Anti-human ROR1 antibody-drug conjugate and uses thereof

Abstract

The application relates to antibody-drug conjugates, pharmaceutical compositions comprising the antibody-drug conjugates, and medicinal uses of the antibody-drug conjugates for treating diseases such as cancer. In one embodiment, the antibody-drug conjugate is formed with an anti-human ROR1 antibody and a camptothecin derivative.

Description

[0001] ANTI-HUMAN ROR1 ANTIBODY-DRUG CONJUGATE AND USES THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The application is based on and claims the benefit of priority from Chinese Application No. 202411030454.3, filed on July 30, 2024, the disclosures of which are incorporated herein by reference in its entirety.

[0004] TECHNICAL FIELD

[0005] The application relates to the biopharmaceutical field, and particularly to an antibody-drug conjugate formed with an anti-human ROR1 antibody and a drug having cell killing effect, and a preparation method and application of the antibody-drug conjugate.

[0006] BACKGROUND

[0007] R0R1 (receptor tyrosine kinase-like orphan receptor 1) is a type I transmembrane protein belonging to RTKs (receptor tyrosine kinases). The ROR1 has a similar structure to members of the RTKs, which contains a cytoplasmic domain having tyrosine kinase activity, a single transmembrane hydrophobic a-helical region and an extracellular domain capable of binding a ligand. The cytoplasmic domain of ROR1 can be divided into 4 domains: a tyrosine kinase-like domain, two serine / threonine-rich domains and a proline-rich domain. The extracellular domain of ROR1 is further divided into three domains: an immunoglobulin-like domain (Ig-Like), a Frizzled domain (FZD), and a Kringle domain (KD). Among these, the FZD contains a site of binding to Wnt5a, thereby recognizing the Wnt5a and participating in the regulation of non- classical Wnt signals; the KD mediates the interaction of ROR1 with other receptors, e.g., ROR2.

[0008] ROR1 plays an important role in the regulation of physiological processes such as cell division, proliferation and migration, by mediating the signal transduction in the Wnt signaling pathway. R0R1 is highly expressed during embryonic and infant development stages with a high level of stem cells, the expression level of ROR1 gradually decreases as organism individuals grow and develop, and it is almost undetectable on the cell surface of normal tissues in the children and adult stages. With research, it has been found that although it is not demonstrated whether the ROR1 is directly functionally associated with the occurrence of tumors, the expression of ROR1 is significantly increased in a variety of hematologic cancers and solid tumors. For example, high levels of ROR1 expression have been detected in hematologic cancers such as B cell Chronic Lymphocytic Leukemia (CLL), non-Hodgkin lymphoma (NHL), lymphoma and myeloid hematologic cancer, and various solid tumors such as kidney cancer, non-small cell lung cancer, pancreatic cancer, lung cancer and skin cancer, which indicates that the ROR1 promises to be a new target with broad-spectrum anticancer potential.

[0009] At present, there are a variety of development directions for drugs targeting ROR1, including small molecule inhibitors, monoclonal antibodies, bispecific antibodies, chimeric antigen receptor T cells, ADCs and the like, wherein monoclonal antibody drugs and ADC drugs have shown faster progress and have achieved significant therapeutic effects. The ROR1 monoclonal antibody drug Cirmtuzumab developed by Onternal Company competitively binds to the FZD domain of the extracellular domain of ROR1 with Wnt5a, thereby blocking the non-classical Wnt signaling pathway and inhibiting growth and metastasis of cancers. CS5001 (formerly LCB 71) developed by a Korean biotechnology company, LegoChem Biosciences through utilizing a prodrug ADC technology (pPBD), has a masking moiety of PBD toxin, which is not toxic in normal tissues, and when the CS5001 is endocytosed by tumor cells, the masking moiety is degraded under enzyme action so that the toxicity of the toxin is restored, to kill tumor cells. Now, CS5001 has entered the clinical stage I. NBE-002 is an ADC drug developed by NBE-Therapeutics and it consists of a humanized antibody and an anthracycline derivative, which shows significant antitumor activity in patient-derived xenograft models of ovarian cancer, breast cancer, lung adenocarcinoma and sarcoma. Currently, research on the NBE-002 against triple-negative breast cancer, lung cancer and ovarian cancer is in the clinical stage I. The ADC drug VLS-101 developed by Velosbio Company is connected to an MMAE toxin via a degradable linker, and the results of clinical stage I show that the VLS-101 has good treatment prospects, and currently, it has entered the clinical stage II, being an ADC drug targeting R0R1 with fastest progress.

[0010] Antibody-drug conjugate (ADC) is a targeting technology that utilizes a linker to bind a toxin or drug to an antibody. When the antibody recognizes the antigen, the ADC molecule is endocytosed by cells, and then it releases the toxin or drug in the cells, resulting in the death of the cells. As compared with antibody drugs, the ADC technology has higher cell killing capability, and as compared with traditional anticancer drugs, it enhances the targeting property so as to greatly reduce toxic and side effects. Focusing on the development of ADC drugs is therefore a powerful approach to enhance tumor therapy. New ADC drugs also need to be developed.

[0011] SUMMARY

[0012] Ligand-Camptothecin Derivative Conjugates

[0013] A first aspect of the application relates to ligand-camptothecin derivative conjugate represented by Formula I, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof, wherein:

[0014] Ab is an antibody targeting human R0R1 or an antigen-binding fragment thereof;

[0015] L2 has the structure of Formula A: wherein Y is a skeleton selected from the group consisting of C1-C6 alkylene, substituted C1-C6 alkylene and C3-C8 cycloalkylene; Ac is a hydrophilic structural unit; the carbon atom at the position of 2 linked to Y has an absolute chirality of R-configuration or S-configuration;

[0016] L3 is present or absent, and when present, L3 is selected from a PEG hydrophilic unit

[0017] 0 , wherein o is an integer selected from 1 to 10,

[0018] L4 is an enzyme digestion unit;

[0019] L5 is a linking unit; in the formula I, the carbon atom at the position of 1 linked to N has an absolute chirality of R-configuration or S-configuration;

[0020] R is selected from the group consisting of hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl;

[0021] Ri is selected from the group consisting of hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxyl, 3- to 7-membered heterocyclyl, substituted 3- to 7-membered heterocyclyl, C6-C10 aryl, substituted C6-C 10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl;

[0022] R2 is selected from the group consisting of hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxyl, 3- to 7-membered heterocyclyl, substituted 3- to 7-membered heterocyclyl, C6-C10 aryl, substituted C6-C 10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl;

[0023] X is -C(O)-CRaRb-(CR3R4)m-O-, -C(O)-CRaRb-(CR3R4)m-NH- or -C(O)-CRaRb-(CR3R4)m-S-, preferably -C(O)-CRaRb-(CR3R4)m-O-, wherein

[0024] Raand Rb each independently are selected from the group consisting of hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C6-C10 aryl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, 3- to 7-membered heterocyclyl, substituted 3- to 7-membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl; or

[0025] Ra, Rb and carbon atoms linked thereto form C3-C8 cycloalkyl, substituted C3-C8 cycloalkyl, 3- to 7-membered heterocyclyl or substituted 3- to 7-membered heterocyclyl;

[0026] R3, R4 each independently are hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, deuterated C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, amino, cyano, nitro, hydroxy C1-C6 alkyl, C3-C8 cycloalkyl, 3- to 7-membered heterocyclyl, substituted 3- to 7- membered heterocyclyl; or

[0027] R3, R4 and carbon atoms linked thereto form C3-C8 cycloalkyl, substituted C3-C8 cycloalkyl,

[0028] 3- to 7-membered heterocyclyl or substituted 3- to 7-membered heterocyclyl, m is selected from the group consisting of 0, 1, 2, 3 and 4; nl, n2 and n3 each independently are any integer of 0 to 10 or any decimal of 0 to 10, and nl, n2 and n3 are not 0 simultaneously, with l<nl+n2+n3<10.

[0029] In some embodiments, L11, L12 and L13 each independently are selected from the group consisting of:

[0030] In some embodiments, L11, L12 and L13 each independently are selected from the group consisting of: wherein:

[0031] Z is carboxyl, phosphoryloxy or -(OCH2CH2)iOCH3, wherein i is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and preferably i is 1, 2, 3, 4, 5, 6, 7 or 8;

[0032] Y’ is a skeleton linking amino group and Z, and it is C1-C6 alkylene or carboxyl-substituted C1-C6 alkylene, preferably methylene, ethylidene, carboxyl-substituted methylene or carboxylsubstituted ethylidene, and further preferably methylene, ethylidene, or carboxyl-substituted methylene.

[0033] In some embodiments, Ac is a residue formed by removing one hydrogen atom from the amino terminus of glycine, (D / L)alanine, (D / L)leucine, (D / L)isoleucine, (D / L)valine, (D / L)phenyl alanine, (D / L)proline, (D / L)(tryptophan, (D / L)serine, (D / L)tyrosine, (D / L()cysteine, (D / L()cy stine, (D / L)arginine, (D / L)histidine. (D / L)methionine, (D / L)asparagine, (D / L)glutamine, (D / L)threonine, (D / L)aspartic acid or (D / L)glutamic acid.

[0034] In some embodiments, Ac is:

[0035] In some embodiments, L4 is a peptide residue composed of amino acids, wherein the amino acids are optionally substituted by one or more substituents selected from the group consisting of deuterium atom, halogen, hydroxyl, cyano, amino, nitro, carboxyl, C1-C6 alkyl, substituted Cl- C6 alkyl, C1-C6 alkoxy and C3-C8 cycloalkyl and substituted C3-C8 cycloalkyl.

[0036] In some embodiments, the peptide residue is a peptide residue formed by one, two or more amino acids selected from the group consisting of phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline (C), serine (S), glutamic acid (E) and aspartic acid (D).

[0037] In some embodiments, the peptide residue is a tetrapeptide residue formed by glycine (G) - glycine (G) - phenylalanine (F) - glycine (G).

[0038] In some embodiments, the peptide residue is -GGFG-.

[0039] In some embodiments, L5 is -NR5(CRgR7)q- or a chemical bond, wherein q is 0, 1, 2, 3, 4, 5 or 6;

[0040] Rs, Re and R7 each independently are selected from the group consisting of hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, 3- to 7-membered heterocyclyl, substituted 3- to 7-membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl.

[0041] In some embodiments, q is 0, 1, 2 or 3. In some embodiments, q is 0, 1 or 2. In some embodiments, q is 0 or 1.

[0042] In some embodiments, R5, Re and R7 each independently are selected from the group consisting of hydrogen atom and C1-C6 alkyl.

[0043] In some embodiments, R5, Re and R7 each independently are selected from the group consisting of hydrogen atom and C1-C4 alkyl.

[0044] In some embodiments, R5, Re and R7 each independently are selected from the group consisting of hydrogen atom, methyl, ethyl, n-propyl and n-butyl.

[0045] In some embodiments, R5, Re and R7 each independently are hydrogen atom.

[0046] In some embodiments, Ra and Rb each independently are selected from the group consisting of hydrogen atom, C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl and C6- C10 aryl C1-C6 alkyl; or Ra, Rb and carbon atoms linked thereto form C3-C8 cycloalkyl.

[0047] In some embodiments, Ra is hydrogen atom or C1-C4 alkyl, Rb is hydrogen atom, C1-C4 alkyl, halogenated C1-C4 alkyl, C3-C6 cycloalkyl C1-C4 alkyl or phenyl C1-C4 alkyl. In some embodiments, R , Rb and carbon atoms linked thereto form C3-C6 cycloalkyl.

[0048] In some embodiments, Ra is hydrogen atom, methyl, ethyl, n-propyl or n-butyl, Rb is hydrogen atom, methyl, ethyl, n-propyl, n-butyl, halogenated methyl, halogenated ethyl, halogenated n-propyl, halogenated n-butyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclopropylethyl, cyclopentylethyl, cyclohexyl ethyl, phenylmethyl, phenylethyl or phenylpropyl.

[0049] In some embodiments, Ra, Rb and carbon atoms linked thereto form cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0050] In some embodiments, Ra is hydrogen atom or methyl, Rb is hydrogen atom, methyl, ethyl, trifluoromethyl, cyclopropylmethyl, or phenylmethyl. In some embodiments, Ra, Rb and carbon atoms linked thereto form cyclopropyl, cyclobutyl or cyclopentyl. y v y y.

[0051] In some embodiments, X is 0 , 0 , preferably, the position shown by the right side wavy line is connected to L5.

[0052] In some embodiments, R3, R4 each independently are hydrogen atom or C1-C6 alkyl.

[0053] In some embodiments, m is 0, 1 or 2. In some embodiments, m is 0 or 1.

[0054] In some embodiments, R is hydrogen atom or C1-C6 alkyl. In some embodiments, R is hydrogen atom or C1-C4 alkyl. In some embodiments, R is hydrogen atom, methyl, ethyl, n-propyl or n-butyl. In some embodiments, R is hydrogen atom or methyl.

[0055] In some embodiments, Ri is hydrogen atom or C1-C6 alkyl. In some embodiments, Ri is Cl- C6 alkyl. In some embodiments, Ri is C1-C4 alkyl. In some embodiments, Ri is methyl, ethyl, n- propyl or n-butyl. In some embodiments, Ri is methyl.

[0056] In some embodiments, R2 is hydrogen atom, halogen or C1-C6 alkyl. In some embodiments, R2 is hydrogen atom, halogen or C1-C4 alkyl. In some embodiments, R2 is halogen. In some embodiments, R2 is fluorine, chlorine, or bromine. In some embodiments, R2 is fluorine.

[0057] In some embodiments, o is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, 0 is 1, 2, 3, 4,

[0058] 5, 6, 7 or 8.

[0059] In some embodiments, Y is C1-C6 alkylene. In some embodiments, Y is C1-C4 alkylene. In some embodiments, Y is methylene, ethylidene, propylidene or butylidene. In some embodiments, Y is methylene.

[0060] In some embodiments, nl, n2 and n3 each independently are any integer of 0 to 8 or any decimal of 0 to 8, and nl, n2 and n3 are not 0 simultaneously, with I<nl+n2+n3<8. In some embodiments, 5<nl+n2+n3<8. In some embodiments, 6<nl+n2+n3<8. In some embodiments, 7<nl+n2+n3<8.

[0061] In some embodiments, the antibody targeting human ROR1 or the antigen-binding fragment thereof comprises:

[0062] (a) the following three complementarity determining regions (CDRs) in heavy chain variable region (VH):

[0063] (i) VH CDR1, having a CDR1 sequence contained in the VH as set forth in SEQ ID NO:1, or having a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR1 sequence contained in the VH;

[0064] (ii) VH CDR2, having a CDR2 sequence contained in the VH as set forth in SEQ ID NO: 1, or having a sequence with substitution, deletion or addition of one or more amino acids (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR2 sequence contained in the VH; and

[0065] (iii) VH CDR3, having a CDR3 sequence contained in the VH as set forth in SEQ ID NO: 1, or having a sequence with substitution, deletion or addition of one or more amino acids (e.g., substitution, deletion or addition of one amino acid) as compared with the CDR3 sequence contained in the VH; and / or

[0066] (b) the following three CDRs in light chain variable region (VL):

[0067] (iv) VL CDR1, having a CDR1 sequence contained in the VL as set forth in SEQ ID NO:5, or having a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR1 sequence contained in the VL;

[0068] (v) VL CDR2, having a CDR2 sequence contained in the VL as set forth in SEQ ID NO: 5, or having a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR2 sequence contained in the VL; and

[0069] (vi) VL CDR3, having a CDR3 sequence contained in the VL as set forth in SEQ ID NO: 5, or having a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR3 sequence contained in the VL.

[0070] In some embodiments, the substitution described in any one of (i) to (vi) is a conservative substitution.

[0071] In some embodiments, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH), and / or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by Kabat, Chothia or IMGT numbering system.

[0072] In some embodiments, the antibody targeting human R0R1 or the antigen-binding fragment thereof comprises:

[0073] CDR1, CDR2 and CDR3 sequences contained in the VH as set forth in SEQ ID NO:1; and / or CDR1, CDR2 and CDR3 sequences contained in the VL as set forth in SEQ ID NO:5.

[0074] In some embodiments, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH) and / or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by Kabat, Chothia or IMGT numbering system.

[0075] In some embodiments, the antibody targeting human R0R1 or the antigen-binding fragment thereof comprises:

[0076] (a) the following three CDRs in heavy chain variable region (VH):

[0077] (i) VH CDR1, which is composed of a sequence as set forth in SEQ ID NO:2,

[0078] (ii) VH CDR2, which is composed of a sequence as set forth in SEQ ID NOG, and

[0079] (iii) VH CDR3, which is composed of a sequence as set forth in SEQ ID NOH; and / or

[0080] (b) the following three CDRs in light chain variable region (VL):

[0081] (iv) VL CDR1, which is composed of a sequence as set forth in SEQ ID NO:6,

[0082] (v) VL CDR2, which is composed of a sequence as set forth in SEQ ID NO:7, and

[0083] (vi) VL CDR3, which is composed of a sequence as set forth in SEQ ID NOG.

[0084] In some embodiments, the VH of the antibody targeting human R0R1 or the antigen-binding fragment thereof comprises: VH CDR1 as set forth in SEQ ID NOG; VH CDR2 as set forth in SEQ ID NOG; and, VH CDR3 as set forth in SEQ ID NO:4; and / or, the VL of the antibody targeting human R0R1 or the antigen-binding fragment thereof comprises: VL CDR1 as set forth in SEQ ID NO:6; VL CDR2 as set forth in SEQ ID NO:7; and VL CDR3 as set forth in SEQ ID NO:8

[0085] In some embodiments, the VH of the antibody targeting human R0R1 or the antigen-binding fragment thereof comprises: VH CDR1 as set forth in SEQ ID NOG; VH CDR2 as set forth in SEQ ID NOG; and VH CDR3 as set forth in SEQ ID NOG; and the VL of the antibody targeting human R0R1 or the antigen-binding fragment thereof comprises: VL CDR1 as set forth in SEQ ID NO:6; VL CDR2 as set forth in SEQ ID NOG; and VL CDR3 as set forth in SEQ ID NOG.

[0086] In some embodiments, the antibody targeting human R0R1 or the antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH), comprising an amino acid sequence selected from the following sequences:

[0087] (i) a sequence as set forth in SEQ ID NO: 1;

[0088] (ii) a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence as set forth in SEQ ID NO: 1

[0089] (iii) a sequence with a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence as set forth in SEQ ID NO: 1; and

[0090] (b) a light chain variable region (VL), comprising an amino acid sequence selected from the following sequences:

[0091] (iv) a sequence as set forth in SEQ ID NO:5;

[0092] (v) a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence as set forth in SEQ ID NO: 5;

[0093] (vi) a sequence with a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence as set forth in SEQ ID NO: 5.

[0094] In some embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0095] In some embodiments, the antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region (VH), comprising a sequence as set forth in SEQ ID NO: 1 or a sequence with a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence as set forth in SEQ ID NO: 1, and a light chain variable region (VL), comprising a sequence as set forth in SEQ ID NO: 5 or a sequence with a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence as set forth in SEQ ID NO:5.

[0096] In some embodiments, the antibody targeting human ROR1 or the antigen-binding fragment thereof comprises: a VH with a sequence as set forth in SEQ ID NO: 1 and a VL with a sequence as set forth in SEQ ID NO:5.

[0097] In some embodiments, the antibody targeting human ROR1 or the antigen-binding fragment thereof is humanized.

[0098] In some embodiments, the antibody targeting human ROR1 or the antigen-binding fragment thereof further comprises a framework region of a human immunoglobulin.

[0099] In some embodiments, the antibody targeting human ROR1 or the antigen-binding fragment thereof further comprises a heavy chain framework region of a human immunoglobulin (e.g., a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline antibody gene), and / or, a light chain framework region of a human immunoglobulin (e.g., a light chain framework region contained in an amino acid sequence encoded by a human light chain germline antibody gene).

[0100] In some embodiments, the heavy chain framework region and / or the light chain framework region optionally comprises one or more (such as, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to murine residues. In some embodiments, the antibody targeting human R0R1 or the antigen-binding fragment thereof comprises: a heavy chain with a sequence as set forth in SEQ ID NO:9 and a light chain with a sequence as set forth in SEQ ID NO: 10.

[0101] In some embodiments, the antibody or the antigen-binding fragment thereof further comprises a constant region derived from a human immunoglobulin.

[0102] In some embodiments, the heave chain of the antibody or the antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (such as IgGl, IgG2, IgG3 or IgG4).

[0103] In some embodiments, the light chain of the antibody or the antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (such as K or Z).

[0104] In some embodiments, the antibody targeting human ROR1 or the antigen-binding fragment thereof are selected from the group consisting of monoclonal antibody, mouse antibody, rabbit antibody, humanized antibody, fully human antibody, chimeric antibody (e.g., human-mouse chimeric antibody), bispecific antibody, multi-specific antibody, single chain antibody, dAb, complementarity determining region fragment, Fv, single chain Fv (scFv), Fd, Fab, Fab', and F(ab')2.

[0105] In some embodiments, the monoclonal antibody includes a non-CDR region, and the non- CDR region is derived from species other than murine, e g., from a human antibody.

[0106] In some embodiments, in Formula I, the linking unit -L11-L2-L3-L4-L5-, -L12-L2-L3-L4-L5- or -L13-L2-L3-L4-L5- each independently is selected from the group consisting of: wherein:

[0107] Ac, o, Rs, Re and R7 are as defined in any one embodiment of the application; the carbon atom at the position of 2 linked to N has an absolute chirality of R-configuration or S-configuration; the position shown by the left side wavy line is connected to the antibody or the antigenbinding fragment thereof, and the position shown by the right side wavy line is connected to X.

[0108] In some embodiments, in Formula I, -L11-L2-L3-L4-L5-, -L12-L2-L3-L4-L5- or -L13-L2-L3-L4-

[0109] L5- each independently is selected from the group consisting of: wherein: Ac, o, Rs, Re and R7 are as defined in any one embodiment of the application; the carbon atom at the position of 2 linked to N has an absolute chirality of R-configuration or S-configuration; the position shown by the left side wavy line is connected to the antibody or the antigenbinding fragment thereof, and the position shown by the right side wavy line is connected to X.

[0110] In some embodiments, the conjugate of Formula I has a structure represented by Formula II: wherein: Ab, Ln, L12, L13, Ac, L3, X, R, Ri, R2, nl, n2, n3 are as defined in any one embodiment of the application; the chiral carbon atom at the position of 1, 2 or 3 has an absolute chirality ofR-configuration or S-configuration.

[0111] In some embodiments, the conjugate of Formula I is selected from the group consisting of:

[0112] 

[0113] 13C1 represents Ab; Ab, nl, n2 and n3 as defined in any one embodiment of the application.

[0114] In some embodiments, the pharmaceutically acceptable salt of the conjugate represented by Formula I includes a salt formed by an acidic functional group in the structural formula with sodium, potassium, calcium or magnesium, or an acetate, a trifluoroacetate, a citrate, an oxalate, a tartrate, a malate, a nitrate, a chloride, a bromide, an iodide, a sulfate, a bisulfate, a phosphate, a lactate, an oleate, an ascorbate, a salicylate, a formate, a glutamate, a methanesulfonate, an ethanesulfonate, a benzenesulfonate or a p-toluenesulfonate formed by a basic functional group in the structural formula with an acid.

[0115] A second aspect of the application relates to a pharmaceutical composition, which comprises the conjugate represented by Formula I, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof according to any one embodiment of the application, and optionally a pharmaceutically acceptable carrier.

[0116] A third aspect of the application relates to a pharmaceutical preparation, which comprises the conjugate represented by Formula I, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof according to any one embodiment of the application.

[0117] A fourth aspect of the application provides use of the conjugate represented by Formula I, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition and / or the pharmaceutical preparation according to any one embodiment of the application in the manufacture of a medicament for treating or preventing a cancer or tumor.

[0118] In some embodiments, the cancer or tumor expresses R0R1.

[0119] In some embodiments, the cancer or tumor is selected from solid tumor and hematologic tumor, such as adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urinary tract cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, breast cancer (e.g., triplenegative breast cancer), rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma and leukemia.

[0120] Linker-Drug Conjugate

[0121] A fifth aspect of the application provides a linker-drug conjugate represented by Formula III- A or Formula III-B, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, 2, L4, L5, X, R, Ri, R2, Ac, L3, m, Ra, Rb are as defined in any one embodiment according to the first aspect of the application; the chiral carbon atom at the position of 1, 2 or 3 has an absolute chirality of R-configuration or S-configuration.

[0122] In some embodiments, the linker-drug conjugate represented by Formula III-A or Formula III-B is selected from the group consisting of:

[0123] wherein: o is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably, o is 1, 2, 3, 4, 5, 6, 7 or 8.

[0124] In some embodiments, the pharmaceutically acceptable salt of the linker-drug conjugate represented by Formula III-A or Formula III-B is a salt formed by an acidic functional group in the structural formula with sodium, potassium, calcium or magnesium, or an acetate, a trifluoroacetate, a citrate, an oxalate, a tartrate, a malate, a nitrate, a chloride, a bromide, an iodide, a sulfate, a bisulfate, a phosphate, a lactate, an oleate, an ascorbate, a salicylate, a formate, a glutamate, a methanesulfonate, an ethanesulfonate, a benzenesulfonate or a p-toluene sulfonate formed by a basic functional group in the structural formula with an acid.

[0125] In some embodiments, in the ligand-camptothecin derivative conjugate represented by Formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, the camptothecin derivative has a structure represented by Formula d: wherein R, Ri, R2, m, Ra, Rb are as defined in any one embodiment according to the first aspect of the application.

[0126] In some embodiments, the compound represented by Formula d is selected from the group consisting of:

[0127]

[0128] The application also relates to a use of the linker-drug conjugate represented by Formula III- A or Formula III-B in the manufacture of an antibody-drug conjugate, in particular in the manufacture of the conjugate represented by Formula I, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof according to the first aspect of the application.

[0129] Preparation Process

[0130] The application also relates to a process for preparing the ligand-camptothecin derivative conjugate represented by Formula I or Formula II, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof according to the first aspect of the application, comprising: conjugating a reduced antibody or an antigen-binding fragment thereof with a linker-drug compound, to obtain the ligand-camptothecin derivative conjugate represented by Formula I or Formula II,

[0131] wherein the chiral carbon atom at the position of 1, 2 or 3 has an absolute chirality of R- configuration or S-configuration;

[0132] Ab, L, Ln, L12, L13, L2, L3, L4, L5, X, R, Ri, R2, nl, n2 or n3 are described as above.

