GIP and GLP-1 dual receptor agonist, pharmaceutical composition, and use

Abstract

A GIP and GLP-1 dual receptor agonist, a pharmaceutical composition, and the use. In particular, the present application relates to a compound represented by formula I, a pharmaceutical composition comprising the compound, and the use of the compound as a GIP and GLP-1 dual receptor agonist in the field of medicine. The compound represented by formula I exhibits excellent GIPR and GLP-lR agonist activity and excellent pharmaceutical activity in reducing blood sugar and controlling body weight, is a therapeutic drug having a clinical application prospect, and can be used for preventing and treating diseases such as diabetes, diabetes complications, obesity, or obesity complications.

Description

GIP and GLP-1 dual receptor agonist, pharmaceutical composition and use

[0001] This disclosure claims priority to the following patent applications: Chinese patent application filed with the China Patent Office on March 25, 2021, with application number 202110321851.6, and Chinese patent application filed with the China Patent Office on May 20, 2021, with application number 202110553745.0; the entire contents of the above patent applications are incorporated by reference into this disclosure. Technical Field

[0002] The present disclosure belongs to the field of medical technology. Specifically, the present disclosure relates to a polypeptide as shown in Formula I, a dual receptor agonist of GIP and GLP-1, a pharmaceutical composition, and use thereof in preventing and / or treating diseases related to metabolic disorders. Background Art

[0003] Obesity and diabetes are growing global health problems, linked to a variety of other diseases, including cardiovascular disease (CVD), obstructive sleep apnea, stroke, peripheral arterial disease, microvascular complications, and osteoarthritis. Therefore, developing new therapies and therapeutic drugs for obesity, diabetes, and their complications is crucial for improving human health.

[0004] Glucose-dependent insulinotropic peptide (GIP) is a 42-amino acid gastrointestinal regulatory peptide that plays a physiological role in glucose homeostasis by stimulating insulin secretion from pancreatic β cells in the presence of glucose and protecting pancreatic β cells. Glucagon-like peptide-1 (GLP-1) is a 37-amino acid peptide that stimulates insulin secretion, protects pancreatic β cells, and inhibits glucagon secretion, gastric emptying, and food intake, leading to weight loss. GIP and GLP-1 are secreted by K cells and L cells of the small intestinal endothelium, respectively, and are known as incretins. Incretin receptor signaling plays a key physiological role in glucose homeostasis.

[0005] Both GIP and GLP-1 produce their corresponding physiological effects by binding to their specific receptors, namely the GIP (GIPR) receptor and the GLP-1 receptor (GLP-1R). Both receptors belong to the G protein-coupled receptor family. In normal physiology, GIP and GLP-1 are secreted from the intestine after a meal. These incretins enhance the physiological response to food, including inducing satiety, promoting insulin secretion, protecting pancreatic beta cells, and disposing of nutrients. Patients with type 2 diabetes have been found to have an impaired incretin response.

[0006] Despite the potential antidiabetic activity of GIP and GLP-1, the administration of GLP-1 analogs has been found to be limited by side effects such as nausea and vomiting, and thus most often, administration fails to achieve full glycemic control and weight loss. GIP alone has very limited glucose-lowering capacity in patients with type 2 diabetes. Both native GIP and GLP-1 are rapidly inactivated by the ubiquitous protease DPP IV and therefore can only be used for short-term metabolic control.

[0007] Diabetics generally develop a tolerance to GIP, and even levels of GIP above physiological levels have little effect on lowering blood sugar. However, this is different with GLP-1, with most diabetic patients maintaining sensitivity to GLP-1. This relationship between GIP and GLP-1 is similar to that of two other important hormones, insulin and leptin: diabetic patients are sensitive to insulin, but obese patients are resistant to leptin. Therefore, while the GLP-1 field has flourished and become the most important class of glucose-lowering drugs, the development of GIP has been very slow. Studies have shown that sensitivity to GIP can be restored if blood sugar is lowered to a certain level, suggesting that co-stimulation of the GLP-1R / GIPR can exert a synergistic hypoglycemic effect. Dual agonists of GIP and GLP-1 receptors may produce even more superior blood sugar-lowering effects and stimulate insulin secretion.

[0008] WO2016111971A1 discloses a GIP / GLP dual agonist, LY3298176. LY3298176 primarily consists of the amino acid sequence of GIP, modified at lysine 20 using a GLP-1 drug modification method. LY3298176 is currently in a Phase II clinical trial for its glucose-lowering and weight-loss efficacy. Results show improved efficacy and tolerability with a low initial dose and minimal subsequent dose escalation. After just eight weeks of treatment, patients with type 2 diabetes demonstrated significant reductions in A1C and body weight.

[0009] The development of novel dual receptor agonists of GIP and GLP-1 to provide safe and effective new treatments for obesity, diabetes and their complications is an important issue that needs to be urgently addressed in this field.

[0010] Summary of the Invention

[0011] Problems to be solved by the invention

[0012] Given the problems existing in the prior art, for example, the therapeutic application prospects of administering GIP or GLP-1 alone as an antidiabetic drug are limited. To this end, the present disclosure provides a novel compound as shown in Formula I, which has agonistic activity at both GLP-1R and GIP receptors. This compound, as a dual agonist of GLP-1 and GIP receptors, exhibits excellent hypoglycemic effects and has good clinical application prospects.

[0013] Solutions for solving problems

[0014] The present disclosure first provides a compound as shown in Formula I or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope-labeled substance or prodrug thereof:

[0015] Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-A 40

[0016] I;

[0017] Among them, A 40 is the following structure connected to Ser via a peptide bond:

[0018] A 40 for

[0019] R1 is -(Z1) m -(Z2) n -C(=O)-(CH2) o -C(=O)-Z3;

[0020] m is 0 or 1;

[0021] If present, Z1 is -C(=O)-(CH2) r -(Z4) p -NH-, wherein p is any integer from 4 to 8, each Z4 is independently -O-CH2-CH2- or -NH-C(=O)-CH2-, and r is 1 or 2;

[0022] n is any integer between 0 and 2;

[0023] If present, each Z2 is independently Where q is any integer between 2 and 5;

[0024] o is any integer between 14 and 18, preferably 18;

[0025] Z3 is hydroxyl or

[0026] In the present disclosure, the amino acid sequence from amino acid 1 to amino acid 39 in the compound of Formula I (Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser) is shown in SEQ ID NO: 1.

[0027] In one embodiment, the compound of the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotopically labeled substance or prodrug thereof, wherein:

[0028] R1 is -C(=O)-(CH2) o -C(=O)-Z3;

[0029] o is any integer between 14 and 18, preferably 18;

[0030] Z3 is hydroxyl or Hydroxyl is preferred.

[0031] In one embodiment, the compound of the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotopically labeled substance or prodrug thereof, wherein:

[0032] R1 is -Z1-Z2-C(=O)-(CH2) o -C(=O)-Z3;

[0033] Z1 is -C(=O)-CH2-(Z4)7-NH-, wherein each Z4 is independently -O-CH2-CH2- or -NH-C(=O)-CH2-, preferably -(Z4)7- is -(O-CH2-CH2)3-(NH-C(=O)-CH2)-(O-CH2-CH2)3- or -(O-CH2-CH2)7-;

[0034] Z2 is Preferred wherein q is any integer between 2 and 5, preferably 2;

[0035] o is any integer between 14 and 18;

[0036] Z3 is hydroxyl or Hydroxyl is preferred.

[0037] In one embodiment, the compound according to the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope-labeled substance or prodrug thereof, is characterized in that A 40 Select from any of the following structures:

[0038]

[0039]

[0040]

[0041]

[0042] The structure shown in any one of the following is preferred:

[0043]

[0044] The present disclosure further provides a compound represented by any one of the following formulae I-1 to I-14, or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope-labeled compound, or prodrug thereof:

[0045]

[0046]

[0047]

[0048]

[0049] The present disclosure further provides a dual receptor agonist of GIP and GLP-1, wherein the dual receptor agonist of GIP and GLP-1 is a compound represented by the following formula I-3 or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof:

[0050]

[0051] The length and specific structure of the fatty side chain of the peptide in the present disclosure do not affect the hypoglycemic effect, but mainly affect the metabolic rate of the compound in the body, that is, the sustained release time of the drug molecule in the body, that is, the total effective time of the drug effect, that is, the compounds I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-11, I-12, I-13, and I-14 of the present disclosure have considerable hypoglycemic effects, and there are similarities in the metabolic rates in the body. Prior art documents WO2016111971A1 and WO2006097537A2 have disclosed the effects of fatty side chains on the metabolism of GLP agonist active agents, and the entire contents of the above patents are incorporated herein by reference.

[0052] The present disclosure further provides a pharmaceutical composition comprising the compound according to the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope-labeled substance or prodrug thereof, or the dual receptor agonist of GIP and GLP-1 according to the present disclosure;

[0053] Optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

[0054] The present disclosure further provides use of the compound according to the present disclosure or its pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug, the dual receptor agonist of GIP and GLP-1 according to the present disclosure, or the pharmaceutical composition according to the present disclosure in the preparation of a medicament for preventing and / or treating diseases related to metabolic disorders;

[0055] Optionally, the metabolic disorder-related disease is diabetes, diabetic complications, obesity, or obesity complications.

[0056] In one embodiment, the compound according to the present disclosure or its pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope-labeled or prodrug, the dual receptor agonist of GIP and GLP-1 according to the present disclosure, or the pharmaceutical composition according to the present disclosure is used to prevent and / or treat diseases related to metabolic disorders;

[0057] Optionally, the metabolic disorder-related disease is diabetes, diabetic complications, obesity, or obesity complications.

[0058] The present disclosure further provides a method for preventing and / or treating diseases related to metabolic disorders, comprising administering to a subject a preventively and / or therapeutically effective amount of the compound according to the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof, the dual receptor agonist of GIP and GLP-1 according to the present disclosure, or the pharmaceutical composition according to the present disclosure;

[0059] Optionally, the metabolic disorder-related disease is diabetes, diabetic complications, obesity, or obesity complications.

