WO2021143810A1 - Polypeptide compound and use thereof - Google Patents

Abstract

Disclosed are a polypeptide compound and the use thereof, dual or triple agonist polypeptide drugs that activate a glucagon-like peptide-1 receptor and optionally activate a glucose-dependent insulinotropic polypeptide receptor and a glucagon receptor, and the use thereof for the treatment of metabolic syndrome.

Description

Polypeptide compound and its application Technical field

The present invention relates to the field of medicine. More specifically, the present invention relates to agonizing the glucagon-like peptide-1 (GLP-1) receptor and optionally agonizing the glucose-dependent insulinotropic polypeptide (GIP) receptor and glucagon GCG receptor double or triple agonist polypeptide drug and its application for the treatment of metabolic syndrome (such as diabetes, obesity, non-alcoholic fatty liver).

Background technique

Type 2 diabetes and obesity are a type of energy metabolism disorders, which continue to become more and more serious health problems in many countries, and cause a wide range of related dangerous diseases (such as cardiovascular and cerebrovascular diseases, kidney diseases, dyslipidemia, liver diseases, Osteoporosis). Type 2 diabetes is characterized by insulin resistance and hyperglycemia. Obesity is the main cause of insulin resistance, and obesity and insulin resistance are the two major causes of non-alcoholic fatty liver disease (NAFLD/NASH). Therefore, there is an increasing need to find safe and effective drugs to treat these metabolic disorders-related diseases.

Secretin is a kind of substance secreted from the intestine after eating stimulation under normal physiological conditions. It can rely on the glucose level to stimulate the pancreatic β-cells to secrete insulin, regulate the homeostasis of glucose levels, and protect the pancreatic β-cells. GLP-1 and GIP are two types of incretins (Glucagon-like peptide-1 and glucose-dependent insulin-releasing polypeptide plasma levels in response to nutrients. Digestion 1995; 56: 117-126.) that are currently discovered.

GLP-1 is expressed in intestinal mucosal L cells by the proglucagon gene. It has a 31-amino acid polypeptide and mainly acts on the GLP-1 receptor (GLP-1 R), which stimulates insulin secretion and inhibits glucagon It secretes and protects pancreatic β-cells. It has the physiological role of regulating blood sugar homeostasis. It can also inhibit food intake and gastric emptying through the central nervous system signaling pathway, increase satiety, and reduce body weight (Glucagon-like peptide-1 7-36 :a physiological incremental in 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 empty of glucose and plasma concentrations of glucagon-like peptide-1. Peptides. 1998; 19:1049-1053.).

Natural GLP-1 is easily degraded by dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase (NEP) that are ubiquitous in plasma, with a half-life of less than 2 min. Exendin-4 (Exendin-4) is a GLP-1 analogue extracted from the salivary glands of African poisonous lizards. It has a stronger GLP-1 receptor agonistic effect and a similar hypoglycemic effect of GLP-1. Compared with natural GLP-1, Exendin-4 has stronger resistance to DPP-4 and NEP, and its half-life and action time in vivo are also longer. Exenatide (trade name

) Is the first GLP-1 drug to be developed and marketed, and it has shown a good clinical effect on diabetes treatment, but it still has greater human immunogenicity and insufficient administration twice a day.

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

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH ₂

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

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG-OH

In order to improve the half-life of GLP-1 and the effect of diabetes treatment, a variety of long-acting GLP-1 analogues 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 Somaglutide (trade name

) And Eli Lilly's dulaglutide (trade name

).

Liraglutide uses C ₁₆ fatty acids to chemically modify the Lys side chain at position 20 of GLP-1 to increase albumin binding capacity and extend its half-life to 13-16h. The amino acid sequence of liraglutide (SEQ ID NO: 3) is as follows:

HAEGTFTSDVSSYLEGQAAK(γGlu-palmitoyl)EFIAWLVRGRG-OH

Somaglutide uses the unnatural amino acid isoaminobutyric acid (Aib) to replace Ala at position 2, which significantly improves DPP-IV resistance. At the same time, the C ₁₈ fatty diacid side chain further enhances the albumin binding force, and the half-life is significantly extended to achieve Dosing once a week. The amino acid sequence of Somaglutide (SEQ ID NO: 4) is as follows:

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

GIP is a 42 amino acid single-chain polypeptide, produced by small intestinal mucosal K cells, and mainly acts on the GIP receptor (GIP R) in pancreatic islet cells and adipocytes. GIP glucose-dependently promotes insulin secretion, enhances the quality of islet β-cells, stimulates insulin secretion, inhibits gastric acid secretion, and slows gastric motility. In addition, 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 has the physiological effects of promoting osteoblast differentiation, inhibiting osteoblast apoptosis, inhibiting bone resorption, and increasing bone mineral density, and plays a role in protecting bone (Glucose-dependent insulinotropic peptide is an integral hormone with osteotropic effects. Mol Cell Endocrinol.2001;177:35–41.;Effects of glucose-dependent insulinotropic peptide on osteoclast function.AmJ Physiol 2007;292:E543–E548.).

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

YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ

GCG is a 29-amino acid polypeptide. It is expressed and secreted by the proglucagon gene in pancreatic islet α-cells. It acts on the glucagon receptor (GCG R) mainly distributed in the liver and kidney to stimulate the decomposition of liver glycogen. , Raise blood sugar, activate lipase, promote fat decomposition, at the same time strengthen fatty acid oxidation, increase the production of ketone bodies. Research results show that GCG has a certain effect on reducing food intake, increasing adipose tissue energy consumption, and reducing body fat (The Metabolic actions of glucagon revised. Nat. Rev. Endocrinol. 2010; 6: 689-697.; Effects of glucagon) on lipolysis and ketogenesis in normal and diabetes men.J.Clin.Invest.1974;53:190–197.). The proper effect of GCG in raising blood sugar can feedback and regulate the effect of insulin and reduce the occurrence of hypoglycemic events.

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

HSQGTFTSDYSKYLDSRRAQDFVQWLMNT

Obesity is a state in which body fat, especially triglycerides, accumulates excessively due to excessive energy intake or abnormal metabolism of the body. Fat accumulation on the pancreas impairs the function of islet cells and aggravates diabetes. Accumulation on the liver can lead to non-alcoholic fatty liver disease. The advantage of the currently marketed GLP-1 analogues is that they have both cardiovascular benefits and weight management effects while lowering blood sugar. However, the clinical maximum weight reduction effects of liraglutide and somaglutide are only 3% and 5%, respectively. Left and right, and gastrointestinal side effects (mainly nausea, vomiting, diarrhea) are more common. Therefore, for more and more obese people, there is still an urgent need for therapeutic drugs with better weight control effects, wider benefits, and greater safety ranges.

According to the physiological effects of GLP-1, GIP and GCG, a number of current studies have found that integrating the effects of two or three of them through multi-target activation can achieve better diabetes and obesity control effects than single-agent therapy. GLP-1/GCG co-agonists are more effective than a single GLP-1 receptor agonist in reducing food intake, reducing body weight, improving glucose tolerance and reducing triglycerides, and the therapeutic effect on obese mice is more significant (Glucagon- like peptide 1/glucagon receptor dual agonism reverses obesity in mice.Diabetes.2009; 58: 2258-2266; A new glucagon and GLP-1 coagonist eliminates obesity in rodents. Nat Chem Biol. 2009; 5: 749-757. Animal studies at Indiana University confirmed that GLP-1/GIP and GLP-1/GIP/GCG co-agonists can significantly reduce blood sugar and body weight in high-fat diet-induced obesity (DIO) mice, and the therapeutic effects are significantly better than acetylation. GLP-1 (Acyl-GLP-1), acetylated GIP (Acyl-GIP) and liraglutide. In addition, it can also reduce plasma cholesterol, body fat and liver fat, and improve a variety of 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 monomeric peptide triagonist corrects obesity and diabetes in rodents. Nat Med. 2015; 21: 27-36.).

There is still a need for new double- or tri-agonistic polypeptide molecules for the prevention or treatment of metabolic abnormalities syndrome, with improved stability or lower mass production costs.

Summary of the invention

The present invention provides a novel polypeptide compound that can have dual or triple agonistic effects. Such polypeptides may have significant GLP-1 R agonistic activity, and may also have GIP R and/or GCG R agonistic activity. They can be used to prevent or treat metabolic syndrome, such as reducing blood sugar, weight, and fat, and have improved stability. sex.

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

R ¹ -His-Aib-X ₃ -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)

in,

R ^{1 is} selected from H, Ac or pGlu;

X _{3 is} selected from His, Gln;

X _{10 is} selected from Tyr, Leu, Lys, Cys or ψ;

X _{12 is} selected from Ile, Arg, Lys, Cys or ψ;

X _{13 is} selected from Tyr, Gln, Lys, Cys or ψ;

X _{14 is} selected from Leu, Lys, Cys or ψ;

X _{15 is} selected from Asp or Glu;

X _{16 is} selected from Glu, Lys, Cys or ψ;

X _{17 is} selected from Arg, Ile, Gln, Glu, Lys, Cys or ψ;

X _{18 is} selected from Ala or Arg;

X _{19 is} selected from Ala, Val or Gln;

X _{20 is} selected from Gln or Arg;

X _{21 is} selected from Asp or Leu;

X _{23 is} selected from Val or Ile;

X _{24 is} selected from Glu or Gln;

X _{27 is} selected from Leu or Lys;

X _{28 is} selected from Ala or Asp;

R ² is NH ₂ or OH, or a pharmaceutically acceptable salt and/or ester thereof;

Wherein, X ₁₀ , X ₁₂ , X ₁₃ , X ₁₄ , X ₁₆ , and X ₁₇ are ψ, and the ψ is Cys or Lys whose side chain is modified by the structure having the following formula II, so The formula Ⅱ mentioned is:

Y-Z(Ⅱ)

(i) When ψ is Lys, Y is selected from the group consisting of Glu (preferably γGlu), AEEAc, GABA, GSEGSEE, and any combination of two and/or more thereof, and its carboxyl end is connected to the ε-amino group of the side chain of Lys Connected, Z is -CO-(CH ₂ ) _m -R ³ , m is an integer between 6-24, R ^{3 is} selected from -CH ₃ or -COOH; Y preferably represents at most 10, or at most 5, or Up to 4, or up to 3, or up to 2, or 1 linker selected from Glu (preferably γGlu), AEEAc, GABA, GSEGSEE, and any combination of two and/or more thereof; for example, Y It can be γGlu, GSEGSEE, AEEAc-AEEAc-γGlu, γGlu-γGlu-AEEAc-AEEAc, γGlu-γGlu-AEEAc-AEEAc-γGlu, or γGlu-GABA-AEEAc-γGlu;

(ii) When ψ is Cys, Y is Y1-Y2, Y1 is selected from acetylglycyl, 3-maleimidopropionyl and any combination thereof, and Y2 is selected from Glu (preferably γGlu), AEEAc and Any combination thereof, and it is connected to the Cys side chain sulfhydryl group through acetylglycyl or 3-maleimidopropionyl, Z is -NH-(CH ₂ ) _m -R ³ , and m is between 6-24 R ^{3 is} selected from -CH ₃ or -COOH; Y1 preferably represents a linker selected from acetylglycyl, 3-maleimidopropionyl and any combination thereof; Y2 preferably represents the most 10, or at most 5, or at most 4, or at most 3, or at most 2, or 1 linker selected from Glu (preferably γGlu), AEEAc and any combination thereof; for example, Y1 can be 3- Maleimido propionyl; Y2 can be γGlu or AEEAc-AEEAc;

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.

