polypeptide compounds and their applications

Abstract

This invention relates to a polypeptide compound and its applications, and more specifically, to a dual or triple agonist polypeptide drug that activates glucagon-like peptide-1 receptor and optionally glucose-dependent insulinotropic peptide receptor and glucagon receptor, and its application in the treatment of metabolic syndrome.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and more specifically, to dual or triple agonist peptide drugs that activate glucagon-like peptide-1 (GLP-1) receptors and optionally glucose-dependent insulinotropic peptide (GIP) and glucagon (GCG) receptors, and their use in the treatment of metabolic syndromes (e.g., diabetes, obesity, non-alcoholic fatty liver disease). Background Technology

[0002] Type 2 diabetes and obesity are metabolic disorders that continue to be a growing health problem in many countries, leading to a wide range of related risk factors, such as cardiovascular disease, kidney disease, dyslipidemia, liver disease, and osteoporosis. Type 2 diabetes is characterized by hyperglycemia due to insulin resistance, and obesity is the leading cause of insulin resistance. Obesity and insulin resistance are also two major contributing factors to non-alcoholic fatty liver disease (NAFLD / NASH). Therefore, there is an increasing need to find safe and effective drug treatments for these metabolic disorders.

[0003] Incretins are substances secreted from the intestines in response to food stimulation under normal physiological conditions. They stimulate pancreatic β-cells to secrete insulin in response to glucose levels, regulate glucose homeostasis, and protect pancreatic β-cells. GLP-1 and GIP are two currently known incretins (Glucagon-like peptide-1 and glucose-dependent insulin-releasing polypeptide plasma levels in response to nutrients. Digestion 1995; 56:117-126.).

[0004] GLP-1 is a 31-amino acid polypeptide expressed in L cells of the intestinal mucosa by the proglucagon gene. It mainly acts on the GLP-1 receptor (GLP-1R), stimulating insulin secretion, inhibiting glucagon secretion, and protecting pancreatic β-cells. It plays a physiological role in regulating blood glucose homeostasis. At the same time, it can inhibit food intake and gastric emptying through central nervous system signaling pathways, increase satiety, and thus reduce weight (Glucagon-like peptide-1 7-36: a physiological incretin man. Lancet. 1987; 2:1300-1304.; Glucagon-like peptide-1 receptor signaling modulates beta cell apoptosis. J Biol Chem. 2003; 278:471-478.; Relation between gastric emptying of glucose and plasma concentrations of glucagon-like peptide-1. Peptides. 1998; 19:1049-1053.).

[0005] Natural GLP-1 is readily degraded by dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase (NEP), which are ubiquitous in plasma, with a half-life of less than 2 minutes. Exendin-4, a GLP-1 analog extracted from the salivary glands of the African venomous lizard, has a stronger GLP-1 receptor agonist effect and similar blood glucose-lowering effects to GLP-1. Compared to natural GLP-1, Exendin-4 is more resistant to DPP-4 and NEP, and has a longer in vivo half-life and duration of action. Exenatide (trade name...) It was the first GLP-1 drug to be developed and marketed, and it has shown good clinical efficacy in the treatment of diabetes. However, it still has significant human immunogenicity and the inadequacy of being administered twice a day.

[0006] The amino acid sequence of Exendin-4 (SEQ ID NO:1) is as follows:

[0007] HGEGFTTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2

[0008] The amino acid sequence of GLP-1(7-37) (SEQ ID NO:2) is as follows:

[0009] HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG-OH

[0010] To improve the half-life of GLP-1 and its efficacy in diabetes treatment, several long-acting GLP-1 analogs have been successfully developed on the market, such as Novo Nordisk's liraglutide (trade name) for the treatment of diabetes and / or obesity. and ) and semaglutide (trade name) ) and Eli Lilly's dulaglutide (brand name) ).

[0011] Liraglutide uses C 16 Fatty acid chemical modification of the 20-position Lys side chain of GLP-1 increases albumin binding affinity and extends the half-life to 13-16 hours. The amino acid sequence of liraglutide (SEQ ID NO:3) is as follows:

[0012] HAEGTFTSDVSSYLEGQAAK(γGlu-palmitoyl)EFIAWLVRGRG-OH

[0013] Semaglutide, by replacing the 2-position Ala with the non-natural amino acid isoaminobutyric acid (Aib), significantly improves DPP-IV resistance, while C 18 The fatty acid side chain further enhances albumin binding affinity, significantly extending the half-life to allow for once-weekly dosing. The amino acid sequence of semaglutide (SEQ ID NO:4) is as follows:

[0014] HAibEGTFTSDVSSYLEGQAAK([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-γGlu-octadecanoic acid acyl)EFIAWLVRGRG-OH.

[0015] GIP is a 42-amino acid single-chain polypeptide produced by K cells in the small intestinal mucosa. It primarily acts on GIP receptors (GIPRs) in pancreatic islet cells and adipocytes. GIP promotes insulin secretion in a glucose-dependent manner, enhances the quality of pancreatic β-cells, stimulates insulin secretion, inhibits gastric acid secretion, and slows gastric motility. It also stimulates the uptake and utilization of fatty acids by adipose tissue cells (Biology of Incretins: GLP-1 and GIP. Gastroenterology. 2007; 132:2131-2157.). GIP also promotes osteoblast differentiation, inhibits osteoblast apoptosis, inhibits bone resorption, and increases bone mineral density, thus playing a protective role in bone function (Glucose-dependent insulinotropic peptide is an integrative hormone with osteotropic effects. Mol Cell Endocrinol. 2001; 177:35-41.; Effects of glucose-dependent insulinotropic peptide on osteoclast function. Am J Physiol 2007; 292:E543-E548.).

[0016] The amino acid sequence of GIP (SEQ ID NO:5) is as follows:

[0017] YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ

[0018] GCG is a 29-amino acid polypeptide expressed and secreted by the proglucagon gene in pancreatic α-cells. It acts on glucagon receptors (GCGR), primarily located in the liver and kidneys, stimulating glycogenolysis, raising blood glucose levels, activating lipases, promoting lipolysis, and simultaneously enhancing fatty acid oxidation, leading to increased ketone body production. Research indicates that GCG has a certain effect on reducing food intake, increasing energy expenditure in adipose tissue, and reducing body fat (The metabolic actions of glucagon revisited. Nat. Rev. Endocrinol. 2010; 6:689-697.; Effects of glucagon onlipolysis and ketogenesis in normal and diabetic men. J. Clin. Invest. 1974; 53:190-197.). Appropriate blood glucose-raising effects of GCG can feedback regulate insulin action, reducing the occurrence of hypoglycemic events.

[0019] The amino acid sequence of GCG (SEQ ID NO:6) is as follows:

[0020] HSQGTFTSDYSKYLDSRRAQDFVQWLMNT

[0021] Obesity is a condition caused by excessive energy intake or metabolic abnormalities, leading to an excessive accumulation of body fat, especially triglycerides. Fat accumulation in the pancreas damages pancreatic islet cell function, worsening diabetes, while accumulation in the liver leads to non-alcoholic fatty liver disease. Currently available GLP-1 analogs offer the advantage of lowering blood sugar while also providing cardiovascular benefits and weight management effects. However, the maximum clinical weight loss effects of liraglutide and semaglutide are only around 3% and 5%, respectively, and gastrointestinal side effects (mainly nausea, vomiting, and diarrhea) are relatively common. Therefore, there is still an urgent need for treatment drugs with better weight control effects, broader benefits, and a wider safety margin for the increasingly widespread obese population.

[0022] Based on the physiological functions of GLP-1, GIP, and GCG, multiple studies have found that multi-target agonist therapy, which integrates the effects of two or all three, can achieve better control of diabetes and obesity than monotherapy. GLP-1 / GCG co-agonists are more effective than single GLP-1 receptor agonists in reducing food intake, lowering body weight, improving glucose tolerance, and reducing triglycerides, and their therapeutic effects are more significant in obese mice (Glucagon-like peptide1 / glucagon receptor dual agonism reverses obesity in mice. Diabetes. 2009; 58:2258-2266; A new glucose and GLP-1 coagonist eliminates obesity in rodents. Nat ChemBiol. 2009; 5:749-757.). Animal studies at Indiana University have confirmed that GLP-1 / GIP and GLP-1 / GIP / GCG co-agonists significantly reduce blood glucose and body weight in high-fat diet-induced obese (DIO) mice, with therapeutic effects significantly superior to acetylated GLP-1 (Acyl-GLP-1), acetylated GIP (Acyl-GIP), and liraglutide. Furthermore, they can reduce plasma cholesterol, body fat, and liver fat, and improve various lipid metabolism indicators, such as triglycerides, leptin, adiponectin, ketone bodies, and FGF-21 (Unimolecular Dual Incretins Maximize Metabolic Benefits in Rodents, Monkeys, and Humans. Sci Transl Med. 2013; 5:209ra151; A rationally designed monomericpeptide triagonist corrects obesity and diabetes in rodents. Nat Med. 2015; 21:27-36.).

[0023] There is still a need for new bi- or tri-agonistic peptide molecules for the prevention or treatment of metabolic syndromes, with improved stability or lower costs for large-scale production. Summary of the Invention

[0024] This invention provides a novel polypeptide compound that can have dual or triple agonistic effects. These polypeptides can exhibit significant GLP-1R agonistic activity, and can simultaneously possess GLP-1R and / or GCG-1R agonistic activity, making them suitable for the prevention or treatment of metabolic disorders, such as lowering blood glucose, weight, and fat, and exhibiting improved stability.

