Oxyntomodulin analogue

Abstract

Provided is an oxyntomodulin analogue. The analogue comprises GCGR and GLP-1R dual agonist activity, improved enzymolysis stability and biological activity, and no adverse reactions. The analogue can be used to prepare medication for treating hyperphagia, obesity and diabetes.

Claims

1. An oxyntomodulin analogue, comprising a parent peptide having the following amino acid sequence (SEQ ID NO: 29): TABLE-US-00010 His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser- Lys-Xaa13-Leu-Asp-Xaa16-Xaa17-Xaa18-Ala-Xaa20- Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29- Xaa30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37- Xaa38-Xaa39-Xaa40-COR.sub.1 wherein, R.sub.1=OH or NH.sub.2; Xaa2=Aib, Ser or D-Ser; Xaa10=Lys or Tyr; Xaa13=Lys or Tyr; Xaa16=Ser, Aib, Lys; Xaa17=Lys or Arg; Xaa18=Arg or Ala; Xaa20=His, Gln or Lys; Xaa21=Asp or Glu; Xaa23=IIe, Leu or Val; Xaa24=Glu or Gln; Xaa27=Met or Leu; Xaa28=Ser, Asn, Asp, or Arg; Xaa29=Ala, Gly, or Thr; Xaa30=Gly; Xaa31=Gly; Xaa32=Pro; Xaa33=Ser; Xaa34=Ser; Xaa35=Gly; Xaa36=Ala; Xaa37=Pro; Xaa38=Pro; Xaa39=Pro; and Xaa40=Ser; in the amino acid sequence of the parent peptide, at least one of Xaa10, Xaa16, Xaa17 or Xaa20 is Lys, the side chain of said at least one Lys or the Lys at position 12 is attached to a lipophilic substituent in such a way that a carboxyl group of the lipophilic substituent forms an amide bond with an amino group of a bridging group, the bridging group is attached to the parent peptide by means of a carboxy group of the amino acid residue of the bridging group which forms an amide bond with the amino group of the side chain of said at least one Lys or the Lys of the parent peptide; the bridging group is Glu-(PEG).sub.m or Asp-(PEG).sub.m or (PEG).sub.m, wherein m is an integer of 2-10; and the lipophilic substituent is an acyl group selected from CH.sub.3(CH.sub.2).sub.nCO or HOOC(CH2).sub.nCO, wherein n is an integer of 10-24.

2. The oxyntomodulin analogue according to claim 1, wherein the parent peptide has an amino acid sequence of (SEQ ID NO: 29): TABLE-US-00011 His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser- Lys-Xaa13-Leu-Asp-Xaa16-Xaa17-Xaa18-Ala-Xaa20- Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29- Xaa30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37- Xaa38-Xaa39-Xaa40-COR.sub.1 wherein, R.sub.1=NH.sub.2; Xaa2=Aib or D-Ser; Xaa10=Lys or Tyr; Xaa13=Lys or Tyr; Xaa16=Ser, Aib, or Lys; Xaa17=Lys or Arg; Xaa18=Arg or Ala; Xaa20=His, Gln or Lys; Xaa21=Asp or Glu; Xaa23=IIe or Val; Xaa24=Glu or Gln; Xaa27=Met or Leu; Xaa28=Asn, Arg, or Asp; Xaa29=Gly or Thr; Xaa30=Gly; Xaa31=Gly; Xaa32=Pro; Xaa33=Ser; Xaa34=Ser; Xaa35=Gly; Xaa36=Ala; Xaa37=Pro; Xaa38=Pro; Xaa39=Pro; and Xaa40=Ser.

3. The oxyntomodulin analogue according to claim 1, wherein that a molecular bridge is formed between the side chains of amino acid residue pairs 12 and 16, 16 and 20, 17 and 21, or 20 and 24.

4. The oxyntomodulin analogue according to claim 2, wherein the amino acid sequence of the parent peptide is a sequence selected from the group consisting of SEQ ID NO.7, SEQ ID NO.10, SEQ ID NO.13, and SEQ ID NO.17.

5. The oxyntomodulin analogue according to claim 1, wherein that-when the position 10, 12, 16, 17, or 20 of the amino acid sequence is Lys, the lipophilic substituent attached to the side chain of Lys is one of the following structures: ##STR00004##

6. A pharmaceutical composition comprising the oxyntomodulin analogue of claim 1.

7. A method of making a drug for treating diabetes, the method comprising preparing the oxyntomodulin analogue of claim 1.

