Long-acting GGG three-target agonist

Abstract

The invention discloses a long-acting GGG three-target agonist, and the compound provided by the invention simultaneously achieves nM-level excellent agonist ability on three targets of GLP-l / GIP / GCGR, and can fully excite corresponding target organ tissue cells. The compound provided by the invention can promote insulin secretion and reduce blood sugar; in addition, ingestion can be inhibited, gastric emptying can be delayed, energy consumption can be increased, and finally the weight reduction effect can be observed. The compound provided by the invention can reduce pancreatic beta-cell apoptosis, increase the quantity of pancreatic beta-cells and improve functions of pancreatic cells. The compound disclosed by the invention can also improve blood fat, reduce liver fat accumulation and inhibit liver inflammation development. The compound or the composition can be used for preventing and / or treating metabolic disorder diseases and related complications thereof. The compound or the composition can be used for treating bone diseases related to endocrine diseases, metabolic disorders, nephropathy and the like, such as osteoporosis and osteoarthritis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a long-acting GGG triple-target agonist. Background Art

[0002] GLP-1R (glucagon-like peptide-1 receptor) agonists have always been a hot research and development topic in the field of diabetes, and have become the non-insulin diabetes drug with the highest market share in the world. Many star products such as dulaglutide and semaglutide have performed well in 2021. Therefore, the continued development of iterative products - multi-target agonists of GLP-1R has also become a hot competitive direction.

[0003] Currently, most of the multi-target agonists under development worldwide are concentrated in GLP-1R / GIPR and GLP-1R / GCGR. Among them, Eli Lilly's Tirzepatide leads the GLP-1R / GIPR field and has been approved by the FDA for marketing in the United States. Among domestic companies, Hausen Pharmaceuticals and Hongyun Huaning have their own products in Phase I clinical trials, and Heze Pharmaceuticals and Borri Bio have also applied for IND respectively.

[0004] In contrast, the competition in GLP-1R / GCGR is more intense. Multinational companies such as AstraZeneca, Eli Lilly, and Boehringer Ingelheim have all made plans, among which AstraZeneca has taken the lead in launching Phase III clinical trials. Innovent Biologics is the fastest-progressing domestic company. IBI362, jointly promoted by it and Eli Lilly, has conducted Phase II clinical trials for the treatment of type 2 diabetes and obesity respectively; in addition, Salubris, Page Pharmaceuticals, Tuwei Anchuang and Hengrui Medicine also have their own products in Phase I clinical trials.

[0005] Not only dual-target, GLP-1R / GIPR / GCGR triple-target agonists may also show stronger efficacy. Eli Lilly and Hanmi Pharmaceutical have advanced their products to Phase II clinical trials. In addition, GLP-1R also has the possibility of combining with GCGR, FGF21R, GLP-2R, etc. to form a single-molecule dual-receptor or triple-receptor agonist.

[0006] Studies have shown that although single glucagon GCG receptor drugs can effectively reduce blood sugar and glycosylated hemoglobin before and after meals in patients with type 2 diabetes, they are also accompanied by adverse reactions such as increased blood lipids and liver transaminases. When GLP-1R and GCGR are developed and used as co-agonists, they can not only lower blood sugar, but also effectively reduce these adverse reactions. The successful development of various GLP-1 / GCG and GLP-1 / GIP dual receptor agonists has also triggered research on single-molecule agonists that simultaneously stimulate these three target receptors. Summary of the invention

[0007] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0008] The present invention provides a GLP1R-GIPR-GCGR triple-target agonist peptide or a pharmaceutically acceptable salt thereof, wherein the agonist peptide or a pharmaceutically acceptable salt thereof comprises an amino acid sequence or consists of an amino acid sequence, and the amino acid sequence general formula I is as follows:

[0009] X1-Aib-QGTFTSDYSI-aMeL-LDK-X 17 -AQ-Aib-X 21 -FX 23 -EYLLE-XX1-R 1 ,

[0010] in,

[0011] X1 is H or Y;

[0012] X 17 is Ψ, Ψ is Lys with modified side chain;

[0013] X 21 is D or A;

[0014] X 23 V or I;

[0015] XX1 is GGPSSGAPPPSKVSRA (SEQ ID NO:1), GGGPSSGAPPPSKVSRA (SEQ ID NO:2), GGPSSGA d PPPSKVSRA(SEQ ID NO:3),GGGPSSGA d PPPSKVSRA (SEQ ID NO: 4), GGPSSGAPPPS (SEQ ID NO: 9), GGGPSSGAPPPS (SEQ ID NO: 10), GGPSSGA d PPPS (SEQ ID NO: 11), or GGGPSSGA d PPPS (SEQ ID NO: 12);

[0016] R1 is NH2 or OH, or a pharmaceutically acceptable salt and / or ester thereof.

[0017] Furthermore, the Ψ is a Lys with a side chain modified by a structure having the following general formula II, wherein the general formula II is: BZ; wherein B is (AEEA or Glu) a -(AEEA or Glu) b -(AEEA or Glu) c, wherein a, b, and c are each independently 0 or 1, and a, b, and c are not 0 at the same time; the carboxyl end of B is connected to the ε-amino group of the side chain of Lys, and Z is -CO-(CH2) m -R 2 , m is an integer between 6 and 24, R 2 Selected from -COOH.

[0018] Further, the general formula II is AEEA-γG1u-CO(CH2) 18 COOH.

[0019] Furthermore, at least one site on the agonist peptide or its pharmaceutically acceptable salt is chemically modified by a fatty acid side chain group.

[0020] Furthermore, the agonist peptide or a pharmaceutically acceptable salt thereof is selected from:

[0021] H-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-DFVEYLLE-GGPSSGAPPPSKVSRA-NH2, named ZG1;

[0022] H-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-AFIEYLLE-GGGPSSGAPPPSKVSRA-NH2, named ZG2;

[0023] Y-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-DFVEYLLE-GGPSSGAPPPS-NH2, named ZG3;

[0024] Y-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-AFIEYLLE-GGGPSSGAPPPS-NH2, named ZG4;

[0025] H-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-DFVEYLLE-GGPSSGA d PPPSKVSRA-NH2, named ZG5;

[0026] H-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-AFIEYLLE-GGGPSSGA d PPPSKVSRA-NH2, named ZG6;

[0027] Y-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-DFVEYLLE-GGPSSGA d PPPS-NH2, named ZG7;

[0028] Y-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-AFIEYLLE-GGGPSSGA d PPPS-NH2, named ZG8.

[0029] The amino acid sequence QGTFTSDYSI was named SEQ ID NO: 5; the amino acid sequence DFVEYLLE was named SEQ ID NO: 6; the amino acid sequence AFIEYLLE was named SEQ ID NO: 7; and the amino acid sequence LDKK was named SEQ ID NO: 8.

[0030] Furthermore, the agonist peptide or its pharmaceutically acceptable salt has a relative activity of at least 30% higher than that of natural GLP-1 in agonist ability of cells transfected with GLP-1 receptors. Furthermore, at least 60% higher. Furthermore, at least 80% higher. Furthermore, at least 100% higher.

[0031] Furthermore, the agonist peptide or its pharmaceutically acceptable salt has a relative activity of at least 100% higher than that of natural GIP in agonizing cells transfected with GIP receptors, and further, at least 150% higher.

[0032] Furthermore, the agonist peptide or its pharmaceutically acceptable salt has a relative activity of at least 10% higher than that of natural GCG in agonizing cells transfected with GCG receptors, and further, at least 30% higher.

[0033] In a specific embodiment of the present invention, the agonist peptide or its pharmaceutically acceptable salt has an agonist ability in cells transfected with GLP-1 receptors, and the relative activity of the agonist peptide or its pharmaceutically acceptable salt on GLP-1 receptor agonism is increased by at least 30%-100% compared with natural GLP-1. Specifically, the relative activity of the agonist peptide or its pharmaceutically acceptable salt on GLP-1 receptor agonism is increased by at least 30%, 60%, 80%, 100%.

