Peptide inhibitor of interleukin-23 receptor and use thereof

Abstract

The present invention relates to a peptide inhibitor of the interleukin-23 receptor and the use thereof. Specifically, the present invention relates to a peptide inhibitor of the interleukin-23 receptor, a stereoisomer or pharmaceutically acceptable salt or solvate thereof, a pharmaceutical composition thereof, and the use of the peptide inhibitor in the treatment or prevention of diseases or conditions including inflammatory bowel disease, Crohn's disease, and psoriasis.

Description

Peptide inhibitors of interleukin-23 receptor and their uses Technical Field

[0001] This disclosure relates to novel peptide inhibitors of the interleukin-23 receptor and their use in treating or preventing a variety of diseases and conditions, including inflammatory bowel disease, Crohn's disease, and psoriasis. Background Technology

[0002] Interleukin-23 (IL-23) cytokines are believed to play a decisive role in the pathogenesis of autoimmune diseases such as multiple sclerosis, asthma, rheumatoid arthritis, psoriasis, and inflammatory bowel disease (IBD). Studies in mouse models of acute and chronic IBD have shown that IL-23R and downstream effector cytokines play a major role in disease pathogenesis. IL-23R is expressed on a variety of adaptive and innate immune cells, including Th17 cells, γδT cells, natural killer (NK) cells, dendritic cells, macrophages, and innate lymphocytes, which are abundant in the intestine. Elevated IL-23R gene expression and protein levels have been found on the intestinal mucosal surface in IBD patients. It is believed that IL-23 mediates this role by promoting the development of pathogenic CD4+ T cell populations that produce IL-6, IL-17, and tumor necrosis factor (TNF).

[0003] The produced IL-23 accumulates in the gut, playing a crucial role in regulating the balance between tolerance and immunity through both T-cell-dependent and T-cell-independent enteritis pathways. This is achieved by acting on helper T cell 1 (Th1) and Th17-related cytokines and by inhibiting regulatory T cell responses in the gut (which are pro-inflammatory). Furthermore, the polymorphism of the IL-23 receptor (IL-23R) has been associated with susceptibility to inflammatory bowel disease (IBD), further establishing the critical role of the IL-23 pathway in intestinal homeostasis.

[0004] Psoriasis is a chronic skin disease affecting 2% to 3% of the general population, and it has been shown to be mediated by a T-cell inflammatory response mechanism. IL-23, one of several interleukins, is considered to play a key role in the pathogenesis of psoriasis, and is believed to maintain chronic autoimmune inflammation by inducing interleukin-17, regulating memory T cells, and activating macrophages. Increased expression of IL-23 and IL-23R has been shown in psoriasis patient tissues, and antibodies neutralizing IL-23 have demonstrated IL-23-dependent inhibition of psoriasis development in animal models of psoriasis.

[0005] IL-23 is a heterodimer composed of a unique p19 subunit and the p40 subunit of IL-12. It is a developmental cytokine of helper T cell 1 (TH1) involved in the production of interferon-γ (IFN-γ). Although both IL-23 and IL-12 contain a p40 subunit, they have different phenotypic properties. For example, IL-12-deficient animals are susceptible to inflammatory autoimmune diseases, while IL-23-deficient animals are resistant, presumably due to a reduced number of CD4+ T cells producing IL-6, IL-17, and TNF in the CNS of IL-23-deficient animals. IL-23 binds to IL-23R, a heterodimer receptor composed of the IL-12Rβ1 and IL-23R subunits. The binding of IL-23 to IL-23R activates Jak-stat signaling molecules, Jak2, Tyk2, and Stat1, Stat3, Stat4, and Stat5, although the activation of Stat4 is substantially weaker and it forms a different DNA-binding Stat complex in response to IL-23 compared to IL-12. IL-23R constitutively binds to Jak2 and binds to Stat3 in a ligand-dependent manner. Compared to IL-12, which primarily acts on naive CD4(+) T cells, IL-23 preferentially acts on memory CD4(+) T cells.

[0006] Therapeutic components that inhibit the IL-23 pathway have been identified for the treatment of IL-23-related diseases. Numerous antibodies that bind to IL-23 or IL-23R have been identified, including ustekinumab (a humanized antibody that binds to IL-23), which is already approved for the treatment of psoriasis. Recently, peptide inhibitors that bind to and inhibit the binding of IL-23 to IL-23R have been identified. Clinical trials of ustekinumab and briakinumab (which target the common p40 subunit) and tildrakizumab, guselkumab, MEDI2070, and BI-655066 (which target the unique p19 subunit of IL-23) in Crohn's disease or psoriasis highlight the potential of blocking IL-23 signaling in the treatment of human inflammatory diseases. While these findings are promising, identifying stable and selective agents that preferentially target the IL-23 pathway in the gut for the treatment of enteritis, including Crohn's disease, ulcerative colitis, and related conditions, remains challenging.

[0007] Therefore, there remains a need in the art for novel therapies targeting the IL-23 pathway, which can be used to treat and prevent IL-23-related diseases, including those related to autoimmunity. Furthermore, compounds and methods that specifically target IL-23R from the intestinal lumen could provide therapeutic benefits to patients with IBD. This invention addresses these needs by providing novel peptide inhibitors that bind to IL-23R to inhibit IL-23 binding and signal transduction and are suitable for oral administration. Summary of the Invention

[0008] This invention discloses a cyclic peptide inhibitor of the interleukin-23 receptor, its stereoisomer or pharmaceutically acceptable salt, or a pharmaceutical composition containing them, and the use of said cyclic peptide inhibitor in the treatment or prevention of diseases including inflammatory bowel disease, Crohn's disease and psoriasis.

[0009] The cyclic peptide compounds of the present invention exhibit protein stability, are stable against plasma proteases, epithelial proteases, gastric and intestinal proteases, lung surface proteases, and intracellular proteases, have specific targeting of IL-23, have a long plasma half-life, and possess good pharmacokinetic and pharmacodynamic characteristics.

[0010] This invention relates to a cyclic peptide compound, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the peptide compound has an amino acid sequence of formula (I), formula (I-1A), formula (I-1), formula (I-2A), or formula (I-2):

[0011] Xa1-HT-Xa4-Xa5-[Pen]-Xa7-[2-Nal]-Xa9-EN-[3-Pal]-Xa 13 (I)(SEQ ID NO: 1), or

[0012] Xa1-HT-Xa4-Xa5-Xa6-Xa7-[2-Nal]-Xa9-EN-[3-Pal]-Xa 13 (I-1A)(SEQ ID NO: 6A) or

[0013] Xa1-HT-Xa4-Xa5-[Pen]-Xa7-[2-Nal]-Xa9-EN-[3-Pal]-Xa 13 (I-1)(SEQ ID NO: 6) or

[0014] Xa1-HT-Xa4-Xa5-Xa6-Xa7-[2-Nal]-Xa9-EN-[3-Pal]-Xa 13 (I-2A)(SEQ ID NO: 7A) or

[0015] Xa1-HT-Xa4-Xa5-[Pen]-Xa7-[2-Nal]-Xa9-EN-[3-Pal]-Xa 13 (I-2)(SEQ ID NO: 7)

[0016] in,

[0017] Xa1 is Pen or (D)Pen;

[0018] Xa4 is Trp(7-methyl) or Trp(7-cyclopropyl);

[0019] Xa5 is either Lys or Gln;

[0020] Xa6 is Pen or (D)Pen;

[0021] Xa7 is or

[0022] Xa9 is Thrp or

[0023] Xa 13 It is either Sarc or does not exist;

[0024] The cyclic peptide compound is cyclized via a disulfide bond between Xa1 and Xa6, or between Xa1 and Pen, and the cyclic peptide compound is optionally linked to a protecting group; in some embodiments, the cyclic peptide compound is cyclized via a Pen-Pen disulfide bond, and the cyclic peptide compound is optionally linked to a protecting group.

[0025] At least one amino acid in the cyclic peptide compound is quaternized and / or conjugated with a modifying group, wherein the modifying group comprises a PEG linker or a quaternary ammonium group;

[0026] In some embodiments, at least one amino acid in the cyclic peptide compound is quaternized and / or conjugated with a modifying group, wherein the modifying group comprises a PEG linker or a quaternary ammonium, and the modifying group comprises at least one ester bond, or the modifying group is connected by forming an ester bond with the side chain of an amino acid in the sequence of formula (I-1A), formula (I-1), or formula (I).

[0027] In some embodiments, the cyclic peptide compound is in Xa1, Xa5, Xa7, E, Xa 13 At least one conjugation of -LR in 3-Pal or T, wherein R is hydrogen, hydroxyl, C 1-6 Alkyl, -COC 1-6 Alkyl, -CO halogenated C 1-6 alkyl;

[0028] Alternatively, the cyclic peptide compound Xa1 is linked to the glutamic acid side chain in the sequence of formula (I) to form a ring via a modifying group, wherein the modifying group comprises a PEG linker;

[0029] The conditions are:

[0030] (1) When Xa4 is Trp(7-cyclopropyl), Xa5 is Gln, and Xa9 is... Furthermore, when the modifying group is attached only at Xa1, the modifying group is not:

[0031] (2) When Xa4 is Trp(7-cyclopropyl), Xa5 is Lys, and Xa9 is... When, and only at Xa5, the modifying group is attached, the modifying group is not:

[0032] In some embodiments, the cyclic peptide compound is in Xa1, Xa5, Xa7, E, Xa 13 At least one of the following is conjugated to -LR: 3-Pal or T, wherein L contains a PEG linker, and R is hydrogen, hydroxyl, C 1-6 Alkyl, -COC 1-6 Alkyl, -CO halogenated C 1-6 Alkyl, C 1-30 Fatty acids, C 1-30 heterofatty acids, C 1-30 Fatty acid esters or C 1-30 heterofatty acid esters;

[0033] In some embodiments, the cyclic peptide compound is in Xa1, Xa5, Xa7, E, Xa 13 At least one of the following 3-Pal or T ligation-LR, wherein L comprises a PEG connector and R is C 1-30 Fatty acids, C 1-30 heterofatty acids, C 1-30 Fatty acid esters or C 1-30 heterofatty acid esters;

[0034] In some embodiments, the cyclic peptide compound is in Xa1, Xa5, Xa7, E, Xa 13 At least one of the following is conjugated to -LR: 3-Pal or T, wherein L contains a PEG linker, and R is hydrogen, hydroxyl, C 1-6 Alkyl, -COC 1-6 Alkyl, -CO halogenated C 1-6 alkyl;

[0035] In some embodiments, the cyclic peptide compound is in Xa1, Xa5, Xa7, E, Xa 13 Or at least one conjugation of -LR in 3-Pal, wherein L contains a PEG linker, and R is hydrogen, hydroxyl, or C. 1-6 Alkyl, -COC 1-6 Alkyl, -CO halogenated C 1-6 Alkyl, C 1-30 Fatty acids or C 1-30 heterofatty acids;

[0036] In some embodiments, the cyclic peptide compound is conjugated with -LR at at least one of Xa1, Xa5, Xa7, E, or Sarc, wherein the L comprises a PEG linker, and R is hydrogen, hydroxyl, or C. 1-6 Alkyl, -COC 1-6 Alkyl, -CO halogenated C 1-6 Alkyl, C 1-30 Fatty acids or C 1-30 heterofatty acids;

[0037] In some embodiments, the cyclic peptide compound is conjugated with -LR at at least one of Xa1, Xa5, or Xa7, wherein the L comprises a PEG linker and R is hydrogen, hydroxyl, or C. 1-6 Alkyl, -COC 1-6 Alkyl, -CO halogenated C 1-6 Alkyl, C 1-30 Fatty acids or C 1-30 heterofatty acids;

[0038] In some embodiments, the cyclic peptide compound Xa1 forms a ring with the glutamate side chain in the sequence of formula (I) via a -L-link;

[0039] In some embodiments, the PEG connector is p is 0-50, q is 1-50;

[0040] In some embodiments, the PEG connector is p is 0-50, q is 1-50;

[0041] In some embodiments, R is hydrogen, hydroxyl, C 1-3 Alkyl, -COC 1-3 Alkyl, -CO halogenated C 1-3 Alkyl, C 1-24 Fatty acids or C 1-24 Heterofatty acids; in some embodiments, R is C 1-24 Fatty acids, C 1-24 heterofatty acids, C 1-24 Fatty acid esters or C 1-24heterofatty acid esters;

[0042] In some implementations, the C 1-30 Fatty acids or C 1-30 The following are heterofatty acids: Where n is 1-15;

[0043] In some implementations, the C 1-20 Fatty acids or C 1-20 The following are heterofatty acids: Where n is 1-15;

[0044] In some implementations, the C 1-24 Fatty acids or C 1-24 The following are heterofatty acids: Where n is 2-15;

[0045] In some implementations, the C 1-24 Fatty acids or C 1-24 The following are heterofatty acids: Where n is 4-12;

[0046] In some implementations, R is Where n is 4-15;

[0047] In some implementations, the C 1-30 Fatty acids or C 1-30 The following are heterofatty acids:

[0048] In some implementations, the C 1-24 Fatty acids or C 1-24 The following are heterofatty acids:

[0049] In some implementations, C 1-30 Fatty acid esters or C 1-30 heterofatty acid esters are

[0050] In some implementations, L is -L1-L2-L3-, where L2 is p is 0-50, q is 1-50;

[0051] In some implementations, L1 and L3 are each independently a key, One or more combinations of R, where each R l Each independently is hydrogen, C 1-3 Alkyl, Halogenated C 1-3 Alkyl or C 3-6 Cycloalkyl groups, with two R atoms on the same carbon atom. l If they are not both hydrogen, m is 0-10;

[0052] In some implementations, L1 is a key, Each R l Each independently is hydrogen, C 1-3 Alkyl, Halogenated C 1-3 Alkyl or C 3-6 Cycloalkyl groups, with two R atoms on the same carbon atom. l Not both being hydrogen, m is an integer from 1 to 10, preferably R. l Each of the following groups is independently hydrogen, methyl, ethyl, propyl, monofluoromethyl, difluoromethyl, trifluoromethyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, and the two R groups on the same carbon atom are... l They are not both hydrogen;

[0053] In some implementations, L3 is L3 is preferred

[0054] In some implementations, L1 and L3 are each independently a key, One or more combinations of the above, where m is 0-6, in some embodiments m is 1-6, in some embodiments m is 0-5, and in some embodiments m is 1-5;

[0055] In some implementations, L is -L1-L2-L3-, where L2 is p is 0-50, q is 1-50; L1 and L3 are each independently a bond, One or more combinations thereof, where m is 0-5, and in some embodiments, m is 1-5;

[0056] In some implementations, L2 is p is 0-50, q is 1-50; in some implementations, p is 1-3, q is 1-2;

[0057] In some implementations, L2 is p is 0-10, q is 1-5;

[0058] In some implementations, L2 is p is 1-6, q is 1-4;

[0059] In some implementations, L2 is p = 1-3, q = 1-2;

[0060] In some implementations, L2 is

[0061] In some implementations, L1 and L3 are each independently a key, One or more combinations thereof;

[0062] In some implementations, L1 is a key, One or more combinations thereof;

[0063] In some implementations, L3 is One or more combinations thereof;

[0064] In some implementations, L3 is

[0065] In some implementations, L is: p = 1-3, q = 1-2, m = 1-3;

[0066] In some implementations, L is:

[0067] In some embodiments, the cyclic peptide compound is in Xa1, Xa5, Xa7, E, Xa 13 At least one of 3-Pal or T is quaternized and / or conjugated with a modifying group, said modifying group comprising quaternary ammonium;

[0068] In some embodiments, the cyclic peptide compound is in Xa1, Xa5, Xa7, E, Xa 13 At least one of 3-Pal is quaternized and / or conjugated with a modifying group, the modifying group comprising a quaternary ammonium;

[0069] In some embodiments, the cyclic peptide compound is quaternized and / or conjugated with a modifying group at least at one of Xa1, Xa5, Xa7, E, or Sarc, the modifying group comprising a quaternary ammonium group;

[0070] In some embodiments, the cyclic peptide compound is quaternized and / or conjugated with a modifying group at at least one of Xa1, Xa5, or Xa7, the modifying group comprising a quaternary ammonium group.

[0071] The present invention also relates to a peptide dimer compound, its stereoisomer or its pharmaceutically acceptable salt, wherein two cyclic peptide compounds are linked together via one or more linkers selected from diethylene glycol, iminodiacetic acid (IDA), β-Ala-iminodiacetic acid (β-Ala-IDA) or polyethylene glycol to form a peptide dimer, said cyclic peptide compound having an amino acid sequence as shown in formula (I).

[0072] In some embodiments, the two cyclic peptide compounds are linked via polyethylene glycol to form a peptide dimer;

[0073] In some embodiments, the two cyclic peptide compounds are via The peptide dimers are linked together, with t ranging from 0 to 99.

[0074] In some embodiments, the two cyclic peptide compounds are via The peptide dimers are formed by linking, with t ranging from 0 to 10.

[0075] The present invention also relates to a cyclic peptide compound, its stereoisomer or its pharmaceutically acceptable salt, wherein the cyclic peptide compound has an amino acid sequence of formula (II-1), (II-2), (II-3), (II-4):

[0076] Pen-HT-[Trp(7-methyl)]-Gln-[Pen]-Xa7-[2-Nal]-[Thp]-EN-[3-Pal]-[Sarc] (II-1)(SEQ ID NO: 2)

[0077] Pen-HT-[Trp(7-methyl)]-Lys-[Pen]-Xa7-[2-Nal]-[Thp]-EN-[3-Pal]-[Sarc] (II-2)(SEQ ID NO: 3)

[0078] Xa1-HT-[Trp(7-methyl)]-Lys-Xa6-Xa7-[2-Nal]-[Thp]-EN-[3-Pal]-[Sarc] (II-3)(SEQ ID NO: 4)

[0079] Xa1-HT-[Trp(7-methyl)]-Lys-Xa6-Xa7-[2-Nal]-Xa9-EN-[3-Pal]-[Sarc] (II-4)(SEQ ID NO: 5)

[0080] In some embodiments, the cyclic peptide compound, its stereoisomer, or its pharmaceutically acceptable salt, has an amino acid sequence of formula (II-2):

[0081] Pen-HT-[Trp(7-methyl)]-Lys-[Pen]-Xa7-[2-Nal]-[Thp]-EN-[3-Pal]-[Sarc] (II-2)(SEQ ID NO: 3)

[0082] Xa1 and Xa6 are Pen or Furthermore, Xa1 and Xa6 are not both Pen;

[0083] Xa7 is or

[0084] Xa9 is

[0085] The cyclic peptide compound is cyclized via Pen-Pen or Xa1-Xa6 disulfide bonds;

[0086] Alternatively, the terminal amino group of the side chain of amino acid Pen, Lys, or Xa7 is condensed and linked with α-hydroxyacetic acid, β-hydroxypropionic acid, 2-methyllactic acid, amino acid, α-hydroxyisobutyric acid, α-hydroxycyclopropionic acid, α-hydroxycyclobutyric acid, α-hydroxycyclopentanoic acid, or 2-cyclopropyl-2-hydroxyacetic acid.

[0087] In some embodiments, alternatively, the terminal amino group of the side chain of amino acids Pen, Gln, Lys, or Xa7 or Xa1 is condensed and linked with α-hydroxyacetic acid, β-hydroxypropionic acid, 2-methyllactic acid, amino acid, or α-hydroxyisobutyric acid.

[0088] The cyclic peptide compound is optionally linked to a protecting group;

[0089] The condition is that the cyclic peptide compound is not...

[0090] In some embodiments, the protecting group is a hydroxyl group, NH2, or C.1-6 Alkyl, -NHC 1-6 Alkyl, -N(C) 1-6 Alkyl)2、-COC 1-6 Alkyl, -CO halogenated C 1-6 Alkyl, Gly, Ser;

[0091] In some embodiments, the protecting group is a hydroxyl group, NH2, or C. 1-3 Alkyl, -NHC 1-3 Alkyl, -N(C) 1-3 Alkyl)2、-COC 1-3 Alkyl or -CO halogenated C 1-3 alkyl;

[0092] In some embodiments, the protecting group is a hydroxyl group, NH2, methyl group, -N(CH3)2, -COCH3, or -COCF3;

[0093] In some embodiments, the protecting group is an amino protecting group, selected from acetyl groups;

[0094] In some embodiments, the terminal amino group of the side chain of amino acids Pen, Gln, Lys, Xa7, or Xa1 is condensed and linked with α-hydroxyacetic acid, β-hydroxypropionic acid, serine, α-hydroxyisobutyric acid, α-hydroxycyclopropionic acid, α-hydroxycyclobutyric acid, α-hydroxycyclopentanoic acid, or 2-cyclopropyl-2-hydroxyacetic acid.

[0095] In some embodiments, the terminal amino group of the side chain of amino acids Pen, Gln, Lys, Xa7, or Xa1 is condensed and linked with α-hydroxyacetic acid, β-hydroxypropionic acid, serine, or α-hydroxyisobutyric acid.

[0096] In some embodiments, the terminal amino group of the side chain of amino acids Pen, Gln, Lys, or Xa7 is condensed and linked with α-hydroxyacetic acid, β-hydroxypropionic acid, or serine.

[0097] In some implementations, n is 4, 5, 6, 7, 8, 9, 10, 11, or 12;

[0098] In some implementations, p is 1, 2, 3, 4, 5, or 6;

[0099] In some implementations, q is 1, 2, 3, or 4;

[0100] In some implementations, m is 0, 1, 2, 3, 4, or 5;

[0101] In some implementations, m is 0, 1, or 2;

[0102] In some implementations, m is 1, 2, 3, 4, or 5;

[0103] In some implementations, t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0104] Specifically, the first embodiment relates to a cyclic peptide compound, its stereoisomer or its pharmaceutically acceptable salt, wherein the cyclic peptide compound has the amino acid sequence of formula (I):

[0105] Xa1-HT-Xa4-Xa5-[Pen]-Xa7-[2-Nal]-Xa9-EN-[3-Pal]-Xa 13 (I)(SEQ ID NO: 1)

[0106] in:

[0107] Xa1 is Pen or (D)Pen;

[0108] Xa4 is Trp(7-methyl) or Trp(7-cyclopropyl);

[0109] Xa5 is either Gln or Lys;

[0110] Xa7 is or

[0111] Xa9 is Thrp or

[0112] Xa 13 It is either Sarc or does not exist;

[0113] The cyclic peptide compound is cyclized via a Pen-Pen disulfide bond, and the cyclic peptide compound is optionally linked to a protecting group;

[0114] At least one amino acid in the cyclic peptide compound is quaternized and / or conjugated with a modifying group, wherein the modifying group comprises a PEG linker or a quaternary ammonium group;

[0115] Alternatively, the cyclic peptide compound Xa1 is linked to the glutamic acid side chain in the sequence of formula (I) to form a ring via a modifying group, wherein the modifying group comprises a PEG linker;

[0116] The conditions are:

[0117] (1) When Xa4 is Trp(7-cyclopropyl), Xa5 is Gln, and Xa9 is... Furthermore, when the modifying group is attached only at Xa1, the modifying group is not:

[0118] (2) When Xa4 is Trp(7-cyclopropyl), Xa5 is Lys, and Xa9 is... When, and only at Xa5, the modifying group is attached, the modifying group is not:

[0119] The second specific embodiment relates to a cyclic peptide compound, its stereoisomer or its pharmaceutically acceptable salt, wherein the cyclic peptide compound has the amino acid sequence of formula (I):

[0120] Xa1-HT-Xa4-Xa5-[Pen]-Xa7-[2-Nal]-Xa9-EN-[3-Pal]-[Sarc](I) (SEQ ID NO: 1)

[0121] in:

[0122] Xa1 is Pen or (D)Pen;

[0123] Xa4 is Trp(7-methyl) or Trp(7-cyclopropyl);

[0124] Xa5 is either Gln or Lys;

[0125] Xa7 is or

[0126] Xa9 is Thrp or

[0127] The cyclic peptide compound is cyclized via a Pen-Pen disulfide bond, and the cyclic peptide compound is optionally linked to a protecting group;

[0128] At least one amino acid in the cyclic peptide compound is quaternized and / or conjugated with a modifying group, wherein the modifying group comprises a PEG linker or a quaternary ammonium group;

[0129] Alternatively, the cyclic peptide compound Xa1 is linked to the glutamic acid side chain in the sequence of formula (I) to form a ring via a modifying group, wherein the modifying group comprises a PEG linker;

[0130] The conditions are:

[0131] (1) When Xa4 is Trp(7-cyclopropyl), Xa5 is Gln, and Xa9 is... Furthermore, when the modifying group is attached only at Xa1, the modifying group is not:

[0132] (2) When Xa4 is Trp(7-cyclopropyl), Xa5 is Lys, and Xa9 is... When, and only at Xa5, the modifying group is attached, the modifying group is not:

[0133] Specifically, in the third embodiment, the cyclic peptide compound is in Xa1, Xa5, Xa7, E, Xa 13 At least one of the following is conjugated to -LR: 3-Pal or T, wherein L contains a PEG linker, and R is hydrogen, hydroxyl, C 1-6 Alkyl, -COC 1-6 Alkyl, -CO halogenated C 1-6 Alkyl, C 1-30 Fatty acids or C 1-30 Mixed fatty acids; alternatively, the cyclic peptide compound Xa1 forms a ring with the glutamic acid side chain in the sequence of formula (I) via a -L-link.

[0134] Specifically, in the fourth embodiment, the cyclic peptide compound is in Xa1, Xa5, Xa7, E, Xa 13 At least one of the following 3-Pal or T ligation-LR, wherein L comprises a PEG connector and R is C 1-30 Fatty acids, C 1-30 heterofatty acids, C 1-30 Fatty acid esters or C 1-30 Hetero-fatty acid esters; alternatively, the cyclic peptide compound Xa1 forms a ring with the glutamic acid side chain in the sequence of formula (I) via a -L-link.

[0135] Specifically, in the fifth embodiment, the cyclic peptide compound is in Xa1, Xa5, Xa7, E, Xa 13 Or at least one conjugation of -LR in 3-Pal, wherein L contains a PEG linker, and R is hydrogen, hydroxyl, or C. 1-6 Alkyl, -COC 1-6 Alkyl, -CO halogenated C 1-6 Alkyl, C 1-30 Fatty acids or C 1-30 Mixed fatty acids; alternatively, the cyclic peptide compound Xa1 forms a ring with the glutamic acid side chain in the sequence of formula (I) via a -L-link.