In one aspect, there is provided a compound having the following formula Ib or a salt or solvate thereof:

R ¹ -His-Aib-X ₃ -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)

in,

R ^{1 is} selected from H, Ac or pGlu;

X _{3 is} selected from His, Gln;

X _{10 is} selected from Leu, Lys, Cys or ψ;

X _{13 is} selected from Tyr or Gln;

X _{14 is} selected from Leu, Lys, Cys or ψ;

X _{15 is} selected from Asp or Glu;

X _{17 is} selected from Arg, Gln or Glu;

X _{19 is} selected from Ala or Val;

X _{20 is} selected from Gln or Arg

X _{21 is} selected from Asp or Leu

X _{23 is} selected from Val or Ile;

X _{24 is} selected from Glu or Gln;

X _{27 is} selected from Leu or Lys

X _{28 is} selected from Ala or Asp;

R ² is NH ₂ or OH, or a pharmaceutically acceptable salt and/or ester thereof;

Wherein, _{one and only one of X 10} and X ₁₄ is ψ, and the ψ is Lys whose side chain is modified by the structure having the following formula II, and the formula II is:

Y-Z(Ⅱ)

Y is selected from Glu (preferably γGlu), AEEAc, GABA, GSEGSEE and any combination of two and/or more thereof, and its carboxyl end is connected to the ε-amino group of the side chain of Lys, Z is -CO-(CH ₂ ) _m -R ³ , m is an integer between 6-24, R ^{3 is} selected from -CH ₃ or -COOH; Y preferably represents at most 10, or at most 5, or at most 4, or at most 3, Or at most 2, or 1 linker selected from Glu (preferably γGlu), AEEAc, GABA, GSEGSEE, and any combination of two and/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

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.

The above compounds with the structure of formula Ib can be prepared by chemical synthesis of polypeptides.

In one aspect, there is provided a compound having the following formula Ic or a salt or solvate thereof:

R ¹ -His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Tyr-X ₁₄ -Glu-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)

in,

R ¹ is H;

X ₁₄ is ψ;

R ² is NH ₂ or OH, or a pharmaceutically acceptable salt and/or ester thereof;

Wherein, X ₁₄ is ψ, and the ψ is Cys whose side chain is modified by the structure having the following formula II, and the formula II is:

Y-Z (Ⅱ)

Y is Y1-Y2, Y1 is selected from acetylglycyl, 3-maleimidopropionyl and any combination thereof, Y2 is selected from Glu (preferably γGlu), AEEAc and any combination thereof, and it passes through the acetyl group Glycyl or 3-maleimidopropionyl is connected to Cys side chain sulfhydryl group, Z is -NH-(CH ₂ ) _m -R ³ , m is an integer between 6-24, R ^{3 is} selected from- CH ₃ or -COOH; Y1 preferably represents a linker selected from acetylglycyl, 3-maleimidopropionyl and any combination thereof; Y2 preferably represents at most 10, or at most 5, or Up to 4, or up to 3, or up to 2, or 1 linker selected from Glu (preferably γGlu), AEEAc and any combination thereof; for example, Y1 can be 3-maleimidopropionyl; Y2 can be γGlu or AEEAc-AEEAc;

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.

The above-mentioned compounds with the structure of Formula Ic can be prepared by chemically synthesizing polypeptides.

In one aspect, there is provided a compound having the following formula Id or a salt or solvate thereof:

R ¹ -His-Aib-X ₃ -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-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R ² (Id)(SEQ ID NO:48)

in,

R ¹ is H;

X _{3 is} selected from His, Gln;

X _{10 is} selected from Tyr or Leu;

X _{12 is} selected from Ile, Arg, or ψ;

X _{13 is} selected from Tyr, Gln, or ψ;

X _{14 is} selected from Leu, or ψ;

X _{16 is} selected from Glu, or ψ;

X _{17 is} selected from Arg, Ile, Gln, or ψ;

X _{18 is} selected from Ala or Arg;

X _{19 is} selected from Ala or Gln;

X _{23 is} selected from Val or Ile;

X _{28 is} selected from Ala or Asp;

R ² is NH ₂ or OH, or a pharmaceutically acceptable salt and/or ester thereof;

Wherein, X ₁₂ , X ₁₃ , X ₁₄ , X ₁₆ , X ₁₇ and only one is ψ, and the ψ is Lys whose side chain is modified by the structure having the following formula II, and the formula II is :

Y-Z(Ⅱ)

Y is selected from Glu (preferably γGlu), AEEAc, GABA, GSEGSEE and any combination of two and/or more thereof, and its carboxyl end is connected to the ε-amino group of the side chain of Lys, Z is -CO-(CH ₂ ) _m -R ³ , m is an integer between 6-24, R ^{3 is} selected from -CH ₃ or -COOH; Y preferably represents at most 10, or at most 5, or at most 4, or at most 3, Or at most 2, or 1 linker selected from Glu (preferably γGlu), AEEAc, GABA, GSEGSEE, and any combination of two and/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;

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.

The above-mentioned compounds with the structure of Formula Id can be prepared by chemical synthesis of polypeptides or biological semi-synthesis.

The biological semi-synthesis refers to a method similar to that disclosed in patents CN201510459093 and US9732137 for producing somaglutide (SEQ ID NO: 4).

The method features: firstly use yeast or E. coli to ferment and express part of the peptide TFTSDVSSYLEGQAAKEFIAWLVRGRG-OH in the somaglutide sequence; then use fatty acid activated ester to react with the side chain NH ₂ of K in the peptide to obtain the peptide. TFTSDVSSYLEGQAAK([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-γE-octadecanoic acid acyl)EFIAWLVRGRG-OH; finally, use the N-terminal overhang Boc-His(Boc )-Aib-Glu(O-tBu)-Gly-OSuc reacts with the α-NH ₂ acylation reaction of the above-mentioned peptide, and removes the Boc protecting group to obtain somaglutide. This method overcomes the cumbersome steps of chemical synthesis, makes full use of the advantages of biological fermentation expression, and is beneficial to the reduction of production costs and large-scale industrial production.

The realization of the above-mentioned technical methods depends on two characteristics of the polypeptide sequence: 1. Lys with no other sites except for the specific site Lys modified by fatty acids in the sequence. If there is an extra Lys site, then there is no guarantee that the fatty acid is specifically linked to the desired modified Lys site; 2. Except for the unnatural amino acid Aib at position 2, there are no other unnatural amino acids after position 5. Otherwise, biological cells such as yeast or E. coli will not be able to express peptides with unnatural amino acids.

Since the above formula Id meets the above two characteristics, it can be prepared by a similar biological semi-synthetic method.

In one aspect, there is provided a compound having the following general formula Ie, or a salt or solvate thereof:

R ¹ -His-Aib-X ₃ -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-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R ² (Ⅰe)(SEQ ID NO:49)

in,

R ¹ is H;

X _{3 is} selected from His, Gln;

X _{10 is} selected from Tyr or Leu;

X _{12 is} selected from Ile, Arg, Lys, Cys or ψ;

X _{13 is} selected from Tyr, Gln, Lys, Cys or ψ;

X _{14 is} selected from Leu, Lys, Cys or ψ;

X _{16 is} selected from Glu, Lys, Cys or ψ;

X _{17 is} selected from Arg, Ile, Gln, Lys, Cys or ψ;

X _{18 is} selected from Ala or Arg;

X _{19 is} selected from Ala or Gln;

X _{23 is} selected from Val or Ile;

X _{28 is} selected from Ala or Asp;

R ² is NH ₂ or OH, or a pharmaceutically acceptable salt and/or ester thereof;

Wherein, X ₁₂ , X ₁₃ , X ₁₄ , X ₁₆ , X ₁₇ and only one is ψ, and the ψ is Lys whose side chain is modified by the structure having the following formula II, and the formula II is :

Y-Z(Ⅱ)

Y is selected from Glu (preferably γGlu), AEEAc, GABA, GSEGSEE and any combination of two and/or more thereof, and its carboxyl end is connected to the ε-amino group of the side chain of Lys, Z is -CO-(CH ₂ ) _m -R ³ , m is an integer between 6-24, R ^{3 is} selected from -CH ₃ or -COOH; Y preferably represents at most 10, or at most 5, or at most 4, or at most 3, Or at most 2, or 1 linker selected from Glu (preferably γGlu), AEEAc, GABA, GSEGSEE, and any combination of two and/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;

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.

In one aspect, the above formula II has the following structure:

When ψ is Lys whose side chain is modified by the structure of formula II, the structure of formula II can be selected from (the term "R" in the following structure is intended to indicate the connection site of the peptide backbone to the formula II, that is Ε-amino group of Lys side chain):

γGlu-CO(CH ₂ ) ₁₄ CH ₃ :

AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ CH ₃ :

γGlu-CO(CH ₂ ) ₁₆ COOH:

AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH:

AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₈ COOH:

GABA-GABA-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH:

γGlu-GABA-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH:

γGlu-γGlu-AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH:

γGlu-γGlu-AEEAc-AEEAc-CO(CH ₂ ) ₁₆ COOH:

GSEGSEE-CO(CH ₂ ) ₁₆ COOH:

When ψ is Cys whose side chain is 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 indicate the connection site of the peptide backbone to the formula II, that is Cys side chain mercapto group):

Acetylglycyl-γGlu-NH(CH ₂ ) ₁₅ CH ₃ :

3-Maleimidopropionyl-γGlu-NH(CH ₂ ) ₁₅ CH ₃ :

3-Maleimidopropionyl-AEEAc-AEEAc-NH(CH ₂ ) ₁₅ CH ₃ :

In one aspect, the compound is selected from:

Compound 1 (SEQ ID NO: 7):

H-Aib-QGTFTSDK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )SKYLEEQAAQDFIEWLLAGGPSSGAPPPS-NH ₂

Compound 2 (SEQ ID NO: 8):

H-Aib-QGTFTSDLSKYK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EEQAAQDFIEWLLAGGPSSGAPPPS-NH ₂

Compound 3 (SEQ ID NO: 9):

N-Pyroglutamyl-H-Aib-QGTFTSDK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )SKYLDERAAQDFVQWLLDGGPSSGAPPPS-NH ₂

Compound 4 (SEQ ID NO: 10):

Ac-H-Aib-QGTFTSDK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )SKYLDERAAQDFVQWLLDGGPSSGAPPPS-NH ₂

Compound 5 (SEQ ID NO: 11):