[0133] In the above aspects of the application and embodiments thereof, the “C1-C6 alkyl” and the “C1-C6 alkyl” in various complex groups involving “C1-C6 alkyl” (such as, “substituted C1-C6 alkyl”, or “deuterated C1-C6 alkyl”) may be replaced with “C1-C20 alkyl”, “C1-C12 alkyl” or “C1-C10 alkyl”; the “C3-C8 cycloalkyl” and the “C3-C8 cycloalkyl” in various complex groups involving “C3-C8 cycloalkyl” may be replaced with “C3-C20 cycloalkyl” or “C3-C10 cycloalkyl”; the “C1-C6 alkoxy” and the “C1-C6 alkoxy” in various complex groups involving “C1-C6 alkoxy” may be replaced with “C1-C20 alkoxy”, “Cl -Cl 2 alkoxy” or “Cl -CIO alkoxy”; the “C6-C10 aryl” and the “C6-C10 aryl” in various complex groups involving “C6-C10 aryl” may be replaced with “C6-C12 aryl”; the “3- to 7-memberedheterocyclyl” and the “3- to 7-membered heterocyclyl” in various complex groups involving “3- to 7-membered heterocyclyl” may be replaced with a “3- to 20- membered heterocyclyl”, a “3- to 12-membered heterocyclyl” or a “3- to 10-membered heterocyclyl”.

[0134] Beneficial Effects

[0135] As compared to existing drugs of the same type, the anti-human R0R1 antibody-drug conjugate as provided by the application, being an antibody-drug conjugate obtained by conjugating a humanized anti-human R0R1 antibody with a camptothecin derivative, has a good molecular stability and good pre-clinical therapeutic effects, and it is expected to have excellent clinical therapeutic effects, with great development values and treatment potential.

[0136] BRIEF DESCRIPTION OF THE DRAWINGS

[0137] FIG. 1 A shows a chromatogram of ADC-6 aggregation detected by SEC-HPLC;

[0138] FIG. IB shows a chromatogram of ADC- 107 aggregation detected by SEC-HPLC;

[0139] FIG. 1C shows a chromatogram of ADC- 108 aggregation detected by SEC-HPLC;

[0140] FIG. 2A shows a chromatogram of drug-to-antibody ratio (DAR) of ADC-6 detected by RP- HPLC;

[0141] FIG. 2B shows a chromatogram of drug-to-antibody ratio (DAR) of ADC- 107 detected by RP-HPLC;

[0142] FIG. 2C shows a chromatogram of drug-to-antibody ratio (DAR) of ADC- 108 detected by RP-HPLC;

[0143] FIG. 3A shows a fitting curve of the relative affinity of ADC-6 and its corresponding antibody 13C1 to RORl;

[0144] FIG. 3B shows a fitting curve of the relative affinity of ADC- 107 and its corresponding antibody 13C9 to R0R1;

[0145] FIG. 3C shows a fitting curve of the relative affinity of ADC- 108 and its corresponding antibody NBE-002 to ROR1 ;

[0146] FIG. 4 A shows in vitro tumor inhibitory activity of ADC-6, ADC- 107 and ADC- 108 in an experimental model of the humanized cell strain RPMI-8226;

[0147] FIG. 4B shows in vitro tumor inhibitory activity of ADC-6, ADC- 107 and ADC- 108 in an experimental model of the humanized cell strain NCI-N87;

[0148] FIG. 4C shows in vitro tumor inhibitory activity of ADC-6, ADC- 107 and ADC- 108 in an experimental model of the humanized cell strain MDA-MB-468;

[0149] FIG. 4D shows in vitro tumor inhibitory activity of ADC-6, ADC-107 and ADC-108 in an experimental model of the humanized cell strain JVM-3;

[0150] FIG. 5 A shows tumor inhibitory effects of ADC-6, ADC- 107 and ADC- 108 in an in vivo NCI-N87 tumor model;

[0151] FIG. 5B shows tumor inhibitory effects of ADC-6, mAb and payload at different doses in an in vivo HCC1187 tumor model;

[0152] FIG. 5C shows tumor inhibitory effects of ADC-6, mAb and payload at different doses in an in vivo NCLN87 tumor model;

[0153] FIG. 5D shows tumor inhibitory effects of ADC-6, mAb and payload at different doses in an in vivo NCLH1975 tumor model; FIG. 5E shows tumor inhibitory effects of ADC-6, mAh and payload at different doses in an in vivo MDA-MB-231 tumor model;

[0154] FIG. 6A shows tumor inhibitory effects of ADC-6 and ADC-107 in an in vivo HEL92.1.7- R0R1 #5A7 tumor model.

[0155] DETAILED DESCRIPTION

[0156] Abbreviations and definitions

[0157] Unless otherwise indicated, the following terms and phrases as used herein are intended to have the following meanings. When brand names are used herein, the brand names, unless otherwise indicated in the context, include the product formulations, the general drugs and the active ingredients of products with the brand names.

[0158] Unless otherwise indicated, the terms used in the claims and specification herein have the following meanings.

[0159] The term “ligand” is a macromolecular compound capable of identifying and binding to an antigen or receptor associated with a target cell. The ligand functions to present a drug to a target cell population binding to the ligand, including, but not being limited to, protein hormone, lectin, growth factor, antibody, or other molecules capable of binding to cells. In an embodiment of the application, the ligand, expressed as Ab, may form a linking bond with a linking unit through a heteroatom in the ligand, preferably is an antibody or an antigen-binding fragment thereof, which is selected from the group consisting of a chimeric antibody, a humanized antibody, fully human antibody and a mouse antibody, preferably a monoclonal antibody.

[0160] In the application, the ligand unit is a targeting agent that specifically binds to a target. The ligand can specifically bind to a cell component or to other target molecules of interest. The target part or the target is usually on the surface of the cell. In some aspects, the ligand unit functions to deliver a drug unit to a specific target cell population interacting with the ligand unit. The ligands include, but are not limited to, proteins, polypeptides and peptides, as well as non-proteins such as sugars. Suitable ligand units include, for example, antibodies such as full-length (intact) antibodies and antigen-binding fragments thereof. In embodiments where the ligand unit is a nonantibody targeting agent, the ligand may be a peptide or a polypeptide, or a non-proteinaceous molecule. Examples of such targeting agents include interferons, lymphokines, hormones, growth factors and colony stimulating factors, vitamins, nutrient transport molecules, or any other cell binding molecules or substances. In some embodiments, a linker is covalently linked to the sulfur atom of the ligand. In one aspect, the sulfur atom is a sulfur atom of a cysteine residue, which forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is a sulfur atom of a cysteine residue which has been introduced into the ligand unit, which forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is a sulfur atom of a cysteine residue that has been introduced into the ligand unit e.g., by site-directed mutagenesis or chemical reaction. In other aspects, the sulfur atom linked to the linker is selected from a cysteine residue that forms an interchain disulfide bond of the antibody and a cysteine residue that has been introduced into the ligand unit (e.g., by site-directed mutagenesis or chemical reaction). In some embodiments, numbering is performed according to the EU index numbering system in Kabat {[Kabat E.A et al, (1991)], Sequences of Proteins of Immunological Interest, fifth edition, NIH publication 91-3242}.

[0161] The “antibody” or “antibody unit” includes within the scope thereof any one part of an antibody structure. This unit may bind, reactively associate, or complex with a receptor, an antigen or other receptor units possessed by the targeted cell population. The antibody may be any protein or proteinaceous molecule, which can bind, complex, or react with a part of the cell population to be treated or to be biologically modified. The antibody constituting the antibody-drug conjugate in the application retain its antigen-binding ability in the originally wild state. Thus, the antibody of the application can specifically bind to an antigen. Involved antigens include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival regulatory factors, cell proliferation regulatory factors, molecules associated with tissue growth and differentiation (e.g., known or foreseen to be functional), lymphokines, cytokines, molecules involved in the regulation of cell circulation, molecules involved in angiogenesis, and molecules associated with angiogenesis (e.g., known or foreseen to be functional). The tumor-associated factor may be a cluster of differentiation factor (e g., a CD protein).

[0162] Antibodies used in the antibody drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known in the art and can be prepared by well-known methods and information for preparing antibodies in the art. In order to develop effective cell-level targets for cancer diagnosis and treatment, researchers seek to find transmembrane or other tumor-associated polypeptides. These targets can be specifically expressed on the surface of one or more cancer cells, but less expressed or not expressed on the surface of one or more non-cancer cells. In general, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells than on the surface of noncancer cells. The identification of the tumor-associated factors can greatly improve the specific targeting characteristic of the antibody-based cancer treatment. For convenience, information related to antigens well known in the art is indicated below, including name, other names, and GenBank accession numbers. Nucleic acid and protein sequences correspondent to the tumor- associated antigens can refer to public databases, such as Genbank. The tumor-associated antigens to which the antibody targets include all variants and congeners of amino acid sequences, having a homology of at least 70%, 80%, 85%, 90% or 95% to the sequences identified in the references, or having biological properties and characteristics completely consistent to the sequences of the tumor-associated antigen in the references.

[0163] The term “inhibit” or “inhibition of’ refers to reducing detectable amount, or completely preventing.

[0164] The term “cancer” refers to the physiological condition or disease characterized by unregulated cell growth. A “tumor” comprises cancerous cells.

[0165] The term “autoimmune disease” is a disease or disorder derived from tissues or proteins of an individual itself.

[0166] The term “drug” refers to a cytotoxic drug, expressed as “d”, being a chemical molecule which, in tumor cells, has a strong ability to destroy normal growth of the tumor cells. The cytotoxic drug can kill tumor cells in principle at a sufficiently high concentration, but due to a lack of the specificity, they will lead to the apoptosis of normal cells while killing tumor cells, resulting in serious side effects. The term includes toxins such as small molecule toxins or enzymatically active toxins which are derived from bacterium, fungus, plant or animal, radioisotopes (e.g., radioactive isotopes of At211, 1131, 1125, Y90, Re186, Re188, Sm153, Bi212, P32and Lu176), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes, preferably toxic drugs. In the application, the term “linker” or “linking fragment” or “linking unit” refers to a chemical structural fragment or bond of which one end is linked to a ligand and the other end is linked to a drug directly or by other linkers.

[0167] The linker, including an extender, a spacer and an amino acid unit, can be synthesized by methods known in the art, for example those described in US2005-0238649A1. The linker may be a “cleavable linker” that facilitates the release of drugs in cells. For example, acid-labile linkers (e.g., hydrazones), protease sensitive (e.g., peptidase-sensitive) linkers, photo-labile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, Cancer Research 52: 127-131, 1992; U.S. patent No.5, 208, 020.

[0168] As used herein, the “linker” or the “linker of an antibody-drug conjugate” can be classified into two classes: non-cleavable linkers and cleavable linkers, according to the mechanisms of drug release in cells. As to the antibody-drug conjugate containing a non-cleavable linker, its drug release mechanism is that after the conjugate binds to an antigen and endocytosed by cells, the antibody is enzymolyzed in lysosomes, to release active molecules consisting of a small-molecule drug, a linker and antibody amino acid residues together. The resulted structural changes in the drug molecule do not reduce its cytotoxicity, but because the active molecules are charged (amino acid residues), they cannot permeate into neighboring cells. Thus, such active drugs cannot kill neighboring tumor cells that do not express the target antigen (antigen negative cells) (bystander effect) (Ducry et al, 2010, Bioconjugate Chem. 21: 5-13). As to the antibody-drug conjugate containing a cleavable linker, its drug release mechanism is that after the conjugate binds to an antigen and endocytosed by cells, it is cleaved in the target cells and releases the active ingredient (the small molecule drug itself). The cleavable linkers are mainly divided into: chemosensitive linkers and enzyme sensitive linkers. The chemosensitive linkers can be selectively cleaved due to differences in the nature of plasma and cytoplasmic or tumor microenvironment, such as pH, and glutathione concentration. The pH sensitive linkers, such as hydrazones, carbonates, acetals, and ketals, are relatively stable in neutral or weakly alkaline environment of blood (pH 7.3-7.5), but will be hydrolyzed in weakly acidic tumor microenvironment (pH of 5.0-6.5) and in lysosomes (pH of 4.5-5.0). Since the acid-cleavable linkers have very limited plasma stability, the antibodydrug conjugates based on such linkers usually have a short half-life (2-3 days). This short half-life limits the use of the pH sensitive linkers in the new generation of antibody-drug conjugates to some extent. Linkers sensitive to glutathione are also called as disulfide linkers. The release of drug is caused based on the difference between high glutathione concentrations (millimolar range) in cells and relatively low glutathione concentrations in blood (micromolar range). This is especially true for a tumor cell, where its low oxygen content leads to an enhanced activity of the reductase, thereby resulting in a higher glutathione concentration. The disulfide bond has a thermodynamical stability, and thus it can have a good stability in plasma. The enzyme labile linkers, e.g., a peptide linker, can better control drug release. The peptide linkers can be efficiently cleaved by a protease in the lysosomes, e.g., cathepsin B. This peptide linkage is considered to be very stable in the plasma circulation due to the unsuitable extracellular pH value and the extracellular inactivity of protease caused by serum protease inhibitors. In view of a high plasma stability and good selectivity and efficiency of intracellular cleavage, the enzyme labile linkers are widely used as cleavable linkers for antibody-drug conjugates.

[0169] The term “antibody-drug conjugate” refers to linking an antibody to a biologically active drug via a stable linking unit. In the application, the “ligand-drug conjugate” is preferably an antibody drug conjugate (ADC), which refers to linking a monoclonal antibody or an antibody fragment to a biologically active toxic drug via a stable linking unit.

[0170] Three-letter codes and one-letter codes for amino acids used in the application are as described in J. boil. Chem. 1968, 243, 3558.

[0171] The term “alkyl” refer to a saturated aliphatic hydrocarbon group, which is a linear or branched group containing 1 to 20 carbon atoms (i.e., “C1-C20 alkyl”), preferably an alkyl group containing 1 to 12 carbon atoms (i.e., “C1-C12 alkyl”), more preferably an alkyl group containing 1 to 10 carbon atoms (i.e., “C1-C10 alkyl”), and most preferably an alkyl group containing 1 to 6 carbon atoms (i.e.,“Cl-C6 alkyl”).Non-limiting examples of the alkyl group include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, sec-butyl, n-pentyl, 1 , 1 -dimethylpropyl,

[0172] 1.2-dimethylpropyl, 2,2-dimethylpropyl, 1 -ethylpropyl, 2-methylbutyl, 3 -methylbutyl, n-hexyl,

[0173] 1-ethyl-2-methylpropyl, 1 , 1 ,2-trimethylpropyl, 1,1 -dimethylbutyl, 1,2-dimethylbutyl, 2,2- dimethylbutyl, 1,3 -dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3 -methylpentyl, 4-methylpentyl,

[0174] 2.3 -dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5 -methylhexyl, 2,3- dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3, 3 -dimethylpentyl, 2-ethylpentyl, 3- ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2- dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl,

[0175] 2-methyl-2-ethylpentyl, 2-methyl-3 -ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3- ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers and the like. The more preferred alkyl group is a lower alkyl containing 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, sec-butyl, n-pentyl, 1 , 1 -dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1- ethylpropyl, 2-methylbutyl, 3 -methylbutyl, n-hexyl, l-ethyl-2-methylpropyl, 1,1,2- trimethylpropyl, 1,1 -dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 -dimethylbutyl, 2- ethylbutyl, 2-methylpentyl, 3 -methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl and the like. The alkyl may be substituted or non-substituted, and when it is substituted, it may be substituted at any useful linking sites, and the substituents are preferably one or more independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.

[0176] The term “substituted alkyl” refers to an alkyl of which hydrogen is substituted with substituents, unless otherwise indicated herein, the substituents of the alkyl may be one or more groups selected from the following group consisting of: -halogen, -OR’, -NR’R”, -SR’, - SiR’R”R”, -OC(O)R’, -C(O)R’, -CO2R’, -CONR’R”, -OC(O)NR’R”, -NR”C(O)R’, -NR’- C(0)NR”R’”, -NR”C(0)2R’, -NH-C(NH2)=NH, -NR’C(NH2)=NH, -NH-C(NH2)=NR’, -S(O)R’, -S(O)2R’, -S(O)2NR’R”, -NR’S(O)2R”, -CN and -NO2, and the number of the substituents may be 0 to (2m’+l), wherein m’ is the total number of the carbon atoms in the group. R’, R” and R’” each independently represent hydrogen, unsubstituted Cus alkyl, unsubstituted C6-C12 aryl (or C6-C10 aryl), C6-C12 aryl (or C6-C10 aryl) substituted with 1 to 3 halogens, unsubstituted Ci-s alkyl, Ci-8 alkoxy or Cns thioalkoxy, or unsubstituted C6-C12 aryl (or C6-C10 aryl)-Ci-4 alkyl. When R’ and R” are linked to the same one nitrogen atom, they together with the nitrogen atom may form a 3-, 4-, 5-, 6- or 7-memebered ring. For example, -NR’R” includes 1-pyrrolidyl and 4- morpholinyl.

[0177] The term “alkylene” refers to a linear or branched aliphatic hydrocarbon group, which has two residues derived from the removal of two hydrogen atoms on the same carbon atom or on two different carbon atoms of the parent alkyl, and which is a linear or branched group containing 1 to 20 carbon atoms, preferably an alkylene group containing 1 to 12 carbon atoms, and more preferably an alkylene group containing 1 to 6 carbon atoms. Non-limiting examples of the alkylene include, but not are limited to, methylene (-CH2-, 1 , 1 -ethylidene(-CH(CH3)-), 1,2- ethylidene (-CH2CH2)-,l,l-propylidene (-CH(CH2CH3)-), 1,2-propylidene (-CH2CH(CH3)-), 1,3- propylidene (-CH2CH2CH2-), 1,4-butylidene (-CH2CH2CH2CH2-) and 1,5-pentylidene (- CH2CH2CH2CH2CH2-) and the like. The alkylene may be substituted or unsubstituted, and when it is substituted, it may be substituted at any useful linking sites, and the substituents are preferably one or more independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.

[0178] The term “alkoxy” refers to -O-(alkyl) and -O-(cycloalkyl), wherein alkyl or cycloalkyl are as defined above. Non-limiting examples of the C1-C6 alkoxy include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy. The alkoxy may be optionally substituted or unsubstituted, and when it is substituted, the subsituents are preferably one or more independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0179] The term “cycloalkyl” refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, and the ring of the cycloalkyl contains 3 to 20 carbon atoms (i.e., “C3- C20 cycloalkyl”), preferably 3 to 12 carbon atoms (i.e., “C3-C12 cycloalkyl”), more preferably 3 to 10 carbon atoms (i.e., “C3-C10 cycloalkyl”), and most preferably 3 to 8 carbon atoms (i.e., “C3- C8 cycloalkyl”). Non-limiting examples of the monocyclic cycloalkyl (e.g., “C3-C8 cycloalkyl”) include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclopentatrienyl, cyclooctyl and the like; the polycyclic cycloalkyl include spiro, fused and bridged cycloalkyl.

[0180] The term “heterocyclyl” refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, which comprises 3 to 20 ring atoms (i.e., “3- to 20- membered heterocyclyl s”), wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, and S(O)m(where m is an integer of 0 to 2), but not include a cyclic moiety of- O-O-, -O-S-, or-S-S-, with the remaining ring atoms being carbon. Preferably, the heterocyclyl contains 3 to 12 ring atoms (i.e., “3- to 12-membered heterocyclyl s”), of which 1 to 4 atoms are heteroatoms. More preferably, the heterocyclyl contains 3 to 10 ring atoms (i.e., “3- to 10- membered heterocyclyls”). Non-limiting examples of monocyclic heterocyclyls (e.g., 3- to 7- membered heterocyclyls) include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclyls include spiro, fused, and bridged heterocyclyls.

[0181] The term “cycloalkylalkyl” refers to an alkyl group substituted by one or more cycloalkyl groups, preferably by one cycloalkyl group, wherein the alkyl group is as defined above, and wherein the cycloalkyl group is as defined above, for example, C3-C8 cycloalkyl C1-C6 alkyl.

[0182] The term “haloalkyl” refers to an alkyl group substituted with one or more halogens, wherein the alkyl group is as defined above, for example, halogenated C1-C6 alkyl.

[0183] The term “deuterated alkyl” refers to an alkyl group substituted with one or more deuterium atoms, wherein the alkyl group is as defined above, for example, deuterated C1-C6 alkyl.

[0184] The term “C6-C12 aryl” refers to the group of a carbocyclic aromatic system having 6 to 12 carbon atoms.

[0185] The term “C6-C10 aryl” refers to the group of a carbocyclic aromatic system having 6-10 carbon atoms, such as phenyl and naphthyl.

[0186] The term “5-10 membered heteroaryl” refers to an aromatic heterocyclic ring, usually is a 5-, 6-, 7-, 8-, 9-, or 10-membered heterocyclic ring having 1 to 3 heteroatoms selected from the group consisting of N, O and S; the ring of the heteroaryl may optionally be further fused or linked to aromatic and non-aromatic carbocyclic rings and heterocyclic rings. Non-limiting examples of the 5- to 10-membered heteroaryls are, for example, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, imidazolyl, thiazolyl, isothiazolyl, thiaoxazolyl, pyrrolyl, phenyl -pyrrolyl, furyl, phenyl- furyl, oxazolyl, isoxazolyl, pyrazolyl, thienyl, benzofuranyl, benzothienyl, 1,3-benzodioxolyl, isoindolinyl, benzimidazolyl, indazolyl, quinolinyl, isoquinolinyl, 1,2,3-triazolyl, 1 -phenyl- 1,2,3 - triazolyl, 2,3-indolinyl, 2,3-dihydrobenzofuranyl, 2,3 -dihydrobenzothienyl, benzopyranyl, 2,3- dihydrobenzoxazinyl, and 2,3-dihydroquinoxalinyl.

[0187] The term “substituted C6-C10 aryl” or “substituted 5- to 10-membered heteroaryl” or “substituted 3- to 7-membered heterocyclyl” refers to an aryl or heteroaryl or heterocyclyl of which hydrogen is substituted by substituents. Unless otherwise indicated herein, the substituents of the aryl or heteroaryl or heterocyclyl may be one or more groups selected from the following group consisting of: -halogen, -OR’, -NR’R”, -SR’, -SiR’R”R’”, -OC(O)R’, -C(O)R’, -CO2R’, - CONR’R”, -OC(O)NR’R”, -NR”C(O)R’, -NR’-C(O)NR”R’”, -NR”C(O)2R’, -NH- C(NH2)=NH, -NR’C(NH2)=NH, -NH-C(NH2)=NR’, -S(O)R’, -S(O)2R’, -S(O)2NR’R”, - NR’ S(O)2R”, -CN and -NO2, and the number of the substituents may be 0 to (2m’+l), wherein m’ is the total number of the carbon atom in the group. R’, R” and R’” each independently represent hydrogen, unsubstituted C1-8 alkyl, unsubstituted C6-C12 aryl (or C6-C10 aryl), C6-C12 aryl (or C6-C10 aryl) substituted with 1 to 3 halogens, unsubstituted Cus alkyl, Cns alkoxy or Ci-s thioalkoxy, or unsubstituted C6-C12 aryl (or C6-C10 aryl)-Ci-4 alkyl. When R’ and R” are linked to the same one nitrogen atom, they together with the nitrogen atom may form a 3-, 4-, 5-, 6- or 7-memebered ring. For example, -NR’R” includes 1-pyrrolidyl and 4-morpholinyl.

[0188] The term “hydroxyl” refers to a -OH group.

[0189] The term “halogen” refers to fluorine, chlorine, bromine or iodine.

[0190] The term “amino” refers to -NH2.

[0191] The term “nitro” refers to -NO2.

[0192] The term “amido” refers to -C(O)N(alkyl) or -C(O)N(cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

[0193] The term “carboxylate group” refers to -C(O)O(alkyl) or-C(O)O(cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

[0194] The application also includes the compounds represented by Formula I in various deuterated forms. Each usable hydrogen atom linked to carbon atoms may be independently substituted with a deuterium atom. Those skilled in the art could synthesize the compounds represented by Formula I in the deuterated form by reference to relevant literatures. Commercially available deuterated starting materials can be used in the preparation of the compounds represented by Formula I in the deuterated form, or they can be synthesized with a deuterated reagent according to conventional techniques, and non-limiting examples of the deuterated reagent includes deuterated borane, trideuterated borane in tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane, and the like.

[0195] The term “antibody” refers to an immunoglobulin, which is a tetrapeptide chain structure formed by linking two identical heavy chains and two identical light chains via an interchain disulfide bond. The amino acid composition and arrangement order in the heavy chain constant region of the immunoglobulin are different, and thus its antigenicity is also different. Accordingly, the immunoglobulins can be classified into five classes, or called as the isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being p, 8, y, a, and e chains, respectively. The same class of Ig can be classified into different subclasses according to the differences in the amino acid composition of the hinge region and in the number and position of the heavy chain disulfide bonds, for example, IgG may be classified into IgGl, IgG2, IgG3 and IgG4. The light chains are classified into K chains and X chains according to the differences in the constant regions. Each class of Ig in the five classes of Ig may have either K chains or chains. The antibody of the application is preferably a specific antibody against cell surface antigens on target cells, preferably an anti-RORl antibody.

[0196] The term “solvate” or “solvate compound” refer to a pharmaceutically acceptable solvate formed by the ligand-drug conjugate of the application with one or more solvent molecules. Nonlimiting examples of the solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0197] The term “drug loading rate” refers to the average amount of cytotoxic drug loaded per antibody in Formula I, and it can also be expressed by a ratio (DAR) of drug to antibody, and the drug loading rate can range from 0 to 12, preferably from 1 to 10, cytotoxic drugs (d) linked to per antibody (Ab). In an embodiment of the application, the drug loading rate is expressed as n (n=nl+n2+n3), which illustratively is a mean value of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The average amount of drugs per ADC molecule after the conjugation reaction can be identified by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA test and HPLC characterization.

[0198] In one embodiment, the cytotoxic drug is conjugated to a thiol group (-SH) of cysteine opened between antibody chains and / or a thiol group (-SH) of site-directed mutated cysteine residue in the antibody by a linking unit, and generally, the number of the drug molecules that can be conjugated to the antibody in the conjugation reaction will be less than or equal to the theoretical maximum.

[0199] The loading of the ligand cytotoxic drug conjugate can be controlled by the following nonlimiting methods, including:

[0200] (1) controlling the molar ratio of linking reagent to monoclonal antibody,

[0201] (2) controlling the reaction time and temperature,

[0202] (3) selecting different reaction reagents.

[0203] For the preparation of conventional drug compositions, see the Chinese Pharmacopoeia.

[0204] The term “pharmaceutically acceptable salt” or “pharmaceutically usable salt” refers to a salt of the ligand-drug conjugate of the application, or a salt of the compound of the application, and the salt has safety and effectiveness for use in a mammal and has required biological activity. The ligand-drug conjugate compound of the application has at least one carboxyl group and thus it can form a salt with a base. Non-limiting examples of the pharmaceutically acceptable salts include sodium, potassium, calcium or magnesium salts, and the like.

[0205] The term “pharmaceutically acceptable salt” or “pharmaceutically usable salt” refers to a salt of the ligand-drug conjugate of the application, or a salt of the compound of the application, and the salt has safety and effectiveness for use in a mammal and has required biological activity. The ligand-drug conjugate compound of the application has at least one amino group, and thus it can form a salt with an acid. Non-limiting examples of the pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, biphosphate, dihydric phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, and p-toluenesulphonate.