[0034] In a specific embodiment of the present invention, the agonist peptide or its pharmaceutically acceptable salt has an agonist ability in cells transfected with GIP receptors, and the relative activity of the agonist peptide or its pharmaceutically acceptable salt on GIP receptor agonism is at least increased by 100%-150% compared with natural GIP.

[0035] In a specific embodiment of the present invention, the agonist peptide or its pharmaceutically acceptable salt has an agonist ability in cells transfected with GCG receptors, and the relative activity of the agonist peptide or its pharmaceutically acceptable salt on GCG receptor agonism is at least increased by 10%-30% compared with natural GCG.

[0036] In a specific embodiment of the present invention, the relative activity of the agonist peptide or its pharmaceutically acceptable salt on 3T3-L1 cells, representative cells of adipose tissue, is increased by at least 60%-100% compared to natural GIP.

[0037] In a specific embodiment of the present invention, the relative activity of the agonist peptide or its pharmaceutically acceptable salt on human primary hepatocytes, which are representative cells of liver tissue, is increased by at least 60%-100% compared to natural GCG.

[0038] In a specific embodiment of the present invention, the peptide backbone of the agonist peptide or its pharmaceutically acceptable salt is chemically modified by a fatty acid side chain group at least at one site. More specifically, the agonist peptide or its pharmaceutically acceptable salt may have a stable peptide α-helical structure and may have enhanced albumin binding ability, that is, the stability of the peptide compound may be improved and the duration of peptide action may be prolonged.

[0039] The present invention provides a method for preparing the aforementioned agonist peptide or a pharmaceutically acceptable salt thereof by solid phase synthesis, wherein the method comprises the following steps:

[0040] 1) Resin pretreatment: Add the resin to a reactor, add DMF, stir and swell with nitrogen, filter and wash;

[0041] 2) Prepare amino acid solution: dissolve Fmoc-Ser-tBu-OH and HOBt in DMF, then add DIC and mix well;

[0042] 3) Deprotection: Add 20% piperidine / DMF solvent to the reaction kettle, stir and wash with nitrogen;

[0043] 4) Coupling reaction: add the amino acid solution prepared in step 2) to the reaction kettle, control the temperature and stir with nitrogen. After the reaction is completed, wash the resin with DMF, repeat step 4), and sequentially couple the corresponding protected amino acids according to the amino acid sequence of the agonist peptide or its pharmaceutically acceptable salt described above until the main chain of the agonist peptide is synthesized;

[0044] 5) Removal of the 17-position Lys side chain protecting group iLEde: Add 8% hydrazine hydrate / DMF solution to the reaction kettle, stir with nitrogen, and after the reaction is complete, remove the solvent by suction filtration and wash;

[0045] 6) Lys side chain modification: Eicosanedioic acid (mon-tBu) -γGlu (α-OtBu) -AEEA-OH and HOBt were dissolved in DMF, and DIC was added to mix to prepare a side chain modification solution, which was added to a reaction kettle, and the temperature was controlled and nitrogen was stirred. After the reaction was completed, the solution was washed with DMF, then with DCM, and then with methyl tert-butyl ether to obtain a peptide resin containing the aforementioned agonist peptide or a pharmaceutically acceptable salt thereof, and then dried.

[0046] Furthermore, the swelling time in step 1) is 30 minutes.

[0047] Furthermore, in the step 2), the dissolution ratio of Fmoc-Ser-tBu-OH in DMF is 5.75 g / 75 mL.

[0048] Furthermore, in the step 2), the dissolution ratio of HOBt in DMF is 2.02 g / 75 mL.

[0049] Furthermore, in the step 2), the dissolution ratio of DIC in DMF is 1.89 g / 75 mL.

[0050] Furthermore, the ratio of the amount of the 20% piperidine / DMF solvent added in step 3) to the amount of the resin added in step 1) is 100 mL / 13.15 g.

[0051] Furthermore, in step 3), the stirring time of nitrogen gas passage is 35 minutes.

[0052] Furthermore, after washing in step 3), a ninhydrin reagent is used to detect whether the resin is positive.

[0053] Furthermore, the ratio of the amount of the amino acid solution added in step 4) to the amount of the resin added in step 1) is 100 mL / 13.15 g.

[0054] Furthermore, in step 4), the temperature range of the temperature-controlled nitrogen stirring is 25-35°C.

[0055] Furthermore, the sign of completion of the reaction in step 4) is that the resin is negative when tested with a ninhydrin reagent.

[0056] Furthermore, the ratio of the amount of the 8% hydrazine hydrate / DMF solution in step 5) to the amount of the resin added in step 1) is 100 mL / 13.15 g.

[0057] Furthermore, the stirring time of nitrogen in step 5) is 1 hour.

[0058] Furthermore, after washing in step 5), a ninhydrin reagent is used to detect whether the resin is positive.

[0059] Furthermore, in the step 6), the dissolution ratio of Eicosanedioic acid (mon-tBu)-γGlu (α-OtBu)-AEEA-OH in DMF is 7.29 g / 150 mL.

[0060] Furthermore, in the step 6), the dissolution ratio of HOBt in DMF is 1.35 g / 150 mL.

[0061] Furthermore, in the step 6), the dissolution ratio of DIC in DMF is 1.30 g / 150 mL.

[0062] Furthermore, the ratio of the amount of the side chain modification solution in step 6) to the amount of the resin in step 1) is 150 mL / 13.15 g.

[0063] Furthermore, in step 6), the temperature range of the temperature-controlled nitrogen stirring is 25-35°C.

[0064] Furthermore, the sign of completion of the reaction in step 6) is that the resin is negative when detected by ninhydrin reagent.

[0065] The present invention provides a post-treatment method after solid phase synthesis of the aforementioned agonist peptide or a pharmaceutically acceptable salt thereof, the post-treatment method comprising the following steps:

[0066] 1) taking the peptide resin containing the agonist peptide or its pharmaceutically acceptable salt finally synthesized by the method described above, adding a cutting liquid, stirring the reaction, filtering, adding the filtrate to ice methyl tert-butyl ether for precipitation, suction filtering, washing the filter cake with methyl tert-butyl ether, and vacuum drying to obtain a crude product;

[0067] 2) The crude product obtained in step 1) is passed through a refining and purification system to obtain a purified product.

[0068] Furthermore, in step 1), the volume ratio of the cutting fluid is TFA / TIS / DTT / H2O=90 / 2.5 / 2.5 / 5.

[0069] Furthermore, the stirring reaction time in step 1) is 3 hours.

[0070] Furthermore, in the step 1), the volume ratio of the filtrate to the glacial methyl tert-butyl ether is 1:5.

[0071] Furthermore, the refining and purification system in step 2) is a C18 reverse phase preparative chromatography system.

[0072] Furthermore, the purity of the purified product in step 2) is not less than 90%.

[0073] The present invention provides a product, which comprises the reagent used in the above-mentioned method and / or the reagent used in the above-mentioned post-treatment method.

[0074] Furthermore, the product includes a kit.

[0075] The present invention provides a pharmaceutical composition, which comprises a therapeutically effective amount or a preventively effective amount of the aforementioned agonist peptide or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.

[0076] Furthermore, the pharmaceutical composition may also include other active pharmaceutical ingredients.

[0077] Furthermore, the pharmaceutical composition is an injection, lyophilized powder, tablet, pill, lozenge, soft capsule, hard capsule, granule, powder, solution, suspension, or syrup.

[0078] Furthermore, the pharmaceutical composition is administered orally, by inhalation or parenterally.

[0079] Furthermore, the parenteral administration is selected from intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous or intradermal injection.

[0080] Furthermore, the therapeutically effective amount or preventively effective amount needs to be administered at least once a week or once every two weeks.

