Preparation of Polypeptides and Uses Thereof

Abstract

The preparation and use of a series of polypeptides are disclosed, specifically, a polypeptide having the sequence shown in formula (II): YAibEGT FTSDY SIAibLD KIAQK AFVKW LIAGG PSSGA PPPS0(II)

Description

[Technical field]

[0001] This application claims priority to Chinese patent application CN2021105946626 filed on May 28, 2021, Chinese patent application CN2021108141169 filed on July 19, 2021, Chinese patent application CN2022101388358 filed on February 15, 2022, and Chinese patent application CN2022105520774 filed on May 18, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the preparation and use of a series of polypeptides, in particular to polypeptides having the sequence shown in formula (II): [Background technology]

[0003] The global prevalence of nonalcoholic fatty liver disease (NAFLD) is as high as 25%, and the global prevalence of nonalcoholic steatohepatitis (NASH) is approximately 3% to 8%. Some patients with NASH further progress to liver sclerosis and liver cancer, which is currently one of the leading causes of end-stage liver disease and liver transplantation. The pathogenesis of NASH is complex, and there is currently no FDA-approved drug for the disease. Several preclinical studies have shown that glucose-dependent insulinotropic polypeptide (GIP) / glucagon-like peptide-1 (GLP-1) dual agonists can be used to treat NASH. Clinical studies of the GLP-1 / GIP dual agonist Tirzepatide, a drug under investigation by Eli Lilly and Company, have shown that it improves related markers such as NASH-related transaminases, indicating its potential as a treatment for NASH.

[0004] GLP-1 agonists can treat NASH through multi-pathway synergistic effects. For example, GLP-1 agonists can reduce the circulating levels of tumor necrosis factor (TNF)-α, interleukin IL-1β, IL-6, CD163 and hsCRP, achieving anti-inflammatory effects. GIP is a polypeptide secreted from neuroendocrine K cells in the small intestine, and based on GLP-1 agonist treatment, it can further alleviate hepatic lipid synthesis, so that GLP-1 / GIP dual agonists have a synergistic effect in the treatment of NASH. Summary of the Invention

[0005] The present invention relates to a compound represented by formula (II): YAibEGT FTSDY SIAibLD KIAQK AFVKW LIAGG PSSGA PPPS0 (II) As shown below: 1) The amino groups on the lysine side chain at positions i and i+3 or the amino groups on the lysine side chain at positions j and j+4 TIFF0007672503000001.tif11170, where i is 17 (i.e., when i is 17, first the isoleucine at position 17 is replaced with lysine, and then the amino group on its side chain is replaced with the amino group on the lysine side chain at position 20). TIFF0007672503000002.tif11170), or where j is 20, and 2) an additional 0 to 2 amino acids of the polypeptide of formula (II) are substituted; Where: The structure of Aib is: TIFF0007672503000003.tif16170, S0 is Selected from TIFF0007672503000004.tif18170, X is Selected from TIFF0007672503000005.tif18170, where "*" represents the position concatenated to X1, X1 is selected from a single bond, -C(=O)-, -OC(=O)- and -N(R1)-C(=O)-; R1 is H and C 1-3 alkyl, X2 is Selected from TIFF0007672503000006.tif38170, m is selected from 2, 3 and 4; n is selected from 15, 16, 17, 18 and 19; p provided a polypeptide having a sequence comprising an alteration selected from 1 and 2.

[0006] The present invention is represented by formula (P): YAibEGT FTSDY SIAibLD KKAQK AFVKW LIAGG PSSGA PPPS0 (P) As shown below: 1) The amino groups on the lysine side chains at positions 17 and 20 Concatenated to TIFF0007672503000007.tif11170, and 2) 0 to 2 amino acids of a polypeptide having a sequence represented by formula (P) are substituted, Where: The structure of Aib is: TIFF0007672503000008.tif16170, S0 is Selected from TIFF0007672503000009.tif18170, X is Selected from TIFF0007672503000010.tif18170, where "*" represents the position concatenated to X1, X1 is selected from a single bond, -C(=O)-, -OC(=O)- and -N(R1)-C(=O)-; R1 is H and C 1-3 alkyl, X2 is Selected from TIFF0007672503000011.tif38170, m is selected from 2, 3 and 4; n is selected from 15, 16, 17, 18 and 19; p provided a polypeptide having a sequence comprising an alteration selected from 1 and 2.

[0007] The present invention relates to a compound represented by formula (II): YAibEGT FTSDY SIAibLD KIAQK AFVKW LIAGG PSSGA PPPS0 (II) As shown below: 1) The amino groups on the lysine side chain at positions i and i+3 or the amino groups on the lysine side chain at positions j and j+4 TIFF0007672503000012.tif11170, where i is 17 and j is 20 (first the isoleucine at position 17 is replaced with lysine, and then the amino group on its side chain is linked to the amino group on the lysine side chain at position 20), and 2) an additional 0 to 2 amino acids of the polypeptide of formula (II) are substituted; Where: The structure of Aib is: TIFF0007672503000013.tif16170, S0 is Selected from TIFF0007672503000014.tif18170, X is Selected from TIFF0007672503000015.tif19170, where "*" represents the position linked to X1, X1 is selected from a single bond, -C(=O)-, -OC(=O)- and -N(R1)-C(=O)-; R1 is H and C 1-3 alkyl, X2 is Selected from TIFF0007672503000016.tif38170, m is selected from 2, 3 and 4; n is selected from 15, 16, 17, 18 and 19; p provided a polypeptide having a sequence comprising an alteration selected from 1 and 2.

[0008] In some aspects of the invention, 0 to 2 amino acids at positions 21, 23 or 24 of the above polypeptides are substituted, with the remaining variables being as defined herein.

[0009] In some aspects of the present invention, the above-mentioned polypeptide is represented by formula (II-1), (II-2), (II-3), (II-4), (III-6), (IV-7), (IV-8), (IV-9) and (IV-10): YAibEGT FTSDY SIAibLD KIAQK AFVKW LIAGG PSSGA PPPS-NH2 (II-1) YAibEGT FTSDY SIAibLD KIAQK EFVKW LIAGG PSSGA PPPS-NH2 (II-2) YAibEGT FTSDY SIAibLD KIAQK AFIKW LIAGG PSSGA PPPS-NH2 (II-3) YAibEGT FTSDY SIAibLD KIAQK AFVKW LLAGG PSSGA PPPS-NH2 (II-4) YAibEGT FTSDY SIAibLD KKAQK AFVEW LIAGG PSSGA PPPS-NH2 (III-6) YAibEGT FTSDY SIAibLD KKAQK AFVQW LIAGG PSSGA PPPS-NH2 (IV-7) YAibEGT FTSDY SIAibLD KKAQK AFVAW LIAGG PSSGA PPPS-NH2 (IV-8) YAibEGT FTSDY SIAibLD KKAQK AFVIW LIAGG PSSGA PPPS-NH2 (IV-9) YAibEGT FTSDY SIAibLD KKAQK EFVEW LIAGG PSSGA PPPS-NH2 (IV-10) As shown below: The amino groups on the lysine side chains at positions 17 and 20 or the amino groups on the lysine side chains at positions 20 and 24 Concatenated into TIFF0007672503000017.tif11170, Where: Aib, X, X1 and X2 have sequences containing modifications as defined herein.

[0010] In some aspects of the invention, R1 above is selected from H, with the remaining variables being as defined herein.

[0011] In some aspects of the invention, X1 above is selected from a single bond, -C(=O)-, -OC(=O)-, and -NH-C(=O)-, with the remainder of the variables being as defined herein.

[0012] In some aspects of the invention, m above is selected from 2, with the remaining variables being as defined herein.

[0013] In some aspects of the invention, n above is selected from 15 and 17, with the remaining variables being as defined herein.

[0014] In some aspects of the present invention, X2 is TIFF0007672503000018.tif60170, and the remaining variables are as defined herein.

[0015] In some aspects of the present invention, X2 is TIFF0007672503000019.tif78170, and the remaining variables are as defined herein.

[0016] In some aspects of the invention, the amino groups on the lysine side chains at positions i and i+3 or the amino groups on the lysine side chains at positions j and j+4 are Link to TIFF0007672503000020.tif11170, TIFF0007672503000021.tif96170, with the remaining variables as defined herein.

[0017] In some aspects of the invention, the amino groups on the lysine side chains at positions i and i+3 or the amino groups on the lysine side chains at positions j and j+4 are Link to TIFF0007672503000022.tif11170, TIFF0007672503000023.tif94170, with the remaining variables as defined herein.

[0018] In some aspects of the invention, TIFF0007672503000024.tif11170 is TIFF0007672503000025.tif157170, and the remaining variables are as defined herein.

[0019] In some aspects of the invention, TIFF0007672503000026.tif11170 is TIFF0007672503000027.tif127170, and the remaining variables are as defined herein.

