POLYPEPTIDE COMPOUND AND APPLICATION THEREOF

Abstract

A polypeptide compound having a structure shown in formula I, or a stereoisomer, mixture or pharmaceutically acceptable salt thereof Experimental results show that the polypeptide compound can effectively exhibit high agonistic activity to GHSR-1a.

##STR00001##

Claims

1. A polypeptide compound having a structure represented by formula I, or a stereoisomer, mixture or pharmaceutically acceptable salt thereof: ##STR00117## wherein R.sub.1 is selected from NR.sub.2R.sub.3, OR.sub.2 and SR.sub.2; and, R.sub.1 is not a D- or L-amino acid; R.sub.2 and R.sub.3 are independently selected from hydrogen, deuterium, a polymer derived from polyethylene glycol, an acyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyclic group, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted aralkyl; W is selected from a single bond, a D-amino acid and an L-amino acid; U.sub.1 is selected from any one of the following structures: ##STR00118## wherein X and Z are independently selected from CHR.sub.4, NR.sub.4, O, S, Se, S?O and O?S?O; R.sub.4, R.sub.7 and R.sub.8 are independently selected from hydrogen, deuterium, amino, a protective group, a polymer derived from polyethylene glycol, an acyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyclic group, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, and R.sub.9CO; Y is selected from halogen, amino, nitro, hydroxyl and cyano; R.sub.5 is selected from NR.sub.2R.sub.3, OR.sub.2 and SR.sub.2; R.sub.6 is selected from hydrogen, deuterium, an acyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyclic group, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted aralkyl; m.sub.1 and m.sub.2 are independently selected from 0, 1, 2 and 3; particularly, when X is N, m.sub.1 is 2, and m.sub.2 is then independently selected from 0, 1 and 3; m.sub.2 is 2, and m.sub.1 is then independently selected from 0, 1 and 3; m.sub.3 and m.sub.4 are independently selected from 0, 1, 2 and 3; n.sub.1, n.sub.2, n.sub.3 and n4 are independently selected from 0, 1, 2 and 3; p is 0, 1, 2, 3, 4 or 5; U.sub.2 is a single bond, or is selected from any one of the following structures, and the carbonyl end of U.sub.2 is linked to W: ##STR00119## R.sub.9 is selected from hydrogen, an acyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyclic group, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted aralkyl.

2. The polypeptide compound according to claim 1, wherein the R.sub.7 and R.sub.8 are selected from hydrogen, C.sub.1-6 alkyl, C.sub.6-14 aryl, C.sub.3-8 cycloalkyl and C.sub.2-10 acyl; the R.sub.1 is selected from NR.sub.2R.sub.3 and OR.sub.2, wherein R.sub.2 and R.sub.3 are independently selected from hydrogen, methyl, ethyl, hexyl, dodecyl and hexadecyl.

3. The polypeptide compound according to claim 1, wherein the W is selected from one or more of a single bond and alanine, arginine, asparagine, cysteine, glutamine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine residues.

4. The polypeptide compound according to claim 1, wherein the U.sub.1 is selected from the following structures: ##STR00120## wherein R.sub.6 is selected from hydrogen, substituted or unsubstituted C.sub.1-6 alkyl, substituted or unsubstituted C.sub.6-14 aryl, and substituted or unsubstituted C.sub.3-8 cycloalkyl; the substituted group is selected from halogen, amino, nitro, hydroxyl, acyl-substituted amino, ureido and guanidino; R.sub.7 and R.sub.8 are independently selected from hydrogen, C.sub.1-6 alkyl, C.sub.6-14 aryl, C.sub.3-8 cycloalkyl and C.sub.2-10 acyl; p is 0, 1, 2, 3, 4 or 5.

5. The polypeptide compound according to claim 4, wherein the R.sub.6 is selected from hydrogen and substituted or unsubstituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl; the substituted group is selected from halogen, amino, nitro, hydroxyl, formamido, acetamido, propionamido, butyramido, ureido and guanidino; R.sub.7 and R.sub.8 are independently selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, formyl, acetyl, propionyl and butyryl; p is 0, 1, 2, 3, 4 or 5.