8. A method of making a drug for reducing blood glucose, the method comprising preparing the oxyntomodulin analogue of claim 1.

9. The oxyntomodulin analogue according to claim 3, wherein, when the position 16 of the amino acid sequence is Lys, the lipophilic substituent attached to the side chain of Lys is one of the following structures: ##STR00005##

10. A pharmaceutical composition comprising the oxyntomodulin analogue of claim 3.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: stimulation curve of Exenatide and Compounds 4, 5, 6, 7 on GLP-IR;

(2) FIG. 2: stimulation curve of Glucagon and Compounds 4, 5, 6, 7 on GCGR;

(3) FIG. 3: Effect of Exenatide (Ex-4), Compounds 4, 5, 6 and 7 on insulin release after oral administration of glucose in male C57B1/6J mice; compared with the control group, p<0.05;

(4) FIG. 4: Effect of Exenatide (Ex-4), Compounds 19, 20, 21, 22, 23 and 24 on insulin release after oral administration of glucose in male C57B1/6J mice; compared with the control group, p<0.05;

(5) FIG. 5: Effect of polypeptide Compounds 4, 5, 6 and 7 on body weight in obese mice, compared with the control group, p<0.05.

DETAILED DESCRIPTION OF THE INVENTION

(6) The embodiments of the present invention will be described in detail hereafter in conjunction with the examples, but the person skilled in the art will appreciate that the following examples are only intended to illustrate the present invention and should not be construed to limit the scope of the present invention. Unless otherwise specified, the examples were carried out according to conventional conditions or the conditions recommended by manufacturers. The reagents or instruments used, the manufacture of which were not specified, were all conventional products can be obtained commercially.

Example 1 Synthesis of Octadecanedioic Acid Mono-Tert-Butyl Ester

(7) Octadecanoic acid (31.4 g, 100 mmol) was suspended in toluene (250 ml), and the mixture was heated to reflux. N,N-dimethylformamide di-tert-butyl acetal (50.9 g, 250 mmol) was added dropwise over 4 hours. The mixture was refluxed overnight. The solvent was removed under vacuum, and at 50 C., the crude material was suspended in DCM/ethyl acetate (500 ml, 1:1) and stirred for 15 minutes. The solid was collected by filtration and triturated in DCM (200 mL), filtered and evaporated under vacuum to give 20 g of crude mono-tert-butyl-hexadecane, which was recrystallized in heptane (200 ml) to give 12.9 g of octadecanedioic acid mono-tert-butyl ester (33%). Except recrystallization, this mono-ester can be purified by chromatography on silica in AcOEt/heptane. .sup.1H-NMR (400 MHz, CDCl.sub.3) : 2.35 (t, 2H), 2.20 (t, 2H), 1.65-1.55 (m, 4H), 1.44 (s, 9H), 1.34-1.20 (m, 22H).

Example 2 Synthesis of Polypeptide Compound

(8) Synthesis of polypeptide compound 1 and polypeptide compound 5 was taken as an example.

(9) Materials:

(10) All amino acids were purchased from NovaBiochem company. Unless otherwise specified, all reagents were analytical grade, purchased from Sigma company. Protein Technologies PRELUDE 6-channel polypeptide synthesizer. Phenomenex Luna C18 preparative column (46 mm250 mm) was used for purification of the polypeptides. High performance liquid chromatography instrument was manufactured by Waters company. MS analysis was determined using Agilent mass spectrometer.

(11) Method:

(12) 1. Synthesis of Polypeptide Compound 1

(13) Structure Sequence:

(14) His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys(PEG.sub.2-PEG.sub.2-Glu-CO(CH.sub.2).sub.16CO.sub.2H)-Ala-Ala-His-Asp-Phe-Val-Glu-Trp-Leu-Leu-Arg-Ala-NH.sub.2

(15) 1a) Main Peptide Chain Assembly:

(16) The following polypeptide in a scale of 0.25 mol was synthesized on a CS336X peptide synthesizer (CS Bio American company) according to Fmoc/t-Bu strategy:

(17) Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Ser(OtBu)-Lys(ivDde)-Ala-Ala-His(Boc)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Leu-Arg(pbf)-Ala-rink amide resin

(18) (1) Step 1: 0.75 g of Rink amide resin was swelled in dichloromethane, and the resin was washed with N,N-dimethylformamide for three times;