[0020] The present invention relates to a compound represented by formula (II): YAibEGT FTSDY SIAibLD KIAQK AFVKW LIAGG PSSGA PPPS0 (II) As shown below: 1) The amino groups on the lysine side chains at positions i and i+3 or the amino groups on the lysine side chains at positions j and j+4 are TIFF0007672503000028.tif11170, where i is 17 and j is 20, 2) 0 to 2 amino acids of the polypeptide represented by formula (II) are substituted, Where: The structure of Aib is: TIFF0007672503000029.tif16170, S0 is Selected from TIFF0007672503000030.tif16170, X is Selected from TIFF0007672503000031.tif17170, where "*" represents the position concatenated to X1, X1 is selected from -C(=O)-, -OC(=O)- and -N(R1)-C(=O)-; R1 is H and C 1-3 alkyl, X2 is Selected from TIFF0007672503000032.tif19170, m is selected from 2, 3 and 4; n provided a polypeptide having a sequence comprising an alteration selected from 15, 16, 17, 18 and 19.

[0021] The present invention relates to a compound represented by formula (II): YAibEGT FTSDY SIAibLD KIAQK AFVKW LIAGG PSSGA PPPS0 (II) As shown below: 1) The amino groups on the lysine side chains at positions i and i+3 or the amino groups on the lysine side chains at positions j and j+4 are TIFF0007672503000033.tif11170, where i is 17 and j is 20, 2) 0 to 1 amino acids of the polypeptide represented by formula (II) are substituted, Where: The structure of Aib is: TIFF0007672503000034.tif16170, S0 is Selected from TIFF0007672503000035.tif18170, X is Selected from TIFF0007672503000036.tif17170, where "*" represents the position concatenated to X1, X1 is selected from -C(=O)-, -OC(=O)- and -N(R1)-C(=O)-; R1 is H and C 1-3 alkyl, X2 is Selected from TIFF0007672503000037.tif19170, m is selected from 2, 3 and 4; n provided a polypeptide having a sequence comprising an alteration selected from 15, 16, 17, 18 and 19.

[0022] In some aspects of the present invention, the above-mentioned polypeptide is represented by formula (II-1), (II-2), (II-3), (II-4), (II-5) and (III-6): YAibEGT FTSDY SIAibLD KIAQK AFVKW LIAGG PSSGA PPPS-NH2 (II-1) YAibEGT FTSDY SIAibLD KIAQK EFVKW LIAGG PSSGA PPPS-NH2 (II-2) YAibEGT FTSDY SIAibLD KIAQK AFIKW LIAGG PSSGA PPPS-NH2 (II-3) YAibEGT FTSDY SIAibLD KIAQK AFVKW LLAGG PSSGA PPPS-NH2 (II-4) YAibEGT FTSDY SIAibLD KKAQK AFVKW LIAGG PSSGA PPPS-NH2 (II-5) YAibEGT FTSDY SIAibLD KKAQK AFVEW LIAGG PSSGA PPPS-NH2 (III-6) As shown below: The amino groups on the lysine side chains at positions 17 and 20 or the amino groups on the lysine side chains at positions 20 and 24 Concatenated into TIFF0007672503000038.tif11170, Where: Aib, X, X1 and X2 have sequences containing modifications as defined herein.

[0023] In some aspects of the invention, R1 above is selected from H, with the remaining variables being as defined herein.

[0024] In some aspects of the invention, X1 above is selected from -C(=O)-, -OC(=O)-, and -NH-C(=O)-, with the remainder of the variables being as defined herein.

[0025] In some aspects of the invention, m above is selected from 2, with the remaining variables being as defined herein.

[0026] In some aspects of the invention, n above is selected from 17, with the remaining variables being as defined herein.

[0027] In some aspects of the present invention, X2 is TIFF0007672503000039.tif19170, and the remaining variables are as defined herein.

[0028] In some aspects of the invention, the amino groups on the lysine side chains at positions i and i+3 or the amino groups on the lysine side chains at positions j and j+4 are Link to TIFF0007672503000040.tif11170, TIFF0007672503000041.tif48170, where Z2FZ3 is AFV, EFV or AFI, and the remaining variables are as defined herein.

[0029] In some aspects of the invention, the amino groups on the lysine side chains at positions i and i+3 or the amino groups on the lysine side chains at positions j and j+4 are Link to TIFF0007672503000042.tif11170, TIFF0007672503000043.tif96170, with the remaining variables as defined herein.

[0030] In some aspects of the invention, TIFF0007672503000044.tif11170 is TIFF0007672503000045.tif72170, and the remaining variables are as defined herein.

[0031] Some aspects of the present invention further comprise any combination of the above variables.

[0032] The present invention relates to a compound represented by the formula Polypeptides shown at TIFF0007672503000046.tif233170TIFF0007672503000047.tif227170TIFF0007672503000048.tif238170 are further provided.

[0033] The present invention further provides the above pharmaceutical composition, which comprises, as an active ingredient, a therapeutically effective amount of the above polypeptide compound or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0034] In some aspects of the present invention, there is provided a use of the above-mentioned polypeptide compound or a pharma- ceutically acceptable salt thereof, or the above-mentioned composition in the preparation of a medicament for treating NAFLD and NASH.

[0035] In some aspects of the present invention, the above-mentioned polypeptide compound or a pharma- ceutically acceptable salt thereof, or the above-mentioned composition is provided, which is administered once every 3 days, once every 4 days, once every week, or once every 2 weeks. Technical effects

[0036] The polypeptide of the present invention has very strong agonist activity against GLP-1R / GIPR, and the compound of the present invention has excellent pharmacokinetic properties, plasma stability and extremely high plasma protein binding ability, and can significantly improve the NAS score in an STZ-NASH mouse model. Definitions and Explanations

[0037] Unless otherwise specified, the following terms and phrases used herein shall have the following meanings: Certain terms or phrases, unless otherwise defined, should not be considered unclear or unclear, but should be understood in their general sense. When a trade name is mentioned herein, it is intended to refer to the corresponding product or its active ingredient.

[0038] As used herein, the term "pharmacologically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, other problem or complication, and commensurate with a reasonable benefit / risk ratio.

[0039] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention prepared from a compound having certain substituents found in the present invention and a relatively non-toxic acid or base. When a compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting such a compound with a sufficient amount of a base in a pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium salts, potassium salts, calcium salts, ammonium salts, organic amine or magnesium salts or similar salts. When a compound of the present invention contains a relatively basic functional group, an acid addition salt can be obtained by contacting such a compound with a sufficient amount of an acid in a pure solution or in a suitable inert solvent. Examples of pharma- ceutically acceptable acid addition salts include inorganic acid salts, including hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, bisulfate, hydroiodic acid, phosphorous acid, and the like, and organic acid salts, including acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanedioic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like, as well as salts of amino acids (such as arginine) and organic acids such as glucuronic acid. Certain compounds of the present invention contain both basic and acidic functional groups and therefore can be converted into either base or acid addition salts.

[0040] The pharma- ceutically acceptable salts of the present invention can be synthesized from parent compounds containing acids or bases using conventional chemical methods. In general, the method for preparing such salts is by reacting the compounds in their free acid or base form with a stoichiometric amount of an appropriate base or acid in water, an organic solvent, or a mixture thereof.

[0041] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that have been subsequently modified, such as hydroxyproline, gamma-carboxyglutamine, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure (e.g., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group) as a naturally occurring amino acid, 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 chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.

[0042] As used herein, A or Ala has the structure TIFF0007672503000049.tif15170 represents alanine, and R or Arg represents TIFF0007672503000050.tif19170 represents arginine, and N or Asn represents the structure TIFF0007672503000051.tif17170 represents asparagine, and D or Asp represents TIFF0007672503000052.tif17170 represents aspartic acid, and C or Cys represents TIFF0007672503000053.tif17170 represents cysteine, and Q or Gln represents TIFF0007672503000054.tif17170 represents glutamine, and E or Glu represents the structure TIFF0007672503000055.tif17170 represents glutamic acid, and G or Gly has the structure TIFF0007672503000056.tif12170 represents glycine, and H or His represents TIFF0007672503000057.tif18170 represents histidine, and I or Ile represents the structure TIFF0007672503000058.tif19170 represents isoleucine, and L or Leu represents TIFF0007672503000059.tif17170 represents leucine, and K or Lys represents leucine, whose structure is TIFF0007672503000060.tif17170 represents lysine, and M or Met represents TIFF0007672503000061.tif17170 represents methionine, and F or Phe represents TIFF0007672503000062.tif17170 represents phenylalanine, and P or Pro represents TIFF0007672503000063.tif17170 represents proline, and S or Ser represents TIFF0007672503000064.tif17170 represents serine, and T or Thr represents the structure TIFF0007672503000065.tif17170 represents threonine, and W or Trp represents TIFF0007672503000066.tif21170 represents tryptophan, and Y or Tyr represents TIFF0007672503000067.tif19170 represents tyrosine, and V or Val represents TIFF0007672503000068.tif17170 represents valine, and Fmoc-AEEA-OH represents Represents TIFF0007672503000069.tif13170.

