Anti-human DLL3 antibody-camptothecin drug conjugate and medical application thereof

Abstract

The invention relates to an antibody-drug conjugate formed by an anti-human DLL3 antibody and a camptothecin drug, and a medical application of the antibody-drug conjugate. The present invention also relates to linker-drug conjugates that can be conjugated with antibodies to form antibody drug conjugates.

Description

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to CN application number 202410529362.3, filed on April 29, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to the field of biopharmaceuticals, specifically to an antibody-drug conjugate formed by an anti-human DLL3 antibody and a camptothecin-based drug, and the pharmaceutical uses of this antibody-drug conjugate. This invention also relates to linker-drug conjugates that can be conjugated with antibodies to form antibody-drug conjugates. Background Technology

[0004] Delta-Like Ligand 3 (or DLL3 for short) is a transmembrane protein that attaches to the cell surface and is a member of the Notch ligand family, encoded by the DLL3 gene.

[0005] The Notch ligand family is a family of transmembrane proteins, including Delta-like Ligand 1 (DLL1), DLL3, DLL4, Jagged1 (JAG1), and Jagged2 (JAG2). These ligands interact with Notch proteins, triggering activation of the Notch signaling pathway. The Notch signaling pathway is a highly conserved intercellular communication system that plays a crucial role in the maintenance of multiple developmental processes and tissues. Activation of the Notch signaling pathway depends on the normal cleavage of Notch proteins and ligand binding. When a ligand binds to a Notch protein, the Notch protein undergoes cleavage, releasing its intracellular domain (NICD). The NICD then enters the cell nucleus and participates in gene transcription regulation. The Notch signaling pathway plays a vital role in embryonic development, organ development, cell proliferation, and differentiation. Abnormal Notch signaling pathway activity is associated with the occurrence and development of various diseases, such as tumors, cardiovascular diseases, and neurological disorders.

[0006] DLL3 expression has been found in embryonic development and certain mature tissues. Studies have shown that DLL3 plays an important role in the development of the nervous system. It is involved in processes such as neuronal directional migration, axonal guidance, and dendritic branching. Furthermore, overexpression of DLL3 has been observed in cancer types such as small cell lung cancer (SCLC), prostate cancer, pancreatic cancer, and esophageal squamous cell carcinoma. DLL3 is highly expressed in SCLC cells, and its expression level is associated with tumor invasiveness, metastatic potential, and treatment resistance.

[0007] Currently, antibody drugs targeting DLL3 mainly include Stemcentrx Inc.'s Rovalpituzumabtesirine (Rova-T) and Amgen's AMG-757. Rova-T's research for SCLC progressed to Phase III clinical trials but ultimately failed. AMG-757 is a bispecific antibody developed by Amgen using the next-generation bispecific antibody platform HLE BiTE and is currently in Phase III clinical trials. Harpoon Therapeutics' triple antibody HPN-328 is currently in Phase II clinical trials. In addition, clinical studies targeting DLL3 include Amgen's CAR-T therapy AMG-119, Nanjing Legend Biotech's CAR-T therapy LB-2102, Boehringer Ingelheim GmbH / Oxford Biotherapeutics Ltd's bispecific antibody OBT620, Memorial Sloan Kettering Cancer Center's monoclonal antibody 89Zr-DFO-SC16.56, Qilu Pharmaceutical's bispecific antibody QLS-31904, Boehringer Ingelheim's bispecific antibody BI-764532, and Phanes Therapeutics Inc's bispecific antibody PT-217, all of which are in Phase I clinical trials.

[0008] Antibody-drug conjugates (ADCs) are a class of biological drugs that link antibodies or antibody fragments to biologically active small molecule toxins through stable chemical linkers. They not only make full use of the high specificity of antibody-antigen binding and the high killing power of cytotoxins, but also effectively avoid the disadvantages of weak therapeutic effects of antibodies and indiscriminate attack of small molecule toxins.

[0009] Mylotarg, the world's first marketed ADC, received accelerated approval from the FDA in May 2000 for the treatment of acute myeloid leukemia (AML) patients who had experienced first-relapse, were over 60 years of age, were CD33-positive, and were unsuitable for cytotoxic chemotherapy. Later studies revealed that Mylotarg treatment caused severe and fatal liver damage, leading Pfizer to voluntarily withdraw it from the market in June 2010. Subsequently, in 2017, Mylotarg was re-approved by the FDA at a reduced dose for the treatment of newly diagnosed CD33-positive AML (children and adults ≥1 month of age) and relapsed or refractory CD33-positive AML (children and adults ≥2 years of age). The recently approved ELAHERE is the first FDA-approved ADC targeting folate receptor alpha (FR-α) for the treatment of relapsed or metastatic cervical cancer. Further development of new ADC drugs is still needed. Summary of the Invention

[0010] Ligand-camptothecin derivative conjugates

[0011] The first aspect of this invention discloses a ligand-camptothecin derivative conjugate as shown in general formula I, its stereoisomers, or pharmaceutically acceptable salts or solvates thereof.

[0012]

[0013] in:

[0014] Ab is an antibody or its antigen-binding fragment that targets human DLL3;

[0015] L 11 L 12 L 13 Each is selected independently from:

[0016]

[0017] L2 has the structure shown in equation A below.

[0018]

[0019] Y is a support selected from C1-C6 alkylene, substituted C1-C6 alkylene, or C3-C8 cycloalkylene; Ac is a hydrophilic structural unit; the second carbon atom attached to Y has absolute chirality in either the R or S configuration;

[0020] L3 may or may not exist; if present, L3 is selected from the PEG hydrophilic unit. Where o is an integer selected from 1 to 10;

[0021] L4 is the enzyme digestion unit;

[0022] L5 is a connection unit;

[0023] X is -C(O)-CR a R b -(CR3R4) m -O-、-C(O)-CR a R b -(CR3R4) m -NH- or -C(O)-CR a R b -(CR3R4) m -S-, preferably -C(O)-CR a R b -(CR3R4) m -O-, where

[0024] Ra and R b Each is independently selected from hydrogen atoms, deuterium atoms, halogens, C1-C6 alkyl groups, deuterated C1-C6 alkyl groups, halogenated C1-C6 alkyl groups, C3-C8 cycloalkyl groups, C3-C8 cycloalkyl C1-C6 alkyl groups, C6-C10 aryl C1-C6 alkyl groups, C1-C6 alkoxy C1-C6 alkyl groups, 3-7 membered heterocyclic groups, substituted 3-7 membered heterocyclic groups, C6-C10 aryl groups, substituted C6-C10 aryl groups, 5-10 membered heteroaryl groups, substituted 5-10 membered heteroaryl groups; or,

[0025] R a R b The carbon atoms attached to them constitute C3-C8 cycloalkyl, substituted C3-C8 cycloalkyl, 3-7 membered heterocyclic group or substituted 3-7 membered heterocyclic group;

[0026] R3 and R4 are each independently a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a halo-C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C1-C6 alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxy C1-C6 alkyl group, a C3-C8 cycloalkyl group, a 3-7 membered heterocyclic group, or a substituted 3-7 membered heterocyclic group; or,

[0027] R3, R4 and the carbon atoms attached thereto constitute C3-C8 cycloalkyl, substituted C3-C8 cycloalkyl, 3-7 membered heterocyclic group or substituted 3-7 membered heterocyclic group;

[0028] m is selected from 0, 1, 2, 3 or 4;

[0029] The first chiral carbon atom bonded to N has absolute chirality, either in the R or S configuration.

[0030] R is selected from 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, 5-10 heteroaryl, substituted 5-10 heteroaryl;

[0031] R1 is selected from 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, 3-7 membered heterocyclic group, substituted 3-7 membered heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, substituted 5-10 membered heteroaryl;

[0032] R2 is selected from 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, 3-7 membered heterocyclic group, substituted 3-7 membered heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, substituted 5-10 membered heteroaryl;

[0033] n1, n2, and n3 are each an independent integer or decimal from 0 to 10, and n1, n2, and n3 are not all 0 at the same time, and 1 ≤ n1 + n2 + n3 ≤ 10.

[0034] In some implementations, L 11 L 12 L 13 Each is selected independently from:

[0035]

[0036] In some implementations, L 11 L 12 L 13 Each is selected independently from:

[0037]

[0038] In some implementations, Ac has a structure as shown in Formula B.

[0039]

[0040] in:

[0041] Z represents a carboxyl group, a phosphoryloxy group, or -(OCH2CH2). i OCH3, where i is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably i is 1, 2, 3, 4, 5, 6, 7 or 8;

[0042] Y' is a support connecting the amino group and Z, and is a C1-C6 alkylene group or a C1-C6 alkylene group substituted with a carboxyl group. It is preferably methylene, ethylene, carboxyl-substituted methylene or carboxyl-substituted ethylene, and more preferably methylene, ethylene or carboxyl-substituted methylene.

[0043] In some embodiments, Ac is a residue formed by removing a hydrogen atom from the amino terminus of glycine, (D / L) alanine, (D / L) leucine, (D / L) isoleucine, (D / L) valine, (D / L) phenylalanine, (D / L) proline, (D / L) tryptophan, (D / L) serine, (D / L) tyrosine, (D / L) cysteine, (D / L) cystine, (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.

[0044] In some implementations, Ac is:

[0045]

[0046] In some implementations, Ac is:

[0047] In some embodiments, L4 is a peptide residue consisting of an amino acid, wherein the amino acid is optionally substituted by one or more substituents selected from the group consisting of deuterium, 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.

[0048] In some embodiments, the peptide residue is a peptide residue formed from one, two or more amino acids selected from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline (C), serine (S), glutamic acid (E) and aspartic acid (D).

[0049] In some embodiments, the peptide residue is a tetrapeptide residue formed from glycine (G)-glycine (G)-phenylalanine (F)-glycine (G).

[0050] In some implementations, L5 is -NR5 (CR6R7). q - or chemical bond, where q is 0, 1, 2, 3, 4, 5 or 6;

[0051] R5, R6, and R7 are each independently selected from hydrogen atoms, deuterium atoms, halogens, C1-C6 alkyl groups, substituted C1-C6 alkyl groups, deuterated C1-C6 alkyl groups, C3-C8 cycloalkyl groups, C3-C8 cycloalkyl C1-C6 alkyl groups, C1-C6 alkoxy C1-C6 alkyl groups, 3-7 membered heterocyclic groups, substituted 3-7 membered heterocyclic groups, C6-C10 aryl groups, substituted C6-C10 aryl groups, 5-10 membered heteroaryl groups, and substituted 5-10 membered heteroaryl groups.

[0052] In some embodiments, q is 0, 1, 2, or 3. In some embodiments, q is 0, 1, or 2, more preferably 0 or 1.

[0053] In some embodiments, R5, R6, and R7 are each independently selected from hydrogen atoms or C1-C6 alkyl groups.

[0054] In some embodiments, R5, R6, and R7 are each independently selected from hydrogen atoms or C1-C4 alkyl groups.

[0055] In some embodiments, R5, R6 and R7 are each independently selected from hydrogen atoms, methyl, ethyl, n-propyl or n-butyl.

[0056] In some implementations, R5, R6, and R7 are each independently selected from hydrogen atoms.

[0057] In some implementations, R a and R b Each is independently selected from hydrogen atom, C1-C6 alkyl, halo-C1-C6 alkyl, C3-C8 cycloalkyl-C1-C6 alkyl, or C6-C10 aryl-C1-C6 alkyl; or R a R b It and the carbon atoms it is attached to form C3-C8 cycloalkyl groups.

[0058] In some implementations, R a R is a hydrogen atom or a C1-C4 alkyl group. b It is a hydrogen atom, a C1-C4 alkyl group, a halo-C1-C4 alkyl group, a C3-C6 cycloalkyl group, a C1-C4 alkyl group, or a phenyl C1-C4 alkyl group, or R a R b It and the carbon atoms it is attached to form C3-C6 cycloalkyl groups.

[0059] In some implementations, R a For hydrogen atoms, methyl, ethyl, n-propyl or n-butyl, R b The following are possible meanings: hydrogen atom, methyl, ethyl, n-propyl, n-butyl, halomethyl, haloethyl, halopropyl or halobutyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclopropylethyl, cyclopentylethyl, cyclohexylethyl, benzyl, phenethyl or phenylpropyl, or R a R b The carbon atoms attached to it form cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl groups.

[0060] In some implementations, R a R is a hydrogen atom or a methyl group. b It can be hydrogen atom, methyl, ethyl, trifluoromethyl, cyclopropylmethyl, benzyl, or R a Rb The carbon atoms attached to it form cyclopropyl, cyclobutyl, or cyclopentyl groups.

[0061] In some implementations, X is

[0062] Preferably, the position indicated by the wavy line on the right is connected to L5.

[0063] In some embodiments, R3 and R4 are each independently a hydrogen atom or a C1-C6 alkyl group. In some embodiments, R3 and R4 are each independently a hydrogen atom or a C1-C4 alkyl group. In some embodiments, R3 and R4 are each independently a hydrogen atom.

[0064] In some implementations, m is 0, 1, or 2.

[0065] In some embodiments, R is a hydrogen atom or a C1-C6 alkyl group. In some embodiments, R is a hydrogen atom or a C1-C4 alkyl group. In some embodiments, R is a hydrogen atom, methyl, ethyl, n-propyl, or n-butyl. In some embodiments, R is a hydrogen atom or a methyl group.

[0066] In some embodiments, R1 is a hydrogen atom or a C1-C6 alkyl group. In some embodiments, R1 is a C1-C6 alkyl group. In some embodiments, R1 is a C1-C4 alkyl group. In some embodiments, R1 is methyl, ethyl, n-propyl, or n-butyl. In some embodiments, R1 is methyl.

[0067] In some embodiments, R2 is a hydrogen atom, a halogen, or a C1-C6 alkyl group. In some embodiments, R2 is a hydrogen atom, a halogen, or a C1-C4 alkyl group. In some embodiments, R2 is a halogen. In some embodiments, R2 is fluorine, chlorine, or bromine. In some embodiments, R2 is fluorine.

[0068] In some implementations, o is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0069] In some embodiments, Y is a C1-C6 alkylene. In some embodiments, Y is a C1-C4 alkylene. In some embodiments, Y is methylene, ethylene, propylene, or butylene. In some embodiments, Y is methylene.

[0070] In some implementations, n1, n2, and n3 are each independently any integer or decimal from 0 to 8, and n1, n2, and n3 are not all 0 at the same time, and 1 ≤ n1 + n2 + n3 ≤ 8. In some implementations, 5 ≤ n1 + n2 + n3 ≤ 10. In some implementations, 6 ≤ n1 + n2 + n3 ≤ 8. In some implementations, 7 ≤ n1 + n2 + n3 ≤ 8.

[0071] In some embodiments, the antibody targeting human DLL3 or its antigen-binding fragment comprises:

[0072] (a) The following three heavy chain variable region (VH) complementarity-determining regions (CDRs):

[0073] (i) VH CDR1, having the sequence of CDR1 contained in VH as shown in SEQ ID NO:1, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of CDR1 contained in VH.

[0074] (ii) VH CDR2, having the sequence of CDR2 contained in VH as shown in SEQ ID NO:1, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of one or two amino acids) compared to the sequence of CDR2 contained in VH; and

[0075] (iii) VH CDR3, having the sequence of CDR3 contained in VH as shown in SEQ ID NO:1, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1 amino acid) compared to the sequence of CDR3 contained in VH.

[0076] and / or

[0077] (b) The following three light chain variable region (VL) CDRs:

[0078] (iv) VL CDR1, having the sequence of CDR1 contained in VL as shown in SEQ ID NO:7, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of CDR1 contained in VL.

[0079] (v) VL CDR2, having the sequence of CDR2 contained in VL as shown in SEQ ID NO:7, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of one or two amino acids) compared to the sequence of CDR2 contained in VL; and

[0080] (vi) VL CDR3, having the sequence of CDR3 contained in VL as shown in SEQ ID NO:7, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to the sequence of CDR3 contained in VL.

[0081] In some implementations, the permutation described in any one of (i)-(vi) is a conservative permutation.

[0082] In some implementations, 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 the Kabat, Chothia, or IMGT numbering system.

[0083] In some embodiments, the antibody targeting human DLL3 or its antigen-binding fragment comprises:

[0084] The sequences of CDR1, CDR2, and CDR3 contained in VH as shown in SEQ ID NO:1; and / or

[0085] The CDR1, CDR2 and CDR3 sequences contained in VL as shown in SEQ ID NO:7.

[0086] In some implementations, 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 the Kabat, Chothia, or IMGT numbering system.

[0087] In some embodiments, the antibody targeting human DLL3 or its antigen-binding fragment comprises:

[0088] (a) The following three heavy chain variable region (VH) CDRs:

[0089] (i) VH CDR1, which consists of the sequence shown in SEQ ID NO:2.

[0090] (ii) VH CDR2, which consists of the sequence shown in SEQ ID NO:3, and

[0091] (iii) VH CDR3, which consists of the sequence shown in SEQ ID NO:4;

[0092] and / or

[0093] (b) The following three light chain variable region (VL) CDRs:

[0094] (iv) VL CDR1, which consists of the sequence shown in SEQ ID NO:8.

[0095] (v)VL CDR2, which consists of the sequence shown in SEQ ID NO:9, and

[0096] (vi)VL CDR3, which consists of the sequence shown in SEQ ID NO:10.

[0097] In some embodiments, the VH of the antibody targeting human DLL3 or its antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO:2; VH CDR2 as shown in SEQ ID NO:3; and VHCDR3 as shown in SEQ ID NO:4; and / or, the VL of the antibody targeting human DLL3 or its antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO:8; VL CDR2 as shown in SEQ ID NO:9; and VL CDR3 as shown in SEQ ID NO:10.

[0098] In some embodiments, the VH of the antibody targeting human DLL3 or its antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO:2; VH CDR2 as shown in SEQ ID NO:3; and VHCDR3 as shown in SEQ ID NO:4; and the VL of the antibody targeting human DLL3 or its antigen-binding fragment thereof comprises: VLCDR1 as shown in SEQ ID NO:8; VL CDR2 as shown in SEQ ID NO:9; and VL CDR3 as shown in SEQ ID NO:10.

[0099] In some embodiments, the antibody targeting human DLL3 or its antigen-binding fragment comprises:

[0100] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0101] (i) The sequence shown in SEQ ID NO: 1;

[0102] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 1; or

[0103] (iii) A sequence having 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% sequence identity with the sequence shown in SEQ ID NO: 1;

[0104] and

[0105] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0106] (iv) The sequence shown in SEQ ID NO: 7;

[0107] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 7; or

[0108] (vi) A sequence having 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% sequence identity with the sequence shown in SEQ ID NO: 7.

[0109] In some implementations, the permutation described in (ii) or (v) is a conservative permutation.