[0136] In the sixth specific embodiment, the cyclic peptide compound is conjugated with -LR at least once in Xa1, Xa5, Xa7, E, or Sarc, wherein L comprises a PEG linker, and R is hydrogen, hydroxyl, or C. 1-6 Alkyl, -COC 1-6 Alkyl, -CO halogenated C 1-6 Alkyl, C 1-30 Fatty acids or C 1-30Mixed fatty acids; alternatively, the cyclic peptide compound Xa1 forms a ring with the glutamic acid side chain in the sequence of formula (I) via a -L-link.

[0137] In a specific seventh embodiment, the cyclic peptide compound is conjugated with -LR at least once in Xa1, Xa5, or Xa7, wherein L comprises a PEG linker, and R is hydrogen, hydroxyl, or C. 1-6 Alkyl, -COC 1-6 Alkyl, -CO halogenated C 1-6 Alkyl, C 1-30 Fatty acids or C 1-30 Mixed fatty acids; alternatively, the cyclic peptide compound Xa1 forms a ring with the glutamic acid side chain in the sequence of formula (I) via a -L-link.

[0138] Specifically, in the eighth implementation scheme, wherein C 1-30 Fatty acids or C 1-30 The following are heterofatty acids: Where n is between 1 and 15.

[0139] Specifically, in the ninth implementation scheme, wherein C 1-24 Fatty acids or C 1-24 The following are heterofatty acids: Where n is 2-15.

[0140] In the specific tenth implementation scheme, L stands for -L1-L2-L3-, where L2 is... p is 0-50, q is 1-50; L1 and L3 are each independent bonds. One or more combinations of these, where m is 0-5;

[0141] The preferred value for L is: p = 1-3, q = 1-2, m = 1-2.

[0142] Specifically, in the eleventh implementation scheme, L stands for -L1-L2-L3-, where L2 is... p is 0-50, q is 1-50; L1 and L3 are each independently a bond, One or more combinations of these, where m is 0-5;

[0143] In some implementations, L is: p is 1-3, q is 1-2.

[0144] Specifically, in the twelfth implementation scheme, L stands for -L1-L2-L3-, where L2 is... p is 0-50, q is 1-50; L1 and L3 are each independently a bond, One or more combinations of these, where m is 0-5;

[0145] In some implementations, L is: p is 1-3, q is 1-2.

[0146] Specifically, in the thirteenth embodiment, the cyclic peptide compound is in Xa1, Xa5, Xa7, E, Xa 13 At least one of 3-Pal or T is quaternized and / or conjugated with a modifying group, said modifying group comprising quaternary ammonium.

[0147] Specifically, in the fourteenth embodiment, the cyclic peptide compound is in Xa1, Xa5, Xa7, E, Xa 13 At least one of 3-Pal is quaternized and / or conjugated with a modifying group, the modifying group comprising a quaternary ammonium.

[0148] In the fifteenth embodiment, the cyclic peptide compound is quaternized and / or conjugated with a modifying group at least at one of Xa1, Xa5, Xa7, E, or Sarc, the modifying group comprising a quaternary ammonium group.

[0149] In a specific sixteenth embodiment, the cyclic peptide compound is quaternized and / or conjugated with a modifying group at least at one of Xa1, Xa5, or Xa7, the modifying group comprising a quaternary ammonium group.

[0150] The seventeenth specific embodiment relates to a peptide dimer compound, its stereoisomer or its pharmaceutically acceptable salt, wherein two cyclic peptide compounds are linked together via one or more linkers selected from diethylene glycol, iminodiacetic acid (IDA), β-Ala-iminodiacetic acid (β-Ala-IDA) or polyethylene glycol to form a peptide dimer, said cyclic peptide compound having an amino acid sequence of formula (I) as described in any of the preceding embodiments.

[0151] Specifically, the eighteenth embodiment relates to a cyclic peptide compound, its stereoisomer or its pharmaceutically acceptable salt, wherein the cyclic peptide compound has an amino acid sequence of formula (II-1), (II-2), (II-3), (II-4):

[0152] Pen-HT-[Trp(7-methyl)]-Gln-[Pen]-Xa7-[2-Nal]-[Thp]-EN-[3-Pal]-[Sarc] (II-1)(SEQ ID NO: 2)

[0153] Pen-HT-[Trp(7-methyl)]-Lys-[Pen]-Xa7-[2-Nal]-[Thp]-EN-[3-Pal]-[Sarc] (II-2)(SEQ ID NO: 3)

[0154] Xa1-HT-[Trp(7-methyl)]-Lys-Xa6-Xa7-[2-Nal]-[Thp]-EN-[3-Pal]-[Sarc] (II-3)(SEQ ID NO: 4)

[0155] Xa1-HT-[Trp(7-methyl)]-Lys-Xa6-Xa7-[2-Nal]-Xa9-EN-[3-Pal]-[Sarc] (II-4)(SEQ ID NO: 5)

[0156] Xa1 and Xa6 are Pen or Furthermore, Xa1 and Xa6 are not both Pen;

[0157] Xa7 is

[0158] Xa9 is

[0159] The cyclic peptide compound is cyclized via Pen-Pen or Xa1-Xa6 disulfide bonds;

[0160] Alternatively, the terminal amino group of the side chain of amino acids Pen, Gln, Lys, Xa7, or Xa1 is condensed and linked with α-hydroxyacetic acid, β-hydroxypropionic acid, 2-methyllactic acid, amino acid, or α-hydroxyisobutyric acid.

[0161] The cyclic peptide compound is optionally linked to a protecting group;

[0162] The condition is that the cyclic peptide compound is not...

[0163] Specifically, the nineteenth embodiment relates to a cyclic peptide compound, its stereoisomer or its pharmaceutically acceptable salt, wherein the cyclic peptide compound has an amino acid sequence of formula (II-1) or (II-2):

[0164] Pen-HT-[Trp(7-methyl)]-Gln-[Pen]-Xa7-[2-Nal]-[Thp]-EN-[3-Pal]-[Sarc] (II-1)(SEQ ID NO: 2)

[0165] Pen-HT-[Trp(7-methyl)]-Lys-[Pen]-Xa7-[2-Nal]-[Thp]-EN-[3-Pal]-[Sarc] (II-2)(SEQ ID NO: 3)

[0166] Xa7 is

[0167] The cyclic peptide compound is cyclized via a Pen-Pen disulfide bond, and the cyclic peptide compound is optionally linked to a protecting group;

[0168] Alternatively, the terminal amino groups of the side chains of amino acids Pen, Gln, Lys, and Xa7 are condensed and linked with α-hydroxyacetic acid, β-hydroxypropionic acid, 2-methyllactic acid, and amino acids.

[0169] The condition is that the cyclic peptide compound is not...

[0170] The twentieth embodiment relates to a cyclic peptide compound, its stereoisomer or its pharmaceutically acceptable salt, wherein the cyclic peptide compound has an amino acid sequence of formula (II-1) or (II-2):

[0171] Pen-HT-[Trp(7-methyl)]-Gln-[Pen]-Xa7-[2-Nal]-[Thp]-EN-[3-Pal]-[Sarc] (II-1)(SEQ ID NO: 2)

[0172] Pen-HT-[Trp(7-methyl)]-Lys-[Pen]-Xa7-[2-Nal]-[Thp]-EN-[3-Pal]-[Sarc] (II-2)(SEQ ID NO: 3)

[0173] Xa7 is

[0174] The cyclic peptide compound is cyclized via a Pen-Pen disulfide bond, and the cyclic peptide compound is optionally linked to a protecting group;

[0175] Alternatively, the terminal amino groups of the side chains of amino acids Pen, Gln, and Lys are condensed and linked with α-hydroxyacetic acid, β-hydroxypropionic acid, and amino acids.

[0176] The condition is that the cyclic peptide compound is not...

[0177] Specifically, the twenty-first embodiment relates to a cyclic peptide compound, its stereoisomer or its pharmaceutically acceptable salt, wherein the cyclic peptide compound has an amino acid sequence of formula (I-1A) or formula (I-1):

[0178] Xa1-HT-Xa4-Xa5-Xa6-Xa7-[2-Nal]-Xa9-EN-[3-Pal]-Xa 13 (I-1A)(SEQ ID NO: 6A)

[0179] Xa1-HT-Xa4-Xa5-[Pen]-Xa7-[2-Nal]-Xa9-EN-[3-Pal]-Xa 13 (I-1)(SEQ ID NO: 6)

[0180] in:

[0181] Xa1 is Pen or (D)Pen;

[0182] Xa4 is Trp(7-methyl) or Trp(7-cyclopropyl);

[0183] Xa5 is either Gln or Lys;

[0184] Xa6 is Pen or (D)Pen;

[0185] Xa7 is or

[0186] Xa9 is Thrp or

[0187] Xa 13 It is either Sarc or does not exist;

[0188] The cyclic peptide compound is cyclized via a disulfide bond between Xa1 and Xa6, or between Xa1 and Pen, and the cyclic peptide compound is optionally linked to a protecting group.

[0189] At least one amino acid in the cyclic peptide compound is quaternized and / or conjugated with a modifying group, wherein the modifying group comprises a PEG linker or a quaternary ammonium group; and the modifying group comprises at least one ester bond, or the modifying group is connected by forming an ester bond with the side chain of an amino acid in the sequence of formula (I-1A) or formula (I-1);

[0190] Furthermore, the cyclic peptide compound has the amino acid sequence of formula (I-1):

[0191] Xa1-HT-Xa4-Xa5-[Pen]-Xa7-[2-Nal]-Xa9-EN-[3-Pal]-Xa 13 (I-1)(SEQ ID NO: 6)

[0192] in:

[0193] Xa1 is Pen or (D)Pen;

[0194] Xa4 is Trp(7-methyl) or Trp(7-cyclopropyl);

[0195] Xa5 is either Gln or Lys;

[0196] Xa7 is or

[0197] Xa9 is Thrp or

[0198] Xa 13 It is either Sarc or does not exist;

[0199] The cyclic peptide compound is cyclized via a Pen-Pen disulfide bond, and the cyclic peptide compound is optionally linked to a protecting group;

[0200] At least one amino acid in the cyclic peptide compound is quaternized and / or conjugated with a modifying group, wherein the modifying group comprises a PEG linker or a quaternary ammonium group; and the modifying group comprises at least one ester bond, or the modifying group is connected to the amino acid side chain in the sequence of formula (I-1) by forming an ester bond;

[0201] Furthermore, the cyclic peptide compound has the amino acid sequence of formula (I-1),

[0202] Xa1 is Pen;

[0203] Xa4 is Trp(7-methyl);

[0204] Xa5 is Gln;

[0205] Xa7 is

[0206] Xa9 is for Thrp;

[0207] Xa 13 For Sarc;

[0208] The cyclic peptide compound is cyclized via a Pen-Pen disulfide bond, and the cyclic peptide compound is optionally linked to a protecting group;

[0209] The cyclic peptide compound contains at least one amino acid conjugated modifying group, wherein the modifying group comprises a PEG linker; and the modifying group contains at least one ester bond, or the modifying group is connected to the amino acid side chain in the sequence of formula (I-1) by forming an ester bond;

[0210] In some embodiments, the protecting group is a hydroxyl group, NH2, or C. 1-3 Alkyl, -NHC 1-3 Alkyl, -N(C) 1-3 Alkyl)2、-COC 1-3 Alkyl or -CO halogenated C 1-3 alkyl;

[0211] In some embodiments, the protecting group is a hydroxyl group, NH2, methyl group, -N(CH3)2, -COCH3, or -COCF3;

[0212] In some embodiments, the carboxyl protecting group is selected from NH2, -N(CH3)2, and the amino protecting group is selected from -COCH3 or -COCF3;

[0213] In some embodiments, the protecting group is an amino protecting group, selected from acetyl groups;

[0214] In some embodiments, the protecting group is an amino protecting group, and the terminal amino protecting group of the side chain of amino acids Pen, (D)Pen, Gln, Lys, Xa7 or Xa1 is selected from α-hydroxyacetic acid, β-hydroxypropionic acid, serine, α-hydroxyisobutyric acid, α-hydroxycyclopropionic acid, α-hydroxycyclobutyric acid, α-hydroxycyclopentanoic acid or 2-cyclopropyl-2-hydroxyacetic acid;

[0215] In some embodiments, the protecting group is an amino protecting group, and the terminal amino protecting group of the side chain of amino acids Pen, Gln, Lys, Xa7 or Xa1 is selected from α-hydroxyacetic acid, β-hydroxypropionic acid, serine or α-hydroxyisobutyric acid.

[0216] In some embodiments, the protecting group is an amino protecting group, and the terminal amino protecting group of the side chain of amino acids Pen, Gln, Lys or Xa7 is selected from α-hydroxyacetic acid, β-hydroxypropionic acid, and serine.

[0217] In some embodiments, the modifying group comprises at least one carboxylic acid ester bond or phosphate ester bond, or the modifying group is connected by forming a carboxylic acid ester bond or phosphate ester bond with the side chain of an amino acid in the sequence (I-1A) or (I-1); in some embodiments, the modifying group comprises at least one carboxylic acid ester bond or phosphate ester bond, or the modifying group is connected by forming a carboxylic acid ester bond [-O(C=O)- or -(C=O)O-] with the carboxyl or hydroxyl group on the side chain of threonine, glutamic acid, or sarcosine in the sequence (I-1A) or (I-1), or the modifying group is connected by forming a phosphate ester bond [-O(P=O)(OH)O-] with the hydroxyl group on the side chain of threonine in the sequence (I-1A) or (I-1).

[0218] Specifically, the twenty-second embodiment relates to a cyclic peptide compound as described in the twenty-first embodiment, its stereoisomer or its pharmaceutically acceptable salt, said cyclic peptide compound being in the form of Xa1, Xa5, Xa7, E, Xa 13 At least one conjugated modifying group in 3-Pal or T, wherein the modifying group is -LR, the L contains a PEG linker, and R is C 1-30 Fatty acids, C 1-30 heterofatty acids, C 1-30 Fatty acid esters or C 1-30 heterofatty acid esters.

[0219] Specifically, the twenty-third embodiment involves a cyclic peptide compound as described in the twenty-second embodiment, its stereoisomer, or its pharmaceutically acceptable salt, wherein R is: Where n is 4-15; in some implementations, n is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; in some implementations, n is 15;

[0220] In some implementations, R is n is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; in some implementations, n is 13 or 15;

[0221] In some implementations, R is

[0222] In some implementations, R is n is 15;

[0223] In some implementations, R is In some implementations, R is

[0224] Specifically, the twenty-fourth embodiment involves a cyclic peptide compound as described in the twenty-two or twenty-three embodiments, its stereoisomer, or its pharmaceutically acceptable salt.

[0225] The L is -L1-L2-L3-, where L2 is p is 0-50, q is 1-50; L1 and L3 are each independently a bond, One or more combinations of R, where each R l Each independently is hydrogen, C 1-3 Alkyl, Halogenated C 1-3 Alkyl or C 3-6 Cycloalkyl groups, with two R atoms on the same carbon atom. l If they are not both hydrogen, m is 0-10;

[0226] In some implementations, L is -L1-L2-L3-, where L2 is p is 0-50, q is 1-50; L1 and L3 are each independently a bond, One or more combinations of the above, where m is 0-6.

[0227] Specifically, the twenty-fifth embodiment relates to a cyclic peptide compound as described in any of the twenty-two to twenty-four embodiments, its stereoisomer, or its pharmaceutically acceptable salt, wherein:

[0228] L1 is the key, Each R l Each independently is hydrogen, C 1-3 Alkyl, Halogenated C 1-3 Alkyl or C 3-6 Cycloalkyl groups, with two R atoms on the same carbon atom. l Not both of them are hydrogen, and m is an integer from 1 to 10;

[0229] L3 is

[0230] In some implementations, R l Each of the following groups is independently hydrogen, methyl, ethyl, propyl, monofluoromethyl, difluoromethyl, trifluoromethyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, and the two R groups on the same carbon atom are... l They are not both hydrogen;

[0231] In some implementations, L3 is

[0232] Specifically, the twenty-sixth embodiment relates to a cyclic peptide compound as described in any one of embodiments twenty-two to twenty-five, its stereoisomer, or its pharmaceutically acceptable salt, wherein L is: p = 1-10, q = 1-2, m = 1-10;

[0233] In some implementations, L is: p = 1-3, q = 1-2, m = 1-3;

[0234] In some implementations, L is: p = 1-3, q = 1-2, m = 1-2;

[0235] In some implementations, L is p is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, q is 1 or 2, and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0236] In some implementations, p is 1 or 2, q is 1 or 2, and m is 2, 7, or 8.

[0237] Specifically, the twenty-seventh embodiment relates to a cyclic peptide compound, its stereoisomer or its pharmaceutically acceptable salt, wherein the cyclic peptide compound has an amino acid sequence of formula (I-2A) or formula (I-2):

[0238] Xa1-HT-Xa4-Xa5-Xa6-Xa7-[2-Nal]-Xa9-EN-[3-Pal]-Xa 13 (I-2A)(SEQ ID NO: 7A)

[0239] Xa1-HT-Xa4-Xa5-[Pen]-Xa7-[2-Nal]-Xa9-EN-[3-Pal]-Xa 13 (I-2)(SEQ ID NO: 7)

[0240] in:

[0241] Xa1 is either Pen or (D)pen;

[0242] Xa4 is Trp(7-methyl) or Trp(7-cyclopropyl);

[0243] Xa5 is either Gln or Lys;

[0244] Xa6 is Pen or (D)Pen;

[0245] Xa7 is or

[0246] Xa9 is Thrp or

[0247] Xa 13 It is either Sarc or does not exist;

[0248] The cyclic peptide compound is cyclized via a disulfide bond between Xa1 and Xa6, or between Xa1 and Pen, and the cyclic peptide compound is optionally linked to a protecting group.

[0249] The cyclic peptide compound is found in Xa1, Xa5, Xa7, E, and Xa... 13 At least one conjugation of -LR in 3-Pal or T, wherein R is hydrogen, hydroxyl, C 1-6 Alkyl, -COC 1-6 Alkyl, -CO halogenated C 1-6 Alkyl group, wherein the L is as described in any of the preceding twenty-four to twenty-six embodiments;

[0250] The conditions are:

[0251] (1) When Xa4 is Trp(7-cyclopropyl), Xa5 is Gln, and Xa9 is... Furthermore, when the modifying group is attached only at Xa1, the modifying group is not:

[0252] (2) When Xa4 is Trp(7-cyclopropyl), Xa5 is Lys, and Xa9 is... When, and only at Xa5, the modifying group is attached, the modifying group is not:

[0253] Furthermore, the cyclic peptide compound has the amino acid sequence of formula (I-2):

[0254] Xa1-HT-Xa4-Xa5-[Pen]-Xa7-[2-Nal]-Xa9-EN-[3-Pal]-Xa 13(I-2)(SEQ ID NO: 7)

[0255] in:

[0256] Xa1 is either Pen or (D)pen;

[0257] Xa4 is Trp(7-methyl) or Trp(7-cyclopropyl);

[0258] Xa5 is either Gln or Lys;

[0259] Xa7 is or

[0260] Xa9 is Thrp or

[0261] Xa 13 It is either Sarc or does not exist;

[0262] The cyclic peptide compound is cyclized via a Pen-Pen disulfide bond, and the cyclic peptide compound is optionally linked to a protecting group;

[0263] The cyclic peptide compound is found in Xa1, Xa5, Xa7, E, and Xa... 13 At least one conjugation of -LR in 3-Pal or T, wherein R is hydrogen, hydroxyl, C 1-6 Alkyl, -COC 1-6 Alkyl, -CO halogenated C 1-6 Alkyl group, wherein L is -L1-L2-L3-, and L2 is... p is 0-50, q is 1-50; L1 and L3 are each independently a bond, One or more combinations of R, where each R l Each independently is hydrogen, C 1-3 Alkyl, Halogenated C 1-3 Alkyl or C 3-6 Cycloalkyl groups, with two R atoms on the same carbon atom. l If they are not both hydrogen, m is 0-10;

[0264] In some implementations, L1 is a key, Each R l Each independently is hydrogen, C 1-3 Alkyl, Halogenated C 1-3 Alkyl or C 3-6 Cycloalkyl groups, with two R atoms on the same carbon atom. l Not both of them are hydrogen, and m is an integer from 1 to 10;

[0265] In some implementations, L3 is

[0266] In some implementations, the R l Each of the following groups is independently hydrogen, methyl, ethyl, propyl, monofluoromethyl, difluoromethyl, trifluoromethyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, and the two R groups on the same carbon atom are... l They are not both hydrogen;

[0267] In some implementations, L3 is

[0268] In some implementations, L is: p = 1-10, q = 1-2, m = 1-10;

[0269] In some implementations, L2 is p is 0-50, q is 1-50; L1 and L3 are each independently a bond, One or more combinations of the above, where m is 0-5; preferably L is: p is 1-3, q is 1-2, and m is 1-3; in some implementations, L is preferably: p = 1-3, q = 1-2, m = 1-2.

[0270] The conditions are:

[0271] (1) When Xa4 is Trp(7-cyclopropyl), Xa5 is Gln, and Xa9 is... Furthermore, when the modifying group is attached only at Xa1, the modifying group is not:

[0272] (2) When Xa4 is Trp(7-cyclopropyl), Xa5 is Lys, and Xa9 is... When, and only at Xa5, the modifying group is attached, the modifying group is not:

[0273] Specifically, in the twenty-eighth embodiment, L in the cyclic peptide compound described in any of the aforementioned twenty-first to twenty-seventh embodiments is: Preferred Preferred

[0274] Specifically, the twenty-ninth embodiment relates to a cyclic peptide compound, its stereoisomer or its pharmaceutically acceptable salt, wherein the cyclic peptide compound has an amino acid sequence of formula (II-2):

[0275] Pen-HT-[Trp(7-methyl)]-Lys-[Pen]-Xa7-[2-Nal]-[Thp]-EN-[3-Pal]-[Sarc](II-2)(SEQ ID NO: 3)

[0276] Xa7 is

[0277] The cyclic peptide compound is cyclized via Pen-Pen disulfide bonds;

[0278] Alternatively, the terminal amino group of the side chain of amino acid Pen, Lys, or Xa7 is condensed and linked with α-hydroxyacetic acid, β-hydroxypropionic acid, 2-methyllactic acid, amino acid, α-hydroxyisobutyric acid, α-hydroxycyclopropionic acid, α-hydroxycyclobutyric acid, α-hydroxycyclopentanoic acid, or 2-cyclopropyl-2-hydroxyacetic acid.

[0279] In some embodiments, alternatively, the terminal amino group of the side chain of amino acid Pen, Lys, or Xa7 is condensed and linked with α-hydroxyacetic acid, β-hydroxypropionic acid, 2-methyllactic acid, amino acid, or α-hydroxyisobutyric acid.

[0280] The cyclic peptide compound is optionally linked to a protecting group;

[0281] The condition is that the cyclic peptide compound is not...

[0282] Specifically, in the thirtieth embodiment, the amino acid sequence of the cyclic peptide compound of formula (I-1) of the present invention has the following structure:

[0283] Glutamic acid, threonine, and sarcosine have side chains that are conjugated with modifying groups. When the sarcosine side chain is not conjugated with modifying groups, its terminal carboxylic acid group is protected by a carboxyl protecting group. In some embodiments, the carboxyl protecting group is selected as an amino group.

[0284] Furthermore, the amino acid sequence of the cyclic peptide compound of formula (I-1) described in this invention has the following structure:

[0285] Glutamic acid side chain conjugation modification group;

[0286] In some embodiments, the modifying group is -LR, where L comprises a PEG linker and R is C. 1-30 Fatty acids, C 1-30 heterofatty acids, C 1-30 Fatty acid esters or C 1-30 heterofatty acid esters;

[0287] In some embodiments, the modifying group is -LR, where L is... p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; q is 1 or 2; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; in some embodiments, p is 1 or 2, q is 1 or 2, and m is 2, 7, or 8; R is... n is 15; in some implementations, R is

[0288] In some embodiments, the modifying group is -LR, where L is... In some implementations, L is, The R is n is 15; in some implementations, R is

[0289] Specifically, in the thirty-first embodiment, the amino acid sequence of the cyclic peptide compound of formula (I-1) of the present invention has the following structure:

[0290] Among them, the cyclic peptide compound contains Pen, lysine, In some embodiments, the cyclic peptide compound has a conjugated modifying group LR at the Pen position; in some embodiments, the cyclic peptide compound has a conjugated modifying group at the lysine residue; in some embodiments, the cyclic peptide compound has a conjugated modifying group at the lysine residue. Addition of modifying groups;

[0291] When the cyclic peptide compound contains unconjugated modifying groups for Pen and lysine, it may optionally be protected by an amino protecting group; in some embodiments, the amino protecting group is selected from acetyl groups.

[0292] The L is selected from In some implementations, L is, The R is n is 15; in some implementations, R is

[0293] In the specific thirty-second embodiment, the compound is selected from one of the structures in Table 1 below:

[0294] Table 1:

[0295] The present invention also relates to a pharmaceutical composition comprising the peptide compound or a pharmaceutically acceptable salt thereof described in any of the foregoing technical solutions, and a pharmaceutically acceptable carrier and / or excipient.

[0296] The present invention also relates to an application of the peptide compound or its pharmaceutically acceptable salt, or the pharmaceutical composition described in any of the foregoing technical solutions, in the preparation of a medicament for the prevention and treatment of diseases or conditions in which IL-23 is overexpressed in diseased tissues of a subject.

[0297] Furthermore, the diseases or conditions that overexpress IL-23 include inflammatory bowel disease, Crohn's disease, and psoriasis.

[0298] The present invention also relates to a pharmaceutical composition or pharmaceutical formulation comprising 1-1500 mg of the peptide compound or a pharmaceutically acceptable salt thereof as described in any of the foregoing technical solutions, and a pharmaceutically acceptable carrier and / or excipient.

[0299] The present invention also relates to a method for treating diseases in mammals or humans, the method comprising administering to a subject a therapeutically effective amount of the peptide compound or a pharmaceutically acceptable salt thereof described in any of the foregoing technical solutions, preferably 1-1500 mg, wherein the disease is preferably inflammatory bowel disease, Crohn's disease, or psoriasis.

[0300] The present invention also provides a composition or pharmaceutical formulation comprising the peptide compound or a pharmaceutically acceptable salt thereof as described in any of the foregoing embodiments, and a pharmaceutically acceptable carrier and / or excipients. The pharmaceutical composition may be in unit dosage form (unit dosage is also referred to as a “dosage strength”).

[0301] Furthermore, the compositions or pharmaceutical preparations of the present invention contain 1-1500 mg of the peptide compound or a pharmaceutically acceptable salt thereof described in any of the foregoing schemes, as well as a pharmaceutically acceptable carrier and / or excipients.

