MULTI-RECEPTOR AGONIST AND MEDICAL USE THEREOF

Abstract

A series of pharmaceutical compositions containing polypeptide dual agonist compounds and pharmaceutically acceptable salts thereof, wherein same have dual agonist effects on a human glucagon-like peptide-1 (GLP-1) receptor and a human blood glucose-dependent insulinotropic polypeptide (GIP) receptor, and can be used for treating non-insulin-dependent diabetes, insulin-dependent diabetes, obesity and other related diseases.

Claims

1. A GLP-1 analog of formula (I) or a pharmaceutically acceptable salt form thereof: TABLE-US-00019 X.sub.1-X.sub.2-Glu-Gly-Thr-Phe-Thr-Ser-Asp-X.sub.10-Ser-X.sub.12-Tyr- Leu-X.sub.15-X.sub.16-X.sub.17-X.sub.18-X.sub.19-X.sub.20-Glu-Phe-X.sub.23-X.sub.24-Trp- Leu-X.sub.27-X.sub.28-X.sub.29-X.sub.30-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser-X.sub.40 (I) wherein: each of X.sub.1, X.sub.2, X.sub.10, X.sub.12, X.sub.15, X.sub.16, X.sub.17, X.sub.18, X.sub.19, X.sub.20, X.sub.27, X.sub.28, X.sub.29 and X.sub.30 is independently a natural amino acid, an unnatural amino acid, or a peptide fragment consisting of a natural amino acid or an unnatural amino acid; and X.sub.23 is Ile or Val; X.sub.24 is Ala, Asn or Gln X.sub.40 is a natural amino acid, an unnatural amino acid, or a peptide fragment consisting of a natural amino acid or an unnatural amino acid, or X.sub.40 is absent.

2. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 1, wherein the GLP-1 analog is modified at both ends thereof to have formula (II): TABLE-US-00020 R.sub.1-X.sub.1-X.sub.2-Glu-Gly-Thr-Phe-Thr-Ser-Asp-X.sub.10-Ser-X.sub.12- Tyr-Leu-X.sub.15-X.sub.16-X.sub.17-X.sub.18-X.sub.19-X.sub.20-Glu-Phe-X.sub.23-X.sub.24- Trp-Leu-X.sub.27-X.sub.28-X.sub.29-X.sub.30-Pro-Ser-Ser-Gly-Ala-Pro- Pro-Pro-Ser-X.sub.40-R.sub.2 (II) wherein: R.sub.1 is H, alkyl, acetyl, formyl, benzoyl, trifluoroacetyl or pGlu; and R.sub.2 is —NH.sub.2 or —OH.

3. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X.sub.1 is Tyr or His; X.sub.2 is Aib or D-Ala; X.sub.10 is Val or Tyr; X.sub.12 is Ser or Ile, X.sub.15 is Asp or Glu; X.sub.16 is Glu, Gly, Lys or Aib; X.sub.17 is Glu, Ile or Gln; X.sub.18 is Ala, Aib or His; X.sub.19 is Ala, Aib or Gln; X.sub.20 is Gln, Glu, Lys or Y1; X.sub.23 is Ile or Val; X.sub.24 is Ala, Asn or Gln; X.sub.27 is Val or Leu; X.sub.28 is Arg or Ala; X.sub.29 is Gly or Gln; X.sub.30 is Gly, Lys or Y1; X.sub.40 is Lys or Y1, or is absent; Y1 is Lys, Orn, Dap, Dab or Cys, in which a side chain of Y1 is coupled to a substituent shown as formula of {[2-(2-amino-ethoxy)-ethoxy]-acetyl}.sub.a-(y-Glu).sub.b-CO—(CH.sub.2).sub.c—COOH; a is an integer between 1-3; b is an integer between 1-2; and c is an integer between 10-30.

4. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X.sub.1 is Tyr or His; X.sub.2 is Aib or D-Ala; X.sub.10 is Val or Tyr; X.sub.12 is Ser or Ile, X.sub.15 is Asp or Glu; X.sub.16 is Glu, Gly, Lys or Aib; X.sub.17 is Glu, Ile or Gln; X.sub.18 is Ala, Aib or His; X.sub.19 is Ala, Aib or Gln; X.sub.20 is Gln, Glu, Lys or Y1; X.sub.23 is Ile or Val; X.sub.24 is Ala, Asn or Gln; X.sub.27 is Val, Ile or Leu; X.sub.28 is Arg or Ala; X.sub.29 is Gly or Gln; X.sub.30 is Gly, Lys or Y1; X.sub.40 is Lys or Y1, or is absent; Y1 is Lys, Orn, Dap, Dab or Cys, in which a side chain of Y1 is coupled to a substituent shown as formula of {[2-(2-amino-ethoxy)-ethoxy]-acetyl}.sub.a-(y-Glu).sub.b-CO—(CH.sub.2).sub.c—COOH; a is an integer between 1-3; b is an integer between 1-2; and c is an integer between 10-30.

5. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X.sub.1 is Tyr; X.sub.2 is Aib; X.sub.10 is Tyr; X.sub.12 is Ile, X.sub.15 is Asp or Glu; X.sub.16 is Lys or Aib; X.sub.17 is Glu or Ile; X.sub.18 is Ala or Aib; X.sub.19 is Ala or Gln; X.sub.20 is Gln, Lys, or Y1; X.sub.23 is Val; X.sub.24 is Asn or Gln; X.sub.27 is Leu; X.sub.28 is Ala; X.sub.29 is Gly or Gln; X.sub.30 is Gly, Lys or Y1; X.sub.40 is Lys or Y1, or is absent; Y1 is Lys, Orn, Dap, Dab or Cys, in which a side chain of Y1 is coupled to a substituent shown as formula of {[2-(2-amino-ethoxy)-ethoxy]-acetyl}.sub.a-(y-Glu).sub.b-CO—(CH.sub.2).sub.c—COOH; a is an integer between 1-3; b is an integer between 1-2; and c is an integer between 10-30.

6. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X.sub.1 is Tyr; X.sub.2 is Aib; X.sub.10 is Tyr; X.sub.12 is Ile, X.sub.15 is Glu; X.sub.16 is Lys; X.sub.17 is Glu; X.sub.18 is Ala or Aib; X.sub.19 is Ala; X.sub.20 is Gln, Lys, or Y1; X.sub.23 is Val; X.sub.24 is Asn; X.sub.27 is Leu; X.sub.28 is Ala; X.sub.29 is Gly; X.sub.30 is Gly, Lys or Y1; X.sub.40 is Lys or Y1, or is absent; Y1 is Lys, Orn, Dap, Dab or Cys, in which a side chain of Y1 is coupled to a substituent shown as formula of {[2-(2-amino-ethoxy)-ethoxy]-acetyl}.sub.a-(y-Glu).sub.b-CO—(CH.sub.2).sub.c—COOH; a is an integer between 1-3; b is an integer between 1-2; and c is an integer between 10-30.

7. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X.sub.1 is Tyr; X.sub.2 is Aib; X.sub.10 is Tyr; X.sub.12 is Ile, X.sub.15 is Glu; X.sub.16 is Lys; X.sub.17 is Ile; X.sub.18 is Ala or Aib; X.sub.19 is Ala; X.sub.20 is Gln, Lys, or Y1; X.sub.23 is Val; X.sub.24 is Asn; X.sub.27 is Leu; X.sub.28 is Ala; X.sub.29 is Gly; X.sub.30 is Gly, Lys or Y1; X.sub.40 is Lys or Y1, or is absent; Y1 is Lys, Orn, Dap, Dab or Cys, in which a side chain of Y1 is coupled to a substituent shown as formula of {[2-(2-amino-ethoxy)-ethoxy]-acetyl}.sub.a-(y-Glu).sub.b-CO—(CH.sub.2).sub.c—COOH; a is an integer between 1-3; b is an integer between 1-2; and c is an integer between 10-30.

8. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X.sub.1 is Tyr; X.sub.2 is Aib; X.sub.10 is Tyr; X.sub.12 is Ile; X.sub.15 is Glu; X.sub.16 is Lys; X.sub.17 is Glu or Ile; X.sub.18 is Ala; X.sub.19 is Ala; X.sub.20 is Gln, Lys, or Y1; X.sub.23 is Val; X.sub.24 is Asn; X.sub.27 is Leu; X.sub.28 is Ala; X.sub.29 is Gly; X.sub.30 is Gly, Lys or Y1; X.sub.40 is Lys or Y1, or is absent; Y1 is Lys, Orn, Dap, Dab or Cys, in which a side chain of Y1 is coupled to a substituent shown as formula of {[2-(2-amino-ethoxy)-ethoxy]-acetyl}.sub.a-(y-Glu).sub.b-CO—(CH.sub.2).sub.c—COOH; a is an integer between 1-3; b is an integer between 1-2; and c is an integer between 10-30.

9. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X.sub.1 is Tyr; X.sub.2 is Aib; X.sub.10 is Tyr; X.sub.12 is Ile; X.sub.15 is Glu; X.sub.16 is Lys; X.sub.17 is Glu or Ile; X.sub.18 is Aib; X.sub.19 is Ala; X.sub.20 is Gln, Lys, or Y1; X.sub.23 is Val; X.sub.24 is Asn; X.sub.27 is Leu; X.sub.28 is Ala; X.sub.29 is Gly; X.sub.30 is Gly, Lys or Y1; X.sub.40 is Lys or Y1, or is absent; Y1 is Lys, Orn, Dap, Dab or Cys, in which a side chain of Y1 is coupled to a substituent shown as formula of {[2-(2-amino-ethoxy)-ethoxy]-acetyl}.sub.a-(y-Glu).sub.b-CO—(CH.sub.2).sub.c—COOH; a is an integer between 1-3; b is an integer between 1-2; and c is an integer between 10-30.

10. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X.sub.1 is Tyr; X.sub.2 is Aib; X.sub.10 is Tyr; X.sub.12 is Ile; X.sub.15 is Glu; X.sub.16 is Lys; X.sub.17 is Glu or Ile; X.sub.18 is Ala or Aib; X.sub.19 is Ala; X.sub.20 is Gln; X.sub.23 is Val; X.sub.24 is Asn; X.sub.27 is Leu; X.sub.28 is Ala; X.sub.29 is Gly; X.sub.30 is Gly, Lys or Y1; X.sub.40 is Lys or Y1, or is absent; Y1 is Lys, Orn, Dap, Dab or Cys, in which a side chain of Y1 is coupled to a substituent shown as formula of {[2-(2-amino-ethoxy)-ethoxy]-acetyl}.sub.a-(y-Glu).sub.b-CO—(CH.sub.2).sub.c—COOH; a is an integer between 1-3; b is an integer between 1-2; and c is an integer between 10-30.

11. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X.sub.1 is Tyr; X.sub.2 is Aib; X.sub.10 is Tyr; X.sub.12 is Ile; X.sub.15 is Glu; X.sub.16 is Lys; X.sub.17 is Glu or Ile; X.sub.18 is Ala or Aib; X.sub.19 is Ala; X.sub.20 is Lys; X.sub.23 is Val; X.sub.24 is Asn; X.sub.27 is Leu; X.sub.28 is Ala; X.sub.29 is Gly; X.sub.30 is Gly, Lys or Y1; X.sub.40 is Lys or Y1, or is absent; Y1 is Lys, Orn, Dap, Dab or Cys, in which a side chain of Y1 is coupled to a substituent shown as formula of {[2-(2-amino-ethoxy)-ethoxy]-acetyl}.sub.a-(y-Glu).sub.b-CO—(CH.sub.2).sub.c—COOH; a is an integer between 1-3; b is an integer between 1-2; and c is an integer between 10-30.

12. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X.sub.1 is Tyr; X.sub.2 is Aib; X.sub.10 is Tyr; X.sub.12 is Ile; X.sub.15 is Glu; X.sub.16 is Lys; X.sub.17 is Glu or Ile; X.sub.18 is Ala or Aib; X.sub.19 is Ala; X.sub.20 is Y1; X.sub.23 is Val; X.sub.24 is Asn; X.sub.27 is Leu; X.sub.28 is Ala; X.sub.29 is Gly; X.sub.30 is Gly, Lys or Y1; X.sub.40 is Lys or Y1, or is absent; Y1 is Lys, Orn, Dap, Dab or Cys, in which a side chain of Y1 is coupled to a substituent shown as formula of {[2-(2-amino-ethoxy)-ethoxy]-acetyl}.sub.a-(y-Glu).sub.b-CO—(CH.sub.2).sub.c—COOH; a is an integer between 1-3; b is an integer between 1-2; and c is an integer between 10-30.

13. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X.sub.1 is Tyr; X.sub.2 is Aib; X.sub.10 is Tyr; X.sub.12 is Ile; X.sub.15 is Glu; X.sub.16 is Lys; X.sub.17 is Glu or Ile; X.sub.18 is Ala or Aib; X.sub.19 is Ala; X.sub.20 is Gln; X.sub.23 is Val; X.sub.24 is Asn; X.sub.27 is Leu; X.sub.28 is Ala; X.sub.29 is Gly; X.sub.30 is Gly, Lys or Y1; X.sub.40 is Lys or Y1, or is absent; Y1 is Lys, in which a side chain of Y1 is coupled to a substituent shown as formula of {[2-(2-amino-ethoxy)-ethoxy]-acetyl}.sub.a-(y-Glu).sub.b-CO—(CH.sub.2).sub.c—COOH; a is an integer between 1-3; b is an integer between 1-2; and c is an integer between 10-30.

14. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 1, characterized in that X.sub.27 is Ile.

15. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 1, wherein X.sub.20, X.sub.30 and X.sub.40 are each independently Y1; Y1 is Lys, Orn, Dap, Dab or Cys, in which a side chain of Y1 is coupled to a substituent shown as formula of {[2-(2-amino-ethoxy)-ethoxy]-acetyl}.sub.a-(y-Glu).sub.b-CO—(CH.sub.2).sub.c—COOH; a is an integer between 1-3; b is an integer between 1-2; and c is an integer between 10-30.

16. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 3, characterized in that, in the definition of Y1, a is 2, b is 1 or 2, and c is 16-20.

17. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 3, characterized in that, in the definition of Y1, a is 2, b is 1 or 2, and c is 16, 18 or 20.

18. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 3, characterized in that X.sub.40 is Y1; Y1 is Lys, in which a side chain of Y1 is coupled to a substituent shown as formula of {[2-(2-amino-ethoxy)-ethoxy]-acetyl}.sub.a-(y-Glu).sub.b-CO—(CH.sub.2).sub.c—COOH; a is 2; b is 1 or 2; and c is 16 or 18.

19. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 3, characterized in that Y1 is Lys which is covalently linked to a fatty acid via the side chain amino group of Lys by forming an amide bond with the fatty acid.

20. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 3, characterized in that Y1 is K (—OEG-OEG-yGlu-C18-OH) or K (—OEG-OEG-yGlu-C20-OH): ##STR00003##

21. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 3, characterized in that Y1 is Lys which is covalently linked to a fatty acid via the c amino group of the C-terminal of Lys by forming an amide bond with the fatty acid.