[0110] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0111] The heavy chain variable region (VH) comprises a sequence having the sequence shown in SEQ ID NO:1 or having 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% sequence identity with the sequence shown in SEQ ID NO:1, and the light chain variable region (VL) comprises a sequence having the sequence shown in SEQ ID NO:7 or having 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% sequence identity with the sequence shown in SEQ ID NO:7.

[0112] In some embodiments, the antibody targeting human DLL3 or its antigen-binding fragment comprises:

[0113] VH having the sequence shown in SEQ ID NO:1 and VL having the sequence shown in SEQ ID NO:7.

[0114] In some embodiments, the antibody targeting human DLL3 or its antigen-binding fragment is humanized.

[0115] In some embodiments, the antibody targeting human DLL3 or its antigen-binding fragment further comprises the framework region of a human immunoglobulin.

[0116] In some embodiments, the antibody targeting human DLL3 or its antigen-binding fragment further comprises a heavy chain framework region of human immunoglobulin (e.g., a heavy chain framework region contained in the amino acid sequence encoded by the human heavy chain germline antibody gene), and / or a light chain framework region of human immunoglobulin (e.g., a light chain framework region contained in the amino acid sequence encoded by the human light chain germline antibody gene).

[0117] In some embodiments, the heavy chain framework region and / or light chain framework region optionally includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) reversion mutations from human residues to mouse residues.

[0118] In some embodiments, the antibody targeting human DLL3 or its antigen-binding fragment comprises:

[0119] Heavy chains having the sequence shown in SEQ ID NO:5 and light chains having the sequence shown in SEQ ID NO:11.

[0120] In some embodiments, the antibody or its antigen-binding fragment further comprises a constant region derived from human immunoglobulins.

[0121] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment includes a heavy chain constant region derived from human immunoglobulins (e.g., IgG1, IgG2, IgG3, or IgG4).

[0122] In some embodiments, the light chain of the antibody or its antigen-binding fragment includes a light chain constant region derived from human immunoglobulins (e.g., κ or λ).

[0123] In some embodiments, the antibody targeting human DLL3 or its antigen-binding fragment comprises:

[0124] The heavy chain constant region having the sequence shown in SEQ ID NO:6 and the light chain constant region having the sequence shown in SEQ ID NO:12.

[0125] In some embodiments, the antibody targeting human DLL3 or its antigen-binding fragment is selected from monoclonal antibodies, mouse antibodies, rabbit antibodies, humanized antibodies, fully human antibodies, chimeric antibodies (e.g., human-mouse chimeric antibodies), bispecific antibodies, multispecific antibodies, single-chain antibodies, dAb, complementarity-determining region fragments, Fv, single-chain Fv (scFv), Fd, Fab, Fab', and F(ab')2.

[0126] In some embodiments, the monoclonal antibody includes a non-CDR region, and the non-CDR region is derived from a species other than rodents, such as from human antibodies.

[0127] In some implementations, in general formula I -L 11 -L2-L3-L4-L5-、-L 12 -L2-L3-L4-L5-or-L 13 -L2-L3-L4-L5- are each independently selected from the following structures:

[0128]

[0129]

[0130] Wherein: Ac, o, R5, R6 and R7 are defined as described in any embodiment of the present invention;

[0131] The carbon atom bonded to N has absolute chirality in either the R or S configuration;

[0132] The position indicated by the wavy line on the left is connected to the antibody or its antigen-binding fragment, and the position indicated by the wavy line on the right is connected to X.

[0133] In some implementations, in general formula I -L 11 -L2-L3-L4-L5-、-L 12 -L2-L3-L4-L5-or-L 13 -L2-L3-L4-L5- are each selected independently from:

[0134]

[0135] Wherein: Ac, o, R5, R6 and R7 are defined as described in any embodiment of the present invention;

[0136] The carbon atom bonded to N has absolute chirality in either the R or S configuration;

[0137] The position indicated by the wavy line on the left is connected to the antibody or its antigen-binding fragment, and the position indicated by the wavy line on the right is connected to X.

[0138] In some embodiments, the coupling shown in general formula I has a structure as shown in general formula II.

[0139]

[0140] Among them: Ab, L 11 L 12 L 13 The definitions of Ac, L3, X, R, R1, R2, n1, n2, and n3 are as described in any embodiment of the present invention;

[0141] Chiral carbon atoms at positions 1, 2, or 3 have either an R-absolute configuration or an S-absolute configuration.

[0142] In some implementations, the coupling agent represented by general formula I is selected from:

[0143]

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[0196]

[0197] Wherein: 18M1 represents Ab; Ab, n1, n2, and n3 are defined as described in any embodiment of the present invention.

[0198] In some embodiments, the pharmaceutically acceptable salt of the conjugate shown in Formula I is a sodium, potassium, calcium, or magnesium salt formed with the acidic functional group in the formula, or an acetate, trifluoroacetate, citrate, oxalate, tartrate, malate, nitrate, chloride, bromide, iodide, sulfate, bisulfate, phosphate, lactate, oleate, ascorbate, salicylate, formate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, or p-toluenesulfonate formed with the basic functional group in the formula.

[0199] Linker-drug conjugate

[0200] A second aspect of the present invention provides a linker-drug conjugate of formula III-A or III-B, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein:

[0201]

[0202] L is selected from

[0203] L2, L4, L5, X, R, R1, R2, Ac, L3, m, R a R bThe definition is as described in any embodiment of the first aspect of the present invention; the chiral carbon atom at position 1, 2 or 3 has an R absolute configuration or an S absolute configuration.

[0204] In some embodiments, the linker-drug conjugate shown in Formula III-A or Formula III-B is selected from:

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215] 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.

[0216] In some embodiments, the pharmaceutically acceptable salt of the linker-drug conjugate shown in Formula III-A or Formula III-B is a sodium, potassium, calcium, or magnesium salt formed with the acidic functional group in the formula, or an acetate, trifluoroacetate, citrate, oxalate, tartrate, malate, nitrate, chloride, bromide, iodide, sulfate, bisulfate, phosphate, lactate, oleate, ascorbate, salicylate, formate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, or p-toluenesulfonate formed with the basic functional group in the formula.

[0217] In some embodiments of the first and second aspects of the present invention, the camptothecin derivative of the ligand-camptothecin derivative conjugate, its stereoisomer, or its pharmaceutically acceptable salt or solvate represented by general formula I has a structure represented by formula d.

[0218]

[0219] Where: R, R1, R2, m, R a Rb The definition is as described in any embodiment of the first aspect of this invention.

[0220] In some embodiments, the compound represented by formula d is selected from the following compounds:

[0221]

[0222]

[0223] Applications of linker-drug conjugates shown in formula III-A or formula III-B

[0224] The third aspect of the invention provides the use of the linker-drug conjugate of formula III-A or III-B in the preparation of antibody-drug conjugates, particularly in the preparation of the conjugate of formula I of the first aspect of the invention, its stereoisomer or pharmaceutically acceptable salt or solvate thereof.

[0225] Pharmaceutical composition or pharmaceutical preparation

[0226] A fourth aspect of the present invention provides a pharmaceutical composition comprising a conjugate of formula I, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug conjugate of formula III-A or III-B, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof, and optionally a pharmaceutically acceptable carrier.

[0227] The fifth aspect of the present invention provides a pharmaceutical formulation comprising a conjugate of formula I of the first aspect of the present invention, its stereoisomer or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug conjugate of formula III-A or III-B of the second aspect of the present invention, its stereoisomer or a pharmaceutically acceptable salt or solvate thereof.

[0228] Medical Use

[0229] The sixth aspect of the invention provides the use of the conjugate of formula I of the first aspect of the invention, its stereoisomer or a pharmaceutically acceptable salt or solvate thereof, or the linker-drug conjugate of formula III-A or III-B of the second aspect of the invention, its stereoisomer or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of the fourth aspect of the invention, or the pharmaceutical preparation of the fifth aspect of the invention, in the preparation of a medicament for treating or preventing cancer or tumors.

[0230] In some implementations, the cancer or tumor expresses DLL3.

[0231] In some embodiments, the cancer or tumor is selected from solid tumors or hematologic malignancies such as adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach 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, glioma multiforme, sarcoma, lymphoma, and leukemia.

[0232] Preparation method

[0233] A seventh aspect of the present invention provides a method for preparing ligand-camptothecin derivative conjugates, stereoisomers thereof, or pharmaceutically acceptable salts or solvates of general formula I or general formula II as described in the first aspect, comprising:

[0234] By coupling reduced antibodies or their antigen-binding fragments with linker-drug compounds, ligand-camptothecin derivative conjugates as shown in general formula I or general formula II are obtained.

[0235]

[0236] The chiral carbon atoms at positions 1, 2, or 3 have absolute chirality in either the R or S configuration;

[0237] Ab、L、L 11 L 12 L 13 L2, L3, L4, L5, X, R, R1, R2, n1, n2 or n3 as described above.

[0238] In the above aspects and embodiments of the present invention

[0239] The “C1-C6 alkyl” and various complex groups involving “C1-C6 alkyl” (e.g., “substituted C1-C6 alkyl”, “deuterated C1-C6 alkyl”) can be replaced with “C1-C20 alkyl”, “C1-C12 alkyl” or “C1-C10 alkyl”.

[0240] "C3-C8 cycloalkyl" and various complex groups involving "C3-C8 cycloalkyl" can be replaced with "C3-C20 cycloalkyl" or "C3-C10 cycloalkyl";

[0241] "C1-C6 alkoxy" and "C1-C6 alkoxy" in various complex groups involving it can be replaced with "C1-C20 alkoxy", "C1-C12 alkoxy" or "C1-C10 alkoxy".

[0242] "C6-C10 aryl" and various complex groups involving it can be replaced with "C6-C12 aryl";

[0243] The term “3-7 membered heterocyclic group” and various complex groups involving it can be replaced with “3-20 membered heterocyclic group”, “3-12 membered heterocyclic group” or “3-10 membered heterocyclic group”.

[0244] Beneficial effects

[0245] The antibody-drug conjugate obtained by conjugating humanized anti-human DLL3 antibody with camptothecin-based drugs provided by this invention has good molecular stability and good preclinical efficacy compared with existing drugs of the same type. It is expected to have excellent clinical therapeutic effects and has great development value and therapeutic potential. Attached Figure Description

[0246] Figure 1 The chromatogram of ADC-6 aggregation detected by SEC-HPLC is shown.

[0247] Figure 2 The chromatogram of RP-HPLC detection of ADC-6 drug-antibody conjugate ratio (DAR) is shown.

[0248] Figure 3 The in vitro tumor-suppressive activity of ADC-6 in an experimental model of the human cell line NCI-H446 DLL3#B5C was demonstrated.

[0249] Figure 4 The in vitro tumor-suppressive activity of ADC-6 in the experimental model of human cell line A375 DLL3#B2G was demonstrated.

[0250] Figure 5 The in vitro tumor-suppressive activity of ADC-6 in the experimental model of human cell line NCI-H2286 DLL3#B6G was demonstrated.

[0251] Figure 6 The in vitro tumor-suppressive activity of ADC-6 in an experimental model of the human tumor cell line NCI-H1339 DLL3#C3G was demonstrated.

[0252] Figure 7 The in vitro tumor-suppressive activity of ADC-6 in an experimental model of the human tumor cell line SHP-77Wild Type was demonstrated.

[0253] Figure 8 The in vitro tumor-suppressive activity of ADC-6 in an experimental model of the human tumor cell line NCI-H460 was demonstrated.

[0254] Figure 9 The in vitro tumor-suppressive activities of ADC-6, ADC-107, and ADC-108 in the experimental model of human cell line A375 DLL3#B2G were demonstrated.

[0255] Figure 10 The in vitro tumor-suppressive activities of ADC-6, ADC-107, and ADC-108 in the experimental model of the human tumor cell line NCI-H1339 DLL3#C3G were demonstrated.

[0256] Figure 11 The in vitro tumor-suppressive activities of ADC-6, ADC-107, and ADC-108 in the experimental model of human cell line NCI-H2286DLL3#B6G were demonstrated.

[0257] Figure 12 The in vitro tumor-suppressive activities of ADC-6, ADC-107, and ADC-108 in the experimental model of the human tumor cell line SHP-77Wild Type were demonstrated.

[0258] Figure 13 The in vitro tumor-suppressive activities of ADC-6, ADC-107, and ADC-108 in the experimental model of the human tumor cell line NCI-H460 were demonstrated.

[0259] Figure 14 The results of in vivo efficacy experiments of ADC-6 in the SHP-77-DLL3#B7G monotumor model are shown.

[0260] Figure 15 The results of in vivo efficacy experiments of ADC-6 in the A375-DLL3#B2G monotumor model are shown.

[0261] Figure 16 The results of in vivo efficacy experiments of ADC-6 in the H446-DLL3#B5C monotumor model are shown.

[0262] Figure 17 The in vivo efficacy results of ADC-6, ADC-107, and ADC-108 in the A375-DLL3#B2G monotumor model are shown.

[0263] Abbreviations and Definitions

[0264] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. When a trademark name is used herein, unless the context otherwise indicates, the trademark name includes the product formulation, generic medicine, and active ingredient of the product for which the trademark name is used.

[0265] Unless otherwise stated, the terms used in the claims and specification herein shall have the following meanings.

[0266] In this invention, the term "ligand" refers to a macromolecular compound that can recognize and bind to antigens or receptors associated with target cells. The role of the ligand is to deliver the drug to the target cell population that has bound it. These ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules that can bind to cells. In embodiments of this invention, the ligand is denoted as Ab. The ligand can form a linker bond with a linker unit via heteroatoms on the ligand, preferably an antibody or its antigen-binding fragment. The antibody is selected from chimeric antibodies, humanized antibodies, fully human antibodies, or murine antibodies; preferably, a monoclonal antibody.

[0267] In this invention, the ligand unit is a targeting agent that specifically binds to a target moiety. The ligand is capable of specifically binding to cellular components or to other target molecules of interest. The target moiety or target is typically located on the cell surface. In some aspects, the ligand unit functions to deliver a drug unit to a specific target cell population with which the ligand unit interacts. 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 their antigen-binding fragments. In embodiments where the ligand unit is a non-antibody targeting agent, it may be a peptide or polypeptide, or a non-protein 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 a sulfur atom of the ligand. In some aspects, the sulfur atom is a sulfur atom of a cysteine ​​residue that forms an interchain disulfide bond in the antibody. In another aspect, the sulfur atom is a sulfur atom of a cysteine ​​residue that has been incorporated into the ligand unit that forms an interchain disulfide bond in the antibody. In another aspect, the sulfur atom is a sulfur atom of a cysteine ​​residue that has been incorporated into the ligand unit (e.g., by site-directed mutagenesis or chemical reaction). In other aspects, the sulfur atom bound to the linker is selected from cysteine ​​residues of interchain disulfide bonds that form antibodies or cysteine ​​residues that have been incorporated into the ligand unit (e.g., by site-directed mutagenesis or chemical reaction). In some embodiments, the EU indexing system is followed as in Kabat {[Kabat EA et al., (1991)] Sequences of proteins of Immunological Interest, 5th edition, NIH Publication 91-3242}.

[0268] In this invention, "antibody" or "antibody unit" within its scope includes any part of an antibody structure. This unit may bind, reactively associate, or chelate a receptor, antigen, or other receptor units present in a target cell population. An antibody can be any protein or protein-like molecule that can bind, chelate, or react with a portion of a cell population to be treated or bioengineered. The antibodies constituting the antibody-drug conjugates of this invention retain their original wild-state antigen-binding capacity. Therefore, the antibodies of this invention can specifically bind to antigens. Antigens involved include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules associated with tissue growth and differentiation (such as those known or anticipated to be functional), lymphokines, cytokines, molecules involved in cell circulation regulation, molecules involved in angiogenesis, and molecules associated with angiogenesis (such as those known or anticipated to be functional). Tumor-associated factors may be cluster differentiation factors (such as CD proteins).

[0269] Antibodies used in antibody-drug conjugates include, but are not limited to, antibodies targeting cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well-known in the industry and can be prepared using well-known antibody preparation methods and information. To develop effective cellular-level targets for cancer diagnosis and treatment, researchers seek transmembrane or other tumor-associated peptides. These targets are specifically expressed on the surface of one or more cancer cells, but rarely or not expressed on the surface of one or more non-cancer cells. Typically, such tumor-associated peptides are more overexpressed on the surface of cancer cells compared to non-cancer cells. Identifying such tumor-associated factors can significantly improve the specific targeting properties of antibody-based cancer therapies. For convenience, information related to antigens well-known in the industry is indicated below, including name, other names, and gene bank accession number. Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in public databases such as GenBank. The tumor-associated antigens targeted by the antibody include all amino acid sequence variants and homologs, and have at least 70%, 80%, 85%, 90%, or 95% homology with the sequences identified in the references, or have biological properties and characteristics that are completely consistent with the tumor-associated antigen sequences in the cited literature.

[0270] In this invention, the term "suppression" or "inhibition" refers to reducing the detectable amount or completely blocking it.

[0271] In this invention, the term "cancer" refers to a physiological condition or disease characterized by disordered cell growth. "Tumor" includes cancer cells.

[0272] In this invention, the term "autoimmune disease" is derived from diseases or disorders affecting an individual's own tissues or proteins.

[0273] In this invention, the term "drug" refers to a cytotoxic drug, denoted as d, which is a chemical molecule that can strongly disrupt the normal growth of tumor cells. Cytotoxic drugs can kill tumor cells at sufficiently high concentrations, but due to their lack of specificity, they can also cause apoptosis of normal cells while killing tumor cells, leading to serious side effects. This term also includes toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, and radioactive isotopes (e.g., At). 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 and Lu 176 Radioactive isotopes), toxic drugs, chemotherapeutic drugs, antibiotics and ribolysins, preferably toxic drugs.

[0274] In this invention, the terms "connector," "connector fragment," or "connector unit" refer to a chemical structural fragment or bond that is connected to a ligand at one end and to a drug at the other end. It can also be connected to other connectors before being connected to a drug.

[0275] The linker, comprising extensions, spacers, and amino acid units, can be synthesized using methods known in the art, such as those described in US2005-0238649A1. The linker can be a “cleavable linker” that facilitates drug release into cells. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive linkers (e.g., peptidase-sensitive linkers), photostable linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Research 52:127-131, 1992); US Patent No. 5,208,020.

