structure of Anisomycin is as follows:

[0009]

[0010] CAS number: 22862-76-6, molecular weight: 265.30, chemical name is (2R, 3S, 4S)-2-[(4-methoxyphenyl)methyl]-3,4-pyrrolidinediol 3-acetate, which is a pyrrolidine antibiotic isolated from Streptomyces griseus. Anisomycin is a protein synthesis inhibitor that shows good inhibitory effects on certain fungi and pathogens. It has been successfully used in the clinic to treat diseases such as vaginitis caused by Trichomonas and dysentery caused by amoeba, and is also widely used to prevent and control fungal diseases of plants. Studies have found that anisomycin can significantly inhibit the reactivity of T lymphocytes, and no cytotoxicity has been found; anisomycin can inhibit the rejection reaction of mouse skin transplantation, and its immunosuppression is higher than that of the immunosuppressive drug cyclosporine A, and its toxicity is lower, so it can be used to develop immunosuppressive drugs to treat autoimmune diseases. In addition, anisomycin can activate p38 MAPK and JNKs. The p38 and JNK signaling pathways are involved in a variety of cell activities, such as cell proliferation and differentiation, apoptosis, etc., suggesting that anisomycin has potential anti-tumor activity and can be used in the development of anti-tumor drugs. The inventor unexpectedly found that anisomycin has the effect of stimulating GLP-1R and can be used as a GLP-1R agonist in medicine, which can promote insulin secretion, lower blood sugar, treat diabetes, and other uses as a GLP-1R agonist. Anisomycin and its derivatives have the effect of stimulating GLP-1R, which has not been reported before. Summary of the invention

[0011] In order to overcome the disadvantages in synthesis and application of GLP-1R receptor agonists in the prior art that they are all large molecular polypeptides, the present invention provides a use of anisomycin in preparing GLP-1R agonists.

[0012] The purpose of the present invention is achieved through the following technical solutions:

[0013] An anisomycin derivative represented by general formula (I) or a pharmaceutically acceptable salt, solvate or stereoisomer thereof,

[0014]

[0015] R1 is selected from hydrogen, optionally substituted C1-6 alkyl, -C(O)R4; R2 is selected from hydrogen, optionally substituted C1-6 alkyl, optionally substituted C1-6 alkoxy, -NH-R4; R3 is selected from hydrogen, optionally substituted C1-6 alkyl, optionally substituted C1-6 alkyl, optionally substituted C6-12 aryl, optionally substituted heteroaryl, -C(O)R4; R4 is selected from H, optionally substituted C1-6 alkyl, optionally substituted C1-6 alkoxy; R5, R6, R7, R8 are independently selected from halogen atoms, nitro, -COOR9, CONHR9, optionally substituted C1-6 alkyl, optionally substituted C1-6 alkoxy; R9 is selected from H, optionally substituted C1-6 alkyl; provided that the compound of general formula (I) is not anisomycin.

[0016] Preferably, the anisomycin derivatives provided by the present invention are the following compounds 1 and 2:

[0017]

[0018] Ra, Rb are independently selected from optionally substituted C1-C6 alkyl, C1-6 alkoxy, C6-C20 aryl.

[0019] The C1-C6 alkyl group is selected from methyl, ethyl, propyl and butyl; the C6-20 aryl group is selected from phenyl, naphthyl and anthracenyl; the optional substitution means that any H atom is replaced by a hydroxyl group, a halogen atom, a nitro group, a C1-4 alkyl group or a C1-4 alkoxy group.

[0020] A GLP-1R agonist, characterized in that it contains at least one of anisomycin, anisomycin derivatives represented by formula (I) or pharmaceutically acceptable salts, solvates and stereoisomers thereof as an active ingredient.