[0025] In one aspect, a compound having the following formula I or a salt or solvate thereof is provided:

[0026] R 1 -His-Aib-X3-Gly-Thr-Phe-Thr-Ser-Asp-X 10 -Ser-X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -Phe-X 23 -X 24 -Trp-Leu-X 27 -X 28 -Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R 2 (Ⅰ)(SEQ ID NO:45)

[0027] in,

[0028] R 1 Selected from H, Ac, or pGlu;

[0029] X3 is selected from His, Gln;

[0030] X 10 Selected from Tyr, Leu, Lys, Cys, or ψ;

[0031] X 12 Selected from Ile, Arg, Lys, Cys, or ψ;

[0032] X 13 Selected from Tyr, Gln, Lys, Cys, or ψ;

[0033] X 14 Selected from Leu, Lys, Cys, or ψ;

[0034] X 15 Selected from Asp or Glu;

[0035] X 16 Selected from Glu, Lys, Cys, or ψ;

[0036] X 17 Selected from Arg, Ile, Gln, Glu, Lys, Cys, or ψ;

[0037] X 18Selected from Ala or Arg;

[0038] X 19 Selected from Ala, Val, or Gln;

[0039] X 20 Selected from Gln or Arg;

[0040] X 21 Selected from Asp or Leu;

[0041] X 23 Selected from Val or Ile;

[0042] X 24 Selected from Glu or Gln;

[0043] X 27 Selected from Leu or Lys;

[0044] X 28 Selected from Ala or Asp;

[0045] R 2 It is NH2 or OH, or a pharmaceutically acceptable salt and / or ester thereof;

[0046] Among them, X 10 X 12 X 13 X 14 X 16 X 17 There is one and only one ψ, and the ψ is a Cys or Lys whose side chain is modified by a structure having the following formula II:

[0047] YZ(Ⅱ)

[0048] (i) When ψ is Lys, Y is selected from Glu (preferably γGlu), AEEAc, GABA, GSEGSEE and any combination of two or more thereof, and its carboxyl terminus is attached to the ε-amino group of the side chain of Lys, and Z is -CO-(CH2). m -R 3 m is an integer between 6 and 24, R 3Selected from -CH3 or -COOH; Y preferably represents a maximum of 10, or a maximum of 5, or a maximum of 4, or a maximum of 3, or a maximum of 2, or 1 linker selected from Glu (preferably γGlu), AEEAc, GABA, GSEGSEE and any combination of two or more thereof; for example, Y can be γGlu, GSEGSEE, AEEAc-AEEAc-γGlu, γGlu-γGlu-AEEAc-AEEAc, γGlu-γGlu-AEEAc-AEEAc-γGlu, or γGlu-GABA-AEEAc-γGlu;

[0049] (ii) When ψ is Cys, Y is Y1-Y2, Y1 is selected from acetylglycyl, 3-maleimide propionyl and any combination thereof, Y2 is selected from Glu (preferably γGlu), AEEAc and any combination thereof, and it is connected to the Cys side chain thiol group through acetylglycyl or 3-maleimide propionyl, and Z is -NH-(CH2). m -R 3 m is an integer between 6 and 24, R 3 The linkage is selected from -CH3 or -COOH; Y1 preferably represents one linkage selected from acetylglycyl, 3-maleimide propionyl, and any combination thereof; Y2 preferably represents a maximum of 10, or a maximum of 5, or a maximum of 4, or a maximum of 3, or a maximum of 2, or one linkage selected from Glu (preferably γGlu), AEEAc, and any combination thereof; for example, Y1 can be 3-maleimide propionyl; Y2 can be γGlu or AEEAc-AEEAc;

[0050] Or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity or identity to the amino acid sequence.

[0051] In one aspect, compounds having the following formula Ib or salts or solvates thereof are provided:

[0052] R 1 -His-Aib-X3-Gly-Thr-Phe-Thr-Ser-Asp-X 10 -Ser-Lys-X 13 -X 14 -X 15 -Glu-X 17 -Ala-X 19 -X 20 -X 21 -Phe-X 23 -X 24 -Trp-Leu-X 27 -X 28-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R 2 (Ⅰb)(SEQ ID NO:46)

[0053] in,

[0054] R 1 Selected from H, Ac, or pGlu;

[0055] X3 is selected from His, Gln;

[0056] X 10 Selected from Leu, Lys, Cys, or ψ;

[0057] X 13 Selected from Tyr or Gln;

[0058] X 14 Selected from Leu, Lys, Cys, or ψ;

[0059] X 15 Selected from Asp or Glu;

[0060] X 17 Selected from Arg, Gln, or Glu;

[0061] X 19 Selected from Ala or Val;

[0062] X 20 Selected from Gln or Arg

[0063] X 21 Selected from Asp or Leu

[0064] X 23 Selected from Val or Ile;

[0065] X 24 Selected from Glu or Gln;

[0066] X 27 Selected from Leu or Lys

[0067] X 28 Selected from Ala or Asp;

[0068] R 2 It is NH2 or OH, or a pharmaceutically acceptable salt and / or ester thereof;

[0069] Among them, X 10 X 14 There is one and only one ψ, and the ψ is a Lys whose side chain is modified by a structure having the following formula II:

[0070] YZ(Ⅱ)

[0071] Y is selected from Glu (preferably γGlu), AEEAc, GABA, GSEGSEE, and any combination of two or more thereof, and its carboxyl terminus is attached to the ε-amino group of the side chain of Lys, and Z is -CO-(CH2). m -R 3 m is an integer between 6 and 24, R 3 Selected from -CH3 or -COOH; Y preferably represents a maximum of 10, or a maximum of 5, or a maximum of 4, or a maximum of 3, or a maximum of 2, or 1 linker selected from Glu (preferably γGlu), AEEAc, GABA, GSEGSEE, and any combination of two or more thereof; for example, Y can be γGlu, GSEGSEE, AEEAc-AEEAc-γGlu, γGlu-γGlu-AEEAc-AEEAc, γGlu-γGlu-AEEAc-AEEAc-γGlu, or γGlu-GABA-AEEAc-γGlu

[0072] Or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity or identity to the amino acid sequence.

[0073] The compounds with the structure of formula Ib described above can be prepared by chemically synthesizing peptides.

[0074] In one aspect, compounds having the following formula Ic or salts or solvates thereof are provided:

[0075] R 1 -His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Tyr-X 14 -Gl u-Glu-Gln-Ala-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Leu-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R 2 (Ⅰc)(SEQ ID NO:47)

[0076] in,

[0077] R 1 For H;

[0078] X 14 For ψ;

[0079] R 2 It is NH2 or OH, or a pharmaceutically acceptable salt and / or ester thereof;

[0080] Among them, X 14 Let ψ be a Cys whose side chain is modified with a structure having the following formula II:

[0081] YZ(Ⅱ)

[0082] Y is Y1-Y2, where Y1 is selected from acetylglycyl, 3-maleimide propionyl, and any combination thereof, Y2 is selected from Glu (preferably γGlu), AEEAc, and any combination thereof, and it is connected to the Cys side chain thiol group through acetylglycyl or 3-maleimide propionyl, and Z is -NH-(CH2). m -R 3 m is an integer between 6 and 24, R 3 The linkage is selected from -CH3 or -COOH; Y1 preferably represents one linkage selected from acetylglycyl, 3-maleimide propionyl, and any combination thereof; Y2 preferably represents a maximum of 10, or a maximum of 5, or a maximum of 4, or a maximum of 3, or a maximum of 2, or one linkage selected from Glu (preferably γGlu), AEEAc, and any combination thereof; for example, Y1 can be 3-maleimide propionyl; Y2 can be γGlu or AEEAc-AEEAc;

[0083] Or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity or identity to the amino acid sequence.

[0084] The compounds with the above-mentioned structure of formula Ic can be prepared by chemically synthesizing peptides.

[0085] In one aspect, compounds having the following formula Ⅰd or their salts or solvates are provided:

[0086] R 1 -His-Aib-X3-Gly-Thr-Phe-Thr-Ser-Asp-X 10 -Ser-X 12 -X 13 -X 14 -Glu-X 16 -X 17 -X 18 -X 19 -Gln-Asp-Phe-X 23 -Glu-Trp-Leu-Leu-X 28 -Gly-Gly-Pro-Se r-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R 2 (Ⅰd)(SEQ ID NO:48)

[0087] in,

[0088] R 1 For H;

[0089] X3 is selected from His, Gln;

[0090] X 10 Selected from Tyr or Leu;

[0091] X 12 Selected from Ile, Arg, or ψ;

[0092] X 13 Selected from Tyr, Gln, or ψ;

[0093] X 14 Selected from Leu, or ψ;

[0094] X 16 Selected from Glu, or ψ;

[0095] X 17 Selected from Arg, Ile, Gln, or ψ;

[0096] X 18 Selected from Ala or Arg;

[0097] X 19 Selected from Ala or Gln;

[0098] X 23 Selected from Val or Ile;

[0099] X 28 Selected from Ala or Asp;

[0100] R 2 It is NH2 or OH, or a pharmaceutically acceptable salt and / or ester thereof;

[0101] Among them, X 12 X 13 X 14 X 16 X 17 There is one and only one ψ, and the ψ is a Lys whose side chain is modified by a structure having the following formula II:

[0102] YZ(Ⅱ)

[0103] Y is selected from Glu (preferably γGlu), AEEAc, GABA, GSEGSEE, and any combination of two or more thereof, and its carboxyl terminus is attached to the ε-amino group of the side chain of Lys, and Z is -CO-(CH2). m -R 3 m is an integer between 6 and 24, R3 Selected from -CH3 or -COOH; Y preferably represents a maximum of 10, or a maximum of 5, or a maximum of 4, or a maximum of 3, or a maximum of 2, or 1 linker selected from Glu (preferably γGlu), AEEAc, GABA, GSEGSEE and any combination of two or more thereof; for example, Y can be γGlu, GSEGSEE, AEEAc-AEEAc-γGlu, γGlu-γGlu-AEEAc-AEEAc, γGlu-γGlu-AEEAc-AEEAc-γGlu, or γGlu-GABA-AEEAc-γGlu;

[0104] Or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity or identity to the amino acid sequence.

[0105] The compounds with the above-mentioned formula Id structure can be prepared by chemical synthesis of peptides or by biological semi-synthesis.

[0106] The aforementioned bio-semi-synthesis refers to the use of methods similar to those disclosed in patents CN201510459093 and US9732137 for the manufacture of semaglutide (SEQ ID NO:4).

[0107] The method is characterized by the following steps: First, a partial peptide segment TFTSDVSSYLEGQAAKEFIAWLVRGRG-OH from the semaglutide sequence is expressed using yeast or E. coli fermentation. Then, an acylation reaction is performed between a fatty acid activated ester and the NH2 side chain of the peptide segment K to obtain the peptide segment TFTSDVSSYLEGQAAK([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-γE-octadecanoyl)EFIAWLVRGRG-OH. Finally, the N-terminal overhang Boc-His(Boc)-Aib-Glu(O-tBu)-Gly-OSuc is used to react with the above peptide segment via α-NH2 acylation, and the Boc protecting group is removed to obtain semaglutide. This method overcomes the cumbersome steps of chemical synthesis, fully utilizes the advantages of biological fermentation expression, and is conducive to reducing production costs and large-scale industrial production.

[0108] The implementation of the above-mentioned technical method relies on two characteristics of the polypeptide sequence: 1. The sequence contains no Lys sites other than the specific Lys site modified by fatty acids. If there are extra Lys sites, it cannot be guaranteed that the fatty acid will specifically attach to the desired Lys site; 2. The sequence contains no non-natural amino acids after position 5, except for the non-natural amino acid Aib at position 2. Otherwise, biological cells such as yeast or E. coli would not be able to express peptides containing non-natural amino acids.

[0109] Since the above formula Ⅰd meets the two characteristics mentioned above, it can be prepared using a similar biosynthetic method.