[0276] Based on the mechanism of intracellular drug release, as used in this paper, "linkers" or "linkers of antibody-drug conjugates" can be divided into two categories: unbreakable linkers and breakable linkers. For antibody-drug conjugates containing unbreakable linkers, the drug release mechanism is as follows: after the conjugate binds to the antigen and is endocytosed by the cell, the antibody is enzymatically cleaved in lysosomes, releasing an active molecule composed of the small molecule drug, the linker, and antibody amino acid residues. This alteration in drug molecular structure does not weaken its cytotoxicity, but because the active molecule is charged (amino acid residues), it cannot penetrate neighboring cells. Therefore, this type of active drug 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). For antibody-drug conjugates containing breakable linkers, the drug release mechanism is as follows: after the conjugate binds to the antigen and is endocytosed by the cell, it cleaves within the target cell, releasing the active ingredient (the small molecule drug itself). Cleavable linkers are mainly classified into chemically sensitive linkers and enzyme-sensitive linkers. Chemically sensitive linkers can selectively cleave due to differences in the properties of plasma and cytoplasm or the tumor microenvironment. These properties include pH value and glutathione concentration. pH-sensitive linkers are relatively stable in the neutral or weakly alkaline environment of blood (pH 7.3-7.5), but will be hydrolyzed in the weakly acidic tumor microenvironment (pH 5.0-6.5) and lysosomes (pH 4.5-5.0), for example, hydrazones, carbonates, acetals, and ketals. Due to the limited plasma stability of acid-cleavable linkers, antibody-drug conjugates based on this type of linker typically have a short half-life (2-3 days). This short half-life limits the application of pH-sensitive linkers in next-generation antibody-drug conjugates to some extent. Glutathione-sensitive linkers are also known as disulfide linkers. Drug release is based on the difference between the high intracellular glutathione concentration (millimolar range) and the relatively low blood glutathione concentration (micromolar range). This is especially true for tumor cells, where low oxygen levels lead to increased reductase activity, resulting in higher glutathione concentrations. Disulfide bonds are thermodynamically stable, thus exhibiting good stability in plasma. Enzyme-unstable linkers, such as peptide linkers, offer better control over drug release. Peptide linkers can be effectively cleaved by lysosomal proteases, such as cathepsin B. These peptide links are considered highly stable in plasma circulation because unsuitable extracellular pH and serum protease inhibitors typically render proteases inactive extracellularly. Due to their high plasma stability and good intracellular cleavage selectivity and efficiency, enzyme-unstable linkers are widely used as cleavable linkers for antibody-drug conjugates.

[0277] In this invention, the term "antibody-drug conjugate" refers to an antibody linked to a biologically active drug via a stable linker unit. In this invention, "ligand-drug conjugate" is preferably an antibody-drug conjugate (ADC), which refers to a monoclonal antibody or antibody fragment linked to a biologically active toxic drug via a stable linker unit.

[0278] The amino acid three-letter codes and single-letter codes used in this invention are as described in J.boil.Chem.1968,243,3558.

[0279] In this invention, the term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain 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., "C1-C6 alkyl"). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 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, 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, 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 their various branched isomers, etc. More preferably, lower alkyl groups containing 1 to 6 carbon atoms are used. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 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, 2,3-dimethylbutyl, etc. Alkyl groups can be substituted or unsubstituted. When substituted, the substituents can be substituted at any usable connection point. The substituents are preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0280] In this invention, the term "substituted alkyl" refers to an alkyl group in which hydrogen is replaced by a substituent group. Unless otherwise stated herein, the substituent group of an alkyl group may be one or more groups selected from the 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"' The groups -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 have substituent numbers from 0 to (2m'+1), where m' is the total number of carbon atoms in the group. R', R”, and R”’ each independently refer to hydrogen, unsubstituted carbon, and unsubstituted carbon. 1-8 Alkyl, unsubstituted C6-C12 aryl (or C6-C10 aryl), C6-C12 aryl (or C6-C10 aryl) substituted with 1-3 halogens, unsubstituted C 1-8 Alkyl, C 1-8 Alkoxy or C 1-8 Thioalkoxy, or unsubstituted C6-C12 aryl (or C6-C10 aryl)-C 1-4 Alkyl groups. When R' and R” are attached to the same nitrogen atom, they can form 3-, 4-, 5-, 6-, or 7-membered rings together with that nitrogen atom. For example, -NR'R” includes 1-pyrrolidinyl and 4-morpholinyl.

[0281] In this invention, the term "alkylene" refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane. It is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably containing 1 to 12 carbon atoms, and more preferably containing 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-pentylene (-CH2CH2CH2CH2-). (e.g., 2CH2CH2CH2CH2-). The alkylene group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable linking point. The substituent is preferably independently selected from one or more substituents chosen from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0282] In this invention, the term "alkoxy" refers to -O-(alkyl) and -O-(cycloalkyl), wherein alkyl or cycloalkyl is defined as described above. Non-limiting examples of C1-C6 alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0283] In this invention, the term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring comprises 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 monocyclic cycloalkyl (e.g., "C3-C8 cycloalkyl") include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl includes spirocyclic, fused-ring, and bridged-ring cycloalkyl.

[0284] In this invention, the term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms (i.e., "3-20 membered heterocyclic group"), wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O). m (in m The ring consists of 2 to 0 heteroatoms, excluding the -OO-, -OS-, or -SS- ring portions, with the remaining ring atoms being carbon. Preferably, it contains 3 to 12 ring atoms (i.e., "3-12 membered heterocyclic groups"), of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms (i.e., "3-10 membered heterocyclic groups"). Non-limiting examples of monocyclic heterocyclic groups (e.g., 3-7 membered heterocyclic groups) include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups.

[0285] In this invention, the term "cycloalkylalkyl" refers to an alkyl group substituted with one or more cycloalkyl groups, preferably substituted with one cycloalkyl group, wherein the alkyl group is as defined above, and the cycloalkyl group is as defined above. For example, C3-C8 cycloalkyl and C1-C6 alkyl.

[0286] In this invention, the term "halogenated alkyl" refers to an alkyl group that is substituted with one or more halogens, wherein the alkyl group is as defined above. For example, halogenated C1-C6 alkyl groups.

[0287] In this invention, the term "deuterated alkyl" refers to an alkyl group that is substituted with one or more deuterium atoms, wherein the alkyl group is as defined above. For example, deuterated C1-C6 alkyl groups.

[0288] In this invention, the term "C6-C12 aryl" refers to a group in a carbocyclic aromatic system having 6-12 carbon atoms.

[0289] In this invention, the term "C6-C10 aryl" refers to a group in a carbocyclic aromatic system having 6-10 carbon atoms, such as phenyl, naphthyl, etc.

[0290] In this invention, the term "5-10-membered heteroaryl" refers to an aromatic heterocycle, typically a 5-, 6-, 7-, 8-, 9-, or 10-membered heterocycle having 1 to 3 heteroatoms selected from N, O, or S; the heteroaryl ring may optionally be further fused or connected to aromatic and non-aromatic carbon rings and heterocycles. Non-limiting examples of the 5-10 membered heteroaryl groups include, for example, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, imidazolyl, thiazolyl, isothiazolyl, thiazolyl, pyrroleyl, phenyl-pyrroleyl, furanyl, phenyl-furanyl, oxazolyl, isoxazolyl, pyrazolyl, thiophene, benzofuranyl, benzothiophene, benzo1,3-dioxolane (benzodioxotrope), isodihydroindolyl, benzimidazolyl, indolyl, quinolinyl, isoquinolinyl, 1,2,3-triazolyl, 1-phenyl-1,2,3-triazolyl, 2,3-dihydroindolyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothiophene, benzopyranyl, 2,3-dihydrobenzoxazinyl, 2,3-dihydroquinoxolinyl, etc.

[0291] In this invention, the terms "substituted C6-C10 aryl", "substituted 5-10 membered heteroaryl", or "substituted 3-7 membered heterocyclic" refer to the substitution of hydrogen atoms in the aryl, heteroaryl, or heterocyclic group by a substituent group. Unless otherwise stated herein, the substituents of the aryl, heteroaryl, or heterocyclic group can be a variety of groups selected from the 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, with the number of substituents ranging from 0 to (2m'+1), where m' is the total number of carbon atoms in the group. R', R”, and R”' each independently refer to hydrogen, unsubstituted carbon, and unsubstituted carbon. 1-8 Alkyl, unsubstituted C6-C12 aryl (or C6-C10 aryl), C6-C12 aryl (or C6-C10 aryl) substituted with 1-3 halogens, unsubstituted C 1-8 Alkyl, C 1-8 Alkoxy or C 1-8 Thioalkoxy, or unsubstituted C6-C12 aryl (or C6-C10 aryl)-C 1-4 Alkyl groups. When R' and R” are attached to the same nitrogen atom, they can form 3-, 4-, 5-, 6-, or 7-membered rings together with that nitrogen atom. For example, -NR'R” includes 1-pyrrolidinyl and 4-morpholinyl.

[0292] In this invention, the term "hydroxyl group" refers to the -OH group.

[0293] In this invention, the term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0294] In this invention, the term "amino" refers to -NH2.

[0295] In this invention, the term "nitro" refers to -NO2.

[0296] In this invention, the term "amide group" refers to -C(O)N(alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

[0297] In this invention, the term "carboxylic acid ester group" refers to -C(O)O(alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

[0298] This invention also includes compounds of general formula I in various deuterated forms. Each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize the deuterated forms of formula I with reference to relevant literature. Commercially available deuterated starting materials can be used to prepare the deuterated forms of formula I, or they can be synthesized using conventional techniques with deuterating reagents. Non-limiting examples of deuterating reagents include: deuterated boranes, trideuterated borane tetrahydrofuran solutions, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane, etc.

[0299] In this invention, the term "antibody" refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in its heavy chain, it can be further divided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ or λ chain. The antibody described in this invention is preferably a specific antibody against cell surface antigens on target cells, and more preferably an anti-DLL-3 antibody.

[0300] In this invention, the term "solvent" or "solvent compound" refers to the ligand-drug conjugate of this invention forming a pharmaceutically usable solvate with one or more solvent molecules. Non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0301] In this invention, the term "drug loading" refers to the average number of cytotoxic drugs loaded onto each antibody in Formula I, or it can be expressed as the ratio of drug amount to antibody amount. The drug loading range can be 0-12, preferably 1-10, cytotoxic drugs (d) linked to each antibody (Ab). In embodiments of this invention, the drug loading is expressed as n, which can be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as exemplarily. The average number of drugs per ADC molecule after the coupling reaction can be identified using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, and HPLC characterization.

[0302] In one embodiment of the present invention, a cytotoxic drug is coupled to an open cysteine ​​thiol group (-SH) and / or a site-mutated cysteine ​​residue (-SH) of an antibody chain via a linker unit. Generally, the number of drug molecules that can be coupled to the antibody in the coupling reaction will be less than or equal to the theoretical maximum value.

[0303] The loading of ligand-cytotoxic drug conjugates can be controlled using the following non-limiting methods, including:

[0304] (1) Control the molar ratio of the ligation reagent and the monoclonal antibody.

[0305] (2) Control the reaction time and temperature.

[0306] (3) Choose different reaction reagents.

[0307] For the preparation of conventional pharmaceutical compositions, please refer to the Chinese Pharmacopoeia.

[0308] In this invention, the terms "pharmaceutical-acceptable salt" or "medicinal salt" refer to the salt of the ligand-drug conjugate of this invention, or the salt of the compound described in this invention, which is safe and effective when used in mammals and has the intended biological activity. The ligand-drug conjugate of this invention contains at least one carboxyl group and can therefore form a salt with a base. Non-limiting examples of pharmaceutically acceptable salts include sodium salts, potassium salts, calcium salts, or magnesium salts, etc.

[0309] In this invention, the term "pharmaceutically acceptable salt" or "medicinal salt" refers to the salt of the antibody-drug conjugate of this invention, or the salt of the compound described in this invention, which is safe and effective when used in mammals and has the desired biological activity. The ligand-drug conjugate of this invention contains at least one amino group and can therefore form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0310] In this invention, the term "acidic amino acid" refers to an amino acid with an isoelectric point less than 7. Acidic amino acid molecules often contain one or more acidic groups such as carboxyl groups, which can effectively ionize into negative ions in their structure, thus increasing their hydrophilicity. Acidic amino acids can be natural or non-natural.

[0311] In this invention, the term "natural amino acid" refers to an amino acid synthesized by a organism. Natural amino acids are generally L-type, but there are a few exceptions, such as glycine, which includes both naturally occurring and organismally synthesized amino acids.

[0312] In this invention, the term "non-natural amino acid" refers to amino acids obtained through synthetic means.

[0313] In this invention, if the chemical name of a compound is inconsistent with its structural formula, the structural formula shall prevail. Detailed Implementation

[0314] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, all percentages, proportions, ratios, or parts are by weight.

[0315] Example 1: Synthesis of Compound M1

[0316]

[0317] In a 5L single-necked flask, N-fluorenemethoxycarbonyl-glycine-glycine (100 g, 282 mmol), lead tetraacetate (175 g, 395 mmol), 2 L of dry tetrahydrofuran, and 670 mL of toluene were added. The mixture was stirred thoroughly, and under nitrogen protection, it was heated to 85 °C for 2.5 h. The reaction was monitored by TLC. After the reactants had reacted completely, the mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to give compound M1 (87 g); LC-MS: [M+NH4] + =386.0.

[0318] Example 2 Synthesis of compound M3

[0319]

[0320] Compound SM-2 (synthesized according to the method disclosed in patent application CN108452321A) (40 g, 96 mmol, 1.0 eq), triethylamine (26.7 mL, 2.0 eq), and toluene (400 mL) were added to a 1000 mL single-necked flask and refluxed at 120 °C for 2 h. The reaction was monitored by TLC until nearly complete. The solvent was removed by rotary evaporation under reduced pressure at 50 °C. The solution was dissolved in ethyl acetate (150 mL) and water (40 mL), and the pH was adjusted to 2-3 with 1 M HCl under stirring in an ice bath. The layers were separated. The aqueous layer was extracted again with ethyl acetate, and the organic layers were combined and dried over anhydrous sodium sulfate. After filtration, the solution was concentrated to obtain a pale yellow oily crude product. The crude product was purified by column chromatography (DCM:MeOH = 40:1) to give compound M2 (26.6 g); LC-MS: [M+H] + =399.3.

[0321] Compound M2 (26.5 g, 60.5 mmol, 1.0 eq), 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 to a 1000 mL single-necked flask. The mixture was reacted at room temperature for 30 min (monitored by TLC), and the insoluble matter was filtered off. The reaction solution was directly purified by preparative liquid chromatography. The preparative solution was concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile, and then lyophilized to obtain compound M3 (31.5 g), yield 64%; LC-MS: [M+H] + =565.1.

[0322] Example 3 Synthesis of compound ent-M3

[0323]

[0324] Following the synthetic route in Example 2, compound ent-M3 (27.8 g) was obtained; LC-MS: [M+H] + =565.2.

[0325] Example 4 Synthesis of Compound 1

[0326]

[0327] Step 1: Compound 1a

[0328] In a 250 mL single-necked flask, compound M1 (6 g, 16.3 mmol), 100 mL THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added. The mixture was stirred and cooled to 0 °C. Benzyl glycolate (5.4 g, 32.6 mmol) was added dropwise. After the addition was complete, the mixture was allowed to warm naturally to room temperature (reaction time approximately 2-4 h), and monitored by TLC. After the reaction was complete, saturated NaHCO3 solution was added, and the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue purified by silica gel column chromatography (PE:EA = 10:1-5:1-1:1) to give compound 1a (4 g), yield 52%; LC-MS: [M+H] + =475.18.

[0329] Step 2: Compound 1b

[0330] In a 25 mL single-necked flask, compound 1a (2 g, 4.2 mmol) and 10 mL of DMF were added. The mixture was stirred at 0 °C, and then DBU (766 mg, 5.04 mmol) was added. The reaction was carried out for 1 h. After the deprotection of Fmoc was completed by TLC monitoring, the reaction solution was obtained and set aside for later use.

[0331] In a separate 25 mL single-necked flask, compound M4 (prepared according to the method disclosed in patent application CN111051330 A) (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. DIPEA (830 μL, 5.04 mmol) was added under ice-water bath conditions, and the mixture was stirred for 30 min. The above reaction solution was then added, and the mixture was allowed to react at room temperature. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative liquid chromatography to obtain the product preparation solution. The preparation solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain solid compound 1b (1.7 g), yield 63%; LCMS: [M+H] + =648.26.

[0332] Step 3: Compound 1c

[0333] Compound 1b (900 mg, 1.39 mmol) was added to a 25 mL single-necked flask, dissolved in 15 mL of DMF, and then 900 mg of 5% Pd / C was added. The reaction was carried out for 2 h. After the reaction was completed, the filtrate containing compound 1c was obtained and used directly in the next step of the reaction without purification.

[0334] Step 4: Compound 1d

[0335] The filtrate containing compound 1c was placed in an ice-water bath, and DIPEA (235 μL, 1.39 mmol) was added, followed by compound M3 (784 mg, 1.39 mmol). The mixture was then allowed to rise to room temperature and reacted for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by preparative HPLC to obtain the preparative solution, which was then lyophilized to obtain compound 1d (504 mg). LC-MS: [M+H] + =804.4.

[0336] Step 5: Compound 1e

[0337] Compound 1d (500 mg, 0.62 mmol), ixotecan 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 to a 50 mL single-necked flask. DIPEA (378 μL, 2.29 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by HPLC to obtain the preparative solution of compound 1e. The preparative solution was lyophilized to obtain compound 1e (210 mg). LC-MS: [M+H] + =1221.6.

[0338] Step 6: Compound 1

[0339] Compound 1e (100 mg, 0.081 mmol), zinc bromide (368 mg, 1.63 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 1 (60 mg); LC-MS: [M+H] + =1065.3.

[0340] Example 5 Synthesis of Compound 2

[0341]

[0342] Compound 2 (51 mg) was obtained by replacing compound M3 with compound ent-M3, following the synthetic route in Example 4; LC-MS: [M+H] + =1065.3.

[0343] Example 6 Synthesis of Compound 3

[0344]

[0345] Step 1: Compound 3a

[0346] In a 250 mL single-necked flask, M1 (6 g, 16.3 mmol), 100 mL THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added. The mixture was stirred and cooled to 0 °C. Benzyl 2-hydroxy-2-methylpropionate (6.3 g, 32.6 mmol) was added dropwise. After the addition was complete, the mixture was allowed to warm naturally to room temperature (reaction time approximately 2-4 h), and monitored by TLC. After the reaction was complete, a saturated NaHCO3 solution was added, and the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to give compound 3a (4.2 g), yield 52%; LC-MS: [M+H] + =503.3.

[0347] Step 2: Compound 3b

[0348] In a 25 mL single-necked flask, compound 3a (2 g, 4.0 mmol) and 10 mL of DMF were added. The mixture was stirred at 0 °C, and then DBU (760 mg, 5.0 mmol) was added. The reaction was carried out for 1 h. After the deprotection of Fmoc was completed by TLC monitoring, the reaction solution was obtained and set aside for later use.

[0349] In a separate 25 mL single-necked 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. DIPEA (823 μL, 5.04 mmol) was added under ice-water bath conditions, and the mixture was stirred for 30 min. The above reaction solution was then added, and the mixture was allowed to react at room temperature. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative liquid chromatography to obtain the product preparation solution. The preparation solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain solid compound 3b (1.4 g), yield 53%; LC-MS: [M+H] + =676.2.