[0206] The term “acidic amino acid” means an amino acid having an isoelectric point of less than 7, and the acidic amino acid molecule often has one or more acidic groups such as carboxyl group, which can be effectively ionized into a negative ion form in the structure to increase the hydrophilicity. The acidic amino acid may be a natural amino acid or an unnatural amino acid.

[0207] The term “natural amino acid” refers to an amino acid synthesized in a living organism. The natural amino acids are generally in an L-form, with a few exceptions, e.g., glycine, including natural and biosynthesized.

[0208] The term “unnatural amino acid” refers to an amino acid obtained by synthetic means.

[0209] If the chemical name of the compound described in the application is inconsistent with the structural formula, the structural formula of the compound shall prevail.

[0210] EXAMPLES

[0211] The application will be further described below with reference to the specific examples. It should be understood that these examples are only for illustrating the application but not for limiting the scope of the application. The test method of which specific conditions are not given in the examples is usually carried out in accordance with the conventional conditions or the conditions recommended by the manufacturer. Unless otherwise indicated, all percentages, proportions, ratios or fractions are by weight.

[0212] Example 1. Synthesis of Compound Ml

[0213] To a 5000 m single-neck flask, N-(9H-fluoren-9-ylmethoxycarbonyl)-glycylglycine (100 g, 282 mmol, 1.0 eq), lead tetraacetate (175 g, 395 mmol, 1.4 eq), 2000 mL of dry tetrahydrofuran and 670 mL of toluene were added, stirred well, and the resulting mixture was heated to 85 °C and reacted for 2.5 h under nitrogen protection, and the reaction was monitored with TLC. After the raw materials were reacted completely, the reaction solution was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by column chromatography to obtain Compound Ml (87 g); LC-MS: [M+NH4]+=386.0.

[0214] Example 2. Synthesis of Compound M3

[0215] To a 1000 mL single-neck flask, Compound SM-2 (synthesized according to the method as disclosed in the patent application CN108452321A) (40 g, 96 mmol, 1.0 eq), trimethylamine (26.7 mL, 2.0 eq), and toluene (400 mL) were added, and the resulting mixture was heated to 120 °C, refluxed and reacted for 2 h, and the reaction was monitored with TLC. When the reaction was substantially completed, the reaction solution was cooled to under 50 °C and the solvent was removed under a reduced pressure. The residue was dissolved with ethyl acetate (150 mL) and water (40 mL), and the obtained solution was adjusted with IM HC1 to have a pH of 2 to 3 under stirring in an ice bath, and separated. The aqueous layer was additionally extracted with ethyl acetate once, and the organic layers were combined and dried under anhydrous sodium sulfate. The resultant solution was fdtrated, the resulting product was concentrated to obtain a pale yellow oily crude product, and the crude product was purified by column chromatography (DCM : MeOH = 40 : 1), to obtain Compound M2 (26.6 g); LC-MS: [M+H]+=399.3.

[0216] To a 1000 mL single-neck flask, Compound M2 (26.5 g, 60.5 mmol, LOeq), pentafluorophenol (12.2 g, 66.5 mmol, 1.1 eq), DCC (13.7 g, 66.5 mmol, 1.1 eq), and THF (300 mL) were added and reacted at room temperature for 30 min (monitored by TLC), and insoluble material was removed by filtration. The reaction solution was directly purified by preparative liquid chromatography, and the preparation solution was concentrated to remove acetonitrile under reduced pressure with a water pump in a water bath at 35 °C, and lyophilized to obtain Compound M3 (31.5g), with a yield of 64%; LC-MS: [M+H]+= 565.1.

[0217] Example 3. Synthesis of Compound C / / Z-M3

[0218] By referring to the synthetic route of Example 2, Compound cz / Z-M3 (27.8 g) was obtained; LC-MS: [M+H]+=565.2.

[0219] Example 4. Synthesis of Compound 1

[0220] First Step: Compound la

[0221] To a 250 mL single-neck flask, Compound Ml (6 g, 16.3 mmol), 100 mL of THF, and p- toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added, stirred and cooled to 0 °C, and then benzyl hydroxyacetate (5.4 g, 32.6 mmol) was dropwise added. Upon completion of the dropwise addition, the resulting mixture was naturally warmed up to room temperature and reacted (for about 2 to 4 h), and the reaction was monitored by TLC. Upon completion of the reaction, a saturated NaHCCL solution was added to the reaction solution, the resulting mixture was extracted with ethyl acetate, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, and the residue was purified by a silica gel column (PE : EA = 10 : 1-5 : 1-1 : 1) to obtain Compound la (4 g), with a yield of 52%; LC-MS: [M+H]+=475.18.

[0222] Second Step: Compound lb To a 25 mL single-neck flask, Compound la (2 g, 4.2 mmol), and 10 mL of DMF were added and stirred at 0 °C, and DBU (766 mg, 5.04 mmol) was added, and then the resulting mixture was reacted for 1 h, and the reaction was monitored with TLC. After the Fmoc de-protection was completed, a reaction solution was obtained, and set aside for use;

[0223] To another 25 mL single-neck flask, Compound M4 (prepared by referring to the method as disclosed in the patent application CN111051330A) (1.73 g, 4.2 mmol), PyBOP (2.61 g, 5.04 mmol), HOBt (680 mg, 5.04 mmol) and 10 mL of DMF were added, and DIPEA (830 pL, 5.04 mmol) was added in an ice bath, and continuously stirred for 30 min, the above obtained reaction solution was added. The resulting mixture was warmed up to room temperature and reacted, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative-HPLC, to obtain a preparation solution of product. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure, to obtain a solid compound lb (1.7 g), with a yield of 63%; LCMS: [M+H]+=648.26.

[0224] Third Step: Compound 1c

[0225] To a 25 mL single-neck flask, Compound lb (900 mg, 1.39 mmol), and 15 mL of DMF were added and dissolved, then 900 mg of 5% Pd / C was added, and then the hydrogenation reaction was carried out for 2 h. Upon completion of the reaction, the reaction solution was filtered to obtain a filtrate containing Compound 1c, which was directly used in the next step reaction without purification.

[0226] Fourth Step: Compound Id

[0227] The filtrate containing Compound 1c was placed in an ice bath, to which DIPEA (235 pL, 1.39 mmol) was added, and then Compound M3 (784 mg, 1.39 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was lyophilized to obtain Compound Id (504 mg); LC-MS: [M+H]+=804.4.

[0228] Fifth Step: Compound le

[0229] To a 50 mL single-neck flask, Compound Id (500 mg, 0.62 mmol), Exatecan mesylate M5 (310 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were added, and then DIPEA (378 pL, 2.29 mmol) were added in an ice bath. The resulting mixture were heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound le, and the preparation solution was lyophilized to obtain Compound le (210 mg); LC-MS: [M+H]+=1221.6.

[0230] Sixth Step: Compound 1

[0231] To a 25 mL single-neck flask, Compound le (100 mg, 0.081 mmol), zinc bromide (368 mg, 1.63 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution of product, and the preparation solution was lyophilized to obtain Compound 1 (60 mg); LC-MS: [M+H]+=1065 3.

[0232] Example 5. Synthesis of Compound 2

[0233] Compound M3 was replaced with Compound C / / Z-M3, and Compound 2 (51 mg) was prepared by referring to the synthetic route of Example 4; LC-MS: [M+H]+=1065.3.

[0234] Example 6. Synthesis of Compound 3

[0235] First Step: Compound 3a

[0236] To a 250 mL single-neck flask, Compound Ml (6 g, 16.3 mmol), 100 mL of THF, and p- toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added, stirred and cooled to 0 °C, and then benzyl 2-hydroxy-2-methylpropionate (6.3 g, 32.6 mmol) was added dropwise. Upon completion of the dropwise addition, the resulting mixture was naturally warmed up to room temperature and reacted (for about 2 to 4 h), and monitored by TLC. Upon completion of the reaction, a saturated NaHCOa solution was added to the reaction solution, the resulting mixture was extracted with ethyl acetate, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, and the residue was purified by a silica gel column (PE: EA=10: 1-5: 1-2:1) to obtain Compound 3a (4.2 g), with a yield of 52%; LC-MS: [M+H]+=503.3.

[0237] Second Step: Compound 3b

[0238] To a 25 mL single-neck flask, Compound 3a (2 g, 4.0 mmol), and 10 mL of DMF were added and stirred at 0 °C, and then DBU (760 mg, 5.0 mmol) was added. The resulting mixture was reacted for 1 h, and the reaction was monitored with TLC. After the Fmoc de-protection was completed, a reaction solution was obtained, and set aside for use.

[0239] To another 25 mL single-neck flask, Compound M4 (1.65 g, 4.0 mmol), PyBOP (2.59 g, 5.0 mmol), HOBt (675 mg, 5.0 mmol) and 10 mL of DMF were added, and then DIPEA (823 pL, 5.04 mmol) was added in an ice bath, and continuously stirred for 30 min, the above obtained reaction solution was added. The resulting mixture was heated to room temperature and reacted, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative-HPLC, to obtain a preparation solution of product. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure, to obtain a solid compound 3b (1.4 g), with a yield of 53%; LC-MS: [M+H]+=676.2.

[0240] Third Step: Compound 3c

[0241] To a 25 mL single-neck flask, Compound 3b (700 mg, 1.04 mmol), and 10 mL of DMF were added and dissolved, and then 700 mg of 5% Pd / C was added, then the hydrogenation reaction was carried out for 1.5 h. Upon completion of the reaction, the reaction solution was filtered to obtain a filtrate containing Compound 3c, which was directly used in the next step reaction without purification.

[0242] Fourth Step: Compound 3d

[0243] The filtrate containing Compound 3c was placed in an ice bath, to which DIPEA (210 pL, 1.25 mmol) was added, and then Compound M3 (704 mg, 1.25 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 3d (486 mg); LC-MS: [M-H]-=830.5.

[0244] Fifth Step: Compound 3e

[0245] To a 50 mL single-neck flask, Compound 3d (300 mg, 0.36 mmol), Exatecan mesylate M5 (180 mg, 0.36 mmol), PyBOP (260 mg, 0.5 mmol), HOBt (67 mg, 0.5 mmol) and 10 mL of DMF were added, and then DIPEA (219.5 pL, 1.33 mmol) were added in an ice bath. The resulting mixture was heated to room temperature and reacted for 3 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 3e, and the preparation solution was lyophilized to obtain Compound 3e (157 mg); LC-MS: [M+H]+=1249.6.

[0246] Sixth Step: Compound 3

[0247] To a 25 mL single-neck flask, Compound 3e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution of product, and the preparation solution was lyophilized to obtain Compound 3 (64 mg); LC-MS: [M+H]+=1093.1.

[0248] Example 7. Synthesis of Compound 4

[0249] Compound M3 was replaced with Compound C / / / -M3, and Compound 4 (60 mg) was prepared by referring to the synthetic route of Example 6; LC-MS: [M+H]+=1093.2.

[0250] Example 8. Synthesis of Compound 5 A

[0251] First Step: Compound 5a

[0252] To a 25 mL single-neck flask, Compound Ml (500 mg, 1.4 mmol, 1.0 eq), p-toluenesulfonic acid monohydrate (26 mg, 0. Immol, 0. leq) and 10 mL of THF were added, stirred well, cooled to 0 °C, and then benzyl / .-lactate (benzyl (S)-(-)-lactate) (1.2 g, 7.0 mmol, 5 eq) was slowly added. After the addition was completed, the resulting mixture was heated to room temperature and reacted, and the reaction was monitored with TLC. After the reaction was completed, a saturated NaHCOa solution was add to the reaction solution, the resulting mixture was extracted with ethyl acetate, dried with anhydrous sodium sulfate, fdtered and concentrated, and the residue was purified by a reverse phase column, to obtain Compound 5a (400 mg).

[0253] LC-MS: [M+NH4]+=506.2.

[0254] 'H NMR(400 Mz, CDC13 / CD3OD):1.39 (3H, d, J = 6.8 Hz), 3.78 (2H, t, J= 4.0 Hz), 4.17- 4.27 (2H, m), 4.42 (2H, d, J= 4.0 Hz), 4.72-4.85 (2H, m), 5.11-5.58 (2H, m), 5.43 (1H, s), 7.06 (1H, t, J= 8.0 Hz), 7.25-7.33 (6H, m), 7.38 (2H, t, J=8.0 Hz), 7.57 (2H, d, J= 8.0 Hz), 7.75 (2H, d, J= 8.0 Hz).

[0255] Second Step: Compound 5b

[0256] To a 25 mL single-neck flask, Compound 5a (400 mg, 0.8 mmol, 1.0 eq) and 4mL of DMF were added, stirred well, cooled to 0 °C, and then DBU (137 mg, 0.9 mmol, 1.1 eq) was added slowly. After completion of the addition was completed, the resulting mixture was heated to room temperature and reacted, and the reaction was monitored with TLC. The reaction was completed, and the reaction solution was expressed as the reaction solution (1).

[0257] To another 25 mL single-neck flask, Compound M4 (372 mg, 0.9 mmol, 1.1 eq), PyBOP (852 mg, 1.6 mmol, 2.0 eq), and 3 mL of DMF were added, and stirred at room temperature for 5 minutes, and then the above reaction solution (1) was added. The resulting mixture was reacted at room temperature, and the reaction was monitored with HPLC. The reaction was completed, and the reaction solution was purified by preparative high performance liquid chromatography, to obtain Compound 5b (326mg); LC-MS: [M+NH4]+=679.2.

[0258] Third Step: Compound 5c

[0259] To a 100 mL single-neck flask, Compound 5b (4.0 g, 6.05 mmol, 1.0 eq), and DMF (60 mL) were added and dissolved, then 5% Pd / C (4 g) was added, and then the hydrogenation reaction was carried out at room temperature for 4 h (the reaction was monitored by HPLC). The Pd / C was removed by filtration, and the filtrate containing Compound 5c were not concentrated and directly placed in an ice bath (about 0 °C), and set aside for use.

[0260] Fourth Step: Compound 5d

[0261] The filtrate containing Compound 5c was placed in an ice bath, to which DIPEA (1.1 mL, 1.1 eq) was added, and then Compound M3 (3.4 g, 6.05 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was lyophilized to obtain Compound 5d (3.15g); LC-MS: [M-H]'816.3.

[0262] Fifth Step: Compound 5e

[0263] To a 100 mL single-neck flask, Compound 5d (2.07 g, 2.53 mmol, 1.0 eq), Exatecan mesylate M5 (1.35 g, 2.53 mmol, 1.0 eq), PyBOP (1.98 g, 3.79 mmol, 1.5 eq), HOBt (0.51 g, 3.79 mmol, 1.5 eq) and DMF (40 mL) were added, and then DIPEA (1.05 mL, 1.5 eq) was added in an ice bath. The resulting mixture was heated to room temperature and reacted for 2 h (the reaction was monitored by HPLC). The reaction solution was directly purified by preparative liquid chromatography, and the resulting preparation solution was concentrated to remove acetonitrile under reduced pressure with a water pump in a water bath at 35 °C, and lyophilized to obtain Compound 5e (1.92 g), with a yield of 61%; LC-MS: [M+H]+=1235.4.

[0264] Sixth Step: Compound 5 A

[0265] To a 100 mL single-neck flask, Compound 5e (1.0 g, 0.8 mmol, 1.0 eq), and 35 mL of nitromethane were added and dissolved, then zinc bromide (3.64 g, 16 mmol, 20.0 eq) was added, and the resulting mixture was reacted in an oil bath at 40 °C (pre-heated for stabilization in advance) for 30 min. The reaction solution was concentrated to remove nitromethane under reduced pressure with a water pump in a water bath at 45°C, to obtain a yellow residue solid (with monitoring by HPLC). The residue solid was purified by preparative liquid chromatography, 0.1% trifluoroacetic acid was added to the flowing phase, and a preparation solution of compound 5A was obtained. The preparation solution was concentrated under reduced pressure with a water pump in a water bath at 35 °C to remove acetonitrile, and lyophilized, to obtain Compound 5 A (786 mg), with a yield of 90%.

[0266] LC-MS: [M+H]+= 1079.4;

[0267] 'H NMR (400 MHz, DMSO-d6) 3 9.39-9.02 (m, 1H), 8.70 (t, J = 6.5 Hz, 1H), 8.64 (t, J = 5.7 Hz, 1H), 8.56 (d, J = 8.8 Hz, 1H), 8.34 (t, J = 5.7 Hz, 1H), 8.16 (d, J = 8.2 Hz, 1H), 8.01 (t, J = 5.5 Hz, 1H), 7.71 (d, J = 10.9 Hz, 1H), 7.30 (s, 1H), 7.28-7.15 (m, 4H), 7.14 (s, 2H), 5.53 (dd, J = 14.5, 6.4 Hz, 1H), 5.49-5.34 (m, 2H), 5.22 (d, J = 18.8 Hz, 1H), 5.09 (d, J = 18.7 Hz, 1H), 5.03 (dd, J = 9.6, 3.9 Hz, 1H), 4 73 (dd, J = 9.9, 6.9 Hz, 1H), 4.59 (dd, J = 10.1, 6.5 Hz, 1H), 4.49 (ddd, J = 13.2, 8.6, 4.4 Hz, 1H), 4.14 (dd, J = 13.3, 6.6 Hz, 2H), 3.93 (s, 2H), 3.84 (dd, J = 16.5, 6.3 Hz, 1H), 3.76 (dd, J = 16.9, 5.7 Hz, 2H), 3.70 (d, J = 5.2 Hz, 2H), 3.60 (dd, J = 16.7, 5.4 Hz, 1H), 3.52 (dd, J = 16.4, 5.1 Hz, 1H), 3.45 (dd, J = 12.8, 10.1 Hz, 1H), 3.25-3.15 (m, 1H), 3.14-3.05 (m, 1H), 3.01 (dd, J = 13.7, 4.1 Hz, 1H), 2.73 (dd, J = 13.5, 9.8 Hz, 1H), 2.54-2.47 (m, 1H), 2.33 (s, 2H), 2.17 (d, J = 5.5 Hz, 2H), 1.91-1.79 (m, 2H), 1.33 (d, J = 6.6 Hz, 2H), 0.87 (t, J = 7.3 Hz, 2H).

[0268] Example 9. Synthesis of Compound 5B

[0269] First Step: Compound 5d-l

[0270] To a 25 mL single-neck flask, Compound 5b (300 mg, 0.45 mmol, 1.0 eq), and DMF (3 mL) were added, stirred and dissolved, 5% Pd / C (300 mg) was added, three replacements of hydrogen gas were carried out, and the hydrogenation reaction was carried out for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was filtered to remove the Pd / C, to obtain a filtrate containing compound 5c. The filtrate was cooled to 0 °C to 5 °C, and then DIPEA (65 mg, 0.5 mmol, 1.1 eq) was added. Then, ent-M3 (255 mg, 0.45 mmol) was added to the filtrate. Upon completion of the addition, the resulting mixture was heated to 20±5 °C and reacted for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the purification was performed by preparative HPLC, and a preparation solution of product was collected and lyophilized to obtain Compound 5d-l (200 mg), with a yield of 54%; LC-MS: [M-H]-=816.3.

[0271] Second Step: Compound 5e-l

[0272] To a 25 mL single-neck flask, Compound 5d-l (200 mg, 0.24 mmol, 1.0 eq), Exatecan mesylate M5 (127 mg, 0.24 mmol, 1.0 eq), PyBOP (187 mg, 0.36 mmol, 1.2eq), HOBt (48 mg, 0.36 mmol, 1.2 eq) and DMF (6 mL) were added and cooled to 0 °C to 5°C in an ice bath, then DIPEA (62 mg, 0.48 mmol, 2.0 eq) was added. Upon completion of the addition, the resulting mixture was heated 20±5 °C and reacted for 2 h, and the reaction was monitored with HPLC until the reaction was completed. The reaction solution was directly purified by preparative HPLC, and a preparation solution of product was collected and lyophilized to obtain Compound 5e-l (162.8 mg); LC-MS: [M+H]+=1235.4. Third Step: Compound 5B

[0273] To a 25 mL single-neck flask, Compound 5e-l (110 mg, 0.089 mmol, 1.0 eq), ZnBr2 (400 mg, 1.78 mmol, 20.0 eq) and CH3NO2 (10 mL) were added in order, and upon completion of the addition, the resulting mixture was heated to 40 °C and reacted for 0.5 h, then the reaction was stopped. The reaction solution was directly dried under reduced pressure at 45 °C, to obtain a yellow solid. By sampling, the reaction was monitored by HPLC. The dried solid was directly purified by preparative HPLC. The preparation solution of product was collected and lyophilized to obtain Compound 5B (73.4 mg), with the field 76.5%; LC-MS: [M+HJ M 079.4.

[0274] Example 10. Preparation of Compound 6A

[0275] Benzyl (S)-(-)-lactate was replaced with benzyl (R)-(+)-lactate, and Compound 6A (71 mg) was prepared by referring to the synthetic route of Example 8; LC-MS:[M+H]+=1079.4.

[0276] Example 11. Preparation of Compound 6B

[0277] Benzyl (S)-(-)-lactate was replaced with benzyl (R)-(+)-lactate, and Compound 6B (59 mg) was prepared by referring to the synthetic route of Example 9; LC-MS: [M+H]+=1079.4.

[0278] Example 12. Preparations of Compounds 7A and 7B

[0279]

[0280] First Step: Compound 7a

[0281] To a 250 mL single-neck flask, Compound Ml (10 g, 27.1 mmol), benzyl 3,3,3- trifluorolactate (prepared according to the method disclosed in the patent application W02020063673A1) (12.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were added, and the resulting mixture was heated to 100 °C and reacted for 4 h. Upon completion of the reaction, the reaction solution was cooled to room temperature, and filtered to remove insoluble substances. The filtrate was concentrated to obtain a crude product. The crude product was purified by silica column chromatography (PE:EA=10: 1-5: 1-2:1), to obtain 5.15 g of Compound 7a, with a yield of 35.1%; LC-MS: [M+H]+=543.17.

[0282] Second Step: Compound 7b

[0283] To a 50 mL single-neck flask, Compound 7a (5 g, 9.2 mmol) and 15 mL of DMF were added and dissolved, then DBU (1.68 g, 11 mmol) were added in an ice bath, and the resulting mixture was reacted for 1 h, the resulting reaction solution was expressed as a reaction solution (1).

[0284] To another 50 mL single-neck flask, Compound M4 (3.8 g, 9.2 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol) and 10 mL of DMF were added and dissolved, then DIPEA (1.82 mL, 11 mmol) were added in an ice bath, the resulting mixture was reacted for 30 min, and then the reaction solution (1) was added. The resulting mixture was heated to room temperature and reacted for 2 h, and the reaction progress was monitored by HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain 4.1 g of solid Compound 7b, with a yield of 62.3%; LC-MS: [M+H]+=716.25.

[0285] Third Step: Compound 7d

[0286] To a 25 mL single-neck flask, Compound 7b (900 mg, 1.26 mmol), and 15 mL of DMF were added and dissolved, then 900 mg of 5% Pd / C were added, the hydrogenation reaction was carried out for 2h. Upon completion of the reaction, the reaction solution was filtered to obtain a filtrate containing Compound 7c. The filtrate was placed in an ice bath, to which DIPEA (228 pL, 1.38 mmol) was added, and then Compound M3 (712 mg, 1.26 mmol) was add. Upon completion of the additions, the mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by preparative high performance liquid chromatography, to obtain a preparation solution. The preparation solution was lyophilized to obtain 525 mg of Compound 7d, with a yield of 47.9%; LC-MS: [M-H]’=870.33.

[0287] Fourth Step: Compound 7e

[0288] To a 50 mL single-neck flask, Compound 7d (500 mg, 0.57 mmol), Exatecan mesylate M5 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were added, and then DIPEA (378 pL, 2.29 mmol) were added in an ice bath. The resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain the preparation solutions of Compound 7e-l and Compound 7e-2. The preparation solutions were lyophilized respectively, to obtain 150 mg of Compound 7e-l, LC-MS: [M+H]+=1289.46; and 220mg of Compound 7e-2,

[0289] To a 25 mL single-neck flask, Compound 7e-l (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 52 mg of Compound 7A; TOF results: 1133.3613.

[0290] To a 25 mL single-neck flask, Compound 7e-2(100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added, and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 63 mg of Compound 7B; TOF result: 1133.3668.

[0291] Example 13. Synthesis of Compounds 8 A and 8B

[0292] First Step: Compound 8d

[0293] To a 25 mL single-neck flask, Compound 7c (900 mg, 1.83 mmol), and 20 mL of DMF were added and dissolved, then DIPEA (303 pL, 1.83 mmol) was added, and then C / / Z-M3 (1034 mg, 1.83 mmol) were added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. After completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography, and the preparation solution was lyophilized to obtain 613 mg of Compound 8d, with a yield of 38.5%; LC-MS: [M-H] =870.32.

[0294] Second Step: Compound 8e-l and Compound 8e-2

[0295] To a 50 mL single-neck flask, Compound 8d (500 mg, 0.57 mmol), Exatecan mesylate M5 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were added, and then DIPEA (378 pL, 2.29 mmol) was added in an ice bath. The resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 8e-l and Compound 8e-2, respectively. The preparation solutions were lyophilized respectively, to obtain 140 mg of Compound 8e-l, and 210 mg of Compound 8e-2. LC-MS of Compound 8e-l : [M+H]+=1289.47; LC-MS of Compound 8e-2: [M+H]+=1289.47.

[0296] Third Step: Compound 8A

[0297] To a 25 mL single-neck flask, Compound 8e-l (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 50 mg of Compound 8 A; TOF result: 1133.3623.

[0298] Fourth Step: Compound 8B

[0299] To a 25 mL single-neck flask, Compound 8e-2 (100 mg, 0.077 mmol), zinc bromide (349 mg,

[0300] 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 58 mg of Compound 8B; TOF result: 1133.3653.

[0301] Example 14. Synthesis of Compound 9A

[0302] First Step: Compound 9a

[0303] To a 250 mL single-neck flask, Compound Ml (6 g, 16.3 mmol), 100 mL of THF, and p- toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added, stirred and cooled to 0 °C, and then benzyl 1 -hydroxy cyclopropanecarboxylate (prepared by referring to the method as disclosed in US20050020645 Al) (6.3 g, 32.6 mmol) was dropwise added. Upon completion of the dropwise addition, the resulting mixture was naturally heated to room temperature and reacted (for about 2h to 4 h), and the reaction was monitored by TLC. Upon completion of the reaction, a saturated NaHCCL solution was added to the reaction solution, the resulting mixture was extracted with ethyl acetate, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, and the residue was purified by a silica column (PE:EA=10:l- 5: 1-2: 1), to obtain Compound 9a (3.7 g), with a yield of 45%; LC-MS: [M+H]+=501.5.

[0304] Second Step: Compound 9b To a 25 mL single-neck flask, Compound 9a (2 g, 4.0 mmol), and 10 mL of DMF were added and stirred at 0 °C, and then DBU (760 mg, 5.0 mmol) were added. The resulting mixture was reacted for 1 h, and the reaction was monitored with TLC. After the Fmoc deprotection was completed, the reaction solution was set aside for use.

