Polypeptide with modified structure and application thereof

Abstract

The invention provides a series of polypeptides containing modified structures, the series of polypeptides act on three targets of GLP-1, GIP and GCG and have the prospect of being developed into drugs for preventing or treating metabolic diseases, and the sequence of the polypeptides is shown as a formula Z-3. The formula Z-3 is as follows: X5X0X6GT FTSDY SIX1X7X8KX9X10X11X0X4FX12X13X14LLX15GG PSSGAPPPS0, and the formula Z-3 is as follows: X5X0X6GT FTSDY SIX1X7X8KX9X10X11LLX15GG;

Description

[0001] This application is a divisional application of the invention patent application with application date of January 16, 2024, application number 202410061582.8, and name “Polypeptides with modified structures and their applications”. Technical Field

[0002] The present invention relates to a series of polypeptides with modified structures and applications thereof. Specifically, the present invention relates to a series of polypeptides with stapled structures, a pharmaceutical composition containing the series of polypeptides and applications thereof in treating metabolic diseases. Background Art

[0003] Glucagon-like peptide-1 (GLP-1) and insulinotropic peptide (GIP) are incretins, and glucagon (GCG) is secreted by pancreatic α cells. The aforementioned substances can directly or indirectly affect human glucose metabolism and / or lipid metabolism. Therefore, compounds that act on GLP-1, GIP, and GCG have the prospect of being developed into drugs for the treatment of metabolic diseases. For example, the compounds (peptides) that have been marketed and act on GLP-1, such as exenatide, liraglutide, and semaglutide, are drugs for the treatment of type 2 diabetes mellitus type 2 (T2DM) and / or obesity.

[0004] However, compounds that act on a single GLP-1, GIP or GCG usually have deficiencies in terms of therapeutic areas, therapeutic effects, metabolic behavior, etc., which in turn affect the drugability of the compound or its clinical application after marketing. Therefore, attempts are made to develop compounds that act on multiple targets in order to intervene from multiple mechanisms of action at the same time and achieve better clinical effects. Tirzepatide, a GLP-1 / GIP dual agonist developed by Eli Lilly, has shown superior effects to semaglutide in head-to-head clinical trials and has been approved for marketing; LY3437943, a GLP-1 / GIP / GCG triple agonist developed by Eli Lilly, has also shown good clinical effects in clinical studies and has good development prospects.

[0005] GLP-1R / GIPR / GCGR multiple agonists can activate the molecular mechanism of blood sugar regulation receptors, affect food intake and satiety through different mechanisms, and play a role in maintaining weight homeostasis. At the same time, it was found during the research process that because GLP-1 receptor agonists can promote the incretin effect by stimulating GLP-1 receptors to produce good hypoglycemic and weight loss effects, they can also treat NASH (non-alcoholic fatty liver disease) through multi-pathway synergistic effects, suggesting that compounds acting on this target or multiple targets have the prospect of being developed into drugs for the prevention and / or treatment of a series of specific diseases related to abnormal glucose metabolism and / or lipid metabolism. Summary of the invention

[0006] The present invention aims to solve the deficiencies of the prior art and provides a series of polypeptides with modified structures and pharmaceutically acceptable salts thereof. The polypeptides in this series show good effects on GLP-1 receptor (GLP-1R), GIP receptor (GIPR) and GCG receptor (GCGR) in in vitro experiments and have good drug development prospects.

[0007] The present invention provides a polypeptide with a modified structure having a polypeptide sequence shown in Formula Z or a pharmaceutically acceptable salt thereof:

[0008] X5X0X6GT X a TSDY SX b X1X7X8KX9X 10 X 11 X0X4FX 12 X 13 X 14 X c X d X 15 GG PSSGA PPPS0

[0009] Formula Z

[0010] in:

[0011] X0 is independently selected from Or Aib or alanine (Ala, A), the structure of Aib is

[0012] X1 is independently selected from α-methyl substituted leucine (α-MeLeu, α-MeL), tyrosine (Tyr, Y) or Aib, wherein the α-MeL has a structure of

[0013] X4 is selected from α-methyl substituted lysine (α-MeLys, α-MeK), D-lysine (dK), L-ornithine (L-Ornithine, L-Orn), alanine (Ala, A), lysine (Lys, K), glutamic acid (Glu, E) or leucine (Leu, L); the structure of the α-methyl substituted lysine is The structure of D-lysine (dK) is The structure of L-Ornithine (L-Ornithine, L-Orn) is

[0014] X5 is selected from tyrosine (Tyr, Y) or histidine (His, H);

[0015] X6 is selected from glutamine (Gln, Q) or histidine (His, H);

[0016] X7 is selected from leucine (Leu, L) or lysine (Lys, K);

[0017] X8 is selected from aspartic acid (Asp, D) or glutamic acid (Glu, E);

[0018] X9 is selected from lysine (Lys, K), glutamine (Gln, Q) or glutamic acid (Glu, E);

[0019] X 10 Selected from alanine (Ala, A) or tyrosine (Tyr, Y);

[0020] X 11 is selected from lysine (Lys, K), glutamine (Gln, Q) or alanine (Ala, A);

[0021] X 12 is selected from valine (Val, V) or isoleucine (Ile, I);

[0022] X 13 Selected from lysine (Lys, K), glutamine (Gln, Q) or glutamic acid (Glu, E);

[0023] X 14 is selected from lysine (Lys, K), tryptophan (Trp, W), tyrosine (Tyr, Y), glutamic acid (Glu, E) or phenylalanine (Phe, F);

[0024] X 15 Selected from lysine (Lys, K) or glutamic acid (Glu, E);

[0025] X a is selected from phenylalanine (Phe, F) or α-methyl substituted phenylalanine (α-MePhe, α-MeF), wherein the structure of α-methyl substituted phenylalanine is

[0026] X b is selected from lysine (Lys, K) or isoleucine (Ile, I);

[0027] X c is selected from leucine (Leu, L) or phenylalanine (Phe, F);

[0028] X d is selected from leucine (Leu, L) or isoleucine (Ile, I);

[0029] S0 is selected from (i.e.: the C-terminal amino acid is optionally amidated to a C-terminal primary amide);

[0030] And the compound (polypeptide) has the following modifications:

[0031] 1) The amino acid side chains at positions i and i+j in the sequence can be modified to form a modified structure (stapled body) similar to a "staple" structure (peptide stapled), wherein i is independently selected from 12, 14, 16, 17, 19, 21, 24 or 25, and j is independently selected from 2, 3, 4, 5 or 7,

[0032] 2) The modification is that the amino or carboxyl groups on the side chains of the two amino acid unit structures are Condensation connected;

[0033] X2 is selected from Wherein, "*" indicates the position connected to X3, and the two ends are the positions connected to the corresponding amino or carboxyl groups in the modified amino acid;

[0034] X3 is selected from

[0035] R1 and R2 are independently selected from

[0036] m is selected from 1, 2 or 3;

[0037] p is selected from 1 or 2;

[0038] n is selected from 8, 9 or 10.

[0039] In some embodiments of the present invention, the polypeptide with a modified structure or a pharmaceutically acceptable salt thereof has a polypeptide sequence as shown in Formula Z-1:

[0040] X5X0X6GT FTSDY SX b X1X7X8KX9X 10 X 11 X0X4FX 12 X 13 X 14 X c X d X 15 GG PSSGA PPPS0

[0041] Formula Z-1

[0042] The compound (polypeptide) has the following modifications:

[0043] 1) The amino acid side chains at positions i and i+j in the sequence can be modified to form a modified structure (stapled body) similar to a "staple" (peptide stapled) structure, wherein i is independently selected from 12, 14, 16, 17, 19, 21, 24 or 25, and j is independently selected from 2, 3, 4, 5 or 7,

[0044] 2) The modification is that the amino or carboxyl groups on the side chains of the two amino acid unit structures are The other variables are as defined in the present invention.

[0045] In some embodiments of the present invention, the polypeptide with a modified structure or a pharmaceutically acceptable salt thereof has a polypeptide sequence as shown in Formula Z-2:

[0046] X5X0X6GT FTSDY SIX1X7X8KX9X 10 X 11 X0X4FX 12 X 13 X 14 LLX 15 GG PSSGA PPPS0

[0047] Formula Z-2

[0048] The compound (polypeptide) has the following modifications:

[0049] 1) The amino acid side chains at positions i and i+j in the sequence can be modified to form a modified structure (stapled body) similar to a "staple" structure, wherein i is independently selected from 12, 14, 16, 17, 19, 21, 24, 25, and j is independently selected from 2, 3, 4, 5, 7,

[0050] 2) The modification is that the amino or carboxyl groups on the side chains of the two amino acid unit structures are The other variables are as defined in the present invention.

[0051] In some embodiments of the present invention, the polypeptide with a modified structure or a pharmaceutically acceptable salt thereof has a polypeptide sequence as shown in Formula Z-3:

[0052] X5X0X6GT FTSDY SIX1X7X8KX9X 10 X 11 X0X4FX 12 X 13 X 14 LLX 15 GG PSSGA PPPS0

[0053] Formula Z-3

[0054] And the polypeptide compound has the following modifications:

[0055] 1) The amino acid side chains at positions i and i+j in the sequence can be modified to form a modified structure (stapled body) similar to a "staple" structure, wherein i is independently selected from 14, 17, 19, 21, 24 or 25, and j is independently selected from 3, 4, 5 or 7,

[0056] 2) The modification is that the amino or carboxyl groups on the side chains of the two amino acid unit structures are Condensation connected;

[0057] X2 is selected from Wherein, "*" indicates the position connected to X3, and the two ends are the positions connected to the corresponding amino or carboxyl groups in the modified amino acid;

[0058] X3 is selected from:

[0059]

[0060] R1 and R2 are independently selected from

[0061] m is selected from 1, 2 or 3;

[0062] p is selected from 1 or 2;

[0063] n is selected from 8, 9 or 10; other variables are as defined in the present invention.

[0064] In some embodiments of the present invention, the polypeptide with a modified structure or a pharmaceutically acceptable salt thereof has a polypeptide sequence as shown in Formula Z-4:

[0065] YX0QGT FTSDY SX b X1LD KKAQX0 X4FX 12 X 13 X 14 X c X d X 15 GG PSSGA PPPS0

[0066] Formula Z-4

[0067] S0 is selected from (i.e.: the C-terminal amino acid is optionally amidated to a C-terminal primary amide);

[0068] And the polypeptide compound has the following modifications:

[0069] 1) The amino acid side chains at positions i and i+j in the sequence can be modified to form a modified structure (stapled body) similar to a "staple" structure, wherein i is independently selected from 12, 17, 21 or 25; j is independently selected from 3, 4 or 7,

[0070] 2) The modification is that the amino or carboxyl groups on the side chains of the two amino acid unit structures are Condensation connected;

[0071] X2 is selected from and; wherein "*" indicates the position connected to X3, and both ends are the positions connected to the corresponding amino or carboxyl groups in the modified amino acid;

[0072] X3 is selected from:

[0073] m is selected from 1, 2 or 3;

[0074] p is selected from 1 or 2;

[0075] n is selected from 8, 9 or 10; other variables are as defined in the present invention.

[0076] In some embodiments of the present invention, the polypeptide with a modified structure or a pharmaceutically acceptable salt thereof has a polypeptide sequence as shown in Formula Z-5:

[0077] YX0QGT X a TSDY SIX1KD KKAQX0 X4FIX 13 Y 15 GG PSSGA PPPS0

[0078] Type Z-5

[0079] And the polypeptide compound has the following modifications:

[0080] 1) The amino acid side chains at positions i and i+j in the sequence can be modified to form a modified structure similar to a "staple" structure, wherein i is independently selected from 14; j is independently selected from 3,

[0081] 2) The modification is that the amino or carboxyl groups on the side chains of the two amino acid unit structures are Condensation connected;

[0082] X2 is selected from Wherein, "*" indicates the position connected to X3, and the two ends are the positions connected to the corresponding amino or carboxyl groups in the modified amino acid;

[0083] X3 is selected from:

[0084] m is selected from 1, 2 or 3;

[0085] p is selected from 1 or 2;

[0086] n is selected from 8, 9 or 10; other variables are as defined in the present invention.

[0087] In some embodiments of the present invention, the polypeptide with a modified structure or a pharmaceutically acceptable salt thereof has a polypeptide sequence as shown in Formula Z-6, Z-7, Z-8, Z-9, Z-10, Z-11, Z-12, and Z-13, respectively:

[0088] X5X0X6GT FTSDY SIX1LX8KKX 10 X 11 X0X4FX 12 X 13 X 14 LLX 15 GG PSSGA PPPS0

[0089] Formula Z-6

[0090] YX0QGT FTSDY SIX1LD KKAX 11 X0X4FIX 13 X 14 LLX 15 GG PSSGA PPPS0

[0091] Formula Z-7

[0092] YX0QGT FTSDY SIX1X7D KKAQX0X4FIX 13 YLLX 15 GG PSSGA PPPS0

[0093] Formula Z-8

[0094] YX0QGT FTSDY SXbX1LD KKAQX0X4FX 12 EX 14 X c XdX 15 GG PSSGA PPPS0

[0095] Formula Z-9

[0096] YX0QGT FTSDY SX b X1LD KKAQX0KFX 12 EX 14 LX d X 15 GG PSSGA PPPS0

[0097] Formula Z-10

[0098] YX0QGT FTSDY SX b X1KD KKAQX0 X4FIEY LLX 15 GG PSSGA PPPS0

[0099] Formula Z-11

[0100] YX0QGT FTSDY SIX1KD KKAQX0X4FIEY LLEGG PSSGA PPPS0

[0101] Type Z-12

[0102] YX0QGT X a TSDY SIX1KD KKAQX0X4FIEY LLX 15 GG PSSGA PPPS0

[0103] Type Z-13

[0104] The compound (polypeptide) has the following modifications:

[0105] 1) The amino acid side chains at positions i and i+j in the sequence can be modified to form a modified structure (stapled body) similar to a "staple" structure, wherein i is independently selected from 12, 14, 16, 17, 19, 21, 24 or 25, and j is independently selected from 2, 3, 4, 5 or 7,

[0106] 2) The modification is that the amino or carboxyl groups on the side chains of the two amino acid unit structures are Condensation connected;

[0107] X2 is selected from Wherein, "*" indicates the position connected to X3, and the two ends are the positions connected to the corresponding amino or carboxyl groups in the modified amino acid;

[0108] X3 is selected from:

[0109]

[0110] R1 and R2 are independently selected from

[0111] m is selected from 1, 2 or 3;

[0112] p is selected from 1 or 2;

[0113] n is selected from 8, 9 or 10; other variables are as defined in the present invention.

[0114] In some embodiments of the present invention, S0 in the above polypeptide sequence is That is, the C-terminal amino acid is amidated to a primary amide, and the other variables are as defined in the present invention.

[0115] In some embodiments of the present invention, the second amino acid X0 in the polypeptide sequence is Aib, and other variables are as defined in the present invention.

[0116] In some embodiments of the present invention, the 20th amino acid X0 in the polypeptide sequence is Aib, and other variables are as defined in the present invention.

[0117] In some embodiments of the present invention, the 2nd and 20th amino acids X0 in the polypeptide sequence are both Aib, and other variables are as defined in the present invention.

[0118] In some embodiments of the present invention, i is 12, j is 4, i+j is 16, and other variables are defined as in the present invention.

[0119] In some embodiments of the present invention, i is 14, j is 3, i+j is 17, and other variables are defined as in the present invention.

[0120] In some embodiments of the present invention, i is 16, j is 3, i+j is 19, and other variables are defined as in the present invention.

[0121] In some embodiments of the present invention, i is 17, j is 4, i+j is 21, and other variables are as defined in the present invention.

[0122] In some embodiments of the present invention, i is 17, j is 7, i+j is 24, and other variables are as defined in the present invention.

[0123] In some embodiments of the present invention, i is 19, j is 5, i+j is 24, and other variables are as defined in the present invention.

[0124] In some embodiments of the present invention, i is 21, j is 7, i+j is 28, and other variables are as defined in the present invention.

[0125] In some embodiments of the present invention, i is 24, j is 4, i+j is 28, and other variables are defined as in the present invention.

[0126] In some embodiments of the present invention, i is 25, j is 3, i+j is 28, and other variables are defined as in the present invention.

[0127] In some embodiments of the present invention, the first amino acid X5 in the polypeptide sequence is Y, and other variables are as defined in the present invention.

[0128] In some embodiments of the present invention, the first amino acid X5 in the polypeptide sequence is H, and other variables are as defined in the present invention.

[0129] In some embodiments of the present invention, the third amino acid X6 in the polypeptide sequence is Q, and other variables are as defined in the present invention.

[0130] In some embodiments of the present invention, the third amino acid X6 in the polypeptide sequence is H, and other variables are as defined in the present invention.

[0131] In some embodiments of the present invention, the sixth amino acid X in the polypeptide sequence a is F, and other variables are as defined in the present invention.

[0132] In some embodiments of the present invention, the sixth amino acid X in the polypeptide sequence a is α-MeF, and other variables are as defined in the present invention.

[0133] In some embodiments of the present invention, the 12th amino acid X in the polypeptide sequence b is 1, and other variables are as defined in the present invention.

[0134] In some embodiments of the present invention, the 12th amino acid X in the polypeptide sequence b is K, and other variables are as defined in the present invention.

[0135] In some embodiments of the present invention, the 13th amino acid X1 in the polypeptide sequence is α-MeL, and other variables are as defined in the present invention.

[0136] In some embodiments of the present invention, the 13th amino acid X1 in the polypeptide sequence is Y, and other variables are as defined in the present invention.

[0137] In some embodiments of the present invention, the 13th amino acid X1 in the polypeptide sequence is Aib, and other variables are as defined in the present invention.

[0138] In some embodiments of the present invention, the 14th amino acid X7 in the polypeptide sequence is L, and other variables are as defined in the present invention.

[0139] In some embodiments of the present invention, the 14th amino acid X7 in the polypeptide sequence is K, and other variables are as defined in the present invention.

[0140] In some embodiments of the present invention, the 15th amino acid X8 in the polypeptide sequence is D, and other variables are as defined in the present invention.

[0141] In some embodiments of the present invention, the 15th amino acid X8 in the polypeptide sequence is E, and other variables are as defined in the present invention.

[0142] In some embodiments of the present invention, the 17th amino acid X9 in the polypeptide sequence is K, and other variables are as defined in the present invention.

[0143] In some embodiments of the present invention, the 17th amino acid X9 in the polypeptide sequence is Q, and other variables are as defined in the present invention.

[0144] In some embodiments of the present invention, the 17th amino acid X9 in the polypeptide sequence is E, and other variables are as defined in the present invention.

[0145] In some embodiments of the present invention, the 18th amino acid X in the polypeptide sequence 10 is A, and other variables are as defined in the present invention.

[0146] In some embodiments of the present invention, the 18th amino acid X in the polypeptide sequence 10 is Y, and other variables are as defined in the present invention.

[0147] In some embodiments of the present invention, the 19th amino acid X in the polypeptide sequence 11 is Q, and other variables are as defined in the present invention.

[0148] In some embodiments of the present invention, the 19th amino acid X in the polypeptide sequence 11 is A, and other variables are as defined in the present invention.

[0149] In some embodiments of the present invention, the 19th amino acid X in the polypeptide sequence 11 is K, and other variables are as defined in the present invention.

[0150] In some embodiments of the present invention, the 21st amino acid X4 in the polypeptide sequence is K, and other variables are as defined in the present invention.

[0151] In some embodiments of the present invention, the 21st amino acid X4 in the polypeptide sequence is A, and other variables are as defined in the present invention.

[0152] In some embodiments of the present invention, the 21st amino acid X4 in the polypeptide sequence is E, and other variables are as defined in the present invention.

[0153] In some embodiments of the present invention, the 21st amino acid X4 in the polypeptide sequence is α-MeK, and other variables are as defined in the present invention.

[0154] In some embodiments of the present invention, the 21st amino acid X4 in the polypeptide sequence is dK, and other variables are as defined in the present invention.

[0155] In some embodiments of the present invention, the 21st amino acid X4 in the polypeptide sequence is L-Orn, and other variables are as defined in the present invention.

[0156] In some embodiments of the present invention, the 21st amino acid X4 in the polypeptide sequence is L, and other variables are as defined in the present invention.

[0157] In some embodiments of the present invention, the 23rd amino acid X in the polypeptide sequence 12 is 1, and other variables are as defined in the present invention.

