Conjugate, method for preparing same, and use thereof

Abstract

The present invention relates to a small molecule compound modified on the basis of exatecan, a conjugate comprising the small molecule compound, a method for preparing same, and use thereof in disease treatment.

Description

Couplings, their preparation methods and applications Technical Field

[0001] This application relates to the field of biomedicine, specifically to a conjugated drug, its preparation method, and the application of the conjugated drug in the treatment of diseases. Background Technology

[0002] Antibody-drug conjugates (ADCs) are drug molecules obtained by conjugating antibodies and small molecule drugs through linkers. They effectively combine the targeting properties of antibodies with the cytotoxic activity of small molecule drugs, achieving targeted tumor killing and demonstrating promising clinical efficacy. In recent years, ADC molecules using the topoisomerase 1 inhibitor Dxd as the payload and GGFG as the cleavable unit (such as DS-8201 and U3-1402) have achieved remarkable clinical efficacy.

[0003] However, these types of ADCs still generally have safety issues such as side effects and toxicity, which to some extent limit their therapeutic window. In the overall structure of an ADC, the drug payload, cleavable units, connecting subunits, the connection method, and their effective combination all play a crucial role in the overall efficacy and safety of the ADC.

[0004] Therefore, developing topozyme inhibitors, cleavable units, linker units, and / or effective combinations thereof through structural modification is of great value for developing ADCs with a wider therapeutic window. Summary of the Invention

[0005] This application provides a conjugate (or coupling compound), or a pharmaceutically acceptable salt thereof, which may have one or more effects selected from the group consisting of: (1) having inhibitory activity against the in vitro proliferation of tumor cells; (2) having targeted inhibition; (3) having a bystander effect; (4) having plasma stability; (5) having in vivo tumor-suppressive effects; (6) having in vivo tumor-targeting ability; and (7) having good in vivo safety.

[0006] In a first aspect, this application provides a conjugate, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein the conjugate comprises the structure of formula (I):

[0007] In some implementations, R 1 It is a hydrogen or hydrocarbon group. Specifically, the R 1Selected from the group consisting of hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl and 3- to 8-membered cycloalkyl.

[0008] In some implementations, R 2 It is a hydrocarbon group containing one nitrogen atom. Furthermore, R 2 It can contain ring structures.

[0009] In a specific implementation, R 2 for Among them, R 21 Selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, and 3- to 8-membered cycloalkyl, R 22 and R 23 Each is independently selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, and 3- to 8-membered cycloalkyl, R 22 Can be used with R 23 Together they form a ring structure, the ring structure comprising R 22 and R 23 Connected carbon atoms, or R 21 Can be used with R 22 , or, R 21 Can be used with R 23 Together they form a ring structure, the ring structure comprising R 21 The bonded nitrogen atom, R 21 R 22 R 23 Each hydrogen atom in the definition can be independently replaced by F, Cl, Br or I.

[0010] In some implementations, R 3 R 4 and R 5 Each contains a carbonyl group and an imino group.

[0011] Furthermore, in R 3 R 4 or R 5 In this context, the carbonyl group and the imino group are separated by one carbon atom. Specifically, R 3 for Among them, R 31 Selected from the group consisting of hydrogen and C1-C6 alkyl groups. Specifically, R 4 for Among them, R 41 Selected from the following group: hydrogen, C1-C6 alkyl and -R 6 -NH-C(=O)-NH2, where R 6 It is a C1-C6 alkylene group. Specifically, R 5 for Among them, R51 It is a C1-C6 alkyl group.

[0012] In the structure of this application, Represents the connection site. Specifically, equation (I) uses the structure shown in equation (I). It can be directly or indirectly linked to a ligand. For example, formula (I) can be derived through the structure shown in formula (I). It is directly connected to the ligand. For example, formula (I) can be derived through the structure shown in formula (I). The ligand is indirectly connected via a connecting subunit (Equation (I) through the structure shown in Equation (I)). It is connected to the connecting subunit, which in turn is connected to the ligand.

[0013] In a preferred embodiment, formula (I) can form a conjugate containing multiple peptide bonds.

[0014] In a specific implementation, the structure shown in formula (I) can be cleaved to release the small molecule drug therein. This cleavage can occur within the subject.

[0015] In a first embodiment of the first aspect, this application also relates to a conjugate comprising a ligand, a linker unit, a "cleavable unit + small molecule drug (load)", or a pharmaceutically acceptable salt thereof. Specifically, the structure of the small molecule drug is contained in the structure shown in formula (I).

[0016] Furthermore, the conjugate has the structure of formula (II-a):

[0017] Where R 1 R 2 R 3 R 4 and R 5 As defined above, L is a connecting subunit, meaning L can connect n. 1 The fragment shown in formula (I) and an AB; AB is a ligand, preferably an antigen-binding protein, an Fc fusion protein, or an Fc fragment, n 1 Let n be an integer selected from 1 to 4. 2 It can be an integer or decimal selected from 1 to 8. For example, n 1 It can be 1, 2, 3, or 4. For example, n 2 It can be 1, 2, 3, 4, 5, 6, 7, or 8. For example, n 2 It is an integer or decimal of 1 to 2, an integer or decimal of 2 to 3, an integer or decimal of 3 to 4, an integer or decimal of 4 to 5, an integer or decimal of 5 to 6, an integer or decimal of 6 to 7, and an integer or decimal of 7 to 8.

[0018] In some specific embodiments, the structure shown in formula (II-a) can be cleaved to release the small molecule drug therein. This cleavage can occur in vivo, in vitro, or in a tumor environment.

[0019] Furthermore, the conjugate has the structure of formula (II-b):

[0020] Among them, R 1 R 2 R 3 R 4 and R 5 As defined above, P 2 For small molecule drugs, nucleic acids or peptides or combinations thereof, L 2 The unit is a cleavable unit, L' is a linker unit, AB is a ligand, preferably an antigen-binding protein, an Fc fusion protein, or an Fc fragment, and n 1 Let n be an integer selected from 1 to 4. 2 It can be an integer or decimal selected from 1 to 8. For example, n 1 It can be 1, 2, 3, or 4. For example, n 2 It can be 1, 2, 3, 4, 5, 6, 7, or 8. For example, n 2 It is an integer or decimal of 1 to 2, an integer or decimal of 2 to 3, an integer or decimal of 3 to 4, an integer or decimal of 4 to 5, an integer or decimal of 5 to 6, an integer or decimal of 6 to 7, and an integer or decimal of 7 to 8.

[0021] In a specific implementation, the structure shown in formula (II-b) can release the small molecule drug within it through cleavage. This cleavage can occur in vivo, in vitro, or in a tumor environment.

[0022] In a preferred embodiment, in formula (I), formula (II-a), or formula (II-b), wherein R 1 It is hydrogen or a C1-C3 alkyl group. More preferably, in formula (I), formula (II-a) or formula (II-b), wherein R is hydrogen or C1-C3 alkyl. 1 It is hydrogen or methyl, and more preferably hydrogen.

[0023] In a preferred embodiment, in formula (I), formula (II-a), or formula (II-b), wherein R 21 It is hydrogen or C1-C3 alkyl, preferably hydrogen.

[0024] In a preferred embodiment, in formula (I), formula (II-a), or formula (II-b), wherein R 22 and R 23 Each is independently selected from the following group: hydrogen, C1-C6 alkyl and 3- to 6-membered cycloalkyl.

[0025] In a preferred embodiment, in formula (I), formula (II-a), or formula (II-b), wherein R 22 Selected from the group consisting of hydrogen and C1-C6 alkyl groups. In a preferred embodiment, in formula (I), formula (II-a), or formula (II-b), wherein R 23 Selected from the group consisting of hydrogen and C1-C6 alkyl groups.

[0026] In a preferred embodiment, in formula (I), formula (II-a), or formula (II-b), wherein R 22 With R 23 The ring structure formed together is a 3-membered cycloalkyl, a 4-membered cycloalkyl, a 5-membered cycloalkyl, or a 6-membered cycloalkyl, more preferably a 4-membered cycloalkyl.

[0027] In some embodiments, in formula (I), formula (II-a) or formula (II-b), wherein R 2 Selected from the following group:

[0028] Among them, each R 21 Independently hydrogen or methyl, optionally, R 2 Each hydrogen atom in the mixture is independently replaced by F, Cl, Br, or I. Represents the connection site.

[0029] More specifically, in equation (I), equation (II-a), or equation (II-b), where R 2 Selected from the following group:

[0030] Among them, each R 21 Independently hydrogen or methyl, optionally, R 2 Each hydrogen atom in the mixture is independently replaced by F, Cl, Br, or I. Represents the connection site.

[0031] In some embodiments, in formula (I), formula (II-a) or formula (II-b), wherein R 2 Selected from the following group:

[0032] Among them, each R 21 Independently hydrogen or methyl, optionally, R 2 Each hydrogen atom in the mixture is independently replaced by F, Cl, Br, or I. Represents the connection site.

[0033] More specifically, in equation (I), equation (II-a), or equation (II-b), where R 2 Selected from the following group:

[0034] Among them, each R 21 Independently hydrogen or methyl, optionally, R 2 Each hydrogen atom in the mixture is independently replaced by F, Cl, Br, or I. Represents the connection site.

[0035] In some embodiments, in formula (I), formula (II-a) or formula (II-b), wherein R 2 Selected from the following group:

[0036] Among them, each R 21 Independently hydrogen or methyl, Represents the connection site. More specifically, in equation (I), equation (II-a), or equation (II-b), where R 2 Selected from the following group:

[0037] Among them, each R 21 Independently hydrogen or methyl, Represents the connection site.

[0038] More specifically, in equation (I), equation (II-a), or equation (II-b), where R 2 for

[0039] In some embodiments, in formula (I), formula (II-a) or formula (II-b), wherein R 21 It is hydrogen.

[0040] In some embodiments, in formula (I), formula (II-a) or formula (II-b), wherein R 31 Selected from the group consisting of: hydrogen, methyl, ethyl, isopropyl, isobutyl, and sec-butyl.

[0041] In some embodiments, in formula (I), formula (II-a) or formula (II-b), wherein R 3 Selected from the following group:

[0042] Represents the connection site.

[0043] More specifically, in equation (I), equation (II-a), or equation (II-b), where R 3 Selected from the following group:

[0044] Represents the connection site.

[0045] More specifically, in equation (I), equation (II-a), or equation (II-b), where R 3 Selected from the following group:

[0046] Represents the connection site.

[0047] In some embodiments, in formula (I), formula (II-a) or formula (II-b), wherein R 41 Selected from the following group: hydrogen, methyl, ethyl, isopropyl, isobutyl, sec-butyl, -(CH2)2-NH-C(=O)-NH2 and -(CH2)3-NH-C(=O)-NH2.

[0048] In some embodiments, in formula (I), formula (II-a) or formula (II-b), wherein R 4 Selected from the following group:

[0049] Represents the connection site.

[0050] More specifically, in equation (I), equation (II-a), or equation (II-b), where R 4 Selected from the following group:

[0051] Represents the connection site.

[0052] In some embodiments, in formula (I), formula (II-a) or formula (II-b), wherein R 51 Selected from the group consisting of: methyl, ethyl, isopropyl, isobutyl, and sec-butyl.

[0053] In some embodiments, in formula (I), formula (II-a) or formula (II-b), wherein R 5 Selected from the following group:

[0054] Represents the connection site.

[0055] More specifically, in equation (I), equation (II-a), or equation (II-b), where R 5 Selected from the following group:

[0056] Represents the connection site.

[0057] In some embodiments, in formula (I), formula (II-a) or formula (II-b), R 5 for R 4 for In other embodiments, in formula (I), formula (II-a) or formula (II-b), R5 for R 4 for

[0058] In some embodiments, in formula (I), formula (II-a) or formula (II-b), R 5 for R 4 for In other embodiments, in formula (I), formula (II-a) or formula (II-b), R 5 for R 4 for

[0059] In some embodiments, in formula (I), formula (II-a) or formula (II-b), wherein R 1 For hydrogen, R 2 for R 3 for R 4 for R 5 for Represents the connection site.

[0060] In some embodiments, in formula (I), formula (II-a), or formula (II-b), the structure of formula (I) is selected from the group consisting of:

[0061] Represents the connection site.

[0062] In some embodiments, in formula (I), formula (II-a), or formula (II-b), the structure of formula (I) is selected from the group consisting of:

[0063] Represents the connection site.

[0064] In some embodiments, in formula (II-a) or formula (II-b), AB is an antibody or its antigen-binding fragment, an Fc fusion protein, or an Fc fragment. The antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, and / or dAb.

[0065] In some embodiments, in formula (II-a) or formula (II-b), AB includes monoclonal antibodies, chimeric antibodies, humanized antibodies, and / or fully human antibodies.

[0066] In some embodiments, in formula (II-a) or formula (II-b), AB binds to targets selected from the group consisting of tumor-associated antigens, tissue-specific antigens, cell surface molecules, extracellular matrix proteins or proteases, and post-translational modified residues.

[0067] More specifically, in formula (II-a) or formula (II-b), where AB can bind to targets selected from the group consisting of: Trop-2, Her2, Her3, Her4, EGF, EGFR, CD2, CD3, CD5, CD7, CD13, CD19, CD20, CD21, CD23, CD30, CD33, CD34, CD38, CD46, CD55, CD59, CD69, CD70, CD71, CD97, CD117, CD123, CD127, CD134, CD137, CD138, CD146, CD147, CD152, CD154, CD1 74. CD195, CD200, CD205, CD212, CD223, CD227, CD253, CD272, CD274, CD276, CD278, CD279, CD309, CD319, CD326, CD340, DR6, Kv1.3, 5E10, M UC1, uPA, MAGE3, MUC16, KLK3, K-ras, Mesothelin, p53, Survivin, G250, PSMA, Endoplasmin, BCMA, GPNMB, EphA2, EphB2, TMEFF2, Integrin beta 6, 5T4, CA9, IGF-1R, Axl, B7H3, B7H4, CDH6, HAVCR1, STEAP-1, STEAP-2, UPK2, CLDN18, CLDN6, CLDN9, c-Met, MICA, LIV-1, ROR1, ADAM9, Stn, DLK-1 and CEACAM-5.

[0068] In specific embodiments, in formula (II-a) or formula (II-b), AB is an antibody that specifically binds to Her2 or its antigen-binding fragment. For example, AB is an antibody that specifically binds to Her2 and has HCDR1 containing the amino acid sequence shown in SEQ ID NO: 35, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 36, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 37, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 32, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 33, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 34. For example, AB is an antibody that specifically binds to Her2 and has VH containing the amino acid sequence shown in SEQ ID NO: 39 and VL containing the amino acid sequence shown in SEQ ID NO: 38. For example, AB is trastuzumab.

[0069] In specific embodiments, in formula (II-a) or formula (II-b), AB is an antibody that specifically binds to Trop2 or its antigen-binding fragment. For example, AB is an antibody that specifically binds to Trop2 and has HCDR1 containing the amino acid sequence shown in SEQ ID NO: 19, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 20, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 21, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 16, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 17, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 18. For example, AB is an antibody that specifically binds to Trop2 and has VH containing the amino acid sequence shown in SEQ ID NO: 23 and VL containing the amino acid sequence shown in SEQ ID NO: 22. For example, AB is sacitrulline.

[0070] In specific embodiments, in formula (II-a) or formula (II-b), AB is an antibody that specifically binds to EGFR or its antigen-binding fragment. For example, AB is an antibody that specifically binds to EGFR and has HCDR1 containing the amino acid sequence shown in SEQ ID NO: 43, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 44, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 45, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 40, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 41, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 42. For example, AB is an antibody that specifically binds to EGFR and has VH containing the amino acid sequence shown in SEQ ID NO: 47 and VL containing the amino acid sequence shown in SEQ ID NO: 46. For example, AB is nimotuzumab.

[0071] In specific embodiments, in formula (II-a) or formula (II-b), AB is an antibody that specifically binds to Claudin18.2 or its antigen-binding fragment. For example, AB is an antibody that specifically binds to Claudin18.2 and has HCDR1 containing the amino acid sequence shown in SEQ ID NO: 27, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 28, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 29, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 24, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 25, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 26. For example, AB is an antibody that specifically binds to Claudin18.2 and has VH containing the amino acid sequence shown in SEQ ID NO: 31 and VL containing the amino acid sequence shown in SEQ ID NO: 30. For example, AB is zobetuximab.

[0072] In specific embodiments, in formula (II-a) or formula (II-b), AB is an antibody that specifically binds to CD20 or its antigen-binding fragment. For example, AB is an antibody that specifically binds to CD20 and has HCDR1 containing the amino acid sequence shown in SEQ ID NO: 51, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 52, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 53, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 48, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 49, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 50. For example, AB is an antibody that specifically binds to CD20 and has VH containing the amino acid sequence shown in SEQ ID NO: 55 and VL containing the amino acid sequence shown in SEQ ID NO: 54. For example, AB is rituximab.

[0073] In some implementations, in formula (II-a) or formula (II-b), where n 2 It can be 2 or 4.

[0074] In some implementations, in formula (II-a) or formula (II-b), where n 2 It is a decimal between 1 and 2 or a decimal between 3 and 4.

[0075] In some embodiments, in formula (II-a), the connecting subunit L has the following structure: Wherein, Sp is a spacer unit containing PEG or methylene, and t 1 The integer is selected from 0 to 4, preferably 0, 1, or 2, more preferably 1 or 2, Brch is a branching unit, and t 2 It is 0 or 1, Cnt is the linker unit connected to the ligand, which contains the functional group after the reaction. When t 1 When it is not 0, Sp is related to R in the structure of equation (II-a). 5 Connection. When t 1 t is 0 2 When Brch is 1, R in the structure of formula (II-a) 5 Connection. When t 1 =0 and t 2 When Cnt is 0, it is similar to R in the structure of equation (II-a). 5 connect.

[0076] In some implementations, in the connecting subunit L, Sp is a structure selected from the group consisting of: -C(=O)-(CH2). s1 -、-C(=O)-(CH2) s1 -(CH2-O-CH2)s2 -、-C(=O)-(CH2) s1 -(CH2-O-CH2) s2 -(CH2) s3 -、-C(=O)-(CH2) s1 -(CH2-O-CH2) s2 -(CH2) s3 -W1-、-C(=O)-(CH2) s1 -(CH2-O-CH2) s2 -(CH2) s3 -W1-(CH2) s4 -(CH2-O-CH2) s5 -(CH2) s6 -、-(CH2) s1 -C(=O)-NH-(CH2-O-CH2) s2 -(CH2) s3 -C(=O)- and -C(=O)-(CH2) s1 -(CH2-O-CH2) s2 -(CH2) s3 -C(=O)-NH-(CH2) s4 - where W1 is s1, s3, s4 and s6 are each independently an integer selected from 0 to 4, and s2 and s5 are each independently an integer selected from 0 to 10, wherein the right side of the above Sp structure is connected to Brch or Cnt.

[0077] In some implementations, in the connecting subunit L, Sp is a structure selected from the group consisting of:

[0078] In some implementations, in the connecting subunit L, Brch is a structure selected from the group consisting of: Where A1 is -(CH2). p1 -NH-C(=O)-(CH2) p2 -or-(CH2) p2 -, B1 is -(CH2) p2 -, C1 is -C(=O)-(CH2) p2 -、-C(=O)-(CH2) p2 -(CH2-O-CH2) p3 -(CH2) p4 -、-C(=O)-(CH2) p2 -(CH2-O-CH2) p3-(CH2) p4 -W2- or -C(=O)-(CH2) p2 -(CH2-O-CH2) p3 -(CH2) p4 -W2-(CH2) p5 -(CH2-O-CH2) p6 -(CH2) p7 -,W2 is The left side of structure A1, the right side of structure B1, and the left side of structure C1 are connected to the N atom in the Brch structure. A2 is -C(=O)-NH-(CH2). p2 -, B2 is -(CH2) p2 -C(=O)-NH-, C2 is -(CH2) p2 - The carbon atoms in the A2 structure, B2 structure, and C2 structure are connected to the carbon atoms in the Brch structure. Brch is connected to Cnt through C1 or C2. Each p1 is independently selected from an integer from 2 to 4, each p2 is independently selected from an integer from 1 to 4, each p4, p5 and p7 is independently selected from an integer from 0 to 4, and each p3 and p6 is independently selected from an integer from 0 to 10.

[0079] More specifically, in the connecting subunit L, where Brch is a structure selected from the following group: The right side of the Brch structure is connected to Cnt.

[0080] In some embodiments, in the linker unit L, Cnt comprises a functional group selected from the group consisting of: maleimide, iodoacetamide, bromoacetamide, thiol, amino, alkyl bromide (e.g., C1-C1). 12 Alkyl bromide), alkyl iodine (e.g., C1-C) 12 Alkyl iodide), acrylamide group, carboxyl group, NHS ester, GDP-fucosylate group, aldehyde group, tetrazinyl group, cycloalkenyl group (e.g., C2-C 12 Cycloalkenyl), linear alkyne (e.g., C3-C) 12 Linear alkynyl) and cycloalkynyl (e.g., C2-C) 12 Cycloaceous yne

[0081] More specifically, in the connecting subunit L, Cnt is a structure selected from the following group: Preferred The right side of the Cnt structure described above is connected to AB.

[0082] In some embodiments, in formula (II-b), the connecting subunit L' has the following structure:

[0083] in,

[0084] Sp' 1 and Sp' 2 Each is an independent spacer unit containing PEG or methylene, t' 1 and t' 2 Each is independently 0 or 1, Brch' is the branching unit, and Cnt' is the linker unit connected to the ligand, which contains a functional group selected from the following group after the reaction: maleimide, iodoacetamide, bromoacetamide, thiol, amino, alkyl bromide (e.g., C1-C). 12 Alkyl bromide), alkyl iodine (e.g., C1-C) 12 Alkyl iodide), acrylamide group, carboxyl group, NHS ester, GDP-fucosylate group, aldehyde group, tetrazinyl group, cycloalkenyl group (e.g., C2-C 12 Cycloalkenyl), linear alkyne (e.g., C3-C) 12 Linear alkynyl) and cycloalkynyl (e.g., C2-C) 12 (Cycloynyl group).

[0085] In some implementations, when t' 1 When Sp' is 1, 1 With the structure of equation (II-b) R 5 Connection. When t' 1 When Brch' is 0, R in the structure of equation (II-b) 5 connect.

[0086] In some implementations, when t' 2 When Sp' is 1, 2 With the structure of equation (II-b) L 2 Connected. When t' 2 When Brch' is 0, L in the structure of formula (II-b) 2 connect.

[0087] In some implementations, Sp' in the connecting subunit L' 1 The structure selected is from the following group: -C(=O)-(CH2) s1’ -(CH2-O-CH2) s2’ -(CH2) s3’ -、-C(=O)-(CH2) s1’ -(CH2-O-CH2) s2’ -(CH2) s3’ -W3-(CH2) s4’ -(CH2-O-CH2) s5’ -(CH2) s6’ -、-(CH2) s1’-C(=O)-NH-(CH2-O-CH2) s2’ -(CH2) s3’ -C(=O)-、-C(=O)-(CH2) s1’ -(CH2-O-CH2) s2’ -(CH2) s3’ -C(=O)-NH-(CH2) s4’ -、 Among them, W3 is s1', s3', s4', s6', s7', and s9' are each independently an integer selected from 0 to 4, and s2', s5', and s8' are each independently an integer selected from 0 to 10, wherein the above Sp' 1 The right side of the structure is connected to Brch' or Cnt'.

[0088] In some implementations, in the connecting subunit L', n1 is 1, and Sp' 1 for

[0089] In some implementations, in the connecting subunit L', n1 is 2, and Sp' 1 for

[0090] In some implementations, in the connecting subunit L', where Sp' 2 The structure is selected from the following group: -C(=O)-(CH2) s1” -、-C(=O)-(CH2) s1” -C(=O)-NH-(CH2) s4” -、-C(=O)-(CH2) s1” -(CH2-O-CH2) s2” -(CH2) s3” -、-C(=O)-(CH2) s1” -(CH2-O-CH2) s2” -(CH2) s3” -W4-(CH2) s4” -(CH2-O-CH2) s5” -(CH2) s6” -、-(CH2) s1” -C(=O)-NH-(CH2-O-CH2) s2” -(CH2) s3” -C(=O)- and -C(=O)-(CH2) s1” -(CH2-O-CH2) s2”-(CH2) s3” -C(=O)-NH-(CH2) s4” -, where W4 is s1”, s3”, s4” and s6” are each independent integers selected from 0 to 4, and s2” and s5” are each independent integers selected from 0 to 10 (or 0 to 8, 0 to 6 or 0 to 4).

[0091] In some implementations, in the connecting subunit L', where Sp' 2 for

[0092] In some implementations, in the connecting subunit L', where Brch' comprises a structure selected from the group consisting of: Where A'1 is -(CH2). p1’ -NH-C(=O)-(CH2) p2’ -or-(CH2) p2’ -, B'1 is -(CH2) p2’ -,C'1 is -C(=O)-(CH2) p2’ -、-C(=O)-(CH2) p2’ -(CH2-O-CH2) p3’ -(CH2) p4’ -、-C(=O)-(CH2) p2’ -(CH2-O-CH2) p3’ -(CH2) p4’ -W'2- or -C(=O)-(CH2) p2’ -(CH2-O-CH2) p3’ -(CH2) p4’ -W'2-(CH2) p5’ -(CH2-O-CH2) p6’ -(CH2) p7’ -, W'2 is The left side of the A'1 structure, the right side of the B'1 structure, and the left side of the C'1 structure are connected to the N atom in the Brch' structure. A'2 is -C(=O)-NH-(CH2). p2’ -, B'2 is -(CH2) p2’ -C(=O)-NH-, C'2 is -(CH2) p2’- The carbon atoms in the A'2 structure (left side), B'2 structure (right side), and C'2 structure (left side) are connected to the carbon atoms in the Brch' structure. Brch' is connected to Cnt' via C'1 or C'2. Each p1' is independently selected from an integer from 2 to 4, each p2' is independently selected from an integer from 1 to 4, each p4', p5', and p7' is independently selected from an integer from 0 to 4, and each p3' and p6' is independently selected from an integer from 0 to 10.

[0093] In some implementations, in the connecting subunit L', Brch' is a structure selected from the group consisting of: The right side of the structure is connected to Cnt'.

[0094] In some implementations, in the connecting subunit L', Cnt' is a structure selected from the group consisting of:

[0095] Preferred

[0096] In some embodiments, AB comprises a sugar chain having GlcNAc, and L or L' is connected to said GlcNAc.

[0097] In some embodiments, in the connecting subunit L of formula (II-a), Cnt is a structure selected from the group consisting of: Preferred The right side of the above structure is connected to GlcNAc.

[0098] In some embodiments, in the connecting subunit L' of formula (II-b), where Cnt' is a structure selected from the group below. Preferred The right side of the above structure is connected to GlcNAc.

[0099] In a specific implementation, in equation (II-a), the connecting subunit L is a structure selected from the following group:

[0100] In a specific implementation, in equation (II-b), the connecting subunit L' is a structure selected from the following group:

[0101] Among them, the lower left side of the structure and L 2 Connected.

[0102] In some implementations, in formula (II-b), where P 2 These are small molecules (such as small molecule drugs), nucleic acids (DNA, RNA, or a combination thereof), and / or polypeptides. In some embodiments, in formula (II-b), where P... 2 Selected from the following group: cytotoxins, agonists, antagonists, antiviral agents, antibacterial agents, radioisotopes or radionuclides, metal chelators, fluorescent dyes, biotin, and combinations thereof. Specifically, in formula (II-b), where P 2 It is a microtubule inhibitor. In some embodiments, in formula (II-b), where P 2 Selected from the following group: MMAE, MMAF and their prodrugs and / or intermediates.

[0103] In some implementations, in formula (II-b), where L 2 It is a dipeptide, tripeptide, or tetrapeptide, galactosyl, glucuronic acid, or phosphate group composed of naturally occurring and / or non-naturally occurring amino acids, and may optionally contain a self-eliminating group (e.g., PAB).

[0104] In some implementations, in formula (II-b), where L 2 for The left side of the structure is connected to P2.

[0105] In a specific embodiment, the conjugate comprising a ligand, a linker subunit, and a "disintegrable unit + load" includes a structure selected from the group consisting of: F-6, F-7, F-8, F-9, F-10, F-11, F-12, F-13, F-14, F-15, F-16, F-18, F-19, F-20, F-21, F-22, F-23, F-24, F-25, F-26, F-27, F-28, F-30, F-31, F-36, F-38, F-39, F-40, F-43, F-44, F-47, F-D1, F-D2, F-D3, F-D4, F-D 5. F-D6, F-D7, F-D8, F-D9, F-D10, F-D11, F-D12, F-D15, F-D16, F-D17, F-D18, F-D19, F-D20, F-D21, F-D22, F-D23, F-D24, F-D25, F-DP1, F-DP2, F-DP3, F-DP4, F-DP5, F-DP6, F-DP7, F-DP8, F-DP9, F-DP10, F-DP11, F-DP12, F-DP13, F-DP14, MC'-1 and DBCO'-1 (the corresponding structure of the numbers is shown in Table 1).

[0106] In a specific embodiment, the conjugate comprising a ligand, a linker subunit, and a "disintegrable unit + load" has the structure shown in (II-a), and includes structures selected from the group consisting of: F-6, F-7, F-8, F-9, F-10, F-11, F-12, F-13, F-14, F-15, F-16, F-18, F-19, F-20, F-21, F-22, F-23, F-24, F-25, F-26, F-27, F-28, F-30, F-31, F-36, F- 38, F-39, F-40, F-43, F-44, F-47, F-D1, F-D2, F-D3, F-D4, F-D5, F-D6, F-D7, F-D8, F-D9, F-D10, F-D11, F-D12, F-D15, F-D16, F-D17, F-D18, F-D19, F-D20, F-D21, F-D22, F-D23, F-D24, F-D25, MC'-1 and DBCO'-1 (the corresponding structure of the numbers is shown in Table 1).

[0107] In a specific embodiment, the conjugate comprising a ligand, a connecting subunit, and a "disintegrable unit + load" has the structure shown in (II-a), and includes structures selected from the group consisting of: F-6, F-7, F-8, F-9, F-10, F-11, F-12, F-13, F-14, F-15, F-16, F-18, F-19, F-20, F-21, F-22, F-23, F-24, F-25, F-26, F-27, F-28, F-30, and F-31. F-36, F-38, F-39, F-40, F-43, F-44, F-47, F-D1, F-D2, F-D3, F-D4, F-D5, F-D6, F-D7, F-D8, F-D9, F-D10, F-D11, F-D12, F-D15, F-D16, F-D17, F-D18, F-D19, F-D20, F-D21, F-D22, F-D23, F-D24, and F-D25 (the corresponding structures are shown in Table 1). In a specific embodiment, the conjugate comprising a ligand, a connecting subunit, and a "disintegrable unit + load" has the structure shown in (II-a) and includes the structure shown in MC'-1 (the corresponding structures are shown in Table 1).

[0108] In a specific embodiment, the conjugate comprising a ligand, a linker subunit, and a “disintegrable unit + load” has the structure shown in (II-a) and includes the structure shown in DBCO'-1 (the numbered structures are shown in Table 1).

[0109] In a specific embodiment, the conjugate comprising a ligand, a small molecule drug and a linker unit has the structure shown in (II-b) and includes structures selected from the group consisting of: F-DP1, F-DP2, F-DP3, F-DP4, F-DP5, F-DP6, F-DP7, F-DP8, F-DP9, F-DP10, F-DP11, F-DP12, F-DP13 and F-DP14 (the corresponding structures are shown in Table 1).

[0110] In some embodiments, in formulas (II-a) and (II-b), AB has the following structure: Wherein, AB' is a ligand, preferably an antigen-binding protein, an Fc fusion protein, or an Fc fragment, and GalX is substituted or unsubstituted galactose. AB is obtained through... Linked to L or L'. Based on the description in this specification and the prior art, those skilled in the art will clearly understand that AB' and AB are both ligands, differing in that AB contains AB' and the glycosylated chain portion shown in the above structural formula. In some embodiments, AB' is the protein portion of AB. In some embodiments, one or more molecules of interest can be further linked to AB via the GalX. For example, in some embodiments, one or more molecules of interest can be linked via a click chemistry reaction.

[0111] In some embodiments, in AB, the GalX has the following structure: Each Rg independently selects the next group:

[0112] Among them, Rg 1 Selected from the following groups: hydrogen, halogens, -OH, -NH2, -SH, -N3, -COOH, -CN, Cl-C 24 Alkyl, C3-C 24 cycloalkyl, C2-C 24 alkenyl, C5-C 24 Cycloalkenyl, C2-C 24 alkynyl group, C7-C 24 Cycloalkynyl, C2-C 24 (Miscellaneous) aryl, C3-C 24 Alkyl (hetero)aryl, C3-C 24 (Hetero)arylalkyl and any combination thereof, wherein the alkyl, the cycloalkyl, the alkenyl, the cycloalkenyl, the ynyl, the cycloynyl, the (hetero)aryl, the alkyl(hetero)aryl, or the (hetero)arylalkyl is optionally oxidized by one or more Rs 4 Replaced and / or optionally by one or more Rs 2 Interruption, where Rs 4Selected from the following groups: halogen, -OH, -NH2, -SH, -N3, -COOH, and -CN, Rs 2 Selected from the following groups: -O-, -S-, Where Rs 3 Selected from the following groups: hydrogen, C1-C 24 Alkyl, C2-C 24 alkenyl, C2-C 24 alkynyl and C3-C 24 Cycloalkyl, tg is 0 or 1,

[0113] Rg 2 Selected from the following groups: C1-C 24 Alkylene, C3-C 24 Cycloalkylene, C2-C 24 alkenyl, C5-C 24 Cycloalkenyl, C2-C 24 Ethyne group, C7-C 24 Cycloethynyl, C2-C 24 (Miscellaneous) aryl, C3-C 24 Alkyl (hetero)arylene and C3-C 24 (Hetero)arylalkylene, wherein the alkylene, the cycloalkylene, the alkenyl, the cycloalkenyl, the alynylene, the cycloynylene, the (hetero)aryl, the alkyl(hetero)aryl, or the (hetero)arylalkylene optionally is affected by one or more Rs 4 Replaced and / or optionally by one or more Rs 2 Interruption, where Rs 4 Selected from the following groups: hydrogen, -OH, -NH2, -SH, -N3, -COOH, and -CN, Rs 2 Selected from the following groups: -O-, -S-, Where Rs 3 Selected from the following groups: hydrogen, C1-C 24 Alkyl, C2-C 24 alkenyl, C2-C 24 alkynyl and C3-C 24 cycloalkyl,

[0114] Rg 3 Selected from the following groups: hydrogen, halogen, -OH, -NH2, -SH, -N3, -COOH, -CN, Cl-C 24 Alkyl, C3-C 24 cycloalkyl, C2-C 24 alkynyl group, C5-C 24 Cycloalkynyl, C2-C 24 alkynyl group, C8-C 24 Cycloalkynyl, C2-C24 (Hetero)aryl and any combination thereof, wherein C1-C 24 Alkyl, C3-C 24 cycloalkyl, C2-C 24 alkynyl group, C5-C 24 Cycloalkynyl, C2-C 24 alkynyl group, C8-C 24 Cycloalkynyl or C2-C 24 (Miscellaneous) aryl groups are optionally bounded by one or more Rs 4 Replace, where Rs 4 Selected from the following groups: hydrogen, -OH, -NH2, -SH, -N3, -COOH, and -CN, Rs 2 Selected from the following groups: -O-, -S-, Where Rs 3 Selected from the following groups: hydrogen, C1-C 24 Alkyl, C2-C 24 alkenyl, C2-C 24 alkynyl and C3-C 24 Cycloalkyl.

[0115] For example, C1-C 24 Alkylenes can be C1-C 20 Alkylene, C1-C 12 Alkylene or C1-C6 alkylene.

[0116] For example, C3-C 24 Cycloalkylene groups can be C3-C 20 Cycloalkylene, C3-C 12 Cycloalkylene or C3-C6 cycloalkylene.

[0117] For example, C2-C 24 The alkenyl group can be C2-C 20 alkenyl, C2-C 12 Alkenyl or C2-C6 alkenyl.

[0118] For example, C5-C 24 Cycloalkenyl groups can be C5-C 20 Cycloalkenyl, C5-C 16 Cycloalkenyl or C5-C 12 Cycloalkenyl groups.

[0119] For example, C2-C 24 The alkynyl group can be C2-C 20 alkyne group, C2-C 12 Alynyl or C2-C6 alynyl.

[0120] For example, C7-C 24 Cycloynyl groups can be C7-C 20Cycloethynyl, C7-C 16 Cycloethynyl or C7-C 12 Cycloacetylenic.

[0121] For example, C2-C 24 (Miscellaneous) arylene groups can be C2-C 20 (Miscellaneous) arylene, C2-C 12 (Hetero)arylene or C2-C6 (hetero)arylene.

[0122] For example, C3-C 24 Alkyl (hetero)arylene groups can be C3-C 20 Alkyl(hetero)arylene, C3-C 12 Alkyl(hetero)arylene or C3-C6 alkyl(hetero)arylene

[0123] For example, C3-C 24 (Hetero)arylalkylene groups can be C3-C 20 (Hetero)arylalkylene, C3-C 12 (Hetero)arylalkylene or C3-C6 (hetero)arylalkylene.

[0124] The heteroatoms of the heteroarylene, alkyl heteroarylene, and heteroarylene alkylene can refer to one or more of N, O, or O.

[0125] In some implementations, in AB, GalX is selected from the group consisting of:

[0126] For information on the application of galactose derivatives in coupling compounds, please refer to PCT international publication text WO2023 / 065137A1. Those skilled in the art can combine existing technologies to apply suitable galactose derivative structures to coupling compounds.

[0127] In some embodiments, the AB further comprises a thiol group, and Cnt or Cnt' comprises a functional group selected from the group consisting of maleimide, haloacetyl, and pyridinedithioyl after the reaction, preferably.

[0128] In some embodiments, the AB is modified to include an aldehyde group, an azide group, a tetrazinyl group, a cycloalkenyl group, a linear alkyne group, or a cycloalkyne group.

[0129] In some embodiments, the AB is modified to include an azide functional group, and Cnt or Cnt' is...

[0130] In some embodiments, the AB or AB' comprises an amino acid sequence selected from the group consisting of: (1) VH: SEQ ID NO:23, VL: SEQ ID NO:22; (2) VH: SEQ ID NO:31, VL: SEQ ID NO:30; (3) VH: SEQ ID NO:39, VL: SEQ ID NO:38; (4) VH: SEQ ID NO:47, VL: SEQ ID NO:46 and (5) VH: SEQ ID NO:55, VL: SEQ ID NO:54.

[0131] In some embodiments, the conjugate has a DAR value of 1.5 to 2, or 3 to 4, or 6 to 8.

[0132] In some embodiments, the conjugate has two DARs, D 1 AR is 1.5 to 2, or 3 to 4, or 6 to 8, D 2 AR is 1.5 to 2 or 3 to 4.

[0133] In some embodiments, the conjugate has a structure selected from those shown in Table 2.

[0134] Secondly, this application also relates to a compound or a pharmaceutically acceptable salt thereof. The compound comprises a structural segment represented by any of the aforementioned formulas (I).

[0135] In some embodiments of the second aspect, the compound has the structure of formula (III-a): Among them, R 1 Selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, and 3- to 8-membered cycloalkyl; R 2 for Among them, R 21 Selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, and 3- to 8-membered cycloalkyl, R 22 and R 23 Each is independently selected from the following group: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C 2- C6 alkynyl and 3- to 8-membered cycloalkyl, R 22 Can be used with R 23 Together they form a ring structure, the ring structure comprising R 22 and R 23 Connected carbon atoms, or R 21 Can be used with R 22 or R 21 Can be used with R 23 Together they form a ring structure, the ring structure comprising R 21The bonded nitrogen atom, R 21 R 22 R 23 Each hydrogen atom in the definition can be independently replaced by F, Cl, Br, or I, R 3 for Among them, R 31 Selected from the following group: hydrogen, C1-C6 alkyl, R 4 for Among them, R 41 Selected from the following group: hydrogen, C1-C6 alkyl and -R 6 -NH-C(=O)-NH2, where R 6 It is a C1-C6 alkylene group, R 5 for Among them, R 51 Selected from the following group: C1-C6 alkyl, n 1 Let Lx be an integer selected from 1 to 4. Wherein, Sp is a spacer unit containing PEG or methylene, Brch is a branching unit, and t 1 The integer is selected from 0 to 4, preferably 0, 1, or 2, more preferably 1 or 2, t 2 It can be 0 or 1, and CntR is a structure selected from the following groups: Maleimide, iodoacetamide, bromoacetamide, thiols, amino groups, alkyl bromides (e.g., C1-C) 12 Alkyl bromide), alkyl iodine (e.g., C1-C) 12 Alkyl iodide), acrylamide group, carboxyl group, NHS ester, GDP-fucosylate group, aldehyde group, tetrazinyl group, cycloalkenyl group (e.g., C2-C 12 Cycloalkenyl), linear alkyne (e.g., C3-C) 12 Linear alkynyl) and cycloalkynyl (e.g., C2-C) 12 Cycloalkynyl), preferably, CntR is Represents the connection site.

[0136] When t 1 When it is not 0, Sp is related to R in the structure of equation (III-a). 5 Connection. When t 1 t is 0 2 When Brch is 1, R in the (III-a) structure 5 Connection. When t 1 =0 and t 2 When CntR is 0, it is similar to R in the structure of equation (III-a). 5 connect.

[0137] In some embodiments, in formula (III-a), Sp is a structure selected from the group consisting of: -C(=O)-(CH2). s1 -、-C(=O)-(CH2) s1 -(CH2-O-CH2) s2 -、-C(=O)-(CH2) s1 -(CH2-O-CH2) s2 -(CH2) s3 -、-C(=O)-(CH2) s1 -(CH2-O-CH2) s2 -(CH2) s3 -W1-、-C(=O)-(CH2) s1 -(CH2-O-CH2) s2 -(CH2) s3 -W1-(CH2) s4 -(CH2-O-CH2) s5 -(CH2) s6 -、-(CH2) s1 -C(=O)-NH-(CH2-O-CH2) s2 -(CH2) s3 -C(=O)- and -C(=O)-(CH2) s1 -(CH2-O-CH2) s2 -(CH2) s3 -C(=O)-NH-(CH2) s4 - where W1 is s1, s3, s4 and s6 are each independently an integer selected from 0 to 4, and s2 and s5 are each independently an integer selected from 0 to 10, wherein the right side of the Sp structure is preferably connected to Brch or CntR.

[0138] In some implementations, in formula (III-a), Sp is a structure selected from the group consisting of:

[0139] In some implementations, in formula (III-a), Brch is a structure selected from the group consisting of: Where A1 is -(CH2). p1 -NH-C(=O)-(CH2) p2 -or-(CH2) p2 -, B1 is -(CH2) p2 -, C1 is -C(=O)-(CH2) p2 -、-C(=O)-(CH2) p2-(CH2-O-CH2) p3 -(CH2) p4 -、-C(=O)-(CH2) p2 -(CH2-O-CH2) p3 -(CH2) p4 -W2- or -C(=O)-(CH2) p2 -(CH2-O-CH2) p3 -(CH2) p4 -W2-(CH2) p5 -(CH2-O-CH2) p6 -(CH2) p7 -,W2 is The left side of structure A1, the right side of structure B1, and the left side of structure C1 are connected to the N atom in the Brch structure. A2 is -C(=O)-NH-(CH2). p2 -, B2 is -(CH2) p2 -C(=O)-NH-, C2 is -(CH2) p2 - The carbon atoms in the A2 structure, B2 structure, and C2 structure are connected to the carbon atoms in the Brch structure. Brch is connected to Cnt through C1 or C2. Each p1 is independently selected from an integer from 2 to 4, each p2 is independently selected from an integer from 1 to 4, each p4, p5 and p7 is independently selected from an integer from 0 to 4, and each p3 and p6 is independently selected from an integer from 0 to 10.