[0060] Effects of the Invention

[0061] The present disclosure provides a novel compound as shown in Formula I, wherein the lysine residue at position 40 is modified with an R1 group. The present disclosure has found through experiments that the compound shown in Formula I has both GIP and GLP-1 polypeptide activities and is a dual receptor agonist for GIP and GLP-1. Compared with the compound LY3298176 modified with the lysine residue at position 20, the compound shown in Formula I exhibits comparable or reduced EC 50 value.

[0062] The side chain attachment position has a significant impact on the compound's hypoglycemic effect. The compound shown in Formula I, as a dual receptor agonist of GIP and GLP-1, exhibits significant hypoglycemic and weight-reducing effects in type 2 diabetes model (db / db) mice, providing a therapeutic drug with great clinical application prospects for the prevention and treatment of metabolic disorder-related diseases. It can be used to prevent and treat diseases such as diabetes, diabetic complications, obesity, or obesity complications.

[0063] In one embodiment, the present disclosure provides a compound as shown in formula I-3. The present disclosure found in cell experiments that the compound as shown in formula I-3 has a lower EC for both GIPR and GLP-1R than LY3298176, a dual receptor agonist of GIP and GLP-1. 50 Compared with the marketed drug semaglutide, the compound represented by formula I-3 has a significantly lower EC 50The values ​​indicate that the compound shown in Formula I-3 exhibits a significantly good activation effect on both GIPR and GLP-1R, and its activity as a dual receptor agonist of GIP and GLP-1 is superior to that of currently developed compounds. In addition, the compound shown in Formula I-3 can effectively reduce the blood glucose content and the content of glycosylated hemoglobin in type 2 diabetes model (db / db) mice, control the weight gain of mice, and also show a better effect in affecting the insulin secretion of mice. The compound shown in Formula I-3 is superior to compound LY3298176 in reducing blood glucose and controlling body weight in type 2 diabetes model mice. The compound shown in Formula I-3 shows important clinical application prospects in the prevention and treatment of metabolic disorders such as diabetes or diabetic complications, and obesity or obesity complications.

[0064] In one embodiment, the compound represented by Formula I-3 (BG128) can cause an acute decrease in body weight and food intake in type 2 diabetes model (db / db) mice, which remain relatively stable in the later stages of administration. The compound represented by Formula I-3 significantly reduces non-fasting blood glucose and fasting blood glucose in db / db mice after the first administration, and reduces peripheral blood insulin and glycosylated hemoglobin levels. Furthermore, the effect of the compound represented by Formula I-3 on non-fasting blood glucose, serum insulin, and weight loss rate in mice is superior to that of the positive drug Tirzepatide at the same dose.

[0065] In one embodiment, the compound shown in Formula I-3 (BG128) has a significant therapeutic effect on STZ-HFD-induced C57BL / 6 diabetic mice with NASH. Each dose group of BG128 can significantly reduce fasting blood glucose, insulin, and peripheral blood HbA1c% in the model mice, and has a certain protective effect on the model mice. At the same time, the compound shown in Formula I-3 improves clinical biochemical indicators related to liver function and blood lipids, reduces liver NAS scores and fibrosis rates, and significantly delays NASH progression and fibrosis. The same dose of the compound shown in Formula I-3 has a better effect than the same dose of the positive drug Tirzepatide. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIG1 shows a mass spectrum of compound BG128;

[0067] FIG2 shows a mass spectrum of compound BGM125;

[0068] Figure 3 shows the blood glucose levels of db / db mice 1, 3, 6, 24, 48, and 72 hours after subcutaneous injection of BG121 (30 nmol / kg), BG128 (30 nmol / kg), BG128 (10 nmol / kg), and solvent control (Control); **, P < 0.01 [BG121 (30 nmol / kg), BG128 (30, 10 nmol / kg) vs. Control].

[0069] Figure 4 shows that db / db mice were subcutaneously injected with BG121 (30 nmol / kg), BG128 (30 nmol / kg), and BG128 (10 nmol / kg) as a solvent control every 3 days, and the blood glucose levels of the mice were measured on days 7, 13, 19, and 25 after the first administration; *, P < 0.05; ***, P < 0.001 [BG121 (30 nmol / kg) vs. Control]; #, P < 0.05 [BG128 (30 nmol / kg) vs. Control];

[0070] Figure 5 shows that db / db mice were subcutaneously injected with BG121 (30 nmol / kg), BG128 (30 nmol / kg), and BG128 (10 nmol / kg) as a solvent control every 3 days. The body weights of the db / db mice were measured on days 4, 7, 10, 13, 16, 19, and 22 after the first administration.

[0071] Figure 6 shows that db / db mice were subcutaneously injected with BG121 (30 nmol / kg), BG128 (30 nmol / kg), and BG128 (10 nmol / kg) as a solvent control every 3 days. The serum glycosylated hemoglobin levels of db / db mice were measured on the 25th day after the first administration; *, P < 0.05;

[0072] FIG7 shows that db / db mice were subcutaneously injected with BG121 (30 nmol / kg), BG128 (30 nmol / kg), and BG128 (10 nmol / kg) as a solvent control every 3 days. The insulin levels of db / db mice were measured on the 25th day after the first administration; ***, P < 0.001, P < 0.01;

[0073] Figure 8 shows the changes in animal body weight during the experimental period in the db / db-ND group, the Tirzepatide-0.15 mg / kg group, the BG128-0.05 mg / kg group, the BG128-0.15 mg / kg group, and the BG128-0.5 mg / kg group;

[0074] Figure 9 shows the changes in body weight growth rates of animals in the db / db-ND group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group;

[0075] Figure 10 shows the changes in food intake of animals in the db / db-ND group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group;

[0076] Figure 11 shows the changes in non-fasting blood glucose in animals in the db / db-ND group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group;

[0077] Figure 12 shows the changes in fasting blood glucose in animals in the db / db-ND group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group; Two-way ANOVA: ***p<0.001 vs.db / db-ND group; **p<0.01 vs.db / db-ND group; *p<0.05 vs.db / db-ND group; in Figure 12 , the bar graphs at the time points of 0, 7, 13, 19, 25, and 31 days represent the b / db-ND group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group, from left to right;

[0078] Figure 13 shows the changes in serum insulin concentrations in animals in the db / db-ND group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group; T-test: *p<0.05 vs.db / db-ND group;

[0079] Figure 14 shows the percentage of HbA1C in anticoagulated whole blood of animals in the db / db-ND, Tirzepatide-0.15 mg / kg, BG128-0.05 mg / kg, BG128-0.15 mg / kg, and BG128-0.5 mg / kg groups; One-way ANOVA: ***p<0.001 vs. db / db-ND group; #p<0.05vs.Tirzepatide-0.15mg / kg; & p<0.05vs.BG128-0.05mg / kg; $ p<0.05 vs. BG128-0.15mg / kg;

[0080] Figure 15 shows the results of HbA1C percentage reduction in animals in the Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group compared with the model group; One-way ANOVA: *** p<0.001 vs. db / db-ND group, ##p<0.001 vs. Tirzepatide-0.15 mg / kg;

[0081] Figure 16 shows the changes in body weight of animals in the normal group, model group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group during the experiment;

[0082] Figure 17 shows the changes in food intake of animals in the normal group, model group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group;

[0083] Figure 18 shows the changes in fasting blood glucose in animals in the normal group, model group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group; in Figure 18 , the bar graphs at each time point before HFD, before administration, 1 wks, 2 wks, 3 wks, 4 wks, 5 wks, 6 wks, 7 wks, and the endpoint represent the normal group, model group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group from left to right;

[0084] Figure 19 shows the insulin changes in animals in the model group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group; One-Way ANOVA: **p<0.05 vs. model group; ***p<0.001 vs. model group;

[0085] Figure 20 shows the percentage changes in % HbA1C in the peripheral blood of animals in the model group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group; One-Way ANOVA: *p<0.05 vs. model group;

[0086] Figure 21 shows the changes in ALT and AST in the blood of animals in the normal group, model group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group;

[0087] Figure 22 shows the changes in TC and TG in the blood of animals in the normal group, model group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group;

[0088] Figure 23 shows the changes in high- and low-density lipoprotein levels in the blood of animals in the normal group, model group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group;

[0089] Figure 24 shows HE staining images of liver tissues of animals in the normal group, model group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group (x400); A: normal control group; B: model group; C: Tirzepatide-0.15 mg / kg; D: BG128-0.05 mg / kg; E: BG128-0.15 mg / kg; F: BG128-0.5 mg / kg;

[0090] Figure 25 shows the changes in inflammatory cell infiltration and fatty degeneration in animals of the normal group, model group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group;

[0091] Figure 26 shows the NAS score results of animals in the normal group, model group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group;

[0092] Figure 27 shows Sirius red staining images of liver tissues of animals in the normal group, model group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group (x200); A: normal control group; B: model group; C: Tirzepatide-0.15 mg / kg; D: BG128-0.05 mg / kg; E: BG128-0.15 mg / kg; F: BG128-0.5 mg / kg;

[0093] FIG28 shows the percentage of fibrosis in the liver tissues of animals in the normal group, model group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group. DETAILED DESCRIPTION

[0094] definition

[0095] Unless stated otherwise, the terms used in this disclosure have the following meanings.

[0096] In the claims and / or description of the present disclosure, the word "a" or "an" or "the" may mean "one", but may also mean "one or more", "at least one" and "one or more than one".

[0097] As used in the claims and the specification, the words "comprising," "having," "including," or "containing" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. At the same time, "comprising," "having," "including," or "containing" can also be closed-ended and exclude additional, unrecited elements or method steps.

[0098] Throughout this application, the term "about" indicates that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0099] As used herein, the term "agonist" refers to a substance (ligand) that activates signaling through a target receptor type. For example, if the GLP-1 receptor is the target receptor, the agonist has GLP-1 receptor activating activity, such as a GLP-1 polypeptide or an analog thereof.

[0100] The term "EC 50 ” refers to the half effective concentration, and in this disclosure, EC 50 The agonist activity of the compound is evaluated. For example, the GIP receptor is used as the target and the EC value of the compound on the GIP receptor is tested. 50 The values ​​are used to evaluate the agonist activity of the compounds on GIP receptors.