[0110] In one aspect, compounds having the following general formula Ie or salts or solvates thereof are provided:

[0111] R 1 -His-Aib-X3-Gly-Thr-Phe-Thr-Ser-Asp-X 10 -Ser-X 12 -X 13 -X 14 -Glu-X 16 -X 17 -X 18 -X 19 -Gln-Asp-Phe-X 23 -Glu-Trp-Leu-Leu-X 28 -Gly-Gly-Pro-Se r-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R 2 (Ⅰe)(SEQ ID NO:49)

[0112] in,

[0113] R 1 For H;

[0114] X3 is selected from His, Gln;

[0115] X 10 Selected from Tyr or Leu;

[0116] X 12 Selected from Ile, Arg, Lys, Cys, or ψ;

[0117] X 13 Selected from Tyr, Gln, Lys, Cys, or ψ;

[0118] X 14 Selected from Leu, Lys, Cys, or ψ;

[0119] X 16 Selected from Glu, Lys, Cys, or ψ;

[0120] X 17 Selected from Arg, Ile, Gln, Lys, Cys, or ψ;

[0121] X 18 Selected from Ala or Arg;

[0122] X 19 Selected from Ala or Gln;

[0123] X 23 Selected from Val or Ile;

[0124] X 28 Selected from Ala or Asp;

[0125] R 2 It is NH2 or OH, or a pharmaceutically acceptable salt and / or ester thereof;

[0126] Among them, X 12 X 13 X 14 X 16 X 17 There is one and only one ψ, and the ψ is a Lys whose side chain is modified by a structure having the following formula II:

[0127] YZ(Ⅱ)

[0128] Y is selected from Glu (preferably γGlu), AEEAc, GABA, GSEGSEE, and any combination of two or more thereof, and its carboxyl terminus is attached to the ε-amino group of the side chain of Lys, and Z is -CO-(CH2). m -R 3 m is an integer between 6 and 24, R 3 Selected from -CH3 or -COOH; Y preferably represents a maximum of 10, or a maximum of 5, or a maximum of 4, or a maximum of 3, or a maximum of 2, or 1 linker selected from Glu (preferably γGlu), AEEAc, GABA, GSEGSEE and any combination of two or more thereof; for example, Y can be γGlu, GSEGSEE, AEEAc-AEEAc-γGlu, γGlu-γGlu-AEEAc-AEEAc, γGlu-γGlu-AEEAc-AEEAc-γGlu, or γGlu-GABA-AEEAc-γGlu;

[0129] Or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity or identity to the amino acid sequence.

[0130] In one respect, the structure of Equation II above is as follows:

[0131] When ψ is Lys with a side chain modified by the structure of Formula II, the structure of Formula II can be selected from (the term "R" in the following structures is intended to represent the linking site of Formula II at the peptide backbone, i.e., the ε-amino group of the Lys side chain):

[0132] γGlu-CO(CH2) 14 CH3:

[0133]

[0134] AEEAc-AEEAc-γGlu-CO(CH2) 16 CH3:

[0135]

[0136] γGlu-CO(CH2) 16 COOH:

[0137]

[0138] AEEAc-AEEAc-γGlu-CO(CH2) 16 COOH:

[0139]

[0140] AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH:

[0141]

[0142] GABA-GABA-AEEAc-γGlu-CO(CH2) 16 COOH:

[0143]

[0144] γGlu-GABA-AEEAc-γGlu-CO(CH2) 16 COOH:

[0145]

[0146] γGlu-γGlu-AEEAc-AEEAc-γGlu-CO(CH2) 16 COOH:

[0147]

[0148] γGlu-γGlu-AEEAc-AEEAc-CO(CH2) 16 COOH:

[0149]

[0150] GSEGSEE-CO(CH2) 16 COOH:

[0151]

[0152] When ψ is a Cys with a side chain modified by the structure of Formula II, the structure of Formula II can be selected from (the term "R" in the following structures is intended to represent the linking site of Formula II at the peptide backbone, i.e., the thiol group of the Cys side chain):

[0153] Acetylglycyl-γGlu-NH(CH2) 15 CH3:

[0154]

[0155] 3-Maleiminopropionyl-γGlu-NH(CH2) 15 CH3:

[0156]

[0157] 3-Maleiminopropionyl-AEEAc-AEEAc-NH(CH2) 15 CH3:

[0158]

[0159] In one respect, the compound is selected from:

[0160] Compound 1 (SEQ ID NO:7):

[0161] H-Aib-QGTFTSDK(γGlu-CO(CH2) 14 CH3)SKYLEEQAAQDFIEW LLAGGPSSGAPPPS-NH2

[0162] Compound 2 (SEQ ID NO:8):

[0163] H-Aib-QGTFTSDLSKYK(γGlu-CO(CH2) 14 CH3)EEQAAQDFIEW LLAGGPSSGAPPPS-NH2

[0164] Compound 3 (SEQ ID NO:9):

[0165] N-pyroglutamyl-H-Aib-QGTFTSDK(γGlu-CO(CH2)) 14 CH3)SKYLDERAAQDFVQWLLDGPSSGAPPPS-NH2

[0166] Compound 4 (SEQ ID NO:10):

[0167] Ac-H-Aib-QGTFTSDK(γGlu-CO(CH2) 14CH3)SKYLDERAAQDFV QWLLDGPSSGAPPPS-NH2

[0168] Compound 5 (SEQ ID NO:11):

[0169] H-Aib-QGTFTSDK(γGlu-CO(CH2) 14 CH3)SKYLEEEAVRLFIEWL KAGGPSSGAPPPS-NH2

[0170] Compound 6 (SEQ ID NO:12):

[0171] H-Aib-HGTFTSDLSKQK(γGlu-CO(CH2) 14 CH3)DERAAQDFVQW LLDGGPSSGAPPPS-NH2

[0172] Compound 7 (SEQ ID NO:13):

[0173] H-Aib-QGTFTSDLSKYC(3-maleimide propionyl-γGlu-NH(CH2)) 15 CH3)EEQAAQDFIEWLLAGPGPSSGAPPPS-NH2

[0174] Compound 8 (SEQ ID NO:14):

[0175] H-Aib-QGTFTSDLSKYC(3-maleimide propionyl-AEEAc-AEEAc-NH(CH2)) 15 CH3)EEQAAQDFIEWLLAGPGPSSGAPPP S-NH2

[0176] Compound 9 (SEQ ID NO:15):

[0177] H-Aib-QGTFTSDLSRYK(γGlu-CO(CH2) 14 CH3)EEQAAQDFIEWL LAGGPSSGAPPPS-NH2

[0178] Compound 10 (SEQ ID NO:16):

[0179] H-Aib-HGTFTSDLSIQK(γGlu-CO(CH2) 14 CH3)EERAAQDFIEWL LDGGPSSGAPPPS-NH2

[0180] Compound 11 (SEQ ID NO:17):

[0181] H-Aib-HGTFTSDLSIQK(γGlu-CO(CH2) 14 CH3)EEQAAQDFIEWL LDGGPSSGAPPPS-NH2

[0182] Compound 12 (SEQ ID NO:18):

[0183] H-Aib-QGTFTSDLSIQK(γGlu-CO(CH2) 14 CH3)EEQAAQDFIEWLLDGGGPSSGAPPPS-NH2

[0184] Compound 13 (SEQ ID NO:19):

[0185] H-Aib-HGTFTSDLSIYK(γGlu-CO(CH2) 14 CH3)EERAQQDFIEWLLDGGGPSSGAPPPS-NH2

[0186] Compound 14 (SEQ ID NO:20):

[0187] H-Aib-HGTFTSDYSIQK(γGlu-CO(CH2) 14 CH3)EEIAAQDFIEWLLDGGGPSSGAPPPS-NH2

[0188] Compound 15 (SEQ ID NO:21):

[0189] H-Aib-HGTFTSDLSIQK(γGlu-CO(CH2) 14 CH3)EEIAQQDFIEWLLDGGGPSSGAPPPS-NH2

[0190] Compound 16 (SEQ ID NO:22):

[0191] H-Aib-HGTFTSDLSK(γGlu-CO(CH2) 14 CH3)YLEERAQQDFIEWLLDGPGPSSGAPPPS-NH2

[0192] Compound 17 (SEQ ID NO:23):

[0193] H-Aib-HGTFTSDLSIK(γGlu-CO(CH2) 14CH3)LEERAQQDFIEWLLDGGGPSSGAPPPS-NH2

[0194] Compound 18 (SEQ ID NO:24):

[0195] H-Aib-HGTFTSDLSIYLEK(γGlu-CO(CH2) 14 CH3)RAQQDFIEWLLDGGGPSSGAPPPS-NH2

[0196] Compound 19 (SEQ ID NO:25):

[0197] H-Aib-HGTFTSDLSIYLEEK(γGlu-CO(CH2) 14 CH3)AQQDFIEWLLDGGGPSSGAPPPS-NH2

[0198] Compound 20 (SEQ ID NO:26):

[0199] H-Aib-HGTFTSDLSIYK(AEEAc-AEEAc-γGlu-CO(CH2) 16 CH3)EERAQQDFIEWLLDGGGPSSGAPPPS-NH2

[0200] Compound 21 (SEQ ID NO:27):

[0201] H-Aib-HGTFTSDLSIQK(AEEAc-AEEAc-γGlu-CO(CH2) 16 COOH)EERAAQDFIEWLLDGGGPSSGAPPPS-NH2

[0202] Compound 22 (SEQ ID NO:28):

[0203] H-Aib-HGTFTSDLSIYK(γGlu-γGlu-AEEAc-AEEAc-CO(CH2) 16 COOH)EERAQQDFIEWLLDGGGPSSGAPPPS-NH2

[0204] Compound 23 (SEQ ID NO:29):

[0205] H-Aib-HGTFTSDYSIQK(AEEAc-AEEAc-γGlu-CO(CH2) 16 COOH)EEIAAQDFVEWLLAGGPSSGAPPPS-NH2

[0206] Compound 24 (SEQ ID NO:30):

[0207] H-Aib-HGTFTSDLSIYK(AEEAc-AEEAc-γGlu-CO(CH2) 16 COOH)EERAQQDFIEWLLDGGGPSSGAPPPS-NH2

[0208] Compound 25 (SEQ ID NO:31):

[0209] H-Aib-QGTFTSDYSIQK(AEEAc-AEEAc-γGlu-CO(CH2) 16 COOH)EERRAQDFIEWLLDGGGPSSGAPPPS-NH2

[0210] Compound 26 (SEQ ID NO:32):

[0211] H-Aib-HGTFTSDLSIYK(γGlu-CO(CH2) 16 COOH)EERAQQDFIEWLLDGGGPSSGAPPPS-NH2

[0212] Compound 27 (SEQ ID NO.33):

[0213] H-Aib-HGTFTSDLSIYK(GSEGSEE-CO(CH2) 16 COOH)EERAQQDFIEWLLDGGGPSSGAPPPS-NH2

[0214] Compound 28 (SEQ ID NO.34):