H-Aib-QGTFTSDK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )SKYLEEEAVRLFIEWLKAGGPSSGAPPPS-NH ₂

Compound 6 (SEQ ID NO: 12):

H-Aib-HGTFTSDLSKQK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )DERAAQDFVQWLLDGGPSSGAPPPS-NH ₂

Compound 7 (SEQ ID NO: 13):

H-Aib-QGTFTSDLSKYC(3-Maleimidopropionyl-γGlu-NH(CH ₂ ) ₁₅ CH ₃ )EEQAAQDFIEWLLAGGPSSGAPPPS-NH ₂

Compound 8 (SEQ ID NO: 14):

H-Aib-QGTFTSDLSKYC(3-Maleimidopropionyl-AEEAc-AEEAc-NH(CH ₂ ) ₁₅ CH ₃ )EEQAAQDFIEWLLAGGPSSGAPPPS-NH ₂

Compound 9 (SEQ ID NO: 15):

H-Aib-QGTFTSDLSRYK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EEQAAQDFIEWLLAGGPSSGAPPPS-NH ₂

Compound 10 (SEQ ID NO: 16):

H-Aib-HGTFTSDLSIQK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EERAAQDFIEWLLDGGPSSGAPPPS-NH ₂

Compound 11 (SEQ ID NO: 17):

H-Aib-HGTFTSDLSIQK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EEQAAQDFIEWL LDGGPSSGAPPPS-NH ₂

Compound 12 (SEQ ID NO: 18):

H-Aib-QGTFTSDLSIQK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EEQAAQDFIEWLLDGGPSSGAPPPS-NH ₂

Compound 13 (SEQ ID NO: 19):

H-Aib-HGTFTSDLSIYK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

Compound 14 (SEQ ID NO: 20):

H-Aib-HGTFTSDYSIQK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EEIAAQDFIEWLLDGGPSSGAPPPS-NH ₂

Compound 15 (SEQ ID NO: 21):

H-Aib-HGTFTSDLSIQK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EEIAQQDFIEWLLDGGPSSGAPPPS-NH ₂

Compound 16 (SEQ ID NO: 22):

H-Aib-HGTFTSDLSK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )YLEERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

Compound 17 (SEQ ID NO: 23):

H-Aib-HGTFTSDLSIK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )LEERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

Compound 18 (SEQ ID NO: 24):

H-Aib-HGTFTSDLSIYLEK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )RAQQDFIEWLLDGGPSSGAPPPS-NH ₂

Compound 19 (SEQ ID NO: 25):

H-Aib-HGTFTSDLSIYLEEK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )AQQDFIEWLLDGGPSSGAPPPS-NH ₂

Compound 20 (SEQ ID NO: 26):

H-Aib-HGTFTSDLSIYK(AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ CH ₃ )EERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

Compound 21 (SEQ ID NO: 27):

H-Aib-HGTFTSDLSIQK(AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH)EERAAQDFIEWLLDGGPSSGAPPPS-NH ₂

Compound 22 (SEQ ID NO: 28):

H-Aib-HGTFTSDLSIYK(γGlu-γGlu-AEEAc-AEEAc-CO(CH ₂ ) ₁₆ COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

Compound 23 (SEQ ID NO: 29):

H-Aib-HGTFTSDYSIQK(AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH)EEIAAQDFVEWLLAGGPSSGAPPPS-NH ₂

Compound 24 (SEQ ID NO: 30):

H-Aib-HGTFTSDLSIYK(AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

Compound 25 (SEQ ID NO: 31):

H-Aib-QGTFTSDYSIQK(AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH)EERRAQDFIEWLLDGGPSSGAPPPS-NH ₂

Compound 26 (SEQ ID NO: 32):

H-Aib-HGTFTSDLSIYK(γGlu-CO(CH ₂ ) ₁₆ COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

Compound 27 (SEQ ID NO.33):

H-Aib-HGTFTSDLSIYK(GSEGSEE-CO(CH ₂ ) ₁₆ COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

Compound 28 (SEQ ID NO.34):

H-Aib-HGTFTSDLSIYK(γGlu-γGlu-AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

Compound 29 (SEQ ID NO.35):

H-Aib-HGTFTSDLSIYK(γGlu-GABA-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

Compound 30 (SEQ ID NO: 36):

H-Aib-HGTFTSDLSIYK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EERAQQDFIEWLLDGGPSSGAPPPS-OH

Compound 31 (SEQ ID NO: 37):

H-Aib-HGTFTSDYSIQK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EEIAAQDFIEWLLDGGPSSGAPPPS-OH

Compound 32 (SEQ ID NO: 38):

H-Aib-HGTFTSDLSIQK(AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH)EERAAQDFIEWLLDGGPSSGAPPPS-OH

Compound 33 (SEQ ID NO: 39):

H-Aib-HGTFTSDLSIQK(AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₈ COOH)EERAAQDFIEWLLDGGPSSGAPPPS-NH ₂

Compound 34 (SEQ ID NO: 40):

H-Aib-HGTFTSDLSIYK (AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₈ COOH) EERAQQDFIEWLLDGGPSSGAPPPS-NH ₂ .

In one aspect, a pharmaceutical composition is provided, which includes an effective amount of the compound or a salt or solvate of any one of the foregoing aspects, and pharmaceutically acceptable excipients, diluents, carriers or excipients.

In one aspect, the pharmaceutical composition is 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.

In one aspect, the pharmaceutical composition is for oral administration, inhalation administration or parenteral administration, and the parenteral administration is selected from the group consisting of intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous or intradermal injection. Medicine.

In one aspect, the pharmaceutical composition is administered at least once a day, once a week, or once a month.

In one aspect, there is provided the use of the compound or its salt or solvate according to any one of the preceding aspects in the preparation of a medicine for use as a GLP-1 receptor agonist, a GIP receptor agonist or a GCG receptor One, two or three of the agonists.

In one aspect, there is provided the use of the compound or its salt or solvate according to any one of the preceding aspects in the preparation of a medicament for the prevention or treatment of metabolic syndrome, the metabolic syndrome is selected from high Glycemia, insulin resistance, impaired 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.

In one aspect, a method for agonizing one, two or three of GLP-1 receptor, GIP receptor or GCG receptor is provided, which comprises administering to an individual in need an effective amount of the compound of the present invention or its Salt or solvate.

In one aspect, there is provided a method of preventing or treating a disease selected from the group consisting of: metabolic syndrome, the metabolic syndrome being selected from hyperglycemia, insulin resistance, glucose intolerance, type 2 diabetes, obesity or Non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH), diabetic nephropathy, diabetic retinopathy, dyslipidemia, osteoporosis; or neurodegenerative disease selected from Alzheimer’s disease Or Parkinson's syndrome, including administering an effective amount of the compound of the present invention or a salt or solvate thereof to an individual in need.

The compound of the present invention can be a double- or tri-agonistic polypeptide molecule, which can be used to prevent or treat abnormal metabolic syndrome and has improved stability.

Description of the drawings:

Figure 1. Plasmid map of pET31b-SEQ ID NO.19.

Figure 2. The electropherogram of the precursor fusion protein of KSI-DDDDK-peptide SEQ ID NO.19 (5-40).

Figure 3. Acute effect of the first dose of compound on blood glucose in DIO mice. After DIO mice were given the first dose of the compound, the non-fasting blood glucose levels at 0, 1, 2, 4, 8, 24, 48, 72 h were measured (the compound administered once a day was normally given the second and third doses at 24 and 72 h Corresponding compound). Data are expressed as mean±SEM, n=8.

Figure 4. The effect of compounds on fasting blood glucose in DIO mice. At the end of the experiment, the animals were fasted for 5 hours and then blood was collected. Compared with the Vehicle control group, the compound significantly reduced the fasting blood glucose of the animals (*p<0.05). Data are expressed as mean±SEM, n=8.

Figure 5. Effects of compounds on oral glucose tolerance in DIO mice. On the 19th day of administration, the glucose tolerance of DIO mice was measured. After the animals were given oral glucose, the blood glucose content at a specific time point within 120 minutes was measured, and the area under the blood glucose-time curve (AUC _0-120min ) was calculated. Figure 5A. Blood glucose-time curve after oral glucose; Figure 5B. Blood glucose-time curve after oral glucose (AUC _0-120min ). Compared with the Vehicle control group, the compound significantly reduced the glucose tolerance of DIO mice after oral administration of glucose (***p<0.001). Data are expressed as mean±SEM, n=8.

Detailed ways

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, this document including definitions shall prevail. The preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

The amino acid sequence of the parent peptide of the polypeptide drug of the present invention may be based on Exendin-4 (SEQ ID NO: 1), GLP-1 (SEQ ID NO: 2), GIP (SEQ ID NO: 5), GCG ( The amino acid sequence of SEQ ID NO: 6) is modified.

The polypeptide compounds of the present invention can be synthesized and modified by those skilled in the art through known technical methods. For example, the peptide sequence backbone of the polypeptide compound of the present invention can be prepared by synthetic methods, recombinant DNA biotechnology and other methods.

The peptide backbone of the compound of the present invention is chemically modified by a fatty acid side chain group at at least one site. Preferably, the compound may have a stable peptide α-helical structure, and may have an enhanced albumin binding capacity, which can achieve improved stability of the peptide compound and prolonged peptide action time.

The compound of the present invention has agonistic activity on GLP-1 receptor, and optionally also has agonistic activity on GIP receptor and/or GCG receptor. The "agonistic activity" means that the compound can stimulate specific receptor cells to produce cAMP, and the cells used can be host cells or pancreatic islets that overexpress GLP-1 receptor or GIP receptor or GCG receptor constructed by those skilled in the art. Tissue cells, fat cells, liver cells, etc. _{The receptor agonistic activity can be measured by the EC 50} value of the compound agonizing the receptor cells to produce cAMP. The EC ₅₀ value is the concentration of the drug required to reach half of the maximum activity (50% activity) of the compound in a specific assay system.

In a specific embodiment, the agonistic activity of a compound can be evaluated by evaluating the relative activity of a specific natural compound. ₅₀ percent relative activity of the particular value of EC EC ₅₀ value of natural compounds in the ratio of the test compound.

Compared with natural GLP-1 (7-37), the relative activity of the compound of the present invention on GLP-1 receptor agonism is at least 0.5%, preferably at least 5%, more preferably at least 50%, even most preferably at least 100% .

Compared with natural GIP, the relative activity of the compound of the present invention on GIP receptor agonism is at least 0.5%, preferably at least 5%, more preferably at least 50%, and even most preferably at least 100%.

Compared with natural GCG, the relative activity of the compound of the present invention on GCG receptor agonism is at least 0.5%, preferably at least 5%, more preferably at least 50%, and even most preferably at least 100%.

Peptide sequence similarity and peptide sequence identity

The structural similarity of two polypeptides can be determined by aligning the residues of two polypeptides (such as the candidate polypeptides described herein and any suitable reference polypeptides) along their sequence lengths to optimize the number of identical amino acids; In order to optimize the number of identical amino acids in the alignment, gaps in any one or two sequences are allowed, but the amino acids in each sequence must still retain its correct order. Where appropriate, the reference polypeptide may be the polypeptide described herein. The candidate polypeptide is a polypeptide that is compared with a reference polypeptide.

