Multiagonists and uses thereof

Abstract

The present invention relates to the pharmaceutical and biological fields, in particular to polypeptide compounds of general formula (I) having triple agonist activity on the glucagon-like peptide-1 receptor (GLP-1 R), the glucose-dependent insulinotropic polypeptide receptor (GIPR) and the glucagon receptor (GCG R) and their use in the treatment of metabolic syndrome.

Description

[Technical Field]

[0001] The present invention relates to a polypeptide compound and its use in pharmaceutical field.More specifically, the present invention relates to a polypeptide compound with triple agonist activity on glucagon-like peptide-1 receptor (GLP-1 R), glucose-dependent insulinotropic polypeptide receptor (GIPR) and glucagon receptor (GCGR), and its use in the treatment of metabolic syndrome. [Background technology]

[0002] Type II diabetes and obesity are increasingly becoming global diseases that affect human health.Obesity is the main cause of many chronic diseases, such as diabetes, hypertension, heart disease, dyslipidemia, fatty liver disease, atherosclerosis, arthritis, stroke, neurodegenerative disease, etc., and diabetes can also lead to cardiovascular and cerebrovascular diseases and other complications.Most of the hypoglycemic drugs currently on the market only have the effect of controlling blood glucose, but cannot improve the weight of obese patients.Some drugs even have the side effect of increasing weight.Therefore, there remains an urgent need to develop a drug that can both lower blood glucose and improve body weight, and contains multiple beneficial effects that can meet the needs of most obese and type II diabetes people.

[0003] Incretin is a kind of polypeptide hormone secreted from the intestine after normal physiological food stimulation.Early research has found that it can stimulate pancreatic islet β-cells to secrete insulin as glucose level rises after a meal, regulate glucose homeostasis, protect pancreatic islet β-cells, suppress appetite and delay gastric emptying, thereby reducing body weight.GLP-1 and GIP are currently found to be two types of pancreatic stimulating hormones.

[0004] GLP-1 is a 31-amino acid polypeptide expressed in intestinal mucosal L cells by the glucagon gene. It primarily acts on the GLP-1 receptor (GLP-1R), stimulating insulin secretion, inhibiting glucagon secretion, protecting pancreatic islet β-cells, and regulating blood glucose homeostasis. It also inhibits food intake and gastric emptying through central nervous system signaling pathways, increasing satiety and thus reducing body weight. Exendin-4 is a GLP-1 analogue extracted from the salivary glands of the African venomous lizard, and exhibits stronger GLP-1 receptor activation and similar GLP-1 effects. Compared to native GLP-1, exendin-4 has stronger resistance to DPP-4 and a longer plasma half-life in the body.

[0005] GIP is a 42-amino acid single-chain polypeptide produced by small intestinal mucosal K cells and primarily acts on GIP receptors (GIPR) in pancreatic islet cells and adipocytes. GIP-dependent insulin secretion enhances pancreatic islet β-cell quality, stimulates insulin secretion, inhibits gastric acid secretion, and slows gastric peristalsis. It also stimulates fatty acid uptake and utilization by adipose tissue cells. GIP also has physiological effects such as promoting osteoblast differentiation, inhibiting osteoblast apoptosis, inhibiting bone resorption, increasing bone mineral density, and protecting bone.

[0006] GCG is a polypeptide containing 29 amino acids, expressed and secreted by the glucagon progenitor gene in pancreatic islet α-cells. It mainly acts on glucagon receptors (GCG R) distributed in the liver and kidney, stimulating liver glycogen breakdown, increasing blood glucose, activating lipase, promoting lipolysis, while inhibiting liver lipogenesis and enhancing fatty acid oxidation. Research results show that GCG has certain effects on reducing food intake, increasing adipose tissue energy expenditure, and reducing body fat. The appropriate effect of GCG in increasing blood glucose can provide feedback for insulin regulation and reduce the occurrence of hypoglycemic events.

[0007] Aiming at the effects of incretin, GLP-1 receptor agonists such as exenatide, lisenatide, liraglutide, dulaglutide, and semaglutide have been successfully developed for the treatment of type II diabetes. In addition, liraglutide has been successfully developed for weight loss, and semaglutide is also undergoing clinical studies for obesity indications. The advantage of GLP-1 analogs is that they can lower blood glucose and also have cardiovascular benefits and weight management effects. However, at present, the weight loss effect of single GLP-1 receptor agonists is still less than 10%, and there are obvious dose-related gastrointestinal side effects (mainly nausea, vomiting, and diarrhea). There remains an urgent need for more effective weight loss therapeutic agents for populations suffering from metabolic diseases with complex pathologies, such as obese type II diabetes, nonalcoholic fatty liver disease / nonalcoholic steatohepatitis (NAFLD / NASH), diabetes, and obesity with cardiovascular risk.

[0008] According to the physiological effects of GLP-1, GIP, and GCG, currently, many studies have confirmed that activating both or all three of these receptors simultaneously can achieve better therapeutic effects on diabetes and obesity than activating only GLP-1R.It has been reported that hyperglycemia, obesity, and insulin resistance hinder the effects of GIP R and its signaling pathway.However, as blood glucose decreases and insulin resistance improves, the role of GIP in promoting insulin secretion and improving pancreatic islet function can be improved.In addition to promoting glycogen breakdown and increasing blood glucose, GCG can also promote fat breakdown, inhibit hepatic fat synthesis, and exert the effects of reducing blood lipids and body weight.However, synergistic inhibition of GLP-1 is required to suppress its hyperglycemic effect. Therefore, lowering blood glucose and improving insulin resistance through the "leading" effect of GLP-1 may further enhance the insulin secretion-promoting function and insulin sensitization synergistic effect of GIP, improve pancreatic islet function, further enhance the lipolytic effect of GCG, improve lipid metabolism, and enhance the weight loss effect.