[0305] To another 25 mL single-neck flask, Compound M4 (1.65 g, 4.0 mmol), PyBOP (2.59 g, 5.0 mmol), HOBt (675 mg, 5.0 mmol) and 10 mL of DMF were added, and then DIPEA (823 pL, 5.04 mmol) was added in an ice bath. The resulting mixture was continuously stirred for 30 min. The above obtained reaction solution was added to the flask, and the resulting mixture was heated to room temperature and reacted, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure, to obtain 1.5 g of solid Compound 9b, with a yield of 56%; LC-MS: [M+H]+=674.7.

[0306] Third Step: Compound 9c

[0307] To a 25 mL single-neck flask, Compound 9b (900 mg, 1.3 mmol), and 10 mL of DMF were added and dissolved, then 900 mg of 5% Pd / C was added, the hydrogenation reaction was carried out for 1.5 h. Upon completion of the reaction, the reaction solution was filtered, to obtain a filtrate containing Compound 9c, which was directly used in the next step reaction without purification.

[0308] Fourth Step: Compound 9d

[0309] The filtrate containing Compound 9c was placed in an ice bath, to which DIPEA (223 pL, 1.3 mmol) was added, and then Compound M3 (750 mg, 1.3 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by a preparative high-performance liquid chromatography, the preparation solution was lyophilized to obtain Compound 9d (529 mg); LC-MS: [M-H]'=828.4.

[0310] Fifth Step: Compound 9e

[0311] To a 50 mL single-neck flask, Compound 9d (500 mg, 0.6 mmol), Exatecan mesylate M5 (300 mg, 0.6 mmol), PyBOP (416 mg, 0.8 mmol), HOBt (108 mg, 0.8 mmol) and 15 mL of DMF were added, and then DIPEA (351 pL, 2.13 mmol) was added in an ice bath. The resulting mixture was heated to room temperature and reacted for 3 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 9e, the preparation solution was lyophilized to obtain Compound 9e (257 mg); LC-MS: [M+H]+=1247.5.

[0312] Sixth Step: Compound 9 A

[0313] To a 25 mL single-neck flask, Compound 9e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 9A (55 mg); LC-MS: [M+H]+=1091.3.

[0314] Example 15. Synthesis of Compound 9B Compound M3 , (44 mg) was prepared by referring to the synthetic route of Example 14; LC-MS: [M+H]+=1091.3.

[0315] Example 16. Synthesis Compound 10A

[0316] First Step: Compound 10a

[0317] To a 250 mL single-neck flask, Compound Ml (6 g, 16.3 mmol), 100 mL of THF, and p- toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added, stirred and cooled to 0 °C, and then benzyl 1 -hydroxymethylpropanecarboxylate (prepared by referring to the method as disclosed in WO2013187496A1) (6.7 g, 32.6 mmol) was dropwise added. Upon completion of the dropwise addition, the resulting mixture was naturally warmed up to room temperature and reacted (for about 2-4 h), and the reaction was monitored by TLC. Upon completion of the reaction, a saturated NaHCCh solution was added to the reaction solution, the resulting mixture was extracted with ethyl acetate, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, and the residue was purified by a silica column (PE:EA=10:l- 5 : 1 -2: 1) to obtain Compound 10a (4.9 g), with a yield of 58%; LC-MS: [M+H]+=515.4.

[0318] Second Step: Compound 10b

[0319] To a 25 mL single-neck flask, Compound 10a (4 g, 7.8 mmol), and 10 mL of DMF were added and stirred at 0 °C, and then DBU (1.2 g, 8.0 mmol) was added. The resulting mixture was reacted for 1 h, and the reaction was monitored with TLC. After the Fmoc de-protection was completed, the reaction solution was set aside for use.

[0320] To another 25 mL single-neck flask, Compound M4 (3.3 g, 8.0 mmol), PyBOP (5.2 g, 10.0 mmol), HOBt (1.35 g, 10.0 mmol) and 10 mL of DMF were added, and the DIPEA (1.65 mL, 10.1 mmol) was added in an ice bath, and continuously stirred for 50 min. The above reaction solution was added to the flask, and the resulting mixture was heated to room temperature and reacted, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with di chloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure, to obtain 2.3 g of solid Compound 10b, with a yield of 42%; LC-MS: [M+H]+=688.8.

[0321] Third Step: Compound 10c

[0322] To a 25 mL single-neck flask, Compound 10b (1.0 g, 1.45 mmol), and 15 mL of DMF was added and dissolved, then 1.0 g of 5% Pd / C was added, and the hydrogenation reaction was carried out for 1.5 h. Upon completion of the reaction, the reaction solution was filtered to obtain a filtrate containing Compound 10c, which was directly used in the next step reaction without purification.

[0323] Fourth Step: Compound lOd

[0324] The filtrate containing Compound 10c was placed in an ice bath, to which DIPEA (258 pL,

[0325] 1.5 mmol) was added, and then Compound M3 (837 mg, 1.45 mmol) was added. Upon completion of the addition, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. After completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound lOd (499 mg); LC-MS: [M-H]' =842.4.

[0326] Fifth Step: Compound lOe

[0327] To a 50 mL single-neck flask, Compound lOd (400 mg, 0.48 mmol), Exatecan mesylate M5 (240 mg, 0.48 mmol), PyBOP (250 mg, 0.48 mmol), HOBt (65 mg, 0.48 mmol) and 15 mL of DMF were added, and then DIPEA (330 pL, 2.0 mmol) was added in an ice bath, the resulting mixture were heated to room temperature and reacted for 3 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound lOe. The preparation solution was lyophilized to obtain Compound lOe (188 mg); LC-MS: [M+H]+=1261.5.

[0328] Sixth Step: Compound 10A

[0329] To a 25 mL single-neck flask, Compound lOe (100 mg, 0.08 mmol), zinc bromide (360 mg,

[0330] 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain CompoundlOA (61 mg); LC-MS: [M+H]+=1105.4.

[0331] Example 17. Synthesis of Compound 10B

[0332] Compound M3 was replaced with Compound ent-M3, and Compound 10B (75 mg) was prepared by referring to the synthetic route of Example 16; LC-MS: [M+H]+=l 105.4.

[0333] Example 18. Synthesis of Compound 11A

[0334] First Step: Compound Ila

[0335] To a 250 mL single-neck flask, Compound Ml (6 g, 16.3 mmol), 100 mL of THF, and p- toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added, stirred and cooled to 0 °C, and then benzyl 1 -hydroxy cyclobutanecarboxylate (synthesized according to the method as disclosed in Journal of Medicinal Chemistry, 2013, 56 (13), 5541-5552) (6.7 g, 32.6 mmol) was dropwise added. Upon completion of the dropwise addition, the resulting mixture was naturally heated to room temperature and reacted (for about 2-4 h), the reaction was monitored by TLC. Upon completion of the reaction, a saturated NaHCCh solution was added to the reaction solution, the resulting mixture was extracted with ethyl acetate, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, fdtered, and concentrated, and the residue was purified by a silica column (PE:EA=10: 1-5: 1-2: 1), to obtain Compound Ila (5.1 g), with a yield of 62%; LC-MS: [M+H]+=515.7.

[0336] Second Step: Compound 11b To a 25 mL single-neck flask, Compound Ila (4 g, 7.8 mmol), and 10 mL of DMF were added and stirred at 0 °C, then DBU (1.2 g, 8.0 mmol) was added, and the resulting mixture was reacted for 1 h. The reaction was monitored with TLC. After the Fmoc de-protection was completed, the reaction solution was set aside for use.

[0337] To another 25 mL single-neck flask, Compound M4 (3.3 g, 8.0 mmol), PyBOP (5.2 g, 10.0 mmol), HOBt (1.35 g, 10.0 mmol) and 10 mL of DMF were added, then DIPEA (1.63 mL, 10.0 mmol) was added in an ice bath, and the resulting mixture was continuously stirred for 40 min. The above reaction solution was added to the flask, and the resulting mixture was heated to room temperature and reacted. The reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure, to obtain 2.3 g of solid Compound 11b, with a yield of 42%; LC-MS: [M+H]+=688.3.

[0338] Third Step: Compound 11c

[0339] To a 25 mL single-neck flask, Compound 11b (2.0 g, 2.9 mmol), and 25 mL of DMF were added and dissolved, 2.0 g of 5% Pd / C were added, and the hydrogenation reaction was carried out for 3 h. Upon completion of the reaction, the reaction solution was filtered, to obtain a filtrate containing Compound 11c, which was directly used in the next step reaction without purification.

[0340] Fourth Step: Compound lid

[0341] The filtrate containing Compound 11c was placed in an ice bath, to which DIPEA (516 pL, 3.0 mmol) was added, and then Compound M3 (1.7 g, 2.9 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution The preparation solution was lyophilized to obtain Compound lid (934 mg); LC-MS: [M-H]' -842.4.

[0342] Fifth Step: Compound lie

[0343] To a 50 mL single-neck flask, Compound lid (800 mg, 0.96 mmol), Exatecan mesylate M5 (480 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (130 mg, 0.96 mmol) and 30 mL of DMF were added, then DIPEA (660 pL, 4.0 mmol) was added in an ice bath, and the resulting mixture was heated to room temperature and reacted for 4 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound lie. The preparation solution was lyophilized to obtain Compound lie (401 mg); LC-MS: [M+H]+=1261.4.

[0344] Sixth Step: Compound 11A

[0345] To a 25 mL single-neck flask, Compound lie (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h. The reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 11A (86 mg); LC-MS: [M+H]+=l 105.4.

[0346] Example 19. Synthesis of Compound 11B Compound M3 w B (50 mg) was prepared by referring to the synthetic route of Example 18. LC-MS: [M+H]+= 1105.4.

[0347] Example 20. Synthesis of Compound 12A

[0348] First Step: Compound 12a

[0349] To a 250 mL single-neck flask, Compound Ml (6 g, 16.3 mmol), 100 mL THF, and p- toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added, stirred and cooled to 0 °C, and then benzyl 1 -hydroxymethylcyclobutanecarboxylate (prepared by referring to the method as disclosed in the patent application W02009011285A1) (7.2 g, 32.6 mmol) was dropwise added Upon completion of the dropwise addition, the resulting mixture was naturally heated to room temperature and reacted (for about 2 h to 4 h), the reaction was monitored by TLC. Upon completion of the reaction, a saturated NaHCCL solution was added to the reaction solution, the resulting mixture was extracted with ethyl acetate, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, and the residue was purified by a silica column (PE:EA=10: 1-5: 1-2: 1), to obtain Compound 12a (4.5 g), with a yield of 52%; LC-MS: [M+H]+=529.4.

[0350] Second Step: Compound 12b

[0351] To a 25 mL single-neck flask, Compound 12a (4 g, 7.6 mmol), and 10 mL of DMF were added and stirred at 0 °C, then DBU (1.2 g, 8.0 mmol) was added, and the resulting mixture was reacted for 1 h. The reaction was monitored with TLC. After the Fmoc de-protection was completed, the reaction solution was set aside for use. To another 25 mL single-neck flask, Compound M4 (3.2 g, 7.6 mmol), PyBOP (4.7 g, 9.0 mmol), HOBt (1.22 g, 9.0 mmol) and 10 mL of DMF were added, and then DIPEA (1.49 mL, 0.9 mmol) was added in an ice bath, and the resulting mixture was continuously stirred for 30 min The above reaction solution was added to the flask, and the resulting mixture was heated to room temperature and reacted, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure, to obtain 2.0 g of solid Compound 12b, with a yield of 37%; LC-MS: [M+H]+=702.8.

[0352] Third Step: Compound 12c

[0353] To a 25 mL single-neck flask, Compound 12b (1.0 g, 1.43 mmol), and 15 mL of DMF were added and dissolved, then 1.0 g of 5% Pd / C were added, and then the hydrogenation reaction was carried out for 1.5 h. Upon completion of the reaction, the reaction solution was filtered, to obtain a filtrate containing Compound 12c, which was directly used in the next step reaction without purification.

[0354] Fourth Step: Compound 12d

[0355] The filtrate containing Compound 12c was placed in an ice bath, to which DIPEA (258 pL,

[0356] 1.5 mmol) was added, and Compound M3 (825 mg, 1.43 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 12d (522 mg); LC-MS: [M-H]' =856.4.

[0357] Fifth Step: Compound 12e

[0358] To a 50 mL single-neck flask, Compound 12d (400 mg, 0.47 mmol), Exatecan mesylate M5 (240 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt (64 mg, 0.47 mmol) and 15 mL of DMF were added, then DIPEA (330 pL, 2.0 mmol) was added in an ice bath, and the resulting mixture was heated to room temperature and reacted for 3 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 12e. The preparation solution was lyophilized to obtain Compound 12e (198 mg); LC-MS: [M+H]+=1275.4.

[0359] Sixth Step: Compound 12A

[0360] To a 25 mL single-neck flask, Compound 12e (100 mg, 0.08 mmol), zinc bromide (360 mg,

[0361] 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain solid Compound 12A (55 mg); LC-MS: [M+H]+=1119.4.

[0362] Example 21. Synthesis of Compound 12B

[0363] Compound M3 was replaced with Compound e«LM3, and Compound 12B (50 mg) was prepared by referring to the synthetic route of Example 20; LC-MS: [M+H]+=l 119.4.

[0364] Example 22. Synthesis of Compound 13 A

[0365] First Step: Compound 13a

[0366] To a 250 mL single-neck flask, Compound Ml (6 g, 16.3 mmol), 100 mL THF, and p- toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added, stirred and cooled to 0 °C, and then benzyl 1 -hydroxy cyclopentanecarboxylate (synthesized according to the method as disclosed in the literation “Journal of Medicinal Chemistry”, 2013, 56(13), 5541-5552) (7.2 g, 32.6 mmol) was dropwise added. Upon completion of the dropwise addition, the resulting mixture was naturally heated to room temperature and reacted (for about 2 h to 4 h), the reaction was monitored by TLC. Upon completion of the reaction, a saturated NaHCCE solution was added to the reaction solution, the resulting mixture was extracted with ethyl acetate, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, and the residue was purified by a silica column (PE:EA=10: 1-5: 1-2: 1), to obtain Compound 13a (4.6 g), with a yield of 53%; LC-MS: [M+H]+=529.5.

[0367] Second Step: Compound 13b To a 25 mL single-neck flask, Compound 13a (4 g, 7.6 mmol), and 10 mL of DMF were added and stirred at 0 °C, then DBU (1.17 g, 7.8 mmol) was added, and then the resulting mixture was reacted for 1 h. The reaction was monitored with TLC, and after the Fmoc de-protection was completed, the reaction solution was set aside for use.

[0368] To another 25 mL single-neck flask, Compound M4 (3.14 g, 7.6 mmol), PyBOP (4.42 g, 8.5 mmol), HOBt (1.15 g, 8.5 mmol) and 10 mL of DMF were added, then DIPEA (1.39 mL, 0.85 mmol) was added in an ice bath, and then the resulting mixture was continuously stirred for 30 min. The above reaction solution was added to the flask, and the resulting mixture was heated to room temperature and reacted, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure, to obtain 2.1 g of solid Compound 13b, with a yield of 39%; LC-MS: [M+H]+=702.8.

[0369] Third Step: Compound 13c

[0370] To a 25 mL single-neck flask, Compound 13b (1.5 g, 1.87 mmol), and 25 mL of DMF were added and dissolved, then 1.5 g of 5% Pd / C were added, and then the hydrogenation reaction was carried out for 3 h. Upon completion of the reaction, the reaction solution was filtered, to obtain a filtrate containing Compound 13c, which was directly used in the next step reaction without purification.

[0371] Fourth Step: Compound 13d

[0372] The filtrate containing Compound 13c was placed in an ice bath, to which DIPEA (333 pL, 1.93 mmol) was added, and then Compound M3 (1.1 g, 1.87 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was lyophilized to obtain Compound 13d (519 mg); LC-MS: [M-H]’ =856.6.

[0373] Fifth Step: Compound 13e

[0374] To a 50 mL single-neck flask, Compound 13d (400 mg, 0.47 mmol), Exatecan mesylate M5 (240 mg, 0.48 mmol), PyBOP (250 mg, 0.48 mmol), HOBt (65 mg, 0.48 mmol) and 15 mL of DMF were added, then DIPEA (330 pL, 2.0 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 4 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 13e. The preparation solution was lyophilized to obtain Compound 13e (187 mg); LC-MS: [M+H]+=1275.5.

[0375] Sixth Step: Compound 13A

[0376] To a 25 mL single-neck flask, Compound 13e (100 mg, 0.08 mmol), zinc bromide (355 mg, 0.16 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 13 A (60 mg); LC-MS: [M+H]+=1119 6.

[0377] Example 23. Synthesis of Compound 13B

[0378] Compound M3 was replaced with Compound en -M3, and Compound 13B (51mg) was prepared by referring to the synthetic route of Example 22; LC-MS: [M+H]+=l 119.6.

[0379] Example 24. Synthesis of Compound 14A

[0380] First Step: Compound 14a

[0381] To a 250 rnL single-neck flask, Compound Ml (6 g, 16.3 mmol), 100 mL of THF, and p- toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added, stirred and cooled to 0 °C, and then benzyl 1 -hydroxymethylcyclopentanecarboxylate (prepared by referring to the method as disclosed in W02009011285A1) (7.6 g, 32.6 mmol) was dropwise added. Upon completion of the dropwise addition, the resulting mixture was naturally heated to room temperature and reacted (for about 2h to 4 h), the reaction was monitored by TLC. Upon completion of the reaction, a saturated NaHCCE solution was added to the reaction solution , the resulting mixture was extracted with ethyl acetate, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, and the residue was purified by a silica column (PE:EA=10:l- 5 : 1 -2: 1), to obtain Compound 14a (4.4 g), with a yield of 49%; LC-MS: [M+H]+=543.6.

[0382] Second Step: Compound 14b

[0383] To a 25 mL single-neck flask, Compound 14a (4 g, 7.4 mmol), and 10 mL of DMF were added and stirred at 0 °C, then DBU (1.2 g, 8.0 mmol) was added, and then the resulting mixture was reacted for 1 h. The reaction was monitored with TLC, and after the Fmoc de-protection was completed, the reaction solution was set aside for use. To another 25 mL single-neck flask, Compound M4 (3.1 g, 7.4 mmol), PyBOP (4.6 g, 8.8 mmol), HOBt (1.19 g, 8.8 mmol) and 10 mL of DMF were added, then DIPEA (1.49 mL, 9.0 mmol) was added in an ice bath, and then the resulting mixture was continuously stirred for 30 min. The above reaction solution was added to the flask, and the resulting mixture was heated to room temperature and reacted, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered, the filtrate was concentrated under reduced pressure, to obtain 2.6 g of solid Compound 14b, with a yield of 49%; LC-MS: [M+H]+=716.4.

[0384] Third Step: Compound 14c

[0385] To a 25 mL single-neck flask, Compound 14b (1.0 g, 1.4 mmol), and 15 mL of DMF were added and dissolved, then 1.0 g of 5% Pd / C were added, and then the hydrogenation reaction was carried out for 1.5 h. Upon completion of the reaction, the reaction solution was filtered, to obtain a filtrate containing Compound 14c, which was directly used in the next step reaction without purification.

[0386] Fourth Step: Compound 14d

[0387] The filtrate containing Compound 14c was placed in an ice bath, to which DIPEA (248 pL,

[0388] 1.5 mmol) was added, and then Compound M3 (808 mg, 1.4 mmol) was added. Upon completion of the additions, the mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 14d (500 mg); LC-MS: [M-H]' =870.5.

[0389] Fifth Step: Compound 14e

[0390] To a 50 mL single-neck flask, Compound 14d (400 mg, 0.46 mmol), Exatecan mesylate M5 (235 mg, 0.46 mmol), PyBOP (245 mg, 0.46 mmol), HOBt (63 mg, 0.46 mmol) and 15 mL of DMF were added, then DIPEA (331 pL, 2.0 mmol) were added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 3 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution of Compound 14e. The preparation solution was lyophilized to obtain Compound 14e (146 mg); LC- MS: [M+H]+=1289.5.

[0391] Sixth Step: Compound 14A

[0392] To a 25 mL single-neck flask, Compound 14e (100 mg, 0.08 mmol), zinc bromide (360 mg,

[0393] 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain solid Compound 14A (52 mg); LC-MS: [M+H]+=1133.4.

[0394] Example 25. Synthesis of Compound 14B

[0395] Compound M3 was replaced with Compound e«CM3, and Compound 14B (48 mg) was prepared by referring to the synthetic route of Example 24; LC-MS: [M+H]+=l 133.4.

[0396] Example 26. Synthesis of Compounds 15A and 15B

[0397] First Step: Compound 15a

[0398] To a 250 mL single-neck flask, CompoundMl (10 g, 27.1 mmol), benzyl 2-hydroxyl-butylate (synthesized by the method as disclosed in the literature “Chemical Communications”, 2019, 55(53), 7699-7702) (10.5 g, 54.3 mmol), and zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were added, and the resulting mixture was heated and reacted at 100 °C for 4 h. Upon completion of the reaction, the reaction solution was cooled to room temperature, and filtered to remove insoluble substances. The filtrate was concentrated to obtain a crude product, and the crude product was purified by silica column chromatography (PE:EA=10: 1-5: 1-2: 1), to obtain 5.67 g of Compound 15a, with a yield of 42%; LC-MS: [M+H]+=503.5.

[0399] Second Step: Compound 15b

[0400] To a 50 mL single-neck flask, Compound 15a (5 g, 9.95 mmol) and 15 mL of DMF were added and dissolved, then DBU (1.68 g, 11 mmol) was added in an ice bath, and then the resulting mixture was reacted for 1 h, the reaction solution was expressed as the reaction solution (1).

[0401] To another 50 mL single-neck flask, Compound M4 (4.1 g, 10.0 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol) and 10 mL of DMF were added and dissolved, then DIPEA (1.82 mL, 11 mmol) was added in an ice bath, the resulting mixture was reacted for 40 min, and then the reaction solution (1) was added. The resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, to obtain 4.6 g of solid Compound 15b, with a yield of 68%; LC-MS: [M+H]+-676.7.

[0402] Third Step: Compound 15d

[0403] To a 25 mL single-neck flask, Compound 15b (2.0 g, 2.96 mmol), and 15 mL of DMF were added and dissolved, then 2.0 g of 5% Pd / C was added, and then the hydrogenation reaction was carried out for 2h. Upon completion of the reaction, the reaction solution was filtered. The filtrate was placed in an ice bath, to which DIPEA (496 pL, 3.0 mmol) was added, and then Compound M3 (1.7 g, 2.96 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 1120.0 mg of Compound 15d, with a yield of 45%; LC-MS: [M-H]'=830.3.

[0404] Fourth Step: Compound 15e-l and Compound 15e-2

[0405] To a 50 mL single-neck flask, Compound 15d (500 mg, 0.60 mmol), Exatecan mesylate M5 (321 mg, 0.60 mmol), PyBOP (469 mg, 0.90 mmol), HOBt (121 mg, 0.90mmol) and 15 mL of DMF were added, then DIPEA (446 pL, 2.7 mmol)was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 15e-l and Compound 15e-2, respectively. The preparation solutions were lyophilized respectively, to obtain 138 mg of Compound 15e-l, LC-MS: [M+HJ - 1249.5; and 140 mg of Compound 15e-2, LC-MS: [M+H]+=1249.5.

[0406] Fifth Step: Compound 15A

[0407] To a 25 mL single-neck flask, Compound 15e-l (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 59 mg of solid Compound 15A; LC-MS: [M+H]+=1093.4.

[0408] Sixth Step: Compound 15B no

[0409] To a 25 mL single-neck flask, Compound 15e-2 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 60 mg of solid Compound 15B; LC-MS: [M+H]+=1093.4.

[0410] Example 27. Synthesis of Compounds 16A and 16B

[0411] Compound M3 was replaced with Compound C / / / -M3, and Compound 16A (55 mg) was prepared by referring to the synthetic route of Example 26; LC-MS: [M+H]+=1093.4.

[0412] Compound M3 was replaced with Compound e«LM3, and Compound 16B (54 mg) was prepared by referring to the synthetic route of Example 26; LC-MS: [M+H]+=1093.4.

[0413] Example 28. Synthesis of Compounds 17A and 17B

[0414] First Step: Compound 17a

[0415] To a 250 mL single-neck flask, Compound Ml (10 g, 27.1 mmol), benzyl 2-hydroxyl-3- phenylpropi onate (synthesized by the method as disclosed in the literature “Nature Communications”, 2020. 11 (1), 56) (14.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were added, and the resulting mixture was heated to 100 °C and reacted for 4 h. Upon completion of the reaction, the reaction solution was cooled to room temperature, and filtered to remove insoluble substances. The filtrate was concentrated to obtain a crude product, and the crude product was purified by silica column chromatography (PE:EA=10:l-5:l-2:l), to obtain 6.13 g of Compound 17a, with a yield of 40%; LC-MS: [M+H]+=565.6.

[0416] Second Step: Compound 17b

[0417] To a 50 mL single-neck flask, Compound 17a (5 g, 8.86 mmol) and 15 mL of DMF were in added and dissolved, then DBU (1.53 g, 10 mmol) were added in an ice bath, and then the resulting mixture was reacted for 1 h, the reaction solution was expressed as the reaction solution (1).

[0418] To another 50 mL single-neck flask, Compound M4 (3.6 g, 8.86 mmol), PyBOP (5.23 g, 10 mmol), HOBt (1.36 g, 10 mmol) and 10 mL of DMF were added and dissolved, then DIPEA (1.65 mL, 10 mmol) was added in an ice bath, and then the resulting mixture was continuously reacted for 30 min, and then the reaction solution (1) was added. The resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain 5.0 g of solid Compound 17b, with a yield of 77%;

[0419] Third Step: Compound 17d

[0420] To a 25 mL single-neck flask, Compound 17b (3.0 g, 4.07 mmol), and 15 mL of DMF were added and dissolved, then 3.0 g of 5% Pd / C was added, and then the hydrogenation reaction was carried out for 2h. Upon completion of the reaction, the reaction solution was filtered, to obtain a filtrate containing Compound 17c. The filtrate was placed in an ice bath, to which DIPEA (744 pL, 4.5 mmol) was added, and then Compound M3 (2.34 g, 4.07 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography to obtain a preparation solution, and the preparation solution was lyophilized to obtain 1.2 g of Compound 17d, with a yield of 33%; LC-MS: [M-H]’=892.4.

[0421] Fourth Step: Compound 17e

[0422] To a 50 mL single-neck flask, Compound 17d (500 mg, 0.56 mmol), Exatecan mesylate M5 (300 mg, 0.56 mmol), PyBOP (438 mg, 0.84 mmol), HOBt (113 mg, 0.84 mmol) and 15 mL of DMF were added, then DIPEA (330 pL, 2.0 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 17e-l and Compound 17e-2, respectively. The preparation solutions were lyophilized respectively to obtain 156 mg of Compound 17e-l, LC-MS: [M+H]+=1311.4; and 150 mg of Compound 17e-2, LC-MS: [M+H]+=1311.7.