[0215] H-Aib-HGTFTSDLSIYK(γGlu-γGlu-AEEAc-AEEAc-γGlu-CO(CH2) 16 COOH)EERAQQDFIEWLLDGGGPSSGAPPPS-NH2

[0216] Compound 29 (SEQ ID NO.35):

[0217] H-Aib-HGTFTSDLSIYK(γGlu-GABA-AEEAc-γGlu-CO(CH2) 16 COOH)EERAQQDFIEWLLDGGGPSSGAPPPS-NH2

[0218] Compound 30 (SEQ ID NO:36):

[0219] H-Aib-HGTFTSDLSIYK(γGlu-CO(CH2) 14 CH3)EERAQQDFIEWLLDGPGPSSGAPPPS-OH

[0220] Compound 31 (SEQ ID NO:37):

[0221] H-Aib-HGTFTSDYSIQK(γGlu-CO(CH2) 14 CH3)EEIAAQDFIEWLLDGPGPSSGAPPPS-OH

[0222] Compound 32 (SEQ ID NO:38):

[0223] H-Aib-HGTFTSDLSIQK(AEEAc-AEEAc-γGlu-CO(CH2) 16 COOH)EERAAQDFIEWLLDGPGPSSGAPPPS-OH

[0224] Compound 33 (SEQ ID NO:39):

[0225] H-Aib-HGTFTSDLSIQK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)EERAAQDFIEWLLDGGGPSSGAPPPS-NH2

[0226] Compound 34 (SEQ ID NO:40):

[0227] H-Aib-HGTFTSDLSIYK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)EERAQQDFIEWLLDGGGPSSGAPPPS-NH2.

[0228] In one aspect, a pharmaceutical composition is provided comprising an effective amount of the compound described in any of the foregoing aspects or a salt or solvate thereof, and a pharmaceutically acceptable excipient, diluent, carrier, or excipient.

[0229] In one aspect, the pharmaceutical composition is an injection or lyophilized powder, tablet, pill, lozenge, soft capsule, hard capsule, granule, powder, solution, suspension, or syrup; or the pharmaceutical composition is in the form of microcapsules, microspheres, nanoparticles, or liposomes.

[0230] In one aspect, the pharmaceutical composition is for oral administration, inhalation administration, or parenteral administration, wherein the parenteral administration is selected from intraperitoneal, intramuscular, intra-arterial, intravenous, subcutaneous, or intradermal injection.

[0231] In one aspect, the pharmaceutical composition is administered at a frequency of at least once a day, once a week, or once a month.

[0232] In one aspect, the use of the compounds described in any of the foregoing aspects or their salts or solvates in the preparation of a medicament, said medicament being used as one, two, or three of a GLP-1 receptor agonist, a GIP receptor agonist, or a GCG receptor agonist.

[0233] In one aspect, the use of the compounds described in any of the foregoing aspects or their salts or solvates in the preparation of a medicament for the prevention or treatment of a metabolic disorder syndrome selected from hyperglycemia, insulin resistance, poor glucose tolerance, type 2 diabetes, obesity or non-alcoholic fatty liver disease / non-alcoholic steatohepatitis (NAFLD / NASH), diabetic nephropathy, diabetic retinopathy, dyslipidemia, osteoporosis; or a neurodegenerative disease selected from Alzheimer's disease or Parkinson's syndrome.

[0234] In one aspect, a method for activating one, two, or three of the GLP-1 receptor, GIP receptor, or GCG receptor is provided, comprising administering an effective amount of the compound of the invention or a salt or solvate thereof to an individual in need.

[0235] In one aspect, methods are provided for the prevention or treatment of diseases selected from: metabolic syndromes, including hyperglycemia, insulin resistance, poor glucose tolerance, type 2 diabetes, obesity or non-alcoholic fatty liver disease / non-alcoholic steatohepatitis (NAFLD / NASH), diabetic nephropathy, diabetic retinopathy, dyslipidemia, osteoporosis; or neurodegenerative diseases, including Alzheimer's disease or Parkinson's syndrome, comprising administering an effective amount of the compound of the present invention or its salt or solvate to an individual in need.

[0236] The compounds of this invention can be bi- or tri-agonistic polypeptide molecules, which can be used to prevent or treat metabolic syndromes and have improved stability. Attached image description:

[0237] Figure 1 Image of the .pET31b-SEQ ID NO.19 plasmid.

[0238] Figure 2Electrophoresis diagram of the precursor fusion protein of KSI-DDDDK-peptide SEQ ID NO.19(5-40).

[0239] Figure 3 Acute effects of the first dose of the compound on blood glucose in DIO mice. Blood glucose levels were measured at 0, 1, 2, 4, 8, 24, 48, and 72 hours after the first dose of the compound in DIO mice (for compounds administered once daily, the second and third doses of the corresponding compound were administered normally at 24 and 72 hours). Data are presented as mean ± SEM, n = 8.

[0240] Figure 4 Effect of the compound on fasted blood glucose in DIO mice. Blood was collected from animals 5 hours after fasting at the experimental endpoint. Compared with the Vehicle control group, the compound significantly reduced fasted blood glucose in the animals (*p<0.05). Data are expressed as mean ± SEM, n=8.

[0241] Figure 5 Effects of the compound on oral glucose tolerance in DIO mice. Glucose tolerance in DIO mice was measured on day 19 after administration of oral glucose. Blood glucose levels were measured at specific time points within 120 minutes after oral glucose administration, and the area under the blood glucose-time curve (AUC) was calculated. 0-120min ). Figure 5 A. Blood glucose-time curve after oral glucose administration; Figure 5 B. Area under the blood glucose-time curve (AUC) after oral glucose administration 0-120min Compared with the Vehicle control group, the compound significantly reduced glucose tolerance in DIO mice after oral glucose administration (***p<0.001). Data are presented as mean ± SEM, n=8. Detailed Implementation

[0242] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the definition herein shall prevail. Preferred methods and materials are described below, but similar or equivalent methods and materials may be used to practice or test the invention. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0243] The parent peptide amino acid sequence of the polypeptide drug of the present invention can be modified based on the amino acid sequences of Exendin-4 (SEQ ID NO:1), GLP-1 (SEQ ID NO:2), GIP (SEQ ID NO:5), and GCG (SEQ ID NO:6) as described above.

[0244] The polypeptide compounds described in this invention can all be synthesized and modified by those skilled in the art using known techniques. For example, the peptide sequence backbone of the polypeptide compounds described in this invention can be prepared by synthetic methods, recombinant DNA biotechnology, and other methods.

[0245] The peptide backbone of the compound of the present invention is chemically modified at least one site by a fatty acid side chain group. Preferably, the compound may have a stable peptide α-helix structure and enhanced albumin binding force, thereby achieving improved stability and prolonged peptide action time.

[0246] The compounds of this invention exhibit agonistic activity against the GLP-1 receptor, and optionally also against the GIP receptor and / or GCG receptor. "Agonistic activity" refers to the compound's ability to stimulate specific receptor cells to produce cAMP. The cells used can be host cells, pancreatic islet cells, adipocytes, hepatocytes, etc., that overexpress the GLP-1 receptor, GIP receptor, or GCG receptor, as constructed by those skilled in the art. The receptor agonistic activity can be achieved by stimulating ECMO (electrode-associated cells) of receptor cells to produce cAMP. 50 Value as a numerical measure. EC 50 The value is the drug concentration required to achieve half (50%) of the compound's maximum activity in a specific assay system.

[0247] In a specific implementation, the agonistic activity of a compound can be evaluated by assessing its relative activity against a specific natural compound. The relative activity is defined as the EC50 of the specific natural compound. 50 Values ​​and EC values ​​of the tested compounds 50 The percentage of the value.

[0248] Compared to natural GLP-1 (7-37), the compounds of the present invention have a relative activity of at least 0.5% to GLP-1 receptor agonism, preferably at least 5%, more preferably at least 50%, and even most preferably at least 100%.

[0249] Compared to natural GIP, the compounds of the present invention exhibit a relative activity of at least 0.5% for stimulating GIP receptors, preferably at least 5%, more preferably at least 50%, and even most preferably at least 100%.

[0250] Compared to natural GCG, the compounds of the present invention exhibit a relative activity of at least 0.5% for activating GCG receptors, preferably at least 5%, more preferably at least 50%, and even most preferably at least 100%.

[0251] Peptide sequence similarity and peptide sequence identity

[0252] The structural similarity of two peptides can be determined by aligning their residues along their sequence lengths (e.g., the candidate peptide described herein and any suitable reference peptide) to optimize the number of identical amino acids. During the alignment, vacancies are allowed in one or both sequences to optimize the number of identical amino acids, but the amino acids in each sequence must still be in the correct order. Where appropriate, the reference peptide may be the peptide described herein. The candidate peptide is the peptide being compared to the reference peptide.

[0253] Pairwise comparison analysis of amino acid sequences can be performed using software packages known in the art. In the comparison of two amino acid sequences, structural similarity can be referred to as a percentage of "identity" or a percentage of "similarity." "Identity" refers to the presence of identical amino acids. "Similarity" refers not only to the presence of identical amino acids but also to the presence of conserved substitutions. The conserved substitutions of amino acids in the polypeptides of this invention can be selected from other members of the amino acid class. For example, it is well known in the field of protein biochemistry that amino acids belonging to a specific size or property (e.g., charge, hydrophobicity, and hydrophilicity) can be substituted by another amino acid without altering the protein's activity, especially in protein regions not directly related to biological activity. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions include, for example, Lys substitution for Arg and vice versa, to maintain a positive charge; Glu substitution for Asp and vice versa, to maintain a negative charge; Ser substitution for Thr to maintain free -OH; and Gln substitution for Asn to maintain free -NH2. Similarly, bioactive analogs of peptides containing the deletion or addition of one or more adjacent or non-adjacent amino acids that do not eliminate the functional activity of the peptide are also considered.

[0254] Therefore, as used herein, references to the polypeptides of the present invention and / or references to one or more SEQ ID NOs may include polypeptides having amino acid sequence similarity to a reference amino acid sequence of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0255] Alternatively, as used herein, references to the polypeptides of the present invention and / or references to one or more SEQ ID NOs may include polypeptides having the following amino acid sequence identity with a reference amino acid sequence: at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0256] The polypeptides of the present invention can be further modified, for example, including polypeptides (or their analogues, such as fragments thereof) chemically or enzymatically derived at one or more constituent amino acids. Such modifications may include, for example, side-chain modifications, main-chain modifications, and N- and C-terminal modifications, such as acetylation, hydroxylation, methylation, amidation, and the attachment of carbohydrate or lipid moieties, cofactors, etc., and combinations thereof. Modified polypeptides of the present invention may retain the biological activity of the unmodified polypeptides, or may exhibit reduced or increased biological activity.