22. The GLP-1 analog of general formula (I) or the pharmaceutically acceptable salt thereof according to claim 1, characterized in that it is selected from the following compounds of numbers 1-83: TABLE-US-00021 Compound number sequence 1 H-HAibEGTFTSDVSSYLEEEAAKEFIAWLVRGGPSSGAPPPSK-NH.sub.2 2 H-YAibEGTFTSDVSSYLEEEAAKEFIAWLVRGGPSSGAPPPSK-NH.sub.2 3 H-YAibEGTFTSDVSSYLEEEHQKEFIAWLVRGGPSSGAPPPSK-NH.sub.2 4 H-YAibEGTFTSDVSSYLEEIHQKEFIAWLVRGGPSSGAPPPSK-NH.sub.2 5 H-HAibEGTFTSDVSSYLEEEAAKEFVNWLLAGGPSSGAPPPSK-NH.sub.2 6 H-YAibEGTFTSDVSSYLEEEAAKEFVNWLLAGGPSSGAPPPSK-NH.sub.2 7 H-YAibEGTFTSDVSIYLEKEAAKEFVNWLLAGGPSSGAPPPSK-NH.sub.2 8 H-YAibEGTFTSDVSIYLEKEAAEEFVNWLLAGGPSSGAPPPSK-NH.sub.2 9 H-YAibEGTFTSDYSIYLEKEAAKEFVNWLLAGGPSSGAPPPSK-NH.sub.2 10 H-YAibEGTFTSDYSIYLEKEAAQEFVNWLLAGGPSSGAPPPSK-NH.sub.2 11 H-YAibEGTFTSDYSIYLEKEAQKEFVNWLLAGGPSSGAPPPSK-NH.sub.2 12 H-YAibEGTFTSDYSIYLEAibEAAKEFVNWLLAGGPSSGAPPPSK-NH.sub.2 13 H-YAibEGTFTSDYSIYLEKIAAKEFVNWLLAGGPSSGAPPPSK-NH.sub.2 14 H-YAibEGTFTSDYSIYLEKIAAQEFVNWLLAGGPSSGAPPPSK-NH.sub.2 15 H-YAibEGTFTSDYSIYLEKIAQKEFVNWLLAGGPSSGAPPPSK-NH.sub.2 16 H-YAibEGTFTSDYSIYLEKEAibAKEFVNWLLAGGPSSGAPPPSK-NH.sub.2 17 H-YAibEGTFTSDYSIYLEKEAAibKEFVNWLLAGGPSSGAPPPSK-NH.sub.2 18 H-YAibEGTFTSDYSIYLEAiblAAQEFVNWLLAGGPSSGAPPPSK-NH.sub.2 19 H-YAibEGTFTSDYSIYLDKEAAKEFVNWLLAGGPSSGAPPPSK-NH.sub.2 20 H-YAibEGTFTSDYSIYLDKIAAQEFVNWLLAGGPSSGAPPPSK-NH.sub.2 21 H-YAibEGTFTSDYSIYLDKIAAQEFVQWLLAGGPSSGAPPPSK-NH.sub.2 22 H-YAibEGTFTSDYSIYLDAibIAAQEFVQWLLAGGPSSGAPPPSK-NH.sub.2 23 H-YAibEGTFTSDYSIYLEKEAAKEFVNWLLAQKPSSGAPPPSK-NH.sub.2 24 H-YAibEGTFTSDYSIYLEKIAAQEFVNWLLAQKPSSGAPPPSK-NH.sub.2 25 H-YAibEGTFTSDYSIYLEKEAAKEFVNWLLAGGPSSGAPPPSK (OEG-OEG-yGlu-C18-OH)-NH.sub.2 26 H-YAibEGTFTSDYSIYLEKEAAKEFVNWLLAGK(OEG-OEG-yGlu-C18-OH) PSSGAPPPS-NH.sub.2 27 H-YAibEGTFTSDYSIYLEKEAAK(OEG-OEG-yGlu-C18-OH)EFVNWLLAG GPSSGAPPPS-NH.sub.2 28 H-YAibEGTFTSDYSIYLEKIAAQEFVNWLLAGGPSSGAPPPSK (OEG-OEG-yGlu-C18-OH)-NH.sub.2 29 H-YAibEGTFTSDYSIYLEKIAAQEFVNWLLAGK(OEG-OEG-yGlu-C18-OH) PSSGAPPPS-NH.sub.2 30 H-YAibEGTFTSDYSIYLEKIAAK(OEG-OEG-yGlu-C18-OH)EFVNWLLAG GPSSGAPPPS-NH.sub.2 31 H-YAibEGTFTSDYSIYLEKEAibAKEFVNWLLAGGPSSGAPPPSK (OEG-OEG-yGlu-C18-OH)-NH.sub.2 32 H-YAibEGTFTSDYSIYLEKEAibAKEFVNWLLAGK(OEG-OEG-yGlu-C18-OH) PSSGAPPPS-NH.sub.2 33 H-YAibEGTFTSDYSIYLEKEAibAK(OEG-OEG-yGlu-C18-OH) EFVNWLLAGGPSSGAPPPS-NH.sub.2 34 H-YAibEGTFTSDYSIYLEKIAQQEFIQWLLAGGPSSGAPPPS-NH.sub.2 35 H-YAibEGTFTSDYSIYLEKIAQQEFIQWLLAQGPSSGAPPPS-NH.sub.2 36 H-YAibEGTFTSDYSIYLEKIAAQEFIQWLLAGGPSSGAPPPS-NH.sub.2 37 H-YAibEGTFTSDYSIYLEGIAAQEFIQWLLAGGPSSGAPPPS-NH.sub.2 38 H-YAibEGTFTSDYSIYLEGIAQQEFIQWLLAGGPSSGAPPPS-NH.sub.2 39 H-YAibEGTFTSDYSIYLEKIAibQQEFIQWLLAGGPSSGAPPPS-NH.sub.2 40 H-YAibEGTFTSDYSIYLEKQAAQEFIQWLLAGGPSSGAPPPS-NH.sub.2 41 H-YAibEGTFTSDYSIYLEKEAAQEFIQWLLAGGPSSGAPPPS-NH.sub.2 42 H-YAibEGTFTSDYSIYLEGQAAQEFIQWLLAGGPSSGAPPPS-NH.sub.2 43 H-YAibEGTFTSDYSIYLEEEAAQEFIQWLLAGGPSSGAPPPS-NH.sub.2 44 H-YAibEGTFTSDYSIYLEGEAAQEFIQWLLAGGPSSGAPPPS-NH.sub.2 45 H-YAibEGTFTSDYSIYLEEIAAQEFIQWLLAGGPSSGAPPPS-NH.sub.2 46 H-YAibEGTFTSDYSIYLEEQAAQEFIQWLLAGGPSSGAPPPS-NH.sub.2 47 H-YAibEGTFTSDYSIYLEKIAAQEFIQWLLAGGPSSGAPPPSK-NH.sub.2 48 H-YAibEGTFTSDYSIYLEKIAAQEFINWLLAGGPSSGAPPPS-NH.sub.2 49 H-YAibEGTFTSDYSIYLEKIAAK(OEG-OEG-yGlu-C18-OH)EFINWLLAG GPSSGAPPPS-NH.sub.2 50 H-YAibEGTFTSDYSIYLEKIAAKEFVNWLLAGK(OEG-OEG-yGlu-C18-OH) PSSGAPPPS-NH.sub.2 51 H-YAibEGTFTSDYSIYLEKIAAKEFVNWLLAGGPSSGAPPPSK (OEG-OEG-yGlu-C18-OH)-NH.sub.2 52 H-YAibEGTFTSDYSIYLEKIAAQEFINWLLAGK(OEG-OEG-yGlu-C18-OH) PSSGAPPPS-NH.sub.2 53 H-YAibEGTFTSDYSIYLEKIAAQEFINWLLAGGPSSGAPPPSK (OEG-OEG-yGlu-C18-OH)-NH.sub.2 54 H-YAibEGTFTSDYSIYLEKIAQK(OEG-OEG-yGlu-C18-OH)EFVNWLLAGGPS SGAPPPS-NH.sub.2 55 H-YAibEGTFTSDYSIYLEKIAQKEFVNWLLAGK(OEG-OEG-yGlu-C18-OH) PSSGAPPPS-NH.sub.2 56 H-YAibEGTFTSDYSIYLEKIAQKEFVNWLLAGGPSSGAPPPSK (OEG-OEG-yGlu-C18-OH)-NH.sub.2 57 H-YAibEGTFTSDYSIYLEKEAibAK(OEG-OEG-yGlu-C18-OH)EFINWLLAGG PSSGAPPPS-NH.sub.2 58 H-YAibEGTFTSDYSIYLEKEAibAKEFINWLLAGK(OEG-OEG-yGlu-C18-OH) PSSGAPPPS-NH.sub.2 59 H-YAibEGTFTSDYSIYLEKEAibAKEFINWLLAGGPSSGAPPPSK (OEG-OEG-yGlu-C18-OH)-NH.sub.2 60 H-YAibEGTFTSDYSIYLDKIAAK(OEG-OEG-yGlu-C18-OH)EFVNWLLAG GPSSGAPPPS-NH.sub.2 61 H-YAibEGTFTSDYSIYLDKIAAQEFVNWLLAGK(OEG-OEG-yGlu-C18-OH) PSSGAPPPS-NH.sub.2 62 H-YAibEGTFTSDYSIYLDKIAAQEFVNWLLAGGPSSGAPPPSK (OEG-OEG-yGlu-C18-OH)-NH.sub.2 63 H-YAibEGTFTSDYSIYLEKEAAKEFINWLLAGGPSSGAPPPS-NH.sub.2 64 H-YAibEGTFTSDYSIYLEKEAAKEFVQWLLAGGPSSGAPPPS-NH.sub.2 65 H-YAibEGTFTSDYSIYLEKEAAKEFIQWLLAGGPSSGAPPPS-NH.sub.2 66 H-YAibEGTFTSDYSIYLEKEAAKEFVAWLLAGGPSSGAPPPS-NH.sub.2 67 H-YAibEGTFTSDYSIYLEKEAAKEFIAWLLAGGPSSGAPPPS-NH.sub.2 68 H-YAibEGTFTSDVSIYLEKIAAQEFVNWLLAGGPSSGAPPPS-NH.sub.2 69 H-YAibEGTFTSDVSIYLDKIAAQEFVNWLLAGGPSSGAPPPS-NH.sub.2 70 H-YAibEGTFTSDYSIYLEEIAAQEFVNWLLAGGPSSGAPPPS-NH.sub.2 71 H-YAibEGTFTSDYSIYLEEEAAKEFVNWLLAGGPSSGAPPPS-NH.sub.2 72 H-YAibEGTFTSDYSIYLEEIAAKEFVNWLLAGGPSSGAPPPS-NH.sub.2 73 H-YAibEGTFTSDYSIYLEEEAAQEFVNWLLAGGPSSGAPPPS-NH.sub.2 74 H-YAibEGTFTSDYSIYLEKIAAQEFVNWLLAGGPSSGAPPPSK (OEG-OEG-yGlu-C20-OH)-NH.sub.2 75 H-YAibEGTFTSDYSIYLEKIAAK(OEG-OEG-yGlu-C20-OH) EFVNWLLAGGPSSGAPPPS-NH.sub.2 76 H-YAibEGTFTSDYSIYLEKEAAKEFINWLLAGGPSSGAPPPSK (OEG-OEG-yGlu-C20-OH)-NH.sub.2 77 H-YAibEGTFTSDYSIYLEEIAAQEFINWLLAGGPSSGAPPPSK (OEG-OEG-yGlu-C20-OH)-NH.sub.2 78 H-YAibEGTFTSDYSIYLEKIAAQEFVQWLLAGGPSSGAPPPS-NH.sub.2 79 H-YAibEGTFTSDYSIYLEKIAAQEFVQWLIAGGPSSGAPPPS-NH.sub.2 80 H-YAibEGTFTSDYSIYLEKIAAQEFVNWLIAGGPSSGAPPPS-NH.sub.2 81 H-YAibEGTFTSDYSIYLEKIAAQEFVQWLLAGGPSSGAPPPSK (OEG-OEG-yGlu-C20-OH)-NH.sub.2 82 H-YAibEGTFTSDYSIYLEKIAAQEFVQWLIAGGPSSGAPPPSK (OEG-OEG-yGlu-C20-OH)-NH.sub.2 83 H-YAibEGTFTSDYSIYLEKIAAQEFVNWLIAGGPSSGAPPPSK (OEG-OEG-yGlu-C20-OH)-NH.sub.2.

