Co-agonist targeting GLP-1r, GIPR and gcgr

Abstract

Provided is a co-agonist targeting GLP-1R, GIPR and GCGR. The co-agonist has a polypeptide sequence of Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{dicarboxylic acid-γ-Glu-(AEEA)n-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2, wherein n is 0, 1 or 2 and the dicarboxylic acid is a C14-C24 dicarboxylic acid.

Description

A co-agonist targeting GLP-1R, GIPR, and GCGR Cross-references

[0001] This application incorporates Chinese Patent Application No. 202411001515.3, filed on May 28, 2024, entitled “A Co-agonist Targeting GLP-1R, GIPR and GCGR”, which is incorporated herein by reference in its entirety. Technical Field

[0002] This application relates to the field of peptide drug technology, and in particular to a co-agonist targeting GLP-1R, GIPR and GCGR. Background Technology

[0003] The problem of obesity in humans is gradually worsening and is closely linked to chronic diseases such as non-alcoholic fatty liver disease (NAFLD).

[0004] 1. The prevalence of obesity:

[0005] For example, according to the "Report on Nutrition and Chronic Diseases of Chinese Residents (2020)," more than half of Chinese adults are overweight or obese, with an overweight rate of 34.3% and an obesity rate of 16.4%.

[0006] Among children under 6 years old, about one in ten are overweight or obese, with overweight and obesity rates of 6.8% and 3.6%, respectively; among adolescents aged 6 to 17, nearly one in five are overweight or obese, with overweight and obesity rates of 11.1% and 7.9%, respectively.

[0007] Regarding gender differences, among children and adolescents, boys have a higher rate of overweight and obesity than girls; while among adults, although men have historically had a lower rate of overweight and obesity than women, this gender difference has been narrowing or even reversing in recent years.

[0008] 2. Socioeconomic factors of obesity:

[0009] The overweight and obesity rate is also higher among people with higher socioeconomic status.

[0010] Overweight and obesity rates are rising faster in rural areas than in urban areas, and the urban-rural gap is gradually narrowing.

[0011] 3. The association between obesity and non-alcoholic fatty liver disease (NAFLD):

[0012] Obesity is a significant risk factor for the development of NAFLD.

[0013] NAFLD is a chronic liver disease characterized by fat accumulation and inflammation in the liver, which can eventually lead to cirrhosis and hepatocellular carcinoma.

[0014] Obesity promotes the development of NAFLD by increasing insulin resistance and inflammation.

[0015] 4. Management and treatment of obesity:

[0016] Currently, for example, in China, only orlistat, liraglutide, smegglutide, and benaglutide have been approved for the treatment of obesity. There are no products for the treatment of NAFLD or obesity combined with NAFLD.

[0017] Glucagon-like peptide-1 (GLP-1) is a hormone primarily produced by L cells in the intestine. It belongs to the incretin family, and its receptor is called the glucagon-like peptide-1 receptor (GLP-1R). GLP-1 receptor agonists (GLP-1RAs) are novel hypoglycemic agents that activate GLP-1R to enhance insulin secretion in a glucose-dependent manner, inhibit glucagon secretion, and delay gastric emptying. This results in reduced food intake through central appetite suppression, thereby lowering blood sugar and promoting weight loss.

[0018] Furthermore, the GLP-1 receptor (GLP-1R) is one of the most important targets for the treatment of type 2 diabetes, and it has been nearly forty years since the target was discovered.

[0019] GIPR (Gastric inhibitory polypeptide receptor) is the receptor for GIP and is a G protein-coupled receptor with seven transmembrane domains. GIP regulates energy metabolism, particularly the incretin effect, through its interaction with GIPR. Studies have shown that GIPR plays a crucial role in regulating insulin secretion, glucose and lipid metabolism, and is essential for maintaining normal metabolic function.

[0020] GCGR protein is a glucagon G protein-coupled receptor that is crucial for blood glucose regulation and actively controls hepatic glucose production. It promotes glycogenolysis and gluconeogenesis, which are essential for the fasting response.

[0021] The three receptors GLP-1R, GIPR, and GCGR proteins play a key role in the treatment of obesity and NAFLD, and multiple receptor agonists that target these three receptors simultaneously also have a synergistic effect.