[0009] With the publication of clinical data on GLP-1 / GIP and GLP-1 / GCG dual agonists, the influence of the distribution of GLP-1 / GIP or GLP-1 / GCG dual agonist activity among different receptors on the clinical therapeutic efficacy of the drug is becoming increasingly clear.

[0010] For example, the results of a phase II clinical study of Lilly's tirzepatide (LY3298176) showed that a 1 mg dose of tirzepatide had little weight loss effect, and its blood glucose lowering effect was significantly lower than that of 1.5 mg of dulaglutide. Only when the dose of tirzepatide was increased to 5 mg could it produce better blood glucose lowering and weight loss effects than 1.5 mg of dulaglutide. This is likely due to tirzepatide's tendency to have stronger GIP receptor agonist activity, while its GLP-1 receptor activity EC 50 is the wild-type GLP-1 activity EC 50In pursuit of maximum efficacy, the dosage of tirzepatide has been significantly increased (up to a maximum dose of 15 mg), which may increase the safety risks of the drug.

[0011] On the other hand, due to the strong hyperglycemic effect of GCG, sufficient GLP-1 activity is required in GLP-1 / GCG receptor dual agonists to balance and suppress the hyperglycemic effect of GCG, thereby ensuring the hypoglycemic effect. The relative activity EC of GLP-1 and GCG receptors in MEDI0382 (cotadutide), a GLP-1 / GCG receptor dual agonist manufactured by AstraZeneca, was 50 The rates of glycemic control were 29% and 12.8%, respectively. Results of a 26-week clinical trial showed that 100-300 μg of cotadutide significantly reduced body weight, blood lipids, and ALT and AST levels compared with liraglutide, with the 300 μg dose demonstrating significantly better results than 1.8 mg of liraglutide. However, there was no significant benefit in reducing HbA1c levels compared with liraglutide in either dose group. Furthermore, as the cotadutide dosage increased, the reduction in glycated hemoglobin in the 300 μg group was actually worse than that in the 200 μg group. This is likely due to the cotadutide molecule's low GLP-1 receptor activity as the dose increased, which is insufficient to suppress the effects of GCG.

[0012] An ideal long-acting GLP-1 / GIP / GCG triple receptor agonist molecule, administered once, twice, or three times a week, should have the highest possible GLP-1R / GIPR activity and relatively controllable GCGR activity, ensuring maximum weight loss and blood glucose lowering effects.CN104902919A and CN111040022A disclose a series of GLP-1 / GIP / GCG R triple agonist molecules based on the structural modification of venomous lizard exopeptide-4 (exendin-4);CN109071624A discloses a series of cyclic peptide molecules combined with long-acting conjugates to form molecules.In addition, WO2015067716A1, WO2019125929A1, and WO2019125938A1 also disclose a polypeptide in which a fatty acid is linked to the side chain of the amino acid at position 17. All of these polypeptides demonstrate a triple activation effect of GLP-1R / GIP / GCG R and have the potential for long-lasting weekly action. The molecules disclosed in the above patent documents cannot simultaneously exhibit sufficiently high activity against GLP-1R / GIP R / GCG R. 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 novel polypeptide molecule having GLP-1 / GIP / GC G R triple activating activity, which can be used in the treatment of type II diabetes, obesity, dyslipidemia, non-alcoholic fatty liver disease / non-alcoholic steatohepatitis, and other related metabolic diseases.

[0015] The present invention relates to a compound of general formula (I): Y-Aib-X3-GT-X6-TSDYSI-X13-LDK-X17-AQ-Aib-AFIE-X25-LLE-X29-X30-PSS-X34-X35-PP-X38-SR1 (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 NH or OH, or a pharmaceutically acceptable salt and / or ester thereof; 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 Glu)c, where a, b, and c are independently 0 or 1, and a, b, and c are not simultaneously zero (as an illustrative example, AEEAc-AEEAc-γGlu), and 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 is selected from -COOH) The present invention provides a GLP-1 / GIP / GCG R triple agonist polypeptide compound, a salt thereof, or a solvate thereof, which has the following structure: DETAILED DESCRIPTION OF THE INVENTION

[0017] In one embodiment of the present invention, the compound of general formula (I) contains 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.

[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] TIFF0007803570000002.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 compound has a relative activity in terms of its ability to activate GLP-1 receptor stably transfected cells compared to native GLP-1, of at least 30%, preferably at least 60%, more preferably at least 80%, and even more preferably at least 100% in terms of its ability to activate the GLP-1 receptor.

[0026] In one embodiment of the present invention, the compound has a relative activity in terms of its ability to activate GLP-1 receptor stably transfected cells compared to native GIP, of at least 100%, more preferably at least 150%, in terms of its ability to activate the GIP receptor.

[0027] In one embodiment of the present invention, the compound has a relative activity in terms of activation of the GCG receptor in stably transfected cells compared to native GCG, of at least 10%, more preferably at least 30%, in terms of activation of the GCG receptor.

[0028] In one embodiment of the present invention, the compound has a relative activity on RIN-m5F cells, representative of pancreatic islet tissue, of at least 60%, preferably at least 80%, and more preferably at least 100%, compared to native GLP-1(7-37).

[0029] In one embodiment of the present invention, the compound has a relative activity of at least 60%, preferably at least 100%, in terms of activation on 3T3-L1 cells, which are representative cells of adipose tissue, compared to native GIP.

[0030] In one embodiment of the present invention, the compound has a relative activity in terms of activation on human primary liver cells representative of liver tissue, compared to native GCG, of at least 60%, preferably at least 100%, in terms of activation on liver cells.

