Staple-containing polypeptides and their applications

Abstract

A series of staple-containing polypeptides and their applications are disclosed, specifically, polypeptides having the sequences shown in formulas (I-1) to (I-5).

Description

[Technical Field]

[0001] This application claims priority from the following application: CN2021114005918 filed November 19, 2021; CN2022106482638 filed on June 8, 2022

[0002] The present disclosure relates to a series of staple-containing polypeptides and uses thereof. Specifically, the present disclosure discloses polypeptides having the sequences shown in formulas (I-1) to (I-5). [Background technology]

[0003] Overweight and obesity are serious health problems facing all humanity. They are often accompanied by other diseases, such as coronary artery disease, hypertension, type 2 diabetes, nonalcoholic fatty liver disease, kidney disease, and certain cancers. The World Health Organization (WHO) defines obesity as one of the top 10 chronic diseases. Obesity, along with hypertension, hyperlipidemia, and hyperglycemia, are known as the "deadly quartet" and are likely to become the leading cause of death in the 21st century. WHO data indicates that the prevalence of obesity in China was approximately 6.2% in 2016. Furthermore, the Lancet published a 2016 survey report on global adult weight status. The survey found that the number of obese adults worldwide has exceeded the number of healthy adults, making China the country with the largest obese population in the world, surpassing the United States. The number of people suffering from diabetes, hypertension, cardiovascular disease, and other diseases caused by overweight and obesity is increasing year by year, and the age groups affected are becoming younger.

[0004] Glucagon (GCG) is a hormone secreted by the pancreas and binds to the glucagon receptor (GCGR) to exert physiological functions. Glucagon promotes blood glucose levels by increasing gluconeogenesis and glycogenolysis. Furthermore, GCG can also reduce fatty acid synthesis and lipolysis in hepatic adipose tissue. Glucagon-like peptide 1 (GLP-1) is a hormone secreted by intestinal L-cells. It can reduce body weight by suppressing appetite and reducing food intake, as well as by increasing energy expenditure and promoting thermogenesis in brown adipose tissue. Induction of GCG activity while maintaining the efficacy of GLP-1 agonists can further enhance insulin secretion from pancreatic β cells, promote brown adipose tissue metabolism, enhance fatty acid β-oxidation in the liver and reduce lipid and cholesterol production, improve cardiomyocyte survival, and accelerate lipid metabolism and reduce fat content in white adipose tissue. The synergistic effect of dual GLP-1 / GCG targets is likely to improve blood glucose levels and reduce weight more effectively than single-targeting. Therefore, research into GLP-1 / GCG dual-targeting drugs in the treatment of obesity and related diseases is of great importance. Summary of the Invention

[0005] The present disclosure provides a compound of the formula: His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu- 1 Lys-Lys-Ala- 1 Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-X2 (I-1 SEQ ID NO: 1 ) His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu- 1 Lys-Lys-Ala-Lys- 1 Lys-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-X2 (I-2 SEQ ID NO: 2 ) His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys- 1 Lys-Ala-Lys- 1 Lys-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-X2 (I-3 SEQ ID NO: 3 ) His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala- 1 Lys-Glu-Phe-Val- 1 Lys-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-X2 (I-4 SEQ ID NO:4 ) His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val- 1 Lys-Trp-Leu-Leu- 1 Lys-Gly-Gly-Pro-Ser-Ser-X2 (I-5 SEQ ID NO:5 ) [In the formula, The structure of Aib is [ka] and; X2 is [ka] Selected from; 1 Lys represents a modified lysine, the modification being such that the amino groups on the two lysine side chains are [ka] which binds to; X is [ka] wherein "*" indicates the position of attachment to X; X0 is [ka] and; m is selected from 2 and 3; n is selected from 8, 9, and 10; p is selected from 1 and 2. The present invention provides a polypeptide having a sequence represented by the formula:

[0006] In some embodiments of the present disclosure, m is 2, and the other variables are as defined herein.

[0007] In some embodiments of the present disclosure, n is 9, and other variables are as defined herein.

[0008] In some embodiments of the present disclosure, p is 1, and the other variables are as defined herein.

[0009] In some embodiments of the present disclosure, X2 is [ka] and other variables are as defined herein.

[0010] In some embodiments of the present disclosure, X0 is [ka] and other variables are as defined herein.

[0011] In some embodiments of the present disclosure, [ka] teeth [ka] and other variables are as defined herein.

[0012] The present disclosure also includes embodiments obtained by combining any of the above variables.

[0013] The present disclosure also provides a polypeptide represented by the formula: [ka]

[0014] The disclosure also provides the use of the polypeptide compound in the manufacture of a medicament for the treatment of a GLP-1R / GCGR-related disorder.

[0015] In some embodiments of the present disclosure, the GLP-1R / GCGR-related disease is selected from obesity and non-alcoholic steatohepatitis (NASH). DETAILED DESCRIPTION OF THE INVENTION

[0016] Technical effects The compounds of the present disclosure have potent agonist activity against GLP-1R / GCGR. The compounds of the present disclosure show excellent weight loss effects in DIO mice. The compounds of the present disclosure show excellent NASH improvement effects in STZ-NASH mice. The compounds of the present disclosure have extremely high plasma protein binding and excellent plasma stability. The compounds of the present disclosure have excellent pharmacokinetic properties.