[0022] The inventors have discovered that the related technology has at least the following problems:

[0023] Compared with single receptor agonist therapy, the high activity of multiple receptor agonists can reduce the dosage and thus reduce adverse reactions to some extent. Summary of the Invention

[0024] The purpose of some embodiments of this application is to provide a triple co-agonist that simultaneously targets GLP-1R, GIPR and GCGR.

[0025] Specifically, the polypeptide sequence of the co-agonist is: Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{dicarboxylic acid-γ-Glu-(AEEA)n-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2, where n is 0, 1, or 2.

[0026] For example, in the above co-activators, the dicarboxylic acid is a C14 to C24 dicarboxylic acid.

[0027] In addition, in the above co-activators, the dicarboxylic acid is selected from any one of the following: C14 dicarboxylic acid, C16 dicarboxylic acid, C18 dicarboxylic acid, C20 dicarboxylic acid, C22 dicarboxylic acid and C24 dicarboxylic acid.

[0028] Furthermore, in the above-mentioned co-agonists, the polypeptide sequence of the co-agonist is selected from any of the following:

[0029] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,12-dodecanedioic acid-γ-Glu-AEEA-Ly s}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2;

[0030] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,14-tetradecanedioic acid-γ-Glu-AEEA-Ly s}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2;

[0031] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,16-Hexadecanedioic acid-γ-Glu-AEEA-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2;

[0032] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,18-Octadecanedioic acid-γ-Glu-AEEA-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2;

[0033] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,18-Octadecanedioic acid-γ-Glu-AEEA-AEEA-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2;

[0034] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,20-Eicosanedioic acid-γ-Glu-AEEA-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2;

[0035] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,22-Docosanedioic acid-γ-Glu-AEEA-Ly s}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2.

[0036] This application provides a pharmaceutical composition comprising: a co-agonist as described above and a pharmaceutically acceptable carrier.

[0037] This application also provides the use of a co-agonist targeting GLP-1R, GIPR, and GCGR in the preparation of a medicament for treating diseases, wherein the polypeptide sequence of the co-agonist is:

[0038] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{Dicarboxylic acid-γ-Glu-(AEEA)n-Lys} -Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2,

[0039] Where n is 0, 1, or 2;

[0040] The disease is selected from any one or a combination of the following: obesity, non-alcoholic fatty liver disease, type 2 diabetes, and hyperlipidemia.

[0041] In addition, in the above-mentioned uses, the dicarboxylic acid is a C14 to C24 dicarboxylic acid.

[0042] In addition, in the above-mentioned uses, the dicarboxylic acid is selected from any one of the following: C14 dicarboxylic acid, C16 dicarboxylic acid, C18 dicarboxylic acid, C20 dicarboxylic acid, C22 dicarboxylic acid and C24 dicarboxylic acid.

[0043] In addition, in the above-described uses, the polypeptide sequence of the co-agonist is selected from any of the following:

[0044] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,12-dodecanedioic acid-γ-Glu-AEEA-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2;

[0045] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,14-tetradecanedioic acid-γ-Glu-AEEA-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2;

[0046] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,16-hexadecanedioic acid-γ-Glu-AEEA-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2;

[0047] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,18-octadecanedioic acid-γ-Glu-AEEA-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2;

[0048] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,18-octadecanedioic acid-γ-Glu-AEEA-AEEA- Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2;

[0049] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,20-eicosanedioic acid-γ-Glu-AEEA-Ly s}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2;

[0050] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,22-Docosanedioic acid-γ-Glu-AEEA-Ly s}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of this application clearer, some embodiments of this application will be described in further detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of this application and are not intended to limit this application.

[0052] The first embodiment of this application relates to a co-agonist targeting GLP-1R, GIPR, and GCGR, wherein the polypeptide sequence of the co-agonist targeting GLP-1R, GIPR, and GCGR is as follows:

[0053] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{Dicarboxylic acid-γ-Glu-(AEEA)n-Lys} -Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2,.

[0054] Where n is 1 or 2.

[0055] In the first embodiment, the dicarboxylic acid is a C14 to C24 dicarboxylic acid.

[0056] In the first embodiment, the dicarboxylic acid is selected from any one of the following: C14 dicarboxylic acid, C16 dicarboxylic acid, C18 dicarboxylic acid, C20 dicarboxylic acid, C22 dicarboxylic acid, and C24 dicarboxylic acid.