(19) (2) Step 2: The procedure reaction was performed using Rink amide resin as carrier, the mixture of benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine at a molar ratio of 1:1:1 as coupling agent, and N, N-dimethylformamide as solvent, condensation reactions were performed to successively attach Fmoc-Arg(pbf)-OH, Fmoc-Leu-OH (2), Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)-OH, Fmoc-His(Boc)-OH, Fmoc-Ala-OH (2), Fmoc-Lys(ivDde)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Phe-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Gly-OH, Fmoc-Gln(OtBu)-OH, Fmoc-D-Ser(t-Bu)-OH and Boc-His(Boc)-OH to obtain Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Ser(OtBu)-Lys(ivDde)-Ala-Ala-His(Boc)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Leu-Arg(pbf)-Ala-rink amide resin, wherein in each condensation reaction, the feeding amount of Fmoc-protected amino acid and the amount of the resin was at a molar ratio of 1:16:1; in each condensation reaction, the amount of benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate and the amount of Fmoc protected amino acid was at a molar ratio of 3:1.

(20) 1b) Removal of 1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)-3-methyl-butyl (ivDde) and Introduction of Lipophilic Substituent

(21) In the solution of N-methylpyrrolidone:dichloromethane=1:1 (volume ratio), the protected peptidyl resin synthesized in 1a) was washed twice, and freshly prepared 2.0% hydrazine hydrate N-methylpyrrolidone solution was added. The reaction mixture was shaken at room temperature for 12.0 minutes, and then filtered. The hydrazine treatment step was repeated twice to obtain:

(22) Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Ser(OtBu)-Lys-Ala-Ala-His(Boc)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Leu-Arg(pbf)-Ala-rink amide resin. Subsequently, the resin was thoroughly washed with dichloromethane and N-methylpyrrolidon. Thereto was added a mixed coupling solution of FmocNH-PEG.sub.2-OH, benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole and diisopropylethylamine in N-methylpyrrolidon, shaken for 3.0 hours, filtrated and washed. The hydrazine treatment was repeated twice to obtain:
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Ser(OtBu)-Lys(Fmoc-PEG.sub.2-OH)-Ala-Ala-His(Boc)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Leu-Arg(pbf)-Ala-rink amide resin. The Fmoc group was removed in piperidine/N, N-dimethylformamide solution, Fmoc-PEG.sub.2-OH coupling reaction was repeated once to obtain:
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Ser(OtBu)-Lys(Fmoc-PEG.sub.2-PEG.sub.2-OH)-Ala-Ala-His(Boc)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Leu-Arg(pbf)-Ala-rink amide resin. The Fmoc group was removed in piperidine/N,N-dimethylformamide solution, then according to the conventional conditions, coupling of Fmoc-Glu-OtBu, tBu mono-protected stearidonic fatty acid was performed sequentially to obtain:
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Ser(OtBu)-Lys(Fmoc-PEG.sub.2-PEG.sub.2-Glu-CO(CH.sub.2).sub.16CO.sub.2tBu)-Ala-Ala-His(Boc)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Leu-Arg(pbf)-Ala-rink amide resin.

(23) 1c) Removal of Polypeptide Full Protection:

(24) Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-A sp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Ser(OtBu)-Lys(Fmoc-PEG.sub.2-PEG.sub.2-Glu-CO(CH.sub.2).sub.16CO.sub.2tBu)-Ala-Ala-His(Boc)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Leu-Arg(pbf)-Ala-rink amide resin was added to a round bottom flask, under ice bath, added with a cutting fluid of TFA/EDT/Phenol/H.sub.2O (88/2/5/5, volume ratio), heated, controlling the temperature of the lysates at 25 C., and reacted for 120 minutes. After filtration, the filter cake was washed with a small amount of trifluoroacetic acid for three times, and the filtrate was combined. The filtrate was slowly poured into ice diethyl ether with stirring, placed on standing for at least 1.0 hours to precipitate completely. The supernatant was removed, and the precipitate was centrifuged and washed with ice diethyl ether for six times to give crude compound:

(25) His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys(PEG.sub.2-PEG.sub.2-Glu-CO(CH.sub.2).sub.16CO.sub.2H)-Ala-Ala-His-Asp-Phe-Val-Glu-Trp-Leu-Leu-Arg-Ala-NH.sub.2.

