Multi-agonist and use thereof

Abstract

To provide a novel polypeptide molecule having GLP-1 / GIP / GCG R triple agonist activity, which can be used in the treatment of type II diabetes, obesity, dyslipidemia, nonalcoholic fatty liver disease / nonalcoholic steatohepatitis, and other related metabolic diseases.SOLUTION: Provided is a novel polypeptide molecule with GLP-1 / GIP / GCG R triple agonist activity, comprising multiple specific sequences, wherein compared to natural GLP-1 / GIP / GCG, the compound has improved relative activity in terms of activation ability of GLP-1 / GIP / GCG receptor stably transfected cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to polypeptide compounds and their uses in the pharmaceutical field. Specifically, the present invention relates to a method for treating glucagon-like peptide-1 receptor (GLP-1R), a glucose-dependent The insulinotropic polypeptide receptor (GIPR), and the glucagon receptor ( A polypeptide compound having triple agonist activity against GCG R, and and its use in the treatment of inflammatory bowel syndrome. [Background technology]

[0002] Type 2 diabetes and obesity are increasingly becoming global diseases affecting human health. Obesity is associated with diabetes, hypertension, heart disease, dyslipidemia, fatty liver disease, and atherosclerosis. Diabetes is the leading cause of many chronic diseases, including atherosclerosis, arthritis, stroke, and neurodegenerative diseases. It can also lead to cardiovascular and cerebrovascular diseases and other complications. Most of the hypoglycemic drugs available only have the effect of controlling blood glucose and do not increase the weight of obese patients. Some drugs even have the side effect of causing weight gain. It can reduce and improve weight loss, and is effective in most obese and type 2 diabetic patients. There remains an urgent need to develop drugs containing multiple beneficial effects that address individual needs. be.

[0003] Incretins are polypeptide hormones secreted from the intestine following normal physiological food stimulation. Early research suggests that it is a type of , stimulates pancreatic islet β-cells to secrete insulin, regulates glucose homeostasis, and It may protect cells, suppress appetite and slow gastric emptying, thereby reducing body weight. GLP-1 and GIP are currently the two types of pancreatic stimulating hormones. It has been found as a type of

[0004] GLP-1 is a polypeptide with 31 amino acids that binds to the intestinal mucosa of L cells. It is expressed by the glucagon gene and acts primarily on the GLP-1 receptor (GLP-1 R). , stimulates insulin secretion, inhibits glucagon secretion, protects pancreatic islet β-cells, and reduces blood glucose levels. It has physiological functions in regulating course homeostasis, while also acting through central nervous system signaling pathways. It inhibits food intake and gastric emptying, increasing satiety and therefore weight loss. Xendin-4 is a GLP-1 analogue extracted from the salivary glands of the African venomous lizard. , it exhibits stronger GLP-1 receptor activation and similar GLP-1 effects. Compared with GLP-1, exendin-4 confers stronger resistance to DPP-4 and and has a longer plasma half-life in the body.

[0005] GIP is a single-chain polypeptide with 42 amino acids produced by the K cells of the small intestinal mucosa. It acts primarily on the GIP receptor (GIPR) in pancreatic islet cells and adipocytes. GIP-dependent insulin secretion enhances islet β-cell quality and increases insulin secretion. It stimulates secretion, inhibits gastric acid secretion, and slows gastric peristalsis. In addition, it has a beneficial effect on adipose tissue cells. GIP also stimulates fatty acid uptake and utilization by osteoblasts. It also has a therapeutic effect, inhibiting osteoblast apoptosis, inhibiting bone resorption, and increasing bone mineral density. and protects bones.

[0006] GCG is a polypeptide containing 29 amino acids and is expressed in pancreatic islet α-cells. It is expressed and secreted by the lucagon progenitor gene, and is distributed in the liver and kidney. It acts primarily on glucagon receptors (GC-R), stimulating liver glycogen breakdown and increasing blood glucose. It increases the cholesterol level, activates lipase, and promotes lipolysis, while inhibiting hepatic fat synthesis and promoting fat Enhances acid oxidation. Research results show that GCG reduces food intake and increases the energy content of adipose tissue. - has a specific effect on increasing fat burning and reducing body fat The moderate effect of GCG in raising blood glucose is related to the feed- ing-chain of insulin. This may provide a boost and reduce the occurrence of hypoglycemic events.

[0007] Exenatide and Lisenatide: Targeting the effects of incretin GLP-1 receptor agonists such as liraglutide, dulaglutide, and semaglutide has been successfully developed for the treatment of type 2 diabetes. In addition, liraglutide has been shown to promote weight loss. Semaglutide has been successfully developed for the treatment of obesity, and semaglutide is also undergoing clinical studies for the treatment of obesity. The advantage of GLP-1 analogues is that they lower blood glucose and improve cardiac function. It may also have vascular benefits and weight management effects. However, at present, no single Weight loss with GLP-1 receptor agonists remains below 10% with no clear dose-related effect. There are gastrointestinal side effects (mainly nausea, vomiting, and diarrhea). Obese type II diabetes, non-alcoholic Fatty liver disease / non-alcoholic steatohepatitis (NAFLD / NASH), diabetes, and heart disease Weight loss in populations with metabolic diseases and complex medical conditions, such as obesity with vascular risk There remains an urgent need for more effective treatments for dementia.

[0008] According to the physiological effects of GLP-1, GIP, and GCG, many studies are currently being conducted. Activating all or all three of these receptors simultaneously is more effective than activating only GLP-1R. It has been confirmed that this method can achieve better therapeutic effects on diabetes and obesity than conventional methods. Hyperglycemia, obesity, and insulin resistance are linked to GIPR and its signaling pathways. However, it has been reported that it reduces blood sugar and inhibits insulin resistance. Promoting insulin secretion and improving pancreatic islet function as resistance improves The role of GIP in promoting glycogen breakdown and increasing blood glucose levels may be improved. In addition to increasing hepatic lipid metabolism, GCG can also promote fat breakdown and inhibit hepatic fat synthesis. However, the synergistic effect of GLP-1 on blood lipids and body weight can be reduced. Inhibition of GLP-1 is required to suppress its hyperglycemic effect. " effect, lowering blood sugar and improving insulin resistance is a GI It can further enhance the insulin secretion-promoting function and insulin sensitization synergistic effect of P, and can also enhance pancreatic islet function It can improve the lipid metabolism, further enhance the lipolytic effect of GCG, and improve the weight loss effect. This may enhance the effect.

[0009] Development of clinical data on GLP-1 / GIP and GLP-1 / GCG dual agonists The clinical therapeutic efficacy of drugs is also influenced by the relationship between different receptors, such as GLP-1 / GIP or The distributional effects of GLP-1 / GCG dual agonist activity are becoming increasingly clear.

[0010] For example, the results of a Phase II clinical study of Lilly's tirzepatide (LY3298176) reported that a 1 mg dose of tirzepatide had little effect on weight loss and its blood glucose lowering effect was The dose of tirzepatide was significantly lower than that of dulaglutide 1.5 mg. Only when increased to 5 mg did it produce a better blood glucose lowering effect than 1.5 mg dulaglutide. This may result in a weight loss effect due to stronger GIP receptor agonist activity. This is probably due to the tendency of tirzepatide to inhibit GLP-1 receptor activity. 50 is wild type GLP-1 active EC 50 This is only about 15% of the total. The dosage of Patid has been significantly increased (up to a maximum dose of 15 mg), which is This may increase safety risks.