[0101] The term "treatment" as used in the context of this disclosure means that after a subject has developed a disease, the subject is exposed to (e.g., administered) a compound of the present disclosure, or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotopically labeled substance, prodrug, or pharmaceutical composition containing the same (hereinafter also referred to as the "pharmaceutical composition of the present disclosure"), thereby alleviating the symptoms of the disease compared to the absence of such exposure. It does not necessarily mean that the symptoms of the disease are completely suppressed. Suffering from a disease means that the body has developed symptoms of the disease.

[0102] The term "prevention" as used in the context of the present disclosure means that before a subject develops a disease, the subject is exposed to (e.g., administered) a compound of the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label, prodrug or pharmaceutical composition of the present disclosure, thereby alleviating the symptoms after developing the disease compared to when the subject does not develop the disease. It does not necessarily mean that the disease must be completely suppressed.

[0103] The terms "individual," "patient," or "subject" as used herein include mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0104] The term "therapeutically effective amount" as used in the context of this disclosure refers to an amount effective to achieve the desired therapeutic outcome at the required dosage and for the required period of time. The therapeutically effective amount of a compound or pharmaceutical composition of the present disclosure can vary depending on a variety of factors such as the disease state, the age, sex, and weight of the individual, and the ability of the immunoadjuvant or pharmaceutical composition to elicit a desired response in the individual.

[0105] The term "pharmaceutically acceptable salt" as used in the context of this disclosure refers to a salt prepared from a compound of the present disclosure with a relatively non-toxic acid or base. When the compound of the present disclosure contains a relatively acidic functional group (e.g., a carboxyl group or a sulfonic acid group), a base addition salt can be obtained by contacting its free form with a sufficient amount of a base in a pure solution or a suitable inert solvent. Non-limiting examples of pharmaceutically acceptable base addition salts include, but are not limited to, sodium salts, potassium salts, ammonium salts, calcium salts, magnesium salts, organic amine salts, or similar salts. When the compound of the present disclosure contains a relatively alkaline functional group (e.g., an amino group or a guanidine group), an acid addition salt can be obtained by contacting its free form with a sufficient amount of an acid in a pure solution or a suitable inert solvent. Non-limiting examples of pharmaceutically acceptable acid addition salts include, but are not limited to, inorganic acid salts (e.g., hydrochloride, hydrobromide, hydroiodide, nitrate, carbonate, bicarbonate, phosphate, monohydrogen phosphate, dihydrogen phosphate, phosphite, sulfate, bisulfate, etc.), organic acid salts (e.g., acetate, propionate, isobutyrate, malonate, succinate, suberate, maleate, fumarate, citrate, tartrate, lactate, mandelate, benzoate, phthalate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, glucuronic acid, etc.), and amino acid salts (e.g., arginine salt, etc.). Specific forms of pharmaceutically acceptable salts can also be found in Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66: 1-19).

[0106] The term "pharmaceutical composition" as used in the context of this disclosure refers to a pharmaceutical composition comprising one or more compounds as shown in Formula I or pharmaceutically acceptable forms thereof (e.g., salts, hydrates, solvates, stereoisomers, tautomers, metabolites, prodrugs, etc.), and other components (e.g., pharmaceutically acceptable excipients).

[0107] The term "pharmaceutically acceptable excipient" as used in the context of this disclosure refers to an auxiliary material widely used in the field of drug production. The main purpose of using excipients is to provide a pharmaceutical composition that is safe to use, stable in nature and / or has specific functionality, and also to provide a method so that after the drug is administered to a subject, the active ingredient can be dissolved at a desired rate, or to promote the effective absorption of the active ingredient in the body of the subject receiving the drug. Pharmaceutically acceptable excipients can be fillers with inertness, or they can be functional ingredients that provide a certain function to the pharmaceutical composition (such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient in the composition). Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesives, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavorings, sweeteners, etc.

[0108] The pharmaceutical compositions of the present disclosure can be prepared using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, pulverizing, encapsulating, embedding and / or lyophilizing processes.

[0109] In the present disclosure, the purpose of using pharmaceutical composition is to promote the administration for organism, is conducive to the absorption of active ingredient, and then brings into play biological activity. Pharmaceutical composition of the present disclosure can be administered in any form, including injection (intra-arterial, intravenous, intramuscular, intraperitoneal, subcutaneous), mucosa, oral (oral solid preparation, oral liquid preparation), rectum, inhalation, implantation, local (such as eye) administration etc. Non-limiting examples of oral solid preparation include but are not limited to powders, capsules, lozenges, granules, tablets etc. Non-limiting examples of liquid preparations for oral or mucosal administration include but are not limited to suspensions, tinctures, elixirs, solutions etc. Non-limiting examples of topical preparations include but are not limited to emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum preparations. Non-limiting examples of parenteral preparations include but are not limited to solutions for injection, dry powders for injection, suspensions for injection, emulsions for injection etc. Pharmaceutical composition of the present disclosure can also be made into controlled release or delayed release dosage forms (such as liposomes or microspheres).

[0110] In the present disclosure, the route of administration can be varied or adjusted in any applicable manner to meet the requirements of the nature of the drug, the convenience of the patient and medical staff, and other relevant factors.

[0111] The term "metabolic disorder" used in the context of the present disclosure may be a disorder of glucose metabolism such as diabetes, diabetic complications, obesity, complications of obesity, etc. Since the association between obesity, diabetes and blood glucose metabolism is well known, these conditions can be, but are not necessarily, separate or mutually exclusive. In some embodiments, diabetes or diabetic complications include insulin resistance, glucose intolerance, elevated fasting blood glucose, prediabetes, type I diabetes, type II diabetes, gestational diabetes, hypertension, dyslipidemia, or a combination thereof. In some embodiments, complications of obesity include inflammation associated with obesity, gallbladder disease associated with obesity, or sleep apnea induced by obesity, or may be selected from the following related conditions: atherogenic dyslipidemia, dyslipidemia, elevated blood pressure, hypertension, a prothrombotic state, and a proinflammatory state, or a combination thereof.

[0112] The compounds disclosed herein, or their pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, metabolites, or prodrugs, or pharmaceutical compositions comprising the same, have excellent dual agonist activity for GIPR and GLP-1R, can effectively lower blood sugar and control weight gain in type 2 diabetes model mice, are used for preventing and / or treating metabolic disorders, and have good clinical applications and medical uses.

[0113] The technical solutions of the present disclosure will be described below in conjunction with specific embodiments. The following embodiments are provided to further illustrate the present disclosure, but are not intended to limit the scope of the present disclosure. It will be apparent to those skilled in the art that various changes and improvements can be made to the specific embodiments of the present disclosure without departing from the spirit and scope of the present disclosure.

[0114] The preparation of the compounds of the present disclosure can be achieved by synthetic methods well known to those skilled in the art, including but not limited to the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art, and preferred embodiments include but are not limited to the examples of the present disclosure. The known starting materials used in this disclosure can be synthesized using methods known in the art or purchased through conventional commercial means.

[0115] The separation and purification of the compounds of the present invention can be achieved by synthetic methods well known to those skilled in the art, including but not limited to column chromatography (CC), high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), etc.

[0116] The amino acids and their abbreviations and English abbreviations in this disclosure are shown in the following table:

[0117]

[0118] Example

[0119] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples (although indicating specific embodiments of the present disclosure) are given for illustrative purposes only, as various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art after reading the detailed description.

[0120] Unless otherwise specified, all reagents and raw materials used in this disclosure can be purchased through commercial channels.

[0121] Example 1 Solid-phase synthesis of target compound BG128 using the Fmoc method

[0122] Compound structure of BG128:

[0123]

[0124] Synthesis steps:

[0125] Fmoc-Rink MBHA Amide resin was used, 20% piperidine / DMF was used to remove Fmoc, HOBT / DIC was used as coupling reagent, DMF was used as reaction solvent, and the reaction was monitored by ninhydrin detection method.

[0126] (1) Sequentially connect the following protected amino acids to the Rink MBHA Amide resin:

[0127] Fmoc-Lys(Alloc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, F moc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)- OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tB u)-OH, Fmoc-Asp(OtBu)OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH, Boc-Tyr(tBu)-OH.

[0128] (2) Removal of protecting groups: Add 3 eq of a solution of Pd(PPh3)4 in CHCl3:AcOH:NMM (18:1:0.5) and react for 2 h. Then wash with chloroform (6*30 ml), 20% HOAc in DCM solution (6*30 ml), DCM (6*30 ml) and DMF (6*30 ml). Monitor with ninhydrin and it is positive. Then, condense Fmoc-AEEEA-OH, Fmoc-AEEEA-OH, Fmoc-Glu-OtBu and mono-tert-butyl eicosadioate in sequence to obtain the fully protected resin of BG128.

[0129] (3) Resin cleavage: 95% TFA / 2.5% water / 2.5% TIS was used, followed by precipitation and washing with icy MTBE. The crude product was purified by HPLC and freeze-dried to obtain the target compound BG128. The MS spectrum of compound BG128 is shown in Figure 1.

[0130] The following abbreviations are used:

[0131] DMF, N,N-dimethylformamide; Fmoc, 9-fluorenyloxycarbonyl; HOBT, N-hydroxybenzotriazole; DIC, N,N-diisopropylcarbodiimide; NMM, N-methylmorpholine; DCM, dichloromethane; Fmoc-AEEEA-OH, [2-[2-[2-(Fmoc-amino)ethoxy]ethoxy]ethoxy]acetic acid; MTBE, methyl tert-butyl ether; TFA, trifluoroacetic acid; TIS, triisopropylsilane.

[0132] Example 2: Solid-phase synthesis of the compound represented by formula I-1 using the Fmoc method

[0133] The structure of the compound shown in I-1:

[0134]

[0135]

[0136] Synthesis steps:

[0137] According to the preparation method shown in Example 1, in step (2), mono-tert-butyl eicosandioate is condensed on the side chain to obtain the compound shown in formula I-1. The MS of the compound shown in formula I-1 is: [M+3] 3+ It is 1508.4.

[0138] Example 3: Solid-phase synthesis of the compound represented by formula I-2 using the Fmoc method

[0139] The structure of the compound shown in I-2:

[0140]

[0141] Synthesis steps:

[0142] According to the preparation method shown in Example 1, in step (2), octadecane dioic acid mono-tert-butyl ester is condensed on the side chain to obtain the compound shown in formula I-2. The MS of the compound shown in formula I-2 is: [M+3] 3+ It is 1499.0.