[0140] In some implementations, in formula (III-a), Brch is a structure selected from the group consisting of: The right side of the Brch structure is preferably connected to CntR.

[0141] In other embodiments of the second aspect, the compound has the structure of formula (III-b):

[0142] Where Lx' is Where R 1 Selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, and 3- to 8-membered cycloalkyl; R 2 for Among them, R 21 Selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, and 3- to 8-membered cycloalkyl, R 22 and R 23 Each is independently selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, and 3- to 8-membered cycloalkyl, R 22Can be used with R 23 Together they form a ring structure, the ring structure comprising R 22 and R 23 Connected carbon atoms, or R 21 Can be used with R 22 or R 21 Can be used with R 23 Together they form a ring structure, the ring structure comprising R 21 The bonded nitrogen atom, R 21 R 22 R 23 Each hydrogen atom in the definition can be independently replaced by F, Cl, Br, or I, R 3 for Among them, R 31 Selected from the following group: hydrogen, C1-C6 alkyl, R 4 for Among them, R 41 Selected from the group consisting of: hydrogen, C1-C6 alkyl, and -R 6 -NH-C(=O)-NH2, where R 6 It is a C1-C6 alkylene group, R 5 for Among them, R 51 Selected from the following group: C1-C6 alkyl, n 1 Sp' is an integer selected from 1 to 4. 1 and Sp' 2 Each is an independent spacer unit containing PEG or methylene, Brch' is a branching unit, and t' 1 and t' 2 Each can be 0 or 1 independently, and Cnt'R contains functional groups selected from the following group:

[0143] Maleimide, iodoacetamide, bromoacetamide, thiols, amino groups, alkyl bromides (e.g., C1-C) 12 Alkyl bromide), alkyl iodine (e.g., C1-C) 12 Alkyl iodide), acrylamide group, carboxyl group, NHS ester, GDP-fucosylate group, aldehyde group, tetrazinyl group, cycloalkenyl group (e.g., C2-C 12 Cycloalkenyl), linear alkyne (e.g., C3-C) 12 Linear alkynyl) and cycloalkynyl (e.g., C2-C) 12 Cycloynyl), preferably, Cnt'R is P 2 For small molecule drugs, nucleic acids or peptides or combinations thereof, L 2 It is a pyrolytic unit. Represents the connection site.

[0144] In some implementations, when t' 1 When Sp' is 1, 1 With the structure of equation (III-b) R 5 Connection. When t' 1 When Brch' is 0, R in the structure of equation (III-b) 5 connect.

[0145] In some implementations, when t' 2 When Sp' is 1, 2 With the structure of equation (III-b) L 2 Connected. When t' 2 When Brch' is 0, L in the structure of formula (III-b) 2 Connection. In some implementations, in formula (III-b), Sp' 1 The structure selected is from the following group: -C(=O)-(CH2) s1’ -(CH2-O-CH2) s2’ -(CH2) s3’ -、-C(=O)-(CH2) s1’ -(CH2-O-CH2) s2’ -(CH2) s3’ -W3-(CH2) s4’ -(CH2-O-CH2) s5’ -(CH2) s6’ -、-(CH2) s1’ -C(=O)-NH-(CH2-O-CH2) s2’ -(CH2) s3’ -C(=O)-、-C(=O)-(CH2) s1’ -(CH2-O-CH2) s2’ -(CH2) s3’ -C(=O)-NH-(CH2) s4’ -、 W3 is s1', s3', s4', s6', s7', and s9' are each independently an integer selected from 0 to 4, and s2', s5', and s8' are each independently an integer selected from 0 to 10 (e.g., 0 to 2, 0 to 4, 0 to 6, or 0 to 8), wherein the aforementioned Sp' 1 The right side of the structure is connected to Brch' or Cnt'R.

[0146] In some implementations, in formula (III-b), Sp' 1 The structure selected from the following group:

[0147] In some implementations, in formula (III-b), where Sp' 2 The structure is selected from the following group: -C(=O)-(CH2) s1” -、-C(=O)-(CH2) s1” -C(=O)-NH-(CH2) s4” -、-C(=O)-(CH2) s1” -(CH2-O-CH2) s2” -(CH2) s3” -、-C(=O)-(CH2) s1” -(CH2-O-CH2) s2” -(CH2) s3” -W4-(CH2) s4” -(CH2-O-CH2) s5” -(CH2) s6” -、-(CH2) s1” -C(=O)-NH-(CH2-O-CH2) s2” -(CH2) s3” -C(=O)- and -C(=O)-(CH2) s1” -(CH2-O-CH2) s2” -(CH2) s3” -C(=O)-NH-(CH2) s4” -, where W4 is s1”, s3”, s4” and s6” are each independently an integer selected from 0 to 4, and s2” and s5” are each independently an integer selected from 0 to 10 (e.g., 0 to 2, 0 to 4, 0 to 6 or 0 to 8).

[0148] In some implementations, in formula (III-b), where Sp' 2 The structure is selected from the following group:

[0149] In some embodiments, in formula (III-b), Brch' comprises a structure selected from the group consisting of: Where A'1 is -(CH2). p1’ -NH-C(=O)-(CH2) p2’ -or-(CH2) p2’ -, B'1 is -(CH2) p2’ -,C'1 is -C(=O)-(CH2) p2’ -、-C(=O)-(CH2) p2’ -(CH2-O-CH2)p3’ -(CH2) p4’ -、-C(=O)-(CH2) p2’ -(CH2-O-CH2) p3’ -(CH2) p4’ -W'2- or -C(=O)-(CH2) p2’ -(CH2-O-CH2) p3’ -(CH2) p4’ -W'2-(CH2) p5’ -(CH2-O-CH2) p6’ -(CH2) p7’ -, W'2 is The left side of the A'1 structure, the right side of the B'1 structure, and the left side of the C'1 structure are connected to the N atom in the Brch' structure. A'2 is -C(=O)-NH-(CH2). p2’ -, B'2 is -(CH2) p2’ -C(=O)-NH-, C'2 is -(CH2) p2’ - The carbon atoms in the A'2 structure, B'2 structure, C'2 structure, and Brch' structure are connected to each other. Brch' is connected to Cnt'R through C'1 or C'2. Each p1' is independently selected from an integer from 2 to 4, each p2' is independently selected from an integer from 1 to 4, each p4', p5', and p7' is independently selected from an integer from 0 to 4, and each p3' and p6' is independently selected from an integer from 0 to 10.

[0150] In some embodiments, in formula (III-b), Brch' is a structure selected from the group consisting of: The right side of the structure is connected to Cnt'R.

[0151] In some embodiments, in formula (III-a) or formula (III-b), where R 1 It is hydrogen or C1-C3 alkyl.

[0152] In some embodiments, in formula (III-a) or formula (III-b), where R 1 It is hydrogen or methyl, preferably hydrogen.

[0153] In some embodiments, in formula (III-a) or formula (III-b), where R 21 It is hydrogen or C1-C3 alkyl, preferably hydrogen.

[0154] In some embodiments, in formula (III-a) or formula (III-b), where R 22 and R 23Each is independently selected from the following group: hydrogen, C1-C6 alkyl and 3- to 6-membered cycloalkyl.

[0155] In some embodiments, in formula (III-a) or formula (III-b), wherein R 22 With R 23 The ring structure formed together is a 3- to 6-membered cycloalkyl group. Furthermore, in formula (III-a) or formula (III-b), where R... 22 With R 23 The ring structure formed together is a 4-membered cycloalkyl group.

[0156] In some embodiments, in formula (III-a) or formula (III-b), where R 2 Selected from the following group: Among them, each R 21 Independently hydrogen or methyl, optionally, R 2 Each hydrogen atom in the mixture is independently replaced by F, Cl, Br, or I. Represents the connection site.

[0157] Furthermore, in equation (III-a) or equation (III-b), where R 2 Selected from the following group: Among them, each R 21 Independently hydrogen or methyl, optionally, R 2 Each hydrogen atom in the mixture is independently replaced by F, Cl, Br, or I. Represents the connection site.

[0158] In some embodiments, in formula (III-a) or formula (III-b), where R 2 Selected from the following group: Among them, each R 21 Independently hydrogen, optionally, R 2 Each hydrogen atom in the mixture is independently replaced by F, Cl, Br, or I. Represents the connection site.

[0159] Furthermore, in equation (III-a) or equation (III-b), where R 2 Selected from the following group: Among them, each R 21 Independently hydrogen or methyl, optionally, R 2Each hydrogen atom in the mixture is independently replaced by F, Cl, Br, or I. Represents the connection site.

[0160] In some embodiments, in formula (III-a) or formula (III-b), where R 2 Selected from the following group: Among them, each R 21 Independently hydrogen or methyl, Represents the connection site.

[0161] Furthermore, in equation (III-a) or equation (III-b), where R 2 Selected from the following group: Among them, each R 21 Independently hydrogen or methyl, Represents the connection site.

[0162] Furthermore, in equation (III-a) or equation (III-b), where R 2 for

[0163] In some embodiments, in formula (III-a) or formula (III-b), where R 21 It is hydrogen.

[0164] In some embodiments, in formula (III-a) or formula (III-b), where R 31 Selected from the group consisting of: hydrogen, methyl, ethyl, isopropyl, isobutyl, and sec-butyl.

[0165] In some embodiments, in formula (III-a) or formula (III-b), where R 3 Selected from the following group: Represents the connection site. Furthermore, in equation (III-a) or (III-b), where R... 3 Selected from the following group: Represents the connection site. Furthermore, in equation (III-a) or (III-b), where R... 3 Selected from the following group: Represents the connection site.

[0166] In some embodiments, in formula (III-a) or formula (III-b), where R 41 Selected from the following group: hydrogen, methyl, ethyl, isopropyl, isobutyl, sec-butyl, -(CH2)2-NH-C(=O)-NH2 and -(CH2)3-NH-C(=O)-NH2.

[0167] In some embodiments, in formula (III-a) or formula (III-b), where R 4 Selected from the following group:

[0168] Represents the connection site.

[0169] Furthermore, in equation (III-a) or equation (III-b), where R 4 Selected from the following group:

[0170] Represents the connection site.

[0171] In some embodiments, in formula (III-a) or formula (III-b), where R 51 Selected from the group consisting of: methyl, ethyl, isopropyl, isobutyl, and sec-butyl.

[0172] In some embodiments, in formula (III-a) or formula (III-b), where R 5 Selected from the following group: Represents the connection site. Furthermore, in equation (III-a) or (III-b), where R... 5 Selected from the following group: Represents the connection site.

[0173] Furthermore, in equation (III-a) or equation (III-b), R 5 for R 4 for In other embodiments, in formula (I), formula (II-a) or formula (II-b), R 5 for R 4 for

[0174] In some embodiments, in formula (III-a) or formula (III-b), where R 1 For hydrogen, R 2 for R 3 for R 4 for R 5 for Represents the connection site.

[0175] In some embodiments, in formula (III-a) or formula (III-b), wherein The structure shown is selected from the following group: Represents the connection site.

[0176] Furthermore, in equations (III-a) and (III-b), where The structures shown are selected from the following group: Represents the connection site.

[0177] In some implementations, in formula (III-b), where P 2 These are small molecules (such as small molecule drugs), nucleic acids (DNA, RNA, or a combination thereof), and / or polypeptides. In some embodiments, in formula (III-b), where P... 2 Selected from the following group: cytotoxins, agonists, antagonists, antiviral agents, antibacterial agents, radioisotopes or radionuclides, metal chelators, fluorescent dyes, biotin, and combinations thereof. Specifically, in formula (III-b), where P 2 It is a microtubule inhibitor. In some embodiments, in formula (III-b), where P 2 Selected from the following group: MMAE, MMAF and their prodrugs and / or intermediates.

[0178] In some implementations, in formula (III-b), where L 2 It is a dipeptide, tripeptide, or tetrapeptide, galactosyl, glucuronic acid, or phosphate group composed of naturally occurring and / or non-naturally occurring amino acids, and may optionally contain a self-eliminating group (e.g., PAB).

[0179] In some implementations, in formula (III-b), where L 2 for The left side of the structure is connected to P2. In some embodiments, the compound of formula (III-a) has a structure selected from the group consisting of: GF-6, GF-7, GF-8, GF-9, GF-10, GF-11, GF-12, GF-13, GF-14, GF-15, GF-16, GF-18, GF-19, GF-20, GF-21, GF-22, GF-23, GF-24, GF-25, GF-26, GF-27, GF-28, GF-30, GF-31, GF-36, GF-38, GF-39, GF-40, G F-43, GF-44, GF-47, GF-D1, GF-D2, GF-D3, GF-D4, GF-D5, GF-D6, GF-D7, GF-D8, GF-D9, GF-D10, GF-D11, GF-D12, GF-D15, GF-D16, GF-D17, GF-D18, GF-D19, GF-D20, GF-D21, GF-D22, GF-D23, GF-D24, GF-D25, MC-1, and DBCO-1 (the corresponding structures are shown in the examples).

[0180] In some embodiments, the compound of formula (III-b) has a structure selected from the group consisting of GF-DP1, GF-DP2, GF-DP3, GF-DP4, GF-DP5, GF-DP6, GF-DP7, GF-DP8, GF-DP9, GF-DP10, GF-DP11, GF-DP12, GF-DP13 and GF-DP14 (the corresponding structures are shown in the examples).

[0181] Thirdly, this application also relates to a compound having the structure of formula (IV). Among them, R 1 Selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, and 3- to 8-membered cycloalkyl; R 2 for Among them, R 21 Selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, and 3- to 8-membered cycloalkyl, R 22 and R 23 Together they form a 4-membered ring structure, the ring structure including R 22 With R 23 bonded carbon atoms, R 21 R 22 R 23 Each hydrogen atom in the definition can be independently replaced by F, Cl, Br, or I, R 3’ For hydrogen or Among them, R 31Selected from the following group: hydrogen, C1-C6 alkyl.

[0182] In some implementations, in formula (IV), where R 1 It is hydrogen or methyl, preferably hydrogen.

[0183] In some implementations, in formula (IV), where R 2 for

[0184] In some implementations, in formula (IV), where R 21 It is hydrogen or methyl, preferably hydrogen.

[0185] In some implementations, in formula (IV), where R 3’ It is hydrogen.

[0186] In some implementations, in formula (IV), where R 3’ for And R 31 It can be hydrogen or methyl.

[0187] In some implementations, the structure of formula (IV) is selected from the group consisting of:

[0188] Furthermore, the structure of formula (IV) is selected from the following group:

[0189] Tp-12-2 and Tp-12-1 are derivatives of Tp-12. In some cases, Tp-12-1 and Tp-12-2 can further release Tp-12 through peptide bond hydrolysis.

[0190] Fourthly, this application also relates to methods for preparing any of the conjugates of the first aspect or pharmaceutically acceptable salts thereof.

[0191] In some embodiments, the method includes contacting any one of the compounds of the second and / or third aspects, or a pharmaceutically acceptable salt thereof, with a ligand. The contact can occur in vivo or in vitro. The contact can occur within any container. The ligand is an antibody or its antigen-binding moiety, or an Fc fusion protein or Fc fragment.

[0192] In a preferred embodiment, the contact can occur in the presence of an enzyme. For example, the contact can occur in the presence of fucosyltransferase.

[0193] For example, the contact is carried out in the presence of galactosyltransferase, fucosyltransferase, and UDP-GalX or a pharmaceutically acceptable salt thereof.

[0194] For example, the contact is carried out in the presence of glycoside endonuclease, galactosyltransferase, fucosyltransferase, and UDP-GalX.

[0195] For example, the contact is carried out in the presence of glycoside endonuclease, α-1,6-fucosidase, galactosyltransferase, fucosyltransferase, and UDP-GalX.

[0196] The fucosyltransferase can be α-1,3-fucosyltransferase or a functional variant or fragment thereof. In some specific embodiments, the fucosyltransferase may comprise the amino acid sequence shown in SEQ ID NO:1.

[0197] The galactosyltransferase can be β-1,4-galactosyltransferase or a functional variant or fragment thereof. In some specific embodiments, the galactosyltransferase may comprise the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:3.

[0198] Endoglycosidases can be Endo S, Endo S2, Endo A, Endo F, Endo M, Endo D, and Endo H, or functional mutants or variants thereof, or any combination thereof. For example, an endoglycosidase can be Endo S. For example, an endoglycosidase can contain an amino acid sequence as shown in SEQ ID NO:4.

[0199] α-1,6-fucosidase can be BfFucH, fucosidase O, Alfc, BKF, fucosidase O, or a functional mutant or variant thereof, or any combination thereof. For example, α-1,6-fucosidase can be Alfc. For example, α-1,6-fucosidase can contain the amino acid sequence shown in SEQ ID NO:5.

[0200] The contact can be carried out in a suitable buffer solution, such as phosphate-buffered saline (e.g., phosphate-buffered saline, Tris-buffered saline), citrate, HEPES, Tris, Tris-HCl, and glycine. Suitable buffers are known in the art.

[0201] The contact can be performed within a suitable temperature range. In some embodiments, it can be performed within a temperature range of about 0°C to about 50°C. In some embodiments, the method can be performed within a temperature range of about 5°C to about 45°C. In some embodiments, the method can be performed within a temperature range of about 20°C to about 40°C. In some embodiments, the method can be performed within a temperature range of about 25°C to about 30°C. For example, the method can be performed at a temperature of about 30°C.

[0202] The contact can be performed within a suitable pH range. In some embodiments, the method can be performed at a pH range of about 4 to about 10. In some embodiments, the method can be performed at a pH range of about 5 to about 9. In some embodiments, the method can be performed at a pH range of about 6 to about 8. In some embodiments, the method can be performed at a pH range of about 7 to about 8, for example, about 7 to about 7.5.

[0203] The contact can occur in the presence of certain metal ions, such as Mn. 2+ and / or Mg 2+ If it exists.

[0204] Fifthly, this application also relates to a pharmaceutical composition. The pharmaceutical composition comprises any conjugate of the first aspect or a pharmaceutically acceptable salt thereof, any compound of the second and / or third aspects or a pharmaceutically acceptable salt thereof, and / or, optionally, a pharmaceutically acceptable carrier.

[0205] In some embodiments, the pharmaceutical composition may also contain other drugs. For example, the other drugs may be drugs that assist the small molecule drug in taking effect.

[0206] Sixthly, this application also relates to the use of any conjugate of the first aspect or a pharmaceutically acceptable salt thereof, any compound of the second and / or third aspects or a pharmaceutically acceptable salt thereof, and / or, the pharmaceutical composition of the fifth aspect, in the preparation of a medicament.

[0207] In a specific embodiment, the drug is used to treat and / or prevent tumors. Further, the tumor includes Her2-positive tumors, Trop2-positive tumors, EGFR-positive tumors, CD20-positive tumors, or Claudin18.2-positive tumors. Furthermore, the tumor is selected from one or more of the following: lung cancer, breast cancer, colorectal cancer, prostate cancer, stomach cancer, liver cancer, pancreatic cancer, ovarian cancer, cervical cancer, thyroid cancer, melanoma, lymphoma, leukemia, brain tumor, bone cancer, kidney cancer, bladder cancer, esophageal cancer, nasopharyngeal carcinoma, melanoma, skin cancer, laryngeal cancer, oral cancer, tongue cancer, gallbladder cancer, bile duct cancer, skin cancer, testicular cancer, uterine cancer, endometrial cancer, renal pelvis cancer, renal cell carcinoma, bladder cancer, glioma, neuroblastoma, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, myeloid leukemia, myelodysplastic syndrome, soft tissue sarcoma, osteosarcoma, liposarcoma, neurofibrosarcoma, angiosarcoma, gastrointestinal stromal tumor, thymoma, thymic carcinoma, pituitary adenoma, retinoblastoma, and glioblastoma.

[0208] Seventhly, this application also relates to a method of treating and / or preventing tumors. The method comprises administering to a subject in need an effective amount of any conjugate of the first aspect or a pharmaceutically acceptable salt thereof, any compound of the second and / or third aspects or a pharmaceutically acceptable salt thereof, and / or, the pharmaceutical composition of the fifth aspect.

[0209] Furthermore, the tumor includes Her2-positive tumors, Trop2-positive tumors, EGFR-positive tumors, or Claudin18.2-positive tumors. In some embodiments, the tumor is selected from one or more of the following: lung cancer, breast cancer, colorectal cancer, prostate cancer, gastric cancer, liver cancer, pancreatic cancer, ovarian cancer, cervical cancer, thyroid cancer, melanoma, lymphoma, leukemia, brain tumor, bone cancer, kidney cancer, bladder cancer, esophageal cancer, nasopharyngeal carcinoma, melanoma, skin cancer, laryngeal cancer, oral cancer, tongue cancer, gallbladder cancer, bile duct cancer, skin cancer, testicular cancer, uterine cancer, endometrial cancer, renal pelvis cancer, renal cell carcinoma, bladder cancer, glioma, neuroblastoma, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, myeloid leukemia, myelodysplastic syndrome, soft tissue sarcoma, osteosarcoma, liposarcoma, neurofibrosarcoma, angiosarcoma, gastrointestinal stromal tumor, thymoma, thymic carcinoma, pituitary adenoma, retinoblastoma, and glioblastoma.

[0210] In this application, the molar ratio of drug molecules to AB or AB' is also known as DAR, or drug-antibody conjugation ratio. It can be understood as the average molar ratio of the drug-drug conjugate obtained after conjugation of a monoclonal antibody molecule to the monoclonal antibody molecule. This ratio can generally be determined using methods such as hydrophobic-interaction chromatography (HIC), reverse-phase HPLC (RP-HPLC), SDS-polyacrylamide gel electrophoresis (SDS-PAGE), liquid chromatography-mass spectrometry (LC-MS), and ultraviolet / visible spectroscopy (UV / Vis).

[0211] In some embodiments, the coupling having the structure of formula (II-a), n 2 *n 1 The numerical value can be used to represent the DAR value. In some implementations, the coupling may contain two or more loads, thus having multiple DAR values ​​(D...). 1 AR, D 2AR….). For example, in some embodiments, couplings having the structure of formula (II-b), n 2 *n 1 The numerical value can be used to represent D 1 AR, n 2 It can represent D 2 AR. In this application, the coupling may contain two or more loads, and in some specific embodiments, D is typically used. 1 AR represents the molar ratio of the eczemab derivative to AB or AB', D 2 AR represents P 2 The molar ratio of AB to AB'.

[0212] For example, in Table 3, Tras-IF-19 has one DAR value of 3.76. Tras-IF-D5 has one DAR value of 7.88, which can also be expressed as 3.94*2. As another example, in Table 3, Tras-II-F-DP2 has two DAR values, where the DAR value corresponding to the eczema derivative is... 1 AR is 1.99, and MMAE corresponds to D. 2 The DAR value is 1.99. The DAR value of Tras-II-F-DP2 can also be expressed as 1.99 + 1.99. For example, in Table 3, Tras-II-F-DP12 has two DAR values, where the DAR value corresponding to the eczema derivative is... 1 AR is 3.92 (which can also be expressed as 1.96*2), and MMAE corresponds to D. 2 The AR is 1.96. The DAR value of Tras-II-F-DP12 can also be expressed as 3.92+1.96.

[0213] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0214] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0215] Figure 1 shows the in vitro enzyme cleavage analysis of the "cleavable unit-load" of this application.

[0216] Figure 2 shows the inhibitory activity of the Her2-targeting conjugate on the in vitro proliferation of NCI-N87 (Her2-positive) cells. Figure 2A shows the in vitro proliferation inhibition curve of the Her2-targeting conjugate on NCI-N87 cells, and Figure 2B shows the IC50 of the Her2-targeting conjugate on the in vitro proliferation inhibition of NCI-N87 cells. 50 value.

[0217] Figure 3 shows the in vitro proliferation inhibition curve of NCI-N87 cells by the CD20-targeting conjugate (negative control conjugate).

[0218] Figure 4 shows the in vitro proliferation inhibition curve of the Her2-targeting conjugate on MDA-MB-468 (Her2-negative) cells.

[0219] Figure 5 shows the inhibitory activity of the Her2-targeting conjugate on the in vitro proliferation of SKBR-3 (Her2-positive) cells. Figure 5A shows the in vitro proliferation inhibition curve of the Her2-targeting conjugate on SKBR-3 cells, and Figure 5B shows the IC50 of the Her2-targeting conjugate on the in vitro proliferation inhibition of SKBR-3 cells. 50 value.

[0220] Figure 6 shows the in vitro inhibitory activity of the Her2-targeting dual-load conjugate on the proliferation of human gastric cancer NCI-N87 (Her2-positive) cells. Figure 6A shows the in vitro proliferation inhibition curve of the Her2-targeting dual-load conjugate on NCI-N87 cells, and Figure 6B shows the IC50 of the Her2-targeting dual-load conjugate on the in vitro proliferation inhibition of NCI-N87 cells. 50 value.

[0221] Figure 7 shows the inhibitory activity of the Her2-targeting dual-load conjugate on the in vitro proliferation of SKBR-3 (Her2-positive) cells. Figure 7A shows the in vitro proliferation inhibition curve of the Her2-targeting dual-load conjugate on SKBR-3 cells, and Figure 7B shows the IC50 of the Her2-targeting dual-load conjugate on the in vitro proliferation inhibition of SKBR-3 cells. 50 value.

[0222] Figure 8 shows the in vitro bystander killing activity assay of Her2-targeting conjugates. Figure 8A shows the bystander killing activity assay results of the DAR4 or DAR2*2 series conjugates at a concentration of 10 nM in a mixed NCI-N87 (Her2-positive) and MDA-MB-468 (Her2-negative) cell model. Figure 8B shows the bystander killing activity assay results of the DAR8 or DAR4*2 series conjugates at a concentration of 10 nM in the same cell model. Figure 8C shows the bystander killing activity assay results of the DAR4 series conjugates. The results of bystander killing tests of the conjugates at a concentration of 5 nM in the mixed cell models of NCI-N87 (Her2 positive) and MDA-MB-468 (Her2 negative) are shown in Figure 8D. The results of bystander killing tests of the DAR4 series conjugates at a concentration of 2.5 nM in the mixed cell models of NCI-N87 (Her2 positive) and MDA-MB-468 (Her2 negative) are shown in Figure 8E. The results of bystander killing tests of the DAR4 series conjugates at a concentration of 5 nM in the mixed cell models of SKBR-3 (Her2 positive) and MDA-MB-468 (Her2 negative) are shown in Figure 8E.

[0223] Figure 9 shows the inhibitory activity of the Trop2-targeting conjugate on the in vitro proliferation of human pancreatic cancer BXPC-3 (Trop2-positive) cells. Figure 9A shows the in vitro proliferation inhibition curve of the conjugate on tumor cells, and Figure 9B shows the IC50 of the conjugate on the in vitro proliferation inhibition of tumor cells. 50 value.

[0224] Figure 10 shows the in vitro inhibitory activity of the Trop2-targeting dual-load conjugate on the proliferation of human pancreatic cancer BXPC-3 (Trop2-positive) cells. Figure 10A shows the in vitro proliferation inhibition curve of the conjugate on tumor cells, and Figure 10B shows the IC50 of the conjugate on the in vitro proliferation inhibition of tumor cells. 50 value.

[0225] Figure 11 shows the inhibitory activity of the EGFR-targeting conjugate on the in vitro proliferation of human lung cancer HCC827 (Trop2-positive) cells. Figure 11A shows the in vitro proliferation inhibition curve of the conjugate on tumor cells, and Figure 11B shows the IC50 of the conjugate on the in vitro proliferation inhibition of tumor cells. 50 value.

[0226] Figure 12 shows the efficacy evaluation results of the Her2-targeting conjugate in the NCI-N87 (Her2 positive) / MDA-MB-468-Luc (Her2 negative) mixed tumor-CDX model. In Figure 12A, the tumor volume of each group of mice changes over time; in Figure 12B, the tumor weight of each group of mice; in Figure 12C, the relative fluorescence intensity of each group; and in Figure 12D, the body weight of each group of mice changes over time.

[0227] Figure 13 shows the efficacy evaluation results of the Her2-targeting conjugate in the JIMT-1 (Her2-positive) CDX model. Figure 13A shows the change in tumor volume over time in each group of mice, Figure 13B shows the tumor weight in each group of mice, and Figure 13C shows the change in body weight over time in each group of mice.

[0228] Figure 14 shows the efficacy evaluation results of the conjugate in the NCI-N87 (Her2 positive, Trop2 positive) / MDA-MB-468-Luc (Her2 negative, Trop2 positive) mixed tumor-CDX model. In Figure 14A, the tumor volume of each group of mice changes over time; Figure 14B, the tumor weight of each group of mice; Figure 14C, the relative fluorescence intensity of each group; and Figure 14D, the body weight of each group of mice changes over time.

[0229] Figure 15 shows the efficacy evaluation results of the Trop2-targeting conjugate in the BXPC-3 (Trop2-positive) CDX model. In Figure 15A, the tumor volume of each group of mice changes over time; in Figure 15B, the tumor weight of each group of mice changes over time; and in Figure 15C, the body weight of each group of mice changes over time.

[0230] Figure 16 shows the efficacy evaluation results of the EGFR-targeting conjugate in the HCC827 (EGFR-positive) CDX model. In Figure 16A, the tumor volume of each group of mice changes over time; in Figure 16B, the tumor weight of each group of mice changes over time; and in Figure 16C, the body weight of each group of mice changes over time.

[0231] Figure 17 shows the efficacy evaluation results of the Her2-targeting dual-load conjugate in the NCI-N87 (Her2 positive) / MDA-MB-468-Luc (Her2 negative) mixed tumor-CDX model. In Figure 17A, the tumor volume of each group of mice changes over time; Figure 17B, the tumor weight of each group of mice; Figure 17C, the relative fluorescence intensity of each group; and Figure 17D, the body weight of each group of mice changes over time.

[0232] Figure 18 shows the in vitro stability study of the conjugate of this application in human plasma.

[0233] Figure 19 shows the pharmacokinetic study of the conjugate in rats.

[0234] Figure 20 shows the acute toxicity study of the conjugate in mice. Figure 20A shows the results of Tras-II-F-D5, Figure 20B shows the results of Tras-IF-D5, Figure 20C shows the results of Tras-IF-19, and Figure 20D shows the results of Tras-II-F-DP2. Detailed Implementation

[0235] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0236] Terminology Definition

[0237] In this application, the term "ligand" generally refers to a macromolecular compound that can recognize and bind to antigens or receptors associated with target cells. The role of a ligand can be to present a drug to a target cell population that has bound the ligand. These ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules that can bind to cells, receptors, and / or antigens. In this application, a ligand may be represented as AB or AB', and the ligand may be an antigen-binding protein. The antigen-binding protein may be an antibody or its antigen-binding fragment, and the antibody may be selected from chimeric antibodies, humanized antibodies, fully human antibodies, or murine antibodies; the antibody may be a monoclonal antibody. For example, the ligand may be an antibody or its antigen-binding fragment targeting a target selected from the group consisting of: HER2, HER3, TROP2, B7-H3, GPC20, CDH6, EGFR, EGFRvIII, AXL, Nectin-4, Tissue factor, TIM-1, PSMA, EpCAM, MUC1, STEAP1, GPNMB, FGF2, FOLR1, c-MET, GFR, AGS-16, Guanylyl cyclase C, Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, ROR1, PD-L1, CD27L, 5T4, Mucin 16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, CEACAM5, SC-16, SLC39A6, Delta-like protein3, Claudin 18.2, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD73, CD74, CD79b, CD138, CD147, CD166, CD223, MUC16, MSLN, ENPP3, SLTRK6, FGFR, LIV-1, Lewis Y, av-integrin, ASCT2, C4.4a, CA-IX, CD324, CD352, CD44v6, CD48a, CLL-1, Cripto, CS1, DPEP3, Ephrin-A2, Ephrin-A4, ETBR, FGFR2, FGFR3, FLT3, GD3, Globo H, GPC3, LAMP-1, LRRC15, Ly6E, MFI2, NOTCH3, p-cadherin, PRLR, and RNF43. In this application, the ligand can also be an Fc fusion protein or an Fc fragment. In some more extensive embodiments, the ligand of this application can be understood as a protein with a linker group, and the structure of formula (I) of this application can be conjugated to the protein using linking methods known in the art or exemplified in this application.In this application, the ligand comprises a protein having the following linking groups: thiol, amino, aldehyde, ketone, GlcNAc, LAcNAc, maleimide, acrylamide, azide, tetrazinyl, cycloalkenyl (e.g., C2-C). 12 Cycloalkenyl), linear alkyne (e.g., C3-C) 12 Linear alkynyl) and cycloalkynyl (e.g., C2-C) 12 (Cycloynyl group).

[0238] In this application, the term "DAR" refers to the Drug-to-Antibody Ratio. DAR is one of the important quality attributes of ADC drugs, directly affecting their efficacy and safety. In this application, the DAR value can be a decimal, reflecting the properties of the ADC composition.

[0239] In this application, the term "alkyl" generally refers to a residue derived from an alkane by removing a hydrogen atom. Alkyl groups can be substituted or unsubstituted, substituted or non-substituted. The term "alkyl" generally refers to a saturated straight-chain or branched aliphatic hydrocarbon group having residues derived from the removal of hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, and can be a straight-chain or branched group containing 1 to 20 carbon atoms, for example, containing 1 to 12 carbon atoms, such as a chain alkyl containing 1 to 6 carbon atoms. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, etc. Alkyl groups can be substituted or unsubstituted, alternative or non-substituted. For example, when substituted, the substituent can be replaced at any usable connection point. The substituent can be independently selected from one or more substituents chosen from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo, for example, it can be hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H or C 1-6 Aliphatic groups. In this application, "C1-C6 alkyl" refers to alkyl groups having 1-6 carbon atoms, including methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl and tert-butyl), pentyl (including n-pentyl, isopentyl, sec-pentyl, tert-pentyl and neopentyl) and / or hexyl (including n-hexyl, isohexyl, sec-hexyl, tert-hexyl and neohexyl).

[0240] In this application, the term "alkylene" generally refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane. It can be a straight-chain or branched group containing 1 to 20 carbon atoms. For example, the term "methylene" can refer to a residue derived from the removal of two hydrogen atoms from a 1-carbon group. The methylene group can be substituted or unsubstituted, substituted or non-substituted; for example, containing 1 to 12 carbon atoms, such as an alkylene group containing 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH2CH2CH2CH2CH2-). The alkylene group can be substituted or unsubstituted, alternative or non-substituted. For example, when substituted, the substituent can be replaced at any usable connection point. The substituent can be independently selected from one or more substituents chosen from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo, for example, it can be hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H or C 1-6 Aliphatic group. The methylene or alkylene group may be substituted or unsubstituted. In this application, "C1-C6 alkylene" refers to an alkylene group having 1-6 (e.g., 1-5, 1-4, 1-3 or 1-2) carbon atoms.

[0241] In this application, the term "alkenyl" generally refers to a straight-chain or branched hydrocarbon group containing one or more double bonds. Exemplary examples of alkenyl groups include allyl, homoallyl, vinyl, crotonyl, butenyl, pentenyl, and hexenyl. "C2-C6 alkenyl" in this application refers to an alkenyl group having 2-6 (e.g., 2-5, 2-4, or 2-3) carbon atoms. Exemplary examples of C2-C6 alkenyl groups having more than one double bond include vinyl, propenyl, butadienyl, pentadienyl, hexadienyl, and hextrienyl, as well as their branched forms. The unsaturated bond (double bond) can be located at any position on the carbon chain. The alkenyl group can be substituted or unsubstituted.

[0242] In this application, the term "alkynyl" generally refers to an unsaturated straight-chain or branched alkynyl group containing one or more carbon-carbon triple bonds, such as ethynyl, 1-propynyl, propynyl, butynyl, etc. Alynyl groups can be substituted or unsubstituted. In this application, "C2-C6 alkynyl" refers to an alkynyl group having 2-6 (e.g., 2-5, 2-4, or 2-3) carbon atoms, including ethynyl, 1-propynyl, propynyl, butynyl, pentynyl, or hexynyl.

[0243] In this application, "alkyl", "alkenyl", "alkynyl", etc., can be linear groups.

[0244] In this application, "3- to 8-membered cycloalkyl" can be 3- to 7-membered cycloalkyl, 3- to 6-membered cycloalkyl, 3- to 5-membered cycloalkyl, or 3- to 4-membered cycloalkyl. In some cases, one or more carbon atoms of the cycloalkyl group may be substituted by one or more heteroatoms selected from O, S, and N.

[0245] In this application, the terms “optional” or “optionally” generally refer to an event or environment described subsequently that may but does not have to occur, and the description includes situations in which the event or environment occurs or does not occur. For example, “optionally alkyl-substituted heterocyclic group” means that an alkyl group may but does not have to be present, and the description can include cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0246] In this application, the term "substituted" generally refers to one or more hydrogen atoms in a group, for example, up to five, or for example, one to three hydrogen atoms, which are independently substituted by the corresponding number of substituents. Substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort.

[0247] In this application, as those skilled in the art will know, terms such as "alkyl," "alkenyl," and "cycloalkyl" may be preceded by an identifier indicating the number of atoms present in the group under specific conditions, for example, C1-C4 alkyl, C3-C7 cycloalkoxy, C1-C4 alkylcarbonylamino, etc., where the subscript number following "C" indicates the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl group having three carbon atoms (e.g., n-propyl, isopropyl); C1-C... 10 In this context, the members of the group can have any number of carbon atoms falling within the range of 1-10.

[0248] One or more hydrogen atoms in the functional group of this application, for example, up to five, or for example, one to three hydrogen atoms, are independently replaced by the corresponding number of substituents. The substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort.

[0249] In this application, the term "antibody" generally refers to a polypeptide or protein that specifically binds to an antigenic epitope or its mimic epitope. It typically refers to all antibodies from all species, including dimer, trimer, and multimer antibodies; bispecific antibodies; chimeric antibodies; fully human antibodies; humanized antibodies; recombinant and modified antibodies; and fragments thereof. The term "antibody" in this application may include an Fc fragment. For example, in this application, the antibody may be an IgG class antibody, such as an IgG1 antibody, IgG2 antibody, IgG3 antibody, or IgG4 antibody. The term "antigen-binding fragment" may refer to one or more fragments of an antibody that maintain its ability to specifically bind to an antigen. Binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-domain antibodies (DABs), scFv (single-chain Fv), etc. For example, fragments of full-length antibodies may be used to perform the antigen-binding function of the antibody. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art. The antibodies may include: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-TROP2 antibody, and anti-Claudin antibody. 18.2 Antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody or anti-Mesothelin antibody, for example, trastuzumab or pertuzumab.

[0250] In this application, the term "Fc fragment" generally refers to the portion of an antibody molecule located at the C-terminus, opposite the antigen-binding site (Fab fragment). Specifically, the Fc fragment consists of the constant regions (CH2 and CH3 domains) of the antibody heavy chain and is a region of the antibody molecule that does not participate in antigen recognition and binding. The Fc fragment plays an important role in antibody function, participating in various biological processes such as complement activation, immune complex clearance, placental transfer, antibody half-life regulation, antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell-mediated phagocytosis (ADCP).

[0251] In this application, the term "Fc fusion protein" generally refers to a fusion protein formed by fusing the Fc fragment with other proteins and / or peptides. The protein and / or peptide fused with the Fc fragment may be a component of an antibody (e.g., a Fab fragment), or may not be a component of an antibody (e.g., a cytokine, growth factor, hormone, receptor protein or ligand, enzyme, antigen, etc.).

[0252] In this application, the term "chimeric antibody" generally refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by murine antibodies. To establish a chimeric antibody, a hybridoma that secretes murine-specific monoclonal antibodies can be created. The variable region gene can then be cloned from the murine hybridoma cells, and the constant region gene of a human antibody can be cloned as needed. The murine variable region gene and the human constant region gene can be linked to form a chimeric gene, which is then inserted into an expression vector. The chimeric antibody molecule can then be expressed in eukaryotic or prokaryotic systems.

[0253] In this application, the term "humanized antibody," also known as a CDR-grafted antibody, generally refers to an antibody generated by grafting a mouse CDR sequence into a human antibody variable region framework, i.e., an antibody generated within a different type of human germline antibody framework sequence. This can overcome the heterologous response induced by chimeric antibodies carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available references. For example, germline DNA sequences of human heavy chain and light chain variable region genes can be found in the VBase human germline sequence database.

[0254] In this application, the terms "fully human antibody," "fully human antibody," or "completely human antibody," also known as "fully human monoclonal antibody," refer to antibodies whose variable and constant regions can both be human-derived, thus eliminating immunogenicity and toxic side effects. The development of monoclonal antibodies has gone through four stages: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies. The antibody or ligand described in this application can be a fully human monoclonal antibody. Related technologies for the preparation of fully human antibodies include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology, etc.

[0255] In this application, the term "Her2" generally refers to human epidermal growth factor receptor 2 (Her2). For example, the term "Her2" refers to any natural Her2 from any human source. The term also covers "full-length" and unprocessed Her2, as well as any form of Her2 derived from cells and processed (e.g., mature protein). The term also covers naturally occurring variants and isotypes of Her2, such as splice variants or allelic variants. For example, Uniprot accession number P04626 provides a description of Her2 and its sequence.

[0256] In this application, the terms "Trop2," "TROP2," and "Trop-2" generally refer to a single-pass transmembrane type I cell membrane protein. In this application, the term "Trop2" may also encompass homologs, variants, and isoforms of Trop2, including splice isoforms. The term "Trop2" also includes proteins having one or more sequences from Trop2 homologs, variants, and isoforms, as well as fragments of those sequences, provided they are variant proteins (including isoforms). Trop2 can be human Trop2. For example, Uniprot accession number P09758 provides a description of Trop2 and its sequence.

[0257] In this application, the term "EGFR" generally refers to the epidermal growth factor receptor. For example, the term "EGFR" refers to any naturally occurring EGFR from any human source. The term also covers "full-length" and unprocessed EGFR, as well as any form of EGFR derived from cells and processed (e.g., mature protein). The term also covers naturally occurring variants and isotypes of EGFR, such as splice variants or allelic variants. For example, Uniprot accession number P00533 provides a description of an EGFR and its sequence.

[0258] In this application, the term "Claudin18.2" generally refers to a transmembrane protein that makes up a tight junction. For example, the term "Claudin18.2" refers to any natural Claudin18.2 from any human source. The term also covers "full-length" and unprocessed Claudin18.2, as well as any form of Claudin18.2 derived from cells and processed (e.g., mature protein). The term also covers naturally occurring variants and isotypes of Claudin18.2, such as splice variants or allelic variants. For example, Uniprot accession number P56856 provides a description of Claudin18.2 and its sequence.

[0259] In this application, the term "self-eliminating group" refers to a group that can spontaneously undergo a chemical change under specific conditions to release the payload (drug). This design allows the linker unit to cleave after drug delivery to target cells via a specific triggering mechanism (such as enzyme catalysis, pH change, etc.), releasing the cytotoxic drug to kill tumor cells. The introduction of a self-eliminating group can increase the stability of ADC drugs and safely release potent cytotoxins at the appropriate time. For example, para-aminobenzoic acid ester (PABC) is a self-eliminating group that exhibits self-cleavage capability, promoting the release of the unmodified payload through a 1,6-elimination process. This self-elimination mechanism helps improve drug efficacy and reduce toxic side effects on normal cells.

