A human amylin analogue and uses thereof

Abstract

The present application provides a human amylin analogue and its application. The human amylin analogue is selected from the compounds shown in the following structural formula or chiral isomers thereof, tautomers thereof, or pharmaceutically acceptable salts, esters or amides thereof: γGlu-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gly-Orn-X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -Arg-Ser-Ser-X 21 -X 22 -X 23 -X 24 -X 25 -Lys-X 27 -X 28 -X 29 -Thr-Glu-Val-Gly-Ser-Asn-Thr-Tyr-NH2, wherein two Cys residues at the 2nd and 7th positions form an intramolecular methylene bridge, and a fatty acid side chain is connected to the epsilon-amino group of the Lys side chain at the 26th position. The human amylin analogue of the present application has good biological activity and bioavailability, and can be used for the treatment of metabolic diseases such as diabetes and obesity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a human pancreatic amylin analogue and its applications. Background Technology

[0002] Diabetes mellitus is a metabolic disorder characterized by hyperglycemia, caused by an absolute or relative deficiency of insulin secretion and decreased sensitivity of target tissues or organs to insulin. It is mainly classified into three types: type 1, type 2, and gestational diabetes mellitus.

[0003] Obesity is a disease caused by excessive accumulation of body fat, which negatively impacts health. According to relevant standards, a BMI (Body Mass Index) of 25.0-29.9 kg / m² is considered healthy. 2 Defined as overweight, 30 kg / m 2 Obesity is defined as the condition defined as above. Obese patients often have complications such as fatty liver disease, dyslipidemia, and hypertension. The disease burden brought about by overweight and obesity should not be ignored.

[0004] Although GLP-1 inhibitors such as semaglutide and telpoxetine have been approved for the treatment of metabolic diseases such as diabetes and obesity, market demand remains unmet. Furthermore, these drugs often suffer from side effects such as muscle loss and gastrointestinal adverse reactions during treatment, which severely limits their clinical application. Amylin analogues have the potential advantage of "fat reduction without muscle loss," thus potentially overcoming a significant therapeutic deficiency of GLP-1 inhibitors.

[0005] Amylin, also known as islet amyloid polypeptide (IAPP), is a peptide hormone composed of 37 amino acids. It is synthesized by pancreatic β-cells and secreted in conjunction with insulin, and is produced from precursor proteins. Amylin participates in metabolic regulation by activating amylin receptors (AMYRS), through mechanisms including delaying gastric emptying, inhibiting glucagon secretion, regulating central satiety, and synergistic effects with insulin. Amylin receptors are a special type of protein heterodimer, formed by the polymerization of a calcitonin receptor (CTR) with three homologous receptor-modifying proteins (RAMP1, RAMP2, and RAMP3). These receptors have become important drug targets for the treatment of diabetes and obesity.

[0006] Natural human amylin is easily and rapidly degraded by enzymes in the body, with an extremely short half-life of less than one hour. Secondly, it is chemically unstable under physiological conditions, has a high tendency to fibrosis, and is prone to misfolding and aggregation, leading to reduced drug stability and even cytotoxicity.

[0007] Introducing methylated amino acids, proline, or salt bridges into polypeptide chains can reduce the tendency to form fibrils and enhance stability. Simultaneously, fatty acid side chain modification can slow renal clearance and prolong half-life. These polypeptide drug modification technologies have been applied to several amylin analogs. Pramlintide is currently the only approved amylin analog for diabetes treatment. Its structure contains three proline substitutions compared to human amylin. Pramlintide exhibits enhanced stability, but it still forms aggregates and precipitates, and its half-life is only 30-50 minutes, requiring two to three injections daily, leading to poor patient compliance. Summary of the Invention

[0008] One object of the present invention is to provide a human pancreatic amylin analogue.

[0009] Another object of the present invention is to provide the application of human pancreatic amylin analogues.

[0010] In a first aspect, the present invention provides a human amylin analogue selected from compounds shown in the following structural formulas or their chiral isomers, tautomers, or pharmaceutically acceptable salts, esters, or amides:

[0011] In this structure, an intramolecular methylene bridge (-S-CH2-S-) is formed between the two Cys residues at positions 2 and 7; and / or

[0012] Lys at position 26 The ε-amino group is covalently attached to the Lys side chain; R1 is selected from carboxyl, carboxylic acid ester, tetrazolium, phosphoric acid, phosphate ester, sulfonic acid, and sulfonate groups; n is any integer from 10 to 24. Among them, X 12 X 13 X 14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29 Each is independently selected from amino acids.

[0013] The human amylin analogue of the present invention is a polypeptide compound (with a modified structure on the peptide chain). As a novel amylin drug, it can exert metabolic regulation by activating amylin receptors and has good biological activity and bioavailability.

[0014] In a second aspect, the present invention provides an amylin receptor agonist comprising the aforementioned human amylin analogue of the present invention.

[0015] Thirdly, the present invention provides the use of the aforementioned human amylin analogue or amylin receptor agonist in the preparation of a medicament for the prevention or treatment of any of the following diseases, said diseases including obesity, diabetes, insulin resistance syndrome, sleep apnea, obesity-induced cartilage degeneration and / or osteoarthritis, cardiovascular disease, large vessel disease, microvascular disease, diabetic cardiomyopathy, coronary heart disease, myocardial infarction, stroke, arteriosclerosis, peripheral artery disease, gallbladder disease, non-alcoholic steatohepatitis, Alzheimer's disease, fatty liver disease, alcoholic liver disease, diabetic nephropathy, renal failure, chronic kidney disease, hypertension, heart failure, eating disorders, cholecystitis, and short bowel syndrome.

[0016] The human amylin analogue of the present invention can exert metabolic regulatory functions such as hypoglycemia and weight regulation by activating amylin receptors, exhibiting good biological activity. In the human amylin analogue of the present invention, an intramolecular methylene bridge (-S-CH2-S-) is formed between the two Cys residues at positions 2 and 7 of the polypeptide sequence, and some Asn residues prone to deamidation are replaced, and methylated amino acids are introduced, resulting in good stability and safety under or near-physiological conditions. In the human amylin analogue of the present invention, the ε-amino group of the Lys side chain at position 26 of the polypeptide sequence is covalently linked to the fatty acid side chain via an amide bond, which can reduce renal clearance and enzymatic degradation of the compound, thereby increasing the plasma half-life and bioavailability in vivo, reducing the frequency of administration, and improving patient compliance. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below. Unless otherwise specified, specific conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0018] Amylin analogues have the potential to treat metabolic diseases such as obesity, diabetes, and hypertension. More than 40 amylin analogues are under investigation, with over 10 in clinical trials. The only approved amylin analogue for diabetes management, prinlantide, has limited clinical efficacy and an extremely short half-life, leading to poor patient compliance due to frequent dosing. Furthermore, many amylin analogues under investigation are in early clinical stages and have gastrointestinal side effects. Therefore, developing amylin analogues with better bioactivity and safety would significantly improve available treatment options. The human amylin analogue provided in this invention is a polypeptide compound and an amylin receptor agonist. It not only possesses the aforementioned efficacy for treating corresponding diseases but also has a longer in vivo half-life and better bioavailability, making it a long-acting amylin drug that improves patient compliance.

[0019] Specifically, in a first aspect, embodiments of the present invention provide a human amylin analogue selected from compounds shown in the following structural formulas or their chiral isomers, tautomers, or pharmaceutically acceptable salts, esters, or amides:

[0020] In this structure, an intramolecular methylene bridge (-S-CH2-S-) is formed between the two Cys residues at positions 2 and 7; and / or

[0021] Lys at position 26 The ε-amino group is covalently attached to the Lys side chain; R1 is selected from any one of carboxyl, carboxylic acid ester, tetrazolium, phosphoric acid, phosphate ester, sulfonic acid, and sulfonate groups; n is any integer from 10 to 24. Among them, X 12 X 13 X 14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29 Each is independently selected from amino acids.

[0022] In some specific embodiments, the amino acid described herein is selected from any one of modified or unmodified amino acids. Optionally, the amino acid may be, for example, any one of Leu, Ala, Aib, Aad, Asp, Glu, Phe, Val, Pro, Asn, Gly, and Ser. Optionally, the modification may be, for example, any one or more of N-Me modification, α-Me modification, 2-F modification, and 4-F modification.

[0023] In some specific embodiments, in the structure of the human pancreatic amylin analogue of the present invention, X 12 It is selected from any one of Leu, N-Me-Leu, and α-Me-Leu.

[0024] In some specific embodiments, in the structure of the human pancreatic amylin analogue of the present invention, X 13 Choose from Ala, N-Me-Ala, and Aib.

[0025] In some specific embodiments, in the structure of the human pancreatic amylin analogue of the present invention, X 14 It is selected from any one of Aad, Asp, Glu, α-Me-Asp, N-Me-Glu and α-Me-Glu.

[0026] In some specific embodiments, in the structure of the human pancreatic amylin analogue of the present invention, X 15 It is selected from any one of Phe, N-Me-Phe, α-Me-Phe, α-Me-Phe(2-F) and α-Me-Phe(4-F).

[0027] In some specific embodiments, in the structure of the human pancreatic amylin analogue of the present invention, X 16 It is selected from any one of Leu, N-Me-Leu, and α-Me-Leu.

[0028] In some specific embodiments, in the structure of the human pancreatic amylin analogue of the present invention, X 17 It is selected from Val, N-Me-Val and α-Me-Val.

[0029] In some specific embodiments, in the structure of the human pancreatic amylin analogue of the present invention, X 21 It is selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn.

[0030] In some specific embodiments, in the structure of the human pancreatic amylin analogue of the present invention, X 22 It is selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn.

[0031] In some specific embodiments, in the structure of the human pancreatic amylin analogue of the present invention, X 23 It is selected from any one of Phe, N-Me-Phe, α-Me-Phe, α-Me-Phe(2-F) and α-Me-Phe(4-F).

[0032] In some specific embodiments, in the structure of the human pancreatic amylin analogue of the present invention, X 24 Choose from any one of Gly, N-Me-Gly, Ala, and Aib; optionally, X 24 Choose from Gly, N-Me-Gly, and Ala.

[0033] In some specific embodiments, in the structure of the human pancreatic amylin analogue of the present invention, X 25 Choose either Pro or α-Me-Pro.