[0143] Example 4: Solid-phase synthesis of the compound represented by formula I-4 using the Fmoc method

[0144] The structure of the compound shown in I-4:

[0145]

[0146] Synthesis steps:

[0147] According to the preparation method shown in Example 1, in step (2), Fmoc-AEEEA-OH, Fmoc-AEEEA-OH, and 3-(N-(20-(tert-butoxy)-20-oxoeicosanoyl)sulfamoyl)propionic acid are sequentially condensed on the side chain to obtain the compound shown in Formula I-4. The MS of the compound shown in Formula I-4 is: [M+3] 3+ It is 1679.6.

[0148] Example 5: Solid-phase synthesis of the compound represented by formula I-5 using the Fmoc method

[0149] The structure of the compound shown in I-5:

[0150]

[0151] Synthesis steps:

[0152] According to the preparation method shown in Example 1, in step (2), Fmoc-NH-PEG4-PA, Fmoc-Glu-OtBu, and octadecane dioic acid mono-tert-butyl ester are sequentially condensed on the side chain to obtain the compound shown in Formula I-5. The MS of the compound shown in Formula I-5 is: [M+3] 3+ 1626.5, [M+4] 4+ It is 1220.1.

[0153] Example 6: Solid-phase synthesis of the compound represented by formula I-6 using the Fmoc method

[0154] The structure of the compound shown in I-6:

[0155]

[0156] Synthesis steps:

[0157] According to the preparation method shown in Example 1, in step (2), Fmoc-NH-PEG8-PA, Fmoc-Glu-OtBu, and octadecane dioic acid mono-tert-butyl ester are sequentially condensed on the side chain to obtain the compound shown in Formula I-6. The MS of the compound shown in Formula I-6 is: [M+3] 3+ 1683.2, [M+4] 4+ It is 1262.7.

[0158] Example 7: Solid-phase synthesis of the compound represented by formula I-7 using the Fmoc method

[0159] The structure of the compound shown in I-7:

[0160]

[0161] Synthesis steps:

[0162] According to the preparation method shown in Example 1, in step (2), Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu-OtBu, and octadecane dioic acid mono-tert-butyl ester are sequentially condensed on the side chain to obtain the compound shown in formula I-7. The MS of the compound shown in formula I-7 is: [M+3] 3+ 1638.8, [M+4] 4+ It is 1229.4.

[0163] Example 8: Solid-phase synthesis of the compound represented by formula I-8 using the Fmoc method

[0164] The structure of the compound shown in I-8:

[0165]

[0166] Synthesis steps:

[0167] According to the preparation method shown in Example 1, in step (2), Fmoc-AEEA-OH, Fmoc-AEEA-OH, and 4-(N-(18-(tert-butoxy)-18-oxooctadecanoyl)sulfamoyl)butanoic acid are sequentially condensed on the side chain to obtain the compound shown in Formula I-8. The MS of the compound shown in Formula I-8 is: [M-3] 3- It is 1643.5.

[0168] Example 9: Solid-phase synthesis of the compound represented by formula I-9 using the Fmoc method

[0169] The structure of the compound shown in I-9:

[0170]

[0171] Synthesis steps:

[0172] According to the preparation method shown in Example 1, in step (2), Fmoc-AEEA-OH, Fmoc-AEEA-OH, and 6-(N-(18-(tert-butoxy)-18-oxooctadecanoyl)sulfamoyl)hexanoic acid are sequentially condensed on the side chain to obtain the compound of formula I-9. The MS of the compound of formula I-9 is: [M-3] 3- It is 1652.9.

[0173] Example 10: Solid-phase synthesis of the compound represented by formula I-10 using the Fmoc method

[0174] The structure of the compound shown in I-10:

[0175]

[0176] Synthesis steps:

[0177] According to the preparation method shown in Example 1, in step (2), Fmoc-AEEA-OH, Fmoc-AEEA-OH, and 4-((18-(tert-butoxy)-N-(3-(tert-butoxy)-3-oxypropyl)-18-oxyoctadecylamide)methyl)benzoic acid are sequentially condensed on the side chain to obtain the compound shown in Formula I-10. The MS of the compound shown in Formula I-10 is: [M+3] 3+ It is 1664.2.

[0178] Example 11: Solid-phase synthesis of the compound represented by formula I-11 using the Fmoc method

[0179] The structure of the compound shown in I-11:

[0180]

[0181] Synthesis steps:

[0182] According to the preparation method shown in Example 1, in step (2), Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu-(Asp(OtBu)-OtBu)-OH, and octadecanediol mono-tert-butyl ester are sequentially condensed on the side chain to obtain the compound shown in formula I-11. The MS of the compound shown in formula I-11 is: [M+3] 3+ It is 1677.2.

[0183] Example 12: Solid-phase synthesis of the compound represented by formula I-12 using the Fmoc method

[0184] The structure of the compound shown in I-12:

[0185]

[0186] Synthesis steps:

[0187] According to the preparation method shown in Example 1, in step (2), Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu-OtBu, and Asp[(OtBu)-OtBu]-octadecane dioic acid are sequentially condensed on the side chain to obtain the compound of formula I-12. The MS of the compound of formula I-12 is: [M+3] 3+ It is 1677.2.

[0188] Example 13: Solid-phase synthesis of the compound represented by formula I-13 using the Fmoc method

[0189] The structure of the compound shown in I-13:

[0190]

[0191] Synthesis steps:

[0192] According to the preparation method shown in Example 1, in step (2), Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosandioate are sequentially condensed on the side chain to obtain the compound shown in formula I-13. The MS of the compound shown in formula I-13 is: [M+3] 3+ It is 1648.1.

[0193] Example 14: Solid-phase synthesis of the compound represented by formula I-14 using the Fmoc method

[0194] The structure of the compound shown in I-14:

[0195]

[0196] Synthesis steps:

[0197] According to the preparation method shown in Example 1, in step (2), Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu-OtBu, Fmoc-Glu-OtBu, and mono-tert-butyl eicosandioate are sequentially condensed on the side chain to obtain the compound shown in formula I-14. The MS of the compound shown in formula I-14 is: [M+3] 3+ It is 1691.2.

[0198] Comparative Example 1: Solid-phase synthesis of target compound BGM125 by Fmoc method

[0199] Compound structure of BGM125:

[0200]

[0201] The amino acid sequence of the polypeptide portion of compound BGM125 is shown in SEQ ID NO: 1.

[0202] Synthesis steps:

[0203] The target peptide was synthesized using the Fmoc method using Fmoc-Rink MBHA Amide resin, 20% piperidine / DMF for Fmoc removal, HOBT / DIC as the coupling reagent, DMF as the reaction solvent, and ninhydrin detection for reaction monitoring:

[0204] (1) Sequentially connect the following protected amino acids to the Rink MBHA Amide resin:

[0205] Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc -Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln (Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH , Fmoc-Asp(OtBu)OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH, Boc-Tyr(tBu)-OH.

[0206] (2) Removal of protecting groups: Add 3 eq of Pd(PPh3)4 in CHCl3:AcOH:NMM (18:1:0.5) and react for 2 h. Then wash with chloroform (6*30 ml), 20% HOAc in DCM solution (6*30 ml), DCM (6*30 ml) and DMF (6*30 ml). Monitor with ninhydrin and it is positive. Then, condense Fmoc-AEEEA-OH, Fmoc-AEEEA-OH, Fmoc-Glu-OtBu and mono-tert-butyl eicosadioate in sequence to obtain the fully protected resin of BGM125.

[0207] (3) Resin cleavage: 95% TFA / 2.5% water / 2.5% TIS was used, followed by precipitation and washing with glacial methyl tert-butyl ether (MTBE). The crude product was purified by HPLC and freeze-dried to obtain the target compound BGM125, which was characterized by MS (see Figure 2).

[0208] Experimental Example 1

[0209] 1. Experimental materials:

[0210] 1.1 Reagents and equipment:

[0211] cAMP detection kit, Cisbio; 1M HEPES, Invitrogen; 1X HBSS, Invitrogen; Casein, Sigma; IBMX, Sigma; GIP (GIP), Tocris;

[0212] Host cells for GLP-1R: HEK293 cell line, deposited in Bioray Biopharmaceuticals (Suzhou) Co., Ltd.

[0213] Host cells for GIP R: CHO cell line, deposited in Bioray Biopharmaceuticals (Suzhou) Co., Ltd.;

[0214] OptiPlate-384 plate, PerkinElmer; 384 echo plate, Labcyte; EnVision, PerkinElmer; Cellular counter, Beckman;

[0215] 1.2 Test compound:

[0216] Table 1 Test compounds for cell experiments

[0217]

[0218] 2. Experimental methods:

[0219] a) Preparation of compound plates:

[0220] (1) Test compounds BG121, BG128, BGM125, and BGM134 were tested at the GIPR target site. Ten 4-fold dilutions were performed for each compound, starting at a concentration of 5 μM, and the dilutions were completed with Bravo. Ten 4-fold dilutions were performed for the reference compound GIP, starting at a concentration of 5 μM, and the dilutions were completed with Bravo.

[0221] (2) Test compounds BG121, BG128, BGM125, and BGM134 were tested at the GLP-1R target site. Ten 4-fold dilutions were performed for each compound, starting at a concentration of 0.5 μM, and dilutions were completed with Bravo. Ten 4-fold dilutions were performed for the reference compound GLP-1(7-37), starting at a concentration of 2 μM, and dilutions were completed with Bravo.

[0222] b) Transfer compounds:

[0223] (1) Use an Echo pipette to transfer 100 nL of compound to an OptiPlate-384 plate.

[0224] (2) Centrifuge the OptiPlate-384 plate at 1000 rpm for 5 seconds.

[0225] c) Preparation of cell suspension

[0226] (1) Take one cryopreserved tube of GLP-1R cells and one cryopreserved tube of GIPR cells and thaw them quickly in 37°C warm water.

[0227] (2) Transfer the cell suspension to a 15 mL centrifuge tube and gently rinse with 10 mL of HBSS.