23. A pharmaceutical composition comprising: 1) a therapeutic amount of the GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 1, and 2) a pharmaceutically acceptable excipient or a pharmaceutical carrier.

24. A method of treating non-insulin-dependent diabete, insulin-dependent diabete or obesity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 23.

25. The method according to claim 24, wherein the pharmaceutical composition is administered simultaneously, separately or successively in combination with one or more reagent(s) selected from the group consisting of metformin, thiazolidinediones, sulfonylurea, dipeptidyl peptidase inhibitors and sodium glucose transporters.

26. The GLP-1 analog or the pharmaceutically acceptable salt thereof according to claim 13, characterized in that Y1 is K (—OEG-OEG-yGlu-C18-OH) or K (—OEG-OEG-yGlu-C20-OH): ##STR00004##

Description

DETAILED DESCRIPTION OF THE INVENTION

[0054] Unless stated to the contrary, the terms used in the specification and claims have the following meanings.

[0055] The amino acid sequence of the present invention comprises standard one-letter or three-letter codes for twenty amino acids. Unless explicitly stated otherwise, the preferred configuration of all amino acid residues in the present invention is L-configuration. In addition, Aib refers to α-aminoisobutyric acid and D-Ala refers to D-alanine.

[0056] The term agonist is defined as a substance that activates the type of receptor in discussion:

[0057] As used in the context of the present invention, the term GLP-1/GIP dual agonist refers to a substance or ligand that can activate both GLP-1 receptor and GIP receptor. In the present invention, the term treatment includes inhibiting, slowing down, stopping or reversing the existing symptoms or the progress or severity of the disease.

[0058] “Natural amino acids” refer to 20 types of conventional amino acids (i.e. Alanine (A), Cysteine (C), Aspartic acid (D), Glutamic acid (E), Phenylalanine (F), Glycine (G), Histidine (H), Isoleucine (I), Lysine (K), Leucine (L), Methionine (M), Asparagine (N), Proline (P), Glutamine (Q), Arginine (R), Serine (S), Threonine (T), Valine (V), Tryptophan (W) and Tyrosine (Y).

[0059] “Unnatural amino acid” refers to an amino acid that is not naturally encoded or found in the genetic code of any organism. They can be, for example, purely synthetic compounds. Examples of unnatural amino acids include, but are not limited to, hydroxyproline, γ-carboxyglutamic acid, O-phosphoserine, azetidine carboxylic acid, 2-aminoadipate, 3-aminoadipate, β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid (Dap), desmosine, 2,2′-diaminopimelic acid, 2,3-diaminopropionic acid (Dab), N-ethylglycine, N-methylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline, norleucine, ornithine (Om), D-omithine, D-arginine, p-aminophenylalanine acid, pentylglycine, pipecolic acid and thioproline. In addition, natural or unnatural amino acids which are chemically modified on C-terminal carboxyl group, N-terminal amino group and/or side chain functional group are also included.

[0060] The term “alkyl” refers to a saturated aliphatic alkyl group, which is a linear or branched chain group comprising 1 to 20 carbon atoms, preferably an alkyl group comprising 1 to 8 carbon atoms, more preferably an alkyl group comprising 1 to 6 carbon atoms, most preferably an alkyl group of 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-Dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers thereof. More preferred are lower alkyl groups comprising 1 to 6 carbon atoms, non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. Alkyl groups may be substituted or unsubstituted. When substituted, substituents can be substituted at any available attachment point. The substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxy, or carboxylate, preferably in the present invention is methyl, ethyl, isopropyl, tert-butyl, haloalkyl, deuterated alkyl, alkoxy-substituted alkyl and hydroxy-substituted alkyl.

[0061] “X is selected from A, B, or C”, “X is selected from the group consisting of A, B and C”, “X is A, B or C”, “X is A, B and C” and other terms all express the same meaning, which means that X can be any one or more of A, B, and C.

[0062] The “modification” of amino acids described in this patent refers to the substitution, addition or deletion of amino acids, including substitution or addition of any of the 20 natural amino acids.

[0063] The term “natural GLP-1” refers to a peptide comprising the human GLP-1 (7-36 or 7-37) sequence, and the term “natural GIP” refers to a peptide comprising the human GIP (1-42) sequence.

[0064] The terms “GLP-1” or “GIP”, if not explained further, refer to natural GLP-1 or natural GIP, respectively.

[0065] The “substitution” of an amino acid described in this patent refers to the substitution of an amino acid residue by a different amino acid residue.

[0066] The term “polyethylene glycol” or “PEG” refers to the mixture of the condensation polymer of ethylene oxide and water, which exists in linear or branched form and is represented by the general formula of H(OCH.sub.2CH.sub.2).sub.nOH, wherein n is no less than 9. Unless otherwise specified, this term includes polyethylene glycol polymers with an average total molecular weight between 5,000 and 40,000 Daltons.

[0067] The term “polyethylene glycol” or “PEG” is used with a numerical suffix to indicate its approximate average molecular weight. For example, PEG-5000 refers to polyethylene glycol having an average molecular weight of about 5000 Daltons.

[0068] The term “PEGylation” or similar terms refer to the modification of compound in natural state by linking a PEG chain to a peptide.

[0069] The term “PEGylated peptide” refers to a peptide in which a PEG chain is covalently bound to the peptide.

[0070] The term “fatty acid” refers to a carboxylic acid with a long fatty acid tail (chain), which can be saturated or unsaturated; in the present invention, the fatty acid refers to a carboxylic acid with C4-C30 linear or branched aliphatic groups.

[0071] The general definition of peptides described in this patent includes peptides with modified amino and carboxyl terminus. For example, amino acid sequence designated as a natural amino acid also includes an amino acid chain comprising a terminal carboxylic acid substituted with an amide group.

[0072] The hydrogen atoms described in the present invention can be replaced by its isotope deuterium, and any hydrogen atom in the exemplary compounds of the present invention can also be replaced by a deuterium atom.

[0073] “Optional” or “optionally” means that the event or circumstance that follows the term may, but does not necessarily, occur; and the description includes the instances in which the event or circumstance does or does not occur. For example, “heterocyclic group optionally substituted by an alkyl group” means that the alkyl group may be (but not necessarily) present, and the description includes the instances where the heterocyclic group is substituted by the alkyl group and the instances where the heterocyclic group is not substituted by the alkyl group.

[0074] “Substituted” refers to one or more hydrogen atoms in a group, preferably up to 5, more preferably 1 to 3 hydrogen atoms independently substituted by a corresponding number of substituents. It is obvious that the substituents are only located at possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, the binding of an amino group or a hydroxyl group having free hydrogen to a carbon atom having an unsaturated bond (e.g., olefinic) may be unstable.

[0075] “Pharmaceutical composition” refers to a mixture comprising one or more compounds described herein or a physiologically/pharmaceutically acceptable salt or produg thereof and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The pharmaceutical composition aims at promoting the administration to an organism, facilitating the absorption of the active ingredient and thereby exerting a biological effect.

[0076] “Pharmaceutically acceptable salt” refers to the salt of the compound of the present invention, which is safe and effective when applied to mammals in vivo, and has expected biological activity.

DETAILED DESCRIPTION OF THE PARTICULAR EMBODIMENTS

[0077] In order to describe the present invention in more detail, this specification provides the following specific embodiments, but the technical solution of the present invention is not limited to the embodiments.