(26) d) Purification of Polypeptide Compound:

(27) The crude product obtained in 1c was dissolved with 5.0% acetic acid solution in acetonitrile: H.sub.2O=1:1 (volume ratio), and purified twice by semipreparative HPLC on a 5.0 m reverse-phase C18-packed 50 mm250 mm column. The column was eluted with 30-60% acetonitrile 0.1% trifluoroacetic acid/H.sub.2O gradient at 40 mL/min for 45.0 minutes, and the fractions containing the peptide were collected, and concentrated to remove acetonitrile, and then lyophilized, to obtain pure product with HPLC purity greater than 95%. The isolated product was analyzed by LC-MS, and the m/z value of protonated molecular ion peak was found to be: 4116.0, and the theoretical value is 4116.6.

(28) 2. Synthesis of polypeptide Compound 5

(29) Structure Sequence:

(30) His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG.sub.2-PEG.sub.2-CO(CH.sub.2).sub.14CH.sub.3)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH.sub.2

(31) 2a) Main Peptide Chain Assembly:

(32) The following polypeptide in a scale of 0.25 mol was synthesized on a CS336X peptide synthesizer (CS Bio American company) according to Fmoc/t-Bu strategy Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(ivDde)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Aib-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala- Pro-Pro-Pro-Ser(t-Bu)-rink amide resin

(33) (1) Step 1: 0.75 g of Rink amide MBHA-LL resin (Novabiochem, loading 0.34 mmol/g) was swelled in dichloromethane (DCM) for 1 hour, and the resin was fully washed with N, N-dimethylformamide (DMF) for three times;

(34) (2) Step 2: The procedure reaction was performed using Rink amide resin as carrier, the mixture of 6-chloro-benzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), organic base N,N-diisopropylethylamine (DIEPA) at a molar ratio of 1:1 as coupling agent, and N, N-dimethylformamide (DMF) as solvent, the condensation reactions were performed to successively link Fmoc-Ser(t-Bu)-OH, Fmoc-Pro-OH (3), Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(t-Bu)-OH (2), Fmoc-Pro-OH, Fmoc-Gly-OH (2), Fmoc-Thr(t-Bu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH (2), Fmoc-Aib-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Lys(ivDde)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Phe-OH, Thr(t-Bu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-D-Ser(t-Bu)-OH, Boc-His(Boc)-OH to obtain Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(ivDde)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Aib-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala- Pro-Pro-Pro-Ser(t-Bu)-rink amide resin. Subsequently, the resin was thoroughly washed with N, N-dimethylformamide (DMF), dichloromethane (DCM), methanol, dichloromethane (DCM), N, N-dimethylformamide (DMF) for three times respectively.

(35) It should be noted that: 1) wherein the feeding amount of the first amino acid FFmoc-Ser(t-Bu)-OH and the amount of the resin was at a molar ratio of 1:16:1; 2) in each of the subsequent condensation reactions, each of the amount of Fmoc protected amino acid, 6-chloro-benzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), organic base N, N-diisopropylethylamine (DIEPA) was excess by 2-8 times, the reaction time was 1-5 hours.

(36) 2b) Removal of 1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)-3-methyl-butyl (ivDde) and Introduction of Lipophilic Substituent

(37) The resin was washed twice in the solution of N,N-dimethylformamide (DMF)/dichloromethane (DCM)=1:1 (volume ratio), and added with freshly prepared 3.0% hydrazine hydrate in N, N-dimethylformamide (DMF). The reaction mixture was shaken at room temperature for 1030 minutes, and then filtered. The hydrazine treatment step was repeated five times to obtain:

(38) Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Aib-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro- Pro-Pro-Ser(t-Bu)-rink amide resin. Subsequently, the resin was thoroughly washed with N, N-dimethylformamide (DMF), dichloromethane (DCM), methanol (Methanol), dichloromethane (DCM), N, N-dimethylformamide (DMF) for three times respectively.

(39) Thereto was added a mixed coupling solution of FmocNH-PEG.sub.2-OH (Quanta BioDesign), 2-(7-azo BTA)-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU), diisopropylethyl amine (DIEPA) of N, N-dimethylformamide (DMF) (5 times excess of each), shaken for 2 hours, and filtrated. Subsequently, the resin was thoroughly washed with N, N-dimethylformamide (DMF), dichloromethane (DCM), methanol, dichloromethane (DCM), N,N-dimethylformamide (DMF) for three times respectively to obtain:

(40) Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(Fmoc-PEG.sub.2)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Aib-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin. Subsequently, the resin was thoroughly washed with N,N-dimethylformamide (DMF), dichloromethane (DCM), methanol, dichloromethane (DCM), N,N-dimethylformamide (DMF) for three times respectively.