Software packages known in the art can be used to perform pairwise comparison analysis of amino acid sequences. In the comparison of two amino acid sequences, the structural similarity can be referred to by the percentage of "identity", or can be referred to by the percentage of "similarity". "Identity" refers to the presence of identical amino acids. "Similarity" refers to the existence of not only identical amino acids, but also conservative substitutions. Conservative substitutions of amino acids in the polypeptides of the present invention can be selected from other members of the class to which the amino acids belong. For example, it is well known in the field of protein biochemistry that an amino acid belonging to a group of amino acids with specific sizes or properties (such as charge, hydrophobicity, and hydrophilicity) can be replaced by another amino acid without changing the activity of the protein, especially if it is not directly related to Protein regions related to biological activity. For example, non-polar (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. The 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, substitution of Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr to maintain free -OH; and Gln for Asn to maintain free -NH ₂ . Similarly, biologically active analogs of polypeptides are also considered, which contain the deletion or addition of one or more adjacent or non-adjacent amino acids that do not eliminate the functional activity of the polypeptide.

Therefore, as used herein, the amino acid sequence referring to the polypeptide of the present invention and/or referring to one or more SEQ ID NOs may include polypeptides having the following amino acid sequence similarity with the 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%.

Alternatively, as used herein, the amino acid sequence referring to the polypeptide of the present invention and/or one or more SEQ ID NOs may include polypeptides having the following amino acid sequence identity with the 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%.

The polypeptide of the present invention may be further modified, for example, including a chemically or enzymatically derived polypeptide (or an analog thereof, such as a fragment thereof) at one or more constituent amino acids. The modification may include, for example, side chain modification, main chain modification, and N- and C-terminal modification, such as acetylation, hydroxylation, methylation, amidation, and attachment of carbohydrate or lipid moieties, cofactors, etc., and their The combination. The modified polypeptide of the present invention may retain the biological activity of the unmodified polypeptide, or may exhibit reduced or increased biological activity.

The effective amount of the compound of the present invention can be combined with at least one pharmaceutically acceptable auxiliary material, diluent, carrier or excipient to form a pharmaceutical composition. "Effective amount of compound" refers to the amount of compound that can produce disease alleviation or treatment, but not produce damaging effects. The effective amount can be appropriately determined by the attending physician according to the patient's disease severity, age, sex, weight, general health status, and the like. In one embodiment, the patient is a mammal. In a preferred embodiment, the mammal is selected from cows, horses, goats, sheep, dogs, chimpanzees, rabbits, mice, rats, monkeys, pigs, and humans. In a more preferred embodiment, the patient is a human.

The compound of the present invention and its pharmaceutical composition can be made into injections or freeze-dried powders, tablets, pills, lozenges, soft capsules, hard capsules, granules, powders, solutions, suspensions or syrups, Preferably, injection or lyophilized powder.

Excipients that can be used in the composition of the present invention such as lubricants, binders, fillers, preservatives, surfactants, colorants, flavoring agents, emulsifiers, suspending agents, diluents, gelling agents, disintegrants Agents, pH adjusters, solubilizers, etc. Those skilled in the art know that these adjuvants can be appropriately selected according to a suitable dosage form, and their content can be changed according to specific needs.

The compound of the present invention can also be loaded into a variety of drug carrier materials (for example, microcapsules, microspheres, nanoparticles, liposomes) or drug delivery devices for use.

In addition, the compound of the present invention can also be used in combination with at least one of the following therapeutic active agents, including anti-diabetic active agents (such as: insulin and its analogs, biguanides, sulfonylureas, thiazolidinediones, α -Glucosidase inhibitor, DPP-4 inhibitor, SGLT2 inhibitor, dual SGLT1/SGLT2 inhibitor, GLP-1 receptor agonist, amylin and its analogs), GIP receptor agonist, GCG receptor Agonist or antagonist, dual GLP-1/GIP receptor agonist, GLP-1/GCG receptor agonist, GIP/GCG receptor agonist, FGF-21 and its analogs, cholecystokinin B (CCKB) And its analogues, PYY(3-36) and its analogues, leptin and its analogues, calcitonin and its analogues, active drugs for regulating blood lipids, PPAR-α, β, δ agonists or regulators, anti- Platelet aggregation active agent, PCSK9 inhibitor, lipase inhibitor, anti-liver fibrosis or liver cirrhosis active agent, anti-inflammatory active agent.

Preferably, the compound of the present invention can promote insulin secretion and lower blood sugar. Preferably, food intake can be suppressed, gastric emptying can be delayed, energy consumption can be increased, and the effect of weight reduction can be observed in the end.

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

Preferably, the compound 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.

Preferably, the compound of the present invention is expected to promote the growth of brain neurons, eliminate neurotoxic substances, inhibit the development of inflammation, and play a neuroprotective effect.

Preferably, the compound or composition of the present invention can be used to prevent and/or treat metabolic disorders and related complications. It is preferably used for the treatment of diabetes, obesity and non-alcoholic fatty liver disease.

Preferably, the compound or composition of the present invention can be used to treat dyslipidemia and related diseases, neurodegenerative diseases (e.g., Parkinson's disease, Alzheimer's disease).

Preferably, the compound or composition of the present invention can be used to treat endocrine diseases, metabolic disorders, kidney diseases, weight loss and other causes-related skeletal diseases, such as osteoporosis, osteoarthritis.

Preferably, the compound or composition of the present invention can be administered by various routes, for example, it can be used for oral administration, inhalation administration or parenteral administration, such as intraperitoneal, intramuscular, and intraarterial administration. , Intravenous, subcutaneous or intradermal injection.

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

The compound of the present invention exhibits better solubility and stability, which is better than natural peptide molecules and liraglutide.

The compound of the present invention has a significant agonistic effect on two or three of GLP-1, GIP, and GCG receptors.

The specific meanings of the abbreviations used in the present invention are as follows:

Aib: Diaminoisobutyric acid

GABA: γ-aminobutyric acid

AEEAc: [2-(2-Amino-ethoxy)-ethoxy]-acetyl

Ac: Acetyl

pGlu: Pyroglutamyl

cAMP: Cyclic Adenosine Phosphate

PEG: polyethylene glycol

Fmoc: Fluorenylmethyloxycarbonyl

Boc: tert-Butoxycarbonyl

DMF: Dimethylformamide

DIC: N,N-Diisopropylcarbodiimide

Boc: tert-Butoxycarbonyl

Trt: Trityl

ivdde: 1-(4,4-Dimethyl-2,6-dioxocyclohexylene)-3-methyl-butyl

t-Bu: tert-butyl

OtBu: tert-butyl ester

TFA: Trifluoroacetic acid

HPLC/MS: High Performance Liquid Chromatography/Mass Spectrometry

HPLC-UV: High Performance Liquid Chromatography-UV

IPTG: Isopropyl-β-D-thiogalactoside

Tris: Tris

DCM: Dichloromethane

THF: Tetrahydrofuran

DIPEA: N,N-Diisopropylethylamine

NMP: N-methylpyrrolidone

PAM: Peptidylglycine α-amidating monooxygenase

MES: Fatty Acid Methyl Ester Sulfonate

HEK-293: Human embryonic kidney cells

GLP-1 R: Glucagon-like peptide-1 receptor

GIPR: Glucose-dependent insulin releasing peptide receptor

GCG R glucagon receptor

PBS: Phosphate buffered solution

FBS: Fetal Bovine Serum

DMEM: Dulbecco's Modified Eagle Medium

HBSS: Hank’s Balanced Salt Solution

HEPES: 4-hydroxyethylpiperazine ethanesulfonic acid

BSA: Bovine Serum Albumin

EC ₅₀ : Half-effect concentration

IBMX: 3-isobutyl-1-methylxanthine.

S.C.: subcutaneous injection

QD: Once a day

Q3D: once every three days

OGTT: Oral glucose tolerance test

TC: total cholesterol

LDL-C: Low-density lipoprotein cholesterol

TG: Triglyceride

HOMA: Insulin Resistance Index

Specific embodiment:

Example 1: Synthesis of peptide compounds:

The intermediates and compounds of the present invention can be synthesized and prepared by various methods known in the art. The following specific examples illustrate the preparation of the compounds of the present invention by chemical synthesis methods. Each specific synthesis step described can be combined with different materials and methods to synthesize multiple corresponding compounds of the present invention or their salts. The reagents and raw materials used are easily available to those of ordinary skill in the art. In particular, the following examples are only used to illustrate the present invention, and should not limit the scope of the present invention in any way.

Material:

The materials and reagents used in the present invention are all purchased from commercial products, and the protective 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-Phe-OH, Fmoc-Arg(pbf )-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(ivdde)-OH , Fmoc-Thr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Aib-OH, Boc-His(Boc)-OH.

In the following, the compound of SEQ ID NO: 12 is taken as an example to illustrate the synthetic preparation method of the compound of the present invention.

method:

(1) Pretreatment of Rink amino resin: Weigh 1g of dry Rink amino resin, soak and swell with DMF for 30 minutes, and remove the solvent.

(2) Removal of protective group Fmoc: Add 20% piperidine/DMF solution to the treated Rink amino resin, and stir for reaction for 20 minutes. During the reaction, the ninhydrin color method was used to monitor the degree of reaction. If the color of the resin changes, it indicates that the Fmoc has been removed successfully. After the completion of the reaction, the solvent was removed by filtration, DMF was added to the reaction system and the resin was washed with stirring for 1 min, and the washing was repeated 3 times.

(3) Coupling reaction (peptide bond formation): Add the configured corresponding Fmoc-protected amino acid solution into the reactor, then add the DIC/DMF solution, and stir for 1 hour. During the reaction, the ninhydrin color method was used to monitor the progress of the reaction. If there is no change in the color of the resin, the coupling was successful. After the completion of the reaction, the solvent was removed by filtration, DMF was added to the reaction system to wash the resin for 1 min, and the washing was repeated 3 times. Repeat the above operation and add the corresponding amino acid solution in sequence until the peptide chain is synthesized. The last amino acid was coupled with Boc-His(Trt)-OH. The Lys at the side chain modification site is replaced with Fmoc-Lys(ivdde)-OH. SEQ ID NO: The sequence of synthesis of the main peptide sequence of 12 compound and amino acid coupling is 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.

(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 the removal is successful, filter to remove the solvent, add DMF to the system, stir and wash for 1 min, filter to remove the solvent, Repeat the washing 4 times.

(5) Lys side chain modification: add the prepared Fmoc-Glu-OtBu solution to the above-mentioned processed resin, add DIC/DMF solution, and stir for 1 hour. After the reaction is completed, filter and wash, add 20% piperidine/DMF solution, stir and react for 20 minutes to remove the Fmoc group. Then add the prepared palmitic acid solution to the resin, add the DIC/DMF solution, and stir and react for 1 hour. After the reaction is completed, the resin is washed 4 times, and the resin is drained.