[0260] In this application, the term "compound" generally refers to a substance having two or more different elements. For example, the compound in this application can be an organic compound, a compound with a molecular weight of less than 500, less than 1000, more than 1000, or more than 10,000 or 100,000. In this application, a compound can also refer to a compound linked by chemical bonds. For example, it can be a compound in which one or more molecules with a molecular weight of less than 1000 are linked by chemical bonds to a biological macromolecule, such as a polysaccharide, protein, nucleic acid, polypeptide, etc. For example, the compound in this application can include a compound in which a protein is linked to one or more molecules with a molecular weight of less than 1000, a compound in which a protein is linked to one or more molecules with a molecular weight of less than 10,000, or a compound in which a protein is linked to one or more molecules with a molecular weight of less than 100,000.

[0261] In this application, the term "conjugate" refers to a compound with or without a linker site. The conjugate may be a product obtained by removing a hydrogen atom from the compound at the linker site. The conjugate may contain one or more linker sites. The conjugate may be linked with other conjugates to form the substance protected in this application, comprising a small molecule drug, a cleavable unit, a ligand, and a linker unit.

[0262] In this application, the term "direct connection" generally refers to a connection between one part and another part without any intermediate parts. For example, equation (I) can be constructed using the structure shown in equation (I). Directly connected to the ligand, meaning there is no intermediate unit (usually a connecting subunit) between formula (I) and the ligand. For example, formula (I) can be constructed using the structure shown in formula (I). By indirectly connecting the subunit to the ligand, formula (I) is derived from the structure shown in formula (I). The linker unit is connected to a ligand, which in turn is connected to a ligand. In this application, the term "conjugate" may also be referred to as a "coupled substance" or "ligand-drug conjugate," typically referring to a ligand linked to a cleavable unit (optional) and a biologically active payload via a linker unit. In this application, "ligand-drug conjugate" can be an antibody-drug conjugate (ADC), where an ADC refers to a monoclonal antibody or antibody fragment linked to a biologically active cytotoxic drug via a stable linker unit.

[0263] In this application, the term "fucosylation" generally refers to the group formed by linking fucoidan or a fucoidan derivative to the target molecule.

[0264] In this application, the term "fucosyltransferase" generally refers to an enzyme or functional fragment or variant thereof capable of transferring fucose or fucose derivatives from a fucose or fucose derivative donor substrate to an acceptor substrate. The term "fucosyltransferase" may include any functional fragment (e.g., its catalytic domain) of the parent enzyme (e.g., wild-type fucosyltransferase) or a functional variant (e.g., a mutant). The fucosyltransferase in this application may be α-1,3-fucosyltransferase or a functional fragment or variant thereof. As in some specific embodiments, the fucosyltransferase may comprise the amino acid sequence shown in SEQ ID NO:1. A detailed technical description of the α-1,3-fucosyltransferase in this application can be found in PCT International Publication WO2023 / 097604.

[0265] In this application, the term "galactosyltransferase" generally refers to an enzyme or its functional fragment or variant capable of transferring D-galactose (Gal) or a D-galactose derivative from a galactose or galactose derivative donor substrate to an acceptor substrate. The galactosyltransferase in this application can be a β-1,4-galactosyltransferase or its functional fragment or variant. In some specific embodiments, the galactosyltransferase may comprise the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:3. In this application, the term "UDP-GalX" generally refers to a uridine diphosphate galactose derivative. Specifically, UDP-GalX is a class of compounds containing a uridine diphosphate (UDP) and a galactose or galactose derivative (GalX) structure. Specifically, UDP-GalX is an important donor substrate in glycosylation reactions, providing galactose or galactose derivative groups during biosynthesis for the synthesis of glycoconjugates containing galactose or galactose derivatives. Typically, the GalX structure in UDP-GalX corresponds to the GalX structure on the ligand. For example, in some embodiments of this application, UDP-GalX can be

[0266] In this application, the term "pharmaceutical composition" generally refers to a mixture containing one or more of the compounds described in this application or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. Pharmaceutical compositions can facilitate administration to organisms, promote the absorption of the active ingredient, and thereby exert its biological activity. Conventional methods for preparing pharmaceutical compositions can be found in the Chinese Pharmacopoeia.

[0267] The term "compound" in this application also includes its tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, or its pharmaceutically acceptable salts, prodrugs, or solvates.

[0268] In this application, Dxd has the following structure: In this application, Extc refers to eczetide, which has the following structure:

[0269] In this application, the terms "pharmaceutically acceptable salt" or "medicinal salt" generally refer to salts of compounds or ligand-drug conjugates of this application, or salts of compounds described in this application, which are safe and / or effective when used in mammals and possess the desired biological activity. The conjugates or compounds of this application can form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, etc. The conjugates or compounds of this application can form salts with bases. Non-limiting examples of pharmaceutically acceptable salts include: sodium salts, potassium salts, ammonium salts, lithium salts, Tris salts, calcium salts, magnesium salts, or zinc salts, etc.

[0270] In this application, the terms "solvent" or "solvent compound" generally refer to the conjugate or compound of this application that forms a pharmaceutically usable solvate with one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0271] In this application, the term "pharmaceutically acceptable carrier" generally refers to a carrier for administering therapeutic agents, such as antibodies or peptides, genes, and other therapeutic agents. This term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition and can be administered without causing excessive toxicity. For example, a pharmaceutically acceptable carrier can be distinguished from a nucleic acid vector used in genetic engineering to contain a target gene. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polyamino acids, amino acid copolymers, lipid aggregates, and inactivated viral particles. These carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in therapeutic compositions may include liquids such as water, saline, glycerol, and ethanol. These carriers may also contain excipients such as wetting agents or emulsifiers, pH buffers, etc.

[0272] The pharmaceutical composition may be in the form of a sterile injectable aqueous or oil suspension for intramuscular and subcutaneous administration. This suspension may be formulated using suitable dispersants or wetting agents and suspending agents as described above, according to known techniques. The sterile injectable formulation may also be a sterile injectable solution or suspension prepared in a non-toxic, parenteral-acceptable diluent or solvent, such as a solution prepared in 1,3-butanediol. Furthermore, a sterile fixative oil may be conveniently used as a solvent or suspension medium. For example, any blended fixative oil, including synthetic mono- or diglycerides of glycerol, may be used. Additionally, fatty acids such as oleic acid may also be used to prepare the injectable formulation.

[0273] In this application, the term "subject" can generally include humans, non-human primates (e.g., monkeys), or other animals, particularly mammals such as cattle, horses, pigs, sheep, goats, dogs, cats, or rodents such as mice and rats. In a particularly preferred embodiment, the subject is a human.

[0274] In this application, the terms "effective amount" and "therapeutic effective amount" are used interchangeably, generally referring to an amount or dose sufficient to produce a desired therapeutic outcome when a pharmaceutical composition comprising one or more of the said conjugates and / or compounds is administered. More specifically, a therapeutic effective amount is an amount sufficient to treat a specified condition, ailment, or disease over a period of time, specifically referring to an amount of a drug (e.g., an antigen-binding peptide provided in this application) or a pharmaceutical composition thereof that improves, alleviates, reduces, and / or delays one or more of its symptoms. In some instances targeting tumors, an effective amount includes an amount sufficient to cause tumor contraction and / or reduce tumor growth rate (e.g., inhibit tumor growth) or prevent or delay the proliferation of other unwanted cells. In some embodiments, an effective amount is an amount sufficient to delay the development of a tumor or cancer. In some embodiments, an effective amount is an amount sufficient to prevent or delay the recurrence of a tumor or cancer. An effective amount may be administered in one or more administrations. In some embodiments, an effective amount of the drug or a pharmaceutical composition thereof may: (1) reduce the number of cancer cells; (2) reduce tumor size; (3) inhibit, delay, slow down, and preferably prevent cancer cells from infiltrating into surrounding organs to a certain extent; (4) inhibit (i.e., slow down or stop) tumor metastasis to a certain extent; (5) inhibit tumor growth; (6) prevent or delay the occurrence and / or recurrence of tumors; and (7) alleviate one or more symptoms associated with cancer to a certain extent. The effective amount may vary depending on the specific antigen-binding peptide used and also on a variety of factors and conditions associated with the patient being treated and the severity of the disease. For example, if the antigen-binding peptide is to be administered in vivo, factors obtained in preclinical animal work, such as the patient's age, weight, and health, as well as dose-response curves and toxicity data, would be among those considered. Determining an effective or therapeutically effective amount of a given pharmaceutical composition is entirely within the capabilities of those skilled in the art.

[0275] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements. The terms "above" and "below" generally refer to situations that include the stated number.

[0276] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0277] Invention Details

[0278] This application relates to a conjugate comprising a small molecule drug (load), a cleavable unit, a linker unit, and a ligand, and / or a pharmaceutically acceptable salt. Those skilled in the art will understand that the conjugate comprising the small molecule drug (load), the cleavable unit, the linker unit, and the ligand may also be referred to as a "ligand-drug conjugate".

[0279] Small molecule drugs

[0280] In this application, a small molecule drug may also be referred to as a payload, which is a molecule that is biologically active in vivo or in vitro. In some embodiments, the small molecule drug has a compound with the structure shown in formula (IV).

[0281] Decomposable unit

[0282] In this application, a cleavable unit generally refers to a structural unit that can be cleaved under certain conditions, such as within a specific pH range, within a specific temperature range, or in the presence of an enzyme. For example, a "cleavable unit" can be an acid-cleavable unit, a redox-cleavable unit, a photoactive cleavable unit, and / or a proteolytic cleavable unit.

[0283] "Load + Decomposable Unit"

[0284] In this application, the "load + cleavable unit" is typically obtained by combining a small molecule drug (load) and a cleavable unit. In some embodiments, the "load + cleavable unit" has the structure shown in formula (I). In some embodiments, the structure shown in formula (I) comprises a small molecule drug with the structure shown in formula (IV) and a cleavable unit. Typically, the "load + cleavable unit" can release the load by cleaving the cleavable unit.

[0285] In this application, the compound represented by formula (IV) can be a product of the cleavage of formula (I), formula (II-a), formula (II-b), formula (III-a), and / or formula (III-b). The cleavage can occur in vivo or in vitro. The inducing conditions for the cleavage can be selected from one or more of the following: pH sensitivity, redox sensitivity, photosensitivity, enzyme cleavage, and radiotherapy response, preferably enzyme cleavage. The cleavage is more preferably carried out in a tumor environment.

[0286] In some embodiments, in formulas (I), (II-a), (II-b), (III-a), and / or (III-b), R 1 For hydrogen, R 2 for R 3 for In some cases, these structures are cleaved by enzymes into Tp-12-2 and trace amounts of Tp-12 (Figure 1).

[0287] In other embodiments, in formulas (I), (II-a), (II-b), (III-a), and / or (III-b), R 1 For hydrogen, R 2 for R 3 for In some cases, these structures are cleaved into Tp-12-1 and Tp-12 by enzyme cleavage (Figure 1).

[0288] Furthermore, in the conjugates protected in this application that include a ligand, a "load + cleavable unit", and a linker unit, the load in the same conjugate molecule can be a compound represented by the same formula (IV), or a compound represented by different formulas (IV), or as shown in P 2 Other small molecule drugs, nucleic acids, or peptides, or combinations thereof. In the conjugates protected in this application comprising a ligand, a "load + cleavable unit," and a linker unit, the "load + cleavable unit" in the same conjugate molecule may be the same structure shown in formula (I), or different structures shown in formula (I), or "P..." 2 +L 2 The combination of "".

[0289] For example, P 2 Other small molecule drugs may be selected from one or more of the following: pyrrolobenzodiazepine (PBD), olritatin (e.g., MMAE or MMAF), maytansine alkaloids (maytansine, DM1 or DM4), docalamycin, enediyne (e.g., calichiomycin), doxorubicin (PNU), pyrrole-based kinesin spindle protein (KSP) inhibitors, calichiomycin, amanita (e.g., α-amanita), etc.

[0290] For example, P 2The nucleic acid can be selected from one or more of the following: antisense oligonucleotides, small interfering RNA, microRNA, small activating RNA, messenger RNA, and RNA aptamers. The target of the nucleic acid may be selected from one or more of the following: HER2, HER3, TROP2, B7-H3, GPC20, CDH6, EGFR, EGFRvIII, AXL, Nectin-4, Tissue factor, TIM-1, PSMA, EpCAM, MUC1, STEAP1, GPNMB, FGF2, FOLR1, c-MET, GFR, AGS-16, Guanylyl cyclase C, Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, ROR1, PD-L1, CD27L, 5T4, Mucin 16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, CEACAM5, SC-16, SLC39A6, Delta-like protein3, Claudin 18.2, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD73, CD74, CD79b, CD138, CD147, CD166, CD223, MUC16, MSLN, ENPP3, SLTRK6, FGFR, LIV-1, Lewis Y, av-integrin, ASCT2, C4.4a, CA-IX, CD324, CD352, CD44v6, CD48a, CLL-1, Cripto, CS1, DPEP3, Ephrin-A2, Ephrin-A4, ETBR, FGFR2, FGFR3, FLT3, GD3, Globo H, GPC3, LAMP-1, LRRC15, Ly6E, MFI2, NOTCH3, p-cadherin, PRLR, and RNF43.

[0291] For example, P 2 The polypeptide may be selected from one or more of the following: salmon calcitonin, somatostatin, human glucagon, oxytocin, octreotide acetate, lanreotide acetate, ticokide acetate, human erythropoietin, human thrombopoietin, human growth hormone, nesiritide, eptifibatide, atebandide, capperidide, glucagon, exenatide, liraglutide, and pramlintide.

[0292] In some embodiments, when the conjugate protected by this application, comprising a ligand, a "load + cleavable unit", and a connecting subunit, contains P 2 At that time, P 2It can synergize with the compound shown in formula (I) to enhance the efficacy of the conjugate and improve effects such as targeting activity or bystander kill effect.

[0293] Connecting subunit

[0294] In this application, the connecting subunit can effectively connect the ligand and the load, or the ligand and a "load + cleavable unit". In this application, the connecting subunit can be composed of a connector unit, a branch unit, and a spacer unit. The connector unit is responsible for connecting the ligand. The connector unit plays a crucial role in the therapeutic process of the ligand-drug conjugate. The stability of the connector unit significantly affects the safety and toxicity of the conjugate. For example, in some cases, connector units based on maleimide and thiol linkages are not stable enough in plasma due to the presence of reverse reactions, leading to premature load detachment and toxic side effects. In contrast, fucosylate-based connectors have better stability in plasma. The branch unit can be used to increase the number of loads or "load + cleavable units" connected to the connecting subunit. The spacer unit can be used to adjust the spatial distance between the "load + cleavable unit" and the connector unit, the structural rigidity, and the hydrophilicity / hydrophobicity of the conjugate.

[0295] In this application, the bonding between the linker unit and the ligand can be formed through the interaction of an electrophilic group and a nucleophilic group, or through the interaction of a nucleophilic group and an electrophilic group. In some embodiments, the nucleophilic group of the ligand includes, but is not limited to, thiol, hydroxyl, acylhydrazine, oxime, hydrazine, thiourea, hydrazide carboxylate, and aryl acylhydrazine groups; the nucleophilic group of the ligand can react with the electrophilic group to form a covalent bond, and the electrophilic group is selected from: active esters, hydrocarbon halides, benzyl halides, aldehydes, ketones, carboxyl groups, and maleimide groups, preferably hydrocarbon halides and maleimide groups, more preferably maleimide groups; the active esters are preferably NHS esters, HOBt esters, haloformates, and acid halides, and the hydrocarbon halides are preferably haloacetamides. In some embodiments, the ligand has an electrophilic group selected from aldehydes, ketones, carboxyl groups, and maleimide groups, preferably maleimide groups; the electrophilic group of the ligand is capable of reacting with a nucleophilic group to form a covalent bond, the nucleophilic group being selected from mercapto, hydroxyl, acylhydrazine, oxime, hydrazine, thiourea, hydrazide ester, and aryl acylhydrazine groups, preferably mercapto.

[0296] In some embodiments, the nucleophilic group of the ligand may also be selected from N-terminal amino groups, side-chain amino groups, side-chain thiol groups, hydroxyl or amino groups of sugars in glycosylated antigen-binding proteins, preferably side-chain thiol groups, more preferably thiol groups of cysteine; the electrophilic group that reacts therewith is selected from active esters, hydrocarbon halides, benzyl halides, aldehydes, ketones, carboxyl groups and maleimide groups, preferably hydrocarbon halides, maleimide groups, more preferably maleimide groups; the active esters are preferably NHS esters, HOBt esters, haloformates, acid halides, and the hydrocarbon halides are preferably haloacetamides.

[0297] In this application, the connection between the ligand and the linker unit can be achieved using glycosyltransferases. For example, a molecule of interest can be linked to a ligand via a glycosyltransfer reaction catalyzed by fucosyltransferase, such as linking the structure shown in formula (III-a) or (III-b) of this application to a protein ligand to form the structure shown in formula (II-a) or (II-b) of this application. For techniques related to the application of fucosyltransferases in the preparation of conjugates, please refer to PCT international publications WO2022037665, WO2023065137, WO2023097604 and WO2023141855.

[0298] In this application, the connection between the ligand and the linker unit can also be achieved through a bioorthogonal reaction. The bioorthogonal functional groups that the ligand may contain are selected from: azide, tetrazinyl, cycloalkenyl, linear alkynyl, and cycloalkynyl. The bioorthogonal functional groups that react with it are selected from: azide, tetrazinyl, cycloalkenyl, linear alkynyl, and cycloalkynyl.

[0299] ligands

[0300] In some implementations, the ligand is typically an antibody or its antigen-binding fragment. The ligand can typically target a specific region within the subject's body after administration, releasing the small molecule drug at that region.

[0301] In some implementations, the ligand may also be other molecules that can target the target region, such as Fc fragments or Fc fusion proteins.

[0302] In some embodiments, in formula (II-a) and / or formula (II-b), the AB may be an antibody or an antigen-binding fragment thereof.Specifically, the AB can be selected from the following group: anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-GPC20 antibody, anti-CDH6 antibody, anti-EGFR antibody, anti-EGFRvIII antibody, anti-AXL antibody, anti-Nectin-4 antibody, anti-Tissue factor antibody, anti-TIM-1 antibody, anti-PSMA antibody, anti-EpCAM antibody, anti-MUC1 antibody, anti-STEAP1 antibody, anti-GPNMB antibody, anti-FGF2 antibody, anti-FOLR1 antibody, anti-c-MET antibody, anti-GFR antibody, anti-AGS-16, anti-Guanylyl cyclase C antibody, anti-Mesothelin antibody, anti-SLC44A4 antibody, anti-PSMA antibody, anti-EphA2 antibody, anti-AGS-5 antibody, anti-GPC-3 antibody, anti-c-KIT antibody, anti-ROR1 antibody, anti-PD-L1 antibody, anti-CD27L antibody, anti-5T4 antibody, anti-Mucin Anti-16 antibody, anti-NaPi2b antibody, anti-STEAP antibody, anti-SLITRK6 antibody, anti-ETBR antibody, anti-BCMA antibody, anti-CEACAM5 antibody, anti-SC-16 antibody, anti-SLC39A6 antibody, anti-Delta-like protein3 antibody, anti-Claudin 18.2 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD45 antibody, anti-CD56 antibody, anti-CD66e antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD74 antibody, anti-CD79b antibody, anti-CD138 antibody, anti-CD147 antibody, anti-CD166 antibody, anti-CD223 antibody, anti-MUC16 antibody, anti-MSLN antibody, anti-ENPP3 antibody, anti-SLTRK6 antibody, anti-FGFR antibody, anti-LIV-1 antibody, anti-Lewis Antibody Y, anti-av-integrin antibody, anti-ASCT2 antibody, anti-C4.4a antibody, anti-CA-IX antibody, anti-CD324 antibody, anti-CD352 antibody, anti-CD44v6 antibody, anti-CD48a antibody, anti-CLL-1 antibody, anti-Cripto antibody, anti-CS1 antibody, anti-DPEP3 antibody, anti-Ephrin-A2 antibody, anti-Ephrin-A4 antibody, anti-ETBR antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FLT3 antibody, anti-GD3 antibody, anti-Globo H antibody, anti-GPC3 antibody, anti-LAMP-1 antibody, anti-LRRC15 antibody, anti-Ly6E antibody, anti-MFI2 antibody, anti-NOTCH3 antibody, anti-p-cadherin antibody, anti-PRLR antibody, and anti-RNF43 antibody, as well as the antigen-binding fragments mentioned above.

[0303] In some embodiments, in formula (II-a) and / or formula (II-b), the heavy chains HCDR1, HCDR2 and HCDR3 and the light chains LCDR1, LCDR2 and LCDR3 of the AB respectively comprise the heavy chains HCDR1, HCDR2 and HCDR3 and the light chains LCDR1, LCDR2 and LCDR3 of the antibody.

[0304] In some embodiments, in formula (II-a) and / or formula (II-b), the heavy chain variable region VH and the light chain variable region VL of the AB respectively comprise the heavy chain variable region VH and the light chain variable region VL of the antibody.

[0305] The ligands described in this application may be protein hormones, lectins, growth factors, antibodies, or other molecules capable of binding to cells, receptors, and / or antigens. For example, the ligands in this application may be antibodies or their antigen-binding fragments.

[0306] In this application, the ligand comprises at least one CDR in the antibody variable region. The CDR in this application may be defined according to Kabat or according to Chothia, and CDR sequences defined in various ways are all included within the scope of protection of this application.

[0307] The antibodies described in this application can be prepared using techniques well-known in the art, such as hybridoma methods, recombinant DNA technology, phage display technology, synthetic techniques, or combinations thereof, or other techniques known in the art. Variants can refer to amino acid sequence mutants of the antibody, as well as covalent derivatives of natural polypeptides, provided they retain biological activity comparable to the natural polypeptide. The difference between amino acid sequence mutants and natural amino acid sequences generally lies in the substitution of one or more amino acids in the natural amino acid sequence, or the deletion and / or insertion of one or more amino acids in the polypeptide sequence. Deletion mutants include fragments of the natural polypeptide and N-terminal and / or C-terminal truncated mutants. Typically, amino acid sequence mutants possess at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% homology to the natural sequence. As is well known to those skilled in the art, antibodies containing a particular amino acid sequence may undergo some common modifications during fermentation, expression, and purification, such as C-terminal lysine removal, methionine oxidation, and disulfide bond opening. The antibodies described in this application that contain specific amino acid sequences also include these modified antibodies, such as those with C-terminal lysine removal, methionine oxidation, disulfide bond opening, etc.

[0308] Because the conjugates provided in this application can target specific cell populations and bind to cell surface-specific proteins (antigens), thereby releasing the drug into the cells in its active form through conjugate endocytosis or drug penetration, the conjugates of this application can be used to treat target diseases. The conjugates of this application can be administered to subjects (e.g., humans) in therapeutically effective amounts via appropriate routes. Subjects requiring treatment may be patients at risk or suspected of having a condition related to the activity or expression level of a specific antigen. Such patients can be identified through routine physical examinations.

[0309] When used for treatment with the conjugate of this application, delivery can be performed using methods conventional in the art. For example, it can be introduced into cells using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the conjugate can be delivered by direct injection or by using an infusion pump. Other methods may include the use of various conjugates and biodegradable transport and carrier systems.

[0310] Conjugate

[0311] The conjugates of this application exhibit excellent tumor cell-targeting killing activity.

[0312] In some cases, in some implementations, in the structure shown in equation (I), R 1 For hydrogen, R 3 ,R 4 ,R 5 When they are the same, and all other conditions are the same, R 2 for (R 21 Conjugates containing hydrogen (C) exhibit excellent targeted killing activity. For example, among the Tras-IF series conjugates 6-19, 29, and 44, Tras-IF-6, Tras-IF-7, Tras-IF-8, Tras-IF-16, Tras-IF-19, and Tras-IF-44 show superior or comparable targeted killing activity at NCI-N87 to the existing technology control conjugates Tras-IF-1 (“load + cleavable unit” is GGFG-Dxd) or Tras-IF-2 (“load + cleavable unit” is GGFG-Extc) (Figure 2). The remaining conjugates show weaker targeted killing activity at NCI-N87 than the positive control conjugates Tras-IF-1 or Tras-IF-2 (Figure 2).

[0313] In some cases, in some implementations, in the structure shown in equation (I), R 1 For hydrogen, when R 2 ,R 4 ,R 5If they are the same, and all other conditions are the same, R 3 for The conjugate compared to R 3 for The conjugates exhibit superior or comparable targeted killing activity. For example, Tras-IF-27 shows superior or comparable targeted killing activity on NCI-N87 compared to Tras-IF-19, and Tras-IF-28 shows superior or comparable targeted killing activity on NCI-N87 compared to Tras-IF-8 (Figure 2). Similarly, Tras-II-F-D7 shows superior or comparable targeted killing activity on NCI-N87 compared to Tras-II-F-D5 (Figure 2).

[0314] In some cases, in some implementations, in the structure shown in equation (I), R 1 For hydrogen, when R 2 ,R 4 ,R 5 When they are the same, and all other conditions are the same, R 3 for The couplings and R 3 for The conjugates exhibit considerable targeted killing activity. For example, Tras-IF-8 and Tras-IF-31 show considerable targeted killing activity on NCI-N87 (Figure 2).

[0315] In some cases, in some implementations, in the structure shown in equation (I), R 1 For hydrogen, under the same conditions, R 21 The targeted killing activity of hydrogen conjugates is significantly better than that of R. 21 These are conjugates of methyl groups. For example, Tras-IF-8 exhibits significantly enhanced targeted killing activity on NCI-N87 compared to Tras-IF-29. Similarly, Tras-IF-28, Tras-IF-33, Tras-IF-36, Tras-IF-34, Tras-IF-31, Tras-IF-32, and Tras-IF-30, compared to Tras-IF-35, all show significantly enhanced targeted killing activity on NCI-N87.

[0316] In some cases, in some implementations, in the structure shown in equation (I), R 1 For hydrogen, R 2 for R 3 for R 4 for R 5 for The conjugates and existing technology control conjugates Tras-IF-1 (“load + cleavable unit” is GGFG-Dxd) or Tras-IF-2 (“load + cleavable unit” is GGFG-Extc) exhibit superior or comparable targeted killing activity on NCI-N87. For example, Tras-IF-19, Tras-IF-27, Tras-IF-39, Tras-IF-40, Tras-IF-43, Tras-IF-47, Tras-II-F-D4, Tras-II-F-D5, Tras-II-F-D7, Tras-II-F-D8, Tras-II-F-D15, Tras-II-F-D18, Tras-II-F-D19, and Tras-II-F... Tras-II-F-D21, Tras-II-F-D22, Tras-IV-F-D5, Tras-VI-F-D5, and Tras-IV-F-Az0-DBCO'-1 all exhibited superior or comparable targeted killing activity compared to the positive control conjugates Tras-IF-1 (“cleavable unit-load” is GGFG-Dxd) and Tras-IF-2 (“cleavable unit-load” is GGFG-Extc) (Figure 2).

[0317] The conjugate of this application exhibits excellent bystander killing activity.

[0318] In some cases, in some implementations, in the structure shown in equation (I), R 1 For hydrogen, R 3 ,R 4 ,R 5 When they are the same, and all other conditions are the same, they contain different R values. 2 The bystander lethality of the coupled structures varies significantly. 2 for The structure of the coupling has optimal bystander lethality. Furthermore, R 2 for Harmony Conjugates of this structure also exhibit good bystander-killing activity. For example, in Figure 8A, Tras-IF-19 shows the best bystander-killing activity, superior to or equivalent to the positive control conjugate Tras-IF-1. Furthermore, Tras-IF-7 and Tras-IF-8 also demonstrate good bystander-killing activity. However, while Tras-IF-6 and Tras-IF-12 exhibit good targeted-killing activity, their bystander-killing activity is relatively poor. Similarly, in Figure 8C, Tras-IF-19 shows the strongest bystander-killing activity, superior to or equivalent to the control conjugate Tras-IF-1. In contrast, although Tras-IF-16 and Tras-IF-44 also exhibit good targeted-killing activity, their bystander-killing activity is significantly reduced compared to Tras-IF-19.

[0319] In some cases, in some implementations, in the structure shown in equation (I), R 1 For hydrogen, R 2 R 3 When all other conditions are the same, R 4 The absence of the structure leads to a significant reduction in the bystander lethal activity of the coupling. For example, in Figure 8C, Tras-IF-41(R 5 for R 4 (structural missing) and Tras-IF-42 (R 5 for R 4 (Structural missing) compared to Tras-IF-19 (R 5 for R 4 for Its bystander lethality is significantly reduced.

[0320] In some cases, in some implementations, in the structure shown in equation (I), R 1 For hydrogen, R 2 R 4 ,R 5 And all other things being equal, R 3 for The couplings compared to R 3 for The conjugates exhibit superior or comparable bystander killing activity. For example, in Figures 8D and 8E, Tras-IF-27 exhibits superior or comparable bystander killing activity compared to Tras-IF-19.

[0321] In some cases, in some implementations, R 1 For hydrogen, R 1 ,R2 ,R 3 ,R 4 And all other things being equal, R 5 for The coupling with R 5 for The conjugates exhibit superior or comparable bystander killing activity. For example, in Figures 8D and 8E, Tras-IF-39 exhibits superior or comparable bystander killing activity compared to Tras-IF-19.

[0322] In some cases, in some implementations, in the structure shown in equation (I), R 1 For hydrogen, R 2 for R 3 for R 4 for R 5 for The conjugates exhibit superior or comparable bystander killing activity compared to the existing control conjugate Tras-IF-1 (“load + cleavable unit” is GGFG-Dxd). For example, in Figure 8E, Tras-IF-19, Tras-IF-27, Tras-IF-39, Tras-IF-40, Tras-IF-43, and Tras-IF-47 all show superior or comparable bystander killing activity compared to the positive control conjugate Tras-IF-1.

[0323] Pharmaceutical Composition

[0324] On the one hand, this application provides a pharmaceutical composition which may contain any conjugate and / or compound described in any one of the claims, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, and may optionally contain a pharmaceutically acceptable carrier.

[0325] In addition to the active compound, the pharmaceutical composition described in this application may contain one or more excipients, which may be selected from the group consisting of fillers (diluents), binders, wetting agents, disintegrants, and excipients. Depending on the method of administration, the composition may contain 0.1 to 99% by weight of the active compound.

[0326] Pharmaceutical compositions containing active ingredients may be in oral forms, such as tablets, sugar lozenges, tablets, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups. Oral compositions may be prepared according to any method known in the art for preparing pharmaceutical compositions, and may contain binders, fillers, lubricants, disintegrants, or pharmaceutically acceptable wetting agents, and may also contain one or more ingredients selected from the group consisting of sweeteners, flavoring agents, coloring agents, and preservatives.

[0327] Aqueous suspensions may contain active substances and excipients suitable for mixing. Aqueous suspensions may also contain one or more preservatives, such as one or more colorants, one or more flavoring agents, and one or more sweeteners. Oil suspensions can be prepared by suspending the active ingredient in vegetable oil. Oil suspensions may contain thickeners. The aforementioned sweeteners and flavoring agents may also be added.

[0328] The pharmaceutical composition may also provide the active ingredient as a dispersible powder or granules for preparing an aqueous suspension, by adding one or more of a water-mixing dispersant, wetting agent, suspending agent, or preservative. Other excipients such as sweeteners, flavoring agents, and coloring agents may also be added. These compositions are preserved by adding antioxidants such as ascorbic acid. The pharmaceutical compositions of this application may also be in the form of an oil-in-water emulsion.

[0329] The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Acceptable solvents or media that can be used include water, Ringer's solution, and isotonic sodium chloride solution. The sterile injectable formulation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase. For example, the active ingredient may be dissolved in a mixture of soybean oil and lecithin. The oil solution may then be treated with a mixture of water and glycerol to form a microemulsion. The injection solution or microemulsion may be injected into the patient's bloodstream via local large-volume injection. Alternatively, the solution and microemulsion may be administered in a manner that maintains a constant circulating concentration of the compound of this application. To maintain such a constant concentration, a continuous intravenous delivery device may be used. For example, the device may be an intravenous infusion pump.

[0330] The pharmaceutical composition may be in the form of a sterile injectable aqueous or oil suspension for intramuscular and subcutaneous administration. This suspension may be formulated using suitable dispersants or wetting agents and suspending agents described above, according to known techniques. The sterile injectable formulation may also be a sterile injectable solution or suspension prepared in a parenteral-acceptable, non-toxic diluent or solvent. Alternatively, a sterile fixative oil may be conveniently used as a solvent or suspension medium.

[0331] The compounds of this application may be administered in suppository form for rectal administration. These pharmaceutical compositions can be prepared by mixing the drug with a suitable, non-irritating excipient that is solid at normal temperatures but liquid in the rectum, and thus dissolves in the rectum to release the drug. Such substances include cocoa butter, glycerin gelatin, hydrogenated vegetable oils, polyethylene glycol of various molecular weights, and mixtures of fatty acid esters of polyethylene glycol.

[0332] As is well known to those skilled in the art, the dosage or effective amount of a drug depends on a variety of factors, including but not limited to: the activity of the specific compound used, the patient's age, the patient's weight, the patient's health status, the patient's behavior, the patient's diet, the timing of administration, the route of administration, the rate of excretion, and the combination of drugs; in addition, the optimal mode of treatment, such as the treatment regimen, the form of the compound described in this application or its tautomers, mesosomes, racemates, enantiomers, diastereomers or mixtures thereof, or pharmaceutically acceptable salts thereof, and / or the daily dosage or type of pharmaceutically acceptable salt of the compound or its tautomers, mesosomes, racemates, enantiomers, diastereomers or mixtures thereof, or pharmaceutically acceptable salts thereof, can be verified based on conventional treatment regimens.

[0333] The pharmaceutical composition of this application may contain a safe and effective amount of the ligand conjugate of this application and a pharmaceutically acceptable carrier. Such carriers may include (but are not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched to the route of administration. The pharmaceutical composition of this application may be formulated as a solution, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. The pharmaceutical composition may be manufactured under aseptic conditions. The dosage of the active ingredient may be a therapeutically effective amount.

[0334] The effective amount of the ligand conjugate described in this application can vary depending on the administration method and the severity of the disease to be treated. The selection of the effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors may include, but are not limited to: pharmacokinetic parameters of the conjugate such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. Generally, satisfactory results can be obtained when the antibody-drug conjugate of this application is administered daily at an appropriate dose. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0335] The compounds of the present application can be administered alone or in combination with other pharmaceutically acceptable therapeutic agents. When administered in combination with other therapeutic agents, the compounds of the present application can be administered either sequentially or concurrently with one or more other therapeutic agents.

[0336] tumor

[0337] This application provides the use of the compound or conjugate of this application, or its tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug, or solvate, and / or the pharmaceutical composition of this application in the preparation of a medicament for the treatment and / or prevention of tumors. For example, the tumor may be selected from tumors associated with the expression of the following target groups: HER2, HER3, TROP2, B7-H3, GPC20, CDH6, EGFR, EGFRvIII, AXL, Nectin-4, Tissue factor, TIM-1, PSMA, EpCAM, MUC1, STEAP1, GPNMB, FGF2, FOLR1, c-MET, GFR, AGS-16, Guanylyl cyclase C, Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, ROR1, PD-L1, CD27L, 5T4, Mucin 16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, CEACAM5, SC-16, SLC39A6, Delta-like protein3, Claudin18.2, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD73, CD74, CD79b, CD138, CD147, CD166, CD223, MUC16, MSLN, ENPP3, SLTRK6, FGFR, LIV-1, Lewis Y, av-integrin, ASCT2, C4.4a, CA-IX, CD324, CD352, CD44v6, CD48a, CLL-1, Cripto, CS1, DPEP3, Ephrin-A2, Ephrin-A4, ETBR, FGFR2, FGFR3, FLT3, GD3, Globo H, GPC3, LAMP-1, LRRC15, Ly6E, MFI2, NOTCH3, p-cadherin, PRLR, and RNF43. For example, tumors associated with the expression of the target include tumors with high expression of the target and / or tumors positive for the target. For example, the tumors include solid tumors and / or hematologic malignancies. For example, the tumors are selected from the group consisting of: breast cancer, ovarian cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphoblastic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, pancreatic cancer, prostate cancer, colorectal cancer, gastric cancer, glioma, and mesothelioma.

[0338] This application provides a method for preventing and / or treating tumors, which may include administering to a subject the compound or conjugate of this application, or its tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, or its pharmaceutically acceptable salts, prodrugs, or solvates, and / or the pharmaceutical composition of this application. For example, the tumor may be selected from tumors associated with the expression of the following target groups: HER2, HER3, TROP2, B7-H3, GPC20, CDH6, EGFR, EGFRvIII, AXL, Nectin-4, Tissue factor, TIM-1, PSMA, EpCAM, MUC1, STEAP1, GPNMB, FGF2, FOLR1, c-MET, GFR, AGS-16, Guanylyl cyclase C, Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, ROR1, PD-L1, CD27L, 5T4, Mucin 16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, CEACAM5, SC-16, SLC39A6, Delta-like protein3, Claudin18.2, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD73, CD74, CD79b, CD138, CD147, CD166, CD223, MUC16, MSLN, ENPP3, SLTRK6, FGFR, LIV-1, Lewis Y, av-integrin, ASCT2, C4.4a, CA-IX, CD324, CD352, CD44v6, CD48a, CLL-1, Cripto, CS1, DPEP3, Ephrin-A2, Ephrin-A4, ETBR, FGFR2, FGFR3, FLT3, GD3, Globo H, GPC3, LAMP-1, LRRC15, Ly6E, MFI2, NOTCH3, p-cadherin, PRLR, and RNF43. For example, the tumors associated with the expression of the target include tumors with high expression of the target and / or tumors positive for the target. For example, the tumors include solid tumors and / or hematologic malignancies. For example, the tumors are selected from the group consisting of: breast cancer, ovarian cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphoblastic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, pancreatic cancer, prostate cancer, colorectal cancer, gastric cancer, glioma, and mesothelioma.

[0339] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the compounds, conjugates, preparation methods and uses of this application, and are not intended to limit the scope of the invention.

[0340] Example

[0341] Example 1: Compound Preparation

[0342] Instruments, materials and reagents

[0343] The structure of the compound was determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR measurements were performed using BRUKER AVANCE III HD spectrometer with deuterated dimethyl sulfoxide (DMSO-D) and deuterated water (D2O) as solvents and tetramethylsilane (TMS) as the internal standard.

[0344] The LC-MS determination was performed using Waters E2695-2489-Qda (manufacturer: Waters, model: E2695-2489).

[0345] HRMS measurements were performed using Waters XEVO G2-XS Q-TOF (Manufacturer: Waters, Model: XEVO G2-XS Q-TOF).

[0346] HPLC preparation was performed using either the Hanbang preparation system (Hanbang NP7010C pump, NU3000 UV detector, NC6100 fraction collector, Nucifera C18M column), the Waters preparation system (Waters 1525 Binary HPLC pump, Waters 2489 UV detector, Waters Fraction Collector III fraction collector, UniSil 10-120C18 Aq column), or the Waters preparation system (Waters 2545 Binary HPLC pump, Waters 2489 UV detector, Waters Fraction Collector III fraction collector, UniSil 10-120C18 Aq column).

[0347] The silica gel plates used for thin-layer chromatography were Leyan HPTLC Silica Gel 60GF254 plates. The eluent system was a dichloromethane and methanol system, and the volume ratio of the solvent was adjusted according to the polarity of the compounds. Small amounts of ammonia and formic acid could also be added for adjustment.

[0348] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art. Unless otherwise specified, the solvents and reagents disclosed herein can be purchased from companies such as Maclean, J&K, Innocare, Bidex, Leyan, Hines and Confuno.

[0349] Unless otherwise specified in the examples, the reaction temperature is room temperature. Room temperature is the optimal reaction temperature, and the temperature range is 20-30℃.

[0350] Example 1.1 Preparation of Tp-1

[0351] Step 1: N-tert-butoxycarbonyl-glycine (52.6 mg, 0.3 mmol), eczemab mesylate (106.3 mg, 0.2 mmol), HATU (114.1 mg, 0.3 mmol), DMF (5 mL), and DIPEA (105 μL, 0.6 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. The product was further purified by a preparative HPLC system to obtain Tp-1a (75 mg, yield 63.3%).

[0352] Step 2: Tp-1a (75 mg, 0.127 mmol), DCM (10 mL), and TFA (2 mL) were added sequentially to the reaction mixture, and the reaction was carried out at room temperature for 5 h. The product was further purified using a preparative HPLC system to obtain Tp-1 (53.1 mg, yield 85.0%). Purity (HPLC-RP): 98.1%. LC-MS: [M+H] + =493.6.

[0353] Example 1.2 Preparation of Tp-2

[0354] Step 1: N-tert-Butoxycarbonyl-L-alanine (28 mg, 0.15 mmol), eczemab mesylate (53 mg, 0.1 mmol), HATU (76 mg, 0.2 mmol), DMF (3 mL), and DIPEA (50 μL, 0.3 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-2a (50 mg, yield 82.6%).

[0355] Step 2: Add Tp-2a (50 mg, 0.083 mmol), TFA (3 mL), and DCM (6 mL) sequentially to the reaction flask, and react at room temperature for 1 h. After the reaction, concentrate the product and further purify it using a preparative HPLC system to obtain Tp-2 (29.3 mg, yield 70.2%). Purity (HPLC-RP): 95.7%. LC-MS: [M+H] +=507.6. 1 H-NMR (400MHz, DMSO-d6) δ8.62(s,1H),7.75(d,J=8.0Hz,1H),7.28(s,1H),5.53(t,J=8.0Hz,1H),5.41(s,2H),5.12(s,2H),3.53 -3.49(m,1H),3.17-3.12(m,2H),2.36(s,3H),2.18-2.12(m,2H),1.90-1.79(m,2H),1.25(d,J=8.0Hz,3H),0.87(t,J=4.0Hz,3H).

[0356] Example 1.3 Preparation of Tp-3

[0357] Step 1: N-tert-butoxycarbonyl-L-2-aminobutyric acid (30 mg, 0.15 mmol), eczemab mesylate (53 mg, 0.1 mmol), HATU (76 mg, 0.2 mmol), DMF (3 mL), and DIPEA (50 μL, 0.3 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was complete, the product was further purified using a preparative HPLC system to obtain Tp-3a (55 mg, yield 88.9%). LC-MS: [M+H] + =621.8.

[0358] Step 2: Add Tp-3a (55 mg, 0.089 mmol), TFA (3 mL), and DCM (6 mL) sequentially to the reaction flask, and react at room temperature for 1 h. After the reaction is complete, concentrate the solution, and take 20% of the sample for further purification using a preparative HPLC system to obtain Tp-3 (8.3 mg, yield 90.0%). Purity (HPLC-RP): 97.8%. LC-MS: [M+H] + =521.7. 1 H-NMR (400MHz, DMSO-d6) δ8.43(s,1H),7.75(d,J=8.0Hz,1H),7.29(s,1H),5.52(s,1H),5.41(s,2H),5.16(q,J=12.0Hz,2H),3.19(t,J=4.0H z,1H),3.15(t,J=4.0Hz,2H),2.37(s,3H),2.22-2.09(m,2H),1.91-1. 80(m,2H),1.66-1.09(m,1H),1.51-1.44(m,1H),0.87(q,J=4.0Hz,6H).