[0158] In some embodiments of the present invention, the 23rd amino acid X in the polypeptide sequence 12 is V, and other variables are as defined in the present invention.

[0159] In some embodiments of the present invention, the 24th amino acid X in the polypeptide sequence 13 is E, and other variables are as defined in the present invention.

[0160] In some embodiments of the present invention, the 24th amino acid X in the polypeptide sequence 13 is K, and other variables are as defined in the present invention.

[0161] In some embodiments of the present invention, the 24th amino acid X in the polypeptide sequence 13 is Q, and other variables are as defined in the present invention.

[0162] In some embodiments of the present invention, the 25th amino acid X in the polypeptide sequence 14 is Y, and other variables are as defined in the present invention.

[0163] In some embodiments of the present invention, the 25th amino acid X in the polypeptide sequence 14 is W, and other variables are as defined in the present invention.

[0164] In some embodiments of the present invention, the 25th amino acid X in the polypeptide sequence 14 is K, and other variables are as defined in the present invention.

[0165] In some embodiments of the present invention, the 25th amino acid X in the polypeptide sequence 14 is E, and other variables are as defined in the present invention.

[0166] In some embodiments of the present invention, the 25th amino acid X in the polypeptide sequence 14 is F, and other variables are as defined in the present invention.

[0167] In some embodiments of the present invention, the 26th amino acid X in the polypeptide sequence c is L, and other variables are as defined in the present invention.

[0168] In some embodiments of the present invention, the 26th amino acid X in the polypeptide sequence c is F, and other variables are as defined in the present invention.

[0169] In some embodiments of the present invention, the 27th amino acid X in the polypeptide sequence d is L, and other variables are as defined in the present invention.

[0170] In some embodiments of the present invention, the 27th amino acid X in the polypeptide sequence d is 1, and other variables are as defined in the present invention.

[0171] In some embodiments of the present invention, the 28th amino acid X in the polypeptide sequence 15is E, and other variables are as defined in the present invention.

[0172] In some embodiments of the present invention, the 28th amino acid X in the polypeptide sequence 15 is K, and other variables are as defined in the present invention.

[0173] In some embodiments of the present invention, the above X3 is selected from:

[0174] Other variables are as defined herein.

[0175] In some embodiments of the present invention, in the above X3 structure, m is selected from 1 or 2, and other variables are as defined in the present invention.

[0176] In some embodiments of the present invention, in the above X3 structure, p is selected from 1, and other variables are as defined in the present invention.

[0177] In some embodiments of the present invention, in the above X3 structure, n is selected from 9, and other variables are as defined in the present invention.

[0178] In some embodiments of the present invention, the above X3 is selected from:

[0179]

[0180] Other variables are as defined in the present invention.

[0181] In some embodiments of the present invention, the above X3 is selected from:

[0182]

[0183] Other variables are as defined herein.

[0184] In some embodiments of the present invention, the above structural unit for:

[0185]

[0186] Other variables are as defined herein.

[0187] In some embodiments of the present invention, the above structural unit for:

[0188]

[0189] Other variables are as defined in the present invention.

[0190] The present invention provides a polypeptide with a modified structure as shown in Formula I or a pharmaceutically acceptable salt thereof:

[0191] Formula I: X5X0X6GTFTSDYSIX1X7X8KX9X 10 X 11 X0X4FX 12 X 13 X 14 LLX 15 GGPSSGAPPPS0

[0192] in:

[0193] X0 is independently selected from Or Aib or alanine (Ala, A), the structure of Aib is

[0194] X1 is independently selected from α-methyl substituted leucine (α-MeLeu, α-MeL) or tyrosine (Tyr, Y), wherein the α-MeL has a structure of

[0195] X4 is selected from α-methyl substituted lysine (α-MeLys, α-MeK), and its structure is Or D-lysine (dK), whose structure is Or L-Ornithine (L-Orn), its structure is or alanine (Ala, A), lysine (Lys, K), or glutamic acid (Glu, E);

[0196] X5 is selected from tyrosine (Tyr, Y) or histidine (His, H);

[0197] X6 is selected from glutamine (Gln, Q) or histidine (His, H);

[0198] X7 is selected from leucine (Leu, L) or lysine (Lys, K);

[0199] X8 is selected from aspartic acid (Asp, D) or glutamic acid (Glu, E);

[0200] X9 is selected from lysine (Lys, K), glutamine (Gln, Q) or glutamic acid (Glu, E);

[0201] X 10 Selected from alanine (Ala, A) or tyrosine (Tyr, Y);

[0202] X 11is selected from lysine (Lys, K), glutamine (Gln, Q) or alanine (Ala, A);

[0203] X 12 is selected from valine (Val, V) or isoleucine (Ile, I);

[0204] X 13 Selected from lysine (Lys, K), glutamine (Gln, Q) or glutamic acid (Glu, E);

[0205] X 14 is selected from lysine (Lys, K), tryptophan (Trp, W), tyrosine (Tyr, Y), glutamic acid (Glu, E) or phenylalanine (Phe, F);

[0206] X 15 Selected from lysine (Lys, K) or glutamic acid (Glu, E);

[0207] S0 is selected from (i.e.: the C-terminal amino acid is optionally amidated to a C-terminal primary amide);

[0208] And the compound has the following modifications:

[0209] 1) The amino acid side chains at positions i and i+j in the sequence can be modified to form a modified structure (stapled body) similar to a "staple" structure, wherein i is independently selected from 12, 14, 16, 17, 19, 21, 24 or 25, and j is independently selected from 2, 3, 4, 5 or 7,

[0210] 2) The modification is that the amino or carboxyl groups on the side chains of the two amino acid unit structures are Condensation connected;

[0211] X2 is selected from Wherein, "*" indicates the position connected to X3, and the two ends are the positions connected to the corresponding amino or carboxyl groups in the modified amino acid;

[0212] X3 is selected from:

[0213]

[0214] R1 and R2 are independently selected from

[0215] m is selected from 1, 2 or 3; p is selected from 1 or 2; n is selected from 8, 9 or 10.

[0216] In some embodiments of the present invention, the above X3 is selected from Other variables are as defined in the present invention.

[0217] In some embodiments of the present invention, the above X3 is selected from Other variables are as defined herein.

[0218] In some embodiments of the present invention, the above X3 is selected from Other variables are as defined herein.

[0219] In some embodiments of the present invention, the above structural unit for Other variables are as defined herein.

[0220] In some embodiments of the present invention, the above structural unit for Other variables are as defined herein.

[0221] The present invention provides a polypeptide with a modified structure as shown in Formula I or a pharmaceutically acceptable salt thereof:

[0222] Formula I: X5X0X6GT FTSDY SIX1X7X8 KX9X 10 X 11 X0X4FX 12 X 13 X 14 LLX 15 GG PSSGA PPPS0

[0223] in:

[0224] X0 can be independently selected from Or Aib or alanine (Ala, A), the structure of Aib is

[0225] X1 can be independently selected from α-methyl substituted leucine (α-MeLeu, α-MeL) or tyrosine (Tyr, Y), wherein the α-MeL has a structure of

[0226] X4 is selected from α-methyl substituted lysine (α-MeLys, α-MeK), and its structure is Or D-lysine (dK), whose structure is Or L-Ornithine (L-Orn), its structure is or alanine (Ala, A), lysine (Lys, K), or glutamic acid (Glu, E);

[0227] X5 is selected from tyrosine (Tyr, Y) or histidine (His, H);

[0228] X6 is selected from glutamine (Gln, Q) or histidine (His, H);

[0229] X7 is selected from leucine (Leu, L) or lysine (Lys, K);

[0230] X8 is selected from aspartic acid (Asp, D) or glutamic acid (Glu, E);

[0231] X9 is selected from lysine (Lys, K), glutamine (Gln, Q) or glutamic acid (Glu, E);

[0232] X 10 Selected from alanine (Ala, A) or tyrosine (Tyr, Y);

[0233] X 11 is selected from lysine (Lys, K), glutamine (Gln, Q) or alanine (Ala, A);

[0234] X 12 is selected from valine (Val, V) or isoleucine (Ile, I);

[0235] X 13 Selected from lysine (Lys, K), glutamine (Gln, Q) or glutamic acid (Glu, E);

[0236] X 14 is selected from lysine (Lys, K), tryptophan (Trp, W), tyrosine (Tyr, Y), glutamic acid (Glu, E) or phenylalanine (Phe, F);

[0237] X 15 Selected from lysine (Lys, K) or glutamic acid (Glu, E);

[0238] S0 is selected from (i.e.: the C-terminal amino acid is optionally amidated to a C-terminal primary amide);

[0239] And the compound has the following modifications:

[0240] 1) The amino acid side chains at positions i and i+j in the sequence can be modified to form a modified structure (stapled body) similar to a "staple" structure, wherein i is independently selected from 12, 14, 16, 17, 19, 21, 24 or 25, and j is independently selected from 2, 3, 4, 5 or 7,

[0241] 2) The modification is that the amino or carboxyl groups on the side chains of the two amino acid unit structures are Condensation connected;

[0242] X2 is selected from Wherein, "*" indicates the position connected to X3, and the two ends are the positions connected to the corresponding amino or carboxyl groups in the modified amino acid;

[0243] X3 is selected from:

[0244]

[0245] m is selected from 1, 2 or 3; p is selected from 1 or 2; n is selected from 8, 9 or 10.

[0246] In some embodiments of the present invention, the polypeptide with a modified structure, the amino acids with the same superscript number in the peptide chain sequence can be modified and connected to form a modified group (staple body) similar to a "staple" structure, that is, the amino acid side chains at positions i and i+j in the sequence can be modified and connected to form a modified structure (staple body) similar to a "staple" structure, wherein i is independently selected from any integer in 10-28, such as 10, 12, 14, 16, 17, 19, 21, 24 or 25, and j is independently selected from any integer in 1-8, such as 1, 2, 3, 4, 5, 7 or 8. The number of the "staple" structure is 1-2. In some embodiments, the first amino acid is at position 25 of the peptide and the second amino acid is at position 28 of the peptide. In some embodiments, the first amino acid is at position 24 of the peptide and the second amino acid is at position 28 of the peptide. In some embodiments, the first amino acid is at position 17 of the peptide and the second amino acid is at position 21 of the peptide. In some embodiments, the first amino acid is at position 16 of the peptide and the second amino acid is at position 19 of the peptide. In some embodiments, the first amino acid is at position 17 of the peptide and the second amino acid is at position 24 of the peptide. In some embodiments, the first amino acid is at position 19 of the peptide and the second amino acid is at position 24 of the peptide. In some embodiments, the first amino acid is at position 21 of the peptide and the second amino acid is at position 28 of the peptide. In some embodiments, the first amino acid is at position 19 of the peptide and the second amino acid is at position 24 of the peptide.

[0247] In some embodiments of the present invention, the polypeptide with a modified structure may have a peptide sequence with at most 0, 1, 2, 3 or 4 amino acid insertions, deletions, modifications or substitutions. For example, isoleucine (I) at position 12 may be replaced by lysine (K); leucine (L) at position 26 or 27 may be replaced by isoleucine (I) or phenylalanine (F); X1 at position 13 may be replaced by X0; X0 at position 20 may be replaced by a natural amino acid alanine (A), etc.

[0248] The present invention provides a series of polypeptide sequences as follows:

[0249] YX0QGT FTSDY SIX1LD KKAQX0AFIE 1 K L1 EGG PSSGA PPPS0

[0250] YX0QGT FTSDY SIX1LD KKAQX0AFI 1 KW LL 1 KGG PSSGA PPPS0

[0251] YX0QGT FTSDY SIX1LD K 1 KAQX0 1 KFIEY LLEGG PSSGA PPPS0

[0252] HX0HGT FTSDY SIYLE KKYAX0EFV 1 KW LL 1 KGG PSSGA PPPS0

[0253] HX0HGT FTSDY SIYLE K 1 KYAX0 1 KFVQW LLEGG PSSGA PPPS0

[0254] YX0QGT FTSDY SIX1LD KKAQX0AFIQ 1 K LL 1 KGG PSSGA PPPS0

[0255] YX0QGT FTSDY SIX1LD 1 KQA 1 QX0AFIE 2 K LL 2 KGG PSSGA PPPS0

[0256] YX0QGT FTSDY SIX1LD KEAQX0AFIE 1 K LL 1 KGG PSSGA PPPS0

[0257] HX0QGT FTSDY SIX1LD KKAQX0AFIE 1 K LL 1 KGG PSSGA PPPS0

[0258] YX0QGT FTSDY SIX1LD KKYQX0AFIE 1 K LL 1 KGG PSSGA PPPS0

[0259] YX0QGT FTSDY SIX1LE KKAQX0AFIE 1 K LL 1 KGG PSSGA PPPS0

[0260] HX0HGT FTSDY SIYLE KKYAX0EFVQ 1 E LL 1 KGG PSSGA PPPS0

[0261] YX0QGT FTSDY SIX1LD KKAQX0AFIE 1 E LL 1 KGG PSSGA PPPS0

[0262] YX0QGT FTSDY SIX1LD KKAQX0AFI 1 EW LL 1 KGG PSSGA PPPS0

[0263] YX0QGT FTSDY SIX1LD K 1 KAQX0 1 KFIEF LLEGG PSSGA PPPS0

[0264] YX0QGT FTSDY SIX1LD K 1 KAQX0 1 EFIEY LLEGG PSSGA PPPS0

[0265] YX0QGT FTSDY SIX1LD K 1 KAQX0AFI 1 KY LLEGG PSSGA PPPS0

[0266] YX0QGT FTSDY SIX1LD K 1 KAQX0 1 KFIEY LLEGG PSSGA PPPS0

[0267] YX0QGT FTSDY SIX1LD KKA 1 KX0AFI 1 KY LLEGG PSSGA PPPS0

[0268] YX0QGT FTSDY SIX1LD KKAQX0 1 KFIEY LL 1 KGG PSSGA PPPS0

[0269] YX0QGT FTSDY SIX1LD K 1 KAQA 1 X4FIEY LLEGG PSSGA PPPS0

[0270] YX0QGT FTSDY SIX1LD K 1 QXd 1 X4FIEY LLEGG PSSGA PPPS0

[0271] YX0QGT FTSDY SIX1LD K 1 QXd 1 X4FIEY LLEGG PSSGA PPPS0

[0272] YX0QGT FTSDY SIX1 1 KD K 1 KAQX0AFIEY LLEGG PSSGA PPPS0

[0273] The superscripts (left superscript) in the sequence refer to the modified amino acid unit structure, and the amino acids with the same superscript number refer to the modified groups (stapled bodies) connected by the modifying groups to form a "staple" structure, such as 1 K and 1 E represents modified lysine and glutamic acid, and the two are connected by a modification group (KE modification); 1 K and 1 K represents a modified lysine, and the two are connected by a modification group (KK modification); 1 K and 1 X4 represents modified lysine and X4, and the two are connected by a modification group (K-X4 modification); wherein X0, X1, X4 and S0 and the modification method are as defined in the present invention.

[0274] The present invention provides a series of polypeptide sequences as follows:

[0275] YX0QGT FTSDY SIX1LD K 1 QXd 1 KFIEF LLEGG PSSGA PPPS0

[0276] YX0QGT FTSDY SIX1LD K 1 QXd 1 KFIEF LIEGG PSSGA PPPS0

[0277] YX0QGT FTSDY SIX1LD K 1 QXd 1 KFVEF LIEGG PSSGA PPPS0

[0278] YX0QGT FTSDY SIX1LD KKAQX0AFIE 1 K F 1 KGG PSSGA PPPS0

[0279] YX0QGT FTSDY SIX1LD KKAQX0 1 KFIEF LL 1 KGG PSSGA PPPS0

[0280] YX0QGT FTSDY SIX0LD K 1 QXd 1 KFIEF LLEGG PSSGA PPPS0

[0281] YX0QGT FTSDY S 1 KX1LD 1 KKAQX0AFIEF LLEGG PSSGA PPPS0

[0282] wherein X0, X1 and S0 and the modification methods are as defined above.

[0283] The present invention provides a series of polypeptide sequences as follows:

[0284] YX0QGT FTSDY SIX1LD K 1 QXd 1 KFIEF LLEGG PSSGA PPPS-NH2

[0285] YX0QGT FTSDY SIX1LD K 1 QXd 1 KFIEF LIEGG PSSGA PPP-NH2

[0286] YX0QGT FTSDY SIX1LD K 1 QXd 1 KFVEF LIEGG PSSGA PPP-NH2

[0287] YX0QGT FTSDY SIX1LD KKAQX0AFIE 1 K F 1 KGG PSSGA PPP-NH2

[0288] YX0QGT FTSDY SIX1LD KKAQX0 1 KFIEF LL 1 KGG PSSGA PPP-NH2

[0289] YX0QGT FTSDY SIX0LD K 1 QXd 1 KFIEF LLEGG PSSGA PPP-NH2

[0290] YX0QGT FTSDY S 1 KX1LD 1 KKAQX0AFIEF LLEGG PSSGA PPP-NH2

[0291] Among them, S0 is (ie, the C-terminal amino acid is amidated to become a C-terminal primary amide), X0, X1 and S0 and the modification method are as defined above.

[0292] The present invention provides a series of polypeptide sequences as follows, wherein part X0 is Aib:

[0293] YX0QGT FTSDY SIX1LD K 1 KAQAib 1 KFIEF LLEGG PSSGA PPPS-NH2

[0294] YX0QGT FTSDY SIX1LD K 1 KAQAib 1 KFIEF LIEGG PSSGA PPP-NH2

[0295] YX0QGT FTSDY SIX1LD K 1 KAQAib 1 KFVEF LIEGG PSSGA PPP-NH2

[0296] YX0QGT FTSDY SIX1LD KKAQAib AFIE 1 K F 1 KGG PSSGA PPP-NH2

[0297] YX0QGT FTSDY SIX1LD KKAQAib 1 KFIEF LL 1 KGG PSSGA PPP-NH2

[0298] YX0QGT FTSDY SIAibLD K 1 KAQAib 1 KFIEF LLEGG PSSGA PPP-NH2

[0299] YX0QGT FTSDY S 1 KX1LD 1 KKAQAib AFIEF LLEGG PSSGA PPP-NH2

[0300] Other variables are as defined above.

[0301] The present invention provides a series of polypeptide sequences as follows, wherein X0 is all Aib, and X1 is α-MeL:

[0302] YAibQGT FTSDY SIX1LD K 1 KAQAib 1 KFIEF LLEGG PSSGA PPPS-NH2

[0303] YAibQGT FTSDY SIX1LD K 1 KAQAib 1 KFIEF LIEGG PSSGA PPP-NH2

[0304] YAibQGT FTSDY SIX1LD K 1 KAQAib 1 KFVEF LIEGG PSSGA PPP-NH2

[0305] YAibQGT FTSDY SIX1LD KKAQAib AFIE 1 K F 1 KGG PSSGA PPP-NH2

[0306] YAibQGT FTSDY SIX1LD KKAQAib 1 KFIEF LL 1 KGG PSSGA PPP-NH2

[0307] YAibQGT FTSDY SIAibLD K 1 KAQAib 1 KFIEF LLEGG PSSGA PPP-NH2

[0308] YAibQGT FTSDY S 1 KX1LD 1KKAQAib AFIEF LLEGG PSSGA PPP-NH2

[0309] Other variables are as defined above.

[0310] The present invention provides a series of polypeptide sequences as follows:

[0311] YX0Q GTFTSDYSIX1 1 KDK 1 KAQX0AFIEY LLEGG PSSGA PPPS-NH2

[0312] in,

[0313] S0 is (i.e., the C-terminal amino acid is amidated to a C-terminal primary amide);

[0314] X0 is independently selected from Or Aib, wherein the structure of Aib is

[0315] X1 is α-methyl substituted leucine (α-MeLeu, α-MeL), and its structure is

[0316] The superscripts in the sequence refer to the modified amino acid unit structure, and the amino acids with the same superscript number refer to the modified groups that are connected by modification to form a "staple" structure, such as 1 K and 1 K represents a modified lysine, and the two are connected by a modifying group (KK modification);

[0317] The modification is that the amino groups or carboxyl groups on the side chains of the two amino acid unit structures are Condensation connected;

[0318] X2 is selected from Wherein, "*" indicates the position connected to X3, and the two ends are the positions connected to the corresponding amino or carboxyl groups in the modified amino acid;

[0319] X3 is selected from

[0320]

[0321] m is selected from 1, 2 or 3; p is selected from 1 or 2; n is selected from 8, 9 or 10.