[0011] On the other hand, due to the strong blood glucose increasing effect of GCG, it is necessary to balance the blood glucose increasing effect of GCG. To suppress and prevent GLP-1 / GCG receptor dual agonists, sufficient GLP-1 / GCG receptor dual agonists were used. P-1 activity is required to ensure the blood glucose lowering effect. Manufactured by AstraZeneca The GLP-1 / GCG receptor dual agonist MEDI0382 (cotadutide) Relative activity EC of GLP-1 and GCG receptors 50 are 29% and 12.8%, respectively. The results of the 26-week clinical trial showed that liraglutide was 100-300 μg / day higher than liraglutide. g dose of cotadutide on body weight, blood lipids, and ALT and AST levels The effect of the 300 μg dose group was greater than that of the 1.8 mg liraglutide. However, in each dose group, the results were significantly better than those of liraglutide. Furthermore, there was no significant benefit in reducing HbA1c with increasing cotadutide dosage. As the dose increased, the reduction in glycated hemoglobin in the 300 μg group was greater than that in the 200 μg group. This is due to the fact that as the dose increases, This is likely due to low GLP-1 receptor activity, which is insufficient to inhibit the effects of GCG. minutes.

[0012] Ideal for long-acting GLP-1 / GIP / G-CSF treatments that occur once, twice, or three times a week. CG triple receptor agonist molecules have the highest possible GLP-1R / GIPR activity and and relatively controllable GCGR activity to ensure maximum weight loss and blood glucose lowering effects CN104902919A and CN111040022A are poisonous toxins. A series of GLP-1 / G-protein complexes based on structural modifications of exopeptide-4 (exendin-4) We disclose an IP / GC G R triple agonist molecule; CN109071624A has long-lasting A series of cyclic peptide molecules are disclosed that can be combined with interacting conjugates to form molecules. In addition, WO2015067716A1, WO2019125929A1, and WO2019125938A1 discloses a method for linking a fatty acid to the side chain of an amino acid at position 17. All of these polypeptides are also disclosed as GLP-1 / GIP / It demonstrates the triple activation effect of GCG R and has the potential for long-lasting action when taken once a week. The molecules disclosed in the patent literature have sufficiently high activity against GLP-1R / GIPR / GCGR. It is not possible to simultaneously exhibit both high and low activity. Summary of the Invention [Problem to be solved by the invention]

[0013] There is still room for further development of the three agonist molecules. [Means for solving the problem]

[0014] In consideration of the above technical conditions, the present invention provides a method for treating type II diabetes, obesity, dyslipidemia, non-alcoholic fatty liver disease, and the like. In the treatment of alcoholic fatty liver disease / non-alcoholic steatohepatitis, and other related metabolic diseases A novel polynucleotide having GLP-1 / GIP / GC-R triple activation activity that can be used A peptide molecule is provided.

[0015] The present invention relates to a compound of general formula (I): Y-Aib-X3-GT-X6-TSDYSI-X13-LDK-X17-AQ-Ai b-AFIE-X25-LLE-X29-X30-PSS-X34-X35-PP-X3 8-SR 1 (I)

[0016] (In the formula, X3 is Q or H; X6 is F, αMeF, or αMeF(2F); X13 is αMeL, F, αMeF, or L; X17 is Ψ; X25 is Y or F; X29 is T, S, G, or Aib; X30 is G, H, R, or Aib; X34 is G or Aib; X35 is A, Q, Aib, or H; X38 is Ac3c or P; R 1 is NH2 or OH, or a pharmaceutically acceptable salt and / or ester thereof. Teru; Ψ is represented by the following general formula (II): YZ(II) and Lys having a side chain modified by Y is (AEEAc or Glu)a-(AEEAc or Glu)b-(AEEAc or or Glu)c, and a, b, and c are independently 0 or 1; and c are not simultaneously zero (as an illustrative example, AEEAc-AEEAc-γGlu). , the carboxyl terminus of Y is connected to the ε-amino on the side chain of Lys; Z is -CO-(CH2) m -R 2 where m is an integer from 6 to 24, and R 2 -COO H) A GLP-1 / GIP / GC G R triple agonist polypeptide compound having the formula: or a solvate thereof. DETAILED DESCRIPTION OF THE INVENTION

[0017] In one embodiment of the present invention, the compound of general formula (I) is Contains at least two specific amino acids: X13 is F or αMeF; X25 is F; X29 is T or S; X30 is H, R, or Aib; X35 is Q, Aib, or H; X38 is Ac3c.

[0018] In one embodiment of the present invention, the general formula (II) is AEEAc-AEEAc -γGlu-CO(CH2) 18 It is COOH.

[0019] In one embodiment of the present invention, the compound is

[0020] [Table 1] TIFF2025143457000002.tif186170

[0021] is selected from.

[0022] In one embodiment of the present invention, the compound is

[0023] [Table 2]

[0024] Further selected from:

[0025] In one embodiment of the present invention, the GLP-1 receptor is In terms of their ability to activate stably transfected cells, the compounds are In terms of activation capacity, it is at least 30%, preferably at least 60%, more preferably Preferably, the relative activity is at least 80%, and more preferably at least 100%.

[0026] In one embodiment of the present invention, a GLP-1 receptor agonist is provided, which is a compound having a higher GLP-1 receptor agonist activity than native GIP. In terms of their ability to activate stable transfected cells, the compounds are believed to be GIP receptor activators. A relative activity in terms of potency of at least 100%, more preferably at least 150% It has.

[0027] In one embodiment of the present invention, a stable GCG receptor is provided as a GCG receptor stabilizing factor compared to native GCG. In terms of the ability to activate transfected cells, the compounds have the ability to activate GCG receptors. have a relative activity of at least 10%, more preferably at least 30% in terms of .

[0028] In one embodiment of the present invention, the pancreatic In terms of activity against RIN-m5F, a representative cell of islet tissue, the compound - at least 60%, preferably at least 80% in terms of activation against m5F cells 0%, more preferably at least 100% relative activity.

[0029] In one embodiment of the present invention, a representative example of adipose tissue GIP is compared with native GIP. In terms of activity against 3T3-L1 cells, the compound In terms of activity, it has a relative activity of at least 60%, preferably at least 100%. do.

[0030] In one embodiment of the present invention, a representative liver tissue GCG is compared to native GCG. In terms of activity on human primary hepatocytes, the compound exhibits activity on hepatocytes. In some respects it has a relative activity of at least 60%, preferably at least 100%.

[0031] The present invention provides an effective amount of a compound according to any of the above aspects, or a salt or solvate thereof. and a pharmaceutical composition comprising a pharmaceutically acceptable excipient, diluent, carrier, or vehicle. Pharmaceutical compositions are further provided.

[0032] In one embodiment, the pharmaceutical composition is in the form of an injection, a lyophilized powder, a tablet, a pill, a lozenge, It may be in the form of a soft capsule, hard capsule, granule, powder, liquid, suspension, or syrup; Alternatively, the drug composition may be in the form of a microcapsule, microsphere, nanoparticle, or is in the form of a liposomal agent.

[0033] In one embodiment, the pharmaceutical composition is for oral, inhaled, or parenteral administration; Parenteral administration is selected from intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous, or intradermal injection administration. can be.

[0034] In one embodiment, the pharmaceutical composition is administered at least once daily, once weekly, once every two weeks, or It is administered once a month.