(6) Post-treatment of peptide resin: Add cutting reagent K to cut the resin. After filtering the filtrate, the filtrate was precipitated and centrifuged to obtain a white solid as the crude product of the target compound.

Crude peptide compound purification:

The obtained crude peptide was purified by a reverse-phase C18 preparative chromatographic column (Shimadzu, Inertsil ODS 20x 250mm 5um), and the sample was purified to a purity greater than 95%. With 95% buffer A (0.065% TFA/H ₂ O) and 5% buffer B (0.05% TFA/acetonitrile) as the starting eluent, gradually increase the proportion of buffer B at a rate of 2%/min to 65%, run continuously for 30 minutes for elution, and collect target peptide components. The purified peptide compound was analyzed and confirmed by analytical HPLC/MS method.

Based on the above synthesis method, the present invention synthesized and characterized the following peptide compound SEQ ID NO: 7-40 (see Table 1).

Table 1. List of synthesized peptide compounds and molecular weight

Example 2 Biological semi-synthesis of peptide compounds:

The following takes the biological semisynthesis of the peptide compound SEQ ID NO. 19 as an example to illustrate the method steps of the biological semisynthesis of the compound of the present invention.

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

Refer to Example 3 of patent CN201711154044 to construct an engineered E. coli strain with a fusion gene. The fusion gene has a gene sequence shaped like an ABC structure, where A is the chaperone protein coding gene, B is the linker peptide coding gene, and C is the tri-agonist peptide SEQ ID NO.19 (5-40) fragment encoding gene. It is subjected to high-density fermentation, induced, and the inclusion body or fusion protein containing the fusion protein is extracted.

The recombinant E. coli strain is preferably obtained by the following steps: cloning the fusion gene in the shape of A-B-C structure into a prokaryotic expression vector, and then transferring the obtained recombinant expression vector into an engineered E. coli strain to obtain a recombinant E. coli strain.

The prokaryotic expression vector is pET31b(+).

The engineered Escherichia coli bacteria is Escherichia coli BL21 (DE3).

The induction is induced by IPTG.

The fusion protein has the following structure, that is, from the N-terminus to the C-terminus, three fragments of the KSI chaperone protein, the connecting peptide and the peptide SEQ ID NO.19 (5-40) precursor are connected.

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

SEQ ID NO.41:

The connecting peptide in the fusion protein is DDDDK.

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

SEQ ID NO.42:

TFTSDLSIYKEERAQQDFIEWLLDGGPSSGAPPPSG-OH

The construction method of the fusion gene and engineered bacteria with the A-B-C structure can refer to the experimental guide in this field (J. Sambrook et al., "Molecular Cloning Experiment Guide" Second Edition, Science Press, 1995).

Design DDDDK-peptide SEQ ID NO.19(5-40) precursor fusion gene fragment, convert these amino acid sequences into nucleotide sequences according to the codon table, and select according to the preference of E. coli codon usage during the conversion process Use more frequent codons, adjust their GC content, remove cis-acting elements and repetitive sequences that affect gene transcription, and optimize them. At the same time, introduce the double stop codon TAATGA at the 3'end of the gene sequence. In order to facilitate gene manipulation, DDDDK-peptide SEQ ID NO.19 (5-40) precursor fusion gene sequence is introduced into the 5'end of the AlwNI restriction site sequence CAGATGCTG, so two amino acids of ML are introduced before the N-terminal extension peptide, in the DDDDK-peptide SEQ ID The 3'end of NO.19(5-40) precursor fusion gene introduces the XhoI restriction site CTCGAG, and the optimized DDDDK-peptide SEQ ID NO. 19(5-40) precursor fusion gene sequence is as SEQ ID NO: 43 shown. The gene sequence was synthesized by a gene synthesis service company, and TA cloned into pUC57 vector.

SEQ ID NO.43:

Use TaKaRa's restriction enzymes AlwNI and XhoI to double digest the plasmid pET-31b(+) (purchased from Invitrogen), and also use AlwNI and XhoI to double digest the recombinant vector pUC57-SEQ ID NO.43 . Connect the digested DNA fragment to pET-31b(+) that has been double digested with the same enzymes. After being verified by sequencing, it is named pET31b-SEQ ID NO.19, as shown in Figure 1.

Competent cells of Escherichia coli BL21 (DE3) (both purchased from Life Technologies) were prepared according to the calcium chloride method provided in the third edition of the "Molecular Cloning Experiment Guide" published by Cold Spring Harbor Laboratory in the United States. 1 μL of the recombinant expression vector pET31b-SEQ ID NO.19 was transformed into E. coli BL21(DE3) competent cells, and the transformation method was also carried out in accordance with the calcium chloride method in the third edition of the "Molecular Cloning Experiment Guide". Spread the transformation solution on LB solid medium supplemented with ampicillin (final concentration of 100μg/ml), and invert the culture at 37°C until a single colony appeared, and the strain library was obtained, named BL21(DE3)/pET31b- SEQ ID NO.19. Use a toothpick to pick a single colony BL21(DE3)/pET31b-SEQ ID NO.19, inoculate it into 50ml LB liquid medium, culture with shaking at 37℃ and 250rpm, when the OD600 of the bacterial solution is 0.5～1.0, take 3～6ml bacterial solution Inoculate to 100ml LB liquid medium, culture with shaking at 37℃, 250rpm until OD ₆₀₀ =0.6～0.8, add IPTG (isopropyl-β-D-thiogalactopyranoside, final concentration 1mmol/L) to start induction , And take 1ml of the induced 2h bacterial solution, centrifuge at 12000rpm for 1 minute, remove the supernatant, and store the bacterial cells at -20°C for later use.

Take out the cells frozen at -20°C, add 5ml 8M urea solution to resuspend the cells, sonicate them in an ice-water mixture for 10 minutes (ultrasound for 3 seconds, stop for 5 seconds, and cycle like this). Take 15μl of crushing solution, add 15μl of loading buffer, mix well and take 10μl for SDS-PAGE (wherein the concentrated gel contains 5% by volume of acrylamide-methylenebisacrylamide (29:1), the separation gel contains volume 15% acrylamide-methylenebisacrylamide (29:1)). The electrophoresis conditions were: the set current of the concentrated gel was 11mA, and the set current of the separation gel was 22mA. After the electrophoresis is over, take out the gel and stain it with Coomassie Brilliant Blue Staining Solution (each liter contains 0.6g Coomassie Brilliant Blue R-250, 450ml ethanol, 100ml glacial acetic acid, and the remainder is purified water) overnight, and use decolorizing solution (each liter contains (250ml ethanol, 80ml glacial acetic acid, the remainder is purified water) decolorize until the background is transparent. The gel was photographed on a transparent background to extract an image (as shown in Figure 2), which was consistent with the theoretical molecular weight of the KSI-DDDDK-peptide SEQ ID NO.19 (5-40) precursor fusion protein of 18.18KDa.

(2) Modified digestion of KSI-DDDDK-peptide SEQ ID NO.19 (5-40) precursor fusion protein

First, referring to Embodiments 3 and 4 of patent CN201711154044, Na-hexadecanoyl-Glu(ONSu)-OBut is synthesized.

Referring again to Example 6 of patent CN201711154044, the constructed engineering bacteria are subjected to high-density fermentation, and the bacteria are broken and washed to obtain inclusion bodies. The 10L fermentation broth finally obtained 0.62kg of inclusion bodies, and the Folin-phenol method showed that the amount of protein per 1g of inclusion bodies was 0.20g. Dissolve 200g of the above fusion protein inclusion body in 1500ml 6mol/L guanidine hydrochloride solution, add 80ml DMSO and 15ml N,N-diisopropylethylamine, mix well, pH 10.8; then add 50mg/ml Nα-ten 80ml of hexaacyl-Glu(ONSu)-OH in DMSO solution, stirred for 3 hours; then add 1700ml 20mmol/L tris solution, and add 900IU lysyl specific endonuclease, dilute hydrochloric acid or Sodium hydroxide solution was used to adjust the pH to 9.0, and after 8 hours of reaction, the pH was adjusted to 7.8.

Load the liquid onto a 500ml Uni NM200 (particle size 200μm, pore diameter 300 angstrom) chromatography column equilibrated with 1500ml equilibrium solvent containing 20mmol/L Tris, 5% isopropanol, pH 7.8, and then equilibrate with 1500ml Solvent equilibration of the chromatography column, followed by elution with a gradient elution solvent containing 20 mmol/LTris, 5% to 40% isopropanol, pH 7.8, and collect the components containing peptide SEQ ID NO. 19 (5-40).

In order to further improve the purity, the collected components were diluted with one-fold volume of purified water, and the sample was loaded to 500ml UniPS40 (particle size 40μm, 500ml UniPS40 (particle size 40μm, 1500ml) containing 20mmol/LTris, 5% isopropanol, and pH 7.0 balance solvent. 500 angstroms (pore size) in the chromatography column, and then equilibrate the chromatography column with 1500 ml of equilibrium solvent. Elution was performed with a gradient elution solvent containing 20 mmol/LTris, 5% to 30% isopropanol pH 7.0, and the fractions containing peptide SEQ ID NO. 19 (5-40) were collected. And lyophilize it into powder.

The sequence of the peptide SEQ ID NO. 19 (5-40) is shown in SEQ ID NO. 44.

SEQ ID NO.44:

TFTSDLSIYK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EERAQQDFIEWLLDGGPSS GAPPPSG-OH

HPLC/MS: m/z=1422.6[M+3H] ³⁺

Calculated molecular weight = 4264.7

(3) Preparation of N-terminal overhang Boc-His(Boc)-Aib-His(Trt)-Gly-Osu

Refer to Example 4 of patent CN201510459093, and respectively couple Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Aib-OH, Boc-His(Boc) on 2-chlorotrityl chloride resin in sequence. -OH, finally add trifluoroethanol-DCM (1:4), stir the resin. The filtrate was collected, the solvent was removed in vacuo, cold ether was added, the precipitate was filtered, washed with ether and dried in vacuo to obtain Boc-His(Boc)-Aib-His(Trt)-Gly-OH.

Then refer to Example 5 of patent CN201510459093, to the anhydrous THF solution of Boc-His(Boc)-Aib-His(Trt)-Gly-OH, add a certain amount of DIPEA and hexafluorophosphate O-(N-succinyl Imino)-N,N,N,N-tetramethyluronium. The reaction solution is added with dichloromethane, washed with water, and the organic phase is dried over magnesium sulfate and dried in vacuum to obtain Boc-His(Boc)-Aib-His(Trt)-Gly-Osu.

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

Referring to Example 8 of patent CN201510459093, the peptide SEQ ID NO.19(5-40) (0.24mmol, 1.0g) was dissolved in 100ml of NMP, and then 300ul DIPEA and 0.4mmol Boc-His(Boc)-Aib-His were added (Trt)-Gly-Osu, stir the reaction overnight. 1000ml of ice-cold ether was added, the precipitate was separated by centrifugation, washed with 500ml of ether, and then dried in vacuo.