[0322] The present invention provides a series of polypeptide sequences as follows:

[0323] YX0QGT FTSDY SIX1LDKKAQX0AFIE 1 K LL 1 EGG PSSGA PPPS-NH2

[0324] YX0QGT FTSDY SIX1LD KKAQX0AFI 1 KW LL 1 KGG PSSGA PPPS-NH2

[0325] YX0QGT FTSDY SIX1LD K 1 KAQX0 1 KFIEY LLEGG PSSGA PPPS-NH2

[0326] HX0HGT FTSDY SIYLE KKYAX0EFV 1 KW LL 1 KGG PSSGA PPPS-NH2

[0327] HX0HGT FTSDY SIYLE K 1 KYAX0 1 KFVQW LLEGG PSSGA PPPS-NH2

[0328] YX0QGT FTSDY SIX1LD KKAQX0AFIQ 1 K LL 1 KGG PSSGA PPPS-NH2

[0329] YX0QGT FTSDY SIX1LD 1 KQA 1 QX0AFIE 2 K LL 2 KGG PSSGA PPPS-NH2

[0330] YX0QGT FTSDY SIX1LD KEAQX0AFIE 1 K LL 1 KGG PSSGA PPPS-NH2

[0331] HX0QGT FTSDY SIX1LD KKAQX0AFIE 1 K LL 1 KGG PSSGA PPPS-NH2

[0332] YX0QGT FTSDY SIX1LD KKYQX0AFIE 1K LL 1 KGG PSSGA PPPS-NH2

[0333] YX0QGT FTSDY SIX1LE KKAQX0AFIE 1 K LL 1 KGG PSSGA PPPS-NH2

[0334] HX0HGT FTSDY SIYLE KKYAX0EFVQ 1 E LL 1 KGG PSSGA PPPS-NH2

[0335] YX0QGT FTSDY SIX1LD KKAQX0AFIE 1 E LL 1 KGG PSSGA PPPS-NH2

[0336] YX0QGT FTSDY SIX1LD KKAQX0AFI 1 EW LL 1 KGG PSSGA PPPS-NH2

[0337] YX0QGT FTSDY SIX1LD K 1 KAQX0 1 KFIEF LLEGG PSSGA PPPS-NH2

[0338] YX0QGT FTSDY SIX1LD K 1 KAQX0 1 EFIEY LLEGG PSSGA PPPS-NH2

[0339] YX0QGT FTSDY SIX1LD K 1 KAQX0AFI 1 KY LLEGG PSSGA PPPS-NH2

[0340] YX0QGT FTSDY SIX1LD K 1 KAQX0 1 KFIEY LLEGG PSSGA PPPS-NH2

[0341] YX0QGT FTSDY SIX1LD KKA 1 KX0AFI 1 KY LLEGG PSSGA PPPS-NH2

[0342] YX0QGT FTSDY SIX1LD KKAQX0 1 KFIEY LL 1 KGG PSSGA PPPS-NH2

[0343] YX0QGT FTSDY SIX1LD K 1 KAQA 1 X4FIEY LLEGG PSSGA PPPS-NH2

[0344] YX0QGT FTSDY SIX1LD K 1 QXd 1 X4FIEY LLEGG PSSGA PPPS-NH2

[0345] YX0QGT FTSDY SIX1LD K 1 QXd 1 X4FIEY LLEGG PSSGA PPPS-NH2

[0346] in,

[0347] S0 is (i.e., the C-terminal amino acid is amidated to a C-terminal primary amide);

[0348] X0 is independently selected from Or Aib, wherein the structure of Aib is

[0349] X1 is α-methyl substituted leucine (α-MeLeu, α-MeL), and its structure is

[0350] X4 can be independently selected from α-methyl substituted lysine (α-MeLys, α-MeK), whose structure is Or D-lysine (dK), whose structure is Or L-Ornithine (L-Orn), its structure is

[0351] The superscripts in the sequence refer to the modified amino acid unit structure. Amino acids with the same superscript number refer to the modification groups connected by the modification groups to form a "staple" structure, such as 1 K and 1 E represents modified lysine and glutamic acid, and the two are connected by a modification group (KE modification); 1 K and 1 K represents a modified lysine, and the two are connected by a modification group (KK modification);1 K and 1 X4 represents modified lysine and X4, and the two are connected by a modifying group (K-X4 modification);

[0352] The modification is that the amino groups or carboxyl groups on the side chains of the two amino acid unit structures are Condensation connected;

[0353] X2 is selected from Wherein, "*" indicates the position connected to X3, and the two ends are the positions connected to the corresponding amino or carboxyl groups in the modified amino acid;

[0354] X3 is selected from

[0355]

[0356] m is selected from 1, 2 or 3; p is selected from 1 or 2; n is selected from 8, 9 or 10.

[0357] The present invention also provides a series of polypeptide sequences as follows:

[0358] YX0QGT FTSDY SIX1LD KKAQX0AFIE 1 K L 1 EGG PSSGA PPPS-NH2

[0359] YX0QGT FTSDY SIX1LD KKAQX0AFI 1 KW LL 1 KGG PSSGA PPPS-NH2

[0360] YX0QGT FTSDY SIX1LD K 1 QXd 1 KFIEY LLEGG PSSGA PPPS-NH2

[0361] HX0HGT FTSDY SIYLE KKYAX0EFV 1 KW LL 1 KGG PSSGA PPPS-NH2

[0362] HX0HGT FTSDY SIYLE K 1 KYAX0 1 KFVQW LLEGG PSSGA PPPS-NH2

[0363] YX0QGT FTSDY SIX1LD KKAQX0AFIQ 1 K L 1KGG PSSGAPPPS-NH2

[0364] YX0QGT FTSDY SIX1LD 1 QQ 1 QXd 2 K L 2 KGG PSSGA PPPS-NH2

[0365] YX0QGT FTSDY SIX1LD KEAQX0AFIE 1 K L 1 KGG PSSGA PPPS-NH2

[0366] HX0QGT FTSDY SIX1LD KKAQX0AFIE 1 K L 1 KGG PSSGA PPPS-NH2

[0367] YX0QGT FTSDY SIX1LD KKYQX0AFIE 1 K L 1 KGG PSSGA PPPS-NH2

[0368] YX0QGT FTSDY SIX1LE KKAQX0AFIE 1 K L 1 KGG PSSGA PPPS-NH2

[0369] HX0HGT FTSDY SIYLE KKYAX0EFVQ 1 E LL 1 KGG PSSGA PPPS-NH2

[0370] YX0QGT FTSDY SIX1LD KKAQX0AFIE 1 E LL 1 KGG PSSGA PPPS-NH2

[0371] YX0QGT FTSDY SIX1LD KKAQX0AFI 1 E W LL 1 KGG PSSGA PPPS-NH2

[0372] in,

[0373] X0 is independently selected from Or Aib, wherein the structure of Aib is

[0374] X1 is α-methyl substituted leucine (α-MeLeu, α-MeL), and its structure is

[0375] The superscripts in the sequence refer to the modified amino acid unit structure. Amino acids with the same superscript number refer to the modification groups connected by the modification groups to form a "staple" structure, such as 1 K and 1 E represents modified lysine and glutamic acid, and the two are connected by a modification group (KE modification); 1 K and 1 K represents modified lysine, and the two are connected by a modifying group (KK modification).

[0376] The modification is that the amino groups or carboxyl groups on the side chains of the two amino acid unit structures are Condensation connected;

[0377] X2 is selected from Wherein, "*" indicates the position connected to X3, and the two ends are the positions connected to the corresponding amino or carboxyl groups in the modified amino acid;

[0378] X3 is selected from

[0379]

[0380] m is selected from 1, 2 or 3; p is selected from 1 or 2; n is selected from 8, 9 or 10.

[0381] In some embodiments of the present invention, the polypeptide with a modified structure has a peptide chain sequence as follows:

[0382] YX0QGT FTSDY SIX1LD KKAQAib AFIE 1 K L 1 EGG PSSGA PPPS-NH2

[0383] YX0QGT FTSDY SIX1LD KKAQAib AFI 1 KW LL 1 KGG PSSGA PPPS-NH2

[0384] YX0QGT FTSDY SIX1LD K 1 KAQAib 1 KFIEY LLEGG PSSGA PPPS-NH2

[0385] HX0HGT FTSDY SIYLE KKYAAib EFV1 KW LL 1 KGG PSSGA PPPS-NH2

[0386] HX0HGT FTSDY SIYLE K 1 KYAAib 1 KFVQW LLEGG PSSGA PPPS-NH2

[0387] YX0QGT FTSDY SIX1LD KKAQAib AFIQ 1 K LL 1 KGG PSSGA PPPS-NH2

[0388] YX0QGT FTSDY SIX1LD 1 KQA 1 QAib AFIE 2 K LL 2 KGG PSSGA PPPS-NH2

[0389] YX0QGT FTSDY SIX1LD KEAQAib AFIE 1 K LL 1 KGG PSSGA PPPS-NH2

[0390] HX0QGT FTSDY SIX1LD KKAQAib AFIE 1 K LL 1 KGG PSSGA PPPS-NH2

[0391] YX0QGT FTSDY SIX1LD KKYQAib AFIE 1 K LL 1 KGG PSSGA PPPS-NH2

[0392] YX0QGT FTSDY SIX1LE KKAQAib AFIE 1 K LL 1 KGG PSSGA PPPS-NH2

[0393] HX0HGT FTSDY SIYLE KKYAAib EFVQ 1 E LL 1 KGG PSSGA PPPS-NH2

[0394] YX0QGT FTSDY SIX1LD KKAQAib AFIE 1 E LL1 KGG PSSGA PPPS-NH2

[0395] YX0QGT FTSDY SIX1LD KKAQAib AFI 1 E W LL 1 KGG PSSGA PPPS-NH2

[0396] Other variables are as defined in the present invention.

[0397] In some embodiments of the present invention, the polypeptide with a modified structure has a peptide chain sequence as follows:

[0398] YX0QGT FTSDY SIX1LD K 1 KAQAib 1 KFIEF LLEGG PSSGA PPPS-NH2

[0399] YX0QGT FTSDY SIX1LD K 1 KAQAib 1 EFIEY LLEGG PSSGA PPPS-NH2

[0400] YX0QGT FTSDY SIX1LD K 1 KAQAib AFI 1 KY LLEGG PSSGA PPPS-NH2

[0401] YX0QGT FTSDY SIX1LD K 1 KAQAib 1 KFIEY LLEGG PSSGA PPPS-NH2

[0402] YX0QGT FTSDY SIX1LD KKA 1 KAib AFI 1 KY LLEGG PSSGA PPPS-NH2

[0403] YX0QGT FTSDY SIX1LD KKAQAib 1 KFIEY LL 1 KGG PSSGA PPPS-NH2

[0404] YX0QGT FTSDY SIX1LD K 1 KAQA 1 X4FIEY LLEGG PSSGA PPPS-NH2

[0405] YX0QGT FTSDY SIX1LD K 1 KAQAib 1 X4FIEY LLEGG PSSGA PPPS-NH2

[0406] YX0QGT FTSDY SIX1LD K 1 KAQAib 1 X4FIEY LLEGG PSSGA PPPS-NH2

[0407] Other variables are as defined herein.

[0408] In some embodiments of the present invention, the polypeptide with a modified structure has a peptide chain sequence as follows:

[0409] YX0QGT FTSDY SIX1 1 KD K 1 KAQAib AFIEY LLEGG PSSGA PPPS-NH2

[0410] Other variables are as defined herein.

[0411] In some embodiments of the present invention, the polypeptide with a modified structure has a peptide chain sequence as shown in any of the following:

[0412] YX0QGTFTSDY SI X1 1 KD K 1 KAQAib EFIEYLLE GG PSSGAPPP S-NH2

[0413] YX0QGTFTSDY SI X1 1 KD K 1 KAQAib AF I EY LLKGG P SSGA PPPS-NH2 Other variables are as defined in the present invention.

[0414] In some embodiments of the present invention, the polypeptide with a modified structure has X0 all being Aib and X1 all being α-MeL, that is, its peptide chain sequence is any of the following:

[0415] YAibQG T FTSDY SIX1 1 KDK 1 KAQAibEFIEY L LEGG PS SG AP PP S-NH2

[0416] YAibQ GTF TSDY SI X1 1 KDK1 KAQAibAFIE YL LKG GPS SG AP PPS-NH2

[0417] Other variables are as defined in the present invention.

[0418] In some embodiments of the present invention, the polypeptide with a modified structure has X0 all being Aib and X1 all being α-MeL, that is, its peptide chain sequence is as follows:

[0419] YAibQGT FTSDY SIX1LD KKAQAib AFIE 1 K L 1 EGG PSSGA PPPS-NH2 (SEQ ID NO: 3)

[0420] YAibQGT FTSDY SIX1LD KKAQAib AFI 1 KW LL 1 KGG PSSGA PPPS-NH2 (SEQ ID NO: 2)

[0421] YAibQGT FTSDY SIX1LD K 1 KAQAib 1 KFIEY LLEGG PSSGA PPPS-NH2 (SEQ ID NO: 1)

[0422] HAibHGT FTSDY SIYLE KKYAAib EFV 1 KW LL 1 KGG PSSGA PPPS-NH2 (SEQ ID NO: 4)

[0423] HAibHGT FTSDY SIYLE K 1 YjA 1 KFVQW LLEGG PSSGA PPPS-NH2 (SEQ ID NO: 5)

[0424] YAibQGT FTSDY SIX1LD KKAQAib AFIQ 1 K L 1 KGG PSSGA PPPS-NH2 (SEQ ID NO: 6)

[0425] YAibQGT FTSDY SIX1LD 1 QQ 1 QAib AFIE 2 K L 2KGG PSSGA PPPS-NH2 (SEQ ID NO: 7)

[0426] YAibQGT FTSDY SIX1LD KEAQAib AFIE 1 K L 1 KGG PSSGA PPPS-NH2 (SEQ ID NO: 8)

[0427] HAibQGT FTSDY SIX1LD KKAQAib AFIE 1 K L 1 KGG PSSGA PPPS-NH2 (SEQ ID NO: 9)

[0428] YAibQGT FTSDY SIX1LD KKYQAib AFIE 1 K L 1 KGG PSSGA PPPS-NH2 (SEQ ID NO: 10)

[0429] YAibQGT FTSDY SIX1LE KKAQAib AFIE 1 K L 1 KGG PSSGA PPPS-NH2 (SEQ ID NO: 11)

[0430] HAibHGT FTSDY SIYLE KKYAAib EFVQ 1 E LL 1 KGG PSSGA PPPS-NH2 (SEQ ID NO: 12)

[0431] YAibQGT FTSDY SIX1LDKKAQAib AFIE 1 E LL 1 KGG PSSGA PPPS-NH2 (SEQ ID NO: 13)

[0432] YAibQGT FTSDY SIX1LD KKAQAib AFI 1 E W LL 1 KGG PSSGA PPPS-NH2 (SEQ ID NO: 14)

[0433] Other variables are as defined in the present invention.

[0434] In some embodiments of the present invention, the polypeptide with a modified structure has X0 all being Aib, and its peptide chain sequence is as follows:

[0435] YAibQGT FTSDY SIX1LD K 1 KAQAib 1 KFIEF LLEGG PSSGA PPPS-NH2

[0436] YAibQGT FTSDY SIX1LD K 1 KAQAib 1 EFIEY LLEGG PSSGA PPPS-NH2

[0437] YAibQGT FTSDY SIX1LD K 1 KAQAib AFI 1 KY LLEGG PSSGA PPPS-NH2

[0438] YAibQGT FTSDY SIX1LD K 1 KAQAib 1 KFIEY LLEGG PSSGA PPPS-NH2

[0439] YAibQGT FTSDY SIX1LD KKA 1 KAIB AFI 1 KY LLEGG PSSGA PPPS-NH2

[0440] YAibQGT FTSDY SIX1LD KKAQAib 1 KFIEY LL 1 KGG PSSGA PPPS-NH2

[0441] YAibQGT FTSDY SIX1LD K 1 KAA 1 X4KFIEY LLEGG PSSGA PPPS-NH2

[0442] YAibQGT FTSDY SIX1LD K 1 KAQAib 1 X4FIEY LLEGG PSSGA PPPS-NH2

[0443] YAibQGT FTSDY SIX1LD K 1 KAQAib 1 X4FIEY LLEGG PSSGA PPPS-NH2

[0444] Other variables are as defined in the present invention.

[0445] In some embodiments of the present invention, the polypeptide with a modified structure, X1 is α-MeL, and its peptide chain sequence is as follows:

[0446] YAibQGT FTSDY SIX1LD K 1 KAQAib 1 KFIEF LLEGG PSSGA PPPS-NH2

[0447] YAibQGT FTSDY SIX1LD K 1 KAQAib 1 EFIEY LLEGG PSSGA PPPS-NH2

[0448] YAibQGT FTSDY SIX1LD K 1 KAQAib AFI 1 KY LLEGG PSSGA PPPS-NH2

[0449] YAibQGT FTSDY SIX1LD K 1 KAQAib 1 KFIEY LLEGG PSSGA PPPS-NH2

[0450] YAibQGT FTSDY SIX1LD KKA 1 KAib AFI 1 KY LLEGG PSSGA PPPS-NH2

[0451] YAibQGT FTSDY SIX1LD KKAQAib 1 KFIEY LL 1 KGG PSSGA PPPS-NH2

[0452] YAibQGT FTSDY SIX1LD K 1 KAQA 1 α-MeKFIEY LLEGG PSSGA PPPS-NH2

[0453] YAibQGT FTSDY SIX1LD K 1 KAQAib 1 d-KFIEY LLEGG PSSGA PPPS-NH2

[0454] YAibQGT FTSDY SIX1LD K 1 KAQAib 1L-OrnFIEY LLEGG PSSGA PPPS-NH2

[0455] Other variables are as defined herein.

[0456] In some embodiments of the present invention, the polypeptide with a modified structure has X0 all being Aib, and its peptide chain sequence is as follows:

[0457] Y Aib QGTFTSDYSI X1 1 KDK 1 KQ Aib

[0458] AFIEYLLEGGPSSGAPPP S-NH2

[0459] Other variables are as defined herein.

[0460] The present invention also provides a series of polypeptides with modified structures or pharmaceutically acceptable salts thereof:

[0461] YX0QGT FTSDY SIX1LD KKAQX0 1 KFVEF LL 1 KGG PSSGA PPPS0

[0462] YX0QGT FTSDY SIX1LD KKAQX0 1 KFVEF LI 1 KGG PSSGA PPPS0

[0463] YX0QGT FTSDY SIX1LD K 1 QXd 1 KFIEF LLKGG PSSGA PPPS0

[0464] YX0QGT FTSDY SIX1LD K 1 QXd 1 KFIKF LLEGG PSSGA PPPS0

[0465] YX0QGT FTSDY S 1 KX1LD 1 KKAQX0KFIEF LLEGG PSSGA PPPS0

[0466] And the compound has the following modifications:

[0467] The modification is that the amino groups or carboxyl groups on the side chains of the two amino acid unit structures are Condensation connected;

[0468] X2 is selected from Wherein, "*" indicates the position connected to X3, and the two ends are the positions connected to the corresponding amino or carboxyl groups in the modified amino acid;

[0469] X3 is selected from

[0470] m is selected from 1, 2 or 3; p is selected from 1, 2; n is selected from 8, 9 or 10, and other variables are as defined in the present invention.

[0471] In some embodiments of the present invention, the polypeptide with a modified structure or a pharmaceutically acceptable salt thereof has a peptide chain sequence as shown below:

[0472] YX0QGT FTSDY SIX1LD KKAQX0 1 KFVEF LL 1 KGG PSSGA PPPS-NH2

[0473] YX0QGT FTSDY SIX1LD KKAQX0 1 KFVEF LI 1 KGG PSSGA PPPS-NH2

[0474] YX0QGT FTSDY SIX1LD K 1 QXd 1 KFIEF LLKGG PSSGA PPPS-NH2

[0475] YX0QGT FTSDY SIX1LD K 1 QXd 1 KFIKF LLEGG PSSGA PPPS-NH2

[0476] YX0QGT FTSDY S 1 KX1LD 1 KKAQX0KFIEF LLEGG PSSGA PPPS-NH2

[0477] , other variables are as defined in the present invention.