[0035] In one embodiment, the pharmaceutical composition comprises an antidiabetic active agent (insulin and its analogs, Guanides, sulfonylureas, thiazolidinediones, α-glucosidase inhibitors, DPP- 4 inhibitors, SGLT2 inhibitors, dual SGLT1 / SGLT2 inhibitors, GLP-1 receptor agonists agonists, amylin and its analogues, GIP receptor agonists, GCG receptor agonists nts or antagonists, GLP-1 / GIP receptor agonists, GLP-1 / GC G receptor agonists, GIP / GCG receptor agonists, FGF-21 and its analogs , cholecystokinin B (CCKB) and its analogs, PYY(3-36) and its analogs analogs, leptin and its analogs, calcitonin and its analogs, lipid-regulating active agents, P PAR-α, β, δ agonists or modulators, anti-platelet aggregation activators, PCSK9 inhibitors therapeutic agents, including lipase inhibitors, anti-liver fibrosis or anti-liver cirrhosis activators, and anti-inflammatory activators; It may also be used in combination with at least one of the following active agents for the treatment of: Antidiabetic active agents include insulin and its analogs, biguanides, sulfonamides, and Nilurea, thiazolidinedione, α-glucosidase inhibitor, DPP-4 inhibitor, SGL T2 inhibitors, dual SGLT1 / SGLT2 inhibitors, GLP-1 receptor agonists, or Includes amylin and its analogs.

[0036] The use of the present invention in the preparation of a drug for promoting insulin secretion and reducing blood glucose any one of the compounds, salts or solvates thereof, or pharmaceutical compositions thereof Species application.

[0037] Inhibits feeding, delays gastric emptying, increases energy expenditure, and weight a compound of the present invention or a salt or solvate thereof in the preparation of a medicament for reducing or the application of any one of the pharmaceutical compositions.

[0038] Reduces islet β-cell apoptosis, increases islet β-cell number, and promotes islet cell proliferation. The use of a compound of the present invention or a salt or solution thereof in the preparation of a medicament for improving cell function. Application of any one of the mediators or any one of the pharmaceutical compositions.

[0039] Improves blood lipids, reduces liver fat accumulation, inhibits the development of liver inflammation, and The present invention relates to the preparation of a drug for preventing and treating non-alcoholic fatty liver disease. any one of the compounds of the present invention or a salt or solvate thereof, or a pharmaceutical composition thereof Use one type.

[0040] Promoting the growth of brain nerves, eliminating neurotoxic substances, inhibiting the onset of inflammation, and The compound of the present invention or a salt or solution thereof in the preparation of a medicament for exerting a protective effect. Use of any one of the mediators or any one of the pharmaceutical compositions.

[0041] for the prevention and / or treatment of metabolic disorders and associated complications, preferably diabetes, obesity a compound of the present invention in the preparation of a medicament for the treatment of non-alcoholic fatty liver disease; or any one of its salts or solvates, or any one of its pharmaceutical compositions use.

[0042] including dyslipidemia and related disorders, as well as Parkinson's disease and Alzheimer's disease A compound of the present invention or a salt thereof in the preparation of a medicament for treating a neurodegenerative disease. or the use of any one of the solvates or any one of the pharmaceutical compositions.

[0043] In the preparation of drugs for treating bone diseases, metabolic disorders, kidney diseases, etc. associated with endocrine disorders any one of the compounds of the present invention or salts or solvates thereof, or pharmaceutical compositions thereof The use of any one of the above compounds in bone diseases includes osteoporosis and osteoarthritis.

[0044] Any one of the compounds of the present invention may be synthesized by solid phase synthesis.

[0045] In one aspect, the present invention provides a compound having the following sequence: Provide the complex: Compound 1 (SEQ ID NO: 1) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AE EAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLETG PSSGAPP-Ac3c-S-NH2 Compound 2 (SEQ ID NO: 2) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AE EAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLETH PSSGAPP-Ac3c-S-NH2 Compound 3 (SEQ ID NO: 3) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AE EAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLESH PSSGAPP-Ac3c-S-NH2 Compound 4 (SEQ ID NO: 4) Y-Aib-QGT-αMeF(2F)-TSDYSIFLDKK(AEEAc-AEE Ac-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLEGGP SSGAPP-Ac3c-S-NH2 Compound 5 (SEQ ID NO: 5) Y-Aib-QGT-αMeF(2F)-TSDYSIFLDKK(AEEAc-AEE Ac-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLEGGP SSGAPP-Ac3c-S-NH2 Compound 6 (SEQ ID NO: 6) Y-Aib-HGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEA c-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYL LE-Aib-HPSSGQPPPS-NH2 Compound 7 (SEQ ID NO: 7) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AE EAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLETG PSSG-Aib-PPPS-NH2 Compound 8 (SEQ ID NO: 8) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AE EAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLETG PSSGQPPPS-NH2 Compound 9 (SEQ ID NO: 9) Y-Aib-QGT-αMeF(2F)-TSDYSIFLDKK(AEEAc-AEE Ac-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLETGP SSGAPP-Ac3c-S-NH2 Compound 10 (SEQ ID NO: 10) Y-Aib-QGT-αMeF(2F)-TSDYSIFLDKK(AEEAc-AEE Ac-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLESHP SSGAPP-Ac3c-S-NH2 Compound 11 (SEQ ID NO: 11) Y-Aib-QGT-αMeF-TSDYSIFLDKK(AEEAc-AEEAc-γ Glu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLETGPSSGA PP-Ac3c-S-NH2 Compound 12 (SEQ ID NO: 12) Y-Aib-QGT-αMeF-TSDYSIFLDKK(AEEAc-AEEAc-γ Glu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLETGPSSGA PPPS-NH2 Compound 13 (SEQ ID NO: 13) Y-Aib-QGT-αMeF-TSDYSIFLDKK(AEEAc-AEEAc-γ Glu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLETGPSSGA PPPS-NH2 Compound 14 (SEQ ID NO: 14) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AE EAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLETG PSSGAPPPS-NH2 Compound 15 (SEQ ID NO: 15) Y-Aib-QGT-αMeF-TSDYSIFLDKK(AEEAc-AEEAc-γ Glu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLESHPSSGA PPPS-NH2 Compound 16 (SEQ ID NO: 16) Y-Aib-QGT-αMeF-TSDYSIFLDKK(AEEAc-AEEAc-γ Glu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLE-Aib-HP SSGAPPPS-NH2 Compound 17 (SEQ ID NO: 17) Y-Aib-QGT-αMeF(2F)-TSDYSIFLDKK(AEEAc-AEE Ac-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLETGP SSGAPPPS-NH2 Compound 18 (SEQ ID NO: 18) Y-Aib-QGT-αMeF(2F)-TSDYSIFLDKK(AEEAc-AEE Ac-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLE-Ai b-HPSSGAPPPS-NH2 Compound 19 (SEQ ID NO: 19) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEA c-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFL LETGPSSGAPPPS-NH2 Compound 20 (SEQ ID NO: 20) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEA c-AEEAc-γGlu-CO(CH2) 18COOH)AQ-Aib-AFIEFL LE-Aib-HPSSGAPPPS-NH2 Compound 21 (SEQ ID NO: 21) Y-Aib-QGT-αMeF(2F)-TSDYSIFLDKK(AEEAc-AEE Ac-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLEGGP SSGQPPPS-NH2 Compound 22 (SEQ ID NO: 22) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEA c-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFL LEGGPSSGQPPPS-NH2 Compound 23 (SEQ ID NO: 23) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEA c-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYL LETGPSSGQPPPS-NH2 Compound 24 (SEQ ID NO: 24) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEA c-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFL LETGPSS-Aib-HPPPS-NH2 Compound 25 (SEQ ID NO: 25) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEA c-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFL LEGRPSS-Aib-HPPPS-NH2 Compound 26 (SEQ ID NO: 26) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AE EAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLESH PSSGAPPPS-NH2 Compound 27 (SEQ ID NO: 27) Y-Aib-QGT-αMeF-TSDYSI-αMeF-LDKK(AEEAc-AE EAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLESH PSSGAPPPS-NH2 Compound 28 (SEQ ID NO: 28) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AE EAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLES- Aib-PSSGAPP-Ac3c-S-NH2 Compound 29 (SEQ ID NO: 29) Y-Aib-QGT-αMeF-TSDYSIFLDKK(AEEAc-AEEAc-γ Glu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLES-Aib-P SSGAPP-Ac3c-S-NH2 Compound 30 (SEQ ID NO: 30) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AE EAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLET- Aib-PSSGAPP-Ac3c-S-NH2 Compound 31 (SEQ ID NO: 31) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEA c-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFL LE-Aib-HPSSGAPP-Ac3c-S-NH2 Compound 32 (SEQ ID NO: 32) Y-Aib-QGT-αMeF(2F)-TSDYSIFLDKK(AEEAc-AEE Ac-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLE-Ai b-HPSSGAPP-Ac3c-S-NH2 Compound 33 (SEQ ID NO: 33) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AE EAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLESH PSS-Aib-HPP-Ac3c-S-NH2 Compound 34 (SEQ ID NO: 34) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEA c-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYL LETGPSSGQPP-Ac3c-S-NH2 Compound 35 (SEQ ID NO: 35) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AE EAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLETG PSSGQPP-Ac3c-S-NH2 Compound 36 (SEQ ID NO: 36) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AE EAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLEGG PSS-Aib-HPPPS-NH2 Compound 37 (SEQ ID NO: 37) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AE EAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLEGR PSS-Aib-HPPPS-NH2 Compound 38 (SEQ ID NO: 38) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEA c-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFL LETGPSS-Aib-HPPPS-NH2 Compound 39 (SEQ ID NO: 39) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEA c-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFL LETRPSS-Aib-HPPPS-NH2 Compound 40 (SEQ ID NO: 40) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEA c-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFL LESHPSS-Aib-HPP-Ac3c-S-NH2 Compound 41 (SEQ ID NO: 41) Y-Aib-QGT-αMeF(2F)-TSDYSILLDKK(AEEAc-AEE Ac-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLESHP SS-Aib-HPP-Ac3c-S-NH2 Compound 42 (SEQ ID NO: 42) Y-Aib-QGT-αMeF-TSDYSILLDKK(AEEAc-AEEAc-γ Glu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLESHPSS-A ib-HPP-Ac3c-S-NH2 Compound 43 (SEQ ID NO: 43) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEA c-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYL LESHPSS-Aib-HPP-Ac3c-S-NH2.