(41) 20% Piperidine/N, N-dimethylformamide (DMF) solution was used to remove the Fmoc group (30 minutes, repeated removal for twice). Thereto is added a mixed coupling solution of Fmoc-PEG-OH, 2-(7-azo BTA)-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU), diisopropylethyl amine (DIEPA) in N, N-dimethylformamide (DMF) was added (5 times excess of each). The coupling reaction produced:

(42) Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(Fmoc-PEG.sub.2-PEG.sub.2)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Aib-Arg (Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin. Subsequently, the resin was thoroughly washed with N, N-dimethylformamide (DMF), dichloromethane (DCM), methanol (Methanol), dichloromethane (DCM), N, N-dimethylformamide (DMF) for three times respectively.

(43) 20% Piperidine/N, N-dimethylformamide (DMF) solution was used to remove the Fmoc group (30 minutes, repeated removal for twice). Mixed coupling solution of hexadecanoic acid (palmitic acid), 2-(7-azo BTA)-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU), diisopropylethyl amine (DIEPA) of N,N-dimethylformamide (DMF) was added (5-fold excess of each). The coupling reaction produced:

(44) Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(PEG.sub.2-PEG.sub.2-C16)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Aib-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin. Subsequently, the resin was thoroughly washed with N,N-dimethylformamide (DMF), dichloromethane (DCM), methanol, dichloromethane (DCM) for three times respectively, and dried under vacuum.

(45) 2c) Removal of Polypeptides Full Protection:

(46) Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-A sp(OtBu)-Lys(PEG.sub.2-PEG.sub.2-C16)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Aib-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin was added with a cutting fluid TFA/Phenol/thioanisole/EDT/H2O (82.5:5:5:2.5:5, volume ratio), heated, controlling the temperature of the lysates at 25 C., and reacted for 2.5 hours. After filtration, the filter cake was washed with a small amount of trifluoroacetic acid for three times, and the filtrate was combined. The filtrate was slowly poured into ice diethyl ether with stirring, placed on standing for more than 2 hours to precipitate completely. The precipitate was centrifuged and washed with ice diethyl ether for three times to give crude compound:

(47) His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG.sub.2-PEG.sub.2-CO(CH.sub.2).sub.14CH.sub.3)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH.sub.2

(48) 2d) Purification of Polypeptide Compound:

(49) The crude product obtained in 2c was dissolved in a solution of acetonitrile (ACN): H.sub.2O=1:1 (volume ratio), and purified by preparative HPLC on a 5.0 m reverse-phase C18-packed 46 mm250 mm column. The column was subjected to gradient elution, with 30% acetonitrile (containing 0.05% trifluoroacetic acid)/H.sub.2O (containing 0.05% trifluoroacetic acid) as a starting, adding the proportion of acetonitrile at a rate of 1.33%/min, at a flow rate of 40 mL/min, for 30 min. The reactions containing the peptide were collected, and lyophilized to obtain pure product with HPLC purity greater than 95% (if the purity does not meet the requirements, HPLC purification can be repeated once). The isolated product was analyzed by LC-MS.

(50) Based on the above synthetic steps, the polypeptide compounds synthesized the present invention comprises (Table 1):

