CN119039417A - Polypeptide compound and application thereof - Google Patents

Abstract

The present invention relates to a polypeptide compound and its application, and more specifically, to a dual or triple agonist polypeptide drug that stimulates the glucagon-like peptide-1 receptor and optionally stimulates the glucose-dependent insulinotropic polypeptide receptor and the glucagon receptor, and its application in treating metabolic syndrome.

Description

Polypeptide compound and application thereof

Technical Field

The present invention relates to the field of medicine, more specifically to dual or triple agonist polypeptide drugs which agonize the glucagon-like peptide-1 (GLP-1) receptor and optionally the glucose-dependent insulinotropic polypeptide (GIP) receptor and glucagon (GCG) receptor and their use for the treatment of metabolic syndrome (e.g. diabetes, obesity, non-alcoholic fatty liver).

Background

Type II diabetes and obesity are a type of energy metabolism disorder disease, continue to become a more and more serious health problem in many countries, and cause a wide range of related dangerous diseases (e.g., cardiovascular and cerebrovascular diseases, kidney diseases, dyslipidemia, liver diseases, osteoporosis). Type 2 diabetes is characterized by hyperglycemia resulting from insulin resistance, obesity being the leading cause of insulin resistance, and obesity and insulin resistance are two major causative factors of nonalcoholic fatty liver disease (NAFLD/NASH). Thus, there is an increasing need to find safe and effective medicaments for the treatment of diseases associated with these metabolic disorders.

The incretin is a substance secreted from intestinal tracts after feeding stimulation under normal physiological conditions, and can stimulate islet beta-cells to secrete insulin, regulate glucose level homeostasis and protect islet beta-cells depending on glucose level. GLP-1 and GIP are two currently discovered incretins (Glucagon-like peptide-1and glucose-dependent insulin-releasing polypeptide plasma levels in response to nutrients.Digestion1995;56:117-126.).

GLP-1 is expressed in intestinal mucosa L cells from a glucagon-pro-gene, has a polypeptide of 31 amino acids, mainly acts on GLP-1 receptor (GLP-1R), stimulates insulin secretion, inhibits glucagon secretion, protects islet beta-cells, has physiological effects of regulating blood glucose homeostasis, and can inhibit ingestion and gastric emptying through central nervous system signaling pathways, increase satiety, thereby reducing body weight (Glucagon-like peptide-1 7-36:a physiological incretin in man.Lancet.1987;2:1300-1304.;Glucagon-like peptide-1receptor signaling modulates beta cell apoptosis.J Biol Chem.2003;278:471-478.;Relation between gastric emptying of glucose and plasma concentrations of glucagon-like peptide-1.Peptides.1998;19:1049-1053.).

Natural GLP-1 is very susceptible to degradation by dipeptidyl peptidase-IV (DPP-IV) and Neutral Endopeptidase (NEP), which are ubiquitous in plasma, with half-lives below 2min. Exendin-4 (Exendin-4) is a GLP-1 analogue extracted from the salivary glands of African lizard, has stronger GLP-1 receptor agonism and similar GLP-1 hypoglycemic effect. Compared with natural GLP-1, exendin-4 has stronger resistance to DPP-4 and NEP, and has longer in vivo half-life and action time. Exenatide (trade name is Exenatide)) For the first development of GLP-1 drugs on the market, the clinic shows better diabetes treatment effect, but the GLP-1 drugs still have the defects of larger human immunogenicity and twice-a-day administration.

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

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂

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

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG-OH

various long-acting GLP-1 analogues, such as liraglutide (trade name) for treating diabetes and/or obesity, such as norand rule of virtue, have been successfully developed on the market with a view to improving half-life of GLP-1 and therapeutic effects on diabetes And) And somalu peptide (trade name)) Dolalundum from Gift (trade name))。

Liraglutide adopts C ₁₆ fatty acid to chemically modify a 20-bit Lys side chain of GLP-1, so that the binding force of albumin is increased, and the half life is prolonged to 13-16h. The amino acid sequence of liraglutide (SEQ ID NO: 3) is as follows:

HAEGTFTSDVSSYLEGQAAK (gamma Glu-palmitoyl) EFIAWLVRGRG-OH

The Somamuno peptide adopts unnatural amino acid iso-aminobutyric acid (Aib) to replace Ala at the 2-position, so that DPP-IV resistance is obviously improved, meanwhile, the C ₁₈ fatty diacid side chain further enhances albumin binding force, and half life is obviously prolonged to the point that once-a-week administration can be realized. The amino acid sequence of the somalupeptide (SEQ ID NO: 4) is as follows:

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

GIP is a 42 amino acid single chain polypeptide produced by small intestine mucosal K cells and acts primarily on GIP receptors (GIPR) in islet cells and adipocytes. GIP glucose-dependent insulinotropic secretion, insulin secretion stimulation, gastric acid secretion inhibition, and gastric motility alleviation. In addition, fatty acid uptake by adipose tissue cells was stimulated (Biology ofIncretins: GLP-1and GIP.Gastroenterology.2007;132:2131-2157.). GIP also has physiological effects of promoting osteoblast differentiation, inhibiting osteoblast apoptosis, inhibiting bone resorption, increasing bone mineral density, and protecting bone (Glucose-dependent insulinotropic peptide is an integrative hormone with osteotropic effects.Mol Cell Endocrinol.2001;177:35-41.;Effects of glucose-dependent insulinotropic peptide on osteoclast function.Am J Physiol 2007;292:E543-E548.).

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

YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ

GCG is a 29 amino acid polypeptide expressed and secreted in islet α -cells by the pro-glucagon gene, acting on glucagon receptor (GCG R) distributed mainly in the liver and kidney, stimulating hepatic glycogenolysis, increasing hyperglycemia, activating lipase, promoting lipolysis, and enhancing fatty acid oxidation, leading to increased ketone body production. The research result shows that GCG has a certain effect (The metabolic actions of glucagon revisited.Nat.Rev.Endocrinol.2010;6:689-697.;Effects of glucagon on lipolysis and ketogenesis in normal and diabetic men.J.Clin.Invest.1974;53:190-197.).GCG on reducing food intake, increasing energy consumption of adipose tissues and reducing body fat, and a proper blood sugar increasing effect can feed back and regulate the action of insulin and reduce the occurrence of hypoglycemia.

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

HSQGTFTSDYSKYLDSRRAQDFVQWLMNT

Obesity is a condition in which body fat, especially triglycerides, accumulate excessively due to excessive energy intake or metabolic abnormality of the body. Fat accumulates in the pancreas, which impairs islet cell function, aggravates diabetes, and accumulates in the liver, which can lead to nonalcoholic fatty liver disease. The GLP-1 analogues on the market at present have the advantages of reducing blood sugar and simultaneously having cardiovascular benefits and weight management effects, but the clinical maximum weight reduction effects of liraglutide and somalunin are only about 3% and 5% respectively, and gastrointestinal side effects (mainly nausea, vomiting and diarrhea) are common. Therefore, there is still an urgent need for therapeutic drugs with better weight control effect, broader benefit and greater safety margin for more and more obese people.

According to the physiological actions of GLP-1, GIP and GCG, a plurality of researches at present find that better diabetes and obesity control effects can be realized by integrating the actions of two or three of the GLP-1, GIP and GCG through multi-target activation compared with single-drug treatment. GLP-1/GCG co-agonists have more obvious effects on reducing ingestion, reducing weight, improving glucose tolerance and reducing triglyceride than single GLP-1 receptor agonists, and the treatment effect on obese mice is more obvious than that of (Glucagon-like peptide1/glucagon receptor dual agonism reverses obesity in mice.Diabetes.2009;58:2258-2266;A new glucagon and GLP-1coagonist eliminates obesity in rodents.Nat ChemBiol.2009;5:749-757.). Indiana university animal research results prove that GLP-1/GIP and GLP-1/GIP/GCG co-agonists can obviously reduce blood glucose and weight of obese (DIO) mice induced by high-fat diet, and the treatment effect is obviously better than that of acetylated GLP-1 (Acyl-GLP-1), acetylated GIP (Acyl-GIP) and liraglutide. In addition, it can reduce plasma cholesterol, body fat and liver fat, and improve various lipid metabolism indexes such as triglyceride, leptin, adiponectin, ketone body 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.NatMed.2015;21:27-36.).

There remains a need for new bi-or tri-agonistic polypeptide molecules for the prevention or treatment of metabolic syndrome with improved stability or lower mass production costs.

Disclosure of Invention

The present invention provides a novel polypeptide compound which can have a dual or triple agonistic effect. Such polypeptides may have significant GLP-1R agonistic activity, and may have both GIP R and/or GCG R agonistic activity, may be used for the prevention or treatment of metabolic syndrome, e.g. lowering blood glucose, body weight, fat, and have improved stability.

In one aspect, there is provided a compound having the formula I:

R¹-His-Aib-X₃-Gly-Thr-Phe-Thr-Ser-Asp-X₁₀-Ser-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-X₁₉-X₂₀-X₂₁-Phe-X₂₃-X₂₄-Trp-Leu-X₂₇-X₂₈-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R²(Ⅰ)(SEQ ID NO:45)

Wherein,

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 one and only one of X ₁₀,X₁₂,X₁₃,X₁₄,X₁₆,X₁₇ is psi, and wherein the psi is Cys or Lys with side chain modified by structure having formula II:

Y-Z(II)

(i) When ψ is Lys, Y is selected from Glu (preferably γglu), AEEAc, GABA, GSEGSEE and any combination of two and/or more thereof, and the carboxy-terminus thereof is linked to the epsilon-amino group of the side chain of Lys, Z is-CO- (CH ₂)_m-R³, m is an integer between 6 and 24, R ³ is selected from-CH ₃ or-COOH; Y preferably represents up to 10, or up to 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 or more thereof, e.g., Y may be γGlu, GSEGSEE, AEeac- γGlu, γGlu-AEeac- γGlu, or γGlu-GABA-AEEAc- γGlu;

(ii) When ψ is Cys, Y is Y1-Y2, Y1 is selected from the group consisting of acetylglycyl, 3-maleimidopropionyl, and any combination thereof, Y2 is selected from the group consisting of Glu (preferably γglu), AEEAc, and any combination thereof, and is linked to Cys side chain thiol by acetylglycyl or 3-maleimidopropionyl, Z is-NH- (CH ₂)_m-R³, m is an integer between 6 and 24, R ³ is selected from-CH ₃ or-COOH, Y1 preferably represents 1 linker selected from the group consisting of acetylglycyl, 3-maleimidopropionyl, and any combination thereof, Y2 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 the group consisting of Glu (preferably γglu), AEEAc, and any combination thereof, e.g., Y1 may be 3-maleimidopropionyl;

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 said amino acid sequence.