[0423] Fifth Step: Compound 17A

[0424] To a 25 mL single-neck flask, Compound 17e-l (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 43 mg of Compound 17A; LC-MS: [M+H]+=l 155.4.

[0425] Sixth Step: Compound 17B

[0426] To a 25 mL single-neck flask, Compound 17e-2 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 40 mg of Compound 17B; LC-MS: [M+H]+=l 155.4.

[0427] Example 29. Synthesis of Compounds 18A and 18B Compound M3 was replaced with Compound C / / / -M3, and Compound 18A (54 mg) was prepared by referring to the synthetic route of Example 28; LC-MS: [M+H]+=l 155.4.

[0428] Compound M3 was replaced with Compound C / / / -M3, and Compound 18B (55 mg) was prepared by referring to the synthetic route of Example 28; LC-MS: [M+H]+=l 155.4.

[0429] Example 30. Synthesis of Compounds 19A and 19B

[0430] First Step: Compound 19a

[0431] To a 250 mL single-neck flask, Compound Ml (10 g, 27.1 mmol), benzyl 2-cyclopropyl-2- hydroxyacetate (prepared by referring to the method as disclosed in WO2020244657A1) (11.2 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were added, and the resulting mixture was heated to 100 °C and reacted for 4 h. Upon completion of the reaction, the reaction solution was cooled to room temperature, and filtered to remove insoluble substances. The filtrate was concentrated to obtain a crude product, and the crude product was purified by silica column chromatography (PE:EA=10: 1-5: 1-2: 1), to obtain 4 97 g of Compound 19a, with a yield of 36%; LC-MS: [M+H]+=515.2.

[0432] Second Step: Compound 1 b

[0433] To a 50 mL single-neck flask, Compound 19a (4 g, 7.8 mmol) and 10 mL of DMF were added and dissolved, then DBU (1.42 g, 9.3 mmol) was added in an ice bath, and then the resulting mixture was reacted for 1 h, the reaction solution was expressed as the reaction solution (1).

[0434] To another 50 mL single-neck flask, Compound M4 (3.2 g, 7.8 mmol), PyBOP (4.5 g, 8.6 mmol), HOBt (1.16 g, 8.6 mmol) and 10 mL of DMF were added and dissolved, then DIPEA (1.65 mL, 10 mmol) was added in an ice bath, and then the resulting mixture was reacted for 30 min, and then the reaction solution (1) was added. The resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, to obtain 4.2 g of solid Compound 19b, with a yield of 78%; LC-MS: [M+H]+=688.3.

[0435] Third Step: Compound 19d

[0436] To a 25 mL single-neck flask, Compound 19b (1000 mg, 1.45 mmol), and 15 mL of DMF were added and dissolved, then 1000 mg of 5% Pd / C was added, and then the hydrogenation reaction was carried out for 2 h. Upon completion of the reaction, the reaction solution was filtered, to obtain a filtrate containing Compound 19c. The filtrate was placed in an ice bath, to which DIPEA (248 pL, 1.5 mmol) was added, and then Compound M3 (720 mg, 1.45 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography to obtain a preparation solution, and the preparation solution was lyophilized to obtain 503 mg of Compound 19d, with a yield of 41%; LC-MS: [M-H]'=842.3.

[0437] Fourth Step: Compounds 19e-l and 19e-2

[0438] To a 50 mL single-neck flask, Compound 19d (500 mg, 0.59 mmol), Exatecan mesylate M5 (317 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.65 mmol) and 10 mL of DMF were added, then DIPEA (292 pL, 1.77 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 19e-l and Compound 19e-2, respectively. The preparation solutions were lyophilized respectively to obtain 112mg of Compound 19e-l, LC-MS: [M+H]+=1261.5; and 131 mg of Compound 19e-2, LC-MS: [M+HJ 1261 5

[0439] Fifth Step: Compound 19A

[0440] To a 25 mL single-neck flask, Compound 19e-l (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 55 mg of Compound 19A; LC-MS: [M+H]+=l 105.4.

[0441] Sixth Step: Compound 19B

[0442] To a 25 mL single-neck flask, Compound 19e-2 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a product preparation solution, and the preparation solution was lyophilized to obtain 58 mg of Compound 19B; LC-MS: [M+H]+=l 105.4.

[0443] Example 31. Synthesis of Compounds 20A and 20B

[0444] First Step: Compound 20a

[0445] To a 250 mL single-neck flask, Compound Ml (10 g, 27.1 mmol), benzyl 2-hydroxyl-3- cyclopropylpropionate (prepared by referring to the method as disclosed in W02020063676A) (12.0 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were added, and the resulting mixture was heated 100 °C and reacted for 4 h. Upon completion of the reaction, the reaction solution was cooled to room temperature, and filtered to remove insoluble substances. The filtrate was concentrated to obtain a crude product, and the crude product was purified by silica column chromatography (PE:EA=10: 1-5: 1-2: 1) to obtain 5.09 g of Compound 20a; LC-MS: [M+H]+=529.2.

[0446] Second Step: Compound 20b

[0447] To a 50 mL single-neck flask, Compound 20a (4 g, 7.6 mmol) and 10 mL of DMF were added and dissolved, then the DBU (1.39 g, 9.1 mmol) was added in an ice bath, and then the resulting mixture was reacted for 1 h, the reaction solution was expressed as the reaction solution (1).

[0448] To another 50 mL single-neck flask, Compound M4 (3.12 g, 7.6 mmol), PyBOP (4.5 g, 8.6 mmol), HOBt (1.16 g, 8.6 mmol) and 10 mL of DMF were added and dissolved, then DIPEA (1.65 mL, 10 mmol) was added in an ice bath, and then the resulting mixture was reacted for 30 min, and then the reaction solution (1) was added. The resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, to obtain 4.5 g of solid Compound 20b, with a yield of 84%; LC-MS: [M+H]+=702.3.

[0449] Third Step: Compound 20d

[0450] To a 25 mL single-neck flask, Compound 20b (1000 mg, 1.42 mmol), and 15 mL of DMF were added and dissolved, then 1000 mg of 5% Pd / C were added, and then the hydrogenation reaction was carried out for 2 h. Upon completion of the reaction, the reaction solution was filtered, to obtain a filtrate containing Compound 20c. The filtrate was placed in an ice bath, to which DIPEA (248 pL, 1.5 mmol) was added, and Exatecan mesylate M5 (708 mg, 1.42 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 443 mg of Compound 20d, with a yield of 36%; LC-MS: [M-H]'=856.4.

[0451] Fourth Step: Compounds 20e-l and 20e-2

[0452] To a 50 mL single-neck flask, Compound 20d (400 mg, 0.47 mmol), Exatecan mesylate M5 (250 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol) and 10 mL of DMF were added, then DIPEA (248 pL, 1.5 mmol) were added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 20e-l and Compound 20e-2, respectively. The preparation solutions were lyophilized respectively to obtain 103mg of Compound 20e-l, LC-MS: [M+HJ -1275.5; and 103 mg of Compound 20e-2, LC-MS: [M+H]+=1275.5.

[0453] Fifth Step: Compound 20 A

[0454] To a 25 mL single-neck flask, Compound 20e-l (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 51 mg of Compound 20A; LC-MS: [M+H]+=l 119.4.

[0455] Sixth Step: Compound 20B

[0456] To a 25 mL single-neck flask, Compound 20e-2(100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 47 mg of solid Compound 20B; LC-MS: [M+H]+=1119.4.

[0457] Example 32. Synthesis of Compound 21

[0458] First Step: Compound SM3-1

[0459] To a 2000 mL single-neck flask, 77087-60-6 (100 g, 458 mmol), maleic acid (53.4 g, 460 mmol), TEA (64 mL, 460 mmol) and 1000 mL of toluene were added, and the resulting mixture was heated to 100 °C and reacted for 5 h. Upon completion of the reaction, the reaction solution was cooled to room temperature, and filtered to remove insoluble substances. The filtrate was concentrated to obtain a crude product, and the crude product was purified by silica column chromatography (PE:EA=100: l-50:l-20:l) to obtain 75.6 g of Compound SM3-1; LC-MS: [M+H]+=299.1.

[0460] Second Step: Compound di- / c / 7-butyl 7?)-2-hydroxypentanedioate

[0461] To a 2000 mL single-neck flask, 172793-31-6 (100 g, 338 mmol) and 1000 mL of water were added, then sodium nitrite (35 g, 507 mmol), concentrated sulfuric acid (32 mL, 35 mmol) were added in order, and then the resulting mixture was slowly heated to room temperature and reacted for 24 h. Upon completion of the reaction, the reaction solution was extracted with 500 mL of ethyl acetate three times, and the organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by silica column chromatography (PE:EA=50: 1-30: 1-2: 1) to obtain 91.2 g of Compound di-Zc / 7-butyl (7^-2-hydroxypentanedioate; LC-MS: [M+H]+=261.4.

[0462] Third Step: Compound SM3

[0463] To a 2000 mL single-neck flask, di- / e / 7-butyl (K)-2-hydroxypentanedioate (50 g, 192 mmol) and 1000 mL of anhydrous tetrahydrofuran were added and cooled in an ice bath to 0 °C, then PPh3(87.7 g, 288 mmol), DEAD (50.2 g, 288 mmol) and Compound SM3-1 (57.3, 192 mmol) were added in order. The resulting mixture was slowly heated to room temperature and reacted for 13 h. Upon completion of the reaction, the reaction solution was filtered to remove insoluble substances. The filtrate was concentrated to obtain a crude product, and the crude product was purified by silica column chromatography (PE:EA=50: 1-30: 1-1 : 1) to obtain 68.6 g of the product.

[0464] The above product was dissolved in 500 mL of methanol and cooled in an ice bath to 0 °C, at this temperature, NaOH (64 mL, 190 mmol, 3M / L) was dropwise added, and then the resulting mixture was reacted for 12 h at this temperature, then HC1 (6 M / L) was added to adjust the pH to be 3. The reaction solution was extracted with 500 mL of dichloromethane five times, dried with anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The obtained crude product was purified by column chromatography (DCM / MeOH=50 / l-20 / l-2 / l), to obtain 50.4 g of Compound SM3; LC-MS: [M-H]’=525.5.

[0465] Fourth Step: Compound M6

[0466] To a 2000 mL single-neck flask, Compound SM3 (50 g, 95 mmol, l.Oeq), pentafluorophenol (19.2 g, 104.5 mmol, 1.1 eq), DCC (21.5 g, 104.5 mmol, 1.1 eq) and THF (600 mL) were added, and the resulting mixture was reacted at room temperature for 1 h (monitored by TLC), and then filtered to remove insoluble substances. The reaction solution was directly purified by preparative liquid chromatography, and the preparation solution was concentrated under reduced pressure with a water pump in a water bath at 35 °C to remove acetonitrile, and lyophilized to obtain Compound M6 (51.9 g), with a yield of 79%; LC-MS: [M+HJ -693.3.

[0467] Fifth Step: Compound 21a

[0468] To a 25 mL single-neck flask, Compound 1c (1 g, 2.36 mmol), and 25 mL of DMF were added and dissolved, then DIPEA (430 pL, 2.6 mmol) was added, and then Compound M6 (1177 mg, 2.36 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography to obtain a preparation solution, and the preparation solution was lyophilized to obtain 555 mg of Compound 21a; LC-MS: [M-H] =931.0.

[0469] Sixth Step: Compound 21b

[0470] To a 100 mL single-neck flask, Compound 21a (500 mg, 0.54 mmol), Exatecan mesylate M5 (285 mg, 0.54 mmol), PyBOP (312 mg, 0.6 mmol), HOBt (81 mg, 0.6 mmol) and 10 mL of DMF were added, then DIPEA (248 pL, 1.5 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 21b, and the preparation solution was lyophilized to obtain 231 mg of Compound 21b; LC-MS: [M+H]+=1349.5.

[0471] Seventh Step: Compound 21

[0472] To a 25 mL single-neck flask, Compound 21b (200 mg, 0.1488 mmol), zinc bromide (665 mg, 2.96 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution of product, and the preparation solution was lyophilized to obtain 103 mg of solid Compound 21; LC-MS: [M+H]+=1137.5.

[0473] Example 33. Synthesis of Compound 22

[0474] Compound M6 and Compound 3c were used as starting materials, and Compound 22 (91 mg) was prepared by referring to the synthetic route of Example 32; LC-MS: [M+H]+=1165.5.

[0475] Example 34. Synthesis of Compounds 23 and 24

[0476] Benzyl (S)-(-)-lactate was replaced with benzyl (R)-(+)-lactate, and Compound 5c’ was prepared by referring to the synthetic route of Example 8.

[0477] Compound M6 and Compound 5c were used as starting materials, and 102 mg of Compound

[0478] 23 was prepared by referring to the synthetic route of Example 32, LC-MS: [M+H]+=1151.4.

[0479] Compound M6 and Compound 5c’ were used as starting materials, and 99 mg of Compound

[0480] 24 was prepared by referring to the synthetic route of Example 32, LC-MS: [M-HJ M 151 .4.

[0481] Example 35. Synthesis of Compounds 25 and 26

[0482] Compound M6 and Compound 7c were used as starting materials, and by referring to the synthetic route of Example 32, 83 mg of Compound 25 was prepared, LC-MS: [M+H]+=1205.7; and 80 mg of Compound 26 was prepared, LC-MS: [M+H]+=1205.7.

[0483] Example 36. Synthesis of Compounds 27 and 28

[0484]

[0485] Compound M6 and Compound 19c were used as starting materials, and by referring to the synthetic route of Example 32, 100 mg of Compound 27 was prepared, LC-MS: [M+H]+=l 177.5; 101 mg of Compound 28 was prepared, LC-MS: [M+H]+=1177.5.

[0486] Example 37. Synthesis of Compound 29

[0487] First Step: Compound SM4-1

[0488] To a 5000 mL single-neck flask, maleic acid (50 g, 431 mmol, l.Oeq), 114559-25-0 (110 g, 431 mmol, 1 eq), TEA (263 g, 2.16 mol, 5 eq) and toluene (2000 mL) were added, and the resulting mixture was heated, refluxed and reacted for 5 h (monitored by TLC), and fdtered to remove insoluble substances The reaction solution was directly evaporated under reduced pressure by a rotation evaporator to remove the solvent, and the residue was purified by a silica column chromatography (PE / EA=50 / l-20 / l-l / l, to obtain Compound SM4-1 (64.7 g), with a yield of 50%; LC-MS: [M+H]+=299.2.

[0489] Second Step: Compound SM4-2 To a 2000 mL single-neck flask, Compound SM4-1 (64 g, 215 mmol), and 1000 mL of DMF were added and dissolved, DIPEA (71 mL, 430 mmol) were added, and then nonaethylene glycol monomethyl ether mesylate (111 5 g, 220 mmol) was added. Upon completion of the addition, the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by a silica column chromatography (PE / EA=50 / l -20 / 1 -1 / 1), to obtain 59.9 g of Compound SM4-2; LC-MS: [M+H]+=709.4.

[0490] Third Step: Compound SM4

[0491] To a 2000 mL single-neck flask, Compound SM4-2 (59 g, 83 mmol), and 1000 mL of MeOH were added and dissolved, then K2CO3 (11.75 g, 85 mmol) was added, and upon completion of the addition, the resulting mixture was reacted at room temperature for 4 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was filtered to remove insoluble substances, and purified by preparative high-performance liquid chromatography to obtain a preparation solution. The preparation solution was concentrated under reduced pressure with a water pump in a water bath at 35 °C to remove acetonitrile, and lyophilized to obtain Compound SM4 (27 g); LC-MS: [M-H] =693.5.

[0492] Fourth Step: Compound M7

[0493] To a 500 mL single-neck flask, Compound SM4 (25 g, 36 mmol, l.Oeq), pentafluorophenol (7.3 g, 40 mmol, 1.1 eq), DCC (8.2 g, 40 mmol, 1.1 eq) and THF (200 mL) were added, and the resulting mixture was reacted at room temperature for 1 h (monitored by TLC), and the reaction solution was filtered to remove insoluble substances, and directly purified by preparative liquid chromatography to obtain a preparation solution. The preparation solution was concentrated under reduced pressure with a water pump in a water bath at 35 °C to remove acetonitrile, and lyophilized to obtain Compound M7 (23.3 g), with a yield of 93%; LC-MS: [M+H]+=695.8.

[0494] Fifth Step: Compound 29a

[0495] To a 25 mL single-neck flask, Compound 1c (1 g, 2.36 mmol), and 25 mL of DMF were added and dissolved, then DIPEA (430 pL, 2.6 mmol) was added, and then Compound M7 (1640 mg, 2.36 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 609 mg of Compound 29a; LC-MS: [M-H] =1098.5.

[0496] Sixth Step: Compound 29b

[0497] To a 100 mL single-neck flask, Compound 29a (500 mg, 0.45 mmol), Exatecan mesylate M5 (240 mg, 0.45 mmol), PyBOP (281 mg, 0.54 mmol), HOBt (73 mg, 0.54 mmol) and 10 mL of DMF were added, then DIPEA (248 pL, 1.5 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 29b, and the preparation solution was lyophilized to obtain 187 mg of Compound 29b; LC-MS: [M+H]+=1517.6.

[0498] Seventh Step: Compound 29

[0499] To a 25 mL single-neck flask, Compound 29b (150 mg, 0.988 mmol), zinc bromide (223 mg, 0.988 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution of product, and the preparation solution was lyophilized to obtain 114 mg of solid

[0500] Compound M7 and Compound 3c were used as starting materials, Compound 30 (125 mg) was prepared by referring to the synthesis route of Example 37; LC-MS: [M+H]+=1445.6.

[0501] Example 39. Synthesis of Compounds 31 and 32

[0502] Compound M7 and Compound 5c were used as starting materials, and by referring to the synthesis route of Example 37, 61 mg of Compound 31 was prepared, LC-MS: [M+H]+=1431.7.

[0503] Compound M7 and Compound 5c’ were used as starting materials, and by referring to the synthesis route of Example 37, 63 mg of Compound 32 was prepared, LC-MS: [M+H]+=1431.7.

[0504] Example 40. Synthesis of Compounds 33 and 34

[0505] Compound M7 and Compound 7c were used as starting materials, and by referring to the synthesis route of Example 37, 60 mg of Compound 33 was prepared, LC-MS: [M+H]+=1485.6; and 58 mg of Compound 34 was prepared, LC-MS: [M+H]+=1485.6.

[0506] Example 41. Synthesis of Compounds 35 and 36

[0507]

[0508] Compound M7 and 19c were used as starting materials, and by referring to the synthesis route of Example 37, 102 mg of Compound 35 was prepared, LC-MS: [M+H]+=1457.8; and 102 mg of

[0509] Compound 36 was prepared, LC-MS: [M+H]+=1457.8.

[0510] Example 42. Synthesis of Compound 37

[0511] First Step: Compound SM5-1

[0512] To a 2000 mL single-neck flask, Compound 16947-84-5 (100 g, 295 mmol, l.Oeq), DIPEA (50 mL, 300 mmol), benzyl bromide (51.3 g, 300 mmol) and THF (1000 mL) were added and reacted at room temperature for 12 h (the reaction was monitored by TLC). The reaction solution was filtered to remove insoluble substances and directly evaporated under reduced pressure by a rotation evaporator to remove the solvent, and the residue was purified by silica column chromatography (PE / EA=50 / 1 -20 / 1 -2 / 1), to obtain Compound SM5-1 (110.1 g), with a yield of 87%; LC-MS: [M+H]+=429.2.

[0513] Second Step: Compound SM5-2

[0514] To a 2000 mL single-neck flask, Compound SM5-1 (100 g, 233.4 mmol, 1.0 eq) and THF (1000 mL) were added, and cooled in an ice bath to 0 °C, and then NaH (37.4 g, 933.5 mmol), and Mel (132.5 g, 933.5 mmol) were added in batch. The resulting mixture was reacted at 0 °C for 24 h (the reaction was monitored by TLC), and then 500 mL of a saturated NH4CI aqueous solution was added to quench the reaction. The reaction solution was extracted with 500 mL of ethyl acetate three times, and the organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was directly evaporated under reduced pressure by a rotation evaporator to remove the solvent, and the residue was purified by silica column chromatography (PE / EA=100 / l-50 / l-10 / l) to obtain Compound SM5-2 (37.1 g); LC-MS: |M+H] 443.3.

[0515] Third Step: Compound SM5 (referring to the literature Org. Lett., 2006, 8, 3387-3390.)

[0516] To a 1000 mL single-neck flask, Compound SM5-2 (35 g, 79 mmol, l.Oeq) and DCE (500 mL) was added, then palladium diacetate (180 mg, 0.8 mmol), I2 (20 g, 79 mmol), and iodobenzene diacetate (40.8 g, 126.4 mmol) were added in order. The resulting mixture was heated to 60 °C and reacted for 40 h (the reaction was monitored by TLC), then 500 mL of a sodium thiosulfate aqueous solution was added to quench the reaction. The reaction solution was extracted with 500 mL of ethyl acetate three times, and the organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was directly evaporated under reduced pressure by a rotation evaporator to remove the solvent, and the residue was purified by silica column chromatography (PE / EA=100 / l-50 / l-10 / l) to obtain Compound SM5 (28 g); LC-MS: [M+H]+=501.3.

[0517] Fourth Step: Compound SM6

[0518] To a 500 mL single-neck flask, Compound SM5 (25 g, 50 mmol, l.Oeq), potassium di- / - butylphosphate (13.66 g, 55 mmol, 1.1 eq), / i-toluenesulfonic acid monohydrate (951 mg, 5 mmol, 0.1 eq) and THF (200 mL) were added and reacted at room temperature for 1 h (the reaction was monitored by TLC). The reaction solution was filtered to remove insoluble substances, and directly purified by preparative high-performance liquid chromatography to obtain a preparation solution, and the preparation solution was concentrated under reduced pressure with a water pump in a water bath at 35 °C to remove acetonitrile, and lyophilized to obtain Compound SM6 (15.1 g), with a yield of 46%; LC-MS: [M+HJ -651.4.

[0519] Fifth Step: Compound SM7

[0520] To a 250 mL single-neck flask, Compound SM6 (15 g, 23 mmol) and 100 mL of DMF were added and dissolved, then 15 g of 5% Pd / C was added in an ice bath, and then the atmosphere in the system was replaced with hydrogen gas three time. The resulting mixture was reacted at room temperature for 12 h, and filtered to remove Pd / C, and the filtrate was evaporated under reduced pressure with an oil pump to remove the solvent, to obtain a crude product, set aside for use.

[0521] To another 250 mL single-neck flask, the above crude product, 100 mL of toluene, triethylamine (6.4 mL, 46 mmol), and maleic anhydride (2.4 g, 24 mmol) were added and dissolved, the resulting mixture was heated to 100 °C and reacted for 2 h. The reaction progress was monitored by HPLC, and after the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with di chloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain 4.2 g of solid Compound SM7, with a yield of 36%; LC-MS: [M+H]+=507.3.

[0522] Sixth Step: Compound M8

[0523] To a 100 mL single-neck flask, Compound SM7 (4 g, 7.9 mmol, l.Oeq), pentafluorophenol (1.6 g, 8.7 mmol, 1.1 eq), DCC (1.8 g, 8.7 mmol, 1.1 eq) and THF (60 mL) were added and reacted at room temperature for 1 h (the reaction was monitored by TLC), and the reaction solution was filtered to remove insoluble substances, and directly purified by preparative high-performance liquid chromatography to obtain a preparation solution, and the preparation solution was concentrated under reduced pressure with a water pump in a water bath at 35 °C to remove acetonitrile, and lyophilized to obtain Compound M8 (3.7 g), with a yield of 70%; LC-MS: [M+H]+=673.2.

[0524] Seventh Step: Compound 37a

[0525] To a 25 mL single-neck flask, Compound 1c (1 g, 2.36 mmol), and 25 mL of DMF were added and dissolved, then DIPEA (430 pL, 2.6 mmol) was added, and then Compound M8 (1.2 g, 2.36 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography to obtain a preparation solution, the preparation solution was lyophilized to obtain 488 mg of Compound 37a; LC-MS: [M-H]’=911.0.

[0526] Eighth Step: Compound 37b

[0527] To a 100 mL single-neck flask, Compound 37a (400 mg, 0.44 mmol), Exatecan mesylate M5 (235 mg, 0.44 mmol), PyBOP (260 mg, 0.5 mmol), HOBt (69 mg, 0.5 mmol) and 10 mL of DMF were added, then DIPEA (218 pL, 1.32 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 37b, and the preparation solution was lyophilized to obtain 201 mg of Compound 37b; LC-MS: [M+H]+=1329.6.

[0528] Ninth Step: Compound 37

[0529] To a 25 mL single-neck flask, Compound 37b (130 mg, 0.098 mmol), zinc bromide (221 mg, 0.98 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 96 mg of Compound 37; LC-MS: [M+H]+=l 117.4.

[0530] Example 43. Synthesis of Compound 38

[0531] Compound M8 and Compound 3c were used as starting materials, and Compound 38 (51 mg) was prepared by referring to the synthesis route of Example 42; LC-MS: [M+H]+=1145.6.

[0532] Example 44. Synthesis of Compounds 39 and 40 Compound M8 and Compound 5c were used as starting materials, and by referring to the synthesis route of Example 42, 57 mg of Compound 39 was prepared, LC-MS: [M+H]+=1131.4.

[0533] Compound M8 and Compound 5c’ were used as starting materials, and 60 mg of Compound 40 was prepared by referring to the synthesis route of Example 42, LC-MS: [M+H]+=l 131.4.

[0534] Example 45. Synthesis of Compounds 41 and 42

[0535] Compound M8 and Compound 7c were used as starting materials, and by referring to the synthesis route of Example 42, 44 mg of Compound 41 was prepared, LC-MS: [M+H]+=l 185.3; and 44 mg of Compound 42 was prepared, LC-MS: [M+HJ 1 185 3.

[0536] Example 46. Synthesis of Compounds 43 and 44

[0537] Compound M8 and Compound 19c were used as starting materials, and by referring to the synthesis route of Example 42, 62 mg of Compound 43 was prepared, LC-MS: [M+H]+=l 157.4; and 59 mg of Compound 44 was prepared, LC-MS: [M+H]+=l 157.4.

[0538] Example 47. Synthesis of Compound 45

[0539] Compound 45 was synthesized according to the method provided in Example 58 of the patent application CN104755494A.

[0540] Example 48. Synthesis of Compound 46

[0541]

[0542] To a 50 mL single-neck flask, Compound Id (500 mg, 0.62 mmol), Compound M9 (310 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were added, then DIPEA (378 pL, 2.29 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 46a (210 mg); LC-MS: [M+H]+=1221.6.

[0543] Second Step: Compound 46

[0544] To a 25 mL single-neck flask, Compound 46a (200 mg, 0.162 mmol), zinc bromide (736 mg, 3.26 mmol) and 10 mL of nitromethane were added and reacted 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 46 (120 mg); LC-MS: [M+H]+=1065.3.

[0545] Example 49. Synthesis of Compound 47

[0546] Compound M3 was replaced with Compound ent-M3, and Compound Id’ was prepared by referring to the synthetic route of Example 4.