6. The polypeptide compound according to claim 1, wherein the U.sub.1 is selected from any one of the following structures: ##STR00121## wherein R.sub.10 and R.sub.11 are independently selected from hydrogen, amino, nitro, hydroxyl, halogen, cyano, aminomethyl, aminoethyl, aminopropyl and aminobutyl.

7. The polypeptide compound according to claim 1, having any one of the following structures, or a stereoisomer, mixture or pharmaceutically acceptable salt thereof: ##STR00122## ##STR00123## ##STR00124## ##STR00125## ##STR00126## ##STR00127## ##STR00128## ##STR00129## ##STR00130## ##STR00131## ##STR00132## ##STR00133## ##STR00134## ##STR00135## ##STR00136## ##STR00137## ##STR00138## ##STR00139## ##STR00140## ##STR00141## ##STR00142## ##STR00143## ##STR00144## ##STR00145##

8. A composition comprising the polypeptide compound according to claim 1, and an acceptable auxiliary agent.

9. A method for treating, preventing, alleviating and/or diagnosing a related disease caused by a disorder mediated by growth hormone secretagogue receptor, or promoting growth and development, comprising administering polypeptide compound according to claim 1 as an agonist for growth hormone secretagogue receptor, or as a health product.

10. The method according to claim 9, wherein the related disease is growth hormone deficiency.

Description

DETAILED DESCRIPTION

[0144] To further illustrate the present invention, the polypeptide compound provided by the present invention and use thereof are described in detail below using examples.

[0145] Those skilled in the art will appreciate that the following examples are only for illustrating the present invention, and should not be construed as limitations to the scope of the present invention. Experimental procedures without specified conditions in the examples are conducted according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used herein without specified manufacturers are conventional products that are commercially available.

[0146] The polypeptide was synthesized using a standard Fmoc solid phase method. Rink Amide resin was selected. The peptide chain extends from the C-terminus to the N-terminus. Protected amino acids include: Fmoc-Apc(Boc)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Orn(Boc)-OH, Fmoc-Phe-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-D-Bal-OH, Fmoc-D-Cit-OH, Fmoc-D-Arg(Pbf)-OH, Boc-D-Aba-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Gly-OH, Fmoc-D-Ala-OH, Fmoc-D-Val-OH, Fmoc-D-Leu-OH, Fmoc-Pro-OH, Fmoc-?-Ala-OH, Fmoc-D-Tle-OH, Fmoc-Tle-OH, Fmoc-D-Nle-OH, Fmoc-Nle-OH, cis-2-(tert-butoxycarbonylamide)-1-cyclopentanecarboxylic acid, Boc-methyl-1-(aminomethyl)cyclobutanecarboxylic acid, 1-N-Boc-3-azetidinecarboxylic acid, Boc-3-aminooxetane-3-carboxylic acid, 1-Boc-D-acridine-2-carboxylic acid, (S)-1-Boc-pyrrolidine-3-carboxylic acid, Boc-2-morpholinecarboxylic acid, (Boc-3-amino-1-adamantane)acetic acid, (1R,3S,4S)-N-Boc-2-azabicyclo[2.2.1]heptane-3-carboxylic acid, and 3-Boc-3-azabicyclo[3.1.0]hexane-1-carboxylic acid. The condensing agent was HBTU/HOBt/DIEA. The deprotecting reagent was piperidine/DMF solution. The crude peptide was dissolved in water and then lyophilized and stored. Separation and purification were carried out by medium-pressure liquid chromatography or high performance liquid chromatography (HPLC). The pure peptide content was greater than 90%. The molecular weight of the peptide sequence was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

Synthesis of the Peptide Sequence

[0147] The synthesis conditions were as follows: [0148] protected amino acids (natural or non-natural): 0.2 M solutions in DMF; [0149] condensing agent: a 0.45 M solution of HBTU/HOBt in DMF; [0150] activating base: a 2 M solution of DIEA in DMF; [0151] deprotecting reagent: a 20% v/v solution of piperidine in DMF.