[0350] Step 3: Compound 3c

[0351] Compound 3b (700 mg, 1.04 mmol) was added to a 25 mL single-necked flask, dissolved in 10 mL of DMF, and then 700 mg of 5% Pd / C was added. The reaction was carried out for 1.5 h. After the reaction was completed, the mixture was filtered to obtain a filtrate containing compound 3c, which was used directly in the next step of the reaction without purification.

[0352] Step 4: Compound 3d

[0353] The filtrate containing compound 3c was placed in an ice-water bath, and DIPEA (210 μL, 1.25 mmol) was added, followed by compound M3 (704 mg, 1.25 mmol). The mixture was then allowed to rise to room temperature for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by preparative HPLC to obtain the preparative solution, which was then lyophilized to give compound 3d (486 mg). LC-MS: [MH] - =830.5.

[0354] Step 5: Compound 3e

[0355] Compound 3d (300 mg, 0.36 mmol), ixotecan 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 to a 50 mL single-necked flask. DIPEA (219.5 μL, 1.33 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 3 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the preparative solution of compound 3e. The preparative solution was lyophilized to obtain compound 3e (157 mg). LC-MS: [M+H] + =1249.6.

[0356] Step 6: Compound 3

[0357] Compound 3e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 3 (64 mg); LC-MS: [M+H] + =1093.1.

[0358] Example 7 Synthesis of Compound 4

[0359]

[0360] By replacing compound M3 with compound ent-M3, and following the synthetic route of Example 6, compound 4 (60 mg) was obtained; LC-MS: [M+H] + =1093.2.

[0361] Example 8 Synthesis of Compound 5A

[0362]

[0363] Step 1: Compound 5a

[0364] Compound M1 (500 mg, 1.4 mmol), p-toluenesulfonic acid monohydrate (26 mg, 0.1 mmol, 0.1 eq), and 10 mL of THF were added to a 25 mL single-necked flask. After stirring, the mixture was cooled to 0 °C, and then L-benzyl lactate ((S)-(-)-benzyl lactate) (1.2 g, 7.0 mmol, 5 eq) was slowly added. After the addition was complete, the mixture was allowed to rise to room temperature. The reaction was monitored by TLC. After the reaction was complete, saturated NaHCO3 solution was added, and the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by reverse-phase column chromatography to obtain compound 5a (400 mg).

[0365] LC-MS: [M+NH4] + =506.2.

[0366] 1 H NMR (400Mz, CDCl3 / CD3OD): 1.39 (3H, d, J = 6.8Hz), 3.78 (2H, t, J = 4.0Hz), 4.17-4.27 (2H, m), 4.42 (2H, d, J = 4.0Hz), 4.72-4.85 (2H, m), 5.11 -5.58(2H,m),5.43(1H,s),7.06(1H,t,J=8.0Hz),7.25-7.33(6H,m),7.38(2H,t,J=8.0Hz),7.57(2H,d,J=8.0Hz),7.75(2H,d,J=8.0Hz).

[0367] Step 2: Compound 5b

[0368] Compound 5a (400 mg, 0.8 mmol, 1.0 eq) and 4 mL of DMF were added to a 25 mL single-necked flask. After stirring until homogeneous, the temperature was lowered to 0 °C, and then DBU (137 mg, 0.9 mmol, 1.1 eq) was slowly added. After the addition was complete, the temperature was raised to room temperature. The reaction was monitored by TLC. Once the reaction was complete, this was recorded as reaction solution ①.

[0369] In a separate 25 mL single-necked 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. The mixture was stirred at room temperature for 5 minutes, and then reaction solution ① was added. The reaction was allowed to proceed at room temperature, and the reaction was monitored by HPLC. After the reaction was complete, the reaction solution was purified by HPLC to obtain compound 5b (326 mg); LC-MS: [M+NH4] + =679.2.

[0370] Step 3: Compound 5c

[0371] Compound 5b (4.0 g, 6.05 mmol, 1.0 eq) was added to a 100 mL single-necked flask and dissolved in 60 mL of DMF. Then, 4 g of 5% Pd / C was added, and the reaction was carried out at room temperature for 4 h (the reaction progress was monitored by HPLC). The Pd / C was filtered off, and the filtrate containing compound 5c was placed directly in an ice-water bath (approximately 0 °C) without concentration for later use.

[0372] Step 4: Compound 5d

[0373] The filtrate containing compound 5c was placed in an ice-water bath, and DIPEA (1.1 mL, 1.1 eq) was added, followed by compound M3 (3.4 g, 6.05 mmol). The mixture was then allowed to rise to room temperature for 2 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by preparative HPLC to obtain the preparative solution, which was then lyophilized to obtain compound 5d (3.15 g). LC-MS: [MH] - =816.3.

[0374] Step 5: Compound 5e

[0375] Compound 5d (2.07 g, 2.53 mmol, 1.0 eq), ixotecan 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 to a 100 mL single-necked flask. DIPEA (1.05 mL, 1.5 eq) was added under ice-water bath conditions, and the mixture was reacted at room temperature for 2 h (monitored by HPLC). The reaction solution was directly purified by preparative liquid chromatography. The preparative solution was concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile, and then lyophilized to give compound 5e (1.92 g), yield 61%; LC-MS: [M+H] + =1235.4.

[0376] Step 6: Compound 5A

[0377] Compound 5e (1.0 g, 0.8 mmol, 1.0 eq) and 35 mL of nitromethane were added to a 100 mL single-necked flask and dissolved. Zinc bromide (3.64 g, 16 mmol, 20.0 eq) was then added, and the mixture was reacted in an oil bath at 40 °C (preheated for stability) for 30 min. The mixture was then concentrated in a water bath at 45 °C under reduced pressure to remove the nitromethane, yielding a yellow solid residue (monitored by HPLC). Preparative liquid chromatography was used for purification. 0.1% trifluoroacetic acid was added to the mobile phase to obtain a preparative solution of compound 5A. This preparative solution was concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile, and then lyophilized to obtain compound 5A (786 mg) in 90% yield.

[0378] LC-MS: [M+H] + =1079.4;

[0379] 1H NMR(400MHz, DMSO-d6)δ9.39-9.02(m,1H),8.70(t,J=6.5Hz,1H),8.64(t,J=5.7Hz,1H),8.56(d,J=8.8Hz,1H),8 .34(t,J=5.7Hz,1H),8.16(d,J=8.2Hz,1H),8.01(t,J=5.5Hz,1H),7.71(d,J=10.9Hz,1H),7.30(s,1H),7.28-7.1 5(m,4H),7.14(s,2H),5.53(dd,J=14.5,6.4Hz,1H),5.49-5.34(m,2H),5.22(d,J=18.8Hz,1H),5.09(d,J=18.7H z,1H),5.03(dd,J=9.6,3.9Hz,1H),4.73(dd,J=9.9,6.9Hz,1H),4.59(dd,J=10.1,6.5Hz,1H),4.49(ddd,J=13.2, 8.6,4.4Hz,1H),4.14(dd,J=13.3,6.6Hz,2H),3.93(s,2H),3.84(dd,J=16.5,6.3Hz,1H),3.76(dd,J=16.9,5.7H z,2H),3.70(d,J=5.2Hz,2H),3.60(dd,J=16.7,5.4Hz,1H),3.52(dd,J=16.4,5.1Hz,1H),3.45(dd,J=12.8,10.1H z,1H),3.25-3.15(m,1H),3.14-3.05(m,1H),3.01(dd,J=13.7,4.1Hz,1H),2.73(dd,J=13.5,9.8Hz,1H),2.54-2 .47(m,1H),2.33(s,2H),2.17(d,J=5.5Hz,2H),1.91-1.79(m,2H),1.33(d,J=6.6Hz,2H),0.87(t,J=7.3Hz,2H).

[0380] Example 9 Synthesis of Compound 5B

[0381]

[0382] Step 1: Compound 5d-1

[0383] Compound 5b (300 mg, 0.45 mmol, 1.0 eq) and DMF (3 mL) were added to a 25 mL single-necked flask and stirred until dissolved. 5% Pd / C (300 mg) was added, and the mixture was purged with hydrogen three times. The hydrogenation reaction was carried out for 2 h, and the reaction was monitored by HPLC until completion. After the reaction, Pd / C was removed by filtration, yielding a filtrate containing compound 5c. The filtrate was cooled to 0-5 °C, and DIPEA (65 mg, 0.5 mmol, 1.1 eq) was added. Then, ent-M3 (255 mg, 0.45 mmol) was added to the filtrate. After the addition was complete, the mixture was heated to 20 ± 5 °C and reacted for 1 h. The reaction was monitored by HPLC until completion. After the reaction, the product was purified by preparative HPLC, collected, and lyophilized to obtain compound 5d-1 (200 mg), yield 54%. LC-MS: [MH] - =816.3.

[0384] Step 2: Compound 5e-1

[0385] Compound 5d-1 (200 mg, 0.24 mmol, 1.0 eq), ixotecan mesylate M5 (127 mg, 0.24 mmol, 1.0 eq), PyBOP (187 mg, 0.36 mmol, 1.2 eq), HOBt (48 mg, 0.36 mmol, 1.2 eq), and DMF (6 mL) were added to a 25 mL single-necked flask. The mixture was cooled to 0–5 °C in an ice-water bath, and DIPEA (62 mg, 0.48 mmol, 2.0 eq) was added. After the addition was complete, the mixture was heated to 20 ± 5 °C and reacted for 2 h. The reaction was monitored by HPLC until it was complete. The reaction solution was directly purified by preparative HPLC, and the product solution was collected and lyophilized to obtain compound 5e-1 (162.8 mg). LC-MS: [M+H] + =1235.4.

[0386] Step 3: Compound 5B

[0387] Compound 5e-1 (110 mg, 0.089 mmol, 1.0 eq), ZnBr2 (400 mg, 1.78 mmol, 20.0 eq), and CH3NO2 (10 mL) were added sequentially to a 25 mL single-necked flask. After the addition was complete, the mixture was heated to 40 °C and reacted for 0.5 h. The reaction mixture was then removed and evaporated to dryness under reduced pressure at 45 °C to obtain a yellow solid. The reaction mixture was monitored by HPLC. The evaporated solid was directly purified by preparative HPLC. The product solution was collected and lyophilized to obtain compound 5B (73.4 mg), with a yield of 76.5%. LC-MS: [M+H] + =1079.4.

[0388] Example 10 Preparation of Compound 6A

[0389]

[0390] By replacing (S)-(-)-benzyl lactate with (R)-(+)-benzyl lactate, and following the synthetic route in Example 8, compound 6A (71 mg) was obtained; LC-MS: [M+H] + =1079.4.

[0391] Example 11 Preparation of Compound 6B

[0392]

[0393] By replacing (S)-(-)-benzyl lactate with (R)-(+)-benzyl lactate, and following the synthetic route of Example 9, compound 6B (59 mg) was obtained; LC-MS: [M+H] + =1079.4.

[0394] Example 12 Preparation of compounds 7A and 7B

[0395]

[0396] Step 1: Compound 7a

[0397] Compound M1 (10 g, 27.1 mmol), benzyl 3,3,3-trifluorolactate (prepared according to the method disclosed in patent application WO2020063673A1) (12.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene were added to a 250 mL single-necked flask. The mixture was heated to 100 °C and reacted for 4 h. After the reaction was complete, the mixture was cooled to room temperature, filtered to remove insoluble matter, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to give 5.15 g of the target compound 7a, with a yield of 35.1%; LC-MS: [M+H] + =543.17.

[0398] Step 2: Compound 7b

[0399] Compound 7a (5g, 9.2mmol) and 15mL DMF were added to a 50mL single-necked flask. After dissolving, DBU (1.68g, 11mmol) was added under an ice-water bath. The reaction was carried out for 1h and recorded as reaction solution ①.

[0400] In a separate 50 mL single-necked 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. After dissolving, DIPEA (1.82 mL, 11 mmol) was added under ice-water bath conditions, and the reaction was continued for 30 min. Reaction solution ① was then added, and the mixture was allowed to react at room temperature for 2 h. The reaction progress was monitored by HPLC. After the reaction was complete, the reaction solution was purified by preparative HPLC to obtain the preparative solution. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over 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.

[0401] Step 3: Compound 7d

[0402] Compound 7b (900 mg, 1.26 mmol) was added to a 25 mL single-necked flask and dissolved in 15 mL of DMF. Then, 900 mg of 5% Pd / C was added, and the mixture was hydrogenated for 2 h. After the reaction was complete, the mixture was filtered to obtain a filtrate containing compound 7c. The filtrate was placed in an ice-water bath, and DIPEA (228 μL, 1.38 mmol) was added, followed by compound M3 (712 mg, 1.26 mmol). The mixture was then allowed to cool to room temperature for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by HPLC to obtain the preparative solution. The preparative solution was lyophilized to obtain 525 mg of compound 7d, with a yield of 47.9%. LC-MS: [MH] - =870.33.

[0403] Step 4: Compound 7e

[0404] Compound 7d (500 mg, 0.57 mmol), ixotecan 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 to a 50 mL single-necked flask. DIPEA (378 μL, 2.29 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain preparative solutions of compounds 7e-1 and 7e-2. The preparative solutions were lyophilized to obtain 150 mg of compound 7e-1. LC-MS: [M+H] + =1289.46; 220 mg compound 7e-2, LC-MS: [M+H] + =1289.46.

[0405] Step 5: Compound 7A

[0406]

[0407] Compound 7e-1 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 52 mg of solid compound 7A; TOF result: 1133.3613.

[0408] Step 6: Compound 7B

[0409]

[0410] Compound 7e-2 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 63 mg of solid compound 7B; TOF result: 1133.3668.

[0411] Example 13 Synthesis of compounds 8A and 8B

[0412]

[0413] Step 1: Compound 8d

[0414] Compound 7c (900 mg, 1.83 mmol) was added to a 25 mL single-necked flask and dissolved in 20 mL of DMF. Then, DIPEA (303 μL, 1.83 mmol) was added, followed by ent-M3 (1034 mg, 1.83 mmol). The mixture was brought to room temperature and reacted for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by preparative HPLC to obtain the preparative solution. The preparative solution was lyophilized to give compound 8d 613 mg, with a yield of 38.5%. LC-MS: [MH] - =870.32.

[0415] Step 2: Compounds 8e-1 and 8e-2

[0416] Compound 8d (500 mg, 0.57 mmol), ixotecan 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 to a 50 mL single-necked flask. DIPEA (378 μL, 2.29 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain preparative solutions of compounds 8e-1 and 8e-2. The preparative solutions were lyophilized to obtain 140 mg of compound 8e-1 and 210 mg of compound 8e-2, respectively. LC-MS of compound 8e-1: [M+H] + =1289.47; LC-MS of compound 8e-2: [M+H] + =1289.47.

[0417] Step 3: Compound 8A

[0418]

[0419] Compound 8e-1 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 50 mg of solid compound 8A; TOF result: 1133.3623.

[0420] Step 4: Compound 8B

[0421]

[0422] Compound 8e-2 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 58 mg of solid compound 8B; TOF result: 1133.3653.

[0423] Example 14 Synthesis of Compound 9A

[0424]

[0425] Step 1: Compound 9a

[0426] In a 250 mL single-necked flask, compound M1 (6 g, 16.3 mmol), 100 mL THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added. The mixture was stirred and cooled to 0 °C. Benzyl 1-hydroxycyclopropanecarboxylate (prepared according to the method disclosed in patent US20050020645 A1) (6.3 g, 32.6 mmol) was added dropwise. After the addition was complete, the mixture was allowed to warm naturally to room temperature (reaction time approximately 2-4 h), and monitored by TLC. After the reaction was complete, a saturated NaHCO3 solution was added, and the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to give compound 9a (3.7 g), yield 45%; LC-MS: [M+H] + =501.5.

[0427] Step 2: Compound 9b

[0428] In a 25 mL single-necked flask, add compound 9a (2 g, 4.0 mmol) and 10 mL DMF, stir at 0 °C, add DBU (760 mg, 5.0 mmol), react for 1 h, and monitor by TLC until Fmoc deprotection is complete. The reaction solution is then set aside for use.

[0429] In a separate 25 mL single-necked 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. DIPEA (823 μL, 5.04 mmol) was added under ice-water bath conditions, and the mixture was stirred for 30 min. The above reaction solution was then added to the reaction flask, and the mixture was allowed to react at room temperature. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative liquid chromatography to obtain the product preparation solution. The preparation solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, 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.

[0430] Step 3: Compound 9c

[0431] Compound 9b (900 mg, 1.3 mmol) was added to a 25 mL single-necked flask, dissolved in 10 mL of DMF, and then 900 mg of 5% Pd / C was added. The hydrogenation reaction was carried out for 1.5 h. After the reaction was completed, the filtrate containing compound 9c was obtained and used directly in the next reaction without purification.

[0432] Step 4: Compound 9d

[0433] The filtrate containing compound 9c was placed in an ice-water bath, and DIPEA (223 μL, 1.3 mmol) was added, followed by compound M3 (750 mg, 1.3 mmol). The mixture was then allowed to rise to room temperature for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by preparative HPLC to obtain the preparative solution, which was then lyophilized to give compound 9d (529 mg). LC-MS: [MH] - =828.4.

[0434] Step 5: Compound 9e

[0435] Compound 9d (500 mg, 0.6 mmol), ixotecan mesylate M5 (300 mg, 0.6 mmol), PyBOP (416 mg, 0.8 mmol), HOBt (108 mg, 0.5 mmol), and 15 mL of DMF were added to a 50 mL single-necked flask. DIPEA (351 μL, 2.13 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 3 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the preparative solution of compound 9e. The preparative solution was lyophilized to obtain compound 9e (257 mg). LC-MS: [M+H] + =1247.5.

[0436] Step 6: Compound 9A

[0437] Compound 9e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 9A (55 mg). LC-MS: [M+H] + =1091.3.

[0438] Example 15 Synthesis of Compound 9B

[0439]

[0440] By replacing compound M3 with compound ent-M3, and following the synthetic route of Example 14, compound 9B (44 mg) was obtained; LC-MS: [M+H] + =1091.3.

[0441] Example 16 Synthesis of Compound 10A

[0442]

[0443] Step 1: Compound 10a

[0444] In a 250 mL single-necked flask, compound M1 (6 g, 16.3 mmol), 100 mL THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added. The mixture was stirred and cooled to 0 °C. Benzyl 1-hydroxymethylcyclopropanecarboxylate (prepared according to the method disclosed in patent WO2013187496A1) (6.7 g, 32.6 mmol) was added dropwise. After the addition was complete, the mixture was allowed to warm naturally to room temperature (reaction time approximately 2-4 h), and monitored by TLC. After the reaction was complete, saturated NaHCO3 solution was added, and the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to give compound 10a (4.9 g), yield 58%; LC-MS: [M+H] + =515.4.