TABLE-US-00004 Serial number Reagent Source 1 Rink-amide ChemMatrix resin Biotage 2 DEPBT GL Biochem 3 Fmoc-Aib-OH GL Biochem 4 Fmoc-L-Lys(Mtt)-OH GL Biochem 5 N,N-Dimethylformamide Sinopharm reagent 6 Dichloromethane Sinopharm reagent 7 Trifluoroacetic acid Sinopharm reagent 8 Triisopropylsilane Sigma-Aldrich 9 Hexafluoroisopropanol Sigma-Aldrich 10 Acetonitrile Merck-Millipore 11 Diisopropylethylamine Sigma-Aldrich 12 Piperidine Merck-Millipore 13 Anhydrous isopropyl ether INEOS Solvents 14 Boc-L-Tyr(tBu)-OH GL Biochem 15 Fmoc—NH-PEG.sub.2-COOH GL Biochem 16 Fmoc-L-Glu-OtBu GL Biochem 17 HOOC-(CH.sub.2).sub.16 —COOtBu GL Biochem

TABLE-US-00005 Serial number Instrument Source 1 H-CLASS Analytical Ultra WATERS Performance Liquid Chromatography 2 Xevo Lquid Chromatography/ WATERS Mass Spectrometry 3 Labconco Multifunctional Thermo-Fisher Freeze Dryer Scientific 4 Prep150 Preparative High WATERS Performance Liquid Chromatography 5 Multi-channel High-Speed Sigma Centrifuge

[0078] 3. Specific Experimental Scheme

[0079] 3.1 Chemical Synthesis of Polypeptide Backbone Compound No. 1

[0080] 3.1.1 Coupling of Fmoc-L-Lys(Boc)-OH to Rink-Amide ChemMatrix Resin

[0081] Rink-amide ChemMatrix resin (Biotage, 0.1 mmol) was weighed and placed into a disposable polypropylene polypeptide synthesis solid-phase reaction tube; DMF (10 ml) was added to swell the resin under nitrogen bubbling for 10 minutes; DMF was removed under vacuum, and DMF (10 ml) was added to wash the resin; the washing was repeated for twice; Fmoc-L-Lys(Boc)-OH (1 mmol), 3-(diethoxyphosphyloxy)-1,2,3-benzotriazin-4-one (DEPBT) (1 mmol) and diisopropylethylamine (DIEA, 2 mmol) were weighed and dissolved by adding DMF (10 ml), and then loaded onto the swollen Rink-amide ChemMatrix resin. The reaction was performed with shaking at room temperature for 2 hours. After the reaction was completed, the resin was washed alternately with DMF and dichloromethane (DCM) for twice, and finally washed with DMF for three times.

[0082] 3.1.2 Removal of Fmoc Protective Group with Fmoc-L-Lys(Boc)-Rink-Amide Resin

[0083] Piperidine/DMF (20%, 10 ml) was added into the solid-phase reaction tube comprising Fmoc-L-Lys(Boc)-Rink amide resin. The reaction was performed with shaking at room temperature for 10 minutes; then piperidine/DMF (20%, 10 ml) was added and shaken at room temperature for 10 minutes, and then removed. After the reaction was completed, the resin was washed with DMF (10 ml) for 4 times.

[0084] 3.1.3 Coupling of Peptide Chain Sequences

[0085] According to the peptide chain sequence of compound No. 1, from the amino terminus to the carboxy terminus (H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Glu-Ala-Al a-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-S er-Lys-NH.sub.2), the amount of amino acid derivatives and condensation reagents and their condensation methods were the same as those used for coupling Fmoc-L-Lys(Boc)-OH onto Rink-amide ChemMatrix resin. The amino acid residues used in the synthesis process were: Fmoc-L-His(Trt)-OH, Fmoc-Aib-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Val-OH, Fmoc-Tyr(tBu)-OH, Fmoc-L-Ala-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ile-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Leu-OH, Fmoc-L-Arg(Pbf)-OH and Fmoc-L-Pro-OH. The condensation of amino acid derivatives and Fmoc deprotection were repeated, finally resulting in the resin peptide comprising the polypeptide sequence of Compound No. 1.

[0086] 3.1.4 Cleavage of Resin Peptides

[0087] The resin peptide resulted from step 3 was washed successively with DMF and DCM for 3 times and then dried under vacuum. Then 10 ml of freshly prepared lysate (trifluoroacetic acid:triisopropylsilane:water=90:5:5, v:v:v) was added. The reaction was performed with shaking at room temperature for 2 hours. Filtration was carried out after the reaction was completed, and the resin was washed for twice with trifluoroacetic acid. The filtrate was pooled, and then a large amount of frozen anhydrous isopropyl ether was added to precipitate a solid. After centrifugation, the supernatant was removed and the crude polypeptide product of Compound No. 1 was obtained.

[0088] 3.1.5 Purification of the Crude Peptides by Reversed Phase Liquid Chromatography

[0089] The crude peptide was dissolved in a mixed solvent comprising 0.1% trifluoroacetic acid and 20% acetonitrile/water, filtered through a 0.22 um membrane, and subjected to isolation by using WATERS Prep150 LC reversed-phase high performance liquid chromatography system. Buffer A (0.10% trifluoroacetic acid, 10% acetonitrile in water) and B (0.1% trifluoroacetic acid, 90% acetonitrile in water). Among them, the chromatographic column was X-SELECT OBD C-18 (WATERS) reversed-phase chromatographic column. During the purification process, the detection wavelength of the chromatography was set to 220 nm, and the flow rate was 20 mL/min. The relevant fractions of the product were collected and lyophilized to obtain the pure polypeptide product of Compound No. 1, with a yield of 20%. The purity and identity of the pure polypeptide product were determined by analytical high performance liquid chromatography and liquid chromatography/mass spectrometry. The purity was 95.38%, and the molecular weight of the compound was 4218.4.

[0090] 3.2 Chemical Synthesis of Compound Nos 2-24, 34-48, 63-73 and 78-80

[0091] The polypeptide compounds of the present invention under the compound Nos 2-24, 34-48, 63-73 and 78-80 were synthesized according to the experimental scheme for compound 1, and the purity and molecular weight of the compounds were determined by analytical ultra-high performance liquid chromatography and liquid chromatography/mass spectrometry. Specifically, as shown in Table 1 below:

TABLE-US-00006 TABLE 1 Compound number Purity Molecular weight 2 95.06% 4244.8 3 97.98% 4369.6 4 96.34% 4353.2 5 96.05% 4178.0 6 95.29% 4203.3 7 95.15% 4227.6 8 95.05% 4230.3 9 95.95% 4293.2 10 95.56% 4292.1 11 96.33% 4350.0 12 95.60% 4249.5 13 96.86% 4277.4 14 95.58% 4277.4 15 97.64% 4333.8 16 95.07% 4307.4 17 97.76% 4307.4 18 96.53% 4233.9 19 95.11% 4278.6 20 96.79% 4262.7 21 98.73% 4276.8 22 96.87% 4233.6 23 95.69% 4434.4 24 96.72% 4419.3 34 95.65% 4233.6 35 96.56% 4304.7 36 96.74% 4176.6 37 96.65% 4105.5 38 95.24% 4162.5 39 95.14% 4247.7 40 95.75% 4191.6 41 95.56% 4192.5 42 95.74% 4120.4 43 96.84% 4193.5 44 95.24% 4121.4 45 96.54% 4177.5 46 95.77% 4192.5 47 97.55% 4304.8 48 95.32% 4162.6 63 97.14% 4178.6 64 96.32% 4178.6 65 95.47% 4193.3 66 96.56% 4122.5 67 98.87% 4136.4 68 96.54% 4085.2 69 95.24% 4071.1 70 96.14% 4150.5 71 96.78% 4166.6 72 95.69% 4150.4 73 96.32% 4165.3 78 95.18% 4162.6 79 96.22% 4162.6 80 97.21% 4148.5

[0092] 3.3 Chemical Synthesis of Compound No. 25 Coupled with Fatty Acid

[0093] 3.3.1 Coupling of Fmoc-L-Lys(Mtt)-OH onto Rink-Amide ChemMatrix Resin

[0094] Rink-amide ChemMatrix resin (Biotage, 0.1 mmol) was weighed and placed into a disposable polypropylene polypeptide synthesis solid-phase reaction tube, DMF (10 ml) was added to swell the resin under nitrogen bubbling for 10 minutes, DMF was removed under vacuum, and DMF (10 ml) was added to wash the resin, the washing was repeated for twice; Fmoc-L-Lys(Mtt)-OH (1 mmol), 3-(diethoxyphosphyloxy)-1,2,3-benzotriazin-4-one (DEPBT) (1 mmol) and diisopropylethylamine (DIEA, 2 mmol) were weighed and dissolved by adding DMF (10 ml), and then loaded onto the swollen Rink-amide ChemMatrix resin. The reaction was performed with shaking at room temperature for 2 hours. After the reaction was completed, the resin was washed alternately with DMF and dichloromethane (DCM) for twice, and finally washed with DMF for three times.

[0095] 3.3.2 Fmoc Deprotection and Peptide Chain Extension

[0096] The Fmoc deprotection from Fmoc-L-Lys(Mtt)-Rink amide ChemMatrix resin and the subsequent peptide chain extension were performed according to the same synthetic method as in Example 1, resulting in a resin peptide comprising Compound No. 25, wherein Boc-L-Tyr(t-Bu)-OH was used as the N-terminal amino acid residue.

[0097] 3.3.3 Mtt Deprotection and Lysine Side Chain Modification of Resin Peptide

[0098] After the above peptide-resin extension was completed, a mixture of hexafluoroisopropanol/dichloromethane (30%, 10 ml) was added, shaken and reacted at room temperature for 45 minutes and then removed. Then a mixture of hexafluoroisopropanol/dichloromethane (30%, 10 ml) was added again, shaken and reacted at room temperature for 45 minutes and then removed. After the reaction was completed, the resin was washed with DMF for 6 times. The additional coupling/deprotection cycle by using Fmoc/tBu solid phase synthesis strategy to extend the side chain of lysine involves Fmoc-NH-PEG2-COOH, Fmoc-L-Glu-OtBu and HOOC—(CH.sub.2).sub.16—COOt-Bu. In all couplings, the reaction was carried out at room temperature, and 1 mmol of amino acid construct, 1 mmol of DEPBT and 2 mmol of DIEA were reacted in DMF for 4 hours.