[0257] The effective amount of the compound described in this invention can be combined with at least one pharmaceutically acceptable excipient, diluent, carrier, or excipient to form a pharmaceutical composition. "Effective amount of compound" refers to an amount of compound that can produce disease relief or therapeutic effects without causing harmful effects. The effective amount can be appropriately determined by the attending physician based on the patient's disease severity, age, sex, weight, general health status, etc. In one embodiment, the patient is a mammal. In a preferred embodiment, the mammal is selected from cattle, horses, goats, sheep, dogs, chimpanzees, rabbits, mice, rats, monkeys, pigs, and humans. In a more preferred embodiment, the patient is a human.

[0258] The compounds and pharmaceutical compositions described in this invention can be formulated as injections or lyophilized powders, tablets, pills, lozenges, soft capsules, hard capsules, granules, powders, solutions, suspensions, or syrups, preferably as injections or lyophilized powders.

[0259] Excipients that can be used in the compositions of this invention include, for example, lubricants, binders, fillers, preservatives, surfactants, colorants, flavoring agents, emulsifiers, suspending agents, diluents, gelling agents, disintegrants, pH adjusters, solubilizers, etc. Those skilled in the art will understand that these excipients can be appropriately selected according to suitable dosage forms, and their content can be changed as needed.

[0260] The compounds described in this invention can also be loaded into various drug carrier materials (e.g., microcapsules, microspheres, nanoparticles, liposomes) or drug delivery devices.

[0261] Furthermore, the compounds described in this invention can also be used in combination with at least one of the following therapeutic agents, including antidiabetic agents (such as insulin and its analogues, biguanides, sulfonylureas, thiazolidinediones, α-glucosidase inhibitors, DPP-4 inhibitors, SGLT2 inhibitors, dual SGLT1 / SGLT2 inhibitors, GLP-1 receptor agonists, amylin and its analogues), GIP receptor agonists, GCG receptor agonists or antagonists, dual GLP-1 / GIP receptor agonists, GLP-1 / GCG receptor agonists, GIP / GCG receptor agonists, FGF-21 and its analogues, cholecystokinin B (CCKB) and its analogues, PYY (3-36) and its analogues, leptin and its analogues, calcitonin and its analogues, lipid-regulating agents, PPAR-α, β, δ agonists or modulators, antiplatelet aggregation agents, PCSK9 inhibitors, lipase inhibitors, anti-hepatic fibrosis or cirrhosis agents, and anti-inflammatory agents.

[0262] Preferably, the compound of the present invention can promote insulin secretion and lower blood sugar. Preferably, it can also inhibit food intake, delay gastric emptying, increase energy consumption, and ultimately result in an observable weight loss effect.

[0263] Preferably, the compound of the present invention can reduce pancreatic β-cell apoptosis, increase the number of pancreatic β-cells, and improve pancreatic islet cell function.

[0264] Preferably, the compounds of the present invention can also improve blood lipids, reduce liver fat accumulation, inhibit the development of liver inflammation, and prevent and treat non-alcoholic fatty liver disease.

[0265] Preferably, the compounds of the present invention are expected to promote the growth of brain neurons, clear neurotoxic substances, inhibit the development of inflammation, and play a neuroprotective role.

[0266] Preferably, the compounds or compositions of the present invention can be used for the prevention and / or treatment of metabolic disorders and their related complications. They are particularly preferred for the treatment of diabetes, obesity, and non-alcoholic fatty liver disease.

[0267] Preferably, the compounds or compositions of the present invention can be used to treat lipid metabolism disorders and related diseases, and neurodegenerative diseases (such as Parkinson's disease and Alzheimer's disease).

[0268] Preferably, the compounds or compositions of the present invention can be used to treat bone diseases related to endocrine disorders, metabolic disorders, kidney disease, weight loss, etc., such as osteoporosis and osteoarthritis.

[0269] Preferably, the compounds or compositions of the present invention can be administered via a variety of routes, such as oral administration, inhalation administration, or parenteral administration, wherein the parenteral administration includes, for example, intraperitoneal, intramuscular, intra-arterial, intravenous, subcutaneous, or intradermal injection.

[0270] Preferably, the compound or composition of the present invention can be administered at least once a day, once a week, or once a month.

[0271] The compounds of this invention exhibit better solubility and stability than natural peptide molecules and liraglutide.

[0272] The compounds of this invention have significant agonistic effects on two or all three of the GLP-1, GIP, and GCG receptors.

[0273] The specific meanings of the abbreviations used in this invention are as follows:

[0274] Aib: Diaminoisobutyric acid

[0275] GABA: γ-aminobutyric acid

[0276] AEEAc: [2-(2-amino-ethoxy)-ethoxy]-acetyl

[0277] Ac: Acetyl group

[0278] pGlu: Pyroglutamyl group

[0279] cAMP: Cyclic adenosine monophosphate

[0280] PEG: Polyethylene Glycol

[0281] Fmoc: fluorenemethyloxycarbonyl

[0282] Boc: tert-Butoxycarbonyl

[0283] DMF: Dimethylformamide

[0284] DIC: N,N-Diisopropylcarbodiimide

[0285] Boc: tert-Butoxycarbonyl

[0286] Trt: Triphenylmethyl

[0287] ivdde: 1-(4,4-dimethyl-2,6-dioxocyclohexyl)-3-methyl-butyl

[0288] t-Bu: tert-butyl

[0289] OtBu: tert-butyl ester

[0290] TFA: Trifluoroacetic acid

[0291] HPLC / MS: High Performance Liquid Chromatography / Mass Spectrometry; HPLC-UV: High Performance Liquid Chromatography-Ultraviolet Spectrometry

[0292] IPTG: Isopropyl-β-D-thiogalactoside

[0293] Tris: Tris(hydroxymethyl)aminomethane

[0294] DCM: Dichloromethane

[0295] THF: Tetrahydrofuran

[0296] DIPEA: N,N-Diisopropylethylamine

[0297] NMP: N-methylpyrrolidone

[0298] PAM: peptidylglycine α-amidylmonoxylase; MES: fatty acid methyl ester sulfonate

[0299] HEK-293: Human embryonic kidney cells

[0300] GLP-1R: Glucagon-like peptide-1 receptor; GIP R: Glucose-dependent insulinotropic peptide receptor; GCG R: Glucagon receptor

[0301] PBS: Phosphate Buffer Solution

[0302] FBS: Fetal bovine serum

[0303] DMEM: Duchenne Modified Eagle Medium

[0304] HBSS: Hank's balanced salt solution; HEPES: 4-hydroxyethylpiperazine ethanesulfonic acid.

[0305] BSA: Bovine serum albumin

[0306] EC 50 Half-number effect concentration

[0307] IBMX: 3-Isobutyl-1-methylxanthine.

[0308] SC: Subcutaneous injection

[0309] QD: Once a day

[0310] Q3D: Once every three days

[0311] OGTT: Oral Glucose Tolerance Test

[0312] TC: Total cholesterol

[0313] LDL-C: Low-density lipoprotein cholesterol

[0314] TG: Triglycerides

[0315] HOMA: Insulin Resistance Index Specific implementation examples:

[0317] Example 1: Synthesis of peptide compounds:

[0318] The intermediates and compounds of this invention can be synthesized using various methods known in the art. The following specific examples illustrate the preparation of the compounds of this invention using chemical synthesis methods. Each specific synthetic step described can employ different combinations of materials and methods to synthesize various corresponding compounds of this invention or their salts. The reagents and raw materials used are readily available to those skilled in the art. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of this invention in any way.

[0319] Material:

[0320] All materials and reagents used in this invention were purchased from commercial products. The protected amino acids used in the entire synthesis process are as follows: Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-P he-OH, Fmoc-Arg(pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Bo c)-OH, Fmoc-Lys(ivdde)-OH, Fmoc-Thr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Aib-OH, Boc-His(Boc)-OH.

[0321] The following uses compound SEQ ID NO:12 as an example to illustrate the synthesis and preparation method of the compound of the present invention.

[0322] method:

[0323] (1) Pretreatment of Rink amino resin: Weigh 1g of dry Rink amino resin, soak it in DMF for 30min to swell, and then remove the solvent.

[0324] (2) Removal of the protecting group Fmoc: Add 20% piperidine / DMF solution to the treated Rink amino resin and stir for 20 min. During the reaction, monitor the degree of reaction using the ninhydrin colorimetric method. If the resin changes color, it indicates that Fmoc removal is successful. After the reaction, filter to remove the solvent, add DMF to the reaction system and stir to wash the resin for 1 min. Repeat the washing 3 times.

[0325] (3) Coupling reaction (peptide bond formation): The prepared Fmoc-protected amino acid solution was added to the reactor, followed by DIC / DMF solution, and the mixture was stirred for 1 hour. The reaction progress was monitored using the ninhydrin colorimetric method; if the resin color did not change, the coupling was successful. After the reaction, the solvent was removed by filtration, and DMF was added to the reaction system to wash the resin for 1 minute. This washing was repeated three times. The above steps were repeated, adding the corresponding amino acid solutions sequentially until the peptide chain synthesis was complete. The last amino acid was coupled using Boc-His(Trt)-OH. The Lys at the side chain modification site was replaced with Fmoc-Lys(ivdde)-OH. SEQ ID The order of amino acid conjugation for the main peptide sequence of compound NO:12 is as follows: Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH (3x), Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH (2x), Fmoc-Pro-OH, Fmoc-Gly-OH (2x), Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH (2x), Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH (2x). ), Fmoc-Arg(pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Lys(ivdde)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Aib-OH, Boc-His(Boc)-OH.

[0326] (4) Removal of ivdde: Add hydrazine / DMF solution to the system to remove the side chain protecting group ivdde of the modified site Lys. After successful removal, filter to remove the solvent, add DMF to the system, stir and wash for 1 min, filter to remove the solvent, and repeat the washing 4 times.

[0327] (5) Lys side chain modification: The prepared Fmoc-Glu-OtBu solution was added to the above-treated resin, followed by DIC / DMF solution, and the mixture was stirred for 1 hour. After the reaction was complete, the resin was filtered and washed, and 20% piperidine / DMF solution was added. The mixture was stirred for 20 minutes to remove the Fmoc groups. Then, the prepared palmitic acid solution was added to the resin, followed by DIC / DMF solution, and the mixture was stirred for 1 hour. After the reaction was complete, the resin was washed four times and dried.

[0328] (6) Post-treatment of peptide resin: Add cleavage reagent K to cleave the resin. Filter the filtrate, precipitate and centrifuge to obtain a white solid, which is the crude target compound.