[0547] Compound Id was replaced with Compound Id’, and Compound 47 (81 mg) was prepared by referring to the synthetic route of Example 48; LC-MS: [M+H]+=1065.3.

[0548] Example 50. Synthesis of Compound 48A

[0549]

[0550] First Step: Compound 48a

[0551] To a 100 mL single-neck flask, Compound 5d (1.66 g, 2.02 mmol, 1.0 eq), Compound M9

[0552] (1.08 g, 2.02 mmol, 1.0 eq), PyBOP (1.58 g, 3.03 mmol, 1.5 eq), HOBt (0.41 g, 3.03 mmol, 1.5 eq) and DMF(40 mL) were added, then DIPEA (0.84 mL, 1.5 eq) was added in an ice bath, and the resulting mixture was heated to room temperature and reacted for 2 h (monitored by HPLC). The reaction solution was directly purified by preparative liquid chromatography, and the preparation solution was concentrated under reduced pressure with a water pump in a water bath at 35 °C to remove acetonitrile, and lyophilized to obtain Compound 48a (1.54 g), with a yield of 61%; LC-MS: [M+H]+=1235.4.

[0553] Second Step: Compound 48A

[0554] To a 100 mL single-neck flask, Compound 48a (1.0 g, 0.8 mmol, 1.0 eq), and 35 mL of nitromethane were added and dissolved, then zinc bromide (3.64 g, 16 mmol, 20.0 eq) was added, and then the resulting mixture was reacted in an oil bath at 40 °C (pre-heated for stabilization in advance) for 30 min. The reaction solution was concentrated to remove nitromethane under reduced pressure with a water pump in a water bath at 45°C, to obtain a yellow residue solid (monitored by HPLC). Through purification by preparative liquid chromatography, a preparation solution was obtained by adding 0.1% trifluoroacetic acid to the flowing phase, and the preparation solution was concentrated under reduced pressure with a water pump in a water bath at 35 °C to remove acetonitrile, and lyophilized to obtain Compound 48A (786 mg) with a yield of 90%.

[0555] Example 51. Synthesis of Compound 48B

[0556] First Step: Compound 48b

[0557] To a 25 mL single-neck flask, Compound 5d-l (200 mg, 0.24 mmol, 1.0 eq), Compound M9 (127 mg, 0.24 mmol, 1.0 eq), PyBOP (187 mg, 0.36 mmol, 1.2eq), HOBt (48 mg, 0.36 mmol, 1.2 eq) and DMF (6 mL) were added and cooled to 0 °C to 5°C in an ice bath, then DIPEA (62 mg, 0.48 mmol, 2.0 eq) was added. Upon completion of the addition, the resulting mixture was heated to 20±5 °C and reacted for 2 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was directly purified by preparative HPLC. The preparation solution was collected and lyophilized to obtain Compound 48b (150.2 mg); LC-MS: [M+H]+=1235.4.

[0558] Second Step: Compound 48B

[0559] To a 25 mL single-neck flask, Compound 48b (100 mg, 0.081 mmol, 1.0 eq), ZnBr2 (364 mg, 1.62 mmol, 20.0 eq) and CH3NO2 (10 mL) were added, and upon completion of the addition, the resulting mixture was heated to 40 °C and reacted for 0.5 h, then the reaction was stopped. The reaction solution was directly dried under reduced pressure at 45 °C, to obtain a yellow solid. By sampling, the reaction was monitored by HPLC. The dried solid was directly purified by preparative HPLC. The preparation solution was collected and lyophilized to obtain Compound 48B (70.0 mg); LC-MS: [M+H]+=1079.4.

[0560] Example 52. Synthesis of Compound 49A referring to the synthetic route of Example 8. Compound 5d was replaced with Compound 5d’, and Compound 49A (71 mg) was prepared by referring to the synthetic route of Example 50; LC- MS: [M+H]+=1079.4.

[0561] Example 53. Preparation of Compound 49B

[0562] Compound 5c was replaced with Compound 5c-l’, and Compound 5d-l’ was prepared by referring to the synthetic route of Example 9. Compound 5d- 1 was replaced with Compound 5d- 1’, and Compound 49B (65 mg) was prepared by referring to the synthetic route of Example 51; LC-MS: [M+H]+= 1079.4.

[0563] Example 54. Synthesis of Compounds 50A and 50B

[0564]

[0565] First Step: Compounds 50a and 50b

[0566] To a 50 mL single-neck flask, Compound 7d (500 mg, 0.57 mmol), Compound M9 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were added, then DIPEA (378 pL, 2.29 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 50a and Compound 50b, respectively. The preparation solutions were lyophilized respectively to obtain 170 mg of Compound 50a, LC-MS: [M+H]+=1289.46; and 202 mg of Compound 50b, LC-MS: [M+H]+=1289.46.

[0567] Second Step: Compound 50A

[0568] To a 25 mL single-neck flask, Compound 50a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 44 mg of Compound 50A.

[0569] Third Step: Compound 50B

[0570] To a 25 mL single-neck flask, Compound 50b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 45 mg of Compound 50B.

[0571] Example 55. Synthesis of Compounds 51A and 5 IB

[0572] First Step: Compound 51a and Compound 51b

[0573] To a 50 mL single-neck flask, Compound 8d (500 mg, 0.57 mmol), Compound M9 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were added, then DIPEA (378 pL, 2.29 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 51a and Compound 51b, respectively. The preparation solutions were lyophilized respectively to obtain 190 mg of Compound 51a, and 186 mg of Compound 51b. LC-MS of Compound 51a: [M+HJ I 289.47; LC-MS of Compound 51b: | M+H] - | 289.47.

[0574] Second Step: Compound 51A

[0575] To a 25 mL single-neck flask, Compound 51a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 39 mg of Compound 51A.

[0576] Third Step: Compound 5 IB

[0577] To a 25 mL single-neck flask, Compound 51b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 60 mg of Compound 51B.

[0578] Example 56. Synthesis of Compound 52A

[0579] First Step: Compound 52a

[0580] To a 50 mL single-neck flask, Compound lid (800 mg, 0.96 mmol), Compound M9 (480 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (130 mg, 0.96 mmol) and 30 mL of DMF were added, then DIPEA (660 pL, 4.0 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 4 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 52a, and the preparation solution was lyophilized to obtain Compound 52a (388 mg); LC-MS: [M+H]+=1261.4.

[0581] Second Step: Compound 52A

[0582] To a 25 mL single-neck flask, Compound 52a (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 52A(79 mg); LC-MS: [M+H]+=1105.4.

[0583] Example 57. Synthesis of Compound 52B

[0584] Compound M3 was replaced with Compound e«LM3, and Compound lid’ was prepared by referring to the synthetic route of Example 18.

[0585] Compound l id was replaced with Compound lid’, and Compound 52B (50 mg) was prepared by referring to the synthetic route of Example 56; LC-MS: [M+H]+=l 105.4.

[0586] Example 58. Synthesis of Compound 53A

[0587] To a 50 mL single-neck flask, Compound 12d (400 mg, 0.47 mmol), Compound M9 (240 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt (64 mg, 0.47 mmol) and 15 mL of DMF were added, then DIPEA (330 pL, 2.0 mmol) was added in an ice bath, and the resulting mixture was heated to room temperature and reacted for 3 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution of Compound 53a, and the preparation solution was lyophilized to obtain Compound 53a (200 mg); LC-MS: [M+H]+=1275.4.

[0588] Second Step: Compound 53A

[0589] To a 25 mL single-neck flask, Compound 53a (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain solid Compound 53A (51 mg); LC-MS: [M+H]+=1119.4.

[0590] Example 59. Synthesis of Compound 53B

[0591] Compound M3 was replaced with Compound cv / / -M3, and Compound 12d’ was prepared by referring to the synthetic route of Example 20.

[0592] Compound 12d was replaced with Compound 12d’, and Compound 53B (50 mg) was prepared by referring to the synthetic route of Example 58; LC-MS: [M+HJ M 1 19.4.

[0593] To a 50 mL single-neck flask, Compound 19d (500 mg, 0.59 mmol), Compound M9 (317 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.65 mmol) and 10 mL of DMF were added, then DIPEA (292 pL, 1.77 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 54a and 54b, respectively. The preparation solutions were lyophilized respectively to obtain 103 mg of Compound 54a, LC-MS: [M+H]+=1261.5; and 111 mg of Compound 54b, LC-MS: [M+H]+=1261.5.

[0594] Second Step: Compound 54A

[0595] To a 25 mL single-neck flask, Compound 54a (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 61 mg of Compound 54A; LC-MS: [M+H]+=l 105.4.

[0596] Third Step: Compound 54B

[0597] To a 25 mL single-neck flask, Compound 54b (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 57 mg of Compound 54B; LC-MS: [M+H]+=l 105.4.

[0598] Example 61. Synthesis of Compounds 55A and 55B

[0599] First Step: Compounds 55a and 55b

[0600] To a 50 mL single-neck flask, Compound 20d (400 mg, 0.47 mmol), Compound M9 (250 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol) and 10 mL of DMF were added, then DIPEA (248 pL, 1.5 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 55a and Compound 55b, respectively. The preparation solutions were lyophilized respectively to obtain 100 mg of Compound 55a, LC-MS: [M+H]+=1275.5; and 101 mg of Compound 55b, LC-MS: [M+H]+=1275.5.

[0601] Second Step: Compound 55A

[0602] To a 25 mL single-neck flask, Compound 55a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 42 mg of solid Compound 55A; LC-MS: [M+H]+=1119.4.

[0603] Third Step: Compound 55B

[0604] To a 25 mL single-neck flask, Compound 55b (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 45 mg of solid Compound 55B; LC-MS: [M+H]+=1119.4.

[0605] Example 62. Synthesis of Compound 56

[0606] Compound M3 was replaced with Compound c7? / -M3, and Compound 20d’ was prepared by referring to the synthetic route of Example 31.

[0607] Compound 20d was replaced with Compound 20d’, and Compound 56 (50 mg) was prepared by referring to the synthetic route of Example 61; LC-MS: [M+H]+=1119.3.

[0608] Example 63. Synthesis of Compound 57

[0609] Compound M3 was replaced with Compound ewLM3, and Compound 20d’ was prepared by referring to the synthetic route of Example 31.

[0610] Compound 20d was replaced with Compound 20d’, and Compound 57 (50 mg) was prepared by referring to the synthetic route of Example 61; LC-MS: [M+H]+=l 119.4.

[0611] Example 64. Synthesis of Compound 58

[0612] First Step: Synthesis of Compound 58a

[0613] To Exatecan mesylate M5 (15 g, 28 mol, prepared according to the method disclosed in EP0737683A1), 400 mL of DMF was added, and the resulting mixture was cooled in an ice bath to 0°C, to which triethylamine was dropwise added to adjust pH to 7 to 8, then benzyl bromide (9.6 g, 56 mmol) was dropwise added in an ice bath, and then the resulting mixture was heated to room temperature (25 °C) and reacted for 1 h. The reaction was monitored with TLC, and upon completion of the reaction, the reaction solution was concentrated under reduced pressure. The obtained crude product was purified by a preparative high performance liquid chromatography (acetonitrile / pure water system). The preparation solution at target peak was collected and concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain about 11 g of yellow solid Compound 58a, with a yield of about 74%, MS m / z: [M+H]+526.3.

[0614] Second Step: Synthesis of Compound 58b

[0615] At room temperature, to a 250 mL single-neck flask, Compound 58a (11 g, 21 mol), and 120 mL of formic acid were added in order and dissolved to obtain a bright yellow solution, to which 30 mL of 40% formaldehyde in water were added. The resulting mixture was heated to 50°C and reacted for 1 h, and the reaction was monitored with TLC. Upon completion of the reaction, the reaction solution was cooled to room temperature, and purified by preparative high performance liquid chromatography (acetonitrile / pure water system). The preparation solution at target peak was collected and concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain about 4.5 g of Compound 58b, in a yellow powder solid, with a yield of about 40%, MS m / z: [M+H]+540.6.

[0616] Third Step: Synthesis of Compound 58: At room temperature, to a 250 mL single-neck flask, Compound 58b (2.3 g, 4.3 mol) and 100 mL of DMF was added and dissolved to obtain a bright yellow solution, to which 2.3 g of 5% Pd / C were added. The atmosphere in the system was replaced with hydrogen gas. The resulting mixture was reacted for 1.5 at room temperature, and the reaction was monitored with HPLC.

[0617] Upon completion of the reaction, the reaction solution was filtered to remove Pd / C, concentrated, and purified by preparative high performance liquid chromatography (acetonitrile / pure water system). The preparation solution at target peak was collected and concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain about 1.0 g of Compound 58, in a yellow powder solid, with a yield of about 52%, MS m / z :[M+H]+450.5.

[0618] Example 65. Synthesis of Compound 59:

[0619] To a 50 mL single-neck flask, Compound Id (500 mg, 0.62 mmol), Compound 58 (279 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were added, then DIPEA (378 pL, 2.29 mmol) was added in an ice bath, and the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 59a (166 mg); LC-MS: [M+H]+=1235.6.

[0620] Second Step: Compound 59

[0621] To a 25 mL single-neck flask, Compound 59a (100 mg, 0.081mmol), zinc bromide (368 mg, 1.63 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 59 (43 mg); LC-MS: [M+H]+= 1079.3.

[0622] Example 66. Synthesis of Compound 60

[0623] Compound M3 was replaced with Compound c -M3, and Compound Id’ was prepared by referring to the synthetic route of Example 4.

[0624] Compound Id was replaced with Compound Id’, and Compound 60 (40 mg) was prepared by referring to the synthetic route of Example 65; LC-MS: [M+H]+=1079.3.

[0625] Example 67. Synthesis of Compound 61

[0626] First Step: Compound 61a

[0627] To a 100 mL single-neck flask, Compound 5d (1.66 g, 2.02 mmol, 1.0 eq), Compound 58 (0.91 g, 2.02 mmol, 1.0 eq), PyBOP (1.58 g, 3.03 mmol, 1.5 eq), HOBt (0.41 g, 3.03 mmol, 1.5 eq) and DMF (40 mL) were added, then DIPEA (0.84 mL, 1.5 eq) was added in an ice bath, and the resulting mixture was heated to room temperature and reacted for 2 h (the reaction was monitored by HPLC). the reaction solution was directly purified by preparative high-performance liquid chromatography to obtain a preparation solution, and the preparation solution was concentrated to remove acetonitrile under reduced pressure with a water pump in a water bath at 35 °C, and lyophilized to obtain Compound 61a (1.21 g); LC-MS: [M+H]+=1249.4.

[0628] Second Step: Compound 61

[0629] To a lOOmLsingle-neck flask, Compound 61a (1.0 g, 0.8 mmol, 1.0 eq), 35 mL of nitromethane were added and dissolved, then zinc bromide (3.64 g, 16 mmol, 20.0 eq) was added, and then the resulting mixture was reacted for 30 min in an oil bath at 40 °C (pre-heated for stabilization in advance). The reaction solution was concentrated to remove nitromethane under reduced pressure with a water pump in a water bath at 45 °C, to obtain a yellow residue solid (monitored by HPLC). Through purification by preparative liquid chromatography, a preparation solution was obtained by adding 0.1% trifluoroacetate to the flowing phase, and the preparation solution was concentrated to remove acetonitrile under reduced pressure with a water pump in a water bath at 35 °C, and lyophilized to obtain Compound 61 (786 mg), LC-MS: [M+H]+=1093.6.

[0630] Example 68. Synthesis of Compound 62

[0631]

[0632] To a 25 mL single-neck flask, Compound 5d-l (200 mg, 0.24 mmol, 1.0 eq), Compound 58 (110.3 mg, 0.24 mmol, 1.0 eq), PyBOP (187 mg, 0.36 mmol, 1.2eq), HOBt (48 mg, 0.36 mmol, 1.2 eq) and DMF (6 mL) were added and cooled to 0 °C to 5°C in an ice bath, then DIPEA (62 mg, 0.48 mmol, 2.0 eq) was added. Upon completion of the addition, the resulting mixture was heated to 20±5 °C and reacted from 2 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was directly purified by preparative HPLC, and a preparation solution was collected and lyophilized to obtain Compound 62a (120.9 mg); LC-MS: [M+H]+=1249.4.

[0633] Second Step: Compound 62

[0634] To a 25 mL single-neck flask, Compound 62a (100 mg, 0.081 mmol, 1.0 eq), ZnBr2 (364 mg, 1.62 mmol, 20.0 eq) and CH3NO2 (10 mL) were added in order, and upon completion of the addition, the resulting mixture was heated to 40 °C and reacted for 0.5 h, then the reaction was stopped. The reaction solution was directly dried under reduced pressure at 45 °C, to obtain a yellow solid, the reaction was monitored by HPLC by sampling. The dried solid was directly purified by preparative HPLC, and a preparation solution was collected and lyophilized to obtain Compound 62 (61 mg); LC-MS: [M+H]+=1093.4.

[0635] Example 69. Preparation of Compound 63

[0636] Compound 5d was replaced with Compound 5d’, and Compound 63 (60 mg) was prepared by referring to the synthetic route of Example 67; LC-MS:[M+H]+=1093.4.

[0637] Example 70. Preparation of Compound 64

[0638] Compound 5d-l was replaced with Compound 5d-l’, and Compound 64 (65 mg) was prepared by referring to the synthetic route of Example 68; LC-MS: [M+H]+=1093.4.

[0639] Example 71. Preparations of Compounds 65A and 65B

[0640] First Step: Compounds 65a and 65b

[0641] To a 50 mL single-neck flask, Compound 7d (500 mg, 0.57 mmol), Compound 58 (256.8 mg, 0.57 mmol), PyBOP (448 mg, 0.86mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were added, then DIPEA (378 pL, 2.29 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 65a and Compound 65b, respectively. The preparation solutions were lyophilized respectively to obtain 155 mg of Compound 65a, LC-MS: [M+H]+=1303.4; and 158 mg of Compound 65b, LC-MS: [M+H]+=1303.6.

[0642] Second Step: Compound 65 A

[0643] To a 25 mL single-neck flask, Compound 65a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 49 mg of solid Compound 65A. Third Step: Compound 65B

[0644] To a 25 mL single-neck flask, Compound 65b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 47 mg of solid Compound 65B.

[0645] First Step: Compound 66a and Compound 66b

[0646] To a 50 mL single-neck flask, Compound 8d (500 mg, 0.57 mmol), Compound 58 (256.8 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were added, then DIPEA (378 pL, 2.29 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 66a and Compound 66b, respectively. The preparation solutions were lyophilized respectively to obtain 160 mg of Compound 66a, and 160 mg of Compound 66b. LC-MS of Compound 66a: [M+H]+=1303.7; LC-MS of Compound 66b: [M+H]+=1303.6.

[0647] Second Step: Compound 66A

[0648] To a 25 mL single-neck flask, Compound 66a (100 mg, 0.077 mmol), zinc bromide (349 mg,

[0649] 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 57 mg of Compound 66A, LC-MS: [M+H]+= 1147.5.

[0650] Third Step: Compound 66B

[0651] To a 25 mL single-neck flask, Compound 66b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 57 mg of Compound 66B, LC-MS: [M+H]+=1147.5.

[0652] Example 73. Synthesis of Compound 67A

[0653] First Step: Compound 67a

[0654] To a 50 mL single-neck flask, Compound lid (800 mg, 0.96 mmol), Compound 58 (432.5 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (130 mg, 0.96 mmol) and 30 mL of DMF were added, then DIPEA (660 pL, 4.0 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 4 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 67a, and the preparation solution was lyophilized to obtain Compound 67a (402 mg); LC-MS: [M+H]+=1275.4.

[0655] Second Step: Compound 67A

[0656] To a 25 mL single-neck flask, Compound 67a (100 mg, 0.78 mmol), zinc bromide (356 mg, 1.57 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 67A (47 mg); LC-MS: [M+H]+=1119.5.

[0657] Example 74. Synthesis of Compound 67B

[0658] Compound l id was replaced with Compound lid’, and Compound 67B (50 mg) was prepared by referring to the synthetic route of Example 73; LC-MS: [M+H]+1119.4.

[0659] Example 75. Synthesis of Compound 68A

[0660] To a 50 mL single-neck flask, Compound 12d (400 mg, 0.47 mmol), Compound 58 (211.7 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt (64 mg, 0.47 mmol) and 15 mL of DMF were added, then DIPEA (330 pL, 2.0 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 3 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 68a, and the preparation solution was lyophilized to obtain Compound 68a (177 mg); LC-MS: [M+H]+=1289.4.

[0661] Second Step: Compound 68A

[0662] To a 25 mL single-neck flask, Compound 68a (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 68A (45 mg); LC-MS: [M+H]+=1133.4.

[0663] Example 76. Synthesis of Compound 68B

[0664] Compound 12d was replaced with Compound 12d’, and Compound 68B (50 mg) was prepared by referring to the synthetic route of Example 75; LC-MS: [M+H]+=l 133.4.

[0665] To a 50 mL single-neck flask, Compound 19d (500 mg, 0.59 mmol), Compound 58 (266 mg,

[0666] 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.65 mmol) and 10 mL of DMF were added, then DIPEA (292 pL, 1.77 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 69a and Compound 69b, respectively. The preparation solutions were lyophilized respectively to obtain 109 mg of Compound 69a, LC-MS: [M+H]+=1275.5; and 111 mg of Compound 69b, LC-MS: [M+H]+=1275.7.

[0667] Second Step: Compound 69A

[0668] To a 25 mL single-neck flask, Compound 69a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 53 mg of Compound 69A; LC-MS: [M+H]+=l 119.4.

[0669] Third Step: Compound 69B

[0670] To a 25 mL single-neck flask, Compound 69b (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 54 mg of Compound 69B; LC-MS: [M+H]+=l 119.4.

[0671] Example 78. Synthesis of Compounds 70A and 70B

[0672] First Step: Compounds 70a and 70b

[0673] To a 50 mL single-neck flask, Compound 20d (400 mg, 0.47 mmol), Compound 58 (211.7 mg, 0.47 mmol), PyBOP (292 mg, 0.56 mmol), HOBt (76 mg, 0.56 mmol) and 10 mL of DMF were added, then DIPEA (248 pL, 1.5 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 70a and Compound 70b, respectively. The preparation solutions were lyophilized respectively to obtain 106 mg of Compound 70a, LC-MS: [M+H]+=1289.5; and 101 mg of Compound 70b, LC-MS: [M+H]+=1289.4.

[0674] Second Step: Compound 70A

[0675] To a 25 mL single-neck flask, Compound 70a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 39 mg of Compound 70A; LC-MS: [M+H]+=l 133.4.

[0676] Third Step: Compound 70B

[0677] To a 25 mL single-neck flask, Compound 70b (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 35 mg of Compound 70B; LC-MS: [M+H]+=l 133.4.

[0678] Example 79. Synthesis of Compound 71

[0679] Compound 20d was replaced with Compound 20d’, and Compound 71 (30 mg) was prepared by referring to the synthetic route of Example 78; LC-MS: [M+HJ 1133 3.

[0680] Example 80. Synthesis of Compound 72

[0681] Compound 20d was replaced with Compound 20d’, and Compound 72 (33 mg) was prepared by referring to the synthetic route of Example 78; LC-MS: [M+H]+=1133.4.

[0682] Example 81. Synthesis of Compound Mil

[0683] To a 100 mL single-neck flask, Compound M3 (11.0 g, 19.5 mmol, l.Oeq), DIPEA (2.8 g, 21.4 mmol, 1.1 eq), 27-amino-4,7,10,13,16,19,22,25-octaoxaheptacosanoic acid (9.7 g, 20.5 mmol, 1.05 eq) and DMF (60 mL) were added and reacted at room temperature for 20 min (the reaction was monitored by TLC). The reaction solution was directly purified by preparative high- performance liquid chromatography to obtain a preparation solution, and the preparation solution was concentrated to remove acetonitrile under reduced pressure with a water pump in a water bath at 35 °C, and lyophilized to obtain Compound M10 (13.2 g), with a yield of 78%; LC-MS: [M+H]+=866.5.

[0684] To a 100 mL single-neck flask, Compound M10 (13.0 g, 15 mmol, l .Oeq), pentafluorophenol (3 g, 16.5 mmol, 1.1 eq), DCC (3.4 g, 16.5 mmol, 1.1 eq) and THF (30 mL) were added and reacted at room temperature for 30 min (the reaction was monitored by TLC), and then the reaction solution was fdtered to remove insoluble substances, and directly purified by preparative high- performance liquid chromatography to obtain a preparation solution, and the preparation solution was concentrated to remove acetonitrile under reduced pressure with a water pump in a water bath at 35 °C, and lyophilized to obtain Compound Mil (14.2g), with a yield of 92%; LC-MS: [M+H]+=1032.5.

[0685] Example 82. Synthesis of Compound 73

[0686] First Step: Synthesis of Compound 73a

[0687] To Compound Mil (1 g, 0.79 mol), 10 mL of DMF was added, then the resulting mixture was cooled in an ice bath to 0°C, to which Compound 1c (334 mg, 0.79 mol) and DIPEA (154 mg, 1.19 mol) were added, and at this condition, the resulting mixture was reacted for 1 h. The reaction was monitored with TLC, upon completion of the reaction, the reaction solution was purified by preparative high performance liquid chromatography (acetonitrile / pure water system). The preparation solution at target peak was collected and concentrated to move acetonitrile under reduced pressure, and lyophilized to obtain about 1.2 g of Compound 73a, MS m / z: [M+H]+=I271.9.

[0688] Second Step: Synthesis of Compound 73b

[0689] To a 25 mL single-neck flask, Compound 73a (1.2 g, 0.94 mmol), Exatecan mesylate M5 (500 mg, 0.94 mmol), PyBOP (625 mg, 1.2 mmol), HOBt (162 mg, 1.2 mmol) and 15 mL ofDMF were added, then DIPEA (310 mg, 2.4 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 73b (709 mg); LC-MS: [M+H]+=1720.8.

[0690]

[0691] Third Step: Synthesis of Compound 73:

[0692] To a 25 mL single-neck flask, Compound 73b (200 mg, 0.116 mmol), zinc bromide (523 mg, 2.32 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 73 (88 mg); LC-MS: [M+H]+= 1532.6.

[0693] Example 83. Synthesis of Compound 74

[0694] Compound M3 was replaced with Compound enZ-M3, and Compound Ml 1 ’ was prepared by referring to the synthetic route of Example 80.

[0695] Compound Mil was replaced with Compound MU’, and Compound 74 (90 mg) was prepared by referring to the synthetic route of Example 82; LC-MS: [M+H]+=l 532.6.

[0696] Example 84. Synthesis of Compound 75

[0697] First Step: Synthesis of Compound 75a

[0698] To Compound Mil (1 g, 0.79 mol), 10 mL of DMF was added, then the resulting mixture was cooled in an ice bath to 0°C, to which Compound 5c (345 mg, 0.79 mol) and DIPEA (154 mg, 1.19 mol) were added, and then the resulting mixture was reacted for 1 h at this condition. The reaction was monitored with TLC, upon completion of the reaction, the reaction solution was purified by preparative high performance liquid chromatography (acetonitrile / pure water system). The preparation solution at target peak was collected and concentrated to remove acetonitrile under reduced pressure, and lyophilized to obtain 0.9 g of Compound 75a, MS m / z: [M+H]+

[0699] = 1285.6.