Example 1

Preparation of Compounds 1-5, 9, 11-35, 39 and 41-80

[0152] 1. Deprotection: 0.23 g (0.1 mmol) of Rink Amide resin was weighed out and placed into a polypeptide synthesis reactor. Then the deprotecting reagent was prepared according to the concentration described above and added to the resin. The mixture was allowed to react at room temperature. The resin was dried under vacuum, and piperidine/DMF was added again. The mixture was allowed to react at room temperature, and then the resin was dried under vacuum and washed with DMF until tests showed it was acceptable. [0153] 2. Condensation reaction: Amino acids and the condensing agent were added to DMF for activation in an ice bath, and the activating base was added for reaction to obtain an activating solution. The activating solution was finally added to the resin. After reaction at room temperature, the resin was colored with 5% ninhydrin color-developing reagent; the color of the resin changed. The solvent was removed under vacuum and the resin was washed with DMF. After tests showed the resin was acceptable, the solvent was removed under vacuum, and the condensation reaction was complete by now. [0154] 3. The deprotection and condensation reaction described above was repeated until peptide chain synthesis was complete; a peptide resin containing a complete polypeptide sequence structure was obtained. [0155] 4. Cleavage of the peptide resin: 1.25 g of the synthesized peptide resin was weighed out, placed into a 250 mL eggplant-shaped flask, cooled in an ice bath and electromagnetically stirred. A cleavage solution was prepared in such an amount that the solution would be added at 10 mL per gram of the peptide resin (cleavage solution (volume percentage):trifluoroacetic acid:thioanisole:water=90:5:5). TFA should be pre-cooled in an ice bath for 30 min or stored beforehand in a refrigerator before use. The prepared cleavage solution was added to the peptide resin in an ice bath. The mixture was electromagnetically stirred, and the resin turned black. The reaction was performed in the ice bath for 30 min, and then the ice bath was removed. The reaction was stirred at room temperature for another 180 min, and the reaction was complete. 200 mL of cold diethyl ether was added with vigorous stirring, and a white crystal precipitated. The stirring was continued for 30 min. The precipitate was collected by filtration using a G4 sand core funnel, washed 3 times with cold diethyl ether, and allowed to dry. 50 mL of double distilled water and 5 mL of acetonitrile were added to fully dissolve the solid. The solution was filtered under vacuum, and the filtrate was lyophilized to give a crude peptide (1.04 g). [0156] 5. Purification of the crude peptide: The crude peptide was purified by medium-pressure or high performance liquid chromatography. The chromatography column was a C18 column. The eluent was acetonitrile, water and a small amount of acetic acid. Specifically, 1.00 g of the crude peptide was weighed out, and 20 mL of water and 5 mL of acetonitrile were added to dissolve the solid; the solution was centrifuged at 5000 rpm for 10 min, and the supernatant was collected for sample injection. The chromatography column was equilibrated beforehand with 200 mL of a solution of 15% acetonitrile/water/0.1% glacial acetic acid. After sample injection, a wash was performed using another 200 mL of a solution of 15% acetonitrile/water/0.1% glacial acetic acid, and the composition of the eluate was determined by high performance liquid chromatography. According to the liquid chromatography results, the acetonitrile content was gradually increased until the main peak of the purified polypeptide was obtained by elution. The eluates were combined and concentrated by rotary evaporation to remove most of the solvent. The purified polypeptide was lyophilized, with a content of greater than.sub.90% as determined by HPLC. The molecular weight was confirmed by MALDI-TOF-MS.

Example 2

Preparation of Compounds 10 and 40

[0157] This example is based on Example 1, and is different from Example 1 in that the last protected amino acid at the N-terminus of the peptide chain is Fmoc-Lys(Alloc)-OH. The method of removing the protective group for its side chain is as follows: triphenylphosphine palladium and phenylsilane (1:10, v/v) were added to the resin that had been dried under vacuum, the mixture was allowed to react under N.sub.2 in a dark place for 3 h, detection showed the color of the resin had changed, and deprotection was complete; after the resin was washed and dried under vacuum, an acetylation reaction was performed: 2 mL of acetic acid and 2 mL of DIEA were added, the mixture was allowed to react for 30 min, and the resin was washed and dried under vacuum. Finally, the Fmoc protective group was removed in the 20% v/v solution of piperidine in DMF, and the product was obtained after cleavage and purification.