[0302] The present invention also provides the use of the peptide compound or a pharmaceutically acceptable salt thereof described in any of the foregoing embodiments in the preparation of a medicament for the prevention and treatment of diseases or conditions in which IL-23 is overexpressed in diseased tissues of a subject. Further, the diseases or conditions in which IL-23 is overexpressed include inflammatory bowel disease, Crohn's disease, and psoriasis.

[0303] The present invention also provides a method for treating diseases in mammals or humans, the method comprising administering to a subject a therapeutically effective amount of the peptide compound or a pharmaceutically acceptable salt thereof as described in any of the foregoing embodiments, wherein the disease is preferably inflammatory bowel disease, Crohn's disease, and psoriasis, and the therapeutically effective amount is preferably 1-1500 mg. In some embodiments, the mammals described in the present invention do not include humans.

[0304] The term "effective amount" or "therapeutic effective amount" as used in this application means that administering a sufficient amount of the compound disclosed in this application will alleviate, to some extent, one or more symptoms of the disease or condition being treated. In some embodiments, the result is a reduction and / or mitigation of the signs, symptoms, or causes of the disease, or any other desired alteration of the biological system. For example, an "effective amount" for therapeutic use is the amount of the compound disclosed in this application or a pharmaceutically acceptable salt thereof required to provide a clinically significant reduction in disease symptoms. Examples of therapeutically effective doses include, but are not limited to, 1-1500 mg, 1-1400 mg, 1-1300 mg, 1-1200 mg, 1-1000 mg, 1-900 mg, 1-800 mg, 1-700 mg, 1-600 mg, 1-500 mg, 1-400 mg, 1-300 mg, 1-250 mg, 1-200 mg, 1-150 mg, 1-125 mg, 1-100 mg, 1-80 mg, 1-60 mg, 1-50 mg, 1-40 mg, 1-25 mg, 1- 20mg, 5-1500mg, 5-1000mg, 5-900mg, 5-800mg, 5-700mg, 5-600mg, 5-500mg, 5-400mg, 5-300mg, 5-250mg, 5-200mg, 5 -150mg, 5-125mg, 5-100mg, 5-90mg, 5-70mg, 5-80mg, 5-60mg, 5-50mg, 5-40mg, 5-30mg, 5-25mg, 5-20mg, 10-1500mg, 10-1000mg, 10-900mg, 10-800mg, 10-700mg, 10-600mg, 10-500mg, 10-450mg, 10-400mg, 10-300mg, 10-250mg, 10-20 0mg, 10-150mg, 10-125mg, 10-100mg, 10-90mg, 10-80mg, 10-70mg, 10-60mg, 10-50mg, 10-40mg, 10-30mg, 10-20mg; 2 0-1500mg, 20-1000mg, 20-900mg, 20-800mg, 20-700mg, 20-600mg, 20-500mg, 20-400mg, 20-350mg, 20-300mg, 20-25 0mg, 20-200mg, 20-150mg, 20-125mg, 20-100mg, 20-90mg, 20-80mg, 20-70mg, 20-60mg, 20-50mg, 20-40mg, 20-30mg;50-1500mg, 50-1000mg, 50-900mg, 50-800mg, 50-700mg, 50-600mg, 50-500mg, 50-400mg, 50-300mg, 50-250mg, 50-200mg, 50-150mg, 50-125mg, 5 0-100mg; 100-1500mg, 100-1000mg, 100-900mg, 100-800mg, 100-700mg, 100-600mg, 100-500mg, 100-400mg, 100-300mg, 100-250mg, 100-200mg;

[0305] In some embodiments, the pharmaceutical composition or formulation of the present invention contains a therapeutically effective amount of the peptide compound of the present invention or a pharmaceutically acceptable salt thereof;

[0306] This invention relates to a pharmaceutical composition or pharmaceutical formulation comprising a therapeutically effective amount of the peptide compound of the invention or a pharmaceutically acceptable salt thereof, along with a carrier and / or excipients. The pharmaceutical composition may be in unit dosage form (the amount of the active ingredient in a unit dosage form is also referred to as a "dosage strength"). In some embodiments, the pharmaceutical composition includes, but is not limited to, 1 mg, 1.25 mg, 2.5 mg, 5 mg, 10 mg, 12.5 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 110 mg, 120 mg, 125 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 23 mg, etc. The peptide compounds of the present invention or pharmaceutically acceptable salts thereof are present in the following amounts: 0 mg, 240 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, and 1500 mg.

[0307] A method for treating a disease in mammals or humans, the method comprising administering to a subject a therapeutically effective amount of the peptide compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient, the therapeutically effective amount preferably being 1-1500 mg, the disease preferably being inflammatory bowel disease, Crohn's disease, and psoriasis.

[0308] A method for treating a disease in mammals or humans. The method comprises administering a pharmaceutically acceptable peptide compound of the present invention or a pharmaceutically acceptable salt thereof, along with a pharmaceutically acceptable carrier and / or excipient, to a subject at a daily dose of 1-1500 mg / day. The daily dose may be a single dose or multiple doses. In some embodiments, the daily dose includes, but is not limited to, 10-1500 mg / day, 20-1500 mg / day, 25-1500 mg / day, 50-1500 mg / day, 75-1500 mg / day, 100-1500 mg / day, 200-1500 mg / day, 10-1000 mg / day, 20-1000 mg / day, 25-1000 mg / day, 50-1000 mg / day, 75-1000 mg / day, 100-1000 mg / day, etc. The daily dose may be 1 mg / day, 200-1000 mg / day, 25-800 mg / day, 50-800 mg / day, 100-800 mg / day, 200-800 mg / day, 25-400 mg / day, 50-400 mg / day, 100-400 mg / day, or 200-400 mg / day. In some embodiments, the daily dose may include, but is not limited to, 1 mg / day, 5 mg / day, 10 mg / day, 20 mg / day, 25 mg / day, 50 mg / day, 75 mg / day, 100 mg / day, 125 mg / day, 150 mg / day, 200 mg / day, 300 mg / day, 400 mg / day, 600 mg / day, 800 mg / day, 1000 mg / day, 1200 mg / day, 1400 mg / day, or 1500 mg / day.

[0309] This invention relates to a kit that may comprise a single-dose or multi-dose composition containing a peptide compound of the invention or a pharmaceutically acceptable salt thereof, wherein the amount of the compound of the invention or its stereoisomer or pharmaceutically acceptable salt is the same as that in the pharmaceutical composition described above.

[0310] In this invention, the amount of the compound of the invention or its stereoisomer or pharmaceutically acceptable salt is converted in each case as a free base.

[0311] "Product specification" refers to the weight of the active pharmaceutical ingredient contained in each vial, tablet, or other unit of preparation.

[0312] the term

[0313] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by one of ordinary skill in the art. Generally, the nomenclature used in conjunction with the chemical, molecular biological, cell and cancer biological, immunological, microbiological, pharmacological, and protein and nucleic acid chemistry described herein, as well as the techniques used in these fields, are well known and commonly used in the art. In case of any discrepancy, the definitions provided herein shall prevail. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient. All patents, published patent applications, and publications cited herein are incorporated herein by reference.

[0314] As used herein, the following terms have the meanings assigned to them unless otherwise stated.

[0315] Throughout this specification, the word “comprising” or variations thereof such as “containing” or “including” should be understood to mean that the said integer (or component) or group of integers (or components) is included, but does not exclude the possibility that any other integers (or components) or group of integers (or components) may be included further.

[0316] In a broad sense, a peptide is a sequence of two or more amino acids linked together by peptide bonds. It should be understood that the term does not imply a polymer of amino acids of a specific length, nor is it intended to imply or distinguish whether a polypeptide is produced using recombinant technology, chemical synthesis, or enzymatic synthesis, or whether it is naturally occurring.

[0317] The peptides provided by the present invention may contain at least 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity. Therefore, the polypeptide provided by the present invention comprises having at least 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the sequence of formula (I).

[0318] "Sequence identity," "identity percentage," "homology percentage," or, for example, "50% identical to...", refers to the degree of sequence similarity based on nucleotide to nucleotide or amino acid to amino acid within a comparison window. Therefore, the "sequence identity percentage" can be calculated as follows: comparing two best-aligned sequences within a comparison window, determining the number of identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the sequence identity percentage. Sequence similarity or sequence identity (the terms are used interchangeably herein) between sequences can be calculated as follows. To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., for optimal alignment, vacancies can be introduced into one or both of the first and second amino acid or nucleic acid sequences; while for comparison purposes, non-homologous sequences can be ignored). In some embodiments, the length of the reference sequence for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position.

[0319] The percentage of identity between two sequences is a function of the number of common positions in the sequences (taking into account the number of gaps that must be introduced for optimal alignment of the two sequences and the length of each gap).

[0320] It should be understood that the peptide sequences disclosed herein are shown from left to right, with the left end of the sequence representing the N-terminus of the peptide and the right end representing the C-terminus. The sequences disclosed herein are those incorporating a "Hy-" portion at the amino terminus (N-terminus) and a "-OH" or "-NH2" portion at the carboxyl terminus (C-terminus). In such cases, and unless otherwise stated, the "Hy-" portion at the N-terminus of the discussed sequence represents a hydrogen atom, corresponding to the presence of a free primary or secondary amino group at the N-terminus, while the "-OH" or "NH2" portion at the C-terminus represents a hydroxyl or amino group, respectively, corresponding to the presence of an amide (CONH2) group at the C-terminus. In the sequences of this invention, the C-terminal "-OH" portion may replace the C-terminal "-NH2" portion, and vice versa.

[0321] The term "protecting group" refers to the protecting group at the free amino and / or carboxyl terminus of an amino acid residue in a polypeptide. Preferred protecting groups include, but are not limited to, acetyl, amide, alkyl groups of 3-20 carbon atoms, Fmoc, t-boc, 9-fluorenacetyl, 1-fluorencarboxyl, 9-fluorencarboxyl, 9-fluorenone-1-carboxyl, benzyloxycarbonyl, xanthonyl (Xan), triphenylmethyl (Trt), 4-methyltriphenylmethyl (Mtt), 4-methoxytriphenylmethyl (Mmt), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), 1,3,5-trimethylbenzene-2-sulfonyl (Mts), 4,4-dimethoxydiphenylmethyl (Mbh), toluenesulfonyl (Tos), and 2,2,5,7,8-pentamethylbenzodihydropyran (chroman)-6-sulfonyl (Pmc). 4-Methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), benzyloxy (BzlO), benzyl (Bzl), benzoyl (Bz), 3-nitro-2-pyridinesulfonyl (sulphenyl)(Npys), 1-(4,4-dimentyl-2,6-diazocyclohexyl)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl (Bom), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-butyl (tBu), acetyl (Ac), benzoyl, benzyloxycarbonyl, propyl, butyl, pentyl, hexyl and trifluoroacetyl (TFA). In some embodiments, the polypeptide of the present invention contains a protecting group coupled to the amino terminus, said amino-terminal protecting group being C 1-6 Alkyl, -COC 1-6 Alkyl or -CO halogenated C 1-6Alkyl group. In some embodiments, the polypeptide of the present invention contains a protecting group coupled to a carboxyl terminus, said carboxyl terminus being NH2 or -NHC. 1-3 Alkyl, -N(C) 1-3 Alkyl)2.

[0322] The term "NH2" may refer to a free amino group present at the amino terminus of a peptide. The term "OH" as used herein may refer to a free carboxyl group present at the carboxyl terminus of a peptide. Additionally, the term "Ac" as used herein refers to acetyl protection formed by acylation of the C-terminus or N-terminus of the peptide. In some peptides shown herein, NH2 at the C-terminus indicates an amino group.

[0323] The term "quaternary ammonium" is used in its common sense in the art. For example, a quaternary ammonium is a substituent containing one or more permanently positively charged nitrogen atoms. A non-limiting example of a quaternary ammonium can be a moiety having four organic substituents on a nitrogen atom, typically described by the formula NR4+X-, where R is any chemically permissible substituent, including acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds, and X is a suitable counterion (or anion), a negatively charged group associated with the positively charged quaternary ammonium to maintain electronic neutrality. Exemplary counterions include halide ions (e.g., F, Cl, Br, I), NO3-, ClO4-, OH-, H2PO4-, HSO4-, sulfonate ions (e.g., methanesulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphorsulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonic-5-sulfonate, ethane-1-sulfonic-2-sulfonate, etc.) and carboxylate ions (e.g., acetate, acetate, propionate, benzoate, glycerate, lactate, tartrate, glycolate, etc.). "Quaternization" refers to the reaction with the aforementioned R groups to form quaternary ammonium compounds. It should be understood that quaternization of amino acids in the cyclic peptide compounds of this invention means that the NH2 residue on the amino acid residue can be quaternized.

[0324] "Fatty acid" refers to a long aliphatic hydrocarbon chain that is fully saturated or contains one or more unsaturated units, and is either straight-chain (i.e., unbranched) or branched, substituted or unsubstituted, and has at least one end a carboxyl group (-COOH), a sulfonic acid group (-S(=O)2(OH)) or a phosphate group (-P(=O)(OH)2). The fatty acid preferably has 1 to 30 carbon atoms, i.e., "C 1-30 "Fatty acids", preferably having 1 to 24 carbon atoms (i.e., C64) 1-24 Fatty acids), more preferably having 1 to 22 carbon atoms (i.e., C64-22 carbon atoms). 1-22Fatty acids). Non-limiting examples include: lauric acid, myristic acid, hard fatty acids, soft fatty acids, arachidic acid, horny acid, palmitic acid, oleic acid, isoleic acid, nervonic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, arachidonic acid, EPA, DPA, DHA, sebacic acid, C12 diacid, C14 diacid, C16 diacid, C18 diacid, C20 diacid, undecanoic acid, pentadecanoic acid, etc. "Heterofatty acids" refer to fatty acids having at least one heteroatom on their straight chain, i.e., one or more carbon atoms on the straight chain of a fatty acid are occupied by at least one heteroatom (e.g., nitrogen, oxygen, P(O)). m and S(O) n The term "fatty acid ester" refers to a compound formed by the esterification of said fatty acid and alcohol, and non-limiting examples include: methyl tridecanoate. "Heterofatty acid ester" refers to a compound formed by the esterification of said heterofatty acid and alcohol.

[0325] Exemplary C 1-24 Fatty acids or C 1-24 heterofatty acids

[0326] "Polyethylene glycol" or "PEG" is a polyether compound with the general formula H-(O-CH2-CH2)n-OH. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on their molecular weight, but PEG tends to refer to oligomers and polymers with a molecular weight less than 20,000 Da. In some embodiments, PEG with a molecular weight of 50 to 2,000 or 100 to 500 is used. Different forms of PEG can also be used, depending on the initiator used in the polymerization process—common initiators are monofunctional methyl ether PEG or methoxylated poly(ethylene glycol) (abbreviated as mPEG). In some embodiments, PEG is PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, or PEG11. In some embodiments, the PEG connector is...

[0327] The term "carboxyl group" refers to -COOH, and "Ac" refers to "acetyl group".

[0328] The term "connector" broadly refers to a chemical structure that can link or combine two peptide monomer subunits to form a dimer.

[0329] The term "dimer" broadly refers to a peptide containing two or more monomeric subunits. Some dimers contain two DRPs. The dimers of this invention include homodimers and heterodimers. The monomeric subunits of the dimer may be linked at their C-terminus or N-terminus, or they may be linked via internal amino acid residues. The monomeric subunits of the dimer may be linked through the same site, or each may be linked through different sites (e.g., C-terminus, N-terminus, or internal sites).

[0330] The term "cyclization" or "forming a peptide ring" refers to a reaction in which a portion of a polypeptide molecule links with another portion of the polypeptide molecule to form a closed ring, or a portion of a polypeptide molecule links with several other portions of the polypeptide molecule to form multiple closed rings, such as by forming a disulfide bridge or other similar bond, or by linker linkage.

[0331] "Derivative" or "analyte" refers to a product derived from a compound by replacing a hydrogen atom or group of atoms with another atom or group of atoms. It should be understood that amino acid analogs of peptide compounds as defined herein are within the scope of this invention. Examples of such suitable modified amino acid derivatives include one or more modifications selected from: N-terminal and / or C-terminal modifications; replacement of one or more amino acid residues with one or more non-natural amino acid residues (e.g., replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of a spacer group; replacement of one or more oxidatively resistant amino acid residues with one or more oxidatively resistant amino acid residues; replacement of one or more amino acid residues with alanine, or replacement of one or more L amino acid residues with one or more D amino acid residues; N-alkylation of one or more amide bonds in a bicyclic peptide ligand; replacement of surrogate bonds. Replace one or more peptide bonds with another chemical group; modify the length of the peptide backbone; replace or substitute the hydrogen on the α carbon of one or more amino acid residues with another chemical group; modify amino acids such as glycine, alanine, phenylalanine, cysteine, lysine, glutamic acid / aspartic acid, and tyrosine with suitable amine, thiol, carboxylic acid, and phenolic reactive reagents to functionalize the amino acids; and introduce or replace amino acids to introduce orthogonal reactivity suitable for functionalization, for example, amino acids carrying azide or alkyne groups are respectively allowed to be functionalized with the alkyne or azide-carrying portions.

[0332] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly or conventionally understood by one of ordinary skill in the art. In chemistry, dashes before or at the end of chemical groups are for convenience; chemical groups may or may not be described with one or more dashes without losing their ordinary meaning. Wavy lines drawn through lines in a structure indicate the attachment point of a group. Dashed lines indicate optional bonds. Unless chemically or structurally required, the order in which chemical groups are written or the connection point of a chemical group to the rest of the molecule does not indicate or imply directionality. For example, the group “-SO2CH2-” is equivalent to “-CH2SO2-” and can be attached in either direction. Similarly, the “arylalkyl” group may, for example, be attached to the rest of the molecule at the aryl or alkyl portion of the group. “Cu-v” or (Cu-Cv) indicates that the following groups have u to v carbon atoms. For example, “C…” 1-6 Both "alkyl" and "C1-C6 alkyl" indicate that an alkyl group has 1 to 6 carbon atoms.

[0333] Throughout this specification, unless naturally occurring amino acids are referred to by their full names (e.g., alanine, arginine, etc.), they are designated by conventional three-letter or one-letter abbreviations (e.g., Ala or A for alanine; Arg or R for arginine, etc.). Unless otherwise stated, the three-letter and one-letter abbreviations for amino acids refer to the L-isomers of the amino acid under discussion. The term "L-amino acid" as used herein refers to the "L" isomer of the peptide, and conversely, the term "D-amino acid" refers to the "D" isomer of the peptide (e.g., Dasp, (D)Asp, or D-Asp; Dphe, (D)Phe, or D-Phe). The amino acid residues in the D isomers may substitute for any L-amino acid residue, provided that the peptide retains the desired function. When referred to using one-letter abbreviations, D-amino acids may be conventionally represented by lowercase letters.

[0334] In the case of uncommon or non-naturally occurring amino acids, unless they are mentioned by their full names (e.g., sarcosine, ornithine, etc.), their residues are usually represented by common three- or four-character codes, including Sar or Sarc (sarcosine, i.e., N-methylglycine), Aib (α-aminoisobutyric acid), Dab (2,4-diaminobutyric acid), Dapa (2,3-diaminopropionic acid), γ-Glu (γ-glutamic acid), Gaba (γ-aminobutyric acid), β-Pro (pyrrolidine-3-carboxylic acid), 8Ado (8-amino-3,6-dioxanoic acid), Abu (2-aminobutyric acid), βhPro (β-homoproline), βhPhe (β-homophenylalanine), and Bip (β,β-diphenylalanine), as well as Ida (iminodiacetic acid).

[0335] Unless otherwise specified, all amino acids are used in the L-configuration.

[0336] The following table lists some common amino acid names and their three-letter and single-letter abbreviations:

[0337] The term "pharmaceutically acceptable" refers to a substance that, within the bounds of normal medical judgment, is suitable for contact with a patient's tissues without causing undue toxicity, irritation, allergic reactions, etc., has a reasonable benefit-risk ratio, and is effective for its intended use.

[0338] The term "pharmaceutically acceptable salt" refers to a salt of the compounds of the present invention that is safe and effective when used in mammals and has the intended biological activity.

[0339] The term "pharmaceutical composition" refers to a composition containing one or more compounds described in this invention, or their physiologically / pharmaceutically acceptable salts or prodrugs, as well as other components such as physiologically / pharmaceutically acceptable carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and enabling it to exert its biological activity.

[0340] The term "pharmaceutically acceptable carrier" refers to substances that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. "Pharmaceutically acceptable carriers" include, but are not limited to, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavoring agents, surfactants, wetting agents, dispersants, disintegrants, stabilizers, solvents, or emulsifiers.

[0341] The terms "administration" or "giving" refer to methods that enable the delivery of a compound or composition to a desired biological site of action. These methods include, but are not limited to, oral or parenteral administration (including intraventricular, intravenous, subcutaneous, intraperitoneal, intramuscular, and intravascular injection or infusion), local administration, and rectal administration. In particular, injection or oral administration.

[0342] As used herein, the term "treatment" includes relieving, reducing, or improving a disease or symptom; preventing other symptoms; improving or preventing underlying metabolic factors of symptoms; inhibiting a disease or symptom, for example, preventing the development of a disease or symptom; reducing a disease or symptom; promoting the remission of a disease or symptom; or causing the symptom of a disease or symptom to cease; and extends to include prevention. "Treatment" also includes achieving therapeutic and / or preventive benefits. A therapeutic benefit refers to the eradication or improvement of the condition being treated. Furthermore, a therapeutic benefit is achieved by eradicating or improving one or more physical symptoms associated with an underlying disease, and an improvement in the patient's condition can be observed even though the patient may still have the underlying disease. A preventive benefit refers to the use of a composition by a patient to prevent the risk of a certain disease, or the use by a patient when experiencing one or more physical symptoms of a disease, even though the disease has not yet been diagnosed.

[0343] The terms "active ingredient," "therapeutic agent," "active substance," or "active agent" refer to a chemical entity that can effectively treat or prevent a target disorder, disease, or symptom. The term "neuropsychiatric disorders" is a collective term for neurological and psychiatric disorders, encompassing both neurological and / or psychiatric conditions.

[0344] For the purposes of pharmaceuticals, pharmaceutical units, or active ingredients, the terms "effective amount," "therapeutic effective amount," or "preventive effective amount" refer to a sufficient quantity of a drug or agent that provides acceptable side effects while achieving the desired therapeutic effect. The determination of the effective amount varies from person to person, depending on the individual's age and general condition, as well as the specific active substance. The appropriate effective amount in a given case can be determined by a person skilled in the art based on routine testing.

[0345] As used herein, “individual” includes both human and non-human animals. Exemplary human individuals include human individuals suffering from a disease (such as the disease described herein) (referred to as patients) or normal individuals. In this invention, “non-human animals” includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock, and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0346] The term "room temperature" refers to a temperature ranging from 10°C to 40°C. In some embodiments, "room temperature" refers to a temperature ranging from 15°C to 30°C; in other embodiments, "room temperature" refers to a temperature ranging from 18°C ​​to 25°C.

[0347] "Equivalent" or its abbreviation "eq" is the equivalent amount of other raw materials required based on the equivalence relationship of a chemical reaction, using the basic raw materials used in each step as a reference (1 equivalent).

[0348] In the examples, the asterisk (*) in the chemical structure indicates a single configuration R or S.

[0349] The following detailed description of the invention is intended to illustrate non-limiting embodiments, enabling other skilled in the art to more fully understand the technical solutions, principles, and practical applications of the invention, so that other skilled in the art can modify and implement the invention in many forms to best suit the requirements of a particular application. Attached Figure Description

[0350] Figure 1 shows the changes in right ear thickness in each group.

[0351] Figure 2 shows the right ear thickness of each group 4 days after administration. Detailed Implementation

[0352] The present invention will be described in detail below through embodiments. Unless otherwise specified, experimental methods under conventional conditions were used in the embodiments. The embodiments are provided to better illustrate the present invention, but should not be construed as limiting the invention to the examples given. Non-essential improvements and adjustments made to the implementation schemes by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0353] Detection method: The structure of the compound was determined by mass spectrometry (MS).

[0354] MS determination was performed using (Agilent 6120B (ESI) and Agilent 6120B (APCI));

[0355] HPLC determinations were performed using an Agilent 1260DAD high-performance liquid chromatograph (Zorbax SB-C18 100×4.6mm, 3.5μM).

[0356] Abbreviations: DCM: dichloromethane; DMF: N,N-dimethylformamide; DIEA: N,N-diisopropylethylamine; MeOH: methanol; TFA: trifluoroacetic acid; DMSO: dimethyl sulfoxide; DIC: N,N'-diisopropylcarbodiimide; HOBT: 1-hydroxybenzotriazole; HOAT: N-hydroxy-7-azabenzotriazole.

[0357] Unless otherwise specified in the examples, standard conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products. Unless otherwise specified, all proportions or percentages used herein are by weight.

[0358] Intermediate 1:

[0359] Step 1: Compound 1a (10.0 g, 73.43 mmol) was dissolved in dichloromethane (100 mL), and TiCl4 (25.5 g, 134.38 mmol) was added dropwise to the above reaction solution. The mixture was cooled to 0 °C. After 5 min, 1,1-dichlorodimethyl ether (9.4 g, 81.51 mmol) was added dropwise, and the mixture was stirred at 0 °C for 3 h. After the reaction was completed, 200 mL of water was added to the reaction system, and the resulting solution was extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to column chromatography (EA:PE = 1:4) to obtain compound 1b (5.58 g, 46.3%).

[0360] LC-MS (ESI): m / z = 163.2 [M+H] + .

[0361] Step 2: Compound 1b (3.0 g, 18.27 mmol), tert-butyl (2-bromoethyl)carbamate (4.9 g, 21.91 mmol), potassium carbonate (5.1 g, 36.54 mmol), and sodium iodide (0.8 g, 5.47 mmol) were dissolved in N,N-dimethylformamide (20 mL) and stirred at 25 °C for 16 h. After the reaction was complete, 100 mL of water was added, and the reaction solution was extracted three times with ethyl acetate (50 mL × 3). The solution was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to column chromatography (EA:PE = 4:1) to obtain compound 1c (4.9 g, 87%).

[0362] LC-MS (ESI): m / z = 306.1 [M+H] + .

[0363] Step 3: Trimethyl benzyloxycarbonyl-α-phosphonoglycine (6.2 g, 18.74 mmol) was dissolved in dichloromethane (100 mL), purged with nitrogen, and DBU (3.1 g, 20.31 mmol) was added and stirred for 30 min. Then, compound 1c (4.8 g, 15.62 mmol) dissolved in dichloromethane (100 mL) was added dropwise to the reaction mixture, and the mixture was stirred overnight. The reaction mixture was diluted with dichloromethane (100 mL) and washed with 5% citric acid aqueous solution (100 mL) and saturated brine (100 mL), then dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. Compound 1d (6.1 g, 76.2%) was purified by silica gel column chromatography (EA:PE = 3:1).