[0046] GLP-1R, GIP R, and GCG R are involved in multiple metabolic pathways in the human body. It has been found to be distributed and expressed in tissues, organs, and cells. GLP-1R, GIP R, and GCG R are co-expressed in pancreatic islet tissue cells. GLP-1R is the most abundant receptor. GIP R was found to be expressed in adipocytes, with the highest GIP R abundance. GLP-1R and GLP-2R in liver cells have the highest abundance of GCG-R. There have also been reports on the expression of GCG R. At the same time, the present inventors have found that some compounds Better EC than wild-type polypeptide in stably transfected cells 50 of However, in the tissue cells, the tissue cells may be more sensitive than the wild-type polypeptide. We were surprised to find that even higher doses were required for sufficient stimulation. This is likely related to the distribution tendency of multi-agonist compounds across multiple receptors. To achieve a large therapeutic effect, the three agonist molecules bind to multiple receptors in tissue cells. and at least effectively activates receptors that are highly abundant in tissue cells. Therefore, it is necessary to be able to detect three types of agonist molecules on various tissue cells. EC 50 is at least as strong as the corresponding wild-type polypeptide GLP-1 / GIP / GCG. should also be good.

[0047] The compounds of the present invention act on three components of GLP-1 / GIP / GCCR. Strong relative activity EC 50 and can sufficiently stimulate the corresponding target organ tissue cells.

[0048] Preferably, the compound of the present invention can promote insulin secretion and reduce blood glucose. Preferably, it may also inhibit feeding, delay gastric emptying, and increase energy expenditure. The amount of serotonin in the blood can be increased and ultimately a weight loss effect can be observed.

[0049] Preferably, the compounds of the present invention can reduce pancreatic islet β-cell apoptosis, It can increase the number of islet cells and improve the function of pancreatic islet cells.

[0050] Preferably, the compounds of the present invention can also improve blood lipids and reduce liver fat accumulation. This can inhibit the development of liver inflammation and prevent and treat non-alcoholic fatty liver disease.

[0051] Preferably, the compounds of the present invention promote the growth of brain nerves, eliminate neurotoxic substances, and reduce inflammation. It is predicted to inhibit the onset of inflammatory bowel disease and play a neuroprotective role.

[0052] Preferably, the compounds or compositions of the present invention inhibit metabolic disorders and associated complications and It can be used to treat and / or cure diabetes, obesity, and non-alcoholic beverages. It is used to treat alcoholic fatty liver disease.

[0053] Preferably, the compounds or compositions of the present invention are used to treat dyslipidemia and related disorders, as well as papain. It can be used to treat neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease.

[0054] Preferably, the compound or composition of the present invention is used to treat bone diseases, metabolic disorders, and the like associated with endocrine disorders. It is used to treat kidney disease, as well as other causes of osteoporosis and osteoarthritis. It is possible.

[0055] The compounds of the present invention have significant stimulatory effects on GLP-1, GIP, and GCG receptors. It has.

[0056] The polypeptide compounds of the present invention can be synthesized by those skilled in the art by known technical methods. For example, the polypeptide sequence backbone of the polypeptide compounds of the present invention may be It can be prepared by a method such as synthesis.

[0057] The peptide backbone of the compounds of the present invention is chemically linked at least at one site by a fatty acid side group. Preferably, the compound has a stable peptide α-helical structure, It is possible to enhance albumin binding ability, improve the stability of peptide compounds, and increase the duration of peptide action. Achieve extension.

[0058] The compounds of the present invention have agonist activity against GLP-1 receptors, GIP receptors, and GCG receptors. The "agonist activity" referred to is the ability to stimulate specific receptor cells. This refers to the ability of a compound to produce cAMP in host cells, pancreatic tissue cells, and adipocytes. Overexpression of GLP-1 receptor, GIP receptor, or GCG receptor in liver cells, etc. Receptor activating activity can be achieved by a compound that stimulates receptor cells. EC50 of cAMP produced by the compound 50 It can be measured by the EC value. 50 The value is To achieve half of the compound's maximum activity (50% activity) in a particular assay system This refers to the drug concentration value required for

[0059] In a specific embodiment, the agonist activity of a compound is measured by the relative activity of a specific natural compound. Relative activity can be assessed by assessing the EC 50 Value Pair EC of test compound 50 It is a percentage of the ratio of values.

[0060] Compared with existing compounds, the GLP-1 / GIP / GCG R provided by the present invention The triple agonist polypeptide molecule has good GLP-1 R, GI P R, and GCG R, and tissue cell relative activity EC 50 It has.

[0061] definition Relative activity EC 50 " refers to the corresponding wild-type positive peptide human GLP-1(7-37) , human GIP, and human GCG EC 50 EC values ​​for compounds of the present invention 50 Specify the ratio of values vinegar.