[0700] Second Step: Synthesis of Compound 75b

[0701] To a 25 mL single-neck flask, Compound 75a (700 mg, 0.54 mmol), Exatecan mesylate M5 (289 mg, 0.54 mmol), PyBOP (313 mg, 0.6 mmol), HOBt (81 mg, 0.6 mmol) and 10 mL of DMF were added, then DIPEA (155 mg, 1.2 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 75b (304 mg); LC-MS: [M+H]+=1734.8.

[0702] Third Step: Synthesis of Compound 75

[0703] To a 25 mL single-neck flask, Compound 75b (200 mg, 0.116 mmol), zinc bromide (523 mg, 2.32 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain solid Compound 75 (96 mg); LC-MS: [M+H]+=l 546.6.

[0704] Example 85. Synthesis of Compound 76

[0705] Compound Mil was replaced with Compound MU’, and Compound 76 (92 mg) was prepared by referring to the synthetic route of Example 84; LC-MS: [M+H]+=l 546.5.

[0706] Example 86. Synthesis of Compound 77

[0707] Compound Mil was replaced with Compound MU’, Compound 5c was replaced with Compound 5c’, and Compound 77 (87 mg) was prepared by referring to the synthetic route of Example 84; LC-MS: [M+HJ -1546.5.

[0708] Example 87. Synthesis of Compound 78

[0709] Compound 5c was replaced with Compound 5c’, and Compound 78 (94 mg) was prepared by referring to the synthetic route of Example 84; LC-MS: [M+H]+=1546.7.

[0710] Example 88. Synthesis of Compounds 79 and 80

[0711] First Step: Synthesis of Compound 79a

[0712] To Compound Mil (1 g, 0.79 mol), 10 mL of DMF was added, then the mixture was cooled in an ice bath to 0°C, to which Compound 20c (377 mg, 0.79 mol) and DIPEA (154 mg, 1.19 mol) were added, and then at this condition, the resulting mixture was reacted for 1 h. The reaction was monitored with TLC, upon completion of the reaction, the reaction solution was purified by a preparative high performance liquid chromatography (acetonitrile / pure water system). The preparation solution at target peak was collected and concentrated to remove acetonitrile under reduced pressure, and lyophilized to obtain about 783 mg of Compound 79a, MS m / z: [M+H]+=1325.8.

[0713] Second Step: Synthesis of Compounds 79b-l and 79b-2

[0714] To a 25 mL single-neck flask, Compound 79a (600 mg, 0.45 mmol), Exatecan mesylate M5 (240 mg, 0.45 mmol), PyBOP (261 mg, 0.5 mmol), HOBt (68 mg, 0.5 mmol) and 10 mL of DMF were added, then DIPEA (130 mg, 1 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 79b- 1 and Compound 79b-2, respectively, and the preparation solutions were lyophilized respectively to obtain Compound 79b-l (124 mg); LC-MS: [M+H]+= 1743.0; and Compound 79b-l (122 mg); LC-MS: [M+H]+= 1743.0.

[0715]

[0716] To a 25 mL single-neck flask, Compound 79b-l (100 mg, 0.057 mmol), zinc bromide (258 mg, 1.15 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 79 (30 mg); LC-MS: [M+H]+= 1586.9.

[0717] To a 25 mL single-neck flask, Compound 79b-2 (100 mg, 0.057 mmol), zinc bromide (258 mg, 1.15 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 80 (33 mg); LC-MS: [M+H]+= 1587.0.

[0718] Example 89. Synthesis of Compound 81

[0719] Compound Mllwas replaced with Compound MU’, and Compound 81 (24 mg) was prepared by referring to the synthetic route of Example 88; LC-MS: [M+H]+=l 586.9.

[0720] Example 90. Synthesis of Compound 82

[0721] Compound Mil was replaced with Compound MU’, and Compound 82 (29 mg) was prepared by referring to the synthetic route of Example 88; LC-MS: [M+H]+=l 586.9.

[0722] Example 91. Expression and purification of antibody

[0723] An antibody (13C 1 , 13C9 or NBE-002) was expressed by using Expi293 suspension cells (Shanghai OPM Biosciences Co., Ltd ). On the day before transfection, the cells were seeded in a shake flask containing OPM-293 CD05 Medium (81075-001, Shanghai OPM Biosciences Co., Ltd.) at a density of about 0.9* 106 / mL and cultured overnight under the conditions of 37 °C, 5% CO2 and 120 rpm in a cell culture shaker. On the next day, the transfection of antibody-expressed plasmid was performed with PEI-MAX, wherein the mass ratio of plasmid to PEI-MAX was 1:3. On the first day after the transfection, OPM-293 Profeed (Shanghai OPM Biosciences Co., Ltd.) was added at 5% (v / v), on the third day after the transfection, OPM-293 Profeed was added again at 5% (v / v), and on the sixth day after the transfection, the supernatant was collected by centrifugation.

[0724] The collected cell-expressed supernatant passed through a Protein A affinity chromatography column (UniMab 50, Suzhou NanoMicro Tech Co., Ltd.) and eluted with 0.05 M sodium acetate (pH3.6). The captured antibody was adjusted to pH7.0 with 1 M Tris-HCl (pH8.8), and impurities, such as multimers, were removed by passing through a gel filtration chromatography column SEC (Superdex 200, GE Company). Antibodies 13C1, 13C9 (i.e., VLS-101 antibody) and NBE-002 were obtained, and their sequence information was shown below:

[0725] Example 92. General Preparation of Antibody-Drug Conjugates

[0726] The antibody prepared in Example 91 was replaced with 20 mM of a NaAc-HAc, pH6.0 buffer, and the antibody was concentrated or diluted to a protein concentration of 5 mg / mL. The linker-payloads prepared in Examples 1-90 were dissolved with N,N-dimethylacetamide (DMA) to 10 mg / mL, and set aside for use

[0727] To open the interchain disulfide bonds of the antibody, tris(2-carboxyethyl)phosphine (TCEP) was first added in a molecular ratio of 15 to 25 times, and at room temperature, the resulting mixture was reacted for 2 h. A linker-payload solution was added in a molecular ratio of 15 to 25 times, and at room temperature, the resulting mixture was reacted for 2 h. After the reactions were completed, unconjugated linker-payload was removed by using a 30 KDa ultrafiltration centrifugal tube, to obtain an antibody-dmg conjugate sample.

[0728] ADC-1 to ADC-106 of the following examples were prepared and obtained by conjugating the compounds (linker-payload, linker-drug) prepared in the examples of the application to Antibody 13C1, with reference to this preparation method, in which the sum of nl, n2 and n3 represents the drug to antibody ratio (DAR) of the ADC.

[0729] Moreover, it should be noted that when the ADC of the application is prepared, the compound (linker-drug) in the ADC is easy to interact with water molecules under the condition of easy hydrolysis when it is conjugated to the antibody, and a hydrolytic ring-opening reaction occurs, and the hydrolysis site is at the maleimide. When multiple linker-drugs are conjugated to the antibody, depending on different hydrolysis degrees, the following cases may occur: (1) the maleimides are not hydrolyzed, i.e., each maleimide is in a closed-ring form o ; (2) the maleimides are not completely hydrolyzed, i.e., a part of the maleimides are in a ring-closed form o , and another part of the maleimides are in a ring-opening form the maleimides are fully hydrolyzed, i.e. all the maleimides are in the ring-opening form or . Hence, in the ADCs prepared in the following examples, nl, n2, n3 are not 0 at the same time, and the sum of nl, n2 and n3 represents the DAR, the specific value of which is determined in Example 202. Meanwhile, those skilled in the art would understand that, as described above, the number of the linker-drugs conjugated to each antibody may be either the same or different, and therefore, the average number of the linker-drugs conjugated to each antibody may be either an integer or a decimal. Accordingly, when the linker-drugs are conjugated to the antibody, the maleimide may be hydrolyzed under the condition of easy hydrolysis, and the hydrolysis degrees may be either the same or different, therefore, nl, n2, and n3 may be either integers or decimals.

[0730] Example 93: ADC-1

[0731] According to the general conjugation method of the application, Compound 2 was conjugated to antibody 13C1, to prepare and obtain ADC- 1.

[0732] Example 94:

[0733] According to the general conjugation method of the application, Compound 1 was conjugated to antibody 13C1, to prepare and obtain ADC-2.

[0734] Example 95:

[0735] According to the general conjugation method of the application, Compound 4 was conjugatedtibody 13C1, to prepare and obtain ADC-3.

[0736] Example 96:

[0737] According to the general conjugation method of the application, Compound 3 was conjugatedtibody 13C1, to prepare and obtain ADC-4.

[0738]

[0739] Example 97: ADC-5

[0740] According to the general conjugation method of the application, Compound 5B was conjugated to antibody 13C1, to prepare and obtain ADC-5.

[0741] According to the general conjugation method of the application, Compound 5A was conjugated to antibody 13C1, to prepare and obtain ADC-6.

[0742]

[0743] According to the general conjugation method of the application, Compound 6B was conjugated to antibody 13C1, to prepare and obtain ADC-7.

[0744] Example 100:

[0745] According to the general conjugation method of the application, Compound 6A was conjugated to antibody 13C1, to prepare and obtain ADC-8.

[0746] Example 101 :

[0747] According to the general conjugation method of the application, Compound 8B was conjugated to antibody 13C1, to prepare and obtain ADC-9.

[0748] Example 102:

[0749] According to the general conjugation method of the application, Compound 7B was conjugated to antibody 13C1, to prepare and obtain ADC-10.

[0750] Example 103:

[0751] According to the general conjugation method of the application, Compound 8A was conjugated to antibody 13C1, to prepare and obtain ADC-11.

[0752] Example 104:

[0753] According to the general conjugation method of the application, Compound 7A was conjugated to antibody 13C1, to prepare and obtain ADC-12.

[0754] Example 105:

[0755] According to the general conjugation method of the application, Compound 9B was conjugated to antibody 13C1, to prepare and obtain ADC-13.

[0756] Example 106:

[0757] According to the general conjugation method of the application, Compound 9A was conjugated to antibody 13C1, to prepare and obtain ADC-14.

[0758] Example 107:

[0759] According to the general conjugation method of the application, Compound 10B was conjugated to antibody 13C1, to prepare and obtain ADC-15.

[0760] Example 108:

[0761] According to the general conjugation method of the application, Compound 10A was conjugated to antibody 13C1, to prepare and obtain ADC-16.

[0762] Example 109:

[0763] According to the general conjugation method of the application, Compound 11B was conjugated to antibody 13C1, to prepare and obtain ADC-17.

[0764] Example 110:

[0765] According to the general conjugation method of the application, Compound 11A was conjugated to antibody 13C1, to prepare and obtain ADC-18.

[0766] Example 111:

[0767] According to the general conjugation method of the application, Compound 12B was conjugated to antibody 13C1, to prepare and obtain ADC-19.

[0768] Example 112:

[0769] According to the general conjugation method of the application, Compound 12A was conjugated to antibody 13C1, to prepare and obtain ADC-20.

[0770] Example 113:

[0771] According to the general conjugation method of the application, Compound 13B was conjugated to antibody 13C1, to prepare and obtain ADC-21.

[0772] Example 114:

[0773] According to the general conjugation method of the application, Compound 13A was conjugated to antibody 13C1, to prepare and obtain ADC-22.

[0774] Example 115:

[0775] According to the general conjugation method of the application, Compound 14B was conjugated to antibody 13C1, to prepare and obtain ADC-23.

[0776] Example 116:

[0777] According to the general conjugation method of the application, Compound 14A was conjugated to antibody 13C1, to prepare and obtain ADC-24.

[0778] Example 117:

[0779] According to the general conjugation method of the application, Compound 16A was conjugated to antibody 13C1, to prepare and obtain ADC-25.

[0780] Example 118:

[0781] According to the general conjugation method of the application, Compound 15A was conjugated to antibody 13C1, to prepare and obtain ADC-26.

[0782] Example 119:

[0783] According to the general conjugation method of the application, Compound 16B was conjugated to antibody 13C1, to prepare and obtain ADC-27.

[0784] Example 120:

[0785] According to the general conjugation method of the application, Compound 15B was conjugated to antibody 13C1, to prepare and obtain ADC-28.

[0786] Example 121 :

[0787] According to the general conjugation method of the application, Compound 18A was conjugated to antibody 13C1, to prepare and obtain ADC-29.

[0788] Example 122:

[0789] According to the general conjugation method of the application, Compound 17A was conjugated to antibody 13C1, to prepare and obtain ADC-30.

[0790] Example 123:

[0791] According to the general conjugation method of the application, Compound 18B was conjugated to antibody 13C1, to prepare and obtain ADC-31.

[0792] Example 124:

[0793] According to the general conjugation method of the application, Compound 17B was conjugated to antibody 13C1, to prepare and obtain ADC-32.

[0794] Example 125:

[0795] According to the general conjugation method of the application, Compound 19A was conjugated to antibody 13C1, to prepare and obtain ADC-33.

[0796] Example 126:

[0797] According to the general conjugation method of the application, Compound 19B was conjugated to antibody 13C1, to prepare and obtain ADC-34.

[0798] Example 127:

[0799] According to the general conjugation method of the application, Compound 20B was conjugated to antibody 13C1, to prepare and obtain ADC-35.

[0800] Example 128:

[0801] According to the general conjugation method of the application, Compound 20A was conjugated to antibody 13C1, to prepare and obtain ADC-36.

[0802] Example 129:

[0803] According to the general conjugation method of the application, Compound 21 was conjugated to antibody 13C1, to prepare and obtain ADC-37.

[0804] Example 130:

[0805] According to the general conjugation method of the application, Compound 22 was conjugated to antibody 13C1, to prepare and obtain ADC-38.

[0806] Example 131 :

[0807] According to the general conjugation method of the application, Compound 23 was conjugated to antibody 13C1, to prepare and obtain ADC-39.

[0808] Example 132:

[0809] According to the general conjugation method of the application, Compound 24 was conjugated to antibody 13C1, to prepare and obtain ADC-40.

[0810] Example 133:

[0811] According to the general conjugation method of the application, Compound 25 was conjugated to antibody 13C1, to prepare and obtain ADC-41.

[0812] Example 134:

[0813] According to the general conjugation method of the application, Compound 26 was conjugated to antibody 13C1, to prepare and obtain ADC-42.

[0814] Example 135:

[0815] According to the general conjugation method of the application, Compound 27 was conjugated to antibody 13C1, to prepare and obtain ADC-43.

[0816] Example 136:

[0817] According to the general conjugation method of the application, Compound 28 was conjugated to antibody 13C1, to prepare and obtain ADC-44.

[0818] Example 137:

[0819] According to the general conjugation method of the application, Compound 29 was conjugated to antibody 13C1, to prepare and obtain ADC-45.

[0820] Example 138:

[0821] According to the general conjugation method of the application, Compound 30 was conjugated to antibody 13C1, to prepare and obtain ADC-46.

[0822] Example 139:

[0823] According to the general conjugation method of the application, Compound 31 was conjugated to antibody 13C1, to prepare and obtain ADC-47.

[0824] Example 140:

[0825] According to the general conjugation method of the application, Compound 32 was conjugated to antibody 13C1, to prepare and obtain ADC-48.

[0826] Example 141 :

[0827] According to the general conjugation method of the application, Compound 34 was conjugated to antibody 13C1, to prepare and obtain ADC-49.

[0828] Example 142:

[0829] According to the general conjugation method of the application, Compound 33 was conjugated to antibody 13C1, to prepare and obtain ADC-50.

[0830] Example 143:

[0831] According to the general conjugation method of the application, Compound 35 was conjugated to antibody 13C1, to prepare and obtain ADC-51.

[0832] Example 144:

[0833] According to the general conjugation method of the application, Compound 36 was conjugated to antibody 13C1, to prepare and obtain ADC-52.

[0834] Example 145:

[0835] According to the general conjugation method of the application, Compound 37 was conjugated to antibody 13C1, to prepare and obtain ADC-53.

[0836] Example 146:

[0837] According to the general conjugation method of the application, Compound 38 was conjugated to antibody 13C1, to prepare and obtain ADC-54.

[0838] Example 147:

[0839] According to the general conjugation method of the application, Compound 39 was conjugated to antibody 13C1, to prepare and obtain ADC-55.

[0840] Example 148:

[0841] According to the general conjugation method of the application, Compound 40 was conjugated to antibody 13C1, to prepare and obtain ADC-56.

[0842] Example 149:

[0843] According to the general conjugation method of the application, Compound 42 was conjugated to antibody 13C1, to prepare and obtain ADC-57.

[0844] Example 150:

[0845] According to the general conjugation method of the application, Compound 41 was conjugated to antibody 13C1, to prepare and obtain ADC-58.

[0846] Example 151 :

[0847] According to the general conjugation method of the application, Compound 43 was conjugated to antibody 13C1, to prepare and obtain ADC-59.

[0848] Example 152:

[0849] According to the general conjugation method of the application, Compound 44 was conjugated to antibody 13C1, to prepare and obtain ADC-60.

[0850] Example 153:

[0851] According to the general conjugation method of the application, Compound 47 was conjugated to antibody 13C1, to prepare and obtain ADC-61.

[0852] Example 154:

[0853] According to the general conjugation method of the application, Compound 46 was conjugated to antibody 13C1, to prepare and obtain ADC-62.

[0854] Example 155:

[0855] According to the general conjugation method of the application, Compound 48B was conjugated to antibody 13C1, to prepare and obtain ADC-63.

[0856] Example 156:

[0857] According to the general conjugation method of the application, Compound 48A was conjugated to antibody 13C1, to prepare and obtain ADC-64.

[0858] Example 157:

[0859] According to the general conjugation method of the application, Compound 49B was conjugated to antibody 13C1, to prepare and obtain ADC-65.

[0860] Example 158:

[0861] Compound 49A and antibody 13C1, according to the general conjugation method of the application, Compound 49A was conjugated to antibody 13C1, to prepare and obtain ADC-66.

[0862] Example 159:

[0863] According to the general conjugation method of the application, Compound 5 IB was conjugated to antibody 13C1, to prepare and obtain ADC-67.

[0864] Example 160:

[0865] According to the general conjugation method of the application, Compound 50B was conjugated to antibody 13C1, to prepare and obtain ADC-68.

[0866] Example 161 :

[0867] According to the general conjugation method of the application, Compound 51A was conjugated to antibody 13C1, to prepare and obtain ADC-69.

[0868] Example 162:

[0869] According to the general conjugation method of the application, Compound 50A was conjugated to antibody 13C1, to prepare and obtain ADC-70.

[0870] Example 163:

[0871] According to the general conjugation method of the application, Compound 53B was conjugated to antibody 13C1, to prepare and obtain ADC-71.

[0872] Example 164:

[0873] According to the general conjugation method of the application, Compound 53A was conjugated to antibody 13C1, to prepare and obtain ADC-72.

[0874] Example 165:

[0875] According to the general conjugation method of the application, Compound 54A was conjugated to antibody 13C1, to prepare and obtain ADC-73.

[0876] Example 166:

[0877] According to the general conjugation method of the application, Compound 54B was conjugated to antibody 13C1, to prepare and obtain ADC-74.

[0878] Example 167:

[0879] According to the general conjugation method of the application, Compound 55B was conjugated to antibody 13C1, to prepare and obtain ADC-75.

[0880] Example 168:

[0881] According to the general conjugation method of the application, Compound 55A was conjugated to antibody 13C1, to prepare and obtain ADC-76.

[0882] Example 169:

[0883] According to the general conjugation method of the application, Compound 57 was conjugated to antibody 13C1, to prepare and obtain ADC-77.

[0884] Example 170:

[0885] According to the general conjugation method of the application, Compound 56 was conjugated to antibody 13C1, to prepare and obtain ADC-78.

[0886] Example 171 :

[0887] According to the general conjugation method of the application, Compound 60 was conjugated to antibody 13C1, to prepare and obtain ADC-79.

[0888] Example 172:

[0889] According to the general conjugation method of the application, Compound 59 was conjugated to antibody 13C1, to prepare and obtain ADC-80.

[0890] Example 173:

[0891] According to the general conjugation method of the application, Compound 62 was conjugated to antibody 13C1, to prepare and obtain ADC-81.

[0892] Example 174:

[0893] According to the general conjugation method of the application, Compound 61 was conjugated to antibody 13C1, to prepare and obtain ADC-82.

[0894] Example 175:

[0895] According to the general conjugation method of the application, Compound 64 was conjugated to antibody 13C1, to prepare and obtain ADC-83.

[0896] Example 176:

[0897] According to the general conjugation method of the application, Compound 63 was conjugated to antibody 13C1, to prepare and obtain ADC-84.

[0898] Example 177:

[0899] According to the general conjugation method of the application, Compound 66B was conjugated to antibody 13C1, to prepare and obtain ADC-85.

[0900] Example 178:

[0901] According to the general conjugation method of the application, Compound 65B was conjugated to antibody 13C1, to prepare and obtain ADC-86.

[0902] Example 179:

[0903] According to the general conjugation method of the application, Compound 66A was conjugated to antibody 13C1, to prepare and obtain ADC-87.

[0904] Example 180:

[0905] According to the general conjugation method of the application, Compound 65A was conjugated to antibody 13C1, to prepare and obtain ADC-88.

[0906] Example 181 :

[0907] According to the general conjugation method of the application, Compound 68B was conjugated to antibody 13C1, to prepare and obtain ADC-89.

[0908] Example 182:

[0909] According to the general conjugation method of the application, Compound 68A was conjugated to antibody 13C1, to prepare and obtain ADC-90.

[0910] Example 183:

[0911] According to the general conjugation method of the application, Compound 69A was conjugated to antibody 13C1, to prepare and obtain ADC-91.

[0912] Example 184:

[0913] According to the general conjugation method of the application, Compound 69B was conjugated to antibody 13C1, to prepare and obtain ADC-92.

[0914] Example 185:

[0915] According to the general conjugation method of the application, Compound 70B was conjugated to antibody 13C1, to prepare and obtain ADC-93.

[0916] Example 186:

[0917] According to the general conjugation method of the application, Compound 70A was conjugated to antibody 13C1, to prepare and obtain ADC-94.

[0918] Example 187:

[0919] According to the general conjugation method of the application, Compound 72 was conjugated to antibody 13C1, to prepare and obtain ADC-95.

[0920] Example 188:

[0921] According to the general conjugation method of the application, Compound 71 was conjugated to antibody 13C1, to prepare and obtain ADC-96.

[0922] Example 189:

[0923] According to the general conjugation method of the application, Compound 74 was conjugated to antibody 13C1, to prepare and obtain ADC-97.

[0924] Example 190:

[0925] According to the general conjugation method of the application, Compound 73 was conjugated to antibody 13C1, to prepare and obtain ADC-98.

[0926] Example 191 :

[0927] According to the general conjugation method of the application, Compound 76 was conjugated to antibody 13C1, to prepare and obtain ADC-99.

[0928] Example 192: ADC- 100

[0929] According to the general conjugation method of the application, Compound 77 was conjugated to antibody 13C1, to prepare and obtain ADC- 100.

[0930]

[0931] Example 193: ADC-101

[0932] According to the general conjugation method of the application, Compound 75 was conjugated to antibody 13C1, to prepare and obtain ADC-101.

[0933] Example 194: ADC- 102

[0934] According to the general conjugation method of the application, Compound 78 was conjugated to antibody 13C1, to prepare and obtain ADC- 102.

[0935]

[0936] Example 195: ADC-103

[0937] According to the general conjugation method of the application, Compound 82 was conjugated to antibody 13C1, to prepare and obtain ADC- 103.

[0938] Example 196: ADC- 104

[0939] According to the general conjugation method of the application, Compound 81 was conjugated to antibody 13C1, to prepare and obtain ADC- 104.

[0940]

[0941] Example 197: ADC-105

[0942] According to the general conjugation method of the application, Compound 79 was conjugated to antibody 13C1, to prepare and obtain ADC- 105.

[0943] Example 198: ADC-106

[0944] According to the general conjugation method of the application, Compound 80 was conjugated to antibody 13C1, to prepare and obtain ADC-106.

[0945]

[0946] Example 199: ADC- 107 (control group)

[0947] According to the general conjugation method of the application, Compound 5A was conjugated to antibody 13C9, to prepare and obtain ADC- 107.

[0948] Example 200: ADC- 108 (control group)

[0949] According to the general conjugation method of the application, Compound 5A was conjugated to antibody NBE-002, to prepare and obtain ADC-108.

[0950]

[0951] Example 201 : Determination of monomer ratio by using an SEC-HPLC method Chromatographic column: Biocore SEC -300 5pm, 4.6x300 mm

[0952] Manufacturer: NanoChrom, Item No.: B213-050030-04630S

[0953] Flowing phase: 50 mM PB+300 mM NaCl+200 mM Arg+5% IPA, pH=6.5

[0954] Table 1 : Parameters of method

[0955] Table 2: Determination results of monomer rate of the ligand-drug conjugates (ADC) disclosed in the application

[0956] The determination results of the monomer rate of the ligand-drug conjugates (ADC) disclosed in the application were shown in Table 2 and FIG. 1A to FIG. 1C. The results showed that as compared to the control ADCs, the ADCs (e.g., ADC-6) disclosed in the application had the excellent property such as low degradation rate, low aggregation rate, and high monomer rate.

[0957] Example 202 Determination of drug to antibody rate (DAR) by using a RP-HPLC method Chromatographic column: Proteomix RP-10004.6 100mm 5pm 1000A

[0958] Manufacturer: Sepax. Item No.: 465950-4610 Table 3 : Parameters of method

[0959] Table 4: Determination results of drug to antibody rate (DAR) of ADC

[0960] The Determination results of drug to antibody rate (DAR) of the ligand-drug conjugates (ADC) disclosed in the application were shown in Table 2 and FIG. 2A to FIG. 2C. The results showed that in the same feed ratio of antibody and linker-payload, the ADCs (such as ADC-6) prepared in the application had a higher DAR value as compared to the control ADC, and the drug concentration at the target site would be significantly improved under the same administration dose of the ADC drugs.

[0961] Example 203 : Plasma stability data of ADC

[0962] A mixed solution of ADC and IgG-depleted plasma was formulated to make the final concentration of the ADC be 0.6 mg / mL, and incubated in a water bath in a 37 °C incubator. The incubation time was set to be 0 day, 3 days, and 7 days, and a non-incubated plasma was set as a control. After incubation, samples were purified and extracted for determination of the drug to antibody ratio (DAR) to evaluate the stability of the ADC in plasma.

[0963] Table 5: Plasma stability data of ADC drugs in the application

[0964] Note: “Without incubation and extraction” corresponds to the DAR values of the ADC which was placed stably 4 °C for 0 day; “Incubation with plasma for 0 day” corresponds to the DAR values determined after the purification and extraction step which was started immediately after the ADC was added into the plasma; “Incubation with plasma for 3 days” corresponds to the DAR values determined after the purification and extraction step which was started after the ADC was added into the plasma at 37 °C for 3 days; “Incubation with plasma for 7 days” corresponds to the DAR values determined after the purification and extraction step which was started after the ADC was added into the plasma at 37 °C for 7 days.