[0081] Furthermore, the other active pharmaceutical ingredients include anti-diabetic active agents, GIP receptor agonists, GCG receptor agonists or antagonists, GLP-1 / GIP receptor agonists, GLP-1 / GCG receptor agonists, GIP / GCG receptor agonists, FGF-21 and its analogs, CCKB and its analogs, PYY and its analogs, leptin and its analogs, calcitonin and its analogs, lipid-regulating active drugs, PPAR-α, β, δ agonists or regulators, anti-platelet aggregation active agents, PCSK9 inhibitors, lipase inhibitors, anti-liver fibrosis or cirrhosis active agents, and anti-inflammatory active agents.

[0082] Furthermore, the diabetes active agents include insulin and its analogs, biguanides, sulfonylureas, thiazolidinediones, X-glucosidase inhibitors, DPP-4 inhibitors, SGLT2 inhibitors, dual SGLT1 / SGLT2 inhibitors, GLP-1 receptor agonists, amylin and its analogs.

[0083] The present invention provides any of the following applications:

[0084] 1) Use of the aforementioned agonist peptide or its pharmaceutically acceptable salt or the aforementioned pharmaceutical composition in the preparation of a drug for promoting insulin secretion and lowering blood sugar.

[0085] 2) Use of the aforementioned agonist peptide or its pharmaceutically acceptable salt or the aforementioned pharmaceutical composition in the preparation of a drug for inhibiting food intake, delaying gastric emptying, increasing energy consumption and reducing body weight.

[0086] 3) Use of the aforementioned agonist peptide or its pharmaceutically acceptable salt or the aforementioned pharmaceutical composition in the preparation of a drug for reducing pancreatic β-cell apoptosis, increasing the number of pancreatic β-cells, and improving pancreatic cell function.

[0087] 4) Use of the aforementioned agonist peptide or its pharmaceutically acceptable salt or the aforementioned pharmaceutical composition in the preparation of a drug for improving blood lipids, reducing liver fat accumulation, and inhibiting the development of liver inflammation.

[0088] 5) Use of the aforementioned agonist peptide or its pharmaceutically acceptable salt or the aforementioned pharmaceutical composition in the preparation of drugs that promote the growth of brain neurons, remove neurotoxic substances, inhibit the development of inflammation, and play a neuroprotective role.

[0089] 6) Use of the aforementioned agonist peptide or its pharmaceutically acceptable salt or the aforementioned pharmaceutical composition in the preparation of a drug for preventing and / or treating metabolic disorders.

[0090] Furthermore, the metabolic disorders include diabetes, obesity, non-alcoholic fatty liver disease, and dyslipidemia.

[0091] 7) Use of the aforementioned agonist peptide or its pharmaceutically acceptable salt or the aforementioned pharmaceutical composition in the preparation of a drug for treating neurodegenerative diseases.

[0092] Furthermore, the neurodegenerative diseases include Parkinson's disease and Alzheimer's disease.

[0093] 8) Use of the aforementioned agonist peptide or its pharmaceutically acceptable salt or the aforementioned pharmaceutical composition in the preparation of a drug for treating bone diseases.

[0094] Furthermore, the bone diseases include bone diseases caused by endocrine diseases, metabolic disorders, and kidney diseases.

[0095] Furthermore, the bone diseases include osteoporosis and osteoarthritis.

[0096] In a specific embodiment of the present invention, the aforementioned agonist peptide or its pharmaceutically acceptable salt can be prepared by solid phase synthesis.

[0097] Beneficial effects of the present invention:

[0098] The compounds of the present invention simultaneously achieve excellent stimulatory ability at the nM level for the three targets of GLP-1 / GIP / GCGR, and can fully stimulate the corresponding target organ tissue cells. The compounds of the present invention can promote insulin secretion and lower blood sugar; they can also inhibit food intake, delay gastric emptying, increase energy consumption, and ultimately observe a weight loss effect. The compounds of the present invention can reduce pancreatic β-cell apoptosis, increase the number of pancreatic β-cells, and improve pancreatic cell function. The compounds of the present invention can also improve blood lipids, reduce liver fat accumulation, and inhibit the development of liver inflammation. The compounds or compositions of the present invention can be used to prevent and / or treat metabolic disorders and their related complications. The compounds or compositions of the present invention can be used to treat bone diseases related to endocrine diseases, metabolic disorders, kidney diseases, etc., such as osteoporosis and osteoarthritis.

[0099] Compared with existing compounds, the GLP-1 / GIP / GCGR triple agonist polypeptide molecule provided by the present invention has better GLP-1R, GIPRs, and GCGR agonist activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 is the staining diagram of glucose consumption of each group of peptide compounds;

[0101] Figure 2 This is the Oil Red O staining image of mouse 3T3-L1;

[0102] Figure 3 It is a graph showing changes in blood sugar levels over time;

[0103] Figure 4 It is a curve chart of the change of mouse weight and time;

[0104] Figure 5 is a graph of CK content in serum of each group of peptide compounds;

[0105] Figure 6 is a graph of the ALT content in serum of each group of peptide compounds;

[0106] Figure 7 It is a statistical chart of the stability of each group of peptide compounds in serum. DETAILED DESCRIPTION

[0107] The amino acids in the compound sequence of the present invention are derived from natural amino acids or related amino acid variants and / or derivatives. The abbreviations and codes of the natural amino acids adopt the general rules well known to people in the industry. The chemical structure of Aib, αMel is as follows:

[0108]

[0109] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as are commonly understood by those skilled in the art. In the event of a conflict, the present document, including definitions, shall prevail. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used to implement or test the present invention. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0110] The compounds of the present invention have agonist activity on GLP-1 receptor, GIP receptor and GCG receptor. The "agonist activity" means that the compounds can stimulate specific receptor cells to produce cAMP. The cells used can be host cells or pancreatic tissue cells, adipocytes, hepatocytes, etc. that overexpress GLP-1 receptor, GIP receptor or GCG receptor constructed by those skilled in the art. The receptor agonist activity can be achieved by using ECs that the compounds can stimulate receptor cells to produce CAMP. 50 The value is used as a measure. 50 The value refers to the drug concentration required to achieve half of the maximal activity (50% activity) of the compound in a specific assay system.

[0111] As used herein, "effective amount" refers to the amount, concentration or dosage of one or more agonist peptides described herein or a pharmaceutically acceptable salt thereof that provides the desired effect in a subject being diagnosed or treated when administered in single or multiple doses to such subject in need thereof. The effective amount can be readily determined by one skilled in the art using known techniques and by observing the results obtained under similar circumstances. In determining the effective amount for an individual, many factors are considered, including, but not limited to, the species of the mammal; its size, age and general health; the specific disease or disorder involved; the extent or severity of the disease or disorder; the response of the individual patient; the mode of administration; the bioavailability characteristics of the administration formulation; the selected dosing regimen; the use of concomitant drug therapy; and other relevant circumstances.

[0112] For a human subject of about 50-70 kg, the unit dose of the pharmaceutical composition of the present invention or combination may contain about 1-1000 mg active ingredient (one or more), or about 1-500 mg, or about 1-250 mg, or about 1-150 mg, or about 0.5-100 mg, or about 1-50 mg active ingredient. The therapeutically effective dose of the compound, pharmaceutical composition, or combination thereof depends on the type, body weight, age, and individual condition of the subject, the disorder or disease treated, or its severity. A physician, clinician, or veterinarian of ordinary skill can easily determine the effective amount of each active ingredient necessary for preventing, treating, or inhibiting the progression of a condition or disease.

[0113] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise", "include" and other variations should be understood to imply the inclusion of the member, integer or step or the group of members, integers or steps, but do not exclude any other member, integer or step or the group of members, integers or steps, but in some embodiments, such other members, integers or steps or the group of members, integers or steps can be excluded, that is, the subject is to include the member, integer or step or the group of members, integers or steps. Unless otherwise indicated herein or clearly contradictory to the context, the terms "one / a kind" and "said" used in the context of describing the present invention (especially in the context of the claims) should be interpreted as covering the singular and plural at the same time. The range of values ​​listed in this article is intended only to be used as a shorthand method for individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into this specification, as if the value is listed separately in this article. Unless otherwise indicated herein or clearly contradictory to the context, all methods described herein can be carried out in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and does not impose any limitation on the scope of the present invention as originally claimed. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the present invention.