[0099] 3.3.4 Cleavage and Purification of the Product

[0100] The resin peptide resulted from the above steps was washed successively with DMF and DCM for twice and then dried under vacuum. Then the freshly prepared lysate was added (trifluoroacetic acid:triisopropylsilane:water=90:5:5, v:v:v). The reaction was performed with shaking at room temperature for 2 hours. Filtration was carried out after the reaction was completed, and the resin was washed for twice with trifluoroacetic acid. The filtrate was pooled, and then a large amount of frozen anhydrous isopropyl ether was added to precipitate a solid. After centrifugation, the supernatant was removed and the crude polypeptide product of Compound No. 25 was obtained.

[0101] 3.3.5 Purification of Compound 25 with Reversed-Phase Liquid Chromatography

[0102] The crude peptide was dissolved in a mixed solvent comprising 0.1% trifluoroacetic acid and 20% acetonitrile/water, filtered through a 0.22 μm membrane, and subjected to isolation by using WATERS Prep150 LC reversed-phase high performance liquid chromatography system. Buffer A (0.10% trifluoroacetic acid, 10% acetonitrile in water) and B (0.1% trifluoroacetic acid, 90% acetonitrile in water). Among them, the chromatographic column was X-SELECT OBD C-18 reversed-phase chromatographic column. During the purification process, the detection wavelength of the chromatography was set to 220 nm, and the flow rate was 20 mL/min. The relevant fractions of the product were collected and lyophilized to obtain the pure polypeptide product of Compound No. 25, with a yield of 18%. The purity and molecular weight of the pure polypeptide product were determined by analytical high performance liquid chromatography and liquid chromatography/mass spectrometry. The purity was 96.23%, and the molecular weight of the compound was 5008.6.

[0103] 3.4 Chemical Synthesis of Compound Nos 26-33 and 49-62

[0104] The polypeptide compounds of the present invention (compound Nos 26-33) were synthesized according to the experimental scheme for compound 25, and the purity and molecular weight of the compounds were determined by analytical high performance liquid chromatography and liquid chromatography/mass spectrometry. Specifically, as shown in Table 2 below:

TABLE-US-00007 TABLE 2 Compound number Purity Molecular weight 26 96.01% 4951.6 27 95.04% 4880.5 28 96.56% 4992.6 29 96.36% 4935.5 30 95.64% 4864.4 31 95.45% 5022.6 32 95.36% 4965.6 33 95.41% 4894.4 49 95.92% 4864.4 50 96.31% 4936.6 51 95.14% 4992.6 52 96.58% 4992.6 53 95.74% 4921.5 54 97.63% 4992.6 55 95.54% 5049.7 56 97.24% 4894.4 57 96.64% 4965.6 58 95.27% 5022.6 59 96.64% 4850.4 60 95.65% 4921.5 61 96.67% 4978.6 62 95.32% 4992.6

[0105] 3.5 Chemical Synthesis of Compound No. 74

[0106] 3.5.1 Coupling of Fmoc-L-Lys(Mtt)-OH onto Rink-Amide ChemMatrix Resin

[0107] Rink-amide ChemMatrix resin (Biotage, 0.1 mmol) was weighed and placed into a disposable polypropylene polypeptide synthesis solid-phase reaction tube, DMF (10 ml) was added to swell the resin under nitrogen bubbling for 10 minutes, DMF was removed under vacuum, and DMF (10 ml) was added to wash the resin, the washing was repeated for twice; Fmoc-L-Lys(Mtt)-OH (1 mmol), 3-(diethoxyphosphyloxy)-1,2,3-benzotriazin-4-one (DEPBT) (1 mmol) and diisopropylethylamine (DIEA, 2 mmol) were weighed and dissolved by adding DMF (10 ml), and then loaded onto the swollen Rink-amide ChemMatrix resin. The reaction was performed with shaking at room temperature for 2 hours. After the reaction was completed, the resin was washed alternately with DMF and dichloromethane (DCM) for twice, and finally washed with DMF for three times.

[0108] 3.5.2 Fmoc Deprotection and Peptide Chain Extension

[0109] The Fmoc deprotection from Fmoc-L-Lys(Mtt)-Rink amide ChemMatrix resin and the subsequent peptide chain extension were performed according to the same synthetic method as in Example 1, resulting in a resin peptide comprising Compound No. 74, wherein Boc-L-Tyr(t-Bu)-OH was used as the N-terminal amino acid residue.

[0110] 3.5.3 Mtt Deprotection and Lysine Side Chain Modification of Resin Peptide

[0111] After the above peptide-resin extension was completed, a mixture of hexafluoroisopropanol/dichloromethane (30%, 10 ml) was added, shaken and reacted at room temperature for 45 minutes and then removed. Then a mixture of hexafluoroisopropanol/dichloromethane (30%, 10 ml) was added again, shaken and reacted at room temperature for 45 minutes and then removed. After the reaction was completed, the resin was washed with DMF for 6 times. The additional coupling/deprotection cycle by using Fmoc/tBu solid phase synthesis strategy to extend the side chain of lysine involves Fmoc-NH-PEG2-COOH, Fmoc-L-Glu-OtBu and HOOC—(CH.sub.2).sub.18—COOt-Bu. In all coupling reactions, the reaction was carried out at room temperature, and 1 mmol of amino acid construct, 1 mmol of DEPBT and 2 mmol of DIEA were reacted in DMF for 4 hours.

[0112] 3.5.4 Cleavage and Purification of the Product

[0113] The resin peptide resulted from the above steps was washed successively with DMF and DCM for twice and then dried under vacuum. Then the freshly prepared lysate was added (trifluoroacetic acid:triisopropylsilane:water=90:5:5, v:v:v). The reaction was performed with shaking at room temperature for 2 hours. Filtration was carried out after the reaction was completed, and the resin was washed for twice with trifluoroacetic acid. The filtrate was pooled, and then a large amount of frozen anhydrous isopropyl ether was added to precipitate a solid. After centrifugation, the supernatant was removed and the crude polypeptide product of Compound No. 74 was obtained.

[0114] 3.5.5 Purification of Compound 74 with Reversed-Phase Liquid Chromatography

[0115] The crude peptide was dissolved in a mixed solvent comprising 0.1% trifluoroacetic acid and 20% acetonitrile/water, filtered through a 0.22 μm membrane, and subjected to isolation by using WATERS Prep150 LC reversed-phase high performance liquid chromatography system. Buffer A (0.1% trifluoroacetic acid, 10% acetonitrile in water) and B (0.1% trifluoroacetic acid, 90% acetonitrile in water). Among them, the chromatographic column was X-SELECT OBD C-18 reversed-phase chromatographic column. During the purification process, the detection wavelength of the chromatography was set to 220 nm, and the flow rate was 20 mL/min. The relevant fractions of the product were collected and lyophilized to obtain the pure polypeptide product of Compound No. 74, with a yield of 18%. The purity and molecular weight of the pure polypeptide product were determined by analytical high performance liquid chromatography and liquid chromatography/mass spectrometry. The purity was 95.14%, and the molecular weight of the compound was 5020.6.

[0116] 3.6 Chemical Synthesis of Compound Nos 75-77 and 81-83

[0117] The polypeptide compounds of the present invention (compound Nos 75-77 and 81-83) were synthesized according to the experimental scheme for compound 74, and the purity and molecular weight of the compounds were determined by analytical high performance liquid chromatography and liquid chromatography/mass spectrometry. Specifically, as shown in Table 3 below:

TABLE-US-00008 TABLE 3 Compound number Purity Molecular weight 75 95.91% 4892.5 76 96.25% 5047.6 77 95.36% 5035.6 81 96.31% 5034.7 82 97.22% 5034.7 83 95.61% 5020.6

Biological Test and Evaluation

[0118] The following test examples are provided to further describe the present invention, but are not intended to limit the scope of the invention.