[0057] In the first embodiment, the polypeptide sequence of the co-agonist is selected from any of the following:

[0058] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,12-dodecanedioic acid-γ-Glu-AEEA-Ly s}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2;

[0059] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,14-tetradecanedioic acid-γ-Glu-AEEA-Ly s}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2;

[0060] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,16-Hexadecanedioic acid-γ-Glu-AEEA-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2;

[0061] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,18-Octadecanedioic acid-γ-Glu-AEEA-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2;

[0062] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,18-Octadecanedioic acid-γ-Glu-AEEA-AEEA-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2;

[0063] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,20-Eicosanedioic acid-γ-Glu-AEEA-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2;

[0064] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,22-Docosanedioic acid-γ-Glu-AEEA-Ly s}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2.

[0065] Compared with the prior art, this embodiment has the following advantages: (1) Multi-target effect: By simultaneously targeting GLP-1 receptor, GIP receptor and glucagon receptor, the triple agonist can exert a more comprehensive physiological effect, including lowering blood sugar, promoting insulin secretion, controlling appetite, regulating metabolic disorders, and controlling fat breakdown; (2) Significant weight loss effect: Due to the synergistic effect between the three targets, the triple agonist has a stronger weight loss potential than single or dual agonists; (3) Improvement of multiple metabolic indicators: The triple agonist can improve multiple metabolic markers related to obesity and non-alcoholic fatty liver disease, including BMI, body fat percentage, glycated hemoglobin, fasting blood glucose, postprandial blood glucose, etc.; (4) Good safety and tolerability: Triple agonists have similar safety and tolerability to other incretin-based therapies; (5) Potential treatment for multiple diseases: Triple agonists are not only expected to be used as a new generation of drugs for the prevention or treatment of diabetes and obesity, but may also be used to prevent or treat non-alcoholic fatty liver disease, hyperlipidemia, Alzheimer's disease and other diseases; (6) Long-lasting effect: Due to the modification of long-chain fatty acids, it has a long half-life in the body, low dosing frequency and good medication compliance; (7) Advantages of chemical synthesis: The preparation of this compound by chemical synthesis has the advantages of reliable method, controllable cost and precise control of amino acid sequence, which is conducive to the large-scale production and application of the drug.

[0066] The second embodiment of this application relates to a method for synthesizing a polypeptide sequence of a co-agonist targeting GLP-1R, GIPR and GCGR. The following synthesis method is only an example, and the steps are conventional operations unless otherwise specified. The raw materials and reaction reagents can be obtained from publicly available commercial sources unless otherwise specified.

[0067] sequence:

[0068] Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,18-octadecanedioic acid-γ-Glu-AEEA-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2 (i.e. T yr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{diacid-C20-γ-Glu-(AEEA)-Ly s}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2).

[0069] 1. Experimental equipment: 0.0001 g electronic balance; vertical reactor (20*500 mm, No. 1 sintered glass core); 50 mL beaker; magnetic stirrer; HPLC semi-preparative unit; 30 cm C18 column; disposable pipettes; nitrogen generator; liquid nitrogen; circulating water vacuum pump; wash bottle; test tubes; long-necked pipettes; freeze dryer; constant temperature heater.

[0070] 1.2 Experimental materials and reagents: RINK resin; anhydrous DCM; DIC, methanol; DCM; DMF; 20% piperidine / DMF; Detection reagent A (5g ninhydrin-100ml anhydrous ethanol), B (analytical grade pyridine); HOBT; acetonitrile, diethyl ether, pure water; TFA; TIS; hydrazine hydrate; diacyl-C20; BOC anhydride; ammonium acetate.

[0071] Amino acids:

[0072] FMOC-Ser(tbu)-OH, FMOC-PRO-OH, FMOC-AIB-OH, FMOC-GLY-OH, FMOC-GLU(OTBU)-OH

[0073] FMOC-LEU-OH, FMOC-TYR(TBU)-OH, FMOC-ILE-OH, FMOC-PHE-OH, FMOC-ALA-OH

[0074] FMOC-GLN(TRT)-OH, FMOC-LYS(DDE)-OH, FMOC-GLU-OTBU, FMOC-α-Me-Leu-OH

[0075] FMOC-ASP(OTBU)-OH, FMOC-THR(TBU)-OH, Fmoc-Peg2-OH; diacid-C20

[0076] 1.3 A vertical reactor (20*500mm, No. 1 sand core) was selected, and the peptide name was marked on the reactor. 2g of RINK resin was weighed using an electronic balance, placed in the reactor, and soaked in DCM for 30 minutes. Then, the resin was washed with DMF once and dried.