[0021] The derivatives include products obtained by substituting some groups or reacting anisomycin as a parent, such as substances substituted by halogen atoms, alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, hydroxyl groups, aryl groups or heteroaryl groups, or products obtained by reacting with other substances having reactive functional groups, such as products of esterification, etherification, amidation, transesterification and the like; or salts thereof, such as quaternary ammonium salts; or deuterated derivatives thereof, wherein the deuterated hydrogen is not particularly limited, and is preferably active hydrogen, such as hydrogen on an amine group or hydrogen on a hydroxyl group.

[0022] Disclosed is a use of at least one of anisomycin, anisomycin derivatives represented by formula (I) or pharmaceutically acceptable salts, solvates and stereoisomers thereof in the preparation of a GLP-1R agonist.

[0023] Furthermore, the present invention provides a use of at least one of anisomycin, anisomycin derivatives represented by formula (I) or pharmaceutically acceptable salts, solvates and stereoisomers thereof in the preparation of drugs for treating diseases mediated by GLP-1R.

[0024] The diseases mediated by the GLP-1R include, but are not limited to, obesity, diabetes, weight loss, lowering of systolic blood pressure, epilepsy, cerebral ischemic disease, Alzheimer's disease, fatty liver, improvement of dyslipidemia, and neuroprotection.

[0025] The present invention also provides the use of at least one of anisomycin, anisomycin derivatives shown in formula (I) or pharmaceutically acceptable salts, solvates, stereoisomers thereof for preparing drugs for treating diabetes, dyslipidemia, metabolic syndrome, hyperinsulinemia, nocturnal hypoglycemia, obesity, reducing body weight, reducing systolic blood pressure, improving Alzheimer's disease, fatty liver, and neuroprotection.

[0026] Preferably, the diabetes is selected from type 2 diabetes.

[0027] The inventors unexpectedly discovered that anisomycin and its derivatives have the effect of GLP-1R agonists and can be used as drugs for treating diabetes. GLP-1 receptors (GLP-1R) stimulate insulin secretion in a glucose concentration-dependent manner, that is, they will only play a role in promoting insulin secretion at a certain concentration of blood sugar, and will not play a role at a lower blood sugar concentration. Therefore, while treating diabetes, it can also avoid the occurrence of hypoglycemia. This is the advantage of GLP-1R agonists as drugs for treating diabetes compared to conventional drugs.

[0028] Furthermore, the present invention provides a GLP-1R agonist comprising at least one of anisomycin, anisomycin derivatives represented by formula (I) or pharmaceutically acceptable salts, solvates and stereoisomers thereof as an active ingredient, wherein the effective dose is 0.5-50 mg / kg, and preferably, the effective dose is 5-30 mg / kg.

[0029] The present invention also provides a pharmaceutical composition having GLP-1R agonist activity, comprising at least one of anisomycin, anisomycin derivatives represented by formula (I) or pharmaceutically acceptable salts, solvates and stereoisomers thereof as an effective active ingredient.

[0030] Furthermore, the present invention provides a pharmaceutical composition for treating or preventing diabetes, comprising at least one of anisomycin, anisomycin derivatives represented by formula (I) or pharmaceutically acceptable salts, solvates and stereoisomers thereof as an effective active ingredient.

[0031] The pharmaceutical composition may also include metformin, SGLT-2 inhibitors, and auxiliary materials.

[0032] The pharmaceutical composition is used in dosage forms well known in the art, such as tablets, capsules, and injections.

[0033] In particular, the present invention provides a method for lowering blood glucose levels, specifically by administering an effective dose of anisomycin or at least one of its derivatives, salts, and solvates.

[0034] The anisomycin and its derivatives of the present invention exert GLP-1R agonist effects in a blood glucose concentration-dependent manner. Therefore, the above method of the present invention can not only treat or prevent diabetes, but also effectively reduce the occurrence of hypoglycemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Effects of anisomycin on insulin secretion in rats.

[0036] Figure 2 Effects of different doses of anisomycin on insulin secretion in rats.

[0037] Figure 3 Effects of anisomycin on insulin secretion in rats under different glucose concentrations.