[0017] Definitions and Terminology Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings: A particular term or phrase should not be considered unclear or ambiguous in the absence of a specific definition, but should be understood in its ordinary sense. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0018] The term "pharmaceutically acceptable" is used herein in reference to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable risk-benefit ratio.

[0019] The term "pharmaceutically acceptable salt" refers to a salt of a compound disclosed herein prepared by reacting a compound having certain substituents disclosed herein with a relatively non-toxic acid or base. When a compound disclosed herein contains a relatively acidic functional group, a base addition salt can be obtained by contacting the compound with a sufficient amount of base in a pure solution or in a suitable inert solvent. When a compound disclosed herein contains a relatively basic functional group, an acid addition salt can be obtained by contacting the compound with a sufficient amount of acid in a pure solution or in a suitable inert solvent. Some specific compounds disclosed herein contain both basic and acidic functional groups and can be converted into either base or acid addition salts.

[0020] The pharmaceutically acceptable salts disclosed herein can be prepared from a parent compound that contains an acidic or basic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base form of the compound with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture thereof.

[0021] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that perform functions similar to those of naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (e.g., an α-carbon bonded to a hydrogen, carboxyl group, amino group, and R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure different from the general chemical structure of an amino acid but perform a similar function to a naturally occurring amino acid.

[0022] As used herein, A or Ala [ka] represents alanine having the structure: [ka] represents arginine having the structure [ka] D or Asp represents asparagine having the structure [ka] represents aspartic acid having the structure [ka] represents a cysteine ​​having the structure: [ka] represents glutamine having the structure [ka] G or Gly represents glutamic acid having the structure [ka] represents glycine having the structure [ka] and I or Ile represents a histidine having the structure [ka] represents isoleucine having the structure [ka] represents leucine having the structure [ka] and M or Met represents a lysine having the structure [ka] represents methionine having the structure [ka] P or Pro represents phenylalanine having the structure [ka] represents a proline having the structure [ka] represents serine having the structure T or Thr [ka] W or Trp represents threonine having the structure [ka] represents tryptophan having the structure [ka] V or Val represents a tyrosine having the structure [ka] represents valine having the structure:

[0023] The term "treating" includes preventing, slowing, arresting, or reversing existing symptoms or the progression or severity of a patient's condition.

[0024] Unless otherwise specified, the term "isomer" is intended to include geometric isomers, cis or trans isomers, stereoisomers, enantiomers, optical isomers, diastereomers, and tautomers.

[0025] The compounds disclosed herein may exist in specific geometric or stereoisomers. The present disclosure contemplates and encompasses all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereoisomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures, such as mixtures enriched in enantiomers or diastereoisomers, all of which are encompassed within the scope disclosed herein. Additional asymmetric carbon atoms may also be present in substituents such as alkyl. All of these isomers and their mixtures are encompassed within the scope disclosed herein.

[0026] Unless otherwise specified, the terms "enantiomers" or "optical isomers" refer to stereoisomers that are mirror images of one another.

[0027] Unless otherwise specified, the terms "cis-trans isomers" or "geometric isomers" result from the inability to freely rotate about a double bond or a single bond between ring-forming carbon atoms.

[0028] Unless otherwise specified, the term "diastereomer" refers to stereoisomers in which there are two or more centers of chirality in the molecules and the molecules are not mirror-images of each other.

[0029] Unless otherwise specified, "(+)" denotes the dextrorotatory isomer, "(-)" denotes the levorotatory isomer, and "(±)" denotes the racemate.

[0030] Unless otherwise specified, [ka] and [ka] indicates the absolute configuration of the stereocenter, [ka] and [ka] indicates the relative configuration of the stereocenters, [ka] teeth, [ka] or [ka] or [ka] teeth, [ka] and [ka] Shows.

[0031] Unless otherwise specified, the terms "enriched in one isomer," "enriched in an isomer," "enriched in one enantiomer," or "enantiomerically enriched" mean less than 100% isomer or enantiomer, and that isomer or enantiomer is present in an amount of 60% or more, or 70% or more, or 80% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, or 99.5% or more, or 99.6% or more, or 99.7% or more, or 99.8% or more, or 99.9% or more.

[0032] Unless otherwise specified, the terms "isomeric excess" or "enantiomeric excess" refer to the difference in relative proportions of two isomers or two enantiomers. For example, if one isomer or enantiomer is present in an amount of 90% and the other isomer or enantiomer is present in an amount of 10%, the isomeric or enantiomeric excess (ee) is 80%.

[0033] Optically active (R)- and (S)-isomers, or D- and L-isomers, can be prepared using chiral synthesis or chiral reagents or other conventional techniques. When obtaining one enantiomer of some compounds disclosed herein, the pure desired enantiomer can be obtained by asymmetric synthesis or derivatization of a chiral auxiliary, followed by separation of the resulting diastereomeric mixture and cleavage of the auxiliary. Alternatively, if the molecule contains a basic (e.g., amino) or acidic (e.g., carboxyl) functional group, the compound can be reacted with an appropriate optically active acid or base to form a diastereomeric salt, which can then be diastereoisomerically resolved by methods conventional in the art to obtain the pure enantiomers. Furthermore, enantiomers and diastereoisomers are typically separated by chromatography using chiral stationary phases, optionally with chemical derivatization (e.g., formation of carbamates from amines).