[0114] As used herein, the term "peptide" generally refers to an amino acid whose polymeric form of any length, which for example comprises about two or more, or about 3 or more, or about 4 or more, or about 6 or more, or about 8 or more, or about 9 or more, or about 10 or more, or about 13 or more, or about 16 or more, or about 21 or more amino acids covalently linked by peptide bonds. Peptide can for example be made up of up to 100 amino acids. As used herein, the term "polypeptide" refers to a large peptide. In one embodiment, the term "polypeptide" refers to a peptide having more than about 100 amino acid residues. The terms "polypeptide" and "protein" are used interchangeably in this article.

[0115] As used herein, the term "amino acid" or "amino acid residue" refers to naturally occurring amino acids, non-natural amino acids that function in a manner similar to the naturally occurring amino acids, amino acid analogs and / or amino acid mimetics, in their D and L stereoisomeric forms, if their structure permits such stereoisomeric forms. Amino acids are referred to herein by their names, their three letter symbols as known in the art, or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0116] As used herein, the terms "naturally occurring", "naturally" when used in conjunction with biological materials (such as nucleic acid molecules, (poly)peptides, host cells, etc.) refer to materials found in nature and have not been manipulated by humans.

[0117] When used in conjunction with amino acids, the terms "naturally occurring" and "natural" refer to the 20 conventional amino acids (i.e., alanine (Ala or A), cysteine ​​(Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y)), as well as selenocysteine, pyrrolysine (PYL), and pyrroline-carboxylysine (PCL).

[0118] As used herein, the term "non-natural amino acid" is intended to refer to an amino acid that is not naturally encoded or found in the genetic code of any organism. It can, for example, be a purely synthetic compound. Examples of non-natural amino acids include, but are not limited to, hydroxyproline, γ-carboxyglutamate, O-phosphoserine, azetidine carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N- Ethylglycine, N-methylglycine, N-ethylasparagine, homoproline, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthylalanine, norvaline, norleucine, ornithine, D-ornithine, D-arginine, D-alanine, p-aminophenylalanine, pentylglycine, pipecolic acid, and thioproline.

[0119] As used herein, the term "amino acid analogue" refers to a compound having the same basic chemical structure as a naturally occurring amino acid. Amino acid analogues include natural amino acids and non-natural amino acids, which are chemically blocked reversibly or irreversibly, or chemically modified, for example, at one or any combination of its C-terminal carboxyl group, its N-terminal amino group and / or its side chain functional groups. Such analogs include, but are not limited to, methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine ​​sulfoxide, S-(carboxymethyl)-cysteine ​​sulfone, aspartic acid-(β methyl ester), N-ethylglycine, alanine carboxamide, homoserine, norleucine and methionine methyl sulfonium.

[0120] The compounds of the present invention can form acid and / or base salts due to the presence of amino and / or carboxyl groups or similar groups. As used herein, the term "salt" or "salts" refers to acid addition salts or base addition salts of the compounds of the present invention. "Salt" particularly includes "pharmaceutically acceptable salts" and "pharmaceutically acceptable salts". The term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the compounds of the present invention and are generally biologically or otherwise desirable. In particular, salts prepared from pharmaceutically acceptable non-toxic bases or acids. When the compound provided by the present invention is an acid, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from inorganic bases include salts such as aluminum, ammonium, calcium, copper (high and low valence), ferric iron, ferrous iron, lithium, magnesium, manganese (high and low valence), potassium, sodium, zinc, etc. Ammonium, calcium, magnesium, potassium and sodium salts are particularly preferred. Pharmaceutically acceptable non-toxic organic bases capable of being derived into salts include primary amines, secondary amines and tertiary amines, as well as cyclic amines and amines containing substituents, such as naturally occurring and synthetic amines containing substituents. Other pharmaceutically acceptable non-toxic organic bases capable of forming salts include ion exchange resins and arginine, betaine, caffeine, choline, N', N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, reduced glucosamine, glucosamine, histidine, halamine, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

[0121] When compound provided by the invention is alkali, can be from pharmaceutically acceptable nontoxic acid, comprise inorganic acid and organic acid, conveniently make its corresponding salt.Such acid comprises inorganic acid and organic acid, for example, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, formic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid and p-toluenesulfonic acid etc.Preferably, inorganic acid is hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid etc., organic acid is citric acid, lactic acid, malic acid, gluconic acid, tartaric acid, adipic acid, acetic acid, succinic acid, fumaric acid, aseptic acid, itaconic acid, methanesulfonic acid or benzenesulfonic acid etc. Since the compound represented by formula (I) is to be used as a medicine, it is preferably used with a certain purity, for example, at least 60% purity, more preferably at least 75% purity, and particularly preferably at least 98% purity (% is weight ratio).

[0122] Pharmaceutically acceptable salts of the present invention can be synthesized by alkaline or acidic moieties by conventional chemical methods. Usually, these salts can be prepared by reacting the free acid form of these compounds with the appropriate alkali (e.g. Na, Ca, Mg or K, hydroxide, carbonate, bicarbonate etc.) of stoichiometric amount, or by reacting the free alkali form of these compounds with the appropriate acid of stoichiometric amount. Such reactions are usually carried out in water or in an organic solvent or in a mixture of the two. Usually, in feasible situations, it is relatively ideal to use non-aqueous media such as ether, ethyl acetate, tetrahydrofuran, toluene, chloroform, methylene dichloride, methanol, ethanol, isopropanol or acetonitrile.

[0123] The term "administering" refers to the administration of compounds disclosed in the present invention that can treat various diseases, or compounds that are not explicitly disclosed but can be converted into compounds disclosed in the present invention in vivo after administration to a subject. Conventional methods for selecting and preparing suitable prodrug derivatives are described in books such as Design of Prodrugs, ed. H. Bundgaard, Elsevier, 1985.

[0124] The compounds of the present invention may contain one or more asymmetric centers and may thus produce diastereomers and optical isomers. The present invention includes all possible diastereomers and racemic mixtures thereof, their substantially pure resolved enantiomers, all possible geometric isomers and pharmaceutically acceptable salts thereof.

[0125] In the present invention, the term "composition" refers to a product comprising a specified amount of each specified component, and any product produced directly or indirectly by a combination of specified amounts of each specified component. Therefore, pharmaceutical compositions containing the compounds of the present invention as active ingredients and methods for preparing the compounds of the present invention are also part of the present invention. In addition, some crystalline forms of the compounds may exist in polymorphic forms, and such polymorphic forms are included in the present invention. In addition, some compounds may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates also fall within the scope of the present invention.

[0126] According to conventional drug mixing techniques, the agonist peptide compound shown in formula I of the present invention, or a drug precursor, or a metabolite, or a pharmaceutically acceptable salt, can be used as an active ingredient and mixed with a drug carrier to form a pharmaceutical composition. The drug carrier can take a variety of forms, depending on the desired mode of administration, for example, oral or injection (including intravenous injection). Therefore, the pharmaceutical composition of the present invention can be a separate unit suitable for oral administration, such as a capsule, cachet or tablet containing a predetermined dose of the active ingredient. Further, the pharmaceutical composition of the present invention can be in the form of a powder, granule, solution, aqueous suspension, non-aqueous liquid, water-in-oil emulsion, or oil-in-water emulsion. In addition, in addition to the common dosage forms mentioned above, the compound shown in formula (I) or its pharmaceutically acceptable salt can also be administered by controlled release and / or delivery device. The pharmaceutical composition of the present invention can be prepared by any pharmaceutical method. Generally, this method includes the step of associating the active ingredient with a carrier constituting one or more necessary ingredients. Generally, the pharmaceutical composition is prepared by uniformly and closely mixing the active ingredient with a liquid carrier or a finely divided solid carrier or a mixture of the two. In addition, the product can be easily prepared into a desired appearance.