[0364] LC-MS(ESI): m / z = 511.1 [M+H] + .

[0365] Step 4: Compound 1d (6.0 g, 11.71 mmol) was dissolved in a mixed solution of methanol (50 mL) and dichloromethane (20 mL), and (+)-1,2-bis(2S,5S)-2,5-diethylcyclobutanphosphine (cyclooctadiene) rhodium trifluoromethanesulfonate (0.6 g, 0.83 mmol) was added. The autoclave was filled with hydrogen gas to a pressure of about 4.0 bar, stirred at room temperature for 3 h, the solid was filtered off, and the reaction solution was concentrated to obtain compound 1e (5.9 g, 98%).

[0366] LC-MS (ESI): m / z = 513.2 [M+H] + .

[0367] Step 5: Dissolve compound 1e (5.9 g, 11.46 mmol) in a mixed solution of methanol (50 mL) and dichloromethane (20 mL), add palladium on carbon (10%, 1 g), stir overnight under hydrogen atmosphere, then filter off the solid, and evaporate the reaction solution to dryness to obtain compound 1f (3.7 g, 84.5%).

[0368] LC-MS (ESI): m / z = 379.4 [M+H] + .

[0369] Step 6: Compound 1f (3.7g, 9.72mmol) was dissolved in tetrahydrofuran (25mL), and lithium hydroxide monohydrate (1.2g, 48.6mmol) and water (25mL) were added. The reaction was stirred overnight at room temperature. Impurities were extracted with ethyl acetate (15mL x 3). The pH of the aqueous phase was then adjusted to neutral with dilute hydrochloric acid (1N). The product precipitated, filtered, and dried to give compound 1g (3.2g, 89.8%).

[0370] LC-MS (ESI): m / z = 365.2 [M+H] + .

[0371] Step 7: Dissolve 1 g (2.7 g, 7.41 mmol) of the compound in a mixed solution of acetonitrile (25 mL) and water (25 mL), add sodium bicarbonate (6.2 g, 74.16 mmol) and 9-fluorenylmethyl-N-succinimide carbonate (3.3 g, 9.63 mmol), and stir the reaction overnight at room temperature. After the reaction is complete, add dilute hydrochloric acid to adjust the pH to neutral. Concentrate under vacuum at 40 °C to remove most of the acetonitrile, filter and collect the solid to obtain the crude product, and separate and purify it using silica gel column chromatography (DCM:MeOH = 10:1) to obtain intermediate 1 (2.7 g, 62.1%).

[0372] LC-MS (ESI): m / z = 587.2 [M+H] + .

[0373] 1 H NMR(400MHz,DMSO-d6)δ12.65(s,1H),7.88(d,2H),7.67(s,2H),7.41(d,2H),7.36-7.24(m,2H),7.03-6.88(m,2H),6.65(d ,1H),4.26-4.09(m,4H),3.91(s,2H),3.28(s,2H),2.99(d,1H),2.90-2.68(m,5H),2.51(s,1H),1.98(s,2H),1.39(s,9H).

[0374] Intermediate 2:

[0375] Step 1: Compound 2a (5 g, 18.36 mmol) was dissolved in tetrahydrofuran (50 mL), and borane dimethyl sulfide solution (3.67 mL, 36.71 mmol, 10 mol / L) was added dropwise at 0 °C. The reaction was stirred at room temperature for 16 h. The reaction was monitored by LCMS until it was complete. Methanol (20 mL) was added to quench the reaction, and the mixture was concentrated under reduced pressure and purified by silica gel column chromatography (MeOH:DCM = 0-10:1) to obtain compound 2b (4.5 g, 94.9%).

[0376] LC-MS (ESI): m / z = 259.2 [M+H] + .

[0377] Step 2: Compounds 2b (3 g, 11.61 mmol) and 2c (2.74 g, 11.61 mmol) were added to dichloromethane (50 mL), followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.67 g, 13.93 mmol). 4-Dimethylaminopyridine (709 mg, 5.8 mmol) was added at 0 °C. The reaction was stirred at room temperature for 16 h. The reaction was monitored by TLC until it was complete. Water (100 mL) and dichloromethane (20 mL) were added. The organic phase was dried and concentrated, and then purified by silica gel column chromatography (MeOH:DCM = 0–10%) to obtain compound 2d (3.5 g, 81.3%).

[0378] LC-MS (ESI): m / z = 477.2 [M+H] + .

[0379] Step 3: Compound 2d (3.5 g, 7.34 mmol) was dissolved in methanol (35 mL), palladium on carbon (350 mg, 10%) was added, and the mixture was stirred for 4 h under hydrogen atmosphere. The reaction was monitored by LCMS until it was complete. After filtering out the palladium on carbon, the mixture was concentrated and purified by silica gel column chromatography (MeOH:DCM = 0-10%) to obtain intermediate 2 (2.8 g, 98.7%).

[0380] LC-MS (ESI): m / z = 387.2 [M+H] + .

[0381] 1 H NMR (400MHz, Chloroform-d) δ4.06(t,2H),2.42-2.29(m,4H),2.20(t,2H),1.73-1.65(m,4H),1.65-1.52(m,4H),1.44(s,9H),1.37-1.24(m,12H).

[0382] Intermediate 3:

[0383] Step 1: A tetrahydrofuran solution of 3a (45 g, 121 mmol) was cooled in an ice bath, and a boron dimethyl sulfide complex (16 mL, 158 mmol) was added dropwise under nitrogen. After addition, the mixture was slowly brought to room temperature and stirred overnight. The reaction was cooled in an ice bath, quenched with saturated sodium carbonate solution (100 mL), and extracted with ethyl acetate (100 mL × 3). The organic layer was concentrated under vacuum, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate (v / v) = 5:1) to give compound 3b (32 g, 74%).

[0384] LC-MS (ESI): m / z = 357.2 [M+H] + .

[0385] Step 2: Dissolve 3b (5.0 g, 14.04 mmol), (S)-2-((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-benzyloxy-5-oxovaleric acid (6.5 g, 14.04 mmol), and 4-(N,N-dimethylaminopyridine) in dichloromethane (100 mL). Add 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride (4.0 g, 21.06 mmol) at 0 °C and allow to rise naturally to room temperature for 16 h. After the reaction is complete, pour the reaction solution into water (50 mL), extract and separate (50 mL × 3), concentrate the organic phase under reduced pressure, and then purify by silica gel column chromatography (EA / PE = 0-30%) to obtain 3c (5.3 g, 47.3%).

[0386] LC-MS (ESI): m / z = 799.2 [M+H] +

[0387] Step 3: Dissolve 3c (5.3g, 6.64mmol) in methanol, add 10% palladium on carbon (1g), react at room temperature for two hours, filter palladium on carbon, wash three times with methanol, and evaporate the solvent to obtain intermediate 3 (4.6g, 99%).

[0388] LC-MS (ESI): m / z = 708.2 [M+H] +

[0389] 1 HNMR(400MHz,DMSO-d6)δ12.19-12.05(m,1H),7.92-7.84(m,2H),7.80-7. 67(m,3H),7.44-7.36(m,2H),7.35-7.29(m,2H),4.33-4.20(m,3H),4.09- 3.98(m,3H),2.34-2.28(m,2H),2.18-2.12(m,2H),2.02-1.91(m,1H),1.8 6-1.74(m,1H),1.58-1.42(m,5H),1.40-1.37(m,10H),1.28-1.17(m,24H).

[0390] Intermediate 4:

[0391] Step 1: A dichloromethane solution (50 mL) of intermediate 1 (35.0 g, 71.93 mmol) was added to a mixed solution of dichloromethane (100 mL) and trifluoroacetic acid (50 mL), and stirred at room temperature for 1 h. After the reaction was completed, the solution was concentrated under reduced pressure to obtain crude intermediate 4a (35.83 g, 100%).

[0392] LC-MS (ESI): m / z = 487.2 [M+H] + .

[0393] Step 2: Intermediate 4a (26.8 g, 55.08 mmol) and 2-acetyl-5,5-dimethyl-1,3-cyclohexanedione (40.2 g, 220.32 mmol) were added to a mixed solution of tetrahydrofuran (210 mL) and glacial acetic acid (42 mL), and stirred at 95 °C for 16 h. After the reaction was completed, the solution was concentrated under reduced pressure and purified by reverse-phase column chromatography (ACN:H2O = 3:2) to obtain intermediate 4 (11.2 g, 45.9%).

[0394] LC-MS (ESI): m / z = 651.3 [M+H] + .

[0395] 1 HNMR(400MHz,DMSO-d6)δ13.46-13.43(m,1H),12.64(s,1H),7.88-7.86(d,2H ),7.68-7.64(m,3H),7.43-7.38(m,2H),7.33-7.27(m,2H),7.01-6.99(m,1H) ,6.69-6.67(d,1H),4.22-4.20(m,2H),4.09-4.03(m,4H),3.82-3.80(m,4H), 3.02-2.97(m,1H),2.84-2.79(m,5H),2.53(s,3H),2.27(s,4H),0.94(s,6H).

[0396] Intermediate 5:

[0397] Step 1: Using compound 5a (10 g, 37.98 mmol) and N-(9-fluorenylmethoxycarbonyl)-L-serine tert-butyl ester (14.56 g, 37.98 mmol) as starting materials, compound 5b (9.9 g, 41.5%) was obtained by referring to the procedure in step 2 of intermediate 2.

[0398] LC-MS (ESI): m / z = 529.2 [M+H] + .

[0399] Step 2: Compound 5b (9.9 g, 15.75 mmol) was dissolved in dichloromethane (100 mL), piperidine (25 mL) was added, and the mixture was stirred at room temperature for 1 h. After dilution with dichloromethane (100 mL), the organic phase was washed with water, dried and concentrated, and purified by silica gel column chromatography (MeOH:DCM = 0-10%) to obtain compound 5c (4.6 g, 71.9%).

[0400] LC-MS (ESI): m / z = 307.2 [M+H] + .

[0401] Step 3: Compound 5c (4.6 g, 11.32 mmol) was dissolved in dichloromethane (50 mL), and triethylamine (3.46 g, 33.95 mmol) and acetic anhydride (2.31 g, 22.63 mmol) were added sequentially. The reaction was stirred at room temperature for 16 h, diluted with dichloromethane (50 mL), washed with water, dried and concentrated the organic phase, and purified by silica gel column chromatography (EtOAc:PE = 0-1:1) to obtain compound 5d (4.6 g, 90.6%).

[0402] LC-MS (ESI): m / z = 349.2 [M+H] + .

[0403] Step 4: Add 4 mL of trifluoroacetic acid to 20 mL of dichloromethane containing compound 5d (2 g, 4.46 mmol), stir at room temperature for 16 h, and directly concentrate to obtain the trifluoroacetate of compound 5e. It can be used directly in the next step of the reaction without purification.

[0404] LC-MS (ESI): m / z = 293.1 [M+H] + .

[0405] Step 5: Dissolve the trifluoroacetate of compound 5e in a mixed solution of water (20 mL) and acetonitrile (20 mL). Slowly add sodium bicarbonate until the pH reaches 7. Then add sodium bicarbonate (2.23 g, 26.5 mmol) and Fmoc-Osu (2.15 g, 6.36 mmol). Stir at room temperature for 2 h. Monitor the reaction for completion by LCMS. Add water (20 mL) and ethyl acetate (20 mL). After separation, retain the aqueous phase. Adjust the pH of the aqueous phase to about 6 using 1 M hydrochloric acid. Extract with ethyl acetate (20 mL × 3). Combine the organic phases, dry to anhydrous sodium sulfate, concentrate under reduced pressure, and then separate and purify using silica gel column chromatography (MeOH:DCM = 0–10%) to obtain intermediate 5 (1.7 g, two-step yield 74.1%).

[0406] LC-MS (ESI): m / z = 515.2 [M+H] + .

[0407] 1 H NMR(400MHz,Chloroform-d)δ7.74(d,2H),7.64-7.50(m,4H),7.38(t,2H),7.29(t,2H),5.68-5.58(m,1H), 4.93-4.82(m,1H),4.53-4.32(m,4H),4.26-3.92(m,3H),3.74-3.49(m,5H),3.44-3.32(m,2H),1.99(s,3H).

[0408] Intermediate 6:

[0409] Step 1: Add 6a (5 mmol, 5 g, sub: 1 mmol / g) and dichloromethane to the reactor. After swelling the resin for 5 min, add Fmoc-AEEA-OH (7.5 mmol, 2.9 g) and N,N-diisopropylethylamine (15 mmol, 2.6 ml) sequentially. React at room temperature for 1 h. Then add methanol (5 ml) and react for 30 min. Drain the solvent and wash the resin three times with DMF to obtain 6b.

[0410] Step 2: Add 20% piperidine / DMF to 6b, react for 30 minutes, then remove the solvent and wash the resin 5 times with DMF to obtain 6c. Monitor the reaction with ninhydrin.

[0411] Step 3: Add Fmoc-Glu-OtBu (10 mmol, 4.25 g), DMF (20 ml), 1-hydroxybenzotriazole (10 mmol, 1.35 g), and N,N'-diisopropylcarbodiimide (10 mmol, 1.57 ml) sequentially to 6c. React at room temperature for 1.5 h. After the reaction is complete as monitored by ninhydrin, the solvent is removed and the resin is washed three times with DMF to obtain 6d.

[0412] Step 4: Add 20% piperidine / DMF to 6d, react for 30 minutes, then remove the solvent and wash the resin 5 times with DMF to obtain 6e. Monitor the reaction with ninhydrin.

[0413] Step 5: Add 10 mmol, 3.7 g, 20 ml, 1-hydroxybenzotriazole (10 mmol, 1.35 g), and N,N'-diisopropylcarbodiimide (10 mmol, 1.57 ml) sequentially to 6e. React at room temperature for 1 h. After the reaction is complete as monitored by ninhydrin, the solvent is removed and the resin is washed 3 times with DMF, 2 times with methanol, 1 time with dichloromethane, and 2 times with methanol. The mixture is then dried to obtain 6f.

[0414] Step 6: Add 70 ml of 30% trifluoroethanol / dichloromethane solution to 6f (7.3 g), react at room temperature for 1 h, filter, collect the filtrate and concentrate under reduced pressure to obtain intermediate 6 (2.6 g, yield 74%).

[0415] 1HNMR(400MHz,DMSO-d6)δ12.42(s,1H),8.03-8.01(m,1H),7.88-7.85(m, 1H),4.07-4.02(m,1H),4.00(s,2H),3.59-3.57(m,2H),3.53-3.50(m,2H ),3.41-3.38(m,2H),3.21-3.16(m,2H),2.17-2.07(m,6H),1.93-1.84(m ,1H),1.78-1.71(m,1H),1.48-1.43(m,4H),1.39(s,18H),1.23(s,24H).

[0416] Intermediate 7:

[0417] Step 1: Compound 7a (5 g, 18.99 mmol) and tert-butyl bromoacetate (4.4 g, 22.79 mmol) were dissolved in acetonitrile (100 mL), potassium carbonate (7.86 g, 56.97 mmol) and sodium iodide (361 mg, 1.9 mmol) were added, and the mixture was stirred at 60 °C for 16 h. After the reaction was completed, water (300 mL) was added, and the mixture was extracted with ethyl acetate (50 mL × 3). The extract was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (EtOAc:PE = 0–1:1) to obtain compound 7b (7 g, 97.7%).

[0418] LC-MS (ESI): m / z = 278.2 [M+H] + .

[0419] Step 2: Add 10 mL of trifluoroacetic acid to dichloromethane (4 g, 10.6 mmol) of compound 7b and stir at room temperature for 16 h. Directly concentrate to obtain trifluoroacetate of compound 7c, which can be used directly in the next step of the reaction without purification.

[0420] LC-MS(ESI): m / z = 222.1 [M+H] + .

[0421] Step 3: Dissolve the trifluoroacetate of compound 7c in a mixed solution of water (40 mL) and acetonitrile (40 mL). Slowly add sodium bicarbonate until the pH reaches 7. Then add sodium bicarbonate (4.45 g, 52.99 mmol) and Fmoc-Osu (4.29 g, 12.72 mmol). Stir at room temperature for 2 h. Monitor the reaction for completion by LCMS. Add water (40 mL) and ethyl acetate (40 mL). After separation, retain the aqueous phase. Adjust the pH of the aqueous phase to about 6 using 1 M hydrochloric acid. Extract with ethyl acetate (40 mL × 3). Combine the organic phases, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and then separate and purify using silica gel column chromatography (MeOH:DCM = 0–10%) to obtain intermediate 7 (4 g, two-step yield 85.1%).

[0422] LC-MS (ESI): m / z = 444.2 [M+H] + .

[0423] 1 H NMR(400MHz,Chloroform-d)δ7.75(d,2H),7.63-7.53(m,2H),7.38(t,2H),7.34-7.27(m,2H),5.46(s,1H ),4.68(s,2H),4.39(d,2H),4.29-4.16(m,3H),3.79-3.70(m,2H),3.69-3.60(m,2H),3.60-3.31(m,4H).

[0424] Intermediate 8:

[0425] Step 1: Using compound 8a (5 g, 12.97 mmol) and tert-butyl 3-hydroxypropionate (1.9 g, 12.97 mmol) as starting materials, compound 8b (4.5 g, 67.5%) was obtained by referring to the procedure in step 2 of intermediate 2.

[0426] LC-MS (ESI): m / z = 514.2 [M+H] + .

[0427] Step 2: Using compound 8b (4.5g, 8.76mmol) as the starting material, the crude product was obtained by referring to the operation method in step 4 of intermediate 2, and then purified by reverse column to obtain intermediate 8 (3g, 74.8%).

[0428] LC-MS (ESI): m / z = 458.1 [M+H] + .

[0429] 1H NMR(400MHz,DMSO-d6)δ12.39(s,1H),7.89(d,2H),7.69(d,2H),7.41(t,2H),7.33(t,2H),4.41- 4.17(m,5H),4.10(s,2H),3.64-3.47(m,4H),3.46-3.37(m,2H),3.19-3.09(m,2H),2.58(t,2H).

[0430] Intermediate 9:

[0431] Step 1: Using compound 9a (11.7 g, 47 mmol) and fluorenemethyloxycarbonyl-L-glutamic acid 1-tert-butyl ester (20 g, 47.0 mmol) as starting materials, compound 9b (25 g, 80.98%) was obtained by referring to the procedure in step 2 of intermediate 2.

[0432] LC-MS (ESI): m / z = 657.2 [M+H] + .

[0433] Step 2: Add trifluoroacetic acid (40 mL) to dichloromethane (200 mL) containing compound 9b (20 g, 30.45 g), stir at room temperature for 16 h, concentrate and then purify by reverse reaction to obtain compound 9c (13.5 g, 88.66%).

[0434] LC-MS (ESI): m / z = 501.2 [M+H] + .

[0435] Step 3: Compound 9c (5g, 9.99mmol) was dissolved in ethanol (50mL), and 2-acetyl-5,5-dimethyl-1,3-cyclohexanedione (4.46g, 29.97mmol) and trifluoroacetic acid (114mg, 1.0mmol) were added. The mixture was stirred at 90℃ for 4h. After the reaction was completed by TLC monitoring, the mixture was concentrated and purified by silica gel column chromatography (MeOH:DCM = 0-10%) to obtain intermediate 9 (4.8g, 72.3%).

[0436] LC-MS (ESI): m / z = 665.2 [M+H] + .

[0437] 1H NMR(400MHz,Chloroform-d)δ13.34(s,1H),7.76(d,2H),7.60(d,2H),7.43-7.36(m,2H),7.34-7.28(m,2H),5.69(d,1H), 4.52-4.35(m,3H),4.28-4.17(m,3H),3.76-3.56(m,10H),2.57(s,3H),2.53-2.37(m,6H),2.34-2.09(m,2H),1.03(s,6H).

[0438] Intermediate 10:

[0439] Step 1: Add 10a (5 mmol, 5 g, sub: 1 mmol / g) and dichloromethane to the reactor. After swelling the resin for 5 min, add Fmoc-AEEA-OL (7.5 mmol, 2.8 g) and N,N-diisopropylethylamine (15 mmol, 2.6 ml) in sequence. React at room temperature for 1 h. Then add methanol (5 ml) and react for 30 min. Drain the solvent and wash the resin three times with DMF to obtain 10b.

[0440] Step 2: Add 20% piperidine / DMF to 10b, react for 30 minutes, then remove the solvent and wash the resin 5 times with DMF to obtain 10c. Monitor the reaction with ninhydrin.

[0441] Step 3: Add Fmoc-AEEA-OH (10 mmol, 3.7 g), DMF (20 ml), 1-hydroxybenzotriazole (10 mmol, 1.35 g), and N,N'-diisopropylcarbodiimide (10 mmol, 1.57 ml) sequentially to 10c. React at room temperature for 1.5 h. After the reaction is complete as monitored by ninhydrin, the solvent is removed and the resin is washed three times with DMF to obtain 10d.

[0442] Step 4: Add 20% piperidine / DMF to 10d, react for 30 minutes, then remove the solvent and wash the resin 5 times with DMF to obtain 10e. Monitor the reaction with ninhydrin.

[0443] Step 5: Add Fmoc-Glu-OtBu (10 mmol, 4.25 g), DMF (20 ml), 1-hydroxybenzotriazole (10 mmol, 1.35 g), and N,N'-diisopropylcarbodiimide (10 mmol, 1.57 ml) sequentially to 10e. React at room temperature for 1.5 h. After the reaction is complete as monitored by ninhydrin, remove the solvent and wash the resin three times with DMF to obtain 10f.

[0444] Step 6: Add 20% piperidine / DMF to 10f, react for 30 minutes, then remove the solvent and wash the resin 5 times with DMF to obtain 10g. Monitor the reaction with ninhydrin.

[0445] Step 7: Add 10g of octadecanoic acid monotert-butyl ester (10mmol, 3.7g), DMF (20ml), 1-hydroxybenzotriazole (10mmol, 1.35g), and N,N'-diisopropylcarbodiimide (10mmol, 1.57ml) sequentially to 10g of the solution. React at room temperature for 1h. After the reaction is complete as monitored by ninhydrin, the solvent is removed and the resin is washed 3 times with DMF, 2 times with methanol, 1 time with dichloromethane, and 2 times with methanol. The solution is then removed and dried to obtain 10h.

[0446] Step 8: Add 30% trifluoroethanol / dichloromethane solution (70 ml) to 10 h (7.1 g), react at room temperature for 1 h, filter, collect the filtrate and concentrate under reduced pressure to obtain intermediate 10 (2.5 g, yield 60%).

[0447] 1 HNMR(400MHz,DMSO-d6)δ8.07-8.04(m,2H),7.93-7.89(m,1H),6.10(s,1H),4 .21-4.19(m,1H),4.14(s,1H),4.07-4.03(m,1H),4.01(s,1H),3.90-3.83(m, 1H),3.64-3.58(m,4H),3.57(s,3H),3.54-3.51(m,2H),3.41-3.35(m,6H),3. 21-3.16(m,2H),2.97-2.93(m,1H),2.17-2.07(m,6H),1.93-1.84(m,1H),1.79 -1.69(m,1H),1.48-1.45(m,4H),1.39(s,18H),1.23(s,24H).

[0448] Intermediate 11:

[0449] Step 1: Compound 11a (5 g, 20.06 mmol) and triethylamine (8.1 g, 80.22 mmol) were added to ultra-dry dichloromethane (50 mL). POCl3 (3.69 g, 24.07 mmol) was added dropwise at -78 °C. After stirring for 1 h, the reaction was detected by TLC to indicate that the starting material had been consumed. A dichloromethane solution (50 mL) of compound 11b (6.63 g, 50.14 mmol) was added dropwise. The mixture was slowly heated to room temperature and stirred for 16 h. After the reaction was complete, water (200 mL) was added. The mixture was extracted three times with dichloromethane (100 mL). The organic phases were combined and concentrated. The mixture was then purified by silica gel column chromatography (EA:PE = 0–100%) to obtain compound 11c (6.5 g, 58%).

[0450] LC-MS (ESI): m / z = 458.1 [M+H] + .

[0451] Step 2: Compound 11c (6.5 g, 11.66 mmol) was dissolved in acetone (80 mL), and lithium bromide (1.2 g, 13.99 mmol) was added. The mixture was stirred at 50 °C for 16 h. The reaction solution was then concentrated and purified by silica gel chromatography (MeOH:DCM = 0–10%) to obtain compound 11d (4.9 g, 96.9%).

[0452] LC-MS (ESI): m / z = 344.2 [M+H] + .

[0453] Step 3: Using compound 11d (4.9g, 11.05mmol) as the starting material, and following the procedure in step 4 of intermediate 4, we obtained compound 11e (crude product, which was directly added to the next step).

[0454] Step 4: Using compound 11e (crude product) as raw material, intermediate 11 (1g, two-step yield 17.36%) was obtained by referring to the operation method of step 5 of intermediate 4.

[0455] LC-MS (ESI): m / z = 510.2 [M+H] + .

[0456] 1 H NMR(400MHz,Chloroform-d)δ7.72(d,2H),7.58-7.50(m,2H),7.39-7.32(m,2H),7.31-7.26(m,3H ),4.63-4.50(m,2H),4.48-4.30(m,2H),4.24-4.06(m,3H),3.69-3.45(m,8H),3.45-3.17(m,3H).

[0457] Intermediate 12:

[0458] Step 1: To a solution of 12a (3.2 g, 31.83 mmol) and benzyl alcohol (4.1 g, 38.19 mmol) in dichloromethane (10 mL), 4-dimethylaminopyridine (0.78 g, 6.39 mmol) was added, and the solution was stirred at 0 °C. Then, N,N'-dicyclohexylcarbodiimide (9.9 g, 47.99 mmol) was added, and the mixture was stirred overnight at room temperature. The precipitated insoluble matter was removed by filtration, and the filtrate was concentrated and purified by silica gel column chromatography (0→70% EA / PE) to give compound 12b (4.5 g, 80%).

[0459] LC-MS(ESI): m / z = 209.2 [M+H] +

[0460] Step 2: 12b (4.5 g, 21.6 mmol), 1-(9H-fluorene-9-yl)-3-oxo-2,7,10-trioxa-4-azadodecane-12-acid (8.4 g, 21.6 mmol), and 4-(N,N-dimethylaminopyridine) (1.3 g, 10.8 mmol) were dissolved in dichloromethane (100 mL). 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride (6.2 g, 32.4 mmol) was added at 0 °C, and the mixture was allowed to rise naturally to room temperature for 16 h. After the reaction was complete, the reaction solution was poured into water (50 mL), extracted, and separated (50 mL × 3). The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (EA / PE = 0-30%) to obtain 12c (3.5 g, 47.3%).