[0034] In some specific embodiments, in the structure of the human pancreatic amylin analogue of the present invention, X 27 It is selected from any one of Leu, N-Me-Leu, and α-Me-Leu.

[0035] In some specific embodiments, in the structure of the human pancreatic amylin analogue of the present invention, X 28 Choose from Ser, Pro, and α-Me-Pro.

[0036] In some specific embodiments, in the structure of the human pancreatic amylin analogue of the present invention, X 29 Choose from Ser, Pro, and α-Me-Pro.

[0037] In this invention, two or more of the multiple features described in the embodiments or implementations can be combined arbitrarily. For example, regarding amino acid residue feature X... 12 X 13 X 14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29 Within the specified constraints, two or more of these features can be combined arbitrarily. Amino acid residue X 12 X 13 X 14 X 15 X16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29 One or more of the features can also be arbitrarily combined with the intramolecular methylene bridge (-S-CH2-S-) feature between the two Cys residues at positions 2 and 7 of the human amylin analog and / or the modification feature of Lys at position 26.

[0038] In some specific embodiments, in the structure of the human amyloid analogue of the present invention, Lys at position 26 is expressed by the formula... The ε-amino group is covalently attached to the Lys side chain; R1 is selected from any one of carboxyl, carboxylic acid ester, tetrazolium, phosphoric acid, phosphate ester, sulfonic acid, and sulfonate groups; n is any integer from 10 to 24. X 12 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 13 Selected from Ala, N-Me-Ala, and Aib; X 14 Selected from any one of Aad, Asp, Glu, α-Me-Asp, N-Me-Glu, and α-Me-Glu; X 15 Selected from any one of Phe, N-Me-Phe, α-Me-Phe, α-Me-Phe(2-F) and α-Me-Phe(4-F); X 16 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 17 Selected from Val, N-Me-Val, and α-Me-Val; X 21 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 22 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 23 Selected from any one of Phe, N-Me-Phe, α-Me-Phe, α-Me-Phe(2-F) and α-Me-Phe(4-F); X 24Choose from any one of Gly, N-Me-Gly, Ala, and Aib; optionally, X 24 Selected from Gly, N-Me-Gly, and Ala; X 25 Selected from either Pro or α-Me-Pro; X 27 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 28 Selected from Ser, Pro, and α-Me-Pro; and / or X 29 Choose from Ser, Pro, and α-Me-Pro.

[0039] In some specific embodiments, the structure of the human pancreatic amylin analogue of the present invention is as follows: It forms an amide bond covalently with the ε-amino group of the Lys side chain at position 26.

[0040] In some specific embodiments, in the structure of the human pancreatic amylin analog of the present invention, R1 is selected from any one of carboxyl, tetrazolium, phosphate, and sulfonic acid groups; n is any integer from 14 to 24; X 12 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 13 Selected from Ala, N-Me-Ala, and Aib; X 14 Selected from any one of Aad, Asp, Glu, α-Me-Asp, N-Me-Glu, and α-Me-Glu; X 15 Selected from any one of Phe, N-Me-Phe, α-Me-Phe, α-Me-Phe(2-F) and α-Me-Phe(4-F); X 16 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 17 Selected from Val, N-Me-Val, and α-Me-Val; X 21 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 22 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 23Selected from any one of Phe, N-Me-Phe, α-Me-Phe, α-Me-Phe(2-F) and α-Me-Phe(4-F); X 24 Choose from any one of Gly, N-Me-Gly, Ala, and Aib; optionally, X 24 Selected from Gly, N-Me-Gly, and Ala; X 25 Selected from either Pro or α-Me-Pro; X 27 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 28 Selected from Ser, Pro, and α-Me-Pro; and / or X 29 Choose from Ser, Pro, and α-Me-Pro.

[0041] In some specific embodiments, in the structure of the human amylin analog of the present invention, R1 is selected from any one of carboxyl, tetrazolium, and phosphate groups; n is 18 or 20; X 12 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 13 Selected from any one of Ala, N-Me-Ala, and Aib; X 14 Selected from any one of Glu, N-Me-Glu, and α-Me-Glu; X 15 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 16 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 17 Selected from Val, N-Me-Val, and α-Me-Val; X 21 Selected from Pro, Asn, and α-Me-Asn; X 22 Selected from either Asn or N-Me-Asn; X 23 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 24 Selected from any one of Gly, N-Me-Gly, and Aib; X 25 Selected from either Pro or α-Me-Pro; X 27 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 28 Selected from Pro; and / or X 29 Selected from either Pro or α-Me-Pro; And when R1 is a carboxyl group and n=18, X 12 X 13 X 14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29 They are not all selected from Leu, Ala, Glu, α-Me-Phe, Leu, Val, Asn, N-Me-Asn, Phe, Gly, Pro, Leu, Pro and Pro.

[0042] In some specific embodiments, in the structure of the human amylin analog of the present invention, R1 is selected from any one of carboxyl, tetrazolium, and phosphate groups; n is 18 or 20; X 12 Selected from either Leu or α-Me-Leu; X 13 Choose either Ala or Aib; X 14 Selected from either Glu or α-Me-Glu; X 15 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 16 Choose from either Leu or N-Me-Leu; X 17 Selected from either Val or α-Me-Val; X 21 Selected from Pro, Asn, and α-Me-Asn; X 22 Selected from either Asn or N-Me-Asn; X 23Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 24 Selected from either Gly or N-Me-Gly; X 25 Selected from Pro; X 27 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 28 Selected from Pro; and / or X 29 Selected from either Pro or α-Me-Pro; And when R1 is a carboxyl group and n=18, X 12 X 13 X 14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29 They are not all selected from Leu, Ala, Glu, α-Me-Phe, Leu, Val, Asn, N-Me-Asn, Phe, Gly, Pro, Leu, Pro and Pro.

[0043] In some specific embodiments, in the structure of the human amylin analog of the present invention, R1 is selected from any one of carboxyl, tetrazolium, and phosphate groups; n is 16, 18, or 20; X 12 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 13 Selected from Ala, N-Me-Ala, and Aib; X 14 Selected from any one of Aad, Asp, Glu, α-Me-Asp, N-Me-Glu, and α-Me-Glu; X 15 Selected from any one of Phe, N-Me-Phe, α-Me-Phe, α-Me-Phe(2-F) and α-Me-Phe(4-F); X 16 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 17Selected from Val, N-Me-Val, and α-Me-Val; X 21 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 22 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 23 Selected from any one of Phe, N-Me-Phe, α-Me-Phe, α-Me-Phe(2-F) and α-Me-Phe(4-F); X 24 Selected from Gly, N-Me-Gly, and Ala; X 25 Selected from either Pro or α-Me-Pro; X 27 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 28 Selected from Ser, Pro, and α-Me-Pro; and / or X 29 Selected from Ser, Pro, and α-Me-Pro; And when R1 is a carboxyl group and n=18, X 12 X 13 X 14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29 They are not all selected from Leu, Ala, Glu, α-Me-Phe, Leu, Val, Asn, N-Me-Asn, Phe, Gly, Pro, Leu, Pro and Pro.

[0044] In some specific embodiments, in the structure of the human amylin analog of the present invention, R1 is selected from any one of carboxyl, tetrazolium, and phosphate groups; n is 16, 18, or 20; X 12 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 13Selected from Ala, N-Me-Ala, and Aib; X 14 Selected from any one of Aad, Asp, Glu, α-Me-Asp, N-Me-Glu, and α-Me-Glu; X 15 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 16 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 17 Selected from Val, N-Me-Val, and α-Me-Val; X 21 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 22 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 23 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 24 Selected from Gly, N-Me-Gly, and Ala; X 25 Selected from either Pro or α-Me-Pro; X 27 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 28 Selected from either Pro or α-Me-Pro; and / or X 29 Selected from either Pro or α-Me-Pro; And when R1 is a carboxyl group and n=18, X 12 X 13 X 14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29They are not all selected from Leu, Ala, Glu, α-Me-Phe, Leu, Val, Asn, N-Me-Asn, Phe, Gly, Pro, Leu, Pro and Pro.

[0045] In some specific embodiments, in the structure of the human amylin analog of the present invention, R1 is selected from any one of carboxyl and phosphate groups; n is 16, 18 or 20; X 12 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 13 Selected from Ala, N-Me-Ala, and Aib; X 14 Selected from any one of Aad, Asp, Glu, N-Me-Glu, and α-Me-Glu; X 15 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 16 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 17 Selected from Val, N-Me-Val, and α-Me-Val; X 21 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 22 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 23 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 24 Selected from Gly, N-Me-Gly, and Ala; X 25 Selected from either Pro or α-Me-Pro; X 27 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 28 Selected from either Pro or α-Me-Pro; and / or X 29 Selected from either Pro or α-Me-Pro; And when R1 is a carboxyl group and n=18, X 12 X 13 X14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29 They are not all selected from Leu, Ala, Glu, α-Me-Phe, Leu, Val, Asn, N-Me-Asn, Phe, Gly, Pro, Leu, Pro and Pro.

[0046] In some specific embodiments, in the structure of the human amylin analog of the present invention, R1 is selected from either a carboxyl group or a phosphate group; n is 18 or 20; X 12 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 13 Selected from Ala, N-Me-Ala, and Aib; X 14 Selected from any one of Glu, N-Me-Glu, and α-Me-Glu; X 15 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 16 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 17 Selected from Val, N-Me-Val, and α-Me-Val; X 21 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 22 Selected from any one of Asn, α-Me-Asn, and N-Me-Asn; X 23 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 24 Selected from Gly, N-Me-Gly, and Ala; X 25 Selected from either Pro or α-Me-Pro; X 27 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 28 Selected from either Pro or α-Me-Pro; and / or X 29 Selected from either Pro or α-Me-Pro; And when R1 is a carboxyl group and n=18, X 12 X 13 X 14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29 They are not all selected from Leu, Ala, Glu, α-Me-Phe, Leu, Val, Asn, N-Me-Asn, Phe, Gly, Pro, Leu, Pro and Pro.