[0228] (3) Centrifuge the tube at 1000 rpm at room temperature for 1 minute.

[0229] (4) Discard the supernatant.

[0230] (5) Gently break up the cells at the bottom and gently rinse with 10 ml of HBSS. Centrifuge to sediment the cells and resuspend the cells in the experimental buffer.

[0231] (6) Use Vi-cell to measure cell density and activity.

[0232] (7) Dilute the GLP-1R cell concentration to 2.0*E using the experimental buffer. 5 / mL.

[0233] (8) Transfer 100 nL of the diluted cell suspension into the OptiPlate-384 plate.

[0234] (9) Incubate at room temperature for 30 minutes.

[0235] d) Add detection reagent:

[0236] (1) Add 10 μL of 800 nM serially diluted cAMP standard to the empty wells of the OptiPlate-384 plate.

[0237] (2) Add 10 μL cAMP detection reagent.

[0238] (3) Cover the OptiPlate-384 plate with TopSeal-A film and incubate at room temperature for 60 minutes. Remove the TopSeal-A film and read the plate on EnVision.

[0239] 3. Experimental results:

[0240] Table 2 GIP receptor cAMP experimental results

[0241] No. Sample name EC 50(nM) 1BG121(LY3298176)1.794 2BG1280.2785 3BGM1250.6839 4BGM134(Semaglutide)>50 RefGIP0.1397

[0242] Table 3 GLP-1 receptor cAMP experimental results

[0243] No. Sample name EC 50 (nM) 1BG121(LY3298176)0.22732BG1280.10973BGM1250.069224BGM134(Semaglutide)0.01347RefGLP-10.005069

[0244] As can be seen from the data in Tables 2 and 3, the compounds disclosed herein have high dual receptor agonist activity for both the GLP-1 receptor and the GIP receptor, and their agonist activity for the GIP receptor is superior to that of the existing dual receptor agonist LY3298176, as well as the GLP-1 receptor agonist semaglutide, and their agonist activity for the GLP-1 receptor is superior to that of LY3298176.

[0245] Experimental Example 2

[0246] 1. Experimental materials:

[0247] 1.1 Solvent

[0248] Name: Citric acid solution;

[0249] Ingredients: 20 mM citric acid pH 7.0;

[0250] Storage conditions: 4°C;

[0251] Supplier: Sangon Bioengineering (Shanghai) Co., Ltd.

[0252] 1.2 Experimental animals

[0253] Strain: BKS mice (db / db mice); Source: Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; License No.: SCXK(Su)2018-0008; Certificate No.: 202100117; Grade: SPF; Number: 32; Gender: male; Age: 8 weeks (at the beginning of modeling); Animal identification: ear tag.

[0254] Rearing conditions: temperature, 22±2℃; humidity, 40-70%; light, artificial lighting to simulate day and night changes; drinking water, free drinking; feed, use mouse breeding feed, fast overnight before fasting blood sugar measurement, and eat freely at other times.

[0255] 1.3 Test compounds

[0256] Table 4 Test compounds in animal experiments

[0257] Name / Abbreviation BG121 (LY3298176) BG128 Specifications 2.69 mg 2.80 mg Assay 96.64% 95.61% Appearance White powder Storage Conditions -20°C -20°C Source Borri Biopharmaceuticals (Suzhou) Co., Ltd. Borri Biopharmaceuticals (Suzhou) Co., Ltd.

[0258] Limited Company

[0259] 1.4 4. Sample preparation method

[0260] Preparation of BG121 (1 mg / ml) stock solution: Weigh a certain amount of BG121, add an appropriate volume of solvent (20 mM citric acid, pH 7.0), aliquot into 100 μl tubes, and freeze at -80°C until use;

[0261] Preparation of BG128 (1 mg / ml) stock solution: Weigh a certain amount of BG128, add an appropriate volume of solvent (20 mM citric acid, pH 7.0), aliquot into 100 μl tubes, and freeze at -80°C until use;

[0262] BG121 (0.0144 mg / ml, for 30 nmol / kg dosing): Take 90 μL of the 1 mg / mL stock solution, add 810 μL of solvent to 10 mL of EP, vortex thoroughly, then add 5333 μL of solvent and vortex thoroughly. Use on the same day.

[0263] BG128 (0.0151 mg / ml, for 30 nmol / kg dosing): Take 90 μL of the 1 mg / mL stock solution, add 810 μL of solvent to 10 mL of EP, vortex thoroughly, then add 5044 μL of solvent and vortex thoroughly. Use on the same day.

[0264] BG128 (0.0050 mg / ml, for 10 nmol / kg administration): Add 2 mL of BG128 (0.0144 mg / ml) to 4 mL of solvent, vortex thoroughly, and use on the same day.

[0265] 2. Experimental design and methods

[0266] 2.1 Experimental design

[0267] 32 SPF male BKS-DB mice (db / db mice) were adaptively raised for 1 week. According to the fasting blood glucose value and body weight of the mice, they were randomly divided into 4 groups: solvent control group, BG121 (30nmol / kg) group, BG128 (30nmol / kg) group, and BG128 (10nmol / kg) group, and were fed with mouse breeding feed. During the experiment, the experimenter needs to observe the physical signs and health status of the experimental animals. Any abnormal manifestations of the animals, such as pain, depression, decreased activity, etc., must be recorded in the original experimental records. If the abnormal manifestations of the experimental animals exceed the provisions of the relevant animal welfare documents, the animals can be euthanized after notification. The experimental design is shown in the following table:

[0268] Table 5 Mouse grouping

[0269] Group No. Group Quantity Compound Concentration (mg / mL) 1 Solvent Control Group 8 Citric Acid-2 BG121 (30 nmol / kg) Group 8 BG121 0.014 43 BG128 (30 nmol / kg) Group 8 BG128 0.015 14 BG128 (10 nmol / kg) Group 8 BG128 0.0050

[0270] 2.2 Experimental methods

[0271] Modeling method: db / db mice develop spontaneous type 2 diabetes after 6 weeks of breeding, with symptoms such as obesity, insulin resistance, and hyperglycemia.

[0272] Administration: The drug is intended to be administered by subcutaneous injection in a clinical setting, with a volume of 10 mL / kg, administered once every 3 days for 22 consecutive days.

[0273] Fasting blood glucose measurement: According to the above animal grouping and dosing, fasting blood glucose and body weight were measured two days before the first dose. 8-week-old male mice with fasting blood glucose levels above 16.8 mmol / L were randomly divided into groups based on blood glucose levels. Fasting blood glucose levels were measured on days 7, 13, 19, and 25 after the first dose (bedding was changed and food was fasted before fasting blood glucose measurement).

[0274] Non-fasting blood glucose measurement: blood glucose was measured at 1, 3, 6, 24, 48, and 72 hours after the first administration;

[0275] Body weight measurement: twice a week;

[0276] Food intake measurement: measured twice a week.

[0277] 2.3 Blood biochemical test

[0278] After 25 days of administration, blood samples were collected by removing the eyeballs, and the serum insulin and glycosylated hemoglobin levels of the mice were measured.

[0279] 2.4 Statistical analysis

[0280] All reported quantitative data were calculated using GraphPad Prism 8.0 software for group mean and standard deviation calculations.

[0281] 3. Experimental results:

[0282] 3.1 Changes in non-fasting blood glucose in db / db mice after single administration of BG121 and BG128

[0283] BG121 (30 nmol / kg), BG128 (30 nmol / kg), and BG128 (10 nmol / kg) were subcutaneously injected into db / db mice, and the changes in non-fasting blood glucose in the mice were measured 1, 3, 6, 24, 48, and 72 h after the first administration. The results showed that the blood glucose levels at each time point after the administration of BG121 (30 nmol / kg) were lower than those in the solvent control group (Control); the administration of BG128 (30 nmol / kg) and BG128 (10 nmol / kg) could reduce blood glucose at each time point after administration, and was dose-dependent (Figure 3).

[0284] 3.2 Changes in fasting blood glucose in db / db mice after administration of BG121 and BG128

[0285] According to the experimental method, BG121, BG128, and a solvent control were subcutaneously administered to db / db mice continuously, and fasting blood glucose levels were measured on days 7, 13, 19, and 25 after the first dose. The results showed that BG121 (30 nmol / kg) and BG121 (10 nmol / kg) significantly reduced fasting blood glucose levels compared to the solvent control on day 25 after the first dose. BG128 (30 nmol / kg) also significantly reduced fasting blood glucose levels on days 19 and 25 after the first dose, with the hypoglycemic effect showing a dose-dependent manner (Figure 4).

[0286] 3.3 Body weight changes of db / db mice after BG121 and BG128 administration

[0287] According to the experimental method, db / db mice were continuously administered BG121, BG128, and a solvent control (Control). The mice were weighed on days 4, 7, 10, 13, 16, 19, and 22 after the first administration. The results showed that the mean body weight of the mice in the BG128 (30 nmol / kg) administration group was lower than that of the solvent control group at each time point after administration; while there was no significant difference in body weight between the BG128 (10 nmol / kg) administration group, the BG121 (30 nmol / kg) administration group, and the solvent control group. The results showed that the body weight of the mice in the BG128 (30 nmol / kg) administration group was significantly lower than that of the solvent control group and the BG121 (30 nmol / kg) administration group (Figure 5).

[0288] 3.4 Effects of BG121 and BG128 on glycosylated hemoglobin in db / db mice

[0289] According to the experimental method, the glycated hemoglobin content in the mouse serum was measured 25 days after administration. The results showed that 25 days after administration, the glycated hemoglobin content in the serum of the mice in the BG128 (30nmol / kg) administration group was lower than that in the solvent control group, while there was no significant difference in the glycated hemoglobin content between the BG128 (10nmol / kg) administration group and the BG121 (30nmol / kg) administration group and the solvent control group. The results showed that the glycated hemoglobin content in the mice in the BG128 (30nmol / kg) administration group was significantly lower than that in the BG121 (30nmol / kg) administration group and the solvent control group (Figure 6).