[0031] The present invention further provides a pharmaceutical composition comprising an effective amount of a compound according to any of the above aspects, or any of its salts or solvates, and a pharmaceutically acceptable excipient, diluent, carrier, or vehicle.

[0032] In one embodiment, the pharmaceutical composition is an injectable solution, lyophilized powder, tablet, pill, lozenge, soft capsule, hard capsule, granule, powder, liquid, suspension, or syrup; alternatively, the drug composition is in the form of a microcapsule, microsphere, nanoparticle, or liposome.

[0033] In one aspect, the pharmaceutical composition is for oral administration, inhalation administration, or parenteral administration, wherein parenteral administration is selected from intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous, or intradermal injection administration.

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

[0035] In one embodiment, the pharmaceutical composition comprises an antidiabetic active agent (such as insulin and its analogs, biguanides, sulfonylureas, thiazolidinediones, α-glucosidase inhibitors, DPP-4 inhibitors, SGLT2 inhibitors, dual SGLT1 / SGLT2 inhibitors, GLP-1 receptor agonists, amylin and its analogs), GIP receptor agonists, GCG receptor agonists or antagonists, GLP-1 / GIP receptor agonists, GLP-1 / GCG receptor agonists, GIP / GCG receptor antagonists, or the like. The antidiabetic activator may also be used in combination with at least one of the following active agents for treatment, including receptor agonists, FGF-21 and analogs thereof, cholecystokinin B (CCKB) and analogs thereof, PYY(3-36) and analogs thereof, leptin and analogs thereof, calcitonin and analogs thereof, lipid-regulating activators, PPAR-α, β, δ agonists or regulators, anti-platelet aggregation activators, PCSK9 inhibitors, lipase inhibitors, anti-liver fibrosis or anti-liver cirrhosis activators, and anti-inflammatory activators. In one embodiment, the antidiabetic activator comprises insulin and analogs thereof, biguanides, sulfonylureas, thiazolidinediones, α-glucosidase inhibitors, DPP-4 inhibitors, SGLT2 inhibitors, dual SGLT1 / SGLT2 inhibitors, GLP-1 receptor agonists, or amylin and analogs thereof.

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

[0037] The use of any one of the compounds of the present invention or its salts or solvates, or any one of the pharmaceutical compositions, in the preparation of a medicament for inhibiting feeding, delaying gastric emptying, increasing energy expenditure, and reducing body weight.

[0038] The use of any one of the compounds of the present invention or its salts or solvates, or any one of the pharmaceutical compositions, in the preparation of a medicament for reducing pancreatic islet β-cell apoptosis, increasing pancreatic islet β-cell number, and improving pancreatic islet cell function.

[0039] Use of any one of the compounds of the present invention or its salts or solvates, or any one of the pharmaceutical compositions, in the preparation of a medicament for improving blood lipids, reducing liver fat accumulation, inhibiting the development of liver inflammation, and preventing and treating non-alcoholic fatty liver disease.

[0040] Use of any one of the compounds of the present invention or its salts or solvates, or any one of the pharmaceutical compositions, in the preparation of a drug for promoting the growth of brain nerves, eliminating neurotoxic substances, inhibiting the onset of inflammation, and exerting neuroprotective effects.

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

[0042] Use of any one of the compounds of the present invention or salts or solvates thereof, or any one of the pharmaceutical compositions, in the preparation of a medicament for treating dyslipidemia and related disorders, and neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease.