[0047] In some specific embodiments, in the structure of the human amylin analog of the present invention, R1 is selected from either a carboxyl group or a phosphate group; n is 18 or 20; X 12 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 13 Choose either Ala or Aib; X 14 Selected from any one of Glu, N-Me-Glu, and α-Me-Glu; X 15 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 16 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 17 Selected from either Val or α-Me-Val; X 21 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 22 Selected from any one of Asn, α-Me-Asn, and N-Me-Asn; X 23 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X24 Selected from either Gly or N-Me-Gly; X 25 Selected from either Pro or α-Me-Pro; X 27 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 28 Selected from either Pro or α-Me-Pro; and / or X 29 Selected from either Pro or α-Me-Pro; And when R1 is a carboxyl group and n=18, X 12 X 13 X 14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29 They are not all selected from Leu, Ala, Glu, α-Me-Phe, Leu, Val, Asn, N-Me-Asn, Phe, Gly, Pro, Leu, Pro and Pro.

[0048] In some specific embodiments, in the structure of the human pancreatic amylin analogue of the present invention, X 12 X 13 X 14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29 As shown in the structure of any of compounds 1-67.

[0049] In some specific embodiments, the amino acid sequence of the peptide chain portion in the structure of the human amylin analogue of the present invention is as shown in the amino acid sequence of any of the structures of compounds 1-67.

[0050] In some specific embodiments, in the human pancreatic amylin analogue of the present invention, the compound is selected from any one of the compounds shown in the following structural formulas: (SEQ ID NO: 2)、 (SEQ ID NO: 3)、 (SEQ ID NO: 4)、 (SEQ ID NO: 5)、 (SEQ ID NO: 6)、 (SEQ ID NO: 7)、 (SEQ ID NO: 8)、 (SEQ ID NO: 9)、 (SEQ ID NO: 10)、 (SEQ ID NO: 11)、 (SEQ ID NO: 12)、 (SEQ ID NO: 13)、 (SEQ ID NO: 14)、 (SEQ ID NO: 15)、 (SEQ ID NO: 16)、 (SEQ ID NO: 17)、 (SEQ ID NO: 18)、 (SEQ ID NO: 19)、 (SEQ ID NO: 20)、 (SEQ ID NO: 21)、 (SEQ ID NO: 22)、 (SEQ ID NO: 23)、 (SEQ ID NO: 24)、 (SEQ ID NO: 25)、 (SEQ ID NO: 26)、 (SEQ ID NO: 27)、 (SEQ ID NO: 28)、 (SEQ ID NO: 29)、 (SEQ ID NO: 30)、 (SEQ ID NO: 31)、 (SEQ ID NO: 32)、 (SEQ ID NO: 33)、 (SEQ ID NO: 34)、 (SEQ ID NO: 35)、 (SEQ ID NO: 36)、 (SEQ ID NO: 37)、 (SEQ ID NO: 38)、 (SEQ ID NO: 39)、 (SEQ ID NO: 40)、 (SEQ ID NO: 41)、 (SEQ ID NO: 42)、 (SEQ ID NO: 43)、 (SEQ ID NO: 44)、 (SEQ ID NO: 45)、 (SEQ ID NO: 46)、 (SEQ ID NO: 47)、 (SEQ ID NO: 48)、 (SEQ ID NO: 49)、 (SEQ ID NO: 50)、

[0051] (SEQ ID NO: 51)、 (SEQ IDNO: 52)、 (SEQ ID NO: 53)、 (SEQ ID NO: 54)、 (SEQ ID NO: 55)、 (SEQ ID NO: 56)、 (SEQ ID NO: 57)、 (SEQ ID NO: 58)、 (SEQ ID NO: 59)、 (SEQ ID NO: 60)、 (SEQ ID NO: 61)、 (SEQ ID NO: 62)、 (SEQ ID NO: 63)、 (SEQ ID NO: 64)、 (SEQ ID NO: 65)、 (SEQ ID NO: 66)、 (SEQ ID NO: 67)、 (SEQ IDNO: 68)、 (SEQ ID NO: 69)、 (SEQ ID NO: 70)、 (SEQ ID NO: 71)、 (SEQ ID NO: 72)、 (SEQ ID NO: 73)、 (SEQ ID NO: 74)、 (SEQ ID NO: 75)、 (SEQ ID NO: 76)、 (SEQ ID NO: 77)、 (SEQ ID NO: 78)、 (SEQ ID NO: 79)、 (SEQ ID NO: 80)、 (SEQ ID NO: 81)、 (SEQ ID NO: 82)、 (SEQ ID NO: 83)、 (SEQ ID NO: 84)、 (SEQ ID NO: 85)、 (SEQ ID NO: 86)、 (SEQ ID NO: 87)、 (SEQ ID NO: 88)、 (SEQ ID NO: 89)、 (SEQ ID NO: 90)、 (SEQ ID NO: 91)、 (SEQ ID NO: 92)、 (SEQ ID NO: 93)、 (SEQ ID NO: 94)、 (SEQ ID NO: 95)、 (SEQ ID NO: 96)、 (SEQ ID NO: 97) (SEQ ID NO: 98) (SEQ ID NO: 99) (SEQ ID NO: 100) (SEQ ID NO: 101) (SEQ ID NO: 102) (SEQ ID NO: 103) (SEQ ID NO: 104) (SEQ IDNO: 105) (SEQ ID NO: 106) (SEQ ID NO: 107)

[0052] (SEQ ID NO: 108) (SEQ ID NO: 109) or (SEQ ID NO: 110); In some specific embodiments, in the structure of the human pancreatic amylin analog of the present invention, the polypeptide compound is selected from any one of the compounds shown in the following structural formulas: (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4) (SEQ ID NO: 5) (SEQ ID NO: 7) (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) (SEQ ID NO: 11)、 (SEQ ID NO: 12)、 (SEQ ID NO: 14)、 (SEQ ID NO: 16)、 (SEQ ID NO: 17)、 (SEQ ID NO: 18)、 (SEQ ID NO: 19)、 (SEQ ID NO: 20)、 (SEQ ID NO: 21)、 (SEQ ID NO: 22)、 (SEQ ID NO: 111)、 (SEQ ID NO: 24)、 (SEQ ID NO: 26)、 (SEQ ID NO: 29)、 (SEQ ID NO: 36)、 (SEQ ID NO: 37)、 (SEQ ID NO: 43)、 (SEQ ID NO: 45)、 (SEQ ID NO: 46)、 (SEQ ID NO: 48)、 (SEQ ID NO: 49)、 (SEQ ID NO: 50)、 (SEQ ID NO: 53)、 (SEQ ID NO: 112)、 (SEQ ID NO: 54)、 (SEQ ID NO: 59)、 (SEQ ID NO: 62)、 (SEQ ID NO: 64)、 (SEQ ID NO: 65)、 (SEQ ID NO: 66)、 (SEQ ID NO: 68)、 (SEQ ID NO: 70)、 (SEQ ID NO: 72)、 (SEQ ID NO: 75)、 (SEQ ID NO: 76)、 (SEQ ID NO: 77)、 (SEQ ID NO: 78)、 (SEQ ID NO: 80)、 (SEQ ID NO: 82)、 (SEQ ID NO: 83)、 (SEQ ID NO: 85)、 (SEQ ID NO: 86)、 (SEQ ID NO: 87)、 (SEQ ID NO: 88)、 (SEQ ID NO: 89)、 (SEQ ID NO: 90)、 (SEQ ID NO: 92)、 (SEQ ID NO: 93) (SEQ ID NO: 94) (SEQ ID NO: 95) (SEQ ID NO: 97) (SEQ ID NO: 98) (SEQ ID NO: 99) (SEQ ID NO: 100) (SEQ ID NO: 101) (SEQ ID NO: 104) (SEQ ID NO: 113) (SEQ ID NO: 106) or

[0053] (SEQ ID NO: 108).

[0054] Furthermore, the present invention also provides a method for preparing the above-mentioned human amylin analog (peptide compound), which can be carried out with reference to the human amylin analog of the present invention and the prior art methods for synthesizing similar compounds. As an example of some specific embodiments of the present invention, specifically, the method for preparing the human pancreatic amylin analogue includes: The peptide chain of the synthetic human amylin analogue (the amino acid sequence of the peptide chain is:

[0055] Among them, X 12 X 13 X 14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29As defined in any of the embodiments of the present invention above), and the ε-amino group of the Lys side chain at position 26 of the peptide chain is coupled with a fatty acid moiety ( ); An intramolecular methylene bridge is formed between two Cys residues at positions 2 and 7 of the peptide chain to obtain the target compound; Selectively, chiral isomers, tautomers, or pharmaceutically acceptable salts, esters, or amides of the compound may be further prepared as needed.

[0056] In some specific embodiments, the peptide chain is synthesized using amino acids with protecting groups as raw materials. In some specific embodiments, the present invention uses SPPS via a peptide synthesis column based on the Fmoc chemical method. The Fmoc-protected amino acids used in the method are all purchased from Shanghai Jier Biochemical Co., Ltd., such as: Fmoc-Glu-OtBu, Fmoc-Cys(Trt)-OH , Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Orn(Boc)-OH, F moc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-α-Me-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-O H, Fmoc-N-Me-Asn(Trt)-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Tyr(tBu )-OH, Fmoc-α-Me-Leu-OH, Fmoc-N-Me-Leu-OH, Fmoc-Aib-OH, Fmoc-N-Me-Ala-OH, Fmoc-α-Me- Glu(OtBu)-OH, Fmoc-N-Me-Glu(OtBu)-OH, Fmoc-Aad(OtBu)-OH, Fmoc-N-Me-Phe-OH, Fmoc-α-Me-Val-OH, Fmoc-N-Me-Val-OH, Fmoc-α-Me-Asn(Trt)-OH, Fmoc-N-Me-Gly-OH, Fmoc-α-Me-Pro-OH, Fmoc-α-Me-Asn(Trt)-OH, Fmoc-N-Me-Gly-OH, Fmoc-Val-OH, Fmoc-α-Me-Phe(2-F)-OH, Fmoc-α-Me-Phe(4-F)-OH, Fmoc-Asp(OtBu)-OH. In some specific embodiments, in the method for preparing the human amylin analog of the present invention, the coupling of each amino acid in the peptide chain is performed according to the following operations: The resin used was Rink Amide-AM Resin crosslinked with 1% DVB from Jiangsu Haipu Functional Materials Co., Ltd., with a mesh size of 100-200 and a degree of substitution of 0.2-0.3 mmol / g. The Fmoc process was employed, using standard side-chain protecting groups, and amino acids were sequentially condensed from the C-terminus to the N-terminus of the peptide chain. Fmoc-Glu-OtBu was used for the synthesis of γGlu at the 1-position of the peptide chain, and Fmoc-Lys(Mtt)-OH was used for the synthesis of Lys at the 26-position of the peptide chain. Rink Amide-AM Resin was added to a reaction vessel, DCM was added, nitrogen was purged, and the mixture was stirred to swell. The mixture was washed 2-3 times with DCM, and the protection was removed using a 20% Pip / DMF solution (containing 0.1M Oxyme) at 35°C. After the reaction was complete, the resin was washed and filtered to obtain the resin to be reacted. Fmoc-Tyr(tBu)-OH and Oxyme were dissolved in NMP, followed by the addition of DIC and thorough mixing. The activated protected amino acid solution was added to the resin to be reacted, stirred under nitrogen at 60°C, filtered and washed, and the reaction was completed to obtain Fmoc-Tyr(tBu)-AM-Resin. The above operation was repeated, sequentially adding the protected amino acids to the main chain. Before each subsequent coupling step, the Fmoc group was removed using 20% ​​Pip / DMF (containing 0.1M Oxyme). All standard amino acid couplings used a 2-fold molar ratio of Fmoc protected amino acid, a 3-fold molar ratio of DIC, and a 3-fold molar ratio of HOBt, coupled under nitrogen protection for 2–3 hours, and the degree of coupling was detected using ninhydrin colorimetric reagent. In some difficult coupling cases (e.g., methylated amino acids or other sterically hindered amino acids), the coupling time was increased or the coupling steps were repeated to achieve a satisfactory coupling level. After assembling the complete peptide sequence, the resin was washed with DCM to remove residual DMF.