Example 3

Preparation of Compounds 6-8 and 36-38

[0158] This example is based on Example 1, and is different from Example 1 in that after the deprotection of the last amino acid at the N-terminus of the peptide chain was complete, 2 mL of acetic acid and 2 mL of DIEA were added, the mixture was allowed to react for 30 min, acetylation capping was performed, and finally the product was obtained after cleavage and purification. The polypeptide compounds prepared using the synthesis methods of the embodiments disclosed herein are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Synthesized polypeptide compounds SEQ Calculated Observed ID M.W. M.W. Purity NO. Structure (g/mol) (g/mol) (%) 1 [00033] 837.01 838.0 90.0 2 [00034] 764.95 765.5 92.7 3 [00035] 764.95 765.6 91.7 4 [00036] 808.01 808.5 93.7 5 [00037] 808.01 808.4 91.3 6 [00038] 879.05 879.5 98.2 7 [00039] 850.05 850.2 98.5 8 [00040] 850.05 850.4 96.4 9 [00041] 836.03 837.0 93.7 10 [00042] 850.05 850.6 95.3 11 [00043] 736.89 737.5 97.3 12 [00044] 750.92 751.7 93.6 13 [00045] 778.97 779.4 95.3 14 [00046] 793 793.7 99.8 15 [00047] 776.96 777.6 94.9 16 [00048] 750.92 751.0 97.1 17 [00049] 793 793.5 92.1 18 [00050] 793 793.3 93.6 19 [00051] 793 793.7 95.2 20 [00052] 793 793.9 94.9 21 [00053] 790.98 791.7 97.4 22 [00054] 790.98 791.9 97.1 23 [00055] 762.93 763.6 94.8 24 [00056] 778.93 779.5 90.8 25 [00057] 762.93 763.6 93.7 26 [00058] 776.96 777.8 93.6 27 [00059] 792.96 793.4 95.4 28 [00060] 871.11 872.0 96.6 29 [00061] 802.99 803.4 92.2 30 [00062] 788.97 789.5 93.7 31 [00063] 839.03 839.7 94.0 32 [00064] 766.96 767.4 95.5 33 [00065] 766.96 767.8 91.2 34 [00066] 810.03 810.6 92.0 35 [00067] 810.03 810.4 93.2 36 [00068] 881.07 882.0 92.8 37 [00069] 852.07 852.9 97.4 38 [00070] 852.07 852.7 93.1 39 [00071] 838.04 838.6 93.8 40 [00072] 852.07 852.8 92.2 41 [00073] 738.91 739.8 92.7 42 [00074] 752.93 753.9 93.5 43 [00075] 780.99 781.4 94.2 44 [00076] 795.02 796.0 96.1 45 [00077] 778.97 779.8 95.0 46 [00078] 752.93 753.4 92.6 47 [00079] 795.02 795.7 93.0 48 [00080] 795.02 795.3 94.3 49 [00081] 795.02 795.9 92.5 50 [00082] 795.02 796.0 90.9 51 [00083] 793 793.7 91.4 52 [00084] 793 793.6 93.8 53 [00085] 764.95 765.7 92.3 54 [00086] 780.94 781.3 92.2 55 [00087] 764.95 765.8 92.7 56 [00088] 778.97 779.4 93.2 57 [00089] 794.97 795.6 92.6 58 [00090] 873.13 874.0 96.4 59 [00091] 805.01 805.7 93.4 60 [00092] 790.98 791.6 92.2 61 [00093] 821.01 821.7 93.5 62 [00094] 821.01 821.2 92.5 63 [00095] 792.96 793.8 92.6 64 [00096] 808.96 809.3 95.0 65 [00097] 792.96 793.6 94.8 66 [00098] 806.99 807.5 93.7 67 [00099] 822.99 823.3 95.3 68 [00100] 901.14 901.8 94.7 69 [00101] 833.02 833.2 93.0 70 [00102] 819 819.3 97.4 71 [00103] 664.82 665.2 93.0 72 [00104] 664.82 665.7 95.8 73 [00105] 636.77 637.6 94.3 74 [00106] 652.77 653.3 94.8 75 [00107] 636.77 637.5 95.7 76 [00108] 650.8 651.1 93.6 77 [00109] 666.8 667.5 95.0 78 [00110] 744.95 745.8 94.7 79 [00111] 676.84 677.7 96.4 80 [00112] 662.81 663.4 93.6

Example 4

Assessment of Agonistic Activity of Polypeptide Compounds for GHSR-1a (IC.SUB.50.)