In one aspect, there is provided a compound having the formula Ib:

R¹-His-Aib-X₃-Gly-Thr-Phe-Thr-Ser-Asp-X₁₀-Ser-Lys-X₁₃-X₁₄-X₁₅-Glu-X₁₇-Ala-X₁₉-X₂₀-X₂₁-Phe-X₂₃-X₂₄-Trp-Leu-X₂₇-X₂₈-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R²(Ⅰb)(SEQ ID NO:46)

Wherein,

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,X₁₄ is psi, and wherein the psi is Lys having a side chain modified by a structure having the formula II:

Y-Z(II)

Y is selected from Glu (preferably γGlu), AEEAc, GABA, GSEGSEE and any combination of two and/or more thereof, and the carboxy terminus of which is linked to the epsilon-amino group of the side chain of Lys, Z is-CO- (CH ₂)_m-R³), m is an integer between 6 and 24, R ³ is selected from-CH ₃ or-COOH; Y preferably represents up to 10, or up to 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 or more thereof, e.g. Y may be γGlu, GSEGSEE, AEeac- γGlu, γGlu-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 said amino acid sequence.

The compound with the structure shown in the formula Ib can be prepared by chemically synthesizing the polypeptide.

In one aspect, there is provided a compound having the formula Ic:

R¹-His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Tyr-X₁₄-Gl u-Glu-Gln-Ala-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Leu-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R²(Ⅰc)(SEQ ID NO:47)

Wherein,

R ¹ is H;

X ₁₄ is psi;

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 having a side chain modified by a structure having the following formula II:

Y-Z(II)

Y is Y1-Y2, Y1 is selected from the group consisting of acetylglycyl, 3-maleimidopropionyl, and any combination thereof, Y2 is selected from the group consisting of Glu (preferably γGlu), AEEAc, and any combination thereof, and is linked to a Cys side chain thiol group by acetylglycyl or 3-maleimidopropionyl, Z is-NH- (CH ₂)_m-R³, m is an integer between 6 and 24, R ³ is selected from the group consisting of-CH ₃ or-COOH, Y1 preferably represents 1 linking group selected from the group consisting of acetylglycyl, 3-maleimidopropionyl, and any combination thereof, Y2 preferably represents at most 10, or at most 5, or at most 4, or at most 3, or at most 2, or 1 linking group selected from the group consisting of Glu (preferably γGlu), AEEAc, and any combination thereof, e.g., Y1 may be 3-maleimidopropionyl;

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 said amino acid sequence.

The compound with the structure shown in the formula Ic can be prepared by chemically synthesizing the polypeptide.

In one aspect, there is provided a compound having the formula Id:

R¹-His-Aib-X₃-Gly-Thr-Phe-Thr-Ser-Asp-X₁₀-Ser-X₁₂-X₁₃-X₁₄-Glu-X₁₆-X₁₇-X₁₈-X₁₉-Gln-Asp-Phe-X₂₃-Glu-Trp-Leu-Leu-X₂₈-Gly-Gly-Pro-Se r-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R²(Ⅰd)(SEQ ID NO:48)

Wherein,

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 one and only one of X ₁₂,X₁₃,X₁₄,X₁₆,X₁₇ is psi, and wherein the psi is Lys having a side chain modified by a structure having the formula II:

Y-Z(II)

Y is selected from Glu (preferably γGlu), AEEAc, GABA, GSEGSEE and any combination of two and/or more thereof, and the carboxy terminus of which is linked to the epsilon-amino group of the side chain of Lys, Z is-CO- (CH ₂)_m-R³), m is an integer between 6 and 24, R ³ is selected from-CH ₃ or-COOH; Y preferably represents up to 10, or up to 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 or more thereof, e.g., Y may be γGlu, GSEGSEE, AEeac- γGlu, γGlu-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 said amino acid sequence.

The compound with the structure shown in the formula Id can be prepared by chemically synthesizing polypeptide or biosynthetically.

The biological semisynthesis refers to a similar method for preparing the sorlutide (SEQ ID NO: 4) disclosed in similar patents CN201510459093 and US 9732137.

The method is characterized in that yeast or escherichia coli is used for fermenting and expressing to obtain a part of peptide TFTSDVSSYLEGQAAKEFIAWLVRGRG-OH in a sorup peptide sequence, fatty acid activated ester is used for carrying out acylation reaction with a side chain NH ₂ of K in the peptide to obtain a peptide TFTSDVSSYLEGQAAK ([ 2- (2-amino-ethoxy) -ethoxy ] -acetyl) 2-gamma E-octadecanoyl) EFIAWLVRGRG-OH, and finally, an N-terminal protruding end Boc-His (Boc) -Aib-Glu (O-tBu) -Gly-OSuc is used for carrying out acylation reaction with alpha-NH ₂ of the peptide, and a Boc protecting group is removed to obtain the sorup peptide. The method overcomes the complicated steps of chemical synthesis, fully utilizes the advantages of biological fermentation expression, and is beneficial to the reduction of production cost and large-scale industrial production.

The implementation of the above technical method depends on two characteristics of the polypeptide sequence 1. Lys of the sequence has no other sites except the specific site Lys modified by fatty acid. If there is a Lys in the excess, it is not guaranteed that the fatty acid will specifically bind to the desired modified Lys site, 2. There are no other unnatural amino acids in the sequence after position 5, except for the unnatural amino acid Aib in position 2. Otherwise, the biological cells such as yeast or E.coli will not express the peptide fragment with the unnatural amino acid.

Since the above formula Id corresponds to both of the above features, it can be prepared in a similar manner by biological semisynthesis.

In one aspect, there is provided a compound having the following formula Ie:

R¹-His-Aib-X₃-Gly-Thr-Phe-Thr-Ser-Asp-X₁₀-Ser-X₁₂-X₁₃-X₁₄-Glu-X₁₆-X₁₇-X₁₈-X₁₉-Gln-Asp-Phe-X₂₃-Glu-Trp-Leu-Leu-X₂₈-Gly-Gly-Pro-Se r-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R²(Ⅰe)(SEQ ID NO:49)

Wherein,

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 one and only one of X ₁₂,X₁₃,X₁₄,X₁₆,X₁₇ is psi, and wherein the psi is Lys having a side chain modified by a structure having the formula II:

Y-Z(II)

Y is selected from Glu (preferably γGlu), AEEAc, GABA, GSEGSEE and any combination of two and/or more thereof, and the carboxy terminus of which is linked to the epsilon-amino group of the side chain of Lys, Z is-CO- (CH ₂)_m-R³), m is an integer between 6 and 24, R ³ is selected from-CH ₃ or-COOH; Y preferably represents up to 10, or up to 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 or more thereof, e.g., Y may be γGlu, GSEGSEE, AEeac- γGlu, γGlu-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 said amino acid sequence.

In one aspect, the above formula II is of the structure:

When ψ is Lys with side chains modified by the structure of formula II, the structure of formula II may be selected (the term "R" in the following structures is intended to denote the attachment site to formula II at the peptide backbone, i.e. the epsilon-amino group of the Lys side chain):

γGlu-CO(CH₂)₁₄CH₃:

AEEAc-AEEAc-γGlu-CO(CH₂)₁₆CH₃:

γGlu-CO(CH₂)₁₆COOH:

AEEAc-AEEAc-γGlu-CO(CH₂)₁₆COOH:

AEEAc-AEEAc-γGlu-CO(CH₂)₁₈COOH:

GABA-GABA-AEEAc-γGlu-CO(CH₂)₁₆ COOH:

γGlu-GABA-AEEAc-γGlu-CO(CH₂)₁₆ COOH:

γGlu-γGlu-AEEAc-AEEAc-γGlu-CO(CH₂)₁₆COOH:

γGlu-γGlu-AEEAc-AEEAc-CO(CH₂)₁₆COOH:

GSEGSEE-CO(CH₂)₁₆COOH:

When ψ is Cys with a side chain modified by the structure of formula II, the structure of formula II may be selected (the term "R" in the following structures is intended to denote the attachment site to formula II at the peptide backbone, i.e. the thiol group of the Cys side chain):

Acetylglycyl- γglu-NH (CH ₂)₁₅CH₃:

3-maleimidopropionyl- γglu-NH (CH ₂)₁₅CH₃:

3-maleimidopropionyl-AEEAc-NH (CH ₂)₁₅CH₃:

in one aspect, the compound is selected from:

Compound 1 (SEQ ID NO: 7):

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

compound 2 (SEQ ID NO: 8):

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

compound 3 (SEQ ID NO: 9):

N-pyroglutamyl-H-Aib-QGTFTSDK (gamma Glu-CO (CH) ₂)₁₄CH₃)SKYLDERAAQDFVQWLL DGGPSSGAPPPS-NH₂

Compound 4 (SEQ ID NO: 10):