[0038] Figure 4 Effects of anisomycin on insulin secretion in rats in the presence or absence of the GLP-1R blocker Exendin (9-39).

[0039] Figure 5 ForteBio Octet molecular interaction assay to detect the affinity of anisomycin to GLP-1R.

[0040] Figure 6 A is the blood glucose change curve over time in oral glucose tolerance test; B is the area under the blood glucose curve. DETAILED DESCRIPTION

[0041] Preparation Example

[0042] Using anisomycin as raw material, different anisomycin derivatives were obtained according to the following synthetic route.

[0043] Compound 1 Ra 1-1 -Ph 1-2 <![CDATA[-C2H5]]> 1-3 -4-Cl-Ph Compound 2 R 2-1 -Ph 2-2 <![CDATA[-C2H5]]> 2-3 <![CDATA[-C3H7]]>

[0044]

[0045] Example 1

[0046] The materials and methods used in the present invention are described below.

[0047] 1.1 Experimental animals

[0048] Male Wistar rats, weighing 180-250 g, were purchased from the Experimental Animal Center of Shanxi Provincial People's Hospital and maintained at 20-22°C with standard rodent food and drinking water. All procedures were in accordance with the Guidelines for the Management and Use of Laboratory Animals of Shanxi Medical University.

[0049] 1.2 Main drugs and reagents

[0050] Anisomycin MCE Corporation glucose Beijing Solable Technology Co., Ltd. DispaseII Amresco Corporation TEA Sigma-Aldrich (Shanghai) Trading Co., Ltd. Histopaque-1077 Sigma-Aldrich (Shanghai) Trading Co., Ltd. Albumin bovine serum(BSA) Beijing Solable Technology Co., Ltd. RPMI 1640 medium American Hydrone Company Collagenase P Roche Pharmaceuticals, Switzerland Fura 2-AM Shanghai Dongren Chemical Technology Co., Ltd. Fetal bovine serum (FBS) Theromo Fisher Technology Hanks buffered saline (HBSS) Boster Biotechnology Co., Ltd. Analytical grade NaCl BBI Life Sciences Ltd. Analytical grade KCl BBI Life Sciences Ltd. <![CDATA[NaHCO3]]> BBI Life Sciences Ltd. HEPES Beijing Solable Technology Co., Ltd. <![CDATA[CaCl2]]> BBI Life Sciences Ltd. <![CDATA[Analytical pure MgCl2]]> BBI Life Sciences Ltd. <![CDATA[KH2PO4]]> BBI Life Sciences Ltd. 100× Penicillin-Streptomycin Mixture Beijing Solable Technology Co., Ltd. DMSO Beijing Solable Technology Co., Ltd. <![CDATA[MgSO4]]> BBI Life Sciences Ltd.

[0051] 2. Main experimental instruments

[0052] Cell culture incubator Eppendorf, Germany Constant temperature oscillator Changzhou Guohua Electric Co., Ltd. Pressure Steam Sterilizer Taiyuan Xingyu Scientific and Educational Instruments and Equipment Manufacturing Co., Ltd. Clean bench Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd. Medical Refrigerator Freezer Aucma Co., Ltd. Fully automatic flake ice machine Changshu Xueke Electric Appliance Co., Ltd. Acidity meter Ohaus Instruments Ltd. Digital speed peristaltic pump Baoding Chuangrui Pump Industry Co., Ltd. Inverted microscope Japan Olympus IX51 VORTEX Vortex Mixer Haimen Qilin Bell Instrument Co., Ltd.

[0053] 3. The isolation and culture of rat pancreatic islet tissue is a routine experimental operation performed by those skilled in the art.

[0054] 4. Data processing and statistics

[0055] The data were processed by SigmaPlot 12.5 software and expressed as Mean ± SEM. Statistical analysis was performed using Student's t-test, one-way ANOVA or paired t-test, and p < 0.05 was considered statistically significant.