[0057] In some specific embodiments, in the preparation method of the human amylin analogue, the fatty acid side chain is linked to the 26-position of the peptide chain via an amide coupling reaction, and triethylamine and diiodomethane are added to introduce a disulfide bond via an acetal reaction.

[0058] In some specific embodiments, the process of coupling the fatty acid moiety to the ε-amino group at position 26 of Lys in the preparation method of the present invention includes: The peptide resin was treated multiple times with 20%-30% HFIP / DCM to selectively remove the Mtt protecting group of the 26-position Lys side chain, exposing the ε-amino group. The fatty acid side chain fragment was activated in DCM using EDC·HCl / HOSu. After activation, DIPEA was added to the deprotected peptide resin at the 26-position Lys side chain for amide coupling. After the reaction, the resin was washed and dried to obtain the modified peptide resin. If the product is a dibenzyl phosphate modified product, it needs to be debenzylated by Pd / C hydrogenation before washing and drying. In some specific embodiments, in the method for preparing human amylin analogs of the present invention, the obtained peptide resin is further cleaved. The synthesized dried peptide resin was cleaved at room temperature for 2-4 hours using 7-12 times the volume of a lysis mixture (TFA:H2O:TIS, 92:4:4 v / v). The resin was discarded, and the combined filtrates were treated with 3-4 times the volume of cold methyl ether (-15℃ to -25℃) to precipitate the crude peptide. The crude peptide / methyl ether suspension was centrifuged at 3500 rpm for about 5 minutes, the supernatant was discarded, and the solid precipitate was washed 2-3 times with methyl ether and then dried under vacuum to obtain the crude peptide.

[0059] In some specific embodiments, the process of preparing intramolecular methylene bridges in the preparation method of the human pancreatic amylin analog of the present invention can be carried out according to the following steps: The crude peptide was dissolved in 20mM-30mM potassium phosphate buffer and ACN. TCEP aqueous solution, Et3N, and CH2I2 were added with stirring, and the reaction was continued for approximately 20 minutes. After the reaction was complete, 0.1% TFA aqueous solution and acetic acid were added to the reaction solution to obtain the crude target compound.

[0060] In some specific embodiments, in the method for preparing the human pancreatic amylin analog of the present invention, the crude target compound is further purified: The crude peptide was dissolved in MQ water containing 40% acetic acid and purified by reversed-phase preparative HPLC (Waters Delta Prep 4000) on a C18 silica column. Elution was performed with a gradually increasing gradient of ACN in MQ water containing 0.1% TFA. Fractions were analyzed by UPLC, and fractions containing the purified target peptide were combined. The resulting solutions were analyzed (UPLC, LC-MS), and the product was aliquoted into glass vials. The vials were capped with a Millipore glass fiber pre-filter. Lyophilization yielded the target compound as a white solid.

[0061] In some specific embodiments, the human amylin analogue of the present invention can be analyzed by LC-MS. Optionally, LC-MS can be performed according to the following method: Mass spectrometry (MS) parameters: Detection instrument: Agilent 6410 triple quadrupole liquid chromatography-mass spectrometry system; Ionization source: +ESI.

[0062] Liquid chromatography parameters: Column: Agilent ZORBAX SB-C18, size: 250×4.6mm, 5μm; Detection wavelength: 195nm; Column temperature: 30℃; Injection volume: 10μL; Flow rate: 1mL / min; Mobile phase A: 0.05% glacial acetic acid; Mobile phase B: acetonitrile.

[0063] Secondly, the present invention also provides an amylin receptor agonist, which includes the aforementioned human amylin analogue of the present invention.

[0064] In some specific embodiments, the amylin receptor agonist of the present invention further includes a pharmaceutically acceptable carrier.

[0065] Thirdly, the present invention also provides the use of the above-mentioned human amylin analogues or amylin receptor agonists in the preparation of medicaments for the prevention or treatment of any of the following diseases, including: obesity, diabetes, insulin resistance syndrome, sleep apnea, obesity-induced cartilage degeneration and / or osteoarthritis, cardiovascular disease, large vessel disease, microvascular disease, diabetic cardiomyopathy, coronary heart disease, myocardial infarction, stroke, arteriosclerosis, peripheral artery disease, gallbladder disease, non-alcoholic steatohepatitis, Alzheimer's disease, fatty liver disease, alcoholic liver disease, diabetic nephropathy, renal failure, chronic kidney disease, hypertension, heart failure, eating disorders, cholecystitis, and short bowel syndrome.

[0066] Furthermore, the names of the compounds or raw materials used in the preparation method provided by this invention, and their corresponding abbreviations, are as follows: Boc: tert-Butoxycarbonyl; Fmoc: 9-fluorenylmethoxycarbonyl; tBu: tert-Butyl; OtBu: tert-Butoxy; Mtt: 4-Methyltriphenyl; Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; Trt: Triphenylmethyl; EDC·HCl: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; HOSu: N-hydroxysuccinimide; TFA: Trifluoroacetic acid; NMP: N-methylpyrrolidone; DIC: Diisopropylcarbodiimide; TIS: Triisopropylsilane; Oxyme: Ethyl 2-oxime cyanoacetate; DCM: Dichloromethane; DMF: N,N-dimethylformamide; Pip: Piperidine; HOBt: 1-hydroxybenzotriazole; DIPEA: N,N-diisopropylethylamine; HFIP: Hexafluoroisopropanol; ACN: Acetonitrile; TCEP: Tris(2-carboxyethyl)phosphine; Et3N: Triethylamine; CH2I2: Diiodomethane; K3PO4: Potassium phosphate.

[0067] Furthermore, in this invention, unless otherwise stated, all natural amino acids are L-amino acids.

[0068] Furthermore, in this invention, when synthesizing the compound peptide chain, the γGlu at position 1 is bonded to a peptide bond using a carboxylic acid at position γ, and the amino acids at other positions are bonded to peptide bonds using a carboxylic acid and an amino group at position α.

[0069] Furthermore, the non-natural amino acids used in the embodiments of the present invention and their corresponding structures are as follows: The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0070] Example 1

[0071] This invention provides a polypeptide compound, hereinafter referred to as Compound 1 (PKWX202507-002), whose structural formula is as follows:

[0072] This invention also provides a method for preparing the above-mentioned polypeptide compound, which is synthesized according to the following synthetic route:

[0073] Specifically, at room temperature, 1 mmol of resin 1-A1 was added to a reaction vessel, followed by 25 mL of DCM. The mixture was stirred under nitrogen for 2 hours until the resin swelled, then the solvent was removed. 30 mL of 20% Pip / DMF (containing 0.1 M Oxyme) solution was added, and the reaction was carried out at 35°C for 30 minutes to remove the Fmoc protecting group, yielding 1-A2. Fmoc-Tyr(tBu)-OH (2 mmol) and Oxyme (3 mmol) were dissolved in 15 mL of NMP, and then 3 mmol of DIC was added. After stirring and activating for 5 minutes, 1-A2 was added, and the reaction was carried out under nitrogen for 3 hours at 60°C. The reaction was detected by ninhydrin colorimetric reagent. After the reaction was complete, the resin was thoroughly washed with DMF and DCM sequentially, and then air-dried to obtain 1-A3. Using 1-A3 as the starting support, standard Fmoc... Solid-phase synthesis cycle (de-Fmoc → amino acid activation coupling → washing), sequentially condensing the following sequences: Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-N-Me-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH. )-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-α-Me-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aal-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-O H, Fmoc-Aal-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Aal-OH, Fmoc-Thr(tBu)-O H, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Glu-OtBu; each round of Fmoc removal uses 20%Pip / DMF (containing 0.1M Oxyme) was stirred at room temperature for 20-30 minutes; amino acid condensation was performed using 2 times molar ratio of Fmoc to protect the amino acids, 3 times molar ratio of DIC and 3 times molar ratio of HOBt, under nitrogen protection at room temperature for 2-3 hours, with ninhydrin monitoring the endpoint to obtain the complete peptide sequence 1-A4.