[0359] Example 1.4 Preparation of Tp-4

[0360] Step 1: N-tert-butoxycarbonyl-L-valine (64 mg, 301.6 μmol), eczemab mesylate (160 mg, 301.6 μmol), HATU (228 mg, 603.2 μmol), DMF (20 mL), and DIPEA (212 μL, 1206.4 μmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-4a (181 mg, yield 94.8%).

[0361] Step 2: Add Tp-4a (181 mg, 285 μmol), DCM (8 mL), and TFA (2 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the solution and further purify the product using a preparative HPLC system to obtain Tp-4 (54.6 mg, yield 35.9%). Purity (HPLC-RP): 96.7%. LC-MS: [M+H] + =535.7. 1 H-NMR (400MHz, DMSO-d6) δ8.48(s,1H),7.73(d,J=8.8Hz,1H),7.28(s,1H),5.51(s,1H),5.40(s,2H),5.14(dd,J=25.1,15.0Hz,2H) ,3.14(t,J=3.8Hz,3H),2.35(s,3H),2.25-2.16(m,1H),2.16-2.07(m,1H),2.00-1.90(m,1H),1.90-1.78(m,2H),0.93-0.78(m,9H).

[0362] Example 1.5 Preparation of Tp-5

[0363] Step 1: N-tert-butoxycarbonyl-L-threonine (16.5 mg, 75.4 μmol), eczemab mesylate (40 mg, 75.4 μmol), HATU (57 mg, 150.8 μmol), DMF (5 mL), and DIPEA (53 μL, 301.6 μmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-5a (42 mg, yield 87.7%).

[0364] Step 2: Add Tp-5a (42 mg, 66 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the product and further purify it using a preparative HPLC system to obtain Tp-5 (31.5 mg, yield 89.0%). Purity (HPLC-RP): 97.8%. 28 H 29 O6FN4[M+H] + The calculated HRMS (ESI+) is 537.21439, and the measured value is 537.22310.

[0365] Example 1.6 Preparation of Tp-6

[0366] Step 1: Add Boc-L-leucine monohydrate (9.4 mg, 37.7 μmol), eczemab mesylate (20 mg, 37.7 μmol), HATU (29 mg, 75.4 μmol), DMF (5 mL), and DIPEA (27 μL, 150.8 μmol) sequentially to the reaction flask, and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain Tp-6a (18 mg, yield 73.5%).

[0367] Step 2: Add Tp-6a (18 mg, 27.7 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the solution and further purify the product using a preparative HPLC system to obtain Tp-6 (2.3 mg, yield 15.1%). Purity (HPLC-RP): 98.7%. LC-MS: [M+H] + =549.7.

[0368] Example 1.7 Preparation of Tp-7

[0369] Step 1: N-tert-Butoxycarbonyl-L-Isoleucine (17.4 mg, 75.4 μmol), eczemab mesylate (40 mg, 75.4 μmol), HATU (57 mg, 150.8 μmol), DMF (5 mL), and DIPEA (53 μL, 301.6 μmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-7a (43 mg, yield 87.9%).

[0370] Step 2: Add Tp-7a (43 mg, 66.3 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 5 h. After the reaction is complete, concentrate the solution and further purify the product using a preparative HPLC system to obtain Tp-7 (33.2 mg, yield 91.2%). Purity (HPLC-RP): 97.1%. LC-MS: [M+H] + =549.7.

[0371] Example 1.8 Preparation of Tp-8

[0372] Step 1: N-tert-butyloxycarbonyl-L-cyclobutylglycine (34 mg, 0.15 mmol), eczemab mesylate (53 mg, 0.1 mmol), HATU (76 mg, 0.2 mmol), DMF (3 mL), and DIPEA (50 μL, 0.3 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-8a (64 mg, yield 99.3%).

[0373] Step 2: Add Tp-8a (64 mg, 0.1 mmol), TFA (3 mL), and DCM (6 mL) sequentially to the reaction flask and react at room temperature for 1 h. After the reaction, concentrate the solution, and take 20% of the sample for further purification using a preparative HPLC system to obtain Tp-8 (9.4 mg, yield 86.9%). Purity (HPLC-RP): 95.9%. LC-MS: [M+H] + =547.7.

[0374] Example 1.9 Preparation of Tp-9

[0375] Step 1: Add Boc-L-cyclopropylglycine (32 mg, 150.8 μmol), eczemab mesylate (80 mg, 150.8 μmol), HATU (114 mg, 301.6 μmol), DMF (10 mL), and DIPEA (106 μL, 603.2 μmol) sequentially to the reaction flask, and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain Tp-9a (84 mg, yield 88.1%).

[0376] Step 2: Add Tp-9a (84 mg, 133 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the solution, and take 12.5% ​​of the sample for further purification using a preparative HPLC system to obtain Tp-9 (7.0 mg, yield 79.2%). Purity (HPLC-RP): 96.5%. LC-MS: [M+H] + =533.6.

[0377] Example 1.10 Preparation of Tp-10

[0378] Step 1: (S)-Boc-2-amino-4,4,4-trifluorobutyric acid (39 mg, 150.8 μmol), eczemab mesylate (80 mg, 150.8 μmol), HATU (114 mg, 301.6 μmol), DMF (10 mL), and DIPEA (106 μL, 603.2 μmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was complete, the product was further purified using a preparative HPLC system to obtain Tp-10a (75 mg, yield 73.5%).

[0379] Step 2: Add Tp-10a (75 mg, 111 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 2 h 40 min. After the reaction is complete, concentrate the sample, and further purify the product using a preparative HPLC system to obtain Tp-10 (2.1 mg, yield 26.3%). Purity (HPLC-RP): 93.6%. LC-MS: [M+H] + =575.6.

[0380] Example 1.11 Preparation of Tp-11

[0381] Step 1: N-tert-Butoxycarbonyl-2-methylalanine (31 mg, 150.8 μmol), eczemab mesylate (80 mg, 150.8 μmol), HATU (114 mg, 301.6 μmol), DMF (10 mL), and DIPEA (106 μL, 603.2 μmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-11a (82 mg, yield 87.6%).

[0382] Step 2: Add Tp-11a (82 mg, 132 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the sample, and further purify the product using a preparative HPLC system to obtain Tp-11 (7.4 mg, yield 86.0%). Purity (HPLC-RP): 98.9%. LC-MS: [M+H] + =521.6.

[0383] Example 1.12 Preparation of Tp-12

[0384] Step 1: Add Boc-1-aminocyclobutanecarboxylic acid (32.3 mg, 0.15 mmol), eczemab mesylate (53.2 mg, 0.1 mmol), HATU (57 mg, 0.15 mmol), DMF (5 mL), and DIPEA (53 μL, 0.3 mmol) sequentially to the reaction flask, and react overnight at room temperature. Further purification of the product using a preparative HPLC system yielded Tp-12a (56 mg, yield 88.6%).

[0385] Step 2: Tp-12a (56 mg, 0.089 mmol), DCM (10 mL), and TFA (2 mL) were added sequentially to the reaction mixture, and the reaction was carried out at room temperature for 4 h. The product was further purified using a preparative HPLC system to obtain Tp-12 (41.1 mg, yield 86.8%). Purity (HPLC-RP): 98.5%. LC-MS: [M+H] + =533.7. 1 H-NMR (400MHz, DMSO-d6): 8.15(s,1H),7.65(d,J=8.0Hz,1H),7.21(s,1H),6.46(s,1H),5.44(s,1H),5.33(t,J=16.0Hz,2H),5.02(q,J=12. 0Hz,2H),3.12-3.11(m,1H),3.09-3.01(m,2H),2.53-2.45(m,2H),2.28(s,3H),2.13-2.05(m,2H),1.92-1.70(m,6H),0.8(t,J=4.0Hz,3H).

[0386] Example 1.13 Preparation of Tp-13

[0387] Step 1: Add Boc-1-aminocycloalkylcarboxylic acid (34.4 mg, 0.1 mmol), eczemab mesylate (53.2 mg, 0.1 mmol), HATU (57 mg, 0.15 mmol), DMF (5 mL), and DIPEA (53 μL, 0.3 mmol) sequentially to the reaction flask, and react overnight at room temperature. Further purification of the product using a preparative HPLC system yielded Tp-13a (48 mg, yield 74.3%).

[0388] Step 2: Tp-13a (48 mg, 0.074 mmol), DCM (10 mL), and TFA (2 mL) were added sequentially to the reaction mixture, and the reaction was carried out at room temperature for 1 h. The product was further purified using a preparative HPLC system to obtain Tp-13 (30.5 mg, yield 75.5%). Purity (HPLC-RP): 95.8%. LC-MS: [M+H] + =547.7.

[0389] Example 1.14 Preparation of Tp-14

[0390] Step 1: (S)-2-((tert-butoxycarbonyl)amino)-2,3-dimethylbutyric acid (35 mg, 0.15 mmol), eczemab mesylate (53 mg, 0.1 mmol), HATU (76 mg, 0.2 mmol), DMF (3 mL), and DIPEA (50 μL, 0.3 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was complete, the product was further purified using a preparative HPLC system to obtain Tp-14a (46 mg, yield 71.1%).

[0391] Step 2: Add Tp-14a (46 mg, 0.071 mmol), TFA (2 mL), and DCM (4 mL) sequentially to the reaction flask, and react at room temperature for 1 h. After the reaction is complete, concentrate to obtain Tp-14 (to be used for the next reaction according to theoretical quantities). LC-MS: [M+H] + =549.8.

[0392] Example 1.15 Preparation of Tp-15

[0393] Step 1: N-tert-butoxycarbonyl-L-proline (8.1 mg, 37.7 μmol), eczemab mesylate (20 mg, 37.7 μmol), HATU (29 mg, 75.4 μmol), DMF (5 mL), and DIPEA (26 μL, 150.8 μmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-15a (18 mg, yield 75.3%).

[0394] Step 2: Add Tp-15a (18 mg, 28.4 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the product and further purify it using a preparative HPLC system to obtain Tp-15 (13.4 mg, yield 88.7%). Purity (HPLC-RP): 97.6%. LC-MS: [M+H] + =533.7.

[0395] Example 1.16 Preparation of Tp-16

[0396] Step 1: Add Boc-N-methyl-L-valine (69.3 mg, 0.3 mmol), eczemab mesylate (106.3 mg, 0.2 mmol), HATU (114 mg, 0.3 mmol), DMF (10 mL), and DIPEA (105 μL, 0.6 mmol) sequentially to the reaction flask, and react overnight at room temperature. Further purification of the product using a preparative HPLC system yielded Tp-16a (123 mg, 94.9% yield).

[0397] Step 2: Tp-16a (123 mg, 0.19 mmol), DCM (20 mL), and TFA (4 mL) were added sequentially to the reaction mixture, and the reaction was carried out at room temperature for 3 hours. The product was then further purified using a preparative HPLC system to obtain Tp-16 (84.2 mg, yield 80.8%). Purity (HPLC-RP): 98.7%. LC-MS: [M+H] + =549.7.

[0398] Example 1.17 Preparation of Tp-17

[0399] Step 1: N-tert-Butoxycarbonyl-N-methyl-L-alanine (30 mg, 0.15 mmol), eczemab mesylate (53 mg, 0.1 mmol), HATU (76 mg, 0.2 mmol), DMF (3 mL), and DIPEA (50 μL, 0.3 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-17a (62 mg, yield 99.9%).

[0400] Step 2: Add Tp-17a (62 mg, 0.1 mmol), TFA (3 mL), and DCM (6 mL) sequentially to the reaction flask, and react at room temperature for 1.5 h. After the reaction is complete, concentrate to obtain Tp-17 (for the next reaction step based on theoretical quantities). LC-MS: [M+H] + =521.7.

[0401] Example 1.18 Preparation of Tp-18

[0402] Step 1: N-tert-Butoxycarbonyl-sarcosine (28 mg, 0.15 mmol), eczemab mesylate (53 mg, 0.1 mmol), HATU (76 mg, 0.2 mmol), DMF (3 mL), and DIPEA (50 μL, 0.3 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-18a (53 mg, yield 87.9%).

[0403] Step 2: Add Tp-18a (53 mg, 0.087 mmol), TFA (3 mL), and DCM (6 mL) sequentially to the reaction flask, and react at room temperature for 1 hour. After the reaction is complete, concentrate to obtain Tp-18 (to be used for the next reaction according to theoretical quantities). LC-MS: [M+H] + =507.7.

[0404] Example 1.19 Preparation of Tp-19

[0405] Step 1: N-Boc-L-tert-leucine (35 mg, 150.8 μmol), eczemab mesylate (80 mg, 150.8 μmol), HATU (114 mg, 301.6 μmol), DMF (10 mL), and DIPEA (106 μL, 603.2 μmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-19a (70 mg, yield 71.4%).

[0406] Step 2: Add Tp-19a (70 mg, 108 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 2 h 15 min. After the reaction is complete, concentrate the sample, and further purify the product using a preparative HPLC system to obtain Tp-19 (7.5 mg, yield 88.7%). Purity (HPLC-RP): 96.7%. LC-MS: [M+H] + =549.6.

[0407] Example 1.20 Preparation of Tp-20

[0408] Step 1: Add eczemab mesylate (200 mg, 0.376 mmol), potassium iodide (31 mg, 0.188 mmol), and anhydrous DMF (10 mL) to the reaction flask. Cool to below 0°C, then add anhydrous triethylamine (366 μL, 2.632 mmol) and benzyl bromide (322 mg, 1.88 mmol) sequentially. Under nitrogen protection, transfer to room temperature and react overnight. After the reaction is complete, further purify the product using a preparative HPLC system to obtain Tp-20a (120 mg, yield 60.7%).

[0409] Step 2: Add Tp-20a (120 mg, 0.228 mmol), formic acid (5.45 mL), and formaldehyde (1.09 mL) to the reaction flask, heat to 50 °C, and react for 1.5 h. After the reaction is complete, concentrate the product and further purify it using a preparative HPLC system to obtain Tp-20b (108 mg, yield 87.7%).

[0410] Step 3: Add Tp-20b (108 mg, 0.2 mmol), DMF (5 mL), and 10% Pd / C (53 mg, 50 μmol) to the reaction flask. React under atmospheric pressure with a hydrogen balloon for 4 hours. After the reaction, further purify the product using a preparative HPLC system to obtain Tp-20 (25 mg, yield 27.8%). Purity (HPLC-RP): 96.7%. LC-MS: [M+H] + =450.6.

[0411] Example 1.21 Preparation of Tp-21

[0412] Step 1: Tp-20 (25 mg, 55.6 μmol), N-tert-butoxycarbonyl-L-valine (18 mg, 83.4 μmol), HATU (42 mg, 111.2 μmol), DMF (5 mL), and DIPEA (39 μL, 222.4 μmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-21a (6 mg, yield 16.7%).

[0413] Step 2: Add Tp-21a (6 mg, 9.2 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the solution and further purify the product using a preparative HPLC system to obtain Tp-21 (3.9 mg, yield 78.0%). Purity (HPLC-RP): 99.1%. LC-MS: [M+H] + =549.7.

[0414] Example 1.22 Preparation of Tp-22

[0415] Step 1: Add Boc-1-aminocyclopropylformic acid (121 mg, 602 μmol), eczemab mesylate (320 mg, 602 μmol), HATU (458 mg, 1204 μmol), DMF (40 mL), and DIPEA (419 μL, 2408 μmol) sequentially to the reaction flask, and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain Tp-22a (326 mg, yield 87.6%).

[0416] Step 2: Add Tp-22a (232 mg, 375 μmol), DCM (12 mL), and TFA (3 mL) to the reaction flask and react at room temperature for 3 hours. After the reaction is complete, concentrate the solution, and take 10% of the sample for further purification using a preparative HPLC system to obtain Tp-22 (13.5 mg, yield 69.6%). Purity (HPLC-RP): 95.0%. LC-MS: [M+H] + =519.7.

[0417] Example 1.23 Preparation of Tp-4-2

[0418] Step 1: Tp-4 (35 mg, 65.5 μmol), N-tert-butoxycarbonyl-L-alanine (19 mg, 98.3 μmol), HATU (50 mg, 131 μmol), DMF (5 mL), and DIPEA (46 μL, 262 μmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-4-2a (39 mg, yield 84.4%).

[0419] Step 2: Add Tp-4-2a (39 mg, 55.3 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the product and further purify it using a preparative HPLC system to obtain Tp-4-2 (16.4 mg, yield 49.0%). Purity (HPLC-RP): 98.1%. LC-MS: [M+H] + =606.6. 1 H-NMR (400MHz, DMSO-d6) δ8.67(d,J=6.5Hz,1H),8.28(d,J=39.5Hz,2H),7.77(d,J=8.8Hz,1H), 7.29(s,1H),5.55-5.48(m,1H),5.42(s,2H),5.18(dd,J=79.6,15.1Hz,2H),4.21(s,1H),3.48(d ,J=4.8Hz,1H),3.22-3.09(m,2H),2.38(s,3H),2.23-2.15(m,1H),2.14-2.04(m,1H),2.03-1.9 3(m,1H),1.93-1.79(m,2H),1.14(d,J=5.4Hz,3H),0.88(t,J=5.8Hz,3H),0.81(q,J=5.4Hz,6H).

[0420] Example 1.24 Preparation of Tp-2-4

[0421] Step 1: (tert-butoxycarbonyl)-L-valine-L-alanine (43.3 mg, 0.15 mmol), eczemab mesylate (53.2 mg, 0.1 mmol), HATU (57 mg, 0.15 mmol), DMF (5 mL), and DIPEA (53 μL, 0.3 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. The product was further purified using a preparative HPLC system to obtain Tp-2-4a (64 mg, yield 90.7%).

[0422] Step 2: Tp-2-4a (64 mg, 0.091 mmol), DCM (10 mL), and TFA (1 mL) were added sequentially to the reaction mixture, and the reaction was carried out at room temperature for 2 h. The product was further purified using a preparative HPLC system to obtain Tp-2-4 (41.8 mg, yield 75.9%). Purity (HPLC-RP): 98.0%. LC-MS: [M+H] + =606.6. 1 H-NMR (400MHz, DMSO-d6) δ8.66(d,J=4.0Hz,1H),8.40(d,J=4.0Hz,1H),8.27(s,1H),7. 76(d,J=8.0Hz,1H),7.28(s,1H),5.55-5.21(m,1H),5.41(s,2H),5.13(q,J=16.0Hz,2H ),4.39-4.34(m,2H),3.21-3.10(m,3H),2.36(s,3H),2.15-2.12(m,2H),1.98-1.90(m, 1H),1.89-1.80(m,2H),1.28(d,J=8.0Hz,3H),0.89-0.86(m,6H),0.81(d,J=8.0Hz,3H).

[0423] Example 1.25 Preparation of Tp-1-4

[0424] Step 1: Tp-1 (42.3 mg, 0.086 mmol), N-tert-butoxycarbonyl-L-valine (28 mg, 0.129 mmol), HATU (49.1 mg, 0.129 mmol), DMF (5 mL), and DIPEA (60 μL, 0.344 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain Tp-1-4a (58 mg, yield 97.6%).

[0425] Step 2: Tp-1-4a (58 mg, 0.084 mmol), DCM (10 mL), and TFA (2 mL) were added sequentially to the reaction mixture, and the reaction was carried out at room temperature for 1 h. The product was further purified using a preparative HPLC system to obtain Tp-1-4 (26.8 mg, yield 54.0%). Purity (HPLC-RP): 98.7%. LC-MS: [M+H] + =592.7. 1H-NMR (400MHz, DMSO-d6) δ8.56(d,J=8.0Hz,1H),8.38(s,1H),8.22(s,1H),7.77(d,J=8.0Hz,1H),7.30(s,1H),5.59-5.55(m,1H),5.42(s,2H),5 .25-5.16(m,2H),3.84-3.74(m,4H),3.17-3.15(m,3H),2.38(s,3H),2.2 0-2.09(m,2H),1.95-1.81(m,3H),0.90-0.83(m,6H),0.82-0.78(m,3H).

[0426] Example 1.26 Preparation of Tp-4-1

[0427] Step 1: Tp-4 (26.7 mg, 0.05 mmol), N-tert-butoxycarbonyl-glycine (13.1 mg, 0.075 mmol), HATU (28.5 mg, 0.075 mmol), DMF (5 mL), and DIPEA (35 μL, 0.2 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain Tp-4-1a (27 mg, yield 78.1%).

[0428] Step 2: Tp-4-1a (27 mg, 0.038 mmol), DCM (10 mL), and TFA (1 mL) were added sequentially to the reaction mixture, and the reaction was carried out at room temperature for 2.5 h. The product was further purified using a preparative HPLC system to obtain Tp-4-1 (18.3 mg, yield 81.5%). Purity (HPLC-RP): 98.4%. LC-MS: [M+H] + =592.6. 1 H-NMR(400MHz,DMSO-d6)δ8.81(d,J=4.0Hz,1H),8.47(s,1H),8.24(s,1H),7.77(d ,J=8.0Hz,1H),7.30(s,1H),5.53-5.49(m,1H),5.42(s,2H),5.18(q,J=16.0Hz,2H) ,4.30-4.28(m,2H),3.49-3.42(m,2H),3.18-3.12(m,2H),2.38(s,3H),2.23-2.17( m,1H),2.13-2.08(m,1H),2.05-1.98(m,1H),1.91-1.81(m,2H),0.90-0.81(m,9H).

[0429] Example 1.27 Preparation of Tp-13-2

[0430] Step 1: Tp-13 (49.2 mg, 0.09 mmol), N-tert-butoxycarbonyl-L-alanine (25.5 mg, 0.135 mmol), HATU (51.3 mg, 0.135 mmol), DMF (5 mL), and DIPEA (63 μL, 0.361 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain Tp-13-2a (47 mg, yield 72.8%).

[0431] Step 2: Tp-13-2a (47 mg, 0.066 mmol), DCM (10 mL), and TFA (2 mL) were added sequentially to the reaction mixture, and the reaction was carried out at room temperature for 1 h. The product was further purified using a preparative HPLC system to obtain TP-13-2 (34.9 mg, yield 85.7%). Purity (HPLC-RP): 97.7%. LC-MS: [M+H] + =618.7. 1 H-NMR (400MHz, DMSO-d6) δ8.34-8.29 (m, 2H), 7.93 (s, 1H), 7.68 (d, J = 12.0Hz, 1H), 7.28 (s,1H),5.47-5.41(m,3H),5.09(q,J=16.0Hz,2H),3.46-3.43(m,1H),3.17-3.11(m,1H ),3.06-3.00(m,1H),2.33(s,3H),2.30-2.25(m,1H),2.11-1.98(m,5H),1.92-1.82(m, 3H),1.75-1.66(m,4H),1.63-1.57(m,1H),1.00(d,J=4.0Hz,3H),0.87(t,J=4.0Hz,3H).

[0432] Example 1.28 Preparation of Tp-12-2

[0433] Step 1: Tp-12 (51.6 mg, 0.097 mmol), N-tert-butoxycarbonyl-L-alanine (27.6 mg, 0.146 mmol), HATU (55.5 mg, 0.146 mmol), DMF (5 mL), and DIPEA (68 μL, 0.39 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain Tp-12-2a (43 mg, yield 63%).

[0434] Step 2: Tp-12-2a (43 mg, 0.06 mmol), DCM (10 mL), and TFA (2 mL) were added sequentially to the reaction mixture, and the reaction was carried out at room temperature for 3 h. The product was further purified using a preparative HPLC system to obtain Tp-12-2 (25.8 mg, yield 71.3%). Purity (HPLC-RP): 98.1%. LC-MS: [M+H] + =604.7. 1 H-NMR (400MHz, DMSO-d6) δ8.73(s,1H),8.19(d,J=4.0Hz,1H),8.06(s,1H),7.73(d,J=8.0Hz,1H),7 .29(s,1H),5.50(q,J=4.0Hz,1H),5.42(t,J=12.0Hz,2H),5.12(q,J=12.0Hz,2H),3.44(q,J=4.0Hz ,1H),3.19-3.13(m,1H),3.10-3.04(m,1H),2.78-2.72(m,1H),2.63-2.57(m,1H),2.36(s,3H),2.2 5-2.19(m,1H),2.11-2.07(m,2H),2.02-1.82(m,5H),1.05(d,J=4.0Hz,3H),0.87(t,J=4.0Hz,3H).

[0435] Example 1.29 Preparation of Tp-16-2

[0436] Step 1: Tp-16 (32.9 mg, 0.06 mmol), N-tert-butoxycarbonyl-L-alanine (17.0 mg, 0.09 mmol), HATU (34.2 mg, 0.09 mmol), DMF (6 mL), and DIPEA (42 μL, 0.241 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain Tp-16-2a (22 mg, yield 51%).

[0437] Step 2: Tp-16-2a (22 mg, 0.03 mmol), DCM (10 mL), and TFA (2 mL) were added sequentially to the reaction mixture, and the reaction was carried out at room temperature for 2 h. The product was further purified using a preparative HPLC system to obtain Tp-16-2 (14.8 mg, yield 79.7%). Purity (HPLC-RP): 98.6%. LC-MS: [M+H] + =620.7. 1H-NMR (400MHz, DMSO-d6) δ9.89(d,J=8.0Hz,1H),8.48(d,J=4.0Hz,1H),8.31(s,1H),7.76(d,J=8.0Hz,1H),7.30(s,1H), 5.54-5.48(m,1H),5.42(t,J=12.0Hz,2H),5.20(s,1H),4.59(d,J=8.0Hz,1H),4.05(d,J=8.0Hz,1H),3.95(s,1H),3.84(q ,J=8.0Hz,1H),3.13-3.04(m,2H),3.01(s,1H),2.79(s,1H),2.37(d,J=8.0Hz,3H),2.33-2.22(m,1H),2.12-2.04(m,2H) ,1.91-1.81(m,2H),1.12(dd,J=16.0,4.0Hz,3H),0.95(dd,J=16.0,4.0Hz,3H),0.87(t,J=8.0Hz,3H),0.81-0.74(m,3H).

[0438] Example 1.30 Preparation of Tp-16-4

[0439] Step 1: Tp-16 (32.9 mg, 0.06 mmol), N-tert-butoxycarbonyl-L-valine (19.5 mg, 0.09 mmol), HATU (34.2 mg, 0.09 mmol), DMF (6 mL), and DIPEA (42 μL, 0.241 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain Tp-16-4a (33 mg, yield 73.6%).

[0440] Step 2: Tp-16-4a (33 mg, 0.044 mmol), DCM (10 mL), and TFA (2 mL) were added sequentially to the reaction mixture, and the reaction was carried out at room temperature for 2 h. The product was further purified using a preparative HPLC system to obtain Tp-16-4 (20.9 mg, yield 73.4%). Purity (HPLC-RP): 97.4%. LC-MS: [M+H] + =648.7. 1H-NMR (400MHz, DMSO-d6) δ8.60(d,J=8.0Hz,1H),8.27(s,1H),7.74(t,J=8.0Hz,1H),7.29(s,1H),5. 54-5.48(m,1H),5.41(t,J=16.0Hz,2H),5.27-5.20(m,1H),5.08(t,J=16.0Hz,1H),4.65(d,J=12.0Hz ,1H),4.13(d,J=8.0Hz,1H),3.68(d,J=4.0Hz,1H),3.13-3.00(m,4H),2.74(s,1H),2.37-2.33(m,3H) ,2.32-2.20(m,1H),2.12-2.02(m,2H),1.92-1.79(m,3H),1.04(d,J=8.0Hz,1H),0.93-0.73(m,15H).

[0441] Example 1.31 Preparation of Tp-16-1

[0442] Step 1: Tp-16 (38.4 mg, 0.07 mmol), N-tert-butoxycarbonyl-glycine (18.4 mg, 0.105 mmol), HATU (39.9 mg, 0.105 mmol), DMF (7 mL), and DIPEA (49 μL, 0.281 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain Tp-16-1a (38 mg, yield 77%).

[0443] Step 2: Tp-16-1a (38 mg, 0.054 mmol), DCM (15 mL), and TFA (2 mL) were added sequentially to the reaction mixture, and the reaction was carried out at room temperature for 2 h. The product was further purified using a preparative HPLC system to obtain Tp-16-1 (18.4 mg, yield 56.3%). Purity (HPLC-RP): 96.4%. LC-MS: [M+H] + =606.7. 1H-NMR(400MHz,DMSO-d6)δ9.17(d,J=8.0Hz,1H),8.71(d,J=8.0Hz,1H),8.24(s,1H),7.80-7.75 (m,1H),7.30(s,1H),5.48-5.45(m,1H),5.42(s,2H),5.25(d,J=16.0Hz,1H),5.09-5.04(m,1H), 4.67(d,J=8.0Hz,1H),3.78-3.69(m,2H),3.18-3.08(m,2H),2.96(s,2H),2.88(s,1H),2.39-2.3 6(m,3H),2.24-2.05(m,3H),1.91-1.82(m,2H),0.90-0.84(m,6H),0.77(dd,J=12.0,4.0Hz,3H).

[0444] Example 1.32 Preparation of Tp-4-4

[0445] Step 1: N-tert-butoxycarbonyl-L-valine (33 mg, 0.15 mmol), Tp-4 (0.1 mmol), HATU (76 mg, 0.2 mmol), DMF (3 mL), and DIPEA (50 μL, 0.3 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-4-4a (48 mg, yield 65.5%).

[0446] Step 2: Add Tp-4-4a (48 mg, 0.065 mmol), TFA (2 mL), and DCM (4 mL) sequentially to the reaction flask, and react at room temperature for 1 h. After the reaction is complete, concentrate the solution, and take 20% of the sample for further purification using a preparative HPLC system to obtain a white solid Tp-4-4 (7 mg, yield 84.4%). Purity (HPLC-RP): 97.3%. LC-MS: [M+H] + =634.8. 1H-NMR (400MHz, DMSO-d6) δ8.63(d,J=4.0Hz,1H),8.17(d,J=8.0Hz,1H),7.76(d,J=8.0H z,1H),7.28(s,1H),5.52-5.49(m,1H),5.41(s,2H),5.16(dd,J=16.0,88.0Hz,1H),4.2 0(t,J=8.0Hz,1H),3.14(t,J=4.0Hz,3H),2.37(s,3H),2.22-2.16(m,1H),2.12-2.06(m ,1H),2.01-1.94(m,2H),1.92-1.81(m,2H),0.89-0.81(m,12H),0.76(d,J=4.0Hz,3H).

[0447] Example 1.33 Preparation of Tp-17-4

[0448] Step 1: N-tert-butoxycarbonyl-L-valine (33 mg, 0.15 mmol), Tp-17 (0.1 mmol), HATU (76 mg, 0.2 mmol), DMF (3 mL), and DIPEA (50 μL, 0.3 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-17-4a (64 mg, yield 89.0%).

[0449] Step 2: Add Tp-17-4a (64 mg, 0.089 mmol), TFA (3 mL), and DCM (6 mL) sequentially to the reaction flask, and react at room temperature for 1.5 h. After the reaction is complete, concentrate the solution, and take 20% of the sample for further purification using a preparative HPLC system to obtain a white solid of Tp-17-4 (11 mg, yield 99.8%). Purity (HPLC-RP): 97.6%. LC-MS: [M+H] + =620.8. 1H-NMR (400MHz, DMSO-d6) δ9.24(d,J=8.0Hz,1H),8.39(d,J=4.0Hz,1H),8.20(s,1H),7.79(d,J=8.0Hz,1H),7.31( d,1H),6.53(s,1H),5.57-5.50(m,1H),5.42(s,2H),5.23(d,J=8.0Hz,1H),5.19(s,1H),4.90(q,J=4.0Hz,1H),3. 49(d,J=4.0Hz,1H),3.14(s,2H),3.03(s,2H),2.78(s,1H),2.40(s,1H),2.39(s,2H),2.13-2.07(m,2H),1.91-1. 82(m,2H),1.78-1.73(m,2H),1.37(d,J=4.0Hz,1H),1.34(d,J=4.0Hz,2H),0.90-0.86(m,4H),0.83-0.78(m,3H).

[0450] Example 1.34 Preparation of Tp-18-4

[0451] Step 1: N-tert-butoxycarbonyl-L-valine (28 mg, 0.13 mmol), Tp-18 (0.087 mmol), HATU (66 mg, 0.174 mmol), DMF (3 mL), and DIPEA (39 μL, 0.261 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-18-4a (48 mg, yield 78.3%).

[0452] Step 2: Add Tp-18-4a (48 mg, 0.068 mmol), TFA (2 mL), and DCM (4 mL) sequentially to the reaction flask, and react at room temperature for 1 h. After the reaction is complete, concentrate the solution, and take 25% of the sample for further purification using a preparative HPLC system to obtain a white solid of Tp-18-4 (8.9 mg, yield 86.4%). Purity (HPLC-RP): 92.4%. LC-MS: [M+H] + =606.8. 1H-NMR (400MHz, DMSO-d6) δ8.64(d,J=4.0Hz,1H),8.22(s,1H),7.76(t,J=8.0Hz,1H),7.29(s,1H),5 .62-5.55(m,1H),5.41(s,2H),5.25-5.12(m,2H),4.20(d,J=12.0Hz,1H),4.02(d,J=16.0Hz,1H),3 .86(s,1H),3.83(s,1H),3.20-3.14(m,2H),3.13(s,2H),2.89(s,1H),2.39(s,1H),2.37(s,2H),2. 20-2.12(m,2H),1.92-1.81(m,3H),0.96(d,J=4.0Hz,2H),0.89-0.84(m,5H),0.76(q,J=4.0Hz,2H).

[0453] Example 1.35 Preparation of Tp-3-2

[0454] Step 1: N-tert-Butoxycarbonyl-L-alanine (20 mg, 0.107 mmol), Tp-3 (0.071 mmol), HATU (54 mg, 0.142 mmol), DMF (3 mL), and DIPEA (35 μL, 0.213 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was complete, the product was further purified using a preparative HPLC system to obtain Tp-3-2a (32 mg, yield 65.2%). LC-MS: [M+H] + =692.7.

[0455] Step 2: Add Tp-3-2a (32 mg, 0.046 mmol), TFA (2 mL), and DCM (4 mL) sequentially to the reaction flask, and react at room temperature for 1.5 h. After the reaction is complete, concentrate the solution, and further purify the product using a preparative HPLC system (33% of the sample) to obtain a white solid of Tp-3-2 (8.7 mg, yield 95.4%). Purity (HPLC-RP): 98.6%. LC-MS: [M+H] + =592.7. 1H-NMR (400MHz, DMSO-d6) δ8.66(d,J=4.0Hz,1H),8.28(s,1H),7.76(d,J=8.0Hz,1H),7.29 (s,1H),5.54-5.50(m,1H),5.41(s,2H),5.15(dd,J=16.0,60.0Hz,2H),4.24(s,1H),3.49 (s,1H),3.15(s,2H),2.38(s,3H),2.19-2.09(m,2H),1.91-1.80(m,2H),1.75-1.69(m,1H ),1.61-1.54(m,1H),1.17(d,J=4.0Hz,3H),0.88(t,J=4.0Hz,3H),0.82(t,J=4.0Hz,3H).

[0456] Example 1.36 Preparation of Tp-8-2

[0457] Step 1: N-tert-butoxycarbonyl-L-alanine (23 mg, 0.12 mmol), Tp-8 (0.08 mmol), HATU (61 mg, 0.16 mmol), DMF (3 mL), and DIPEA (40 μL, 0.24 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-8-2a (51 mg, yield 88.9%).

[0458] Step 2: Add Tp-8-2a (51 mg, 0.071 mmol), TFA (3 mL), and DCM (6 mL) sequentially to the reaction flask and react at room temperature for 1 h. After the reaction is complete, concentrate the solution, and take 20% of the sample for further purification using a preparative HPLC system to obtain a white solid of Tp-8-2 (6.9 mg, yield 78.6%). Purity (HPLC-RP): 95.7%. LC-MS: [M+H] + =618.8. 1H-NMR (400MHz, DMSO-d6) δ8.66(d,J=8.0Hz,1H),8.26(d,J=8.0Hz,2H),7.79(d,J=8.0Hz,1H),7.30( s,1H),5.52-5.48(m,1H),5.43(s,2H),5.16(dd,J=16.0,84.0Hz,2H),4.32(t,J=8.0Hz,1H),3.50(s ,1H),3.16(t,J=4.0Hz,2H),2.63-2.59(m,1H),2.40(s,3H),2.19-2.16(m,1H),2.10-2.04(m,1H),1 .93-1.81(m,3H),1.79-1.71(m,3H),1.66-1.63(m,1H),1.17(d,J=4.0Hz,3H),0.88(t,J=8.0Hz,3H).

[0459] Example 1.37 Preparation of Tp-14-2

[0460] Step 1: N-tert-Butoxycarbonyl-L-alanine (20 mg, 0.106 mmol), Tp-14 (0.071 mmol), HATU (54 mg, 0.142 mmol), DMF (3 mL), and DIPEA (35 μL, 0.213 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-14-2a (51 mg, yield 99.9%).

[0461] Step 2: Add Tp-14-2a (51 mg, 0.071 mmol), TFA (3 mL), and DCM (6 mL) sequentially to the reaction flask, and react at room temperature for 1 h. After the reaction is complete, concentrate the solution, and take 20% of the sample for further purification using a preparative HPLC system to obtain a white solid of Tp-14-2 (6.4 mg, yield 72.8%). Purity (HPLC-RP): 97.5%. LC-MS: [M+H] + =620.8. 1H-NMR(400MHz,DMSO-d6)δ7.94(d,J=8.0Hz,1H),7.75(s,1H),7.69(d,J=8.0 Hz,1H),7.22(s,1H),5.43-5.40(m,1H),5.36(s,2H),5.19(dd,J=16.0,188. 0Hz,2H),3.12-3.01(m,4H),2.32(s,3H),2.14-2.05(m,2H),2.01-1.94(m,1 H),1.85-1.74(m,2H),1.29(s,3H),0.84-0.79(m,9H),0.61(d,J=8.0Hz,3H).

[0462] Example 1.38 Preparation of Tp-2-2

[0463] Step 1: N-tert-Butoxycarbonyl-alanylalanine (15 mg, 56.4 μmol), eczemab mesylate (30 mg, 56.4 μmol), HATU (43 mg, 112.8 μmol), DMF (5 mL), and DIPEA (39 μL, 225.6 μmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-2-2a (22 mg, yield 57.6%).

[0464] Step 2: Add Tp-2-2a (22 mg, 32.5 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the solution and further purify the product using a preparative HPLC system to obtain Tp-2-2 (17.3 mg, yield 92.0%). Purity (HPLC-RP): 96.1%. LC-MS: [M+H] + =578.7. 1H-NMR (400MHz, DMSO-d6) δ8.66(d,J=6.9Hz,1H),8.32(d,J=32.2Hz,2H),7.79(d,J=8.7 Hz,1H),7.30(s,1H),5.57-5.51(m,1H),5.42(s,2H),5.17(dd,J=37.7,15.0Hz,2H),4.3 9-4.28(m,2H),3.49(d,J=4.8Hz,2H),3.21-3.13(m,2H),2.39(s,3H),2.17-2.11(m,2H) ,1.92-1.81(m,2H),1.28(d,J=5.6Hz,3H),1.18(d,J=5.5Hz,3H),0.88(t,J=5.8Hz,3H).

[0465] Example 1.39 Preparation of Tp-12-1

[0466] Step 1: Tp-12 (156 mg, 0.292 mmol), N-tert-butoxycarbonyl-glycine (77 mg, 0.438 mmol), HATU (222 mg, 0.584 mol), DMF (15 mL), and DIPEA (203 μL, 1.168 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-12-1a (177 mg, yield 87.6%).

[0467] Step 2: Add Tp-12-1a (177 mg, 0.257 mmol), DCM (12 mL), and TFA (3 mL) to the reaction flask and react at room temperature for 3 hours. After the reaction is complete, concentrate the sample, and further purify the product using a preparative HPLC system to obtain Tp-12-1 (15.6 mg, 77.2%). Purity (HPLC-RP): 96.0%. LC-MS: [M+H] + =590.8. 1H-NMR (400MHz, DMSO-d6) δ8.94(s,1H),8.34(d,J=7.0Hz,1H),8.08(s,1H),7.65(d,J=8. 7Hz,1H),7.28(s,1H),5.50-5.43(m,1H),5.40(s,2H),5.02(dd,J=99.4,15.1Hz,2H),3.3 4(s,2H),3.18-3.10(m,1H),3.08-2.99(m,1H),2.74-2.64(m,2H),2.32(s,3H),2.25-2,1 7(m,1H),2.15-2.03(m,3H),1.98-1.91(m,2H),1.89-1.80(m,2H),0.87(t,J=5.8Hz,3H).

[0468] Example 1.40 Preparation of Tp-6-2

[0469] Step 1: Tp-6 (37.2 mg, 67.8 μmol), N-tert-butoxycarbonyl-L-alanine (19 mg, 101.7 μmol), HATU (52 mg, 135.6 μmol), DMF (5 mL), and DIPEA (47 μL, 271.2 μmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-6-2a (46 mg, yield 94.3%).

[0470] Step 2: Add Tp-6-2a (46 mg, 63.9 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the solution and further purify the product using a preparative HPLC system to obtain Tp-6-2 (32 mg, yield 80.8%). Purity (HPLC-RP): 96.6%. LC-MS: [M+H] + =620.7. 1H-NMR (400MHz, DMSO-d6) δ8.72(d,J=6.7Hz,1H),8.41(d,J=4.5Hz,1H),8.31(s,1H),7.73(d,J= 8.7Hz,1H),7.28(s,1H),5.52-5.46(m,1H),5.42(s,2H),5.11(dd,J=26.9,15.0Hz,2H),4.38(s ,1H),3.59(s,1H),3.21-3.08(m,2H),2.36(s,3H),2.18-2.08(m,2H),1.92-1.80(m,2H),1.66- 1.56(m,2H),1.56-1.48(m,1H),1.20(d,J=5.3Hz,3H),0.91-0.86(m,6H),0.84(d,J=5.0Hz,3H).

[0471] Example 1.41 Preparation of Tp-7-2

[0472] Step 1: Tp-7 (37.2 mg, 67.8 μmol), N-tert-butoxycarbonyl-L-alanine (19 mg, 101.7 μmol), HATU (52 mg, 135.6 μmol), DMF (5 mL), and DIPEA (47 μL, 271.2 μmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-7-2a (44 mg, yield 90.2%).

[0473] Step 2: Add Tp-7-2a (44 mg, 61.1 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the product and further purify it using a preparative HPLC system to obtain Tp-7-2 (11.6 mg, 30.6%). Purity (HPLC-RP): 97.4%. LC-MS: [M+H] + =620.7. 1H-NMR (400MHz, DMSO-d6) δ8.64(d,J=6.4Hz,1H),8.30(s,1H),8.22(d,J=6.0Hz,1H),7.79(d,J=8.7Hz,1H),7.3 0(s,1H),5.51-5.46(m,1H),5.42(s,2H),5.21(dd,J=84.4,15.0Hz,2H),4.19(t,J=5.6Hz,1H),3.48(d,J=5.1H z,1H),3.15(t,J=4.5Hz,2H),2.39(s,3H),2.25-2.17(m,1H),2.13-2.04(m,1H),1.93-1.80(m,2H),1.77-1.69 (m,1H),1.47-1.38(m,1H),1.12(d,J=5.5Hz,3H),1.09-0.98(m,1H),0.87(t,J=5.8Hz,3H),0.80-0.74(m,6H).

[0474] Example 1.42 Preparation of Tp-9-2

[0475] Step 1: Tp-9 (61.9 mg, 116.4 μmol), DMF (7 mL), N-tert-butoxycarbonyl-L-alanine (33 mg, 174.6 μmol), HATU (89 mg, 232.8 μmol), and DIPEA (81 μL, 465.6 μmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-9-2a (76 mg, yield 92.8%).