[0034] The compounds disclosed herein may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compounds. For example, the compounds may contain tritium ( 3 H), iodine-125( 125 I), or C-14( 14 In another example, hydrogen may be substituted with deuterium to form a deuterated pharmaceutical. The deuterium-carbon bond is stronger than a normal hydrogen-carbon bond. Compared to non-deuterated pharmaceuticals, deuterated pharmaceuticals offer the advantages of reduced toxic side effects, improved drug stability, enhanced efficacy, and a longer biological half-life of the drug. All variations in the isotopic composition of the compounds disclosed herein, whether radioactive or not, are included within the scope of this disclosure.

[0035] When the direction of the linking group is not indicated, the direction of the linking is arbitrary. For example, [ka] When the linking group L is -MW-, -MW- is attached to ring A and ring B in the same direction as reading from left to right, [ka] and is attached to ring A and ring B in the reverse reading order from left to right, [ka] Combinations of linking groups, substituents, and / or variables thereof are permissible only if such combinations result in stable compounds.

[0036] Unless otherwise specified, if a group has one or more bondable sites, one or more sites of the group can be bonded to other groups by chemical bonds. If the bonding position of the chemical bond is variable and there is an H atom at a bondable site, when the bondable site with an H atom is bonded to a chemical bond, the number of H atoms at that site decreases as the number of bonded chemical bonds increases, and the group becomes a group of the corresponding valence. The chemical bond between that site and other groups is [ka] For example, the straight solid line of -OCH3 indicates that the group is bonded to another group via the oxygen atom of that group, [ka] The straight dashed line indicates that the group is bonded to another group via both ends of the nitrogen atom of the group, [ka] The wavy lines indicate that the group is attached to another group through the 1- and 2-carbon atoms of the phenyl group.

[0037] Unless otherwise specified, "C 1-3 The term "alkyl" is used to refer to a straight or branched chain saturated hydrocarbon group composed of 1 to 3 carbon atoms. 1-3 Alkyl groups include C 1-2 and C2-3 Included are alkyl groups and the like. They may be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (e.g., methenyl). C 1-3 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (such as n-propyl and isopropyl), and the like.

[0038] The structure of the compounds disclosed herein can be confirmed by conventional methods known to those skilled in the art. Where the present disclosure relates to the absolute configuration of a compound, the absolute configuration can be confirmed by conventional techniques in the art, such as single crystal X-ray diffraction (SXRD). In single crystal X-ray diffraction (SXRD), diffraction intensity data of a grown single crystal is collected using a Bruker D8 venture diffractometer in φ / ω scan mode with CuKα radiation as the source. After collecting the relevant data, the crystal structure is further analyzed by a direct method (Shelxs97) to confirm the absolute configuration.

[0039] The compounds disclosed herein can be prepared by a variety of synthetic methods known to those skilled in the art, including the embodiments listed below, embodiments formed by combining the embodiments listed below with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples disclosed herein.

[0040] The solvents used in this disclosure are commercially available.

[0041] The following abbreviations are used in this disclosure: aq stands for aqueous; eq stands for equivalent; DCM stands for dichloromethane; PE stands for petroleum ether; DMSO stands for dimethyl sulfoxide; MeOH stands for methanol; Boc stands for tert-butoxycarbonyl, an amine protecting group; Dde stands for (4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl, an amino acid side chain protecting group; rt stands for room temperature; O / N stands for overnight; THF stands for tetrahydrofuran; BocO stands for di-tert-butyl dicarbonate; TFA stands for trifluoroacetic acid; DIEA stands for diisopropylethylamine; DMF stands for N,N-dimethylformamide; HBTU stands for O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; and HOBT stands for 1-hydroxybenzotriazole. HOAT stands for 1-hydroxy-7-azabenzotriazole. DIC stands for N,N'-diisopropylcarbodiimide. DBU stands for 1,8-diazabicyclo[5.4.0]undec-7-ene. PhSiH3 stands for phenylsilane. Pd(PPh3)4 stands for tetrakis(triphenylphosphine)palladium. AEEA stands for 2-(2-(2-aminoethoxy)ethoxy)acetic acid. DIEA stands for diisopropylethylamine.

[0042] Compounds are named according to naming conventions common in the art or by ChemDraw® software; commercially available compounds are named using the supplier's catalog name.

[0043] Detailed Description of the Invention The present disclosure will be described in detail below using examples. However, these examples are not intended to have any adverse limitations on the present disclosure. The present disclosure is described in detail herein, and embodiments are also disclosed herein. It will be apparent to those skilled in the art that various changes and modifications may be made to the embodiments disclosed herein without departing from the technical idea and scope disclosed herein. [Example]

[0044] Example 1 [ka]

[0045] Synthesis of intermediate 1 [ka] 1. Resin Binding to Intermediate 1 1.1 5.0 g of 2-chlorotrityl chloride resin (2-CTC resin, substitution degree S=1.00 mmol / g) and 1.93 g of Fmoc-AEEA-OH were weighed and added to a reaction column, and DCM (40 mL) was further added. Next, DIEA (3.5 mL) was added to the reaction column, and nitrogen was bubbled through the system for 2 hours. Next, MeOH (5 mL) was added to the reaction column, and nitrogen was bubbled through the system for an additional 30 minutes. The reaction column was purged until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time), and the reaction column was purged until no more liquid flowed out.