[0043] The term "treatment" includes preventing, slowing, halting or reversing the progression or severity of an existing condition or disease.

[0044] Unless otherwise specified, the term "isomer" is intended to include geometric isomers, cis-trans isomers, stereoisomers, enantiomers, optical isomers, diastereomers and tautomers.

[0045] The compounds of the present invention can exist in specific geometric or stereoisomeric forms. The present invention contemplates all of these compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, such as enantiomer- or diastereomer-enriched mixtures, and all of these mixtures are within the scope of the present invention. Substituents such as alkyl may have additional asymmetric carbon atoms. All of these isomers and mixtures thereof are within the scope of the present invention.

[0046] Unless otherwise specified, the terms "enantiomers" or "optical isomers" refer to stereoisomers that are mirror images of each other.

[0047] Unless otherwise noted, the term "cis-trans isomers" or "geometric isomers" refers to isomers resulting from the inability to freely rotate about a double bond or a single bond of a ring-forming carbon atom.

[0048] Unless otherwise indicated, the term "diastereomer" means a stereoisomer whose molecules have two or more centers of chirality and which are not mirror-image related to each other.

[0049] Unless otherwise specified, "(+)" denotes dextrorotatory, "(-)" denotes levorotatory, and "(±)" denotes racemic.

[0050] Unless otherwise noted, TIFF0007672503000070.tif5170, which represents the absolute configuration of one stereocenter, TIFF0007672503000071.tif6170 shows the relative configuration of stereocenters, TIFF0007672503000072.tif6170, or Represents TIFF0007672503000073.tif6170.

[0051] Unless otherwise specified, the terms "enriched in one isomer," "enriched in one enantiomer," or "enantiomerically enriched" mean that the content of one isomer or enantiomer is less than 100% and is 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.

[0052] Unless otherwise specified, the term "isomeric excess" or "enantiomeric excess" refers to the relative percentage difference between two isomers or two enantiomers. For example, if one isomer or enantiomer is present at 90% and the other isomer or enantiomer at 10%, then the isomeric or enantiomeric excess (ee value) is 80%.

[0053] Optically active (R)- and (S)-isomers, as well as D- and L-isomers, can be prepared using chiral synthesis, chiral reagents, or other conventional techniques. A single enantiomer of a compound of the invention can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary is cleaved to provide the desired pure enantiomer. Alternatively, if the molecule contains a basic (e.g., amino) or acidic (e.g., carboxy) functional group, the diastereomeric salt can be formed with an appropriate optically active acid or base, followed by separation of the diastereomers by conventional methods known in the art, and then recovery to provide the pure enantiomers. Separation of enantiomers and diastereomers is also typically accomplished by chromatographic methods using chiral stationary phases, and optionally in combination with chemical derivatization methods (e.g., carbamate formation from amines).

[0054] The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, tritium ( 3 H), iodine-125( 125 I) or C-14( 14 Compounds can be labeled with radioisotopes such as C). For example, hydrogen can be replaced with deuterium to form a deuterated drug, and the bond between deuterium and carbon is stronger than the bond between normal hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs have the advantages of reduced toxicity and side effects, increased stability of the drug, enhanced therapeutic effect, and extended biological half-life of the drug. All isotopic constitutional changes of the compounds of the present invention, whether radioactive or not, are all within the scope of the present invention.

[0055] In the case of the linking groups listed, if the linking direction is not indicated, the linking direction is arbitrary, for example When the linking group L in TIFF0007672503000074.tif13170 is -MW-, -MW- links ring A and ring B in the same direction as the reading order from left to right. You can construct TIFF0007672503000075.tif16170 and concatenate rings A and B in the opposite left-to-right reading order: TIFF0007672503000076.tif16170 Combinations of the above linking groups, substituents and / or variants thereof are permissible only if such combinations result in stable compounds.

[0056] Unless otherwise specified, when a group has one or more linkable sites, any one or more of the sites of the group can be linked to other groups via chemical bonds. If the linking mode of the chemical bond is non-directional and there is an H atom at the linkable site, when the chemical bond is linked, the number of H atoms at the site is reduced according to the number of linked chemical bonds to form a group of the corresponding valence. The chemical bond between the above site and other groups is as follows: For example, the linear solid bond -OCH3 represents a bond to another group via the oxygen atom of that group, The straight dashed bond in TIFF0007672503000078.tif8170 represents a bond between the nitrogen atom of the group and other groups at both ends. The wavy lines in TIFF0007672503000079.tif13170 represent bonds to other groups via the 1st and 2nd carbon atoms in the phenyl group.

[0057] Unless otherwise specified, "C 1-3 The term "alkyl" is used to denote a linear or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms. 1-3 Alkyl is C 1-2 and C 2-3 alkyl, etc., and may be monovalent (e.g., methyl), divalent (e.g., methylene) or polyvalent (e.g., methylidyne). 1-3 Examples of alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl) groups, and the like.

[0058] The structure of the compound of the present invention can be confirmed by conventional methods known to those skilled in the art, and if the present invention relates to the absolute configuration of a compound, its absolute configuration can be confirmed by conventional technical means in the art. For example, in the case of single crystal X-ray diffraction (SXRD), the diffraction intensity data is collected in φ / ω scanning mode using a Bruker D8 venture diffractometer with CuKα radiation as a light source for the cultivated single crystal, and after collecting the relevant data, the crystal structure is further analyzed by a direct method (Shelxs97) to confirm the absolute configuration.

[0059] The compounds of the present invention can be prepared by various synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments in combination with other chemical synthetic methods, and equivalent alternative methods known to those skilled in the art, and preferred embodiments include, but are not limited to, the examples of the present invention.

[0060] The solvents used in the present invention can be obtained commercially.

[0061] In the present invention, the following abbreviations are used: aq stands for water, eq stands for equivalent, DCM stands for dichloromethane, PE stands for petroleum ether, DMSO stands for dimethylsulfoxide, MeOH stands for methanol, BOC stands for tert-butoxycarbonyl, an amine protecting group, rt stands for room temperature, O / N stands for overnight, THF stands for tetrahydrofuran, Boc2O stands for di-tert-butyl dicarbonate, rt-butyl ether, TFA represents trifluoroacetic acid, DIEA represents diisopropylethylamine, DMF represents N,N-dimethylformamide, HBTU represents benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluorophosphate, HOBT represents 1-hydroxybenzotriazole, HOAT represents 1-hydroxy-7-azabenzotriazole, DIC represents N,N'-diisopropylcarbodiimide, DBU represents 1,8-diazabicyclo[5.4.0]undec-7-ene, PhSiH3 represents phenylsilane, and Pd(PPh3)4 represents tetrakis(triphenylphosphine)palladium. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] The present invention will be described in detail below with reference to examples, but is not intended to have any adverse limitations to the present invention. In this specification, the present invention is described in detail, but specific embodiments thereof are also disclosed, and it is clear to those skilled in the art that various changes and improvements can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention. Intermediate A-1 TIFF0007672503000080.tif119170Step 1: Resin fixation 1.1 Weigh out 4 g of chloro(o-chlorophenyl)diphenylmethane resin (degree of substitution S = 1.00 mmol / g) and 1.54 g of A-1_1 and place them in a reaction column, add DCM (25 mL) thereto, then add 3 mL of N,N-diisopropylethylamine to the reaction column and bubble nitrogen gas for 2 hours, then add 4 mL of MeOH to the reaction column and continue to bubble nitrogen gas for 30 minutes, drain the waste liquid until no liquid comes out, add DMF (50 mL) and wash 5 times, 1 minute each time, and drain the waste liquid until no liquid comes out. 1.2 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. Step 2: Amino acid coupling 2.1 A-1_1 coupling 1. Weigh out A-1_1 (3.0 eq) and add it to the above resin, add DIEA (6.00 eq) and 20 mL of DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid has dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 2. The reaction was carried out at 25°C for 20 minutes, and ninhydrin was used for detection. The resin was colorless and transparent. 3. The reaction solution was removed and washed with DMF five times for 1 minute each time, and the waste liquid was drained until no more liquid came out. 2.2 A-1_a coupling 1. 20% piperidine / DMF (50 mL) was placed in a reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no more liquid was produced. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out A-1_a (3.0 eq) and add it to the above resin, add DIEA (3.00 eq) and 20 mL of DMF to the reaction column, and then aerate with nitrogen gas. After the amino acid has dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed five times with DMF (50 mL) for 1 minute each time, and the waste liquid was drained until no more liquid came out. 2.3 A-1_b coupling 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, the waste liquid was drained until no liquid came out, and DMF (50 mL) was added to wash 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out A-1_b (3.0 eq) and add it to the above resin, add DIEA (6.00 eq) and 20 mL of DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid has dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF five times for 1 minute each time, and the waste liquid was drained until no more liquid came out. Step 3: Cleavage and drying of crude peptide 3.1 The cutting solution was prepared in the following volumes: TIFF0007672503000081.tif241703.2 60 mL of the prepared cleavage solution was poured into a reactor containing dried peptide resin, the reactor was vented for 20 minutes, filtered, and the filtrate was added to a flask. This operation was repeated twice, and the cleavage solutions collected in the two operations were spin-dried to obtain A-1. Intermediate A-2 Intermediate A-2 was obtained by referring to the synthesis of intermediate A-1 in TIFF0007672503000082.tif27170. Intermediate A-3 Intermediate A-3 was obtained by referring to the synthesis of intermediate A-1 in TIFF0007672503000083.tif28170. Intermediate A-4 Intermediate A-4 was obtained by referring to the synthesis of intermediate A-1 in TIFF0007672503000084.tif28170.