[0127] Specifically, the drug carrier used in the present invention can be, for example, a solid carrier, a liquid carrier or a gas carrier. Solid carriers include, but are not limited to, lactose, gypsum powder, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid. Liquid carriers include, but are not limited to, syrup, peanut oil, olive oil and water. Gas carriers include, but are not limited to, carbon dioxide and nitrogen. When preparing oral pharmaceutical preparations, any pharmaceutically convenient medium can be used. For example, water, ethylene glycol, oils, alcohols, flavor enhancers, preservatives, colorants, etc. can be used for oral liquid preparations such as suspensions, elixirs and solutions; and carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, adhesives, disintegrants, etc. can be used for oral solid preparations such as powders, capsules and tablets. Considering the ease of administration, tablets and capsules are preferred for oral preparations, and solid pharmaceutical carriers are used here. Optionally, tablet coating can use standard aqueous preparation or non-aqueous preparation technology.

[0128] The abbreviations used in the present invention have the following specific meanings:

[0129] Aib: a-amino isobutyric acid;

[0130] αMeL: α-methyl leucine;

[0131] AEEA: [2-(2-amino-ethoxy)-ethoxy]-acetyl;

[0132] cAMP: cyclic adenosine monophosphate;

[0133] Fmoc: fluorenylmethoxycarbonyl;

[0134] Boc: tert-butyloxycarbonyl;

[0135] DMF: dimethylformamide;

[0136] HOAt: N-hydroxy-7-azabenzotriazole;

[0137] Trt: trityl;

[0138] iLEde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyl-butyl;

[0139] tBu: tert-butyl;

[0140] OtBu: tert-butyloxy;

[0141] TFA: trifluoroacetic acid;

[0142] TIS: triisopropylsilane;

[0143] DCM: dichloromethane;

[0144] DIPEA: N,N-diisopropylethylamine;

[0145] A d : D-Alanine;

[0146] GLP-1: glucagon-like peptide-1;

[0147] GIP: glucose-dependent insulinotropic peptide;

[0148] GCG: glucagon;

[0149] GGG0: Eli Lilly's investigational product LY3437943.

[0150] Example 1 Synthesis of peptide compounds

[0151] The reagents and raw materials used in the present invention are easily available to those of ordinary skill in the art, and those skilled in the art can prepare the agonist peptides of the present invention and their pharmaceutically acceptable salts in a synthetic manner not limited to the embodiments of the present invention. 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.

[0152] 1. Experimental Materials

[0153] The materials and reagents used in the present invention were purchased from commercial products. The protected amino acids used in the entire synthesis process are as follows: Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmo-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Aib-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(iLEde)-OH, Fmoc-α-Me-Leu-OH, Fmoc-Ile-OH, Boc-Tyr(tBu)-OH, Eicosanedioic acid(mon-tBu)-γGlu(α-OtBu)-AEEA-OH.

[0154] 2. Taking ZG1 as an example, the synthesis and preparation method of the agonist peptide in the present invention is described (the preparation of other agonist peptides in the present invention only requires replacing the synthesis sequence of amino acid raw materials)

[0155] 1) Resin pretreatment: Weigh 13.15g of Rink Amide AM Resin into a reactor, add 100mL of DMF, and stir to swell for 30 minutes under nitrogen. When swelling is complete, remove the solvent by filtration, and wash the resin with DMF 3 times, 100mL each time;

[0156] 2) Prepare amino acid solution: weigh 5.75 g of Fmoc-Ser(tBu)-OH and 2.02 g of HOBt, dissolve them in 75 mL of DMF, then add 1.89 g of DIC and mix well;

[0157] 3) Deprotection: Add 100 mL of 20% piperidine / DMF solvent to the reactor, stir and react for 35 min under nitrogen. After the reaction, wash the resin with DMF 6 times, 100 mL each time. After washing, take the resin and test it with ninhydrin reagent, and the resin is positive;

[0158] 4) Coupling reaction: Add the prepared amino acid solution to the reactor, control the temperature at 25-35°C, and stir the reaction under nitrogen. Monitor the progress of the reaction with ninhydrin reagent until the resin shows negative, indicating that the reaction is complete. After the reaction is completed, wash the resin with DMF 3 times, 100 mL each time. Repeat the above steps and sequentially couple the corresponding protected amino acids according to the sequence of ZG1 peptide until the peptide main chain is synthesized;

[0159] 5) Removal of the 17-position Lys side chain protecting group iLEde: Add 100 mL of 8% hydrazine hydrate / DMF solution to the reaction kettle, stir and react for 1 hour with nitrogen, and when the reaction is complete, remove the solvent by suction filtration, repeat the operation once, and then wash the resin with DMF 12 times, 100 mL each time. After washing, take the resin and test it with ninhydrin reagent, and the resin is positive;

[0160] 6) Lys side chain modification: Weigh 7.29g of Eicosanedioic acid (mon-tBu) -γGlu (α-OtBu) -AEEA-OH and 1.35g of HOBt, dissolve them in 150mL of DMF, then add 1.30g of DIC, mix well and add to the reactor, control the temperature at 25-35°C, and stir with nitrogen to react. The reaction progress was monitored with ninhydrin reagent until the resin showed negative, indicating that the reaction was complete. After the reaction was completed, the resin was washed 4 times with DMF, 2 times with DCM, and 3 times with methyl tert-butyl ether, each time 100mL. The resin was dried and set aside for use;

[0161] 7) Peptide resin cleavage: Prepare a cleavage solution according to TFA / TIS / DTT / H2O=90 / 2.5 / 2.5 / 5, then add the dried peptide resin to the cleavage solution and stir to react for 3 hours. When the reaction is complete, filter, add the filtrate to 5 volumes of ice methyl tert-butyl ether for precipitation, filter, wash the filter cake with methyl tert-butyl ether 3 times, and vacuum dry to obtain a crude product;

[0162] 8) Purification: The crude product was purified by C18 reverse phase preparative chromatography system to obtain a refined product of ZG1 with a purity of not less than 90%, and the compound was analyzed by analytical HPLC and LC-MS.

[0163] 3. Preparation results

[0164] After the preparation was completed, the molecular weight test results of each agonist peptide compound were shown in Table 1, and the results showed that the prepared compounds were all correct.

[0165] Table 1 List of synthetic agonist peptide compounds and molecular weight

[0166]

[0167] Example 2 Test of Glucose Consumption of Peptide Compounds in Human HepG2 Cell Insulin Resistance Model

[0168] 1. Experimental Materials

[0169] The materials and reagents used in this test were purchased from commercial products, including the basal culture medium for HepG2 cells (MEM+10% FBS+0.38 mg / ml G418+1% P / S).

[0170] 2. Experimental methods and results

[0171] Taking compounds ZG1 and ZG2 as examples, the glucose consumption test is performed as follows:

[0172] Take HepG2 cells in the logarithmic growth phase, remove the original culture medium, add serum-free culture medium, starve for 24 hours, add 10 - 6 mol / L insulin, culture for 48h to establish insulin resistance model. Discard the original culture medium, replace the culture medium (without FBS), add peptide compounds and incubate for 24h, and measure the glucose content of each group according to the instructions of glucose staining kit. The peptide concentration used is 50nmol / L. The results are as follows Figure 1 Compared with the model group, the GGG0 group (50nmol / L), the ZG1 group (50nmol / L) and the ZG2 group (50nmol / L) all increased glucose consumption, indicating that ZG1 / 2 has significant in vitro glucose-lowering ability, and its glucose-lowering ability is stronger than that of GGG0.

[0173] Example 3 Peptide compound functional activity test on mouse 3T3-L1 adipocytes

[0174] 1. Experimental Materials

[0175] The materials and reagents used in this test were purchased from commercial products, including the basal culture medium for mouse 3T3-L1 cells (DMEM, 10% FBS, 1% P / S).