[0290] 3.5 Effects of BG121 and BG128 on serum insulin in db / db mice

[0291] According to the experimental method, the insulin content in the mouse serum was measured 25 days after administration. The results showed that 25 days after administration, the insulin content in the serum of mice in the BG128 (30nmol / kg) and BG128 (10nmol / kg) administration groups was significantly lower than that in the solvent control group, and was dose-dependent; while there was no significant difference in insulin content between the BG121 (30nmol / kg) administration group and the solvent control group (Figure 7). This experiment proves that the compounds of the present invention do not affect the secretion of insulin when exerting the effect of lowering blood sugar, and also confirms that BG128 is more effective than BG121 in exerting the effect of lowering blood sugar on insulin.

[0292] Experimental Example 3: Testing the inhibitory effect of BG128 on hyperglycemia in db / db mice

[0293] 1. Experimental Materials

[0294] 1.1 Experimental Animals

[0295] Fifty-five SPF male db / db mice, 6-8 weeks old, were purchased from Zhejiang Weitonglihua Laboratory Animal Technology Co., Ltd.

[0296] 1.2 The information of test and reference products is shown in the following table

[0297] Table 6 Information of test and reference products

[0298]

[0299] 1.3 Preparation of test and reference products

[0300] According to the dosing regimen in Table 7, a certain amount of Tirzepatide and BG128 were weighed and added to a solvent (pH=7.0 20 mM sodium citrate buffer) to prepare the corresponding concentration. The mixture was prepared once every 7 days and stored in aliquots at 2-8°C in the dark.

[0301] 2. Experimental methods

[0302] 2.1 Trial Grouping

[0303] Before administration, the animals were divided into groups based on their body weight and fasting blood glucose: db / db-ND group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group, with 11 animals in each group; see Table 7 for details.

[0304] 2.2 Dosage regimen

[0305] The day of the first administration was recorded as d1, and the time was recorded analogously; Tirzepatide and BG128 were administered once every three days, at the time points of d1, d4, d7, d10, d13, d16, d19, d22, d25, and d28, for a total of 10 administrations; see Table 7 for details.

[0306] Table 7 Trial groups and dosing regimen

[0307]

[0308] 2.3 Detection indicators

[0309] Body weight: Weigh the animals before administration, once every three days, and continue until the end of the experiment.

[0310] 2.3.1 Animal food intake

[0311] The animals' food intake was recorded starting three days before dosing and continued until the end of the experiment. The remaining amount was weighed before dosing each time.

[0312] 2.3.2 Animal blood glucose

[0313] 1) Non-fasting blood glucose test: 0h (pre), 1h, 2h, 3h, 6h, 24h, 48h, and 72h after the first administration;

[0314] 2) Fasting blood glucose: The test dates were d-1 (before grouping), d7, d13, d19, d25, and d31. The animals were fasted for 6 hours, and the fasting blood glucose was tested before drug administration.

[0315] 2.3.3 Blood biochemical tests

[0316] Three days after the last administration, the animals were weighed and fasted for 6 hours, and their fasting blood glucose was measured. After anesthesia, a non-anticoagulated whole blood aliquot was collected from the orbital venous plexus. The blood was allowed to stand at room temperature for 30 minutes, centrifuged at 5000 rpm for 5 minutes, and the serum was separated and stored at -80°C for serum insulin measurement. An anticoagulated whole blood aliquot was collected for HbA1c measurement. Serum insulin was measured using an ELISA assay.

[0317] 2.4 Data Analysis

[0318] Data were compiled in Excel 2017 and Prism 6.0 and presented as mean ± SEM (standard error of the mean). One-way ANOVA was used for analysis, with Tukey's test for significance between groups. Two-group comparisons were performed using a two-tailed T-test. Significant differences were considered between the two groups when p < 0.05. When a few significant differences were detected within the groups, GraphPad Prism 6.0 was used to identify outliers and exclude discrepant values.

[0319] 3. Results

[0320] 3.1 Body weight and weight gain rate

[0321] Results of animal body weight and weight growth rates (Figures 8 and 9) show that during the first dosing period, the weight of animals in each treatment group showed a significant downward trend, with the extent of weight loss in the BG128-treated groups positively correlated with the dose. After day 3, the weight of animals in each treatment group resumed steady growth. During the later stages of dosing, there were no significant differences in weight changes among the groups (p>0.05).

[0322] As shown in Figures 8 and 9, at the same dose, the weight loss rate of animals in the BG128-treated group was better than that in the Tirzepatide-treated group, that is, the therapeutic effect of BG128 was better than that of the positive control Tirzepatide.

[0323] 3.2 Food intake

[0324] The results of the changes in animal food intake (Figure 10) showed that compared with the db / db-ND group, the food intake of animals in each drug-treated group decreased significantly after the first dose, among which the decrease in food intake in the groups treated with different doses of BG128 was positively correlated with the dose; after d4, the food intake of animals in each drug-treated group resumed growth and fluctuated within a small range, maintaining relative stability.

[0325] As shown in Figure 10, at the same dose, the food intake of the BG128-treated group decreased significantly more than that of the positive control group Tirzepatide.

[0326] 3.3 Non-fasting blood glucose after the first dose

[0327] The results of non-fasting blood glucose monitoring of animals (Figure 11) showed that after the first dose, the non-fasting blood glucose of animals in the positive drug Tirzepatide-0.15 mg / kg group decreased rapidly within 6 hours after the first dose and gradually recovered and increased within 6-72 hours. The non-fasting blood glucose of animals in the BG128-treated group decreased rapidly within 24 hours after the first dose. The low-dose group (0.05 mg / kg) and the medium-dose group (0.15 mg / kg) gradually recovered and increased within 24-72 hours after administration, and the high-dose group (0.5 mg / kg) maintained a relatively low blood glucose level within 24-72 hours after administration. Compared with the db / db-ND group, the Tirzepatide-0.15 mg / kg group and the various doses of BG128 showed a significant inhibitory effect on blood glucose within 72 hours of the first dose. The inhibitory effect of BG128 was dose-dependent and superior to that of the positive drug Tirzepatide-0.15 mg / kg group at the same dose.

[0328] 3.4 Fasting blood glucose

[0329] Fasting blood glucose monitoring results (Figure 12, Table 8) showed that compared with the db / db-ND group, the Tirzepatide-0.15 mg / kg group and the various BG128 dose groups significantly decreased fasting blood glucose at different dosing periods (p<0.05). At the endpoint, fasting blood glucose levels in the BG128-dose groups were significantly lower than those in the db / db-ND group, demonstrating a dose-dependent trend. The high-dose BG128 group exhibited a more potent blood glucose-suppressing effect than the active drug Tirzepatide-0.15 mg / kg group.

[0330] Table 8: Changes in fasting blood glucose in animals (mmol / L) (Mean±SD)

[0331]

[0332] a: *p<0.05vs.db / db-ND,by Two way-ANOVA,Tukey's test

[0333] b: ** p<0.01vs.db / db-ND,by Two way-ANOVA,Tukey's test

[0334] c: *** p<0.001vs.db / db-ND,by Two way-ANOVA,Tukey's test

[0335] 3.5 Serum insulin

[0336] The results of animal serum insulin test (Figure 13, Table 9) showed that the serum insulin concentrations of animals in the Tirzepatide-0.15 mg / kg group and the various doses of BG128 groups were reduced, and the reduction in serum insulin in the various doses of BG128 groups was statistically significant compared with the model group (p = or <0.05).

[0337] As shown in FIG13 , the decrease in serum insulin concentration in the equal-dose BG128 group was significantly greater than that in the Tirzepatide-0.15 mg / kg group.

[0338] Table 9: Changes in serum insulin (nIU / mL) (Mean±SD)

[0339]

[0340] a: * p<0.05vs.db / db-ND,by T-test analysis

[0341] 3.6 Whole blood HbA1C percentage (%)

[0342] The results of whole blood HbA1C% (Figure 14, Table 10) showed that compared with the db / db-ND group, the anticoagulated whole blood HbA1C percentage of animals in the Tirzepatide-0.15 mg / kg group and various doses of BG128 groups was significantly reduced (p<0.001). The anticoagulated whole blood HbA1C% of the Tirzepatide-0.15 mg / kg group, the BG128-0.05 mg / kg group, the BG128-0.15 mg / kg group and the BG128-0.5 mg / kg group decreased by 1.88%, 1.80%, 1.83% and 2.54%, respectively, showing a dose-dependent trend.

[0343] As shown in Figure 15, BG128 and Tirzepatide showed comparable effects at equal doses. The reduction in the high-dose BG128 group was significantly greater than that in the Tirzepatide-0.15 mg / kg group and the BG128-0.15 mg / kg group (p<0.01).

[0344] Table 10: Changes in Glycated Hemoglobin (Mean ± SEM)

[0345]

[0346] a: *** p<0.001 vs.db / db-ND,by One-way ANOVA,Tukey's test

[0347] b:#p<0.05vs.Tirzepatide-0.15mg / kg,by One-way ANOVA,Tukey's test

[0348] c: ## p<0.01vs.Tirzepatide-0.15mg / kg,by One-way ANOVA,Tukey's test

[0349] d: & p<0.05vs.BG128-0.05mg / kg,by One-way ANOVA,Tukey's test

[0350] e: $ p<0.05vs.BG128-0.15mg / kg,by One-way ANOVA,Tukey's test

[0351] 4. Conclusion

[0352] The db / db mouse, derived from the inbred C57BL / KsJ strain and characterized by an autosomal recessive genetic disorder, is a model of type 2 diabetes. Under the conditions of this experiment, blood glucose levels in the animals were greater than 16.7 mmol / L before grouping. Treatment with the positive control, Tirzepatide, significantly suppressed hyperglycemia in the db / db mice and reduced peripheral blood insulin and glycated hemoglobin levels, demonstrating the stability and reliability of this model.

[0353] BG128 caused an acute decrease in body weight and food intake in db / db mice, which remained relatively stable in the later stages of dosing. BG128 significantly reduced non-fasting blood glucose levels after the first dose, surpassing the efficacy of an equivalent dose of Tirzepatide. It also significantly suppressed fasting blood glucose in db / db mice and reduced peripheral blood insulin and glycated hemoglobin levels.

[0354] Experimental Example 4: Testing the efficacy of BG128 in the STZ-HFD diet-induced diabetic and NASH model in male C57BL / 6 mice

[0355] 1. Test materials

[0356] 1.1 The information of test and reference products is shown in Table 11 below.