[0056] Specific operations

[0057] 5.1 Effects of anisomycin on insulin secretion in rats

[0058] Preparation before the experiment: 1) The experiment was divided into 4 groups, and the Eppendorf tubes were numbered (7 in each group); 2) KRBH solution was prepared, incubated in an incubator for 30 min, and the pH was adjusted to 7.4 with NaOH; 3) The samples to be tested were prepared: 2.8 mmol / L glucose solution (2.8G), 2.8G+10μM anisomycin, 11.1 mmol / L glucose solution (11.1G), and 11.1G+10μM anisomycin.

[0059] Experimental steps: 1) Add 500μL 2.8G to each Ep tube, pick 5 islets (uniform size, smooth edges) under a stereo microscope and put them into the Ep tube, and incubate them in an incubator for 30 minutes; 2) Use a pipette to aspirate the supernatant and discard it, be careful not to aspirate the islets, then add 500μL 2.8G, 2.8G+10μM anisomycin, 11.1G, 11.1G+10μM anisomycin to each group in turn, and incubate them in an incubator for 30 minutes; 3) Use a pipette to aspirate the supernatant into the pre-marked Ep tube, mix well, seal, and store at 4°C. Insulin radioimmunoassay to detect insulin content in each group.

[0060] The experimental results are as follows Figure 1 As shown in the figure, the effect of anisomycin on insulin secretion in rats, where + anisomycin means adding a certain amount of anisomycin, and the following figures have the same meaning. Rat islets were incubated with anisomycin (10 μM) under low glucose (2.8G) and high glucose (11.1G) conditions. n=7.***P<0.001. Under basal glucose concentration (2.8G), anisomycin did not play a role in promoting insulin secretion; under high glucose concentration (11.1G), anisomycin had a significant effect in promoting insulin secretion. This shows that anisomycin promotes insulin secretion under high glucose conditions.

[0061] 5.2 Effects of different doses of anisomycin on insulin secretion in rats

[0062] Preparation before the experiment: 1) The experiment was divided into 4 groups, and the Ep tubes were numbered (7 in each group); 2) The samples to be tested were prepared: 2.8 mmol / L glucose solution (2.8G), 11.1 mmol / L glucose solution (11.1G), 11.1G+0.1μM anisomycin, 11.1G+1μM anisomycin, and 11.1G+10μM anisomycin.

[0063] Experimental steps: 1) 2.8G pre-incubation for 30 minutes; 2) Take out the Eppendorf tube, discard the supernatant, add 500μL 11.1G, 11.1G+0.1μM anisomycin, 11.1G+1μM anisomycin, 11.1G+10μM anisomycin to each group, and incubate in the incubator for 30 minutes; 3) Pipette the supernatant into the pre-marked Eppendorf tube, mix well, seal, and store at 4°C. Insulin radioimmunoassay was used to detect the insulin content in each group.

[0064] The experimental results are as follows Figure 2 As shown, the effect of different doses of anisomycin on insulin secretion in rats. At a concentration of 11.1mmol / L glucose (11.1G), rat islets were incubated with different concentrations of anisomycin (0.1μM, 1μM, 10μM). n=7.*P<0.05;***P<0.001. Compared with the administration of 11.1mmol / L glucose (11.1G) alone, the insulin secretion-promoting effect of anisomycin gradually increased with the increase of anisomycin concentration, and when the concentration of anisomycin was 1μM and 10μM, it had a significant insulin secretion-promoting effect. Therefore, anisomycin has a concentration-dependent insulin secretion-promoting effect at high glucose (11.1G).

[0065] 5.3 Effects of anisomycin on insulin secretion in rat pancreatic islets at different glucose concentrations

[0066] Preparation before the experiment: 1) The experiment was divided into 3 groups, and the Ep tubes were numbered (7 in each group); 2) Prepared the samples to be tested: 2.8mmol / L glucose solution (2.8G), 2.8G+10μM anisomycin, 11.1mmol / L glucose solution (11.1G), 11.1G+10μM anisomycin, 16.7mmol / L glucose solution (16.7G), 16.7G+10μM anisomycin.