[0046] 1.2 20% piperidine / DMF (100 mL) was added to the reaction column, and nitrogen was bubbled through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (100 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0047] 2. Amino acid coupling 2.1 Coupling of Fmoc-AEEA-OH 1. Fmoc-AEEA-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (30 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0048] 2. The mixture was allowed to react for 20 minutes at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0049] 3. The reaction solution was aspirated. The reaction column was washed with DMF five times (1 minute each time) and drained until no more liquid came out.

[0050] 2.2 Coupling of Fmoc-Glu-OtBu 1. 20% piperidine / DMF (100 mL) was added to the reaction column and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0051] 2. Fmoc-Glu-OtBu (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (30 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0052] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0053] 4. The reaction solution was aspirated. The reaction column was washed with DMF (100 mL) five times (1 minute each time) and drained until no more liquid came out.

[0054] 2.3 Coupling of 20-(tert-butoxy)-20-oxoicosanoic acid 1. 20% piperidine / DMF (100 mL) was added to the reaction column and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (100 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin and turned blue.

[0055] 2. 20-(tert-butoxy)-20-oxoicosanoic acid (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (30 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0056] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0057] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (1 minute each time) and drained until no more liquid came out.

[0058] 3. Cleavage and Drying of Crude Peptides 3.1. Cleavage solution was prepared according to the following volumes: HFIP / DCM=20 / 80

[0059] 3.2. 100 mL of the prepared cleavage solution was poured into a reactor containing dried peptide resin. The reactor was bubbled for 20 minutes. The mixture was filtered, and the filtrate was added to the flask. This procedure was repeated twice. The two collected cleavage solutions were evaporated to dryness on a rotary evaporator to obtain 4.3 g of crude peptide.

[0060] Synthesis of WX001 1. Resin Bonding 1.1 1.43 g of 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-methyldiphenylmethylamine resin (Rink Amide MBHA Resin, substitution degree Sub = 0.28 mmol / g) was weighed and added to a reaction column. Next, DCM (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 2 hours. The reaction column was emptied until no more liquid flowed out. DMF (50 mL) was added five times (1 minute each time) to wash the column, and the column was emptied until no more liquid flowed out.

[0061] 1.2 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0062] 2. Amino acid coupling 2.1 Coupling of Fmoc-Gly-OH 1. Fmoc-Gly-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was then added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0063] 2. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0064] 3. The reaction liquid was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0065] 2.2 Coupling of Fmoc-Ser(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0066] 2. Fmoc-Ser(tBu)-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was then added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0067] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0068] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0069] 2.3 Coupling of Fmoc-Ser(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0070] 2. Fmoc-Ser(tBu)-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was then added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0071] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0072] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (1 minute each time) and drained until no more liquid came out.

[0073] 2.4 Coupling of Fmoc-Pro-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0074] 2. Fmoc-Pro-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0075] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0076] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0077] 2.5 Coupling of Fmoc-Gly-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with chloranil, which gave a blue color.

[0078] 2. Fmoc-Gly-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0079] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with chloranil and was colorless and transparent.

[0080] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0081] 2.6 Coupling of Fmoc-Gly-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0082] 2. Fmoc-Gly-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0083] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0084] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0085] 2.7 Coupling of Fmoc-Glu(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0086] 2. Fmoc-Glu(OtBu)-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0087] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0088] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0089] 2.8 Coupling of Fmoc-Leu-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0090] 2. Fmoc-Leu-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0091] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0092] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0093] 2.9 Coupling of Fmoc-Leu-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0094] 2. Fmoc-Leu-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0095] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0096] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0097] 2.10 Coupling of Fmoc-Trp(Boc)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0098] 2. Fmoc-Trp(Boc)-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0099] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0100] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0101] 2.11 Coupling of Fmoc-Glu(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0102] 2. Fmoc-Glu(OtBu)-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0103] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0104] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0105] 2.12 Coupling of Fmoc-Val-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0106] 2. Fmoc-Val-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0107] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0108] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0109] 2.13 Coupling of Fmoc-Phe-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0110] 2. Fmoc-Phe-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0111] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0112] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0113] 2.14 Coupling of Fmoc-Glu(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0114] 2. Fmoc-Glu(OtBu)-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0115] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0116] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0117] 2.15 Coupling of Fmoc-Lys(Dde)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0118] 2. Fmoc-Lys(Dde)-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was then added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0119] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0120] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0121] 2.16 Coupling of Fmoc-Ala-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0122] 2. Fmoc-Ala-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0123] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0124] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0125] 2.17 Coupling of Fmoc-Lys(Boc)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0126] 2. Fmoc-Lys(Boc)-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0127] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0128] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0129] 2.18 Coupling of Fmoc-Lys(Alloc)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0130] 2. Fmoc-Lys(Alloc)-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0131] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0132] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0133] 2.19 Coupling of Fmoc-Glu(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0134] 2. Fmoc-Glu(OtBu)-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0135] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0136] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0137] 2.20 Coupling of Fmoc-Asp(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0138] 2. Fmoc-Asp(OtBu)-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0139] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0140] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0141] 2.21 Coupling of Fmoc-Leu-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0142] 2. Fmoc-Leu-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0143] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0144] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0145] 2.22 Coupling of Fmoc-Tyr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0146] 2. Fmoc-Tyr(tBu)-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0147] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0148] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0149] 2.23 Coupling of Fmoc-Lys(Boc)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0150] 2. Fmoc-Lys(Boc)-OH (6.0 eq) was weighed and added to the resin. DIEA (12.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HATU (5.70 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0151] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0152] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0153] 2.24 Coupling of Fmoc-Ser(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0154] 2. Fmoc-Ser(tBu)-OH (6.0 eq) was weighed and added to the resin. DIEA (12.0 eq) was added, and DMF (10 mL) was then added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HATU (5.70 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0155] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0156] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0157] 2.25 Coupling of Fmoc-Tyr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0158] 2. Fmoc-Tyr(tBu)-OH (6.0 eq) was weighed and added to the resin. DIEA (12.0 eq) was added, and DMF (10 mL) was then added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HATU (5.70 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0159] 3. The mixture was allowed to react for 2 hours at 25°C. The resin was tested with ninhydrin and showed a blue color.