The above-mentioned crude dry powder was dissolved in TFA-triisopropylsilane-water (95:2.5:2.5, 200ml) and reacted with stirring for 2 hours, and then the solution was concentrated in vacuo to about 20ml. Then add 500ml of ice-cold ether, stand at 2-8°C for 6-8h to form a precipitate, and centrifuge to precipitate. The precipitate was washed with cold ether and dried under vacuum. The collected dry powder is the crude powder of the peptide SEQ ID NO. 19 (1-40). Then dissolve the dry powder in 500ml pH 7.0 phosphate buffer containing 5% acetonitrile, purify it with a preparative C8 silica gel packed column, inject the peptide sample solution into the processed packed column, and then perform gradient washing according to the following purification method Take off.

The purification method is as follows:

Flow rate: 10.0ml/min; wavelength 214nm

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

Phase B, pure acetonitrile

Gradient: 0～10min 5%～10% Phase B

10～100min 10%～70% Phase B

Perform HPLC/MS detection on the collected liquid containing peptide sample components. The HPLC method is as follows:

Detection wavelength 214nm, flow rate 1.0ml/min;

Mobile phase A, water containing 0.065% TFA; mobile phase B, acetonitrile containing 0.05% TFA;

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

The sequence of the peptide SEQ ID NO. 19 (1-40) is shown below.

SEQ ID NO.19 (1-40):

H-Aib-HGTFTSDLSIYK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EERAQQDFIEWLLDGGPSSGAPPPSG-OH

HPLC/MS: m/z=1561.6[M+3H] ³⁺

Calculated molecular weight = 4681.2

(5) Amidation preparation of SEQ ID NO.19

Peptidylglycine α-amidating monooxygenase (PAM) (purchased from Wuhan Yunclone Technology Co., Ltd.) was used to catalytically cleave the C-terminal PPPSG-OH structure of the peptide SEQ ID NO.19 (1-40) sequence. The C-terminal structure is the amidation product _{of PPPS-NH 2.}

Add 200mg (0.04mmol) peptide SEQ ID NO.19 (1-40) to 80mL containing 100mM MES/KOH (pH 6.0), 30mM KI, 30mM KCl, 1μM copper sulfate, 100ug/mL catalase, 1% (v/v) ethanol, 0.001% (v/v) TritonX-100, 10mM ascorbate solution system, and then add 5ug/mLPAM, incubate in 37℃ water bath for 30min, after the reaction is completed, add 6 to the reaction system %(V/v)TFA terminates the reaction. After the reaction is completed, the reaction solution is diluted with 5% acetonitrile-containing pH 7.0 phosphate buffer by one volume. With reference to the HPLC purification method prepared in SEQ ID NO. 19 (1-40) in step (4), the obtained sample is purified to a purity greater than 95%. The purified samples were characterized and confirmed by analytical HPLC/MS methods, and the peptide compound with the sequence structure SEQ ID NO.19 was obtained.

SEQ ID NO.19:

H-Aib-HGTFTSDLSIYK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

HPLC/MS: m/z=1542.3[M+3H] ³⁺

Calculated molecular weight = 4623.2

SEQ ID NO. 15-40 peptide compounds can all be semi-synthesized by the above method, wherein the peptide sequence molecules with C-terminal amidation can be prepared by amidation after the semi-synthesis.

Example 3 Compound stability test:

The test compound was dissolved in a newly prepared PBS solution at 1 mg/ml, adjusted to pH 7.4, and filtered with a 0.22 μm sterile filter. Aspirate the compound solution and analyze the peak area of 10 μL injection by HPLC-UV. The result of this analysis is the initial point (T ₀ ) of the stability test of the compound.

The compound sample solution for stability test was placed in a 25° C. thermostat, sealed and protected from light for 7 days. After the process, the sample solution was centrifuged at 4500 rpm for 10 min, the supernatant solution was gently aspirated, and the peak area of the 10 μL injection was analyzed by HPLC-UV. This analysis result was the end point (T ₇ ) of the stability test of the compound.

By comparing _{the target peak area of the compound measured at T 0} and T ₇ and the peak area of related impurities, the remaining peptide amount of the measured peptide is calculated. Calculated as follows:

Remaining peptide (%)=(T ₇ main peak area/T ₀ main peak area)×100

The stability of the peptide compound was evaluated by comparing the remaining amount of peptide of the peptide compound, and the measurement results are shown in Table 2.

Table 2. Results of stability analysis of peptide compounds

SEQ ID NO:	Remaining peptide (%)
7	83.76
12	100.53
13	81.80
14	54.99
15	98.62

16	93.17
17	89.87
18	96.93
19	89.08
20	99.64
twenty one	98.40
27	98.12

Example 4 Determination of the activity of peptide compounds on GLP-1/GIP/GCG receptors:

By measuring the cAMP signal response of HEK-293 cells stably overexpressing human GLP-1, GIP, and GCG receptors, the agonistic activity of the peptide compounds on the corresponding receptors was determined. The intracellular cAMP content was determined using Cisbio Corp.'s kit based on HTRF (homogeneous time-resolved fluorescence) technology.

Culture HEK-293 cells stably overexpressing human GLP-1, GIP, and GCG receptors in DMEM complete medium containing 10% FBS and 2mM L-glutamine. When the cells grow to 80-90% density, Digest with 0.025% trypsin, terminate the digestion with complete medium and gently blow the cell mass into individual cells. Centrifuge the cell liquid at 1000 rpm for 5 min at room temperature, discard the supernatant, and use 1×HBSS (20mM HEPES, 0.1% BSA, 250μM IBMX). ) Resuspend the cells at a cell density of 1.0×10 ⁵ /mL.

Add 10μL of cell suspension to each well of a 384-well plate. The compound to be tested was dissolved in 1×PBS buffer, and diluted 4 times in steps from 100000-0.02nM, and a total of 12 concentration point compound solutions were prepared. Use an automatic dispenser to add 100 nL of the prepared compound solution to the corresponding cell suspension of the 384-well plate, rotate and shake at 1000 rpm for 1 min to make the mixture uniform, and then incubate at room temperature for 60 min. After the drug incubation is completed, add 10 μL of the detection reagent in the kit to each well, and then incubate at room temperature for 60 minutes. The plate was placed in the EnVision multifunctional microplate reader (PerkinElmer) to measure the fluorescence reading at 665/615nm. Response curve and _IC50 values _are calculated EC - making compound concentration using GraphPad Prism5 mapping software.

Use natural wild-type human GLP-1, GIP, GCG as a positive control for the receptor agonistic effect of the test compound. For GLP-1 receptor cells, calculate the _{ratio of the EC 50} value of the _{test compound to the EC 50} value of human GLP-1 The percentage is used as the relative activity (%) to evaluate the GLP-1 receptor agonistic activity of the test compound.

For GIP receptor cells, the percentage value ₅₀ and the human GIP EC ₅₀ values as a ratio of the relative activity (%) Evaluation test compound GIP receptor agonistic activity of the test compound is calculated by EC.

GCG for recipient cells, the test compound is calculated by EC as relative activity (%) to assess the percentage of ₅₀ and EC ₅₀ values of the ratio of the value of the GCG GCG human receptor agonistic activity of the test compound.

Table 3. Average EC ₅₀ value and relative activity of peptide compounds

Rel.A: Relative activity; NT: not test; n≥4: Each group has at least 4 independent test data.

Example 5: Rat pharmacokinetics (PK) study of peptide compounds

SD rats received the compound 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), Marglutide (50nmol/kg) was administered by subcutaneous injection. After administration, animals in SEQ ID NO: 19 and liraglutide group were administered at 0.25, 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 56h Blood was collected through the jugular vein at the time point, SEQ ID NO: 39, SEQ ID NO: 40, Semaglutide group animals at 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96h time points The blood was collected through the jugular vein, the blood was processed to obtain the plasma sample, and the sample was analyzed by LC-MS/MS. The blood drug concentration-time curve was analyzed using Phoenix WinNonlin version 6.3 software (non-compartmental model), and the PK parameters and half-life were calculated.

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

Table 4: Rat pharmacokinetic (PK) parameters of test compounds

Example 6: Study on the efficacy of the compound of SEQ ID NO: 36, SEQ ID NO. 37, SEQ ID NO: 38 on diet-induced obesity (DIO) mice

Mice with high-fat diet induced obesity (DIO) have obvious metabolic syndrome characteristics similar to humans, such as obesity, elevated blood sugar, insulin resistance, and dyslipidemia. In C57 BL/6J DIO mice, the effects of the compound of the present invention on the body weight, food intake, blood glucose and blood lipids of DIO mice were studied.

5-week-old male C57 BL/6J mice (purchased from Shanghai Slack Laboratory Animal Company) were raised in a pathogen-free clean environment (controlled temperature 20-24℃, relative humidity 30-70%), 12 hours of light/12 Circulate in the dark for hours, feed with normal feed, 4 animals per cage, and acclimatize for 2 weeks. Obesity of mice was induced by feeding a high-fat diet (60kcal% calories from fat). After 16 weeks of feeding on a high-fat diet, the DIO mice weighed 45-55g and their blood glucose ranged from 8-12 mM. The DIO mice were measured according to body weight and fasting blood glucose. Randomization was performed (n=8), so that the animals in each group had a similar average body weight and blood glucose. After grouping, one animal in each cage was reared for one week, during which time each animal was given a vehicle (1×PBS, 5ml/kg) by subcutaneous injection (S.C.) for 3 days to make the animals pre-adapted to the operation process.

After the animal pre-adaptation is finished, the animal is given a vehicle control, a certain dose of semaglutide or the compound of the present invention by subcutaneous injection according to the experimental group. The compound is dissolved in 1×PBS, and the dosage is 5ml/kg. The administration operation is at 9 in the morning: Starting at 00, Vehicle, SEQ ID NO: 36 and SEQ ID NO: 37 are administered once a day (QD), and Semaglutide and SEQ ID NO: 38 are administered once every three days (Q3D) for 22 days. When the first dose of the compound was administered for the first time, blood was collected by tail-tipping without anesthesia, and t=0h before administration and t=1, 2, 4, 8, 24, 48, 72h after administration were measured with blood glucose meter. Changes in blood glucose (without restricting food and drinking), to evaluate the compound's acute hypoglycemic effect on DIO mice. During the entire experimental study, the weight and food intake of the animals were measured before administration every day. By comparing with the initial body weight and food intake of the same animal before administration, the percentage of animal weight change (%) and the cumulative food intake were calculated to evaluate the compound's effect on body weight and food intake. The effect of changes in food intake.

On the 19th day of administration (Day 19), the effect of the compound on the glucose tolerance of DIO mice was determined. After the administration was completed in the morning, the animals were fasted for 5 hours (without drinking water), the weight of the mice and the blood glucose after fasting were measured as the basic blood glucose for the glucose tolerance test (t=0), and then DIO was given by oral gavage. Mouse glucose solution (2g/kg, 5ml/kg), blood sampling without anesthesia, tail-tip stubbing, blood glucose level measurement at t=15, 30, 60, 90, 120min after glucose solution is given, and the animal will resume eating after the last blood sampling is completed . Take mmol/L as the unit of blood sugar.