[0119] 1. Experimental Reagents

TABLE-US-00009 Serial number Reagent Source 1 DMEM/F12 Gibco 11330032 2 Casein Sigma C3400-500G 3 3-Isobutyl-1-methylxanthine Sigma I7018-250MG 4 cAMP-Gs Dynamic kit-20,000 Cisbio 62AM4PEC tests 5 Corning ® 384 well microplate, Sigma CLS4514-50EA low volume 6 96V-bottom plate (PS) Axygen WIPP02280 7 Countess ® Cell Counting Invitrogen C10228 Chamber Slides 8 puromycin ThermoFisher A1113803 9 Hygromycin B Sigma A1720 10 PBS Gibco 10010023 11 0.25% Trypsin-EDTA(1X), Phenol ThermoFisher 25200-114 Red 12 Gibco ™ Fetal Bovine Serum, ThermoFisher 10099-141 Qualified, Australia Origin 13 glucose Sigma G8270-100G

[0120] 2. Experimental Instrument

TABLE-US-00010 Serial number Instrument Source 1 CO.sub.2 incubator Thermo 311 2 Biosafety cabinet Shanghai BOXUN BSC-1300IIA2 3 Refrigerated centrifuge Eppendorf 5702R 4 Haier double-door household Haier BCD-268TN refrigerator 5 Cell counter Life Technologies CountessII 6 Medicine storage box Haier hyc-940 7 Minus 20 degrees Celsius Haier DW-25L262 refrigerator 8 Refrigerated centrifuge 5810R Eppendorf 5810R 9 Automatic dispenser (Multidrop) Thermo 5840300 10 Microplate reader BioTek H1MFD 11 CO.sub.2 bacteria incubator Shanghai BOXUN BC-J80S 12 ACCU-CHEK Active Roche

[0121] 3. Test Example

[0122] 3.1. Evaluation of the Agonistic Activity of the Test Compounds for the Glucagon-Like Peptide-1 Receptor (GLP-1R)

[0123] 3.1.1 The Purpose of the Experiment:

[0124] The purpose of this test example is to measure the agonistic activity of the numbered compounds for the glucagon-like peptide-1 receptor (GLP-1R).

[0125] 3.1.2 Experimental Method:

[0126] The frozen CHO-K1/GLP-1R/CRE-luc stably transfected cell line was taken out from the liquid nitrogen tank, quickly thawed in a 37° C. water bath, and re-suspended in DMEM/F12 medium (Gibco Cat #11330032). After centrifugation, the cells were washed once, resuspended in the experimental buffer (DMEM/F12 medium comprising 0.1% casein (Sigma Cat #C3400)), and the cell density was adjusted with the experimental buffer. The cells were plated at a density of 2500 cells/5 μL/well in a 384-well plate (Sigma Cat #CLS4514), and then 2.5 μL of IBMX working solution (Sigma Cat #17018) prepared with buffer solution (the final concentration of IBMX was 0.5 mM) and 2.5 μL of the gradient diluted polypeptide sample were added into each well, centrifuged at 1000 rpm for 1 min, shaken for 30 seconds to mix well, and incubated at room temperature for 30 minutes. Cisbio cAMP-Gs Dynamic kit (Cisbio Cat #62AM4PEC) was used for detection. cAMP-d2 and Anti-cAMP-Eu.sup.3+-Cryptate were diluted with 20-fold cAMP Lysis & Detection Buffer respectively, and mixed well. 5 μL of the diluted cAMP-d2 solution was added into each well, and then 5 μL of the diluted Anti-cAMP-Eu.sup.3+-Cryptate solution was added into each well, shaken for 30 seconds to mix well, and incubated at room temperature for 1 hour in the dark.

[0127] 3.1.3 Method for Processing Experimental Data:

[0128] HTRF signals were read with Biotek Synergy H1 microplate reader, with the excitation wavelength of 320 nm, and emission wavelength of 620 nm and 665 nm. The signal ratio (665 nm/620 nm*10,000) was calculated, and the signal ratio was nonlinear fitted to the sample concentration with four-parameter equation in GraphPad Prism 6, resulting in the EC.sub.50 values. The particular data are shown in Table 4 below.

[0129] 3.2. Evaluation of the Agonistic Activity of the Test Compounds for the Glucose-Dependent Insulinotropic Peptide Receptor (GIPR)

[0130] 3.2.1 The Purpose of the Experiment:

[0131] The agonistic activity of the test compounds for the glucose-dependent insulinotropic peptide receptor (GIPR).

[0132] 3.2.2 Experimental Method:

[0133] Wild-type CHO-K1 cells were collected, and the cell suspension was adjusted to an appropriate density, plated in a 6-well plate at 2 mL per well, and placed in a 37° C., 5% CO.sub.2 incubator for adhesion overnight. The mixture for transfection (hGIPR plasmid, Fugene HD (Promega Cat #E2311), OptiMEM (Gibco Cat #31985070)) was mixed well, placed at room temperature for 15 minutes, and was added at 100 μL to the corresponding cell well. After transfection of CHO-K1 cells for 24 h, hGIPR was over-expressed on the cell surface. The cells in 6-well plate were collected after the transient transfection, and were washed once with the experimental buffer (DMEM/F12 medium (Gibco Cat #11330032) comprising 0.1% casein (Sigma Cat #C3400)), and the cell density was adjusted with the experimental buffer. The cells were plated at a density of 5000 cells/5 μL/well in a 384-well plate (Sigma Cat #CLS4514), and then 2.5 μL of IBMX working solution (Sigma Cat #17018) prepared with buffer solution (the final concentration of IBMX was 0.5 mM) and 2.5 μL of the gradient diluted polypeptide sample were added into each well, centrifuged at 1000 rpm for 1 min, shaken for 30 seconds to mix well, and incubated at room temperature for 30 minutes. Cisbio cAMP-Gs Dynamic kit (Cisbio Cat #62 AM4PEC) was used for detection. cAMP-d2 and Anti-cAMP-Eu3+-Cryptate were diluted with 20-fold cAMP Lysis & Detection Buffer respectively, and mixed well. 5 μL of the diluted cAMP-d2 solution was added into each well, and then 5 μL of the diluted Anti-cAMP-Eu3+-Cryptate solution was added into each well, shaken for 30 seconds to mix well, and incubated at room temperature for 1 hour in the dark.

[0134] 3.2.3 Method for Processing Experimental Data:

[0135] HTRF signal was read with Biotek Synergy H1 microplate reader, with the excitation wavelength of 320 nm, and emission wavelength of 620 nm and 665 nm. The signal ratio (665 nm/620 nm*10,000) was calculated, and the signal ratio was nonlinear fitted to the sample concentration with four-parameter equation in GraphPad Prism 6, resulting in the EC.sub.50 values. The particular data are shown in Table 4 below.

[0136] Agonistic activity of the polypeptide backbone compounds for human GLP-1R and human GIPR receptors.

TABLE-US-00011 TABLE 4 Activity for human GLP-1R Activity for human GIPR Compound (EC.sub.50 nM) (EC.sub.50 nM) Natural 0.007 N/A GLP-1 Natural GIP N/A 0.003 Semaglutide 0.025 >10 1 0.005 >10 2 0.017 3.5 3 0.578 0.91 4 0.43  >10 5 0.004 >10 6 0.128 2.1 7 0.016 0.16 8 0.070 0.12 9 0.011 0.018 10 0.097 0.006 11 0.080 0.014 12 0.013 0.081 13 0.013 0.017 14 0.008 0.006 15 0.015 0.009 16 0.007 0.018 17 0.010 0.057 18 0.022 0.012 19 0.024 0.020 20 0.027 0.008 21 0.043 0.015 22 0.051 0.018 23 0.059 0.020 24 0.083 0.008 63 0.010 0.020 64 0.020 0.033 65 0.013 0.027 66 0.026 0.054 67 0.025 0.017 68 0.025 0.047 69 0.052 0.120 70 0.017 0.017 71 0.017 0.042 72 0.017 0.030 73 0.069 0.021 78 0.008 0.012 79 0.012 0.015 80 0.008 0.008

[0137] Experimental Conclusion:

[0138] By designing and studying the polypeptide skeleton/backbone, the present invention has a strong agonist activity and has the potential for better treatment of metabolic diseases than many GLP-1/GIP receptor double agonists in this field. Agonistic activity of polypeptide compounds coupled to fatty acid for human GLP-1R and human GIPR receptors.

TABLE-US-00012 TABLE 5 Fatty acid Activity for Activity for modification Fatty acid human GLP-1R human GIPR Compound site chain length (EC.sub.50 nM) (EC.sub.50 nM) Natural / / 0.007 N/A GLP-1 Natural GIP / / N/A 0.003 Semaglutide 20 18 0.025 >10 25 40 18 0.031 0.059 27 20 18 0.12 0.11 28 40 18 0.074 0.020 29 30 18 0.240 0.015 30 20 18 0.058 0.021 60 20 18 0.20 0.020 62 40 18 0.32 0.045 74 40 20 0.068 0.046 75 20 20 0.20 0.16 83 40 20 0.028 0.032

[0139] Experimental Conclusion:

[0140] The invention found that the activity changes of different peptide skeletons after conjugated to fatty acids are not the same, and the peptide skeleton of the present invention can still maintain favorable activity on GLP-1 and GIP receptors after coupling to fatty acid modification.

[0141] 3.3 Stability of the Polypeptide Backbone and Polypeptide Compound Coupled to Fatty Acid

[0142] The stability in plasma is very important for therapeutic polypeptide agents, because polypeptide agents are likely to be sensitive to polypeptide hydrolase and proteolytic enzymes in plasma. The instability of the polypeptides in plasma will affect the half-life and efficacy thereof.

[0143] 3.3.1 The Purpose of the Experiment:

[0144] The purpose of this experiment is to test the stability of the numbered compounds in plasma. In order to compare the stability of the numbered compounds with the compounds in the prior art, the preferred polypeptide backbone compounds 023 (H23) and 024 (H24) and the preferred modified compound 089 (H89) taught in the patent application WO2012/167744 were also tested in this experiment.