[0445] Step 2: Compound 10b

[0446] In a 25 mL single-necked flask, add compound 10a (4 g, 7.8 mmol) and 10 mL DMF, stir at 0 °C, add DBU (1.2 g, 8.0 mmol), react for 1 h, and monitor by TLC until Fmoc deprotection is complete. The reaction solution is then set aside for use.

[0447] In a separate 25 mL single-necked 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. DIPEA (1.65 mL, 10.1 mmol) was added under ice-water bath conditions, and the mixture was stirred for 50 min. The reaction solution was then added to the reaction flask, and the mixture was allowed to react at room temperature. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the product preparation solution. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, 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.

[0448] Step 3: Compound 10c

[0449] Compound 10b (1.0 g, 1.45 mmol) was added to a 25 mL single-necked flask, dissolved in 15 mL of DMF, and then 1.0 g of 5% Pd / C was added. The hydrogenation reaction was carried out for 1.5 h. After the reaction was completed, the filtrate containing compound 10c was obtained and used directly in the next reaction without purification.

[0450] Step 4: Compound 10d

[0451] The filtrate containing compound 10c was placed in an ice-water bath, and DIPEA (258 μL, 1.5 mmol) was added, followed by compound M3 (837 mg, 1.45 mmol). The mixture was then brought to room temperature and reacted for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by preparative HPLC to obtain the preparative solution, which was then lyophilized to give compound 10d (499 mg). LC-MS: [MH] - =842.4.

[0452] Step 5: Compound 10e

[0453] Compound 10d (400 mg, 0.48 mmol), ixotecan mesylate M5 (240 mg, 0.48 mmol), PyBOP (250 mg, 0.48 mmol), HOBt (104 mg, 0.48 mmol), and 15 mL of DMF were added to a 50 mL single-necked flask. DIPEA (330 μL, 2.0 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 3 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the preparative solution of compound 10e. The preparative solution was lyophilized to obtain compound 10e (188 mg). LC-MS: [M+H] + =1261.5.

[0454] Step 6: Compound 10A

[0455] Compound 10e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 10A (61 mg); LC-MS: [M+H] + =1105.4.

[0456] Example 17 Synthesis of Compound 10B

[0457]

[0458] By replacing compound M3 with compound ent-M3, and following the synthetic route of Example 16, compound 10B (75 mg) was obtained; LC-MS: [M+H] + =1105.4.

[0459] Example 18 Synthesis of Compound 11A

[0460]

[0461] Step 1: Compound 11a

[0462] In a 250 mL single-necked flask, compound M1 (6 g, 16.3 mmol), 100 mL THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added. The mixture was stirred and cooled to 0 °C. 1-hydroxycyclobutane carboxylic acid benzyl ester (synthesized according to the method published in the Journal of Medicinal Chemistry, 2013, 56(13), 5541-5552) (6.7 g, 32.6 mmol) was added dropwise. After the addition was complete, the mixture was allowed to rise naturally to room temperature (for approximately 2-4 hours), and the reaction was monitored by TLC. After the reaction was complete, saturated NaHCO3 solution was added, and the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to give compound 11a (5.1 g), yield 62%; LC-MS: [M+H] + =515.7.

[0463] Step 2: Compound 11b

[0464] In a 25 mL single-necked flask, add compound 11a (4 g, 7.8 mmol) and 10 mL DMF, stir at 0 °C, add DBU (1.2 g, 8.0 mmol), react for 1 h, and monitor by TLC until Fmoc deprotection is complete. The reaction solution is then set aside for use.

[0465] In a separate 25 mL single-necked 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. DIPEA (1.63 mL, 10.0 mmol) was added under ice-water bath conditions, and the mixture was stirred for 40 min. The reaction solution was then added to the reaction flask, and the mixture was allowed to react at room temperature. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative liquid chromatography to obtain the product preparation solution. The preparation solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, 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.

[0466] Step 3: Compound 11c

[0467] Compound 11b (2.0 g, 2.9 mmol) was added to a 25 mL single-necked flask, dissolved in 25 mL of DMF, and then 2.0 g of 5% Pd / C was added. The reaction was carried out for 3 h. After the reaction was completed, the filtrate containing compound 11c was obtained and used directly in the next step of the reaction without purification.

[0468] Step 4: Compound 11d

[0469] The filtrate containing compound 11c was placed in an ice-water bath, and DIPEA (516 μL, 3.0 mmol) was added, followed by compound M3 (1.7 g, 2.9 mmol). The mixture was then allowed to rise to room temperature for 2 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by preparative HPLC to obtain the preparative solution, which was then lyophilized to obtain compound 11d (934 mg). LC-MS: [MH] - =842.4.

[0470] Step 5: Compound 11e

[0471] Compound 11d (800 mg, 0.96 mmol), ixotecan mesylate M5 (480 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol), and 30 mL of DMF were added to a 50 mL single-necked flask. DIPEA (660 μL, 4.0 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 4 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the preparative solution of compound 11e. The preparative solution was lyophilized to obtain compound 11e (401 mg). LC-MS: [M+H] + =1261.4.

[0472] Step 6: Compound 11A

[0473] Compound 11e (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol), and 10 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 11A (86 mg); LC-MS: [M+H] + =1105.4.

[0474] Example 19 Synthesis of Compound 11B

[0475]

[0476] By replacing compound M3 with compound ent-M3, and following the synthetic route of Example 18, compound 11B (50 mg) was obtained. LC-MS: [M+H] + 1105.4.

[0477] Example 20 Synthesis of Compound 12A

[0478]

[0479] Step 1: Compound 12a

[0480] In a 250 mL single-necked flask, compound M1 (6 g, 16.3 mmol), 100 mL THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added. The mixture was stirred and cooled to 0 °C. 7.2 g (32.6 mmol) of 1-hydroxymethylcyclobutane carboxylic acid benzyl ester (prepared according to the method disclosed in patent application WO2009011285A1) was added dropwise. After the addition was complete, the mixture was allowed to warm naturally to room temperature (reaction time approximately 2-4 h), and monitored by TLC. After the reaction was complete, a saturated NaHCO3 solution was added, and the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to give compound 12a (4.5 g), yield 52%; LC-MS: [M+H] + =529.4.

[0481] Step 2: Compound 12b

[0482] In a 25 mL single-necked flask, add compound 12a (4 g, 7.6 mmol) and 10 mL DMF, stir at 0 °C, add DBU (1.2 g, 8.0 mmol), react for 1 h, and monitor by TLC until Fmoc deprotection is complete. The reaction solution is then set aside for use.

[0483] In a separate 25 mL single-necked 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. DIPEA (1.49 mL, 0.9 mmol) was added under ice-water bath conditions, and the mixture was stirred for 30 min. The reaction solution was then added to the reaction flask, and the mixture was allowed to react at room temperature. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative liquid chromatography to obtain the product preparation solution. The preparation solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, 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.

[0484] Step 3: Compound 12c

[0485] Compound 12b (1.0 g, 1.43 mmol) was added to a 25 mL single-necked flask, dissolved in 15 mL of DMF, and then 1.0 g of 5% Pd / C was added. The hydrogenation reaction was carried out for 1.5 h. After the reaction was completed, the filtrate containing compound 12c was obtained and used directly in the next step of the reaction without purification.

[0486] Step 4: Compound 12d

[0487] The filtrate containing compound 12c was placed in an ice-water bath, and DIPEA (258 μL, 1.5 mmol) was added, followed by compound M3 (825 mg, 1.43 mmol). The mixture was then allowed to reach room temperature and reacted for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by preparative HPLC to obtain the preparative solution, which was then lyophilized to obtain compound 12d (522 mg). LC-MS: [MH] - =856.4.

[0488] Step 5: Compound 12e

[0489] Compound 12d (400 mg, 0.47 mmol), ixotecan mesylate M5 (240 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt (101 mg, 0.47 mmol), and 15 mL of DMF were added to a 50 mL single-necked flask. DIPEA (330 μL, 2.0 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 3 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the preparative solution of compound 12e. The preparative solution was lyophilized to obtain compound 12e (198 mg). LC-MS: [M+H] + =1275.4.

[0490] Step 6: Compound 12A

[0491] Compound 12e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 12A (55 mg); LC-MS: [M+H] + =1119.4.

[0492] Example 21 Synthesis of Compound 12B

[0493]

[0494] By replacing compound M3 with compound ent-M3, and following the synthetic route of Example 20, compound 12B (50 mg) was obtained; LC-MS: [M+H] + =1119.4.

[0495] Example 22 Synthesis of Compound 13A

[0496]

[0497] Step 1: Compound 13a

[0498] In a 250 mL single-necked flask, compound M1 (6 g, 16.3 mmol), 100 mL THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added. The mixture was stirred and cooled to 0 °C. 1-hydroxycyclopentanecarboxylic acid benzyl ester (synthesized according to the method published in the Journal of Medicinal Chemistry, 2013, 56(13), 5541-5552) (7.2 g, 32.6 mmol) was added dropwise. After the addition was complete, the mixture was allowed to rise naturally to room temperature (for approximately 2-4 hours), and the reaction was monitored by TLC. After the reaction was complete, saturated NaHCO3 solution was added, and the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to give compound 13a (4.6 g), yield 53%; LC-MS: [M+H] + =529.5.

[0499] Step 2: Compound 13b

[0500] In a 25 mL single-necked flask, add compound 13a (4 g, 7.6 mmol) and 10 mL DMF, stir at 0 °C, add DBU (1.17 g, 7.8 mmol), react for 1 h, and monitor by TLC until Fmoc deprotection is complete. Set the reaction solution aside for later use.

[0501] In a separate 25 mL single-necked 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. DIPEA (1.39 mL, 0.85 mmol) was added under ice-water bath conditions, and the mixture was stirred for 30 min. The reaction solution was then added to the reaction flask, and the mixture was allowed to react at room temperature. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative liquid chromatography to obtain the product preparation solution. The preparation solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, 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.

[0502] Step 3: Compound 13c

[0503] Compound 13b (1.5 g, 1.87 mmol) was added to a 25 mL single-necked flask, dissolved in 25 mL of DMF, and then 1.5 g of 5% Pd / C was added. The reaction was carried out for 3 h. After the reaction was completed, the filtrate containing compound 13c was obtained and used directly in the next step of the reaction without purification.

[0504] Step 4: Compound 13d

[0505] The filtrate containing compound 13c was placed in an ice-water bath, and DIPEA (333 μL, 1.93 mmol) was added, followed by compound M3 (1.1 g, 1.87 mmol). The mixture was then allowed to rise to room temperature for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by preparative HPLC to obtain the preparative solution, which was then lyophilized to obtain compound 13d (519 mg). LC-MS: [MH] - =856.6.

[0506] Step 5: Compound 13e

[0507] Compound 13d (400 mg, 0.47 mmol), ixotecan mesylate M5 (240 mg, 0.48 mmol), PyBOP (250 mg, 0.48 mmol), HOBt (103 mg, 48 mmol), and 15 mL of DMF were added to a 50 mL single-necked flask. DIPEA (330 μL, 2.0 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 4 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the preparative solution of compound 13e. The preparative solution was lyophilized to obtain compound 13e (187 mg). LC-MS: [M+H] + =1275.5.

[0508] Step 6: Compound 13A

[0509] Compound 13e (100 mg, 0.08 mmol), zinc bromide (355 mg, 0.16 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 13A (60 mg); LC-MS: [M+H] + =1119.6.

[0510] Example 23 Synthesis of Compound 13B

[0511]

[0512] By replacing compound M3 with compound ent-M3, and following the synthetic route of Example 22, compound 13B (51 mg) was obtained; LC-MS: [M+H] + =1119.6.

[0513] Example 24 Synthesis of Compound 14A

[0514]

[0515] Step 1: Compound 14a

[0516] In a 250 mL single-necked flask, compound M1 (6 g, 16.3 mmol), 100 mL THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added. The mixture was stirred and cooled to 0 °C. 7.6 g (32.6 mmol) of 1-hydroxymethylcyclopentanecarboxylic acid benzyl ester (synthesized according to the method disclosed in patent WO2009011285A1) was added dropwise. After the addition was complete, the mixture was allowed to warm naturally to room temperature (for approximately 2-4 hours), and the reaction was monitored by TLC. After the reaction was complete, a saturated NaHCO3 solution was added, and the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to give compound 14a (4.4 g), yield 49%; LC-MS: [M+H] + =543.6.

[0517] Step 2: Compound 14b

[0518] In a 25 mL single-necked flask, add compound 14a (4 g, 7.4 mmol) and 10 mL DMF, stir at 0 °C, add DBU (1.2 g, 8.0 mmol), react for 1 h, and monitor by TLC until Fmoc deprotection is complete. The reaction solution is then set aside for use.

[0519] In a separate 25 mL single-necked 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. DIPEA (1.49 mL, 9.0 mmol) was added under ice-water bath conditions, and the mixture was stirred for 30 min. The reaction solution was then added to the reaction flask, and the mixture was allowed to react at room temperature. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative liquid chromatography to obtain the product preparation solution. The preparation solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and 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.

[0520] Step 3: Compound 14c

[0521] Compound 14b (1.0 g, 1.4 mmol) was added to a 25 mL single-necked flask, dissolved in 15 mL of DMF, and then 1.0 g of 5% Pd / C was added. The reaction was carried out for 1.5 h. After the reaction was completed, the mixture was filtered to obtain a filtrate containing compound 14c, which was used directly in the next step of the reaction without purification.

[0522] Step 4: Compound 14d

[0523] The filtrate containing compound 14c was placed in an ice-water bath, and DIPEA (248 μL, 1.5 mmol) was added, followed by compound M3 (808 mg, 1.4 mmol). The mixture was then allowed to rise to room temperature for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by preparative HPLC to obtain the preparative solution, which was then lyophilized to obtain compound 14d (500 mg). LC-MS: [MH] - =870.5.

[0524] Step 5: Compound 14e

[0525] Compound 14d (400 mg, 0.46 mmol), ixotecan mesylate M5 (235 mg, 0.46 mmol), PyBOP (245 mg, 0.46 mmol), HOBt (99 mg, 0.46 mmol), and 15 mL of DMF were added to a 50 mL single-necked flask. DIPEA (331 μL, 2.0 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 3 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the preparative solution of compound 14e. The preparative solution was lyophilized to obtain compound 14e (146 mg). LC-MS: [M+H] + =1289.5.

[0526] Step 6: Compound 14A

[0527] Compound 14e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 14A (52 mg); LC-MS: [M+H] + =1133.4.

[0528] Example 25 Synthesis of Compound 14B

[0529]

[0530] By replacing compound M3 with compound ent-M3, and following the synthetic route of Example 24, compound 14B (48 mg) was obtained; LC-MS: [M+H] + =1133.4.

[0531] Example 26 Synthesis of compounds 15A and 15B

[0532]

[0533] Step 1: Compound 15a

[0534] Compound M1 (10 g, 27.1 mmol), benzyl 2-hydroxybutyrate (prepared according to the method published in Chemical Communications, 2019, 55(53), 7699-7702) (10.5 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene were added to a 250 mL single-necked flask. The mixture was heated to 100 °C and reacted for 4 h. After the reaction was complete, the mixture was cooled to room temperature, filtered to remove insoluble matter, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to give compound 15a 5.67 g, yield 42%; LC-MS: [M+H] + =503.5.

[0535] Step 2: Compound 15b

[0536] Compound 15a (5g, 9.95mmol) and 15mL DMF were added to a 50mL single-necked flask. After dissolving, DBU (1.68g, 11mmol) was added under an ice-water bath. The reaction was carried out for 1h and recorded as reaction solution ①.

[0537] In a separate 50 mL single-necked 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. After dissolving completely, DIPEA (1.82 mL, 11 mmol) was added under ice-water bath conditions, and the reaction was continued for 40 min. Then, reaction solution ① was added, and the mixture was allowed to react at room temperature for 2 h. The reaction progress was monitored by HPLC. After the reaction was complete, the reaction solution was purified by preparative HPLC to obtain the preparative solution. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain solid compound 15c 4.6 g, yield 68%; LC-MS: [M+H] + =676.7.

[0538] Step 3: Compound 15d

[0539] 15b (2.0 g, 2.96 mmol) was added to a 25 mL single-necked flask and dissolved in 15 mL of DMF. Then, 2.0 g of 5% Pd / C was added, and the reaction was hydrogenated for 2 h. After the reaction was complete, the mixture was filtered, and the filtrate was placed in an ice-water bath. DIPEA (496 μL, 3.0 mmol) was added, followed by compound M3 (1.7 g, 2.96 mmol). The mixture was then allowed to cool to room temperature for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by preparative HPLC to obtain the preparative solution. The preparative solution was lyophilized to give compound 15d 1120.0 mg, yield 45%. LC-MS: [MH] - =830.3.

[0540] Step 4: Compound 15e-1 and Compound 15e-2

[0541] Add 15d (500 mg, 0.60 mmol), ixotecan mesylate M5 (321 mg, 0.60 mmol), PyBOP (469 mg, 0.90 mmol), HOBt (121 mg, 0.90 mmol), and 15 mL of DMF to a 50 mL single-necked flask. Add DIPEA (446 μL, 2.7 mmol) under ice-water bath conditions, and react at room temperature for 2 h. After the reaction is complete as monitored by HPLC, the reaction solution is purified by preparative HPLC to obtain preparative solutions of compounds 15e-1 and 15e-2. The preparative solutions are lyophilized to obtain 138 mg of compound 15e-1. LC-MS: [M+H] + =1249.5; 140 mg compound 15e-2, LC-MS: [M+H] + =1249.5.

[0542] Step 5: Compound 15A

[0543]

[0544] Compound 15e-1 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 59 mg of solid compound 15A; LC-MS: [M+H] + =1093.4.

[0545] Step 6: Compound 15B

[0546]

[0547] Compound 15e-2 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 60 mg of solid compound 15B; LC-MS: [M+H] + =1093.4.

[0548] Example 27 Synthesis of compounds 16A and 16B

[0549]

[0550] By replacing compound M3 with compound ent-M3, and following the synthetic route of Example 26, compound 16A (55 mg) was obtained; LC-MS: [M+H] + =1093.4.

[0551]

[0552] By replacing compound M3 with compound ent-M3, and following the synthetic route of Example 26, compound 16B (54 mg) was obtained; LC-MS: [M+H] + =1093.4.

[0553] Example 28 Synthesis of compounds 17A and 17B

[0554]

[0555] Step 1: Compound 17a

[0556] Compound M1 (10 g, 27.1 mmol), benzyl 2-hydroxy-3-phenylpropionate (synthesized by the method published in 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 to a 250 mL single-necked flask. The mixture was heated to 100 °C and reacted for 4 h. After the reaction was complete, the mixture was cooled to room temperature, filtered to remove insoluble matter, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to give compound 17a 6.13 g, yield 40%; LC-MS: [M+H] + =565.6.