[0074] The resin was washed four times with DCM (20 mL) to remove residual DMF. 60 mL of 30% HFIP / DCM solution was added to peptide resin 1-A4 (1 mmol) and treated repeatedly (10 min × 3 times) to selectively remove the Mtt protecting group from the 26-position Lys side chain. The resin was then washed with DCM and dried. Subsequently, 1-B1 (1.2 mmol), HOSu (1.5 mmol), and EDC·HCl (1.5 mmol) were added to a DCM (10 mL) solution, and the mixture was stirred at room temperature for 30 min. TLC was used for observation. After the reaction was complete, DIPEA (2.5 mmol) and the previously de-Mtt-treated 1-A4 resin were added to the activation solution, and the reaction was carried out under nitrogen protection with stirring at room temperature for 5 hours. After the reaction was completed, the intermediate was washed with DCM and dried. The intermediate was then transferred to a hydrogenation flask, and a DCM / MeOH (20 mL) mixed solvent was added. 10% Pd / C was used as a catalyst, and H2 was bubbled through at atmospheric pressure. The mixture was stirred at room temperature for 12 hours. Pd / C was removed by filtration, the resin was washed with DCM, and dried under vacuum to obtain 1-A5. 1-A5 was then cleaved at room temperature for 3 hours using 10 times its volume of a lysis mixture (TFA:H2O:TIS, 92:4:4 v / v). The resin was filtered off and precipitated with 3 times its volume of methyl ether (-20 °C). The precipitate was then centrifuged at 3500 rpm for 5 minutes, the supernatant was removed, and the solid was washed three times with methyl ether and dried under vacuum to obtain 1-A6. 1-A6 (5 mmol) was dissolved in 10 mL of potassium phosphate buffer (30 mM) and 5 mL of ACN. TCEP aqueous solution (30 μM, 20 mmol), Et3N (50 mmol), and CH2I2 (30 mmol) were added under stirring at room temperature, and the reaction was stirred for 20 minutes. After the reaction was completed, 0.1% TFA aqueous solution and acetic acid were added to the reaction solution to obtain 1-A7. 1-A7 was purified by reverse phase and lyophilized to obtain the target compound 1.

[0075] Compound 1 was characterized by: LC-MS

[0076] Calculated mass: (M+3) / 3=1521.41, (M+4) / 4=1141.31, (M+5) / 5=913.25.

[0077] Measured mass: (M+3) / 3=1521.36, (M+4) / 4=1141.39, (M+5) / 5=913.20.

[0078] Example 2-67

[0079] Examples 2-67 each provide a polypeptide compound. The preparation methods for Examples 2-67 are the same as those provided in Example 1, except that in the step of coupling the side chain to the full-sequence peptide resin, different side chain raw materials with different R1 groups are directly selected for the reaction when R1 is different. For example, when R1 is a carboxylic acid group and n=18, the full-sequence peptide resin is continuously treated multiple times with 20% HFIP. Subsequently, the side chain raw material is added to the DCM solution. HOSu and EDC·HCl were used, and the reaction was observed by TLC. After the reaction was complete, DIPEA and full-sequence peptide resin were added. After the reaction was completed, the mixture was washed and dried to obtain the target intermediate compound.

[0080] The structural formula and characterization data of the obtained polypeptide compound 2-67 are as follows: Example 2: Compound 2 (PKWX202507-003), its structure is as follows:

[0081] LC-MS

[0082] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0083] Measured mass: (M+3) / 3=1509.02, (M+4) / 4=1132.19, (M+5) / 5=905.90.

[0084] Example 3: Compound 3 (PKWX202507-004), its structure is as follows:

[0085] LC-MS

[0086] Calculated mass: (M+3) / 3=1521.41, (M+4) / 4=1141.31, (M+5) / 5=913.25.

[0087] Measured mass: (M+3) / 3=1521.44, (M+4) / 4=1141.44, (M+5) / 5=913.29.

[0088] Example 4: Compound 4 (PKWX202507-005), its structure is as follows:

[0089] LC-MS

[0090] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0091] Measured mass: (M+3) / 3=1509.17, (M+4) / 4=1132.13, (M+5) / 5=905.76.

[0092] Example 5: Compound 5 (PKWX202507-006), its structure is as follows:

[0093] LC-MS

[0094] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0095] Measured mass: (M+3) / 3=1509.12, (M+4) / 4=1132.00, (M+5) / 5=905.98.

[0096] Example 6: Compound 6 (PKWX202507-007), its structure is as follows:

[0097] LC-MS

[0098] Calculated mass: (M+3) / 3=1521.41, (M+4) / 4=1141.31, (M+5) / 5=913.25.

[0099] Measured mass: (M+3) / 3=1521.53, (M+4) / 4=1141.30, (M+5) / 5=913.31.

[0100] Example 7: Compound 7 (PKWX202507-008), its structure is as follows:

[0101] LC-MS

[0102] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0103] Measured mass: (M+3) / 3=1508.98, (M+4) / 4=1132.19, (M+5) / 5=905.93.

[0104] Example 8: Compound 8 (PKWX202507-009), its structure is as follows:

[0105] LC-MS

[0106] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0107] Measured mass: (M+3) / 3=1509.16, (M+4) / 4=1132.20, (M+5) / 5=905.77.

[0108] Example 9: Compound 9 (PKWX202507-010), its structure is as follows:

[0109] LC-MS

[0110] Calculated mass: (M+3) / 3=1521.41, (M+4) / 4=1141.31, (M+5) / 5=913.25.

[0111] Measured mass: (M+3) / 3=1521.49, (M+4) / 4=1141.44, (M+5) / 5=913.38.

[0112] Example 10: Compound 10 (PKWX202507-011), its structure is as follows:

[0113] LC-MS

[0114] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0115] Measured mass: (M+3) / 3=1509.13, (M+4) / 4=1132.01, (M+5) / 5=905.97.

[0116] Example 11: Compound 11 (PKWX202507-012), its structure is as follows:

[0117] LC-MS

[0118] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0119] Measured mass: (M+3) / 3=1509.22, (M+4) / 4=1132.17, (M+5) / 5=905.89.

[0120] Example 12: Compound 12 (PKWX202507-013), its structure is as follows:

[0121] LC-MS

[0122] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0123] Measured mass: (M+3) / 3=1509.07, (M+4) / 4=1132.13, (M+5) / 5=905.94.

[0124] Example 13: Compound 13 (PKWX202507-014), its structure is as follows:

[0125] LC-MS

[0126] Calculated mass: (M+3) / 3=1521.41, (M+4) / 4=1141.31, (M+5) / 5=913.25.

[0127] Measured mass: (M+3) / 3=1521.38, (M+4) / 4=1141.43, (M+5) / 5=913.21.

[0128] Example 14: Compound 14 (PKWX202507-015), its structure is as follows:

[0129] LC-MS

[0130] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0131] Measured mass: (M+3) / 3=1508.99, (M+4) / 4=1132.05, (M+5) / 5=905.93.

[0132] Example 15: Compound 15 (PKWX202507-016), its structure is as follows:

[0133] LC-MS

[0134] Calculated mass: (M+3) / 3=1521.41, (M+4) / 4=1141.31, (M+5) / 5=913.25.

[0135] Measured mass: (M+3) / 3=1521.36, (M+4) / 4=1141.44, (M+5) / 5=913.18.

[0136] Example 16: Compound 16 (PKWX202507-017), its structure is as follows:

[0137] LC-MS

[0138] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0139] Measured mass: (M+3) / 3=1509.19, (M+4) / 4=1132.12, (M+5) / 5=905.94.

[0140] Example 17: Compound 17 (PKWX202507-018), its structure is as follows:

[0141] LC-MS

[0142] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0143] Measured mass: (M+3) / 3=1509.19, (M+4) / 4=1132.12, (M+5) / 5=905.94.

[0144] Example 18: Compound 18 (PKWX202507-019), its structure is as follows:

[0145] LC-MS

[0146] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0147] Measured mass: (M+3) / 3=1509.14, (M+4) / 4=1132.10, (M+5) / 5=905.96.

[0148] Example 19: Compound 19 (PKWX202507-020), its structure is as follows:

[0149] LC-MS

[0150] Calculated mass: (M+3) / 3=1517.43, (M+4) / 4=1138.32, (M+5) / 5=910.86.

[0151] Measured mass: (M+3) / 3=1517.38, (M+4) / 4=1138.27, (M+5) / 5=910.89.

[0152] Example 20: Compound 20 (PKWX202507-021), its structure is as follows:

[0153] LC-MS

[0154] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0155] Measured mass: (M+3) / 3=1509.17, (M+4) / 4=1132.10, (M+5) / 5=905.74.

[0156] Example 21: Compound 21 (PKWX202507-022), its structure is as follows:

[0157] LC-MS

[0158] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0159] Measured mass: (M+3) / 3=1509.16, (M+4) / 4=1132.00, (M+5) / 5=905.81.

[0160] Example 22: Compound 22 (PKWX202507-023), its structure is as follows:

[0161] LC-MS

[0162] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0163] Measured mass: (M+3) / 3=1509.16, (M+4) / 4=1132.09, (M+5) / 5=905.92.

[0164] Example 23: Compound 23 (PKWX202507-024), its structure is as follows:

[0165] LC-MS

[0166] Calculated mass: (M+3) / 3=1499.09, (M+4) / 4=1124.57, (M+5) / 5=899.85.

[0167] Measured mass: (M+3) / 3=1499.15, (M+4) / 4=1124.51, (M+5) / 5=899.92.

[0168] Example 24: Compound 24 (PKWX202507-025), its structure is as follows:

[0169] LC-MS

[0170] Calculated mass: (M+3) / 3=1503.42, (M+4) / 4=1127.82, (M+5) / 5=902.45.

[0171] Measured mass: (M+3) / 3=1503.40, (M+4) / 4=1127.77, (M+5) / 5=902.36.

[0172] Example 25: Compound 25 (PKWX202507-026), its structure is as follows:

[0173] LC-MS

[0174] Calculated mass: (M+3) / 3=1503.42, (M+4) / 4=1127.82, (M+5) / 5=902.45.

[0175] Measured mass: (M+3) / 3=1503.38, (M+4) / 4=1127.75, (M+5) / 5=902.36.

[0176] Example 26: Compound 26 (PKWX202507-027), its structure is as follows:

[0177] LC-MS

[0178] Calculated mass: (M+3) / 3=1503.42, (M+4) / 4=1127.82, (M+5) / 5=902.45.

[0179] Measured mass: (M+3) / 3=1503.33, (M+4) / 4=1127.79, (M+5) / 5=902.49.

[0180] Example 27: Compound 27 (PKWX202507-028), its structure is as follows:

[0181] LC-MS

[0182] Calculated mass: (M+3) / 3=1503.42, (M+4) / 4=1127.82, (M+5) / 5=902.45.

[0183] Measured mass: (M+3) / 3=1503.46, (M+4) / 4=1127.91, (M+5) / 5=902.52.