[0160] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0161] 2.26 Coupling of Fmoc-Asp(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0162] 2. Fmoc-Asp(OtBu)-OH (6.0 eq) was weighed and added to the resin. HOAT (6.0 eq) was added, and DMF (10 mL) was then added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid and HOAT were dissolved, DIC (6.0 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0163] 3. The mixture was allowed to react for 1 hour at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0164] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0165] 2.27 Coupling of Fmoc-Ser(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0166] 2. Fmoc-Ser(tBu)-OH (6.0 eq) was weighed and added to the resin. DIEA (12.0 eq) was added, and DMF (10 mL) was then added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HATU (5.70 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0167] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0168] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0169] 2.28 Coupling of Fmoc-Thr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0170] 2. Fmoc-Thr(tBu)-OH (6.0 eq) was weighed and added to the resin. DIEA (12.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HATU (5.70 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0171] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0172] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0173] 2.29 Coupling of Fmoc-Phe-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0174] 2. Fmoc-Phe-OH (6.0 eq) was weighed and added to the resin. DIEA (12.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HATU (5.70 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0175] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0176] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0177] 2.30 Coupling of Fmoc-Thr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0178] 2. Fmoc-Thr(tBu)-OH (6.0 eq) was weighed and added to the resin. DIEA (12.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HATU (5.70 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0179] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0180] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0181] 2.31 Coupling of Fmoc-Gly-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0182] 2. Fmoc-Gly-OH (3.0 eq) was weighed and added to the resin. DIEA (6.00 eq) was added, and DMF (10 mL) was then added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HATU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0183] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0184] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0185] 2.32 Coupling of Fmoc-Gln(Trt)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0186] 2. Fmoc-Gln(Trt)-OH (6.0 eq) was weighed and added to the resin. HOBT (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid and HOBT were dissolved, DIC (6.0 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0187] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0188] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0189] 2.33 Coupling of Fmoc-Aib-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0190] 2. Fmoc-Aib-OH (3.0 eq) was weighed and added to the resin. DIEA (6.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HATU (2.85 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0191] 3. The mixture was allowed to react overnight at 25° C. The resin was tested with ninhydrin and was colorless and transparent.

[0192] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0193] 2.34 Coupling of Boc-His(Trt)-OH 1. 20% piperidine / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with chloranil, which gave a blue color. 2. Boc-His(Trt)-OH (6.0 eq) was weighed and added to the resin. DIEA (12.0 eq) was added, and DMF (10 mL) was then added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HATU (5.70 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0194] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with chloranil and was colorless and transparent.

[0195] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0196] 2.35 Alloc Removal 1. PhSiH3 (10.0 eq) and DCM (10 mL) were added to a reaction column and the system was sparged with nitrogen. Next, Pd(PPh3)4 (0.1 eq) was added and the system was sparged with nitrogen for 20 minutes. The mixture was reacted twice and purged from the reaction column until no liquid flowed out.

[0197] 2. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0198] 2.36 Coupling of Fmoc-Ida-OH 1. Fmoc-Ida-OH (6.0 eq) was weighed and added to the resin. DIEA (12.0 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (5.70 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0199] 2. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0200] 3. The reaction liquid was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0201] 2.37 Coupling of Intermediate 1 1. 10% DBU / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 20 minutes. The reaction column was drained until no more liquid flowed out. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0202] 2. Intermediate 1 (1.50 eq) was weighed and added to the resin. DIEA (3.00 eq) was added, and DMF (10 mL) was further added to the reaction column. Nitrogen was blown into the reaction column. After the amino acid was dissolved, HBTU (1.45 eq) was added. The nitrogen was adjusted so that it was blown evenly through the resin.