[0062] The amino acids in the compound sequences of the present invention may be naturally occurring amino acids or related amino acid variants, and The abbreviations and symbols of the natural amino acids referred to are known to those skilled in the art. Based on well-known general rules: Aib, α-MeF, α-MeF(2F), α- The chemical structures of MeL and Ac3c are as follows:

[0063] [ka]

[0064] Unless otherwise defined, all technical and scientific terms used herein Terms have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, definitions The present document, including the accompanying drawings, shall prevail. Preferred methods and materials are described below, but the present invention The present invention may be practiced using methods and materials similar or equivalent to those described herein. The materials, methods, and examples disclosed herein are illustrative only. , are not intended to be limiting.

[0065] Specific meanings of the abbreviations used in this invention are as follows:

[0066] Aib: α-aminoisobutyric acid α-MeF: α-methylphenylalanine α-MeF(2F): α-methyl-2-fluorophenylalanine α-MeL: α-methylleucine Ac3c: 1-aminocyclopropanecarboxylic acid AEEAc: [2-(2-aminoethoxy)-ethoxy]-acetyl cAMP: adenosine cyclophosphate Fmoc: fluorene methoxycarbonyl Boc: tert-butoxycarbonyl DMF: dimethylformamide HBTU: Benzotriazole-N,N,N',N'-tetramethylurea hexafluoro diphosphate Trt: triphenylmethyl ivdde: 1-(4,4-dimethyl-2,6-dioxocyclohexyl)-3-methyl butyl tBu: tertbutyl OtBu: oxygen tertbutyl TFA: Trifluoroacetic acid Tis: Triisopropylsilane HPLC / MS: High-performance liquid chromatography / mass spectrometry HPLC-UV: High-performance liquid chromatography UV IPTG: Isopropyl-β-D-thiogalactoside Tris: Trishydroxymethylaminomethane aminomethane) DCM: dichloromethane THF: tetrahydrofuran DIPEA: N,N-diisopropylethylamine NMP: N-methylpyrrolidone HEK-293: Human Embryonic Kidney Cells CHO: Chinese hamster ovary cells GLP-1: glucagon-like peptide-1 GIP: glucose-dependent insulinotropic polypeptide GCG: Glucagon GLP-1 R: glucagon-like peptide-1 receptor GIP R: glucose-dependent insulinotropic polypeptide receptor GCG R: glucagon receptor NAFLD: Non-alcoholic fatty liver disease NASH: Nonalcoholic steatohepatitis DMEM: Du Improved Eagle's Medium FBS: fetal bovine blood FCS: fetal calf serum P / S: Penicillin / Streptomycin PBS: phosphate buffer solution HBSS: Hanks' Buffered Salt Solution EC 50 : Half-effective concentration IBMX: 3-isobutyl-1-methylxanthine. [Example]

[0067] The following embodiments and examples are provided to further illustrate the present invention, but by no means It is not intended to limit the effective scope of the present invention. Example 1

[0068] Synthesis of peptide compounds The intermediates and compounds of the present invention can be synthesized by various methods known in the art. The following specific embodiments are described below with reference to chemical methods for preparing the compounds of the present invention. Each specific synthetic step described is illustrated using different materials and methods. The methods may be used in combination to synthesize various corresponding compounds or salts of the present invention. The reagents and raw materials required are readily available to those skilled in the art. The embodiments are intended only to illustrate the invention and should in no way limit the scope of the invention. isn't it.

[0069] material: All materials and reagents used in this invention were purchased commercially and were used throughout the synthesis process. The protected amino acids used in are as follows: Fmoc-Ser(tBu )-OH, Fmoc-Ac3c-OH, Fmoc-Pro-OH, Fmoc-Ala-O H, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu -OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ph e-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(ivdde)-OH, Fmoc-α-MeLeu-OH, Fmoc- Thr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-α-MePh e-OH, Fmoc-α-MePhe(2F)-OH, Fmoc-Aib-OH, Boc -Tyr(tBu)-OH.

[0070] The synthesis and preparation methods of the compounds of the present invention are illustrated below using compound 10 as an example (as well as others). (Preparation of this compound simply requires changing the synthetic order of the amino acid raw materials.)

[0071] (1) Rink Amide MBHA Resin Pretreatment: 1 g of dry Rink Amide MBHA resin (substituted Weigh out 10 ml of DMF (sodium stearate = 0.28 mmol / g) and add it to the reaction column. The mixture was then sparged with nitrogen gas for 30 minutes. The solvent was removed and another 10 ml of DMF was added. The solvent was removed by washing three times for 1 minute each time.

[0072] (2) Removal of the protecting group Fmoc: 20% piperidine / DMF solution (10 ml) was added to the treated The mixture was added to the Link Amino Resin and left to react for 20 minutes under a nitrogen atmosphere. The reaction was monitored using the ninhydrin colorimetric method, and when the resin turned blue, indicates successful removal of Fmoc, and filtration is performed to remove the post-reaction solvent and remove DMF from the reaction system. The resin was washed for 1 minute by adding 100 ml of HCl to the solution, and this was repeated 6 times.

[0073] (3) Coupling reaction (peptide bond formation): The corresponding Fmoc-protected amino acid solution ( 3.0 equiv.) was added to the reactor, followed by DIEA (6.0 equiv.) and 5 ml of DMSO. F was added to the reaction column, nitrogen gas was blown in, and after dissolving the amino acid, HBTU (2.85 equivalents) was added. The nitrogen gas was adjusted so that the resin was uniformly bubbled, and the reaction was carried out at 25°C for 30 minutes. During this time, the extent of the reaction was monitored by using the ninhydrin colorimetric method. If the oil was colorless and transparent, it indicated successful coupling. After the reaction was complete, The solvent was removed by filtration. DMF was added to the reaction system, and the resin was stirred and washed for 1 minute. The above steps were repeated six times until peptide chain synthesis was completed. The corresponding amino acid solutions were added in order. The last amino acid was Boc-Tyr(tBu)-O By coupling H and detecting the colorless and transparent resin with tetrachlorobenzoquinone, Therefore, it was determined that the coupling was complete. Compound 10 was added to the synthesis of the main peptide sequence. The amino acid coupling sequences were Fmoc-Ser(tBu)-OH, Fmoc-Ac3 c-OH, Fmoc-Pro-OH(2×), Fmoc-Ala-OH, Fmoc-Gl y-OH, Fmoc-Ser(tBu)-OH(2×), Fmoc-Pro-OH, Fm oc-Gly-OH(2×), Fmoc-Glu(OtBu)-OH, Fmoc-Leu -OH(2×), Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fm oc-Ile-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-A ib-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc- Lys(ivdde)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp( OtBu)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Il e-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, F moc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-T hr(tBu)-OH, Fmoc-α-MeF(2F)-OH, Fmoc-Thr(tB u)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc- Aib-OH, Boc-Tyr(tBu)-OH.

[0074] (4) Removal of the side chain protecting group ivdde from Lys: 3% hydrazine hydrate / DMF solution (10 ml) was added to the reaction column, and the column was left to react under nitrogen gas for 20 minutes to form the modified portion. The side chain protecting group ivdde was removed from Lys. The blue color of the resin indicated completion. After successful removal, the solvent was removed and DMF was added to the system to wash for 1 minute. The solvent was removed and six washes were performed thereon.