[0176] 2. Experimental methods and results

[0177] Taking compounds ZG1 and ZG2 as examples, lipid droplet formation inhibition test was performed as follows:

[0178] Mouse 3T3-L1 cells in the logarithmic growth phase were taken, and IBMX (0.5mM), dexamethasone (1μmol / L), and insulin (10μg / mL) were added to the basal culture medium to prepare an adipogenic induction medium. The medium was changed every 2 days, and the induction was continued for 8 days to establish an induced adipogenic model. The model cells were grouped into a control group, a GGG0 group (50nmol / L), a ZG1 group (50nmol / L), and a ZG2 group (50nmol / L). Then, peptide compounds were added to each group, and Oil Red O staining was performed after 24 hours. The peptide concentration used was 50nmol / L. The results are shown in Figure 2 Compared with the control group, the GGG0 group (50nmol / L), the ZG1 group (50nmol / L) and the ZG2 group (50nmol / L) could reduce the formation of lipid droplets, indicating that ZG1 / 2 has significant lipid-lowering ability in vitro.

[0179] Example 4: Test of the hypoglycemic efficacy of peptide compounds on mice

[0180] 1. Experimental Materials

[0181] Wild-type C57 female mice were 12 weeks old.

[0182] 2. Experimental methods and results

[0183] Taking compounds ZG1 and ZG2 as examples, an in vivo hypoglycemic test was conducted, as follows:

[0184] 12-week-old C57 mice were divided into vehicle group, GGG0 group (15nmol / kg), ZG1 group (15nmol / kg) and ZG2 group (15nmol / kg), and then injected subcutaneously with different peptide compounds once, and injected subcutaneously with 2g / kg glucose 12 hours later, and blood was collected from the tail tip at 0, 15, 30, 60, and 120 minutes for blood glucose detection. N = 3. Results are shown in Figure 3 Compared with the Vehicle group, the GGG0 group (15nmol / kg), ZG1 group (15nmol / kg) and ZG2 group (15nmol / kg) could all reduce blood sugar. The blood sugar-lowering ability of ZG1 / 2 in vivo is comparable to that of GGG0.

[0185] Example 5: Test of the lipid-lowering efficacy of peptide compounds in mice

[0186] 1. Experimental Materials

[0187] The animals were 16-week-old wild-type C57 female mice.

[0188] 2. Experimental methods and results

[0189] Taking compounds ZG1 and ZG2 as examples, an in vivo lipid-lowering test was conducted, as follows:

[0190] Establish high-fat induced obese mice. 16-week-old C57 female mice were grouped into high-fat model combined with normal diet groups, and fed with high-fat feed and normal feed respectively for 8 consecutive weeks to establish high-fat model mice. Then, 24-week-old C57 female high-fat model mice were grouped into Vehicle group, GGG0 group (15nmol / kg), ZG1 group (15nmol / kg) and ZG2 group (15nmol / kg). Each group received a subcutaneous injection of the peptide compound once every 3 days, and the body weight was measured every 3 days, N=3. The results are shown in Figure 4 Compared with the Vehicle group, the GGG0 group (15nmol / kg), the ZG1 group (15nmol / kg) and the ZG2 group (15nmol / kg) could all reduce the body weight of the model mice.

[0191] Example 6 Safety Evaluation of Peptide Compounds in Mice

[0192] 1. Experimental Materials

[0193] All materials and reagents used in this evaluation were purchased from commercial products. Wild-type C57 female mice were 12 weeks old.

[0194] 2. Experimental methods and results

[0195] The following is an example of compound ZG1 and ZG2 to evaluate the safety of mice, as follows:

[0196] Wild-type C57 female mice aged 12 weeks were divided into vehicle group, GGG0 group (15nmol / kg), ZG1 group (15nmol / kg) and ZG2 group (15nmol / kg). Each group was subcutaneously injected with peptide compound once every 3 days, 15nmol / kg. Blood was collected from the tail tip 21 days after injection for serum creatine kinase (CK) and alanine aminotransferase (ALT) detection, N = 3. The results are shown in Figure 5 and 6 Compared with the Vehicle group, there were no significant differences in the levels of creatine kinase (CK) and alanine aminotransferase (ALT) in serum among the GGG0 group (15nmol / kg), the ZG1 group (15nmol / kg) and the ZG2 group (15nmol / kg), indicating that ZG1 / 2 has good safety in vivo in mice.

[0197] Example 7 Evaluation of Serum Stability of Peptide Compounds

[0198] 1. Experimental Materials

[0199] All materials and reagents used in this evaluation were purchased from commercial products. Wild-type C57 female mice were 12 weeks old.

[0200] 2. Experimental methods and results

[0201] The following is an example of compound ZG1 and ZG2 to evaluate the stability of mouse serum, as follows:

[0202] Six 12-week-old wild-type C57 female mice were taken, whole blood was collected by enucleation, and the upper serum was collected after centrifugation at 1500rpm for 10 minutes. They were divided into 0min group, 15min group, 30min group, 45min group and 60min group. 2.2mmol / L peptide compound was added to each group at one time, and samples were collected at the above time points after thorough mixing. The content of peptide compound was detected by HPLC method, N=3. The results are shown in Figure 7Compared with the 0min group, the detection value of GGG0 decreased by 45.1% after 60min incubation in serum, while the detection value of ZG1 decreased by 12.4% after 60min incubation in serum, and the detection value of ZG2 decreased by 11.5% after 60min incubation in serum, indicating that ZG1 / 2 has good stability in serum.

[0203] Example 8 EC50 test of peptide compounds

[0204] 1. Experimental Materials

[0205] The materials and reagents used in this test were purchased from commercial products.

[0206] 2. Experimental methods

[0207] The following is a cell EC50 test using compounds ZG1, ZG2, ZG3 and ZG4 as examples, as follows:

[0208] 1) Construction of cell model

[0209] HEK293 cells that highly express human GLP1R, GIPR and GCGR were constructed (by transfecting human GLP1R, GIPR and GCGR).

[0210] The specific steps are as follows:

[0211] A. Construction of plasmids for overexpressing human GLP-1, GIP and GCG receptors respectively;

[0212] B. Use Lip3000 transfection reagent for transfection to obtain target cells with high expression of GLP-1, GIP and GCG receptors;

[0213] C. Perform cell verification and detect whether the transfection is successful through RT-qPCR experiment;

[0214] D. Extract Total RNA of cells before and after transfection, perform RT-qPCR reaction, and analyze the results;

[0215] E. After successful cell transfection, drug (peptide compound) testing is performed.

[0216] 2) Peptide compound testing

[0217] A. HEK293 cell culture: HEK293 cells were cultured in a medium containing DMEM + 10% FBS + 1% penicillin + 1% streptomycin at 37°C and 5% CO2.

[0218] B. When the cells are in the logarithmic growth phase, remove the culture medium and wash the cells with PBS without phosphorus and magnesium;

[0219] C. Protease treatment with Accutase;

[0220] D. Wash the separated cells and resuspend them in assay buffer to measure the cell density;

[0221] E. The cell suspension was adjusted to 4×10 5 Cells / ml were seeded in 96-well plates;

[0222] F, adding test peptide chemicals;

[0223] G, room temperature, incubate for 30 minutes;

[0224] H. Use cAMP Gs kit for determination;

[0225] I. Add lysis buffer, dilute HTRF reagent, and incubate for 1 hour;

[0226] J. Measured at 665 / 620nm;

[0227] K. Use the ELISA method to draw a standard curve and then calculate the EC50 value of the peptide.

[0228] 3. Experimental results

[0229] The experimental results are shown in Table 2. The EC50 values ​​of ZG1, ZG2, ZG3 and ZG4 for the agonist effects on the three targets of GLP1R, GIPR and GCGR are all better than those of GGG0.