[0478] In some embodiments of the present invention, the polypeptide with a modified structure or a pharmaceutically acceptable salt thereof, wherein part X0 is Aib, that is, its peptide chain sequence is as follows:

[0479] YAibQGT FTSDY SIX1LD KKAQX0 1KFVEF LL 1 KGG PSSGA PPPS-NH2

[0480] YAibQGT FTSDY SIX1LD KKAQX0 1 KFVEF LI 1 KGG PSSGA PPPS-NH2

[0481] YAibQGT FTSDY SIX1LD K 1 QXd 1 KFIEF LLKGG PSSGA PPPS-NH2

[0482] YAibQGT FTSDY SIX1LD K 1 QXd 1 KFIKF LLEGG PSSGA PPPS-NH2

[0483] YAibQGT FTSDY S 1 KX1LD 1 KKAQX0KFIEF LLEGG PSSGA PPPS-NH2

[0484] Other variables are as defined herein.

[0485] In some embodiments of the present invention, the polypeptide with a modified structure or a pharmaceutically acceptable salt thereof, wherein X0 is all Aib, and X1 is α-MeL, that is, its peptide chain sequence is as follows:

[0486] YAibQGT FTSDY SIX1LD KKAQAib 1 KFVEF LL 1 KGG PSSGA PPPS-NH2

[0487] YAibQGT FTSDY SIX1LD KKAQAib 1 KFVEF LI 1 KGG PSSGA PPPS-NH2

[0488] YAibQGT FTSDY SIX1LD K 1 KAQAib 1 KFIEF LLKGG PSSGA PPPS-NH2

[0489] YAibQGT FTSDY SIX1LD K 1 KAQAib 1KFIKF LLEGG PSSGA PPPS-NH2

[0490] YAibQGT FTSDY S 1 KX1LD 1 KKAQAib KFIEF LLEGG PSSGA PPPS-NH2

[0491] Other variables are as defined in the present invention.

[0492] In some embodiments of the present invention, the polypeptide with a modified structure or a pharmaceutically acceptable salt thereof has a peptide chain sequence as shown below:

[0493] YX0QGT FTSDY SIX1 1 KD K 1 KAQX0AFIQY LLEGG PSSGA PPPS-NH2

[0494] , other variables are as defined in the present invention.

[0495] In some embodiments of the present invention, the polypeptide with a modified structure or a pharmaceutically acceptable salt thereof, the X0 portion is Aib ， The peptide chain sequence is as follows:

[0496] YAib QGTFTSDY SIX1 1 KD K 1 KAQX0AFIQY LLEGG PSSGA PPPS-NH2

[0497] , other variables are as defined in the present invention.

[0498] In some embodiments of the present invention, the polypeptide with a modified structure or a pharmaceutically acceptable salt thereof, X0 is all Aib, X1 is α-MeL, and its peptide chain sequence is as follows:

[0499] YAib QGTFTSDY SIX1 1 KD K 1 KAQAib AFIQY LLEGG PSSGA PPPS-NH2

[0500] , other variables are as defined in the present invention.

[0501] In some embodiments of the present invention, the above m is selected from 1 or 2, and other variables are as defined in the present invention.

[0502] In some embodiments of the present invention, the above p is selected from 1, and other variables are as defined in the present invention.

[0503] In some embodiments of the present invention, the above n is selected from 9, and other variables are as defined in the present invention.

[0504] In some embodiments of the present invention, the above X3 is selected from

[0505]

[0506] Other variables are as defined herein.

[0507] In some embodiments of the present invention, the above structural unit for

[0508] Other variables are as defined herein.

[0509] In some embodiments of the present invention, the above structural unit for Other variables are as defined herein.

[0510] In some embodiments of the present invention, the above structural unit for Other variables are as defined herein.

[0511] In some embodiments of the present invention, the above structural unit for Other variables are as defined herein.

[0512] In some embodiments of the present invention, the above structural unit for Other variables are as defined herein.

[0513] In some embodiments of the present invention, the above structural unit for Other variables are as defined herein.

[0514] In some embodiments of the present invention, the above structural unit

[0515] Other variables are as defined herein.

[0516] In some embodiments of the present invention, the above structural unit for

[0517] Other variables are as defined herein.

[0518] In some embodiments of the present invention, the above structural unit for

[0519] Other variables are as defined in the present invention.

[0520] In some embodiments of the present invention, the "staple" structure (staple complex) in the above-mentioned polypeptide with a modified structure is any of the following, and other variables are as defined in the present invention:

[0521]

[0522]

[0523] In some embodiments of the present invention, the "staple" structure (staple complex) in the above-mentioned polypeptide with a modified structure is any of the following, and other variables are as defined in the present invention:

[0524]

[0525]

[0526] In some embodiments of the present invention, the "staple" structure (staple complex) in the above-mentioned polypeptide with a modified structure is any of the following, and other variables are as defined in the present invention:

[0527]

[0528]

[0529] In some embodiments of the present invention, the "staple" structure (staple complex) in the above-mentioned polypeptide with a modified structure is any of the following, and other variables are as defined in the present invention:

[0530]

[0531] In some embodiments of the present invention, the "staple" structure (staple complex) in the above-mentioned polypeptide with a modified structure is any of the following, and other variables are as defined in the present invention:

[0532]

[0533] In some embodiments of the present invention, the "staple" structure (staple complex) in the above-mentioned polypeptide with a modified structure is any of the following, and other variables are as defined in the present invention:

[0534]

[0535] In some embodiments of the present invention, the "staple" structure (staple body) in the above-mentioned polypeptide with a modified structure is as follows, and other variables are as defined in the present invention:

[0536]

[0537] In some specific embodiments of the present invention, the structure of the polypeptide with the modified structure is shown in Table 1 below, and the structure of Aib is The α-MeL is α-methyl substituted leucine, and its structure is The α-MeK is α-methyl substituted lysine, and its structure is Other variables are as defined in the present invention, for example, the superscripts in the sequence refer to modified amino acid unit structures, and amino acids with the same superscript number refer to modification groups connected by modification groups to form a "staple"-like structure, etc.

[0538] Table 1 Peptides with modified structures

[0539]

[0540]

[0541]

[0542] In some specific embodiments of the present invention, the structure of the polypeptide with the modified structure is shown in Table 2 below, and the structure of Aib is The α-MeL is α-methyl substituted leucine, and its structure is The α-MeK is α-methyl substituted lysine, and its structure is The dK is D-lysine, and its structure is The L-Ornithine or L-Orn is L-ornithine, and its structure is Other variables are as defined in the present invention, for example, the superscripts in the sequence refer to modified amino acid unit structures, and amino acids with the same superscript number refer to modification groups connected by modification groups to form a "staple" (stapled body) structure, etc.

[0543] Table 2 Peptides with modified structures

[0544]

[0545]

[0546]

[0547] In some specific embodiments of the present invention, the structure of the polypeptide with the modified structure is shown in Table 3 below, and the structure of Aib is The α-MeL is α-methyl substituted leucine, and its structure is The α-MeK is α-methyl substituted lysine, and its structure is Other variables are as defined in the present invention, for example, the superscripts in the sequence refer to modified amino acid unit structures, and amino acids with the same superscript number refer to modification groups connected by modification groups to form a "staple"-like structure, etc.

[0548] Table 3 Peptides with modified structures

[0549]

[0550]

[0551] In some specific embodiments of the present invention, X1 is α-MeL, and the structure of the polypeptide with the modified structure is shown in Table 4 below, and the structure of Aib is The α-MeL is α-methyl substituted leucine, and its structure is Other variables are as defined in the present invention, for example, the superscripts in the sequence refer to modified amino acid unit structures, and amino acids with the same superscript number refer to modification groups connected by modification groups to form a "staple"-like structure, etc.

[0552] Table 4 Peptides with modified structures

[0553]

[0554]

[0555] In some specific embodiments of the present invention, X1 is α-MeL, and the structure of the polypeptide with the modified structure is shown in Table 5 below:

[0556] Table 5 Peptides with modified structures

[0557]

[0558]

[0559]

[0560] In some specific embodiments of the present invention, X1 is α-MeL, and the structure of the polypeptide with the modified structure is shown in Table 6 below:

[0561] Table 6 Peptides with modified structures

[0562]

[0563]

[0564]

[0565] In some embodiments of the present invention, the "staple" structure (staple body) in the above-mentioned polypeptide with a modified structure is as follows, and other variables are as defined in the present invention:

[0566]

[0567]

[0568] In some specific embodiments of the present invention, X1 is α-MeL, and the structure of the polypeptide with the modified structure is shown in Table 7 below:

[0569] Table 7 Peptides with modified structures

[0570]

[0571]

[0572] In some embodiments of the present invention, the polypeptide with a modified structure or a pharmaceutically acceptable salt thereof has a peptide chain sequence as shown below:

[0573] YX0QGT FTSDY SIX1 1 KD K 1 KAQX0KFIEY LLEGG PSSGA PPPS-NH2

[0574] YX0QGT FTSDY SIX1 1 KD K 1 KAQX0LFIEY LLEGG PSSGA PPPS-NH2

[0575] YX0QGT a-MeFTSDY SIX1 1 KD K 1 KAQX0AFIEY LLEGG PSSGA PPPS-NH2

[0576] YX0QGT a-MeFTSDY SIX1 1 KD K 1 KAQX0AFIEY LLKGG PSSGA PPPS-NH2

[0577] , other variables are as defined in the present invention.

[0578] In some embodiments of the present invention, the polypeptide with a modified structure or a pharmaceutically acceptable salt thereof, the X0 portion is Aib, and the peptide chain sequence thereof is as follows:

[0579] YAibQGT FTSDY SIX1 1 KD K 1KAQX0KFIEY LLEGG PSSGA PPPS-NH2

[0580] YAibQGT FTSDY SIX1 1 KD K 1 KAQX0LFIEY LLEGG PSSGA PPPS-NH2

[0581] YAibQGT a-MeFTSDY SIX1 1 KD K 1 KAQX0AFIEY LLEGG PSSGA PPPS-NH2

[0582] YAibQGT a-MeFTSDY SIX1 1 KD K 1 KAQX0AFIEY LLKGG PSSGA PPPS-NH2

[0583] , other variables are as defined in the present invention.

[0584] In some embodiments of the present invention, the polypeptide with a modified structure or a pharmaceutically acceptable salt thereof, X0 is all Aib, X1 is α-MeL, and its peptide chain sequence is as follows:

[0585] YAibQGT FTSDY SIX1 1 KD K 1 KAQAib KFIEY LLEGG PSSGA PPPS-NH2

[0586] YAibQGT FTSDY SIX1 1 KD K 1 KAQAib LFIEY LLEGG PSSGA PPPS-NH2

[0587] YAibQGT a-MeFTSDY SIX1 1 KD K 1 KAQAib AFIEY LLEGG PSSGA PPPS-NH2

[0588] YAibQGT a-MeFTSDY SIX1 1 KD K 1 KAQAib AFIEY LLKGG PSSGA PPPS-NH2

[0589] , other variables are as defined in the present invention.

[0590] In some embodiments of the present invention, the "staple" structure (staple body) in the above-mentioned polypeptide with a modified structure is as follows, and other variables are as defined in the present invention:

[0591]

[0592]

[0593] In some specific embodiments of the present invention, the structures of polypeptide compounds with modified structures are shown in Table 8 below:

[0594] Table 8 Peptides with modified structures

[0595]

[0596]

[0597]

[0598] The present invention also provides the above-mentioned pharmaceutical composition, comprising as active ingredients a therapeutically effective amount of the above-mentioned polypeptide compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0599] In some embodiments of the present invention, the above-mentioned polypeptide compound or its pharmaceutically acceptable salt or the above-mentioned composition is used in the preparation of a drug for preventing or treating metabolic diseases and related diseases.

[0600] In some embodiments of the present invention, the above-mentioned polypeptide compound or its pharmaceutically acceptable salt or the above-mentioned composition is used in the preparation of a drug for preventing or treating diabetes and related diseases.

[0601] In some embodiments of the present invention, the above-mentioned polypeptide compound or its pharmaceutically acceptable salt or the above-mentioned composition is used in the preparation of a drug for preventing or treating obesity and related diseases.

[0602] In some embodiments of the present invention, the above-mentioned polypeptide compound or a pharmaceutically acceptable salt thereof or the above-mentioned composition is used in the preparation of a drug for preventing or treating NASH (non-alcoholic steatohepatitis) and related diseases.

[0603] Technical Effects

[0604] The polypeptide of the present invention has strong in vitro agonist activity on GLP-1R / GIPR / GCGR, and the compound of the present invention has excellent in vitro and in vivo pharmacokinetic properties; the compound of the present invention has obvious weight loss, blood sugar lowering and blood lipid lowering effects.

[0605] Definition and Description

[0606] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

[0607] Unless otherwise specified, the “polypeptide”, “peptide” and “polypeptide compound” mentioned in the present invention can be understood by those skilled in the art to have the same meaning in combination with the context.

[0608] Unless otherwise specified, the "polypeptide sequence" and "peptide chain" mentioned in the present invention can be understood by those skilled in the art to have the same meaning in combination with the context.

[0609] The “staple”, “staple”, “stapled body” and “staple” mentioned in the present invention, unless otherwise specified, can be understood by those skilled in the art as having the same meaning in combination with the context.

[0610] Unless otherwise specified, the example numbers of the compounds in the present invention remain consistent with the order of the compound numbers, that is, the compounds indicated by "Example 1" and "Compound 1" are compounds with the same structure.

[0611] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0612] The term "pharmaceutically acceptable salt" refers to salts of compounds of the invention, prepared from compounds of the invention having specific substituents with relatively nontoxic acids or bases. When the compounds of the invention contain relatively acidic functional groups, base addition salts can be obtained by contacting such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When the compounds of the invention contain relatively basic functional groups, acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, trifluoroacetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid and pamoic acid and the like; also include salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and acidic functional groups, and thus can be converted into either base or acid addition salts.

[0613] Pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, the preparation method of such salts is: in water or an organic solvent or a mixture of the two, these compounds in free acid or base form are reacted with a stoichiometric amount of an appropriate base or acid to prepare.

[0614] "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 are later modified, e.g., hydroxyproline, γ-carboxyglutamate, 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 naturally occurring amino acids, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds whose structure is different from the general amino acid chemical structure, but function similarly to naturally occurring amino acids.

[0615] The natural amino acids and their various expressions described herein are well known to those skilled in the art, and their specific corresponding situations are shown in the following table:

[0616]

[0617]

[0618]

[0619] Some other unnatural amino acids, such as 2-aminoisobutyric acid (Aib), etc., correspond to the following table:

[0620]

[0621] The term "treating" includes inhibiting, slowing, halting or reversing the progression or severity of existing symptoms or disorders.

[0622] Unless otherwise stated, the term "isomer" is intended to include geometric isomers, cis-trans isomers, stereoisomers, enantiomers, optical isomers, diastereomers and tautomers.

[0623] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All of these isomers and their mixtures are included within the scope of the present invention.

[0624] Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of one another.

[0625] Unless otherwise indicated, the term "cis-trans isomers" or "geometric isomers" arises from the inability of a ring to rotate freely about double bonds or single bonds of ring carbon atoms.

[0626] Unless otherwise indicated, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and that are not mirror images of each other.

[0627] Unless otherwise indicated, "(+)" indicates dextrorotatory, "(-)" indicates levorotatory, and "(±)" indicates racemic.

[0628] Unless otherwise specified, the key is a solid wedge. and dotted wedge key To indicate the absolute configuration of a stereocenter, use a straight solid bond. and straight dashed key To indicate the relative configuration of a stereocenter, use a wavy line Indicates a wedge-shaped solid key or dotted wedge key Or use a wavy line Represents a straight solid bond or straight dashed key

[0629] Unless otherwise indicated, the terms "enriched in one isomer", "isomerically enriched", "enriched in one enantiomer" or "enantiomerically enriched" mean that the content of one isomer or enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

[0630] Unless otherwise indicated, the term "isomer excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80%.

[0631] Optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is usually accomplished by using chromatography, which uses a chiral stationary phase and is optionally combined with a chemical derivatization method (for example, a carbamate is generated from an amine).

[0632] The compounds of the invention may contain unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound. For example, the compounds may be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125( 125 I) or C-14( 14C). For another example, deuterium can be used to replace hydrogen to form a deuterated drug. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have the advantages of reducing toxic side effects, increasing drug stability, enhancing efficacy, and extending the biological half-life of drugs. All isotopic composition changes of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.

[0633] When the listed linking group does not indicate its linking direction, its linking direction is arbitrary, for example, The connecting group L is -MW-, in which case -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form You can also connect ring A and ring B in the opposite direction of the reading order from left to right to form Combinations of linkers, substituents, and / or variations thereof are permissible only if such combinations result in stable compounds.

[0634] Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of the group can be connected to other groups through chemical bonds. When the chemical bond connection mode is non-positional and there are H atoms at the connectable sites, when the chemical bonds are connected, the number of H atoms at the site will decrease with the number of connected chemical bonds to become a group with the corresponding valence. The chemical bond connecting the site to other groups can be a straight solid bond. Straight dotted key or wavy line For example, the straight solid bond in -OCH3 indicates that it is connected to other groups through the oxygen atom in the group; The straight dashed bond in the group indicates that the two ends of the nitrogen atom in the group are connected to other groups; The wavy lines in the phenyl group represent the connection to other groups through the carbon atoms at positions 1 and 2 in the phenyl group.

[0635] Unless otherwise specified, the term “C 1-3 "Alkyl" is used to refer to a straight or branched chain saturated hydrocarbon group consisting of 1 to 3 carbon atoms. 1-3 Alkyl groups include C 1-2 and C 2-3 Alkyl, etc.; it may be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine). 1-3 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

[0636] The structure of the compound of the present invention can be confirmed by conventional methods known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data of the cultured single crystal using a Bruker D8venture diffractometer, the light source is CuKα radiation, and the scanning mode is: After scanning and collecting relevant data, the crystal structure is further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0637] The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention.

[0638] The materials and solvents used in the present invention are commercially available.

[0639] Compounds are named according to the conventional nomenclature in the art or using The software names were used, and commercially available compounds were named using the supplier's catalog names.

[0640] In the present invention, compound groups or reagents known to those skilled in the art can be represented by abbreviations, including but not limited to compound groups: tert-butyloxycarbonyl (BOC), benzyl (Bn), p-toluenesulfonic acid (Tos), benzyloxycarbonyl (Cbz), fluorenylmethyloxycarbonyl (Fmoc), p-toluenesulfonyl (Ts), allyloxycarbonyl (Alloc), etc.; including but not limited to conventional reagents: dimethyl sulfoxide (DMSO), dichloromethane (DCM), N,N-dimethylformamide (DMF), tetrahydrofuran (THF), trifluoroacetic acid (TFA), N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxy-7-azabenzotriazole (HOAT), tetrakistriphenylphosphine palladium (Pd(PPh3)4); Fmoc-AEEA-OH: Eicosanedioic acid monotert-butyl ester: N-Boc-N'-Fmoc-Lys-OH: 19-(bis(benzyloxy)phosphoryl)nonadecanoic acid: etc. DETAILED DESCRIPTION

[0641] The present invention is described in detail below by way of examples, but it is not intended to impose any adverse limitations on the present invention. The present invention has been described in detail herein, and its specific embodiments are also disclosed therein. It will be apparent 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. The specific synthesis steps described in the present invention also include new intermediates and methods that can be used to synthesize the polypeptide compounds of the present invention or their pharmaceutically acceptable salts. In the specific synthesis steps of the following examples, those of ordinary skill in the art can combine in different ways to prepare the polypeptide compounds of the present invention or their salts.

[0642] In general, the series of polypeptides with modified structures described in the present invention are prepared by a polypeptide solid phase synthesis method, starting from MBHA resin, using the Fmoc-strategy, and condensing protected amino acids one by one from the C-terminus to the N-terminus according to the peptide sequence (including condensation, deprotection, elution cycle, and removal of side chain protection to form a lactam ring), cleavage, filtration and concentration, precipitation, filtration and drying to obtain a branched cyclic peptide cleavage crude product, which is then purified, concentrated and freeze-dried to obtain a finished product.

[0643] Example 1

[0644]

[0645] The peptide resin was synthesized by a conventional solid phase synthesis process, with nitrogen bubbling reaction in a solid phase reaction column with a sintered sieve plate at the bottom, MBHA Resin was used as the starting resin, conventional Fmoc protected amino acids were added, a DIC / HOBt system was used as the condensing agent, DMF was used as the main reaction solvent, and a 20% PIP-DMF solution was used as the Fmoc removal solvent, and condensation was performed one by one from the C-terminus to the N-terminus of the peptide main chain. After the main chain synthesis was completed, the -Mtt on the ε-amino group of Fmoc-Lys(Mtt)-OH at position A-23 was removed, and then the side 1 to side 5 ((2-2-oxoethyl)-glycine, AEEA, AEEA, γGlu, mono-tert-butyl eicosandioic acid) were condensed in sequence, and then the ε-amino protecting group of Lys at position A-19 and the carboxyl protecting group of (2-2-oxoethyl)-glycine at position 1 were removed, and then a condensing agent was added to form a lactam ring to obtain the target peptide resin.