[0075] (5) Lys side chain modification: AEEAc (2.0 equivalents) was added to the resin. 4.00 equivalents) was added, and 5 ml of DMF was added to the reaction column. After the amino acid was dissolved, HBTU (1.9 equiv.) was added. The mixture was adjusted to foam uniformly and reacted at 25°C for 1 hour. The reaction mixture was colored and clear, indicating a complete reaction. The mixture was washed six times with DMF (10 ml). 20% piperidine / DMF (10 ml) was added to the reaction mixture. The column was loaded and nitrogen gas was blown over it for 20 minutes to remove the Fmoc group. The solution was washed 6 times with 1 minute each time, and the solvent was removed. Following the procedure, AEEAc, Fmoc-Glu(OtBu)-OH, and C 20 Monote The side chain modification was completed by sequential addition of rt butyl ester to carry out the coupling reaction. Finally, the resin was shrunk with MeOH (10 ml) for 3 min each time, the solvent was drained, and the resin was poured off and dried and ready for use.

[0076] (6) Post-treatment of peptide resin: The above dried peptide resin was treated with the prepared cleavage reagent (95 % TFA: 2.5% Ti: 2.5% H2O) was added and shaken on a shaking table for 2.5 hours. The resin was then cleaved. Filtration was performed to obtain the filtrate, which was then added to 10 volumes of ice-cold Isopropyl Alcohol. The mixture was added to isopropyl ether, centrifuged, and washed five times with isopropyl ether. The crude peptide was obtained by drying in vacuo.

[0077] (7) Purification of crude peptide compounds: The obtained peptide powder was dissolved in 50% acetonitrile / H2O solution and purified by reversed-phase C18 A preparative column (Shimadzu Corporation, Inertsil ODS 20 × 250 mm 5 μm) was used. The initial eluent was 95% buffer A (0.1% TFA / H2O) and the 5 % Buffer B (0.075% TFA / acetonitrile) was used, and the proportion of Buffer B was 75% The purification was run continuously for 30 min for elution with increasing concentrations of 0.01 to 0.10, and the target peptide constructs were analyzed. The purified peptide compounds were analyzed by analytical HPLC / MS. It was confirmed that the purity of the obtained peptide compound was at least 95%.

[0078] [Table 3] TIFF2025143457000006.tif255154TIFF2025143457000007.tif255155TIFF2025143457000008.tif131170

[0079] Example 2 Peptides against stable transgenic cells of human GLP-1 / GIP / GCG receptors Compound activity testing First, HEK-293 cells stably overexpressing human GLP-1 or GCG receptors were used. CHO cells stably overexpressing the GIP receptor were separately constructed. By measuring the AMP signal response level, the activity of individual compounds on the corresponding receptors can be determined. The agonist activity of the compounds was determined. The intracellular cAMP content was measured by HTRF (homogeneous time-domain reflection). Based on the fluorescence resolved chromatographic technique, the kit is available from Cisbio Corp. and measured.

[0080] Frozen cells stably overexpressing human GLP-1, GIP, or GCG receptors For rapid thawing and recovery, place the cell solution in a 37°C water bath. The mixture was transferred to 10 ml of HBSS and centrifuged at 1000 rpm for 5 minutes at room temperature. Resuspend the cells in 1x HBSS (containing 0.1% casein and 250 μM IBMX). , cell density 1.0 x 10 5 The concentration was adjusted to 1 / mL.

[0081] 10 μL of the cell suspension was added to each well in a 384-well plate. The compound of interest is dissolved in 1x PBS buffer to prepare a stock solution, which is then diluted 3x. The compound solutions were diluted stepwise with 12 concentrations in total. Using a 384-well plate, 100 nL of the prepared compound solution was added to the 384-well plate. Add to the appropriate cell suspension and mix evenly by rotating and shaking at 1000 rpm for 1 minute. After drug incubation was complete, Add 10 μL of detection reagent to each well and incubate at room temperature for 60 minutes. The plates were analyzed using an EnVision multifunction enzyme reader (PerkinElmer). mer) and readouts were taken at 665 / 615 nm. Use sm5 plotting software to generate compound concentration-response curves and calculate EC5 The 0 (nM) value was calculated.

[0082] As a positive control for the receptor activating effect of test compounds, natural wild-type human GLP-1 (7 -37), GIP, and GCG were used to investigate the GLP-1 receptor in human G EC of LP-1(7-37) 50 EC value vs. the test compound 50 By calculating the ratio of the values The relative activity (%) of the test compound was evaluated based on the results. Regarding GIP receptor cells, EC of human GIP 50 EC value vs. the test compound 50 Ratio of values The percentage of the rate was calculated to evaluate the relative activity (%) of the test compound. Regarding GCG receptor cells, human GCG EC 50 EC value vs. the test compound 50 Value Pa The relative activity (%) of the test compounds was evaluated by calculating the percentage of the activity.

[0083] [Table 4] TIFF2025143457000010.tif51170

[0084] The data in the table show that all compounds stabilize human GLP-1 / GIP / GCG receptors. This indicates that it can exhibit high relative activity in cells.

[0085] Example 3 Functional activity test of peptide compounds against rat pancreatic islet tumor RIN-m5F cells The RIN-m5F cell line, derived from rat pancreatic islet tissue, expresses endogenous GLP-1 / GIP / GC G receptors are predominantly expressed in RIN-m5F, with GLP-1 receptors being the most abundant. The cAMP levels produced by the compounds on the cells were measured. Frozen RIN-m5F cells were placed in a 37°C water bath for rapid thawing and recovery. The cell solution was transferred to 10 ml of HBSS for resuspension and incubated at 1000 x g for 5 min at room temperature. The supernatant was discarded and the cells were resuspended in 1x HBSS (0.1% casein, 250 μL). Resuspend the cells in 100 mL of PBS containing IBMX to a cell density of 1.0 x 10 5 The concentration was adjusted to 1 / mL. 10 μL of the cell suspension was added to each well in a 384-well plate. The compound of interest is dissolved in 1x PBS buffer to prepare a stock solution, which is then diluted 3x. The compound solutions were diluted stepwise with 12 concentrations in total. Using a 384-well plate, 100 nL of the prepared compound solution was added to the 384-well plate. Add to the appropriate cell suspension and mix evenly by rotating and shaking at 1000 rpm for 1 minute. After drug incubation was complete, 10 μL of detection reagent was added to each well and incubation was carried out for 60 minutes. The pellet was placed in an EnVision multifunction enzyme reader (PerkinElmer) and read. The data were acquired at 665 / 615 nm. GraphPad Prism 5 plotting was used. Compound concentration-effect curves were generated and calculations were performed using RT-PCR software.

[0086] [Table 5]

[0087] The compounds of the present invention in the table exhibit high relative activity in pancreatic islet tumor cells RIN-m5F. possible.

[0088] Example 4 Functional activity test of peptide compounds on 3T3-L1 adipocytes Mouse 3T3-L1 precursor adipocytes can be induced to differentiate into mature adipocytes. 4 x 10 3T3-L1 cells 4 Directly into 96-well plates at cell density of 1000 / ml The cells were inoculated and cultured in DMEM medium containing 10% FCS and 1% P / S under 5% CO The mixture was incubated in an incubator at 37°C. Afterwards, 20% FBS, 0.5mM IBMX, 0.4ug / mL dexamethasone, and 3T3-L1 adipocytes were differentiated using DMEM differentiation medium containing 5ug / mL insulin. After 5 days of induction and culture, the cells were cultured in 20% FBS, 4 μg / ml insulin. Induction and culture were performed using DMEM medium containing 10 μM rosiglitazone and 10 μM rosiglitazone. This was continued for 3 days. Then, DMEM medium containing 10% FBS was used for 2-3 days. The culture was continued to induce mature 3T3-L1 adipocytes. Induced differentiation and maturation of 3T3-L1 adipocytes, as well as adipose tissue cells, resulted in abundant The compound-induced GIP receptor-expressing 3T3-L1 adipocytes cAMP levels were determined in this experiment. Test compound solutions were added in a 3-fold concentration gradient. The solution was added to the mature 3T3- L1 adipocytes were induced and incubated at room temperature for 60 minutes. Afterwards, 10 μL of the detection reagent provided in the kit was added to each well and incubated at room temperature for another 60 minutes. The plates were then read using an EnVision multifunction enzyme reader (PerkinElmer). r) and read at 665 / 615 nm. Compound concentration-effect curves were generated using Rism 5 plotting software. The calculation was carried out.