Ac-H-Aib-QGTFTSDK(γGlu-CO(CH₂)₁₄CH₃)SKYLDERAAQDFV QWLLDGGPSSGAPPPS-NH₂

compound 5 (SEQ ID NO: 11):

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

compound 6 (SEQ ID NO: 12):

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

compound 7 (SEQ ID NO: 13):

H-Aib-QGTFTSDLSKYC (3-maleimidopropionyl-gamma Glu-NH (CH) ₂)₁₅CH₃)EEQAAQDFIEWLLAGGPSSGAPPPS-NH₂

Compound 8 (SEQ ID NO: 14):

H-Aib-QGTFTSDLSKYC (3-maleimidopropionyl-AEEAc-AEEAc-NH (CH) ₂)₁₅CH₃)EEQAAQDFIEWLLAGGPSSGAPPP S-NH₂

Compound 9 (SEQ ID NO: 15):

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

compound 10 (SEQ ID NO: 16):

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

compound 11 (SEQ ID NO: 17):

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

Compound 12 (SEQ ID NO: 18):

H-Aib-QGTFTSDLSIQK(γGlu-CO(CH₂)₁₄CH₃)EEQAAQDFIEWLLDGGPSSGAPPPS-NH₂

compound 13 (SEQ ID NO: 19):

H-Aib-HGTFTSDLSIYK(γGlu-CO(CH₂)₁₄CH₃)EERAQQDFIEWLLDGGPSSGAPPPS-NH₂

compound 14 (SEQ ID NO: 20):

H-Aib-HGTFTSDYSIQK(γGlu-CO(CH₂)₁₄CH₃)EEIAAQDFIEWLLDGGPSSGAPPPS-NH₂

compound 15 (SEQ ID NO: 21):

H-Aib-HGTFTSDLSIQK(γGlu-CO(CH₂)₁₄CH₃)EEIAQQDFIEWLLDGGPSSGAPPPS-NH₂

Compound 16 (SEQ ID NO: 22):

H-Aib-HGTFTSDLSK(γGlu-CO(CH₂)₁₄CH₃)YLEERAQQDFIEWLLDGGPSSGAPPPS-NH₂

compound 17 (SEQ ID NO: 23):

H-Aib-HGTFTSDLSIK(γGlu-CO(CH₂)₁₄CH₃)LEERAQQDFIEWLLDGGPSSGAPPPS-NH₂

compound 18 (SEQ ID NO: 24):

H-Aib-HGTFTSDLSIYLEK(γGlu-CO(CH₂)₁₄CH₃)RAQQDFIEWLLDGGPSSGAPPPS-NH₂

compound 19 (SEQ ID NO: 25):

H-Aib-HGTFTSDLSIYLEEK(γGlu-CO(CH₂)₁₄CH₃)AQQDFIEWLLDGGPSSGAPPPS-NH₂

Compound 20 (SEQ ID NO: 26):

H-Aib-HGTFTSDLSIYK(AEEAc-AEEAc-γGlu-CO(CH₂)₁₆CH₃)EERAQQDFIEWLLDGGPSSGAPPPS-NH₂

compound 21 (SEQ ID NO: 27):

H-Aib-HGTFTSDLSIQK(AEEAc-AEEAc-γGlu-CO(CH₂)₁₆COOH)EERAAQDFIEWLLDGGPSSGAPPPS-NH₂

compound 22 (SEQ ID NO: 28):

H-Aib-HGTFTSDLSIYK(γGlu-γGlu-AEEAc-AEEAc-CO(CH₂)₁₆COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH₂

compound 23 (SEQ ID NO: 29):

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

Compound 24 (SEQ ID NO: 30):

H-Aib-HGTFTSDLSIYK(AEEAc-AEEAc-γGlu-CO(CH₂)₁₆COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH₂

compound 25 (SEQ ID NO: 31):

H-Aib-QGTFTSDYSIQK(AEEAc-AEEAc-γGlu-CO(CH₂)₁₆COOH)EERRAQDFIEWLLDGGPSSGAPPPS-NH₂

Compound 26 (SEQ ID NO: 32):

H-Aib-HGTFTSDLSIYK(γGlu-CO(CH₂)₁₆COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH₂

Compound 27 (SEQ ID No. 33):

H-Aib-HGTFTSDLSIYK(GSEGSEE-CO(CH₂)₁₆COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH₂

compound 28 (SEQ ID No. 34):

H-Aib-HGTFTSDLSIYK(γGlu-γGlu-AEEAc-AEEAc-γGlu-CO(CH₂)₁₆COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH₂

Compound 29 (SEQ ID No. 35):

H-Aib-HGTFTSDLSIYK(γGlu-GABa-aEEAc-γGlu-CO(CH₂)₁₆COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH₂

compound 30 (SEQ ID NO: 36):

H-Aib-HGTFTSDLSIYK(γGlu-CO(CH₂)₁₄CH₃)EERAQQDFIEWLLDGGPSSGAPPPS-OH

compound 31 (SEQ ID NO: 37):

H-Aib-HGTFTSDYSIQK(γGlu-CO(CH₂)₁₄CH₃)EEIAAQDFIEWLLDGGPSSGAPPPS-OH

compound 32 (SEQ ID NO: 38):

H-Aib-HGTFTSDLSIQK(AEEAc-AEEAc-γGlu-CO(CH₂)₁₆COOH)EERAAQDFIEWLLDGGPSSGAPPPS-OH

compound 33 (SEQ ID NO: 39):

H-Aib-HGTFTSDLSIQK(AEEAc-AEEAc-γGlu-CO(CH₂)₁₈COOH)EERAAQDFIEWLLDGGPSSGAPPPS-NH₂

compound 34 (SEQ ID NO: 40):

H-Aib-HGTFTSDLSIYK(AEEAc-AEEAc-γGlu-CO(CH₂)₁₈COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH₂.

in one aspect, there is provided a pharmaceutical composition comprising an effective amount of a compound of any one of the preceding aspects, or a salt or solvate thereof, and a pharmaceutically acceptable adjuvant, diluent, carrier or excipient.

In one aspect, the pharmaceutical composition is in the form of an injection or lyophilized powder, tablet, pill, lozenge, soft capsule, hard capsule, granule, powder, solution, suspension or syrup, or the pharmaceutical composition is in the form of a microcapsule, microsphere, nanoparticle or liposome.

In one aspect, the pharmaceutical composition is for oral administration, inhaled administration or parenteral administration selected from intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous or intradermal injection administration.

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

In one aspect, there is provided the use of a compound of any one of the preceding aspects, or a salt or solvate thereof, in the manufacture of a medicament for use as one, two or three of a GLP-1 receptor agonist, a GIP receptor agonist or a GCG receptor agonist.

In one aspect, there is provided the use of a compound of any one of the preceding aspects, or a salt or solvate thereof, in the manufacture of a medicament for the prevention or treatment of a metabolic abnormality syndrome selected from hyperglycemia, insulin resistance, glucose intolerance, type II 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 parkinsonism.

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

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

The compounds of the invention may be dual or tri-agonistic polypeptide molecules, are useful for the prevention or treatment of metabolic syndrome, and have improved stability.

Drawings

FIG. 1 shows a plasmid map of pET31b-SEQ ID NO. 19.

FIG. 2 shows an electrophoretogram of the KSI-DDDDK-peptide SEQ ID NO.19 (5-40) precursor fusion protein.

Figure 3 acute effect of first dose compound on DIO mouse blood glucose. DIO mice were assayed for 0,1, 2, 4, 8, 24, 48, 72h non-fasting blood glucose levels after administration of the first dose of compound (compounds administered once a day were normally administered at 24 and 72h for the second and third doses of the corresponding compounds). Data are expressed as mean ± SEM, n=8.

Figure 4 effect of compounds on fasting blood glucose in DIO mice. Animals at the end of the experiment were fasted for 5h and bled, and the compounds significantly reduced the fasting blood glucose of the animals compared to the Vehicle control group (< p < 0.05). Data are expressed as mean ± SEM, n=8.

Figure 5 effect of compounds on DIO mice oral glucose tolerance. DIO mice glucose tolerance was determined on day 19 of dosing, blood glucose levels were determined at specific time points over 120min after oral glucose administration to animals, and the area under the blood glucose-time curve (AUC _0-120min) was calculated. Fig. 5A, graph of blood glucose versus time after oral administration of glucose, and fig. 5B, area under the graph of blood glucose versus time after oral administration of glucose (AUC _0-120min). The compounds significantly reduced glucose tolerance (×p < 0.001) in DIO mice following glucose oral administration compared to the Vehicle control group. Data are expressed as mean ± SEM, n=8.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. 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 parent peptide amino acid sequence of the polypeptide drug can be modified based on the amino acid sequences of Exendin-4 (SEQ ID NO: 1), GLP-1 (SEQ ID NO: 2), GIP (SEQ ID NO: 5) and GCG (SEQ ID NO: 6) as described above.

The polypeptide compounds of the present invention may be synthesized and modified by methods known to those skilled in the art. For example, the peptide sequence backbone of the polypeptide compounds of the present invention may be prepared by methods such as synthesis, recombinant DNA biotechnology, and the like.

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

The compounds of the invention have agonistic activity at the GLP-1 receptor and optionally at the GIP receptor and/or the GCG receptor. By "agonistic activity" is meant that the compound agonizes a particular receptor cell to produce cAMP, and the cell used may be a host cell or islet tissue cell, adipocyte, hepatocyte, etc. that is constructed by one of skill in the art to overexpress the GLP-1 receptor or GIP receptor or GCG receptor. The receptor agonistic activity may be measured as the EC ₅₀ value of the compound for agonizing the receptor cells to produce cAMP. EC ₅₀ values are the drug concentration values required to achieve half the maximum activity (50% activity) of a compound in a particular assay system.