[0063] Example 1 TIFF0007672503000085.tif54170

[0064] Step 1: 1.34 g of 4-(2',4'-dimethoxyphenyl-fluorenemethoxycarbonyl-aminomethyl)-phenoxyacetamide-methylbenzhydrylamine resin (degree of substitution Sub=0.3 mmol / g) was weighed and placed in a reaction column, DMF (50 mL) was added to the reaction column, nitrogen gas was passed through for 2 hours, and the waste liquid was discharged until no liquid was discharged, DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was discharged until no liquid was discharged.

[0065] Step 2: 20% piperidine / DMF (50 mL) was added to the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no more liquid came out. DMF (50 mL) was added to wash 5 times, 1 minute each time, and the waste liquid was drained until no more liquid came out. Ninhydrin was used for detection, and the resin appeared blue.

[0066] Step 3: Amino acid coupling

[0067] 3.1 Coupling of Fmoc-Ser(tBu)-OH 1. Weigh out Fmoc-Ser(tBu)-OH (3.0 eq) and add it to the above resin, add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin swelled evenly. 2. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 3. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0068] 3.2 Coupling of Fmoc-Pro-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Pro-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0069] 3.3 Coupling of Fmoc-Pro-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Chloranil was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Pro-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, chloranil was used for detection, and the resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF five times for 1 minute each time, and the waste liquid was drained until no more liquid came out.

[0070] 3.4 Coupling of Fmoc-Pro-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Chloranil was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Pro-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, chloranil was used for detection, and the resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0071] 3.5 Coupling of Fmoc-Ala-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Chloranil was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ala-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, chloranil was used for detection, and the resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0072] 3.6 Coupling of Fmoc-Gly-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Gly-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0073] 3.7 Coupling of Fmoc-Ser(tBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ser(tBu)-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would swell evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0074] 3.8 Coupling of Fmoc-Ser(tBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ser(tBu)-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would swell evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0075] 3.9 Coupling of Fmoc-Pro-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Pro-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0076] 3.10 Coupling of Fmoc-Gly-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Chloranil was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Gly-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, chloranil was used for detection, and the resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0077] 3.11 Coupling of Fmoc-Gly-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Gly-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0078] 3.12 Coupling of Fmoc-Ala-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ala-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0079] 3.13 Coupling of Fmoc-Ile-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ile-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0080] 3.14 Coupling of Fmoc-Leu-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Leu-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0081] 3.15 Coupling of Fmoc-Trp(Boc)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Trp(Boc)-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0082] 3.16 Coupling of Fmoc-Lys(Dde)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Lys(Dde)-OH (3.0 eq) and add it to the above resin, add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0083] 3.17 Coupling of Fmoc-Val-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Val-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0084] 3.18 Coupling of Fmoc-Phe-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Phe-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0085] 3.19 Coupling of Fmoc-Ala-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ala-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0086] 3.20 Coupling of Fmoc-Lys(Alloc)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Lys(Alloc)-OH (2.0 eq) and add it to the above resin, then add HOBT (2.00 eq) and 10 mL DMF to the reaction column, and after the amino acid and HOBT are dissolved, add DIC (2.00 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0087] 3.21 Coupling of Fmoc-Gln(Trt)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Gln(Trt)-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0088] 3.22 Coupling of Fmoc-Ala-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ala-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0089] 3.23 Coupling of Fmoc-Ile-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ile-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0090] 3.24 Coupling of Fmoc-Lys(Boc)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Lys(Boc)-OH (2.0 eq) and add it to the above resin, add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0091] 3.25 Coupling of Fmoc-Asp(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Asp(OtBu)-OH (6.0 eq) and add it to the above resin, add DIEA (12.0 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HATU (5.70 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0092] 3.26 Coupling of Fmoc-Leu-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Leu-OH (6.0 eq) and add it to the resin above, then add DIEA (12.0 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (5.70 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0093] 3.27 Coupling of Fmoc-Aib-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Aib-OH (6.0 eq) and add it to the resin above, then add DIEA (12.0 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (5.70 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 2 hours, and ninhydrin was used for detection; the resin appeared blue. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0094] 3.28 Coupling of Fmoc-Ile-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ile-OH (6.0 eq) and add it to the above resin, then add HOAT (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid and HOAT are dissolved, add DIC (6.00 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 1 hour, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0095] 3.29 Coupling of Fmoc-Ser(tBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ser(tBu)-OH (6.0 eq) and add it to the resin above, then add DIEA (12.0 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (5.70 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0096] 3.30 Coupling of Fmoc-Tyr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Tyr(tBu)-OH (6.0 eq) and add it to the resin above, then add DIEA (12.0 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (5.70 eq). The nitrogen gas was adjusted so that the resin swelled evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0097] 3.31 Coupling of Fmoc-Asp(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Asp(OtBu)-OH (6.0 eq) and add it to the above resin, add DIEA (12.0 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HATU (5.70 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0098] 3.32 Coupling of Fmoc-Ser(tBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ser(tBu)-OH (6.0 eq) and add it to the resin above, then add DIEA (12.0 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (5.70 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0099] 3.33 Coupling of Fmoc-Thr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Thr(tBu)-OH (6.0 eq) and add it to the resin above, then add DIEA (12.0 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (5.70 eq). The nitrogen gas was adjusted so that the resin would swell evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0100] 3.34 Coupling of Fmoc-Phe-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Phe-OH (6.0 eq) and add it to the resin above, then add DIEA (12.0 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (5.70 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0101] 3.35 Coupling of Fmoc-Thr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Thr(tBu)-OH (6.0 eq) and add it to the resin above, then add DIEA (12.0 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (5.70 eq). The nitrogen gas was adjusted so that the resin would swell evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0102] 3.36 Coupling of Fmoc-Gly-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Gly-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0103] 3.37 Coupling of Fmoc-Glu(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Glu(OtBu)-OH (6.0 eq) and add it to the resin above. Add HOBT (6.00 eq) and 10 mL DMF to the reaction column. Bubble nitrogen gas. After the amino acid and HOBT were dissolved, add DIC (6.00 eq). The nitrogen gas was adjusted so that the resin swelled evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0104] 3.38 Coupling of Fmoc-Aib-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Aib-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out overnight at 25°C, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0105] 3.39 Coupling of Boc-Tyr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Chloranil was used for detection, and the resin appeared blue. 2. Weigh out Boc-Tyr(tBu)-OH (6.0 eq) and add it to the resin above, then add DIEA (12.0 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (5.70 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, chloranil was used for detection, and the resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0106] 3.40 De-Alloc 1. PhSiH3 (10.0 eq) and DCM (10 mL) were placed in a reaction column, and nitrogen gas was passed through it. Then, Pd(PPh3)4 (0.1 eq) was added and nitrogen gas was passed through it for 20 minutes. The reaction was carried out twice, and the waste liquid was discharged until no more liquid was produced. 2. Wash with DMF 5 times (50 mL each time, 1 min each time) and drain the waste liquid until no more liquid came out.

[0107] 3.41 Coupling of Fmoc-Ida-OH 1. Weigh out Fmoc-Ida-OH (4.0 eq) and add it to the above resin, add DIEA (8.00 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HBTU (3.80 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 2. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 3. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0108] 3.42 Coupling of intermediate A-1 1. 10% DBU / DMF (50mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Chloranil was used for detection, and the resin appeared blue. 2. Weigh out 1.50 eq of intermediate A-1 and add it to the resin. Then add 3.00 eq of DIEA and 10 mL of DMF to the reaction column. After the amino acid was dissolved, add 1.45 eq of HBTU. The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0109] 3.43 De-Dde 1. Add 3% hydrazine / DMF (50mL) to the reaction column, pass nitrogen gas through it for 15 minutes, drain the waste liquid, add DMF (50mL) to wash 5 times, 1 minute each time, drain the waste liquid until no liquid comes out. Ninhydrin was used for detection, and the resin appeared blue.

[0110] 3.44 Amide ring closure 1. DIEA (3.0 eq) was added to the DMF solution of the above resin, and then HATU (1.5 eq) dissolved in DMF was slowly dropped into the reaction column, and nitrogen gas was passed through. The nitrogen gas was adjusted so that the resin swelled uniformly. 2. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 3. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out. 4. The resin was shrunk with MeOH (50 mL) for 3 min each time, drained until no more liquid came out, and the resin was removed, dried, and stored as a spare.