[0089] [Table 6]

[0090] The compounds of the present invention can exhibit high relative activity against adipocytes 3T3-L1.

[0091] Example 5 Functional activity testing of peptide compounds on human primary hepatocytes In this experiment, the cAMP levels produced by the compounds in human primary hepatocytes were measured. Human primary hepatocytes were purchased from Lonza (cell product number HUCPG). . Frozen human primary liver cells were placed in a 37°C water bath for rapid thawing and recovery. The cell solution was transferred to 10 ml of HBSS for resuspension and incubated at 1000 rpm for 5 minutes at room temperature. The supernatant was discarded and the cells were resuspended in 1x HBSS (0.1% casein, 250 μM Resuspend the cells in PBS containing IBMX to a cell density of 1.0 x 10 5 The concentration was adjusted to 1 / mL. 10 μL of the cell suspension was added to each well in a 384-well plate. The compound of interest is dissolved in 1x PBS buffer to prepare a stock solution, which is then diluted 3x. The compound solutions were diluted stepwise with 12 concentrations in total. Using a 384-well plate, 100 nL of the prepared compound solution was added to the 384-well plate. Add to the appropriate cell suspension and mix evenly by rotating and shaking at 1000 rpm for 1 minute. After drug incubation was complete, 10 μL of detection reagent was added to each well and incubation was carried out for 60 minutes. The pellet was placed in an EnVision multifunction enzyme reader (PerkinElmer) and read. The data were acquired at 665 / 615 nm. GraphPad Prism 5 plotting was used. Compound concentration-effect curves were generated and calculations were performed using RT-PCR software.

[0092] [Table 7]

[0093] The compounds of the present invention in the table may exhibit high relative activity against liver cells.

[0094] Example 6 In vivo pharmacodynamics High-fat diet-induced obesity (DIO) mice are all highly obese, similar to the human body. The major metabolic syndrome is characterized by obesity, elevated blood sugar, insulin resistance, and The body weight, food intake, and The effects of the compounds of the present invention on blood glucose and lipids were investigated. Five-week-old male C57BL / 6J mice (Shanghai Slake Experiment) (purchased from Central Animal Company) were kept under a 12-hour light / 12-hour in a pathogen-free and clean environment (temperature 20-24°C and They were housed in cages of four animals and kept at 30-70% relative humidity for two weeks. After an adaptation period of 10 min, the rats were fed a normal diet. Obesity was induced in mice by feeding them a high-fat diet for 16 weeks. After feeding, the weight of DIO mice reached 41–55 g, and the blood glucose range was 8–12 mmHg. ol / L. The animals in each group were then placed in a 100-mL bolus, with the aim of achieving similar mean body weights and blood glucose levels. DIO mice were randomly assigned to groups (n=6 / group) according to their body weight and fasting blood glucose. After grouping, one animal was kept in each cage for one week. , each animal was injected subcutaneously (SC) with vehicle (1×PBS, 5 ml / kg). It was pre-adapted to the experimental work process. After preparing the animals, vehicle control or compound was administered to groups of animals by subcutaneous injection. The substance was dissolved in 1x PBS, the dosage was 5ml / kg, and the administration started at 9:00am. The study was started and continued once every 3 days (Q3D) for 15 days. Animal body weight and food intake were measured daily and compared with the initial body weight and food intake of the same animals before treatment. The percentage (%) change in animal weight and cumulative food intake were calculated to determine body weight and The effect of the compounds on changes in food intake was assessed. At the end of the experiment (day 15), mice were weighed and then subjected to tail injury without anesthesia. Fasting blood glucose was measured by collecting blood from the tail tip of the rats. After the experiment, the animals were anesthetized with CO2 and euthanized. Blood was then collected from the heart and plasma was extracted by centrifugation. The plasma was used to measure total cholesterol (TC), low-density lipoprotein (LDLP), and LDL-C, triglycerides (TG), and blood insulin levels The liver was separated for homogenization and then centrifuged to measure the amount of liver The supernatant was obtained for determination of triglyceride content in the liver.

[0095] All results are expressed as mean ± SEM and the results were analyzed using GraphPad Prism 5 software. The data were analyzed by one-way ANOVA using software, followed by comparison with the vehicle control group. Comparisons were followed by Dunnett's post hoc test. Differences at the p<0.05 level were considered statistically significant. Well done.

[0096] [Table 8]

[0097] [Table 9]

[0098] From the results in Tables 6-1 and 6-2, the compounds of the present invention have significant effects on body weight reduction. It is clear that this has an effect.

[0099] [Table 10]

[0100] [Table 11]

[0101] From the results in Tables 7-1 and 7-2, it can be seen that the compounds of the present invention can lower blood lipids. It can be seen that it has a significant therapeutic effect on

[0102] [Table 12]

[0103] [Table 13]

[0104] The results in Tables 8-1 and 8-2 show that the compounds of the present invention can reduce blood glucose and blood insulin levels. It can be seen that this has a significant therapeutic effect on reducing the level of vasoconstriction.

[0105] [Table 14]

[0106] [Table 15]

[0107] The results in Tables 9-1 and 9-2 show that the compounds of the present invention lower liver triglycerides. It is clear that the compound has a significant therapeutic effect on reducing blood pressure.

[0108] Example 7 Pharmacokinetic (PK) study of the compound in rats Male SD rats (7-9 weeks old, 220-250 g, 3 rats / group) were treated with 30 nmol 1 / kg of compound was administered subcutaneously. After administration, the rats were treated at 0.25, 2, 4, 8, 12, 16, and 24 Blood was collected via the jugular vein at 48, 96, 120, and 144 hours. The liquid was processed to obtain plasma samples, which were analyzed using LC-MS / MS. The inter-phase curve was generated using Phoenix WinNonlin version 6.3 software (non-convertible). The pharmacokinetic parameters and half-life were calculated using a compartmental model.

[0109] The PK parameters calculated using the above methods are shown in Table 10.

[0110] [Table 16]

[0111] Table 10 shows that the individual compounds exhibit an extended pharmacokinetic distribution.

Claims

1. General formula (I): Y-Aib-X3-GT-X6-TSDYSI-X13-LDK-X17-AQ-Aib -AFIE-X25-LLE-X29-X30-PSS-X34-X35-PP-X38 -S-R １ (I) (In the formula, X3 is Q or H; X6 is F, αMeF, or αMeF(2F); X13 is αMeL, F, αMeF, or L; X17 is Ψ; X25 is Y or F; X29 is T, S, G, or Aib; X30 is G, H, R, or Aib; X34 is G or Aib; X35 is A, Q, Aib, or H; X38 is Ac3c or P; R １ is NH ２ or OH, or a pharmaceutically acceptable salt and / or ester thereof le; Ψ is represented by the following general formula (II): Y-Z (II) and Lys having a side chain modified by Y is (AEEAc or Glu) ａ -(AEEAc or Glu)b-(AEEAc or or Glu) ｃ a, b, and c are independently 0 or 1; , and c is not zero at the same time, and the carboxyl terminus of Y is the ε-amino acid of the side chain of Lys. is connected to; Z is -CO-(CH ２ ) ｍ -R ２ m is an integer from 6 to 24, and R ２ Ha-COOH (selected from A GLP-1 / GIP / GCGR triple agonist polypeptide compound having the formula: is a salt or solvate thereof.