[0230] Table 2 EC50 values ​​of tested peptide compounds

[0231] GLP1R(nM) GIPR(nM) GCGR(nM) Testing GGG0 0.95±0.05 0.13±0.02 8.01±0.53 Test ZG1 0.85±0.07 0.10±0.02 6.81±0.51 Testing ZG2 0.82±0.69 0.08±0.02 6.75±0.33 Testing ZG3 0.87±0.05 0.06±0.02 5.88±0.34 Testing ZG4 0.77±0.07 0.06±0.02 5.59±0.39

[0232] Example 9 PK testing of peptide compounds

[0233] 1. Experimental Materials

[0234] The materials and reagents used in this test were purchased from commercial products.

[0235] 2. Experimental methods

[0236] The following is an in vivo PK test in rats using compounds ZG1, ZG2, ZG3 and ZG4 as examples, as follows.

[0237] The animals were randomly divided into 2 groups using a computer system, with 3 animals in each group, all of which were female. The dosage and grouping information are shown in Table 3:

[0238] Table 3

[0239]

[0240] The specific contents are as follows:

[0241] 1) Administration route: intravenous injection;

[0242] 2) Dosage frequency and duration: single dose;

[0243] 3) Dosage method: The animal is injected with the corresponding dose of the test article through the tail vein. The dosage is calculated based on the most recently obtained body weight. The injection is completed within 1 minute;

[0244] 4) Blood sample collection: Whole blood (about 0.4 mL) was collected from the jugular vein of the animals for pharmacokinetic analysis;

[0245] 5) Sampling time: The blood sampling time points for all animals were 2 min, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h before each administration and after the end of administration;

[0246] 6) Sample processing: The centrifuge tubes were coated with K2EDTA, and 4 μL of aprotinin aqueous solution (100 μg / mL) and 4 μL of ethanolic PMSF solution (100 mM) were added to each tube and stored in an ice bath; the tubes were centrifuged at 3000 g and 4°C for 10 min within 1 hour, and 160 μL of plasma was taken out after centrifugation and transferred to a new labeled centrifuge tube (2 μL of DMSO was added to the tube in advance), and stored below -60°C. After sample collection, the Watson LIMS 7.5 system was used for sample reception and management;

[0247] 7) Biological sample analysis: The feasibility-verified LC-MS / MS method was used to determine the content of Sell lipidated polypeptides in plasma samples. The analysis method is briefly described as follows: Verapamil was used as the internal standard, and the protein precipitation method was used in the treatment process. The specific method is as follows: 1) Take 50 μL of sample, add 5 μL of phosphoric acid-water solution (10:90, v / v), 5 μL of internal standard working solution (0.5 ng / mL) and 150 μL of acetonitrile, vortex to mix, and centrifuge for 10 minutes (4000g, 2-8°C). 2) Take all the supernatant and filter it through a phospholipid removal plate. The filtrate was diluted 2 times with ultrapure water and then injected for analysis. Use ACQUITY UPLC Protein BEH C4 ( 2.1×50mm, 1.7μm) chromatographic column was used for chromatographic separation, and the running time of each needle was 5 minutes. The aqueous mobile phase was water-formic acid (1000:2), and the organic phase was acetonitrile-methanol-formic acid (800:200:2). The linear range of peptides was 50-15000ng / mL, and the lower limit of quantification was 50ng / mL;

[0248] The non-compartmental model method (NCA) of WinNonlin8.0 software was used to calculate the pharmacokinetic parameters of the drug administration group, including AUC (area under the plasma concentration-time curve), C0, t1 / 2 (elimination half-life), Vd (apparent distribution volume), Cl (clearance rate), MRT (mean residence time), etc.

[0249] 8) Data processing and statistical analysis: Microsoft EXCEL was used to describe the experimental data using mean (Mean), standard deviation (SD), and coefficient of variation (CV%).

[0250] 3. Experimental results

[0251] The results of metabolic kinetic parameters are shown in Table 4. The results show that the in vivo PK test results of ZG1, ZG2, ZG3 and ZG4 in rats, such as T1 / 2 (elimination half-life), Vd (apparent distribution volume), Cl (clearance rate), and MRT (mean residence time), all performed well.

[0252] Table 4 Metabolic kinetic results

[0253] Group Dose(mg / kg) T1 / 2(h) Vd(mL / kg) Cl(mL / hr / kg) MRT0-48h(h) ZG1 12 9.56 137 6.8 10.2 ZG2 12 10.02 142 6.6 11.1 ZG3 12 9.98 133 6.5 10.4 ZG5 12 9.43 129 6.4 10.6

[0254] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A GLP1R-GIPR-GCGR triple-target agonist peptide or a pharmaceutically acceptable salt thereof, characterized in that: The agonist peptide or a pharmaceutically acceptable salt thereof comprises an amino acid sequence or consists of an amino acid sequence, and the amino acid sequence general formula I is as follows: X1-Aib-QGTFTSDYSI-aMeL-LDK-X 17 -AQ-Aib-X 21 -FX 23 -EYLLE-XX1-R 1 , in, X1 is H or Y; X 17 is Ψ, Ψ is Lys with modified side chain; X 21 is D or A; X 23 V or I; XX1 is GGPSSGAPPPSKVSRA, GGGPSSGAPPPSKVSRA, GGPSSGA d PPPSKVSRA, GGGPSSGA d PPPSKVSRA, GGPSSGAPPPS, GGGPSSGAPPPS, GGPSSGA d PPPS, or GGGPSSGA d PPPS; R1 is NH2 or OH, or a pharmaceutically acceptable salt and / or ester thereof; Preferably, the Ψ is a Lys with a side chain modified by a structure having the following general formula II, wherein the general formula II is: BZ; wherein B is (AEEA or Glu) a -(AEEA or Glu) b -(AEEA or Glu) c , wherein a, b, and c are each independently 0 or 1, and a, b, and c are not 0 at the same time; the carboxyl end of B is connected to the ε-amino group of the side chain of Lys, and Z is -CO-(CH2) m -R 2 , m is an integer between 6 and 24, R 2 Selected from -COOH; Preferably, the general formula II is AEEA-γG1u-CO(CH2) 18 COOH; Preferably, at least one site on the agonist peptide or a pharmaceutically acceptable salt thereof is chemically modified with a fatty acid side chain group.

2. The agonist peptide or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The agonist peptide or a pharmaceutically acceptable salt thereof is selected from: H-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-DFVEYLLE-GGPSSGAPPPSKVSRA-NH2; H-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-AFIEYLLE-GGGPSSGAPPPSKVSRA-NH2; Y-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-DFVEYLLE-GGPSSGAPPPS-NH2; Y-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-AFIEYLLE-GGGPSSGAPPPS-NH2; H-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-DFVEYLLE-GGPSSGA d PPPSKVSRA-NH2; H-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-AFIEYLLE-GGGPSSGA d PPPSKVSRA-NH2; Y-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-DFVEYLLE-GGPSSGA d PPPS-NH2: Y-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-AFIEYLLE-GGGPSSGA d PPPS-NH2。 3. The agonist peptide or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The agonist peptide or its pharmaceutically acceptable salt has an agonist ability in cells transfected with GLP-1 receptors, and compared with natural GLP-1, the relative activity of the agonist peptide or its pharmaceutically acceptable salt on GLP-1 receptor agonism is increased by at least 30%, preferably at least 60%, preferably at least 80%, preferably at least 100%; Preferably, the agonist peptide or its pharmaceutically acceptable salt has a relative activity of at least 100% higher, preferably at least 150% higher, in terms of the agonist ability of cells transfected with GIP receptors compared to natural GIP; Preferably, the agonist peptide or its pharmaceutically acceptable salt has a relative activity of at least 10% higher, preferably at least 30% higher, on the agonist ability of cells transfected with GCG receptors compared to natural GCG; Preferably, the agonist peptide or its pharmaceutically acceptable salt on the adipose tissue representative cell 3T3-L1, compared with natural GIP, the relative activity of the agonist peptide or its pharmaceutically acceptable salt on 3T3-L1 cells is increased by at least 60%, preferably, at least 100%; Preferably, the relative activity of the agonist peptide or its pharmaceutically acceptable salt on human primary hepatocytes, which are representative cells of liver tissue, is increased by at least 60%, preferably, by at least 100%, compared to natural GCG.