(51) TABLE-US-00008 TABLE 1 Structure of polypeptide compounds synthesized in the examples of the present invention Polypeptide Theoretical Observed (SEQ ID NO.) Sequence Mass Mass 1 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- 4114.1 4116.0 Lys-Tyr-Leu-Asp-Ser-Lys(PEG.sub.2-PEG.sub.2-Glu-CO(CH.sub.2).sub.16CO.sub.2H)- Ala-Ala-His-Asp-Phe-Val-Glu-Trp-Leu- Leu-Arg-Ala-NH.sub.2 2 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- 4175.2 4176.9 Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Lys(PEG.sub.2-PEG.sub.2- Glu-CO(CH.sub.2).sub.16CO.sub.2H)-Glu-Phe-Ile-Glu-Trp-Leu- Leu-Arg-Ala-NH.sub.2 3 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- 4892.4 4894.8 Lys-Tyr-Leu-Asp-Ser-Lys(PEG.sub.2-PEG.sub.2-Glu-CO(CH.sub.2).sub.16CO.sub.2H)- Ala-Ala-His-Asp-Phe-Val-Glu-Trp-Leu- Leu-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser-NH.sub.2 4 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- 4953.5 4955.1 Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Lys(PEG.sub.2-PEG.sub.2- Glu-CO(CH.sub.2).sub.16CO.sub.2H)-Glu-Phe-Ile-Glu-Trp-Leu- Leu-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser-NH.sub.2 5 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG.sub.2- 4890.5 4891.5 PEG.sub.2-CO(CH.sub.2).sub.14CH.sub.3)-Ser-Lys-Tyr-Leu-Asp-Aib- Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met- Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro- Ser-NH.sub.2 6 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG.sub.2- 4948.5 4949.6 PEG.sub.2-CO(CH.sub.2).sub.16CO.sub.2H)-Ser-Lys-Tyr-Leu-Asp- Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu- Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser-NH.sub.2 7 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG.sub.2- 5019.5 5020.6 PEG.sub.2-Glu-CO(CH.sub.2).sub.14CH.sub.3)-Ser-Lys-Tyr-Leu-Asp- Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu- Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser-NH.sub.2 8 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG.sub.2- 5077.6 5078.5 PEG.sub.2-Glu-CO(CH.sub.2).sub.16CO.sub.2H)-Ser-Lys-Tyr-Leu- Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp- Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro- Pro-Pro-Ser-NH.sub.2 9 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- 5026.5 5027.6 Lys-Tyr-Leu-Asp-Lys(PEG.sub.2-PEG.sub.2-CO(CH.sub.2).sub.16CO.sub.2H)- Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu- Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser-NH.sub.2 10 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- 5097.6 5098.4 Lys-Tyr-Leu-Asp-Lys(PEG.sub.2-PEG.sub.2-Glu-CO(CH.sub.2).sub.14CH.sub.3)- Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu- Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro- Pro-Pro-Ser-NH.sub.2 11 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- 5155.6 5156.8 Lys-Tyr-Leu-Asp-Lys(PEG.sub.2-PEG.sub.2-Glu-CO(CH.sub.2).sub.16CO.sub.2H)- Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp- Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro- Pro-Pro-Ser-NH.sub.2 12 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- 4955.5 4956.6 Lys-Tyr-Leu-Asp-Aib-Lys(PEG.sub.2-PEG.sub.2-CO(CH.sub.2).sub.16CO.sub.2H)- Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu- Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser-NH.sub.2 13 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- 5026.5 5027.6 Lys-Tyr-Leu-Asp-Aib-Lys(PEG.sub.2-PEG.sub.2-Glu-CO(CH.sub.2).sub.14CH.sub.3)- Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu- Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro- Pro-Pro-Ser-NH.sub.2 14 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- 5084.5 5085.6 Lys-Tyr-Leu-Asp-Aib-Lys(PEG.sub.2-PEG.sub.2-Glu-CO(CH.sub.2).sub.16CO.sub.2H)- Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp- Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro- Pro-Pro-Ser-NH.sub.2 15 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- 4890.5 4891.6 Lys(PEG.sub.2-PEG.sub.2-CO(CH.sub.2).sub.14CH.sub.3)-Lys-Leu-Asp-Aib- Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met- Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser-NH.sub.2 16 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- 4948.5 4949.6 Lys(PEG.sub.2-PEG.sub.2-CO(CH.sub.2).sub.16CO.sub.2H)-Lys-Leu-Asp- Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu- Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser-NH.sub.2 17 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- 5019.5 5020.6 Lys(PEG.sub.2-PEG.sub.2-Glu-CO(CH.sub.2).sub.14CH.sub.3)-Lys-Leu-Asp- Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu- Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro- Pro-Pro-Ser-NH.sub.2 18 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- 5077.6 5078.3 Lys(PEG.sub.2-PEG.sub.2-Glu-CO(CH.sub.2).sub.16CO.sub.2H)-Lys-Leu- Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp- Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro- Pro-Pro-Ser-NH.sub.2 19 His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG.sub.2- 4945.5 4946.9 PEG.sub.2-Glu-CO(CH.sub.2).sub.14CH.sub.3)-Ser-Lys-Tyr-Leu-Asp- Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu- Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro- Ser-NH.sub.2 20 His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG.sub.2- 5002.6 5003.8 PEG.sub.2-Glu-CO(CH.sub.2).sub.16CO.sub.2H)-Ser-Lys-Tyr-Leu-Asp- Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu- Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro- Ser-NH.sub.2 21 His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG.sub.2- 4901.8 4903.3 PEG.sub.2-Glu-CO(CH.sub.2).sub.14CH.sub.3)-Ser-Lys-Tyr-Leu-Asp- Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu- Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro- Ser-NH.sub.2 22 His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG.sub.2- 4958.9 4960.3 PEG.sub.2-Glu-CO(CH.sub.2).sub.16CO.sub.2H)-Ser-Lys-Tyr-Leu-Asp- Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu- Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro- Ser-NH.sub.2 23 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- 4769.2 4770.3 Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Lys(PEG.sub.2-PEG.sub.2- CO(CH.sub.2).sub.14CH.sub.3)-Glu-Phe-Ile-Glu-Trp-Leu-Leu- Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser- NH.sub.2 24 His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- 4897.5 4898.6 Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Lys(PEG.sub.2-PEG.sub.2- Glu-CO(CH.sub.2).sub.14CH.sub.3)-Glu-Phe-Ile-Glu-Trp-Leu- Leu-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro- Ser-NH.sub.2