2. The compounds of general formula (I) contain at least two specific amino acids at the following positions: ： X13 is F or αMeF; X25 is F; X29 is T or S; X30 is H, R, or Aib; X35 is Q, Aib, or H; X38 is Ac3c The polypeptide compound according to claim 1, or a salt or solvate thereof. 。

3. The general formula (II) is AEEAc-AEEAc-γGlu-CO(CH ２ ) １８ COOH The polypeptide compound according to claim 1, or a salt or solution thereof, medium.

4. The compound is 【Table 1】 The polypeptide compound or its salt according to claim 1 is also selected from the group consisting of or solvates.

5. The compound is 【Table 2】 The polypeptide compound or compounds thereof according to claim 4, characterized in that it is further selected from A salt or solvate of

6. Activation capacity of GLP-1 receptor stably transfected cells compared to native GLP-1 In this respect, the compound has at least 3 activity levels in terms of its ability to activate the GLP-1 receptor. 0%, preferably at least 60%, more preferably at least 80%, even more preferably The polypeptide of claim 1 has a relative activity of at least 100%. A thiazide compound or a salt or solvate thereof.

7. In terms of the activation ability of GIP receptor stably transfected cells compared to native GIP In this case, the compound is at least 100% or more effective in terms of its ability to activate the GIP receptor.

2. The method according to claim 1, characterized in that it preferably has a relative activity of at least 150%. A polypeptide compound or a salt or solvate thereof.

8. In terms of activation ability of GCG receptor stably transfected cells compared to native GCG In this case, the compound has at least 10% or more of the ability to activate the GCG receptor.

2. The polynucleotide of claim 1, preferably having a relative activity of at least 30%. A peptide compound or a salt or solvate thereof.

9. Observation of RIN-m5F, a representative cell of pancreatic islet tissue, compared with native GLP-1(7-37) In this respect, the compound exhibits at least 60% potency in activating RIN-m5F cells. %, preferably at least 80%, more preferably at least 100% relative activity. The polypeptide compound according to claim 1, or a salt or solvate thereof, thing.

10. In comparison with native GIP, the compound is at least 60%, preferably at least 60%, in terms of the ability to activate 3T3-L1 cells.

2. The polypeptide compound of claim 1, wherein both have a relative activity of 100%. or a salt or solvate thereof.

11. In terms of human primary liver cells representative of liver tissue compared to native GCG, the compound is at least 60%, preferably at least 10% in terms of liver cell activation ability.

10. The polypeptide compound of claim 1, characterized in that it has a relative activity of 0% or its salts or solvates.

12. A compound according to any one of claims 1 to 11, or any of its salts or solvates. an effective amount of any one of the compounds of the present invention and a pharmaceutically acceptable excipient, diluent, carrier, or vehicle; Pharmaceutical compositions.

13. The pharmaceutical compositions are available in the form of injections, freeze-dried powders, tablets, pills, lozenges, soft capsules, hard capsules, and the like. capsules, granules, powders, liquids, suspensions, or syrups; alternatively, the pharmaceutical composition The composition may be in the form of a microcapsule, microsphere, nanoparticle, or liposome.

13. The pharmaceutical composition according to claim 12, characterized in that it is in the form

14. The pharmaceutical composition may be administered orally, by inhalation, or parenterally, with parenteral administration being intraperitoneal administration. The administration is selected from intracavitary, intramuscular, intraarterial, intravenous, subcutaneous, or intradermal injections. The pharmaceutical composition according to claim 12.

15. Administered at least once daily, weekly, biweekly, or monthly The pharmaceutical composition according to claim 12, characterized in that

16. The pharmaceutical composition is used in combination with at least one therapeutically active agent, The substance may be an antidiabetic activator, a GIP receptor agonist, a GCG receptor agonist or an agonist. antagonist, GLP-1 / GIP receptor agonist, GLP-1 / GCG receptor agonist GIP / GCG receptor agonists, FGF-21 and its analogs, cholecystokinin nin B (CCKB) and its analogs, PYY(3-36) and its analogs, leptin and analogs thereof, calcitonin and analogs thereof, lipid-regulating active agents, PPAR-α, β , delta agonists or modulators, antiplatelet aggregation activators, PCSK9 inhibitors, lipase inhibitors an anti-inflammatory agent, an anti-fibrotic or anti-cirrhotic agent, or an anti-inflammatory agent. The pharmaceutical composition according to claim 12.

17. Antidiabetic active agents include insulin and its analogs, biguanides, sulfonylureas, thiazolinones, and thiazolinones. Azolidinediones, α-glucosidase inhibitors, DPP-4 inhibitors, SGLT2 inhibitors, Dual SGLT1 / SGLT2 inhibitors, GLP-1 receptor agonists, or amylin and 17. The pharmaceutical composition of claim 16, comprising:

18. In the preparation of a drug for promoting insulin secretion and reducing blood glucose, 12. A compound according to any one of claims 1 to 11, or a salt or solvate thereof, 18. Use of a pharmaceutical combination according to any one of claims 12 to 17.

19. Inhibits feeding, delays gastric emptying, increases energy expenditure, and weight A compound according to any one of claims 1 to 11 in the preparation of a medicament for reducing or a salt or solvate thereof, or any one of claims 12 to 17 Use of pharmaceutical combinations.

20. Decreasing islet β-cell apoptosis, increasing islet β-cell number, and 12. A method according to claim 1 in the preparation of a medicament for improving cellular function. or a salt or solvate thereof, or a compound of any one of claims 12 to 17 Use of the described pharmaceutical combinations.

21. Improves blood lipids, reduces liver fat accumulation, inhibits the development of liver inflammation, and In the preparation of a drug for preventing and treating non-alcoholic fatty liver disease, Item 12. The compound according to any one of items 1 to 11, or a salt or solvate thereof, or 18. Use of a pharmaceutical combination according to any one of claims 12 to 17.

22. Promoting the growth of brain nerves, eliminating neurotoxic substances, inhibiting the onset of inflammation, and 12. The composition according to any one of claims 1 to 11 in the preparation of a drug for exerting a protective effect. or a salt or solvate thereof, or a compound of any one of claims 12 to 17 Use of the described pharmaceutical combinations.

23. for the prevention and / or treatment of metabolic disorders and associated complications, preferably diabetes, obesity 2. The method of claim 1 for preparing a drug for the treatment of non-alcoholic fatty liver disease. 11 or a salt or solvate thereof, or 18. Use of a pharmaceutical combination according to any one of claims 1 to 17.

24. Dyslipidemia and related disorders, as well as diseases including Parkinson's and Alzheimer's A compound according to any one of claims 1 to 11 in the preparation of a medicament for treating a degenerative disease. or a salt or solvate thereof, or any one of claims 12 to 17.

2. Use of a pharmaceutical combination as described in claim 1.

25. Endocrine disorders related to bone disease, metabolic disorders, kidney disease, osteoporosis and osteoarthritis 12. A method for the preparation of a medicament for treating a bone disease comprising administering to a subject a compound according to any one of claims 1 to 11. or a salt or solvate thereof, or any one of claims 12 to 17.

2. Use of a pharmaceutical combination as described in claim 1.

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