[0329] Crude peptide compound purification:

[0330] The obtained crude peptide was purified using a reverse-phase C18 preparative column (Shimadzu, Inertsil ODS 20x 250mm 5µm) to a purity greater than 95%. Using 95% buffer A (0.065% TFA / H2O) and 5% buffer B (0.05% TFA / acetonitrile) as the initial eluent, the proportion of buffer B was gradually increased to 65% at a rate of 2% / min, and elution was performed continuously for 30 min. The target peptide fraction was collected. The purified peptide compound was confirmed by analytical HPLC / MS.

[0331] Based on the above synthesis method, the following peptide compounds SEQ ID NO:7-40 were synthesized and characterized (see Table 1).

[0332] Table 1. List of synthesized peptide compounds and their molecular weights

[0333]

[0334]

[0335]

[0336] Example 2: Biosemi-synthesis of peptide compounds:

[0337] The following describes the method steps for the biosynthesis of the compound of the present invention using the biosynthesis of peptide compound SEQ ID NO.19 as an example.

[0338] (1) Construction of genetically engineered bacteria containing KSI-DDDDK-SEQ ID NO.19(5-40) precursor

[0339] Referring to Example 3 of patent CN201711154044, an engineered strain of *E. coli* with a fusion gene was constructed. The fusion gene has a gene sequence resembling an ABC structure, where A is a chaperone protein encoding gene, B is a linker peptide encoding gene, and C is a tri-agonist peptide encoding fragment SEQ ID NO.19(5-40). This strain was then subjected to high-density fermentation and induction to extract inclusion bodies or the fusion protein itself.

[0340] The recombinant Escherichia coli strain is preferably obtained through the following steps: cloning a fusion gene with an ABC structure into a prokaryotic expression vector, and then transforming the resulting recombinant expression vector into engineered Escherichia coli bacteria to obtain a recombinant Escherichia coli strain.

[0341] The prokaryotic expression vector is pET31b(+).

[0342] The engineered Escherichia coli strain mentioned is Escherichia coli BL21(DE3).

[0343] The induction was performed via IPTG.

[0344] The fusion protein has the following structure, consisting of three fragments from the N-terminus to the C-terminus: a KSI chaperone protein, a linker peptide, and a peptide precursor SEQ ID NO. 19 (5-40).

[0345] The amino acid sequence of the fusion protein is shown in SEQ ID NO.41.

[0346] SEQ ID NO.41:

[0347] MHTPEHITAVVQRFVAALNAGDLDGIVALFADDATVEDPVGSEPRSGTAAIREFYANSLKLPLAVELTQEVRAVANEAAFTVSFEYQGRKTVVAPIDHFRFNGAGKVVSIRALFGEKNIHACQMLDDDDKTFTSDLSIYKEERAQQDFIEWLLDGGPSSGAPPPSG-OH

[0348] The linker peptide in the fusion protein is DDDDK.

[0349] The precursor sequence of the peptide SEQ ID NO.19(5-40) of the fusion protein is shown in SEQ ID NO.42.

[0350] SEQ ID NO.42:

[0351] TFTSDLSIYKEERAQQDFIEWLLDGGGPSSGAPPPSG-OH

[0352] The construction method of the fusion gene and engineered bacteria with an ABC-like structure can be referred to from the experimental guide in this field (J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Science Press, 1995).

[0353] The DDDDK-peptide SEQ ID NO.19(5-40) precursor fusion gene fragment was designed. Based on the codon table, these amino acid sequences were converted into nucleotide sequences. During the conversion process, codons with higher usage frequency were selected according to the codon usage preferences of *E. coli*, and the GC content was adjusted. Cis-acting elements and repetitive sequences affecting gene transcription were removed for optimization. Simultaneously, a double stop codon TAATGA was introduced at the 3' end of the gene sequence. To facilitate gene manipulation, the AlwNI restriction site sequence CAGATGCTG was introduced at the 5' end of the DDDDK-peptide SEQ ID NO.19(5-40) precursor fusion gene sequence. Therefore, two amino acids, ML, were introduced before the N-terminal extension peptide. An XhoI restriction site CTCGAG was introduced at the 3' end of the DDDDK-peptide SEQ ID NO.19(5-40) precursor fusion gene. The optimized DDDDK-peptide SEQ ID NO.19(5-40) precursor fusion gene sequence is shown in SEQ ID NO:43. The gene sequence was synthesized by a gene synthesis service company and cloned into the pUC57 vector.

[0354] SEQ ID NO.43:

[0355] CAGATGCTGGATGACGATGACAAAACCTTCACCTCTGACCTGTCTATCTACAAAGAAGAACGTGCTCAGCAGGACTTCATCGAATGGCTGCTGGACGGTGGTCCGTCTTCTGGTGCTCCGCCGCCGTCTGGTTAATGACTCGAG

[0356] The plasmid pET-31b(+) (purchased from Invitrogen) was double-digested using the restriction endonucleases AlwNI and XhoI from TaKaRa. The recombinant vector pUC57-SEQ ID NO.43 was also double-digested using AlwNI and XhoI. The digested DNA fragment was ligated into the double-digested pET-31b(+), and after sequencing verification, it was named pET31b-SEQ ID NO.19. Figure 1 As shown.

[0357] Following the calcium chloride method described in the third edition of *Molecular Cloning: A Laboratory Manual* published by Cold Spring Harbor Laboratory, competent *E. coli* BL21(DE3) cells (all purchased from Life Technologies) were prepared. 1 μL of the recombinant expression vector pET31b-SEQ ID NO.19 was transformed into *E. coli* BL21(DE3) competent cells, using the same calcium chloride method as described in the third edition of *Molecular Cloning: A Laboratory Manual*. The transformation solution was plated onto LB agar medium supplemented with ampicillin (final concentration 100 μg / ml) and incubated at 37°C inverted until single colonies appeared. The resulting bacterial strain library was named BL21(DE3) / pET31b-SEQ ID NO.19. Use a toothpick to pick a single colony of BL21(DE3) / pET31b-SEQ ID NO.19 and inoculate it into 50 ml of LB liquid medium. Incubate at 37°C with shaking at 250 rpm. When the OD600 of the bacterial culture is 0.5–1.0, take 3–6 ml of the bacterial culture and inoculate it into 100 ml of LB liquid medium. Incubate at 37°C with shaking at 250 rpm until the OD600 reaches 0.5–1.0. 600 When the concentration of the bacterial culture reaches 0.6 to 0.8, add IPTG (isopropyl-β-D-thiopyranogalactopyranoside, final concentration 1 mmol / L) to start induction. Take 1 ml of the induction culture after 2 hours, centrifuge at 12000 rpm for 1 minute, remove the supernatant, and store the bacterial cells at -20℃ for later use.

[0358] Remove the bacterial cells frozen at -20℃, resuspend them in 5 ml of 8M urea solution, and sonicate them in an ice-water mixture for 10 minutes (sonicate for 3 seconds, pause for 5 seconds, repeat). Take 15 μl of the lysate, add 15 μl of loading buffer, mix thoroughly, and then take 10 μl for SDS-PAGE (the stacking gel contains 5% acrylamide-methylenebisacrylamide (29:1) by volume, and the separating gel contains 15% acrylamide-methylenebisacrylamide (29:1) by volume). Electrophoresis conditions: stacking gel current set at 11 mA, separating gel current set at 22 mA. After electrophoresis, remove the gel and stain it overnight with Coomassie Brilliant Blue solution (containing 0.6g Coomassie Brilliant Blue R-250, 450ml ethanol, 100ml glacial acetic acid, and the remainder purified water). Destain with destaining solution (containing 250ml ethanol, 80ml glacial acetic acid, and the remainder purified water) until the background is transparent. Take an image of the gel against a transparent background to extract the image (e.g., ...). Figure 2 (As shown), this is consistent with the theoretical molecular weight of 18.18 kDa of the precursor fusion protein of KSI-DDDDK-peptide SEQ ID NO.19(5-40).

[0359] (2) Modification and enzymatic digestion of KSI-DDDDK-peptide SEQ ID NO.19(5-40) precursor fusion protein

[0360] First, referring to embodiments 3 and 4 of patent CN201711154044, Nα-hexadecanoyl-Glu(ONSu)-OBut was synthesized.

[0361] Referring to Example 6 of patent CN201711154044, the constructed engineered bacteria were subjected to high-density fermentation. The cells were broken up and washed to obtain inclusion bodies. 0.62 kg of inclusion bodies were obtained from 10 L of fermentation broth. The Folin-phenol method determined that each 1 g of inclusion body contained 0.20 g of protein. 200 g of the above fusion protein inclusion bodies were dissolved in 1500 ml of 6 mol / L guanidine hydrochloride solution, and 80 ml of DMSO and 15 ml of N,N-diisopropylethylamine were added. The mixture was stirred until the pH was 10.8. Then, 80 ml of DMSO solution containing 50 mg / ml Nα-hexadecyl-Glu(ONSu)-OH was added, and the mixture was stirred for 3 hours. Afterward, 1700 ml of 20 mmol / L tris(hydroxymethyl)aminomethane solution was added, along with 900 IU of lysine-specific endonuclease. The pH was adjusted to 9.0 with dilute hydrochloric acid or sodium hydroxide solution. After reacting for 8 hours, the pH was adjusted to 7.8.

[0362] The liquid was loaded onto a 500 mL Uni NM200 (200 μm particle size, 300 Å pore size) chromatography column equilibrated with 1500 mL of equilibration solvent containing 20 mmol / L Tris, 5% isopropanol, and pH 7.8. The column was then equilibrated with another 1500 mL of equilibration solvent. Elution was then performed with a gradient elution solvent containing 20 mmol / L Tris, 5%–40% isopropanol, and pH 7.8. The fraction containing peptide SEQ ID NO. 19 (5-40) was collected.

[0363] To further improve purity, the collected fraction was diluted with an equal volume of purified water and loaded onto a 500 mL Uni PS40 (40 μm particle size, 500 Å pore size) chromatography column equilibrated with 1500 mL of a equilibration solvent containing 20 mmol / L Tris, 5% isopropanol, and pH 7.0. The column was then equilibrated with another 1500 mL of equilibration solvent. Elution was then performed using a gradient elution solvent containing 20 mmol / L Tris, 5%–30% isopropanol, and pH 7.0, collecting the fraction containing peptide SEQ ID NO. 19 (5-40). This fraction was then lyophilized into a powder.

[0364] The peptide SEQ ID NO.19(5-40) sequence is shown in SEQ ID NO.44.

[0365] SEQ ID NO.44:

[0366] TFTSDLSIYK(γGlu-CO(CH2)14 CH3)EERAQQDFIEWLLDGGPSS GAPPPSG-OH

[0367] HPLC / MS: m / z = 1422.6 [M+3H] 3+

[0368] The calculated molecular weight is 4264.7.