[0043] Use of any one of the compounds of the present invention or salts or solvates thereof, or any one of the pharmaceutical compositions thereof, in the preparation of a medicament for treating bone diseases, metabolic disorders, renal diseases, etc. associated with endocrine disorders, wherein bone diseases include 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, or a salt or solvent complex thereof: Compound 1 (SEQ ID NO: 1) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLETGPSSGAPP-Ac3c-S-NH2 Compound 2 (SEQ ID NO: 2) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLETHPSSGAPP-Ac3c-S-NH2 Compound 3 (SEQ ID NO: 3) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLESHPSSGAPP-Ac3c-S-NH2 Compound 4 (SEQ ID NO: 4) Y-Aib-QGT-αMeF(2F)-TSDYSIFLDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18COOH)AQ-Aib-AFIEYLLEGGPSSGAPP-Ac3c-S-NH2 Compound 5 (SEQ ID NO: 5) Y-Aib-QGT-αMeF(2F)-TSDYSIFLDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLEGGPSSGAPP-Ac3c-S-NH2 Compound 6 (SEQ ID NO: 6) Y-Aib-HGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLE-Aib-HPSSGQPPPS-NH2 Compound 7 (SEQ ID NO: 7) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLETGPPSSG-Aib-PPPS-NH2 Compound 8 (SEQ ID NO: 8) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLETGPSSGQPPPS-NH2 Compound 9 (SEQ ID NO: 9) Y-Aib-QGT-αMeF(2F)-TSDYSIFLDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLETGPSSGAPP-Ac3c-S-NH2 Compound 10 (SEQ ID NO: 10) Y-Aib-QGT-αMeF(2F)-TSDYSIFLDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLESHPSSGAPP-Ac3c-S-NH2 Compound 11 (SEQ ID NO: 11) Y-Aib-QGT-αMeF-TSDYSIFLDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLETGPSSGAPP-Ac3c-S-NH2 Compound 12 (SEQ ID NO: 12) Y-Aib-QGT-αMeF-TSDYSIFLDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLETGPSSGAPPPS-NH2 Compound 13 (SEQ ID NO: 13) Y-Aib-QGT-αMeF-TSDYSIFLDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLETGPSSGAPPPS-NH2 Compound 14 (SEQ ID NO: 14) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLETGPSSGAPPPS-NH2 Compound 15 (SEQ ID NO: 15) Y-Aib-QGT-αMeF-TSDYSIFLDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLESHPSSGAPPS-NH2 Compound 16 (SEQ ID NO: 16) Y-Aib-QGT-αMeF-TSDYSIFLDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLE-Aib-HPSSGAPPS-NH2 Compound 17 (SEQ ID NO: 17) Y-Aib-QGT-αMeF(2F)-TSDYSIFLDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLETGPSSGAPPPS-NH2 Compound 18 (SEQ ID NO: 18) Y-Aib-QGT-αMeF(2F)-TSDYSIFLDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLE-Aib-HPSSGAPPS-NH2 Compound 19 (SEQ ID NO: 19) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLETGPSSGAPPPS-NH2 Compound 20 (SEQ ID NO: 20) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLE-Aib-HPSSGAPPS-NH2 Compound 21 (SEQ ID NO: 21) Y-Aib-QGT-αMeF(2F)-TSDYSIFLDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLEGGPSSGQPPPS-NH2 Compound 22 (SEQ ID NO: 22) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLEGGPSSGQPPPS-NH2 Compound 23 (SEQ ID NO: 23) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLETGPSSGQPPPS-NH2 Compound 24 (SEQ ID NO: 24) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLETGPSS-Aib-HPPPS-NH2 Compound 25 (SEQ ID NO: 25) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLEGRPSS-Aib-HPPPS-NH2 Compound 26 (SEQ ID NO: 26) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLESHPSSGAPPS-NH2 Compound 27 (SEQ ID NO: 27) Y-Aib-QGT-αMeF-TSDYSI-αMeF-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLESHPSSGAPPS-NH2 Compound 28 (SEQ ID NO: 28) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-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-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLES-Aib-PSSGAPP-Ac3c-S-NH2 Compound 30 (SEQ ID NO: 30) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-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(AEEAc-AEEAc-γGlu-CO(CH2) 18COOH)AQ-Aib-AFIEFLLE-Aib-HPSSGAPP-Ac3c-S-NH2 Compound 32 (SEQ ID NO: 32) Y-Aib-QGT-αMeF(2F)-TSDYSIFLDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLE-Aib-HPSSGAPP-Ac3c-S-NH2 Compound 33 (SEQ ID NO: 33) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLESHPSS-Aib-HPP-Ac3c-S-NH2 Compound 34 (SEQ ID NO: 34) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLETGPSSGQPP-Ac3c-S-NH2 Compound 35 (SEQ ID NO: 35) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLETGPSSGQPP-Ac3c-S-NH2 Compound 36 (SEQ ID NO: 36) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLEGGPSS-Aib-HPPPS-NH2 Compound 37 (SEQ ID NO: 37) Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLEGRPSS-Aib-HPPPS-NH2 Compound 38 (SEQ ID NO: 38) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLETGPSS-Aib-HPPPS-NH2 Compound 39 (SEQ ID NO: 39) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLETRPSS-Aib-HPPPS-NH2 Compound 40 (SEQ ID NO: 40) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLESHPSS-Aib-HPP-Ac3c-S-NH2 Compound 41 (SEQ ID NO: 41) Y-Aib-QGT-αMeF(2F)-TSDYSILLDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLESHPSS-Aib-HPP-Ac3c-S-NH2 Compound 42 (SEQ ID NO: 42) Y-Aib-QGT-αMeF-TSDYSILLDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEFLLESHPSS-Aib-HPP-Ac3c-S-NH2 Compound 43 (SEQ ID NO: 43) Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2) 18 COOH)AQ-Aib-AFIEYLLESHPSS-Aib-HPP-Ac3c-S-NH2.

[0046] GLP-1R, GIP R and GCG R are found to be distributed and expressed in multiple metabolic tissues, organs and cells in human body.For example, GLP-1R, GIP R and GCG R are simultaneously expressed in pancreatic islet tissue cells, and GLP-1R is the most abundant receptor.For example, GLP-1R and GIP R are found to be expressed in adipocytes, with the highest GIP R abundance.There are also reports of GLP-1R and GCG R being expressed in liver cells, with the highest GCG R abundance.At the same time, the present inventors have found that some compounds have better EC than wild-type polypeptides in single-receptor stable transfected cells. 50 Although the three agonist molecules may have the activity of the above-mentioned tissue cells, it was surprising to find that, compared with wild-type polypeptides, higher doses are required to fully stimulate the tissue cells in the aforementioned tissue cells.This is probably related to the distribution tendency of multi-agonist compounds across multiple receptors.Therefore, in order to achieve maximum therapeutic effect, it is required that the three agonist molecules can fully activate multiple receptors in tissue cells, or at least can effectively activate receptors with high abundance in tissue cells.Therefore, the EC of the three agonist molecules on various tissue cells is 50 should be at least better than the corresponding wild-type polypeptide GLP-1 / GIP / GCG.

[0047] The compounds of the present invention have strong relative activity EC of the three components of GLP-1 / GIP / GCCR. 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, and preferably also inhibit food intake, delay gastric emptying, increase energy expenditure, and ultimately observe a weight loss effect.

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

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

[0051] Preferably, the compounds of the present invention are expected to promote brain nerve growth, eliminate neurotoxic substances, inhibit the development of inflammation, and play a neuroprotective role.

[0052] Preferably, the compound or composition of the present invention can be used to prevent and / or treat metabolic disorders and related complications, and is preferably used to treat diabetes, obesity, and non-alcoholic fatty liver disease.

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

[0054] Preferably, the compounds or compositions of the present invention can be used to treat bone diseases, metabolic disorders, kidney diseases, and other causes such as osteoporosis and osteoarthritis associated with endocrine disorders.

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

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

[0057] The peptide backbone of the compound of the present invention is chemically modified at at least one site with a fatty acid side group. Preferably, the compound has a stable peptide α-helical structure, which can enhance albumin binding ability, improve the stability of the peptide compound, and extend the duration of peptide action.