[0965] The determination results of plasma stability of the ADCs were shown in Table 5, and the results showed that the ADCs (e.g., ADC-6) as disclosed in the application exhibited excellent plasma stability, and not show a great change in the DAR value.

[0966] Example 204: Relative affinity data of ADC

[0967] The ADC-6 retained the affinity of the corresponding original anti-human ROR1 antibody 13C1 to R0R1. The relative affinities of Antibody 13C1 and the ADC-6, Antibody 13C9 and the ADC-107, and Antibody NBE-002 and the ADC-108 to the R0R1 were compared by an ELISA method.

[0968] The method for detecting the relative binding activity of R0R1 comprises the following specific steps:

[0969] The antigen was adjusted to an appropriate concentration with PBS and added into an ELISA plate, and incubated overnight at 4 °C for coating. After coating, the plate was washed and blocked at 37 °C for 1 h. After blocking, the plate was washed, and the test sample was gradiently diluted acording to an appropriate concentration and added to the blocked ELISA plate for incubation. The rabbit anti-human antibody was diluted to an appropriate concentration with a buffer, and added to the plate for incubation. Then a chromogenic solution was added and reacted for 7 minutes, a stop solution was added thereto to terminate the reaction. The plate was read at 450 nm, a four-parameter logistic fit on the results were performed, EC50 values were calculated, to further obtain the results of relative binding activity.

[0970] Table 6: Relative affinity of ADC molecules to antibody

[0971] As shown in FIG. 3A, FIG. 3B and FIG. 3C, the affinity of the ADC after conjugation remained similar to that of the corresponding antibody, with no significant difference in EC50 values, and the corresponding P values ranged from 50% to 150%, indicating that the conjugation of the toxin to the antibody did not significantly affect the affinity of the antibody to the antigen.

[0972] Example 205 In Vitro Efficacy Tests

[0973] Various human tumor cell lines RPML8226, NCI-N87, MDA-MB-468, and JVM-3 were used as in vitro cell models to evaluate the killing effects of bioactive molecules antibody drug conjugates (ADC) on in vitro tumor cells. A certain number of tumor cells were seeded into a 96- well culture plate. After the cells adhere to the wall, gradient-diluted test samples were added into the well plate with an initial concentration of the antibody or ADC of 500 nM and a 7-fold dilution for a total of 8 test concentrations, with three replicates of each test concentration. The sample was incubated at 37 °C with 5% CO2 for 5 days, and the cell viability was detected by using MTS. The cell viability was calculated using the formula: Cell viability = (Test group - blank group) / (Negative control group - blank group)* 100%, and Graph Pad Prism 9.0 statistical software was used to fit the curve by means of a four-parameter model, with the X axis as the molar concentration (nM) of the tested sample and the Y axis as the cell viability (%), then the half-maximal inhibitory concentration (IC50) values of the tested samples were calculated to evaluate the killing activity of the tested samples on in-vitro tumor cell.

[0974] Table 7: Statistic Table of the IC50 (nM) of the tested samples in an in-vitro cell model

[0975] The test results were shown in Table 7 and FIG. 4Ato FIG. 4D, showing that in the above in- vitro cell models, the ADCs (such as ADC-6) disclosed in the application had killing effects on tumor cells, which were superior to those of ADC-107 and ADC-108.

[0976] Example 206 In Vivo Efficacy 1 of ADC tested using an NCI-N87 model

[0977] The application utilized a BALB / c-nu mice subcutaneously inoculated with human tumor cell line (NCI-N87) as an experimental model to evaluate the in vivo efficacy of ADC. The BALB / c- nu mice were inoculated subcutaneously with a certain quantity of a mixed suspension of tumor cell. When the tumor volume grown to about 200 mm3, the mice were administered vehicle solution, ADC-6, ADC- 107 and ADC- 108 at the same doses via the tail vein once a week for four doses. The observation continued for 42 days, and the tumors were measured twice a week, to evaluate the inhibitory effect of the ADC drugs on tumor growth.

[0978] The results were shown in FIG. 5A, indicating that in the xenograft models of human tumor cell line (NCI-N87) expressing R0R1, the ADC-6 had significant tumor inhibitory effects at the same dose, the tumor inhibitory effects of which were superior to those of ADC- 107 and ADC- 108.

[0979] Example 207 In Vivo Efficacy of ADC tested using an HCC1187 model

[0980] A BALB / c-nu mice subcutaneously inoculated with human tumor cell line (HCC1187) were utilized as an experimental model to evaluate the in vivo efficacy of ADC. The BALB / c-nu mice were inoculated subcutaneously with a certain quantity of a mixed suspension of tumor cell. When the tumor volume grown to about 170 mm3, the mice were administered vehicle solution, naked antibody 13C1 (5 mg / kg), payload (compound 5A, 0.133 mg / kg, equivalent to the dose of payload in 5 mg / kg ADC), and ADC-6 (0.2 mg / kg, 1 mg / kg, 5 mg / kg) via the tail vein once a week for two doses. The observation continued for 14 days, and the tumors were measured twice a week, to evaluate the inhibitory effect of the ADC drug on tumor growth.

[0981] The results were shown in FIG. 5B, indicating that in the xenograft models of human tumor cell line (HCC1187) expressing ROR1, the ADC-6 had significant tumor inhibitory effects with a dose-dependent manner, and the inhibitory effect on tumor of the ADC-6 was superior to that of the naked antibody and payload alone.

[0982] Example 208 In Vivo Efficacy 2 of ADC tested using an NCI-N87 model

[0983] A BALB / c-nu mice subcutaneously inoculated with human tumor cell line (NCI-N87) were utilized as an experimental model to evaluate the in vivo efficacy of ADC. The BALB / c-nu mice were inoculated subcutaneously with a certain quantity of a mixed suspension of tumor cell. When the tumor volume grown to about 160 mm3, the mice were administered vehicle solution, naked antibody 13C1 (20 mg / kg), payload (compound 5A, 0.533 mg / kg, equivalent to the dose of payload in 20 mg / kg ADC), and ADC-6 (5 mg / kg, 10 mg / kg, 20 mg / kg) via the tail vein once a week for four doses. The observation continued for 28 days, and the tumors were measured twice a week, to evaluate the inhibitory effects of the ADC drug on tumor growth.

[0984] The results were shown in FIG. 5C, indicating that in the xenograft models of human tumor cell line (NCI-N87) expressing ROR1, the ADC-6 had significant tumor inhibitory effects with a dose-dependent manner, and the inhibitory effect on tumor of the ADC-6 was superior to that of the naked antibody and payload alone.

[0985] Example 209 In Vivo Efficacy of ADC tested using an NCI-H1975 model BALB / c-nu mice subcutaneously inoculated with human tumor cell line (NCI-H1975) were utilized as an experimental model to evaluate the in vivo efficacy of ADC. The BALB / c-nu mice were inoculated subcutaneously with a certain quantity of a mixed suspension of tumor cell. When the tumor volume grown to about 170 mm3, the mice were administered vehicle solution, naked anti-antibody 13C1 (20 mg / kg), payload (compound 5A, 0.533 mg / kg, equivalent to the dose of payload in 20 mg / kg ADC), and ADC-6 (5 mg / kg, 10 mg / kg, 20 mg / kg) via the tail vein once a week, for four doses. The observation continued for 28 days, and the tumors were measured twice a week, to evaluate the inhibitory effects of the ADC drug on tumor growth.

[0986] The results were shown in FIG. 5D, indicating that in the xenograft models of human tumor cell line (NCI-H1975) expressing R0R1, the ADC-6 had significant tumor inhibitory effects with a dose-dependent manner, and the inhibitory effect on tumor of the ADC-6 was superior to that of the naked antibody and payload alone.

[0987] Example 210 In Vivo Efficacy of ADC tested using an MDA-MB-231 model

[0988] A BALB / c-nu mice subcutaneously inoculated with human tumor cell line (MDA-MB-231) were utilized as an experimental model to evaluate the in vivo efficacy of ADC. The BALB / c-nu mice were inoculated subcutaneously with a certain quantity of a mixed suspension of tumor cell. When the tumor volume grown to about 150 mm3, the mice were administered vehicle, naked antiantibody 13C1 (5 mg / kg), a payload (compound 5A, 0.133 mg / kg, equivalent to the dose of payload in 5 mg / kg ADC), and ADC-6 (0.2 mg / kg, 1 mg / kg, 5 mg / kg) via the tail vein once a week, for four doses. The observation continued for 28 days, and the tumors were measured twice a week, to evaluate the inhibitory effects of the ADC drug on tumor growth.

[0989] The results were shown in FIG. 5E, indicating that in the xenograft model of human tumor cell line (MDA-MB-231) expressing R0R1, the ADC-6 had significant tumor inhibitory effects with a dose-dependent manner, and the inhibitory effect on tumor of the ADC-6 was superior to that of the naked antibody and payload alone.

[0990] Example 211 In Vivo Efficacy of ADC tested using an HEL92.1.7-ROR1 #5A7 model

[0991] A NOD Scid female mice subcutaneously inoculated with human erythrocytic leukemia cells HEL92.1.7-ROR1 #5A7 were utilized as an experimental model to evaluate the in vivo efficacy of ADC. The right scapular regions of NOD Scid mice were inoculated subcutaneously with a certain quantity of a mixed suspension of tumor cell. When the tumor volume grown to about 173 mm3, the mice were randomly divided into three groups: the vehicle control group, the ADC-06 (4 mg / kg) treatment group, and the ADC- 107 (4 mg / kg) treatment group, with 5 mice per group, and drug administration begins on day 0. The mice in all groups were administered at 10 mL / kg body weight via the tail vein once a week, for 4 consecutive weeks. All groups are observed on the 28thday (D28) after grouping and treatment. Tumor volume is measured twice a week to evaluate the inhibitory effect of the ADC drugs on tumor growth.

[0992] The results were shown in FIG. 6A, indicating that in the xenograft model of human tumor cell line (HEL92.1.7-ROR1 #5A7) expressing ROR1, compared to the vehicle control group, the ADC-06 (4 mg / kg) treatment group showed a significant inhibitory effect on tumor. At the same dosage, the inhibitory effect on tumor of the ADC-06 treatment group was superior to that of the ADC- 107 treatment group.

Claims

CLAIMSWhat is claimed is:

1. A ligand-camptothecin derivative conjugate represented by Formula I, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof,wherein:Ab is an antibody targeting human R0R1 or an antigen-binding fragment thereof;Ln, L12 and L13 each independently are selected from the group consisting of: o 0L2 has the structure represented by Formula A,wherein Y is a skeleton selected from the group consisting of C1-C6 alkylene, substituted C1-C6 alkylene and C3-C8 cycloalkylene; Ac is a hydrophilic structural unit; the carbon atom at the position of 2 linked to Y has an absolute chirality of R-configuration or S-configuration;L3 is present or absent, and when present, L3 is selected from a PEG hydrophilic unit0 , wherein o is an integer selected from 1 to 10, 4 is an enzyme digestion unit;L5 is a linking unit; in the formula I, the carbon atom at the position of 1 linked to N has an absolute chirality of R-configuration or S-configuration;R is selected from the group consisting of hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl;Ri is selected from the group consisting of hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxyl, 3- to 7-membered heterocyclyl, substituted 3- to 7-membered heterocyclyl, C6-C10 aryl, substituted C6-C 10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl;R2 is selected from the group consisting of hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxyl, 3- to 7-membered heterocyclyl, substituted 3- to 7-membered heterocyclyl, C6-C10 aryl, substituted C6-C 10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl;X is -C(O)-CRaRb-(CR3R4)m-O-, -C(O)-CRaRb-(CR3R4)m-NH- or -C(O)-CRaRb-(CR3R4)m-S-, preferably -C(O)-CRaRb-(CR3R4)m-O-, whereinRaand Rbeach independently are selected from the group consisting of hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C6-C10 aryl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, 3- to 7-membered heterocyclyl, substituted 3- to 7-membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl; orRa, Rband carbon atoms linked thereto form C3-C8 cycloalkyl, substituted C3-C8 cycloalkyl, 3- to 7-membered heterocyclyl or substituted 3- to 7-membered heterocyclyl;R3, R4 each independently are hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, deuterated C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, amino, cyano, nitro, hydroxy C1-C6 alkyl, C3-C8 cycloalkyl, 3- to 7-membered heterocyclyl, substituted 3- to 7- membered heterocyclyl; orR3, R4and carbon atoms linked thereto form C3-C8 cycloalkyl, substituted C3-C8 cycloalkyl, 3- to 7-membered heterocyclyl or substituted 3- to 7-membered heterocyclyl; m is selected from the group consisting of 0, 1, 2, 3 and 4; nl, n2 and n3 each independently are any integer of 0 to 10 or any decimal of 0 to 10, and nl, n2 and n3 are not 0 simultaneously, with l<nl+n2+n3<10.

2. The conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein Ln, 12 and LI3each independently are selected from the group consisting of:Ln, L12 and L13 each independently are selected from the group consisting of:

3. The conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof according to claim 1 or 2, wherein Ac has a structure of Formula B,wherein:Z is carboxyl, phosphoryloxy or -(OCH2CH2)iOCH3, wherein i is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and preferably i is 1, 2, 3, 4, 5, 6, 7 or 8;Y’ is a skeleton linking amino group and Z, and is C1-C6 alkylene or carboxyl-substituted C1-C6 alkylene, preferably methylene, ethylidene, carboxyl-substituted methylene or carboxylsubstituted ethylidene, and further preferably methylene, ethylidene, or carboxyl-substituted methylene; orAc is a residue formed by removing one hydrogen atom from the amino terminus of glycine, (D / L)alanine, (D / L)leucine, (D / L)isoleucine, (D / L)valine, (D / L)phenyl alanine, (D / L)proline, (D / L)(tryptophan, (D / L)serine, (D / L)tyrosine, (D / L()cysteine, (D / L()cy stine, (D / L)arginine, (D / L)histidine. (D / L)methionine, (D / L)asparagine, (D / L)glutamine, (D / L)threonine, (D / L)aspartic acid or (D / L)glutamic acid ; preferably, Ac is:

4. The conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-3, wherein L4 is a peptide residue composed of amino acids, wherein the amino acids are optionally substituted by one or more substituents selected from the group consisting of deuterium atom, halogen, hydroxyl, cyano, amino, nitro, carboxyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy and C3-C8 cycloalkyl and substituted C3-C8 cycloalkyl; preferably, the peptide residue is a peptide residue formed by one, two or more amino acids selected from the group consisting of phenylalanine (F), glycine (G), valine (V), lysine (K),citrulline (C), serine (S), glutamic acid (E) and aspartic acid (D); more preferably, the peptide residue is a tetrapeptide residue formed by glycine (G) -glycine (G) - phenylalanine (F) - glycine (G).

5. The conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-4, wherein L5 is -NRs(CR6R7)q- or a chemical bond, wherein q is 0, 1, 2, 3, 4, 5 or 6, preferably 0, 1, 2 or 3, further preferably 0, 1 or 2, and even further preferably 0 or 1;R5, Re and R7 each independently are selected from the group consisting of hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, 3- to 7-membered heterocyclyl, substituted 3- to 7-membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl; preferably, R5, Re and R7 each independently are selected from the group consisting of hydrogen atom and C1-C6 alkyl; further preferably, Rs, Re and R7 each independently are selected from the group consisting of hydrogen atom and C1-C4 alkyl; further preferably, Rs, Re and R7 each independently are selected from the group consisting of hydrogen atom, methyl, ethyl, n-propyl and n-butyl; more preferably, Rs, Re and R7 each independently are hydrogen atom.

6. The conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-5, characterized by one or more of the following items: i) Ra and Rb each independently are selected from the group consisting of hydrogen atom, C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl and C6-C10 aryl C1-C6 alkyl; or Ra, Rb and carbon atoms linked thereto form C3-C8 cycloalkyl; preferably, Rais hydrogen atom or C1-C4 alkyl, Rb is hydrogen atom, C1-C4 alkyl, halogenated C1-C4 alkyl, C3-C6 cycloalkyl C1-C4 alkyl or phenyl C1-C4 alkyl, or Ra, Rb and carbon atoms linked thereto form C3-C6 cycloalkyl; preferably, Ra is hydrogen atom, methyl, ethyl, n-propyl or n-butyl, Rb is hydrogen atom, methyl, ethyl, n-propyl, n-butyl, halogenated methyl, halogenated ethyl, halogenated n-propyl, halogenated n-butyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclopropylethyl, cyclopentylethyl, cyclohexylethyl, phenylmethyl, phenylethyl or phenylpropyl, or Ra, Rb and carbon atoms linked thereto form cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; preferably, Ra is hydrogen atom or methyl, Rb is hydrogen atom, methyl, ethyl, trifluoromethyl, cyclopropylmethyl, or phenylmethyl, or Ra, Rb and carbon atoms linked thereto form cyclopropyl, cyclobutyl or cyclopentyl; preferably,preferably, the position shown by the right side wavy line is connected to Ls; ii) R3, R4 each independently are hydrogen atom or C1-C6 alkyl; iii) m is 0, 1 or 2, further preferably 0 or 1; iv) R is hydrogen atom or C1-C6 alkyl; preferably, R is hydrogen atom or C1-C4 alkyl;preferably, R is hydrogen atom, methyl, ethyl, n-propyl or n-butyl; preferably, R is hydrogen atom or methyl; v) Ri is hydrogen atom or C1-C6 alkyl; preferably, Ri is C1-C6 alkyl; preferably, Ri is Cl- C4 alkyl; preferably, Ri is methyl, ethyl, n-propyl or n-butyl; preferably, Ri is methyl; vi) R2 is hydrogen atom, halogen or C1-C6 alkyl; preferably, R2 is hydrogen atom, halogen or C1-C4 alkyl; preferably, R2 is halogen; preferably, R2 is fluorine, chlorine, or bromine; preferably, R2 is fluorine; vii) o is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably o is 1, 2, 3, 4, 5, 6, 7 or 8; viii) Y is C1-C6 alkylene; preferably, Y is C1-C4 alkylene; preferably, Y is methylene, ethylidene, propylidene or butylidene; preferably, Y is methylene; ix) nl, n2 and n3 each independently are any integer of 0 to 8 or any decimal of 0 to 8, and nl, n2 and n3 are not 0 simultaneously, with I<nl+n2+n3<8; preferably, 5<nl+n2+n3<8; preferably, 6<nl+n2+n3<8; preferably, 7<nl+n2+n3<8.

7. The conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-6, wherein the antibody targeting human ROR1 or the antigen-binding fragment thereof comprises:(a) the following three complementarity determining regions (CDRs) in heavy chain variable region (VH):(i) VH CDR1, having a CDR1 sequence contained in the VH as set forth in SEQ ID NO:1, or having a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR1 sequence contained in the VH;(ii) VH CDR2, having a CDR2 sequence contained in the VH as set forth in SEQ ID NO: 1, or having a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR2 sequence contained in the VH; and(iii) VH CDR3, having a CDR3 sequence contained in the VH as set forth in SEQ ID NO: 1, or having a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of one amino acid) as compared with the CDR3 sequence contained in the VH; and / or(b) the following three CDRs in light chain variable region (VL):(iv) VL CDR1, having a CDR1 sequence contained in the VL as set forth in SEQ ID NO:5, or having a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR1 sequence contained in the VL;(v) VL CDR2, having a CDR2 sequence contained in the VL as set forth in SEQ ID NO: 5, or having a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR2 sequence contained in the VL; and(vi) VL CDR3, having a CDR3 sequence contained in the VL as set forth in SEQ ID NO: 5, or having a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR3 sequence contained in the VL; preferably, the substitution described in any one of (i) to (vi) is a conservative substitution; preferably, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH),and / or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by Kabat, Chothia or IMGT numbering system.

8. The conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-7, wherein the antibody targeting human R0R1 or the antigen-binding fragment thereof comprises:CDR1, CDR2 and CDR3 sequences contained in the VH as set forth in SEQ ID NO: 1; and / or CDR1, CDR2 and CDR3 sequences contained in the VL as set forth in SEQ ID NO:5; preferably, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH) and / or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by Kabat, Chothia or IMGT numbering system; preferably, the antibody targeting human R0R1 or the antigen-binding fragment thereof comprises:(a) the following three CDRs in heavy chain variable region (VH):(i) VH CDR1, which is composed of a sequence as set forth in SEQ ID NO:2,(ii) VH CDR2, which is composed of a sequence as set forth in SEQ ID NO:3, and(iii) VH CDR3, which is composed of a sequence as set forth in SEQ ID NO:4; and / or(b) the following three CDRs in light chain variable region (VL):(iv) VL CDR1, which is composed of a sequence as set forth in SEQ ID NO:6,(v) VL CDR2, which is composed of a sequence as set forth in SEQ ID NO:7, and(vi) VL CDR3, which is composed of a sequence as set forth in SEQ ID NO:8; preferably, the VH of the antibody targeting human R0R1 or the antigen-binding fragment thereof comprises: VH CDR1 as set forth in SEQ ID NO:2; VH CDR2 as set forth in SEQ ID NO:3; and, VH CDR3 as set forth in SEQ ID NO:4; and / or, the VL of the antibody targeting human R0R1 or the antigen-binding fragment thereof comprises: VL CDR1 as set forth in SEQ ID NO:6; VL CDR2 as set forth in SEQ ID NO:7; and, VL CDR3 as set forth in SEQ ID NO:8; preferably, the VH of the antibody targeting human R0R1 or the antigen-binding fragment thereof comprises: VH CDR1 as set forth in SEQ ID NO:2; VH CDR2 as set forth in SEQ ID NO:3; and VH CDR3 as set forth in SEQ ID NO:4; and the VL of the antibody targeting human R0R1 or the antigen-binding fragment thereof comprises: VL CDR1 as set forth in SEQ ID NO:6; VL CDR2 as set forth in SEQ ID NO:7; and VL CDR3 as set forth in SEQ ID NO:8; preferably, the antibody targeting human R0R1 or the antigen-binding fragment thereof comprises:(a) a heavy chain variable region (VH), comprising an amino acid sequence selected from the following sequences:(i) a sequence as set forth in SEQ ID NO: 1;(ii) a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence as set forth in SEQ ID NO: 1 ;(iii) a sequence with a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence as set forth in SEQ ID NO: 1; and(b) a light chain variable region (VL), comprising an amino acid sequence selected from the following sequences:(iv) a sequence as set forth in SEQ ID NO:5;(v) a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence as set forth in SEQ ID NO: 5;(vi) a sequence with a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence as set forth in SEQ ID NO: 5; preferably, the substitution described in (ii) or (v) is a conservative substitution; preferably, the antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region (VH), comprising a sequence as set forth in SEQ ID NO: 1 or a sequence with a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence as set forth in SEQ ID NO: 1, and a light chain variable region (VL), comprising a sequence as set forth in SEQ ID NO:5 or a sequence with a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence as set forth in SEQ ID NO:5; preferably, the antibody targeting human R0R1 or the antigen-binding fragment thereof comprises: a VH with a sequence as set forth in SEQ ID NO:1 and a VL with a sequence as set forth in SEQ ID NO:5.

9. The conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-8, wherein the antibody targeting human ROR1 or the antigen-binding fragment thereof is humanized; preferably, the antibody targeting human R0R1 or the antigen-binding fragment thereof further comprises a framework region of a human immunoglobulin; preferably, the antibody targeting human R0R1 or the antigen-binding fragment thereof further comprises a heavy chain framework region of a human immunoglobulin (e.g., a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline antibody gene), and / or, a light chain framework region of a human immunoglobulin (e.g., a light chain framework region contained in an amino acid sequence encoded by a human light chain germline antibody gene); preferably, the heavy chain framework region and / or the light chain framework region optionally comprises one or more (such as, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to murine residues; preferably, the antibody targeting human R0R1 or the antigen-binding fragment thereof comprises: a heavy chain with a sequence as set forth in SEQ ID NO: 9 and a light chain with a sequence as set forth in SEQ ID NO: 10; preferably, the antibody or the antigen-binding fragment thereof further comprises a constant region derived from a human immunoglobulin; preferably, the heave chain of the antibody or the antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (such as IgGl, IgG2, IgG3 or IgG4); preferably, the light chain of the antibody or the antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (such as K or ); preferably, the antibody targeting human ROR1 or the antigen-binding fragment thereof isselected from the group consisting of monoclonal antibody, mouse antibody, rabbit antibody, humanized antibody, fully human antibody, chimeric antibody (e.g., human-mouse chimeric antibody), bispecific antibody, multi-specific antibody, single chain antibody, dAb, complementarity determining region fragment, Fv, single chain Fv (scFv), Fd, Fab, Fab', and F(ab')2; preferably, the monoclonal antibody includes a non-CDR region, and the non-CDR region is derived from species other than murine, e g., from a human antibody.

10. The conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-9, wherein the linking unit -L11-L2-L3-L4-L5-, -L12-L2-L3-L4-L5- or -L13-L2-L3-L4-L5- each independently is selected from the group consisting of:y is selected from the group consisting of:wherein Ac, o, R5, Re and R7 are as defined in any one of claims 1-9; the carbon atom at the position of 2 linked to N has an absolute chirality of R-configuration or S-configuration; the position shown by the left side wavy line is connected to the antibody or the antigenbinding fragment thereof, and the position shown by the right side wavy line is connected to X.

11. The conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-10, wherein the conjugate has a structure represented by Formula II:wherein: Ab, Ln, L12, L13, Ac, L3, X, R, Ri, R2, nl, n2, n3 are as defined in any one of claims 1 to 10; the chiral carbon atom at the position of 1, 2 or 3 has an absolute chirality of R-configuration or S-configuration.

12. The conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-11, wherein the conjugate is selected from the group consisting of:TilADC-104wherein:13C1 represents Ab; Ab, nl, n2 and n3 are as defined in any one of claims 1-11.

13. The conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-12, wherein the pharmaceutically acceptable salt includes a salt formed by an acidic functional group in the structural formula with sodium, potassium, calcium or magnesium, or an acetate, a trifluoroacetate, a citrate, an oxalate, a tartrate, a malate, a nitrate, a chloride, a bromide, an iodide, a sulfate, a bisulfate, a phosphate, a lactate, an oleate, an ascorbate, a salicylate, a formate, a glutamate, a methanesulfonate, an ethanesulfonate, a benzenesulfonate or a p-toluenesulfonate formed by a basic functional group in the structural formula with an acid.

14. A pharmaceutical composition, comprising the conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-12, and optionally a pharmaceutically acceptable carrier.

15. A pharmaceutical preparation, comprising the conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-12.

16. Use of the conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-12 or the pharmaceutical composition according to claim 14 and / or the pharmaceutical preparation according to claim 15 in the manufacture of a medicament for treating or preventing a cancer or tumor; preferably, the cancer or tumor expresses R0R1; more preferably, the cancer or tumor is selected from the group consisting of solid tumor and hematologic tumor, such as adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urinary tract cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, breast cancer (e g., triple-negative breast cancer), rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma and leukemia.

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