[0476] Step 2: Add Tp-9-2a (76 mg, 108 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the solution, and take 12.5% ​​of the sample for further purification using a preparative HPLC system to obtain Tp-9-2 (7.8 mg, yield 95.7%). Purity (HPLC-RP): 97.4%. LC-MS: [M+H] + =604.6. 1H-NMR (400MHz, DMSO-d6) δ8.68(d,J=6.8Hz,1H),8.44(d,J=4.7Hz,1H),8.34(s,1H),7.73(d,J=8.7Hz ,1H),7.28(s,1H),5.56-5.49(m,1H),5.41(s,2H),5.17(dd,J=34.2,15.2Hz,2H),3.82(t,J=5.5Hz,1 H),3.51(d,J=3.5Hz,1H),3.21-3.07(m,2H),2.34(s,3H),2.19-2.12(m,2H),1.93-1.80(m,2H),1.19 (d,J=5.4Hz,4H),0.88(t,J=5.8Hz,3H),0.50(d,J=6.4Hz,2H),0.42-0.36(m,1H),0.29-0.22(m,1H).

[0477] Example 1.43 Preparation of Tp-10-2

[0478] Step 1: Tp-10 (55.9 mg, 97.3 μmol), DMF (7 mL), N-tert-butoxycarbonyl-L-alanine (28 mg, 146 μmol), HATU (74 mg, 194.6 μmol), and DIPEA (68 μL, 389.2 μmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-10-2a (69 mg, yield 95.0%).

[0479] Step 2: Add Tp-10-2a (69 mg, 92.5 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the sample, and further purify the product using a preparative HPLC system to obtain Tp-10-2 (8 mg, yield 93.8%). Purity (HPLC-RP): 98.2%. LC-MS: [M+H] + =646.6. 1H-NMR (400MHz, DMSO-d6) δ8.78(d,J=6.8Hz,1H),7.74(d,J=8.7Hz,1H),7.28(s,1H),5.55-5. 50(m,1H),5.41(s,2H),5.13(dd,J=28.1,15.0Hz,2H),4.66(dd,J=7.5,3.0Hz,1H),3.40(q,J =5.5Hz,1H),3.18-3.08(m,2H),2.90-2.78(m,1H),2.74-2.61(m,1H),2.36(s,3H),2.23-2.1 5(m,1H),2.15-2.05(m,1H),1.93-1.78(m,2H),1.14(d,J=5.6Hz,3H),0.87(t,J=5.8Hz,3H).

[0480] Example 1.44 Preparation of Tp-11-2

[0481] Step 1: Tp-11 (60.2 mg, 115.5 μmol), DMF (7 mL), N-tert-butoxycarbonyl-L-alanine (33 mg, 173.3 μmol), HATU (88 mg, 231 μmol), and DIPEA (80 μL, 462 μmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was complete, the product was further purified using a preparative HPLC system to obtain Tp-11-2a (48 mg, yield 60.0%).

[0482] Step 2: Add Tp-11-2a (48 mg, 69.4 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the sample, and further purify the product using a preparative HPLC system to obtain Tp-11-2 (5.3 mg, yield 77.4%). Purity (HPLC-RP): 97.8%. LC-MS: [M+H] + =592.6. 1H-NMR(400MHz,DMSO-d6)δ8.29(s,1H),8.23(d,J=6.8Hz,1H),8.18(s,2H),7.76(d, J=8.8Hz,1H),7.30(s,1H),5.51-5.45(m,1H),5.42(s,2H),5.18(dd,J=38.6,15.1H z,2H),3.39(d,J=5.2Hz,1H),3.19-3.03(m,3H),2.38(s,3H),2.13-2.05(m,2H),1. 92-1.81(m,2H),1.45(d,J=9.2Hz,6H),1.02(d,J=5.4Hz,3H),0.87(t,J=5.8Hz,3H).

[0483] Example 1.45 Preparation of Tp-19-2

[0484] Step 1: Tp-19 (50.7 mg, 92.6 μmol), DMF (6 mL), N-tert-butoxycarbonyl-L-alanine (26 mg, 138.9 μmol), HATU (70 mg, 185.2 μmol), and DIPEA (65 μL, 370.4 μmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was complete, the product was further purified using a preparative HPLC system to obtain Tp-19-2a (64 mg, yield 96.0%).

[0485] Step 2: Add Tp-19-2a (64 mg, 88.9 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 2 h. After the reaction is complete, concentrate the solution, and take 14.3% of the sample for further purification using a preparative HPLC system to obtain Tp-19-2 (6.5 mg, yield 82.6%). Purity (HPLC-RP): 97.8%. LC-MS: [M+H] + =620.7. 1H-NMR (400MHz, DMSO-d6) δ8.64(d,J=6.2Hz,1H),8.26(d,J=6.6Hz,1H),7.76(d,J=8.7Hz,1 H),7.29(s,1H),5.51-5.48(m,1H),5.41(s,2H),5.21(dd,J=116.2,15.1Hz,2H),4.23(d,J= 6.3Hz,2H),3.58(d,J=4.5Hz,1H),3.19-3.10(m,2H),2.38(s,3H),2.30-2.22(m,1H),2.11 -2.02(m,1H),1.92-1.81(m,2H),1.13(d,J=5.4Hz,3H),0.88(t,J=5.8Hz,3H),0.84(s,9H).

[0486] Example 1.46 Preparation of Tp-15-2

[0487] Step 1: Add Boc-acetaminophen (22 mg, 75.4 μmol), eczemab mesylate (40 mg, 75.4 μmol), HATU (57 mg, 150.8 μmol), DMF (5 mL), and DIPEA (53 μL, 301.6 μmol) sequentially to the reaction flask, and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain Tp-15-2a (36 mg, yield 67.9%).

[0488] Step 2: Add Tp-15-2a (36 mg, 51.2 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the solution and further purify the product using a preparative HPLC system to obtain Tp-15-2 (17.4 mg, yield 56.3%). Purity (HPLC-RP): 98.5%. LC-MS: [M+H] + =604.7. 1H-NMR (400MHz, DMSO-d6) δ8.58(d,J=6.9Hz,1H),7.74(d,J=8.6Hz,1H),7.28(s,1H),5.51-5. 44(m,1H),5.41(s,2H),5.10(dd,J=19.8,14.9Hz,2H),4.45(d,J=5.0Hz,1H),4.32(t,J=3.4H z,1H),3.84(s,1H),3.68(d,J=5.0Hz,1H),3.57-3.48(m,1H),3.24-3.08(m,2H),2.36(s,3H) ,2.20-2.08(m,3H),2.07-1.98(m,1H),1.95-1.78(m,4H),1.16(s,3H),0.87(t,J=5.8Hz,3H).

[0489] Example 1.47 Preparation of Tp-15-4

[0490] Step 1: N-tert-butoxycarbonyl-L-valine-L-proline (24 mg, 75.4 μmol), eczemab mesylate (40 mg, 75.4 μmol), HATU (57 mg, 150.8 μmol), DMF (5 mL), and DIPEA (53 μL, 301.6 μmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was complete, the product was further purified using a preparative HPLC system to obtain Tp-15-4a (40 mg, yield 72.5%).

[0491] Step 2: Add Tp-15-4a (40 mg, 54.7 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the solution and further purify the product using a preparative HPLC system to obtain Tp-15-4 (24.8 mg, yield 71.7%). Purity (HPLC-RP): 97.8%. LC-MS: [M+H] + =632.7. 1H-NMR (400MHz, DMSO-d6) δ8.49(d,J=7.0Hz,1H),7.76(d,J=8.8Hz,1H),7.28(s,1H),5.52-5. 46(m,1H),5.41(s,2H),5.14(dd,J=20.5,15.0Hz,2H),4.30(t,J=5.7Hz,1H),3.71-3.62(m,1H ),3.52(d,J=7.5Hz,1H),3.44-3.39(m,2H),3.31(s,1H),3.21-3.11(m,2H),2.37(s,3H),2.20 -2.07(m,3H),2.06-1.98(m,1H),1.95-1.72(m,5H),0.91-0.84(m,6H),0.77(d,J=5.3Hz,2H).

[0492] Example 1.48 Preparation of Tp-22-2

[0493] Step 1: Tp-22 (19.4 mg, 37.5 μmol), DMF (5 mL), N-tert-butoxycarbonyl-L-alanine (10.6 mg, 56.3 μmol), HATU (29 mg, 75 μmol), and DIPEA (26 μL, 150 μmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-22-2a (24 mg, yield 92.7%).

[0494] Step 2: Add Tp-22-2a (24 mg, 34.8 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the solution and further purify the product using a preparative HPLC system to obtain Tp-22-2 (11.9 mg, 58.0%). Purity (HPLC-RP): 98.6%. LC-MS: [M+H] + =590.7. 1H-NMR (400MHz, DMSO-d6) δ8.49(s,1H),8.29(d,J=8.7Hz,1H),7.67(d,J=8.7Hz,1H),7.29(s,1H) ,6.54(s,1H),5.58-5.50(m,1H),5.42(dd,J=13.2,15.2Hz,2H),5.07(dd,J=173.4,15.2Hz,2H),3 .26(d,J=4.5Hz,1H),3.25-3.23(m,1H),3.23-3.16(m,1H),2.35(s,3H),2.17-2.08(m,2H),1.92 -1.80(m,2H),1.57-1.49(m,1H),1.46-1.38(m,1H),1.02(d,J=5.2Hz,4H),0.87(t,J=5.8Hz,4H).

[0495] Example 1.49 Preparation of Tp-22-1

[0496] Step 1: Tp-22 (19.4 mg, 37.5 μmol), DMF (5 mL), N-tert-butoxycarbonyl-glycine (9.9 mg, 56.3 μmol), HATU (29 mg, 75 μmol), and DIPEA (26 μL, 150 μmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain Tp-22-1a (16 mg, yield 63.2%).

[0497] Step 2: Add Tp-22-1a (16 mg, 23.7 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the solution and further purify the product using a preparative HPLC system to obtain Tp-22-1 (5.7 mg, 41.9%). Purity (HPLC-RP): 95.9%. LC-MS: [M+H] + =576.7. 1H-NMR(400MHz,DMSO-d6)δ8.66(s,1H),8.52(d,J=6.7Hz,1H),8.11(s,1H),7.7 1(d,J=8.7Hz,1H),7.30(s,1H),6.53(s,1H),5.61-5.52(m,1H),5.43(s,2H),5. 14(dd,J=182,15.3Hz,2H),3.27-3.02(m,5H),2.37(s,3H),2.22-2.09(m,2H), 1.94-1.79(m,2H),1.56-1.45(m,2H),1.07-0.92(m,2H),0.88(t,J=5.7Hz,3H).

[0498] Example 1.50 Preparation of GF-Az1

[0499] Step 1: Add amino-diethylene glycol-azide (520 mg, 4 mmol), DMSO (10 mL), and triethylamine (556 μL, 4 mmol) to reaction flask 1 and stir. Then add 1-(2-azidoethoxy)-2-bromoethane (386 mg, 2 mmol) and DMSO (10 mL) to reaction flask 2 and stir to dissolve. Add the solution from reaction flask 2 to reaction flask 1 in portions and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system to obtain GF-Az1a (350 mg, yield 72.3%).

[0500] Step 2: Add GF-Az1a (350 mg, 1.44 mmol), succinic anhydride (432 mg, 4.32 mmol), and DMF (15 mL) to the reaction flask and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain GF-Az1b (309 mg, yield 62.6%).

[0501] Step 3: Add GF-Az1b (309 mg, 0.9 mmol), DCM (20 mL), N-hydroxysuccinimide (311 mg, 2.7 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1035 mg, 5.4 mmol) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, add DCM (20 mL) and H2O (40 mL) for extraction and washing. Wash the DCM phase once more with saturated brine (40 mL), dry, filter, and concentrate to obtain GF-Az1c (proceed to the next reaction according to the theoretical amount).

[0502] Step 4: Add H2O (15 mL), THF (15 mL), GDP-FAm (prepared according to Example 1 of patent "CN114990175B" "A method for synthesizing GDP-FAm") solution (aqueous solution / 100 mM, 13.5 mL), GF-Az1c solution (THF solution / 50 mM, 18 mL), and sodium bicarbonate solution (aqueous solution / 200 mM, 9 mL) to the reaction flask, and react overnight at room temperature. After the reaction is complete, concentrate to remove THF, and then further purify the product using a preparative HPLC system to obtain GF-Az1 (646.2 mg, yield 77.2%). 28 H 45 O 19 N 13 P2[MH] - The calculated HRMS (ESI-) is 928.23571, and the measured value is 928.25625. 1 H-NMR(400MHz,D2O)δ8.01(s,1H),5.78(t,J=3.9Hz,1H),4.78(t,J=6.4Hz,1H),4.39-4.36(m,1H),4.22-4.17(m,1H),4.09-4.03(m,2H), 3.75-3.68(m,1H),3.63-3.48(m,12H),3.48-3.37(m,4H),3.36-3.25(m,5H),3.17-3.09(m,1H),2.68-2.57(m,2H),2.38(q,J=5.1Hz,2H).

[0503] Example 1.51 Preparation of GF-Az2

[0504] Step 1: Add 2-[2-(2-azidoethoxy)ethoxy]ethylamine (348 mg, 2 mmol), DMSO (5 mL), and triethylamine (278 μL, 2 mmol) to reaction flask 1 and stir. Then add 1-azido-14-bromo-3,6,9,12-tetraoxotetradecane (326 mg, 1 mmol) and DMSO (5 mL) to reaction flask 2 and stir to dissolve. Add the solution from reaction flask 2 to reaction flask 1 in portions and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system to obtain GF-Az2a (180 mg, yield 42.9%).

[0505] Step 2: Add GF-Az2a (180 mg, 0.429 mmol), succinic anhydride (129 mg, 1.287 mmol), and DMF (10 mL) to the reaction flask and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain GF-Az2b (196 mg, yield 87.9%).

[0506] Step 3: Add GF-Az2b (196 mg, 0.377 mmol), DCM (15 mL), N-hydroxysuccinimide (130 mg, 1.131 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (434 mg, 2.262 mmol) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, add DCM (20 mL) and H2O (20 mL) for extraction and washing. Wash the DCM phase once more with saturated brine (20 mL), dry, filter, and concentrate to obtain GF-Az2c (proceed to the next reaction according to the theoretical amount).

[0507] Step 4: Add H₂O (7.5 mL), THF (7.5 mL), GDP-FAm solution (100 mM aqueous solution, 7.54 mL), GF-Az₂c solution (50 mM THF solution, 7.54 mL), and sodium bicarbonate solution (200 mM aqueous solution, 3.77 mL) to the reaction flask, and react overnight at room temperature. After the reaction is complete, concentrate to remove THF, and then further purify the product using a preparative HPLC system to obtain GF-Az₂ (217 mg, yield 52.0%). 36 H 61 O 23 N 13 P2[MH] - The calculated HRMS (ESI-) is 1104.34057, and the measured value is 1104.36144. 1 H-NMR(400MHz,D2O)δ8.04(s,1H),5.78(t,J=3.8Hz,1H),4.78(t,J=6.3Hz,1H),4.39-4.36(m,1H),4.22-4.17(m,1H),4.09-4.03(m,2H), 3.75-3.68(m,1H),3.65-3.47(m,28H),3.47-3.37(m,4H),3.37-3.25(m,5H),3.19-3.09(m,1H),2.66-2.55(m,2H),2.38(q,J=5.5Hz,2H).

[0508] Example 1.52 Preparation of GF-Az3

[0509] Step 1: Dissolve amino-diethylene glycol-azide (260 mg, 2 mmol) and triethylamine (278 μL, 2 mmol) in DMSO (3 mL). Slowly add N-(2-bromoethyl)carbamate tert-butyl ester (223 mg, 1 mmol) dissolved in DMSO (5 mL) with stirring for 2 hours. React overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain GF-Az3a (61.8 mg, yield 22.6%).

[0510] Step 2: Add GF-Az3a (61.8 mg, 0.226 mmol), azide-ethylene glycol-acrylic acid-succinimide (127.5 mg, 0.226 mmol), triethylamine (157 μL, 1.13 mmol), and DMSO (5 mL) to the reaction flask, and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system to obtain GF-Az3b (84.9 mg, yield 52%).

[0511] Step 3: Add GF-Az3b (84.9 mg, 0.118 mmol), TFA (0.5 mL), and DCM (5 mL) to the reaction flask and react at room temperature for 1 hour. After the reaction is complete, evaporate the DCM to obtain GF-Az3c (proceed to the next reaction according to the theoretical amount).

[0512] Step 4: Add GF-Az3c (74.4 mg, 0.118 mmol), succinic anhydride (35.4 mg, 0.354 mmol), triethylamine (17 μL, 0.118 mmol), and DMF (4 mL) to the reaction flask and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain GF-Az3d (27.6 mg, yield 32.4%).

[0513] Step 5: Add GF-Az3d (27.6 mg, 0.0382 mmol), DCM (5 mL), N-hydroxysuccinimide (13.2 mg, 0.115 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (43.9 mg, 0.229 mmol) to the reaction flask and react at room temperature for 2 h. After the reaction is complete, add DCM (10 mL) and H2O (10 mL) for extraction and washing. Wash the DCM phase once more with saturated brine (10 mL), dry, filter, and concentrate to obtain GF-Az3e (proceed to the next reaction according to the theoretical amount).

[0514] Step Six: Add H₂O (1 mL), THF (1 mL), GDP-FAm solution (100 mM aqueous solution, 400 μL), GF-Az₃e solution (THF solution / 50 mM, 800 μL), and sodium bicarbonate solution (200 mM aqueous solution, 400 μL) to the reaction flask, and react overnight at room temperature. After the reaction is complete, concentrate to remove THF, and then further purify the product using a preparative HPLC system to obtain GF-Az₃ (28.5 mg, yield 54.5%). 45 H 78 O 27 N 14 P2[MH] - The calculated HRMS (ESI-) is 1307.45633, and the measured value is 1307.46021.

[0515] Example 1.53 Preparation of GF-Az4

[0516] Step 1: Add GF-Az4a (63.4 mg, 0.2 mmol) (prepared according to the synthesis method of compound 3 in Example 2 of patent "CN118541380A"), azido-octaethylene glycol-acrylic acid-succinimide (112.8 mg, 0.2 mmol), triethylamine (140 μL, 1 mmol), and DMSO (5 mL) to the reaction flask, and react overnight at room temperature. After the reaction is complete, the product is further purified by a preparative HPLC system to obtain GF-Az4b (102.6 mg, yield 67.0%).

[0517] Step 2: Add GF-Az4b (102.6 mg, 0.134 mmol), TFA (1 mL), and DCM (10 mL) to the reaction flask and react at room temperature for 3 hours. Monitor with TLC. Dry the solvent by rotary evaporation without further purification to obtain crude GF-Az4c (to be used in the next reaction according to theoretical quantities).

[0518] Step 3: Dissolve crude GF-Az4c in 5 mL of N,N-dimethylformamide, and add succinic anhydride (32.1 mg, 0.32 mmol) with stirring. React overnight at room temperature, and further purify using a preparative HPLC system to obtain GF-Az4d (67.7 mg, 44.2% in two steps).

[0519] Step 4: Add GF-Az4d (67.7 mg, 0.09 mmol), DCM (5 mL), N-hydroxysuccinimide (30.4 mg, 0.26 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (101.2 mg, 0.53 mmol) to the reaction flask and react at room temperature for 2 h. After the reaction is complete, extract three times with dichloromethane, collect the organic layer, dry it with anhydrous sodium sulfate, and evaporate to dryness to obtain crude GF-Az4e (to proceed to the next reaction according to the theoretical amount).

[0520] Step 5: Add H2O (5 mL), DMF (5 mL), GDP-FAm solution (100 mM aqueous solution, 1.8 mL), GF-Az4e solution (tetrahydrofuran / 50 mM, 1.8 mL), and sodium bicarbonate solution (200 mM aqueous solution, 0.9 mL) to the reaction flask, and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system to obtain GF-Az4 (62.4 mg, yield 51.3% in two steps). 47 H 82 O 28 N 14 P2[MH] - The calculated HRMS (ESI-) is 1351.48255, and the measured value is 1351.50303.

[0521] Example 1.54 Preparation of GF-Az5

[0522] Step 1: Add Fmoc-O-tert-butyl-L-glutamic acid (200 mg, 0.47 mmol), N-hydroxysuccinimide (70 mg, 0.611 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (117 mg, 0.611 mmol), and DCM (15 mL) to the reaction flask, and react at room temperature for 3.5 h. After the reaction is complete, add H2O (15 mL) for extraction and washing. Extract the aqueous phase again with DCM (15 mL), combine the DCM phases, dry, filter, and concentrate to obtain GF-Az5a (proceed to the next reaction according to the theoretical amount).

[0523] Step 2: Add GF-Az5a (0.47 mmol), HATU (89 mg, 0.235 mmol), 2-[2-(2-azidoethoxy)ethoxy]ethylamine (98 mg, 0.564 mmol), DMF (15 mL), and DIPEA (246 μL, 1.41 mmol) to the reaction flask and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain GF-Az5b (150 mg, yield 54.9%).

[0524] Step 3: Add GF-Az5b (150 mg, 0.258 mmol), DMF (10 mL), and piperidine (1 mL) to the reaction flask and react at room temperature for about 1 hour. After the reaction is complete, further purify the product using a preparative HPLC system to obtain GF-Az5c (85 mg, yield 91.7%).

[0525] Step 4: Add GF-Az5c (85 mg, 0.236 mmol), azido-tetraethylene glycol-succinimide ester (110 mg, 0.283 mmol), DMF (6 mL), and DIPEA (123 μL, 0.708 mmol) to the reaction flask and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system to obtain GF-Az5d (110 mg, yield 73.3%).

[0526] Step 5: Add GF-Az5d (110 mg, 0.174 mmol), DCM (6 mL), and TFA (2 mL) to the reaction flask and react at room temperature for about 4 hours. After the reaction is complete, concentrate the product and further purify it using a preparative HPLC system to obtain GF-Az5e (83 mg, yield 83.0%).

[0527] Step Six: Add GF-Az5e (83 mg, 0.144 mmol), N-hydroxysuccinimide (50 mg, 0.432 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (166 mg, 0.864 mmol), and DCM (10 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, add H2O (10 mL) for extraction and washing. Wash the DCM phase once more with saturated brine (10 mL), dry, filter, and concentrate to obtain GF-Az5f (proceed to the next reaction according to the theoretical amount).

[0528] Step 7: Add H₂O (2 mL), THF (2 mL), GDP-FAm solution (aqueous solution / 72 mM, 2 mL), sodium bicarbonate solution (aqueous solution / 288 mM, 1 mL), and GF-Az5f solution (THF solution / 72 mM, 2 mL) to the reaction flask, and react overnight at room temperature. After the reaction is complete, the product is further purified using a preparative HPLC system to obtain GF-Az5 (65.4 mg, yield 39.1%). 38 H 64 O 24 N 14 P2[MH] - The calculated HRMS (ESI-) is 1161.36149, and the measured value is 1161.36481.

[0529] Example 1.55 Preparation of GF-Az6

[0530] The compound was prepared by referring to the synthesis method of compound 13 in Example 3 of patent "CN118541380A".

[0531] Example 1.56 Preparation of GF-Ayl1

[0532] H₂O (16 mL), THF (16 mL), GDP-FAm (200 mg, 0.33 mmol), propargyl-N-hydroxy ester (112.5 mg, 0.5 mmol), and sodium hydroxide solution (0.5 M, 1.98 mL) were added to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, THF was removed by concentration, and the product was further purified by an ion exchange preparation system to obtain GF-Ayl1 (206 mg, yield 87.1%). 22 H 32 O 17 N6P2[MH] - The calculated HRMS (ESI-) is 713.12264, and the measured value is 713.13072. 1 H-NMR(400MHz,D2O)δ8.01(s,1H),5.83(d,J=4.0Hz,1H),4.83(t,J=8.0Hz,1H),4.66(s, 1H),4.43(q,J=4.0Hz,1H),4.26-4.25(m,1H),4.13-4.12(m,2H),4.08(d,2H),3.78(d,J =4.0Hz,1H),3.70(t,J=4.0Hz,2H),3.62-3.59(m,1H),3.58-3.55(m,1H),3.52-3.45(m, 2H),3.21-3.17(m,1H),2.77(t,J=4.0Hz,1H),2.45(t,J=4.0Hz,2H),3.39-3.27(m,1H).

[0533] Example 1.57 Preparation of GF-Ayl2

[0534] Step 1: Add 2-(2-propynoxy)ethylamine (99 mg, 1 mmol), DMSO (2.5 mL), and triethylamine (139 μL, 1 mmol) to reaction flask 1 and stir. Then add 3-(2-bromoethoxy)-1-propyne (82 mg, 0.5 mmol) and DMSO (2.5 mL) to reaction flask 2 and stir to dissolve. Add the solution from reaction flask 2 to reaction flask 1 in portions and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system to obtain GF-Ayl2a (37 mg, yield 40.8%).

[0535] Step 2: Add GF-Ayl2a (37 mg, 0.204 mmol), succinic anhydride (61 mg, 0.612 mmol), and DMF (5 mL) to the reaction flask and react overnight at room temperature. After the reaction is complete, the product is further purified by a preparative HPLC system to obtain GF-Ayl2b (52 mg, yield 90.6%).

[0536] Step 3: Add GF-Ayl2b (52 mg, 0.185 mmol), DCM (5 mL), N-hydroxysuccinimide (64 mg, 0.555 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (212 mg, 1.11 mmol) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, add DCM (20 mL) and H2O (20 mL) for extraction and washing. Wash the DCM phase once more with saturated brine (20 mL), dry, filter, and concentrate to obtain GF-Ayl2c (to proceed to the next reaction according to the theoretical amount).

[0537] Step 4: Add H₂O (3.7 mL), THF (3.7 mL), GDP-FAm solution (100 mM aqueous solution, 3.7 mL), GF-Ayl₂c solution (50 mM THF solution, 3.7 mL), and sodium bicarbonate solution (200 mM aqueous solution, 1.85 mL) to the reaction flask, and react overnight at room temperature. After the reaction is complete, concentrate to remove THF, and then further purify the product using a preparative HPLC system to obtain GF-Ayl₂ (84.2 mg, yield 52.5%). 30 H 43 O 19 N7P2[MH] - The calculated HRMS (ESI-) is 866.20107, and the measured value is 866.20584.

[0538] Example 1.58 Preparation of GF-Ayl3

[0539] Step 1: Add 2-(2-(propyn-1-yloxy)ethoxy)ethylamine (286 mg, 2 mmol), DMSO (5 mL), and triethylamine (278 μL, 2 mmol) to reaction flask 1 and stir. Then add propynylene-tetraethylene glycol-bromine (295 mg, 1 mmol) and DMSO (5 mL) to reaction flask 2 and stir to dissolve. Add the solution from reaction flask 2 to reaction flask 1 in portions and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system to obtain GF-Ayl3a (128 mg, yield 35.9%).

[0540] Step 2: Add GF-Ayl3a (171 mg, 0.478 mmol), succinic anhydride (144 mg, 1.434 mmol), and DMF (5 mL) to the reaction flask and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system to obtain GF-Ayl3b (195 mg, yield 89.0%).

[0541] Step 3: Add GF-Ayl3b (195 mg, 0.426 mmol), DCM (15 mL), N-hydroxysuccinimide (147 mg, 1.278 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (490 mg, 2.556 mmol) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, add DCM (20 mL) and H2O (20 mL) for extraction and washing. Wash the DCM phase once more with saturated brine (20 mL), dry, filter, and concentrate to obtain GF-Ayl3c (to proceed to the next reaction according to the theoretical amount).

[0542] Step 4: Add H₂O (8.5 mL), THF (8.5 mL), GDP-FAm solution (100 mM aqueous solution, 8.52 mL), GF-Ayl3c solution (50 mM THF solution, 8.52 mL), and sodium bicarbonate solution (200 mM aqueous solution, 4.26 mL) to the reaction flask, and react overnight at room temperature. After the reaction is complete, concentrate to remove THF, and then further purify the product using a preparative HPLC system to obtain GF-Ayl3 (378.8 mg, yield 85.1%). 38 H 59 O 23 N7P2[MH] - The calculated HRMS (ESI-) is 1042.30648, and the measured value is 1042.31324.

[0543] Example 1.59 Preparation of GF-MAz1

[0544] The compound was prepared according to the synthesis method of compound 27 in Example 5 of patent "CN118541380A".

[0545] Example 1.60 Preparation of GF-MAz2

[0546] Step 1: Add [4-[(S)-2-[(S)-2-(FMOC-amino)-3-methylbutyrylamino]propionylamino]benzyl]carbonate [(4-nitrophenyl)] ester (136 mg, 0.2 mmol), HOAT (30 mg, 0.22 mmol), DMF (6 mL), 2,6-dimethylpyridine (100 μL, 0.8 mmol), methylaurestatin E (144 mg, 0.2 mmol), and DIPEA (100 μL, 0.6 mmol) sequentially to the reaction flask, and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain GF-MAz2a (209 mg, yield 83.1%).

[0547] Step 2: GF-MAz2a (209 mg, 0.166 mmol), DMF (6 mL), and piperidine (1.5 mL) were added sequentially to the reaction flask, and the reaction was carried out at room temperature for 2 h. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain GF-MAz2b (76 mg, yield 44.2%).

[0548] Step 3: GF-MAz2b (76 mg, 0.073 mmol), succinic anhydride (22 mg, 0.22 mmol), and DMF (3 mL) were added sequentially to the reaction flask, and the reaction was carried out at room temperature for 3 h. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain GF-MAz2c (70 mg, yield 84.4%). LC-MS: [MH] - =1135.95.

[0549] Step 4: N-tert-butoxycarbonyl-L-glutamic acid-5-methyl ester (130 mg, 0.5 mmol), 2-[2-(2-azidoethoxy)ethoxy]ethylamine (87 mg, 0.5 mmol), HATU (285 mg, 0.75 mmol), DMF (3 mL), and DIPEA (248 μL, 1.5 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified using a preparative HPLC system to obtain GF-MAz2d (166 mg, yield 79.6%).

[0550] Step 5: Add GF-MAz2d (19 mg, 0.046 mmol), DCM (4 mL), and TFA (1 mL) sequentially to the reaction flask, and react at room temperature for 1 h. After the reaction is complete, concentrate to obtain GF-MAz2e (to proceed to the next reaction according to the theoretical amount).

[0551] Step Six: GF-MAz2c (35 mg, 0.031 mmol), GF-MAz2e (0.046 mmol), HATU (23 mg, 0.062 mmol), DMF (3 mL), and DIPEA (15 μL, 0.092 mmol) were added sequentially to the reaction flask, and the reaction was carried out at room temperature for 2.5 h. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain GF-MAz2f (43 mg, yield 96.6%).

[0552] Step 7: GF-MAz2f (43 mg, 0.03 mmol), THF (1.5 mL), H2O (1 mL), and lithium hydroxide (4 mg, 0.15 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain GF-MAz2g (35.8 mg, yield 84.0%). 70 H 111 O 18 N 13 [MH] - The calculated HRMS (ESI-) is 1420.80973, and the measured value is 1420.81280.

[0553] Step 8: Add GF-MAz2g (35.8mg, 0.025mmol), N-hydroxysuccinimide (6mg, 0.05mmol), DCM (4mL), DMF (2mL), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (19mg, 0.1mmol) sequentially to the reaction flask, and react at room temperature for 2.5h. After the reaction is complete, concentrate and remove DCM to obtain GF-MAz2h (for the next reaction based on theoretical amounts).

[0554] Step 9: GF-MAz2h (0.025 mmol), DMF (3 mL), H2O (3 mL), GDP-FAm (30 mg, 0.05 mmol), and DIPEA (41 μL, 0.25 mmol) were added sequentially to the reaction flask, and the reaction was carried out at room temperature for 2.5 h. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain GF-MAz2 (15.8 mg, yield 63.0%). The calculated HRMS (ESI-) is 1002.94251, and the measured value is 1002.94563.

[0555] Example 1.61 Preparation of GF-MAz3

[0556] Step 1: Fmoc-O-tert-butyl-L-glutamic acid (255 mg, 0.6 mmol), 2-[2-(2-azidoethoxy)ethoxy]ethylamine (125.4 mg, 0.72 mmol), HATU (342.3 mg, 0.9 mmol), DMF (15 mL), and DIPEA (210 μL, 1.2 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified using a preparative HPLC system to obtain GF-MAz3a (298 mg, yield 85.4%).

[0557] Step 2: GF-MAz3a (298 mg, 0.51 mmol), DMF (15 mL), and piperidine (4 mL, 40.5 mmol) were added sequentially to the reaction mixture, and the reaction was carried out at room temperature for 3 hours. The product was further purified by a preparative HPLC system to obtain GF-MAz3b (170 mg, yield 92.8%).

[0558] Step 3: GF-MAz3b (27.7 mg, 0.077 mmol), OSu-Suc-vc-PAB-MMAE (prepared according to the synthetic method of compound OSu-Suc-vc-PAB-MMAE in Example 6 of patent "CN118541380A") (92.4 mg, 0.07 mmol), DMF (10 mL), and DIPEA (86 μL, 0.49 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by a preparative HPLC system to obtain GF-MAz3c (91 mg, yield 83.1%).

[0559] Step 4: GF-MAz3c (91 mg, 0.058 mmol), DCM (15 mL), and TFA (2 mL) were added sequentially to the reaction mixture, and the reaction was carried out at room temperature for 2.5 hours. The product was further purified by a preparative HPLC system to obtain GF-MAz3d (39 mg, yield 44.6%).

[0560] Step 5: Add GF-MAz3d (39 mg, 0.026 mmol), N-hydroxysuccinimide (9 mg, 0.078 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (29.9 mg, 0.156 mmol), DCM (10 mL), and DMF (2 mL) to the reaction mixture sequentially, and react overnight at room temperature. After the reaction is complete, concentrate to obtain GF-MAz3e (to proceed to the next reaction according to the theoretical amount).

[0561] Step Six: Add H₂O (1040 μL), DMF (2080 μL), GDP-FAm solution (100 mM aqueous solution, 520 μL), GF-MAz3e solution (50 mM DMF solution, 520 μL), and sodium bicarbonate solution (200 mM aqueous solution, 1040 μL) to the reaction flask, and react overnight at room temperature. After the reaction is complete, concentrate to remove THF, and then further purify the product using a preparative HPLC system to obtain GF-MAz3 (19.3 mg, yield 35.5%). 89 H 141 O 33 N 21 P2[MH] - The calculated HRMS (ESI-) is 2093.94367, and the measured value is 2093.94600.

[0562] Example 1.62 Preparation of GF-1

[0563] Step 1: (S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecanoic acid (400 mg, 0.944 mmol), DMF (15 mL), H₂O (10 mL), propargyl-N-hydroxy ester (256 mg, 1.132 mmol), and DIPEA (494 μL, 2.832 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was complete, the product was further purified using a preparative HPLC system to obtain GF-1a (497 mg, yield 98.6%). LC-MS: [MH] - =532.3.

[0564] Step 2: GF-1a (497 mg, 0.932 mmol), eczemab mesylate (495 mg, 0.932 mmol), HATU (709 mg, 1.864 mmol), DMF (35 mL), and DIPEA (649 μL, 3.728 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by preparative HPLC to obtain GF-1b (830 mg, yield 93.7%). LC-MS: [M+H] + =951.7. 1 H-NMR (400MHz, DMSO-d6) δ8.65(t,J=5.3Hz,1H),8.52(d,J=7.1Hz,1H),8.32(t,J=4.6 Hz,1H),8.21(t,J=4.6Hz,1H),8.14(d,J=6.4Hz,1H),8.01(t,J=4.5Hz,1H),7.76(d,J =8.7Hz,1H),7.30(s,1H),7.27-7.20(m,4H),7.20-7.14(m,1H),6.53(s,1H),5.64-5. 56(m,1H),5.42(dd,J=14.8,13.2Hz,2H),5.17(dd,J=21.6,15.1Hz,2H),4.65(d,J=5.3 Hz,2H),4.50-4.46(m,1H),4.09(d,J=1.9Hz,2H),4.03(s,2H),3.75-3.71(m,2H),3.6 9(d,J=4.6Hz,2H),3.64(t,J=5.2Hz,3H),3.41(t,J=1.9Hz,2H),3.22-3.17(m,1H),3. 17-3.12(m,1H),3.03(dd,J=11.1,3.6Hz,1H),2.78(dd,J=11.0,7.8Hz,1H),2.40(t,J =5.2Hz,2H),2.37(s,3H),2.25-2.13(m,2H),1.93-1.79(m,2H),0.87(t,J=5.8Hz,3H).

[0565] Step 3: Add H2O (4.5 mL) and DMSO (4.5 mL) to the reaction flask and stir. Then add GF-Az0 (prepared according to the synthesis method of compound GDP-FAmAz in Example 11 of patent "CN118119633A") solution (aqueous solution / 50 mM, 1800 μL), CuSO4 solution (aqueous solution / 50 mM, 540 μL), TBTA solution (DMSO solution / 50 mM, 1080 μL), GF-1b solution (DMSO solution / 25 mM, 1800 μL), and sodium ascorbate solution (aqueous solution / 50 mM, 2700 μL), and finally add DMSO (4.5 mL). React overnight at room temperature. After the reaction is complete, the product is further purified by a preparative HPLC system and lyophilized to obtain a white solid GF-1 (29.9 mg, yield 40.6%). Purity (HPLC-RP): 91.3%. 66 H 78 O 28 FN 17 P2[MH] - The calculated HRMS (ESI-) is 1636.45887, and the measured value is 1636.43122. 1H-NMR(400MHz,D2O)δ7.75(s,2H),6.99(s,1H),6.96-6.84(m,3H),6.81(d,J=8.1Hz,1 H),6.74(d,J=5.8Hz,2H),5.50(d,J=3.7Hz,1H),5.34(s,1H),5.23(d,J=12.4Hz,1H),5 .07(d,J=13.0Hz,1H),5.01(s,2H),4.77(t,J=4.8Hz,1H),4.57(d,J=8.2Hz,1H),4.39 (s,3H),4.29(s,2H),4.23(t,J=5.7Hz,1H),4.17(d,J=12.7Hz,1H),4.10(s,1H),4.05( d,J=13.8Hz,3H),3.68(s,3H),3.64-3.56(m,5H),3.53(d,J=4.7Hz,1H),3.50-3.46(m ,1H),3.44(d,J=6.0Hz,1H),3.40(d,J=10.2Hz,1H),3.14(dd,J=11.0,7.8Hz,1H),2.90 -2.76(m,1H),2.65(d,J=6.6Hz,2H),2.57(s,3H),2.49-2.41(m,1H),2.38(t,J=4.4Hz ,2H),2.12(s,1H),1,94(s,1H),1.80(s,3H),1.75-1.60(m,2H),0.76(t,J=5.6Hz,3H).

[0566] Example 1.63 Preparation of GF-2

[0567] Step 1: N-[(1,1-dimethylethoxy)carbonyl]glycylglycyl-L-phenylalanyl-glycine (49 mg, 0.113 mmol), eczemab mesylate (40 mg, 0.075 mmol), HATU (57 mg, 0.15 mmol), DMF (3 mL), and DIPEA (37 μL, 0.225 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain GF-2a (45 mg, yield 70.0%).

[0568] Step 2: Add GF-2a (45 mg, 0.053 mmol), TFA (2 mL), and DCM (4 mL) sequentially to the reaction flask, and react at room temperature for 1 hour. After the reaction is complete, concentrate to obtain GF-2b (to proceed to the next reaction according to the theoretical amount).

[0569] Step 3: GF-2b (0.053 mmol), propargyl-N-hydroxy ester (18 mg, 0.079 mmol), DMF (3 mL), and DIPEA (26 μL, 0.158 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by preparative HPLC to obtain GF-2c (33 mg, yield 72.1%). LC-MS: [M+H] + =864.7.

[0570] Step 4: Add DMSO (2558 μL) to the reaction flask and stir. Then add GF-2c solution (DMSO solution / 9.56 mM, 2 mL), GF-Az0 solution (aqueous solution / 50 mM, 765 μL), copper sulfate solution (aqueous solution / 50 mM, 382 μL), TBTA solution (DMSO solution / 50 mM, 765 μL), and sodium ascorbate solution (aqueous solution / 50 mM, 1.53 mL) sequentially. React overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system, and lyophilize to obtain a white solid of GF-2 (21.2 mg, yield 71.5%). Purity (HPLC-RP): 94.9%. 63 H 73 O 26 FN 16 P2[MH] - The calculated HRMS (ESI-) is 1549.42684, and the measured value is 1549.43923.

[0571] Example 1.64 Preparation of GF-3

[0572] Step 1: N-[(1,1-dimethylethoxy)carbonyl]glycylglycyl-L-phenylalanyl-glycine (87.2 mg, 0.2 mmol), N-hydroxysuccinimide (69 mg, 0.6 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (230 mg, 1.2 mmol), DMF (5 mL), and DCM (10 mL) were added sequentially to the reaction flask. The reaction was allowed to proceed overnight at room temperature. After the reaction was complete, the mixture was quenched with water, extracted three times with DCM, and the organic layer was collected, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain a pale yellow oil, GF-3a (to be used for the next reaction according to theoretical quantities).

[0573] Step 2: GF-3a (106.6 mg, 0.2 mmol), 4-aminobutyric acid (22.7 mg, 0.22 mmol), DMF (5 mL), and DIPEA (105 μL, 0.6 mmol) were added sequentially to the reaction flask, and the reaction was carried out at room temperature for 5 h. The product was further purified using a preparative HPLC system to obtain GF-3b (63 mg, two-step yield 60.4%). LC-MS: [M+H] + =522.7.

[0574] Step 3: GF-3b (63 mg, 0.12 mmol), eczemab mesylate (53.2 mg, 0.1 mmol), HATU (57 mg, 0.15 mmol), DMF (5 mL), and DIPEA (53 μL, 0.3 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. The product was further purified using a preparative HPLC system to obtain GF-3c (81 mg, yield 86.3%).

[0575] Step 4: Add GF-3c (81 mg, 0.086 mmol), DCM (10 mL), and TFA (3 mL) sequentially to the reaction mixture and react at room temperature for 1.5 h. After the reaction is complete, concentrate to obtain GF-3d (to proceed to the next reaction in the theoretical amount).

[0576] Step 5: GF-3d (37.7 mg, 0.045 mmol), propargyl-N-hydroxy ester (20.3 mg, 0.09 mmol), DMF (5 mL), and DIPEA (32 μL, 0.184 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-3e (34 mg, yield 79.7%). LC-MS: [M+H] + =950.0.

[0577] Step Six: Add H₂O (350 μL) and DMSO (2.75 mL) to the reaction flask and stir. Then add GF-AzO solution (50 mM aqueous solution, 400 μL), CuSO₄ solution (50 mM aqueous solution, 100 μL), TBTA solution (DMSO solution, 50 mM, 200 μL), GF-3e solution (DMSO solution, 12.5 mM, 800 μL), and sodium ascorbate solution (50 mM aqueous solution, 400 μL) sequentially. React overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid GF-3 (6.7 mg, yield 20.5%). Purity (HPLC-RP): 96.7%. 67 H 80 FN 17 O27 P2[MH] - The calculated HRMS (ESI-) is 1634.47961, and the measured value is 1634.48550.