[0461] LC-MS (ESI): m / z = 576.2 [M+H] +

[0462] Step 3: Dissolve 12c (3.5g, 6.08mmol) in methanol, add 10% palladium on carbon (1g), react at room temperature for 1 hour, filter palladium on carbon, wash three times with methanol, and evaporate the solvent to obtain intermediate 12 (0.986g, 34%).

[0463] LC-MS (ESI): m / z = 486.2 [M+H] +

[0464] 1HNMR(400MHz,DMSO-d6)δ12.19-12.05(m,1H),7.89-7.87(m,2H),7.70-7.67( m,2H),7.43-7.41(m,2H),7.35-7.32(m,2H),6.29-6.25(s,1H),4.31-4.29(m, 1H),4.23-4.19(m,1H),3.57-3.56(m,1H),3.52-3.51(m,1H),3.43-3.41(m,2H ),3.17-3.11(m,2H),2.62-2.61(m,1H),1.82-1.79(m,1H),1.49-1.47(m,6H).

[0465] Intermediate 14:

[0466] Step 1: Compound 14a (10 g, 40.11 mmol) was added to dioxane (100 mL, 4 M) and stirred at room temperature for 2 h. LCMS detected that the reaction of the starting material was complete and the mixture was directly concentrated for the next step of the reaction (7.4 g, 99%).

[0467] LC-MS (ESI): m / z = 150.1 [M+H] + .

[0468] Step 2: Compound 14b (7.4 g, 39.86 mmol) was dissolved in ethanol (100 mL), and triethylamine (8.1 g, 79.72 mmol) and 2-acetyl-5,5-dimethyl-1,3-cyclohexanedione (8.72 g, 47.83 mmol) were added. The mixture was stirred at room temperature for 2 h, concentrated, and purified by silica gel column chromatography (EA:PE = 0–100%) to obtain compound 14c (10 g, 80.1%).

[0469] LC-MS (ESI): m / z = 314.2 [M+H] + .

[0470] Step 3: Under a nitrogen atmosphere, phosphorus trichloride (2 g, 14.56 mmol) was added to anhydrous tetrahydrofuran (20 mL), and the temperature was lowered to -5 °C. Compound 14d (5 g, 14.65 mmol) was dissolved in anhydrous tetrahydrofuran (30 mL), and pyridine (1.15 g, 14.65 mmol) was added. The prepared solution was slowly added dropwise to the above phosphorus trichloride tetrahydrofuran solution, and the solution temperature was controlled not to exceed 5 °C. After stirring continuously for 1 h, the raw materials were consumed as monitored by LCMS. 2,6-Dimethylpyridine (3.14 g, 29.29 mmol) and compound 14c (4.6 g, 14.65 mmol) were added sequentially, and stirring was continued for 2 h. After the reaction was completed, water (200 mL) was added, and the mixture was extracted three times with ethyl acetate (50 mL). The organic phases were combined and concentrated, and purified by silica gel column chromatography (MeOH:DCM = 0-10%) to obtain compound 14e (4 g, 39.0%).

[0471] LC-MS (ESI): m / z = 701.2 [M+H] + .

[0472] Step 4: Compound 14e (2g, 2.85mmol) was dissolved in a mixed solution of pyridine and water (22mL, v / v = 10:1), iodine (2.17g, 8.56mmol) was added and the mixture was stirred at room temperature for 1h. After the reaction was completed, a saturated sodium sulfite solution was added to quench the reaction. The pyridine was concentrated after filtration and purified by reverse-phase column chromatography to obtain intermediate 14 (500mg, 24.4%).

[0473] LC-MS (ESI): m / z = 717.2 [M+H] + .

[0474] Intermediate 18:

[0475] Step 1: Compound 18a (39.6 g, 114 mmol) was dissolved in N,N-dimethylformamide (200 mL). Sodium hydride (wt = 60%) (4.9 g, 123 mmol) was slowly added at 0 °C, and the mixture was stirred for 10 min. Tert-butyl bromoacetate (18.5 g, 95 mmol) was added to the reaction solution, and the mixture was stirred at 25 °C for 1 h. After the reaction was completed, ethyl acetate (500 mL × 3) was added for extraction. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 4:1) to obtain compound 18b (19.6 g, 44%).

[0476] 1H NMR (400MHz, CDCl3) δ7.46(dt,6H),7.31-7.26(m,6H),7.22-7.20(m,3H),4.04(s,2H),3.75-3.65(m,6H),3.25(t,2H),1.46(s,9H).

[0477] Step 2: Compound 18b (18.0 g, 39 mmol) was dissolved in tetrahydrofuran (200 mL), and lithium dimethylsilylamino (1.0 M solution in THF, 89.49 mmol) was slowly added at -78 °C. The mixture was stirred for 1 h, and methyl iodoform (16.6 g, 117 mmol) was added to the reaction solution and stirred for 16 h. After the reaction was complete, ethyl acetate (300 mL × 3) was added for extraction. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dry column chromatography for purification (petroleum ether / ethyl acetate = 4:1) to give 18c (18.2 g, 95%).

[0478] 1 H NMR (400MHz, CDCl3) δ7.46(dt,6H),7.31-7.25(m,6H),7.24-7.18(m,3H),3.68(dd,4H),3.58(dd,2H),3.23(t,2H),1.46(s,9H),1.39(s,6H).

[0479] Step 3: Compound 18c (8.8 g, 18 mmol) was dissolved in a mixed solution of methanol (44 mL) and tetrahydrofuran (44 mL) under nitrogen protection. p-Toluenesulfonic acid (336 mg, 1.8 mmol) was added, and the mixture was stirred at 30 °C for 2 h. The mixture was monitored by TLC. The solution was concentrated to dryness, and extracted with ethyl acetate (200 mL) and water (200 mL). The ethyl acetate phase was concentrated to dryness to give crude product 18d (4.3 g, 96%).

[0480] Step 4: Compound 18d (4.3 g, 17 mmol) was dissolved in dichloromethane (50 mL), triethylamine (8.7 g, 86 mmol) was added, and methanesulfonic anhydride (3.5 g, 20 mmol) was added dropwise at 0 °C. The mixture was stirred at room temperature for 2 h. The reaction was monitored by TLC (phosphomolybdic acid). The mixture was extracted with dichloromethane (100 mL) and water (100 mL), and the dichloromethane phase was concentrated to dryness. The solution was purified by column chromatography (petroleum ether / ethyl acetate = 4:1) to give 18e (3.5 g, 72%).

[0481] Step 5: Compound 18e (3.5 g, 13 mmol) was dissolved in DMF (35 mL), sodium azide (1.7 g, 26 mmol) was added, and the mixture was stirred at 80 °C for 18 h. The mixture was monitored by TLC (ninhydrin). Extraction was performed with ethyl acetate (100 mL) and water (100 mL), and the ethyl acetate phase was concentrated to dryness to give 18f (2.7 g, 78%).

[0482] Step 6: Compound 18f (2.7 g, 10 mmol) was dissolved in methanol (35 mL), and 10% palladium on carbon (270 mg) was added. The mixture was stirred at room temperature for 1 h. The mixture was monitored by TLC (ninhydrin was observed). The mixture was filtered, the filter cake was washed with methanol, and the filtrates were combined and concentrated to dryness to give 18 g (2.4 g, 96%).

[0483] LC-MS (ESI): m / z = 248.1 [M+H] +

[0484] Step 7: 18 g (2.4 g, 9.7 mmol) of the compound was dissolved in acetonitrile (24 mL) and water (24 mL), and Fmoc-Osu (3.2 g, 9.7 mmol) and sodium bicarbonate (5.1 g, 48.5 mmol) were added. The mixture was stirred at room temperature for 2 h. The reaction solution was slowly neutralized with 1 N hydrochloric acid, and extracted with ethyl acetate (100 mL). The ethyl acetate phase was concentrated to dryness and purified by column chromatography (petroleum ether / ethyl acetate = 4:1) to give 18 h (4.2 g, 92%).

[0485] LC-MS(ESI):m / z=414.1[M+H-56] +

[0486] Step 8: Compound 18h ​​(4.2 g, 8.9 mmol) was added to a mixed solution of dichloromethane (60 mL) and trifluoroacetic acid (20 mL) and stirred at room temperature for 2 h. The solution was concentrated to dryness and purified by column chromatography (dichloromethane / methanol = 10:1) to give intermediate 18 (3.0 g, 81%).

[0487] LC-MS (ESI): m / z = 414.2 [M+H] +

[0488] Intermediate 19:

[0489] Step 1: Add 6a (1 mmol, 1 g, sub: 1 mmol / g) and dichloromethane to the reactor. After swelling the resin for 5 min, add intermediate 18 (3 mmol, 1.2 g) and N,N-diisopropylethylamine (6 mmol, 1.0 ml) in sequence. React at room temperature for 1 h. Then add methanol (5 ml) and react for 30 min. Drain the solvent and wash the resin three times with DMF to obtain 19b.

[0490] Step 2: Add 20% piperidine / DMF to 19b, react for 30 minutes, then remove the solvent and wash the resin 5 times with DMF to obtain 19c. Monitor the reaction with ninhydrin.

[0491] Step 3: Add Fmoc-AEEA-OH (4 mmol, 1.5 g), DMF (20 ml), 1-hydroxybenzotriazole (4 mmol, 0.54 g), and N,N'-diisopropylcarbodiimide (4 mmol, 0.62 ml) sequentially to 19c. React at room temperature for 1.5 h. After the reaction is complete as monitored by ninhydrin, the solvent is removed and the resin is washed three times with DMF to obtain 19d.

[0492] Step 4: Add 20% piperidine / DMF to 19d, react for 30 minutes, then remove the solvent and wash the resin 5 times with DMF to obtain 19e. Monitor the reaction with ninhydrin.

[0493] Step 5: Add Fmoc-Glu-OtBu (4 mmol, 1.7 g), DMF (20 ml), 1-hydroxybenzotriazole (4 mmol, 0.54 g), and N,N'-diisopropylcarbodiimide (4 mmol, 0.62 ml) sequentially to 19e. React at room temperature for 1.5 h. After the reaction is complete as monitored by ninhydrin, remove the solvent and wash the resin three times with DMF to obtain 19f.

[0494] Step 6: Add 20% piperidine / DMF to 19f, react for 30 minutes, then remove the solvent and wash the resin 5 times with DMF to obtain 19g. Monitor the reaction with ninhydrin.

[0495] Step 7: Add 19g of octadecanoic acid monotert-butyl ester (4mmol, 1.48g), DMF (20ml), 1-hydroxybenzotriazole (4mmol, 0.54g), and N,N'-diisopropylcarbodiimide (4mmol, 0.62ml) sequentially. React at room temperature for 1.5h. After the reaction is complete as monitored by ninhydrin, the solvent is removed and the resin is washed 3 times with DMF, 2 times with methanol, 1 time with dichloromethane, and 2 times with methanol. The mixture is then dried to obtain 19h.

[0496] Step 8: Add 30% trifluoroethanol / dichloromethane solution (32 ml) to 19h (3.2 g), react at room temperature for 1 h, filter, collect the filtrate and concentrate under reduced pressure to obtain intermediate 19 (1.0 g).

[0497] LCMS m / z = 874.5 [M+H] + .

[0498] Intermediate 20:

[0499] Step 1: Add 6a (1 mmol, 1 g, sub: 1 mmol / g) and dichloromethane to the reactor. After swelling the resin for 5 min, add Fmoc-Pen(Trt)-OH (3 mmol, 1.2 g) and N,N-diisopropylethylamine (6 mmol, 1.0 ml) in sequence. React at room temperature for 1 h. Then add methanol (5 ml) and react for 30 min. Drain the solvent and wash the resin three times with DMF to obtain 20b.

[0500] Step 2: Add 20% piperidine / DMF to 20b, react for 30 minutes, then remove the solvent and wash the resin 5 times with DMF to obtain 20c. Monitor the reaction with ninhydrin.

[0501] Step 3: Add DMF (20 mL), DIPEA (1 mL), and acetic anhydride (1 mL) sequentially to 20c. React at room temperature for 0.5 h. After the reaction is complete by monitoring with ninhydrin, remove the solvent and wash the resin 3 times with DMF, 2 times with methanol, 1 time with dichloromethane, and 2 times with methanol. Remove the solvent and dry to obtain 20d.

[0502] Step 4: Add 30% trifluoroethanol / dichloromethane solution (30 ml) to 20d (3.0 g), react at room temperature for 1 h, filter, collect the filtrate and concentrate under reduced pressure to obtain intermediate 20 (1.0 g).

[0503] Intermediate 21:

[0504] Step 1: Compound 21a (72.53 g, 346.89 mmol), 2-(2-BOC-aminoethoxy)ethanol (20.00 g, 97.44 mmol), and tetrabutylammonium chloride (4.87 g, 17.54 mmol) were dissolved in toluene (500 mL). Under a nitrogen atmosphere, 50% NaOH (160 mL) was slowly added dropwise at 0 °C. After the addition was complete, the mixture was moved to room temperature and stirred overnight. After the reaction was complete, ethyl acetate (500 mL × 3) was added for extraction. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified to obtain compound 21b (12.5 g, 38%).

[0505] LC-MS(ESI):m / z=234.3[M-100+H] + .

[0506] Step 2: Compound 21b (12.5 g, 37.49 mmol) was dissolved in dichloromethane (150 mL), and trifluoroacetic acid (30 mL) was added to the reaction solution at 0 °C. The mixture was stirred at room temperature for 1 h. After the reaction was completed, the solution was concentrated under reduced pressure to obtain crude compound 21c (6.64 g, 76%).

[0507] LC-MS (ESI): m / z = 178.2 [M+H] + .

[0508] Step 3: Compound 21c (6.64 g, 28.46 mmol) was dissolved in methanol (100 mL). Sodium bicarbonate (23.91 g, 284.60 mmol) and 9-fluorenylmethyl-N-succinimide carbonate (19.20 g, 56.92 mmol) were added to the above reaction solution at 25 °C, and the mixture was stirred at room temperature for 16 h. After the reaction was completed, ethyl acetate (500 mL × 3) was added for extraction. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified to obtain compound 21d (6.7 g, 59%).

[0509] LC-MS(ESI): m / z = 400.0 [M+H] + .

[0510] Step 4: Compound 21d (1.4 g, 3.50 mmol) and D-lactic acid(+)-tert-butyl ester (0.62 g, 4.21 mmol) were added to dichloromethane (15 mL), followed by EDCI (0.66 g, 3.50 mmol) and DMAP (0.21 g, 1.75 mmol). The reaction mixture was reacted at room temperature for 16 h. The reaction solution was then concentrated under reduced pressure and purified by silica gel column chromatography to obtain compound 21e (1.4 g, 76%).

[0511] LC-MS (ESI): m / z = 528.2 [M+H] + .

[0512] Step 5: Add 21e (1.4g, 2.65mmol) to dichloromethane (15mL), then add trifluoroacetic acid (3mL), react at room temperature for 3h, and directly concentrate the reaction solution under reduced pressure and separate and purify it by silica gel column chromatography to obtain intermediate 21 (1.0g, 80%).

[0513] LC-MS (ESI): m / z = 472.2 [M+H] + .

[0514] 1H NMR(400MHz, CDCl3)δ7.76(d,2H),7.63-7.54(m,2H),7.39(t,2H),7.34-7.27(m,2H),5.25-4.99(m,3H),4.49-4.36(m,2H),4.28 -4.18(m,1H),4.17-4.07(m,1H),3.84-3.72(m,1H),3.70-3.55(m,4H),3.50-3.33(m,2H),1.60-1.50(m,3H),1.48-1.41(m,3H).

[0515] Intermediate 22:

[0516] Step 1: Compound 21d (1.4 g, 3.50 mmol) and L-tert-butyl lactate (0.62 g, 4.21 mmol) were added to dichloromethane (15 mL), followed by EDCI (0.66 g, 3.50 mmol) and DMAP (0.21 g, 1.75 mmol). The reaction mixture was reacted at room temperature for 16 h. The reaction solution was then concentrated under reduced pressure and purified by silica gel column chromatography to obtain compound 22b (1.4 g, 76%).

[0517] LC-MS (ESI): m / z = 528.2 [M+H] + .

[0518] Step 2: Add 22b (1.4g, 2.65mmol) to dichloromethane (15mL), then add trifluoroacetic acid (3mL), react at room temperature for 3h, and directly concentrate the reaction solution under reduced pressure and separate and purify it by silica gel column chromatography to obtain intermediate 22 (1.0g, 80%).

[0519] LC-MS (ESI): m / z = 472.2 [M+H] + .

[0520] 1 H NMR(400MHz, CDCl3)δ7.75(d,2H),7.66-7.58(m,2H),7.42(t,2H),7.34-7.29(m,2H),5.23-4.99(m,3H),4.47-4.35(m,2H),4.26 -4.19(m,1H),4.15-4.07(m,1H),3.81-3.72(m,1H),3.70-3.55(m,4H),3.50-3.34(m,2H),1.60-1.52(m,3H),1.49-1.41(m,3H).

[0521] Intermediate 23:

[0522] Step 1: Compound 23a (10.0 g, 118.88 mmol) and selenium dioxide (15.8 g, 146.67 mmol) were added to pyridine (100 mL), and the mixture was reacted at 110 °C for 1 h, then cooled to 90 °C and reacted for another 4 h. After the reaction was complete, the mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. Ethyl acetate (50 mL) was added, followed by 2 mol / L sodium hydroxide aqueous solution (100 mL). The organic phase was separated, and the aqueous phase was extracted with ethyl acetate (2 × 50 mL). The aqueous phase was then adjusted to pH 4-5 with hydrochloric acid, extracted with ethyl acetate (2 × 100 mL), and the organic phase was concentrated under reduced pressure to obtain compound 23b (10 g, 73.72%).

[0523] 1 H NMR (400MHz, CDCl3) δ11.64(s,1H),1.66-1.55(m,1H),1.09-1.03(m,2H),0.96-0.89(m,2H).

[0524] Step 2: Compound 23b (10 g, 87.64 mmol) was added to dichloromethane (100 ml), followed by DMF (0.5 ml). Oxaloyl chloride (22 g, 175 mmol) was slowly added dropwise at 0 °C, and the reaction was maintained at 0 °C for 1 h. The reaction solution was then concentrated to dryness under reduced pressure, followed by the addition of dichloromethane (100 ml), tert-butanol (20 ml), and pyridine (17.3 g, 219 mmol) at 0 °C. The reaction was maintained at room temperature for 16 h. The reaction solution was then concentrated under reduced pressure and purified by silica gel column chromatography to obtain 23c (11 g, 73.7%).

[0525] 1 H NMR (400MHz, CDCl3) δ2.69-2.63(m,1H),1.57(s,9H),1.23-1.18(m,2H),1.14-1.08(m,2H).

[0526] Step 3: Compound 23c (10 g, 58.75 mmol) was added to methanol (100 ml), and sodium borohydride (2.82 g, 76.4 mmol) was added in portions. The mixture was reacted at room temperature for 1 h. Water (300 ml) was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 80 ml). The organic phase was washed with saturated sodium chloride solution, and the organic phase was concentrated under reduced pressure to obtain 23d (8 g, 79.06%).

[0527] 1H NMR (400MHz, CDCl3) δ3.68(d,1H),2.37(s,1H),1.50(s,9H),1.11-1.00(m,1H),0.54-0.44(m,3H),0.44-0.36(m,1H).

[0528] Step 4: Compound 23d (4 g, 23.2 mmol) and Fmoc-AEEA-OH (8.9 g, 23.2 mmol) were added to dichloromethane (60 ml), followed by EDCI (8.8 g, 46.4 mmol) and DMAP (1.4 g, 11.6 mmol). The reaction mixture was reacted at room temperature for 16 h. The reaction solution was then concentrated under reduced pressure and purified by silica gel column chromatography to obtain 23e (7.5 g, 59.8%).

[0529] LC-MS (ESI): m / z = 540.2 [M+H] + .

[0530] Step 5: Add 23e (6.5g, 12.05mmol) to dichloromethane (50ml), then add trifluoroacetic acid (20ml), react at room temperature for 3h, and directly concentrate the reaction solution under reduced pressure and separate and purify it by silica gel column chromatography to obtain intermediate 23 (4.5g, 77.3%).

[0531] LC-MS (ESI): m / z = 484.2 [M+H] + .

[0532] 1 H NMR(400MHz, CDCl3)δ7.75(d,2H),7.59(d,2H),7.39(t,2H),7.30(t,2H),6.70(s,1H),4.52-4.32(m ,3H),4.28-4.17(m,3H),3.78-3.20(m,8H),1.28-1.23(m,1H),0.73-0.54(m,3H),0.49-0.39(m,1H).

[0533] Intermediate 24:

[0534] Step 1: Add 23d (3g, 17.42mmol) to 30ml of 4mol / L dioxane hydrochloride solution and react at room temperature for 3h. Concentrate the reaction solution under reduced pressure to obtain intermediate 24 (2g, 98.8%).

[0535] LC-MS (ESI): m / z = 117.1 [M+H] + .

[0536] 1 H NMR (400MHz, DMSO-d6) δ12.40(s,1H),5.05(s,1H),3.52(d,1H),1.08-0.98(m,1H),0.42-0.26(m,4H).

[0537] Intermediate 25:

[0538] Step 1: Compound 25a (1 g, 8.6 mmol) was dissolved in DMF, and benzyl bromide (1.77 g, 10.34 mmol) and potassium carbonate (2.37 g, 17.2 mmol) were added. The mixture was stirred overnight at room temperature. After the reaction was complete, the solution was diluted with ethyl acetate, extracted with water, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated, and rapidly purified by silica gel column chromatography (0→70% EA / PE) to give compound 25b (1.5 g, 85%).

[0539] LCMS m / z = 207.1 [M+H] +

[0540] Step 2: Compound 25b (1.5 g, 7.28 mmol) and [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (5.61 g, 14.56 mmol) were added to dichloromethane (50 mL), followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.26 g, 14.56 mmol) and 4-dimethylaminopyridine (1.78 g, 14.56 mmol). The reaction was stirred at room temperature for 2 h. The reaction was monitored by TLC until it was complete. Water (100 mL) and dichloromethane (20 mL) were added, and the organic phase was dried and concentrated. The organic phase was then purified by silica gel column chromatography (MeOH:DCM = 0–10:1) to obtain compound 25c (3.01 g, 72%).

[0541] LCMS m / z = 574.2 [M+H] +

[0542] Step 3: Dissolve compound 25c (3.01 g, 5.25 mmol) in methanol (35 mL), add palladium on carbon (0.3 g, 10%), stir for 16 h under hydrogen atmosphere, monitor the reaction completion by LCMS, filter out palladium on carbon and concentrate to obtain crude compound 25d, which can be used directly in the next step.

[0543] LCMS m / z = 262.1 [M+H] +

[0544] Step 4: Dissolve the crude compound 25d from the previous step in a mixed solution of acetonitrile (25 mL) and water (25 mL), add sodium bicarbonate (2.21 g, 26.25 mmol) and 9-fluorenylmethyl-N-succinimide carbonate (3.54 g, 10.5 mmol), and stir the reaction overnight at room temperature. After the reaction is complete, add dilute hydrochloric acid to adjust the pH to neutral, concentrate under vacuum at 40 °C to remove most of the acetonitrile, filter and collect the solid to obtain the crude product, and separate and purify it using silica gel column chromatography (DCM:MeOH = 10:1) to obtain intermediate 25 (2.43 g, 96%).

[0545] LCMS m / z = 484.2[M+H] + .

[0546] Intermediate 26:

[0547] Step 1: Compound 26a (5.0 g, 38.46 mmol), potassium carbonate (10.6 g, 76.92 mmol), and benzyl bromide (7.9 g, 46.15 mmol) were added to DMF (50 mL) and reacted overnight at room temperature. After filtration, the filtrate was extracted with ethyl acetate (30 mL × 1), the organic phase was washed with water (30 mL × 3), washed with saturated brine (300 mL × 1), dried over anhydrous sodium sulfate, filtered, concentrated, and then subjected to silica gel column chromatography (EA:PE = 1:6) to obtain compound 26b (7.0 g, 82.7%).

[0548] 1 H NMR (400MHz, CDCl3) δ7.42-7.25(m,5H),5.20(s,2H),3.07(s,1H),2.15-2.00(m,2H),1.95-1.70(m,6H).

[0549] Step 2: Compound 26b (2.2 g, 10 mmol), 2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (7.7 g, 20 mmol) and EDCI (3.8 g, 20 mmol) were added to DMF (30 mL), followed by DMAP (2.4 g, 20 mmol). The mixture was reacted at room temperature for 4 h, extracted with ethyl acetate (30 mL × 1), washed with water (30 mL × 3), washed with saturated brine (300 mL × 1), dried over anhydrous sodium sulfate, filtered, concentrated, and then subjected to silica gel column chromatography (MeOH:DCM = 1:10) to obtain compound 26c (5.0 g, 85.1%).

[0550] LC-MS (ESI): m / z = 588.2 [M+H] + .

[0551] Step 3: Compound 26c (5.0 g, 8.5 mmol) was dissolved in ethyl acetate (50 mL), palladium on carbon (10%, 2.5 g) was added, and hydrogenation was carried out at room temperature for 1 hour. After filtration, the filtrate was concentrated and then subjected to silica gel column chromatography (MeOH:DCM = 1:10) to obtain intermediate 26 (2.6 g, 61.6%).

[0552] LC-MS (ESI): m / z = 498.2 [M+H] + .

[0553] Intermediate 27:

[0554] Step 1: Compound 27a (20 g, 75.96 mmol) and DIEA (29.4 g, 227.8 mmol) were dissolved in DCM (200 mL). HATU (30.33 g, 79.76 mmol) and dimethylhydroxylamine hydrochloride (11.11 g, 91.15 mmol) were added at 0 °C. After stirring at room temperature for 1 h, the reaction was detected as complete by TLC. Saturated sodium chloride solution (200 mL) was added. Extraction was performed with dichloromethane (100 mL × 2), and the organic layer was washed with saturated brine. After drying with anhydrous sodium sulfate and filtering, the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate / petroleum ether = 0–50%) to give compound 27b (21 g, 90.2%).

[0555] LC-MS (ESI): m / z = 207.1 [M+H] + .