[0369] (3) Preparation of N-terminal protrusion Boc-His(Boc)-Aib-His(Trt)-Gly-Osu

[0370] Referring to Example 4 of patent CN201510459093, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Aib-OH, and Boc-His(Boc)-OH were sequentially coupled onto a 2-chlorotriphenylmethyl chloride resin. Finally, trifluoroethanol-DCM (1:4) was added, and the resin was stirred. The filtrate was collected, the solvent was removed under vacuum, cold diethyl ether was added, the precipitate was filtered off, washed with diethyl ether, and then dried under vacuum to obtain Boc-His(Boc)-Aib-His(Trt)-Gly-OH.

[0371] Then, referring to Example 5 of patent CN201510459093, a certain amount of DIPEA and O-(N-succinimide)-N,N,N,N-tetramethylureaium hexafluorophosphate were added to an anhydrous THF solution of Boc-His(Boc)-Aib-His(Trt)-Gly-OH. The reaction solution was then added to dichloromethane, washed with water, and the organic phase was dried under vacuum after being dried with magnesium sulfate to obtain Boc-His(Boc)-Aib-His(Trt)-Gly-Osu.

[0372] (4) Preparation of peptide SEQ ID NO.19(1-40)

[0373] Referring to Example 8 of patent CN201510459093, peptide SEQ ID NO.19(5-40) (0.24 mmol, 1.0 g) was dissolved in 100 ml of NMP, followed by the addition of 300 μL of DIPEA and 0.4 mmol of Boc-His(Boc)-Aib-His(Trt)-Gly-Osu. The mixture was stirred and reacted overnight. 1000 ml of ice-cold diethyl ether was added, the precipitate was separated by centrifugation, washed with 500 ml of diethyl ether, and then dried under vacuum.

[0374] The crude powder was dissolved in TFA-triisopropylsilane-water (95:2.5:2.5, 200 ml) and stirred for 2 hours. The solution was then concentrated under vacuum to approximately 20 ml. 500 ml of ice-cold diethyl ether was added, and the mixture was allowed to stand at 2-8°C for 6-8 hours to form a precipitate. The precipitate was then centrifuged. The precipitate was washed with cold diethyl ether and dried under vacuum. The collected powder was the crude powder of peptide SEQ ID NO.19(1-40). The powder was then dissolved in 500 ml of pH 7.0 phosphate buffer containing 5% acetonitrile and purified using a preparative C8 silica gel column. The peptide sample solution was injected into the prepared column, and gradient elution was performed according to the purification method described below.

[0375] The purification method is as follows:

[0376] Flow rate: 10.0 ml / min; wavelength: 214 nm

[0377] Mobile phase: Phase A, 95% 0.1M phosphate buffer + 5% acetonitrile, pH 6.5;

[0378] Phase B, pure acetonitrile

[0379] Gradient: 0–10 min, 5%–10% B phase

[0380] 10–100 min, 10%–70% B phase

[0381] The collected solution containing peptide components was analyzed by HPLC / MS. The HPLC method is as follows:

[0382] The detection wavelength was 214 nm, and the flow rate was 1.0 ml / min.

[0383] Mobile phase A is water containing 0.065% TFA; mobile phase B is acetonitrile containing 0.05% TFA.

[0384] Gradient: 0.01 min 15% B phase

[0385] 25.00 min 75% B phase

[0386] 25.01 min 95% B phase

[0387] 31.00 min 95% B phase

[0388] 31.01 min 15% B phase

[0389] 40.00 min 15% B phase

[0390] The components with a purity of not less than 90% were mixed together and freeze-dried to obtain peptide SEQ ID NO.19(1-40) dry powder.

[0391] The peptide SEQ ID NO.19(1-40) sequence is shown below.

[0392] SEQ ID NO.19(1-40):

[0393] H-Aib-HGTFTSDLSIYK(γGlu-CO(CH2) 14 CH3)EERAQQDFIEWL LDGGPSSGAPPPSG-OH

[0394] HPLC / MS: m / z = 1561.6 [M + 3H] 3+

[0395] The calculated molecular weight is 4681.2.

[0396] (5) Preparation of SEQ ID NO.19 by amidation

[0397] The C-terminal PPPSG-OH structure of peptide SEQ ID NO.19(1-40) was catalytically cleaved using peptidylglycine α-amidyl monooxygenase (PAM) (purchased from Wuhan Yunclone Technology Co., Ltd.) to obtain an amidated product with a C-terminal structure of PPPS-NH2.

[0398] 200 mg (0.04 mmol) of peptide SEQ ID NO.19 (1-40) was added to 80 mL of a solution containing 100 mM MES / KOH (pH 6.0), 30 mM KI, 30 mM KCl, 1 μM copper sulfate, 100 μg / mL catalase, 1% (v / v) ethanol, 0.001% (v / v) Triton X-100, and 10 mM ascorbate. Then, 5 μg / mL LPAM was added, and the mixture was incubated in a 37°C water bath for 30 min. After the reaction was complete, 6% (v / v) TFA was added to terminate the reaction. After the reaction was complete, the reaction solution was diluted by one volume with pH 7.0 phosphate buffer containing 5% acetonitrile. Following the HPLC purification method used in step (4) for SEQ ID NO.19 (1-40), the obtained sample was purified to a purity greater than 95%. The purified sample was characterized and confirmed by analytical HPLC / MS to obtain a peptide compound with the sequence structure SEQ ID NO.19.

[0399] SEQ ID NO.19:

[0400] H-Aib-HGTFTSDLSIYK(γGlu-CO(CH2) 14 CH3)EERAQQDFIEWL LDGGPSSGAPPPS-NH2

[0401] HPLC / MS: m / z = 1542.3 [M + 3H] 3+

[0402] The calculated molecular weight is 4623.2.

[0403] The peptide compounds in SEQ ID NO.15-40 can all be biosynthesized using the above method, and the C-terminal amidated polypeptide sequences can all be prepared by amidation after biosynthesis.

[0404] Example 3: Compound stability test:

[0405] The compound to be tested was dissolved in freshly prepared PBS solution at a concentration of 1 mg / ml, adjusted to pH 7.4, and filtered through a 0.22 μm sterile filter. The peak area of ​​a 10 μL injection was analyzed by HPLC-UV, and this result was taken as the initial point (T0) for the stability test of the compound.

[0406] The compound sample solution for stability testing was placed in a 25°C incubator and stored in a sealed, light-protected environment for 7 days. After this process, the sample solution was centrifuged at 4500 rpm for 10 min, and the supernatant was gently aspirated. The peak area of ​​10 μL of the injection was analyzed by HPLC-UV. This analytical result is the endpoint (T7) of the compound stability test.

[0407] The remaining peptide amount is calculated by comparing the target peak area and related impurity peak areas of the compound at times T0 and T7. The calculation formula is as follows:

[0408] Remaining peptides (%) = (T7 main peak area / T0 main peak area) × 100

[0409] The stability of peptide compounds was assessed by comparing the remaining amount of peptides in the compounds. The results are shown in Table 2.

[0410] Table 2. Results of stability analysis of peptide compounds

[0411]

[0412]

[0413] Example 4: Determination of the activity of peptide compounds against GLP-1 / GIP / GCG receptors:

[0414] The agonistic activity of peptide compounds on their respective receptors was determined by measuring the cAMP signaling response in HEK-293 cells stably overexpressing human GLP-1, GIP, and GCG receptors. Intracellular cAMP levels were measured using a Cisbio Corp. kit based on HTRF (homogeneous time-resolved fluorescence) technology.

[0415] HEK-293 cells stably overexpressing human GLP-1, GIP, and GCG receptors were cultured in DMEM complete medium containing 10% FBS and 2 mM L-glutamine. When the cells reached 80-90% confluence, they were digested with 0.025% trypsin. Digestion was stopped by adding complete medium, and the cell clusters were gently dispersed into single cells. The cell suspension was centrifuged at 1000 rpm for 5 min at room temperature, the supernatant was discarded, and the cells were resuspended in 1×HBSS (20 mM HEPES, 0.1% BSA, 250 μM IBMX) at a cell density of 1.0 × 10⁶ cells / year. 5 / mL.

[0416] Add 10 μL of cell suspension to each well of a 384-well plate. Dissolve the test compound in 1×PBS buffer and serially dilute 4-fold from 100,000 to 0.02 nM to prepare 12 concentration points. Using an automated dispensing device, add 100 nL of the prepared compound solution to the corresponding cell suspension in each well of the 384-well plate, shake at 1000 rpm for 1 min to mix thoroughly, and then incubate at room temperature for 60 min. After drug incubation, add 10 μL of the assay reagent from the kit to each well and incubate at room temperature for another 60 min. Place the plate in an EnVision microplate reader (PerkinElmer) to measure fluorescence readings at 665 / 615 nm. Use GraphPadPrism5 software to construct compound concentration-effect curves and calculate EC5. 50 value.

[0417] Natural wild-type human GLP-1, GIP, and GCG were used as positive controls for the receptor agonist effect of the test compound. For GLP-1 receptor cells, the EC50 of the test compound was calculated. 50 EC value of human GLP-1 50 The percentage of the ratio is used as the relative activity (%) to assess the GLP-1 receptor agonist activity of the test compound.

[0418] For GIP receptor cells, the EC of the test compound was calculated. 50 Value and people GIP EC 50 The percentage of the ratio is used as the relative activity (%) to assess the GIP receptor agonist activity of the test compound.

[0419] For GCG receptor cells, the EC of the test compound was calculated. 50 Value and human GCG EC 50 The percentage of the ratio is used as the relative activity (%) to assess the GCG receptor agonist activity of the test compound.

[0420] Table 3. Average EC50 of peptide compounds 50 Value and relative activity

[0421]

[0422]

[0423] Rel.A: Relative activity; NT: nottest; n≥4: Each group has at least 4 independent test data.

[0424] Example 5: Rat pharmacokinetic (PK) study of peptide compounds

[0425] SD rats were administered compounds SEQ ID NO:19 (30, 100 nmol / kg), SEQ ID NO:39 (50 nmol / kg), SEQ ID NO:40 (50 nmol / kg), liraglutide (30 nmol / kg), and semaglutide (50 nmol / kg) subcutaneously. Blood samples were collected via jugular vein from rats in the SEQ ID NO:19 and liraglutide groups at 0.25, 0.5, 1, 2, 4, 8, 12, 24, 36, 48, and 56 hours post-administration. Blood samples were collected via jugular vein from rats in the SEQ ID NO:39, SEQ ID NO:40, and semaglutide groups at 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, and 96 hours post-administration. After processing, plasma samples were obtained and analyzed using LC-MS / MS with Phoenix WinNonlin. The 6.3 version of the software (non-compartmental model) analyzes the blood drug concentration-time curve and calculates PK parameters and half-life.

[0426] The PK parameters calculated using the above method are shown in Table 4.

[0427] Table 4: Rat pharmacokinetic (PK) parameters of the tested compounds

[0428]

[0429] Example 6: Pharmacological study of compounds SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38 on diet-induced obese (DIO) mice.