[0077] 1.4 Weigh 0.6 mmol of FMOC-Ser(tbu)-OH (C-terminus) and HOBT and add them to the reactor, along with 20 mL of LDM and 0.6 mmol of DIC. Bubble the reaction under nitrogen for 1.5 h. After the reaction is complete, wash the reactor four times, add 0.6 mmol of acetic anhydride and 20 mL of DCM mixture, and then add 0.6 mmol of DIEA and react for 30 min.

[0078] 1.5 Washing: After the liquid in the reactor is drained by a circulating water vacuum pump, DMF is added to the reactor through a wash bottle. The volume of the reagent should be about 3 times the volume of the resin to completely immerse the resin in the solution. Wash for 30 seconds, and then drain the liquid in the reactor by a circulating water vacuum pump for about 30 seconds. Repeat this operation 4 times.

[0079] 1.6 De-Fmoc: Add 20% piperidine / DMF solution to the reactor using a wash bottle. The volume of the reagent should be about 3 times the volume of the resin to ensure that the resin is completely immersed in the solution. Bubble the reaction with nitrogen for 20 minutes.

[0080] 1.7 Washing: Refer to 1.5, and replace the industrial-grade DMF with analytical-grade DMF during the fifth wash;

[0081] 1.8 Resin Detection: Use a long-necked pipette to take 10-20 resin particles from the reactor and place them at the bottom of the test tube. Then, use a dropper to add two drops of test reagent A and two drops of test reagent B to the test tube, ensuring that the resin and test reagents are in full contact. Then, place the test tube in a constant temperature of 100℃ for 2 minutes and observe the resin color. If the resin develops color, it indicates that 1.6 Fmoc removal is successful. If no color develops, repeat steps 1.6-1.8.

[0082] 1.9 Coupling: Weigh 2 mmol FMOC-PRO-OH (C-terminal second position) and 2 mmol HOBT into a 50 mL beaker, dissolve them thoroughly with 20 mL DMF, then add 2 mmol DIC, stir magnetically for 5 min, add to the dried resin, and react under nitrogen bubbling for 1 h.

[0083] 1.10 Resin testing: Refer to 1.8 and observe the resin color. If there is no color, it indicates that the connection is complete, and proceed to step 1.11; if there is color, repeat step 1.9.

[0084] 1.11 Washing, same as 1.5.

[0085] 1.12 Repeat steps 1.6-1.11, coupling the remaining amino acids sequentially until peptide coupling is complete (see 1.6), followed by washing (see 1.5).

[0086] 1.13 BOC blocking: Add 2 mmol BOC anhydride and 20 mL DCM mixture, then add 2 mmol DIEA and react for 30 min, then wash (same as 1.5).

[0087] 1.14 Side chain modification: dde was removed using a DMF solution containing 1% hydrazine hydrate. 20 ml was added each time, with each addition lasting 15 min, repeated 3 times. Coupling of FMOC-PEG2-OH, FMOC-Glu-Otbu, and diabetes-C20 was performed according to procedures 1.6-1.11.

[0088] 1.15 Resin drying: After washing the resin three times with methanol, it is dried into dry granules.

[0089] 2. Cleavage of peptides from resin

[0090] 2.1 Preparation of cutting reagent

[0091] Prepare the cutting reagent (using 100ml of cutting reagent as an example).

[0092] The formula is: 95mL TFA + 2mL LEDT + 2mL Tis + 1mL H2O, for later use. The general preparation amount is 1g resin plus 10mL cutting reagent, and 1mL of cutting reagent is washed with 10mL ether.

[0093] 3.2 Preparation for Settlement

[0094] Pre-cool the peptide in anhydrous diethyl ether at -20°C (pre-cooling time ≥ 2h). Precipitate the peptide with 10mL of diethyl ether using 1ml of the cleavage reagent.

[0095] 3.3 Peptide cleavage

[0096] After the resin was dried, it was weighed and then cut with cutting fluid for 2 hours. Then it was centrifuged and settled with diethyl ether.

[0097] 4. Peptide purification

[0098] Preparation: Chromatographic column: 30*250mm Daisogel 8μm

[0099] Mobile phase: A: 0.1% TFA in water; B: 0.1% TFA in acetonitrile

[0100] Flow rate: 15 ml / min

[0101] The sample was loaded via pump A, and then a 10% acetonitrile solution was run for 5 minutes to begin gradient elution.