[0556] Step 2: Compound 27b (21 g, 68.63 mmol) was dissolved in ultra-dry tetrahydrofuran (210 mL). Under nitrogen protection, the temperature was lowered to -10 °C, and a solution of methyl magnesium bromide in 2-methyltetrahydrofuran (70 mL, 3 mol / L) was slowly added dropwise. The reaction was detected as complete by TLC. The reaction was quenched by adding saturated ammonium chloride solution (200 mL). The mixture was extracted with ethyl acetate (100 mL × 2), and the organic layer was washed with saturated brine. After drying with anhydrous sodium sulfate and filtering, the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate / petroleum ether = 0–50%) to give compound 27c (11.6 g, 64.8%).

[0557] LC-MS (ESI): m / z = 162.1 [M+H] + .

[0558] Step 3: Compound 27c (11.6 g, 44.39 mmol) was dissolved in methanol, and sodium borohydride (3.28 g, 88.78 mmol) was added at 0 °C. The reaction was detected as complete by TLC. The methanol was concentrated under reduced pressure, and then water (100 mL) was added. The mixture was extracted with ethyl acetate (100 mL x 2), and the organic layer was washed with saturated brine. After drying with anhydrous sodium sulfate and filtering, the mixture was directly concentrated under reduced pressure to obtain compound 27d (10.5 g, 89.9%).

[0559] LC-MS (ESI): m / z = 164.2 [M+H] + .

[0560] Step 4: Using compound 27d (10.5 g, 39.92 mmol) and fluorenemethoxycarbonyl-L-glutamic acid 1-tert-butyl ester (17 g, 39.92 mmol) as starting materials, compound 27e (12 g, 44.9%) was obtained by referring to the procedure in step 2 of intermediate 2.

[0561] LC-MS (ESI): m / z = 571.2 [M+H] + .

[0562] Step 5: Add trifluoroacetic acid (10 mL) to dichloromethane (50 mL) containing compound 27e (5 g, 7.45 mmol), stir at room temperature for 16 h, concentrate and then purify by reverse reaction to obtain compound 27f (2.7 g, 70.4%).

[0563] LC-MS (ESI): m / z = 515.2 [M+H] + .

[0564] Step 6: Compound 27f (2.7 g, 2.25 mmol) was dissolved in ethanol (27 mL), and 2-acetyl-5,5-dimethyl-1,3-cyclohexanedione (1.43 g, 7.87 mmol) and trifluoroacetic acid (60 mg, 0.5 mmol) were added. The mixture was stirred at 90 °C for 2 h. After the reaction was completed by TLC monitoring, the mixture was concentrated and purified by silica gel column chromatography (MeOH:DCM = 0–10%) to obtain intermediate 27 (3.4 g, 95.5%).

[0565] LC-MS (ESI): m / z = 679.2 [M+H] + .

[0566] Intermediate 28:

[0567] Step 1: Compound 28a (10.0 g, 98 mmol), potassium carbonate (17.6 g, 127 mmol), and benzyl bromide (18.4 g, 108 mmol) were added to DMF (160 mL) and reacted overnight at room temperature. After filtration, the filtrate was extracted with ethyl acetate (300 mL × 1), the organic phase was washed with water (300 mL × 3), washed with saturated brine (300 mL × 1), dried over anhydrous sodium sulfate, filtered, concentrated, and then subjected to silica gel column chromatography (EA:PE = 1:6) to obtain compound 28b (15 g, 79.8%).

[0568] Step 2: Compound 28b (3g, 15.6mmol), 2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (7.7g, 20mmol), and EDCI (3.8g, 20mmol) were added to DMF (30mL), followed by DMAP (2.4g, 20mmol). The mixture was reacted at room temperature for 4h, extracted with ethyl acetate (30mL × 1), washed with water (30mL × 3), washed with saturated brine (300mL × 1), dried over anhydrous sodium sulfate, filtered, concentrated, and then subjected to silica gel column chromatography (MeOH:DCM = 1:10) to obtain compound 28c (7.1g, 81.3%).

[0569] LC-MS (ESI): m / z = 560.2 [M+H] + .

[0570] Step 3: Dissolve compound 28c (6g) in ethyl acetate (60mL), add palladium on carbon (1.2g, 10%), stir for 1h under hydrogen atmosphere, filter after reaction, concentrate and separate and purify using silica gel column chromatography to obtain intermediate 28 (4g, 79.5%).

[0571] LC-MS (ESI): m / z = 470.1 [M+H] + .

[0572] Intermediate 29:

[0573] Step 1: Compound 29a (10 g, 37.7 mmol) and pyridinium chlorochromate (12.2 g, 56.5 mmol) were added to dichloromethane (150 ml) and reacted at room temperature for 16 h. After the reaction was completed, the mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography to obtain 29b (8 g, 80.6%).

[0574] Step 2: Compound 29c (10 g, 44.82 mmol) and triphenylphosphine (14.11 g, 53.79 mmol) were added to acetonitrile (150 ml) and reacted at 85 °C for 35 h. After cooling to room temperature, the reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography to obtain 29d (20 g, 92%).

[0575] Step 3: 29d (20g, 41mmol) was added to dry THF (150ml), purged with N2, and cooled to 0℃. NaHMDS (41mmol) was slowly added, and the mixture was stirred at 0℃ for 20min. Then, 29b (7.2g, 27mmol) in tetrahydrofuran solution (30ml) was slowly added dropwise, and the reaction was maintained at 0℃ for 1h. Water (100ml) was added, and the mixture was extracted with ethyl acetate (100ml*2). The organic phases were combined, washed with saturated sodium chloride solution, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain 29e (5.5g, 52.5%).

[0576] Step 4: Add 5.5 g (14.2 mmol) of 29e to 70 ml of ethyl acetate, add 10% Pd / C (1 g), and react at room temperature for 16 h. Filter, and concentrate the filtrate under reduced pressure to obtain 5.5 g (99.5%).

[0577] Step 5: 29f (5.4 g, 13.8 mmol), dibenzyl phosphite (4 g, 15.2 mmol), tetraethylammonium iodide (3.56 g, 13.8 mmol), and cesium carbonate (9 g, 27.7 mmol) were added to DMF (60 ml) and reacted at 60 °C for 16 h. After cooling to room temperature, water (300 ml) was added, and the mixture was extracted with ethyl acetate (100 ml × 3). The organic phase was washed with saturated sodium chloride solution, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain 29 g (4 g, 50.62%).

[0578] Step 6: Add 29 g (4 g, 7 mmol) and lithium hydroxide (0.84 g, 35 mmol) to a mixed solvent of methanol (30 ml) and water (10 ml), and react at room temperature for 3 h. Concentrate under reduced pressure to remove most of the methanol, and adjust the pH to 4-5 with 6 mol / L hydrochloric acid. A large amount of solid precipitates out. Filter, wash the solid with water, and dry to obtain intermediate 29 (3.7 g, 94.82%).

[0579] LC-MS (ESI): m / z = 559.4 [M+H] + .

[0580] 1H NMR(400MHz,DMSO-d6)δ12.07(s,1H),7.41-7.26(m,10H),5.04-4.92(m,4 H),2.17(t,2H),1.83-1.70(m,2H),1.50-1.39(m,4H),1.29-1.18(m,28H).

[0581] Intermediate 30:

[0582] Step 1: Compound 30a (2.68 g, 6.29 mmol) and hexaethylene glycol-tert-butyloxycarbonyl (2 g, 5.24 mmol) were added to dichloromethane (30 ml), followed by EDCI (2.01 g, 10.48 mmol) and DMAP (0.24 g, 2 mmol). The reaction mixture was reacted at room temperature for 16 h. The reaction solution was then concentrated under reduced pressure and purified by silica gel column chromatography to obtain 30b (3.6 g, 87%).

[0583] LC-MS (ESI): m / z = 789.4 [M+H] + .

[0584] Step 2: Add 30b (3.6g, 4.57mmol) to dichloromethane (40ml), then add trifluoroacetic acid (12ml), react at room temperature for 3h, and directly concentrate the reaction solution under reduced pressure to obtain crude product 30c (4g).

[0585] LC-MS (ESI): m / z = 633.3 [M+H] + .

[0586] Step 3: 30c (4g), 2-(1-hydroxyethylidene)-5,5-dimethylcyclohexane-1,3-dione (1.67g, 9.14mmol), and triethylamine (923mg, 9.14mmol) were added to ethanol (40ml), and the mixture was reacted at room temperature for 3h. The reaction solution was then concentrated under reduced pressure and purified by silica gel column chromatography to obtain intermediate 30 (2.6g, 71.39%).

[0587] LC-MS (ESI): m / z = 797.5 [M+H] + .

[0588] Intermediate 31:

[0589] Step 1: Dissolve 18 g (0.6 g, 2.4 mmol) of compound in tetrahydrofuran (10 mL), cool in an ice bath, and slowly add lithium aluminum hydride (0.17 g, 4.4 mmol). React at room temperature for 2 h. After the reaction is complete, add water (0.2 mL), 15% sodium hydroxide solution (0.2 mL), and water (0.2 mL) sequentially. Filter and concentrate to obtain crude compound 31a, which can be directly used in the next step.

[0590] LCMS m / z = 178.1 [M+H] + .

[0591] Step 2: Dissolve the crude compound 31a from the previous step in dichloromethane (10 mL), add DDE-OH (0.8 g, 4.4 mmol), stir overnight at room temperature, monitor the reaction by TLC until it is complete, quench with water, extract with dichloromethane, collect the organic phase, concentrate the organic phase, and purify by silica gel column chromatography to obtain compound 31b (0.5 g, 67%).

[0592] LCMS m / z = 342.2[M+H] + .

[0593] Step 3: Using compound 31b (0.5 g, 1.47 mmol) and fluorenemethoxycarbonyl-L-glutamic acid 1-tert-butyl ester (1.25 g, 2.94 mmol) as starting materials, compound 31c (0.8 g, 80%) was obtained by referring to the procedure in step 2 of intermediate 2.

[0594] LCMS m / z = 749.4 [M+H] + .

[0595] Step 4: Compound 31c (0.8 g, 1.07 mmol) was placed in a 50 ml round-bottom flask, and dichloromethane (9 ml) and trifluoroacetic acid (3 ml) were added. The mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS until it was complete. The intermediate 31 (0.7 g, 95%) was then purified by reverse chromatography.

[0596] LCMS m / z = 693.3 [M+H] + .

[0597] Intermediate 32:

[0598] Step 1: Compound 32a (5 g, 22.73 mmol) was dissolved in dichloromethane, and triethylamine (9.18 g, 90.92 mmol) and p-toluenesulfonyl chloride (6.48 g, 30.1 mmol) were added. The mixture was stirred at room temperature for 3 h. After the reaction was complete, the mixture was diluted with water, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated, and rapidly purified by silica gel column chromatography to obtain compound 32b (8.3 g, 97%).

[0599] LCMS m / z = 375.2 [M+H] + .

[0600] Step 2: Compound 32c (1 g, 5.75 mmol) was placed in a 50 ml round-bottom flask, and 4N hydrogen chloride-1,4-dioxane (20 ml) was added. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the mixture was directly concentrated to obtain compound 32d, which was used directly in the next step.

[0601] Step 3: Compound 32d from the previous step was placed in a 50 ml round-bottom flask, dissolved in dichloromethane (20 ml), and triethylamine (0.7 g, 6.9 mmol) and DDE-OH (1.57 g, 8.63 mmol) were added. The mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was diluted with water, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated, and rapidly purified by silica gel column chromatography to obtain compound 32e (1.2 g, 88%).

[0602] LCMS m / z = 239.2 [M+H] + .

[0603] Step 4: Compound 32b (1 g, 2.67 mmol) and compound 32e (0.64 g, 2.67 mmol) were added to N,N-dimethylformamide (10 mL), and cesium carbonate (1.74 g, 5.34 mmol) was added. The reaction was stirred in an oil bath at 100 °C for 3 h. The reaction was monitored by TLC until it was complete. Water was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic phase was collected, concentrated, and then purified using a silica gel column to obtain compound 32f (0.5 g, 43%).

[0604] LCMS m / z = 441.3[M+H] + .

[0605] Step 5: Compound 32f (1g, 2.27mmol) was dissolved in tetrahydrofuran (10mL), tetrabutylammonium fluoride (5mL) was added, and the mixture was stirred at room temperature for 2h. The reaction was monitored by LCMS until it was complete. The organic phase was concentrated and then purified by silica gel column chromatography to obtain compound 32g (0.7g, 95%).

[0606] LCMS m / z = 327.2 [M+H] + .

[0607] Step 6: Using compound 32g (1g, 3.07mmol) and fluorenemethoxycarbonyl-L-glutamic acid 1-tert-butyl ester (2.61g, 6.14mmol) as starting materials, compound 32h (1.95g, 87%) was obtained by referring to the procedure in step 2 of intermediate 2.

[0608] LCMS m / z = 734.4 [M+H] + .

[0609] Step 7: Dissolve compound 32h (1g, 1.36mmol) in dichloromethane, add iodomethane (0.97g, 6.8mmol), stir overnight at room temperature, monitor the reaction completion by LCMS, and directly concentrate to obtain compound 32i for the next step.

[0610] LCMS m / z = 748.4 [M] + .

[0611] Step 8: Place compound 32i in a 50ml round-bottom flask, add dichloromethane (9ml) and trifluoroacetic acid (3ml), stir at room temperature for 1h, monitor the reaction completion by LCMS, and then purify by reverse process to obtain intermediate 32 (0.8g, 85%).

[0612] LCMS m / z = 692.4 [M] + .

[0613] Intermediate 33:

[0614] Step 1: Using compound 27b (12 g, 39.17 mmol) and cyclopropylmagnesium bromide (40 mL, 1 M in THF) as starting materials, compound 33a (8 g, 71.1%) was obtained by referring to the procedure in step 2 of intermediate 27.

[0615] LC-MS (ESI): m / z = 288.2 [M+H] + .

[0616] Step 2: Using compound 33a (8g, 27.84mmol) as the starting material, compound 33b (8g, 99.3%) was obtained by referring to the procedure in step 3 of intermediate 27.

[0617] LC-MS (ESI): m / z = 290.2 [M+H] + .

[0618] Step 3: Using compound 33b (7 g, 24.19 mmol) and fluorenemethyloxycarbonyl-L-glutamic acid 1-tert-butyl ester (10.29 g, 24.19 mmol) as starting materials, compound 33c (5 g, 29.7%) was obtained by referring to the procedure in step 4 of intermediate 27.

[0619] LC-MS (ESI): m / z = 697.2 [M+H] + .

[0620] Step 4: Using compound 33c (3.8 g, 5.45 mmol) as the starting material, compound 33d (500 mg, 17.0%) was obtained by following the procedure in step 5 of intermediate 27.

[0621] LC-MS (ESI): m / z = 541.2 [M+H] + .

[0622] Step 5: Using compound 33d (200 mg, 0.37 mmol) as the starting material, intermediate 33 (140 mg, 53.7%) was obtained by referring to the procedure in step 5 of intermediate 27.

[0623] LC-MS (ESI): m / z = 705.2 [M+H] + .

[0624] Intermediate 34:

[0625] Step 1: Using compound 27b (30.0 g, 98.04 mmol) and isopropyl magnesium bromide (100 mL, 2 mol / L) as starting materials, compound 34a (9 g, 31.8%) was obtained by referring to the procedure in step 2 of intermediate 27.

[0626] LC-MS (ESI): m / z = 290.3 [M+H] + .

[0627] Step 2: Using compound 34a (9.0 g, 31.14 mmol) and sodium borohydride (2.4 g, 62.28 mmol) as raw materials, compound 34b (8.5 g, 93.8%) was obtained by referring to the procedure in step 3 of intermediate 27.

[0628] LC-MS (ESI): m / z = 292.3 [M+H] + .

[0629] Step 3: Compound 34b (4.0 g, 13.74 mmol) and fluorenemethoxycarbonyl-L-glutamic acid 1-tert-butyl ester (11.7 g, 27.48 mmol) were dissolved in dichloromethane (100 mL), DCC (5.6 g, 27.48 mmol) and DMAP (3.3 g, 27.48 mmol) were added, and the mixture was reacted at room temperature for 1 hour. Then, water (200 mL) was added, and the organic layer was washed with dichloromethane (100 mL x 2) and saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, concentrated, and then subjected to silica gel column chromatography (MeOH:DCM = 1:10) to obtain compound 34c (6.0 g, 62.7%).

[0630] LC-MS (ESI): m / z = 699.3 [M+H] + .

[0631] Step 4: Using compound 34c (6.0 g, 8.59 mmol) as the starting material, compound 34d (4.5 g, 96.7%) was obtained by following the procedure in step 5 of intermediate 27.

[0632] LC-MS (ESI): m / z = 543.3 [M+H] + .

[0633] Step 5: Using compound 34d (4.5 g, 8.3 mmol) and 2-acetyl-5,5-dimethyl-1,3-cyclohexanedione (2.27 g, 12.45 mmol) as starting materials, intermediate 34 (2.5 g, 42.7%) was obtained by referring to the procedure in step 6 of intermediate 27.

[0634] LC-MS (ESI): m / z = 707.2 [M+H] + .

[0635] Intermediate 37:

[0636] Step 1: Using compound 37a (5 g, 24.39 mmol) and 1-bromopinazone (8.73 g, 48.78 mmol) as starting materials, compound 37b (2.1 g, 28%) was obtained by following the procedure in Step 1 of intermediate 21.

[0637] LCMS m / z = 304.2[M+H] +

[0638] Step 2: Add compound 37b (2.1g, 6.93mmol) to a 50ml round-bottom flask, add trifluoroacetic acid / dichloromethane = 1 / 3 (20ml), stir at room temperature for 1h, and after the reaction is complete, directly evaporate to dryness to obtain crude compound 37c for the next step.

[0639] LCMS m / z = 204.2[M+H] +

[0640] Step 3: Using the crude product from the previous step as a raw material, dissolve it in tetrahydrofuran (20 ml), cool it in an ice bath, add lithium aluminum hydride (0.53 g, 13.86 mmol), and stir at room temperature for 1 h. After the reaction is complete, add water (1 ml), 15% sodium hydroxide aqueous solution (1 ml), and water (1 ml) sequentially. After filtration, collect the filtrate and directly evaporate to dryness to obtain the crude product of compound 37d for the next step.

[0641] LCMS m / z = 206.2[M+H] +

[0642] Step 4: Using compound 37d and DDE-OH (1.26 g, 6.93 mmol) as starting materials, compound 37e (2 g, two-step yield 78%) was obtained by referring to the procedure in step 2 of intermediate 31.

[0643] LCMS m / z = 370.3 [M+H] +

[0644] Step 5: Using compound 37e (2 g, 5.41 mmol) and fluorenemethoxycarbonyl-L-glutamic acid 1-tert-butyl ester (2.3 g, 5.41 mmol) as starting materials, compound 37f (3.1 g, 77%) was obtained by referring to the procedure in step 3 of intermediate 31.

[0645] LCMS m / z = 777.4 [M+H] +

[0646] Step 6: Using compound 37f (3.1 g, 4.14 mmol) as the starting material, compound 37 (2.83 g, 95%) was obtained by referring to the procedure in step 4 of intermediate 31.

[0647] LCMS m / z = 721.4 [M+H] +

[0648] Intermediate 39:

[0649] Step 1: Compound 21d (1.6 g, 4.00 mmol) was dissolved in tetrahydrofuran (15 mL), and 1 M boranetetrahydrofuran solution (6 mL, 6.00 mmol) was added dropwise under ice bath conditions. The mixture was stirred at room temperature for 40 min. After the reaction was complete, 50 mL of water was added to the reaction system, and the resulting solution was extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to column chromatography (EA:PE = 0–100%) to give compound 39a (1.2 g, 77.7%).

[0650] LC-MS (ESI): m / z = 386.1 [M+H] + .

[0651] Step 2: Compound 39a (1g, 2.59mmol) was added to a mixed solution of piperidine and dichloromethane (10mL, 1:1), stirred at room temperature for 16h, concentrated after the reaction was complete, and lyophilized with 10mL of water to remove residual piperidine, yielding crude compound 39b, which was directly used in the next step of the reaction.

[0652] LC-MS (ESI): m / z = 164.1 [M+H] + .

[0653] Step 3: Using compound 39b (crude product) as raw material, compound 39c (580 mg, two-step yield 67.9%) was obtained by referring to the operation method in step 5 of intermediate 35.

[0654] LC-MS (ESI): m / z = 328.2 [M+H] + .

[0655] Step 4: Using compound 39c (580 mg, 1.76 mmol) and fluorenemethoxycarbonyl-L-glutamic acid 1-tert-butyl ester (750 mg, 1.76 mmol) as starting materials, compound 39d (850 mg, 65.7%) was obtained by referring to the procedure in step 2 of intermediate 2.

[0656] LC-MS (ESI): m / z = 735.5 [M+H] + .

[0657] Step 5: Compound 39d (850 mg, 1.16 mmol) was added to a solution of dioxane chloride (10 mL, 4 M) and stirred at room temperature for 16 h. After the reaction was completed, the mixture was concentrated and purified by reverse-phase column chromatography to obtain intermediate 39 (720 mg, 91.6%).

[0658] LC-MS (ESI): m / z = 679.4 [M+H] + .

[0659] Intermediate 40:

[0660] Step 1: Compound 30a (1.0 g, 2.3 mmol) and polyethylene glycol-tert-butyloxycarbonyl (1.2 g, 2.3 mmol) were added to dichloromethane (20 mL), followed by EDCI (0.54 g, 2.8 mmol) and DMAP (0.12 g, 0.92 mmol). The reaction mixture was reacted at room temperature for 16 h. The reaction solution was then concentrated under reduced pressure and purified by silica gel column chromatography to obtain 40b (2.0 g, 92%).

[0661] Step 2: Add 40b (2.0g, 2.17mmol) to dichloromethane (30ml), then add trifluoroacetic acid (10ml), react at room temperature for 3h, and directly concentrate the reaction solution under reduced pressure to obtain crude product 40c (3.8g).

[0662] LC-MS (ESI): m / z = 765.3 [M+H] + .

[0663] Step 3: 40c (3.8 g), 2-(1-hydroxyethylidene)-5,5-dimethylcyclohexane-1,3-dione (1.8 g, 9.8 mmol), and triethylamine (0.99 g, 9.8 mmol) were added to ethanol (40 ml), and the mixture was reacted at room temperature for 3 h. The reaction solution was then concentrated under reduced pressure and purified by silica gel column chromatography to obtain intermediate 40 (1.2 g, 59.5%) (two-step yield).

[0664] LC-MS (ESI): m / z = 929.5 [M+H] + .

[0665] Intermediate 41:

[0666] Step 1: Compound 41a (1 g, 5.56 mmol) and fluorenemethoxycarbonylsarcosine (3.46 g, 11.11 mmol) were added to dichloromethane (50 mL), followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.13 g, 11.11 mmol) and 4-dimethylaminopyridine (1.36 g, 11.11 mmol). The reaction was stirred at room temperature for 2 h. The reaction was monitored by TLC until it was complete. Water (100 mL) and dichloromethane (20 mL) were added, and the organic phase was dried and concentrated. The organic phase was then purified by silica gel column chromatography to obtain 41b (1.97 g, 75% yield).

[0667] LCMS m / z = 474.2 [M+H] +

[0668] Step 2: Compound 41b (1.97 g, 4.16 mmol) was dissolved in methanol (35 mL), palladium on carbon (0.3 g, 10%) was added, and the mixture was stirred for 16 h under hydrogen atmosphere. The reaction was monitored by LCMS until it was complete. After filtering out the palladium on carbon, the mixture was concentrated and purified by silica gel column chromatography to obtain intermediate 41 (1.3 g, yield 81%).

[0669] LCMS m / z = 384.2[M+H] +

[0670] Intermediate 42:

[0671] Step 1: Compound 41a (800 mg, 4.44 mmol) and Fmoc-Thr(tBu)-OH (1.77 g, 4.44 mmol) were added to dichloromethane (20 mL), followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.0 g, 5.33 mmol) and 4-dimethylaminopyridine (217 mg, 1.78 mmol). The mixture was stirred at room temperature for 16 h. The reaction was monitored by TLC until it was complete. Water (50 mL) and dichloromethane (20 mL) were added, and the organic phase was dried and concentrated. The organic phase was then purified by silica gel column chromatography to obtain 42b (2.34 g, yield 94.4%).

[0672] LCMS m / z = 504.2[M+H-56] +

[0673] Step 2: Compound 42b (2.34 g, 4.18 mmol) was dissolved in methanol (30 mL), and palladium on carbon (0.25 g, 10%) was added. The mixture was stirred for 16 h under hydrogen atmosphere. The reaction was monitored by LCMS until it was complete. After filtering out the palladium on carbon, the mixture was concentrated and purified by silica gel column chromatography to obtain intermediate 42 (1.2 g, yield 61.2%).

[0674] LCMS m / z = 414.2[M+H-56] +

[0675] Intermediate 43:

[0676] Step 1: A solution of compound 29f (5 g, 12.78 mmol) in 1-propanol (20 mL) and methanol (10 mL) and a solution of sodium sulfite (3.23 g, 25.63 mmol) in water (20 mL) were added to a reactor, and the mixture was heated to 95 °C and reacted for 16 h. The mixture was cooled to room temperature, and tetrahydrofuran (80 mL) was added. The mixture was stirred in an ice bath for 10 min, filtered, and the solid was washed with a small amount of tetrahydrofuran solution. The solid was added to water (50 mL), followed by an aqueous solution of sodium hydroxide (2 g, 50 mmol), and the mixture was heated to 100 °C and reacted for 3 h. The mixture was cooled to room temperature, the pH was adjusted to 1 with an aqueous solution of sulfuric acid, and the mixture was refluxed for 10 min. The mixture was cooled to room temperature, filtered, and the solid was washed with water and dried to give intermediate 41 (3 g, yield: 62%).

[0677] LC-MS (ESI): m / z = 377.2 [MH] - .

[0678] 1H NMR (400MHz, DMSO-d6) δ2.44-2.31(m,2H),2.20-2.16(m,2H),1.59-1.44(m,4H),1.32-1.18(m,28H).

[0679] Example 8:

[0680] Step 1: The synthesis of 8B employs standard Fmoc chemical methods:

[0681] 1. Add Rink Amide MBHA Resin (1 mmol, 1.4 g, sub: 0.7 mmol / g) and dichloromethane solvent to the reactor, allow it to swell for 30 min, add 20% piperidine / DMF, and mix for 30 min.

[0682] 2. Drain and rinse five times with DMF.

[0683] 3. Add the Fmoc-Sar-OH protected amino acid solution, mix for 30 seconds, then add the coupling reagent, bubble with nitrogen for 1.5 hours, and monitor the reaction with ninhydrin.