[0578] Example 1.65 Preparation of GF-4

[0579] Step 1: Tert-butyl carbamate (64 mg, 0.1 mmol), HOAT (15 mg, 0.11 mmol), DMF (3 mL), 2,6-dimethylpyridine (50 μL, 0.4 mmol), eczemab mesylate (53 mg, 0.1 mmol), and DIPEA (50 μL, 0.3 mmol) were added sequentially to the reaction flask. The reaction was carried out overnight at room temperature. After the reaction was complete, the product was further purified using a preparative HPLC system to obtain GF-4a (86 mg, yield 91.7%). LC-MS: [M+H] + =941.6.

[0580] Step 2: Add GF-4a (86 mg, 0.091 mmol), TFA (3 mL), and DCM (6 mL) sequentially to the reaction flask, and react at room temperature for 2 hours. After the reaction is complete, concentrate to obtain GF-4b (to be used for the next reaction according to the theoretical amount). LC-MS: [M+H] + =841.7.

[0581] Step 3: GF-4b (0.045 mmol), propargyl-N-hydroxy ester (15 mg, 0.068 mmol), DMF (3 mL), and DIPEA (22 μL, 0.136 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by preparative HPLC to obtain GF-4c (28.4 mg, yield 66.4%). LC-MS: [M+H] + =951.9.

[0582] Step 4: Add DMSO (1700 μL) to the reaction flask and stir. Then add GF-4c solution (DMSO solution / 7.47 mM, 1 mL), GF-Az0 solution (water solution / 50 mM, 449 μL), copper sulfate solution (water solution / 50 mM, 150 μL), TBTA solution (DMSO solution / 50 mM, 299 μL), and sodium ascorbate solution (water solution / 50 mM, 598 μL) in sequence. React overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system, and lyophilize to obtain a white solid of GF-4 (6.7 mg, yield 54.8%). Purity (HPLC-RP): 99.2%. 67 H 82 O 27 FN 17 P2[MH] - The calculated HRMS (ESI-) is 1636.49526, and the measured value is 1636.52232.

[0583] Example 1.66 Preparation of GF-5

[0584] Step 1: Add Tp-2-2 (32.4 mg, 56.1 μmol), DMF (5 mL), propargyl-N-hydroxy ester (19 mg, 84.2 μmol), and DIPEA (39 μL, 224.4 μmol) to the reaction flask and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain GF-5a (36.8 mg, yield 95.3%). LC-MS: [M+H] + =688.6.

[0585] Step 2: Add 1 mL of H₂O and 1 mL of DMSO to the reaction flask and stir. Then add GF-AzO solution (50 mM aqueous solution, 400 μL), CuSO₄ solution (50 mM aqueous solution, 120 μL), TBTA solution (DMSO solution, 50 mM, 240 μL), GF-5a solution (DMSO solution, 12.5 mM, 800 μL), and sodium ascorbate solution (50 mM aqueous solution, 600 μL) in sequence. Finally, add 1 mL of DMSO and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid GF-5 (5.4 mg, yield 39.1%). Purity (HPLC-RP): 92.0%. 54 H 65 O 24 FN 14 P2[MH] -The calculated HRMS (ESI-) is 1373.36826, and the measured value is 1373.37253.

[0586] Example 1.67 Preparation of GF-6

[0587] Step 1: N-tert-butoxycarbonyl-alanylalanine (5.5 mg, 0.021 mmol), Tp-2-2 (8.3 mg, 0.014 mmol), HATU (11 mg, 0.028 mmol), DMF (3 mL), and DIPEA (7 μL, 0.042 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain GF-6a (10.7 mg, yield 93.3%).

[0588] Step 2: Add GF-6a (10.7 mg, 0.013 mmol), TFA (2 mL), and DCM (4 mL) sequentially to the reaction flask, and react at room temperature for 1.5 h. After the reaction is complete, concentrate to obtain GF-6b (to proceed to the next reaction according to the theoretical amount).

[0589] Step 3: GF-6b (0.013 mmol), propargyl-N-hydroxy ester (4.4 mg, 0.0195 mmol), DMF (3 mL), and DIPEA (6.5 μL, 0.039 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by preparative HPLC to obtain GF-6c (8 mg, yield 74.2%). LC-MS: [M+H] + =830.7.

[0590] Step 4: Add DMSO (1807 μL) and H2O (324 μL) to the reaction flask and stir. Then add GF-6c solution (DMSO solution / 4 mg, 1 mL), GF-Az0 solution (aqueous solution / 50 mM, 193 μL), copper sulfate solution (aqueous solution / 50 mM, 97 μL), TBTA solution (DMSO solution / 50 mM, 193 μL), and sodium ascorbate solution (aqueous solution / 50 mM, 386 μL) in sequence, and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system, and lyophilize to obtain a white solid of GF-6 (3.7 mg, yield 50.6%). Purity (HPLC-RP): 99.4%. The calculated HRMS (ESI-) is 757.21761, and the measured value is 757.21731.

[0591] Example 1.68 Preparation of GF-7

[0592] Step 1: N-tert-butoxycarbonyl-alanylalanine (18 mg, 0.068 mmol), Tp-3-2 (0.045 mmol), HATU (34 mg, 0.09 mmol), DMF (3 mL), and DIPEA (22 μL, 0.135 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was complete, the product was further purified using a preparative HPLC system to obtain GF-7a (35 mg, yield 93.4%). LC-MS: [M+H] + =834.6.

[0593] Step 2: Add GF-7a (35 mg, 0.042 mmol), TFA (2 mL), and DCM (4 mL) sequentially to the reaction flask, and react at room temperature for 1 h. After the reaction is complete, concentrate to obtain GF-7b (to be used for the next reaction according to the theoretical amount). LC-MS: [M+H] + =734.7.

[0594] Step 3: GF-7b (0.042 mmol), propargyl-N-hydroxy ester (14 mg, 0.063 mmol), DMF (3 mL), and DIPEA (21 μL, 0.126 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by preparative HPLC to obtain GF-7c (33 mg, yield 93.2%). LC-MS: [M+H] + =844.7. 1H-NMR (400MHz, DMSO-d6) δ8.36(d,J=4.0Hz,1H),8.16(d,J=8.0Hz,1H),8.01(d,J=8.0Hz,1H),7.83(d,J=4.0Hz,1H),7.78(d,J=8.0Hz,1H),7.72( d,J=4.0Hz,1H),7.30(s,1H),6.52(s,1H),5.53-5.49(m,1H),5.42(s,2H) ),5.17(dd,J=12.0,76.0Hz,2H),4.21-4.12(m,3H),4.09(d,2H),3.95-3 .89(m,1H),3.62(t,J=4.0Hz,2H),3.41(t,J=4.0Hz,1H),3.16(t,J=4.0H z,2H),2.39(s,4H),2.22-2.18(m,1H),2.09-2.07(m,1H),1.91-1.82(m, 2H),1.75-1.68(m,1H),1.62-1.55(m,1H),1.23(s,1H),1.17(q,J=4.0Hz ,5H),1.12(d,J=4.0Hz,3H),0.87(t,J=4.0Hz,3H),0.82(t,J=4.0Hz,3H).

[0595] Step 4: Add DMSO (2511 μL) to the reaction flask and stir. Then add GF-7c solution (DMSO solution / 9.78 mM, 2 mL), GF-Az0 solution (aqueous solution / 50 mM, 782 μL), copper sulfate solution (aqueous solution / 50 mM, 360 μL), TBTA solution (DMSO solution / 50 mM, 782 μL), and sodium ascorbate solution (aqueous solution / 50 mM, 1565 μL) in sequence. React overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system, and lyophilize to obtain a white solid of GF-7 (24.4 mg, yield 81.5%). Purity (HPLC-RP): 97.1%. 61 H 77 O 26 FN 16 P2[MH] - The calculated HRMS (ESI-) is 1529.45814, and the measured value is 1529.48579. 1H-NMR(400MHz,D2O)δ7.88(s,1H),7.16(s,1H),7.08(d,J=8.0Hz,1H),5.72 (s,1H),5.38-5.34(m,2H),5.24(d,J=8.0Hz,1H),5.14(s,2H),4.84(d,J=16 .0Hz,2H),4.58(s,1H),4.49(s,2H),4.41(s,1H),4.20-4.18(m,2H),4.13(s ,2H),3.96(q,J=4.0Hz,1H),3.87(q,J=4.0Hz,1H),3.79(s,1H),3.68-3.63( m,3H),3.60-3.57(m,1H),3.54-3.49(m,2H),3.27-3.23(m,1H),3.07-3.01( m,1H),2.89(s,2H),2.81(s,1H),2.44-2.38(m,2H),2.22(s,2H),2.01(s,2H ),1.92-1.86(m,2H),1.81(m,2H),1.76-1.70(m,1H),1.22-1.14(m,3H),1.1 1-1.07(m,3H),1.00-0.96(m,1H),0.92-0.87(m,4H),0.82(t,J=8.0Hz,2H).

[0596] Example 1.69 Preparation of GF-8

[0597] Step 1: Tp-4 (267.1 mg, 0.5 mmol), N-tert-butoxycarbonyl-alanylalanine (195.1 mg, 0.75 mmol), HATU (285.2 mg, 0.75 mmol), DMF (20 mL), and DIPEA (348 μL, 2.0 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-8a (308 mg, yield 79.3%).

[0598] Step 2: Add GF-8a (308 mg, 0.4 mmol), DCM (25 mL), and TFA (4 mL) sequentially to the reaction mixture and react at room temperature for 1 hour. After the reaction is complete, concentrate to obtain GF-8b (to proceed to the next reaction in the theoretical amount).

[0599] Step 3: GF-8b (270.5 mg, 0.4 mmol), N-tert-butoxycarbonyl-L-alanine (113.5 mg, 0.6 mmol), HATU (228.1 mg, 0.6 mmol), DMF (15 mL), and DIPEA (279 μL, 1.6 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified using a preparative HPLC system to obtain GF-8c (327 mg, two-step yield 96.5%).

[0600] Step 4: Add GF-8c (327 mg, 0.386 mmol), DCM (25 mL), and TFA (4 mL) sequentially to the reaction mixture and react at room temperature for 1.5 h. After the reaction is complete, concentrate to obtain GF-8d (to proceed to the next reaction in the theoretical amount).

[0601] Step 5: GF-8d (288.5 mg, 0.386 mmol), propargyl-N-hydroxy ester (130.3 mg, 0.579 mmol), DMF (15 mL), and DIPEA (336 μL, 1.93 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-8e (270.1 mg, two-step yield 81.6%). LC-MS: [M+H] + =858.7. 1H-NMR (400MHz, DMSO-d6) δ8.32(d,J=8.0Hz,1H),8.20(d,J=4.0Hz,1H),7.99(d,J=8.0Hz,1H),7.76(dd,J=16.0,8.0Hz,2H),7.48(d,J=8.0Hz,1H),7. 28(s,1H),6.54(s,1H),5.49-5.46(m,1H),5.41(s,2H),5.26(d,J=16.0Hz ,1H),5.03(d,J=12.0Hz,1H),4.18-4.11(m,3H),4.09(d,J=4.0Hz,2H),3.6 9-3.65(m,1H),3.61(t,J=4.0Hz,2H),3.40(t,J=4.0Hz,1H),3.18-3.07(m ,2H),2.52-2.51(m,1H),2.39(t,J=4.0Hz,1H),2.36(s,2H),2.26-2.20(m, 1H),2.11-2.04(m,1H),2.02-1.95(m,1H),1.90-1.81(m,2H),1.17(d,J=4 .0Hz,3H),1.12(d,J=8.0Hz,3H),1.08(d,J=8.0Hz,3H),0.88-0.80(m,9H).

[0602] Step Six: Add H₂O (5.25 mL) and DMSO (41.25 mL) to the reaction flask and stir. Then add GF-AzO solution (50 mM aqueous solution, 6 mL), CuSO₄ solution (50 mM aqueous solution, 1.5 mL), TBTA solution (DMSO solution (50 mM, 3 mL), GF-8e solution (DMSO solution (25 mM, 6 mL), and sodium ascorbate solution (50 mM aqueous solution, 6 mL) in sequence and react overnight at room temperature. After the reaction is complete, the product is further purified by a preparative HPLC system and lyophilized to obtain a white solid (178.8 mg, yield 77.2%). Purity (HPLC-RP): 96.2%. 62 H 79 FN 16 O 26 P2[MH] - The calculated HRMS (ESI-) is 1543.47379, and the measured value is 1543.52996. 1H-NMR(400MHz,D2O)δ7.82(s,1H),7.29(d,J=8.0Hz,1H),7.07(s,1H),7.0 0(d,J=12.0Hz,1H),5.72(s,1H),5.30-5.27(s,2H),5.16(d,J=12.0Hz,1H) ,5.06(s,2H),4.79(s,1H),4.71(s,2H),4.51(s,1H),4.41(s,2H),4.34(s ,1H),4.15(s,1H),4.07(s,2H),4.01(d,J=8.0Hz,1H),3.85-3.76(m,2H),3 .72(s,1H),3.64-3.57(m,3H),3.52-3.50(m,1H),3.45-3.42(m,2H),3.20 -3.15(m,1H),2.87-2.80(m,2H),2.60-2.59(m,2H),2.38-2.29(m,2H),2.2 3-2.11(m,2H),2.05-1.98(m,1H),1.95(s,3H),1.76-1.68(m,2H),1.07(d, J=8.0Hz,3H),0.96(d,J=4.0Hz,3H),0.83-0.79(m,6H),0.74-0.71(m,6H).

[0603] Example 1.70 Preparation of GF-9

[0604] Step 1: Tp-7 (42.2 mg, 0.077 mmol), N-tert-butoxycarbonyl-alanylalanine (30.2 mg, 0.116 mmol), HATU (44.1 mg, 0.116 mmol), DMF (5 mL), and DIPEA (54 μL, 0.31 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-9a (39 mg, yield 64.1%).

[0605] Step 2: Add GF-9a (39 mg, 0.05 mmol), DCM (10 mL), and TFA (1 mL) sequentially to the reaction mixture and react at room temperature for 2 hours. After the reaction is complete, concentrate to obtain GF-9b (to proceed to the next reaction in the theoretical amount).

[0606] Step 3: GF-9b (34.5 mg, 0.05 mmol), N-tert-butoxycarbonyl-L-alanine (14.2 mg, 0.075 mmol), HATU (28.5 mg, 0.075 mmol), DMF (5 mL), and DIPEA (35 μL, 0.2 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified using a preparative HPLC system to obtain GF-9c (39 mg, two-step yield 90.5%).

[0607] Step 4: Add GF-9c (39 mg, 0.045 mmol), DCM (10 mL), and TFA (1.5 mL) sequentially to the reaction mixture and react at room temperature for 2 hours. After the reaction is complete, concentrate to obtain GF-9d (to proceed to the next reaction in the theoretical amount).

[0608] Step 5: GF-9d (34.3 mg, 0.045 mmol), propargyl-N-hydroxy ester (30.4 mg, 0.135 mmol), DMF (5 mL), and Et3N (32 μL, 0.23 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-9e (32 mg, two-step yield 81.6%). 45 H 54 FN7O 10 [M+H] + The calculated HRMS (ESI+) is 872.39890, and the measured value is 872.39793.

[0609] Step Six: Add H₂O (355 μL) and DMSO (1435 μL) to the reaction flask and stir. Then add GF-AzO solution (50 mM aqueous solution, 200 μL), CuSO₄ solution (50 mM aqueous solution, 20 μL), TBTA solution (DMSO solution, 50 mM, 40 μL), GF-9e solution (DMSO solution, 12.5 mM, 400 μL), and sodium ascorbate solution (50 mM aqueous solution, 50 μL) sequentially. React overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid GF-9 (1.2 mg, yield 15.4%). Purity (HPLC-RP): 96.7%. 63 H 81 FN 16 O 26 P2[MH] - The calculated HRMS (ESI-) is 1557.48944, and the measured value is 1557.49287.

[0610] Example 1.71 Preparation of GF-10

[0611] Step 1: Tp-6 (34 mg, 0.062 mmol), N-tert-butoxycarbonyl-alanylalanine (24.2 mg, 0.093 mmol), HATU (35.4 mg, 0.093 mmol), DMF (5 mL), and DIPEA (44 μL, 0.253 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-10a (43 mg, yield 87.7%).

[0612] Step 2: Add GF-10a (43 mg, 0.054 mmol), DCM (10 mL), and TFA (1 mL) sequentially to the reaction mixture and react at room temperature for 2 hours. After the reaction is complete, concentrate to obtain GF-10b (to proceed to the next reaction in the theoretical amount).

[0613] Step 3: GF-10b (34.5 mg, 0.05 mmol), N-tert-butoxycarbonyl-L-alanine (14.2 mg, 0.075 mmol), HATU (28.5 mg, 0.075 mmol), DMF (5 mL), and DIPEA (35 μL, 0.2 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified using a preparative HPLC system to obtain GF-10c (38 mg, two-step yield 88.2%).

[0614] Step 4: Add GF-10c (38 mg, 0.045 mmol), DCM (10 mL), and TFA (1.5 mL) sequentially to the reaction mixture and react at room temperature for 2 hours. After the reaction is complete, concentrate to obtain GF-10d (to proceed to the next reaction in the theoretical amount).

[0615] Step 5: GF-10d (34.3 mg, 0.045 mmol), propargyl-N-hydroxy ester (30.4 mg, 0.135 mmol), DMF (5 mL), and Et3N (32 μL, 0.23 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-10e (35 mg, two-step yield 89.3%). 45 H 54 FN7O 10 [M+H] + The calculated HRMS (ESI+) is 872.39890, and the measured value is 872.39793.

[0616] Step Six: Add H₂O (355 μL) and DMSO (1435 μL) to the reaction flask and stir. Then add GF-AzO solution (50 mM aqueous solution, 200 μL), CuSO₄ solution (50 mM aqueous solution, 20 μL), TBTA solution (DMSO solution, 50 mM, 40 μL), GF-10e solution (DMSO solution, 12.5 mM, 400 μL), and sodium ascorbate solution (50 mM aqueous solution, 50 μL) in sequence and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid GF-10 (1.7 mg, yield 21.8%). Purity (HPLC-RP): 93.7%. 63 H 81 FN 16 O 26 P2[MH] - The calculated HRMS (ESI-) is 1557.48944, and the measured value is 1557.49287.

[0617] Example 1.72 Preparation of GF-11

[0618] Step 1: N-tert-butoxycarbonyl-alanylalanine (22 mg, 0.085 mmol), Tp-8-2 (0.057 mmol), HATU (43 mg, 0.114 mmol), DMF (3 mL), and DIPEA (28 μL, 0.171 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain GF-11a (48 mg, yield 96.9%).

[0619] Step 2: Add GF-11a (48 mg, 0.056 mmol), TFA (2 mL), and DCM (4 mL) sequentially to the reaction flask, and react at room temperature for 1 hour. After the reaction is complete, concentrate to obtain GF-11b (to proceed to the next reaction according to the theoretical amount).

[0620] Step 3: GF-11b (0.056 mmol), propargyl-N-hydroxy ester (19 mg, 0.083 mmol), DMF (3 mL), and DIPEA (28 μL, 0.168 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by preparative HPLC to obtain GF-11c (34 mg, yield 69.9%). LC-MS: [M+H] + =870.8. 1H-NMR (400MHz, DMSO-d6) δ8.39(d,J=8.0Hz,1H),8.18(d,J=4.0Hz,1H),8.02(d,J=4.0Hz,1H),7.84(d,J=8.0Hz,1H),7.78(d,J=8.0H z,1H),7.68(d,J=8.0Hz,1H),7.30(s,1H),6.52(s,1H),5.50-5.48(m,1H),5.43(s,2H),5.15(dd,J=16.0,96.0Hz,2H),4.26(t,J=8.0 Hz,3H),4.22-4.15(m,2H),4.09(s,2H),3.91-3.86(m,1H),3.62(t,J=4.0Hz,2H),3.40(s,1H),3.15(s,2H),2.64-2.59(m,1H),2.39 (s,4H),2.21-2.17(m,1H),2.08-2.04(m,1H),1.91-1.82(m,3H),1.75(s,3H),1.64(s,1H),1.18-1.11(m,8H),0.88(t,J=4.0Hz,3H).

[0621] Step 4: Add DMSO (2511 μL) to the reaction flask and stir. Then add GF-11c solution (DMSO solution / 9.78 mM, 2 mL), GF-Az0 solution (aqueous solution / 50 mM, 782 μL), copper sulfate solution (aqueous solution / 50 mM, 360 μL), TBTA solution (DMSO solution / 50 mM, 782 μL), and sodium ascorbate solution (aqueous solution / 50 mM, 1565 μL) in sequence. React overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system, and lyophilize to obtain a white solid of GF-11 (22.9 mg, yield 75.2%). Purity (HPLC-RP): 98.5%. 63 H 79 O 26 FN 16 P2[MH] - The calculated HRMS (ESI-) is 1555.47379, and the measured value is 1555.50585. 1H-NMR(400MHz,D2O)δ7.88(s,1H),7.15(s,1H),7.06(d,J=8.0Hz,1H),5.73(s,1 H),5.37-5.31(m,1H),5.25-5.22(m,1H),5.14(s,2H),4.87(s,1H),4.80(s,1H), 4.76-4.75(m,1H),4.68(s,1H),4.64(s,1H),4.58(s,1H),4.49(s,2H),4.41(s,1 H),4.21(s,1H),4.19(s,1H),4.13(s,2H),4.02-3.90(m,2H),3.80(d,1H),3.68- 3.64(m,2H),3.60-3.58(m,1H),3.54-3.50(m,2H),3.28-3.23(m,1H),3.00-2.96 (m,1H),2.88-2.84(m,2H),2.76-2.68(m,1H),2.44-2.38(m,2H),2.19(s,2H),2. 01(s,3H),1.89(s,1H),1.83-1.82(m,3H),1.72(s,2H),1.21-1.13(m,3H),1.09- 1.08(m,2H),0.98(d,J=8.0Hz,1H),0.89(d,J=8.0Hz,2H),0.82(t,J=8.0Hz,2H).

[0622] Example 1.73 Preparation of GF-12

[0623] Step 1: Add Tp-9-2 (57.1 mg, 94.5 μmol), DMF (7 mL), N-tert-butoxycarbonyl-alanylalanine (37 mg, 141.8 μmol), HATU (72 mg, 189 μmol), and DIPEA (66 μL, 378 μmol) to the reaction flask and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain GF-12a (70 mg, yield 87.5%).

[0624] Step 2: Add GF-12a (70 mg, 82.7 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 hours. After the reaction is complete, concentrate to obtain GF-12b (to proceed to the next reaction according to the theoretical amount).

[0625] Step 3: Add GF-12b (61.7 mg, 82.7 μmol), DMF (5 mL), propargyl-N-hydroxy ester (28 mg, 124.1 μmol), and DIPEA (58 μL, 330.8 μmol) to the reaction flask and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain GF-12c (62.0 mg, yield 87.6%). LC-MS: [M+H] + =856.7.

[0626] Step 4: Add 1.5 mL of H₂O and 1.5 mL of DMSO to the reaction flask and stir. Then add GF-AzO solution (50 mM aqueous solution, 600 μL), CuSO₄ solution (50 mM aqueous solution, 180 μL), TBTA solution (DMSO solution, 50 mM, 360 μL), GF-12c solution (DMSO solution, 12.5 mM, 1200 μL), and sodium ascorbate solution (50 mM aqueous solution, 900 μL) in sequence. Finally, add 1.5 mL of DMSO and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid GF-12 (15.1 mg, yield 65.4%). Purity (HPLC-RP): 91.5%. 62 H 77 O 26 FN 16 P2[MH] - The calculated HRMS (ESI-) is 1541.45814, and the measured value is 1541.47149.

[0627] Example 1.74 Preparation of GF-13

[0628] Step 1: N-tert-butoxycarbonyl-alanylalanine (15 mg, 0.058 mmol), Tp-14-2 (0.039 mmol), HATU (30 mg, 0.078 mmol), DMF (3 mL), and DIPEA (19 μL, 0.117 mmol) were added sequentially to the reaction flask, and the reaction was carried out at room temperature for 3 h. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain GF-13a (19 mg, yield 56.6%).

[0629] Step 2: Add GF-13a (19 mg, 0.023 mmol), TFA (2 mL), and DCM (4 mL) sequentially to the reaction flask, and react at room temperature for 1 hour. After the reaction is complete, concentrate to obtain GF-13b (to proceed to the next reaction according to the theoretical amount).

[0630] Step 3: GF-13b (0.023 mmol), propargyl-N-hydroxy ester (8 mg, 0.034 mmol), DMF (3 mL), and DIPEA (11 μL, 0.069 mmol) were added sequentially to the reaction flask, and the reaction was carried out at room temperature for 3 h. After the reaction was completed, the product was further purified by preparative HPLC to obtain GF-13c (17.1 mg, yield 85.4%). LC-MS: [M+H] + =872.9.

[0631] Step 4: Add DMSO (232 μL) to the reaction flask and stir. Then add GF-13c solution (DMSO solution / 4.91 mM, 2 mL), GF-Az0 solution (water solution / 50 mM, 393 μL), copper sulfate solution (water solution / 50 mM, 196 μL), TBTA solution (DMSO solution / 50 mM, 393 μL), and sodium ascorbate solution (water solution / 50 mM, 786 μL) in sequence. React overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid of GF-13 (6.8 mg, yield 44.4%). Purity (HPLC-RP): 96.3%. The calculated HRMS (ESI-) is 778.24108, and the measured value is 778.24012.

[0632] Example 1.75 Preparation of GF-14

[0633] Step 1: Add Tp-10-2 (51.2 mg, 79.3 μmol), DMF (6 mL), N-tert-butoxycarbonyl-alanylalanine (31 mg, 119 μmol), HATU (60 mg, 158.6 μmol), and DIPEA (55 μL, 317.2 μmol) to the reaction flask and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system to obtain GF-14a (68 mg, yield 96.6%).

[0634] Step 2: Add GF-14a (68 mg, 76.6 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 hours. After the reaction is complete, concentrate to obtain GF-14b (to proceed to the next reaction in the theoretical amount).

[0635] Step 3: Add GF-14b (60.3 mg, 76.6 μmol), DMF (5 mL), propargyl-N-hydroxy ester (26 mg, 114.9 μmol), and DIPEA (53 μL, 306.4 μmol) to the reaction flask and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain GF-14c (56.8 mg, yield 82.6%). LC-MS: [M+H] + =898.7.

[0636] Step 4: Add 1.5 mL of H₂O and 1.5 mL of DMSO to the reaction flask and stir. Then add GF-AzO solution (50 mM aqueous solution, 600 μL), CuSO₄ solution (50 mM aqueous solution, 180 μL), TBTA solution (50 mM DMSO solution, 360 μL), GF-14c solution (12.5 mM DMSO solution, 1200 μL), and sodium ascorbate solution (50 mM aqueous solution, 900 μL) in sequence. Finally, add 1.5 mL of DMSO and react overnight at room temperature. After the reaction is complete, the product is further purified by a preparative HPLC system and lyophilized to obtain a white solid GF-14 (18.6 mg, yield 78.2%). Purity (HPLC-RP): 91.3%. 61 H 74 O 26 F4N 16 P2[MH] - The calculated HRMS (ESI-) is 1583.42988, and the measured value is 1583.44763.

[0637] Example 1.76 Preparation of GF-15

[0638] Step 1: Add Tp-19-2 (47.2 mg, 76.2 μmol), DMF (6 mL), N-tert-butoxycarbonyl-alanylalanine (30 mg, 114.3 μmol), and DIPEA (53 μL, 304.8 μmol) to the reaction flask and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system to obtain GF-15a (52 mg, yield 79.1%).

[0639] Step 2: Add GF-15a (52 mg, 60.3 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 hours. After the reaction is complete, concentrate to obtain GF-15b (to proceed to the next reaction according to the theoretical amount).

[0640] Step 3: Add GF-15b (46 mg, 60.3 μmol), DMF (5 mL), propargyl-N-hydroxy ester (20 mg, 90.5 μmol), and DIPEA (42 μL, 241.2 μmol) to the reaction flask and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain GF-15c (45.6 mg, yield 86.7%). LC-MS: [M+H] + =872.8.

[0641] Step 4: Add 1.5 mL of H₂O and 1.5 mL of DMSO to the reaction flask and stir. Then add GF-AzO solution (50 mM aqueous solution, 600 μL), CuSO₄ solution (50 mM aqueous solution, 180 μL), TBTA solution (DMSO solution, 50 mM, 360 μL), GF-15c solution (DMSO solution, 12.5 mM, 1200 μL), and sodium ascorbate solution (50 mM aqueous solution, 900 μL) in sequence. Finally, add 1.5 mL of DMSO and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid of GF-15 (20.6 mg, yield 88.0%). Purity (HPLC-RP): 97.5%. 63 H 81 O 26 FN 16 P2[MH] - The calculated HRMS (ESI-) is 1557.48944, and the measured value is 1557.50095.

[0642] Example 1.77 Preparation of GF-16

[0643] Step 1: Add Tp-11-2 (34.3 mg, 57.8 μmol), DMF (5 mL), N-tert-butoxycarbonyl-alanylalanine (23 mg, 86.7 μmol), HATU (44 mg, 115.6 μmol), and DIPEA (40 μL, 231.2 μmol) to the reaction flask and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system to obtain GF-16a (31 mg, yield 64.3%).

[0644] Step 2: Add GF-16a (31 mg, 37.2 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 hours. After the reaction is complete, concentrate to obtain GF-16b (to proceed to the next reaction in the theoretical amount).

[0645] Step 3: Add GF-16b (27.3 mg, 37.2 μmol), DMF (5 mL), propargyl-N-hydroxy ester (13 mg, 55.8 μmol), and DIPEA (26 μL, 148.8 μmol) to the reaction flask and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain GF-16c (19.1 mg, yield 60.8%). LC-MS: [M+H] + =844.8.

[0646] Step 4: Add 0.5 mL of H₂O and 0.5 mL of DMSO to the reaction flask and stir. Then add GF-AzO solution (50 mM aqueous solution, 200 μL), CuSO₄ solution (50 mM aqueous solution, 60 μL), TBTA solution (50 mM DMSO solution, 120 μL), GF-16c solution (12.5 mM DMSO solution, 400 μL), and sodium ascorbate solution (50 mM aqueous solution, 300 μL) in sequence. Finally, add 1 mL of DMSO and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid GF-16 (5.5 mg, yield 71.4%). Purity (HPLC-RP): 91.0%. 61 H 77 O 26 FN 16 P2[MH] - The calculated HRMS (ESI-) is 1529.45814, and the measured value is 1529.46177.

[0647] Example 1.78 Preparation of GF-17

[0648] Step 1: Add Tp-15-2 (40.3 mg, 66.8 μmol), N-tert-butoxycarbonyl-alanylalanine (21 mg, 80.2 μmol), HATU (51 mg, 133.6 μmol), DMF (5 mL), and DIPEA (47 μL, 267.2 μmol) to the reaction flask and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system to obtain GF-17a (40 mg, yield 70.8%).

[0649] Step 2: Add GF-17a (40 mg, 47.3 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for approximately 3 hours and 50 minutes. After the reaction is complete, concentrate to obtain GF-17b (to proceed to the next reaction in the theoretical amount).

[0650] Step 3: Add GF-17b (35.3 mg, 47.3 μmol), DMF (5 mL), propargyl-N-hydroxy ester (16 mg, 71 μmol), and DIPEA (33 μL, 189.2 μmol) to the reaction flask and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain GF-17c (35 mg, yield 86.4%). LC-MS: [M+H] + =856.8.

[0651] Step 4: Add H2O (0.5 mL) and DMSO (0.5 mL) to the reaction flask and stir. Then add GF-Az0 solution (50 mM aqueous solution, 150 μL), CuSO4 solution (50 mM aqueous solution, 60 μL), TBTA solution (DMSO solution, 50 mM, 120 μL), GF-17c solution (DMSO solution, 12.5 mM, 400 μL), and sodium ascorbate solution (50 mM aqueous solution, 300 μL) in sequence. Finally, add DMSO (0.5 mL) and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid GF-17 (7.1 mg, yield 92.2%). Purity (HPLC-RP): 96.0%. 62 H 77 O 26 FN 16 P2[MH] - The calculated HRMS (ESI-) is 1541.45814, and the measured value is 1541.47953.

[0652] Example 1.79 Preparation of GF-18

[0653] Step 1: Tp-13-2 (24.8 mg, 0.04 mmol), N-tert-butoxycarbonyl-alanylalanine (15.6 mg, 0.06 mmol), HATU (22.8 mg, 0.06 mmol), DMF (10 mL), and DIPEA (28 μL, 0.16 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-18a (24 mg, yield 69.8%).

[0654] Step 2: Add GF-18a (24 mg, 0.028 mmol), DCM (10 mL), and TFA (2 mL) sequentially to the reaction mixture and react at room temperature for 1.5 h. After the reaction is complete, concentrate to obtain GF-18b (to proceed to the next reaction in the theoretical amount).

[0655] Step 3: GF-18b (21.3 mg, 0.028 mmol), propargyl-N-hydroxy ester (12.6 mg, 0.056 mmol), DMF (5 mL), and DIPEA (20 μL, 0.112 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified using a preparative HPLC system to obtain GF-18c (18 mg, two-step yield 73.9%). LC-MS: [M+H] + =870.8.

[0656] Step 4: Add 1.5 mL of H₂O and 1.5 mL of DMSO to the reaction flask and stir. Then add GF-AzO solution (50 mM aqueous solution, 600 μL), CuSO₄ solution (50 mM aqueous solution, 180 μL), TBTA solution (DMSO solution, 50 mM, 360 μL), GF-18c solution (DMSO solution, 12.5 mM, 1200 μL), and sodium ascorbate solution (50 mM aqueous solution, 900 μL) in sequence. Finally, add 1.5 mL of DMSO and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid of GF-18 (15.4 mg, yield 65.8%). Purity (HPLC-RP): 96.0%. 63 H 79 O 26 FN 16 The calculated HRMS (ESI-) of P2[MH]- is 1555.47324, and the measured value is 1555.47355.

[0657] Example 1.80 Preparation of GF-19

[0658] Step 1: Tp-12-2 (585.2 mg, 0.97 mmol), N-tert-butoxycarbonyl-alanylalanine (379.8 mg, 1.46 mmol), HATU (555.2 mg, 1.46 mmol), DMF (30 mL), and DIPEA (676 μL, 3.88 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-19a (727 mg, yield 88.7%).

[0659] Step 2: GF-19a (727 mg, 0.86 mmol), DCM (50 mL), and TFA (10 mL) were added sequentially to the reaction mixture, and the reaction was carried out at room temperature for 2 h. The product was further purified by a preparative HPLC system to obtain GF-19b (520 mg, yield 81.1%).

[0660] Step 3: GF-19b (149 mg, 0.2 mmol), propargyl-N-hydroxy ester (67.5 mg, 0.3 mmol), DMF (10 mL), and DIPEA (105 μL, 0.6 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified using a preparative HPLC system to obtain GF-19c (53 mg, yield 31%). LC-MS: [M+H] + =856.7. 1 H NMR (400MHz, DMSO-d6)8.10-8.08(m,2H),7.93(d,J=4.0Hz,1H),7.72(d,J=8.0Hz,1H),7.65-7.62(m,2H),7.29(s,1H),6.52(m,1H), 5.53-5.49(m,1H),5.41(t,J=16.0Hz,2H),5.10(q,J=16.0Hz,2H),4.02(t,J=4.0Hz,1H),3.98-3.93(m,1H),3.90-3.78(m,2H),3.53- 3.45(m,2H),3.40(t,J=4.0Hz,2H),3.20-3.14(m,1H),3.10-3.04(m,1H),2.76-2.71(m,1H),2.68-2.63(m,1H),2.37(s,3H),2.28-2 .21(m,2H),2.14-1.99(m,4H),1.97-1.90(m,2H),1.88-1.81(m,2H),1.11(d,J=8.0Hz,3H),0.99(d,J=4.0Hz,3H),0.91-0.85(m,6H).

[0661] Step 4: Add H2O (350 μL) and DMSO (3150 μL) to the reaction flask and stir. Then add GF-Az0 solution (50 mM aqueous solution, 400 μL), CuSO4 solution (50 mM aqueous solution, 100 μL), TBTA solution (DMSO solution, 50 mM, 200 μL), GF-19c solution (DMSO solution, 25 mM, 400 μL), and sodium ascorbate solution (50 mM aqueous solution, 400 μL) in sequence and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid of GF-19 (9.3 mg, yield 60.3%). Purity (HPLC-RP): 95.6%. 62 H 77 FN 16 O 26 P2[MH] -The calculated HRMS (ESI-) is 1541.45814, and the measured value is 1541.46345. 1 H-NMR (400MHz, D2O) δ7.79(s,1H),7.68(d,J=8.0Hz,1H),7.27(s,1H),7.08(d,J=8.0Hz,1H),5.76(s,1H),5.50(s,1H),5.31(q,J=12.0Hz,2H ),5.09(s,2H),4.86(s,1H),4.59(s,1H),4.41(s,1H),4.33-4.30(m,3 H),4.22(s,1H),4.13(s,1H),4.07-4.01(m,1H),3.79(m,1H),3.71(q, J=4.0Hz,1H),3.64-3.58(m,2H),3.55-3.48(m,3H),3.43-3.35(m,2H) ,3.26-3.20(m,1H),3.04-3.01(m,1H),2.91-2.81(m,2H),2.64(s,2H) ,2.40-2.32(m,1H),2.20-2.05(m,8H),1.98-1.89(m,2H),1.86-1.80( m,2H),1.19(d,J=8.0Hz,3H),0.97(d,J=4.0Hz,3H),0.86-0.82(m,6H).

[0662] Example 1.81 Preparation of GF-20

[0663] Step 1: GF-19b (22.4 mg, 0.03 mmol), succinic anhydride (9 mg, 0.09 mmol), and DMF (5 mL) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by a preparative HPLC system to obtain GF-20a (18.7 mg, yield 73.7%).

[0664] Step 2: GF-20a (18.7 mg, 0.022 mmol), N-hydroxysuccinimide (7.6 mg, 0.066 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (25.3 mg, 0.132 mmol), DCM (2 mL), and DMF (5 mL) were added sequentially to the reaction mixture, and the mixture was reacted at room temperature for 2 hours. After the reaction was completed, the mixture was concentrated to obtain GF-20b (which was then used for the next reaction according to the theoretical amount).

[0665] Step 3: Add H2O (1540 μL) and DMF (1760 μL) to the reaction flask and stir. Then add GDP-FAm solution (50 mM aqueous solution, 440 μL), NaHCO3 solution (200 mM aqueous solution, 220 μL), and GF-20b (DMF solution / 50 mM, 440 μL) sequentially and react overnight at room temperature. Adjust the pH to 4.13 with 0.2 M HCl and stir at room temperature for 6 h. After the reaction, further purify the product using a preparative HPLC system, and lyophilize to obtain a white solid of GF-20 (1.1 mg, two-step yield 3.5%). Purity (HPLC-RP): 97.8%. 58 H 72 FN 13 O 25 P2[MH] - The calculated HRMS (ESI-) is 1430.41488, and the measured value is 1430.44836.

[0666] Example 1.82 Preparation of GF-21

[0667] Step 1: GF-19b (22.4 mg, 0.03 mmol), polyethylene glycol propionate-succinimide ester (23.5 mg, 0.06 mmol), DMF (5 mL), and DIPEA (16 μL, 0.092 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-21a (15.5 mg, yield 50.6%).

[0668] Step 2: GF-21a (15.5 mg, 0.015 mmol), N-hydroxysuccinimide (5.2 mg, 0.045 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (17.3 mg, 0.09 mmol), DCM (2 mL), and DMF (5 mL) were added sequentially to the reaction mixture, and the mixture was reacted at room temperature for 2 hours. After the reaction was completed, the mixture was concentrated to obtain GF-21b (which was used for the next reaction according to the theoretical amount).

[0669] Step 3: Add H2O (1050 μL) and DMF (1200 μL) to the reaction flask and stir. Then add GDP-FAm solution (50 mM aqueous solution, 300 μL), NaHCO3 solution (200 mM aqueous solution, 150 μL), and GF-21b (DMF solution / 50 mM, 300 μL) sequentially and react overnight at room temperature. Adjust the pH to 4.1 with 0.2 M HCl and stir at room temperature for 7 h. After the reaction, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid of GF-21 (16.9 mg, two-step yield 70.1%). Purity (HPLC-RP): 98.0%. 66 H 88 FN 13 O 29 P2[MH] - The calculated HRMS (ESI-) is 1606.51974, and the measured value is 1606.55451.

[0670] Example 1.83 Preparation of GF-22

[0671] Step 1: GF-19b (18.6 mg, 0.025 mmol), propynyl-polyethylene glycol-succinimide acrylate (13.4 mg, 0.0375 mmol), DMF (5 mL), and DIPEA (13 μL, 0.075 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-22a (17.4 mg, yield 70.5%). LC-MS: [M+H] + =988.9.

[0672] Step 2: Add H2O (350 μL) and DMSO (2750 μL) to the reaction flask and stir. Then add GF-Az0 solution (50 mM aqueous solution, 400 μL), CuSO4 solution (50 mM aqueous solution, 100 μL), TBTA solution (DMSO solution, 50 mM, 200 μL), GF-22a solution (DMSO solution, 12.5 mM, 800 μL), and sodium ascorbate solution (50 mM aqueous solution, 400 μL) sequentially. React overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system, and lyophilize to obtain a white solid of GF-22 (10.9 mg, yield 65.1%). Purity (HPLC-RP): 99.8%. 68 H 89 FN 16 O 29 P2[MH] -The calculated HRMS (ESI-) is 1673.53679, and the measured value is 1673.54433. 1 H-NMR(400MHz,D2O)δ7.94(s,1H),7.70(d,J=8.0Hz,1H),7.27(s,1H),7.13(d, J=8.0Hz,1H),5.80(s,1H),5.50(s,1H),5.32(q,J=12.0Hz,2H),5.16(s,2H),4 .88(s,1H),4.82(d,J=16.0Hz,1H),4.60(s,1H),4.54(s,1H),4.42(s,1H),4.3 4(d,J=12.0Hz,1H),4.22(s,1H),4.15(s,2H),4.02(q,J=4.0Hz,1H),3.81-3.80 (m,1H),3.73-3.65(m,2H),3.61-3.47(m,11H),3.45-3.38(m,3H),3.38-3.30( m,3H),3.29-3.21(m,1H),3.07-3.04(m,1H),2.91-2.80(m,2H),2.75-2.67(m, 1H),2.65(s,2H),2.41-2.33(m,1H),2.29-2.03(m,8H),2.02-1.92(m,2H),1.8 9-1.76(m,2H),1.20(d,J=8.0Hz,3H),1.01(d,J=4.0Hz,3H),0.86-0.76(m,6H).

[0673] Example 1.84 Preparation of GF-23

[0674] Step 1: GF-19b (18.6 mg, 0.025 mmol), alkynyl-succinimide ester (7.8 mg, 0.0375 mmol), DMF (5 mL), and DIPEA (13 μL, 0.075 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-23a (17.3 mg, yield 82.4%). LC-MS: [M+H] + =840.7.

[0675] Step 2: Add H2O (350 μL) and DMSO (2750 μL) to the reaction flask and stir. Then add GF-AzO solution (50 mM aqueous solution, 400 μL), CuSO4 solution (50 mM aqueous solution, 100 μL), TBTA solution (DMSO solution, 50 mM, 200 μL), GF-23a solution (DMSO solution, 12.5 mM, 800 μL), and sodium ascorbate solution (50 mM aqueous solution, 400 μL) in sequence and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid of GF-23 (8.1 mg, yield 53.1%). Purity (HPLC-RP): 98.9%. 62 H 77 FN 16 O 25 P2[MH] - The calculated HRMS (ESI-) is 1525.46323, and the measured value is 1525.46897.