[0557] Step 2: Compound 17b

[0558] Compound 17a (5g, 8.86mmol) and 15mL DMF were added to a 50mL single-necked flask. After dissolving, DBU (1.53g, 10mmol) was added under an ice-water bath. The reaction was carried out for 1h and recorded as reaction solution ①.

[0559] In a separate 50 mL single-necked 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. After dissolving completely, DIPEA (1.65 mL, 10 mmol) was added under ice-water bath conditions, and the reaction was continued for 30 min. Reaction solution ① was then added, and the mixture was allowed to react at room temperature for 2 h. The reaction progress was monitored by HPLC. After the reaction was complete, the reaction solution was purified by preparative HPLC to obtain the preparative solution. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 5.0 g of solid compound 17b, with a yield of 77%. LC-MS: [M+H] + =738.3.

[0560] Step 3: Compound 17d

[0561] Compound 17b (3.0 g, 4.07 mmol) was added to a 25 mL single-necked flask, dissolved in 15 mL of DMF, and then 3.0 g of 5% Pd / C was added. The mixture was hydrogenated for 2 h. After the reaction was complete, the solution was filtered to obtain a filtrate containing compound 17c. The filtrate was placed in an ice-water bath, and DIPEA (744 μL, 4.5 mmol) was added, followed by compound M3 (2.34 g, 4.07 mmol). The mixture was then allowed to cool to room temperature for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by preparative HPLC to obtain a preparative solution. The preparative solution was lyophilized to give 1.2 g of compound 17d (33% yield). LC-MS: [MH] - =892.4.

[0562] Step 4: Compound 17e

[0563] Compound 17d (500 mg, 0.56 mmol), ixotecan 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 to a 50 mL single-necked flask. DIPEA (330 μL, 2.0 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain preparative solutions of compounds 17e-1 and 17e-2. The preparative solutions were lyophilized to obtain 156 mg of compound 17e-1. LC-MS: [M+H] +=1311.4; 150 mg compound 17e-2, LC-MS: [M+H] + =1311.7.

[0564] Step 5: Compound 17A

[0565]

[0566] Compound 17e-1 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to give 43 mg of solid compound 17A; LC-MS: [M+H] + =1155.4.

[0567] Step 6: Compound 17B

[0568]

[0569] Compound 17e-2 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 40 mg of solid compound 17B; LC-MS: [M+H] + =1155.4.

[0570] Example 29 Synthesis of compounds 18A and 18B

[0571]

[0572] By replacing compound M3 with compound ent-M3, and following the synthetic route of Example 28, compound 18A (54 mg) was obtained; LC-MS: [M+H] + =1155.4.

[0573]

[0574] By replacing compound M3 with compound ent-M3, and following the synthetic route of Example 28, compound 18B (55 mg) was obtained; LC-MS: [M+H] + =1155.4.

[0575] Example 30 Synthesis of compounds 19A and 19B

[0576]

[0577] Step 1: Compound 19a

[0578] Compound M1 (10 g, 27.1 mmol), 2-cyclopropyl-2-hydroxyacetic acid benzyl ester (prepared according to the method disclosed in patent WO2020244657A1) (11.2 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene were added to a 250 mL single-necked flask. The mixture was heated to 100 °C and reacted for 4 h. After the reaction was complete, the mixture was cooled to room temperature, filtered to remove insoluble matter, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to give compound 19a 4.97 g, yield 36%; LC-MS: [M+H] + =515.2.

[0579] Step 2: Compound 19b

[0580] Compound 19a (4 g, 7.8 mmol) and 10 mL of DMF were added to a 50 mL single-necked flask. After dissolving, DBU (1.42 g, 9.3 mmol) was added under an ice-water bath. The reaction was carried out for 1 h and recorded as reaction solution ①.

[0581] In a separate 50 mL single-necked 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. After dissolving, DIPEA (1.65 mL, 10 mmol) was added under ice-water bath conditions, and the reaction was continued for 30 min. Reaction solution ① was then added, and the mixture was allowed to react at room temperature for 2 h. The reaction progress was monitored by HPLC. After the reaction was complete, the reaction solution was purified by preparative HPLC to obtain the preparative solution. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over 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.

[0582] Step 3: Compound 19d

[0583] Compound 19b (1000 mg, 1.45 mmol) was added to a 25 mL single-necked flask and dissolved in 15 mL of DMF. Then, 1000 mg of 5% Pd / C was added, and the mixture was hydrogenated for 2 h. After the reaction was complete, the mixture was filtered to obtain a filtrate containing compound 19c. The filtrate was placed in an ice-water bath, and DIPEA (248 μL, 1.5 mmol) was added, followed by compound M3 (720 mg, 1.45 mmol). The mixture was then allowed to cool to room temperature for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by preparative HPLC to obtain a preparative solution. The preparative solution was lyophilized to give 503 mg of compound 19d (yield 41%). LC-MS: [MH] - =842.3.

[0584] Step 4: Compounds 19e-1 and 19e-2

[0585] Compound 19d (500 mg, 0.59 mmol), ixotecan mesylate M5 (317 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol), and 10 mL of DMF were added to a 50 mL single-necked flask. DIPEA (292 μL, 1.77 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain preparative solutions of compounds 19e-1 and 19e-2. The preparative solutions were lyophilized to obtain 112 mg of compound 19e-1. LC-MS: [M+H] + =1261.5; 131 mg compound 19e-2, LC-MS: [M+H] + =1261.5.

[0586] Step 5: Compound 19A

[0587]

[0588] Compound 19e-1 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 55 mg of solid compound 19A; LC-MS: [M+H] + =1105.4.

[0589] Step 6: Compound 19B

[0590]

[0591] Compound 19e-2 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 58 mg of solid compound 19B; LC-MS: [M+H] + =1105.4.

[0592] Example 31 Synthesis of compounds 20A and 20B

[0593]

[0594] Step 1: Compound 20a

[0595] Compound M1 (10 g, 27.1 mmol), benzyl 2-hydroxy-3-cyclopropylpropionate (synthesized according to the method disclosed in patent WO2020063676A) (12.0 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene were added to a 250 mL single-necked flask. The mixture was heated to 100 °C and reacted for 4 h. After the reaction was complete, the mixture was cooled to room temperature, filtered to remove insoluble matter, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to give compound 20a 5.09 g; LC-MS: [M+H] + =529.2.

[0596] Step 2: Compound 20b

[0597] Compound 20a (4g, 7.6mmol) and 10mL DMF were added to a 50mL single-necked flask. After dissolving, DBU (1.39g, 9.1mmol) was added under an ice-water bath. The reaction was carried out for 1h and recorded as reaction solution ①.

[0598] In a separate 50 mL single-necked 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. After dissolving, DIPEA (1.65 mL, 10 mmol) was added under ice-water bath conditions, and the reaction was continued for 30 min. Reaction solution ① was then added, and the mixture was allowed to react at room temperature for 2 h. The reaction progress was monitored by HPLC. After the reaction was complete, the reaction solution was purified by preparative HPLC to obtain the preparative solution. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over 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.

[0599] Step 3: Compound 20d

[0600] Compound 20b (1000 mg, 1.42 mmol) was added to a 25 mL single-necked flask, dissolved in 15 mL of DMF, and then 1000 mg of 5% Pd / C was added. The mixture was hydrogenated for 2 h. After the reaction was complete, the solution was filtered to obtain a filtrate containing compound 20c. The filtrate was placed in an ice-water bath, and DIPEA (248 μL, 1.5 mmol) was added, followed by ixotecan mesylate M5 (708 mg, 1.42 mmol). The mixture was then allowed to reach room temperature and reacted for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by preparative HPLC to obtain a preparative solution. The preparative solution was lyophilized to give 443 mg of compound 20d (36% yield). LC-MS: [MH] - =856.4.

[0601] Step 4: Compounds 20e-1 and 20e-2

[0602] Compound 20d (400 mg, 0.47 mmol), eczemammonium methanesulfonate 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 to a 50 mL single-necked flask. DIPEA (248 μL, 1.5 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain preparative solutions of compounds 20e-1 and 20e-2. The preparative solutions were lyophilized to obtain 103 mg of compound 20e-1. LC-MS: [M+H] + =1275.5; 103 mg compound 20e-2, LC-MS: [M+H] + =1275.5.

[0603] Step 5: Compound 20A

[0604]

[0605] Compound 20e-1 (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 20A 51 mg; LC-MS: [M+H] + =1119.4.

[0606] Step 6: Compound 20B

[0607]

[0608] Compound 20e-2 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to give 47 mg of solid compound 20B; LC-MS: [M+H] + =1119.4.

[0609] Example 32 Synthesis of Compound 21

[0610]

[0611] Step 1: Compound SM3-1

[0612] 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 to a 2000 mL single-necked flask, and the mixture was heated to 100 °C and reacted for 5 h. After the reaction was complete, the mixture was cooled to room temperature, filtered to remove insoluble matter, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 100:1-50:1-20:1) to give compound SM3-1 75.6 g; LC-MS: [M+H] + =299.1.

[0613] Step 2: Compound (R)-2-hydroxy-1,5-pentanoic acid tert-butyl ester

[0614] Add 100 g (338 mmol) of 172793-31-6 and 1000 mL of water to a 2000 mL single-necked flask, followed by sodium nitrite (35 g, 507 mmol) and concentrated sulfuric acid (32 mL, 35 mmol). Slowly heat to room temperature and react for 24 h. After the reaction is complete, extract three times with 500 mL of ethyl acetate. Dry the organic phase with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to remove the solvent, yielding the crude product. Purify the crude product by silica gel column chromatography (PE:EA = 50:1-30:1-2:1) to give 91.2 g of compound (R)-2-hydroxy-1,5-pentanoic acid tert-butyl ester; LC-MS: [M+H] + =261.4.

[0615] Step 3: Compound SM3

[0616] (R)-2-hydroxy-1,5-glutarate tert-butyl ester (50 g, 192 mmol) and 1000 mL of anhydrous tetrahydrofuran were added to a 2000 mL single-necked flask. The mixture was cooled to 0 °C in an ice-water bath, and then PPh3 (87.7 g, 288 mmol), DEAD (50.2 g, 288 mmol), and compound SM3-1 (57.3 g, 192 mmol) were added sequentially. The mixture was slowly heated to room temperature and reacted for 13 h. After the reaction was complete, insoluble matter was removed by filtration, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 50:1-30:1-1:1) to obtain 68.6 g of the product.

[0617] The above product was dissolved in 500 mL of methanol and cooled to 0°C in an ice-water bath. NaOH (64 mL, 190 mmol, 3 M / L) was added dropwise at this temperature, and the reaction was maintained at this temperature for 12 h. Then, HCl (6 M / L) was added to adjust the pH to 3. The mixture was extracted five times with 500 mL of dichloromethane, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (DCM / MeOH = 50 / 1-20 / 1-2 / 1) to give compound SM3 50.4 g; LC-MS: [MH] - =525.5.

[0618] Step 4: Compound M6

[0619] Compound SM3 (50 g, 95 mmol, 1.0 eq), 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 to a 2000 mL single-necked flask. The mixture was reacted at room temperature for 1 h (monitored by TLC), and the insoluble matter was filtered off. The reaction solution was directly purified by preparative liquid chromatography. The preparative solution was concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile, and then lyophilized to give compound M6 (51.9 g), yield 79%; LC-MS: [M+H] + =693.3.

[0620] Step 5: Compound 21a

[0621] Compound 1c (1 g, 2.36 mmol) was added to a 25 mL single-necked flask and dissolved in 25 mL of DMF. Then, DIPEA (430 μL, 2.6 mmol) was added, followed by compound M6 (1177 mg, 2.36 mmol). The mixture was brought to room temperature and reacted for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by preparative HPLC to obtain the preparative solution, which was then lyophilized to give compound 21a 555 mg. LC-MS: [MH] - =931.0.

[0622] Step 6: Compound 21b

[0623] Compound 21a (500 mg, 0.54 mmol), eczemamycin mesylate M5 (285 mg, 0.54 mmol), PyBOP (239 mg, 0.6 mmol), HOBt (239 mg, 0.6 mmol), and 10 mL of DMF were added to a 100 mL single-necked flask. DIPEA (248 μL, 1.5 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the preparative solution of compound 21b. The preparative solution was lyophilized to obtain 231 mg of compound 21b. LC-MS: [M+H] + =1349.5.

[0624] Step 7: Compound 21

[0625] Compound 21b (200 mg, 0.1488 mmol), zinc bromide (665 mg, 2.96 mmol), and 10 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 21b (103 mg). LC-MS: [M+H] + =1137.5.

[0626] Example 33 Synthesis of Compound 22

[0627]

[0628] Starting with compounds M6 and 3c, and following the synthetic route of Example 32, compound 22 (91 mg) was obtained; LC-MS: [M+H] + =1165.5.

[0629] Example 34 Synthesis of compounds 23 and 24

[0630]

[0631] Replacing (S)-(-)-benzyl lactate with (R)-(+)-benzyl lactate, and following the synthetic route of Example 8, compound 5c' was obtained. Using compounds M6 and 5c as starting materials, and following the synthetic route of Example 32, 102 mg of compound 23 was obtained. LC-MS: [M+H] + =1151.4; Using compounds M6 and 5c' as starting materials, 99 mg of compound 24 was prepared according to the synthetic route in Example 32. LC-MS: [M+H] + =1151.4.

[0632] Example 35 Synthesis of compounds 25 and 26

[0633]

[0634] Starting with compounds M6 and 7c, and following the synthetic route of Example 32, 83 mg of compound 25 was obtained. LC-MS: [M+H] + =1205.7; 80 mg of compound 26 was obtained, LC-MS: [M+H] + =1205.7.

[0635] Example 36 Synthesis of compounds 27 and 28

[0636]

[0637] Starting with compounds M6 and 19c, and following the synthetic route of Example 32, 100 mg of compound 27 was obtained. LC-MS: [M+H] + =1177.5; 101 mg of compound 28 was obtained, LC-MS: [M+H] + =1177.5.

[0638] Example 37 Synthesis of Compound 29

[0639]

[0640] Step 1: Compound SM4-1

[0641] In a 5000 mL single-necked flask, maleic acid (50 g, 431 mmol, 1.0 eq), 114559-25-0 (110 g, 431 mmol, 1 eq), TEA (263 g, 2.16 mol, 5 eq), and toluene (2000 mL) were added. The mixture was heated to reflux for 5 h (monitored by TLC). Insoluble matter was filtered off. The reaction solution was directly removed by rotary distillation under reduced pressure. The residue was subjected to silica gel column chromatography (PE / EA = 50 / 1-20 / 1-1 / 1) to give compound SM4-1 (64.7 g), yield 50%; LC-MS: [M+H] + =299.2.

[0642] Step 2: Compound SM4-2

[0643] Compound SM4-1 (64 g, 215 mmol) was added to a 2000 mL single-necked flask and dissolved in 1000 mL of DMF. Then, DIPEA (71 mL, 430 mmol) was added, followed by nonylene glycol monomethyl ether methanesulfonate (111.5 g, 220 mmol). The mixture was brought to room temperature and reacted for 2 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by silica gel column chromatography (PE / EA = 50 / 1-20 / 1-1 / 1) to obtain 59.9 g of the product; LC-MS: [M+H] + =709.4.

[0644] Step 3: Compound SM4

[0645] Compound SM4-2 (59 g, 83 mmol) was added to a 2000 mL single-necked flask and dissolved in 1000 mL of MeOH. Then, K2CO3 (11.75 g, 85 mmol) was added, and the mixture was allowed to react at room temperature for 4 h. The reaction was monitored by HPLC until completion. Insoluble matter was removed by filtration. The reaction solution was directly purified by preparative liquid chromatography. The preparative solution was concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile, and then lyophilized to obtain compound SM4 (27 g). LC-MS: [MH] — =693.5.

[0646] Step 4: Compound M7

[0647] In a 500 mL single-necked flask, compound SM4 (25 g, 36 mmol, 1.0 eq), pentafluorophenol (7.3 g, 40 mmol, 1.1 eq), DCC (8.2 g, 40 mmol, 1.1 eq), and THF (200 mL) were added. The mixture was reacted at room temperature for 1 h (monitored by TLC), and the insoluble matter was filtered off. The reaction solution was directly purified by preparative liquid chromatography. The preparative solution was concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile, and then lyophilized to give compound M7 (23.3 g), yield 93%; LC-MS: [M+H] + =695.8.

[0648] Step 5: Compound 29a

[0649] Compound 1c (1 g, 2.36 mmol) was added to a 25 mL single-necked flask and dissolved in 25 mL of DMF. Then, DIPEA (430 μL, 2.6 mmol) was added, followed by compound M7 (1640 mg, 2.36 mmol). The mixture was brought to room temperature and reacted for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by preparative HPLC to obtain the preparative solution, which was then lyophilized to yield 609 mg of the product. LC-MS: [MH] - =1098.5.

[0650] Step 6: Compound 29b

[0651] Compound 29a (500 mg, 0.45 mmol), eczemamycin mesylate M5 (240 mg, 0.45 mmol), PyBOP (215 mg, 0.54 mmol), HOBt (215 mg, 0.54 mmol), and 10 mL of DMF were added to a 100 mL single-necked flask. DIPEA (248 μL, 1.5 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the preparative solution of compound 29b. The preparative solution was lyophilized to obtain 187 mg of compound 29b. LC-MS: [M+H] + =1517.6.

[0652] Step 7: Compound 29

[0653] Compound 29b (150 mg, 0.988 mmol), zinc bromide (223 mg, 0.988 mmol), and 10 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 29b (114 mg). LC-MS: [M+H] + =1417.9.

[0654] Example 38 Synthesis of Compound 30

[0655]

[0656] Using compounds M7 and 3c as starting materials, and following the synthetic route of Example 37, compound 30 (125 mg) was obtained; LC-MS: [M+H] + =1445.6.

[0657] Example 39 Synthesis of compounds 31 and 32

[0658]

[0659] Starting with compounds M7 and 5c, and following the synthetic route of Example 37, 61 mg of compound 31 was obtained. LC-MS: [M+H] + =1431.7; Using compounds M7 and 5c' as starting materials, following the synthetic route of Example 37, 63 mg of compound 32 was obtained. LC-MS: [M+H] + =1431.7.

[0660] Example 40 Synthesis of compounds 33 and 34

[0661]

[0662] Starting with compounds M7 and 7c, and following the synthetic route of Example 37, 60 mg of compound 33 was obtained. LC-MS: [M+H] + =1485.6; 58 mg of compound 34 was obtained, LC-MS: [M+H] + =1485.6.

[0663] Example 41 Synthesis of compounds 35 and 36

[0664]

[0665] Starting with compounds M7 and 19c, and following the synthetic route of Example 37, 102 mg of compound 35 was obtained. LC-MS: [M+H] + =1457.8; 102 mg of compound 36 was obtained, LC-MS: [M+H] + =1457.8.