[0684] 4. Drain and rinse three times with DMF.

[0685] 5. Add 20% piperidine / DMF and mix for 30 minutes.

[0686] 6. Drain and rinse five times with DMF.

[0687] 7. Add the Fmoc-protected amino acid solution, mix for 30 seconds, then add the coupling reagent, bubble with nitrogen for 1.5 hours, and monitor the reaction with ninhydrin.

[0688] 8. Drain and rinse three times with DMF.

[0689] 9. Repeat steps 5-8 for the next amino acid coupling.

[0690] 10. In the final step, the resin was washed twice with MeOH, once with DCM, and twice with MeOH. After vacuum drying, peptide resin 8B was obtained and used directly in the next reaction.

[0691] Step 2: Add 50 ml of lysis buffer (91% trifluoroacetic acid + 4% triisopropylsilane + 3% 1,2-ethylenedithiol + 2% water) to a 100 ml reaction flask, stir well, then add peptide resin 8B and stir at room temperature for 2 hours. Filter the resin to obtain the filtrate, add the filtrate to 300 ml of methyl tert-butyl ether (pre-cooled to 0°C), a white flocculent precipitate will form, centrifuge (3 min at 3000 rpm). Wash the white precipitate three times with methyl tert-butyl ether, and vacuum dry to obtain a white solid crude peptide 8C, which can be used directly in the next reaction.

[0692] Step 3: In a 2L reaction flask, add water (750ml), acetonitrile (250ml), and 8C (2g) sequentially. After stirring evenly, slowly add iodine / acetonitrile solution (0.1mol / L) until the reaction solution turns pale yellow. Quench with ascorbic acid and perform HPLC purification. Separation method: 1. Instrument: Waters 2767 preparative HPLC; Column: SunFire@PrepC18 (19mm×250mm). 2. Filter the sample through a 0.45μm filter to prepare the sample solution. 3. Preparative chromatographic conditions: a. Mobile phase A, B composition: A: 0.1% trifluoroacetic acid / H2O, B: CH3CN; b. Gradient elution: mobile phase content 5%-45%; c. Flow rate: 12ml / min; d. Elution time: 30min. Lyophilization yielded compound 8 (180mg, purity 98%).

[0693] LCMS m / z = 884.5 [M / 3 + H] + 663.6 [M / 4+H] + .

[0694] Example 9:

[0695] Using the above-mentioned material list as raw materials, compound 9 (185 mg, purity 97%) was synthesized using the method of compound 8.

[0696] LCMS m / z = 888.5 [M / 3 + H] + 666.7 [M / 4+H] + .

[0697] Example 10:

[0698] Using the above-mentioned materials as raw materials, compound 10 (170 mg, 99% purity) was synthesized using the method described for compound 8. LCMS m / z = 1191.1 [M / 2 + H] + 794.3 [M / 3+H] + 596.0 [M / 4+H]+ .

[0699] Example 11:

[0700] Using the above-mentioned materials as raw materials, compound 11 (18 mg, 99% purity) was synthesized using the method described for compound 8. LCMS m / z = 960.3 [M / 2 + H] + 640.5 [M / 3+H] + 480.7 [M / 4+H] + .

[0701] Example 12:

[0702] Using the above-mentioned material list as raw materials, compound 12 (280 mg, 99% purity) was synthesized using the method of compound 8.

[0703] LCMS m / z = 1025.0 [M / 2 + H] + 683.6 [M / 3+H] + .

[0704] Example 13:

[0705] Using the above-mentioned material list as raw materials, compound 13 (110 mg, purity 98%) was synthesized using the method of compound 8.

[0706] LCMS m / z = 823.0 [M / 3 + H] + .

[0707] Example 14:

[0708] Using the above-mentioned material list as raw materials, compound 14 (249 mg, purity 98%) was synthesized using the method of compound 8.

[0709] LCMS m / z = 989.2[M / 2 + H] + 659.9 [M / 3+H] + .

[0710] Example 18:

[0711] After step 14 of the synthesis, Dde was removed with 2% hydrazine hydrate / DMF (twice, 30 min each time), dried under vacuum, and washed three times with DMF. Then the next amino acid was added. Using the above-mentioned material list as raw materials, compound 18 (138 mg, 99% purity) was synthesized using the method of compound 8.

[0712] LCMS m / z = 1347.2[M / 2 + H] + 898.6 [M / 3+H] + .

[0713] Example 19:

[0714] Using the above-mentioned material list as raw materials, compound 19 (180 mg, purity 98%) was synthesized using the method of compound 8.

[0715] LCMS m / z = 1255.4 [M / 2 + H] + 837.3 [M / 3+H] + .

[0716] Example 20:

[0717] Using the above-mentioned material list as raw materials, compound 20 (50 mg, purity 91%) was synthesized using the method of compound 8.

[0718] LCMS m / z = 922.0 [M / 3 + H] + 691.8 [M / 4+H] + .

[0719] Example 21:

[0720] Using the above-mentioned material list as raw materials, compound 21 (400 mg, 99%) was synthesized using the method of compound 8.

[0721] LCMS m / z = 996.3[M / 2 + H] + .

[0722] Example 22:

[0723] Using the above-mentioned material list as raw materials, compound 22 (138 mg, 94%) was synthesized using the method of compound 8, and the deprotection time of intermediate 7 was 2 min.

[0724] LCMS m / z = 898.4 [M / 3 + H]+ 674.0 [M / 4+H] + .

[0725] Example 23:

[0726] Using the above-mentioned material list as raw materials, compound 23 (20 mg, 95%) was synthesized using the method of compound 8. The deprotection time of intermediate 7 and subsequent amino acids was 2 min.

[0727] LCMS m / z = 912.1 [M / 3 + H] + .

[0728] Example 24:

[0729] Using the above-mentioned material list as raw materials, compound 24 (214 mg, 99%) was synthesized using the method of compound 8.

[0730] LCMS m / z = 10¹⁰.³[M / ² + H] + 674.0 [M / 3+H] + .

[0731] Example 25:

[0732] Compound 25 (160 mg, 98%) was synthesized using the method described for compound 8, based on the above-mentioned material list. LCMS m / z = 916.9 [M / 3+H] + 688.0 [M / 4+H] + .

[0733] Example 26:

[0734] Using the above-mentioned material list as raw materials, compound 26 (255 mg, 98%) was synthesized using the method of compound 8.

[0735] LCMS m / z = 1017.2[M / 2 + H] + 678.5 [M / 3+H] + .

[0736] Example 27:

[0737] Using the above-mentioned material list as raw materials, compound 27 (40 mg, 94%) was synthesized using the method of compound 8.

[0738] LCMS m / z = 903.2[M / 3 + H] + 1354.3 [M / 2+H] + .

[0739] Example 28:

[0740] Step 1: The synthesis of 28B employs standard Fmoc chemical methods:

[0741] 28A (2 mmol, 2 g, sub: 1 mmol / g) and dichloromethane were added to the reactor. After the resin swelled for 5 min, Fmoc-Sar-OH (3 mmol, 0.93 g) and N,N-diisopropylethylamine (6 mmol, 1 ml) were added sequentially. The reaction was carried out at room temperature for 1 h. Then methanol (5 ml) was added and the reaction was carried out for 30 min. The solvent was dried and the resin was washed three times with DMF.

[0742] Add 20% piperidine / DMF and mix for 30 minutes.

[0743] Drain and rinse five times with DMF.

[0744] Add the Fmoc-protected amino acid solution, mix for 30 seconds, then add the coupling reagent, bubble under nitrogen for 1.5 hours, and monitor the reaction with ninhydrin.

[0745] Drain and rinse three times with DMF

[0746] The next amino acid coupling repeats steps 2-5.

[0747] In the final step, the resin was washed twice with MeOH, once with DCM, and twice with MeOH. After vacuum drying, peptide resin 28B (6.5 g) was obtained and used directly in the next reaction.

[0748] Step 2: Add 30% trifluoroethanol / dichloromethane solution (70 ml) to 28B (6.5 g), react at room temperature for 1 h, filter, collect the filtrate and concentrate under reduced pressure to obtain 28C (2.1 g).

[0749] Step 3: 28C (2.0 g, 0.63 mmol), intermediate 10 (0.63 g, 0.76 mmol), and dichloromethane (20 ml) were added sequentially to a 50 ml reaction flask. After stirring and dissolving, 1-hydroxybenzotriazole (102 mg, 0.76 mmol), N,N'-diisopropylcarbodiimide (120 ml, 0.76 mmol), and 4-dimethylaminopyridine (15.4 mg, 0.126 mmol) were added sequentially. The reaction was carried out at room temperature for 16 h. LCMS showed that most of the starting material had reacted completely. The organic phase was washed once with water and once with saturated brine. After drying with anhydrous sodium sulfate, the solution was concentrated under reduced pressure to obtain 28D (2.5 g, 0.63 mmol).

[0750] Step 4: Add 20 ml of lysis buffer (91% trifluoroacetic acid + 4% triisopropylsilane + 3% 1,2-ethylenedithiol + 2% water) to a 50 ml reaction flask, stir well, then add 28D (2.5 g, 0.63 mmol), and stir at room temperature for 2 hours. Filter through resin to obtain the filtrate, add the filtrate to 300 ml of methyl tert-butyl ether (pre-cooled to 0°C), a white flocculent precipitate forms, centrifuge (3 min at 3000 rpm). Wash the white precipitate three times with methyl tert-butyl ether, and vacuum dry to obtain a white solid crude peptide 28E (0.9 g, 0.34 mmol), which can be used directly in the next reaction.

[0751] Step 5: In a 1L reaction flask, add water (350ml), acetonitrile (150ml), and 28E (0.9g, 0.34mmol) sequentially. After stirring evenly, slowly add iodine / acetonitrile solution (0.1mol / L) until the reaction solution turns pale yellow. Quench with ascorbic acid and perform HPLC purification. Separation method: 1. Instruments: Waters 2767 preparative HPLC; Column: SunFire@PrepC18 (19mm×250mm). 2. Filter the sample through a 0.45μm filter to prepare the sample solution. 3. Preparative chromatographic conditions: a. Mobile phase A, B composition: A: 0.1% trifluoroacetic acid / H2O, B: CH3CN; b. Gradient elution: mobile phase content 5%-45%; c. Flow rate: 12ml / min; d. Elution time: 30min, retention time: 18min. Lyophilization yields compound 28.

[0752] LCMS m / z = 888.8 [M / 3 + H] + 666.8 [M / 4+H] +

[0753] Example 29:

[0754] Using the above-mentioned material list as raw materials, compound 29 (420 mg, 96%) was synthesized using the method of compound 8.

[0755] LCMS m / z = 888.5 [M / 3 + H] + 666.8 [M / 4+H] + .

[0756] Example 31:

[0757] Using the above-mentioned material list as raw materials, compound 31 (200 mg, purity 98%) was obtained by the method of compound 8.

[0758] LCMS m / z = 920.5 [M / 3 + H] + .

[0759] Example 32:

[0760] Using the above-mentioned material list as raw materials, compound 32 (100 mg, purity 98%) was synthesized using the method of compound 8.

[0761] LCMS m / z = 908.1 [M / 3 + H] + .

[0762] Example 33:

[0763] Using the above-mentioned material list as raw materials, compound 33 (400 mg, purity 98%) was synthesized using the method of compound 8.

[0764] LCMS m / z = 1003.5[M / 2 + H] + 669.3 [M / 3+H] + .

[0765] Example 42:

[0766] Using the above-mentioned material list as raw materials, compound 42 (60 mg, 93.8%) was synthesized using the method of compound 8.

[0767] LCMS m / z = 655.1 [M / 3 + H] + 981.9 [M / 2+H] + .

[0768] Example 43:

[0769] Using the above-mentioned material list as raw materials, compound 43 (15 mg, 94.4%) was synthesized using the method of compound 8.

[0770] LCMS m / z = 915.3 [M / 3 + H] + 1372.2 [M / 2+H] + .

[0771] Example 44

[0772] Step 1: Using the above material list as raw materials, compound 44B is obtained using the method of compound 8.

[0773] Step 2: Add 50 ml of lysis buffer (2% trifluoroacetic acid + 98% dichloromethane) to a 100 ml reaction flask, stir well, then add peptide resin 44B (3 g), stir at room temperature for 0.5 hours, filter the resin to obtain filtrate, lyse the peptide resin again using the above lysis method, collect the filtrates obtained from the two lysis processes, wash twice with water, wash once with saturated brine, dry with anhydrous sodium sulfate, and vacuum dry to obtain compound 44C (1.2 g).

[0774] Step 3: Compound 44C (600 mg, 0.19 mmol), intermediate 19 (500 mg, 0.60 mmol), dichloromethane (20 mL), DIC (37 mg, 3 mmol), HOBT (40 mg, 3 mmol), DMAP (4 mg, 0.04 mmol), and DIEA (100 mg, 0.4 mmol) were added to a 250 mL flask. The mixture was reacted at room temperature for 16 h. LC-MS showed that the reaction was halfway complete. The mixture was washed twice with water, once with saturated brine, dried over anhydrous sodium sulfate, and then dried under vacuum to obtain compound 44D (1.2 g).

[0775] Step 4: Add 20 ml of lysis buffer (91% trifluoroacetic acid + 4% triisopropylsilane + 3% 1,2-ethylenedithiol + 2% water) to a 100 ml reaction flask, stir well, then add compound 44D (1.2 g), and stir at room temperature for 1 hour. Filter through resin to obtain the filtrate, add the filtrate to 200 ml of methyl tert-butyl ether (pre-cooled to 0°C), a white flocculent precipitate forms, centrifuge (3 min at 3000 rpm). Wash the white precipitate three times with methyl tert-butyl ether, and vacuum dry to obtain a white solid crude peptide compound 44E (600 mg).

[0776] Step 5: In a 2L reaction flask, add water (210mL), acetonitrile (90mL), and compound 44E (600mg, 0.22mmol) sequentially. After stirring thoroughly, slowly add iodine / acetonitrile solution (0.1mol / L) until the reaction solution turns pale yellow. Quench with ascorbic acid and perform HPLC purification. Separation method: 1. Instruments: Waters 2767 preparative HPLC; column: SunFire@PrepC18 (19mm×250mm). 2. Filter the sample through a 0.45μm filter to prepare the sample solution. 3. Preparative chromatographic conditions: a. Mobile phase A, B composition: A: 0.1% trifluoroacetic acid / H2O, B: CH3CN; b. Gradient elution: mobile phase content 5%-45%; c. Flow rate: 12ml / min; d. Elution time: 30min, retention time: 14min. Lyophilization yielded compound 44 (22mg, 99% purity).

[0777] LCMS m / z = 902.8 [M / 3 + H] + 1353.6 [M / 2+H] + .

[0778] Example 45:

[0779] Using the above-mentioned material list as raw materials, compound 45 (513 mg, purity 99.07%) was synthesized using the method of compound 8.

[0780] LCMS m / z = 910.6 [M / 3 + H] + .

[0781] Example 46:

[0782] Using the above-mentioned material list as raw materials, compound 46 (10 mg, purity 94.46%) was synthesized using the method of compound 8.

[0783] LCMS m / z = 907.4 [M / 3 + H] + .

[0784] Example 47:

[0785] Using the above-mentioned material list as raw materials, compound 47 (0.8g, purity 99.59%) was synthesized using the method of compound 8.

[0786] LCMS m / z = 673.2[M / 3 + H] + .

[0787] Example 48:

[0788] Using the above-mentioned material list as raw materials, compound 48 (0.77 g, purity 98.43%) was synthesized using the method of compound 8.

[0789] LCMS m / z = 668.9 [M / 3 + H] + .

[0790] Example 49:

[0791] Using the above-mentioned material list as raw materials, compound 49 (60 mg, 99.8%) was synthesized using the method of compound 8.

[0792] LCMS m / z = 1016.2[M / 2 + H] + .

[0793] Example 50:

[0794] Using the above-mentioned material list as raw materials, compound 50 (65 mg, 99.8%) was synthesized using the method of compound 8.

[0795] LCMS m / z = 893.5 [M / 3 + H] + 1339.7 [M / 2+H] + .

[0796] Example 51:

[0797] Using the above-mentioned material list as raw materials, compound 51 (30 mg, 98.5%) was synthesized using the method of compound 8.

[0798] LCMS m / z = 916.8 [M / 3 + H] + 1374.7 [M / 2+H] + .

[0799] Example 52:

[0800] Using the above-mentioned material list as raw materials, compound 52 (200 mg, purity 99.4%) was synthesized using the method of compound 8.

[0801] LCMS m / z = 1009.5 [M / 2 + H] + .

[0802] Example 53:

[0803] Using the above-mentioned material list as raw materials, compound 53 (50 mg, purity 99.7%) was synthesized using the method of compound 8.

[0804] LCMS m / z = 910.9[M / 3 + H] + .

[0805] Example 54:

[0806] Using the above-mentioned material list as raw materials, compound 54 (200 mg, purity 99.8%) was synthesized using the method of compound 8.

[0807] LCMS m / z = 996.7 [M / 2 + H] + .

[0808] Example 55:

[0809] Using the above-mentioned material list as raw materials, compound 55 (150 mg, purity 99.7%) was synthesized using the method of compound 8.

[0810] LCMS m / z = 996.7 [M / 2 + H] + .

[0811] Example 56:

[0812] Using the above-mentioned material list as raw materials, compound 56 (120 mg, purity 94.6%) was synthesized using the method of compound 8.

[0813] LCMS m / z = 908.0 [M / 3 + H] + .

[0814] Example 57:

[0815] Using the above-mentioned material list as raw materials, compound 57 (150 mg, purity 98.3%) was synthesized using the method of compound 8.

[0816] LCMS m / z = 908.1 [M / 3 + H] + .

[0817] Example 58:

[0818] Step 1: Compound 29 (150 mg, 0.056 mmol) was dissolved in methanol (10 mL), and p-toluenesulfonic acid (3.67 mL, 0.028 mmol) was added. The mixture was stirred at room temperature for 72 h to prepare and purify compound 58 (25 mg, 98% purity).

[0819] LC-MS (ESI): m / z = 898.2 [M / 3 + H] + .

[0820] Example 59:

[0821] Using the above-mentioned material list as raw materials, compound 59 (60 mg, purity 98.4%) was synthesized using the method of compound 8.

[0822] LCMS m / z = 909.9 [M / 3 + H] + .

[0823] Example 60:

[0824] Using the above-mentioned material list as raw materials, compound 60 (50 mg, purity 99.7%) was synthesized using the method of compound 8.

[0825] LCMS m / z = 976.7 [M / 3 + H] + .

[0826] Example 61:

[0827] Using the above-mentioned material list as raw materials, compound 61 (30 mg, 95.0%) was synthesized using the method of compound 8.

[0828] LCMS m / z = 903.5[M / 3 + H] + 1354.7 [M / 2+H] + .

[0829] Example 62:

[0830] Using the above-mentioned material list as raw materials, compound 62 (0.5g, purity 99%) was synthesized using the method of compound 8.

[0831] LCMS m / z = 898.0 [M / 3 + H] + .

[0832] Example 63:

[0833] Using the above-mentioned material list as raw materials, compound 63 (0.5g, purity 97.6%) was synthesized using the method of compound 8.

[0834] LCMS m / z = 897.9 [M / 3 + H] + .

[0835] Example 64:

[0836] Using the above-mentioned material list as raw materials, compound 64 (11 mg, 91.57%) was synthesized using the method of compound 8.

[0837] LCMS m / z = 902.2[M / 3+H] + 1352.8 [M / 2+H] + .

[0838] Example 65:

[0839] Using the above-mentioned material list as raw materials, compound 65 (80 mg, 94.5%) was synthesized using the method of compound 8.

[0840] LCMS m / z = 898.8 [M / 3 + H] + .

[0841] Example 66:

[0842] Using the above-mentioned material list as raw materials, compound 66 (60 mg, 95.11%) was synthesized using the method of compound 8.

[0843] LCMS m / z = 902.8 [M / 3 + H] + 1353.6 [M / 2+H] + .

[0844] Example 67:

[0845] Using the above-mentioned material list as raw materials, compound 67 (4 mg, 98.3%) was synthesized using the method of compound 8.

[0846] LCMS m / z = 893.5 [M / 3 + H] + .

[0847] Example 68:

[0848] Using the above-mentioned material list as raw materials, compound 68 (0.5g, purity 95.78%) was synthesized using the method of compound 8.

[0849] LCMS m / z = 907.6 [M / 3 + H] + .

[0850] Example 70:

[0851] Using the above-mentioned material list as raw materials, compound 70 (18 mg, purity 98.38%) was synthesized using the method of compound 8.

[0852] LCMS m / z = 919.9 [M / 3 + H] + .

[0853] Example 71:

[0854] Using the above-mentioned material list as raw materials, compound 71 (80 mg, 97.95%) was synthesized using the method of compound 8.

[0855] LCMS m / z = 924.9 [M / 3 + H] + 1386.6 [M / 2+H] + .

[0856] Example 72:

[0857] Using the above-mentioned material list as raw materials, compound 72 (0.11 g, purity 93.57%) was synthesized using the method of compound 8.

[0858] LCMS m / z = 799.0 [M / 4 + H] + 1065.1 [M / 3+H] + .

[0859] Example 73:

[0860] Using the above-mentioned material list as raw materials, compound 73 (0.5g, purity 98.67%) was synthesized using the method of compound 8.

[0861] LCMS m / z = 1550.4 [M / 2 + H] + .

[0862] Example 74:

[0863] Using the above-mentioned material list as raw materials, compound 74 (0.5g, purity 99.75%) was synthesized using the method of compound 8.

[0864] LCMS m / z = 929.2 [M / 3 + H] + .

[0865] Example 75:

[0866] Using the above-mentioned material list as raw materials, compound 75 (50 mg, purity 97%) was synthesized using the method of compound 8.

[0867] LCMS m / z = 929.1 [M / 3 + H] + .

[0868] Example 76:

[0869] Using the above-mentioned material list as raw materials, compound 76 (80 mg, 95.0%) was synthesized using the method of compound 8.

[0870] LCMS m / z = 752.5 [M / 4 + H] + 1002.8 [M / 3+H] + .

[0871] Example 77:

[0872] Using the above-mentioned material list as raw materials, compound 77 (50 mg, 95% purity) was synthesized using the method of compound 8.

[0873] LCMS m / z = 783.5 [M / 4 + H] + 1044.3 [M / 3+H] + .

[0874] Example 78:

[0875] Using the above-mentioned material list as raw materials, compound 78 (90 mg, 99.45%) was synthesized using the method of compound 8.

[0876] LCMS m / z = 768.2[M / 4+H] +1023.8 [M / 3+H] + .

[0877] Example 79:

[0878] Using the above-mentioned material list as raw materials, compound 79 (170 mg, purity 96.66%) was synthesized using the method of compound 8.

[0879] LCMS m / z = 919.5 [M / 3 + H] + .

[0880] Example 80:

[0881] Using the above-mentioned material list as raw materials, compound 80 (290 mg, purity 97.30%) was synthesized using the method of compound 8.

[0882] LCMS m / z = 914.8 [M / 3 + H] + .

[0883] Example 81:

[0884] Using the above-mentioned material list as raw materials, compound 81 (210 mg, 96%) was synthesized using the method of compound 8.

[0885] LCMS m / z = 910.1 [M / 3 + H] + 1364.4 [M / 2+H] + .

[0886] Example 82:

[0887] Using the above-mentioned material list as raw materials, compound 82 (160 mg, 97%) was synthesized using the method of compound 8.

[0888] LCMS m / z = 879.2 [M / 3 + H] + 1318.2 [M / 2+H] + .

[0889] Biological testing evaluation

[0890] The present invention will be further described and explained below with reference to test examples, but these embodiments are not intended to limit the scope of the present invention.

[0891] 1. IL-23α / IL-12β & IL-23R binding test experiment

[0892] The inhibition of IL-23α / IL-12β & IL-23R binding by the compounds was tested using the TR-FRET method. Solutions of protein IL-23α / IL-12β (ACRO, Cat#ILB-H52W5) and IL-23R (ACRO, Cat#ILR-H82F3) were prepared in reaction buffer PPI (PerkinElmer, Cat#61DB10RDF). The final concentrations of IL-23α / IL-12β and IL-23R in the reaction mixture were both 0.3 nM. 0.1 μL of the analyte diluted in DMSO was transferred to a 384-well plate (Grenier, Cat#784075) using a nano-pipette system (Echo 655) and centrifuged at 1000 rpm for 1 minute. 2.5 μL of IL-23α / IL-12β solution was transferred to the 384-well plate and centrifuged at 1000 rpm for 1 minute, then incubated at 25°C for 60 minutes. 2.5 μL of IL-23R solution was transferred to the 384-well plate and centrifuged at 1000 rpm for 1 minute. 5 μL of the Streptavidin-Tb cryptate (PerkinElmer, Cat#610SATLA) and Anti 6HIS-d2 (PerkinElmer, Cat#61HISDLB) detection mixture was transferred to the 384-well plate and centrifuged at 1000 rpm for 1 minute, then incubated at 25°C for 60 minutes. Finally, the HTRF signal (Ratio) was read using a BMG high-throughput drug screening multi-mode microplate reader. (665 / 620nm). The IC50 (half-maximum inhibitory concentration) value of the compound was obtained using the four-parameter nonlinear fitting formula (Y=Bottom+(Top-Bottom) / (1+10^((LogIC50-X)*HillSlope))) in GraphPad Prism software.

[0893] Table 1

[0894] Conclusion: The compounds of the present invention, such as the compounds in the examples, have a significant inhibitory effect on the binding of IL-23α / IL-12β and IL-23R.

[0895] 2. IL-23R reporter gene assay

[0896] The aim of this assay was to evaluate the ability of the compound to inhibit the binding of IL23p19 and IL23R in a reporter gene system. HEK-blue IL23 reporter gene cell line (Invivogen, hkb-il23) was cultured in DMEM + 10% FBS + 100 μg / mL Normocin medium. When the cell density reached 80%-90%, cells were seeded at 5000 cells / well in 384-well plates and cultured overnight at 37°C and 5% CO2. The stock solution of the compound was then diluted in DMSO, and 40 nmol of the diluted solution was transferred to 384-well plates via Echo. The plates were incubated at 37°C and 5% CO2 for 0.5 h. 40 nmol / well of rhIL23 (R&D, 1290-IL) was added to the 384-well cell culture plates to a final concentration of 1 ng / mL, and the plates were incubated at 37°C and 5% CO2 for 24 h. 18 μL of Quanti-Blue was then added. TM The solution was added to a new 384-well plate, and 2 μL / well of cell culture supernatant was transferred to the 384-well plate prepared in step 6. The plate was incubated at 37°C and 5% CO2 for 1 hour. Absorbance values ​​were read on a BMG spectrophotometer from 620 to 655 nM. The binding affinity of the compound was evaluated using the following formula, and IC50 was fitted using Graphpad. 50 .