[0111] 4. Cleavage and Drying of Crude Peptide

[0112] 4.1 The cleavage solution was prepared in the following volumes: TIFF0007672503000086.tif30170

[0113] 4.2 The dried peptide resin was placed in the prepared cleavage solution, shaken on a shaker for 2.5 hours, filtered, the filtrate was added to 10 times the amount of ice-cold isopropyl ether, centrifuged, and washed with isopropyl ether five times. The crude peptide was obtained by vacuum drying for 2 hours, and purified to obtain polypeptide compound WX-001. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1228.6, and the detected value was 1228.7.

[0114] Example 2 Polypeptide WX-002 was obtained by referring to the synthesis of TIFF0007672503000087.tif54170WX-001. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1243.1, and the detected value was 1242.8.

[0115] Example 3 Polypeptide WX-003 was obtained by referring to the synthesis of TIFF0007672503000088.tif57170WX-001. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1232.1, and the detected value was 1232.2.

[0116] Example 4 Polypeptide WX-004 was obtained by referring to the synthesis of TIFF0007672503000089.tif57170WX-001. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1228.6, and the detected value was 1228.2.

[0117] Example 5 TIFF0007672503000090.tif57170

[0118] Step 1: 1.34g of 4-(2',4'-dimethoxyphenyl-fluorenemethoxycarbonyl-aminomethyl)-phenoxyacetamide-methylbenzhydrylamine resin (degree of substitution Sub=0.3mmol / g) was weighed and placed in a reaction column, DMF (50mL) was added to the reaction column, nitrogen gas was passed through for 2 hours, and the waste liquid was discharged until no liquid was discharged, DMF (50mL) was added and washed 5 times, 1 minute each time, and the waste liquid was discharged until no liquid was discharged.

[0119] Step 2: 20% piperidine / DMF (50 mL) was added to the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no more liquid came out. DMF (50 mL) was added to wash 5 times, 1 minute each time, and the waste liquid was drained until no more liquid came out. Ninhydrin was used for detection, and the resin appeared blue.

[0120] Step 3: Amino acid coupling

[0121] 3.1 Coupling of Fmoc-Ser(tBu)-OH 1. Weigh out Fmoc-Ser(tBu)-OH (3.0 eq) and add it to the above resin, add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin swelled evenly. 2. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 3. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0122] 3.2 Coupling of Fmoc-Pro-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Pro-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out. 5. Step 3.2 was repeated to complete the coupling of the following amino acids: TIFF0007672503000091.tif233170

[0123] 3.40 De-Alloc 1. PhSiH3 (10.0 eq) and DCM (10 mL) were placed in a reaction column, and nitrogen gas was passed through it. Then, Pd(PPh3)4 (0.1 eq) was added and nitrogen gas was passed through it for 20 minutes. The reaction was carried out twice, and the waste liquid was discharged until no more liquid was produced. 2. Wash with DMF 5 times (50 mL each time, 1 min each time) and drain the waste liquid until no more liquid came out.

[0124] 3.41 Coupling of Fmoc-Ida-OH 1. Weigh out Fmoc-Ida-OH (4.0 eq) and add it to the above resin, add DIEA (8.00 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HBTU (3.80 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 2. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 3. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0125] 3.42 Coupling of intermediate A-1 1. 10% DBU / DMF (50mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Chloranil was used for detection, and the resin appeared blue. 2. Weigh out 1.50 eq of intermediate A-1 and add it to the resin. Then add 3.00 eq of DIEA and 10 mL of DMF to the reaction column. After the amino acid was dissolved, add 1.45 eq of HBTU. The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0126] 3.43 De-Dde 1. 3% hydrazine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 15 minutes, the waste liquid was drained, and DMF (50 mL) was added to wash 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue.

[0127] 3.44 Amide ring closure 1. DIEA (3.0 eq) was added to the DMF solution of the above resin, and then HATU (1.5 eq) dissolved in DMF was slowly dropped into the reaction column, and nitrogen gas was passed through. The nitrogen gas was adjusted so that the resin swelled uniformly. 2. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 3. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out. 4. The resin was shrunk with MeOH (50 mL) for 3 min each time, drained until no more liquid came out, and the resin was removed, dried, and stored as a spare.

[0128] 4. Cleavage and Drying of Crude Peptide

[0129] 4.1 The cutting solution was prepared in the following volumes: TIFF0007672503000092.tif30170

[0130] 4.2 The dried peptide resin was placed in the prepared cleavage solution, shaken on a shaker for 2.5 hours, filtered, and the filtrate was added to 10 times the amount of ice-cold isopropyl ether, centrifuged, and washed five times with isopropyl ether. The crude peptide was obtained by vacuum drying for 2 hours, and purified to obtain polypeptide WX-005. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1232.6, and the detected value was 1232.5.

[0131] Example 6 Polypeptide WX-006 was obtained by referring to the synthesis of TIFF0007672503000093.tif51170WX-001. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1232.4, and the detected value was 1232.4.

[0132] Example 7 Polypeptide WX-007 was obtained by referring to the synthesis of TIFF0007672503000094.tif50170WX-001. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1218.1, and the detected value was 1218.3.

[0133] Example 8 Polypeptide WX-008 was obtained by referring to the synthesis of TIFF0007672503000095.tif51170WX-001. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1228.6, and the detected value was 1228.6.

[0134] Example 9 TIFF0007672503000096.tif52170

[0135] Step 1: 1.34g of 4-(2',4'-dimethoxyphenyl-fluorenemethoxycarbonyl-aminomethyl)-phenoxyacetamide-methylbenzhydrylamine resin (degree of substitution Sub=0.3mmol / g) was weighed and placed in a reaction column, DMF (50mL) was added to the reaction column, nitrogen gas was passed through for 2 hours, and the waste liquid was discharged until no liquid was discharged, DMF (50mL) was added and washed 5 times, 1 minute each time, and the waste liquid was discharged until no liquid was discharged.

[0136] Step 2: 20% piperidine / DMF (50 mL) was added to the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no more liquid came out. DMF (50 mL) was added to wash 5 times, 1 minute each time, and the waste liquid was drained until no more liquid came out. Ninhydrin was used for detection, and the resin appeared blue.

[0137] Step 3: Amino acid coupling

[0138] 3.1 Coupling of Fmoc-Ser(tBu)-OH 1. Weigh out Fmoc-Ser(tBu)-OH (3.0 eq) and add it to the above resin, add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin swelled evenly. 2. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 3. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0139] 3.2 Coupling of Fmoc-Pro-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Pro-OH (3.0 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out. 5. Step 3.2 was repeated to complete the coupling of the following amino acids: TIFF0007672503000097.tif233170

[0140] 3.40 De-Alloc 1. PhSiH3 (10.0 eq) and DCM (10 mL) were placed in a reaction column, and nitrogen gas was passed through it. Then, Pd(PPh3)4 (0.1 eq) was added and nitrogen gas was passed through it for 20 minutes. The reaction was carried out twice, and the waste liquid was discharged until no more liquid was produced. 2. Wash with DMF 5 times (50 mL each time, 1 min each time) and drain the waste liquid until no more liquid came out.

[0141] 3.41 Coupling of Fmoc-Ida-OH 1. Weigh out Fmoc-Ida-OH (4.0 eq) and add it to the above resin, add DIEA (8.00 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HBTU (3.80 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 2. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 3. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0142] 3.42 Coupling of intermediate A-1 1. 10% DBU / DMF (50mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Chloranil was used for detection, and the resin appeared blue. 2. Weigh out 1.50 eq of intermediate A-1 and add it to the resin. Then add 3.00 eq of DIEA and 10 mL of DMF to the reaction column. After the amino acid was dissolved, add 1.45 eq of HBTU. The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0143] 3.43 De-Dde 1. 3% hydrazine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 15 minutes, the waste liquid was drained, and DMF (50 mL) was added to wash 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue.

[0144] 3.44 Amide ring closure 1. DIEA (3.0 eq) was added to the DMF solution of the above resin, and then HATU (1.5 eq) dissolved in DMF was slowly dropped into the reaction column, and nitrogen gas was passed through. The nitrogen gas was adjusted so that the resin swelled uniformly. 2. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 3. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out. 4. The resin was shrunk with MeOH (50 mL) for 3 min each time, drained until no more liquid came out, and the resin was removed, dried, and stored as a spare.

[0145] 4. Cleavage and Drying of Crude Peptide

[0146] 4.1 The cleavage solution was prepared in the following volumes: TIFF0007672503000098.tif30170

[0147] 4.2 The dried peptide resin was placed in the prepared cleavage solution, shaken on a shaker for 2.5 hours, filtered, and the filtrate was added to 10 times the amount of ice-cold isopropyl ether, centrifuged, and washed five times with isopropyl ether. The crude peptide was obtained by vacuum drying for 2 hours, and purified to obtain polypeptide WX-009. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1247.1, and the detected value was 1247.2.