[0666] Example 42 Synthesis of Compound 37

[0667]

[0668] Step 1: Compound SM5-1

[0669] In a 2000 mL single-necked flask, compound 16947-84-5 (100 g, 295 mmol, 1.0 eq), DIPEA (50 mL, 300 mmol), benzyl bromide (51.3 g, 300 mmol), and THF (1000 mL) were added. The mixture was reacted at room temperature for 12 h (monitored by TLC). Insoluble matter was filtered off. The solvent was removed by direct rotary distillation under reduced pressure. The residue was subjected to silica gel column chromatography (PE / EA = 50 / 1-20 / 1-2 / 1) to give compound SM5-1 (110.1 g), yield 87%; LC-MS: [M+H] + =429.2.

[0670] Step 2: Compound SM5-2

[0671] In a 2000 mL single-necked flask, compound SM5-1 (100 g, 233.4 mmol, 1.0 eq) and THF (1000 mL) were added. The mixture was cooled to 0 °C in an ice-water bath. NaH (37.4 g, 933.5 mmol) and MeI (132.5 g, 933.5 mmol) were added in portions, and the reaction was maintained at 0 °C for 24 h (monitored by TLC). The reaction was quenched by adding 500 mL of saturated NH4Cl aqueous solution. The mixture was extracted three times with 500 mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was directly removed by rotary distillation under reduced pressure to remove the solvent. The residue was subjected to silica gel column chromatography (PE / EA = 100 / 1-50 / 1-10 / 1) to give compound SM5-2 (37.1 g). LC-MS: [M+H] + =443.3.

[0672] Step 3: Compound SM5 (refer to Org. Lett., 2006, 8, 3387-3390.)

[0673] In a 1000 mL single-necked flask, compound SM5-2 (35 g, 79 mmol, 1.0 eq) and DCE (500 mL) were added, followed by palladium diacetate (180 mg, 0.8 mmol), I2 (20 g, 79 mmol), and iodobenzene diacetate (40.8 g, 126.4 mmol). The mixture was heated to 60 °C and reacted for 40 h (monitored by TLC). The reaction was quenched with 500 mL of saturated sodium thiosulfate aqueous solution. The mixture was extracted three times with 500 mL of dichloromethane. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was directly removed by rotary distillation under reduced pressure to remove the solvent. The residue was subjected to silica gel column chromatography (PE / EA = 100 / 1-50 / 1-10 / 1) to give compound SM5 (28 g); LC-MS: [M+H] + =501.3.

[0674] Step 4: Compound SM6

[0675] In a 500 mL single-necked flask, compound SM5 (25 g, 50 mmol, 1.0 eq), potassium di-tert-butyl phosphate (13.66 g, 55 mmol, 1.1 eq), p-toluenesulfonic acid monohydrate (951 mg, 5 mmol, 0.1 eq), and THF (200 mL) were added. The mixture was reacted at room temperature for 1 h (monitored by TLC). Insoluble matter was filtered off. The reaction solution was directly purified by preparative liquid chromatography. The preparative solution was concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile, and then lyophilized to give compound SM6 (15.1 g), yield 46%; LC-MS: [M+H] + =651.4.

[0676] Step 5: Compound SM7

[0677] Add compound SM6 (15g, 23mmol) and 100mL DMF to a 250mL single-necked flask. After dissolving, add 15g of 5% Pd / C under an ice-water bath. Replace the atmosphere in the system with hydrogen three times. React at room temperature for 12h. Filter to remove Pd / C. Remove the solvent by rotary evaporation under reduced pressure using an oil pump to obtain the crude product for later use.

[0678] In a separate 250 mL single-necked flask, add the above crude product and 100 mL of toluene, triethylamine (6.4 mL, 46 mmol), and maleic anhydride (2.4 g, 24 mmol). After dissolving completely, raise the temperature to 100 °C and react for 2 h. Monitor the reaction progress by HPLC. After the reaction is complete, purify the reaction solution by preparative high-performance liquid chromatography to obtain the preparative solution. Extract the preparative solution with dichloromethane, wash with saturated sodium chloride solution, dry with anhydrous sodium sulfate, filter, and concentrate to obtain 4.2 g of solid compound SM7, yield 36%; LC-MS: [M+H] + =507.3.

[0679] Step 6: Compound M8

[0680] In a 100 mL single-necked flask, compound SM7 (4 g, 7.9 mmol, 1.0 eq), 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. The mixture was reacted at room temperature for 1 h (monitored by TLC), and the insoluble matter was filtered off. The reaction solution was directly purified by preparative liquid chromatography. The preparative solution was concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile, and then lyophilized to give compound M8 (3.7 g), yield 70%; LC-MS: [M+H] + =673.2.

[0681] Step 7: Compound 37a

[0682] Compound 1c (1 g, 2.36 mmol) was added to a 25 mL single-necked flask and dissolved in 25 mL of DMF. Then, DIPEA (430 μL, 2.6 mmol) was added, followed by compound M8 (1.2 g, 2.36 mmol). The mixture was brought to room temperature and reacted for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by preparative HPLC to obtain the preparative solution, which was then lyophilized to give compound 37a 488 mg. LC-MS: [MH] - =911.0.

[0683] Step 8: Compound 37b

[0684] Compound 37a (400 mg, 0.44 mmol), eczemamycin mesylate M5 (235 mg, 0.44 mmol), PyBOP (199 mg, 0.5 mmol), HOBt (69 mg, 0.5 mmol), and 10 mL of DMF were added to a 100 mL single-necked flask. DIPEA (218 μL, 1.32 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the preparative solution of compound 37b. The preparative solution was lyophilized to obtain 201 mg of compound 37b. LC-MS: [M+H] + =1329.6.

[0685] Step 9: Compound 37

[0686] Compound 37b (130 mg, 0.098 mmol), zinc bromide (221 mg, 0.98 mmol), and 10 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to give solid compound 37b 96 mg; LC-MS: [M+H] + =1117.4.

[0687] Example 43 Synthesis of Compound 38

[0688]

[0689] Starting with compounds M8 and 3c, and following the synthetic route of Example 42, compound 38 (51 mg) was obtained; LC-MS: [M+H] + =1145.6.

[0690] Example 44 Synthesis of compounds 39 and 40

[0691]

[0692] Starting with compounds M8 and 5c, and following the synthetic route of Example 42, 57 mg of compound 39 was obtained. LC-MS: [M+H] + =1131.4; Using compounds M8 and 5c' as starting materials, 60 mg of compound 40 was prepared according to the synthetic route in Example 42. LC-MS: [M+H] + =1131.4.

[0693] Example 45 Synthesis of compounds 41 and 42

[0694]

[0695] Starting with compounds M8 and 7c, and following the synthetic route of Example 42, 44 mg of compound 41 was obtained. LC-MS: [M+H] + =1185.3; 44 mg of compound 42 was obtained. LC-MS: [M+H] + =1185.3.

[0696] Example 46 Synthesis of compounds 43 and 44

[0697]

[0698] Starting with compounds M8 and 19c, and following the synthetic route of Example 42, 62 mg of compound 43 was obtained. LC-MS: [M+H] + =1157.4; 59 mg of compound 44 was obtained, LC-MS: [M+H] + =1157.4.

[0699] Example 47 Synthesis of Compound 46

[0700]

[0701] Step 1: Compound 46a

[0702] Compound 1d (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 to a 50 mL single-necked flask. DIPEA (378 μL, 2.29 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the preparative solution. The preparative solution was lyophilized to obtain compound 46a (210 mg). LC-MS: [M+H] + =1221.6.

[0703] Step 2: Compound 46

[0704] Compound 46a (200 mg, 0.162 mmol), zinc bromide (736 mg, 3.26 mmol), and 10 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 46 (120 mg). LC-MS: [M+H] + =1065.3.

[0705] Example 48 Synthesis of Compound 47

[0706]

[0707] Compound 1d' was obtained by replacing compound M3 with compound ent-M3 and following the synthetic route of Example 4. Compound 47 (81 mg) was obtained by replacing compound 1d with compound 1d' and following the synthetic route of Example 47; LC-MS: [M+H] + =1065.3.

[0708] Example 49 Synthesis of Compound 48A

[0709]

[0710] Step 1: Compound 48a

[0711] Compound 5d (1.66 g, 2.02 mmol, 1.0 eq), compound M9 (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 to a 100 mL single-necked flask. DIPEA (0.84 mL, 1.5 eq) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h (monitored by HPLC). The reaction solution was directly purified by preparative treatment. The preparative solution was concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile, and then lyophilized to obtain compound 48a (1.54 g), yield 61%. LC-MS: [M+H] + =1235.4.

[0712] Step 6: Compound 48A

[0713] Compound 48a (1.0 g, 0.8 mmol, 1.0 eq) and 35 mL of nitromethane were added to a 100 mL single-necked flask and dissolved. Zinc bromide (3.64 g, 16 mmol, 20.0 eq) was then added, and the mixture was reacted in an oil bath at 40 °C (preheated for stability) for 30 min. The mixture was then concentrated in a water bath at 45 °C under reduced pressure to remove the nitromethane, yielding a yellow solid residue (monitored by HPLC). This residue was purified by preparative liquid chromatography by adding 0.1% trifluoroacetic acid to the mobile phase to obtain the preparative solution. The preparative solution was concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile, and then lyophilized to obtain compound 48A (786 mg) in 90% yield.

[0714] Example 50 Synthesis of Compound 48B

[0715]

[0716] Step 1: Compound 48b

[0717] Compound 5d-1 (200 mg, 0.24 mmol, 1.0 eq), compound M9 (127 mg, 0.24 mmol, 1.0 eq), PyBOP (187 mg, 0.36 mmol, 1.2 eq), HOBt (48 mg, 0.36 mmol, 1.2 eq), and DMF (6 mL) were added to a 25 mL single-necked flask. The mixture was cooled to 0–5 °C in an ice-water bath, and DIPEA (62 mg, 0.48 mmol, 2.0 eq) was added. After the addition was complete, the mixture was heated to 20 ± 5 °C and reacted for 2 h. The reaction was monitored by HPLC until it was complete. The reaction solution was directly purified by preparative HPLC, and the product solution was collected and lyophilized to obtain compound 48b (150.2 mg). LC-MS: [M+H] + =1235.4.

[0718] Step 2: Compound 48B

[0719] 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 sequentially to a 25 mL single-necked flask. After addition, the mixture was heated to 40 °C and reacted for 0.5 h. The reaction was then stopped, and the reaction solution was directly evaporated to dryness under reduced pressure at 45 °C to obtain a yellow solid. The reaction was monitored by HPLC. The evaporated solid was directly purified by preparative HPLC, and the product preparation solution was collected and lyophilized to obtain compound 48B (70.0 mg). LC-MS: [M+H] + =1079.4.

[0720] Example 51 Synthesis of Compound 49A

[0721]

[0722] Compound 5d' was synthesized by replacing compound 5c' with compound 5c', following the route of Example 8. Compound 49A (71 mg) was obtained by replacing compound 5d' with compound 5d', following the route of Example 49; LC-MS: [M+H] + =1079.4.

[0723] Example 52 Preparation of Compound 49B

[0724]

[0725] Compound 5d-1' was synthesized by replacing compound 5c with compound 5c-1', following the synthetic route of Example 9. Compound 49B (65 mg) was then synthesized by replacing compound 5d-1 with compound 5d-1', following the synthetic route of Example 50. LC-MS: [M+H] + =1079.4.

[0726] Example 53 Synthesis of Compounds 50A and 50B

[0727]

[0728] Step 1: Compound 50a and Compound 50b

[0729] Compound 7d (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 to a 50 mL single-necked flask. DIPEA (378 μL, 2.29 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain preparative solutions of compounds 50a and 50b. The preparative solutions were lyophilized to obtain 170 mg of compound 50a. LC-MS: [M+H] + =1289.46; 202 mg compound 50b, LC-MS: [M+H] + =1289.46.

[0730] Step 2: Compound 50A

[0731]

[0732] Compound 50a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 44 mg of solid compound 50A.

[0733] Step 3: Compound 50B

[0734]

[0735] Compound 50b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 45 mg of solid compound 50B.

[0736] Example 54 Synthesis of Compounds 51A and 51B

[0737]

[0738] Step 1: Compound 51a and Compound 51b

[0739] 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 to a 50 mL single-necked flask. DIPEA (378 μL, 2.29 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain preparative solutions of compounds 51a and 51b. These preparative solutions were lyophilized to yield 190 mg of compound 51a and 186 mg of compound 51b, respectively. LC-MS of compound 51a: [M+H] + =1289.47; LC-MS of compound 51b: [M+H] + =1289.47.

[0740] Step 2: Compound 51A

[0741]

[0742] Compound 51a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 39 mg of solid compound 51A.

[0743] Step 3: Compound 51B

[0744]

[0745] Compound 51b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 60 mg of solid compound 51B.

[0746] Example 55 Synthesis of Compound 52A

[0747]

[0748] Step 1: Compound 52a

[0749] Compound 11d (800 mg, 0.96 mmol), compound M9 (480 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol), and 30 mL of DMF were added to a 50 mL single-necked flask. DIPEA (660 μL, 4.0 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 4 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the preparative solution of compound 52a. The preparative solution was lyophilized to obtain compound 52a (388 mg). LC-MS: [M+H] + =1261.4.

[0750] Step 2: Compound 52A

[0751] Compound 52a (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol), and 10 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 52A (79 mg); LC-MS: [M+H] + =1105.4.

[0752] Example 56 Synthesis of Compound 52B

[0753]

[0754] Replacing compound M3 with compound ent-M3 and following the synthetic route of Example 18, compound 11d' was obtained. Replacing compound 11d with compound 11d' and following the synthetic route of Example 55, compound 52B (50 mg) was obtained. LC-MS: [M+H] + 1105.4.

[0755] Example 57 Synthesis of Compound 53A

[0756]

[0757] Step 1: Compound 53a

[0758] Compound 12d (400 mg, 0.47 mmol), compound M9 (240 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt (101 mg, 0.47 mmol), and 15 mL of DMF were added to a 50 mL single-necked flask. DIPEA (330 μL, 2.0 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 3 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the preparative solution of compound 53a. The preparative solution was lyophilized to obtain compound 53a (200 mg). LC-MS: [M+H] + =1275.4.

[0759] Step 2: Compound 53A

[0760] Compound 53a (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 53A (51 mg); LC-MS: [M+H] + =1119.4.

[0761] Example 58 Synthesis of Compound 53B

[0762]

[0763] Replacing compound M3 with compound ent-M3 and following the synthetic route of Example 20, compound 12d' was obtained. Replacing compound 12d with compound 12d' and following the synthetic route of Example 57, compound 53B (50 mg) was obtained; LC-MS: [M+H] + =1119.4.

[0764] Example 59 Synthesis of Compounds 54A and 54B

[0765]

[0766] Step 1: Compound 54a and Compound 54b

[0767] Compound 19d (500 mg, 0.59 mmol), compound M9 (317 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol), and 10 mL of DMF were added to a 50 mL single-necked flask. DIPEA (292 μL, 1.77 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain preparative solutions of compounds 54a and 54b. The preparative solutions were lyophilized to obtain 103 mg of compound 54a. LC-MS: [M+H] + =1261.5; 111 mg compound 54b, LC-MS: [M+H] + =1261.5.

[0768] Step 2: Compound 54A

[0769]

[0770] Compound 54a (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 61 mg of solid compound 54A; LC-MS: [M+H] + =1105.4.

[0771] Step 3: Compound 54B

[0772]

[0773] Compound 54b (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 57 mg of solid compound 54B; LC-MS: [M+H] + =1105.4.

[0774] Example 60 Synthesis of Compounds 55A and 55B

[0775]

[0776] Step 1: Compound 55a and Compound 55b

[0777] 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 to a 50 mL single-necked flask. DIPEA (248 μL, 1.5 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain preparative solutions of compounds 55a and 55b. The preparative solutions were lyophilized to obtain 100 mg of compound 55a. LC-MS: [M+H] + =1275.5; 101 mg compound 55b, LC-MS: [M+H] + =1275.5.

[0778] Step 2: Compound 55A

[0779]

[0780] Compound 55a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 42 mg of solid compound 55A; LC-MS: [M+H] + =1119.4.

[0781] Step 3: Compound 55B

[0782]

[0783] Compound 55b (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 45 mg of solid compound 55B; LC-MS: [M+H] + =1119.4.

[0784] Example 61 Synthesis of Compound 56

[0785]

[0786] Compound 20d' was obtained by replacing compound M3 with compound ent-M3 and following the synthetic route of Example 31. Compound 56 (50 mg) was obtained by replacing compound 20d with compound 20d' and following the synthetic route of Example 60; LC-MS: [M+H] + =1119.3.

[0787] Example 62 Synthesis of Compound 57

[0788]

[0789] Compound 20d' was obtained by replacing compound M3 with compound ent-M3 and following the synthetic route of Example 31. Compound 57 (50 mg) was obtained by replacing compound 20d with compound 20d' and following the synthetic route of Example 60; LC-MS: [M+H] + =1119.4.

[0790] Example 63 Synthesis of Compound 58

[0791]

[0792] Step 1: Synthesis of compound 58a

[0793] 400 mL of DMF was added to ixotecan mesylate M5 (15 g, 28 mol, prepared according to the method disclosed in patent application "EP0737683A1"). The mixture was cooled to 0 °C in an ice-water bath, and triethylamine was added dropwise to adjust the pH to 7-8. Benzyl bromide (9.6 g, 56 mmol) was then added dropwise in an ice-water bath. The mixture was heated to room temperature (25 °C) and reacted for 1 hour. The reaction was monitored by TLC until complete. The reaction solution was concentrated under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography (acetonitrile / pure water system). The target peak was collected, and after removing acetonitrile under reduced pressure, the product was lyophilized to give approximately 11 g of compound 58a as a yellow solid, with a yield of approximately 74%. MS m / z: [M+H] + 526.3.

[0794] Step 2: Synthesis of compound 58b

[0795] At room temperature, compound 58a (11 g, 21 mol) was added sequentially to a 250 mL single-necked flask and dissolved in 120 mL of formic acid. 30 mL of formaldehyde (40% aqueous solution) was added to the resulting bright yellow solution. The mixture was heated to 50 °C and reacted for 1 hour. The reaction was monitored by TLC until complete. After cooling to room temperature, the reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile / pure water system). The target peak was collected, and after removing acetonitrile under reduced pressure, the solution was lyophilized to obtain approximately 4.5 g of compound 58b as a yellow powder, with a yield of approximately 40%. MS m / z: [M+H] + 540.6.