[0203] 3. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0204] 4. The reaction solution was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0205] 2.38 Dde Removal 1. 3% hydrazine hydrate / DMF (50 mL) was added to the reaction column, and nitrogen was sparged through the system for 15 minutes. The reaction column was drained. DMF (50 mL) was added and washed five times (1 minute each time). The reaction column was drained until no more liquid flowed out. The resin was tested with ninhydrin, which gave a blue color.

[0206] 2.39 Amide Ring Closure 1. DIEA (3.0 eq) was added to the DMF solution of the resin. Next, HATU (1.5 eq) dissolved in DMF was slowly added dropwise to the reaction column, and nitrogen was blown into the system. The nitrogen was adjusted so that it was blown uniformly through the resin.

[0207] 2. The mixture was allowed to react for 0.5 hours at 25°C. The resin was tested with ninhydrin and was colorless and transparent.

[0208] 3. The reaction liquid was aspirated. The reaction column was washed with DMF five times (50 mL each time, 1 minute each time) and drained until no more liquid came out.

[0209] 4. The resin was shrunk with MeOH (50 mL) for 3 minutes each time. The reaction column was drained until no more liquid flowed. The resin was poured out and dried for later use.

[0210] 3. Cleavage and Drying of Crude Peptides 3.1. Cleavage solution was prepared according to the following volumes: TFA / triisopropylsilane / H2O / 3-mercaptopropionic acid = 90 / 2.5 / 2.5 / 5

[0211] 3.2 The dried peptide resin was added to the prepared cleavage solution. The mixture was shaken on a shaker for 2.5 hours and then filtered. The filtrate was added to 10 volumes of ice-cold isopropyl ether. The mixture was centrifuged, washed five times with isopropyl ether, and dried under vacuum for 2 hours to obtain the crude peptide. The crude peptide was separated and purified by preparative HPLC (purification step: mobile phase acetonitrile / water (40% / 60%), 0.075% TFA; salt conversion step: mobile phase acetonitrile / water (20% / 80%), 0.01% ammonium acetate) to obtain polypeptide WX001. The molecular weight of the polypeptide was confirmed by ESI-MS, with a calculated value of 4660.1 and an observed value of 4660.2.

[0212] Example 2 [ka] Polypeptide WX002 was obtained by reference to the synthesis of WX001. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value was 4659.2, and the measured value was 4659.0.

[0213] Example 3 [ka] Polypeptide WX003 was obtained by reference to the synthesis of WX001. The molecular weight of the polypeptide was confirmed by ESI-MS, with a calculated value of 4659.2 and an actual value of 4659.3.

[0214] Example 4 [ka] Polypeptide WX004 was obtained by reference to the synthesis of WX001. The molecular weight of the polypeptide was confirmed by ESI-MS, with a calculated value of 4659.2 and an actual value of 4659.6.

[0215] Example 5 [ka] Polypeptide WX005 was obtained by reference to the synthesis of WX001. The molecular weight of the polypeptide was confirmed by ESI-MS, with a calculated value of 4658.3 and an actual value of 4658.7.

[0216] Biological Test Data Experimental Example 1: Test of in vitro agonist activity against GLP-1R / GIPR / GCGR A: Main materials: (I) Cell line The cell lines were constructed by Shanghai WuXi AppTec. See Table 1 for details. [Table 1]

[0217] (II) Reagents and Consumables See Table 2 for details. [Table 2]

[0218] (III) Equipment See Table 3 for details. [Table 3]

[0219] B. Method i) Experimental materials The experimental buffers are shown in Table 4. [Table 4]

[0220] The preparation of experimental reagents is shown in Table 5. [Table 5]

[0221] ii) Experimental method a) Preparation of compound plates: Compounds to be tested were diluted 10-point 4-fold using a Bravo at a starting concentration of 30 μM.

[0222] b) Compound transport: 1) 100 nL of compound was transferred to an OptiPlate-384 plate using Echo. 2) The OptiPlate-384 plate was centrifuged at 1000 rpm for 5 seconds.

[0223] c) Preparation of cell suspension 1) The GLP-1R / GIPR / GCGR cell cryopreservation tube was quickly placed in 37°C warm water to thaw. 2) The cell suspension was transferred to a 15 mL centrifuge tube and gently washed with 10 mL of HBSS. 3) The tube was centrifuged at 1000 rpm at room temperature for 1 minute. 4) The supernatant was discarded. 5) The cells at the bottom were gently dispersed and washed gently with 10 mL of HBSS. The cells were centrifuged and finally resuspended in the experimental buffer. 6) Cell density and viability were measured using Vi-cell. 7) The concentration of GLP-1R / GIPR / GCGR cells was adjusted to 2.0*10 in the experimental buffer. 5 / mL. 8) 100 nL of the diluted cell suspension was transferred to an OptiPlate-384 plate. 9) The plate was incubated at room temperature for 30 minutes.

[0224] d) Addition of experimental reagents: 1) 10 μL of 800 nM serially diluted cAMP standard solution was added to an empty well of an OptiPlate-384 plate. 2) 10 μL of cAMP experimental reagent was added. 3) The OptiPlate-384 plate was covered with TopSeal-A film and incubated at room temperature for 60 minutes. The TopSeal-A was removed and the plate was read on EnVision.