[0676] Example 1.85 Preparation of GF-24

[0677] Step 1: GF-19b (25 mg, 0.034 mmol), succinimide azidoacetate (10 mg, 0.05 mmol), DMF (3 mL), and DIPEA (17 μL, 0.1 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified using a preparative HPLC system to obtain GF-24a (14.6 mg, yield 52.5%). LC-MS: [M+H] + =829.8.

[0678] Step 2: Add H2O (177 μL) and DMSO (1765 μL) to the reaction flask and stir. Then add GF-Ayl1 solution (50 mM aqueous solution, 235 μL), CuSO4 solution (50 mM aqueous solution, 118 μL), TBTA solution (DMSO solution, 50 mM, 235 μL), GF-24a solution (DMSO solution, 5.88 mM, 1 mL), and sodium ascorbate solution (50 mM aqueous solution, 470 μL) in sequence and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid of GF-24 (7.4 mg, yield 81.6%). Purity (HPLC-RP): 93.8%. The calculated HRMS (ESI-) is 770.22543, and the measured value is 770.22877.

[0679] Example 1.86 Preparation of GF-25

[0680] Step 1: GF-19b (25 mg, 0.034 mmol), azide-polyethylene glycol-succinimide ester (13 mg, 0.05 mmol), DMF (3 mL), and DIPEA (17 μL, 0.1 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified using a preparative HPLC system to obtain GF-25a (12.3 mg, yield 41.4%). LC-MS: [M+H] + =887.8.

[0681] Step 2: Add H2O (352 μL) and DMSO (1815 μL) to the reaction flask and stir. Then add GF-Ayl1 solution (50 mM aqueous solution, 185 μL), CuSO4 solution (50 mM aqueous solution, 93 μL), TBTA solution (DMSO solution, 50 mM, 185 μL), GF-25a solution (DMSO solution, 4.63 mM, 1 mL), and sodium ascorbate solution (50 mM aqueous solution, 370 μL) in sequence and react overnight at room temperature. After the reaction is complete, the product is further purified by preparative HPLC system and lyophilized to obtain a white solid of GF-25 (2.8 mg, yield 37.8%). Purity (HPLC-RP): 92.5%. The calculated HRMS (ESI-) is 799.24636, and the measured value is 799.24916.

[0682] Example 1.87 Preparation of GF-26

[0683] Step 1: GF-19b (25 mg, 0.034 mmol), azido-tetraethylene glycol-succinimide ester (19 mg, 0.05 mmol), DMF (3 mL), and DIPEA (17 μL, 0.1 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified using a preparative HPLC system to obtain GF-26a (15.4 mg, yield 45.1%). LC-MS: [M+H] + =1019.9.

[0684] Step 2: Add H2O (294 μL) and DMSO (1798 μL) to the reaction flask and stir. Then add GF-Ayl1 solution (50 mM aqueous solution, 202 μL), CuSO4 solution (50 mM aqueous solution, 101 μL), TBTA solution (DMSO solution, 50 mM, 202 μL), GF-26a solution (DMSO solution, 5.04 mM, 1 mL), and sodium ascorbate solution (50 mM aqueous solution, 403 μL) in sequence and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid of GF-26 (2.7 mg, yield 30.9%). Purity (HPLC-RP): 97.2%. The calculated HRMS (ESI-) is 865.28569, and the measured value is 865.29016.

[0685] Example 1.88 Preparation of GF-27

[0686] Step 1: Add Tp-12-1 (131 mg, 0.222 mmol), N-tert-butoxycarbonyl-alanylalanine (87 mg, 0.333 mmol), HATU (169 mg, 0.444 mmol), DMF (15 mL), and DIPEA (155 μL, 0.888 mmol) to the reaction flask and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system to obtain GF-27a (170 mg, yield 91.9%).

[0687] Step 2: Add GF-27a (170 mg, 0.204 mmol), DCM (12 mL), and TFA (3 mL) to the reaction flask and react at room temperature for 3 hours. After the reaction is complete, concentrate to obtain GF-27b (to proceed to the next reaction in the theoretical amount).

[0688] Step 3: Add GF-27b (74.7 mg, 0.102 mmol), propargyl-N-hydroxy ester (34 mg, 0.153 mmol), DMF (10 mL), and DIPEA (71 μL, 0.408 mmol) to the reaction flask and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain GF-27c (76 mg, yield 88.5%). LC-MS: [M+H] + =842.8. 1H-NMR (400MHz, DMSO-d6) δ8.20(s,1H),8.05(d,J=5.1Hz,1H),8.01(d,J=5.4Hz,1H),7.99(t,J=4.4Hz,1H),7.77(d,J=7.1Hz,1H),7.74(d,J=8.8 Hz,1H),7.30(s,1H),6.53(s,1H),5.53(q,J=5.0Hz,1H),5.42(dd,J=17.1,13.0Hz,2H),5.14(dd,J=57.7,15.2Hz,2H),4.06(d,J=1.9Hz,2H),4.0 3(t,J=5.6Hz,1H),3.97-3.90(m,1H),3.59-3.54(m,3H),3.41(t,J=1.9H z,2H),3.21-3.17(m,1H),3.11-3.04(m,1H),2.78-2.70(m,1H),2.69-2. 61(m,1H),2.38(s,3H),2.34-2.20(m,3H),2.13-2.05(m,3H),1.98-1.90 (m,2H),1.90-1.79(m,2H),0.93(t,J=5.3Hz,6H),0.86(t,J=5.8Hz,3H).

[0689] Step 4: Add 2 mL of H₂O and 2 mL of DMSO to the reaction flask and stir. Then add GF-AzO solution (50 mM aqueous solution, 1000 μL), CuSO₄ solution (50 mM aqueous solution, 240 μL), TBTA solution (DMSO solution, 50 mM, 480 μL), GF-27c solution (DMSO solution, 12.5 mM, 1600 μL), and sodium ascorbate solution (50 mM aqueous solution, 1200 μL) in sequence. Finally, add 2 mL of DMSO and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid of GF-27 (25.5 mg, yield 83.3%). Purity (HPLC-RP): 99.8%. 61 H 75 O 26 FN 16 P2[MH] - The calculated HRMS (ESI-) is 1527.44249, and the measured value is 1527.45272. 1H-NMR (400MHz, D2O) δ7.67 (s, 1H), 7.20 (s, 1H), 6.93 (d, J = 8.3Hz, 1H), 5.56 (s, 1H),5.43-5.34(m,1H),5.23(dd,J=55.2,13.0Hz,2H),4.93(t,J=16.1Hz,2H),4 .76(s,1H),4.48(t,J=3.6Hz,1H),4.30(t,J=2.8Hz,1H),4.24(dd,J=15.8,10. 2Hz,2H),4.17-4.07(m,2H),4.02(s,2H),3.76-3.65(m,3H),3.63-3.54(m,2H), 3.54-3.47(m,2H),3.42(t,J=4.5Hz,1H),3.37(t,J=4.5Hz,2H),3.12(dd,J=11 .0,7.8Hz,1H),2.97-2.87(m,1H),2.83-2.71(m,2H),2.57(s,5H),2.34-2.25(m ,1H),2.22-2.06(m,3H),1.97(s,4H),1.91-1.84(m,1H),1.80-1.68(m,2H),1.4 9-1.34(m,1H),1.20-1.11(m,1H),0.89(d,J=5.8Hz,5H),0.78(t,J=5.8Hz,3H).

[0690] Example 1.89 Preparation of GF-28

[0691] Step 1: Tp-4-1 (41.4 mg, 0.07 mmol), N-tert-butoxycarbonyl-alanylalanine (27.3 mg, 0.105 mmol), HATU (39.9 mg, 0.105 mmol), DMF (5 mL), and DIPEA (49 μL, 0.281 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-28a (38 mg, yield 65.1%).

[0692] Step 2: Add GF-28a (38 mg, 0.046 mmol), DCM (15 mL), and TFA (2 mL) sequentially to the reaction mixture and react at room temperature for 1 hour. After the reaction is complete, concentrate to obtain GF-28b (to proceed to the next reaction in the theoretical amount).

[0693] Step 3: GF-28b (33.7 mg, 0.046 mmol), propargyl-N-hydroxy ester (31 mg, 0.138 mmol), DMF (5 mL), and DIPEA (40 μL, 0.23 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified using a preparative HPLC system to obtain GF-28c (32 mg, two-step yield 82.5%). LC-MS: [M+H] + =834.7.

[0694] Step 4: Add H2O (175 μL) and DMSO (1375 μL) to the reaction flask and stir. Then add GF-Az0 solution (50 mM aqueous solution, 200 μL), CuSO4 solution (50 mM aqueous solution, 50 μL), TBTA solution (DMSO solution, 50 mM, 100 μL), GF-28c solution (DMSO solution, 12.5 mM, 400 μL), and sodium ascorbate solution (50 mM aqueous solution, 200 μL) in sequence and react overnight at room temperature. After the reaction is complete, the product is further purified by preparative HPLC system and lyophilized to obtain a white solid of GF-28 (3.4 mg, yield 44.4%). Purity (HPLC-RP): 96.6%. 61 H 77 FN 16 O 26 P2[MH] - The calculated HRMS (ESI-) is 1529.45814, and the measured value is 1529.45377.

[0695] Example 1.90 Preparation of GF-29

[0696] Step 1: Tp-16-2 (16.2 mg, 0.026 mmol), N-tert-butoxycarbonyl-alanylalanine (10.1 mg, 0.039 mmol), HATU (9.9 mg, 0.026 mmol), DMF (5 mL), and DIPEA (18 μL, 0.103 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-29a (11 mg, yield 49.1%).

[0697] Step 2: Add GF-29a (11 mg, 0.0128 mmol), DCM (10 mL), and TFA (1 mL) sequentially to the reaction mixture and react at room temperature for 2 hours. After the reaction is complete, concentrate to obtain GF-29b (to proceed to the next reaction in the theoretical amount).

[0698] Step 3: GF-29b (9.7 mg, 0.0128 mmol), propargyl-N-hydroxy ester (8.6 mg, 0.0384 mmol), DMF (5 mL), and DIPEA (11 μL, 0.063 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified using a preparative HPLC system to obtain GF-29c (10.1 mg, two-step yield 90.6%). LC-MS: [M+H] + =872.8.

[0699] Step 4: Add H2O (175 μL) and DMSO (1375 μL) to the reaction flask and stir. Then add GF-Az0 solution (50 mM aqueous solution, 200 μL), CuSO4 solution (50 mM aqueous solution, 50 μL), TBTA solution (DMSO solution, 50 mM, 100 μL), GF-29c solution (DMSO solution, 12.5 mM, 400 μL), and sodium ascorbate solution (50 mM aqueous solution, 200 μL) sequentially. React overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system, and lyophilize to obtain a white solid of GF-29 (3.8 mg, yield 48.8%). Purity (HPLC-RP): 97.5%. 63 H 81 FN 16 O 26 P2[MH] - The calculated HRMS (ESI-) is 1557.48944, and the measured value is 1557.49287.

[0700] Example 1.91 Preparation of GF-30

[0701] Step 1: Tp-1-4 (39.6 mg, 0.067 mmol), N-tert-butoxycarbonyl-alanylalanine (26 mg, 0.1 mmol), HATU (38 mg, 0.1 mmol), DMF (5 mL), and DIPEA (47 μL, 0.27 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-30a (46 mg, yield 82.4%).

[0702] Step 2: GF-30a (46 mg, 0.055 mmol), DCM (15 mL), and TFA (2 mL) were added sequentially to the reaction mixture, and the reaction was carried out at room temperature for 1.5 h. After the reaction was completed, the mixture was concentrated to obtain GF-30b (which was used for the next reaction according to the theoretical amount). LC-MS: [M+H] + =844.6.

[0703] Step 3: GF-30b (40.3 mg, 0.055 mmol), propargyl-N-hydroxy ester (37.1 mg, 0.165 mmol), DMF (5 mL), and DIPEA (48 μL, 0.276 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified using a preparative HPLC system to obtain GF-30c (38 mg, two-step yield 81.9%).

[0704] Step 4: Add H2O (350 μL) and DMSO (2750 μL) to the reaction flask and stir. Then add GF-Az0 solution (50 mM aqueous solution, 400 μL), CuSO4 solution (50 mM aqueous solution, 100 μL), TBTA solution (DMSO solution, 50 mM, 200 μL), GF-30c solution (DMSO solution, 12.5 mM, 800 μL), and sodium ascorbate solution (50 mM aqueous solution, 400 μL) sequentially. React overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid GF-30 (9.1 mg, yield 59.5%). Purity (HPLC-RP): 93.2%. 61 H 77 FN 16 O 26 P2[MH] - The calculated HRMS (ESI-) is 1529.45814, and the measured value is 1529.35769.

[0705] Example 1.92 Preparation of GF-31

[0706] Step 1: N-tert-butoxycarbonyl-alanylalanine (20 mg, 0.078 mmol), Tp-4-4 (0.052 mmol), HATU (40 mg, 0.104 mmol), DMF (3 mL), and DIPEA (26 μL, 0.156 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain GF-31a (40 mg, yield 87.9%).

[0707] Step 2: GF-31a (40 mg, 0.046 mmol), TFA (2 mL), and DCM (4 mL) were added sequentially to the reaction flask, and the reaction was carried out at room temperature for 1 h. After the reaction was completed, the product was concentrated and further purified by a preparative HPLC system to obtain GF-31b (24.1 mg, yield 68.0%).

[0708] Step 3: GF-31b (24.1 mg, 0.031 mmol), propargyl-N-hydroxy ester (10.6 mg, 0.047 mmol), DMF (3 mL), and DIPEA (15 μL, 0.093 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by preparative HPLC to obtain GF-31c (14 mg, yield 50.9%). LC-MS: [M+H] + =886.8.

[0709] Step 4: Add DMSO (2819 μL) and pure water (367 μL) to the reaction flask and stir. Then add GF-31c (8 mg, 0.009 mmol), GF-Az0 solution (aqueous solution / 50 mM, 181 μL), copper sulfate solution (aqueous solution / 50 mM, 90 μL), TBTA solution (DMSO solution / 50 mM, 181 μL), and sodium ascorbate solution (aqueous solution / 50 mM, 362 μL) in sequence, and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system, and lyophilize to obtain a white solid of GF-31 (8.2 mg, yield 57.7%). Purity (HPLC-RP): 96.0%. The calculated HRMS (ESI-) is 785.24891, and the measured value is 785.25240.

[0710] Example 1.93 Preparation of GF-32

[0711] Step 1: Tp-16-4 (13.8 mg, 0.0213 mmol), N-tert-butoxycarbonyl-alanylalanine (8.3 mg, 0.032 mmol), HATU (8.1 mg, 0.0213 mmol), DMF (5 mL), and DIPEA (15 μL, 0.086 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-32a (10 mg, yield 52.8%).

[0712] Step 2: Add GF-32a (10 mg, 0.0112 mmol), DCM (10 mL), and TFA (1 mL) sequentially to the reaction mixture and react at room temperature for 2 hours. After the reaction is complete, concentrate to obtain GF-32b (to proceed to the next reaction in the theoretical amount).

[0713] Step 3: GF-32b (8.8 mg, 0.0112 mmol), propargyl-N-hydroxy ester (7.6 mg, 0.0336 mmol), DMF (5 mL), and DIPEA (10 μL, 0.057 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified using a preparative HPLC system to obtain GF-32c (5.4 mg, two-step yield 53.6%). LC-MS: [M+H] + =900.7.

[0714] Step 4: Add H2O (175 μL) and DMSO (1375 μL) to the reaction flask and stir. Then add GF-Az0 solution (50 mM aqueous solution, 200 μL), CuSO4 solution (50 mM aqueous solution, 50 μL), TBTA solution (DMSO solution, 50 mM, 100 μL), GF-32c solution (DMSO solution, 12.5 mM, 400 μL), and sodium ascorbate solution (50 mM aqueous solution, 200 μL) in sequence and react overnight at room temperature. After the reaction is complete, the product is further purified by preparative HPLC system and lyophilized to obtain a white solid GF-32 (6.1 mg, yield 76.9%). Purity (HPLC-RP): 98.1%. 65 H 85 FN 16 O 26 P2[MH] - The calculated HRMS (ESI-) is 1585.52074, and the measured value is 1585.53392.

[0715] Example 1.94 Preparation of GF-33

[0716] Step 1: Tp-16-1 (40.6 mg, 0.067 mmol), N-tert-butoxycarbonyl-alanylalanine (26 mg, 0.1 mmol), HATU (25.5 mg, 0.067 mmol), DMF (5 mL), and DIPEA (47 μL, 0.27 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-33a (31 mg, yield 54.6%).

[0717] Step 2: Add GF-33a (31 mg, 0.037 mmol), DCM (15 mL), and TFA (2 mL) sequentially to the reaction mixture and react at room temperature for 2 hours. After the reaction is complete, concentrate to obtain GF-33b (to proceed to the next reaction in the theoretical amount).

[0718] Step 3: GF-33b (27.7 mg, 0.037 mmol), propargyl-N-hydroxy ester (25 mg, 0.111 mmol), DMF (5 mL), and DIPEA (33 μL, 0.189 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified using a preparative HPLC system to obtain GF-33c (29 mg, two-step yield 91.4%). LC-MS: [M+H] + =858.7.

[0719] Step 4: Add H2O (175 μL) and DMSO (1375 μL) to the reaction flask and stir. Then add GF-Az0 solution (50 mM aqueous solution, 200 μL), CuSO4 solution (50 mM aqueous solution, 50 μL), TBTA solution (DMSO solution, 50 mM, 100 μL), GF-33c solution (DMSO solution, 12.5 mM, 400 μL), and sodium ascorbate solution (50 mM aqueous solution, 200 μL) in sequence and react overnight at room temperature. After the reaction is complete, the product is further purified by preparative HPLC system and lyophilized to obtain a white solid GF-33 (5.3 mg, yield 68.6%). Purity (HPLC-RP): 95.7%. 62 H 79 FN 16 O 26 P2[MH] - The calculated HRMS (ESI-) is 1543.47379, and the measured value is 1543.48974.

[0720] Example 1.95 Preparation of GF-34

[0721] Step 1: N-tert-butoxycarbonyl-alanylalanine (28 mg, 0.107 mmol), Tp-17-4 (0.071 mmol), HATU (54 mg, 0.142 mmol), DMF (3 mL), and DIPEA (35 μL, 0.213 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain GF-34a (26 mg, yield 42.5%).

[0722] Step 2: Add GF-34a (26 mg, 0.03 mmol), TFA (2 mL), and DCM (4 mL) sequentially to the reaction flask, and react at room temperature for 1 hour. After the reaction is complete, concentrate to obtain GF-34b (to proceed to the next reaction according to the theoretical amount).

[0723] Step 3: GF-34b (0.03 mmol), propargyl-N-hydroxy ester (10 mg, 0.045 mmol), DMF (3 mL), and DIPEA (15 μL, 0.09 mmol) were added sequentially to the reaction flask, and the reaction was carried out at room temperature for 4.5 h. After the reaction was completed, the product was further purified by preparative HPLC to obtain GF-34c (23.3 mg, yield 89.2%). LC-MS: [M+H] + =872.8.

[0724] Step 4: Add DMSO (1734 μL) and pure water (68 μL) to the reaction flask and stir. Then add GF-34c (DMSO solution / 6.66 mM, 1 mL), GF-Az0 solution (aqueous solution / 50 mM, 266 μL), copper sulfate solution (aqueous solution / 50 mM, 133 μL), TBTA solution (DMSO solution / 50 mM, 266 μL), and sodium ascorbate solution (aqueous solution / 50 mM, 533 μL) in sequence, and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system, and lyophilize to obtain a white solid of GF-34 (5.3 mg, yield 51.1%). Purity (HPLC-RP): 96.6%. 63 H 81 O 26 FN 16 P2[MH] - The calculated HRMS (ESI-) is 1557.48944, and the measured value is 1557.50095.

[0725] Example 1.96 Preparation of GF-35

[0726] Step 1: N-tert-butoxycarbonyl-alanylalanine (31 mg, 0.12 mmol), Tp-18-4 (0.08 mmol), HATU (61 mg, 0.16 mmol), DMF (3 mL), and DIPEA (40 μL, 0.24 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain GF-35a (60 mg, yield 88.5%).

[0727] Step 2: Add GF-35a (60 mg, 0.07 mmol), TFA (3 mL), and DCM (6 mL) sequentially to the reaction flask, and react at room temperature for 1 hour. After the reaction is complete, concentrate to obtain GF-35b (to proceed to the next reaction in the theoretical amount).

[0728] Step 3: GF-35b (0.07 mmol), propargyl-N-hydroxy ester (23 mg, 0.105 mmol), DMF (3 mL), and DIPEA (35 μL, 0.21 mmol) were added sequentially to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by preparative HPLC to obtain GF-35c (47 mg, yield 78.3%). LC-MS: [M+H] + =858.8.

[0729] Step 4: Add DMSO (1563 μL) to the reaction flask and stir. Then add GF-35c (DMSO solution / 10.84 mM, 1 mL), GF-Az0 solution (aqueous solution / 50 mM, 437 μL), copper sulfate solution (aqueous solution / 50 mM, 217 μL), TBTA solution (DMSO solution / 50 mM, 437 μL), and sodium ascorbate solution (aqueous solution / 50 mM, 867 μL) sequentially. React overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system, and lyophilize to obtain a white solid of GF-35 (10.7 mg, yield 63.9%). Purity (HPLC-RP): 91.9%. 62 H 79 O 26 FN 16 P2[MH] - The calculated HRMS (ESI-) is 1543.47379, and the measured value is 1543.49778.

[0730] Example 1.97 Preparation of GF-36

[0731] Step 1: Add Tp-2-4 (36 mg, 59.5 μmol), N-tert-butoxycarbonyl-alanylalanine (16 mg, 59.5 μmol), HATU (45 mg, 119 μmol), DMF (5 mL), and DIPEA (41 μL, 238 μmol) to the reaction flask and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system to obtain GF-36a (45 mg, yield 89.3%).

[0732] Step 2: Add GF-36a (45 mg, 53.1 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 2 h 20 min. After the reaction is complete, concentrate to obtain GF-36b (to proceed to the next reaction according to the theoretical amount).

[0733] Step 3: Add GF-36b (39.7 mg, 53.1 μmol), DMF (5 mL), propargyl-N-hydroxy ester (18 mg, 79.7 μmol), and DIPEA (37 μL, 212.4 μmol) to the reaction flask and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain GF-36c (24 mg, yield 52.7%). LC-MS: [M+H] + =858.7.

[0734] Step 4: Add 1 mL of H₂O and 1 mL of DMSO to the reaction flask and stir. Then add GF-AzO solution (50 mM aqueous solution, 225 μL), CuSO₄ solution (50 mM aqueous solution, 90 μL), TBTA solution (DMSO solution, 50 mM, 180 μL), GF-36c solution (DMSO solution, 12.5 mM, 600 μL), and sodium ascorbate solution (50 mM aqueous solution, 450 μL) in sequence. React overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid of GF-36 (8.7 mg, yield 75.0%). Purity (HPLC-RP): 94.0%. 62 H 79 O 26 FN 16 P2[MH] - The calculated HRMS (ESI-) is 1543.47379, and the measured value is 1543.47365.

[0735] Example 1.98 Preparation of GF-37

[0736] Step 1: Add Tp-15-4 (41.1 mg, 65.1 μmol), N-tert-butoxycarbonyl-alanylalanine (20 mg, 78.1 μmol), HATU (50 mg, 130.2 μmol), DMF (5 mL), and DIPEA (45 μL, 260.4 μmol) to the reaction flask and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system to obtain GF-37a (40 mg, yield 70.3%).

[0737] Step 2: Add GF-37a (40 mg, 45.8 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for approximately 3.5 h. After the reaction is complete, concentrate to obtain GF-37b (to proceed to the next reaction in the theoretical amount).

[0738] Step 3: Add GF-37b (35.4 mg, 45.8 μmol), DMF (5 mL), propargyl-N-hydroxy ester (15 mg, 68.7 μmol), and DIPEA (32 μL, 183.2 μmol) to the reaction flask and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain GF-37c (37 mg, yield 91.4%). LC-MS: [M+H] + =884.7.

[0739] Step 4: Add 0.5 mL of H₂O and 0.5 mL of DMSO to the reaction flask and stir. Then add GF-AzO solution (50 mM aqueous solution, 150 μL), CuSO₄ solution (50 mM aqueous solution, 60 μL), TBTA solution (50 mM DMSO solution, 120 μL), GF-37c solution (12.5 mM DMSO solution, 400 μL), and sodium ascorbate solution (50 mM aqueous solution, 300 μL) in sequence. Finally, add 0.5 mL of DMSO and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid of GF-37 (5.0 mg, yield 63.3%). Purity (HPLC-RP): 97.4%. 64 H 81 O 26 FN 16 P2[MH] - The calculated HRMS (ESI-) is 1569.48944, and the measured value is 1569.51459.

[0740] Example 1.99 Preparation of GF-38

[0741] Step 1: Add Tp-4-2 (34.3 mg, 56.7 μmol), (tert-butoxycarbonyl)-L-valine-L-alanine (25 mg, 85.1 μmol), HATU (43 mg, 113.4 μmol), DMF (5 mL), and DIPEA (40 μL, 226.8 μmol) to the reaction flask and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system to obtain GF-38a (46 mg, yield 92.6%).

[0742] Step 2: Add GF-38a (46 mg, 52.5 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 6 h 20 min. After the reaction is complete, concentrate to obtain GF-38b (to proceed to the next reaction according to the theoretical amount).

[0743] Step 3: Add GF-38b (40.7 mg, 52.5 μmol), propargyl-N-hydroxy ester (18 mg, 78.8 μmol), DMF (5 mL), and DIPEA (37 μL, 210 μmol) to the reaction flask and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain GF-38c (39.4 mg, yield 84.7%). LC-MS: [M+H] + =886.6.

[0744] Step 4: Add H2O (0.5 mL) and DMSO (0.5 mL) to the reaction flask and stir. Then add GF-Az0 solution (50 mM aqueous solution, 150 μL), CuSO4 solution (50 mM aqueous solution, 60 μL), TBTA solution (DMSO solution, 50 mM, 120 μL), GF-38c solution (DMSO solution, 12.5 mM, 400 μL), and sodium ascorbate solution (50 mM aqueous solution, 300 μL) in sequence. Finally, add DMSO (0.5 mL) and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid of GF-38 (4.1 mg, yield 51.9%). Purity (HPLC-RP): 97.1%. 64 H 83 O 26 FN 16 P2[MH] - The calculated HRMS (ESI-) is 1571.50509, and the measured value is 1571.51033.

[0745] Example 1.100 Preparation of GF-39

[0746] Step 1: Tp-12-2 (30.2 mg, 0.05 mmol), (tert-butoxycarbonyl)-L-valine-L-alanine (21.6 mg, 0.075 mmol), HATU (28.6 mg, 0.075 mmol), DMF (5 mL), and DIPEA (36 μL, 0.210 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-39a (22 mg, yield 50.4%).

[0747] Step 2: Add GF-39a (22 mg, 0.025 mmol), DCM (10 mL), and TFA (2 mL) sequentially to the reaction mixture and react at room temperature for 2 hours. After the reaction is complete, concentrate to obtain GF-39b (to proceed to the next reaction in the theoretical amount).

[0748] Step 3: GF-39b (19.3 mg, 0.025 mmol), propargyl-N-hydroxy ester (8.4 mg, 0.0375 mmol), DMF (5 mL), and DIPEA (13 μL, 0.075 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified using a preparative HPLC system to obtain GF-39c (17.6 mg, yield 79.7%). LC-MS: [M+H] + =884.8.

[0749] Step 4: Add H2O (350 μL) and DMSO (2750 μL) to the reaction flask and stir. Then add GF-Az0 solution (50 mM aqueous solution, 400 μL), CuSO4 solution (50 mM aqueous solution, 100 μL), TBTA solution (DMSO solution, 50 mM, 200 μL), GF-39c solution (DMSO solution, 12.5 mM, 800 μL), and sodium ascorbate solution (50 mM aqueous solution, 400 μL) in sequence and react overnight at room temperature. After the reaction is complete, the product is further purified by a preparative HPLC system and lyophilized to obtain a white solid of GF-39 (10.5 mg, yield 66.9%). Purity (HPLC-RP): 99.1%. 64 H 81 FN 16 O 26 P2[MH] - The calculated HRMS (ESI-) is 1569.48944, and the measured value is 1569.49026.

[0750] Example 1.101 Preparation of GF-40

[0751] Step 1: Tp-12-1 (126.5 mg, 0.215 mmol), (tert-butoxycarbonyl)-L-valine-L-alanine (93 mg, 0.323 mmol), HATU (163 mg, 0.43 mmol), DMF (15 mL), and DIPEA (150 μL, 0.86 mmol) were added to the reaction flask, and the reaction was carried out overnight at room temperature. After the reaction was completed, the product was further purified by a preparative HPLC system to obtain GF-40a (97 mg, yield 52.4%).

[0752] Step 2: Add GF-40a (97 mg, 0.113 mmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 hours. After the reaction is complete, concentrate to obtain GF-40b (to proceed to the next reaction in the theoretical amount).

[0753] Step 3: Add GF-40b (42.9 mg, 56.5 μmol), propargyl-N-hydroxy ester (19 mg, 84.8 μmol), DMF (5 mL), and DIPEA (39 μL, 226 μmol) to the reaction flask and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain GF-40c (21.2 mg, yield 43.1%). LC-MS: [M+H] + =870.8. 1 H-NMR(400MHz,DMSO-d6)δ8.19(d,J=4.8Hz,1H),8.17(s,1H),8.03(t,J=4.4H z,1H),7.83(d,J=6.4Hz,1H),7.74(d,J=8.7Hz,1H),7.71(d,J=7.1Hz,1H),7.2 9(s,1H),6.53(s,1H),5.52(q,J=6.3Hz,1H),5.41(dd,J=22.7,13.0Hz,2H),5 .12(dd,J=72.3,15.2Hz,2H),4.07(d,J=1.9Hz,2H),3.97-3.87(m,2H),3.66-3 .54(m,4H),3.41(t,J=1.8Hz,1H),3.25-3.18(m,1H),3.11-3.03(m,1H),2.80 -2.73(m,1H),2.68-2.62(m,1H),2.38(s,3H),2.34(t,J=5.2Hz,2H),2.16-2.0 5(m,3H),1.98-1.90(m,2H),1.89-1.80(m,2H),1.77-1.69(m,1H),0.92(d,J=5 .6Hz,3H),0.86(t,J=5.8Hz,3H),0.62(d,J=5.4Hz,3H),0.57(d,J=5.4Hz,3H).

[0754] Step 4: Add 1.5 mL of H₂O and 1.5 mL of DMSO to the reaction flask and stir. Then add GF-AzO solution (50 mM aqueous solution, 450 μL), CuSO₄ solution (50 mM aqueous solution, 180 μL), TBTA solution (DMSO solution, 50 mM, 360 μL), GF-40c solution (DMSO solution, 12.5 mM, 1200 μL), and sodium ascorbate solution (50 mM aqueous solution, 900 μL) in sequence. Finally, add 1.5 mL of DMSO and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid GF-40 (20.7 mg, yield 88.5%). Purity (HPLC-RP): 98.1%. 63 H 79 O 26 FN 16 P2[MH] - The calculated HRMS (ESI-) is 1555.47379, and the measured value is 1555.48324. 1 H-NMR(400MHz,D2O)δ7.69(s,1H),7.23(s,1H),6.97(d,J=8.3Hz,1H),5.58(d,J=4 .0Hz,1H),5.43-5.36(m,1H),5.23(dd,J=63.8,13.0Hz,2H),4.98(dd,J=17.0,13. 4Hz,2H),4.77(t,J=5.8Hz,1H),4.50(t,J=4.1Hz,1H),4.32(t,J=3.1Hz,1H),4.24 (dd,J=13.7,10.1Hz,2H),4.18-4.09(m,2H),4.03(s,2H),3.77-3.67(m,3H),3.57- 3.48(m,3H),3.44(d,J=6.2Hz,1H),3.42-3.33(m,3H),3.14(dd,J=11.2,7.8Hz,1H ),2.98-2.90(m,1H),2.84-2.74(m,2H),2.57(s,5H),2.36-2.26(m,1H),2.20-2.12 (m,2H),2.11-2.05(m,1H),1.99(s,5H),1.79-1.70(m,2H),1.57-1.47(m,1H),1.2 0-1.09(m,1H),0.85(d,J=5.7Hz,3H),0.77(t,J=5.8Hz,3H),0.38(d,J=5.1Hz,6H).

[0755] Example 1.102 Preparation of GF-41

[0756] Step 1: Tp-12 (50.6 mg, 0.095 mmol), N-tert-butoxycarbonyl-alanylalanine (37.2 mg, 0.143 mmol), HATU (54.4 mg, 0.143 mmol), DMF (5 mL), and DIPEA (50 μL, 0.287 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-41a (55 mg, yield 74.8%).

[0757] Step 2: GF-41a (55 mg, 0.071 mmol), DCM (15 mL), and TFA (3 mL) were added sequentially to the reaction mixture, and the reaction was carried out at room temperature for 1.5 h. The product was further purified by a preparative HPLC system to obtain GF-41b (43 mg, yield 89.8%).

[0758] Step 3: GF-41b (43 mg, 0.064 mmol), propargyl-N-hydroxy ester (28.8 mg, 0.128 mmol), DMF (5 mL), and DIPEA (34 μL, 0.195 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified using a preparative HPLC system to obtain GF-41c (44 mg, yield 87.7%). LC-MS: [M+H] + =785.6.

[0759] Step 4: Add H2O (350 μL) and DMSO (3150 μL) to the reaction flask and stir. Then add GF-Az0 solution (50 mM aqueous solution, 400 μL), CuSO4 solution (50 mM aqueous solution, 100 μL), TBTA solution (DMSO solution, 50 mM, 200 μL), GF-41c solution (DMSO solution, 25 mM, 400 μL), and sodium ascorbate solution (50 mM aqueous solution, 400 μL) in sequence and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid GF-41 (10 mg, yield 68%). Purity (HPLC-RP): 95.0%. 59 H 72 FN 15 O 25 P2[MH] - The calculated HRMS (ESI-) is 1470.42103, and the measured value is 1470.43171.

[0760] Example 1.103 Preparation of GF-42

[0761] Step 1: Tp-12 (46.3 mg, 0.087 mmol), (tert-butoxycarbonyl)-L-valine-L-alanine (37.5 mg, 0.13 mmol), HATU (49.4 mg, 0.13 mmol), DMF (5 mL), and DIPEA (46 μL, 0.264 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified by preparative HPLC to obtain GF-42a (65 mg, yield 93.1%).

[0762] Step 2: GF-42a (65 mg, 0.081 mmol), DCM (10 mL), and TFA (2 mL) were added sequentially to the reaction mixture, and the reaction was carried out at room temperature for 2 h. The product was further purified by a preparative HPLC system to obtain GF-42b (44 mg, yield 77.3%).

[0763] Step 3: GF-42b (44 mg, 0.063 mmol), propargyl-N-hydroxy ester (28.4 mg, 0.126 mmol), DMF (5 mL), and DIPEA (33 μL, 0.189 mmol) were added sequentially to the reaction mixture, and the reaction was carried out overnight at room temperature. The product was further purified using a preparative HPLC system to obtain GF-42c (43 mg, yield 84%). LC-MS: [M+H] + =813.7.

[0764] Step 4: Add H2O (350 μL) and DMSO (3150 μL) to the reaction flask and stir. Then add GF-Az0 solution (50 mM aqueous solution, 400 μL), CuSO4 solution (50 mM aqueous solution, 100 μL), TBTA solution (DMSO solution, 50 mM, 200 μL), GF-42c solution (DMSO solution, 25 mM, 400 μL), and sodium ascorbate solution (50 mM aqueous solution, 400 μL) in sequence and react overnight at room temperature. After the reaction is complete, the product is further purified by a preparative HPLC system and lyophilized to obtain a white solid GF-42 (7.2 mg, yield 48%). Purity (HPLC-RP): 94.5%. 61 H 76 FN 15 O 25 P2[MH] - The calculated HRMS (ESI-) is 1498.45233, and the measured value is 1498.49689.

[0765] Example 1.104 Preparation of GF-43

[0766] Step 1: Add Tp-12-2 (30.2 mg, 50 μmol), Boc-L-valine-L-citrulline (28 mg, 75 μmol), HATU (38 mg, 100 μmol), DMF (5 mL), and DIPEA (35 μL, 200 μmol) to the reaction flask and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system to obtain GF-43a (38 mg, yield 79.2%).

[0767] Step 2: Add GF-43a (38 mg, 39.6 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the product and further purify it using a preparative HPLC system to obtain GF-43b (25 mg, yield 73.3%).

[0768] Step 3: Add GF-43b (25 mg, 29.1 μmol), propargyl-N-hydroxy ester (10 mg, 43.7 μmol), DMF (5 mL), and DIPEA (20 μL, 116.4 μmol) to the reaction flask and react overnight at room temperature. After the reaction, further purify the product using a preparative HPLC system to obtain GF-43c (17 mg, yield 60.3%). LC-MS: [M+H] + =970.2.

[0769] Step 4: Add H2O (750 μL) and DMSO (750 μL) to the reaction flask and stir. Then add GF-Az0 solution (50 mM aqueous solution, 300 μL), CuSO4 solution (50 mM aqueous solution, 90 μL), TBTA solution (DMSO solution, 50 mM, 180 μL), GF-43c solution (DMSO solution, 12.5 mM, 600 μL), and sodium ascorbate solution (50 mM aqueous solution, 450 μL) in sequence. Finally, add DMSO (750 μL) and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system and lyophilize to obtain a white solid GF-43 (9.8 mg, yield 79.0%). Purity (HPLC-RP): 99.1%. 67 H 87 O 27 FN 18 P2[MH] - The calculated HRMS (ESI-) is 1655.53745, and the measured value is 1655.54949.

[0770] Example 1.105 Preparation of GF-44

[0771] Step 1: Add Tp-22-2 (23.9 mg, 40.5 μmol), DMF (5 mL), N-tert-butoxycarbonyl-alanylalanine (16 mg, 60.8 μmol), HATU (31 mg, 81 μmol), and DIPEA (28 μL, 162 μmol) to the reaction flask and react overnight at room temperature. After the reaction is complete, further purify the product using a preparative HPLC system to obtain GF-44a (30 mg, yield 89.0%).

[0772] Step 2: Add GF-44a (30 mg, 36.1 μmol), DCM (4 mL), and TFA (1 mL) to the reaction flask and react at room temperature for 3 h. After the reaction is complete, concentrate the product and further purify it using a preparative HPLC system. Lyophilize the product to obtain GF-44b (23.3 mg, yield 88.3%).

[0773] Step 3: Add GF-44b (23.3 mg, 31.8 μmol), propargyl-N-hydroxy ester (10.7 mg, 47.7 μmol), DMF (5 mL), and DIPEA (22 μL, 127.2 μmol) to the reaction flask and react overnight at room temperat...

Claims

1. A conjugate, or a pharmaceutically acceptable salt thereof, said conjugate comprising the structure of formula (I): in, R 1 Selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl and 3- to 8-membered cycloalkyl; R 2 for in, R 21 Selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, and 3- to 8-membered cycloalkyl. R 22 and R 23 Each is independently selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, and 3- to 8-membered cycloalkyl. R 22 Can be used with R 23 Together they form a ring structure, the ring structure comprising R 22 and R 23 Connected carbon atoms, or R 21 Can be used with R 22 or R 21 Can be used with R 23 Together they form a ring structure, the ring structure comprising R 21 Connected nitrogen atoms, R 21 R 22 R 23 Each hydrogen atom in the definition can be independently replaced by F, Cl, Br, or I. R 3 for Among them, R 31 Selected from the following group: hydrogen and C1-C6 alkyl groups, R 4 for Among them, R 41 Selected from the following group: hydrogen, C1-C6 alkyl and -R 6 -NH-C(=O)-NH2, where R 6 It is a C1-C6 alkylene group. R 5 for Among them, R 51 It is a C1-C6 alkyl group. Represents the connection site.

2. The conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the conjugate comprises the structure of formula (II-a): in, R 1 R 2 R 3 R 4 and R 5 As defined in equation (I) L is the connecting sub-unit. AB is the ligand, preferably an antigen-binding protein, an Fc fusion protein, or an Fc fragment. n 1 Integers selected from 1 to 4 n 2 It can be an integer or decimal selected from 1 to 8.

3. The conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, having the structure of formula (II-b): in, R 1 R 2 R 3 R 4 and R 5 As defined in equation (I), P 2 These are small molecule drugs, nucleic acids, or peptides, or combinations thereof. L 2 It is a pyrolytic unit. L' is the connecting sub-unit. AB is the ligand, preferably an antigen-binding protein, an Fc fusion protein, or an Fc fragment. n 1 Integers selected from 1 to 4 n 2 It can be an integer or decimal selected from 1 to 8.

4. The conjugate according to any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein R 1 It is hydrogen or C1-C3 alkyl.

5. The conjugate according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, wherein R 1 It is hydrogen or methyl, preferably hydrogen.

6. The conjugate according to any one of claims 1-5, or a pharmaceutically acceptable salt thereof, wherein R 21 It is hydrogen or C1-C3 alkyl, preferably hydrogen.

7. The conjugate according to any one of claims 1-6, or a pharmaceutically acceptable salt thereof, wherein R 22 and R 23 Each is independently selected from the following group: hydrogen, C1-C6 alkyl and 3- to 6-membered cycloalkyl.

8. The conjugate according to any one of claims 1-6, or a pharmaceutically acceptable salt thereof, wherein R 22 With R 23 The ring structure formed together is a 3- to 6-membered cycloalkyl group.

9. The conjugate according to claim 8, or a pharmaceutically acceptable salt thereof, wherein R 22 With R 23 The ring structure formed together is a 4-membered cycloalkyl group.

10. The conjugate according to any one of claims 1-9, or a pharmaceutically acceptable salt thereof, wherein R 2 Selected from the following group: in, Each R 21 Independently hydrogen or methyl, optionally, R 2 Each hydrogen atom in the mixture is independently replaced by F, Cl, Br, or I. Represents the connection site.

11. The conjugate according to claim 10, or a pharmaceutically acceptable salt thereof, wherein R 2 Selected from the following group: in, Each R 21 Independently hydrogen or methyl, optionally, R 2 Each hydrogen atom in the mixture is independently replaced by F, Cl, Br, or I. Represents the connection site.

12. The conjugate according to any one of claims 1-11, or a pharmaceutically acceptable salt thereof, wherein R 2 Selected from the following group: in, Each R 21 Independently hydrogen or methyl, optionally, R 2 Each hydrogen atom in the mixture is independently replaced by F, Cl, Br, or I. Represents the connection site.

13. The conjugate according to claim 12, or a pharmaceutically acceptable salt thereof, wherein R 2 Selected from the following group: in, Each R 21 Independently hydrogen or methyl, optionally, R 2 Each hydrogen atom in the mixture is independently replaced by F, Cl, Br, or I. Represents the connection site.

14. The conjugate according to any one of claims 1-13, or a pharmaceutically acceptable salt thereof, wherein R 2 Selected from the following group: in, Each R 21 Independently hydrogen or methyl, Represents the connection site.