When the end of the experiment was reached (Day 22), the last dosing operation was performed in the morning. After the dosing was completed, fasting was performed for 5 hours, and then the fasting blood glucose of the animals was measured by blood sampling from the tail tip without anesthesia. After blood collection, the _{animals were anesthetized with CO 2} and sacrificed. The blood was collected from the heart and the plasma was separated by centrifugation. The plasma was used to determine plasma total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), Insulin (calculated HOMA-IR) content. The liver is separated for homogenization, and the supernatant of the homogenate is centrifuged to determine the triglyceride content of the liver.

All result values are expressed in Mean±SEM, and the results are analyzed using GraphPad Prism 5 software's One-way ANOVA and Dunnett's post-hoc test compared with the Vehicle control group. The difference is considered to be statistically significant at the p<0.05 level.

Table 5. Effects of compounds on body weight changes and cumulative food intake of DIO mice

***p<0.001, compared with Vehicle control group (One-way ANOVA, Dunnett’s). The results are expressed as the mean ± SEM of 8 animals.

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

**p<0.01, ***p<0.001, compared with Vehicle control group (One-way ANOVA, Dunnett’s). The results are expressed as the mean ± SEM of 8 animals.

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

*p<0.05, **p<0.01, ***p<0.001, compared with Vehicle control group (One-way ANOVA, Dunnett’s). Mouse HOMA-IR=[(fasting insulin(mIU/L)×fastingglucose(mmol/l)]/22.5. The result is expressed as the mean±SEM of 8 animals.

Example 7: Pharmacodynamic study of the compounds of SEQ ID NO: 19, SEQ ID NO: 39, SEQ ID NO: 40 on diet-induced obesity (DIO) mice

According to the experimental group (n=6), the animal vehicle control Vehicle (1×PBS) and a certain dose of the compound of the present invention were given by subcutaneous injection, SEQ ID NO: 19, SEQ ID NO: 39, SEQ ID NO: 40, and the compound was dissolved in 1×PBS , The dosage is 5ml/kg, the dosing operation starts at 9:00 in the morning, the compound SEQ ID NO: 19 is administered once a day (QD), the compound SEQ ID NO: 39, SEQ ID NO: 40 every three days Dosing once (Q3D) for 14 days. During the entire experimental study, the weight and food intake of the animals were measured before administration every day. By comparing with the initial body weight and food intake of the same animal before administration, the percentage of animal weight change (%) and the cumulative food intake were calculated to evaluate the compound's effect on body weight and food intake. The effect of changes in food intake.

At 21:00 in the evening on Day14, the animal feed of each group was withdrawn, and the animals were fasted overnight for 12h (cannot help water). At the end of the experiment (Day 15), the animals were weighed at 9:00 in the morning, and the animals were measured by blood sampling by tail-tip pruning without anesthesia. Fasting blood sugar. Then the _{animals were anesthetized with CO 2} and sacrificed. Blood was collected from the heart and the plasma was centrifuged. The plasma was used to determine plasma total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), and insulin content ( Calculate HOMA-IR). The liver was separated for homogenization, and the supernatant of the homogenate was centrifuged to determine the content of liver triglycerides.

All result values are expressed in Mean±SEM, and the results are analyzed using GraphPad Prism 5 software's One-way ANOVA and Dunnett's post-hoc test compared with the Vehicle control group. The difference is considered to be statistically significant at the p<0.05 level.

Table 8. Effects of compounds on body weight changes and cumulative food intake of DIO mice

***p<0.001, compared with Vehicle control group (One-way ANOVA, Dunnett’s). The results are expressed as the mean ± SEM of 6 animals.

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

**p<0.01, ***p<0.001, compared with Vehicle control group (One-way ANOVA, Dunnett’s). The results are expressed as the mean ± SEM of 6 animals.

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

*p<0.05, **p<0.01, ***p<0.001, compared with Vehicle control group (One-way ANOVA, Dunnett’s). Mouse HOMA-IR=[(fasting insulin(mIU/L)×fastingglucose(mmol/l)]/22.5. The result is expressed as the mean±SEM of 6 animals.

Certain features of the present invention have been illustrated and described herein, but those skilled in the art will think of many modifications, substitutions, alterations and equivalents. Therefore, it should be understood that the appended claims are intended to cover all such modifications and changes that fall within the true spirit of the present invention.

Claims (17)

1. The compound having the following general formula I or its salt or solvate:

   R ¹ -His-Aib-X ₃ -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)

   in,

   R ^{1 is} selected from H, Ac or pGlu;

   X _{3 is} selected from His, Gln;

   X _{10 is} selected from Tyr, Leu, Lys, Cys or ψ;

   X _{12 is} selected from Ile, Arg, Lys, Cys or ψ;

   X _{13 is} selected from Tyr, Gln, Lys, Cys or ψ;

   X _{14 is} selected from Leu, Lys, Cys or ψ;

   X _{15 is} selected from Asp, Lys, Cys or Glu;

   X _{16 is} selected from Glu, Lys, Cys or ψ;

   X _{17 is} selected from Arg, Ile, Gln, Glu, Lys, Cys or ψ;

   X _{18 is} selected from Ala or Arg;

   X _{19 is} selected from Ala, Val or Gln;

   X _{20 is} selected from Gln or Arg;

   X _{21 is} selected from Asp or Leu;

   X _{23 is} selected from Val or Ile;

   X _{24 is} selected from Glu or Gln;

   X _{27 is} selected from Leu or Lys;

   X _{28 is} selected from Ala or Asp;

   R ² is NH ₂ or OH, or a pharmaceutically acceptable salt and/or ester thereof;

   Wherein, X ₁₀ , X ₁₂ , X ₁₃ , X ₁₄ , X ₁₆ , and X ₁₇ are ψ, and the ψ is Cys or Lys whose side chain is modified by the structure having the following formula II, so The formula Ⅱ mentioned is:

   Y-Z (Ⅱ)

   (i) When ψ is Lys, Y is selected from Glu, AEEAc, GABA, GSEGSEE and any combination of two and/or more thereof, and its carboxyl terminal is connected to the ε-amino group of the side chain of Lys, and Z is -CO-(CH ₂ ) _m -R ³ , m is an integer between 6-24, and R ^{3 is} selected from -CH ₃ or -COOH;

   (ii) When ψ is Cys, Y is Y1-Y2, Y1 is selected from acetylglycyl, 3-maleimidopropionyl and any combination thereof, Y2 is selected from Glu, AEEAc and any combination thereof, And it is connected to Cys side chain sulfhydryl through acetylglycyl or 3-maleimidopropionyl, Z is -NH-(CH ₂ ) _m -R ³ , and m is an integer between 6-24, R ^{3 is} selected from -CH ₃ or -COOH;

   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.
2. A compound of the following general formula Ib or its salt or solvate:

   R ¹ -His-Aib-X ₃ -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)

   in,

   R ^{1 is} selected from H, Ac or pGlu;

   X _{3 is} selected from His, Gln;

   X _{10 is} selected from Leu, Lys, Cys or ψ;

   X _{13 is} selected from Tyr or Gln;

   X _{14 is} selected from Leu, Lys, Cys or ψ;

   X _{15 is} selected from Asp or Glu;

   X _{17 is} selected from Arg, Gln or Glu;

   X _{19 is} selected from Ala or Val;

   X _{20 is} selected from Gln or Arg;

   X _{21 is} selected from Asp or Leu;

   X _{23 is} selected from Val or Ile;

   X _{24 is} selected from Glu or Gln;

   X _{27 is} selected from Leu or Lys;

   X _{28 is} selected from Ala or Asp;

   R ² is NH ₂ or OH, or a pharmaceutically acceptable salt and/or ester thereof;

   Wherein, _{one and only one of X 10} and X ₁₄ is ψ, and the ψ is Lys whose side chain is modified by the structure having the following formula II, and the formula II is:

   Y-Z (Ⅱ)

   Y is selected from Glu, AEEAc, GABA, GSEGSEE, and any combination of two and/or more thereof, and its carboxyl end is connected to the ε-amino group of the side chain of Lys, and Z is -CO-(CH ₂ ) _m- R ³ , m is an integer between 6-24, and R ^{3 is} selected from -CH ₃ or -COOH;

   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.
3. A compound having the following general formula Ic or its salt or solvate:

   R ¹ -His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Tyr-X ₁₄ -Glu-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)

   in,

   R ¹ is H;

   X ₁₄ is ψ;

   R ² is NH ₂ or OH, or a pharmaceutically acceptable salt and/or ester thereof;

   Wherein, X ₁₄ is ψ, and the ψ is Cys whose side chain is modified by the structure having the following formula II, and the formula II is:

   Y-Z (Ⅱ)

   Y is Y1-Y2, Y1 is selected from acetylglycyl, 3-maleimidopropionyl and any combination thereof, Y2 is selected from Glu, AEEAc and any combination thereof, and it is selected from acetylglycyl or 3-maleimido propionyl is connected to the side chain mercapto group of Cys, Z is -NH-(CH ₂ ) _m -R ³ , m is an integer between 6-24, R ^{3 is} selected from -CH _{3 or-} COOH;

   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.
4. The compound having the following general formula Id or its salt or solvate:

   R ¹ -His-Aib-X ₃ -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-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R ² (Ⅰd)

   in,

   R ¹ is H;

   X _{3 is} selected from His, Gln;

   X _{10 is} selected from Tyr or Leu;

   X _{12 is} selected from Ile, Arg, or ψ;

   X _{13 is} selected from Tyr, Gln, or ψ;

   X _{14 is} selected from Leu, or ψ;

   X _{16 is} selected from Glu, or ψ;

   X _{17 is} selected from Arg, Ile, Gln, or ψ;

   X _{18 is} selected from Ala or Arg;

   X _{19 is} selected from Ala or Gln;

   X _{23 is} selected from Val or Ile;

   X _{28 is} selected from Ala or Asp;

   R ² is NH ₂ or OH, or a pharmaceutically acceptable salt and/or ester thereof;

   Wherein, X ₁₂ , X ₁₃ , X ₁₄ , X ₁₆ , X ₁₇ and only one is ψ, and the ψ is Lys whose side chain is modified by the structure having the following formula II, and the formula II is :

   Y-Z (Ⅱ)

   Y is selected from Glu, AEEAc, GABA, GSEGSEE, and any combination of two and/or more thereof, and its carboxyl end is connected to the ε-amino group of the side chain of Lys, and Z is -CO-(CH ₂ ) _m- R ³ , m is an integer between 6-24, and R ^{3 is} selected from -CH ₃ or -COOH;

   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.
5. The compound having the following general formula Ie or its salt or solvate:

   R ¹ -His-Aib-X ₃ -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-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R ² (Ⅰe)

   in,

   R ¹ is H;