[0145] 3.3.2 Experimental Method:

[0146] Five microliters of samples with concentrations of 20, 50, 100, 200, 500, 1000, 2000, 5000, 10000 ng/ml were added into 45 microliters of SD rat plasma, and the compound contents were detected by LC-MS, and a standard curve was plotted. 5 microliters of the polypeptide solution with a concentration of 1 mg/ml was added into 45 microliters of SD rat plasma. Five samples were prepared for each test compound, and 1 sample was taken out at 0 min, 30 min, 60 min, 120 min, and 240 min respectively. The content of the residual compounds was detected by LC-MS. The content at 0 min was used as control (100%), and the relative contents of the residual compound in the sample at other time points were calculated. The LC-MS method for the detection of the compounds was as follows: 5% acetonitrile solution was prepared as solution A, and 95% acetonitrile solution was prepared as solution B; 15 microliters of samples were injected at a flow rate of 0.6 ml/min, the time duration and the solution gradient ratio were as shown in the following table; the content of the compound was detected with Raptor Biphenyl 2.7 micron detection column.

TABLE-US-00013 Time duration (minutes) A (%) B (%) 0.20 95.0 5.00 1.70 5.00 95.0 2.00 5.00 95.0 2.01 95.0 5.00 2.50 95.0 5.00

[0147] 3.3.3 Experimental Results:

[0148] 1) Through the above experimental methods, the stability data of the polypeptide backbone in plasma are shown in Table 6:

TABLE-US-00014 TABLE 6 Compound Relative content of the residual compounds in plasma (%) number 0 min 30 min 60 min 120 min 240 min 14 100.00 106.04 107.00 112.14 102.16 63 100.00 99.31 98.56 109.18 108.30 H23 100.00 86.75 88.43 93.56 84.61 H24 100.00 99.22 93.19 89.30 85.56

[0149] 3.3.4 Experimental Conclusion:

[0150] Through research, it is found that the compounds of the present invention can maintain the stability of plasma content (relative content>95%), indicating that the compounds of the present invention have desirable druggability and have favorable potential for the treatment of diseases. The plasma stability of the compounds of the present invention is superior to that of the compounds H23 and H24 in the prior art.

[0151] 2) Through the above experimental methods, the plasma stability data of the polypeptides coupled to fatty acid are shown in Table 7:

TABLE-US-00015 TABLE 7 Compound Relative content of the residual compounds in plasma (%) number 0 min 30 min 60 min 120 min 240 min 74 100.00 95.54 104.74 100.10 91.16 75 100.00 101.51 92.07 84.74 57.83 H89 100.00 87.94 88.44 90.56 81.97

[0152] Experimental Conclusion:

[0153] It is found that compound 74 of the present invention is more stable in plasma at a time point of 4 hour (relative content>90%) than compound 75 and the prior art compound H89.

[0154] 3.4 Pharmacokinetics of the Polypeptides Coupled to Fatty Acids in Mice

[0155] Plasma stability is one of the factors that affect the pharmacokinetics of polypeptide agents. The in vivo pharmacokinetics of the polypeptide agents is also affected by factors such as their absorption and clearance in the body.

[0156] 3.4.1 The Purpose of the Experiment:

[0157] The purpose of this experiment is to study the in vivo pharmacokinetics in mice (plasma) of the numbered compounds administered by single intravenous injection into Balb/c mice as the test animals.

[0158] 3.4.2 Experimental Method:

[0159] Male Balb/c mice weighing 18-30 grams and 7-9 weeks old were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. The numbered compounds were prepared with 20 mM citrate buffer (pH=7.0), and then were injected into the mice via tail vein at a dose of 30 nanomoles per kilogram of body weight. 0.2 ml of the blood was sampled at time points of 0 hour, 0.083 hour, 0.25 hour, 0.5 hour, 1 hour, 2 hour, 4 hour, 6 hour, 8 hour, 24 hour and 32 hour. The collected mouse blood was centrifuged at 6000 rpm for 6 minutes at a temperature of 4° C. to isolate plasma. The content of the numbered compound in mouse plasma was detected by the experimental method of Test example 3.3.

[0160] 3.4.3 Experimental Results:

[0161] Through the above experimental method, the particular data are shown in Table 8:

TABLE-US-00016 TABLE 8 PK parameters Unit Compound 28 Compound 74 T.sub.1/2 h 4.7 19.5 AUCInf h*ng/mL 9500 30698

[0162] 3.4.4 Experimental Conclusion:

[0163] Through research, it is found that the compounds of the present invention have favorable pharmacokinetic characteristics in mice, indicating that they have advantages in the treatment of diseases.

[0164] 3.5 Pharmacokinetics of the Polypeptides Coupled to Fatty Acids in Rats

[0165] 3.5.1 The Purpose of the Experiment:

[0166] In order to further study the pharmacokinetics of the compounds of the present invention, this experiment is to study the in vivo pharmacokinetics in rats (plasma) of the numbered compounds administered by single subcutaneous injection into SD rats as the test animals.

[0167] 3.5.2 Experimental Method:

[0168] Male SD rats weighing 150-300 grams were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. The numbered compounds were prepared with 20 mM citrate buffer (pH=7.0), and then were injected into the rat via subcutaneous injection at a dose of 50 nanomoles per kilogram of body weight. 0.2 ml of the blood was sampled at time points of 0 hour, 0.5 hour, 1 hour, 2 hour, 4 hour, 6 hour, 8 hour, 24 hour, 32 hour, 48 hour, 72 hour, 96 hour and 120 hour. The collected rat blood was centrifuged at 6000 rpm for 6 minutes at a temperature of 4° C. to isolate plasma. The content of the numbered compound in rat plasma was detected by the experimental method of Test example 3.3.

[0169] 3.5.3 Experimental Results:

[0170] Through the above experimental method, the particular data are shown in Table 9:

TABLE-US-00017 TABLE 9 PK parameters Unit Compound 74 T.sub.1/2 h 15.9 AUCInf h*ng/mL 17673

[0171] 3.5.4 Experimental Conclusion:

[0172] Through research, it is found that the compounds of the present invention have favorable pharmacokinetic characteristics in murine, indicating that they have advantages in the treatment of diseases.

[0173] 3.6 In Vivo Efficacy of Polypeptides Coupled to Fatty Acid

[0174] 3.6.1 The Purpose of the Experiment:

[0175] In order to test the effect of subcutaneous administration of the numbered compounds on regulating the blood glucose in diet-induced obesity mice.

[0176] 3.6.2 Experimental Method:

[0177] High fat food-induced obesity C57BL/6 mice (male, weighing 35-55 grams and 10-12 weeks old) were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. The diet-induced obesity C57BL/6 mice were subcutaneously injected with the numbered compounds (3 nanomol/kg body weight), and then the mice were fasted but freely access to water. After 18 hours, glucose solution with the concentration of 0.2 g/ml was injected intraperitoneally at a dose of 2 g/kg body weight. According to the experimental design, blood was sampled from the tail of the mouse at time points of 0 min, 15 min, 30 min, 60 min, and 120 min to measure the blood glucose levels. Specifically, mouse was physically fixed and the tail was exposed, the tip of tail was cut off, and squeezed until bleeding. The first drop of blood was discarded, and then the blood glucose level was measured with ACCU-CHEK Active (Roche). The area under curve (AUC) of the blood glucose was calculated based on the result of each point.

[0178] 3.6.3 Experimental Results:

[0179] Through the above experimental method, the particular data are shown in Table 10:

TABLE-US-00018 TABLE 10 Test Blood glucose (mmol/L, Mean ± SD) AUC compound Dose 0 min 15 min 30 min 60 min 120 min (mmol/L .Math. hr) Placebo 9.2 ± 0.8 25.0 ± 3.1 31.6 ± 2.0 30.7 ± 1.9 25.3 ± 5.4 54.9 ± 5.2 74 3 nmol/kg 3.8 ± 0.8  9.6 ± 1.0  6.6 ± 1.1  4.8 ± 0.6  3.3 ± 0.5 10.6 ± 1.1 H89 3 nmol/kg 6.9 ± 1.2 20.3 ± 3.9 24.0 ± 4.4 21.2 ± 4.4 14.0 ± 3.3   37.8 ± 6.6*** Semaglutide 3 nmol/kg 4.5 ± 0.5 12.7 ± 1.5  9.9 ± 0.4  7.4 ± 1.4  5.3 ± 0.4  15.6 ± 1.7** ***significant difference compared to the blood glucose AUC of compound 74, P = 0.0001. **significant difference compared to the blood glucose AUC of compound 74, P = 0.002.

[0180] 3.6.4 Experimental Conclusion:

[0181] In this experiment, the compounds of the present invention show a significant hypoglycemic effect at a dose of 3 nanomol/kg body weight, and the AUC of the blood glucose for compound 74 group was decreased by more than 80% compared to that of placebo. The blood glucose AUC is significantly different, when compared to those of compound H89 and semaglutide that have GLP-1 activity in the prior art.