[0225] C. Experimental Results The experimental results are shown in Table 6. [Table 6]

[0226] Conclusion: The compounds of the present disclosure have very potent agonist activity for GLP-1R / GCGR, but no agonist activity for GIPR.

[0227] Experimental Example 2: Drug efficacy study in DIO mice - in vivo drug efficacy evaluation A. Experimental Objective The weight loss effect of the test compounds in DIO mice was examined.

[0228] B. Experimental Procedure 1. Upon arrival at the facility, DIO mice were housed in an animal room under strictly controlled environmental conditions. The temperature and humidity of the room were maintained at 20–24°C and 30–70%. The temperature and humidity of the room were monitored in real time using a thermo-hygrometer, and the temperature and humidity were recorded twice daily (once in the morning and once in the afternoon). Lighting in the animal room was controlled by an electronic time-controlled lighting system. The lights were on for 12 hours and off for 12 hours each day (lights on at 7:00 AM and off at 7:00 PM). During the experiment, animals were housed in individual cages, each with its own toy. During the experiment, animals had free access to food (growth / breeding diet for rats and mice) and water.

[0229] 2. Animals in each group were subcutaneously injected with the vehicle or the test compound (10 nmol / kg), at 9:30 a.m. The administration was once every three days, and the administration cycle was 21 days.

[0230] C. Experimental Results The experimental results are shown in Table 7. [Table 7]

[0231] Conclusion: The compounds of the present disclosure exhibit excellent weight loss effects in DIO mice.

[0232] Experimental Example 3. Plasma protein binding (PPB) test A. Experimental Objective The binding of the test compounds to human / mouse plasma albumin was examined.

[0233] B. Experimental Procedure 1. Matrix preparation: On the day of the experiment, plasma was thawed in cold water and centrifuged at 3220 rpm for 5 minutes to remove any clots. The pH of the resulting plasma was measured and adjusted to 7.4±0.1 with 1% phosphoric acid or 1N sodium hydroxide, if necessary.

[0234] 2. Test Compound Dilution Procedure: Test compounds were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions at concentrations of 10 mM and 2 mM, respectively. A 40 μM working solution was prepared by diluting 2 μL of the 2 mM stock solution with 98 μL of DMSO. A 400 μM working solution of the control compound was prepared by diluting 10 μL of the stock solution with 240 μL of DMSO. The loading matrix was prepared by thoroughly mixing 5 μL of the compound working solution with 995 μL of blank matrix at a ratio of 1:200.

[0235] 3. Analysis step 3.1 For residue measurements, 30 μL of loading matrix (n=2) was transferred to a sample collection plate to prepare time 0 (T0) samples. The samples were immediately combined with the corresponding blank buffer to a final volume of 60 μL. The volume ratio of plasma to buffer in each well was 1:1. Next, 480 μL of stop solution containing 4% H3PO4 and internal standard in 60 μL of H2O was added to the T0 samples of the test compound. They were then stored at 2-8 °C with the other samples for further processing.

[0236] 3.2 The remaining plasma samples were preincubated in a carbon dioxide incubator at 37±1°C for 30 minutes. The protein-free sample (F sample) and the matrix-loaded sample (230 μL) were all transferred to polycarbonate tubes (n=2) and ultracentrifuged at 37°C, 155,000×g (35,000 rpm) for 4 hours.

[0237] 3.3 To prepare T samples (test samples), additional matrix-containing samples were transferred to another 96-well plate (sample incubation plate) and incubated at 37°C for 4 hours.

[0238] 3.4 After centrifugation, 30 μL of the protein-free sample and 30 μL of the T sample were transferred from the second layer of supernatant (below the upper layer) to a new sample collection plate. Each sample was mixed with the corresponding blank buffer or matrix to a final volume of 60 μL with a matrix:buffer volume ratio of 1:1. 60 μL of 4% H3PO4 aqueous solution and 480 μL of stop solution (containing internal standard) were added to all samples. The mixtures were centrifuged at 4000 rpm for 20 minutes, and 100 μL of supernatant from each sample was analyzed by LC-MS / MS.

[0239] C. Experimental Results The experimental results are shown in Table 8. [Table 8] NOTE: NA indicates that plasma protein binding is so high that free drug is not detectable at normal plasma protein concentrations.

[0240] Conclusion: The compounds of the present disclosure have extremely high plasma protein binding.

[0241] Experimental Example 4: Plasma stability test (PLS) A. Experimental Objective The stability of the test compound in normal mouse plasma was examined.

[0242] B. Experimental Procedure 1. Before the experiment, the clotted frozen plasma was thawed in a water bath at 37°C. The plasma was centrifuged at 4000 rpm for 5 minutes. Any clots were removed. The pH was adjusted to 7.4±0.1.

[0243] 2. Preparation of test compound solution: Test compounds were dissolved in DMSO to prepare 100 μM solutions.

[0244] 3. 98 μL of blank control plasma was added to 2 μL of test compound solution (100 μM) to make the final concentration of the mixed solution 2 μM. The mixed solution was incubated in a water bath at 37°C.