[0148] Example 10 TIFF0007672503000099.tif59170

[0149] Step 1: 1.08g of 4-(2',4'-dimethoxyphenyl-fluorenemethoxycarbonyl-aminomethyl)-phenoxyacetamide-methylbenzhydrylamine resin (degree of substitution Sub=0.37mmol / g) was weighed and placed in a reaction column, DMF (50mL) was added to the reaction column, nitrogen gas was passed through for 2 hours, and the waste liquid was discharged until no liquid was discharged, DMF (50mL) was added and washed 5 times, 1 minute each time, and the waste liquid was discharged until no liquid was discharged.

[0150] Step 2: 20% piperidine / DMF (50 mL) was added to the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no more liquid came out. DMF (50 mL) was added to wash 5 times, 1 minute each time, and the waste liquid was drained until no more liquid came out. Ninhydrin was used for detection, and the resin appeared blue.

[0151] Step 3: Amino acid coupling

[0152] 3.1 Coupling of Fmoc-Ser(tBu)-OH 1. Weigh out Fmoc-Ser(tBu)-OH (3.00 eq) and add it to the above resin, add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin swelled evenly. 2. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 3. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0153] 3.2 Coupling of Fmoc-Pro-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Pro-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0154] 3.3 Coupling of Fmoc-Pro-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Chloranil was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Pro-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, chloranil was used for detection, and the resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0155] 3.4 Coupling of Fmoc-Pro-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Chloranil was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Pro-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, chloranil was used for detection, and the resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0156] 3.5 Coupling of Fmoc-Ala-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Chloranil was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ala-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would swell evenly. 3. The reaction was carried out at 25°C for 0.5 hours, chloranil was used for detection, and the resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0157] 3.6 Coupling of Fmoc-Gly-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Gly-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would swell evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0158] 3.7 Coupling of Fmoc-Ser(tBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ser(tBu)-OH (3.00 eq) and add it to the above resin, add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0159] 3.8 Coupling of Fmoc-Ser(tBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ser(tBu)-OH (3.00 eq) and add it to the above resin, add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0160] 3.9 Coupling of Fmoc-Pro-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Pro-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0161] 3.10 Coupling of Fmoc-Gly-Gly-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Chloranil was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Gly-Gly-OH (3.00 eq) and add it to the above resin, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, chloranil was used for detection, and the resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0162] 3.11 Coupling of Fmoc-Ala-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ala-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would swell evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0163] 3.12 Coupling of Fmoc-Ile-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ile-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin swelled evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0164] 3.13 Coupling of Fmoc-Leu-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Leu-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0165] 3.14 Coupling of Fmoc-Trp(Boc)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Trp(Boc)-OH (3.00 eq) and add it to the above resin, add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0166] 3.15 Coupling of Fmoc-Glu(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Glu(OtBu)-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0167] 3.16 Coupling of Fmoc-Val-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Val-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0168] 3.17 Coupling of Fmoc-Phe-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Phe-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0169] 3.18 Coupling of Fmoc-Ala-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ala-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would swell evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0170] 3.19 Coupling of Fmoc-Lys(Alloc)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Lys(Alloc)-OH (3.00 eq) and add it to the above resin, add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0171] 3.20 Coupling of Fmoc-Gln(Trt)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Gln(Trt)-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0172] 3.21 Coupling of Fmoc-Ala-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ala-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HBTU (2.85 eq). The nitrogen gas was adjusted so that the resin would swell evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0173] 3.22 Coupling of Fmoc-Lys(Dde)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Lys(Dde)-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (2.85 eq). The nitrogen gas was adjusted so that the resin would swell evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0174] 3.23 Coupling of Fmoc-Lys(Boc)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Lys(Boc-OH (3.00 eq) and add it to the above resin. Add DIEA (6.00 eq) and 10 mL DMF to the reaction column. Bubble nitrogen gas. After the amino acid is dissolved, add HATU (2.85 eq). The nitrogen gas was adjusted so that the resin swelled evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0175] 3.24 Coupling of Fmoc-Asp(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Asp(OtBu)-OH (3.00 eq) and add it to the above resin, add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HATU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0176] 3.25 Coupling of Fmoc-Leu-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Leu-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0177] 3.26 Coupling of Fmoc-Aib-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Aib-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 2 hours, and ninhydrin was used for detection; the resin appeared blue. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0178] 3.27 Coupling of Fmoc-Ile-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ile-OH (3.00 eq) and add it to the above resin, add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HATU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 1 hour, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0179] 3.28 Coupling of Fmoc-Ser(tBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Ser(tBu)-OH (3.00 eq) and add it to the above resin, add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HATU (2.85 eq). The nitrogen gas was adjusted so that the resin swelled evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0180] 3.29 Coupling of Fmoc-Tyr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Tyr(tBu)-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0181] 3.30 Coupling of Fmoc-Asp(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Asp(OtBu)-OH (3.00 eq) and add it to the above resin, add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HATU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0182] 3.31 Coupling of Fmoc-Thr(tBu)-SerPsi(Me,Me)Pro-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Thr(tBu)-SerPsi(Me,Me)Pro-OH (2.00 eq) and add it to the above resin, then add DIEA (4.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (1.90 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0183] 3.32 Coupling of Fmoc-Phe-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Phe-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0184] 3.33 Coupling of Fmoc-Thr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Thr(tBu)-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0185] 3.34 Coupling of Fmoc-Gly-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Gly-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (2.85 eq). The nitrogen gas was adjusted so that the resin would swell evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0186] 3.35 Coupling of Fmoc-Glu(OtBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Glu(OtBu)-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (2.85 eq). The nitrogen gas was adjusted so that the resin would swell evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0187] 3.36 Coupling of Fmoc-Aib-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out Fmoc-Aib-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out overnight at 25°C, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0188] 3.37 Coupling of Boc-Tyr(tBu)-OH 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Chloranil was used for detection, and the resin appeared blue. 2. Weigh out Boc-Tyr(tBu)-OH (3.00 eq) and add it to the resin above, then add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (2.85 eq). The nitrogen gas was adjusted so that the resin would swell evenly. 3. The reaction was carried out at 25°C for 0.5 hours, chloranil was used for detection, and the resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0189] 3.38 De-Alloc 1. PhSiH3 (10.0 eq) and DCM (20 mL) were placed in a reaction column, and nitrogen gas was passed through it. Then, Pd(PPh3)4 (0.10 eq) was added and nitrogen gas was passed through it for 20 minutes. The reaction was carried out twice, and the waste liquid was discharged until no more liquid was produced. 2. Wash with DMF 5 times (50 mL each time, 1 min each time) and drain the waste liquid until no more liquid came out.

[0190] 3.39 Coupling of Fmoc-Glu-OAll 1. Weigh out Fmoc-Glu-OAll (3.00 eq) and add it to the above resin, add DIEA (6.00 eq) and 10 mL DMF to the reaction column, and then pass nitrogen gas through the column. After the amino acid is dissolved, add HATU (2.85 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 2. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 3. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0191] 3.40 Coupling of intermediate A-1 1. 20% piperidine / DMF (50 mL) was placed in the reaction column, nitrogen gas was bubbled through for 20 minutes, and the waste liquid was drained until no liquid came out. DMF (50 mL) was added and washed 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue. 2. Weigh out intermediate A-1 (1.50 eq) and add it to the above resin, then add DIEA (3.00 eq) and 10 mL DMF to the reaction column, and after the amino acid is dissolved, add HATU (1.45 eq). The nitrogen gas was adjusted so that the resin would expand evenly. 3. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 4. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out.

[0192] 3.41 De-Alloc 1. PhSiH3 (10.0 eq) and DCM (20 mL) were placed in a reaction column, and nitrogen gas was passed through it. Then, Pd(PPh3)4 (0.10 eq) was added and nitrogen gas was passed through it for 20 minutes. The reaction was carried out twice, and the waste liquid was discharged until no more liquid was produced. 2. Wash with DMF 5 times (50 mL each time, 1 min each time) and drain the waste liquid until no more liquid came out.

[0193] 3.42 De-Dde 1. 3% hydrazine hydrate / DMF (50 mL) was placed in a reaction column, nitrogen gas was bubbled through for 15 minutes, the waste liquid was drained, and DMF (50 mL) was added to wash 5 times, 1 minute each time, and the waste liquid was drained until no liquid came out. Ninhydrin was used for detection, and the resin appeared blue.

[0194] 3.42 Amide ring closure 1. DIEA (3.00 eq) was added to the DMF solution of the above resin, and then HATU (1.50 eq) dissolved in DMF was slowly dropped into the reaction column, and nitrogen gas was passed through. The nitrogen gas was adjusted so that the resin swelled uniformly. 2. The reaction was carried out at 25°C for 0.5 hours, and ninhydrin was used for detection. The resin was colorless and transparent. 3. The reaction solution was removed and washed with DMF 5 times (50 mL each time, 1 minute each time), and the waste liquid was drained until no more liquid came out. 4. The resin was shrunk with MeOH (50 mL) for 3 min each time, drained until no more liquid came out, and the resin was removed, dried, and stored as a spare.