[0430] Mice induced to be obese (DIO) by a high-fat diet exhibit significant metabolic syndrome characteristics similar to those in humans, including obesity, elevated blood glucose, insulin resistance, and dyslipidemia. The effects of the compounds of this invention on body weight, food intake, blood glucose, and blood lipids were investigated in C57BL / 6J DIO mice.

[0431] Five-week-old male C57 BL / 6J mice (purchased from Shanghai Slack Laboratory Animal Company) were housed in a pathogen-free, clean environment (controlled temperature 20-24℃, relative humidity 30-70%), with a 12-hour light / 12-hour dark cycle, fed a normal diet, with four animals per cage, for two weeks of acclimatization. Obesity was induced in the mice by feeding a high-fat diet (60 kcal% from fat). After 16 weeks of high-fat diet feeding, DIO mice reached a weight of 45-55 g and a blood glucose range of 8-12 mM. Based on weight and fasting blood glucose, the DIO mice were randomly divided into groups (n=8) to ensure that each group had similar average weight and blood glucose. After grouping, one animal per cage was housed for one week, during which each animal was subcutaneously injected (SC) with the solvent Vehicle (1×PBS, 5 ml / kg) for 3 days to allow pre-acclimatization to the procedure.

[0432] After animal preconditioning, animals were subcutaneously injected with a solvent control, a specific dose of semaglutide, or the compound of this invention, dissolved in 1×PBS, at a dose of 5 ml / kg. Administration began at 9:00 AM. Vehicles (SEQ ID NO:36 and SEQ ID NO:37) were administered once daily (QD), while semaglutide and SEQ ID NO:38 were administered once every three days (Q3D), for a total of 22 days. At the first administration of the compound, blood was collected by tail amputation without anesthesia, and blood glucose changes were measured using a glucometer before administration (t=0h) and at t=1, 2, 4, 8, 24, 48, and 72h after administration (without restriction of food or water intake) to assess the acute hypoglycemic effect of the compound on DIO mice. Throughout the study, animal body weight and food intake were measured daily before administration. The percentage change in body weight (%) and cumulative food intake were calculated by comparing the animal's initial body weight and food intake with those of the same animal before administration to assess the effect of the compound on changes in body weight and food intake.

[0433] On day 19, the effect of the compound on glucose tolerance in DIO mice was determined. After administration in the morning, animals were fasted for 5 hours (without water restriction). Body weight and post-fasting blood glucose levels were measured as the baseline blood glucose for the glucose tolerance test (t=0). DIO mice were then administered glucose solution (2 g / kg, 5 ml / kg) orally via gavage. Blood glucose levels were measured at t=15, 30, 60, 90, and 120 mins after tail dislocation without anesthesia. Animals were allowed to eat after the last blood collection. Blood glucose levels are expressed in mmol / L.

[0434] At the experimental endpoint (Day 22), the final drug administration was performed in the morning. After drug administration, animals were fasted for 5 hours, and then blood was collected via tail tip amputation without anesthesia to measure their fasted blood glucose levels. After blood collection, animals were anesthetized with CO2 and euthanized. Blood was then collected via the heart, and plasma was separated by centrifugation. The plasma was used to measure total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), and insulin levels (to calculate HOMA-IR). The liver was separated, homogenized, and the supernatant was collected by centrifugation to determine the liver triglyceride content.

[0435] All results are expressed as mean ± SEM. Results were analyzed using one-way ANOVA with GraphPad Prism 5 software and followed by Dunnett's post-hoc test comparing the results to the vehicle control group. Differences were considered statistically significant at the p-level < 0.05.

[0436] Table 5. Effects of compounds on body weight change and cumulative food intake in DIO mice

[0437]

[0438] ***p<0.001, compared with the vehicle control group (One-way ANOVA, Dunnett's). Results are expressed as mean ± SEM of 8 animals.

[0439] Table 6. Effects of compounds on plasma TC, LDL-C, and TG in DIO mice

[0440]

[0441] **p<0.01, ***p<0.001, compared with the vehicle control group (One-way ANOVA, Dunnett's). Results are expressed as mean ± SEM of 8 animals.

[0442] Table 7. Effects of compounds on liver TG and HOMA-IR in DIO mice

[0443]

[0444] *p<0.05, **p<0.01, ***p<0.001, compared with the vehicle control group (One-way ANOVA, Dunnett's). MouseHOMA-IR = [(fasting insulin (mIU / L) × fasting glucose (mmol / L)] / 22.5. Results are expressed as mean ± SEM of 8 animals.

[0445] Example 7: Pharmacodynamic study of compounds SEQ ID NO:19, SEQ ID NO:39, and SEQ ID NO:40 on diet-induced obese (DIO) mice.

[0446] According to the experimental group (n=6), animals were subcutaneously injected with a solvent control vehicle (1×PBS) and a certain dose of the compounds of this invention, SEQ ID NO:19, SEQ ID NO:39, and SEQ ID NO:40, dissolved in 1×PBS. The dosage was 5 ml / kg, and the administration began at 9:00 AM. Compound SEQ ID NO:19 was administered once daily (QD), and compounds SEQ ID NO:39 and SEQ ID NO:40 were administered every three days (Q3D) for 14 days. Throughout the experimental study, animal body weight and food intake were measured before each administration. The percentage change in body weight (%) and cumulative food intake were calculated by comparing with the initial body weight and food intake of the same animals before administration to evaluate the effect of the compounds on changes in body weight and food intake.

[0447] At 21:00 on Day 14, the feed for all groups of animals was removed, and they were fasted overnight for 12 hours (water was not prohibited). At 9:00 AM on Day 15, the animals were weighed, and their fasted blood glucose was measured by blood collection via tail tip amputation without anesthesia. The animals were then anesthetized with CO2 and euthanized. Blood was collected from the heart, and plasma was separated by centrifugation. The plasma was used to measure total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), and insulin levels (to calculate HOMA-IR). The liver was separated, homogenized, and the supernatant was collected by centrifugation to measure liver triglyceride levels.

[0448] All results are expressed as mean ± SEM. Results were analyzed using one-way ANOVA with GraphPad Prism 5 software and followed by Dunnett's post-hoc test comparing the results to the vehicle control group. Differences were considered statistically significant at the p-level < 0.05.

[0449] Table 8. Effects of compounds on body weight change and cumulative food intake in DIO mice

[0450]

[0451]

[0452] ***p<0.001, compared with the vehicle control group (One-way ANOVA, Dunnett's). Results are expressed as mean ± SEM of 6 animals.

[0453] Table 9. Effects of compounds on plasma TC, LDL-C, and TG in DIO mice

[0454]

[0455] **p<0.01, ***p<0.001, compared with the vehicle control group (One-way ANOVA, Dunnett's). Results are expressed as mean ± SEM of 6 animals.

[0456] Table 10. Effects of compounds on liver TG and HOMA-IR in DIO mice

[0457]

[0458] *p<0.05, **p<0.01, ***p<0.001, compared with the vehicle control group (One-way ANOVA, Dunnett's). Mouse HOMA-IR = [(fasting insulin (mIU / L) × fasting glucose (mmol / L)] / 22.5. Results are expressed as mean ± SEM of 6 animals.

[0459] While certain features of the invention have been set forth and described herein, many modifications, substitutions, alterations, and equivalents will occur to those skilled in the art. Therefore, it should be understood that the appended claims are intended to cover all such modifications and alterations falling within the true spirit and scope of the invention.

Claims

1. A compound selected from the group consisting of: Compound 1 (SEQ ID NO: 1): Compound 2 (SEQ ID NO: 2): Compound 3 (SEQ ID NO: 3): Compound 4 (SEQ ID NO: 4): Compound 5 (SEQ ID NO: 5): Compound 6 (SEQ ID NO: 6): Compound 7 (SEQ ID NO: 7): Compound 8 (SEQ ID NO: 8): Compound 9 (SEQ ID NO: 9): Compound 10 (SEQ ID NO: 10): Compound 11 (SEQ ID NO: 11): Compound 12 (SEQ ID NO: 12): Compound 13 (SEQ ID NO: 19): Compound 14 (SEQ ID NO: 20): Compound 15 (SEQ ID NO: 21): Compound 16 (SEQ ID NO: 22): Compound 17 (SEQ ID NO: 23): Compound 18 (SEQ ID NO: 24): Compound 19 (SEQ ID NO: 25): Compound 20 (SEQ ID NO: 26): Compound 21 (SEQ ID NO: 27): Compound 22 (SEQ ID NO: 28): Compound 23 (SEQ ID NO: 29): Compound 24 (SEQ ID NO: 30): Compound 25 (SEQ ID NO: 31): Compound 26 (SEQ ID NO: 32): Compound 27 (SEQ ID NO: 33): Compound 28 (SEQ ID NO: 34): Compound 29 (SEQ ID NO: 35): Compound 30 (SEQ ID NO: 36): Compound 31 (SEQ ID NO: 37): or a salt thereof, or a hydrate or solvate thereof.

2. A pharmaceutical composition comprising an effective amount of a compound according to claim 1, or a salt thereof, and a pharmaceutically acceptable adjuvant. H-Aib-HGTFTSDLSIYK(γGlu-CO(CH2) 14 CH3)EERAQQDFIEWLLDGGPSSGAPPPS-NH2; 3. The pharmaceutical composition according to claim 2, wherein the adjuvant is selected from the group consisting of a diluent, a carrier or an excipient. H-Aib-HGTFTSDLSIYK(γGlu-CO(CH2) 14 CH3)EERAQQDFIEWLLDGGPSSGAPPPS-OH; 4. The pharmaceutical composition according to claim 2, which is an injection or a lyophilized powder, a tablet, a pill, a lozenge, a soft capsule, a hard capsule, a granule, a powder, a solution, a suspension or a syrup; or which is a microcapsule or a microsphere. H-Aib-HGTFTSDYSIQK (yGlu-CO(CH2) 14 CH3)EEIAAQDFIEWLLDGGPSSGAPPPS-OH.

5. The pharmaceutical composition according to claim 2, which is in the form of a nanoparticle or a liposome.

6. The pharmaceutical composition according to claim 2, which is for oral administration, inhalation administration or parenteral administration selected from the group consisting of intraperitoneal, intramuscular, intra-arterial, intravenous, subcutaneous or intradermal injection administration.

7. The pharmaceutical composition according to claim 2, which is administered at a frequency of at least once a day, once a week or once a month.

8. Use of a compound according to claim 1, or a salt thereof, for the preparation of a medicament for the prevention or treatment of a metabolic abnormality syndrome selected from the group consisting of hyperglycemia, obesity or non-alcoholic fatty liver disease.

9. A method for preparing a compound according to claim 1, or a salt thereof, which is a chemical synthesis method.

10. A method for preparing a compound according to claim 1, or a salt thereof, which is a biological semi-synthesis method. ​ ​ ​

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