[0245] 4. 100 μL of H3PO4 solution and 800 μL of stop solution (a solution of 200 ng / mL tolbutamide and 200 ng / mL labetalol in 100% methanol) were added at each time point (0, 10, 30, 60, and 120 min), respectively, to precipitate proteins and mixed thoroughly.

[0246] 5. Samples were centrifuged at 4000 rpm for 20 minutes. 100 μL of supernatant was removed from each well for LC-MS / MS analysis.

[0247] C. Experimental Results The experimental results are shown in Table 9. [Table 9]

[0248] Conclusion: The compounds of the present disclosure have excellent plasma stability.

[0249] Experimental Example 5: Pharmacokinetic evaluation of compounds in mice A. Experimental Objective The pharmacokinetics of the compound was studied in C57BL / 6 mice.

[0250] B. Experimental Procedure The pharmacokinetic properties of compounds were tested in rodents after subcutaneous injection using standard protocols. During the experiment, candidate compounds were formulated into clear solutions and administered to mice via a single subcutaneous injection (SC, 0.048 mpk). The vehicle for subcutaneous injection was citrate buffer (20 mM, pH = 7). Whole blood was collected and plasma was prepared. Drug concentrations were analyzed using an LC-MS / MS method, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software.

[0251] C. Experimental Results The experimental results are shown in Table 10. [Table 10]

[0252] Conclusion: The compounds of the present disclosure have excellent pharmacokinetic properties in mice.

[0253] Experimental Example 6: Pharmacokinetic evaluation of compounds in cynomolgus monkeys A. Experimental Objective The pharmacokinetics of the compound was studied in cynomolgus monkeys.

[0254] B. Experimental Procedure The pharmacokinetic properties of compounds in mammals after subcutaneous injection were tested using standard protocols. During the experiment, candidate compounds were formulated into clear solutions and administered to cynomolgus monkeys via a single subcutaneous injection (SC, 0.02 mpk). The vehicle for subcutaneous injection was citrate buffer (20 mM, pH = 7). Whole blood was collected and plasma was prepared. Drug concentrations were analyzed using an LC-MS / MS method, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software.

[0255] C. Experimental Results The experimental results are shown in Table 11. [Table 11]

[0256] Conclusion: Compounds of the present disclosure have excellent pharmacokinetic properties in monkeys.

[0257] Experimental Example 7: Verification of in vivo efficacy in STZ-NASH mouse model A. Experimental Objective The efficacy of the test substance will be verified in a NASH model induced by streptozotocin (STZ) and high-fat diet (HFD) using C57BL / 6 mice.

[0258] B. Experimental Procedure Modeling method: Newborn mice were subcutaneously injected with STZ (200 μg / mouse) within 48 hours after birth. After 4 weeks of lactation, animals with fasting blood glucose levels >12 mmol / L were selected and fed an HFD for 6 consecutive weeks to finally establish the NASH model. Another 8 animals were not injected with STZ or fed an HFD (normal control group).

[0259] Dosing regimen: Dosing began one week after HFD feeding, with the first day of dosing designated as Day 1. Mice were then injected subcutaneously every two days for five consecutive weeks.

[0260] Detection at the end of the experiment: Pathological examination was performed using hematoxylin and eosin staining and picrosirius red staining.

[0261] C. Experimental Results The experimental results are shown in Table 13. [Table 12]

[0262] Conclusion: The compounds of the present disclosure can significantly improve NAS scores in the STZ-NASH mouse model.

Claims

1. The following sequence: His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu- １ Lys-Lys-Ala- １ Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-X ２ (I-1) His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu- １ Lys-Lys-Ala-Lys- １ Lys-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-X ２ (I-2) His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys- １ Lys-Ala-Lys- １ Lys-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-X ２ (I-3) His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala- １ Lys-Glu-Phe-Val- １ Lys-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-X ２ (I-4) or His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val- １ Lys-Trp-Leu-Leu- １ Lys-Gly-Gly-Pro-Ser-Ser-X ２ (I-5) [In the sequence, The structure of Aib is 【Chemistry 1】 and X ２ teeth, 【Chemistry 2】 Selected from: １ Lys represents a modified lysine, the modification being such that the amino groups on the two lysine side chains are 【Transformation 3】 which binds to X is 【Chemistry 4】 wherein "*" represents X ０ indicates the position of binding to X ０ teeth, 【Transformation 5】 and m is selected from 2 and 3; n is selected from 8, 9, and 10; p is selected from 1 and 2. A polypeptide represented by the formula:

2. The following definitions: i) m is 2; ii) n is 9; iii) p is 1; iv) X ２ but 【Transformation 6】 is; v) X ０ but 【Transformation 7】 is; vi) X is 【Transformation 8】 That is, The polypeptide of claim 1, which satisfies one or more of the following conditions: 【Request Item 3】 【Chemistry 9】 but 【Chemistry 10】 The polypeptide of claim 1,

4. A polypeptide represented by the following formula: 【Chemistry 11】

5. A pharmaceutical composition for treating a GLP-1R / GCGR-related disease, comprising the polypeptide according to any one of claims 1 to 4.

6. The pharmaceutical composition according to claim 5, wherein the GLP-1R / GCGR-related disease is obesity or non-alcoholic steatohepatitis.

7. A drug comprising the polypeptide according to any one of claims 1 to 4.

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