[0067] Experimental steps: 1) 2.8G pre-incubation for 30min; 2) Take out the Ep tube, discard the supernatant, add 500μL 2.8G, 11.1G, 16.7G to each group in turn, and incubate in the incubator for 30min; 3) Pipette the supernatant into the pre-marked Ep tube, mix, seal, and store at 4℃, and add 500μL 2.8G+10μM anisomycin, 11.1G+10μM anisomycin, and 16.7G+10μM anisomycin in the order of the previous round of drug addition, and incubate in the incubator for 30min; 4) Take out the Ep tube, aspirate the supernatant into the numbered Ep tube, mix, seal, and store at 4℃. Insulin (INS) Radioimmunoassay to detect insulin content in each group.

[0068] The experimental results are as follows Figure 3 As shown in the figure, the effect of anisomycin on insulin secretion in rats under different glucose concentrations. The rat islets were incubated with 10 μm anisomycin at the concentrations of 2.8 mmol / L glucose solution (2.8G), 11.1 mmol / L glucose solution (11.1G) and 16.7 mmol / L glucose solution (16.7G). n=7,*p<0.5,**p<0.01,***p<0.001. It can be seen that at the basal glucose concentration (2.8G), anisomycin had no effect on promoting insulin secretion; but at the concentration of 11.1 mmol / L glucose solution (11.1G), anisomycin significantly promoted insulin secretion; with the increase of glucose concentration, at the concentration of 16.7 mmol / L glucose solution (16.7G), anisomycin could still further promote insulin secretion, and there was a significant difference. Therefore, anisomycin has a glucose concentration-dependent insulin secretion-promoting effect. It can be explained that anisomycin can also avoid the occurrence of hypoglycemia when treating or preventing diabetes due to its dependence on blood glucose concentration.

[0069] Example 2 The role of GLP-1R in anisomycin regulating insulin secretion in rats

[0070] 1.1 Test reagents

[0071]

[0072] 1.2 Main experimental instruments

[0073] PCR instrument Bio-rad, USA Gel imaging system Bio-rad, USA Western blot vertical electrophoresis system Bio-rad, USA Western blot semi-dry transfer system Bio-rad, USA Octet RE96E Molecular Interaction Analyzer Fortebio Balance Shanghai Tianmei Scientific Instrument Co., Ltd. Pipette eppendorf Research plus set SSA Chip Fortebio Acidity meter Ohaus Instruments Ltd. Digital speed peristaltic pump Baoding Chuangrui Pump Industry Co., Ltd. VORTEX Vortex Mixer Haimen Qilin Bell Instrument Co., Ltd.

[0074] 2.1 Role of GLP-1R in anisomycin-regulated insulin secretion

[0075] Preparation before the experiment: 1) The experiment was divided into 4 groups, and the Ep tubes were numbered (7 in each group); 2) Prepared the samples to be tested: 2.8mmol / L glucose solution (2.8G), 11.1mmol / L glucose solution (11.1G), 11.1G+100nM Exendin (9-39), 11.1G+10μM anisomycin, 11.1G+100nM Exendin (9-39)+10μM anisomycin.

[0076] Experimental steps: 1) 2.8G pre-incubation for 30 minutes; 2) Take out the Eppendorf tube, discard the supernatant, add 500μL 11.1G, 11.1G+100nM Exendin (9-39), 11.1G+10μM anisomycin, 11.1G+100nM Exendin (9-39)+10μM anisomycin to each group, and incubate in the incubator for 30 minutes; 3) Pipette the supernatant into the pre-labeled Eppendorf tube, mix well, seal, and store at 4°C. Insulin radioimmunoassay was used to detect the insulin content in each group.