[0159] Screening for GHSR active compounds was accomplished by recombinant expression of the receptor. The use of recombinant expression of GHSR provides several advantages; for example, the receptor can be expressed in a determined cell system, so that it is easier to distinguish between the reactions of the compounds with GHSR and the reactions with other receptors. For example, cell lines such as HEK293, COS7 and CHO that normally express GHSR without using expression vectors can be used to express GHSR, and the same cell lines without expression vectors are used as controls.

[0160] The activity of GHSR-1a can be measured using different techniques, for example, by detecting the change in the intracellular conformation of GHSR, the change in G-protein coupling activity, and/or the change in intracellular messengers. Techniques such as measuring intracellular Ca.sup.2+ are preferably used to measure the activity of GHSR-1a. Examples of techniques known in the art that can be used to measure Ca.sup.2+ include the use of FLIPR? calcium ion assay kits, among others. The FLIPR? calcium ion assay kits use a calcium ion sensitive indicator and a masking dye to ensure that a researcher carries out high-sensitivity fluorescent screening for G protein-coupled receptors, ion channels and other calcium ion sensitive targets. This experiment used FLIPR calcium 6 assay kits and FLIPR calcium 6-QF assay kits.

1. Process

1.1. Cell Culture and Reagent Preparation

[0161] a) Cell line: Flp In-CHO-GHSR Stable Pool; [0162] b) Complete medium: F12K+10% fetal bovine serum+1?penicillin-streptomycin (PS)+600 ?g/mL hygromycin B; [0163] c) Cell seeding medium: F12K+10% fetal bovine serum. [0164] d) Assay buffer: 1? HBSS+20 mM HEPES. [0165] e) 10? component A: Assay buffer and component A were left at room temperature (RT), 10 mL of buffer was added to component A, and the mixture was vortexed for 1-2 min and stored at ?20? C.

1.2. Compound Management

[0166] a) Compound stock solutions: the powders from in-house synthesis were made into 10 mM stock solutions in DMSO according to the standard protocol. [0167] b) Compound storage: all compounds in DMSO were stored in room temperature desiccators for short-term storage (at most 4 months). The remaining compounds were left at ?20 ? C. for long-term storage.

1.3. Agonist Activity Assay

[0168] a) Flp In-CHO-GHSR Stable Pool cells were cultured in complete medium. [0169] b) The cells were placed in 25 lbs/inch cell seeding medium in a 384-well cell culture plate (Corning, 3764) at 7k cells/well and cultured overnight at 37? C. with 5% CO.sub.2. [0170] c) 20? component A was thawed at room temperature, diluted with assay buffer to 2?, and left at RT. [0171] d) The Petri dish was taken out of the incubator and equilibrated at room temperature for 10 min. The medium was changed to apricot buffer. After the final wash, 20 ?L of buffer was kept in each well, 20 ?L of 2? component A was then added to each well, and the plate was incubated at 37? C. for 3-5 s. [0172] e) 10 ?L of 5? compound was added to the 384-well cell culture plate, and data collection was immediately performed using FLIPR Tetra.

2. Data Analysis

[0173] 1) Z factor=1?3?(SD.sub.Max+SD.sub.Min)/(Mean.sub.Max?Mean.sub.Min); [0174] 2) CV.sub.Max=(SD.sub.Max/Mean.sub.Max)?100%; [0175] 3) CV.sub.Min=(SD.sub.Min/Mean.sub.Min)?100%; [0176] 4) S/B=Singal/Background; [0177] 5) Calculation formula for IC.sub.50: [0178] Y=Bottom+(Top?Bottom)/(1+10{circumflex over ()}((LogIC.sub.50?X)?HillSlope)) [0179] X: log value of compound concentration; Y: Activation % or Inhibition %

[0180] According to the method described above, the activity results are shown in Table 2.