[0184] Example 28: Compound 28 (PKWX202507-029), its structure is as follows:

[0185] LC-MS

[0186] Calculated mass: (M+3) / 3=1503.42, (M+4) / 4=1127.82, (M+5) / 5=902.45.

[0187] Measured mass: (M+3) / 3=1503.44, (M+4) / 4=1127.93, (M+5) / 5=902.38.

[0188] Example 29: Compound 29 (PKWX202507-030), its structure is as follows:

[0189] LC-MS

[0190] Calculated mass: (M+3) / 3=1515.75, (M+4) / 4=1137.06, (M+5) / 5=909.85.

[0191] Measured mass: (M+3) / 3=1515.68, (M+4) / 4=1137.12, (M+5) / 5=909.90.

[0192] Example 30: Compound 30 (PKWX202507-031), its structure is as follows:

[0193] LC-MS

[0194] Calculated mass: (M+3) / 3=1503.42, (M+4) / 4=1127.82, (M+5) / 5=902.45.

[0195] Measured mass: (M+3) / 3=1503.39, (M+4) / 4=1127.89, (M+5) / 5=902.52.

[0196] Example 31: Compound 31 (PKWX202507-032), its structure is as follows:

[0197] LC-MS

[0198] Calculated mass: (M+3) / 3=1515.75, (M+4) / 4=1137.06, (M+5) / 5=909.85.

[0199] Measured mass: (M+3) / 3=1515.68, (M+4) / 4=1137.13, (M+5) / 5=909.82.

[0200] Example 32: Compound 32 (PKWX202507-033), its structure is as follows:

[0201] LC-MS

[0202] Calculated mass: (M+3) / 3=1511.77, (M+4) / 4=1134.08, (M+5) / 5=907.46.

[0203] Measured mass: (M+3) / 3=1511.76, (M+4) / 4=1134.01, (M+5) / 5=907.53.

[0204] Example 33: Compound 33 (PKWX202507-034), its structure is as follows:

[0205] LC-MS

[0206] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0207] Measured mass: (M+3) / 3=1509.13, (M+4) / 4=1132.04, (M+5) / 5=905.77.

[0208] Example 34: Compound 34 (PKWX202507-035), its structure is as follows:

[0209] LC-MS

[0210] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0211] Measured mass: (M+3) / 3=1509.19, (M+4) / 4=1132.09, (M+5) / 5=905.79.

[0212] Example 35: Compound 35 (PKWX202507-036), its structure is as follows:

[0213] LC-MS

[0214] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0215] Measured mass: (M+3) / 3=1509.03, (M+4) / 4=1132.15, (M+5) / 5=905.95.

[0216] Example 36: Compound 36 (PKWX202507-037), its structure is as follows:

[0217] LC-MS

[0218] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0219] Measured mass: (M+3) / 3=1509.04, (M+4) / 4=1132.12, (M+5) / 5=905.97.

[0220] Example 37: Compound 37 (PKWX202507-038), its structure is as follows:

[0221] LC-MS

[0222] Calculated mass: (M+3) / 3=1521.41, (M+4) / 4=1141.31, (M+5) / 5=913.25.

[0223] Measured mass: (M+3) / 3=1521.53, (M+4) / 4=1141.35, (M+5) / 5=913.21.

[0224] Example 38: Compound 38 (PKWX202507-039), its structure is as follows:

[0225] LC-MS

[0226] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0227] Measured mass: (M+3) / 3=1509.00, (M+4) / 4=1132.18, (M+5) / 5=905.84.

[0228] Example 39: Compound 39 (PKWX202507-040), its structure is as follows:

[0229] LC-MS

[0230] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0231] Measured mass: (M+3) / 3=1509.12, (M+4) / 4=1132.19, (M+5) / 5=905.79.

[0232] Example 40: Compound 40 (PKWX202507-041), its structure is as follows:

[0233] LC-MS

[0234] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0235] Measured mass: (M+3) / 3=1509.03, (M+4) / 4=1132.16, (M+5) / 5=905.84.

[0236] Example 41: Compound 41 (PKWX202507-042), its structure is as follows:

[0237] LC-MS

[0238] Calculated mass: (M+3) / 3=1509.09, (M+4) / 4=1132.07, (M+5) / 5=905.85.

[0239] Measured mass: (M+3) / 3=1509.17, (M+4) / 4=1132.11, (M+5) / 5=905.78.

[0240] Example 42: Compound 42 (PKWX202507-043), its structure is as follows:

[0241] LC-MS

[0242] Calculated mass: (M+3) / 3=1521.41, (M+4) / 4=1141.31, (M+5) / 5=913.25.

[0243] Measured mass: (M+3) / 3=1521.36, (M+4) / 4=1141.12, (M+5) / 5=913.29.

[0244] Example 43: Compound 43 (PKWX202507-044), its structure is as follows:

[0245] LC-MS

[0246] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0247] Measured mass: (M+3) / 3=1514.02, (M+4) / 4=1135.72, (M+5) / 5=908.91.

[0248] Example 44: Compound 44 (PKWX202507-045), its structure is as follows:

[0249] LC-MS

[0250] Calculated mass: (M+3) / 3=1526.09, (M+4) / 4=1144.82, (M+5) / 5=916.05.

[0251] Measured mass: (M+3) / 3=1526.00, (M+4) / 4=1144.85, (M+5) / 5=915.94.

[0252] Example 45: Compound 45 (PKWX202507-046), its structure is as follows:

[0253] LC-MS

[0254] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0255] Measured mass: (M+3) / 3=1514.15, (M+4) / 4=1135.77, (M+5) / 5=908.98.

[0256] Example 46: Compound 46 (PKWX202507-047), its structure is as follows:

[0257] LC-MS

[0258] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0259] Measured mass: (M+3) / 3=1514.17, (M+4) / 4=1135.90, (M+5) / 5=908.79.

[0260] Example 47: Compound 47 (PKWX202507-048), its structure is as follows:

[0261] LC-MS

[0262] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0263] Measured mass: (M+3) / 3=1514.14, (M+4) / 4=1135.85, (M+5) / 5=908.74.

[0264] Example 48: Compound 48 (PKWX202507-049), its structure is as follows:

[0265] LC-MS

[0266] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0267] Measured mass: (M+3) / 3=1514.16, (M+4) / 4=1135.70, (M+5) / 5=908.81.

[0268] Example 49: Compound 49 (PKWX202507-050), its structure is as follows:

[0269] LC-MS

[0270] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0271] Measured mass: (M+3) / 3=1514.21, (M+4) / 4=1135.88, (M+5) / 5=908.89.

[0272] Example 50: Compound 50 (PKWX202507-051), its structure is as follows:

[0273] LC-MS

[0274] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0275] Measured mass: (M+3) / 3=1514.16, (M+4) / 4=1135.74, (M+5) / 5=908.77.

[0276] Example 51: Compound 51 (PKWX202507-052), its structure is as follows:

[0277] LC-MS

[0278] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0279] Measured mass: (M+3) / 3=1514.07, (M+4) / 4=1135.93, (M+5) / 5=908.78.

[0280] Example 52: Compound 52 (PKWX202507-053), its structure is as follows:

[0281] LC-MS

[0282] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0283] Measured mass: (M+3) / 3=1514.16, (M+4) / 4=1135.94, (M+5) / 5=908.89.

[0284] Example 53: Compound 53 (PKWX202507-054), its structure is as follows:

[0285] LC-MS

[0286] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0287] Measured mass: (M+3) / 3=1514.22, (M+4) / 4=1135.76, (M+5) / 5=908.95.

[0288] Example 54: Compound 54 (PKWX202507-055), its structure is as follows:

[0289] LC-MS

[0290] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0291] Measured mass: (M+3) / 3=1514.19, (M+4) / 4=1135.87, (M+5) / 5=908.72.

[0292] Example 55: Compound 55 (PKWX202507-056), its structure is as follows:

[0293] LC-MS

[0294] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0295] Measured mass: (M+3) / 3=1514.13, (M+4) / 4=1135.72, (M+5) / 5=908.95.

[0296] Example 56: Compound 56 (PKWX202507-057), its structure is as follows:

[0297] LC-MS

[0298] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0299] Measured mass: (M+3) / 3=1513.96, (M+4) / 4=1135.77, (M+5) / 5=908.80.

[0300] Example 57: Compound 57 (PKWX202507-058), its structure is as follows:

[0301] LC-MS

[0302] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0303] Measured mass: (M+3) / 3=1514.16, (M+4) / 4=1135.91, (M+5) / 5=908.82.

[0304] Example 58: Compound 58 (PKWX202507-059), its structure is as follows:

[0305] LC-MS

[0306] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0307] Measured mass: (M+3) / 3=1514.01, (M+4) / 4=1135.74, (M+5) / 5=908.92.

[0308] Example 59: Compound 59 (PKWX202507-060), its structure is as follows:

[0309] LC-MS

[0310] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0311] Measured mass: (M+3) / 3=1513.98, (M+4) / 4=1135.79, (M+5) / 5=908.93.

[0312] Example 60: Compound 60 (PKWX202507-061), its structure is as follows:

[0313] LC-MS

[0314] Calculated mass: (M+3) / 3=1526.09, (M+4) / 4=1144.82, (M+5) / 5=916.05.

[0315] Measured mass: (M+3) / 3=1526.17, (M+4) / 4=1144.89, (M+5) / 5=916.15.

[0316] Example 61: Compound 61 (PKWX202507-062), its structure is as follows:

[0317] LC-MS

[0318] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0319] Measured mass: (M+3) / 3=1514.22, (M+4) / 4=1135.86, (M+5) / 5=908.78.

[0320] Example 62: Compound 62 (PKWX202507-063), its structure is as follows:

[0321] LC-MS

[0322] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0323] Measured mass: (M+3) / 3=1514.15, (M+4) / 4=1135.96, (M+5) / 5=908.79.

[0324] Example 63: Compound 63 (PKWX202507-064), its structure is as follows:

[0325] LC-MS

[0326] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0327] Measured mass: (M+3) / 3=1514.03, (M+4) / 4=1135.76, (M+5) / 5=908.95.

[0328] Example 64: Compound 64 (PKWX202507-065), its structure is as follows:

[0329] LC-MS

[0330] Calculated mass: (M+3) / 3=1514.09, (M+4) / 4=1135.82, (M+5) / 5=908.86.