[0646] The specific steps are as follows:

[0647] Step 1: Resin coupling Fmoc-RinkLinker

[0648] 1. Take MBHA Resin (S = 0.49mmol / g) 10.2g (5mmol) and add it to the solid phase reaction column, add DCM 70mL and let it stand for 20min to fully swell the resin, drain the DCM, add 60mL DMF and wash the resin by bubbling nitrogen, repeat the above steps once, and drain the solvent. Weigh Fmoc-Rink Linker 10.8g (20mmol, 4.0eq) and dissolve it in 40mL DMF and add it to the above resin, add DIC 2.78g (22mmol, 4.4eq) and add 10mL DMF to the reaction column, bubbling nitrogen, and add HOBt 2.70g (20mmol, 4.0eq) after it is completely dissolved, and continue to bubbling nitrogen for reaction;

[0649] 2. React at 25°C for more than 2 hours. If the sample is negative for ninhydrin test (resin is colorless or light yellow), the reaction is terminated.

[0650] 3. Draw off the reaction liquid, wash with DMF (300 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[0651] Step 2: Fmoc-Ser(tBu)-OH coupling

[0652] 1. Add 20% piperidine / DMF (100 mL) to the reaction column, bubbling nitrogen for 10 min, drain until no liquid flows out, add 20% piperidine / DMF (100 mL) to the reaction column again, continue bubbling nitrogen for 5 min, take a sample for ninhydrin detection, and the resin is blue;

[0653] 2. Weigh 7.66g (4.0eq) of Fmoc-Ser(tBu)-OH, 2.78g (4.4eq) of DIC and 2.70g (4.0eq) of HOBt, dissolve them in 60mL of DMF and add them to the reaction column. Blow nitrogen and adjust the nitrogen to make the resin swell evenly.

[0654] 3. React in an environment of 25°C for 2.0 hours, take samples for ninhydrin detection, and the resin is colorless and transparent;

[0655] 4. Draw off the reaction liquid, wash with DMF (300 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[0656] Main chain amino acid coupling steps

[0657] Referring to the method and amount of Fmoc-Ser(tBu)-OH coupling in step 2, the amino acid sequence is sequentially connected, and the order and amino acids containing protective groups used are as follows:

[0658]

[0659]

[0660] Step 3. Side-chain coupling

[0661] 1. Add 300 mL of DMF and 300 mL of DCM to the reaction column in sequence to cross-wash the resin 4 times. After draining the solvent, add 300 mL of 20% hexafluoroisopropanol (HFIP)-DCM solution and react twice with nitrogen bubbling, each time for 30 min;

[0662] 2. Drain the waste until no liquid flows out, take a sample for ninhydrin testing, and the resin will be blue;

[0663] 3. Add 300 mL of DCM and 300 mL of DMF in sequence to cross-wash the resin 4 times and drain until no liquid flows out.

[0664] 4. Weigh 7.9 g (4.0 eq) of Fmoc-(2-(allyloxy)-2-oxoethyl)glycine (Fmoc-Ida(OAll)-OH), 2.78 g (4.4 eq) of DIC and 2.70 g (4.0 eq) of HOBt, dissolve them in 100 mL of DMF and add them to the reaction column. Add nitrogen and adjust the nitrogen to make the resin swell evenly.

[0665] 5. React in an environment of 25°C for 2.0 hours, take samples for ninhydrin detection, and the resin is colorless and transparent;

[0666] 6. Draw off the reaction liquid, wash with DMF (300 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[0667] The remaining branch coupling steps

[0668] Referring to the method and feed equivalents of Fmoc-Ser(tBu)-OH coupling in step 2, the following compounds were added in sequence:

[0669] Amino acids containing protecting groups used Remark Fmoc-AEEA-OH Fmoc-AEEA-OH Fmoc-Glu-OtBu Eicosanedioic acid monotert-butyl ester

[0670] Step 4: Ring closure

[0671] 1. Add 300 mL of DMF and 300 mL of DCM to the reaction column in sequence, cross-wash the resin 4 times, and drain until no liquid flows out;

[0672] 2. Add 400 ml of the mixed solvent at a volume ratio of DCM: CH3CN: NMP: DEA = 3:3:2:2, and then add 40.58 g (0.1 eq) of Pd(PPh3), and react for 10 min with nitrogen bubbling, and drain until no liquid flows out;

[0673] 3. Repeat the above steps 1 to 2 times;

[0674] 4. Take samples for ninhydrin testing, the resin is blue;

[0675] 5. Add 300 mL of DMF and 300 mL of DCM in sequence and cross-wash the resin 8 times;

[0676] 6. Dissolve 2.78g (4.4eq) of DIC and 2.70g (4.0eq) of HOBt in 150mL of DMF, add to the reaction column, react for 48h with nitrogen, take a sample for ninhydrin detection, and the resin is colorless;

[0677] 7. Add 300 mL of DMF and 300 mL of DCM in sequence and cross-wash the resin 8 times;

[0678] 8. Add 200 mL of MeOH and 300 mL of DCM in sequence to cross-wash the resin 4 times;

[0679] 9. Take out the resin and vacuum dry it until the weight stops decreasing. Weigh it to obtain 17.8 g of peptide resin;

[0680] The intermediate (peptide resin) has the following structural formula:

[0681] Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-a-MeLeu-Leu-Asp(OtBu)-Lys(Boc)-Lys 17 -Ala-Gln(Trt)-Aib-Lys 21 -Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-Leu-Glu(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Linker-MBHA Resin(17,21(Nε(Na-OC-(CH2) 18 -COOtBu)-Glu(ε-OtBu)-AEEA-AEEA)-(2-2-oxoethyl)glycine))

[0682] Step 5. Peptide resin cleavage

[0683] Prepare 250ml of cleavage reagent, the reagent formula is trifluoroacetic acid (TFA), purified water (H2O), triisopropylsilane (TIS), 1,2-ethanedithiol (EDT), phenol (ArOH), by volume ratio TFA:H2O:EDT:TIS:ArOH = 86.5:5:2.5:1:5, and precool to 10±3°C after preparation. Slowly add the resin to the cleavage reagent under stirring, and continue to stir until the temperature of the reaction system is stable; then control the temperature at 18±3°C and continue to stir the reaction for 4h. Filter out the lysate, concentrate it to a viscous state, and precipitate it with methyl tert-butyl ether 12-15 times the volume of the concentrated solution. After filtering out the precipitate, wash it, and dry it under reduced pressure at room temperature to obtain 7.9g of crude cyclopeptide.

[0684] Step 6. Chromatographic purification

[0685] The above 7.9 g crude cyclopeptide was dissolved in 10% acetonitrile and 10% acetic acid aqueous solution, and then purified by high performance liquid chromatography using C18 silica gel matrix filler, and the fractions with purity > 90% were collected and combined. The purification chromatographic system is listed as follows:

[0686] Process Water Phase Organic phase gradient One-time purification 0.2% TFA Acetonitrile B% (0-70min): 41%-51% Secondary purification <![CDATA[35mmol / L K2HPO4(pH 7.4)]]> Acetonitrile B% (0-50min): 33%-44% Salt conversion 0.6mmol / L NaOH Acetonitrile B％：45％

[0687] Step 7. Concentration and freeze drying

[0688] The fraction obtained in the previous step was concentrated to remove acetonitrile, filtered through a 0.22 μm microporous filter membrane, loaded into a hanging bottle freeze dryer, and freeze-dried to obtain 0.93 g (purity: 94.97%) of the target polypeptide product; the molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1258.7, and the detected value was 1258.7.

[0689] Example 2

[0690]

[0691] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 2 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1249.7, and the detected value was 1249.4.

[0692] Example 3

[0693]

[0694] Step 1: Resin coupling Fmoc-Rink Linker

[0695] 1. Take 10.2g (5mmol) of MBHA Resin (S = 0.49mmol / g) and add it to the solid phase reaction column. Add 70mL of DCM and let it stand for 20min to fully swell the resin. Drain the DCM, add 60mL of DMF and wash the resin by bubbling nitrogen. Repeat the above steps once and drain the solvent. Weigh 10.8g (20mmol, 4.0eq) of Fmoc-Rink Linker and dissolve it in 40mL of DMF and add it to the above resin. Add 2.78g (22mmol, 4.4eq) of DIC and add 10mL of DMF to the reaction column. Blow nitrogen. After it is completely dissolved, add 2.70g (20mmol, 4.0eq) of HOBt and continue to bubble nitrogen for reaction.

[0696] 2. React at 25°C for more than 2 hours. If the sample is negative for ninhydrin test (resin is colorless or light yellow), the reaction is terminated.

[0697] 3. Draw off the reaction liquid, wash with DMF (300 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[0698] Step 2: Fmoc-Ser(tBu)-OH coupling

[0699] 1. Add 20% piperidine / DMF (100 mL) to the reaction column, bubbling nitrogen for 10 min, drain until no liquid flows out, add 20% piperidine / DMF (100 mL) to the reaction column again, continue bubbling nitrogen for 5 min, take a sample for ninhydrin detection, and the resin is blue;

[0700] 2. Weigh 7.66g (4.0eq) of Fmoc-Ser(tBu)-OH, 2.78g (4.4eq) of DIC and 2.70g (4.0eq) of HOBt, dissolve them in 60mL of DMF and add them to the reaction column. Blow nitrogen and adjust the nitrogen to make the resin swell evenly.

[0701] 3. React in an environment of 25°C for 2.0 hours, take samples for ninhydrin detection, and the resin is colorless and transparent;

[0702] 4. Draw off the reaction liquid, wash with DMF (300 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[0703] Main chain amino acid coupling steps

[0704] Referring to the method and amount of Fmoc-Ser(tBu)-OH coupling in step 2, the amino acid sequence is sequentially connected, and the order and amino acids containing protective groups used are as follows:

[0705]

[0706]

[0707] Step 3. Side-chain coupling

[0708] 1. Add 300 mL of DMF and 300 mL of DCM to the reaction column in sequence to cross-wash the resin 4 times. After draining the solvent, add 300 mL of 20% hexafluoroisopropanol (HFIP)-DCM solution and react twice with nitrogen bubbling, each time for 30 min;

[0709] 2. Drain the waste until no liquid flows out, take a sample for ninhydrin testing, and the resin will be blue;

[0710] 3. Add 300 mL of DCM and 300 mL of DMF in sequence to cross-wash the resin 4 times and drain until no liquid flows out.

[0711] 4. Weigh 9.05 g (4.0 eq) of Alloc-Lys(Fmoc)-OH, 2.78 g (4.4 eq) of DIC and 2.70 g (4.0 eq) of HOBt, dissolve them in 100 mL of DMF, add them to the reaction column, and blow nitrogen. Adjust the nitrogen to make the resin blow evenly.

[0712] 5. React in an environment of 25°C for 2.0 hours, take samples for ninhydrin detection, and the resin is colorless and transparent;

[0713] 6. Draw off the reaction liquid, wash with DMF (300 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[0714] The remaining branch coupling steps

[0715] Refer to the Fmoc-Ser(tBu)-OH coupling method and feed equivalents in step 2 and add the following compounds in sequence:

[0716] Amino acids containing protecting groups used Remark Fmoc-AEEA-OH Fmoc-Glu-OtBu Eicosanedioic acid monotert-butyl ester

[0717] Step 4: Ring closure

[0718] 1. Add 300 mL of DMF and 300 mL of DCM to the reaction column in sequence, cross-wash the resin 4 times, and drain until no liquid flows out;

[0719] 2. Prepare 400 mL of a mixed solvent with a volume ratio of DCM: CH3CN: NMP: DEA = 3:3:2:2, and add it to the reaction column. Then add 40.58 g (0.1 eq) of Pd(PPh3), and react with nitrogen bubbling for 10 min, and drain until no liquid flows out;

[0720] 3. Repeat the above steps 1 to 2 times;

[0721] 4. Take samples for ninhydrin testing, the resin is blue;

[0722] 5. Add 300 mL of DMF and 300 mL of DCM in sequence and cross-wash the resin 8 times;

[0723] 6. Dissolve 2.78g (4.4eq) of DIC and 2.70g (4.0eq) of HOBt in 150mL of DMF, add to the reaction column, react for 48h with nitrogen, take a sample for ninhydrin detection, and the resin is colorless;

[0724] 7. Add 300 mL of DMF and 300 mL of DCM in sequence and cross-wash the resin 8 times;

[0725] 8. Add MeOH 200 mL and DCM 300 mL in sequence to cross-wash the resin 4 times;

[0726] 9. Take out the resin and vacuum dry it until the weight stops decreasing. Weigh it to obtain 19.2 g of peptide resin;

[0727] The intermediate (peptide resin) has the following structural formula:

[0728] Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu )-Ile-a-MeLeu-Leu-Asp(OtBu)-Lys(Boc)-Lys(Boc)-Ala-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Lys 25 -Leu-Leu-Glu 28 -Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Linker-MBHA Resin(25,28(Nε(COOH-(CH2) 18 -COOtBu)-Glu(ε-OtBu)-AEEA)-(2-2-oxoethyl)glycine))

[0729] Step 5. Peptide resin cleavage

[0730] Prepare 250mL of cleavage reagent, the reagent formula is trifluoroacetic acid (TFA), purified water (H2O), triisopropylsilane (TIS), 1,2-ethanedithiol (EDT), phenol (ArOH), by volume ratio TFA:H2O:EDT:TIS:ArOH=86.5:5:2.5:1:5, and precool to 10±3°C after preparation. Slowly add the resin to the cleavage reagent under stirring, and continue stirring until the temperature of the reaction system is stable; then control the temperature at 18±3°C and continue stirring the reaction for 4h. Filter the lysate, concentrate it to a viscous state, and precipitate it with methyl tert-butyl ether 12-15 times the volume of the concentrated solution. After filtering the precipitate, wash it, and dry it under reduced pressure at room temperature to obtain 8.2g of crude cyclopeptide.

[0731] Step 6. Chromatographic purification

[0732] The above 8.2 g cyclopeptide crude product was dissolved in 10% acetonitrile and 10% acetic acid aqueous solution, and then purified by high performance liquid chromatography using C18 silica gel matrix filler, and the fractions with purity > 90% were collected and combined. The purification chromatographic system is listed as follows:

[0733] Process Water Phase Organic phase gradient One-time purification 0.2% TFA Acetonitrile B% (0-70min): 41%-51% Secondary purification <![CDATA[35mmol / L K2HPO4(pH7.4)]]> Acetonitrile B% (0-50min): 33%-44% Salt conversion 0.6mmol / L NaOH Acetonitrile B％：45％

[0734] Step 7. Concentration and freeze drying

[0735] The fraction obtained in the previous step was concentrated to remove acetonitrile, filtered through a 0.22 μm microporous filter membrane, loaded into a hanging bottle freeze dryer, and freeze-dried to obtain 1.01 g (purity: 94.97%) of the finished product shown in Example 3; the molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1202.6, and the detected value was 1202.6.

[0736] Example 4

[0737]

[0738] Referring to the synthesis of the polypeptide of Example 1, the polypeptide of Example 4 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1277.7, and the detected value was 1277.6.

[0739] Example 5

[0740]

[0741] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 5 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1277.7, and the detected value was 1277.5.

[0742] Example 6

[0743]

[0744] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 6 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1122.4, and the detected value was 1122.5.

[0745] Example 7

[0746]

[0747] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 7 was obtained, wherein the protecting group amino acids used in the branched chain coupling were Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosanedioate. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1250.2, and the detected value was 1250.2.

[0748] Example 8

[0749]

[0750] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 8 was obtained, wherein the protecting amino acids used in the branched chain coupling were Fmoc-Glu(OAll)-OH, Fmoc-AEEA-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosanedioate. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1225.9, and the detected value was 1226.0.

[0751] Example 9

[0752]

[0753] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 9 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1218.4, and the detected value was 1218.3.

[0754] Example 10

[0755]

[0756] Referring to the synthesis of the polypeptide in Example 3, the polypeptide shown in Example 10 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1225.9 and the detected value was 1224.2.

[0757] Embodiment 11

[0758]

[0759] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 11 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1244.2, and the detected value was 1242.7.

[0760] Example 12

[0761]

[0762] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 12 was obtained, wherein the protecting group amino acids used in the branched chain coupling were Fmoc-Glu-OAll, Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosanedioate. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1262.2, and the detected value was 1262.2.

[0763] Example 13

[0764]

[0765] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 13 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1244.2, and the detected value was 1244.2.

[0766] Embodiment 14

[0767]

[0768] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 14 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1258.5, and the detected value was 1258.4.

[0769] Embodiment 15

[0770]

[0771] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 15 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1258.7 and the detected value was 1258.6.

[0772] Example 16

[0773]

[0774] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 16 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1258.7, and the detected value was 1258.6.

[0775] Embodiment 17

[0776]

[0777] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 17 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1255.2, and the detected value was 1255.1.

[0778] Embodiment 18

[0779]

[0780] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 18 was obtained, wherein the protecting amino acids used in the branched chain coupling were Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosanedioate. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1235.7, and the detected value was 1234.7.

[0781] Embodiment 19

[0782]

[0783] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 19 was obtained, wherein the protecting group amino acids used in the branched chain coupling were Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosanedioate. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1250.0, and the detected value was 1249.0.

[0784] Embodiment 20

[0785]

[0786] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 20 was obtained, wherein the protecting group amino acids used in the branched chain coupling were Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosanedioate. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1250.2, and the detected value was 1249.2.

[0787] Embodiment 21

[0788]

[0789] The peptide resin was synthesized by a conventional solid phase synthesis process, with nitrogen bubbling reaction in a solid phase reaction column with a sintered sieve plate at the bottom, MBHA Resin was used as the starting resin, conventional Fmoc protected amino acids were fed, a DIC / HOBt system was used as the condensing agent, DMF was used as the main reaction solvent, and a 20% PIP-DMF solution was used as the Fmoc removal solvent, and condensation was performed one by one from the C-terminus to the N-terminus of the peptide main chain. After the main chain synthesis was completed, the -Mtt on the ε-amino group of Fmoc-Lys (Mtt) -OH at position A-26 was removed, and then the side 1 to side 4 positions ((2-2-oxoethyl) -glycine, AEEA, γGlu, mono-tert-butyl eicosandioic acid) were condensed in sequence, and then the ε-amino protecting group of Lys at position A-23 and the carboxyl protecting group of (2-2-oxoethyl) -glycine at position 1 were removed, and then a condensing agent was added to form a lactam ring to obtain the target peptide resin.

[0790] The specific steps are as follows:

[0791] Step 1: Resin activation and deprotection

[0792] 1. Take 15g (5.1mmol) of MBHA Resin (S=0.34mmol / g) and add it to the solid phase reaction column. Add 225ml of DCM and let it stand for 20min to make the resin fully swell. Drain the DCM, add 150ml of DMF and wash the resin by nitrogen bubbling. Repeat the above steps once and drain the solvent. Add 20% piperidine / DMF (150mL) to the reaction column, bubbling nitrogen for 5min, drain until no liquid flows out, add 20% piperidine / DMF (150mL) to the reaction column again and continue bubbling nitrogen for 15min. Take a sample of ninhydrin for detection. The resin is gray-red. Drain the reaction liquid and wash it with DMF (150mL) 7 times. Adjust pH=7, 1min each time, drain until no liquid flows out.

[0793] Step 2: Fmoc-Ser(tBu)-OH coupling

[0794] 1. Weigh 5.75g (3.0eq) of Fmoc-Ser(tBu)-OH, 2.52g (4.0eq) of DIC and 2.03g (3.0eq) of HOBt, dissolve them in 80mL of DMF and add them to the reaction column. Blow nitrogen and adjust the nitrogen to make the resin swell evenly.

[0795] 2. React in an environment of 25°C for 2.0h, take samples for ninhydrin detection, the resin is colorless and transparent;

[0796] 3. Draw off the reaction liquid, wash with DMF (150 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[0797] Main chain amino acid coupling steps

[0798] Referring to the method and amount of Fmoc-Ser(tBu)-OH coupling in step 2, the amino acid sequence is sequentially connected, and the order and amino acids containing protective groups used are as follows:

[0799]

[0800]

[0801] The AA-11 peptide was obtained.