[0102] Time B.conc

[0103] 0 10%

[0104] 5 35%

[0105] 45 75%

[0106] Prepare and collect the sample peaks for detection, with an analytical purity greater than 50%.

[0107] II. Preparation:

[0108] Column: 30*250mm Daisogel 8 microns

[0109] Mobile phase: A: Water (15 mmol ammonium acetate / L) B: Acetonitrile

[0110] Flow rate: 15 ml / min

[0111] The sample was loaded onto pump A, and then pump A was run for 5 minutes to equilibrate before starting the gradient.

[0112] Time B.conc

[0113] 0 10%

[0114] 5 35%

[0115] 45 75%

[0116] Prepare and collect the sample peaks for detection, with an analytical purity greater than 70%.

[0117] 5. Sample freeze-drying

[0118] The sample was transferred into a lyophilization bottle, frozen in liquid nitrogen, and then freeze-dried in a freeze dryer for 24 hours to obtain the sample containing the target polypeptide sequence.

[0119] Those skilled in the art will understand that the above embodiments are specific embodiments for implementing this application, and in practical applications, various changes can be made to them in form and detail without departing from the spirit and scope of this application.

Claims

1. A co-agonist targeting GLP-1R, GIPR and GCGR, wherein, The polypeptide sequence of the co-agonist is as follows: Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{Dicarboxylic acid-γ-Glu-(AEEA)n-Lys} -Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2, Where n is 0, 1, or 2.

2. The co-agonist of claim 1, wherein, The dicarboxylic acid is a C14 to C24 dicarboxylic acid.

3. The co-agonist of claim 2, wherein, The dicarboxylic acid is selected from any one of the following: C14 dicarboxylic acid, C16 dicarboxylic acid, C18 dicarboxylic acid, C20 dicarboxylic acid, C22 dicarboxylic acid, and C24 dicarboxylic acid.

4. The co-agonist as described in claim 1, wherein, The polypeptide sequence of the co-agonist is selected from any of the following: Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,12-dodecanedioic acid-γ-Glu-AEEA-Ly s}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2; Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,14-tetradecanedioic acid-γ-Glu-AEEA-Ly s}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2; Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,16-hexadecanedioic acid-γ-Glu-AEEA-Ly s}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2; Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,18-octadecanedioic acid-γ-Glu-AEEA-Ly s}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2; Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,18-octadecanedioic acid-γ-Glu-AEEA-AEEA- Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2; Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,20-eicosanedioic acid-γ-Glu-AEEA-Ly s}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2; Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,22-Docosanedioic acid-γ-Glu-AEEA-Ly s}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2.

5. A pharmaceutical composition, wherein, It comprises: the co-agonist as described in claim 1 and a pharmaceutically acceptable carrier.

6. Use of a co-agonist targeting GLP-1R, GIPR, and GCGR in the preparation of a medicament for treating a disease, wherein, The polypeptide sequence of the co-agonist is as follows: Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{Dicarboxylic acid-γ-Glu-(AEEA)n-Lys} -Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2, Where n is 0, 1, or 2; The disease is selected from any one or a combination of the following: obesity, non-alcoholic fatty liver disease, type 2 diabetes, and hyperlipidemia.

7. The use according to claim 6, wherein, The dicarboxylic acid is a C14 to C24 dicarboxylic acid.

8. The use according to claim 7, wherein, The dicarboxylic acid is selected from any one of the following: C14 dicarboxylic acid, C16 dicarboxylic acid, C18 dicarboxylic acid, C20 dicarboxylic acid, C22 dicarboxylic acid, and C24 dicarboxylic acid.

9. The use according to claim 6, wherein, The polypeptide sequence of the co-agonist is selected from any of the following: Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,12-dodecanedioic acid-γ-Glu-AEEA-Ly s}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2; Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,14-tetradecanedioic acid-γ-Glu-AEEA-Ly s}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2; Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,16-hexadecanedioic acid-γ-Glu-AEEA-Ly s}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2; Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,18-octadecanedioic acid-γ-Glu-AEEA-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2; Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,18-octadecanedioic acid-γ-Glu-AEEA-AEEA-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2; Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,20-eicosanedioic acid-γ-Glu-AEEA-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2; Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{1,22-docosanedioic acid-γ-Glu-AEEA-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-{Aib}-Pro-Pro-Pro-Ser-NH2。