Example 3. In Vitro Activity Determination of GCGR and GLP-IR

(52) Stimulation effect on glucagon-like peptide 1 receptor (GLP-1 receptor) and glucagon receptor was determined using cAMP assay kit manufactured by the Cisbo company, meanwhile the dose-effect curves of positive control compound glucagon like peptide 1 (GLP-1), Exenatide and Glucagon were detected.

(53) 5000 of HEK-293 cells, which are used in CRE-luciferase system and express human GCGR or GLP-1R stably, were inoculated into 384 well plates according to 98 L DMEM/10% FBS media/well. On the second day of inoculation, 2 L of sample to be tested was gradient transferred, and the cells were mixed and incubated for 12 hours. Before the cells are added to obtain dose-response curve (EC.sub.50 values were determined from the curve), usually the compound to be tested was prepared into 10 diluted solutions containing from 0.005 nm to 100.0 nM, and Exenatide standard (purchased from Hangzhou Paitai Biochemical Technology Co., Ltd., purity >98%, Exenatide aceteate, CAS No.: 141732-76-5) was prepared into 10 standard solutions of 0.005 nm to 100.0 nm, and Glucagon standard (purchased from Sigma-Aldrich company, product No. and specifications: 1294036-2X2.94MG, CAS No.: 16941-32-5) was prepared into 10 standard solutions of 0.01 nm to 100 nm. After incubation, 10.0 L of luciferase reagent was added directly to each plate and mixed gently for 2 minutes. The plates were placed into an Elmer Perkin plate reader for reading. Concentration curve of the compound was made by drawing software Prism 5, and the EC.sub.50 value was calculated.

(54) Compared with oxyntomodulin purchased from Shanghai Yinggong Industrial Co., Ltd., product No.: Es-1240), the compounds of the present invention have higher relative GLP-1R selectivity and have higher potency on glucagon receptor and GLP-1 receptor.

(55) TABLE-US-00009 TABLE 2 Average EC.sub.50 values of the representative polypeptide compounds of the present invention GLP-1R GCGR Compound (EC.sub.50, nM) (EC.sub.50, nM) GLP-1R/GCGR Exenatide 0.02 >100 <0.0004 Glucagon 3.1 0.032 1000 Oxyntomodulin 2.5 6.2 0.4 1 0.23 0.54 0.4 2 0.39 2.8 0.1 3 0.59 1.02 0.6 4 0.13 0.39 0.3 5 0.06 0.43 0.1 6 0.10 0.33 0.3 7 0.14 0.34 0.4 8 0.20 0.18 1.1 9 0.52 0.18 2.9 10 0.35 0.11 3.3 11 0.15 0.18 0.8 12 0.32 0.15 2.1 13 0.44 0.16 2.8 14 0.40 0.19 2.1 15 1.5 0.6 2.5 16 0.60 0.2 3 17 0.68 0.58 1.2 18 0.76 0.18 4.2 19 0.14 0.56 0.25 20 0.09 0.48 0.18 21 0.15 0.66 0.23 22 0.06 0.32 0.19 23 0.13 0.43 0.30 24 0.16 0.58 0.28

(56) FIG. 1: stimulation curve of Exenatide and Compounds 4, 5, 6, 7 on GLP-IR;

(57) FIG. 2: stimulation curve of Glucagon and Compounds 4, 5, 6, 7 on GCGR;

Example 4 Pharmacokinetic Analysis of Compounds 4, 5, 6, 7, 19, 20, 21, 22, 23, 24 and Ex-4