[0802] Step 3: Deprotection of AA-11 peptide

[0803] 1. Add DMF (80 mL) to the synthesized AA-11 peptide and activate it for 10 min. Drain until no liquid flows out. Add 20% piperidine / DMF (80 mL) to the reaction column and react with nitrogen bubbling for 10 min. Drain until no liquid flows out. Add 20% piperidine / DMF (80 mL) to the reaction column again and continue nitrogen bubbling for 10 min. Take a sample of ninhydrin for detection. The resin is yellow with a slant to ink color. Draw off the reaction liquid and wash it 8 times with DMF (100 mL). Adjust the pH to about 7 for 1 min each time. Drain until no liquid flows out.

[0804] Step 4: Fmoc-Glu(OtBu)-OH coupling

[0805] 1. Weigh 3.04g (3.0eq) of Fmoc-Glu(OtBu)-OH, 1.20g (4.0eq) of DIC and 0.965g (3.0eq) of HOBt, dissolve them in 50mL of DMF and add them to the reaction column. Blow nitrogen and adjust the nitrogen to make the resin swell evenly.

[0806] 2. React in an environment of 25°C for 3.0 hours, take samples for ninhydrin detection, and the resin is colorless and transparent;

[0807] 3. Draw off the reaction liquid, wash with DMF (80 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[0808] Main chain amino acid coupling steps

[0809] Referring to the method and order of the coupling of Fmoc-Glu(OtBu)-OH in step 4, the amino acid sequence is sequentially connected. The order and the amino acids containing protective groups used are as follows:

[0810] Amino acids containing protecting groups used Remark Fmoc-Glu(OtBu)-OH. AA-12 peptide Fmoc-Leu-OH Fmoc-Leu-OH FmocTyr(tBu)-OH Fmoc-Glu(OtBu)-OH Fmoc-Ile-OH Fmoc-Phe-OH Fmoc-Ala-OH Fmoc-Aib-OH Fmoc-Gln(Trt)-OH HPLC monitoring Fmoc-Ala-OH Fmoc-Lys(Alloc)-OH Fmoc-Lys(Boc)-OH Fmoc-Asp(OtBu)-OH Fmoc-Lys(Mtt)-OH Fmoc-α-Me-Leu-OH HPLC monitoring Fmoc-Ile-OH HPLC monitoring Fmoc-Ser(tBu)-OH Fmoc-Tyr(tBu)-OH Fmoc-Asp(OtBu)-OH HPLC monitoring Fmoc-Ser(tBu)-OH Fmoc-Thr(tBu)-OH Fmoc-Phe-OH Fmoc-Thr(tBu)-OH Fmoc-Gly-OH Fmoc-Gln(Trt)-OH Boc-Tyr(tBu)-Aib-OH HPLC monitoring

[0811] Step 5. Side-chain coupling

[0812] 1. Add 300 mL of DCM to the reaction column to wash the resin 4 times, drain the solvent, add 80 ml of 20% hexafluoroisopropanol (HFIP)-DCM solution, and bubble nitrogen for 5 times, each time for 30 min;

[0813] 2. Drain the waste until no liquid flows out, take a sample for ninhydrin testing, and the resin will be gray-green;

[0814] 3. Add 80 mL of DCM and 80 mL of DMF in sequence to cross-wash the resin 4 times and drain until no liquid flows out.

[0815] 4. Weigh 4.7 g (5.0 eq) of Fmoc-(2-(allyloxy)-2-oxoethyl)glycine (Fmoc-Ida(OAll)-OH), 1.67 g (6.0 eq) of DIC and 1.49 g (5.0 eq) of HOBt, dissolve them in 100 mL of DMF and add them to the reaction column. Add nitrogen and adjust the nitrogen to make the resin swell evenly.

[0816] 5. After reacting for 5.0 hours at 25°C, sample ninhydrin for testing, and the resin is colorless and transparent;

[0817] 6. Draw off the reaction liquid, wash with DMF (80 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[0818] The remaining branch coupling steps

[0819] Referring to the coupling method and feed equivalents of Fmoc-Glu(OtBu)-OH in step 4, the following compounds were added in sequence:

[0820] Amino acids containing protecting groups used Remark Fmoc-AEEA-OH HPLC monitoring Fmoc-Glu-OtBu Eicosanedioic acid monotert-butyl ester

[0821] Step 6. Cyclization

[0822] 1. Add 80 mL of DMF and 80 mL of DCM to the reaction column in sequence, cross-wash the resin 4 times, and drain until no liquid flows out;

[0823] 2. Prepare 300 mL of a mixed solvent with a volume ratio of DCM: CH3CN: NMP: DEA = 3:3:2:2 and add it to the reaction column, then add 40.74 g (0.27 eq) of Pd(PPh3), react with nitrogen bubbling for 10 min, and drain until no liquid flows out;

[0824] 3. Take samples for ninhydrin testing, the resin is yellow with a purple tint;

[0825] 4. Add 80 mL of DMF and 80 mL of DCM in sequence and cross-wash the resin 8 times;

[0826] 5. Dissolve 1.67g (6.0eq) of DIC and 1.49g (5.0eq) of HOBt in 80mL of DMF, add to the reaction column, react for 22h with nitrogen, take a sample for ninhydrin detection, and the resin is colorless;

[0827] 6. Add 80 mL of DMF and 80 mL of DCM in sequence and cross-wash the resin 8 times;

[0828] 7. Add 80 mL of MeOH and 80 mL of DCM in sequence to cross-wash the resin 4 times;

[0829] 8. Take out the resin and vacuum dry it until the weight stops decreasing. Weigh it to obtain 19.3 g of peptide resin;

[0830] The intermediate (peptide resin) has the following structural formula:

[0831] Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-a-MeLeu-Lys 14 -Asp(OtBu)-Lys(Boc)-Lys 17 -Ala-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-Leu-Glu(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Linker-MBHA Resin(14,17(Nε(HOOC-(CH2) 18-COOtBu)-Glu(ε-OtBu)-AEEA)-(2-2-oxoethyl)glycine))

[0832] Step 7. Peptide resin cleavage

[0833] Prepare 289mL of cleavage reagent, the reagent formula is trifluoroacetic acid (TFA), purified water (H2O), triisopropylsilane (TIS), 3-(triphenylmethylthio) propionic acid (Mpa(Trt)-OH, MRP), phenol (ArOH), by volume ratio TFA:H2O:Mpa(Trt)-OH:TIS:ArOH=82.5:2.5:5:5:5, and precool to 10±3°C after preparation. Slowly add the resin to the cleavage reagent under stirring, and continue to stir until the temperature of the reaction system is stable; then control the temperature at 18±3°C and continue to stir the reaction for 4h. Filter the lysate, concentrate it to a viscous state, and precipitate it with methyl tert-butyl ether 12-15 times the volume of the concentrated solution. After filtering the precipitate, wash it, and dry it under reduced pressure at room temperature to obtain 7.5g of crude cyclopeptide.

[0834] Step 8. Chromatographic purification

[0835] The above 2.1 g crude cyclopeptide was dissolved in 5% acetonitrile and 2% aqueous ammonia solution, and then purified by high performance liquid chromatography using C8 silica gel matrix filler. The fractions with purity > 90% were collected and combined. The purification chromatographic system is listed as follows:

[0836]

[0837]

[0838] Step 9. Concentration and freeze drying

[0839] The fraction obtained in the previous step was concentrated to remove acetonitrile, filtered through a 0.22 μm microporous filter membrane, loaded into a hanging bottle freeze dryer, and freeze-dried to obtain 0.133 g (purity: 94.2%) of the target polypeptide product; the molecular weight of the polypeptide was confirmed by ESI-MS, the calculated value [M+4H] / 4 was 1211.9, and the detected value was 1210.9.

[0840] Embodiment 22

[0841]

[0842] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 22 was obtained, wherein the protecting amino acids used in the branched chain coupling were Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosanedioate. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1246.2, and the detected value was 1245.1.

[0843] Embodiment 23

[0844]

[0845] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 23 was obtained, wherein the protecting group amino acids used in the branched chain coupling were Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosanedioate. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1246.2, and the detected value was 1245.2.

[0846] Embodiment 24

[0847]

[0848] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 24 was obtained, wherein the protecting group amino acids used in the branched chain coupling were Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosanedioate. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1242.7, and the detected value was 1241.6.

[0849] Embodiment 25

[0850]

[0851] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 25 was obtained, wherein the protecting group amino acids used in the branched chain coupling were Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosanedioate. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1235.4, and the detected value was 1234.3.

[0852] Embodiment 26

[0853]

[0854] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 26 was obtained, wherein the protecting group amino acids used in the branched chain coupling were Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosanedioate. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1246.0, and the detected value was 1250.0.

[0855] Embodiment 27

[0856]

[0857] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 27 was obtained, wherein the protecting group amino acids used in the branched chain coupling were Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosanedioate. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1235.7, and the detected value was 1234.6.

[0858] Embodiment 28

[0859]

[0860] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 28 was obtained, wherein the protecting group amino acids used in the branched chain coupling were Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosanedioate. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1235.7, and the detected value was 1238.6.

[0861] Embodiment 29

[0862]

[0863] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 29 was obtained, wherein the protecting group amino acids used in the branched chain coupling were Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosanedioate. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1242.5, and the detected value was 1241.5.

[0864] Embodiment 30

[0865]

[0866] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 30 was obtained, wherein the protecting amino acids used in the branched chain coupling were Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and eicosanedioic acid mono-tert-butyl ester. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1242.5, and the detected value was 1241.6.

[0867] Embodiment 31

[0868]

[0869] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 31 was obtained, wherein the protecting group amino acids used in the branched chain coupling were Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosanedioate. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1246.0, and the detected value was 1244.9.

[0870] Embodiment 32

[0871]

[0872] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 32 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1218.2, and the detected value was 1217.4.

[0873] Embodiment 33

[0874]

[0875] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 33 was obtained, wherein the protecting group amino acids used in the branched chain coupling were Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosanedioate. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1246.0, and the detected value was 1245.9.

[0876] Embodiment 34

[0877]

[0878] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 34 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1218.2, and the detected value was 1218.1.

[0879] Embodiment 35

[0880]

[0881] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 35 was obtained, wherein the protecting group amino acids used in the branched chain coupling were Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosanedioate. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1250.0, and the detected value was 1250.0.

[0882] Embodiment 36

[0883]

[0884] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 36 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1222.2, and the detected value was 1222.1.

[0885] Embodiment 37

[0886]

[0887] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 37 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1226.4, and the detected value was 1226.3.

[0888] Embodiment 38

[0889]

[0890] A similar synthesis process was used as the polypeptide in Example 21 above, using MBHA Resin as the starting resin, conventional Fmoc-protected amino acids as feed materials, a DIC / HOBt system as the condensing agent, DMF as the main reaction solvent, and 20% PIP-DMF solution as the Fmoc removal solvent. Condensation was carried out one by one from the C-terminus to the N-terminus of the peptide main chain. After the main chain synthesis was completed, the -Mtt on the ε-amino group of Fmoc-Lys(Mtt)-OH at position A-26 was removed, and then positions 1 to 4 ((2-2-oxoethyl)-glycine, AEEA, γGlu, mono-tert-butyl eicosadioate) were condensed in sequence, and then the ε-amino protecting group of Lys at position A-23 and the carboxyl protecting group of (2-2-oxoethyl)-glycine at position 1 were removed, and then a condensing agent was added to form a lactam ring to obtain the target peptide resin.

[0891] The specific steps are as follows:

[0892] Step 1: Resin activation and deprotection

[0893] 1. Take 15g (5.1mmol) of MBHA Resin (S=0.34mmol / g) and add it to the solid phase reaction column. Add 225mL of DCM and let it stand for 20min to fully swell the resin. Drain the DCM, add 150mL of DMF and wash the resin by nitrogen bubbling. Repeat the above steps once and drain the solvent. Add 20% piperidine / DMF (150mL) to the reaction column, bubbling nitrogen for 10min, drain until no liquid flows out, add 20% piperidine / DMF (150mL) to the reaction column again and continue bubbling nitrogen for 10min. Take a sample of ninhydrin for detection. The resin is gray-red. Drain the reaction solution and wash it 8 times with DMF (150mL). Adjust pH=7, 1min each time, drain until no liquid flows out.

[0894] Step 2: Fmoc-Ser(tBu)-OH coupling

[0895] 1. Weigh 5.86g (3.0eq) of Fmoc-Ser(tBu)-OH, 2.57g (4.0eq) of DIC and 2.07g (3.0eq) of HOBt, dissolve them in 80mL of DMF and add them to the reaction column. Blow nitrogen and adjust the nitrogen to make the resin swell evenly.

[0896] 2. React in an environment of 25°C for 2.0h, take samples for ninhydrin detection, the resin is colorless and transparent;

[0897] 3. Draw off the reaction liquid, wash with DMF (150 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[0898] Main chain amino acid coupling steps

[0899] Referring to the method and amount of Fmoc-Ser(tBu)-OH coupling in step 2, the amino acid sequence is sequentially connected, and the order and amino acids containing protective groups used are as follows:

[0900] Amino acids containing protecting groups used Remark Fmoc-Pro-OH Fmoc-Pro-OH Fmoc-Pro-OH Fmoc-Ala-OH Fmoc-Gly-OH Fmoc-Ser(tBu)-OH Fmoc-Ser(tBu)-OH Fmoc-Pro-OH Fmoc-Gly-OH Fmoc-Gly-OH HPLC detection synthesis

[0901] The AA-11 peptide was obtained.

[0902] Step 3: Deprotection of AA-11 peptide

[0903] 1. Add DMF (150 mL) to the synthesized AA-11 peptide and activate it for 10 min. Drain until no liquid flows out. Add 20% piperidine / DMF (150 mL) to the reaction column and react with nitrogen bubbling for 10 min. Drain until no liquid flows out. Add 20% piperidine / DMF (150 mL) to the reaction column again and continue nitrogen bubbling for 10 min. Take a sample of ninhydrin for detection. The resin is dark yellow. Draw off the reaction solution and wash it 8 times with DMF (150 mL). Adjust the pH to about 7 for 1 min each time. Drain until no liquid flows out.

[0904] Step 4: Fmoc-Lys(Boc)-OH coupling

[0905] 1. Weigh 7.17 g (3.0 eq) of Fmoc-Lys(Boc)-OH, 2.57 g (4.0 eq) of DIC and 2.07 g (3.0 eq) of HOBt, dissolve them in 150 mL of DMF, add them to the reaction column, and blow nitrogen. Adjust the nitrogen to make the resin blow evenly.

[0906] 2. React in an environment of 25°C for 3.0 hours, take samples for ninhydrin detection, and the resin is colorless and transparent;

[0907] 3. Draw off the reaction liquid, wash with DMF (150 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[0908] Main chain amino acid coupling steps

[0909] Referring to the Fmoc-Lys(Boc)-OH coupling method and feeding order in step 4, the amino acid sequence is connected in sequence, and the order and amino acids containing protective groups used are as follows:

[0910]

[0911]

[0912] Step 5. Side-chain coupling

[0913] 1. Add 300 mL of DCM to the reaction column to wash the resin 4 times, drain the solvent, add 150 mL of 20% hexafluoroisopropanol (HFIP)-DCM solution, and bubble nitrogen for 5 times, each time for 30 min;

[0914] 2. Drain the waste until no liquid flows out, take a sample for ninhydrin testing, and the resin will be gray-green;

[0915] 3. Add 150 mL of DCM and 150 mL of DMF in sequence to cross-wash the resin 4 times and drain until no liquid flows out.

[0916] 4. Weigh 10.08 g (5.0 eq) of Fmoc-Ida(Oall)-OH, 3.86 g (6.0 eq) of DIC and 3.45 g (5.0 eq) of HOBt, dissolve them in 150 mL of DMF, add them to the reaction column, and blow nitrogen. Adjust the nitrogen to make the resin blow evenly.

[0917] 5. React for 3.0 hours in an environment of 25°C, take samples for ninhydrin detection, and the resin is colorless and transparent;

[0918] 6. Draw off the reaction liquid, wash with DMF (180 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[0919] The remaining branch coupling steps

[0920] Referring to the coupling method and feed equivalents of Fmoc-Ida(Oall)-OH in step 5, the following compounds were added in sequence:

[0921] Amino acids containing protecting groups used Remark Fmoc-AEEA-OH Fmoc-Glu-OtBu Eicosanedioic acid monotert-butyl ester HPLC monitoring

[0922] Step 6. Cyclization

[0923] 1. Add 250 mL of DMF and 250 mL of DCM to the reaction column in sequence, cross-wash the resin 4 times, and drain until no liquid flows out;

[0924] 2. Prepare 300 mL of a mixed solvent with a volume ratio of DCM: CH3CN: NMP: DEA = 3:3:2:2 and add it to the reaction column, then add 41.59 g (0.27 eq) of Pd(PPh3), react with nitrogen bubbling for 30 min, and drain until no liquid flows out;

[0925] 3. Take samples for ninhydrin testing, the resin is purple-red;

[0926] 4. Add 250 mL of DMF and 250 mL of DCM in sequence and cross-wash the resin 8 times;

[0927] 5. Dissolve 3.86g (6.0eq) of DIC and 3.45g (5.0eq) of HOBt in 250mL of DMF, add to the reaction column, react for 22 hours with nitrogen, take a sample of ninhydrin for detection, and the resin is extremely light purple;

[0928] 6. Add 250 mL of DMF and 250 mL of DCM in sequence and cross-wash the resin 8 times;

[0929] 7. Add 200 mL of MeOH and 200 mL of DCM in sequence to cross-wash the resin 4 times;

[0930] 8. Take out the resin and vacuum dry it until the weight stops decreasing. Weigh it to obtain 32.5 g of peptide resin;

[0931] The intermediate (peptide resin) has the following structural formula:

[0932] Nε(Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-a-MeLeu-Lys 14 -Asp(OtBu)-Lys(Boc)-Lys 17 -Ala-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Linker-MBHA Resin(14,17(Nε(HOOC-(CH2) 18 -COOtBu)-Glu(ε-OtBu)-(AEEA)-(2-2-oxoethyl)glycine))

[0933] Step 7. Peptide resin cleavage

[0934] Prepare 500mL of cleavage reagent, the reagent formula is trifluoroacetic acid (TFA), purified water (H2O), triisopropylsilane (TIS), 3-(triphenylmethylthio) propionic acid (Mpa(Trt)-OH), phenol (ArOH), by volume ratio TFA:H2O:Mpa(Trt)-OH:TIS:ArOH = 82.5:2.5:5:5:5, and precool to 10±3°C after preparation. Slowly add the resin to the cleavage reagent under stirring, and continue stirring until the temperature of the reaction system is stable; then control the temperature at 18±3°C and continue stirring the reaction for 4h.

[0935] The lysate was filtered out and concentrated to a viscous state, and methyl tert-butyl ether in an amount 12-15 times the volume of the concentrated solution was used to precipitate it. The precipitate was filtered out, washed, and dried under reduced pressure at room temperature to obtain 9.8 g of a crude cyclopeptide.

[0936] Step 8. Chromatographic purification

[0937] 1 / 6 of the above 9.8g crude cyclopeptide was dissolved in 5% acetonitrile and 2% aqueous ammonia solution, and then purified by high performance liquid chromatography using C18 silica gel matrix filler, and the fractions with purity > 90% were collected and combined. The purification chromatographic system is listed as follows:

[0938] Process Water Phase Organic phase gradient One-time purification 0.2% TFA Acetonitrile B%(0-50min):30%-55% Secondary purification 50mmol / L diammonium hydrogen phosphate Acetonitrile B% (0-50min): 31%-65% Three purifications 0.2mmol / L sodium hydroxide Acetonitrile B% (0-50min): 41%-55%

[0939] Step 9. Concentration and freeze drying

[0940] The fraction obtained in the previous step was concentrated to remove acetonitrile, filtered through a 0.22 μm microporous filter membrane, loaded into a hanging bottle freeze dryer, and freeze-dried to obtain 144 mg of the target polypeptide product (purity: 96.69%); the molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4] / 4 was 1211.6, and the detected value was 1211.5.

[0941] Embodiment 39

[0942]

[0943] Referring to the synthesis of the polypeptide in Example 21, the polypeptide shown in Example 39 was obtained, wherein the protecting group amino acids used in the branched chain coupling were Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and eicosanedioic acid mono-tert-butyl ester. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1239.7, and the detected value was 1239.7.