[0058] The compounds of the present invention have agonist activity for GLP-1 receptor, GIP receptor, and GCG receptor. The "agonist activity" referred to refers to the ability of a compound to stimulate specific receptor cells to produce cAMP, which can be constructed by those skilled in the art to overexpress GLP-1 receptor, GIP receptor, or GCG receptor in host cells, pancreatic tissue cells, adipocytes, liver cells, etc. Receptor activating activity is determined by the EC 200 of cAMP produced by a compound stimulating receptor cells. 50 It can be measured by the EC value. 50 The value refers to the drug concentration value required to achieve half of the maximum activity (50% activity) of a compound in a particular measurement system.

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

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

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

[0062] The amino acids in the compound sequences of the present invention are derived from natural amino acids or related amino acid variants and / or derivatives. The abbreviations and symbols of the natural amino acids referred to are based on general rules familiar to those skilled in the art. The chemical structures of Aib, α-MeF, α-MeF(2F), α-MeL, and Ac3c are as follows:

[0063] [ka]

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present document, including definitions, shall prevail. Preferred methods and materials are described below, but the present invention can be carried out or tested using methods and materials similar or equivalent to those described herein. The materials, methods, and examples disclosed herein are merely illustrative and 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 hexafluorophosphate Trt: triphenylmethyl ivdde: 1-(4,4-dimethyl-2,6-dioxocyclohexyl)-3-methyl-butyl tBu: tert butyl 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: Trihydroxymethylaminomethane 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 in no way 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 and prepared by various methods known in the art. The following specific embodiments illustrate the use of chemical synthesis methods to prepare the compounds of the present invention. Each of the specific synthesis steps described can be combined using different materials and methods to synthesize various corresponding compounds or salts of the present invention. The reagents and raw materials used are easily available to those skilled in the art. Specifically, the following embodiments are intended only to illustrate the present invention and should not limit the scope of the present invention in any way.

[0069] material: All materials and reagents used in this invention were purchased commercially, and the protected amino acids used throughout the synthesis process are as follows: Fmoc-Ser(tBu)-OH, Fmoc-Ac3c-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-I le-OH, Fmoc-Phe-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-MePhe-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 (preparation of other compounds requires only changing the synthetic order of the amino acid starting materials).

[0071] (1) Rink Amide MBHA Resin Pretreatment: 1 g of dry Rink Amide MBHA resin (degree of substitution S = 0.28 mmol / g) was weighed and added to a reaction column. 10 ml of DMF was added and nitrogen gas was blown into the column for 30 minutes. The solvent was removed, and another 10 ml of DMF was added to wash the column three times, each time for 1 minute, and the solvent was removed.

[0072] (2) Removal of the Fmoc protecting group: 20% piperidine / DMF solution (10 ml) was added to the treated Rink amino resin and allowed to react for 20 minutes under a nitrogen atmosphere. During this time, the degree of reaction was monitored using the ninhydrin colorimetric method. When the resin turned blue, it indicated successful removal of Fmoc. Filtration was performed to remove the solvent after the reaction, and DMF was added to the reaction system to wash the resin for 1 minute. 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 DMF was added to the reaction column. Nitrogen gas was blown through the column, and after dissolving the amino acid, HBTU (2.85 equiv.) was added. The nitrogen gas was adjusted to bubble evenly through the resin, and the reaction was carried out at 25 °C for 30 min. During this time, the reaction progress was monitored by ninhydrin colorimetry. A colorless and transparent resin indicated successful coupling. After the reaction was completed, the solvent was removed by filtration. DMF was added to the reaction system, and the resin was stirred and washed for 1 min. This process was repeated six times. The above steps were repeated, with the corresponding amino acid solutions added in order, until peptide chain synthesis was complete. Boc-Tyr(tBu)-OH was coupled to the last amino acid, and the colorless and transparent resin was detected with tetrachlorobenzoquinone to determine completion of the coupling. Lys at the side chain modification site was replaced with Fmoc-Lys(ivdde)-OH.The sequence of additional amino acid couplings in the synthesis of the main peptide sequence of compound 10 was Fmoc-Ser(tBu)-OH, Fmoc-Ac3c-OH, Fmoc-Pro-OH (2x), Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH (2x), Fmoc-Pro-OH, Fmoc-Gly-OH (2x), Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH (2x), Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Aib-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-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-A sp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-α-MeF(2F)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Aib-OH, and Boc-Tyr(tBu)-OH.

[0074] (4) Removal of the side chain protecting group ivdde from Lys: A 3% hydrazine hydrate / DMF solution (10 ml) was added to the reaction column and allowed to react under nitrogen gas for 20 minutes to remove the side chain protecting group ivdde from the modified site Lys. The blue color of the ninhydrin detection resin indicated complete removal. After successful removal, the solvent was removed, and DMF was added to the system to wash for 1 minute, and the solvent was removed, where washing was performed six times.

[0075] (5) Lys side chain modification: AEEAc (2.0 equivalents) was added to the resin. DIEA (4.00 equivalents) was added, and 5 ml of DMF was added to the reaction column. Nitrogen gas was blown through the column, and after the amino acid was dissolved, HBTU (1.9 equivalents) was added. The nitrogen gas was adjusted to bubble evenly through the resin, and the reaction was carried out at 25°C for 1 hour. The ninhydrin-detected resin was colorless and transparent, indicating a completed reaction. The reaction solution was removed, and the column was washed with DMF (10 ml) six times, with each wash lasting 1 minute. 20% piperidine / DMF (10 ml) was added to the reaction column, and nitrogen gas was blown through the column for 20 minutes to remove the Fmoc group. DMF (10 ml) was added, and the column was washed six times, with each wash lasting 1 minute, and the solvent was removed. Following the above steps, AEEAc, Fmoc-Glu(OtBu)-OH, and C 20 The mono-tertbutyl ester was added successively to carry out the coupling reaction to complete the side chain modification. 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, ready for use.