[0331] Measured mass: (M+3) / 3=1514.18, (M+4) / 4=1135.79, (M+5) / 5=908.89.

[0332] Example 65: Compound 65 (PKWX202507-066), its structure is as follows:

[0333] LC-MS

[0334] Calculated mass: (M+3) / 3=1521.77, (M+4) / 4=1141.58, (M+5) / 5=913.46.

[0335] Measured mass: (M+3) / 3=1521.71, (M+4) / 4=1141.67, (M+5) / 5=913.53.

[0336] Example 66: Compound 66 (PKWX202507-067), its structure is as follows:

[0337] LC-MS

[0338] Calculated mass: (M+3) / 3=1499.74, (M+4) / 4=1125.06, (M+5) / 5=900.25.

[0339] Measured mass: (M+3) / 3=1499.68, (M+4) / 4=1125.19, (M+5) / 5=900.28.

[0340] Example 67: Compound 67 (PKWX202507-068), its structure is as follows:

[0341] LC-MS

[0342] Calculated mass: (M+3) / 3=1518.77, (M+4) / 4=1139.33, (M+5) / 5=911.66.

[0343] Measured mass: (M+3) / 3=1518.64, (M+4) / 4=1139.42, (M+5) / 5=911.78.

[0344] Test Example 1: hCTR / hAMYR1 agonist activity assay

[0345] 1. Experimental Methods: This experiment used UM-UC-3 cells (human bladder transitional cell carcinoma) that stably expressed human calcitonin receptor (hCTR) or human amylin 1 receptor (hAMYR1). The hCTR / hAMYR1 receptor activity of the compounds of this invention was evaluated by detecting cAMP content using the HTRF method.

[0346] 2. Experimental procedure: 1) Eloralintide (prepared according to the method described in patent CN116917312A) and the compound of the present invention were formulated into appropriate gradient working concentrations using detection buffer, with a total of 10 dilutions, each concentration in duplicate.

[0347] 2) After digesting and centrifuging UM-UC-3 cells that stably express hCTR or hAMYR1, resuspend them in detection buffer and count them. Seed 2000 cells per well in a 384-well cell plate containing the working concentration of the test compound and incubate at 37°C and 5% CO2 for 30 min.

[0348] 3) After incubation, follow the manufacturer's recommendations and the protocol to measure cAMP levels using Revvity's cAMP kit. Add 10 μL of Anti-cAMP-cryptate working solution and 10 μL of cAMP-d2 working solution to each well. Incubate the cell plate at room temperature in the dark for 1 h.

[0349] 4) After incubation, the fluorescence signals at 665 nm and 620 nm wavelengths were read using a microplate reader. EC was calculated by curve fitting using GraphPadPrism.50 value.

[0350] 3. The experimental results are shown in Table 1: Table 1 Results of agonist activity tests on the compounds

[0351] 4. Conclusion: The compounds provided in the embodiments of the present invention have strong agonistic effects on hCTR and hAMYR1, and the agonistic activity of some of the compounds on hCTR and hAMYR1 is more than 20 times higher than that of Eloralintide.

[0352] Test Example 2: Acute Effects of Food Intake and Body Weight on Normal SD Rats

[0353] 1. Experimental objective: The effects of the compounds provided in the embodiments of this invention on acute food intake and body weight in normal Sprague Dawley (SD) rats were investigated and compared with Eloralintide.

[0354] 2. Animal selection: Adult male SD rats were selected as experimental animals, and the animals were acclimatized for at least one week before the start of the study.

[0355] 3. Grouping of experimental animals and drug intervention measures: Animals were randomly divided into groups of six. Each animal was then administered a single subcutaneous injection, either the solvent, 30 nmol / kg of eloralintide, or 30 nmol / kg of the compound of this invention.

[0356] 4. Measurement of food intake and body weight: Throughout the study, the animals had free access to food and water. Food intake and body weight were recorded at 24, 48, 72, and 96 hours after feeding. Data processing was performed using GraphPadPrism.

[0357] 5. The experimental results are shown in Table 2: Table 2. Cumulative food intake change rate (%, compared to the solvent group) and body weight change rate (%, compared to the solvent group) of SD rats at 96 h.

[0358] Note: "-" indicates a decrease; 6. Conclusion: During the experiment, the rats were in good health and mental condition. The compounds provided in the embodiments of this invention all exhibited good acute food intake inhibition and weight loss in normal rats. Specifically, the compounds provided in the embodiments of this invention showed better acute food intake inhibition and weight loss than Eloralintide in normal rats.

[0359] Test Example 3: Evaluation of the efficacy of diet-induced obese rats.

[0360] 1. Experimental objective: The effects of the compounds provided in the embodiments of this invention on food intake and body weight in diet-induced obese Sprague Dawley (SD) rats were investigated and compared with Eloralintide.

[0361] 2. Animal selection: Five-week-old male SD rats were selected as experimental animals, and the animals were acclimatized for at least one week before the start of the study.

[0362] 3. Diet-induced obesity in rats: After the animals had adapted, the rats were fed a high-fat diet to induce weight gain.

[0363] 4. Grouping of experimental animals and drug intervention measures: Animals were randomly divided into groups of six based on their body weight. Each animal was administered a solvent, 30 nmol / kg of eloralintide, or 30 nmol / kg of the compound of this invention via subcutaneous injection every three days at 16:00. The first administration was on Day 1, with subsequent administrations on Day 4, Day 7, Day 10, and Day 13. Throughout the experiment, the rats continued to be fed a high-fat diet.

[0364] 5. Measurement of food intake and body weight: Throughout the study, the animals had free access to food and water. Food intake and body weight were recorded from Day 1 to Day 13. GraphPadPrism was used for data processing.

[0365] 6. The experimental results are shown in Table 3: Table 3. Changes in food intake (%, compared to the solvent group) and body weight (%, compared to the solvent group) of SD rats on Day 13

[0366] Note: "-" indicates a decrease; 6. Conclusion: The compounds provided in this invention can effectively suppress food intake and reduce body weight in a diet-induced obese rat model, and their effects are superior to those of Eloralintide, demonstrating good potential for treating obesity and overweight diseases.

[0367] Test Example 4: Blood Glucose Changes in ZDF Rats After Acute Feeding

[0368] 1. Experimental objective: The study investigated the changes in blood glucose levels after acute feeding of the compounds provided in the embodiments of the present invention in Zucker Diabetic Fatty (ZDF) rats and compared them with Eloralintide.

[0369] 2. Selection of animal models: ZDF rats were selected as the experimental animals, as they are one of the most commonly used and classic models for studying type 2 diabetes and its complications. The animals underwent at least one week of acclimatization before the start of the study.

[0370] 3. Grouping of experimental animals and drug intervention measures: ZDF rats were randomly divided into groups of six. Each animal was then given a single subcutaneous injection of either the solvent, 30 nmol / kg of eloralintide, or 30 nmol / kg of the compound of this invention, according to its group.

[0371] 4. Blood glucose measurement: Throughout the study, the animals had free access to food and water. Tail blood samples were collected from ZDF rats before administration and at 12 h, 24 h, and 48 h after administration, and blood glucose levels (mmol / L) were measured using a glucometer.

[0372] 5. The experimental results are shown in Table 4: Table 4. Changes in glucose levels (%, compared to baseline) in ZDF rats

[0373] Note: "-" indicates a decrease; 6. Conclusion: The compound of this invention, after a single subcutaneous injection, showed a superior hypoglycemic effect compared to Eloralintide in ZDF rats.

[0374] Test Example 5: Pharmacokinetic (PK) Test in SD Rats

[0375] 1. Experimental objective: The pharmacokinetic parameters of the compounds provided in the embodiments of this invention in Sprague Dawley (SD) rats were studied and compared with those of Eloralintide.

[0376] 2. Animal selection: Adult male SD rats were selected as experimental animals, and the animals were acclimatized for at least one week before the start of the study.

[0377] 3. Grouping of experimental animals and drug intervention measures: Animals were randomly divided into groups of six. Each animal was then given a single subcutaneous injection of either 30 nmol / kg of Eloralintide or 30 nmol / kg of the compound of this invention, according to its group.

[0378] 4. Blood sample collection and analysis: Throughout the study, animals had free access to food and water. Venous blood samples were collected before administration and at 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, and 96 h after administration. Blood samples were placed on ice, and plasma was separated by centrifugation within 1 h and analyzed using LC-MS / MS.

[0379] 5. The experimental results are shown in Table 5: Table 5 Pharmacokinetic parameters of SD rats

[0380] Note: T 1 / 2 Tmax represents the half-life; C represents the time to reach the maximum plasma concentration; max Represents the maximum plasma drug concentration; AUC 0-∞ The area under the blood drug concentration-time curve represents the time from 0 to infinity.

[0381] 6. Conclusion: During the experiment, the rats were in good health and mental condition. The compound provided in this invention exhibits favorable pharmacokinetic characteristics in SD rats after a single subcutaneous injection, T... 1 / 2 It is superior to Eloralintide and can be used for the development of long-acting formulations.

[0382] Compounds 1-67 of this invention can serve as active ingredients in amylin-based drugs, exerting metabolic regulatory effects by activating amylin receptors. Specifically, they can lower blood glucose and regulate body weight, exhibiting good biological activity and bioavailability. Furthermore, an intramolecular methylene bridge (-S-CH2-S-) is formed between the two Cys residues at positions 2 and 7 of the polypeptide sequence of the human amylin analog of this invention, replacing some of the Asn residues prone to deamidation and introducing methylated amino acids, resulting in better stability and safety under or near-physiological conditions. Simultaneously, the ε-amino group of the Lys side chain at position 26 is covalently linked to the fatty acid side chain via an amide bond, reducing renal clearance and enzymatic degradation of the compound, thereby increasing plasma half-life and bioavailability, reducing dosing frequency, and improving patient compliance.

[0383] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A human amylin analogue selected from compounds shown in the following structural formulas or their chiral isomers, tautomers, or pharmaceutically acceptable salts, esters, or amides: in, An intramolecular methylene bridge (-S-CH2-S-) is formed between the two Cys residues at positions 2 and 7 in the structure; and / or Lys at position 26 The ε-amino group is covalently attached to the Lys side chain; R1 is selected from carboxyl, carboxylic acid ester, tetrazolium, phosphoric acid, phosphate ester, sulfonic acid, and sulfonate groups; n is any integer from 10 to 24. Among them, X 12 X 13 X 14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29 Each is independently selected from amino acids.