[0077] The experimental results are as follows Figure 4 As shown, it indicates the role of GLP-1R in anisomycin regulating insulin secretion. At a concentration of 11.1mmol / L glucose solution (11.1G), rat islets were incubated with 10μm anisomycin and 100nM Exendin (9-39). n=7, **p<0.01, ***p<0.001. Exendin (9-39) is a specific competitive GLP-1R antagonist that can block the agonistic effect of GLP-1R agonists on GLP-1R. At a concentration of 11.1mmol / L glucose solution (11.1G), Exendin (9-39) blocked the insulin secretion-promoting effect of anisomycin, indicating that the insulin secretion-promoting effect of anisomycin is related to the stimulation of GLP-1R.

[0078] 2.2 ForteBio Octet molecular interaction detection of anisomycin affinity for GLP-1R

[0079] Expression of cell-free GLP-1R protein: 1) Construction of rat GLP-1R vector: Use PCR amplification technology to obtain the sequence fragment of rat GLP-1R, recover the GLP-1R rat gene fragment by agarose gel, clone the target gene GLP-1R into the vector pEX-3, double-digest pEX-3 with EcoRI and BamHI, perform electrophoresis, and recover the vector pEX-3; use Entry OneStep Cloning Kit was used to clone the amplified fragment into the pEX-3 vector; the recombinant ligation product was transformed into competent cells, clone colonies were picked, a small amount of plasmid was extracted, and double digestion with EcoRI and BamHI was used for identification, electrophoresis was performed, and positive clones were picked; after sequencing and verification of the recombinant plasmid, a large amount of extraction was performed, and the bacterial solution corresponding to the positive clone was sequenced, and the remaining bacterial solution was stored in glycerol; the sequencing result was compared with the target gene sequence, and after verification, the glycerol bacterial solution was inoculated with LB culture medium, and a large amount of plasmid was extracted to obtain a sufficient amount of recombinant plasmid; 2) Expression of cell-free GLP-1R protein: Hangzhou Jinao Biotechnology Z1 high-fidelity DNA polymerase G-POL-001 or Z2 ultra-fidelity DNA polymerase G-POL-002 was used to amplify the target gene. In order to improve the success rate of protein expression, E. coli dialysis cell-free protein expression system, TOB dialysis cell-free protein expression system and Yeast dialysis cell-free protein expression system were used for expression, and finally the expression results were observed by SDS-PAGE.

[0080] GLP-1R protein purification and concentration: 1) Purify the target protein using a Histrap column 2) Obtain the purified sample and perform SDS-PAGE to observe the purification results.

[0081] ForteBio Octet molecular interaction experiment steps: 1) Biotinylate the GLP-1R protein at a molar ratio of 1:1, add an appropriate amount of biotinylation reagent and react in a 30°C water bath for 2 hours. After the reaction, use a gravity desalting column to remove excess biotin and elute with PBS for SSA chip solidification; 2) Dissolve the compound with DMSO, dilute it 100 times with PBST after complete dissolution, and then dilute it with 1% DMSO+PBST to 21.2μM, 42.4μM, 84.8μM, 169.5μM, and 339μM respectively for subsequent detection; 3) Add the above samples and reagents to the sample plate in order; 4) Set the program and perform the detection.

[0082] K on K is the binding rate constant, which represents the speed of intermolecular binding; off is the dissociation rate constant, which represents the speed of molecular dissociation; Kd is the dissociation constant, which reflects the affinity of the compound to the target. The smaller the value, the stronger the affinity. Kd = K off / K on For small molecules (<2 kDa), Kd values ​​are between 10 -4 -10 -7 M, indicating a strong affinity. Figure 5 As shown, the ForteBio Octet molecular interaction results showed that anisomycin Kd = 2.41×10-04 M, indicating that anisomycin has a strong affinity for GLP-1R.