[0076] (6) Post-treatment of peptide resin: The prepared cleavage reagent (95% TFA: 2.5% Ti: 2.5% HO) was added to the dried peptide resin and cleaved by shaking on a shaking table for 2.5 hours. The filtrate was obtained by filtration, added to 10 volumes of ice-cold isopropyl ether, centrifuged, and washed five times with isopropyl ether. The crude peptide was obtained by vacuum drying for 2 hours.

[0077] (7) Purification of crude peptide compounds: The obtained peptide powder was dissolved in 50% acetonitrile / HO solution and purified using a reversed-phase C18 preparative column (Shimadzu, Inertsil ODS 20 x 250 mm, 5 μm). The initial eluent was 95% Buffer A (0.1% TFA / HO) and 5% Buffer B (0.075% TFA / acetonitrile), and the percentage of Buffer B was gradually increased to 75%. The purification was run continuously for 30 minutes to recover the target peptide components. The purified peptide compounds were analyzed and confirmed by analytical HPLC / MS. The purity of the obtained peptide compounds was at least 95%.

[0078] [Table 3] TIFF0007803570000006.tif255154TIFF0007803570000007.tif255155TIFF0007803570000008.tif131170

[0079] Example 2 Activity testing of peptide compounds against stable transgenic cells of human GLP-1 / GIP / GCG receptors First, HEK-293 cells stably overexpressing human GLP-1 or GCG receptors and CHO cells stably overexpressing GIP receptors were constructed separately.The agonist activity of each compound on the corresponding receptors was determined by measuring the cAMP signal response level of the above cells.The intracellular cAMP content was measured using a reagent kit available from Cisbio Corp. based on HTRF (homogeneous time-resolved fluorescence) technology.

[0080] Frozen cells stably overexpressing human GLP-1, GIP, or GCG receptors 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 centrifuged at 1000 rpm for 5 minutes at room temperature. The supernatant was discarded, and the cells were resuspended in 1x HBSS (containing 0.1% casein and 250 μM IBMX) to a cell density of 1.0 x 105 The concentration was adjusted to 1 / mL.

[0081] 10 μL of cell suspension was added to each well in a 384-well plate. The compounds to be tested were dissolved in 1× PBS buffer to prepare stock solutions, which were then serially diluted 3-fold to prepare a total of 12 concentration points of compound solutions. Using the ECHO liquid transfer system, 100 nL of the prepared compound solution was added to the corresponding cell suspension in the 384-well plate, spun and shaken at 1000 rpm for 1 minute to mix evenly, and then incubated at room temperature for 60 minutes. After drug incubation was completed, 10 μL of detection reagent was added to each well, and incubation was performed at room temperature for 60 minutes in the kit. The plate was placed in an EnVision multifunction enzyme reader (PerkinElmer), and readings were taken at 665 / 615 nm. Compound concentration-response curves were generated using GraphPad Prism 5 plotting software, and EC 50 (nM) values ​​were calculated.

[0082] The EC values ​​of human GLP-1(7-37) were measured for GLP-1 receptor cells using native wild-type human GLP-1(7-37), GIP, and GCG as positive controls for the receptor activation effects of test compounds. 50 EC value vs. the test compound 50 The relative activity (%) of the test compound was evaluated by calculating the ratio of the values. Regarding GIP receptor cells, EC of human GIP 50 EC value vs. the test compound 50 The relative activity (%) of the test compound was evaluated by calculating the percentage of the ratio of the values. Regarding GCG receptor cells, human GCG EC 50 EC value vs. the test compound 50 The relative activity (%) of the test compounds was evaluated by calculating the percentage of the values.

[0083] [Table 4] TIFF0007803570000010.tif51170

[0084] The data in the table show that all compounds can exhibit high relative activity in human GLP-1 / GIP / GCG receptor stable 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, primarily expresses endogenous GLP-1 / GIP / GCG receptors, with GLP-1 receptors being the most abundant. This experiment measured the cAMP levels produced by compounds in RIN-m5F cells. 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 centrifuged at 1000 rpm for 5 minutes at room temperature. The supernatant was discarded, and the cells were resuspended in 1x HBSS (containing 0.1% casein, 250 μM IBMX) to a cell density of 1.0 x 10 5 The concentration was adjusted to 1 / mL. 10 μL of cell suspension was added to each well of a 384-well plate. The compounds to be tested were dissolved in 1× PBS buffer to prepare stock solutions, which were then serially diluted 3-fold to prepare a total of 12 concentration points of compound solutions. Using the ECHO liquid transfer system, 100 nL of the prepared compound solution was added to the corresponding cell suspension in the 384-well plate, spun and shaken at 1000 rpm for 1 minute to mix evenly, and then incubated at room temperature for 60 minutes. After drug incubation was completed, 10 μL of detection reagent was added to each well, and incubation was carried out for 60 minutes. The plate was placed in an EnVision multifunction enzyme reader (PerkinElmer), and readings were taken at 665 / 615 nm. Compound concentration-effect curves were generated and calculations were performed using GraphPad Prism 5 plotting software.