In particular embodiments, the agonistic activity of a compound can be assessed by relative activity of a particular natural compound. The relative activity is the percentage of the ratio of the EC ₅₀ value of a particular natural compound to the EC ₅₀ value of the test compound.

The compounds of the present invention have a relative GLP-1 receptor agonism activity of at least 0.5%, preferably at least 5%, more preferably at least 50%, even most preferably at least 100% compared to native GLP-1 (7-37).

The compounds of the present invention have a relative activity at agonism of the GIP receptor of at least 0.5%, preferably at least 5%, more preferably at least 50%, even most preferably at least 100% compared to native GIP.

The compounds of the invention have a relative activity towards GCG receptor agonism of at least 0.5%, preferably at least 5%, more preferably at least 50%, even most preferably at least 100% compared to native GCG.

Polypeptide sequence similarity and polypeptide sequence identity

Structural similarity of two polypeptides can be determined by aligning the residues of the two polypeptides (e.g., the candidate polypeptides and any suitable reference polypeptides described herein) along their sequence length to optimize the number of identical amino acids, allowing gaps in either or both sequences in the alignment to optimize the number of identical amino acids, but the amino acids of each sequence must still be in their correct order. Where appropriate, the reference polypeptide may be a polypeptide as described herein. The candidate polypeptide is a polypeptide that is compared to a reference polypeptide.

Pairing comparison analysis of amino acid sequences can be performed using software packages known in the art. In the comparison of two amino acid sequences, structural similarity may be referred to by a percentage of "identity" or by a percentage of "similarity". "identity" means the presence of the same amino acid. "similarity" means that not only the same amino acids are present, but also conservative substitutions are present. Conservative substitutions of amino acids in the polypeptides of the invention may 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 amino acids belonging to a group of amino acids having a specific size or property (e.g., charge, hydrophobicity, and hydrophilicity) can be substituted with another amino acid without altering the activity of the protein, particularly in regions of the protein that are not directly associated with biological activity. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions include, for example, lys 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. Likewise, biologically active analogs of polypeptides are contemplated which contain deletions or additions of one or more contiguous or non-contiguous amino acids that do not abrogate the functional activity of the polypeptide.

Thus, as used herein, references to a polypeptide of the invention and/or to the amino acid sequence of one or more SEQ ID NOs may include polypeptides having 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% amino acid sequence similarity to a reference amino acid sequence.

Or as used herein, references to a polypeptide of the invention and/or to the amino acid sequence of one or more SEQ ID NOs may include a polypeptide having 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% amino acid sequence identity to a reference amino acid sequence.

The polypeptides of the invention may be further modified, for example, including polypeptides chemically or enzymatically derived at one or more constituent amino acids (or analogs thereof, e.g., fragments thereof). Such modifications may include, for example, side chain modifications, backbone modifications, and N-and C-terminal modifications, such as acetylation, hydroxylation, methylation, amidation, and attachment of carbohydrate or lipid moieties, cofactors, and the like, and combinations thereof. The modified polypeptides of the invention may retain the biological activity of the unmodified polypeptide, or may exhibit reduced or increased biological activity.

The effective amount of the compound can be combined with at least one pharmaceutically acceptable auxiliary material, diluent, carrier or excipient to form a pharmaceutical composition. An "effective amount" of a compound is an amount of the compound that is capable of producing a disease modifying or therapeutic effect without producing a damaging effect. The effective amount can be suitably determined by the attending physician according to the severity of the disease, age, sex, weight, general health, etc., of the patient. In one embodiment, the patient is a mammal. In a preferred embodiment, the mammal is selected from the group consisting of a cow, horse, goat, sheep, dog, chimpanzee, rabbit, mouse, rat, monkey, pig and human. In a more preferred embodiment, the patient is a human.

The compound and the pharmaceutical composition thereof can be prepared into injection or freeze-dried powder, tablets, pills, troches, soft capsules, hard capsules, granules, powder, solution, suspension or syrup, and injection or freeze-dried powder is preferred.

Adjuvants which can be used in the compositions according to the invention are, for example, lubricants, binders, fillers, preservatives, surfactants, colorants, flavoring agents, emulsifiers, suspending agents, diluents, gelling agents, disintegrants, pH-adjusting agents, solubilizing agents and the like. Those skilled in the art will appreciate that these excipients may be appropriately selected according to the appropriate dosage form and their content may be varied as desired.

The compounds of the present invention may also be used loaded into a variety of drug carrier materials (e.g., microcapsules, microspheres, nanoparticles, liposomes) or drug delivery devices.

In addition, the compounds of the invention may be used in combination with at least one therapeutically active agent, including antidiabetic agents (e.g., insulin and analogs thereof, biguanides, sulfonylureas, thiazolidinediones, alpha-glucosidase inhibitors, DPP-4 inhibitors, SGLT2 inhibitors, dual SGLT1/SGLT2 inhibitors, GLP-1 receptor agonists, amylin and analogs thereof), GIP receptor agonists, GCG receptor agonists or antagonists, dual GLP-1/GIP receptor agonists, GLP-1/GCG receptor agonists, GIP/GCG receptor agonists, FGF-21 and analogs thereof, cholecystokinin B (CCKB) and analogs thereof, PYY (3-36) and analogs thereof, leptin and analogs thereof, calcitonin and analogs thereof, lipid modulating active agents, PPAR-alpha, beta, delta agonists or modulators, antiplatelet aggregation active agents, PCSK9 inhibitors, lipase inhibitors, anti-hepatic fibrosis or cirrhosis active agents, anti-inflammatory agents.

Preferably, the compounds of the present invention promote insulin secretion and lower blood glucose. Preferably, ingestion may also be inhibited, gastric emptying may be delayed, energy expenditure may be increased, and finally weight reduction effects may be observed.

Preferably, the compounds of the present invention can reduce islet beta-cell apoptosis, increase islet beta-cell number, and improve islet cell function.

Preferably, the compound of the invention can also improve blood fat, reduce liver fat accumulation, inhibit liver inflammation development and prevent and treat nonalcoholic fatty liver diseases.

Preferably, the compounds of the present invention are expected to promote brain neuron growth, scavenge neurotoxic substances, inhibit inflammation development, and act as neuroprotection.

Preferably, the compounds or compositions of the present invention are useful for the prevention and/or treatment of metabolic disorders and their associated complications. Preferably for the treatment of diabetes, obesity and nonalcoholic fatty liver disease.

Preferably, the compounds or compositions of the invention are useful for the treatment of dyslipidemia and related diseases, neurodegenerative diseases (e.g. parkinson's disease, alzheimer's disease).

Preferably, the compounds or compositions of the present invention are useful for the treatment of bone diseases associated with endocrine disorders, metabolic disorders, kidney disease, weight loss, etc., such as osteoporosis, osteoarthritis.

Preferably, the compounds or compositions of the present invention may be administered by a variety of routes, for example, may be used for oral administration, inhalation administration or parenteral administration, for example, intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous or intradermal injection administration.

Preferably, the compounds or compositions of the present invention may be administered at least once a day, once a week, or once a month.

The compound of the invention has better solubility and stability, and is superior to natural peptide molecules and liraglutide.

The compound has obvious excitation effect on two or three of GLP-1, GIP and GCG receptors.

The abbreviations used in the present invention have the following specific meanings:

Aib diaminoisobutyric acid

GABA gamma-aminobutyric acid

AEeac [2- (2-amino-ethoxy) -ethoxy ] -acetyl

Ac, acetyl

PGlu pyroglutamyl group

CAMP cyclic adenosine monophosphate

PEG polyethylene glycol

Fmoc fluorenylmethoxycarbonyl

Boc

DMF dimethylformamide

DIC: N is a number of the N, N-diisopropylic acid carbodiimides

Boc

Trt trityl radical

Ivdde 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) -3-methyl-butyl

T-Bu t-butyl

OtBu t-butyl ester

TFA trifluoroacetic acid

HPLC/MS high performance liquid chromatography/mass spectrometry HPLC-UV high performance liquid chromatography-ultraviolet

IPTG isopropyl-beta-D-thiogalactoside

Tris-Tris-hydroxymethyl aminomethane

DCM: dichloromethane

THF tetrahydrofuran

DIPEA N, N-diisopropylethylamine

NMP N-methylpyrrolidone

PAM-peptide acyl glycine alpha amidated monooxygenase MES-fatty acid methyl ester sulfonate

HEK-293 human embryonic kidney cells

GLP-1R glucagon-like peptide-1 receptor GIP R glucose-dependent insulinotropic peptide receptor GCG R glucagon receptor

PBS phosphate buffer solution

FBS (bovine serum) fetal bovine serum

DMEM Du modified Eagle Medium

HBSS Hank's Balanced salt solution HEPES 4-hydroxyethylpiperazine ethanesulfonic acid

BSA bovine serum albumin

EC ₅₀ half-maximal effect concentration

IBMX 3-isobutyl-1-methylxanthine.

S.C. subcutaneous injection

QD once daily

Q3D once every three days

OGTT oral glucose tolerance test

TC Total cholesterol

LDL-C, low Density lipoprotein cholesterol

TG triglycerides

HOMA insulin resistance index

Specific examples:

Example 1 peptide compound synthesis:

Both the intermediates and compounds of the present invention can be prepared synthetically by a variety of methods known in the art. The chemical synthesis of the compounds of the invention is illustrated in the following specific examples. Each of the specific synthetic steps described may be combined in different materials and manners to synthesize a plurality of corresponding compounds of the invention or salts thereof. The reagents and starting materials used are readily available to those of ordinary skill in the art. In particular, the following examples are only illustrative of the invention and should not be construed as limiting the scope of the invention in any way.

Materials:

The materials and reagents used in the invention are purchased from commercial products, and the protective amino acids used in the whole 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.

The synthetic preparation of the compounds of the invention will now be described by way of example with reference to the compound SEQ ID NO 12.