2. The human amylin analogue according to claim 1, wherein: X 12 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 13 Selected from any one of Ala, N-Me-Ala, and Aib; X 14 Selected from any one of Aad, Asp, Glu, α-Me-Asp, N-Me-Glu, and α-Me-Glu; X 15 Selected from any one of Phe, N-Me-Phe, α-Me-Phe, α-Me-Phe(2-F) and α-Me-Phe(4-F); X 16 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 17 Selected from Val, N-Me-Val, and α-Me-Val; X 21 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 22 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 23 Selected from any one of Phe, N-Me-Phe, α-Me-Phe, α-Me-Phe(2-F) and α-Me-Phe(4-F); X 24 Choose from any one of Gly, N-Me-Gly, Ala, and Aib; optionally, X 24 Selected from Gly, N-Me-Gly, and Ala; X 25 Selected from either Pro or α-Me-Pro; X 27 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 28 Selected from Ser, Pro, and α-Me-Pro; and / or X 29 Selected from Ser, Pro, and α-Me-Pro; Optionally, R1 is selected from any one of carboxyl, tetrazolium, phosphate, and sulfonic acid groups; n is any integer from 14 to 24; Optionally, R1 is selected from any one of carboxyl, tetrazolium, and phosphate groups; n is any integer from 16 to 20.

3. The human amylin analogue according to claim 1 or 2, wherein, R1 is selected from any one of carboxyl, tetrazolium, and phosphate groups; n is 16, 18, or 20. X 12 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 13 Selected from any one of Ala, N-Me-Ala, and Aib; X 14 Selected from any one of Aad, Asp, Glu, α-Me-Asp, N-Me-Glu, and α-Me-Glu; X 15 Selected from any one of Phe, N-Me-Phe, α-Me-Phe, α-Me-Phe(2-F) and α-Me-Phe(4-F); X 16 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 17 Selected from Val, N-Me-Val, and α-Me-Val; X 21 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 22 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 23 Selected from any one of Phe, N-Me-Phe, α-Me-Phe, α-Me-Phe(2-F) and α-Me-Phe(4-F); X 24 Choose from any one of Gly, N-Me-Gly, Ala, and Aib; optionally, X 24 Selected from Gly, N-Me-Gly, and Ala; X 25 Selected from either Pro or α-Me-Pro; X 27 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 28 Selected from Ser, Pro, and α-Me-Pro; and / or X 29 Selected from Ser, Pro, and α-Me-Pro; And when R1 is a carboxyl group and n=18, X 12 X 13 X 14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29 They are not all selected from Leu, Ala, Glu, α-Me-Phe, Leu, Val, Asn, N-Me-Asn, Phe, Gly, Pro, Leu, Pro and Pro.

4. The human amylin analogue according to claim 1 or 2, wherein, R1 is selected from any one of carboxyl, tetrazolium, and phosphate groups; n is 16, 18, or 20. X 12 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 13 Selected from any one of Ala, N-Me-Ala, and Aib; X 14 Selected from any one of Aad, Asp, Glu, α-Me-Asp, N-Me-Glu, and α-Me-Glu; X 15 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 16 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 17 Selected from Val, N-Me-Val, and α-Me-Val; X 21 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 22 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 23 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 24 Choose from any one of Gly, N-Me-Gly, Ala, and Aib; optionally, X 24 Selected from Gly, N-Me-Gly, and Ala; X 25 Selected from either Pro or α-Me-Pro; X 27 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 28 Selected from either Pro or α-Me-Pro; and / or X 29 Selected from either Pro or α-Me-Pro; And when R1 is a carboxyl group and n=18, X 12 X 13 X 14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29 They are not all selected from Leu, Ala, Glu, α-Me-Phe, Leu, Val, Asn, N-Me-Asn, Phe, Gly, Pro, Leu, Pro and Pro; Optionally, R1 is selected from any one of carboxyl, tetrazolium, and phosphate groups; n is 18 or 20; X 12 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 13 Selected from any one of Ala, N-Me-Ala, and Aib; X 14 Selected from any one of Glu, N-Me-Glu, and α-Me-Glu; X 15 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 16 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 17 Selected from Val, N-Me-Val, and α-Me-Val; X 21 Selected from Pro, Asn, and α-Me-Asn; X 22 Selected from either Asn or N-Me-Asn; X 23 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 24 Selected from any one of Gly, N-Me-Gly, and Aib; X 25 Selected from either Pro or α-Me-Pro; X 27 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 28 Selected from Pro; and / or X 29 Selected from either Pro or α-Me-Pro; And when R1 is a carboxyl group and n=18, X 12 X 13 X 14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29 They are not all selected from Leu, Ala, Glu, α-Me-Phe, Leu, Val, Asn, N-Me-Asn, Phe, Gly, Pro, Leu, Pro and Pro; Optionally, R1 is selected from any one of carboxyl, tetrazolium, and phosphate groups; n is 18 or 20; X 12 Selected from either Leu or α-Me-Leu; X 13 Choose either Ala or Aib; X 14 Selected from either Glu or α-Me-Glu; X 15 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 16 Choose from either Leu or N-Me-Leu; X 17 Selected from either Val or α-Me-Val; X 21 Selected from Pro, Asn, and α-Me-Asn; X 22 Selected from either Asn or N-Me-Asn; X 23 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 24 Selected from either Gly or N-Me-Gly; X 25 Selected from Pro; X 27 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 28 Selected from Pro; and / or X 29 Selected from either Pro or α-Me-Pro; And when R1 is a carboxyl group and n=18, X 12 X 13 X 14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29 They are not all selected from Leu, Ala, Glu, α-Me-Phe, Leu, Val, Asn, N-Me-Asn, Phe, Gly, Pro, Leu, Pro and Pro.

5. The human amylin analogue according to claim 1 or 2, wherein, R1 is selected from either a carboxyl group or a phosphate group; n is 16, 18, or 20. X 12 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 13 Selected from Ala, N-Me-Ala, and Aib; X 14 Selected from any one of Aad, Asp, Glu, N-Me-Glu, and α-Me-Glu; X 15 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 16 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 17 Selected from Val, N-Me-Val, and α-Me-Val; X 21 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 22 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 23 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 24 Selected from Gly, N-Me-Gly, and Ala; X 25 Selected from either Pro or α-Me-Pro; X 27 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 28 Selected from either Pro or α-Me-Pro; and / or X 29 Selected from either Pro or α-Me-Pro; And when R1 is a carboxyl group and n=18, X 12 X 13 X 14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29 They are not all selected from Leu, Ala, Glu, α-Me-Phe, Leu, Val, Asn, N-Me-Asn, Phe, Gly, Pro, Leu, Pro and Pro.

6. The human amylin analogue according to claim 1 or 2, wherein, R1 is selected from either a carboxyl group or a phosphate group; n is 18 or 20. X 12 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 13 Selected from Ala, N-Me-Ala, and Aib; X 14 Selected from any one of Glu, N-Me-Glu, and α-Me-Glu; X 15 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 16 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 17 Selected from Val, N-Me-Val, and α-Me-Val; X 21 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 22 Selected from any one of Asn, α-Me-Asn, and N-Me-Asn; X 23 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 24 Selected from Gly, N-Me-Gly, and Ala; X 25 Selected from either Pro or α-Me-Pro; X 27 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 28 Selected from either Pro or α-Me-Pro; and / or X 29 Selected from either Pro or α-Me-Pro; And when R1 is a carboxyl group and n=18, X 12 X 13 X 14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29 They are not all selected from Leu, Ala, Glu, α-Me-Phe, Leu, Val, Asn, N-Me-Asn, Phe, Gly, Pro, Leu, Pro and Pro.

7. The human amylin analogue according to claim 1 or 2, wherein, R1 is selected from either a carboxyl group or a phosphate group; n is 18 or 20. X 12 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 13 Choose either Ala or Aib; X 14 Selected from any one of Glu, N-Me-Glu, and α-Me-Glu; X 15 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 16 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 17 Selected from either Val or α-Me-Val; X 21 Selected from any one of Pro, Asn, α-Me-Asn, and N-Me-Asn; X 22 Selected from any one of Asn, α-Me-Asn, and N-Me-Asn; X 23 Selected from any one of Phe, N-Me-Phe, and α-Me-Phe; X 24 Selected from either Gly or N-Me-Gly; X 25 Selected from either Pro or α-Me-Pro; X 27 Selected from Leu, N-Me-Leu, and α-Me-Leu; X 28 Selected from either Pro or α-Me-Pro; and / or X 29 Selected from either Pro or α-Me-Pro; And when R1 is a carboxyl group and n=18, X 12 X 13 X 14 X 15 X 16 X 17 X 21 X 22 X 23 X 24 X 25 X 27 X 28 and X 29 They are not all selected from Leu, Ala, Glu, α-Me-Phe, Leu, Val, Asn, N-Me-Asn, Phe, Gly, Pro, Leu, Pro and Pro.

8. The human amylin analogue according to claim 1 or 2, wherein, The compound is selected from any one of the compounds shown in the following structural formulas: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or ； Preferably, the compound is selected from any one of the compounds shown in the following structural formulas: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or 。 9. An amylin receptor agonist comprising the human amylin analogue as described in any one of claims 1-8.

10. The use of a human amylin analogue according to any one of claims 1-8 or an amylin receptor agonist according to claim 9 in the preparation of a medicament for the prevention or treatment of any of the following diseases, wherein, The diseases mentioned include any one of the following: obesity, diabetes, insulin resistance syndrome, sleep apnea, obesity-induced cartilage degeneration and / or osteoarthritis, cardiovascular disease, large vessel disease, microvascular disease, diabetic cardiomyopathy, coronary heart disease, myocardial infarction, stroke, arteriosclerosis, peripheral artery disease, gallbladder disease, non-alcoholic steatohepatitis, Alzheimer's disease, fatty liver disease, alcoholic liver disease, diabetic nephropathy, renal failure, chronic kidney disease, hypertension, heart failure, eating disorders, cholecystitis, and short bowel syndrome.

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