4. A solid phase synthesis method for preparing the agonist peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: 1) Resin pretreatment: Add the resin to a reactor, add DMF, stir and swell with nitrogen, filter and wash; 2) Prepare amino acid solution: dissolve Fmoc-Ser-tBu-OH and HOBt in DMF, then add DIC and mix well; 3) Deprotection: Add 20% piperidine / DMF solvent to the reaction kettle, stir and wash with nitrogen; 4) Coupling reaction: add the amino acid solution prepared in step 2) to the reaction kettle, control the temperature and stir with nitrogen, after the reaction is completed, wash the resin with DMF, repeat step 4), and sequentially couple the corresponding protected amino acids according to any one of claims 1 to 3 or the pharmaceutically acceptable salt thereof until the main chain of the agonist peptide is synthesized; 5) Removal of the 17-position Lys side chain protecting group iLEde: Add 8% hydrazine hydrate / DMF solution to the reaction kettle, stir with nitrogen, and after the reaction is complete, remove the solvent by suction filtration and wash; 6) Lys side chain modification: Eicosanedioic acid (mon-tBu) -γGlu (α-OtBu) -AEEA-OH and HOBt were dissolved in DMF, and DIC was added and mixed to prepare a side chain modification solution, which was added to a reaction kettle, and the temperature was controlled and nitrogen was stirred. After the reaction was completed, the solution was washed with DMF, then with DCM, and then with methyl tert-butyl ether to obtain a peptide resin containing the agonist peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, and then dried.

5. The method according to claim 4, characterized in that The swelling time in step 1) is 30 min; Preferably, in step 2), the dissolution ratio of Fmoc-Ser-tBu-OH in DMF is 5.75 g / 75 mL; Preferably, in step 2), the dissolution ratio of HOBt in DMF is 2.02 g / 75 mL; Preferably, in step 2), the dissolution ratio of DIC in DMF is 1.89 g / 75 mL; Preferably, the ratio of the amount of 20% piperidine / DMF solvent added in step 3) to the amount of the resin added in step 1) is 100 mL / 13.15 g; Preferably, the stirring time of nitrogen in step 3) is 35 min; Preferably, after washing in step 3), the resin is tested using a ninhydrin reagent to determine whether it is positive; Preferably, the ratio of the amount of the amino acid solution added in step 4) to the amount of the resin added in step 1) is 100 mL / 13.15 g; Preferably, the temperature range of the temperature-controlled nitrogen stirring in step 4) is 25-35° C.; Preferably, the sign of completion of the reaction in step 4) is that the resin is negative when tested with a ninhydrin reagent; Preferably, the ratio of the amount of 8% hydrazine hydrate / DMF solution in step 5) to the amount of the resin added in step 1) is 100 mL / 13.15 g; Preferably, the stirring time of nitrogen in step 5) is 1 hour; Preferably, after washing in step 5), the resin is tested using a ninhydrin reagent to determine whether it is positive; Preferably, in step 6), the dissolution ratio of Eicosanedioic acid (mon-tBu)-γGlu (α-OtBu)-AEEA-OH in DMF is 7.29 g / 150 mL; Preferably, in step 6), the dissolution ratio of HOBt in DMF is 1.35 g / 150 mL; Preferably, in step 6), the dissolution ratio of DIC in DMF is 1.30 g / 150 mL; Preferably, the ratio of the amount of the side chain modification solution in step 6) to the amount of the resin in step 1) is 150 mL / 13.15 g; Preferably, the temperature range of the temperature-controlled nitrogen stirring in step 6) is 25-35° C.; Preferably, the sign of completion of the reaction in step 6) is that the resin is negative when tested with a ninhydrin reagent.

6. A post-treatment method for the agonist peptide or a pharmaceutically acceptable salt thereof after solid phase synthesis according to claim 4 or 5, characterized in that: The post-processing method comprises the following steps: 1) taking the peptide resin containing the agonist peptide or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 3 finally synthesized by the method of claim 4 or 5, adding a cutting liquid, stirring the reaction, filtering, adding the filtrate to ice methyl tert-butyl ether for precipitation, suction filtering, washing the filter cake with methyl tert-butyl ether, and vacuum drying to obtain a crude product; 2) The crude product obtained in step 1) is passed through a refining and purification system to obtain a purified product.

7. The post-processing method according to claim 6, characterized in that: The volume ratio of the cutting solution in step 1) is TFA / TIS / DTT / H2O=90 / 2.5 / 2.5 / 5; Preferably, the stirring reaction time in step 1) is 3 hours; Preferably, in step 1), the volume ratio of the filtrate to glacial methyl tert-butyl ether is 1:5; Preferably, the refining and purification system in step 2) is a C18 reverse phase preparative chromatography system; Preferably, the purity of the purified product in step 2) is not less than 90%.

8. A product, characterized in that The product comprises the reagent used in the method of claim 4 or 5, and / or the reagent used in the post-treatment method of claim 6 or 7; Preferably, the product comprises a kit.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a therapeutically effective amount or a preventively effective amount of the agonist peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, and a pharmaceutically acceptable excipient; Preferably, the pharmaceutical composition may further include other active pharmaceutical ingredients; Preferably, the pharmaceutical composition is an injection, lyophilized powder, tablet, pill, lozenge, soft capsule, hard capsule, granule, powder, solution, suspension, or syrup; Preferably, the pharmaceutical composition is administered orally, by inhalation or parenterally; Preferably, the parenteral administration is selected from intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous or intradermal injection administration; Preferably, the therapeutically effective amount or the preventively effective amount needs to be administered at least once a week or once every two weeks; Preferably, the other active pharmaceutical ingredients include antidiabetic agents, GIP receptor agonists, GCG receptor agonists or antagonists, GLP-1 / GIP receptor agonists, GLP-1 / GCG receptor agonists, GIP / GCG receptor agonists, FGF-21 and its analogs, CCKB and its analogs, PYY and its analogs, leptin and its analogs, calcitonin and its analogs, lipid-regulating drugs, PPAR-α, β, δ agonists or regulators, antiplatelet aggregation agents, PCSK9 inhibitors, lipase inhibitors, anti-liver fibrosis or cirrhosis agents, and anti-inflammatory agents; Preferably, the diabetes active agent includes insulin and its analogs, biguanides, sulfonylureas, thiazolidinediones, X-glucosidase inhibitors, DPP-4 inhibitors, SGLT2 inhibitors, dual SGLT1 / SGLT2 inhibitors, GLP-1 receptor agonists, amylin and its analogs.

10. Applications as described in any of the following: 1) Use of the agonist peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 8 in the preparation of a drug for promoting insulin secretion and lowering blood sugar; 2) Use of the agonist peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 8 in the preparation of a drug for inhibiting food intake, delaying gastric emptying, increasing energy expenditure, and reducing body weight; 3) Use of the agonist peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 8 in the preparation of a drug for reducing pancreatic β-cell apoptosis, increasing the number of pancreatic β-cells, and improving pancreatic cell function; 4) Use of the agonist peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 8 in the preparation of a drug for improving blood lipids, reducing liver fat accumulation, and inhibiting the development of liver inflammation; 5) Use of the agonist peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 8 in the preparation of a drug for promoting the growth of brain neurons, removing neurotoxic substances, inhibiting the development of inflammation, and playing a neuroprotective role; 6) Use of the agonist peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for preventing and / or treating metabolic disorders; Preferably, the metabolic disorder diseases include diabetes, obesity, non-alcoholic fatty liver disease, and dyslipidemia; 7) Use of the agonist peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for treating a neurodegenerative disease; Preferably, the neurodegenerative diseases include Parkinson's disease and Alzheimer's disease; 8) Use of the agonist peptide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 8 in the preparation of a drug for treating bone diseases; Preferably, the bone disease includes bone diseases caused by endocrine diseases, metabolic disorders, and kidney diseases; Preferably, the bone disease includes osteoporosis and osteoarthritis.

Images