The method comprises the following steps:

(1) Pretreatment of Rink amino resin, namely weighing 1g of dried Rink amino resin, soaking and swelling the dried Rink amino resin in DMF (dimethyl formamide) for 30min, and pumping out the solvent.

(2) Removing the protecting group Fmoc, namely adding 20% piperidine/DMF solution into the treated Rink amino resin, and stirring for reaction for 20min. The extent of reaction was monitored during the reaction using ninhydrin color development, and if the resin developed a color change, this indicated that Fmoc removal was successful. After the reaction, the solvent was removed by filtration, DMF was added to the reaction system and the resin was washed with stirring for 1min, and washing was repeated 3 times.

(3) Coupling reaction (peptide bond formation) the prepared corresponding Fmoc protected amino acid solution was added to the reactor, followed by DIC/DMF solution and stirred for 1h. The progress of the reaction was monitored during the reaction using ninhydrin chromogenic method, indicating successful coupling if the resin had no change in color. After the reaction was completed, the solvent was removed by filtration, and the resin was washed with DMF under stirring for 1min, followed by repeated washing 3 times. Repeating the above steps, and sequentially adding corresponding amino acid solution until peptide chain synthesis is completed. The last amino acid was coupled with Boc-His (Trt) -OH. Lys of the side chain modification site was replaced with Fmoc-Lys (ivdde) -OH. The sequence of the main peptide sequence of the compound SEQ ID NO. 12 is that the amino acid coupling is added in sequence 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) And ivdde, adding hydrazine/DMF solution into the system to remove the side chain protecting group ivdde of the modification site Lys, filtering to remove the solvent after the removal is successful, adding DMF into the system, stirring and washing for 1min, filtering to remove the solvent, and repeating the washing for 4 times.

(5) Lys side chain modification, namely adding the prepared Fmoc-Glu-OtBu solution into the treated resin, adding DIC/DMF solution, and stirring for reaction for 1h. After the reaction was completed, the mixture was washed by filtration, a 20% piperidine/DMF solution was added, and the reaction was stirred for 20 minutes to remove Fmoc groups. Then adding the prepared palmitic acid solution into the resin, adding DIC/DMF solution, and stirring for reaction for 1h. After the reaction was completed, the resin was washed 4 times and the resin was drained.

(6) And (3) peptide resin post-treatment, namely adding a cutting reagent K to cut the resin. And (3) precipitating and centrifuging the filtered filtrate to obtain a white solid which is a crude product of the target compound.

Purification of crude peptide compound:

The crude peptide obtained was purified by reversed phase C18 preparative chromatography (Shimadzu, inertsil ODS 20X 250mm 5 um) and the sample was purified to a purity of greater than 95%. Starting with 95% buffer A (0.065% TFA/H ₂ O) and 5% buffer B (0.05% TFA/acetonitrile), the ratio of buffer B was gradually increased to 65% at a rate of 2%/min, and the elution was continued for 30min, and the target peptide fraction was collected. The purified peptide compounds were confirmed by analytical HPLC/MS analysis.

Based on the above synthesis method, the present invention synthesizes the following Table peptide compounds SEQ ID NOs 7 to 40 and performs characterization (see Table 1).

TABLE 1 list of synthetic peptide compounds and molecular weights

Example 2 biological half-synthesis of peptide compounds:

The method steps of the biosynthesis of the compounds according to the invention are described below by way of example with regard to the biological semisynthesis of the peptide compound SEQ ID NO. 19.

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

Referring to patent CN201711154044, example 3, an E.coli engineering strain is constructed with a fusion gene having a gene sequence shaped as a-B-C structure, wherein A is a chaperonin encoding gene, B is a connecting peptide encoding gene, and C is a fragment encoding gene of the tri-agonist peptide SEQ ID NO.19 (5-40). And (3) carrying out high-density fermentation and induction on the mixture, and extracting inclusion bodies containing fusion proteins or fusion proteins.

The recombinant escherichia coli strain is preferably obtained by cloning fusion genes with the structure of A-B-C into a prokaryotic expression vector, and transferring the obtained recombinant expression vector into escherichia coli engineering bacteria to obtain the recombinant escherichia coli strain.

The prokaryotic expression vector is pET31b (+).

The engineering bacterium of the escherichia coli is escherichia coli BL21 (DE 3).

The induction is induced by IPTG.

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

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

SEQ ID NO.41:

MHTPEHITAVVQRFVAALNAGDLDGIVALFADDATVEDPVGSEPRSGTAAIREFYANSLKLPLAVELTQEVRAVANEAAFAFTVSFEYQGRKTVVAPIDHFRFNGAGKVVSIRALFGEKNIHACQMLDDDDKTFTSDLSIYKEERAQQDFIEWLLDGGPSSGAPPPSG-OH

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 as SEQ ID NO. 42.

SEQ ID NO.42:

TFTSDLSIYKEERAQQDFIEWLLDGGPSSGAPPPSG-OH

The construction method of the fusion gene with the structure of A-B-C and the engineering bacteria can be referred to from the experimental guidelines in the field (J. Sam Brookfield et al, second edition of molecular cloning experimental guidelines, scientific Press, 1995).

Designing a DDDDK-peptide SEQ ID NO.19 (5-40) precursor fusion gene fragment, converting the amino acid sequences into nucleotide sequences according to codon usage preference of escherichia coli, selecting codons with higher usage frequency in the conversion process, adjusting GC content, removing cis-acting elements and repeated sequences influencing gene transcription to obtain the DDDDK-peptide SEQ ID NO.19 (5-40) precursor fusion gene fragment, introducing a double-termination codon TAATGA at the 3' end of the gene sequence, introducing an AlwNI enzyme digestion site sequence CAGATGCTG at the 5' end of the DDDDK-peptide SEQ ID NO.19 (5-40) precursor fusion gene sequence for facilitating gene operation, introducing ML two amino acids before extending the peptide at the N end, introducing an XhoI enzyme digestion site CTCGAG at the 3' end of the DDK-peptide SEQ ID NO.19 (5-40) precursor fusion gene sequence after optimization is shown as SEQ ID NO. 43. The gene sequence was synthesized by Gene Synthesis service, TA cloned into pUC57 vector.

SEQ ID NO.43:

CAGATGCTGGATGACGATGACAAAACCTTCACCTCTGACCTGTCTATCTACAAAGAAGAACGTGCTCAGCAGGACTTCATCGAATGGCTGCTGGACGGTGGTCCGTCTTCTGGTGCTCCGCCGCCGTCTGGTTAATGACTCGAG

Plasmid pET-31b (+) (purchased from Invitrogen) was double digested with AlwNI and XhoI, which were restriction enzymes of TaKaRa, and recombinant vector pUC57-SEQ ID NO.43 was also double digested with AlwNI and XhoI. The digested DNA fragment was ligated to pET-31b (+) digested with the same enzyme, and after sequencing was confirmed to be correct, it was designated as pET31b-SEQ ID NO.19, as shown in FIG. 1.

Competent cells of E.coli BL21 (DE 3) (all from Life Technologies) were prepared according to the calcium chloride method provided in the third edition of molecular cloning laboratory Manual, published in Cold spring harbor laboratory, U.S.A.. 1 mu L of recombinant expression vector pET31b-SEQ ID NO.19 is transformed into competent cells of escherichia coli BL21 (DE 3), and the transformation method is carried out according to the third edition of calcium chloride method of molecular cloning experiment guidelines. The transformation solutions were respectively spread on LB solid medium supplemented with ampicillin (final concentration: 100. Mu.g/ml), and cultured in an inverted manner at 37℃until single colonies appeared, thus obtaining a strain pool designated BL21 (DE 3)/pET 31b-SEQ ID NO.19. Picking single colony BL21 (DE 3)/pET 31b-SEQ ID NO.19 by using a toothpick, inoculating to 50ml of LB liquid culture medium, shaking and culturing at 37 ℃ and 250rpm, inoculating 3-6 ml of bacterial liquid to 100ml of LB liquid culture medium when the bacterial liquid OD600 = 0.5-1.0, shaking and culturing at 37 ℃ and 250rpm until OD ₆₀₀ = 0.6-0.8, adding IPTG (isopropyl-beta-D-thiopyran galactoside with the final concentration of 1 mmol/L), starting induction, taking and inducing 2h of bacterial liquid 1ml, centrifuging at 12000rpm for 1 minute, removing supernatant, and storing the bacterial body at-20 ℃ for standby.

The frozen cells were removed at-20℃and resuspended in 5ml of 8M urea solution and sonicated in an ice-water mixture for 10 minutes (3 seconds with sonication, 5 seconds stopped, and the cycle thus repeated). 15. Mu.l of the crushed solution was added to 15. Mu.l of the loading buffer, and after thoroughly mixing, 10. Mu.l of SDS-PAGE was performed (wherein the concentrated gel contained 5% by volume of acrylamide-methylene bisacrylamide (29:1), and the separated gel contained 15% by volume of acrylamide-methylene bisacrylamide (29:1)). The electrophoresis condition is that the concentration gel setting current is 11mA and the separation gel setting current is 22mA. After the electrophoresis was completed, the gel was taken out, stained overnight with coomassie brilliant blue staining solution (0.6 g coomassie brilliant blue R-250,450ml ethanol, 100ml glacial acetic acid, balance purified water) and destained with destaining solution (250 ml ethanol, 80ml glacial acetic acid, balance purified water) until the background was clear. The gel was photographed against a clear background to extract an image (as shown in FIG. 2) which corresponds to the theoretical molecular weight of 18.18kDa for the KSI-DDDDK-peptide SEQ ID NO.19 (5-40) precursor fusion protein.

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

Referring first to embodiments 3,4 of patent CN201711154044, nα -hexadecyl-Glu (ONSu) -OBut is synthesized.