[0357] Table 11 Information of test and reference products

[0358] Name Test substance (BG128) Reference substance (Tirzepatide) Content 79.0% 87.4%

[0359] Physical state: White solid Storage conditions: Shade from light, seal tightly, and store below -20°C Provided by: Borri Biopharmaceuticals (Suzhou) Co., Ltd. Borri Biopharmaceuticals (Suzhou) Co., Ltd.

[0360] 1.2 Preparation of test and reference products

[0361] According to the dosing regimen, a certain amount of Tirzepatide and BG128 were weighed and added to a solvent (pH=7.0 20 mM sodium citrate buffer) to prepare the corresponding concentration.

[0362] 1.3 Test system

[0363] 1.3.1 Experimental Animal Information

[0364] Species and strain: C57BL / 6

[0365] Level: SPF level

[0366] Number and gender: 30 pregnant female mice

[0367] Supplier: Shanghai Jihui Experimental Animal Breeding Co., Ltd.

[0368] 1.3.2 Animal husbandry and management

[0369] Animals were housed in a barrier system with a controlled room temperature of 20°C to 26°C and a humidity of 40% to 70%. Animals were kept in the morning and afternoon with free access to water and normal feeding.

[0370] 2. Experimental Design

[0371] 2.1 Modeling Method

[0372] Newborn male mice were subcutaneously injected with STZ (100 μg / mouse) within 48 hours of birth. After being breastfed by mothers for four weeks, the fasting blood glucose of the mice was measured. A fasting blood glucose value greater than or equal to 12 mmol / L was used as the standard. 65 diabetic male mice were selected as experimental animals and fed with HFD for 8 weeks to establish the STZ+HFD-induced diabetic NASH model in male C57BL / 6 mice.

[0373] 2.2 Trial Grouping

[0374] Ten newborn male mice were selected and not injected with STZ. After being breastfed by mother mice for four weeks, they were separated into cages and fed with normal maintenance diet to serve as the normal control group. After one week of HFD diet, the fasting blood glucose of the animals was tested. The animals were randomly divided into groups based on fasting blood glucose and body weight, namely: normal control group, model group, Tirzepatide-0.15 mg / kg group, BG128-0.05 mg / kg group, BG128-0.15 mg / kg group, and BG128-0.5 mg / kg group. The details are shown in Table 12.

[0375] 2.3 Dosage regimen

[0376] The drug was administered on the first day of the second week of HFD feeding and the test was terminated after 7 consecutive weeks of administration. The specific dosing schedule is shown in Table 12.

[0377] Table 12: Experimental groups and dosing regimens

[0378] Group number STZ+HFD compound dosing regimen Group-110 NO Vehicle subcutaneously administered at 10 mL / kg once every three days Group-213 Yes Vehicle subcutaneously administered at 10 mL / kg once every three days Group-313 Yes Tirzepatide subcutaneously administered at 10 mL / kg, 0.15 mg / kg once every three days Group-413 Yes BG128 subcutaneously administered at 10 mL / kg, 0.05 mg / kg once every three days Group-513 Yes BG128 subcutaneously administered at 10 mL / kg, 0.15 mg / kg once every three days Group-613 Yes BG128 subcutaneously administered at 10 mL / kg, 0.5 mg / kg once every three days

[0379] 2.4 Detection indicators

[0380] 2.4.1 General Observations

[0381] Observation began at the start of the trial and continued once a day until the end of the trial;

[0382] Observation content: Observe the animal's death or dying, mental state, behavioral activities, fecal characteristics, eating and drinking conditions, etc.

[0383] 2.4.2 Animal weight

[0384] The animals were weighed starting from the time of HFD feeding and every three days until the end of the experiment.

[0385] 2.4.3 Animal food intake

[0386] The animals' food intake was recorded from the beginning of the HFD feeding period until the end of the experiment. The remaining amount was weighed before each administration.

[0387] 2.4.4 Animal fasting blood glucose

[0388] Fasting blood glucose was measured using a Roche blood glucose meter after 4 weeks of lactation (i.e., before HFD feeding), before administration, and 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks after administration and at the end of the experiment.

[0389] 2.4.5 Clinical biochemical testing

[0390] Three days after the last administration, the animals were weighed and fasted for 6 h. All experimental animals were anesthetized with intraperitoneal injection of sodium pentobarbital, and non-anticoagulated whole blood was collected through the orbital venous plexus, allowed to stand at room temperature for 30 min, centrifuged at 5000 rpm for 5 min, and serum was separated and frozen at -80°C for biochemical testing. EDTA-K2 anticoagulated whole blood was collected through the orbital venous plexus and placed on wet ice for HbA1c determination.

[0391] Serum samples were taken from -80°C and assayed for serum ALT, AST, TG, TC, HDL-c, LDL-c, and whole blood HbA1c using a Hitachi 7060 fully automated biochemical analyzer. Insulin was also assayed using an enzyme-linked immunosorbent assay kit. The assay parameters are shown in Table 13 below:

[0392] Table 13

[0393] Parameter Unit Alanine aminotransferase (ALT) U / L Aspartate aminotransferase (AST) U / L Triglyceride (TG) mmol / L Total cholesterol (TC) mmol / L High-density lipoprotein (HDL-c) mmol / L Low-density lipoprotein (LDL-c) mmol / L Glycated hemoglobin (HbA1c) % Insulin nIU / ml

[0394] 2.4.6 Liver NAS Analysis

[0395] The livers were dissected according to the procedure and perfused with pre-cooled saline. The livers were removed, photographed, weighed, and fixed in 10% formalin for pathological hematoxylin and eosin staining. Hematoxylin and eosin staining was performed on paraffin sections of all animal livers according to the KCI pathological staining standard SOP. The stained sections were scanned panoramically using a NanoZoomer Digital Pathology (S210, Hamamaci, Japan) scanner. Different fields of view were then selected for observation at different magnifications. Scoring was performed for different liver lobes in the sections. The scoring criteria are shown in Table 14.

[0396] Table 14: NAS scoring criteria for nonalcoholic steatohepatitis

[0397]

[0398]

[0399] 2.4.7 Liver fibrosis analysis

[0400] The liver was dissected according to the procedure and perfused with pre-cooled saline. The liver was removed, photographed and weighed, and then fixed in 10% formalin for pathological Sirius red (SR) staining. SR staining of liver paraffin sections was performed according to the KCI pathology staining standard SOP. The stained sections were scanned panoramically using a NanoZoomer Digital Pathology (S210, Hamamaci, Japan) scanner. The SR-stained sections of the panoramic scans were analyzed for liver fibrosis area using VIS7.0 software, and the percentage of liver fibrosis area to the entire section area was calculated.

[0401] 3. Data Analysis

[0402] Data were analyzed using Office Excel 2013 and GraphPad Prism 6.0 and presented as mean ± SEM. One-way and two-way ANOVA analyses were performed using Tukey's or Dunnett's methods, as appropriate, for intergroup significance comparisons. Two-tailed T-tests were used for comparisons between two groups. A p < 0.05 value was considered significant between the two groups.

[0403] 4. Results

[0404] 4.1 Changes in animal population

[0405] The NASH model animals induced by STZ+HFD have a certain mortality rate. The clinical manifestations before the death of the animals were white limbs and reduced activity. This manifestation is due to reduced blood circulation caused by metabolic disorders, which is a normal manifestation of this model and is consistent with historical background data. The animals in the Tirzepatide-0.15mg / kg, BG128-0.15mg / kg and BG128-0.50mg / kg groups were well tolerated, indicating that the positive controls Tirzepatide and BG128 have a certain protective effect on the model animals. In the experimental design, 3 animals were added to each group (except the normal control group), and all animals that survived to the end of the experiment were included in the data statistics. The specific situation is shown in Table 15.

[0406] Table 15: Animal population

[0407]

[0408] Note: -: Not applicable.

[0409] 4.2 Animal weight

[0410] Animal weight changes (Figure 16) show that after the first dose, the weight of animals in each treatment group showed a significant downward trend. The extent of weight loss in the different BG128 dose groups was positively correlated with the dose. After day 12, the weight of animals in each treatment group resumed steady growth, but overall was significantly lower than that of the model group. In the later stages of treatment, there were no significant differences in weight changes among the treatment groups (p>0.05).

[0411] 4.3 Food intake

[0412] The results of changes in animal food intake (Figure 17) show that compared with the normal group, the animals in each group consumed less high-fat feed than the normal group. Throughout the experimental period, there was no statistically significant difference in food intake between the model group and the various treatment groups (p>0.05). As shown in Figure 17, the food intake of animals treated with BG128 at the same dose was lower than that of animals treated with Trizepatide at the same dose.

[0413] 4.4 Fasting blood glucose

[0414] Fasting blood glucose testing revealed that compared to the normal control group, the model animals had significantly elevated fasting blood glucose levels before HFD administration. After one week of HFD administration, the model animals' blood glucose levels were significantly higher than before HFD feeding. From the start of drug administration to the end of the study, fasting blood glucose levels in all treatment groups were significantly lower than those in the model group (p<0.001) (Table 16, Figure 18). At the end of the study, fasting blood glucose levels in all BG128 groups were significantly lower than those in the db / db-ND group, demonstrating a dose-dependent decrease. BG128 and Tirzepatide were comparable in efficacy at the same dose levels.

[0415] Table 16: Fasting blood glucose (mmol / L) (Mean ± SEM)

[0416]

[0417] a: *** p<0.001 vs. model group, by Two-way-ANOVA, Tukey's test

[0418] b: # p<0.05vs.BG128-0.05mg / kg,by Two way-ANOVA,Tukey's test

[0419] c: ### p<0.001vs.BG128-0.05mg / kg,by Two way-ANOVA,Tukey's test

[0420] d: $$$ p<0.001vs.BG128-0.15mg / kg,by Two way-ANOVA,Tukey's test

[0421] 4.5 insulin

[0422] Insulin changes (Figure 19) showed that serum insulin concentrations decreased in the Tirzepatide-0.15 mg / kg group and in the BG128 groups at all doses. The BG128-0.15 mg / kg and BG128-0.5 mg / kg groups showed statistically significant differences in serum insulin compared to the db / db-ND group (p<0.05). BG128 was more effective than Tirzepatide at the same dose.