TABLE-US-00002 TABLE 2 The activity (IC.sub.50) of polypeptide compounds for GHSR-1a SEQ ID NO. IC.sub.50 (nM) 1 149.7 2 3.5 3 17.2 4 1119.0 5 >1000 6 >1000 7 935.2 8 >1000 9 101.3 10 223.2 11 20.3 12 53.7 13 43.5 14 33.2 15 2.3 16 0.7 17 31.1 18 23.5 19 42.6 20 57.4 21 23.0 22 2.3 23 3.3 24 5.4 25 18.1 26 19.5 27 32.8 28 54.1 29 13.3 30 13.4 31 20.8 32 2.4 33 3.1 34 78.4 35 102.3 36 122.1 37 291.3 38 400.5 39 272.2 40 510.3 41 31.4 42 40.1 43 22.3 44 13.5 45 5.1 46 2.8 47 10.7 48 12.1 49 59.4 50 67.2 51 3.2 52 3.8 53 11.2 54 13.9 55 20.0 56 5.5 57 13.4 58 103.9 59 16.8 60 22.4 61 20.5 62 30.3 63 27.0 64 38.1 65 34.3 66 12.0 67 23.3 68 330.2 69 40.2 70 39.7 71 3.3 72 4.0 73 15.9 74 17.3 75 12.4 76 3.9 77 13.6 78 78.4 79 33.1 80 35.1 Ghrelin (control) 43.1

[0181] As can be seen from the results in Table 2, the polypeptide compounds provided by the present invention showed agonistic activity for GHSR-1a.

Example 5

Inhibition of Cytochrome P450 Oxidase by Polypeptide Compounds

[0182] Human liver microsomes containing cytochrome P450 (0.253 mg/mL protein) were incubated with test compounds (0.05-50 ?M), CYPs substrates (10 ?M paracetamol, 5 ?M diclofenac, 30 ?M mephenytoin, 5 ?M dextromethorphan hydrobromide and 2 ?M midazolam) and 1.0 mM NADP at 37? C. for 10 min. Naphthoflavone, sulfaphenazole, N-3-benzylnirvanol, quinidine and ketoconazole were used as reference inhibitors. The results are shown in Table 3.

TABLE-US-00003 TABLE 3 The inhibitory activity (IC.sub.50) of compounds against cytochrome P450 CYP isoenzyme Com- CYPs pound 1A2 (?M) 2D6 (?M) 3A4 (?M) 2C9 (?M) 2C19 (?M) 2 >50 >50 20.0 >50 >50 3 >50 >50 >50 >50 >50 11 >50 >50 33.4 >50 >50 12 >50 >50 >50 >50 >50 13 >50 >50 >50 >50 >50 14 >50 >50 >50 >50 >50 15 >50 31.1 8.82 >50 >50 16 >50 35.4 19.8 >50 >50 21 >50 >50 >50 >50 >50 22 >50 >50 >50 >50 >50 23 >50 >50 >50 >50 >50 24 >50 >50 >50 >50 >50 25 >50 >50 >50 >50 >50 26 >50 >50 >50 >50 >50 30 >50 >50 >50 >50 >50 32 >50 24.8 2.55 >50 >50 33 >50 >50 >50 >50 >50 45 >50 >50 >50 >50 >50 46 >50 >50 >50 >50 >50 47 >50 >50 >50 >50 >50 48 >50 >50 >50 >50 >50 51 >50 >50 >50 >50 >50 52 >50 >50 >50 >50 >50 53 >50 >50 >50 >50 >50 54 >50 >50 >50 >50 >50 56 >50 >50 >50 >50 >50 71 >50 >50 >50 >50 >50 72 >50 >50 >50 >50 >50 76 >50 >50 >50 >50 >50 77 >50 >50 >50 >50 >50 80 >50 >50 >50 >50 >50

[0183] As can be seen from the results in Table 3, the inhibitory IC.sub.50 values of the polypeptide compounds provided by the present invention against cytochrome P450 oxidase are all greater than .sub.50 ?M.

Example 6

Preparation and Activity Assays of Polypeptide Compounds 81-84

[0184] Compounds 81-84 shown in Table 4 below were prepared using the same method as 1-80 in the examples described above, and the activity (IC.sub.50) of these compounds for GHSR was measured using the method described in Example 4. The results are shown in Table 4.

TABLE-US-00004 TABLE 4 The activity (IC.sub.50) of polypeptide compounds for GHSR SEQ ID IC.sub.50 NO. Structure (nM) 81 [00113] 7.2 82 [00114] 9.0 83 [00115] 12.2 84 [00116] 6.7 Ghrelin 43.1 (control)

[0185] As can be seen from the results in Table 4, the addition of additional amino acids to the C-termini of the pentapeptide compounds of compounds 1-80 did not significantly affect their agonistic activity for GHSR.

[0186] The above description of the examples is only intended to facilitate the understanding of the method of the present invention and its core concepts. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.