   X _{3 is} selected from His, Gln;

   X _{10 is} selected from Tyr or Leu;

   X _{12 is} selected from Ile, Arg, Lys, Cys or ψ;

   X _{13 is} selected from Tyr, Gln, Lys, Cys or ψ;

   X _{14 is} selected from Leu, Lys, Cys or ψ;

   X _{16 is} selected from Glu, Lys, Cys or ψ;

   X _{17 is} selected from Arg, Ile, Gln, Lys, Cys or ψ;

   X _{18 is} selected from Ala or Arg;

   X _{19 is} selected from Ala or Gln;

   X _{23 is} selected from Val or Ile;

   X _{28 is} selected from Ala or Asp;

   R ² is NH ₂ or OH, or a pharmaceutically acceptable salt and/or ester thereof;

   Wherein, X ₁₂ , X ₁₃ , X ₁₄ , X ₁₆ , X ₁₇ and only one is ψ, and the ψ is Lys whose side chain is modified by the structure having the following formula II, and the formula II is :

   Y-Z (Ⅱ)

   Y is selected from Glu, AEEAc, GABA, GSEGSEE, and any combination of two and/or more thereof, and its carboxyl end is connected to the ε-amino group of the side chain of Lys, and Z is -CO-(CH ₂ ) _m- R ³ , m is an integer between 6-24, and R ^{3 is} selected from -CH ₃ or -COOH;

   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.
6. The compound according to any one of the preceding claims 1-5, wherein the Y-Z structure of formula (II) of the modified ψ side chain is as follows:

   (i) When ψ is Lys whose side chain is modified by the structure of Formula II, the structure of Formula II is selected from:

   γGlu-CO(CH ₂ ) ₁₄ CH ₃ :

   

   AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ CH ₃ :

   

   γGlu-CO(CH ₂ ) ₁₆ COOH:

   

   AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH:

   

   AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₈ COOH:

   

   GABA-GABA-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH:

   

   γGlu-GABA-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH:

   

   γGlu-γGlu-AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH:

   

   γGlu-γGlu-AEEAc-AEEAc-CO(CH ₂ ) ₁₆ COOH:

   

   GSEGSEE-CO(CH ₂ ) ₁₆ COOH:

   

   Wherein R is the ε-amino group of the side chain of Lys;

   (ii) When ψ is Cys whose side chain is modified by the structure of Formula II, the structure of Formula II is selected from:

   Acetylglycyl-γGlu-NH(CH ₂ ) ₁₅ CH ₃ :

   

   3-Maleimidopropionyl-γGlu-NH(CH ₂ ) ₁₅ CH ₃ :

   

   3-Maleimidopropionyl-AEEAc-AEEAc-NH(CH ₂ ) ₁₅ CH ₃ :

   

   Where R is the mercapto group of the Cys side chain.
7. The compound according to claim 1 or 2, which is selected from:

   Compound 1 (SEQ ID NO: 7):

   H-Aib-QGTFTSDK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )SKYLEEQAAQDFIEWLLAGGPSSGAPPPS-NH ₂

   Compound 2 (SEQ ID NO: 8):

   H-Aib-QGTFTSDLSKYK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EEQAAQDFIEWLLAGGPSSGAPPPS-NH ₂

   Compound 3 (SEQ ID NO: 9):

   N-Pyroglutamyl-H-Aib-QGTFTSDK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )SKYLDERAAQDFVQWLLDGGPSSGAPPPS-NH2

   Compound 4 (SEQ ID NO: 10):

   Ac-H-Aib-QGTFTSDK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )SKYLDERAAQDFVQWLLDGGPSSGAPPPS-NH ₂

   Compound 5 (SEQ ID NO: 11):

   H-Aib-QGTFTSDK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )SKYLEEEAVRLFIEWLKAGGPSSGAPPPS-NH ₂

   Compound 6 (SEQ ID NO: 12):

   H-Aib-HGTFTSDLSKQK (γGlu-CO(CH ₂ ) ₁₄ CH ₃ )DERAAQDFVQWLLDGGPSSGAPPPS-NH ₂ .
8. The compound according to claim 1 or 3, which is selected from:

   Compound 7 (SEQ ID NO: 13):

   H-Aib-QGTFTSDLSKYC(3-Maleimidopropionyl-γGlu-NH(CH ₂ ) ₁₅ CH ₃ )EEQAAQDFIEWLLAGGPSSGAPPPS-NH ₂

   Compound 8 (SEQ ID NO: 14):

   H-Aib-QGTFTSDLSKYC (3-maleimidopropionyl-AEEAc-AEEAc-NH(CH ₂ ) ₁₅ CH ₃ )EEQAAQDFIEWLLAGGPSSGAPPPS-NH ₂ .
9. The compound according to claim 1 or 4, which is selected from:

   Compound 9 (SEQ ID NO: 15):

   H-Aib-QGTFTSDLSRYK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EEQAAQDFIEWLLAGGPSSGAPPPS-NH ₂

   Compound 10 (SEQ ID NO: 16):

   H-Aib-HGTFTSDLSIQK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EERAAQDFIEWLLDGGPSSGAPPPS-NH ₂

   Compound 11 (SEQ ID NO: 17):

   H-Aib-HGTFTSDLSIQK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EEQAAQDFIEWLLDGGPSSGAPPPS-NH ₂

   Compound 12 (SEQ ID NO: 18):

   H-Aib-QGTFTSDLSIQK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EEQAAQDFIEWLLDGGPSSGAPPPS-NH ₂

   Compound 13 (SEQ ID NO: 19):

   H-Aib-HGTFTSDLSIYK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

   Compound 14 (SEQ ID NO: 20):

   H-Aib-HGTFTSDYSIQK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EEIAAQDFIEWLLDGGPSSGAPPPS-NH ₂

   Compound 15 (SEQ ID NO: 21):

   H-Aib-HGTFTSDLSIQK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EEIAQQDFIEWLLDGGPSSGAPPPS-NH ₂

   Compound 16 (SEQ ID NO: 22):

   H-Aib-HGTFTSDLSK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )YLEERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

   Compound 17 (SEQ ID NO: 23):

   H-Aib-HGTFTSDLSIK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )LEERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

   Compound 18 (SEQ ID NO: 24):

   H-Aib-HGTFTSDLSIYLEK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )RAQQDFIEWLLDGGPSSGAPPPS-NH ₂

   Compound 19 (SEQ ID NO: 25):

   H-Aib-HGTFTSDLSIYLEEK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )AQQDFIEWLLDGGPSSGAPPPS-NH ₂

   Compound 20 (SEQ ID NO: 26):

   H-Aib-HGTFTSDLSIYK(AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ CH ₃ )EERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

   Compound 21 (SEQ ID NO: 27):

   H-Aib-HGTFTSDLSIQK(AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH)EERAAQDFIEWLLDGGPSSGAPPPS-NH ₂

   Compound 22 (SEQ ID NO: 28):

   H-Aib-HGTFTSDLSIYK(γGlu-γGlu-AEEAc-AEEAc-CO(CH ₂ ) ₁₆ COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

   Compound 23 (SEQ ID NO: 29):

   H-Aib-HGTFTSDYSIQK(AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH)EEIAAQDFVEWLLAGGPSSGAPPPS-NH ₂

   Compound 24 (SEQ ID NO: 30):

   H-Aib-HGTFTSDLSIYK(AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

   Compound 25 (SEQ ID NO: 31):

   H-Aib-QGTFTSDYSIQK(AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH)EERRAQDFIEWLLDGGPSSGAPPPS-NH ₂

   Compound 26 (SEQ ID NO: 32):

   H-Aib-HGTFTSDLSIYK(γGlu-CO(CH ₂ ) ₁₆ COOH)EERAQQDFIE WLLDGGPSSGAPPPS-NH ₂

   Compound 27 (SEQ ID NO.33):

   H-Aib-HGTFTSDLSIYK(GSEGSEE-CO(CH ₂ ) ₁₆ COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

   Compound 28 (SEQ ID NO.34):

   H-Aib-HGTFTSDLSIYK(γGlu-γGlu-AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

   Compound 29 (SEQ ID NO.35):

   H-Aib-HGTFTSDLSIYK(γGlu-GABA-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH ₂

   Compound 30 (SEQ ID NO: 36):

   H-Aib-HGTFTSDLSIYK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EERAQQDFIEWLLDGGPSSGAPPPS-OH

   Compound 31 (SEQ ID NO: 37):

   H-Aib-HGTFTSDYSIQK(γGlu-CO(CH ₂ ) ₁₄ CH ₃ )EEIAAQDFIEWLLDGGPSSGAPPPS-OH

   Compound 32 (SEQ ID NO: 38):

   H-Aib-HGTFTSDLSIQK(AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₆ COOH)EERAAQDFIEWLLDGGPSSGAPPPS-OH

   Compound 33 (SEQ ID NO: 39):

   H-Aib-HGTFTSDLSIQK(AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₈ COOH)EERAAQDFIEWLLDGGPSSGAPPPS-NH ₂

   Compound 34 (SEQ ID NO: 40):

   H-Aib-HGTFTSDLSIYK (AEEAc-AEEAc-γGlu-CO(CH ₂ ) ₁₈ COOH) EERAQQDFIEWLLDGGPSSGAPPPS-NH ₂ .
10. A pharmaceutical composition comprising an effective amount of the compound of any one of the preceding claims, or a salt or solvate thereof, and pharmaceutically acceptable excipients, diluents, carriers or excipients.
11. The pharmaceutical composition according to claim 10, which is an injection or lyophilized powder, tablet, pill, lozenge, soft capsule, hard capsule, granule, powder, solution, suspension Or a syrup; or the pharmaceutical composition is in the form of microcapsules, microspheres, nanoparticles or liposomes.
12. The pharmaceutical composition according to claim 10, which is used for oral administration, inhalation administration or parenteral administration, and the parenteral administration is selected from the group consisting of intraperitoneal, intramuscular, intraarterial, and intravenous , Subcutaneous or intradermal injection.
13. The pharmaceutical composition according to claim 10, which is administered at a frequency of at least once a day, once a week, or once a month.
14. Use of the compound or its salt or solvate of any one of claims 1-9 in the preparation of a medicament for use as a GLP-1 receptor agonist, GIP receptor agonist or GCG receptor agonist One, two or three kinds of.
15. Use of the compound or its salt or solvate of any one of claims 1-9 in the preparation of a medicament for the prevention or treatment of abnormal metabolic syndrome, which is selected from the group consisting of hyperglycemia, Insulin resistance, glucose intolerance, type 2 diabetes, obesity or non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH), diabetic nephropathy, diabetic retinopathy, dyslipidemia, osteoporosis; or neurodegeneration The neurodegenerative disease is selected from Alzheimer's disease or Parkinson's syndrome.
16. The method for preparing the compound of any one of claims 1-9, which is a chemical synthesis method.
17. The method for preparing the compound according to claim 4 or 9, said method is a semi-synthetic biological method.

Images