(58) Male CD1 mice (Nanjing University model animal research center) were respectively administrated by intravenous (IV) or subcutaneous injection (SC) with Compound 4, 5, 6, 7, 19, 20, 21, 22, 23 and 24 prepared according to the above examples and commercially available Exenatide pure product (Ex-4). Within 0-24 hours after administration the animals were bled at different times, and the plasma of each sample was collected and analyzed by LC-MS/MS determination method. Pharmacokinetic parameters of the drugs were calculated by methods of model dependence (for data obtained from IV) and model independence (data obtained from SC). By IV administration route, the elimination half-life of the compounds 4, 5, 6, 7, 19, 20, 21, 22, 23, and 24 was approximately 4-6 hours, and peak time (T.sub.max) was about 12 hours, and the elimination half-life of Ex-4 was about 0.5 hours. By SC administration route, the peak time of the compounds 4, 5, 6, 7, 19, 20, 21, 22, 23 and 24 was about more than 12 hours, and the elimination half-life of Ex-4 was about 2 hours. The administration of compounds 4, 5, 6, 7, 19, 20, 21, 22, 23 and 24 by IV or SC route showed no clinical adverse effect.

Example 5 Effect of Compounds 4, 5, 6, 7, 19, 20, 21, 22, 23, 24 and Ex-4 on Oral Glucose Tolerance

(59) 12-16 week-old male C57B1/6J mice (Nanjing University model animal research center) were randomly divided into groups 8 in each group, according to similar blood glucose (assessed by the blood samples obtained from the tail tip). After fasting (for 6 hours), the animals were administrated, and about 4 hours later, initial blood samples (fasting blood glucose level) were taken. Subsequently, the animals were administrated with an oral dose of glucose, and then put back into their cages (t=0). Blood glucose was measured at t=15 minutes, t=30 minutes, t=60 minutes, t=90 minutes and t=120 minutes. And then the animals were administrated with an oral dose of glucose again, and blood glucose was tracked until 8 hours. Polypeptides 4, 5, 6, 7, 19, 20, 21, 22, 23 and 24 (the dose of each compound was 250 g/kg) significantly reduced the oral glucose tolerance. The software GraphPadPrism was used to process data to make the blood glucose change line chart and calculate the area under the curve to get the AUC diagram (FIGS. 3 and 4).

(60) Compared with the carrier (PBS), the AUC of Exenatide (250 g/kg) in the first OGTT curve period (1-120 min) was significantly reduced (P<0.05), but in the next three OGTT curve periods (120-480 min), the AUC was close to that of the control group (P>0.05). Compared with the carrier (PBS), the AUC of polypeptide 4, polypeptide 5, polypeptide 6, polypeptide 7, polypeptide 19, polypeptide 20, polypeptide 21, polypeptide 22, polypeptide 23 and polypeptide 24 was significantly decreased in all four OGTT curve periods (0-480 min) (P<0.05), the experimental results suggest that polypeptide 4, polypeptide 5, polypeptide 6, polypeptide 7, polypeptide 19, polypeptide 20, polypeptide 21, polypeptide 22, polypeptide 23 and polypeptide 24 have long-lasting hypoglycemic effect.

Example 6 Effect of Compounds 5, 6, 7 on Body Weight in Obese Mice, Body Weight Reduction Research

(61) Forty 25-week-old DIO mice (Nanjing University model animal research center) were randomly divided into five groups (compound 4, 5, 6, 7, and normal saline group), 8 in each group, having no difference in the basis weight of each group. The mice group was respectively injected subcutaneously daily with compound 4 (250 g/kg), compound 5 (250 g/kg), compound 6 (250 g/kg), compound 7 (250 g/kg) and normal saline and then weighed. Compared with the normal saline group, during day 11 to day 30, the body weight in the compound 4, 5, 6, 7 groups were significantly lower than that of the normal saline group (P<0.05), having weight loss of about 20% (FIG. 5).

INDUSTRIAL APPLICATION

(62) The OXM analogues of the present invention show GCGR and GLP-1R dual agonist activity, long half-life, and have high enzymolysis stability, high biological activity, and no adverse reactions. The compounds of the present invention can be synthesized with high yield, have good stability, are can be easily produced on a large scale with low cost, and can be used to prepare medication for treating hyperphagia, obesity and overweight, high cholesterol and diabetes.

(63) Above detailed description of the invention does not limit the present invention, various changes and modifications may be made by the person skilled in this art in accordance with the present invention, without departing from the spirit and scope of the present invention, and are within the scope of the appended claims of the present invention.