[0944] Embodiment 40

[0945]

[0946] Referring to the synthesis of the polypeptide in Example 21, the polypeptide shown in Example 40 was obtained, wherein the protecting amino acids used in the branched chain coupling were Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosanedioate. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1254.2, and the detected value was 1254.1.

[0947] Embodiment 41

[0948]

[0949] Referring to the synthesis of the polypeptide in Example 38, the polypeptide shown in Example 41 was obtained, wherein the protecting amino acids used in the branched chain coupling were Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and eicosanedioic acid mono-tert-butyl ester. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1239.4, and the detected value was 1239.3.

[0950] Embodiment 42

[0951]

[0952] Referring to the synthesis of the polypeptide of Example 21, the polypeptide of Example 42 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1211.6, and the detected value was 1211.5.

[0953] Embodiment 43

[0954]

[0955] MBHA Resin was used as the starting resin, conventional Fmoc-protected amino acids were added, a DIC / HOBt system was used as the condensing agent, DMF was used as the main reaction solvent, and a 20% PIP-DMF solution was used as the Fmoc removal solvent. Condensation was performed one by one from the C-terminus to the N-terminus of the peptide main chain. After the main chain synthesis was completed, the -Mtt on the ε-amino group of Fmoc-Lys(Mtt)-OH at position A-26 was removed, and then the side 1 to side 4 positions ((2-2-oxoethyl)-glycine, AEEA, γGlu, 19-(bis(benzyloxy)phosphoryl)nonadecanoic acid) were condensed in sequence, and then the ε-amino protecting group of Lys at position A-23 and the carboxyl protecting group of (2-2-oxoethyl)-glycine at position 1 were removed, and then a condensing agent was added to form a lactam ring to obtain the target peptide resin.

[0956] The specific steps are as follows:

[0957] Step 1: Resin activation and deprotection

[0958] 1. Take 15g (5.1mmol) of MBHA Resin (S=0.34mmol / g) and add it to the solid phase reaction column. Add 225mL of DCM and let it stand for 20min to fully swell the resin. Drain the DCM, add 150ml of DMF and wash the resin by nitrogen bubbling. Repeat the above steps once and drain the solvent. Add 20% piperidine / DMF (150mL) to the reaction column, bubbling nitrogen for 10min, drain until no liquid flows out, add 20% piperidine / DMF (150mL) to the reaction column again and continue bubbling nitrogen for 10min. Take a sample of ninhydrin for detection. The resin is gray-red. Drain the reaction liquid and wash it 8 times with DMF (150mL). Adjust pH=7, 1min each time, drain until no liquid flows out.

[0959] Step 2: Fmoc-Ser(tBu)-OH coupling

[0960] 1. Weigh 5.86g (3.0eq) of Fmoc-Ser(tBu)-OH, 2.57g (4.0eq) of DIC and 2.07g (3.0eq) of HOBt, dissolve them in 80mL of DMF and add them to the reaction column. Blow nitrogen and adjust the nitrogen to make the resin swell evenly.

[0961] 2. React in an environment of 25°C for 2.0h, take samples for ninhydrin detection, the resin is colorless and transparent;

[0962] 3. Draw off the reaction liquid, wash with DMF (150 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[0963] Main chain amino acid coupling steps

[0964] Referring to the method and amount of Fmoc-Ser(tBu)-OH coupling in step 2, the amino acid sequence is sequentially connected, and the order and amino acids containing protective groups used are as follows:

[0965] Amino acids containing protecting groups used Remark Fmoc-Pro-OH Fmoc-Pro-OH Fmoc-Pro-OH Fmoc-Ala-OH Fmoc-Gly-OH Fmoc-Ser(tBu)-OH Fmoc-Ser(tBu)-OH Fmoc-Pro-OH Fmoc-Gly-OH Fmoc-Gly-OH HPLC detection synthesis

[0966] The AA-11 peptide was obtained.

[0967] Step 3: Deprotection of AA-11 peptide

[0968] 1. The synthesized AA-11 peptide was activated by adding DMF (150 mL) for 10 min, and then drained until no liquid flowed out. 20% piperidine / DMF (150 mL) was added to the reaction column, and nitrogen bubbling reaction was carried out for 10 min, and the waste was discharged until no liquid flowed out. 20% piperidine / DMF (150 mL) was added to the reaction column again, and nitrogen bubbling reaction was continued for 10 min. The resin was sampled for ninhydrin detection, and the resin was dark yellow; the reaction liquid was removed, and washed with DMF (150 mL) 8 times, and the pH was adjusted to about 7, each time for 1 min, and the waste was discharged until no liquid flowed out.

[0969] Step 4: Fmoc-Glu(tBu)-OH coupling

[0970] 1. Weigh 6.51 g (3.0 eq) of Fmoc-Glu(tBu)-OH, 2.57 g (4.0 eq) of DIC and 2.07 g (3.0 eq) of HOBt, dissolve them in 150 mL of DMF and add them to the reaction column. Blow nitrogen and adjust the nitrogen to make the resin swell evenly.

[0971] 2. React in an environment of 25°C for 3.0 hours, take samples for ninhydrin detection, and the resin is colorless and transparent;

[0972] 3. Draw off the reaction liquid, wash with DMF (150 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[0973] Main chain amino acid coupling steps

[0974] Referring to the method and order of feeding of Fmoc-Glu(tBu)-OH coupling in step 4, the amino acid sequence is connected in sequence, and the order and amino acids containing protective groups used are as follows:

[0975] Amino acids containing protecting groups used Remark Fmoc-Leu-OH AA-13 peptide Fmoc-Leu-OH Fmoc-Tyr(tBu)-OH Fmoc-Glu(OtBu)-OH Fmoc-Ile-OH Fmoc-Phe-OH Fmoc-Ala-OH Fmoc-Aib-OH Fmoc-Gln(Trt)-OH HPLC monitoring Fmoc-Ala-OH Fmoc-Lys(Alloc)-OH Fmoc-Lys(Boc)-OH Fmoc-Asp(OtBu)-OH Fmoc-Lys(Mtt)-OH Fmoc-α-Me-Leu-OH HPLC monitoring Fmoc-Ile-OH HPLC monitoring Fmoc-Ser(tBu)-OH Fmoc-Tyr(tBu)-OH Fmoc-Asp(OtBu)-OH Fmoc-Ser(tBu)-OH Fmoc-Thr(tBu)-OH Fmoc-Phe-OH Fmoc-Thr(tBu)-OH Fmoc-Gly-OH Fmoc-Gln(Trt)-OH Boc-Tyr(tBu)-Aib-OH HPLC monitoring

[0976] Step 5. Side-chain coupling

[0977] 1. Add 300 mL of DCM to the reaction column to wash the resin 4 times, drain the solvent, add 150 mL of 20% hexafluoroisopropanol (HFIP)-DCM solution, and bubbling nitrogen for 5 times, each time for 30 min;

[0978] 2. Drain the waste until no liquid flows out, take a sample for ninhydrin testing, and the resin will be gray-green;

[0979] 3. Add 150 mL of DCM and 150 mL of DMF in sequence to cross-wash the resin 4 times and drain until no liquid flows out.

[0980] 4. Weigh 10.08 g (5.0 eq) of Fmoc-Ida(Oall)-OH, 3.86 g (6.0 eq) of DIC and 3.45 g (5.0 eq) of HOBt, dissolve them in 150 mL of DMF and add them to the reaction column. Blow nitrogen and adjust the nitrogen to make the resin swell evenly.

[0981] 5. React for 3.0 hours in an environment of 25°C, take samples for ninhydrin detection, and the resin is colorless and transparent;

[0982] 6. Draw off the reaction liquid, wash with DMF (180 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[0983] The remaining branch coupling steps

[0984] Referring to the coupling method and feed equivalents of Fmoc-Ida(Oall)-OH in step 5, the following compounds were added in sequence:

[0985] Amino acids containing protecting groups used Remark Fmoc-AEEA-OH Fmoc-Glu-OtBu 19-(bis(benzyloxy)phosphoryl)nonadecanoic acid HPLC monitoring

[0986] Step 6. Cyclization

[0987] 1. Add 250 mL of DMF and 250 mL of DCM to the reaction column in sequence, cross-wash the resin 4 times, and drain until no liquid flows out;

[0988] 2. Prepare 300 mL of a mixed solvent with a volume ratio of DCM: CH3CN: NMP: DEA = 3:3:2:2 and add it to the reaction column, then add 41.59 g (0.27 eq) of Pd(PPh3), react with nitrogen bubbling for 30 min, and drain until no liquid flows out;

[0989] 3. Take samples for ninhydrin testing, the resin is yellow with a purple tint;

[0990] 4. Add 250 mL of DMF and 250 mL of DCM in sequence and cross-wash the resin 8 times;

[0991] 5. Dissolve 3.86g (6.0eq) of DIC and 3.45g (5.0eq) of HOBt in 250mL of DMF, add to the reaction column, react for 23h with nitrogen, take a sample for ninhydrin detection, and the resin is colorless;

[0992] 6. Add 250 mL of DMF and 250 mL of DCM in sequence and cross-wash the resin 8 times;

[0993] 7. Add 200 mL of MeOH and 200 mL of DCM sequentially to cross-wash the resin 4 times;

[0994] 8. Take out the resin and vacuum dry it until the weight stops decreasing, then weigh it to obtain 33 g of peptide resin;

[0995] The intermediate (peptide resin) has the following structural formula:

[0996] Nε(Tyr(tBu)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-a-MeLeu-Lys 14 -Asp(OtBu)-Lys(Boc)-Lys 17 -Ala-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-Leu-Glu(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Linker-MBHA Resin(14,17(Nε(HOOC-(CH2) 18 -PO(OBn)2)-Glu(ε-OtBu)-(AEEA)-(2-2-oxoethyl)glycine))

[0997] Step 7. Peptide resin cleavage

[0998] Prepare 500mL of cleavage reagent, the reagent formula is trifluoroacetic acid (TFA), purified water (H2O), triisopropylsilane (TIS), 3-(triphenylmethylthio) propionic acid (Mpa(Trt)-OH), phenol (ArOH), according to the volume ratio of TFA:H2O:Mpa(Trt)-OH:TIS:ArOH=82.5:2.5:5:5:5, and precool to 10±3°C after preparation. Slowly add the resin to the cleavage reagent under stirring, and continue to stir until the temperature of the reaction system is stable; then control the temperature at 18±3°C and continue to stir the reaction for 8h. Filter the lysate, concentrate it to a viscous state, and precipitate it with methyl tert-butyl ether 12-15 times the volume of the concentrated solution. After filtering the precipitate, wash it and dry it under reduced pressure at room temperature to obtain 8.8g of crude cyclopeptide.

[0999] Step 8. Chromatographic purification

[1000] 2.0 g of the above crude cyclic peptide was taken out and dissolved in 5% acetonitrile and 2% ammonia aqueous solution, and then purified by high performance liquid chromatography using C18 silica gel matrix filler, and the fractions with purity > 90% were collected and combined. The purification chromatographic system is listed as follows:

[1001] Process Water Phase Organic phase gradient One-time purification 0.2% TFA Acetonitrile B%(0-50min):30%-55% Secondary purification 50mmol / L diammonium hydrogen phosphate Acetonitrile B% (0-50min): 31%-65% Three purifications 0.2mmol / L sodium hydroxide Acetonitrile B% (0-50min): 41%-55%

[1002] Step 9. Concentration and freeze drying

[1003] The fraction obtained in the previous step was concentrated to remove acetonitrile, filtered through a 0.22 μm microporous filter membrane, loaded into a hanging bottle freeze dryer, and freeze-dried to obtain 28 mg of the target polypeptide product (purity: 97.67%); the molecular weight of the polypeptide was confirmed by ESI-MS, with a calculated value [M] of 1220.9 and a detected value [M+H] of 1219.8.

[1004] Embodiment 44

[1005]

[1006] Referring to the synthesis of the polypeptide of Example 1, the polypeptide of Example 44 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1259.2, and the detected value was 1258.0.

[1007] Embodiment 45

[1008]

[1009] Referring to the synthesis of the polypeptide of Example 21, the polypeptide of Example 45 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1248.2, and the detected value was 1248.1.

[1010] Embodiment 46

[1011]

[1012] Referring to the synthesis of the polypeptide in Example 1, the polypeptide shown in Example 46 was obtained, wherein the protecting amino acids used in the branched chain coupling were Fmoc-Ida(Oall)-OH, Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu-OtBu, and 19-(bis(benzyloxy)phosphoryl)nonadecanoic acid, respectively. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M] was 5067.61, and the detected value was 5066.61.

[1013] Embodiment 47

[1014]

[1015] Referring to the synthesis of the polypeptide of Example 21, the polypeptide of Example 47 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M] was 4900.56 and the detected value was 4900.62.

[1016] Embodiment 48

[1017]

[1018] Referring to the synthesis of the polypeptide of Example 21, the polypeptide of Example 48 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M] was 4885.56 and the detected value was 4885.50.

[1019] Embodiment 49

[1020]

[1021] Referring to the synthesis of the polypeptide of Example 21, the polypeptide of Example 49 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M] was 4857.51 and the detected value was 4857.55.

[1022] Embodiment 50

[1023]

[1024] Referring to the synthesis of the polypeptide of Example 21, the polypeptide of Example 50 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M] was 5002.59 and the detected value was 5002.62.

[1025] Embodiment 51

[1026]

[1027] Referring to the synthesis of the polypeptide of Example 21, the polypeptide of Example 51 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M] was 4856.57 and the detected value was 4856.48.

[1028] Embodiment 52

[1029]

[1030] MBHA Resin was used as the starting resin, conventional Fmoc-protected amino acids were added, the condensation agent used was a DIC / HOBt system, DMF was used as the main reaction solvent, and a 20% PIP-DMF solution was used as the Fmoc removal solvent. The peptide main chain was condensed one by one from the C-terminus to the N-terminus. After the main chain synthesis was completed, the -Mtt on the ε-amino group of Fmoc-Lys(Mtt)-OH at position A-26 was removed, and then the side 1 to side 4 positions ((2-2-oxoethyl)-glycine, AEEA, N-Boc-N'-Fmoc-Lys-OH, γGlu, mono-tert-butyl eicosandioic acid) were condensed in sequence, and then the ε-amino protecting group of Lys at position A-23 and the carboxyl protecting group of (2-2-oxoethyl)-glycine at position 1 were removed, and then a condensation agent was added to form a lactam ring to obtain the target peptide resin.

[1031] The specific steps are as follows:

[1032] Step 1: Resin activation and deprotection

[1033] 1. Take 15g (5.1mmol) of MBHA Resin (S=0.34mmol / g) and add it to the solid phase reaction column. Add 225mL of DCM and let it stand for 20min to fully swell the resin. Drain the DCM, add 150mL of DMF and wash the resin by nitrogen bubbling. Repeat the above steps once and drain the solvent. Add 20% piperidine / DMF (150mL) to the reaction column, bubbling nitrogen for 10min, drain until no liquid flows out, add 20% piperidine / DMF (150mL) to the reaction column again and continue bubbling nitrogen for 10min. Take a sample of ninhydrin for detection. The resin is gray-red. Drain the reaction solution and wash it 8 times with DMF (150mL). Adjust pH=7, 1min each time, drain until no liquid flows out.

[1034] Step 2: Fmoc-Ser(tBu)-OH coupling

[1035] 1. Weigh 5.86g (3.0eq) of Fmoc-Ser(tBu)-OH, 2.57g (4.0eq) of DIC and 2.07g (3.0eq) of HOBt, dissolve them in 80mL of DMF and add them to the reaction column. Blow nitrogen and adjust the nitrogen to make the resin swell evenly.

[1036] 2. React in an environment of 25°C for 2.0h, take samples for ninhydrin detection, the resin is colorless and transparent;

[1037] 3. Draw off the reaction liquid, wash with DMF (150 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[1038] Main chain amino acid coupling steps

[1039] Referring to the method and amount of Fmoc-Ser(tBu)-OH coupling in step 2, the amino acid sequence is sequentially connected, and the order and amino acids containing protective groups used are as follows:

[1040] Amino acids containing protecting groups used Remark Fmoc-Pro-OH Fmoc-Pro-OH Fmoc-Pro-OH Fmoc-Ala-OH Fmoc-Gly-OH Fmoc-Ser(tBu)-OH Fmoc-Ser(tBu)-OH Fmoc-Pro-OH Fmoc-Gly-OH Fmoc-Gly-OH HPLC detection synthesis

[1041] The AA-11 peptide was obtained.

[1042] Step 3: Deprotection of AA-11 peptide

[1043] 1. Add DMF (150 mL) to the synthesized AA-11 peptide and activate it for 10 min. Drain until no liquid flows out. Add 20% piperidine / DMF (150 mL) to the reaction column, bubbling nitrogen for 10 min, and drain until no liquid flows out. Add 20% piperidine / DMF (150 mL) to the reaction column again and continue bubbling nitrogen for 10 min. Take a sample of ninhydrin for detection. The resin is dark yellow. Draw off the reaction solution and wash it 8 times with DMF (150 mL). Adjust the pH to about 7, each time for 1 min, and drain until no liquid flows out.

[1044] Step 4: Fmoc-Glu(tBu)-OH coupling

[1045] 1. Weigh 6.51 g (3.0 eq) of Fmoc-Glu(tBu)-OH, 2.57 g (4.0 eq) of DIC and 2.07 g (3.0 eq) of HOBt, dissolve them in 150 mL of DMF and add them to the reaction column. Blow nitrogen and adjust the nitrogen to make the resin swell evenly.

[1046] 2. React in an environment of 25°C for 3.0 hours, take samples for ninhydrin detection, and the resin is colorless and transparent;

[1047] 3. Draw off the reaction liquid, wash with DMF (150 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[1048] Main chain amino acid coupling steps

[1049] Referring to the method and order of feeding of Fmoc-Glu(tBu)-OH coupling in step 4, the amino acid sequence is connected in sequence, and the order and amino acids containing protective groups used are as follows:

[1050]

[1051]

[1052] Step 5. Side-chain coupling

[1053] 1. Add 300 mL of DCM to the reaction column to wash the resin 4 times, drain the solvent, add 150 mL of 20% hexafluoroisopropanol (HFIP)-DCM solution, and bubbling nitrogen for 5 times, each time for 30 min;

[1054] 2. Drain the waste until no liquid flows out, take a sample for ninhydrin testing, and the resin will be gray-green;

[1055] 3. Add 150 mL of DCM and 150 mL of DMF in sequence to cross-wash the resin 4 times and drain until no liquid flows out.

[1056] 4. Weigh 10.08 g (5.0 eq) of Fmoc-Ida(Oall)-OH, 3.86 g (6.0 eq) of DIC and 3.45 g (5.0 eq) of HOBt, dissolve them in 150 mL of DMF and add them to the reaction column. Blow nitrogen and adjust the nitrogen to make the resin swell evenly.

[1057] 5. React for 3.0 hours in an environment of 25°C, take samples for ninhydrin detection, and the resin is colorless and transparent;

[1058] 6. Draw off the reaction liquid, wash with DMF (180 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[1059] The remaining branch coupling steps

[1060] Referring to the coupling method and feed equivalents of Fmoc-Ida(Oall)-OH in step 5, the following compounds were added in sequence:

[1061] Amino acids containing protecting groups used Remark Fmoc-AEEA-OH N-Boc-N'-Fmoc-Lys-OH Fmoc-Glu-OtBu Eicosanedioic acid monotert-butyl ester HPLC monitoring

[1062] Step 6. Cyclization

[1063] 1. Add 250 mL of DMF and 250 mL of DCM to the reaction column in sequence, cross-wash the resin 4 times, and drain until no liquid flows out;

[1064] 2. Prepare 300 mL of a mixed solvent with a volume ratio of DCM: CH3CN: NMP: DEA = 3:3:2:2 and add it to the reaction column, then add 41.59 g (0.27 eq) of Pd(PPh3), react with nitrogen bubbling for 30 min, and drain until no liquid flows out;

[1065] 3. Take samples for ninhydrin testing, the resin is yellow with a purple tint;

[1066] 4. Add 250 mL of DMF and 250 mL of DCM in sequence and cross-wash the resin 8 times;

[1067] 5. Dissolve 3.86g (6.0eq) of DIC and 3.45g (5.0eq) of HOBt in 250mL of DMF, add to the reaction column, react for 22h with nitrogen, take a sample for ninhydrin detection, and the resin is colorless;

[1068] 6. Add 250 mL of DMF and 250 mL of DCM in sequence and cross-wash the resin 8 times;

[1069] 7. Add 200 mL of MeOH and 200 mL of DCM in sequence to cross-wash the resin 4 times;

[1070] 8. Take out the resin and vacuum dry it until the weight stops decreasing. Weigh it to obtain 25.5 g of peptide resin;

[1071] The intermediate (peptide resin) has the following structural formula:

[1072] Nε(Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-a-MeLeu-Lys 14 -Asp(OtBu)-Lys(Boc)-Lys 17 -Ala-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-Leu-Glu(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Linker-MBHA Resin(14,17(Nε(HOOC-(CH2) 18 -COOtBu)–Boc(Lys)-Glu(ε-OtBu)-(AEEA)-(2-2-oxoethyl)glycine))

[1073] Step 7. Peptide resin cleavage

[1074] Prepare 500mL of cleavage reagent, the reagent formula is trifluoroacetic acid (TFA), purified water (H2O), triisopropylsilane (TIS), 3-(triphenylmethylthio) propionic acid (Mpa(Trt)-OH), phenol (ArOH), according to the volume ratio of TFA:H2O:Mpa(Trt)-OH:TIS:ArOH = 82.5:2.5:5:5:5, and precool to 10±3°C after preparation. Slowly add the resin to the cleavage reagent under stirring, and continue to stir until the temperature of the reaction system is stable; then control the temperature at 18±3°C and continue to stir and react for 4h. Filter the lysate, concentrate it to a viscous state, and precipitate it with methyl tert-butyl ether 12-15 times the volume of the concentrated solution. After filtering the precipitate, wash it and dry it under reduced pressure at room temperature to obtain 8.6g of crude cyclopeptide.