Referring to the embodiment 6 of the patent CN201711154044, the constructed engineering bacteria are taken for high-density fermentation, and the thallus is crushed and washed to obtain inclusion bodies. The 10L fermentation liquor finally obtains 0.62kg of inclusion bodies, and the Folin-phenol method detection shows that the protein amount of each 1g of inclusion bodies is 0.20g. 200g of the fusion protein inclusion body is taken and dissolved in 1500ml of 6mol/L guanidine hydrochloride solution, 80ml of DMSO and 15ml of N, N-diisopropylethylamine are added, the mixture is uniformly mixed, the pH value is 10.8, 80ml of DMSO solution containing 50mg/ml of N alpha-hexadecyl-Glu (ONSu) -OH is added, stirring is carried out for 3 hours, 1700ml of 20mmol/L tris (hydroxymethyl) aminomethane solution is added, 900IU of lysyl specific endonuclease is added, the pH value is adjusted to 9.0 by dilute hydrochloric acid or sodium hydroxide solution, and the pH value is adjusted to 7.8 after the reaction is carried out for 8 hours.

And the liquid was loaded onto 500ml Uni NM200 (particle size 200 μm, pore size 300 angstrom) column equilibrated with 1500ml equilibration solvent containing 20mmol/L Tris,5% isopropyl alcohol, pH7.8, followed by equilibration of the column with 1500ml equilibration solvent followed by elution with a gradient elution solvent containing 20mmol/LTris,5% -40% isopropyl alcohol pH7.8, and fractions containing peptide SEQ ID NO.19 (5-40) were collected.

To further increase purity, the above collected fractions were diluted with one volume of purified water and applied to 500ml Uni PS40 (particle size 40 μm, pore size 500 angstrom) column equilibrated with 1500ml of equilibration solvent containing 20mmol/LTris,5% isopropyl alcohol, pH7.0 followed by equilibration of the column with 1500ml equilibration solvent. And eluting with a gradient elution solvent containing 20mmol/LTris and 5% -30% isopropanol with pH of 7.0, and collecting the component containing the peptide SEQ ID NO.19 (5-40). And freeze-dried to a powder.

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

SEQ ID NO.44:

TFTSDLSIYK(γGlu-CO(CH₂)₁₄CH₃)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

Referring to patent CN201510459093, example 4, fmoc-Gly-OH, fmoc-His (Trt) -OH, fmoc-Aib-OH, boc-His (Boc) -OH were coupled in sequence to 2-chlorotrityl chloride resin, respectively, and trifluoroethanol-DCM (1:4) was added to stir the resin. The filtrate was collected, the solvent was removed in vacuo, cold diethyl ether was added, the precipitate was filtered off, washed with diethyl ether and dried in vacuo to give Boc-His (Boc) -Aib-His (Trt) -Gly-OH.

Then referring again to patent CN201510459093, example 5, to a solution of Boc-His (Boc) -Aib-His (Trt) -Gly-OH in anhydrous THF was added an amount of DIPEA and O- (N-succinimidyl) -N, N-tetramethyluronium hexafluorophosphate. Dichloromethane is added into the reaction solution, water is used for washing, and the organic phase is dried by magnesium sulfate and then is 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 patent CN201510459093, example 8, peptide SEQ ID NO 19 (5-40) (0.24 mmol,1.0 g) was dissolved in 100ml NMP, and then 300ul DIPEA and 0.4mmol Boc-His (Boc) -Aib-His (Trt) -Gly-Osu were added and the reaction stirred overnight. 1000ml of ice-cold diethyl ether was added, the precipitate was centrifuged, washed with 500ml of diethyl ether and dried under vacuum.

The crude dry powder was dissolved in TFA-triisopropylsilane-water (95:2.5:2.5, 200 ml) and stirred for 2 hours, then the solution was concentrated to about 20ml in vacuo. Then adding ice-cold diethyl ether 500ml, standing for 6-8h at 2-8 ℃ to form precipitate, and centrifuging the precipitate. The precipitate was washed with cold diethyl ether and dried under vacuum. The collected dry powder is the peptide SEQ ID NO.19 (1-40) coarse powder. The dry powder was then dissolved in 500ml of a pH 7.0 phosphate buffer containing 5% acetonitrile, purified by a preparative C8 silica gel packed column, and the peptide sample solution was injected into the treated packed column, followed by gradient elution by reference to the purification method described below.

The purification method is as follows:

The flow rate is 10.0ml/min, and the wavelength is 214nm

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

Phase B, pure acetonitrile

Gradient of 0-10 min 5-10% B phase

10-100 Min 10-70% of phase B

The collection of peptide-containing sample components was subjected to HPLC/MS detection by the following HPLC method:

The detection wavelength is 214nm, and the flow rate is 1.0ml/min;

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

gradient 0.01min 15% phase B

25.00Min 75% phase B

25.01Min 95% phase B

31.00Min 95% phase B

31.01Min 15% phase B

40.00Min 15% phase B

Mixing the above materials with purity not lower than 90%, and lyophilizing to obtain 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(CH₂)₁₄CH₃)EERAQQDFIEWL LDGGPSSGAPPPSG-OH

HPLC/MS:m/z=1561.6[M+3H]³⁺

Calculated molecular weight = 4681.2

(5) Amidation preparation of SEQ ID NO.19

The C-terminal PPPSG-OH structure of the sequence of peptide SEQ ID NO.19 (1-40) was subjected to catalytic cleavage with peptidyl glycine alpha-amidating monooxygenase (PAM) (available from Wuhan cloud cloning technologies Co., ltd.) to obtain an amidated product with a C-terminal structure of PPPS-NH ₂.

200Mg (0.04 mmol) of peptide SEQ ID NO.19 (1-40) was added to 80mL of a solution system containing 100mM MES/KOH (pH 6.0), 30mM KI,30mM KCl,1. Mu.M copper sulfate, 100ug/mL catalase, 1% (v/v) ethanol, 0.001% (v/v) Triton X-100, 10mM ascorbate, and 5ug/mLPAM was further added, incubated in a 37℃water bath for 30min, and after completion of the reaction, 6% (v/v) TFA was added to the reaction system to terminate the reaction. After completion of the reaction, the reaction mixture was diluted by one volume with a phosphate buffer solution of pH 7.0 containing 5% acetonitrile. The resulting sample was purified to a purity of greater than 95% by reference to the HPLC purification method prepared in step (4) with SEQ ID No.19 (1-40). And (3) characterizing the purified sample by an analytical HPLC/MS method to confirm the sample, thereby obtaining the peptide compound with the sequence structure of SEQ ID NO. 19.

SEQ ID NO.19:

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

HPLC/MS:m/z=1542.3[M+3H]³⁺

Calculated molecular weight = 4623.2

The peptide compounds of SEQ ID NO.15-40 can be biosynthesized by the above method, wherein the polypeptide sequence molecules with amidated C-terminal can be prepared by amidation after biosynthesized.

Example 3 stability test of compound:

The test compound was dissolved at 1mg/ml in freshly prepared PBS solution, adjusted to pH7.4 and filtered through a 0.22 μm sterile filter. The compound solution was aspirated and the peak area of 10. Mu.L of injectate was analyzed by HPLC-UV, which analysis result was the initial point of the compound stability test (T ₀).

The compound sample solution subjected to the stability test was placed in a 25 ℃ incubator and stored in a sealed light-proof state for 7 days. After the end of this procedure, the sample solution was centrifuged at 4500rpm for 10min, the supernatant solution was gently aspirated and 10. Mu.L of the peak area of the injectant was analyzed by HPLC-UV, the analysis result being the endpoint of the compound stability test (T ₇).

The residual peptide amount of the measured peptide was calculated by comparing the target peak area of the compound and the peak area of the relevant impurity at the time of T ₀ and T ₇. The calculation formula is as follows:

Residual 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 stability analysis results of peptide compounds

Example 4 determination of the Activity of peptide Compounds at GLP-1/GIP/GCG receptor:

The agonistic activity of the peptide compounds on the corresponding receptors was determined by measuring cAMP signaling responses of HEK-293 cells stably overexpressing human GLP-1, GIP, GCG receptors. Intracellular cAMP content was determined using a kit of Cisbio corp.

HEK-293 cells stably expressing human GLP-1, GIP and GCG receptors were cultured in DMEM complete medium containing 10% FBS and 2mM L-glutamine, and when the cells were grown to 80-90% density, they were digested with 0.025% trypsin, the complete medium was terminated and the cell mass was gently blown off into individual cells, the cell sap was centrifuged at 1000rpm for 5min at room temperature, the supernatant was discarded, and the cells were resuspended with 1 XHBSS (20mM HEPES,0.1%BSA,250. Mu.m IBMX) at a cell density of 1.0X10 ⁵/mL.

10. Mu.L of cell suspension was added to each well of 384 well plates. The compound to be tested is dissolved in 1 XPBS buffer solution, diluted stepwise from 100000-0.02nM by 4 times, and compound solutions of 12 concentration points are prepared in total. 100nL of the prepared compound solution is respectively added into the corresponding cell suspension of the 384-well plate by using an automatic liquid dispenser, and the mixture is uniformly mixed by rotating and shaking for 1min at 1000rpm, and then the mixture is incubated for 60min at room temperature. After the completion of the drug incubation, 10. Mu.L of the detection reagent in the kit was added to each well, and the wells were incubated at room temperature for 60 minutes. The plates were placed in an EnVision multifunctional microplate reader (Perkinelmer) to determine fluorescence readings at 665/615 nm. Compound concentration-effect curves were made using GraphPadPrism5 mapping software and EC ₅₀ values were calculated.

GLP-1 receptor agonism of the test compound is assessed for GLP-1 receptor cells by calculating the percentage of the ratio of the EC ₅₀ value of the test compound to the EC ₅₀ value of human GLP-1 as relative activity (%) using native wild-type human GLP-1, GIP, GCG as positive controls for the receptor agonism of the test compound.