[0195] 4. Cleavage and Drying of Crude Peptide

[0196] 4.1 The cleavage solution was prepared in the following volumes: TIFF0007672503000100.tif30170

[0197] 4.2 The dried peptide resin was placed in the prepared cleavage solution, shaken on a shaker for 2.5 hours, filtered, and the filtrate was added to 10 times the amount of ice-cold isopropyl ether, centrifuged, and washed five times with isopropyl ether. The crude peptide was obtained by vacuum drying for 2 hours and purified. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value was 1236.1, and the detected value was 1236.0.

[0198] Example 11 Polypeptide WX-011 was obtained by referring to the synthesis of TIFF0007672503000101.tif66170WX-010. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1236.1, and the detected value was 1236.0.

[0199] Example 12 Polypeptide WX-012 was obtained using intermediate A-2 by referring to the synthesis of TIFF0007672503000102.tif57170WX-001. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1225.6, and the detected value was 1225.7.

[0200] Example 13 Polypeptide WX-013 was obtained using intermediate A-3 by referring to the synthesis of TIFF0007672503000103.tif64170WX-001. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1264.9, and the detected value was 1264.6.

[0201] Example 14 Polypeptide WX-014 was obtained using intermediate A-4 by referring to the synthesis of TIFF0007672503000104.tif59170WX-001. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1257.9, and the detected value was 1257.8.

[0202] Biological Test Data

[0203] Test Example 1: In vitro GLP-1R / GIPR agonist activity test

[0204] A:Main materials: 1) Cell line This cell line was constructed by Wuxi APPTEC (Shanghai) Co., Ltd. For details, see Table 1 below. Table 1 Cell line information TIFF0007672503000105.tif191702) Reagents and Consumables Table 2. Reagent and consumable information TIFF0007672503000106.tif741703) Equipment Table 3 Equipment information TIFF0007672503000107.tif39170

[0205] B. Method 1) Experimental materials Experimental buffer Table 4. Buffer information TIFF0007672503000108.tif55170 Preparation of assay reagents Table 5. Assay Reagent Information TIFF0007672503000109.tif291702) Experimental method a) Preparation of compound plates: Test compounds were diluted 4-fold in 10-point runs from an initial concentration of 30 μM, performed by Bravo. b) Transport of compounds: 1) 100 nL of compound was transferred to an OptiPlate-384 plate using the Echo. 2) The OptiPlate-384 plate was centrifuged at 1000 rpm for 5 seconds. c) Preparation of cell suspension 1) One of the cryopreserved vials of GLP-1R / GIPR cells was rapidly thawed in 37°C warm water. 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 room temperature at 1000 rpm for 1 minute. 4) The supernatant was discarded. 5) The cells at the bottom were gently dispersed, then gently washed with 10 mL HBSS, the cells were centrifuged to sediment, and finally the cells were resuspended in the experiment buffer. 6) Cell density and viability were measured using Vi-cell. 7) GLP-1R / GIPR cells at a concentration of 2.0*10 5 The solution was diluted with the experimental buffer to give a concentration of 1 / mL. 8) 100 nL of the diluted cell suspension was transferred to an OptiPlate-384 plate. 9) Incubate at room temperature for 30 minutes. d) Addition of Assay Reagents: 1) 10 μL of 800 nM gradient diluted cAMP standard solution was added to an empty well of an OptiPlate-384 plate. 2) 10 μL of cAMP assay 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 read on EnVision.

[0206] C. Experimental Results The experimental results are shown in Table 6. Table 6. In vitro GLP-1R / GIPR agonist activity test results TIFF0007672503000110.tif92170 Conclusion: The compounds of the present invention exhibit very strong agonist activity against GLP-1R / GIPR.

[0207] Test Example 2: Evaluation of the pharmacokinetic profile of the compound in rats A. Purpose of the experiment The purpose was to study the pharmacokinetic profile of the compound in SD rats. B. Experimental Procedure The pharmacokinetic properties of the compounds in rodents after subcutaneous injection were tested using standard protocols. In the experiments, the candidate compounds were formulated in a clear solution and administered to rats by a single subcutaneous injection (SC, 0.048 mpk). The injection vehicle was citrate buffer (20 mM, pH=7). Whole blood was collected and prepared as plasma, the drug concentrations were analyzed by LC-MS / MS method, and the pharmacokinetic parameters were calculated with Phoenix WinNonlin software. C. Experimental Results The experimental results are shown in Table 7. Table 7. Pharmacokinetic profile study results in rats TIFF0007672503000111.tif28170 Conclusion: The compounds of the present invention have excellent pharmacokinetic properties in rats.

[0208] Test Example 3: Evaluation of the pharmacokinetic profile of the compound in mice A. Purpose of the experiment The purpose was to study the pharmacokinetic profile of the compound in C57BL / 6 mice. B. Experimental Procedure The pharmacokinetic properties of the compounds in rodents after subcutaneous injection were tested using standard protocols. In the experiments, the candidate compounds were formulated in a clear solution and administered to rats by subcutaneous injection (SC, 0.048mpk). The subcutaneous injection vehicle was citrate buffer (20mM, pH=7). Whole blood was collected and prepared as plasma, the drug concentrations were analyzed by LC-MS / MS method, and the pharmacokinetic parameters were calculated with Phoenix WinNonlin software. C. Experimental Results The experimental results are shown in Table 8. Table 8. Pharmacokinetic profile test results in mice TIFF0007672503000112.tif28170 Conclusion: The compounds of the present invention have excellent pharmacokinetic properties in mice.

[0209] Test Example 4: Evaluation of the pharmacokinetic profile of a compound in cynomolgus monkeys A. Purpose of the experiment The purpose was to study the pharmacokinetic profile of the compound in cynomolgus monkeys. B. Experimental Procedure The pharmacokinetic properties of compounds in mammals after intravenous and subcutaneous administration were tested using standard protocols. In the experiments, candidate compounds were formulated in clear solutions and administered to cynomolgus monkeys by a single subcutaneous injection (SC, 0.02 mpk). The subcutaneous injection vehicle was citrate buffer (20 mM, pH=7). Whole blood was collected and prepared as plasma, the drug concentrations were analyzed by LC-MS / MS method, and the pharmacokinetic parameters were calculated with Phoenix WinNonlin software. C. Experimental Results The experimental results are shown in Table 9. Table 9. Pharmacokinetic profile study results in cynomolgus monkeys TIFF0007672503000113.tif28170 Conclusion: Compounds of the invention have excellent pharmacokinetic properties in monkeys.

[0210] Test Example 5: Plasma stability test (PLS) A. Purpose of the experiment The purpose was to study the stability of the test compound in the plasma of normal mice. 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 min, and blood clots, if any, were removed, and the pH was adjusted to 7.4±0.1. 2. Preparation of test compound solution: Dilute with DMSO to prepare a 100 μM solution. 3. 98 μL of blank control plasma was added to 2 μL of a test compound solution (100 μM) so that the final concentration of the mixture became 2 μM, and this was incubated under water bath conditions at 37°C. 4. At each time point (0, 10, 30, 60 and 120 min), 100 μL H3PO4 solution and 800 μL stop solution (200 ng / mL tolbutamide and 200 ng / mL labetalol in 100% methanol) were added respectively to precipitate proteins and mixed thoroughly. 5. The samples were centrifuged at 4000 rpm for 20 minutes, and 100 μL of the supernatant was taken from each well for LC-MS / MS analysis. C. Experimental Results The experimental results are shown in Table 10. Table 10 PLS test results TIFF0007672503000114.tif12170 Conclusion: The compounds of the present invention have excellent plasma stability.