[0423] 4.6 HbA1C%

[0424] Peripheral blood HbA1c% changes (Table 17) showed that after administration, HbA1c% in the model group increased significantly compared to the normal control group. HbA1c% decreased to varying degrees in both the Tirzepatide and BG128 dose groups, with the BG128-0.5 mg / kg group showing a significant decrease compared to the model group (p < 0.05). Using the model group's HbA1c% as a baseline, HbA1c% in the Tirzepatide and BG128 dose groups decreased by 0.83%, 0.97%, 0.66%, and 1.04%, respectively (Figure 20). BG128 and Tirzepatide showed comparable effects at the same dose levels.

[0425] Table 17: Changes in Glycated Hemoglobin (Mean ± SEM)

[0426]

[0427]

[0428] a: * p<0.05 vs. model group, by One-way-ANOVA, Tukey's test

[0429] 4.7 Liver function and lipid-related biochemical indicators

[0430] Results of liver function and lipid-related biochemical indicators were shown (Table 18, Figures 21-23). ​​Compared with the model group, ALT and AST levels decreased in the Tirzepatide-0.15 mg / kg and various BG128 dose groups, with significant differences in the decreases in the BG128-0.15 mg / kg and 0.5 mg / kg dose groups (Figure 21). Total cholesterol and triglycerides were significantly reduced in the Tirzepatide-0.15 mg / kg and various BG128 dose groups (Figure 22). Low-density lipoprotein cholesterol was significantly reduced in the Tirzepatide-0.15 mg / kg and various BG128 dose groups, while high-density lipoprotein cholesterol remained unchanged (Figure 23). BG128 was more effective than Tirzepatide at the same dose.

[0431] Table 18: Clinical biochemical test results (Mean ± SEM)

[0432]

[0433] a: * p<0.05 vs. model group, by One-way ANOVA, Tukey's test

[0434] b: ** p<0.01 vs. model group, by One-way ANOVA, Tukey's test

[0435] c: *** p<0.001 vs. model group, by One-way ANOVA, Tukey's test

[0436] d: && p<0.01vs. model group; e: &&& p<0.01 vs. model group, by T-test

[0437] 4.8 Liver Histopathology

[0438] 4.8.1 NAS Evaluation

[0439] H&E staining was used to evaluate liver pathological changes. In the model group, hepatocyte fatty degeneration was manifested as vacuolar degeneration of hepatocytes, which was widely distributed in the liver lobes; interlobular inflammation was manifested as scattered focal inflammatory cell infiltration within the hepatic lobules; no ballooning lesions were observed (Table 19, Figure 24). In Figure 24, A: normal control group; B: model group; C: Tirzepatide-0.15mg / kg; D: BG128-0.05mg / kg; E: BG128-0.15mg / kg; F: BG128-0.5mg / kg. In Figure A, it can be seen that the hepatocytes are neatly arranged and the cell structure is intact. In Figure B, hepatocyte fatty degeneration (yellow arrow) and inflammatory cell infiltration (black arrow) can be seen. In Figures C, D, E, and F, different degrees of inflammatory cell infiltration and reduction of hepatocyte fatty degeneration can be seen.

[0440] According to the NAS scoring criteria listed in Table 14, hepatocyte fatty degeneration, interlobular inflammation and NAS score in the model group were significantly higher than those in the normal group (P<0.001).

[0441] Compared with the model group, the scores of hepatic inflammatory cell infiltration and hepatocyte steatosis in the Tirzepatide-0.15 mg / kg group and the BG128 groups at various doses were significantly reduced ( FIG. 25 ).

[0442] Compared with the model group, the total NAS scores of the Tirzepatide-0.15 mg / kg and various doses of BG128 groups were significantly reduced (p<0.001) ( FIG26 ).

[0443] Table 19: Histopathological results (Mean ± SEM)

[0444]

[0445] a: *** p<0.001 vs. model group, by One-way ANOVA, Tukey's test

[0446] 4.8.2 Liver fibrosis evaluation

[0447] Sirius red staining revealed fibrosis primarily distributed in the portal vein, central vein, and intercellular regions (Figure 27). Figure 27 (A): Normal control group; B: Model group; C: Tirzepatide 0.15 mg / kg; D: BG128 0.05 mg / kg; E: BG128 0.15 mg / kg; F: BG128 0.5 mg / kg). Panel A shows no fibrosis between hepatocyte cords, while panels B, C, D, E, and F show red fibers between hepatocyte cords.

[0448] Semi-quantitative analysis of liver fibrosis showed (Table 20) that the fibrosis rate in the model group was 1.32±0.02%, significantly higher than that in the normal control group (p<0.001). The fibrosis rates in the BG128 groups at all doses were significantly lower than those in the model group (Table 20, Figure 28).

[0449] Table 20: Percent fibrosis (Mean ± SEM)

[0450]

[0451] a: * p<0.05vs. model group, by T-test

[0452] b: ** p<0.01 vs. model group, by T-test

[0453] c: *** p<0.001vs. model group, by T-test

[0454] 5. Conclusion

[0455] In this experiment, STZ-HFD diet was used to induce a diabetic and NASH model in male C57BL / 6 mice. Fasting blood glucose and HbA1c% in the model group increased significantly, and serum biochemical indicators related to liver function and blood lipids were significantly improved. At the same time, significant changes in liver tissue pathology indicators were caused, including hepatocellular fatty lesions, increased interlobular inflammation, elevated NAS scores, and aggravated liver fibrosis, which are similar to the clinical characteristics of human hyperglycemia and NASH, and also indicate that this model is stable and reliable.

[0456] BG128 demonstrated a significant therapeutic effect in the STZ-HFD-induced C57BL / 6 model of diabetes and NASH. All BG128 doses significantly reduced fasting blood glucose, insulin, and HbA1c%, and exhibited a dose-dependent protective effect in the model animals. BG128 significantly improved clinical biochemical parameters related to liver function and lipid profiles, reduced liver NAS scores and fibrosis rates, and significantly slowed NASH progression and fibrosis progression. Overall, BG128 demonstrated superior efficacy compared to the control drug Tirzepatide at an equivalent dose.

[0457] All technical features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may also be replaced by other features having the same, equivalent, or similar functions. Therefore, unless otherwise specified, each feature disclosed is merely an example of a series of equivalent or similar features.

[0458] In addition, from the above description, those skilled in the art can easily understand the key features of the present disclosure from this disclosure. Without departing from the spirit and scope of the present disclosure, many modifications can be made to the invention to adapt to various different purposes and conditions of use. Therefore, such modifications are also intended to fall within the scope of the appended claims.

Claims

1. A compound as shown in Formula I or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope-labeled substance or prodrug thereof: Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-A 40 I； in ， A 40 is the following structure connected to Ser via a peptide bond: R 1 is - (Z 1 ) m - (Z 2 ) n - C(=O) - (CH 2 ) o - C(=O) - Z 3 ; m is 0 or 1; If present, Z 1 -C(=O)-(CH 2 ) r -(Z 4 ) p -NH-, where p is any integer from 4 to 8, and each Z 4 Each is independently -O-CH 2 -CH 2 - or -NH-C(=O)-CH 2 -, r is 1 or 2; n is any integer between 0 and 2; If present, each Z 2 Each independently Where q is any integer between 2 and 5; o is any integer between 14 and 18; Z 3 Hydroxyl or 2. The compound according to claim 1 or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope-labeled substance or prodrug thereof, It is characterized in that R 1 -C(=O)-(CH 2 ) o -C(=O)-Z 3 ; o is any integer between 14 and 18, preferably 18; Z 3 Hydroxyl or Hydroxyl is preferred.

3. The compound according to claim 1 or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope-labeled substance or prodrug thereof, It is characterized in that R 1 is -Z 1 -Z 2 -C(=O)-(CH 2 ) o -C(=O)-Z 3 ; Z 1 -C(=O)-CH 2 -(Z 4 ) 7 -NH-, where each Z 4 Each is independently -O-CH 2 -CH 2 - or -NH-C(=O)-CH 2 -, preferably -(Z 4 ) 7 - is -(O-CH 2 -CH 2 ) 3 -(NH-C(=O)-CH 2 )-(O-CH 2 -CH 2 ) 3 -or-(O-CH 2 -CH 2 ) 7 -; Z 2 for Best Wherein q is any integer between 2 and 5, preferably 2; o is any integer between 14 and 18, preferably 18; Z 3 Hydroxyl or Hydroxyl is preferred.

4. A compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope-labeled substance or prodrug thereof, It is characterized in that A 40 Select from any of the following structures: Preferably, the structure shown in any of the following items:

5. A compound represented by any one of the following formulae I-1 to I-14 or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope-labeled substance or prodrug thereof:

6. A dual receptor agonist of GIP and GLP-1, It is characterized in that The dual receptor agonist of GIP and GLP-1 is a compound shown in the following formula I-3 or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof:

7. A pharmaceutical composition comprising a compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope-labeled substance or prodrug thereof, or a dual receptor agonist of GIP and GLP-1 according to claim 6; Optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

8. Use of the compound according to any one of claims 1 to 5 or its pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope-labeled substance or prodrug, the dual receptor agonist of GIP and GLP-1 according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for preventing and / or treating diseases related to metabolic disorders; Optionally, the metabolic disorder-related disease is diabetes, diabetic complications, obesity, or obesity complications.

9. A compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope-labeled substance or prodrug thereof, a dual receptor agonist of GIP and GLP-1 according to claim 6, or a pharmaceutical composition according to claim 7, for use in preventing and / or treating diseases related to metabolic disorders; Optionally, the metabolic disorder-related disease is diabetes, diabetic complications, obesity, or obesity complications.

10. A method for preventing and / or treating metabolic disorder-related diseases, comprising administering to a subject a preventive and / or therapeutically effective amount of a compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof, a dual receptor agonist of GIP and GLP-1 according to claim 6, or a pharmaceutical composition according to claim 7; Optionally, the metabolic disorder-related disease is diabetes, diabetic complications, obesity, or obesity complications.