[0086] [Table 5]

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

[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. 3T3-L1 cells were cultured at 4 × 10 4 Cells were seeded directly into 96-well plates at a cell density of 100 / ml and cultured in DMEM medium containing 10% FCS and 1% P / S at 37°C in a 5% CO2 incubator. After the cells had fully filled and were in contact with each other, 3T3-L1 adipocyte differentiation was induced using DMEM differentiation medium containing 20% ​​FBS, 0.5 mM IBMX, 0.4 μg / mL dexamethasone, and 5 μg / mL insulin. After 5 days of induction and culture, induction and culture were continued for 3 days using DMEM medium containing 20% ​​FBS, 4 μg / mL insulin, and 10 μM rosiglitazone. Mature 3T3-L1 adipocytes were then induced using DMEM medium containing 10% FBS for 2-3 days. Induced differentiation and maturation of 3T3-L1 adipocytes may express abundant GIP receptors, similar to adipose tissue cells. The cAMP levels produced by compounds on 3T3-L1 adipocytes were determined in this experiment. The test compound solution was serially diluted according to a 3-fold concentration gradient to obtain a total of 10 serial concentration solutions. The solution was added to induce mature 3T3-L1 adipocytes and incubated at room temperature for 60 minutes. After drug incubation, 10 μL of detection reagent from the kit was added to each well and incubated at room temperature for another 60 minutes. The plate was placed in an EnVision multifunction enzyme reader (PerkinElmer) to measure readings at 665 / 615 nm. Compound concentration-effect curves were generated and calculations were performed using GraphPad Prism 5 plotting software.

[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 The cAMP levels produced by the compounds on human primary hepatocytes were determined in this experiment. 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 centrifuged at 1000 rpm at room temperature for 5 minutes. The supernatant was discarded, and the cells were resuspended in 1x HBSS (containing 0.1% casein and 250 μM IBMX) to a cell density of 1.0 x 10 5 The concentration was adjusted to 1 / mL. 10 μL of cell suspension was added to each well of a 384-well plate. The compounds to be tested were dissolved in 1× PBS buffer to prepare stock solutions, which were then serially diluted 3-fold to prepare a total of 12 concentration points of compound solutions. Using the ECHO liquid transfer system, 100 nL of the prepared compound solution was added to the corresponding cell suspension in the 384-well plate, spun and shaken at 1000 rpm for 1 minute to mix evenly, and then incubated at room temperature for 60 minutes. After drug incubation was completed, 10 μL of detection reagent was added to each well, and incubation was carried out for 60 minutes. The plate was placed in an EnVision multifunction enzyme reader (PerkinElmer), and readings were taken at 665 / 615 nm. Compound concentration-effect curves were generated and calculations were performed using GraphPad Prism 5 plotting 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 obese (DIO) mice are characterized by obesity, elevated blood glucose, insulin resistance, and dyslipidemia, all of which are very serious metabolic syndromes similar to those in humans. The effects of the compounds of the present invention on body weight, food intake, blood glucose, and lipids in C57BL / 6J DIO mice were investigated. Five-week-old male C57BL / 6J mice (purchased from Shanghai Slake Experimental Animal Company) were housed in a pathogen-free and clean environment (temperature 20-24°C and relative humidity 30-70%) with a 12-hour light / 12-hour dark cycle. They were fed a normal diet with four animals per cage and a two-week adaptation period. Obesity was induced in mice by feeding them a high-fat diet (60 kcal from fat). After 16 weeks of high-fat diet feeding, the body weight of DIO mice reached 41-55 g, and their blood glucose range reached 8-12 mmol / L. DIO mice were randomly divided into groups (n = 6 / group) according to their body weight and fasting blood glucose, so that animals in each group had similar average body weight and blood glucose levels. After grouping, one animal was kept in each cage for one week, during which time each animal was subcutaneously (SC) injected with vehicle (1x PBS, 5 ml / kg) to pre-adapt to the experimental procedure. After preparing the animals, vehicle control or compound was administered to the animal groups by subcutaneous injection. The compound was dissolved in 1x PBS, and the dosage was 5 ml / kg. The administration started at 9:00 am and was carried out once every three days (Q3D) for 15 days. Throughout the experimental study, animal body weight and food intake were measured daily before administration. The percentage change (%) in animal body weight and cumulative food intake were calculated compared with the initial body weight and food intake of the same animal before administration to evaluate the effect of the compound on changes in body weight and food intake. At the end of the experiment (day 15), the mice were weighed, and fasting blood glucose was measured by collecting blood from the injured tail without anesthesia. After blood collection, the animals were anesthetized with CO2 and euthanized. Blood was then collected from the heart, and plasma was separated by centrifugation. The plasma was used to measure total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), and blood insulin content. The liver was separated by homogenization and then centrifuged to obtain the supernatant for determining triglyceride content in the liver.

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

[0096] [Table 8]

[0097] [Table 9]

[0098] The results in Tables 6-1 and 6-2 show that the compounds of the present invention have significant effects on weight loss.

[0099] [Table 10]

[0100] [Table 11]

[0101] The results in Tables 7-1 and 7-2 show that the compounds of the present invention have significant therapeutic effects on lowering blood lipids.

[0102] [Table 12]

[0103] [Table 13]

[0104] The results in Tables 8-1 and 8-2 show that the compounds of the present invention have significant therapeutic effects on lowering blood glucose and blood insulin.

[0105] [Table 14]

[0106] [Table 15]

[0107] The results in Tables 9-1 and 9-2 show that the compounds of the present invention have significant therapeutic effects on lowering hepatic triglycerides.

[0108] Example 7 Pharmacokinetic (PK) study of the compound in rats Male SD rats (220-250 g, 3 rats / group) aged 7-9 weeks were subcutaneously injected with 30 nmol / kg of compound. Blood was collected via the jugular vein at 0.25, 2, 4, 8, 12, 16, 24, 48, 96, 120, and 144 hours after administration. Blood was processed to obtain plasma samples, which were analyzed using LC-MS / MS. Blood concentration-time curves were analyzed using Phoenix WinNonlin version 6.3 software (non-compartmental model), and PK parameters and half-lives were calculated.