[1075] Step 8. Chromatographic purification

[1076] The above 8.6 cyclopeptide crude product was dissolved in 5% acetonitrile and 2% ammonia aqueous solution, and then purified by high performance liquid chromatography using C18 silica gel matrix filler, and the fractions with purity > 90% were collected and combined. The purification chromatographic system is listed as follows:

[1077] Process Water Phase Organic phase gradient One-time purification 0.2% TFA Acetonitrile B%(0-50min):30%-55% Secondary purification 50mmol / L diammonium hydrogen phosphate Acetonitrile B% (0-50min): 31%-65% Three purifications 0.2mmol / L sodium hydroxide Acetonitrile B% (0-50min): 41%-55%

[1078] Step 9. Concentration and freeze drying

[1079] The fraction obtained in the previous step was concentrated to remove acetonitrile, filtered through a 0.22 μm microporous filter membrane, loaded into a hanging bottle freeze dryer, and freeze-dried to obtain 1.1 g (purity: 99.12%) of the target polypeptide product; the molecular weight of the polypeptide was confirmed by ESI-MS, the calculated value [M] was 4972.62, and the detected value was 4971.42.

[1080] Embodiment 53

[1081]

[1082] Referring to the synthesis of the polypeptide of Example 52, the polypeptide of Example 53 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M] was 4985.61 and the detected value was 4985.42.

[1083] Embodiment 54

[1084]

[1085] Referring to the synthesis of the polypeptide of Example 52, the polypeptide of Example 54 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M] was 4970.65 and the detected value was 4969.65.

[1086] Biological test data

[1087] Study 1: In vitro GLP-1R / GIPR / GCGR agonist activity test

[1088] Method 1:

[1089] A: Main materials:

[1090] 1) Cell lines

[1091] This cell line was constructed by Pharmaron. See the table below for details.

[1092] Target Host cells clone GLP-1R HEK293 N / A GCGR HEK293 N / A GIPR CHO N / A

[1093] 2) Reagents and consumables

[1094] name batch. Part Number factory cAMP Detection Kit 3053757 TRF0263 PerkinElmer 1M HEPES 2458415 15630-080 Gibco Hanks Balanced Salt Solution (HBSS) 2193007 14025-076 Gibco Bovine serum albumin (BSA) 2888494 CR84-100 PerkinElmer 3-Isobutyl-1-methylxanthine (IBMX) 102433079 I5879 Sigma TrypLE 2492769 12604-021 Invitrogen 96-well plate 1363672 249944 Thermo CulturPlate-384 8213-22291 6007680 PerkinElmer

[1095] 3) Instruments

[1096]

[1097] B. Methods

[1098] Ⅰ) Experimental Materials

[1099] Assay buffer

[1100]

[1101] Preparation of detection reagents

[1102] <![CDATA[ Reagents ]]> <![CDATA[ Storage concentration ]]> <![CDATA[ volume ]]> <![CDATA[ Final concentration ]]> Cell lysis buffer 1x 9.73mL ≈1x Eu-cAMP Solution 50x 200μL 1x Ulight-anti-cAMP solution 150x 66.7μL 1x

[1103] II) Experimental methods

[1104] a) Preparation of compound plates:

[1105] The test compound was diluted 5-fold at 10 points, with the initial concentration being 200 nM.

[1106] b) Preparation of cell suspension

[1107] 1) The cell lines were cultured in complete medium at 37°C and 5% CO2.

[1108] 2) After TrypLE digestion, the cells were resuspended in the experimental buffer and inoculated into the CulturPlate-384 cell culture plate at a density of 2000 cells per well and a volume of 15 μL per well.

[1109] c) Agonist activity assay

[1110] 1) Freeze-thaw Eu-cAMP tracer and dilute it 50 times with detection buffer; freeze-thaw Ulight-anti-cAMP and dilute it 150 times with detection buffer.

[1111] 2) Add 5 μL of the test compound to each experimental well of the CulturPlate-384 cell culture plate, centrifuge at 200 g for 30 seconds, and stand at 37°C for 30 minutes.

[1112] 3) Add 10 μL of Eu-cAMP tracer to each experimental well, and then add 10 μL of Uligh-anti-cAMP to each experimental well.

[1113] 4) Centrifuge the reaction plate at 200 g for 30 seconds at room temperature and let it stand at 25°C for 1 hour.

[1114] 5) Data were collected using Envision 2105, excitation light: 340 nm, emission light: 665 nm and 615 nm.

[1115] C Experimental Results

[1116]

[1117]

[1118] Conclusion: The polypeptide compound of the present invention has a strong agonist activity on GLP-1R / GIPR / GCGR, which is better than or not worse than the activity of LY3437943 as a whole. In particular, the introduction of the "staple" structure and position has an effect on the activity of the polypeptide. Furthermore, the experimental results obtained in repeated experiments and testing other compounds have a consistent trend. In the best case, compared with the activity of the reference substance LY3437943, the agonist activity of the compound of the present invention on GLP-1R is 0.05 to 5 times that of LY3437943, the agonist activity on GIPR is 0.1 to 5 times that of LY3437943; the agonist activity on GCGR is 0.2 to 5 times that of LY3437943.

[1119] In summary, the compounds of the present invention have superior in vitro GLP-1R / GIPR / GCGR agonist activity, and are generally better than or not worse than the reference substance LY3437943.

[1120] Study 2: Pharmacokinetic properties of the compound in cynomolgus monkeys

[1121] A. Purpose of the experiment

[1122] Pharmacokinetics of test compounds in cynomolgus monkeys

[1123] B. Experimental Procedure

[1124] Non-juvenile male cynomolgus monkeys were used, 3 per compound; whole blood was collected at 1h, 2h, 4h, 8h, 16h, 24h, 48h, 72h, 120h, 168h and 240h after administration, and plasma was prepared. The drug concentration was analyzed by LC-MS / MS method, and the pharmacokinetic parameters were calculated using Phoenix WinNonlin software.

[1125] C. Experimental Results

[1126] The experimental results are shown in the following table.

[1127] Results of cynomolgus monkey pharmacokinetic tests

[1128]

[1129] Conclusion: In the pharmacokinetic properties of cynomolgus monkeys, the half-life of the compound of the present invention is T 1 / 2 The half-life T is greater than 70h, which is significantly better than that of LY-3437943. 1 / 2 and exposure, and its overall advantage is not inferior to LY-3437943.

[1130] Furthermore, in repeated experiments, the experimental results obtained by testing other compounds showed the same trend. In a better case, compared with the reference substance LY3437943, the half-life T1 / 2 and exposure value AUC INF It is 1.1 to 2.5 times that of LY3437943, and its overall properties are better than LY3437943.

[1131] In addition, in further studies, the compounds of the present application were tested for pharmacokinetic properties in C57BL / 6 mice and SD rats. The experimental results obtained were consistent with the pharmacokinetic properties in cynomolgus monkeys, both showing better pharmacokinetic properties in mice and rats, and better or equivalent half-life T compared to LY-3437943 compound. 1 / 2 and exposure.

[1132] Experiment 3: Human kidney S9 metabolic stability test

[1133] A. Purpose of the experiment

[1134] Evaluate the metabolic stability of compounds in the human kidney to determine whether the drug will be degraded by renal metabolic enzymes, thereby affecting its efficacy and pharmacokinetics.

[1135] B. Experimental Procedure

[1136] 1. Prepare relevant solutions.

[1137] 2.a) With cofactors (NADPH & UDPGA): NADPH and UDPGA were added during the incubation. The final concentrations of KS9 Fractions, NADPH, and UDPGA were 1 mg / mL, 1 mM, and 2 mM, respectively. b) Without cofactors (NADPH & UDPGA): H2O was added to the culture medium. The final concentration of KS9 Fractions was 1 mg / mL. The mixture was preheated at 37°C for 10 min.

[1138] 3. Add 20 μM control compound or 100 μM test compound solution to start the reaction. This study used 7-Hydroxycoumarin and Raloxifene as positive controls. The final concentration of the positive control compound was 0.2 μM, and the final concentration of the test compound was 1 μM. The culture medium was cultured in a 37°C water bath.

[1139] 4. Take aliquots of the reaction solution at 0.5, 5, 15, 30 and 60 min, add acetonitrile to stop the reaction, centrifuge the sample for 30 min, take the supernatant and mix it with ultrapure water, and then use it for LC-MS / MS analysis.

[1140] 5. Data Analysis: All calculations were performed using Microsoft Excel. Peak areas were determined and slope values ​​were calculated from the extracted ion chromatograms.

[1141] C. Experimental Results

[1142] Table 1 Results of the metabolic stability test of kidney S9

[1143]

[1144] Conclusion: The compounds of the present invention have excellent in vitro pharmacokinetic properties.

[1145] Experiment 4: Pharmacodynamic study of the compound in the DIO obese mouse model

[1146] Experimental purpose: To evaluate the effects of repeated administration of the test substance (compound) on body weight and glucose and lipid metabolism.

[1147] In this experiment, mice induced from 4-5 weeks of age on a high-fat diet (Research Diets: D12492) for 8 weeks were used as test animals. The animals were grouped according to their body weight and fasting blood glucose levels. The average body weight of the model animals was 47.2±0.3g, and the average body weight of the control animals was 27.9±0.3g. The average fasting blood glucose of the model animals was 8.8±0.2mmol / L, and the average fasting blood glucose of the control animals was 6.0±0.2mmol / L. There were 6 animals in each group. They were given drugs immediately after grouping. All animals were given drugs by subcutaneous injection, once every 3 days, for 22 consecutive days. Both the normal control group and the model group were given solvent (20mM citric acid buffer), and the body weight and food intake were tested twice a week. Fasting blood glucose was tested on D10 and D20 after drug administration, OGTT test was performed on D20, blood was collected on D23 to test liver function indicators (ALT, AST, ALP), serum lipid metabolism indicators (TC, TG, HDL, LDL), liver tissue was taken to calculate the organ index, liver lipid metabolism indicators (liver TC, TG), and liver tissue was subjected to pathological examination (HE, oil red) to evaluate the effects of the test substances on body weight and glucose and lipid metabolism in the obese mouse model induced by a high-fat diet.

[1148] The experimental data of each group of animals were described by mean ± standard error (X ± SEM). P < 0.05 was considered statistically significant. All statistical analyses were performed using Graphpad 8.0 software.

[1149] Effects of Table 2 Compounds on Body Weight Changes in DIO Mice

[1150] Compound dose D22 Weight change rate (%) Control - 3.40±1.08 Model - 3.26±1.96 Example 7 10nmol / kg -35.68±2.00 Example 9 10nmol / kg -25.85±2.05 Embodiment 21 10nmol / kg -32.78±2.97 LY3437943 10nmol / kg -29.19±2.94

[1151] Conclusion: From the results in the table, the compounds of the present invention have obvious weight loss effects, and show a weight loss effect equivalent to or better than LY3437943. In addition, the experimental results obtained by testing other compounds of the present invention show that in terms of weight loss effect, they show an effect equivalent to or better than LY3437943.

[1152] Effects of Table 1 Compounds on Fasting Blood Glucose Changes in DIO Mice

[1153]

[1154] Conclusion: As can be seen from the table, the compounds of the present invention have obvious hypoglycemic effects, and show a hypoglycemic effect comparable to or better than LY3437943. In addition, the experimental results obtained by testing other compounds of the present invention show that in terms of hypoglycemic effect, they show an effect comparable to or better than LY3437943.

[1155] Effects of Table 2 on the changes of liver-to-body ratio in DIO mice

[1156]

[1157] Conclusion: From the results in the table, the compounds of the present invention have a significant effect of improving the liver-to-body ratio, and show a hypoglycemic effect comparable to or even better than LY3437943. In addition, the experimental results obtained by testing other compounds of the present invention show that in terms of improving the liver-to-body ratio, they show an effect comparable to or better than LY3437943.

[1158] Effects of Table 2 on changes in liver parameters of DIO mice

[1159]

[1160] Conclusion: From the results in the table, the compounds of the present invention have a significant effect on lowering liver triglycerides. In addition, the experimental results obtained by testing other compounds of the present invention show that they have an effect equivalent to or better than LY3437943 in lowering liver triglycerides.

[1161] Further, referring to a similar experimental scheme, the compounds of Example 38 and Example 52 were tested, and the effects on the body weight changes of DIO mice were as follows:

[1162] Effects of Table 2 Compounds on Body Weight Changes in DIO Mice

[1163] Compound dose D22 Weight change rate (%) Control - -1.90±2.67 Model - -0.85±1.75 Embodiment 38 10nmol / kg -29.87±1.30 Embodiment 52 10nmol / kg -30.26±2.80 LY3437943 10nmol / kg -26.32±1.91

[1164] Conclusion: From the results in the table, the compounds of Example 38 and Example 52 of the present invention also have obvious weight loss effects, and show weight loss effects comparable to or better than LY3437943.

[1165] In addition, the pharmacodynamics of the compounds of the present application were further studied in an IPGTT test. The results showed that compared with the vehicle group, the compounds of the present invention showed excellent hypoglycemic ability, which showed a hypoglycemic effect comparable to or better than that of LY3437943.

[1166] In summary, the present application newly designs and synthesizes a series of polypeptide compounds with modified structures. The polypeptide has strong in vitro agonist activity on GLP-1R / GIPR / GCGR, excellent in vitro and in vivo pharmacokinetic properties, better stability properties, and has obvious weight loss, hypoglycemic and hypolipidemic effects. The polypeptide compound of the present invention has a drug development prospect as a long-acting GLP-1 / GIP / GCG multi-agonist.

Claims

1. A polypeptide with a modified structure and a pharmaceutically acceptable salt thereof, wherein the polypeptide sequence is shown in Formula Z-3: X5X0X6GT FTSDY SIX1X7X8 KX9X 10 X 11 X0 X4FX 12 X 13 X 14 LLX 15 GGPSSGAPPPS0 Formula Z-3 in: X5 is selected from tyrosine (Tyr, Y) or histidine (His, H); X0 is independently selected from or Aib or alanine (Ala, A); X6 is selected from glutamine (Gln, Q) or histidine (His, H); X1 can be independently selected from α-methyl substituted leucine (α-MeLeu, α-MeL) or tyrosine (Tyr, Y); X7 is selected from leucine (Leu, L) or lysine (Lys, K); X8 is selected from aspartic acid (Asp, D) or glutamic acid (Glu, E); X9 is selected from lysine (Lys, K), glutamine (Gln, Q) or glutamic acid (Glu, E); X 10 Selected from alanine (Ala, A) or tyrosine (Tyr, Y); X 11 Selected from lysine (Lys, K), glutamine (Gln, Q) or alanine (Ala, A); X4 is selected from α-methyl substituted lysine (α-MeLys, α-MeK), D-lysine (dK), L-ornithine (L-Ornithine, L-Orn), alanine (Ala, A), lysine (Lys, K) or glutamic acid (Glu, E); X 12 is selected from valine (Val, V) or isoleucine (Ile, I); X 13 Selected from lysine (Lys, K), glutamine (Gln, Q) or glutamic acid (Glu, E); X 14 is selected from lysine (Lys, K), tryptophan (Trp, W), tyrosine (Tyr, Y), glutamic acid (Glu, E) or phenylalanine (Phe, F); X 15 Selected from lysine (Lys, K) or glutamic acid (Glu, E); S0 is selected from (i.e.: the C-terminal amino acid is optionally amidated to a C-terminal primary amide); And the polypeptide compound has the following modifications: 1) The amino acid side chains at positions i and i+j in the sequence can be modified to form a stapled body, wherein i is independently selected from 14, 17, 19, 21, 24 or 25, and j is independently selected from 3, 4, 5 or 7, 2) The modification is that the amino or carboxyl groups on the side chains of the two amino acid unit structures are Condensation connected; X2 is selected from Wherein, "*" indicates the position connected to X3, and the two ends are the positions connected to the corresponding amino or carboxyl groups in the modified amino acid; X3 is selected from: R1 and R2 are independently selected from m is selected from 1, 2 or 3; p is selected from 1 or 2; n is selected from 8, 9 or 10.

2. The polypeptide and pharmaceutically acceptable salt thereof according to claim 1, characterized in that: In the polypeptide sequence, S0 is 3. The polypeptide and pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The second amino acid X0 in the polypeptide sequence is selected from Aib.

4. The polypeptide and pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The 20th amino acid X0 in the polypeptide sequence is selected from Aib or A.

5. The polypeptide and pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, characterized in that: The polypeptide sequence is selected from the following sequences: YAibQGT FTSDY SIα-MeLLD KKAQAib KFIEYLLEGPSSGAPPPS-NH2 (SEQ ID NO: 1) YAibQGT FTSDY SIα-MeLLD KKAQAib AFIKW LLKGGPSSGAPPPS-NH2 (SEQ ID NO: 2) NO: 3) HAibHGT FTSDYSIYLE KKYAAib EFVKW LLKGGPSSGAPPPS-NH2 (SEQ ID NO: 4) HAibHGT FTSDY SIYLE KKYAAib KFVQW LLEGGPSSGAPPPS-NH2 (SEQ ID NO: 5) YAibQGT FTSDY SIα-MeLLD KKAQAib KFIEF LLEGGPSSGAPPPS-NH2 (SEQ ID NO: 15) YAibQGT FTSDY SIα-MeLLD KKAQAib EFIEY LLEGGPSSGAPPPS-NH2 (SEQ ID NO: 16) NO: 17) YAibQGT FTSDYSIα-MeLLD KKAQAib KFIEY LLEGGPSSGAPPPS-NH2 (SEQ ID NO: 18) YAibQGT FTSDY SIα-MeLLD KKAKAib AFIKY LLEGGPSSGAPPPS-NH2 (SEQ ID NO: 19) YAibQGT FTSDY SIα-MeLLDKKAQAib KFIEY LLKGGPSSGAPPPS-NH2(SEQ ID NO: 20) YAibQGT FTSDY SIα-MeLLD KKAQAα-MeKFIEYLLEGPGPSSGAPPPS-NH2 (SEQ ID NO: 21) YAibQGT FTSDY SIα-MeLLD KKAQAib d-KFIEY LLEGGPSSGAPPPS-NH2 (SEQ ID NO: 22) YAibQGT FTSDY SIα-MeLLD KKAQAib L-OrnFIEY LLEGGPSSGAPPPS-NH2 (SEQ ID NO: 23) YAibQGT FTSDY SIα-MeLKD KKAQAib AFIEYLLEGPSSGAPPPS-NH2 (SEQ ID NO: 24).

6. The polypeptide and pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X3 is selected from: More preferably:

7. The polypeptide and pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Structural unit Selected from: More preferably:

8. The polypeptide and pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The staple structure is selected from:

9. The polypeptide compound represented by the following formula and its pharmaceutically acceptable salt:

10. The polypeptide and pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, characterized in that: The salt can be a salt formed with the following acids: inorganic acids include hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, and phosphorous acid; organic acids include acetic acid, trifluoroacetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, methanesulfonic acid, pamoic acid, arginine, and glucuronic acid.

11. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the polypeptide according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof.

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