[0211] Test Example 6: Plasma Protein Binding Test (PPB) A. Purpose of the experiment The purpose was to study the binding ability of the test compounds to human / mouse plasma albumin. B. Experimental Procedure 1. Preparation of matrix (preparation of blank matrix): On the day of the experiment, thaw the plasma in cold water and centrifuge at 3220 rpm for 5 minutes to remove any clots. The pH of the obtained plasma was measured and adjusted to 7.4±0.1 with 1% phosphoric acid or 1N sodium hydroxide as necessary. 2. Dilution process of test compounds: Test compounds were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions with concentrations of 10 mM and 2 mM, respectively. 2 μL of stock solution (2 mM) was diluted with 98 μL of DMSO to prepare a standard solution of 40 μM. 10 μL of stock solution was diluted with 240 μL of DMSO to prepare a standard solution of 400 μM of the control compound. The compound standard solution (5 μL) was mixed evenly with the blank matrix (995 μL) in a ratio of 1:200 to prepare the loading matrix. 3. Analysis process 3.1 Equal amounts of 30 μL loading matrix (n=2) were transferred to a sample collection plate to prepare test time 0 (T0) samples for residue measurement. Samples were immediately combined with the corresponding blank buffer to a final volume of 60 μL, such that the volume ratio of plasma (loading matrix) to buffer was 1:1 in each well. Then, 60 μL of 4% H3PO4 in H2O and 480 μL of stop solution containing the internal standard were added to the T0 samples of the test compounds, respectively. They were then stored at 2-8 °C for further processing along with the other samples. 3.2 The remaining plasma samples were pre-incubated for 30 min in a carbon dioxide incubator at 37 ± 1°C. Protein-free samples (F samples) were prepared and transferred together with the matrix-loaded samples (230 μL) into polycarbonate tubes (n = 2) and ultracentrifuged at 37°C and 155,000 × g (35,000 rpm) for 4 h. 3.3 To prepare T samples (test samples), one additional matrix-containing sample was transferred to a single 96-well plate (sample incubation plate) and incubated at 37° C. for 4 hours. 3.4 After centrifugation was completed, 30 μL of protein-free sample (F sample) and 30 μL of T sample were taken from the second layer of the supernatant (below the upper layer) and transferred to a new sample collection plate. Each sample was mixed with the corresponding blank buffer or matrix (blank matrix) to a final volume of 60 μL, with a matrix (blank matrix):buffer volume ratio of 1:1. All samples were added with 60 μL of 4% H3PO4 aqueous solution and 480 μL of stop solution (containing internal standard). The mixture was centrifuged at 4000 rpm for 20 min, and 100 μL of supernatant was taken from each sample for LC-MS / MS analysis. C. Experimental Results The experimental results are shown in Table 11. Table 11 PPB test results TIFF0007672503000115.tif12170Note: NA indicates that plasma protein binding capacity was so high that free drug was not detectable at normal plasma protein concentrations. Conclusion: The compounds of the present invention show extremely high plasma protein binding capacity.

[0212] Test Example 7: Verification of efficacy in STZ-NASH mouse model A. Purpose of the experiment The purpose was to verify the efficacy of the test compound in STZ-HFD diet-induced NASH model in C57BL / 6 mice. B. Experimental Procedure Modeling method: Newborn mice were subcutaneously injected with STZ (200ug / mouse) within 48 hours after birth, and after 4 weeks of lactation, animals with fasting plasma glucose >12mmol / L were selected and continuously fed HFD for 6 weeks, finally establishing the NASH model. Another 8 animals were selected without STZ injection and HFD feeding (normal control group). Administration schedule: One week after HFD feeding, the first administration day was designated Day 1, and then administration was started by subcutaneous injection every two days for five weeks. Experimental endpoint test: pathological HE staining and SR staining. C. Experimental Results The experimental results are shown in Table 12. Table 12. Animal pathological endpoints in the STZ-NASH model TIFF0007672503000116.tif41170 Conclusion: The compounds of the present invention can significantly improve NAS scores in STZ-NASH mouse model.

Claims

1. Represented by formula (II), YAibEGT FTSDY SIAibLD KIAQK AFVKW LIAGG PSSGA PPPS ０ (II) As shown below: 1) The amino acid at position i is replaced with lysine (K), and then the amino group on the lysine (K) is combined with the amino group on the side chain of the lysine (K) at position i+3 to form a compound of the formula or the amino groups on the lysine (K) side chain at positions j and j+4 are linked to a group represented by the formula wherein i is 17 and j is 20; and 2) an additional 0 to 2 amino acids of the polypeptide of the sequence shown in formula (II) are substituted; Where: The amino acid at position i is replaced with a lysine (K), and then the amino group on the lysine (K) is combined with the amino group on the side chain of the lysine (K) at position i+3 to form the compound of the formula When linked to a group represented by the formula (II), 0 to 2 amino acids at positions 21 or 24 of the polypeptide of formula (II) are substituted, where the amino acid at position 21 is substituted with glutamic acid (E), the amino acid at position 24 is substituted with glutamic acid (E), glutamine (Q) or alanine (A), or the amino acids at positions 21 and 24 are both substituted with glutamic acid (E); The amino groups on the side chain of lysine (K) at positions j and j+4 are represented by the formula When linked to a group represented by the formula (II), 0 to 1 amino acids at position 21 of the polypeptide of formula (II) are substituted, wherein the amino acid at position 21 is substituted with glutamic acid (E); where the structure of Aib is of the formula is a group represented by S ０ teeth, and X is selected from the group consisting of wherein "*" represents X １ represents the position connected to １ represents a single bond, -C(=O)-, -O-C(=O)-, and -N(R １ )-C(=O)-; R １ is H and C １－３ alkyl; X ２ teeth, wherein m is selected from 2, 3 and 4; n is selected from 15, 16, 17, 18 and 19; and p is selected from 1 and 2.

2. The polypeptide sequence is shown in formula (P): YAibEGT FTSDY SIAibLD KKAQK AFVKW LIAGG PSSGA PPPS ０ (P) As shown below: 1) The amino groups on the side chains of lysine (K) at positions 17 and 20 are of the formula and 2) 0 to 2 amino acids at positions 21 or 24 of the polypeptide of formula (P) are substituted, where the amino acid at position 21 is substituted with glutamic acid (E), the amino acid at position 24 is substituted with glutamic acid (E), glutamine (Q) or alanine (A), or the amino acids at positions 21 and 24 are both substituted with glutamic acid (E); 2. The polypeptide of claim 1, wherein Aib, S 0 , X, X 1 and X 2 are as defined in claim 1.

3. As follows: 1) The amino groups on the side chains of lysine (K) at positions 20 and 24 are of the formula and 2) 0 to 1 amino acid at position 21 of the polypeptide of formula (II) is substituted, wherein the amino acid at position 21 is substituted with glutamic acid (E); 2. The polypeptide of claim 1, wherein Aib, S 0 , X, X 1 and X 2 are as defined in claim 1.

4. Represented by formulas (II-1), (II-2), (III-6), (IV-7), (IV-8) and (IV-10), YAibEGT FTSDY SIAibLD KIAQK AFVKW LIAGG PSSGA PPPS-NH ２ (II-1) YAibEGT FTSDY SIAibLD KIAQK EFVKW LIAGG PSSGA PPPS-NH ２ (II-2) YAibEGT FTSDY SIAibLD KKAQK AFVEW LIAGG PSSGA PPPS-NH ２ (III-6) YAibEGT FTSDY SIAibLD KKAQK AFVQW LIAGG PSSGA PPPS-NH ２ (IV-7) YAibEGT FTSDY SIAibLD KKAQK AFVAW LIAGG PSSGA PPPS-NH ２ (IV-8) YAibEGT FTSDY SIAibLD KKAQK EFVEW LIAGG PSSGA PPPS-NH ２ (IV-10) The amino groups on the side chains of lysines (K) at positions 17 and 20 or the amino groups on the side chains of lysines (K) at positions 20 and 24 are represented by the formula: wherein Aib, X, X １ and X ２ 2. The polypeptide of claim 1 having a sequence which includes a modification as defined in claim 1.

5. R １ The polypeptide according to any one of claims 1 to 4, wherein

6. X １ The polypeptide according to any one of claims 1 to 4, wherein is selected from the group consisting of a single bond, -C(=O)-, -O-C(=O)-, and -NH-C(=O)-.

7. The polypeptide according to any one of claims 1 to 4, wherein m is 2.

8. The polypeptide according to any one of claims 1 to 4, wherein n is selected from 15 and 17.

9. The polypeptide according to any one of claims 1 to 4, wherein p is 2.

10. X ２ teeth, The polypeptide according to any one of claims 1 to 4, wherein the polypeptide is selected from the group consisting of:

11. X ２ teeth, The polypeptide according to claim 10, selected from the group consisting of:

12. The amino groups on the lysine (K) side chain at positions i and i+3 or the amino groups on the lysine (K) side chain at positions j and j+4 are represented by the formula and linking to a group represented by the formula The polypeptide according to any one of claims 1 to 4, which forms a group represented by the formula:

13. The amino groups on the lysine (K) side chain at positions i and i+3 or the amino groups on the lysine (K) side chain at positions j and j+4 are represented by the formula and linking to a group represented by the formula The polypeptide according to any one of claims 1 to 4, which forms a group represented by the formula:

14. formula The group represented by The polypeptide according to any one of claims 1 to 4, wherein the polypeptide is selected from the group consisting of:

15. formula The group represented by 15. The polypeptide of claim 14, selected from the group consisting of:

16. A polypeptide represented by the following formula:

17. A pharmaceutical composition comprising, as an active ingredient, a therapeutically effective amount of the polypeptide according to any one of claims 1 to 4 or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

18. Use of a polypeptide described in any one of claims 1 to 4 or a pharma- ceutically acceptable salt thereof, or a composition described in claim 17, in the preparation of a pharmaceutical for treating nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH).

19. The polypeptide according to any one of claims 1 to 4 or a pharma- ceutically acceptable salt thereof or the composition according to claim 17, which is administered once every 3 days, once every 4 days, once every week, or once every 2 weeks.

Images