15. The conjugate according to claim 14, or a pharmaceutically acceptable salt thereof, wherein R 2 Selected from the following group: in, Each R 21 Independently hydrogen or methyl, Represents the connection site.

16. The conjugate according to any one of claims 1-15, or a pharmaceutically acceptable salt thereof, wherein R 21 It is hydrogen.

17. The conjugate according to any one of claims 1-16, or a pharmaceutically acceptable salt thereof, wherein R 31 Selected from the group consisting of: hydrogen, methyl, ethyl, isopropyl, isobutyl, and sec-butyl.

18. The conjugate according to any one of claims 1-17, or a pharmaceutically acceptable salt thereof, wherein R 3 Selected from the following group: Represents the connection site.

19. The conjugate according to claim 18, or a pharmaceutically acceptable salt thereof, wherein R 3 Selected from the following group: Represents the connection site.

20. The conjugate according to claim 18, or a pharmaceutically acceptable salt thereof, wherein R 3 Selected from the following group: Represents the connection site.

21. The conjugate according to any one of claims 1-20, or a pharmaceutically acceptable salt thereof, wherein R 41 Selected from the following group: hydrogen, methyl, ethyl, isopropyl, isobutyl, sec-butyl, -(CH2)2-NH-C(=O)-NH2 and -(CH2)3-NH-C(=O)-NH2.

22. The conjugate according to any one of claims 1-21, or a pharmaceutically acceptable salt thereof, wherein R 4 Selected from the following group: Represents the connection site.

23. The conjugate according to claim 22, or a pharmaceutically acceptable salt thereof, wherein R 4 Selected from the following group: Represents the connection site.

24. The conjugate according to any one of claims 1-23, or a pharmaceutically acceptable salt thereof, wherein R 51 Selected from the group consisting of: methyl, ethyl, isopropyl, isobutyl, and sec-butyl.

25. The conjugate according to any one of claims 1-24, or a pharmaceutically acceptable salt thereof, wherein R 5 Selected from the following group: Represents the connection site.

26. The conjugate according to claim 25, or a pharmaceutically acceptable salt thereof, wherein R 5 Selected from the following group: Represents the connection site.

27. The conjugate according to any one of claims 1-26, or a pharmaceutically acceptable salt thereof, wherein R 1 For hydrogen, R 2 for R 3 for R 4 for R 5 for Represents the connection site.

28. The conjugate according to any one of claims 1-27, or a pharmaceutically acceptable salt thereof, wherein the structure of formula (I) is selected from the group consisting of: Represents the connection site.

29. The conjugate according to any one of claims 1-27, or a pharmaceutically acceptable salt thereof, wherein the structure of formula (I) is selected from the group consisting of: Represents the connection site.

30. The conjugate according to any one of claims 1-29, or a pharmaceutically acceptable salt thereof, wherein AB is an antibody or an antigen-binding fragment thereof, an Fc fusion protein, or an Fc fragment.

31. The conjugate according to any one of claims 1-30, or a pharmaceutically acceptable salt thereof, wherein AB comprises a monoclonal antibody, a chimeric antibody, a humanized antibody, and / or a fully human antibody.

32. The conjugate of any one of claims 1-31, or a pharmaceutically acceptable salt thereof, wherein the AB binding targets are selected from the group consisting of tumor-associated antigens, tissue-specific antigens, cell surface molecules, extracellular matrix proteins or proteases, and post-translational modified residues.

33. The conjugate according to any one of claims 1-32, or a pharmaceutically acceptable salt thereof, wherein the AB binding targets are selected from the group consisting of: Trop-2, Her2, Her3, Her4, EGF, EGFR, CD2, CD3, CD5, CD7, CD13, CD19, CD20, CD21, CD23, CD30, CD33, CD34, CD38, CD46, CD55, CD59, CD69, CD70, CD71, CD97, CD117, CD123, CD127, CD134, CD137, CD138, CD146, CD147, CD152, CD15 4. CD174, CD195, CD200, CD205, CD212, CD223, CD227, CD253, CD272, CD274, CD276, CD278, CD279, CD309, CD319, CD326, CD340, DR6, Kv1.3, 5E1 0. MUC1, uPA, MAGE3, MUC16, KLK3, K-ras, Mesothelin, p53, Survivin, G250, PSMA, Endoplasmin, BCMA, GPNMB, EphA2, EphB2, TMEFF2, Integrin beta 6, 5T4, CA9, IGF-1R, Axl, B7H3, B7H4, CDH6, HAVCR1, STEAP-1, STEAP-2, UPK2, CLDN18, CLDN6, CLDN9, c-Met, MICA, LIV-1, ROR1, ADAM9, Stn, DLK-1 and CEACAM-5.

34. The conjugate according to any one of claims 1-33, or a pharmaceutically acceptable salt thereof, wherein AB is an antibody that specifically binds to Her2 or an antigen-binding fragment thereof.

35. The conjugate according to any one of claims 1-33, or a pharmaceutically acceptable salt thereof, wherein AB is an antibody that specifically binds to Trop2 or an antigen-binding fragment thereof.

36. The conjugate according to any one of claims 1-33, or a pharmaceutically acceptable salt thereof, wherein AB is an antibody that specifically binds to EGFR or an antigen-binding fragment thereof.

37. The conjugate according to any one of claims 1-33, or a pharmaceutically acceptable salt thereof, wherein AB is an antibody that specifically binds to Claudin18.2 or an antigen-binding fragment thereof.

38. The conjugate according to any one of claims 1-37, or a pharmaceutically acceptable salt thereof, wherein n 2 It can be 2 or 4.

39. The conjugate according to any one of claims 2, 4-38, or a pharmaceutically acceptable salt thereof, wherein the linker unit L has the following structure: in, Sp is a spacer unit containing PEG or methylene, t 1 The integer is selected from 0 to 4, preferably 0, 1 or 2, Brch is the branching unit, and t 2 It is 0 or 1. Cnt is a linker unit that connects to a ligand and contains a functional group after the reaction.

40. The conjugate according to claim 39, or a pharmaceutically acceptable salt thereof, wherein Sp is a structure selected from the group consisting of -C(=O)-(CH2). s1 -、-C(=O)-(CH2) s1 -(CH2-O-CH2) s2 -、-C(=O)-(CH2) s1 -(CH2-O-CH2) s2 -(CH2) s3 -、-C(=O)-(CH2) s1 -(CH2-O-CH2) s2 -(CH2) s3 -W1-、-C(=O)-(CH2) s1 -(CH2-O-CH2) s2 -(CH2) s3 -W1-(CH2) s4 -(CH2-O-CH2) s5 -(CH2) s6 -、-(CH2) s1 -C(=O)-NH-(CH2-O-CH2) s2 -(CH2) s3 -C(=O)- and -C(=O)-(CH2) s1 -(CH2-O-CH2) s2 -(CH2) s3 -C(=O)-NH-(CH2) s4 -,in, W1 is s1, s3, s4 and s6 are each independently an integer selected from 0 to 4, and s2 and s5 are each independently an integer selected from 0 to 10, wherein the right side of the above Sp structure is connected to Brch or Cnt.

41. The conjugate according to claim 39 or 40, or a pharmaceutically acceptable salt thereof, wherein Sp is a structure selected from the group consisting of:

42. The conjugate according to any one of claims 39-41, or a pharmaceutically acceptable salt thereof, wherein Brch is a structure selected from the group consisting of: in, A1 is -(CH2) p1 -NH-C(=O)-(CH2) p2 -or-(CH2) p2 -, B1 is -(CH2) p2 -, C1 is -C(=O)-(CH2) p2 -、-C(=O)-(CH2) p2 -(CH2-O-CH2) p3 -(CH2) p4 -、-C(=O)-(CH2) p2 -(CH2-O-CH2) p3 -(CH2) p4 -W2- or -C(=O)-(CH2) p2 -(CH2-O-CH2) p3 -(CH2) p4 -W2-(CH2) p5 -(CH2-O-CH2) p6 -(CH2) p7 -,W2 is The left side of structure A1, the right side of structure B1, and the left side of structure C1 are connected to the N atom in the Brch structure. A2 is -C(=O)-NH-(CH2). p2 -, B2 is -(CH2) p2 -C(=O)-NH-, C2 is -(CH2) p2 - The carbon atoms in the A2 structure, B2 structure, and C2 structure are connected to the carbon atoms in the Brch structure. Brch is connected to Cnt through C1 or C2. Each p1 is independently selected from an integer from 2 to 4, each p2 is independently selected from an integer from 1 to 4, each p4, p5 and p7 is independently selected from an integer from 0 to 4, and each p3 and p6 is independently selected from an integer from 0 to 10.

43. The conjugate according to claim 42, or a pharmaceutically acceptable salt thereof, wherein Brch is a structure selected from the group consisting of: The right side of the Brch structure is connected to Cnt.

44. The conjugate according to any one of claims 39-43, or a pharmaceutically acceptable salt thereof, wherein Cnt comprises a functional group selected from the group consisting of maleimide, iodoacetamide, bromoacetamide, thiol, amino, alkyl bromide, alkyl iodide, acrylamide, carboxyl, NHS ester, GDP-fucosylate, aldehyde, tetraazinyl, cycloalkenyl, azide, linear alkyne, and cycloalkyne.

45. The conjugate according to claim 44, or a pharmaceutically acceptable salt thereof, wherein Cnt is a structure selected from the group consisting of: Preferred The right side of the Cnt structure described above is connected to AB.

46. ​​The conjugate according to claim 3, or a pharmaceutically acceptable salt thereof, wherein the linker unit L' has the following structure: in, Sp' 1 and Sp' 2 Each is an independent spacer unit containing PEG or methylene, t' 1 and t' 2 Each is independently either 0 or 1, and Brch' is a branching unit. Cnt' is a linker unit connected to a ligand, which contains a functional group selected from the group consisting of maleimide, iodoacetamide, bromoacetamide, thiol, amino, alkyl bromide, alkyl iodide, acrylamide, carboxyl, NHS ester, GDP-fucosylate, aldehyde, tetraazinyl, cycloalkenyl, azide, linear alkyne, and cycloalkyne.

47. The conjugate according to claim 46, or a pharmaceutically acceptable salt thereof, Sp' 1 The structure selected is from the following group: -C(=O)-(CH2) s1’ -(CH2-O-CH2) s2’ -(CH2) s3’ -、-C(=O)-(CH2) s1’ -(CH2-O-CH2) s2’ -(CH2) s3’ -W3- (CH2) s4’ -(CH2-O-CH2) s5’ -(CH2) s6’ -、-(CH2) s1’ -C(=O)-NH-(CH2-O-CH2) s2’ -(CH2) s3’ -C(=O)-、-C(=O)-(CH2) s1’ -(CH2-O-CH2) s2’ -(CH2) s3’ -C(=O)-NH-(CH2) s4’ -、 in, W3 is s1', s3', s4', s6', s7', and s9' are each independently an integer selected from 0 to 4, and s2', s5', and s8' are each independently an integer selected from 0 to 10, wherein the above Sp' 1 The right side of the structure is connected to Brch' or Cnt'.

48. The conjugate according to any one of claims 46-47, or a pharmaceutically acceptable salt thereof, Sp' 1 The structure selected from the following group:

49. The conjugate according to any one of claims 46-48, or a pharmaceutically acceptable salt thereof, wherein Sp' 2 The structure is selected from the following group: -C(=O)-(CH2) s1” -、-C(=O)-(CH2) s1” -C(=O)-NH-(CH2) s4” -、-C(=O)-(CH2) s1” -(CH2-O-CH2) s2” -(CH2) s3” -、-C(=O)-(CH2) s1” -(CH2-O-CH2) s2” -(CH2) s3” -W4-(CH2) s4” -(CH2-O-CH2) s5” -(CH2) s6” -、-(CH2) s1” -C(=O)-NH-(CH2-O-CH2) s2” -(CH2) s3” -C(=O)- and -C(=O)-(CH2) s1” -(CH2-O-CH2) s2” -(CH2) s3” -C(=O)-NH-(CH2) s4” -,in, W4 is s1”, s3”, s4” and s6” are each independent integers selected from 0 to 4, and s2” and s5” are each independent integers selected from 0 to 10.

50. The conjugate according to any one of claims 46-49, or a pharmaceutically acceptable salt thereof, wherein Sp' 2 The structure selected from the following group:

51. The conjugate according to any one of claims 46-50, or a pharmaceutically acceptable salt thereof, wherein Brch' comprises a structure selected from the group consisting of: in, A'1 is -(CH2) p1’ -NH-C(=O)-(CH2) p2’ -or-(CH2) p2’ -, B'1 is -(CH2) p2’ -,C'1 is -C(=O)-(CH2) p2’ -、-C(=O)-(CH2) p2’ -(CH2-O-CH2) p3’ -(CH2) p4’ -、-C(=O)-(CH2) p2’ -(CH2-O-CH2) p3’ -(CH2) p4’ -W'2- or -C(=O)-(CH2) p2’ -(CH2-O-CH2) p3’ -(CH2) p4’ -W'2-(CH2) p5’ -(CH2-O-CH2) p6’ -(CH2) p7’ -, W'2 is The left side of the A'1 structure, the right side of the B'1 structure, and the left side of the C'1 structure are connected to the N atom in the Brch' structure. A'2 is -C(=O)-NH-(CH2). p2’ -, B'2 is -(CH2) p2’ -C(=O)-NH-, C'2 is -(CH2) p2’ - The carbon atoms in the A'2 structure (left side), B'2 structure (right side), and C'2 structure (left side) are connected to the carbon atoms in the Brch' structure. Brch' is connected to Cnt' via C'1 or C'2. Each p1' is independently selected from an integer from 2 to 4, each p2' is independently selected from an integer from 1 to 4, each p4', p5', and p7' is independently selected from an integer from 0 to 4, and each p3' and p6' is independently selected from an integer from 0 to 10.

52. The conjugate according to any one of claims 46-51, or a pharmaceutically acceptable salt thereof, wherein Brch' is a structure selected from the group consisting of: The right side of the structure is connected to Cnt'.

53. The conjugate according to any one of claims 46-52, or a pharmaceutically acceptable salt thereof, wherein Cnt' is a structure selected from the group consisting of: Preferred 54. The conjugate according to any one of claims 2-53, or a pharmaceutically acceptable salt thereof, wherein AB comprises a sugar chain having GlcNAc, and L or L' is linked to said GlcNAc.

55. The conjugate according to claim 54, or a pharmaceutically acceptable salt thereof, wherein Cnt is a structure selected from the group consisting of: Cnt is preferred, The right side of the structure is connected to GlcNAc.

56. The conjugate according to claim 54, or a pharmaceutically acceptable salt thereof, wherein Cnt' is a structure selected from the group consisting of... Preferred The right side of the structure is connected to GlcNAc.

57. The conjugate according to any one of claims 2, 4-56, or a pharmaceutically acceptable salt thereof, wherein L is a structure selected from the group consisting of:

58. The conjugate according to any one of claims 3-56, or a pharmaceutically acceptable salt thereof, wherein L' is a structure selected from the group consisting of:

59. The conjugate according to any one of claims 3-58, or a pharmaceutically acceptable salt thereof, wherein P 2 Selected from the following group: cytotoxins, agonists, antagonists, antiviral agents, antibacterial agents, radioisotopes or radionuclides, metal chelators, fluorescent dyes, biotin, and combinations thereof.

60. The conjugate according to any one of claims 3-59, or a pharmaceutically acceptable salt thereof, wherein P 2 It is a microtubule inhibitor.

61. The conjugate according to any one of claims 3-60, or a pharmaceutically acceptable salt thereof, wherein P 2 Selected from the following group: MMAE, MMAF and their prodrugs and / or intermediates.

62. The conjugate according to any one of claims 3-61, or a pharmaceutically acceptable salt thereof, wherein L 2 It is a dipeptide, tripeptide, or tetrapeptide, galactosyl, glucuronic acid, or phosphate group composed of naturally occurring and / or non-naturally occurring amino acids, and may optionally contain a self-eliminating group.

63. The conjugate according to any one of claims 3-62, or a pharmaceutically acceptable salt thereof, wherein L 2 for The left side of the structure and P 2 Connected.

64. The conjugate according to any one of claims 2-63, or a pharmaceutically acceptable salt thereof, comprising the following structures selected from Table 1: F-6, F-7, F-8, F-9, F-10, F-11, F-12, F-13, F-14, F-15, F-16, F-18, F-19, F-20, F-21, F-22, F-23, F-24, F-25, F-26, F-27, F-28, F-30, F-31, F-36, F-38, F-39, F-40, F-43, F-44, F-47, F-D1, F-D2, F-D3, F-D4 F-D5, F-D6, F-D7, F-D8, F-D9, F-D10, F-D11, F-D12, F-D15, F-D16, F-D17, F-D18, F-D19, F-D20, F-D21, F-D22, F-D23, F-D24, F-D25, F-DP1, F-DP2, F-DP3, F-DP4, F-DP5, F-DP6, F-DP7, F-DP8, F-DP9, F-DP10, F-DP11, F-DP12, F-DP13, F-DP14, MC'-1, and DBCO'-1, the above structures are obtained through Connect to AB.

65. The conjugate according to any one of claims 2-64, or a pharmaceutically acceptable salt thereof, wherein AB has the following structure: and in, AB' is the ligand, preferably an antigen-binding protein, an Fc fusion protein, or an Fc fragment; GalX is substituted or unsubstituted galactose; AB is obtained through... Connect with L or L'.

66. The conjugate according to claim 65, wherein the GalX has the following structure: Each Rg independently selects the next group: in, Rg 1 Selected from the following groups: hydrogen, halogens, -OH, -NH2, -SH, -N3, -COOH, -CN, Cl-C 24 Alkyl, C3-C 24 cycloalkyl, C2-C 24 alkenyl, C5-C 24 Cycloalkenyl, C2-C 24 alkynyl group, C7-C 24 Cycloalkynyl, C2-C 24 (Miscellaneous) aryl, C3-C 24 Alkyl (hetero)aryl, C3-C 24 (Hetero)arylalkyl and any combination thereof, wherein the alkyl, the cycloalkyl, the alkenyl, the cycloalkenyl, the ynyl, the cycloynyl, the (hetero)aryl, the alkyl(hetero)aryl, or the (hetero)arylalkyl is optionally oxidized by one or more Rs 4 Replaced and / or optionally by one or more Rs 2 Interruption, where Rs 4 Selected from the following groups: halogen, -OH, -NH2, -SH, -N3, -COOH, and -CN, Rs 2 Selected from the following groups: -O-, -S-, Where Rs 3 Selected from the following groups: hydrogen, C1-C 24 Alkyl, C2-C 24 alkenyl, C2-C 24 alkynyl and C3-C 24 cycloalkyl, tg is 0 or 1. Rg 2 Selected from the following groups: C1-C 24 Alkylene, C3-C 24 Cycloalkylene, C2-C 24 alkenyl, C5-C 24 Cycloalkenyl, C2-C 24 Ethyne group, C7-C 24 Cycloethynyl, C2-C 24 (Miscellaneous) aryl, C3-C 24 Alkyl (hetero)arylene and C3-C 24 (Hetero)arylalkylene, wherein the alkylene, the cycloalkylene, the alkenyl, the cycloalkenyl, the alynylene, the cycloynylene, the (hetero)aryl, the alkyl(hetero)aryl, or the (hetero)arylalkylene optionally is affected by one or more Rs 4 Replaced and / or optionally by one or more Rs 2 Interruption, where Rs 4 Selected from the following groups: hydrogen, -OH, -NH2, -SH, -N3, -COOH, and -CN, Rs 2 Selected from the following groups: -O-, -S-, Where Rs 3 Selected from the following groups: hydrogen, C1-C 24 Alkyl, C2-C 24 alkenyl, C2-C 24 alkynyl and C3-C 24 cycloalkyl, Rg 3 Selected from the following groups: hydrogen, halogen, -OH, -NH2, -SH, -N3, -COOH, -CN, Cl-C 24 Alkyl, C3-C 24 cycloalkyl, C2-C 24 alkynyl group, C5-C 24 Cycloalkynyl, C2-C 24 alkynyl group, C8-C 24 Cycloalkynyl, C2-C 24 (Hetero)aryl and any combination thereof, wherein C1-C 24 Alkyl, C3-C 24 cycloalkyl, C2-C 24 alkynyl group, C5-C 24 Cycloalkynyl, C2-C 24 alkynyl group, C8-C 24 Cycloalkynyl or C2-C 24 (Miscellaneous) aryl groups are optionally bounded by one or more Rs 4 Replace, where Rs 4 Selected from the following groups: hydrogen, -OH, -NH2, -SH, -N3, -COOH, and -CN, Rs 2 Selected from the following groups: -O-, -S-, Where Rs 3 Selected from the following groups: hydrogen, C1-C 24 Alkyl, C2-C 24 alkenyl, C2-C 24 alkynyl and C3-C 24 Cycloalkyl.

67. The conjugate according to any one of claims 65-66, wherein GalX is selected from the group consisting of:

68. The conjugate according to any one of claims 2-67, or a pharmaceutically acceptable salt thereof, wherein AB comprises a thiol group, and Cnt or Cnt' comprises a functional group selected from the group consisting of maleimide, haloacetyl, and pyridinedithioyl, preferably.

69. The conjugate according to any one of claims 2-67, or a pharmaceutically acceptable salt thereof, wherein AB is modified to comprise an aldehyde, azide, tetrazinyl, cycloalkenyl, linear alkyne, or cycloalkyne group.

70. The conjugate of claim 69, or a pharmaceutically acceptable salt thereof, wherein AB is modified to include an azide functional group, and Cnt or Cnt' is...

71. The conjugate according to any one of claims 2-70, or a pharmaceutically acceptable salt thereof, wherein AB or AB' comprises an amino acid sequence selected from the group consisting of: (1) VH: SEQ ID NO: 23, VL: SEQ ID NO: 22; (2) VH: SEQ ID NO: 31, VL: SEQ ID NO: 30; (3) VH: SEQ ID NO: 39, VL: SEQ ID NO: 38; (4) VH: SEQ ID NO: 47, VL: SEQ ID NO: 46 and (5) VH: SEQ ID NO:55, VL: SEQ ID NO:

54.

72. A composition comprising the conjugate according to any one of claims 1-71, or a pharmaceutically acceptable salt thereof, wherein: 1) It has one type of DAR, and the DAR is 1.5 to 2, or 3 to 4, or 6 to 8; or 2) It has two types of DAR, D 1 AR is 1.5 to 2, or 3 to 4, or 6 to 8, D 2 AR is 1.5 to 2 or 3 to 4.

73. A compound, or a pharmaceutically acceptable salt thereof, said compound having the structure of formula (III-a): in, R 1 Selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl and 3- to 8-membered cycloalkyl; R 2 for in, R 21 Selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, and 3- to 8-membered cycloalkyl. R 22 and R 23 Each is independently selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6, C2-C6 ynyl, and 3- to 8-membered cycloalkyl. R 22 Can be used with R 23 Together they form a ring structure, the ring structure comprising R 22 and R 23 Connected carbon atoms, or R 21 Can be used with R 22 or R 21 Can be used with R 23 Together they form a ring structure, the ring structure comprising R 21 Connected nitrogen atoms, R 21 R 22 R 23 Each hydrogen atom in the definition can be independently replaced by F, Cl, Br, or I. R 3 for Among them, R 31 Selected from the following group: hydrogen, C1-C6 alkyl, R 4 for Among them, R 41 Selected from the following group: hydrogen, C1-C6 alkyl and -R 6 -NH-C(=O)-NH2, where R 6 It is a C1-C6 alkylene group. R 5 for Among them, R 51 Selected from the following group: C1-C6 alkyl, n 1 Integers selected from 1 to 4 Lx is in, Sp is a spacer unit containing PEG or methylene. Brch is a branching unit. t 1 The integer is selected from 0 to 4, preferably 0, 1 or 2. t 2 It is 0 or 1. CntR is a structure selected from the following groups: Maleimide, iodoacetamide, bromoacetamide, thiol, amino, alkyl bromide, alkyl iodide, acrylamide, carboxyl, NHS ester, aldehyde, tetraazinyl, cycloalkenyl, azide, linear alkynyl, and cycloalkynyl, preferably, CntR is Represents the connection site.

74. The compound according to claim 73, or a pharmaceutically acceptable salt thereof, wherein Sp is a structure selected from the group consisting of -C(=O)-(CH2). s1 -、-C(=O)-(CH2) s1 -(CH2-O-CH2) s2 -、-C(=O)-(CH2) s1 -(CH2-O-CH2) s2 -(CH2) s3 -、-C(=O)-(CH2) s1 -(CH2-O-CH2) s2 -(CH2) s3 -W1-、-C(=O)-(CH2) s1 -(CH2-O-CH2) s2 -(CH2) s3 -W1-(CH2) s4 -(CH2-O-CH2) s5 -(CH2) s6 -、-(CH2) s1 -C(=O)-NH-(CH2-O-CH2) s2 -(CH2) s3 -C(=O)- and -C(=O)-(CH2) s1 -(CH2-O-CH2) s2 -(CH2) s3 -C(=O)-NH-(CH2) s4 -,in, W1 is s1, s3, s4 and s6 are each independently an integer selected from 0 to 4, and s2 and s5 are each independently an integer selected from 0 to 10, wherein the right side of the Sp structure is connected to Brch or CntR.

75. The compound according to claim 73 or 74, or a pharmaceutically acceptable salt thereof, wherein Sp is a structure selected from the group consisting of:

76. The compound according to any one of claims 73-75, or a pharmaceutically acceptable salt thereof, wherein Brch is a structure selected from the group consisting of: in, A1 is -(CH2) p1 -NH-C(=O)-(CH2) p2 -or-(CH2) p2 -, B1 is -(CH2) p2 -, C1 is -C(=O)-(CH2) p2 -、-C(=O)-(CH2) p2 -(CH2-O-CH2) p3 -(CH2) p4 -、-C(=O)-(CH2) p2 -(CH2-O-CH2) p3 -(CH2) p4 -W2- or -C(=O)-(CH2) p2 -(CH2-O-CH2) p3 -(CH2) p4 -W2-(CH2) p5 -(CH2-O-CH2) p6 -(CH2) p7 -,W2 is The left side of structure A1, the right side of structure B1, and the left side of structure C1 are connected to the N atom in the Brch structure. A2 is -C(=O)-NH-(CH2). p2 -, B2 is -(CH2) p2 -C(=O)-NH-, C2 is -(CH2) p2 - The carbon atoms in the A2 structure, B2 structure, and C2 structure are connected to the carbon atoms in the Brch structure. Brch is connected to Cnt through C1 or C2. Each p1 is independently selected from an integer from 2 to 4, each p2 is independently selected from an integer from 1 to 4, each p4, p5 and p7 is independently selected from an integer from 0 to 4, and each p3 and p6 is independently selected from an integer from 0 to 10.

77. The conjugate according to any one of claims 73-76, or a pharmaceutically acceptable salt thereof, wherein Brch is a structure selected from the group consisting of: The right side of the Brch structure is connected to CntR.

78. A compound, or a pharmaceutically acceptable salt thereof, said compound having the structure of formula (III-b): in, Lx' is in R 1 Selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl and 3- to 8-membered cycloalkyl; R 2 for in, R 21 Selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, and 3- to 8-membered cycloalkyl. R 22 and R 23 Each is independently selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, and 3- to 8-membered cycloalkyl. R 22 Can be used with R 23 Together they form a ring structure, the ring structure comprising R 22 and R 23 Connected carbon atoms, Or, R 21 Can be used with R 22 or R 21 Can be used with R 23 Together they form a ring structure, the ring structure comprising R 21 Connected nitrogen atoms, R 21 R 22 R 23 Each hydrogen atom in the definition can be independently replaced by F, Cl, Br, or I. R 3 for Among them, R 31 Selected from the following group: hydrogen, C1-C6 alkyl, R 4 for Among them, R 41 Selected from the group consisting of: hydrogen, C1-C6 alkyl, and -R 6 -NH-C(=O)-NH2, where R 6 It is a C1-C6 alkylene group. R 5 for Among them, R 51 Selected from the following group: C1-C6 alkyl, n 1 Integers selected from 1 to 4 Sp' 1 and Sp' 2 Each is an independent spacer unit containing either PEG or methylene. Brch' is a branching unit. t' 1 and t' 2 Each is independently either 0 or 1. Cnt'R contains functional groups selected from the following group: Maleimide, iodoacetamide, bromoacetamide, thiol, amino, alkyl bromide, alkyl iodide, acrylamide, carboxyl, NHS ester, aldehyde, tetraazinyl, cycloalkenyl, azide, linear alkynyl, and cycloalkynyl, preferably, Cnt'R is P 2 These are small molecule drugs, nucleic acids, or peptides, or combinations thereof. L 2 It is a pyrolytic unit. Represents the connection site.

79. The compound according to claim 78, or a pharmaceutically acceptable salt thereof, Sp' 1 The structure selected is from the following group: -C(=O)-(CH2) s1’ -(CH2-O-CH2) s2’ -(CH2) s3’ -、-C(=O)-(CH2) s1’ -(CH2-O-CH2) s2’ -(CH2) s3’ -W3-(CH2) s4’ -(CH2-O-CH2) s5’ -(CH2) s6’ -、-(CH2) s1’ -C(=O)-NH-(CH2-O-CH2) s2’ -(CH2) s3’ -C(=O)-、-C(=O)-(CH2) s1’ -(CH2-O-CH2) s2’ -(CH2) s3’ -C(=O)-NH-(CH2) s4’ -、 in, W3 is s1', s3', s4', s6', s7', and s9' are each independently an integer selected from 0 to 4, and s2', s5', and s8' are each independently an integer selected from 0 to 10, wherein the above Sp' 1 The right side of the structure is connected to Brch' or Cnt'R.

80. The compound according to claim 78 or 79, or a pharmaceutically acceptable salt thereof, Sp' 1 The structure selected from the following group:

81. The conjugate according to any one of claims 78-80, or a pharmaceutically acceptable salt thereof, wherein Sp' 2 The structure is selected from the following group: -C(=O)-(CH2) s1” -、-C(=O)-(CH2) s1” -C(=O)-NH-(CH2) s4” -、-C(=O)-(CH2) s1” -(CH2-O-CH2) s2” -(CH2) s3” -、-C(=O)-(CH2) s1” -(CH2-O-CH2) s2” -(CH2) s3” -W4-(CH2) s4” -(CH2-O-CH2) s5” -(CH2) s6” -、-(CH2) s1” -C(=O)-NH-(CH2-O-CH2) s2” -(CH2) s3” -C(=O)- and -C(=O)-(CH2) s1” -(CH2-O-CH2) s2” -(CH2) s3” -C(=O)-NH-(CH2) s4” -,in, W4 is s1”, s3”, s4” and s6” are each independent integers selected from 0 to 4, and s2” and s5” are each independent integers selected from 0 to 10.

82. The compound according to any one of claims 78-81, or a pharmaceutically acceptable salt thereof, wherein Sp' 2 The structure is selected from the following group:

83. The compound according to any one of claims 78-82, or a pharmaceutically acceptable salt thereof, wherein Brch' comprises a structure selected from the group consisting of: in, A'1 is -(CH2) p1’ -NH-C(=O)-(CH2) p2’ -or-(CH2) p2’ -, B'1 is -(CH2) p2’ -,C'1 is -C(=O)-(CH2) p2’ -、-C(=O)-(CH2) p2’ -(CH2-O-CH2) p3’ -(CH2) p4’ -、-C(=O)-(CH2) p2’ -(CH2-O-CH2) p3’ -(CH2) p4’ -W'2- or -C(=O)-(CH2) p2’ -(CH2-O-CH2) p3’ -(CH2) p4’ -W'2-(CH2) p5’ -(CH2-O-CH2) p6’ -(CH2) p7’ -, W'2 is The left side of the A'1 structure, the right side of the B'1 structure, and the left side of the C'1 structure are connected to the N atom in the Brch' structure. A'2 is -C(=O)-NH-(CH2). p2’ -, B'2 is -(CH2) p2’ -C(=O)-NH-, C'2 is -(CH2) p2’ - The carbon atoms in the A'2 structure, B'2 structure, C'2 structure, and Brch' structure are connected to each other. Brch' is connected to Cnt'R through C'1 or C'2. Each p1' is independently selected from an integer from 2 to 4, each p2' is independently selected from an integer from 1 to 4, each p4', p5', and p7' is independently selected from an integer from 0 to 4, and each p3' and p6' is independently selected from an integer from 0 to 10.

84. The compound according to any one of claims 78-83, or a pharmaceutically acceptable salt thereof, wherein Brch' is a structure selected from the group consisting of: The right side of the structure is connected to Cnt'R.

85. The compound according to any one of claims 73-84, or a pharmaceutically acceptable salt thereof, wherein R 1 It is hydrogen or C1-C3 alkyl.

86. The compound according to any one of claims 73-85, or a pharmaceutically acceptable salt thereof, wherein R 1 It is hydrogen or methyl, preferably hydrogen.

87. The compound according to any one of claims 73-86, or a pharmaceutically acceptable salt thereof, wherein R 21 It is hydrogen or C1-C3 alkyl, preferably hydrogen.

88. The compound according to any one of claims 73-87, or a pharmaceutically acceptable salt thereof, wherein R 22 and R 23 Each is independently selected from the following group: hydrogen, C1-C6 alkyl and 3- to 6-membered cycloalkyl.

89. The compound according to any one of claims 73-88, or a pharmaceutically acceptable salt thereof, wherein R 22 With R 23 The ring structure formed together is a 3- to 6-membered cycloalkyl group.

90. The compound of claim 89, or a pharmaceutically acceptable salt thereof, wherein R 22 With R 23 The ring structure formed together is a 4-membered cycloalkyl group.

91. The compound according to any one of claims 73-90, or a pharmaceutically acceptable salt thereof, wherein R 2 Selected from the following group: in, Each R 21 Independently hydrogen or methyl, optionally, R 2 Each hydrogen atom in the mixture is independently replaced by F, Cl, Br, or I. Represents the connection site.

92. The compound according to claims 78-91, or a pharmaceutically acceptable salt thereof, wherein R 2 Selected from the following group: in, Each R 21 Independently hydrogen or methyl, optionally, R 2 Each hydrogen atom in the mixture is independently replaced by F, Cl, Br, or I. Represents the connection site.

93. The compound according to any one of claims 73-92, or a pharmaceutically acceptable salt thereof, wherein R 2 Selected from the following group: in, Each R 21 Independently hydrogen, optionally, R 2 Each hydrogen atom in the mixture is independently replaced by F, Cl, Br, or I. Represents the connection site.

94. The compound of claim 93, or a pharmaceutically acceptable salt thereof, wherein R 2 Selected from the following group: in, Each R 21 Independently hydrogen or methyl, optionally, R 2 Each hydrogen atom in the mixture is independently replaced by F, Cl, Br, or I. Represents the connection site.

95. The compound according to any one of claims 73-94, or a pharmaceutically acceptable salt thereof, wherein R 2 Selected from the following group: in, Each R 21 Independently hydrogen or methyl, Represents the connection site.

96. The compound of claim 95, or a pharmaceutically acceptable salt thereof, wherein R 2 Selected from the following group: in, Each R 21 Independently hydrogen or methyl, Represents the connection site.

97. The compound according to any one of claims 73-96, or a pharmaceutically acceptable salt thereof, wherein R 21 It is hydrogen.

98. The compound according to any one of claims 73-91, or a pharmaceutically acceptable salt thereof, wherein R 31 Selected from the group consisting of: hydrogen, methyl, ethyl, isopropyl, isobutyl, and sec-butyl.

99. The compound according to any one of claims 73-98, or a pharmaceutically acceptable salt thereof, wherein R 3 Selected from the following group: Represents the connection site.

100. The compound of claim 99, or a pharmaceutically acceptable salt thereof, wherein R 3 Selected from the following group: Represents the connection site.

101. The compound of claim 99, or a pharmaceutically acceptable salt thereof, wherein R 3 Selected from the following group: Represents the connection site.

102. The compound according to any one of claims 73-101, or a pharmaceutically acceptable salt thereof, wherein R 41 Selected from the following group: hydrogen, methyl, ethyl, isopropyl, isobutyl, sec-butyl, -(CH2)2-NH-C(=O)-NH2 and -(CH2)3-NH-C(=O)-NH2.

103. The compound according to any one of claims 73-102, or a pharmaceutically acceptable salt thereof, wherein R 4 Selected from the following group: Represents the connection site.

104. The compound of claim 103, or a pharmaceutically acceptable salt thereof, wherein R 4 Selected from the following group: Represents the connection site.

105. The compound according to any one of claims 73-104, or a pharmaceutically acceptable salt thereof, wherein R 51 Selected from the group consisting of: methyl, ethyl, isopropyl, isobutyl, and sec-butyl.

106. The compound according to any one of claims 73-105, or a pharmaceutically acceptable salt thereof, wherein R 5 Selected from the following group: Represents the connection site.

107. The compound of claim 106, or a pharmaceutically acceptable salt thereof, wherein R 5 Selected from the following group: Represents the connection site.

108. The compound according to any one of claims 73-107, or a pharmaceutically acceptable salt thereof, wherein R 1 For hydrogen, R 2 for R 3 for R 4 for R 5 for Represents the connection site.

109. The compound according to any one of claims 73-108, or a pharmaceutically acceptable salt thereof, wherein The structures shown are selected from the following group: Represents the connection site.

110. The compound according to any one of claims 73-109, or a pharmaceutically acceptable salt thereof, wherein The structures shown are selected from the following group: Represents the connection site.

111. The compound according to any one of claims 78-110, or a pharmaceutically acceptable salt thereof, wherein P 2 Selected from the following group: cytotoxins, agonists, antagonists, antiviral agents, antibacterial agents, radioisotopes or radionuclides, metal chelators, fluorescent dyes, biotin, and combinations thereof.

112. The compound according to any one of claims 78-111, or a pharmaceutically acceptable salt thereof, wherein P 2 It is a microtubule inhibitor.

113. The compound according to any one of claims 78-112, or a pharmaceutically acceptable salt thereof, wherein P 2 Selected from the following group: MMAE, MMAF and their prodrugs and / or intermediates.

114. The compound according to any one of claims 78-113, or a pharmaceutically acceptable salt thereof, wherein L 2 It is a dipeptide, tripeptide, or tetrapeptide, galactosyl, glucuronic acid, or phosphate group composed of naturally occurring and / or non-naturally occurring amino acids, and may optionally contain a self-eliminating group.

115. The compound according to any one of claims 78-114, or a pharmaceutically acceptable salt thereof, wherein L 2 for The left side of the structure and P 2 Connected.

116. The compound according to any one of claims 73-77, 85-110, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure selected from the group consisting of: GF-6, GF-7, GF-8, GF-9, GF-10, GF-11, GF-12, GF-13, GF-14, GF-15, GF-16, GF-18, GF-19, GF-20, GF-21, GF-22, GF-23, GF-24, GF-25, GF-26, GF-27, GF-28, GF-30, GF-31, GF- 36. GF-38, GF-39, GF-40, GF-43, GF-44, GF-47, GF-D1, GF-D2, GF-D3, GF-D4, GF-D5, GF-D6, GF-D7, GF-D8, GF-D9, GF-D10, G F-D11, GF-D12, GF-D15, GF-D16, GF-D17, GF-D18, GF-D19, GF-D20, GF-D21, GF-D22, GF-D23, GF-D24, GF-D25, MC-1 and DBCO-1.

117. The compound according to any one of claims 78-115, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure selected from the group consisting of GF-DP1, GF-DP2, GF-DP3, GF-DP4, GF-DP5, GF-DP6, GF-DP7, GF-DP8, GF-DP9, GF-DP10, GF-DP11, GF-DP12, GF-DP13 and GF-DP14.

118. A compound, or a pharmaceutically acceptable salt thereof, said compound having the structure of formula (IV). in, R 1 Selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl and 3- to 8-membered cycloalkyl; R 2 for in, R 21 Selected from the group consisting of: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, and 3- to 8-membered cycloalkyl. R 22 and R 23 Together they form a 4-membered ring structure, the ring structure including R 22 With R 23 Connected carbon atoms, R 21 R 22 R 23 Each hydrogen atom in the definition can be independently replaced by F, Cl, Br, or I. R 3’ For hydrogen or Among them, R 31 Selected from the following group: hydrogen, C1-C6 alkyl.

119. The compound of claim 118, or a pharmaceutically acceptable salt thereof, wherein R 1 It is hydrogen or methyl, preferably hydrogen.

120. The compound according to any one of claims 118 or 119, or a pharmaceutically acceptable salt thereof, wherein R 2 for 121. The compound according to any one of claims 118 or 119, or a pharmaceutically acceptable salt thereof, wherein R 21 It is hydrogen or methyl, preferably hydrogen.

122. The compound according to any one of claims 118-121, or a pharmaceutically acceptable salt thereof, wherein R 3’ It is hydrogen.

123. The compound according to any one of claims 118-121, or a pharmaceutically acceptable salt thereof, wherein R 3’ for And R 31 It can be hydrogen or methyl.

124. The compound according to any one of claims 118-123, or a pharmaceutically acceptable salt thereof, wherein the structure of formula (IV) is selected from the group consisting of:

125. The compound according to any one of claims 118-124, or a pharmaceutically acceptable salt thereof, wherein the structure of formula (IV) is selected from the group consisting of:

126. A method for preparing the conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-71, the composition of claim 72, the method comprising contacting the compound or a pharmaceutically acceptable salt thereof according to any one of claims 73-117 with a ligand.

127. The method of claim 126, wherein the contact is carried out in the presence of fucosyltransferase.

128. The method according to any one of claims 126-127, wherein the contact is carried out in the presence of galactosyltransferase, fucosyltransferase, and UDP-GalX or a pharmaceutically acceptable salt thereof.

129. The method according to any one of claims 126-128, wherein the contact is carried out in the presence of glycoside endonuclease, galactosyltransferase, fucosyltransferase and UDP-GalX.

130. A pharmaceutical composition comprising the conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1-71, the composition of claim 72, the compound or a pharmaceutically acceptable salt thereof as described in any one of claims 73-117, 118-125, and optionally a pharmaceutically acceptable carrier.

131. Use of the conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1-71, the composition of claim 72, the compound or a pharmaceutically acceptable salt thereof as described in any one of claims 73-117, 118-125, and / or the pharmaceutical composition of claim 130 in the preparation of a medicament for the treatment and / or prevention of tumors.

132. A method of treating and / or preventing tumors, the method comprising administering to a subject in need an effective amount of any one of the conjugates of claims 1-71 or a pharmaceutically acceptable salt thereof, the composition of claim 72, any one of claims 73-117, 118-125, or a pharmaceutically acceptable salt thereof, and / or the pharmaceutical composition of claim 130.

133. The method according to claim 132, wherein the tumor includes lung cancer, breast cancer, colorectal cancer, prostate cancer, gastric cancer, liver cancer, pancreatic cancer, ovarian cancer, cervical cancer, thyroid cancer, melanoma, lymphoma, leukemia, brain tumor, bone cancer, kidney cancer, bladder cancer, esophageal cancer, nasopharyngeal carcinoma, melanoma, skin cancer, laryngeal cancer, oral cancer, tongue cancer, gallbladder cancer, bile duct cancer, skin cancer, testicular cancer, uterine cancer, endometrial cancer, renal pelvis cancer, renal cell carcinoma, bladder cancer, glioma, neuroblastoma, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, myeloid leukemia, myelodysplastic syndrome, soft tissue sarcoma, osteosarcoma, liposarcoma, neurofibrosarcoma, angiosarcoma, gastrointestinal stromal tumor, thymoma, thymic carcinoma, pituitary adenoma, retinoblastoma, and / or glioblastoma.

Images