[0083] It can be seen from the data of Example 1 and Example 2 that anisomycin can effectively promote insulin secretion at higher blood sugar concentrations, and has a certain blood sugar concentration dependence; and under the action of GLP-1R antagonists, this effect of promoting insulin secretion is reduced or disappears, indicating that GLP-1R mediates the effect of anisomycin in promoting insulin secretion. The present invention also proves that anisomycin and GLP-1R have obvious interactions and strong affinity through ForteBio Octet molecular interaction detection. The above data can fully illustrate that anisomycin is an effective small molecule GLP-1R agonist.

[0084] Example 3 Effect of anisomycin on blood glucose in C57BL / 6 mice

[0085] 1. Materials and Methods

[0086] 1.1 Experimental animals

[0087] Male C57BL / 6 mice, weighing 18-22 g, were purchased from the Experimental Animal Center of Shanxi Provincial People's Hospital and maintained at 20-22°C with standard rodent food and drinking water. All procedures were in accordance with the Guidelines for the Management and Use of Laboratory Animals of Shanxi Medical University.

[0088] 1.2 Main drugs and reagents

[0089] Anisomycin MCE Corporation glucose Beijing Solable Technology Co., Ltd. DMSO Beijing Solable Technology Co., Ltd. PEG-200 Beijing Solable Technology Co., Ltd.

[0090] 1.3 Main experimental instruments

[0091] Animal balance Shanghai Fikosu Tools Co., Ltd. FreeStyle Blood Glucose Meter and Blood Glucose Test Strips Abbott Diabetes Care UK Low temperature ultra-high speed centrifuge Eppendorf, Germany Fully automatic flake ice machine Changshu Xueke Electric Appliance Co., Ltd. Digital speed peristaltic pump Baoding Chuangrui Pump Industry Co., Ltd. Micro Vortex Mixer Shanghai Huxi Analytical Instrument Factory Co., Ltd. Magnetic stirrer Shanghai Silo Instrument Co., Ltd.

[0092] 1.4 Data processing and statistics

[0093] The data were processed by SigmaPlot 12.5 and expressed as Mean ± SEM. Statistical analysis was performed using Student's t-test, one-way ANOVA or paired t-test, and P < 0.05 was considered statistically significant.

[0094] 2 Experimental results

[0095] 2.1 Oral glucose tolerance test (OGTT)

[0096] Drug preparation: Anisomycin was dissolved in DMSO, PEG200 was added, and finally diluted with normal saline (DMSO: PEG200: normal saline = 1:4:5).

[0097] After 1 week of adaptive feeding, 21 SPF C57BL / 6 mice aged 6 weeks were randomly divided into the following 3 groups: control group: intraperitoneal injection of solvent (DMSO: PEG200: saline = 1: 4: 5); 5mg / kg anisomycin group: intraperitoneal injection of 5mg / kg anisomycin solution; 15mg / kg anisomycin group: intraperitoneal injection of 15mg / kg anisomycin solution, the administration volume was 0.1ml / 10g, 7 mice in each group, free access to water and food. Before the experiment, mice in each group were fasted but not watered for 12h. On the day of the experiment, mice in each group were gavaged with 40% glucose solution (2mg / kg) after administration. Blood was collected from the tail vein at 0min, 15min, 30min, 60min, 90min, and 120min after gavage. Blood glucose levels were measured with a blood glucose meter, and the area under the blood glucose curve (AUC) was calculated.

[0098] The experimental results are as follows Figure 6 As shown, A represents the blood glucose change curve over time in the oral glucose tolerance test (OGTT); B represents the blood glucose area under the curve. It can be seen that compared with the control group, the tolerance to glucose load in the anisomycin administration group increased to varying degrees after oral glucose administration, among which 15 mg / kg anisomycin had a more obvious hypoglycemic effect than 5 mg / kg anisomycin (n=7, *p<0.05, **p<0.01, ***p<0.001).

[0099] Example 4 Effects of anisomycin derivatives on insulin secretion in rats

[0100] Compounds 1-1, 1-2, 2-1 and 2-2 were prepared in Preparation Examples.