[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): [___]______________________ ____________________________________________ _______________________________________________ [33], １ () (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; 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 Glu) ｃ wherein a, b, and c are independently and separately 0 or 1, and a, b, and c are not simultaneously zero, and the carboxyl terminus of Y is connected to the ε-amino of the side chain of Lys; Z is -CO-(CH ２ ) ｍ -R ２ m is an integer from 6 to 24, and R ２ is selected from —COOH; Contains 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, and the general formula (II) is AEEAc-AEEAc-γGlu-CO(CH ２ ) １８ COOH, and the compound is Table 1 is selected from.) 1. A GLP-1 / GIP / GCG R triple agonist polypeptide compound having the formula:

2. The compound is a GLP-1 / GIP / GCG R triple agonist polypeptide compound of claim 1 or a salt or solvate thereof, having the formula: Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH2)18COOH)AQ-Aib-AFIEYLLESHPSSGAPP-Ac3c-S-NH2 (compound 3).

3. 10. A pharmaceutical composition comprising an effective amount of a compound of claim 1 or any of its salts or solvates, and a pharmaceutically acceptable diluent, carrier, or excipient.

4. The pharmaceutical composition described in claim 3, comprising an effective amount of Y-Aib-QGT-αMeF-TSDYSI-αMeL-LDKK(AEEAc-AEEAc-γGlu-CO(CH 2 ) 18 COOH)AQ-Aib-AFIEYLLESHPSSGAPP-Ac3c-S-NH 2 (compound 3) or a salt or solvate thereof.

5. 5. The pharmaceutical composition according to claim 3 or 4, wherein the pharmaceutical composition is in the form of an injection, a lyophilized powder, a tablet, a pill, a lozenge, a soft capsule, a hard capsule, a granule, a powder, a liquid, a suspension, or a syrup; alternatively, the pharmaceutical composition is in the form of a microcapsule, a microsphere, a nanoparticle, or a liposome.

6. 5. The pharmaceutical composition according to claim 3 or 4, wherein the pharmaceutical composition is used for oral administration, inhalation administration, or parenteral administration, wherein the parenteral administration is selected from intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous, or intradermal injection administration.

7. 5. The pharmaceutical composition according to claim 3 or 4, which is administered at least once daily, once weekly, once every two weeks, or once monthly.

8. 5. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is used in combination with at least one therapeutically active substance, the therapeutically active substance comprising an antidiabetic activator, a GIP receptor agonist, a GCG receptor agonist or antagonist, a GLP-1 / GIP receptor agonist, a GLP-1 / GCG receptor agonist, a GIP / GCG receptor agonist, FGF-21 and analogs thereof, cholecystokinin B (CCKB) and analogs thereof, PYY(3-36) and analogs thereof, leptin and analogs thereof, calcitonin and analogs thereof, a lipid-regulating activator, a PPAR-α, β, δ agonist or modulator, an anti-platelet aggregation activator, a PCSK9 inhibitor, a lipase inhibitor, an anti-fibrotic or anti-cirrhotic activator, or an anti-inflammatory activator.

9. 9. The pharmaceutical composition according to claim 8, wherein the antidiabetic active agent comprises insulin and its analogs, biguanides, sulfonylureas, thiazolidinediones, α-glucosidase inhibitors, DPP-4 inhibitors, SGLT2 inhibitors, dual SGLT1 / SGLT2 inhibitors, GLP-1 receptor agonists, or amylin and its analogs.

10. 10. Use of a compound according to claim 1 or 2, or a salt or solvate thereof, or a pharmaceutical composition according to any one of claims 3 to 9, in the preparation of a medicament for promoting insulin secretion and reducing blood glucose.

11. Use of a compound according to claim 1 or 2 or a salt or solvate thereof, or a pharmaceutical composition according to any one of claims 3 to 9, in the preparation of a medicament for inhibiting feeding, delaying gastric emptying, increasing energy expenditure, and reducing body weight.

12. Use of a compound according to claim 1 or 2, or a salt or solvate thereof, or a pharmaceutical composition according to any one of claims 3 to 9, in the preparation of a medicament for reducing pancreatic islet β-cell apoptosis, increasing pancreatic islet β-cell number, and improving pancreatic islet cell function.

13. Use of a compound according to claim 1 or 2 or a salt or solvate thereof, or a pharmaceutical composition according to any one of claims 3 to 9, in the preparation of a drug for improving blood lipids, reducing liver fat accumulation, inhibiting the development of liver inflammation, and preventing and treating non-alcoholic fatty liver disease.

14. Use of a compound according to claim 1 or 2 or a salt or solvate thereof, or a pharmaceutical composition according to any one of claims 3 to 9, in the preparation of a drug for promoting the growth of cranial nerves, eliminating neurotoxic substances, inhibiting the onset of inflammation, and exerting neuroprotective effects.

15. 10. Use of a compound according to claim 1 or 2, or a salt or solvate thereof, or a pharmaceutical composition according to any one of claims 3 to 9, in the preparation of a medicament for the prevention and / or treatment of metabolic disorders and associated complications.

16. Use of a compound according to claim 1 or 2, or a salt or solvate thereof, or a pharmaceutical composition according to any one of claims 3 to 9, in the preparation of a medicament for the treatment of diabetes, obesity, or non-alcoholic fatty liver disease.

17. Use of a compound according to claim 1 or 2, or a salt or solvate thereof, or a pharmaceutical composition according to any one of claims 3 to 9, in the preparation of a medicament for treating dyslipidemia and related disorders, and neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease.

18. Use of a compound according to claim 1 or 2 or a salt or solvate thereof, or a pharmaceutical composition according to any one of claims 3 to 9, in the preparation of a medicament for treating bone diseases including bone diseases associated with endocrine disorders, metabolic disorders, kidney diseases, osteoporosis and osteoarthritis.

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