For GIP receptor cells, GIP receptor agonistic activity of the test compound was evaluated by calculating the percentage of the ratio of EC ₅₀ value of the test compound to EC ₅₀ value of human GIP as relative activity (%).

For GCG receptor cells, the GCG receptor agonistic activity of the test compound was evaluated by calculating the percentage of the ratio of the EC ₅₀ value of the test compound to the EC ₅₀ value of human GCG as relative activity (%).

TABLE 3 average EC ₅₀ values and relative Activity of peptide Compounds

Each group has at least 4 independent detection data, and Rel.A. RELATIVEACTIVITY, NT nottest, n.gtoreq.4.

EXAMPLE 5 rat Pharmacokinetic (PK) study of peptide Compounds

SD rats received subcutaneous injections of 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), and cable Ma Lutai (50 nmol/kg), and after administration, SEQ ID NO:19 and liraglutide group animals were blood collected via jugular vein at time points 0.25, 0.5, 1,2, 4, 8, 12, 24, 36, 48, 56h, and SEQ ID NO:39, SEQ ID NO:40, cable Ma Lutai group animals were blood collected via jugular vein at time points 0.25, 0.5, 1,2, 4, 8, 12, 24, 48, 72, 96h, and plasma samples were obtained after blood treatment, and analyzed for plasma concentration-time curves using model Phoenix WinNonlin version software (non-chamber model), PK parameters and half-life were calculated.

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

TABLE 4 rat Pharmacokinetic (PK) parameters for test compounds

EXAMPLE 6 pharmacodynamic studies of compounds SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 on diet-induced obese (DIO) mice

Mice induced to be obese (DIO) with a high fat diet have significant metabolic syndrome characteristics similar to human body such as obesity, elevated blood glucose, insulin resistance, dyslipidemia, etc. The effect of the compounds of the present invention on DIO mice body weight, feeding, blood glucose and blood lipid etc. was studied in C57BL/6J DIO mice.

Male C57 BL/6J mice (purchased from Shanghai Laike laboratory animal Co.) of 5 weeks old were kept in a pathogen free clean environment (controlled temperature 20-24 ℃ C., relative humidity 30-70%) with 12 hours light/12 hours dark cycle, fed with normal feed, 4 animals per cage, adapted for 2 weeks. Mice were induced to be obese by feeding a high fat diet (60 kcal% calories from fat) which, after 16 weeks of feeding, reached DIO mice weighing 45-55g with a blood glucose range of 8-12mM, and DIO mice were randomly grouped (n=8) according to body weight and fasting blood glucose so that each group had a near average body weight and blood glucose. One animal per cage was fed one week after grouping, during which time each animal was given Vehicle (1×pbs,5 ml/kg) by subcutaneous injection (s.c.) for 3 days to pre-adapt the animals to the course of the procedure.

After the animal pre-adaptation was completed, animals were given Vehicle control, a dose of cable Ma Lutai or a compound of the invention, dissolved in 1 XPBS, at a dose of 5ml/kg, starting at 9:00 a.m., once daily (QD) for Vehicle, SEQ ID NO:36 and SEQ ID NO:37, and once every three days (Q3D) for cable Ma Lutai and SEQ ID NO:38 for 22 days according to experimental group subcutaneous injection. On the first administration of the first dose of compound, the acute hypoglycemic effect of the compound on DIO mice was assessed by taking blood without anesthesia from the tail of the tail, measuring with a glucometer t=0 h before administration and t=1, 2, 4, 8, 24, 48, 72h after administration (without limiting feeding and drinking). Animal body weight and food intake were measured daily throughout the trial study, and the effect of the compound on body weight and food intake changes was assessed by calculating the percent (%) change in animal body weight and cumulative food intake as compared to the initial body weight and food intake of the same animal prior to dosing.

On Day 19 of dosing (Day 19), the effect of the compound on DIO mouse glucose tolerance was determined. Animals were fasted for 5h (without limiting drinking water) after the morning dosing was completed, mice body weight and blood glucose after fasted were measured as basal blood glucose (t=0) for glucose tolerance testing, DIO mice glucose solution (2 g/kg,5 ml/kg) was then administered by oral gavage, and blood glucose levels at t=15, 30,60,90,120min after glucose solution administration were measured without anesthesia tail-spike blood sampling, and animals were fed back after the last blood sampling was completed. mmol/L is taken as the unit of blood glucose.

When the experimental end point (Day 22) was reached, the last administration operation was performed in the morning, and after the completion of the administration, fasting was performed for 5 hours, and then fasting blood glucose of the animals was measured by blood sampling without anesthesia tail tip and tail. After completion of the blood collection, the animals were anesthetized with CO ₂ and sacrificed, and then blood was collected by heart, and plasma was centrifuged for measuring total plasma cholesterol (TC), low density lipoprotein cholesterol (LDL-C), triglyceride (TG), insulin (calculation of HOMA-IR) content. The liver is separated for homogenization, and the supernatant of the homogenization is centrifugally taken and used for measuring the triglyceride content of the liver.

All results values are expressed as mean±sem and the results were tested post-hoc by Dunnett's using One-factor analysis of variance (One-wayANOVA) with GRAPHPAD PRISM software followed by comparison with the veccle control group. Differences at p <0.05 level were considered statistically significant.

TABLE 5 influence of Compounds on DIO mice weight change, cumulative food intake

* P <0.001, compared to the Vehicle control group (One-wayANOVA, dunnett's). Results are expressed as mean ± SEM of 8 animals.

TABLE 6 influence of Compounds on DIO mouse plasma TC, LDL-C, TG

* P <0.01, p <0.001, compared to the Vehicle control group (One-wayANOVA, dunnett's). Results are expressed as mean ± SEM of 8 animals.

TABLE 7 influence of Compounds on DIO mouse liver TG and HOMA-IR

* P <0.05, < p <0.01, < p <0.001, compared to the Vehicle control group (One-way ANOVA, dunnett's). MouseHOMA-IR= [ (fasting insulin (mIU/L). Times. fastingglucose (mmol/L) ]/22.5. The results are expressed as mean.+ -. SEM for 8 animals.

EXAMPLE 7 pharmacodynamic studies of compounds SEQ ID NO:19, SEQ ID NO:39, SEQ ID NO:40 on diet-induced obese (DIO) mice

Animals were subcutaneously dosed according to the experimental group (n=6) with Vehicle control Vehicle (1×pbs), with doses of compound of the invention SEQ ID No. 19, SEQ ID No. 39, SEQ ID No. 40, compound dissolved in 1×pbs, at a dose of 5ml/kg, dosing was performed starting at 9:00 a.m., compound SEQ ID No. 19 was dosed once daily (QD), compound of the invention SEQ ID No. 39, SEQ ID No. 40 was dosed once every three days (Q3D) for 14 days. Animal body weight and food intake were measured daily throughout the trial study, and the effect of the compound on body weight and food intake changes was assessed by calculating the percent (%) change in animal body weight and cumulative food intake as compared to the initial body weight and food intake of the same animal prior to dosing.

Animals were fed out at Day14 night 21:00, fasted overnight for 12 hours (no water forbidden), and after weighing animals at experimental endpoint (Day 15) 9:00 a morning, fasted blood glucose was measured by non-anesthetized tail tip tail blood sampling. Animals were then anesthetized with CO ₂ and sacrificed, blood was collected by heart, and plasma was isolated by centrifugation and used to determine plasma Total Cholesterol (TC), low density lipoprotein cholesterol (LDL-C), triglycerides (TG), insulin content (calculation of HOMA-IR). The liver is separated for homogenization, and the supernatant of the homogenization is centrifugally taken and used for measuring the triglyceride content of the liver.

All results values are expressed as mean±sem and the results were tested post-hoc by Dunnett's using One-factor analysis of variance (One-wayANOVA) with GRAPHPAD PRISM software followed by comparison with the veccle control group. Differences at p <0.05 level were considered statistically significant.

TABLE 8 influence of Compounds on DIO mice weight change, cumulative food intake

* P <0.001, compared to the Vehicle control group (One-wayANOVA, dunnett's). Results are expressed as mean ± SEM of 6 animals.

TABLE 9 influence of Compounds on DIO mouse plasma TC, LDL-C, TG

* P <0.01, p <0.001, compared to the Vehicle control group (One-wayANOVA, dunnett's). Results are expressed as mean ± SEM of 6 animals.

TABLE 10 influence of Compounds on DIO mouse liver TG and HOMA-IR

* P <0.05, < p <0.01, < p <0.001, compared to the Vehicle control group (One-way ANOVA, dunnett's). The result was expressed as mean.+ -. SEM of 6 animals using HOMA-IR= [ (fasting insulin (mIU/L). Times. fastingglucose (mmol/L) ]/22.5.

Certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims (10)

1. A compound selected from the group consisting of:

Compound 1 (SEQ ID NO: 7):

H-Aib-QGTFTSDK(γGlu-

CO(CH₂)₁₄CH₃)SKYLEEQAAQDFIEWLLAGGPSSGAPPPS-NH₂

compound 2 (SEQ ID NO: 8):

H-Aib-QGTFTSDLSKYK(γGlu-

CO(CH₂)₁₄CH₃)EEQAAQDFIEWLLAGGPSSGAPPPS-NH₂

compound 5 (SEQ ID NO: 11):

H-Aib-QGTFTSDK(γGlu-

CO(CH₂)₁₄CH₃)SKYLEEEAVRLFIEWLKAGGPSSGAPPPS-NH₂

compound 6 (SEQ ID NO: 12):

H-Aib-HGTFTSDLSKQK(γGlu-

CO(CH₂)₁₄CH₃)DERAAQDFVQWLLDGGPSSGAPPPS-NH₂

compound 7 (SEQ ID NO: 13):

H-Aib-QGTFTSDLSKYC (3-maleimidopropionyl-gamma Glu-NH (CH) ₂)₁₅CH₃)EEQAAQDFIEWLLAGGPSSGAPPPS-NH₂

Compound 8 (SEQ ID NO: 14):

H-Aib-QGTFTSDLSKYC (3-maleimidopropionyl-AEEAc-AEEAc-NH (CH) ₂)₁₅CH₃)EEQAAQDFIEWLLAGGPSSGAPPPS-NH₂

Compound 9 (SEQ ID NO: 15):

H-Aib-QGTFTSDLSRYK(γGlu-

CO(CH₂)₁₄CH₃)EEQAAQDFIEWLLAGGPSSGAPPPS-NH₂

compound 10 (SEQ ID NO: 16):

H-Aib-HGTFTSDLSIQK(γGlu-

CO(CH₂)₁₄CH₃)EERAAQDFIEWLLDGGPSSGAPPPS-NH₂

compound 11 (SEQ ID NO: 17):

H-Aib-HGTFTSDLSIQK(γGlu-

CO(CH₂)₁₄CH₃)EEQAAQDFIEWLLDGGPSSGAPPPS-NH₂

Compound 12 (SEQ ID NO: 18):

H-Aib-QGTFTSDLSIQK(γGlu-CO(CH₂)₁₄CH₃)EEQAAQDFIEWLLDGGPSSGAPPPS-NH₂

compound 13 (SEQ ID NO: 19):

H-Aib-HGTFTSDLSIYK(γGlu-CO(CH₂)₁₄CH₃)EERAQQDFIEWLLDGGPSSGAPPPS-NH₂

compound 14 (SEQ ID NO: 20):

H-Aib-HGTFTSDYSIQK(γGlu-CO(CH₂)₁₄CH₃)EEIAAQDFIEWLLDGGPSSGAPPPS-NH₂

compound 15 (SEQ ID NO: 21):

H-Aib-HGTFTSDLSIQK(γGlu-CO(CH₂)₁₄CH₃)EEIAQQDFIEWLLDGGPSSGAPPPS-NH₂

Compound 16 (SEQ ID NO: 22):

H-Aib-HGTFTSDLSK (gamma Glu-CO (CH ₂)₁₄CH₃)YLEERAQQDFIEWLLDGGPSSGAPPPS-NH₂ Compound 17 (SEQ ID NO: 23):

H-Aib-HGTFTSDLSIK (gamma Glu-CO (CH ₂)₁₄CH₃)LEERAQQDFIEWLLDGGPSSGAPPPS-NH₂ Compound 18 (SEQ ID NO: 24):

H-Aib-HGTFTSDLSIYLEK(γGlu-CO(CH₂)₁₄CH₃)RAQQDFIEWLLDGGPSSGAPPPS-NH₂

compound 19 (SEQ ID NO: 25):

H-Aib-HGTFTSDLSIYLEEK(γGlu-CO(CH₂)₁₄CH₃)AQQDFIEWLLDGGPSSGAPPPS-NH₂

Compound 20 (SEQ ID NO: 26):

H-Aib-HGTFTSDLSIYK(AEEAc-AEEAc-γGlu-CO(CH₂)₁₆CH₃)EERAQQDFIEWLLDGGPSSGAPPPS-NH₂

compound 21 (SEQ ID NO: 27):

H-Aib-HGTFTSDLSIQK(AEEAc-AEEAc-γGlu-CO(CH₂)₁₆COOH)EERAAQDFIEWLLDGGPSSGAPPPS-NH₂ Compound 22 (SEQ ID NO: 28):

H-Aib-HGTFTSDLSIYK(γGlu-γGlu-AEEAc-AEEAc-CO(CH₂)₁₆COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH₂ Compound 24 (SEQ ID NO: 30):

H-Aib-HGTFTSDLSIYK(AEEAc-AEEAc-γGlu-CO(CH₂)₁₆COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH₂

compound 25 (SEQ ID NO: 31):

H-Aib-QGTFTSDYSIQK(AEEAc-AEEAc-γGlu-CO(CH₂)₁₆COOH)EERRAQDFIEWLLDGGPSSGAPPPS-NH₂

Compound 26 (SEQ ID NO: 32):

H-Aib-HGTFTSDLSIYK(γGlu-

CO(CH₂)₁₆COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH₂

compound 28 (SEQ ID No. 34):

H-Aib-HGTFTSDLSIYK(γGlu-γGlu-AEEAc-AEEAc-γGlu-CO(CH₂)₁₆COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH₂

Compound 29 (SEQ ID No. 35):

H-Aib-HGTFTSDLSIYK(γGlu-GABa-aEEAc-γGlu-CO(CH₂)₁₆COOH)EERAQQDFIEWLLDGGPSSGAPPPS-NH₂

compound 30 (SEQ ID NO: 36):

H-Aib-HGTFTSDLSIYK(γGlu-

CO(CH₂)₁₄CH₃)EERAQQDFIEWLLDGGPSSGAPPPS-OH

compound 31 (SEQ ID NO: 37):

H-Aib-HGTFTSDYSIQK(γGlu-

CO(CH₂)₁₄CH₃)EEIAAQDFIEWLLDGGPSSGAPPPS-OH。

2. the compound of claim 1, selected from the group consisting of:

Compound 1 (SEQ ID NO: 7):

H-Aib-QGTFTSDK(γGlu-

CO(CH₂)₁₄CH₃)SKYLEEQAAQDFIEWLLAGGPSSGAPPPS-NH₂

compound 6 (SEQ ID NO: 12):

H-Aib-HGTFTSDLSKQK(γGlu-

CO(CH₂)₁₄CH₃)DERAAQDFVQWLLDGGPSSGAPPPS-NH₂

compound 7 (SEQ ID NO: 13):

H-Aib-QGTFTSDLSKYC (3-maleimidopropionyl-gamma Glu-NH (CH) ₂)₁₅CH₃)EEQAAQDFIEWLLAGGPSSGAPPPS-NH₂

Compound 9 (SEQ ID NO: 15):

H-Aib-QGTFTSDLSRYK(γGlu-CO(CH₂)₁₄CH₃)EEQAAQDFIEWLLAGGPSSGAPPPS-NH₂

compound 10 (SEQ ID NO: 16):

H-Aib-HGTFTSDLSIQK(γGlu-CO(CH₂)₁₄CH₃)EERAAQDFIEWLLDGGPSSGAPPPS-NH₂

compound 11 (SEQ ID NO: 17):

H-Aib-HGTFTSDLSIQK(γGlu-CO(CH₂)₁₄CH₃)EEQAAQDFIEWLLDGGPSSGAPPPS-NH₂

Compound 12 (SEQ ID NO: 18):

H-Aib-QGTFTSDLSIQK(γGlu-CO(CH₂)₁₄CH₃)EEQAAQDFIEWLLDGGPSSGAPPPS-NH₂

compound 13 (SEQ ID NO: 19):

H-Aib-HGTFTSDLSIYK(γGlu-CO(CH₂)₁₄CH₃)EERAQQDFIEWLLDGGPSSGAPPPS-NH₂

compound 14 (SEQ ID NO: 20):

H-Aib-HGTFTSDYSIQK(γGlu-CO(CH₂)₁₄CH₃)EEIAAQDFIEWLLDGGPSSGAPPPS-NH₂

compound 15 (SEQ ID NO: 21):

H-Aib-HGTFTSDLSIQK(γGlu-CO(CH₂)₁₄CH₃)EEIAQQDFIEWLLDGGPSSGAPPPS-NH₂

compound 21 (SEQ ID NO: 27):

H-Aib-HGTFTSDLSIQK(AEEAc-AEEAc-γGlu-CO(CH₂)₁₆COOH)EERAAQDFIEWLLDGGPSSGAPPPS-NH_{2 .}

3. the compound of claim 1, selected from the group consisting of:

compound 13 (SEQ ID NO: 19):

H-Aib-HGTFTSDLSIYK(γGlu-CO(CH₂)₁₄CH₃)EERAQQDFIEWLLDGGPSSGAPPPS-NH₂

compound 30 (SEQ ID NO: 36):

H-Aib-HGTFTSDLSIYK(γGlu-CO(CH₂)₁₄CH₃)EERAQQDFIEWLLDGGPSSGAPPPS-OH

compound 31 (SEQ ID NO: 37):

H-Aib-HGTFTSDYSIQK(γGlu-

CO(CH₂)₁₄CH₃)EEIAAQDFIEWLLDGGPSSGAPPPS-OH 。

4. a pharmaceutical composition comprising an effective amount of a compound of any one of the preceding claims, or a salt or solvate thereof, and a pharmaceutically acceptable adjuvant, diluent, carrier or excipient.

5. The pharmaceutical composition according to claim 4, which is in the form of an injection or a lyophilized powder, a tablet, a pill, a lozenge, a soft capsule, a hard capsule, a granule, a powder, a solution, a suspension or a syrup, or in the form of a microcapsule, a microsphere, a nanoparticle or a liposome.

6. The pharmaceutical composition according to claim 4 for oral administration, inhaled administration or parenteral administration selected from intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous or intradermal injection administration.

7. The pharmaceutical composition of claim 4, which is administered at a frequency of at least once a day, once a week, or once a month.

8. Use of a compound according to any one of claims 1 to 3, or a salt or solvate thereof, for the manufacture of a medicament for the prevention or treatment of a metabolic abnormality syndrome selected from hyperglycemia, insulin resistance, glucose intolerance, type II 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 parkinsonism.

9. A process for preparing a compound according to any one of claims 1 to 3, which is a chemical synthesis process.

10. A process for preparing the compound of claim 3, said process being a biological semisynthetic process.