[0897] Inhibition rate calculation formula:

[0898] DMSO control group mean

[0899] Mean value of the group without IL23 stimulation

[0900] IC 50 The calculation formula is as follows: Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope))

[0901] X: Compound concentration

[0902] Y: Compound inhibition rate

[0903] Conclusion: The compounds of the present invention, such as the compounds in the examples, have a significant inhibitory effect on the binding of IL23p19 and IL23R.

[0904] 3. IL-23-stimulated pSTAT3 detection assay in PBMCs

[0905] Frozen human PBMCs were thawed and seeded onto plates pre-coated with CD3 antibody, at a density of 1 × 10^6 cells per well. CD28 antibody was then added to the plates, and the cells were incubated at 37°C and 5% CO2 for 5 days. On day 5, after 4 hours of FBS starvation stimulation, cells were seeded onto 96-well plates at a density of 100 K cells per well. The diluted compound was transferred to 96-well cell culture plates and incubated at 37°C and 5% CO2 for 1 hour. rhIL23 (R&D, 1290-IL) was added to the cell culture plates, and the plates were incubated at 37°C and 5% CO2 for 30 minutes. Cells in the wells were lysed on ice for 30 minutes with lysis buffer containing 1 × PHOSstop solution, followed by centrifugation at 1000 rpm for 1 minute. The supernatant was then transferred to 96-well ELISA plates, and pSTAT3 ELISA was performed according to the kit (CST, 7300CA) instructions. The absorbance was read at 450 nM on a PHERAstar FSX (BMG LRBTECH) spectrophotometer. The inhibition rate of the compound was evaluated using the following formula, and IC50 was fitted using Graphpad. 50 .

[0906] Inhibition rate calculation formula:

[0907] DMSO control group mean

[0908] Mean value of the group without IL23 stimulation

[0909] IC 50 The calculation formula is as follows: Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope))

[0910] X: Compound concentration

[0911] Y: Compound inhibition rate.

[0912] Conclusion: The compounds of the present invention, such as the compounds in the examples, have a significant inhibitory effect on STAT3 phosphorylation.

[0913] 4. Pharmacokinetic assays in mice

[0914] 4.1 Experimental animals: Male Balb / c mice, 20–25 g, 6 mice / compound. Purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

[0915] 4.2 Experimental Design: On the day of the experiment, Balb / c mice were randomly divided into groups according to their body weight. They were fasted for 12-14 hours before administration but allowed free access to water. They were fed 4 hours after administration.

[0916] Table 2. Dosage Information Note: Intravenous administration solvent: PBS; Gavage administration solvent: PBS

[0917] Blood samples of 0.06 mL were collected via the orbital cavity before and after isoflurane anesthesia, placed in EDTAK2 centrifuge tubes, and centrifuged at 5000 rpm for 10 min at 4°C to collect plasma. Blood collection time points for both the intravenous and gavage groups were 0, 5, 15, 30 min, 1, 2, 4, 6, 8, 24, 48, and 72 h. All samples were stored at -80°C before analysis and quantitative analysis was performed using LC-MS / MS.

[0918] Table 3. Pharmacokinetic parameters of the tested compounds in mouse plasma -: Not applicable. Note: Blood samples for compounds 14, 21, 24, 26, 31, and 32 were collected up to 24 hours prior. Compound 32 was administered via IV (0.89 mg / kg) and Ig (8.9 mg / kg).

[0919] Conclusion: The compounds of the present invention, such as the compounds in the examples, have favorable pharmacokinetic characteristics in mice.

[0920] 5. Rat pharmacokinetic test

[0921] 5.1 Experimental animals: Male SD rats, approximately 220g, 6-8 weeks old, 6 rats / compound. Purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

[0922] 5.2 Experimental Design: On the day of the experiment, SD rats were randomly divided into groups according to their body weight. They were fasted for 12-14 hours before drug administration but allowed free access to water. They were fed 4 hours after drug administration.

[0923] Table 4. Dosage Information Note: Intravenous administration solvent: PBS; Gavage administration solvent: PBS

[0924] Blood samples of 0.15 ml were collected via the orbital cavity before and after isoflurane anesthesia, placed in EDTAK2 centrifuge tubes, and centrifuged at 5000 rpm for 10 min at 4°C to collect plasma. Blood collection time points for both the intravenous and gavage groups were 0, 5, 15, 30 min, 1, 2, 4, 6, 8, 24, 48, and 72 h. All samples were stored at -80°C before analysis and quantitative analysis was performed using LC-MS / MS.

[0925] Table 5. Pharmacokinetic parameters of the tested compounds in rat plasma -: Not applicable. Note: Blood samples for compounds 31, 32, and 62 were collected up to 24 hours prior.

[0926] Conclusion: The compounds of the present invention, such as the compounds in the examples, have good pharmacokinetic characteristics in rats.

[0927] 6. Pharmacokinetics of Beagle Dogs

[0928] 6.1 Experimental animals: Male beagle dogs, weighing approximately 8–11 kg, 6 dogs per compound, purchased from Beijing Mars Biotechnology Co., Ltd.

[0929] 6.2 Experimental Methods: On the day of the experiment, beagles were randomly grouped according to their body weight. They were fasted for 12–14 hours prior to administration but allowed free access to water. Food was given 4 hours after administration.

[0930] Table 6. Dosage Information Note: Intravenous administration solvent: Saline; Gavage administration solvent: Saline

[0931] Blood samples (1 ml) were collected via jugular or limb veins before and after drug administration and placed in EDTAK2 centrifuge tubes. Plasma was collected by centrifugation at 5000 rpm and 4°C for 10 min. Blood collection time points for both the intravenous and gavage groups were: 0, 5, 15, 30 min, 1, 2, 4, 6, 8, 10, 12, 24, 48, 72, 96, 120, and 144 h. All samples were stored at -80°C before analysis and quantitative analysis was performed using LC-MS / MS.

[0932] Table 7. Pharmacokinetic parameters of the tested compounds in canine plasma -: Not applicable. Note: Blood samples for Compound 29 were collected up to 24 hours prior.

[0933] Conclusion: The compounds of the present invention, such as the compounds in the examples, have good pharmacokinetic characteristics in dogs.

[0934] 7. Pharmacokinetics in monkeys

[0935] 7.1 Experimental animals: Male cynomolgus monkeys, 3–5 kg, 3–6 years old, 5 animals / compound. Purchased from Suzhou Xishan Biotechnology Co., Ltd.

[0936] 7.2 Experimental Methods: On the day of the experiment, monkeys were randomly divided into groups according to their body weight. They were fasted for 14–18 hours before administration but allowed free access to water. They were fed 4 hours after administration.

[0937] Table 8. Dosage Information Note: Intravenous administration solvent: Saline; Gavage administration solvent: Saline

[0938] Blood samples of 1.0 mL were collected from venous sites in the extremities before and after drug administration and placed in EDTAK2 centrifuge tubes. Plasma was collected by centrifugation at 5000 rpm and 4°C for 10 min. Blood collection time points for the venous group were: 0, 5 min, 15 min, 30 min, 1, 2, 4, 6, 8, 10, 12, 24, 48, 72, 96, 120, and 144 h. All samples were stored at -80°C before analysis and quantitative analysis was performed using LC-MS / MS.

[0939] Table 9. Pharmacokinetic parameters of the tested compounds in monkey plasma -:not applicable.

[0940] Note: For compound 29, blood was collected for control compound 1 up to 96 hours later.

[0941] The control compound 1 is Example 4 in patent WO2023288019A2.

[0942] Conclusion: The compounds of the present invention, such as the compounds in the examples, have good pharmacokinetic characteristics in monkeys.

[0943] 8. Plasma stability test

[0944] This experiment used plasma from five species—human, monkey, dog, rat, and mouse—to evaluate the plasma stability of the compounds.

[0945] Plasma samples with a concentration of 1000 ng / mL were prepared and aliquoted into EP tubes at time points of 0 h and 6 h. For the 0 h samples, acetonitrile solution containing the internal standard was added directly, while for the 6 h samples, the solution was added after incubation at 37°C for the corresponding time. The concentration of the analyte in the samples was determined using LC-MS / MS. The residual rate was calculated as the ratio of the peak area of ​​the analyte to the internal standard in the time point samples to the peak area of ​​the sample at time zero.

[0946] 9. Stability test of gastrointestinal fluids

[0947] 9.1. Solution Preparation

[0948] Preparation of dilute hydrochloric acid: Measure 23.4 ml of hydrochloric acid and dilute it with water to 1000 ml.

[0949] Preparation of artificial gastric juice: Take 1.64 ml of dilute hydrochloric acid, add about 80 ml of water and 1 g of pepsin, shake well, and then dilute with water to 100 ml.

[0950] To prepare 0.1 mol / L NaOH: Weigh 0.4 g of NaOH and dissolve it in 100 ml of water.

[0951] Preparation of artificial intestinal fluid: Dissolve 0.68g of potassium dihydrogen phosphate in 50ml of water, and adjust the pH to 6.8 with 0.1mol / L sodium hydroxide solution; dissolve 1g of pancreatic enzyme in an appropriate amount of water, mix the two solutions, and dilute with water to 1000ml to obtain the final solution.

[0952] 9.2. Sample Preparation

[0953] Take approximately 12.5 mg of the sample and dissolve it in a 25 ml volumetric flask. Add artificial gastric fluid (or artificial intestinal fluid) to dissolve and dilute to the mark.

[0954] 9.3. Analytical Methods

[0955] Instrument model: Agilent 1260 Infinity; Mobile phase A: 10 mmol / L K₂HPO₄; Mobile phase B: Acetonitrile; Column: Phenomenex Gemini @ 3um C18 150*4.6mm; Wavelength: 224nm; Column temperature: 30℃; Sample tray temperature: 37℃; Injection time: 35min; Injection volume: 10ul; Injection method: Gradient injection; Gradient method. The gradient mobile phase is shown in the table.

[0956] 9.4. Sampling, Testing and Results

[0957] Sampling and testing: First, test the blank solution (artificial gastric fluid or artificial intestinal fluid), then take the prepared sample (freshly prepared before use) and put it into the injection tray for immediate injection, and then inject the sample once at each time.

[0958] Conclusion: The compounds of the present invention, such as the compounds in the examples, have good stability in artificial gastrointestinal fluids.

[0959] 10. hERG potassium ion channel function test

[0960] Experimental platform: Electrophysiological manual patch-clamp system

[0961] Cell line: Chinese hamster ovary (CHO) cell line stably expressing hERG potassium channels

[0962] Experimental Methods: CHO (Chinese Hamster Ovary) cells stably expressing the hERG potassium channel were used to record hERG potassium channel currents at room temperature using whole-cell patch-clamp technique. Glass microelectrodes were fabricated from glass electrode blanks (BF150-86-10, Sutter) using a stretching device. The tip resistance after perfusion with electrode fluid was approximately 2-5 MΩ. The glass microelectrodes were inserted into the amplifier probe to connect to the patch-clamp amplifier. Clamping voltage and data recording were controlled and recorded using pClamp 10 software via computer, with a sampling frequency of 10 kHz and a filtering frequency of 2 kHz. After obtaining whole-cell recordings, the cells were clamped at -80 mV. The step voltage to induce hERG potassium current (IhERG) was applied from -80 mV with a 2-second depolarization voltage to +20 mV, then repolarized to -50 mV, held for 1 second, and then returned to -80 mV. This voltage stimulation was applied every 10 seconds. After confirming that the hERG potassium current was stable (at least 1 minute), the drug delivery process began. Each compound was administered for at least 1 minute at each test concentration, and at least 2 cells were tested for each concentration (n≥2).

[0963] Data processing: Data analysis and processing were performed using pClamp 10, GraphPad Prism 5, and Excel software. The degree of inhibition of hERG potassium current (the peak hERG tail current induced at -50mV) by different compound concentrations was calculated using the following formula:

[0964] Inhibition%=[1-(I / Io)]×100%

[0965] Where Inhibition% represents the percentage of inhibition of hERG potassium current by the compound, and I and Io represent the amplitude of hERG potassium current before and after drug administration, respectively.

[0966] The IC50 of the compound was calculated using GraphPad Prism 5 software by fitting the following equation:

[0967] Y=Bottom+(Top-Bottom) / (1+10^((LogIC50-X)*HillSlope))

[0968] Where X is the Log value of the detected concentration of the test sample, Y is the inhibition percentage at the corresponding concentration, and Bottom

[0969] Top and Minimum and Maximum Suppression Percentages, respectively.

[0970] Conclusion: The compounds of the present invention, such as the compounds in the examples, do not inhibit hERG.

[0971] 11. CYP enzyme inhibition test

[0972] The aim of this study was to evaluate the effects of test substances on the activities of five isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) of human liver microsomal cytochrome P450 (CYP) using an in vitro assay system. Specific probe substrates for CYP450 isoenzymes were co-incubated with human liver microsomes and different concentrations of the test substances. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) was added to initiate the reaction. After the reaction, the metabolites produced by the specific substrates were quantitatively detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS) after sample processing. Changes in CYP enzyme activity were measured, IC50 values ​​were calculated, and the inhibitory potential of the test substances against each CYP enzyme isoform was evaluated. Under the test conditions, the incubation concentration ranged from 0 to 30 μM.

[0973] Conclusion: The compounds of the present invention, such as the compounds in the examples, do not inhibit CYP enzymes.

[0974] 12. Liver microsomal stability test

[0975] This experiment used liver microsomes from five genera—human, canine, rat, and mouse—as in vitro models to evaluate the metabolic stability of the test substance.

[0976] At 37°C, 1 μM of the test substance was co-incubated with microsomal protein and coenzyme NADPH. The reaction was terminated by adding ice-cold acetonitrile containing internal standard after a certain time (5, 10, 20, 30, 60 min). The concentration of the test substance in the sample was detected by LC-MS / MS. T1 / 2 was obtained by using the ln value of the drug residue in the incubation system and the incubation time. The intrinsic clearance rate of liver microsomes CLint(mic) and intrinsic clearance rate of liver CLint(Liver) were further calculated.

[0977] Conclusion: The compounds of the present invention, such as the compounds in the examples, have good liver microsomal stability.

[0978] 13. CaCO2 Permeability Test

[0979] The experiment used monolayers of Caco-2 cells, incubated in triple parallel in 96-well Transwell plates. A transport buffer solution (HBSS, 10 mM HEPES, pH 7.4±0.05) containing the compound of the present invention (2 μM) or the control compound (2 μM), digoxin (10 μM), naldolol (2 μM), and metoprolol (2 μM) was added to the dosing well on the apical or basal side. A transport buffer solution containing DMSO was added to the corresponding receiving well. After incubation at 37±1 °C for 2 hours, the cell plate was removed, and appropriate amounts of sample were transferred from the apical and basal sides to new 96-well plates. Acetonitrile containing an internal standard was then added to precipitate the protein. The samples were analyzed using LC MS / MS to determine the concentrations of the compound of the present invention and the control compound. The concentration data were used to calculate the apparent permeability coefficients for transport from the apical to the basal side of the monolayer cells, and from the basal side to the apical side. The integrity of the monolayer cells after 2 hours of incubation was evaluated by the leakage of fluorescein.

[0980] Conclusion: The permeability of the compounds of the present invention, such as compound 29 of Example, is 2.5 times that of control compound 1.

[0981] 14. Pharmacodynamic testing of the test substance in an IL-23-induced rat skin inflammation model

[0982] SD rats (purchased from Sichuan Vital River Laboratory Animal Co., Ltd.) were grouped according to ear thickness 3 days before modeling (D-3). A 7-day intragastric administration period was conducted (D-2 to D4). These groups were designated as G1-... Groups G1-G2 were divided into four groups: G2-Model, G3-Compound 29-3 mg / kg / day, G4-Compound 29-10 mg / kg / day, and G5-JNJ-77242113-10 mg / kg / day. Animals were administered the drug via gavage for 6 consecutive days (first administration recorded as Day-2, last administration as Day-4). G6-IL-23 antibody (Guselkumab) was administered twice daily at 25 mg / kg on Days 0 and 2. Groups G1-G2 received a blank solvent. During the experiment, 1-1.5 hours after administration on Days 0-3, animals were anesthetized with isoflurane via inhalation using a respiratory anesthesia machine. Subsequently, 10 μL of a 0.5 mg / mL IL-23 (R&D, 11349-IL) solution was injected intradermally into the right ear to establish the model. Each group was injected with an equal volume of PBS solution. On Days 3, 0, 1, 2, 3, and 4, the right ear thickness of the participating animals was measured using a digital micrometer. Based on the measured ear thickness data, the average right ear thickness and its rate of change were statistically analyzed for each group. At the experimental endpoint, the inhibition rate of ear thickness growth in each treatment group was calculated using the Model group and the Sham group as references, to evaluate and compare the efficacy of the test compound, the control JNJ-77242113, and the anti-IL-23 antibody (Guselkumab) in an IL-23-induced skin inflammation model in SD rats. The significance of the experimental endpoint was analyzed using a one-way ANOVA method in Graphpad Prism. All groups were compared with the G2-Model group; **** represents p < 0.0001.

[0983] Table 10 Animal grouping and dosing design Note: JNJ-77242113 is SEQ ID NO:104 in WO2021146441A1.

[0984] The experimental results are shown in Figures 1 and 2. Analysis of the results indicates that compound 29 effectively inhibited ear thickness growth in rats in a dose-dependent manner. At the same dose, 10 mg / kg of compound 29 showed a superior inhibitory effect on ear thickness growth compared to the control compound JNJ-77242113. Compared to the anti-IL-23 antibody, compound 29 also exhibited a better ear thickness inhibition rate at 10 mg / kg than the control antibody.

[0985] 15. PD Study of the Test Drug in an Imiquimod-Induced Rat Model

[0986] IL-17A is a major downstream cytokine of IL-23. This study used IL-17A as a PD indicator to investigate the inhibitory effect of the test drug on IL-17A in an orally administered rat model.

[0987] SD rats (purchased from Vital River) were randomly divided into three groups based on body weight and back skin thickness. Starting Day 0, imiquimod (Sichuan Mingxin Pharmaceutical Co., Ltd.) was applied to the back skin of the rats daily in the afternoon to sensitize them for 4 days. Starting Day 2, the rats were administered the test drug by gavage once daily for 7 days. Detailed grouping and administration protocols are shown in Table 11. The final administration was given on the morning of Day 4. Whole blood samples were collected at 0h, 1h, 4h, 8h, 24h, and 48h after administration, and heparin sodium was used for anticoagulation.

[0988] Whole blood samples were diluted 1:4 with RPMI-1640 and transferred to 96-well plates (240 μL). After incubation at 37°C for 30 minutes, the plates were stimulated for 24 hours with IL-23 and IL-1β at final concentrations of 40 ng / mL and 40 ng / mL, respectively. The supernatant was then collected, and the IL-17A content in the supernatant was determined using an ELISA method. Graphpad Prism was used for plotting. The average IL-17A concentration per animal in the Vehicle group was used as the Control group. The relative content of the compound in the Control group corresponding to the average IL-17A concentration at each time point was calculated as follows:

[0989] IL-17A relative concentration % = (Average IL-17A concentration in the treated group / Average IL-17A concentration in the control group) * 100%

[0990] Data analysis: Two-way ANOVA was used, and... Methods: Significance analysis was performed to compare the effects of two drugs on IL-17A secretion levels at the same time point.

[0991] According to PD research results, all compound administrations can effectively inhibit the secretion of IL-17A.

[0992] Table 11 Grouping Information Table

[0993] The test results for IL-17A are shown in the table below:

[0994] Table 12 Detection results of IL-17A BLOQ: Below the detection limit.

[0995] The relative IL-17A content results are shown in the table below:

[0996] Table 13 Relative Content of IL-17A

[0997] Conclusion: Based on the PD inhibition results, compound 29, at the same dose of 10 mg / kg as JNJ-77242113, showed lower relative concentrations (%) of IL-17A at 24 hr and 48 hr after multiple administrations compared to the control compound JNJ-77242113.

Claims

A cyclic peptide compound, stereoisomer thereof, or pharmaceutically acceptable salt thereof, wherein the cyclic peptide compound has an amino acid sequence of Formula (I-1A) or Formula (I-1): wherein: Xa1-H-T-Xa4-Xa5-Xa6-Xa7-[2-Nal]-Xa9-E-N-[3-Pal]-Xa 13 (I-1A)(SEQ ID NO: 6A) Xa1-H-T-Xa4-Xa5-[Pen]-Xa7-[2-Nal]-Xa9-E-N-[3-Pal]-Xa 13 (I-1)(SEQ ID NO: 6) Xa1 is Pen or (D)Pen; Xa4 is Trp(7-methyl) or Trp(7-cyclopropyl); Xa5 is Gin or Lys; Xa6 is Pen or (D)Pen; the cyclic peptide compound is cyclized via a disulfide bond between Xa1 and Xa6, or Xa1 and Pen, and the cyclic peptide compound is optionally linked to a protecting group; Xa7 is or Xa9 is Thp or Xa 13 Sarc or absent; at least one amino acid in the cyclic peptide compound is quaternized and / or conjugated with a modifying group, wherein the modifying group comprises a PEG linker or a quaternary ammonium; and the modifying group comprises at least one ester bond, or the modifying group is linked by forming an ester bond with a side chain of an amino acid in the sequence of Formula (I-1A), Formula (I-1). the cyclic peptide compound is conjugated with a modifying group at at least one of Xa1, Xa5, Xa7, E, Xa13, 3-Pal, or T, the modifying group is -L-R, L comprises a PEG linker, and R is a C1-30 aliphatic acid, a C1-30 heteroaliphatic acid, a C1-30 aliphatic ester, or a C1-30 heteroaliphatic ester. The cyclic peptide compound according to claim 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that, p is 0-50, and q is 1-50; The cyclic peptide compound according to claim 2, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The R is: wherein n is 4-15; preferably R is The cyclic peptide compound according to claim 3, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The L is -L1-L2-L3-, wherein L2 is The cyclic peptide compound of claim 4, stereoisomer thereof, or pharmaceutically acceptable salt thereof, wherein: L1and L3are each independently a bond, in combination, wherein each R l each independently is hydrogen, C 1-3 alkyl, haloC 1-3 alkyl or C 3-6 cycloalkyl, and two R l are not simultaneously hydrogen, m is 0-10. A cyclic peptide compound, stereoisomer thereof, or pharmaceutically acceptable salt thereof, wherein the cyclic peptide compound has an amino acid sequence of Formula (I-2A) or Formula (I-2): wherein: L1is a bond, wherein each R l each independently is hydrogen, C 1-3 alkyl, haloC 1-3 alkyl or C 3-6 cycloalkyl, and two R l are not simultaneously hydrogen, m is an integer from 1 to 10, preferably R l each independently is hydrogen, methyl, ethyl, propyl, monofluoromethyl, difluoromethyl, trifluoromethyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, and two R l are not simultaneously hydrogen; L3 is Preferably, L3 is The cyclic peptide compound according to claim 5, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The L is: p is 1-10, q is 1-2, m is 1-10; preferably Xa1 is Pen or (D)Pen; Xa1-H-T-Xa4-Xa5-Xa6-Xa7-[2-Nal]-Xa9-E-N-[3-Pal]-Xa 13 (I-2A)(SEQ ID NO: 7A) Xa1-H-T-Xa4-Xa5-[Pen]-Xa7-[2-Nal]-Xa9-E-N-[3-Pal]-Xa 13 (I-2) (SEQ ID NO: 7) Xa4 is Trp(7-methyl) or Trp(7-cyclopropyl); Xa5 is Gin or Lys; Xa6 is Pen or (D)Pen; the cyclic peptide compound is cyclized via a disulfide bond between Xa1 and Xa6, or Xa1 and Pen, and the cyclic peptide compound is optionally linked to a protecting group; provided that: Xa7 is or Xa9 is Thp or Xa 13 Sarc or null; A cyclic peptide compound, stereoisomer thereof, or pharmaceutically acceptable salt thereof, wherein the cyclic peptide compound has an amino acid sequence of Formula (II-2): Pen-H-T-[Trp(7-methyl)]-Lys-[Pen]-Xa7-[2-Nal]-[Thp]-E-N-[3-Pal]-[Sarc] (II-2) (SEQ ID NO: 3) said cyclic peptide compound is conjugated at at least one of Xa1, Xa5, Xa7, E, Xa 13 3-Pal or T to -L-R, said R being hydrogen, hydroxyl, C 1-6 alkyl, -COCH 1-6 2alkyl, -COhaloC 1-6 alkyl, said L being as described in any one of claims 4-6; the cyclic peptide compound is cyclized via a Pen-Pen disulfide bond; (1) when Xa4 is Trp (7-cyclopropyl), Xa5 is Gin, Xa9 is and only when the modifying group is conjugated at Xa1, the modifying group is not: (2) when Xa4 is Trp (7-cyclopropyl), Xa5 is Lys, Xa9 is at the time, and only when the modifying group is conjugated at Xa5, the modifying group is not: alternatively, the side chain terminal amino group of amino acid Pen, Lys, or Xa7 is conjugated with an alpha-hydroxyacetic acid, beta-hydroxypropionic acid, 2-methyl lactic acid, an amino acid, alpha-hydroxyisobutyric acid, alpha-hydroxycyclopropanoic acid, alpha-hydroxycyclobutanoic acid, alpha-hydroxycyclopentanoic acid, or 2-cyclopropyl-2-hydroxyacetic acid; the cyclic peptide compound is optionally linked to a protecting group; Xa7 is wherein the compound is selected from one of the structures in Table I. ​ ​ with the proviso that the cyclic peptide compound is not The compound according to any one of claims 1 to 8, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or characterized in that, ​ A pharmaceutical composition comprising a compound according to any one of claims 1-9, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient. A pharmaceutical composition according to claim 10, comprising 1-1500 mg of a compound according to any one of claims 1-9, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient. Use of a compound according to any one of claims 1-9, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 10 or 11, for the manufacture of a medicament for the prevention and treatment of a disease or disorder in a subject in which IL-23 is overexpressed in a diseased tissue. Use according to claim 12, wherein the disease or disorder in which IL-23 is overexpressed is selected from the group consisting of inflammatory bowel disease, Crohn's disease and psoriasis. A method for the treatment of a disease in a mammal or human, said method comprising administering to the subject a therapeutically effective amount, preferably 1-1500 mg, of a compound according to any one of claims 1-9, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, said disease preferably being selected from the group consisting of inflammatory bowel disease, Crohn's disease and psoriasis.

Images