[0101] Preparation before the experiment: 1) The experiment was divided into 10 groups, and the Eppendorf tubes were numbered (7 in each group); 2) KRBH solution was prepared, incubated in an incubator for 30 min, and the pH was adjusted to 7.4 with NaOH; 3) The samples to be tested were prepared: 2.8 mmol / L glucose solution (2.8G), 2.8G+10μM compound 1-1, 2.8+10μM compound 1-2, 2.8G+10μM compound 2-1, 2.8G+10μM compound 2-2, 11.1 mmol / L glucose solution (11.1G), 11.1G+10μM compound 1-1, 11.1G+10μM compound 1-2, 11.1G+10μM compound 2-1, 11.1G+10μM compound 2-2.

[0102] Experimental steps: 1) Add 500 μL 2.8G to each Eppendorf tube, pick 5 islets (uniform size, smooth edges) under a stereomicroscope and put them into the Eppendorf tube, and incubate them in an incubator for 30 minutes; 2) Use a pipette to aspirate the supernatant and discard it, being careful not to aspirate the islets, and then add 500 μL of 2.8G to each group. 2.8G, 2.8G+10μM compound 1-1, 2.8+10μM compound 1-2, 2.8G+10μM compound 2-1, 2.8G+10μM compound 2-2, 11.1G, 11.1G+10μM compound 1-1, 11.1G+10μM compound 1-2, 11.1G+10μM compound 2-1, 11.1G+10μM compound 2-2, incubate in an incubator for 30min; 3) use a pipette to aspirate the supernatant into a pre-marked Ep tube, mix well, seal, and store at 4°C. Insulin radioimmunoassay to detect insulin content in each group.

[0103] The experimental results are shown in Table 1, the effects of anisomycin derivatives on insulin secretion in rats. Compared with the 2.8G group, each anisomycin derivative (10μM) had no effect on promoting insulin secretion under low glucose (2.8G). Compared with the 11.1G group, each anisomycin derivative (10μM) had a significant effect on promoting insulin secretion under high glucose (11.1G). (n=7. **P<0.01, *P<0.05)

[0104] Table 1

[0105]

[0106]

[0107] It can be seen from the data in Table 1 that, in addition to the obvious effect of anisomycin in promoting insulin secretion under high blood sugar concentration, some anisomycin derivatives also have similar physiological activities. Although the present application specifically implements a limited number of derivatives, that is, certain groups are substituted, the essence of the present invention is to creatively discover that anisomycin and its derivatives with the same parent nucleus have the effect of promoting insulin secretion under high blood sugar concentration, and this effect is exerted through GLP-1R stimulation, indicating that this type of compound is an effective small molecule GLP-1R agonist, and the protection of the compound should not be limited to the above-mentioned specific derivatives.

Claims

1. Use of Compound 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating diabetes; the structural formula of Compound 1 is as follows: Compound 1; Ra is independently selected from -ph, -C2H5, -C3H7.

2. Use of compound 2 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating diabetes; the structural formula of compound 2 is as follows: Compound 2; Rb is independently selected from -ph, -C2H5, -C3H7.

3. Use of anisomycin or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating diabetes.

4. The use according to any one of claims 1 to 3, characterized in that: The diabetes is type 2 diabetes.

5. The use according to claim 3, characterized in that: Anisomycin or a pharmaceutically acceptable salt thereof is used as an effective ingredient, and the dosage is 0.5-50 mg / kg.

6. The use according to claim 5, characterized in that Anisomycin or a pharmaceutically acceptable salt thereof is used as an effective ingredient, and the dosage is 5-30 mg / kg.

7. Use of a pharmaceutical composition in the preparation of a drug for treating diabetes; the pharmaceutical composition contains anisomycin or a pharmaceutically acceptable salt thereof as an effective active ingredient.

8. The use according to claim 7, characterized in that The pharmaceutical composition also includes metformin, an SGLT-2 inhibitor and excipients.

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