[0796] Step 3: Synthesis of Compound 58

[0797] Compound 58b (2.3 g, 4.3 mol) was added to a 250 mL single-necked flask at room temperature and dissolved in 100 mL of DMF. 2.3 g of 5% Pd / C was added to the resulting bright yellow solution. The atmosphere in the system was purged with hydrogen, and the reaction was maintained at room temperature for 1.5 hours. The reaction was monitored by HPLC until complete. Pd / C was removed by filtration. The resulting reaction solution was concentrated and purified by preparative high-performance liquid chromatography (acetonitrile / pure water system). The target peak was collected, and after removing acetonitrile under reduced pressure, the solution was lyophilized to obtain approximately 1.0 g of compound 58 as a yellow powder, with a yield of approximately 52%. MS m / z: [M+H] + 450.5.

[0798] Example 64 Synthesis of Compound 59

[0799]

[0800] Step 1: Compound 59a

[0801] Compound 1d (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 to a 50 mL single-necked flask. DIPEA (378 μL, 2.29 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the preparative solution. The preparative solution was lyophilized to obtain compound 59a (166 mg). LC-MS: [M+H] + =1235.6.

[0802] Step 2: Compound 59

[0803] Compound 59a (100 mg, 0.081 mmol), zinc bromide (368 mg, 1.63 mmol), and 10 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 59 (43 mg). LC-MS: [M+H] + =1079.3.

[0804] Example 65 Synthesis of Compound 60

[0805]

[0806] Compound 1d' was obtained by replacing compound M3 with compound ent-M3 and following the synthetic route of Example 4. Compound 60 (40 mg) was obtained by replacing compound 1d with compound 1d' and following the synthetic route of Example 64; LC-MS: [M+H] + =1079.3.

[0807] Example 66 Synthesis of Compound 61

[0808]

[0809] Step 1: Compound 61a

[0810] 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 to a 100 mL single-necked flask. DIPEA (0.84 mL, 1.5 eq) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h (monitored by HPLC). The reaction solution was directly purified by preparative liquid chromatography. The preparative solution was concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile, and then lyophilized to obtain compound 61a (1.21 g). LC-MS: [M+H] + =1249.4.

[0811] Step 2: Compound 61

[0812] Compound 61a (1.0 g, 0.8 mmol, 1.0 eq) and 35 mL of nitromethane were added to a 100 mL single-necked flask and dissolved. Zinc bromide (3.64 g, 16 mmol, 20.0 eq) was then added. The mixture was reacted in an oil bath at 40 °C (preheated for stability) for 30 min. The mixture was then concentrated in a water bath at 45 °C under reduced pressure to remove the nitromethane, yielding a yellow solid residue (monitored by HPLC). This residue was purified by preparative liquid chromatography by adding 0.1% trifluoroacetic acid to the mobile phase. The preparative solution was then concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile, and lyophilized to obtain compound 61a (786 mg). LC-MS: [M+H] + =1093.6.

[0813] Example 67 Synthesis of Compound 62

[0814]

[0815] Step 1: Compound 62a

[0816] Compound 5d-1 (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, 1.2 eq), HOBt (48 mg, 0.36 mmol, 1.2 eq), and DMF (6 mL) were added to a 25 mL single-necked flask. The mixture was cooled to 0–5 °C in an ice-water bath, and DIPEA (62 mg, 0.48 mmol, 2.0 eq) was added. After the addition was complete, the mixture was heated to 20 ± 5 °C and reacted for 2 h. The reaction was monitored by HPLC until it was complete. The reaction solution was directly purified by preparative HPLC, and the product solution was collected and lyophilized to obtain compound 62a (120.9 mg). LC-MS: [M+H] + =1249.4.

[0817] Step 2: Compound 62

[0818] 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 sequentially to a 25 mL single-necked flask. After the addition was complete, the mixture was heated to 40 °C and reacted for 0.5 h. The reaction mixture was then stopped, and the reaction solution was directly evaporated to dryness under reduced pressure at 45 °C to obtain a yellow solid. The reaction was monitored by HPLC. The evaporated solid was directly purified by preparative HPLC, and the product preparation solution was collected and lyophilized to obtain compound 62 (61 mg). LC-MS: [M+H] + =1093.4.

[0819] Example 68 Preparation of Compound 63

[0820]

[0821] By replacing compound 5d with compound 5d', and following the route in Example 66, compound 63 (60 mg) was obtained; LC-MS: [M+H] + =1093.4.

[0822] Example 69 Preparation of Compound 64

[0823]

[0824] By replacing compound 5d-1 with compound 5d-1', and following the synthetic route of Example 67, compound 64 (65 mg) was obtained; LC-MS: [M+H] + =1093.4.

[0825] Example 70 Preparation of Compounds 65A and 65B

[0826]

[0827] Step 1: Compound 65a and Compound 65b

[0828] Compound 7d (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 to a 50 mL single-necked flask. DIPEA (378 μL, 2.29 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain preparative solutions of compounds 65a and 65b. The preparative solutions were lyophilized to obtain 155 mg of compound 65a. LC-MS: [M+H] + =1303.4; 158 mg compound 65b, LC-MS: [M+H] + =1303.6.

[0829] Step 2: Compound 65A

[0830]

[0831] Compound 65a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the preparative solution, which was then lyophilized to obtain 49 mg of solid compound 65A.

[0832] Step 3: Compound 65B

[0833]

[0834] 65B (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the preparative solution, which was then lyophilized to obtain 47 mg of solid compound 65B.

[0835] Example 71 Synthesis of Compounds 66A and 66B

[0836]

[0837] Step 1: Compound 66a and Compound 66b

[0838] 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 to a 50 mL single-necked flask. DIPEA (378 μL, 2.29 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain preparative solutions of compounds 66a and 66b. These preparative solutions were lyophilized to obtain 160 mg of compound 66a and 160 mg of compound 66b, respectively. LC-MS of compound 66a: [M+H] + =1303.7; LC-MS of compound 66b: [M+H] + =1303.6.

[0839] Step 2: Compound 66A

[0840]

[0841] Compound 66a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 57 mg of solid compound 66A. LC-MS: [M+H] + =1147.5.

[0842] Step 3: Compound 66B

[0843]

[0844] Compound 66b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 57 mg of solid compound 66B. LC-MS: [M+H] + =1147.5.

[0845] Example 72 Synthesis of Compound 67A

[0846]

[0847] Step 1: Compound 67a

[0848] Compound 11d (800 mg, 0.96 mmol), compound 58 (432.5 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol), and 30 mL of DMF were added to a 50 mL single-necked flask. DIPEA (660 μL, 4.0 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 4 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the preparative solution of compound 67a. The preparative solution was lyophilized to obtain compound 67a (402 mg). LC-MS: [M+H] + =1275.4.

[0849] Step 2: Compound 67A

[0850] Compound 67a (100 mg, 0.78 mmol), zinc bromide (356 mg, 1.57 mmol), and 10 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 67A (47 mg); LC-MS: [M+H] + =1119.5.

[0851] Example 73 Synthesis of Compound 67B

[0852]

[0853] By replacing compound 11d with compound 11d' and following the synthetic route of Example 72, compound 67B (50 mg) was obtained. LC-MS: [M+H] + 1119.4.

[0854] Example 74 Synthesis of Compound 68A

[0855]

[0856] Step 1: Compound 68a

[0857] Compound 12d (400 mg, 0.47 mmol), compound 58 (211.7 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt (101 mg, 0.47 mmol), and 15 mL of DMF were added to a 50 mL single-necked flask. DIPEA (330 μL, 2.0 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 3 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the preparative solution of compound 68a. The preparative solution was lyophilized to obtain compound 68a (177 mg). LC-MS: [M+H] + =1289.4.

[0858] Step 2: Compound 68A

[0859] Compound 68a (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 68A (45 mg); LC-MS: [M+H] + =1133.4.

[0860] Example 75 Synthesis of Compound 68B

[0861]

[0862] By replacing compound 12d with compound 12d' and following the synthetic route in Example 74, compound 68B (50 mg) was obtained; LC-MS: [M+H] + =1133.4.

[0863] Example 76 Synthesis of Compounds 69A and 69B

[0864]

[0865] Step 1: Compounds 69a and 69b

[0866] Compound 19d (500 mg, 0.59 mmol), compound 58 (266 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol), and 10 mL of DMF were added to a 50 mL single-necked flask. DIPEA (292 μL, 1.77 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain preparative solutions of compounds 69a and 69b. The preparative solutions were lyophilized to obtain 109 mg of compound 69a. LC-MS: [M+H] + =1275.5; 111 mg compound 69b, LC-MS: [M+H] + =1275.7.

[0867] Step 2: Compound 69A

[0868]

[0869] Compound 69a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 53 mg of solid compound 69A; LC-MS: [M+H] + =1119.4.

[0870] Step 3: Compound 69B

[0871]

[0872] Compound 69b (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 54 mg of solid compound 69B; LC-MS: [M+H] + =1119.4.

[0873] Example 77 Synthesis of Compounds 70A and 70B

[0874]

[0875] Step 1: Compound 70a and Compound 70b

[0876] Compound 20d (400 mg, 0.47 mmol), compound 58 (211.7 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol), and 10 mL of DMF were added to a 50 mL single-necked flask. DIPEA (248 μL, 1.5 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain preparative solutions of compounds 70a and 70b. The preparative solutions were lyophilized to obtain 106 mg of compound 70a. LC-MS: [M+H] + =1289.5; 101 mg compound 70b, LC-MS: [M+H] + =1289.4.

[0877] Step 2: Compound 70A

[0878]

[0879] Compound 70a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 39 mg of solid compound 70A; LC-MS: [M+H] + =1133.4.

[0880] Step 3: Compound 70B

[0881]

[0882] Compound 70b (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 35 mg of solid compound 70B; LC-MS: [M+H] + =1133.4.

[0883] Example 78 Synthesis of Compound 71

[0884]

[0885] By replacing compound 20d with compound 20d' and following the synthetic route in Example 77, compound 71 (30 mg) was obtained; LC-MS: [M+H]+ =1133.3.

[0886] Example 79 Synthesis of Compound 72

[0887]

[0888] By replacing compound 20d with compound 20d' and following the synthetic route in Example 77, compound 72 (33 mg) was obtained; LC-MS: [M+H] + =1133.4.

[0889] Example 80 Synthesis of compound M11

[0890]

[0891] In a 100 mL single-necked flask, compound M3 (11.0 g, 19.5 mmol, 1.0 eq), DIPEA (2.8 g, 21.4 mmol, 1.1 eq), 27-amino-4,7,10,13,16,19,22,25-octaoxaheptacosanonic acid (9.7 g, 20.5 mmol, 1.05 eq), and DMF (60 mL) were added and reacted at room temperature for 20 min (monitored by TLC). The reaction solution was directly purified by preparative liquid chromatography. The preparative solution was concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile, and then lyophilized to obtain compound M10 (13.2 g), yield 78%; LC-MS: [M+H] + =866.5.

[0892] In a 100 mL single-necked flask, compound M10 (13.0 g, 15 mmol, 1.0 eq), 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. The mixture was reacted at room temperature for 30 min (monitored by TLC), and the insoluble matter was filtered off. The reaction solution was directly purified by preparative liquid chromatography. The preparative solution was concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile, and then lyophilized to obtain compound M11 (14.2 g), with a yield of 92%. LC-MS: [M+H] + =1032.5.

[0893] Example 81 Synthesis of Compound 73

[0894]

[0895] Step 1: Synthesis of compound 73a

[0896] 10 mL of DMF was added to compound M11 (1 g, 0.79 mol), and the mixture was cooled to 0 °C in an ice-water bath. Compound 1c (334 mg, 0.79 mol) and DIPEA (154 mg, 1.19 mol) were then added. The reaction was maintained under these conditions for 1 h. The reaction was monitored by TLC until complete. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile / pure water system). The target peak was collected, and after removing acetonitrile under reduced pressure, the solution was lyophilized to obtain approximately 1.2 g of compound 73a. MS m / z: [M+H] + =1271.9.

[0897] Step 2: Synthesis of compound 73b

[0898] Compound 73a (1.2 g, 0.94 mmol), ixotecan mesylate M5 (500 mg, 0.94 mmol), PyBOP (625 mg, 1.2 mmol), HOBt (162 mg, 1.2 mmol), and 15 mL of DMF were added to a 25 mL single-necked flask. DIPEA (310 mg, 2.4 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the preparative solution. The preparative solution was lyophilized to obtain compound 73b (709 mg). LC-MS: [M+H] + =1720.8.

[0899] Step 3: Synthesis of Compound 73

[0900] Compound 73b (200 mg, 0.116 mmol), zinc bromide (523 mg, 2.32 mmol), and 10 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 73 (88 mg); LC-MS: [M+H] + =1532.6.

[0901] Example 82 Synthesis of Compound 74

[0902]

[0903] Replacing compound M3 with compound ent-M3 and following the synthetic route of Example 80, compound M11' was obtained. Replacing compound M11 with compound M11' and following the synthetic route of Example 81, compound 74 (90 mg) was obtained; LC-MS: [M+H] + =1532.6.

[0904] Example 83 Synthesis of Compound 75

[0905]

[0906] Step 1: Synthesis of compound 75a

[0907] 10 mL of DMF was added to compound M11 (1 g, 0.79 mol), and the mixture was cooled to 0 °C in an ice-water bath. Compound 5c (345 mg, 0.79 mol) and DIPEA (154 mg, 1.19 mol) were then added. The reaction was maintained under these conditions for 1 h. The reaction was monitored by TLC until complete. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile / pure water system). The target peak was collected, and after removing acetonitrile under reduced pressure, the solution was lyophilized to obtain compound 75a 0.9 g, MS m / z: [M+H]. + =1285.6.

[0908] Step 2: Synthesis of compound 75b

[0909] Compound 75a (700 mg, 0.54 mmol), ixotecan 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 to a 25 mL single-necked flask. DIPEA (155 mg, 1.2 mmol) was added under ice-water bath conditions, and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the preparative solution. The preparative solution was lyophilized to obtain 75b (304 mg). LC-MS: [M+H] + =1734.8.

[0910] Step 3: Synthesis of Compound 75

[0911] 75b (200 mg, 0.116 mmol), zinc bromide (523 mg, 2.32 mmol), and 10 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 75 (96 mg); LC-MS: [M+H] + =1546.6.

[0912] Example 84 Synthesis of Compound 76

[0913]

[0914] By replacing compound M11 with compound M11' and following the synthetic route of Example 83, compound 76 (92 mg) was obtained; LC-MS: [M+H] + =1546.5.

[0915] Example 85 Synthesis of Compound 77

[0916]

[0917] By replacing compound M11 with compound M11' and compound 5c with compound 5c', following the synthetic route of Example 83, compound 77 (87 mg) was obtained; LC-MS: [M+H] + =1546.5.

[0918] Example 86 Synthesis of Compound 78

[0919]

[0920] By replacing compound 5c with compound 5c' and following the synthetic route in Example 83, compound 78 (94 mg) was obtained; LC-MS: [M+H] + =1546.7.

[0921] Example 87 Synthesis of Compounds 79 and 80

[0922]

[0923] Step 1: Synthesis of compound 79a

[0924] 10 mL of DMF was added to compound M11 (1 g, 0.79 mol), and the mixture was cooled to 0 °C in an ice-water bath. Compound 20c (377 mg, 0.79 mol) and DIPEA (154 mg, 1.19 mol) were then added. The reaction was maintained under these conditions for 1 h. The reaction was monitored by TLC until complete. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile / pure water system). The target peak was collected, and after removing acetonitrile under reduced pressure, the solution was lyophilized to obtain 783 mg of compound 79a. MS m / z: [M+H] + =1325.8.

[0925] Step 2: Synthesis of compounds 79b-1 and 79b-2

[0926] Add 79a (600 mg, 0.45 mmol), ixotecan mesylate M5 (240 mg, 0.45 mmol), PyBOP (261 mg, 0.5 mmol), HOBt (68 mg, 0.5 mmol), and 10 mL of DMF to a 25 mL single-necked flask. Add DIPEA (130 mg, 1 mmol) under ice-water bath conditions and allow the mixture to react at room temperature for 2 h. After the reaction is complete as monitored by HPLC, the reaction solution is purified by preparative HPLC to obtain preparative solutions of compounds 79b-1 and 79b-2. Lyophilize the preparative solutions to obtain compound 79b-1 (124 mg). LC-MS: [M+H] + =1743.0; compound 79b-1 (122 mg) was obtained; LC-MS: [M+H] + =1743.0.

[0927] Step 3: Synthesis of Compound 79

[0928]

[0929] Compound 79b-1 (100 mg, 0.057 mmol), zinc bromide (258 mg, 1.15 mmol), and 10 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain solid compound 79 (30 mg). LC-MS: [M+H] + =1586.9.

[0930] Step 4: Synthesis of Compound 80

[0931]

[0932] Compound 79b-2 (100 mg, 0.057 mmol), zinc bromide (258 mg, 1.15 mmol), and 10 mL of nitromethane were added to a 25 mL single-necked flask, and the mixture was reacted at 40 °C for 1 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to give solid compound 80 (33 mg). LC-MS: [M+H] + =1587.0.

[0933] Example 88 Synthesis of Compound 81

[0934]

[0935] By replacing compound M11 with compound M11' and following the synthetic route of Example 87, compound 81 (24 mg) was obtained; LC-MS: [M+H] + =1586.9.

[0936] Example 89 Synthesis of Compound 82

[0937]

[0938] By replacing compound M11 with compound M11' and following the synthetic route of Example 87, compound 82 (29 mg) was obtained; LC-MS: [M+H] + =1586.9.

[0939] Example 90: Antibody Expression and Purification

[0940] Expand the culture of Expi293 suspension cells. One day before transfection, cells were seeded at a specific density on OPM-293CD05Medium and cultured overnight in a cell culture shaker at 37°C, 5% CO2, and 120 rpm. The following day, transfection with the antibody expression plasmid was performed using PEI-MAX. OPM-293ProFeed was added on the first and third days after transfection, and the supernatant was collected by centrifugation on the sixth day post-transfection.

[0941] The supernatant was initially purified using a Protein A affinity chromatography column, followed by fine purification using hydroxyapatite (CHT) to remove impurities such as polymers, yielding antibody 18M1. The sequences of its heavy and light chain variable regions are shown below:

[0942] 18M1

[0943] SEQ ID No:1 (Heavy chain variable region)

[0944] QVQLVESGGGLVQPGGSLRLSCAASGFDFNTYYMTWVRQAPGKGLEWVGVIYASGGTYYATWAKGRFTISKSKNTMYLQMNSLRAEDTAVYYCARAYPDSGDGLDIWGQGTLVTVSS

[0945] SEQ ID No:2 (Heavy chain variable region CDR1)

[0946] TYYMT

[0947] SEQ ID No:3 (Heavy chain variable region CDR2)

[0948] VIYASGGTYYATWAKG

[0949] SEQ ID No:4 (Heavy chain variable region CDR3)

[0950] AYPDSGDGLDI

[0951] SEQ ID No:5 (Heavy chain)

[0952] QVQLVESGGGLVQPGGSLRLSCAASGFDFNTYYMTWVRQAPGKGLEWVGVIYASGGTYYATWAKGRFTISKSKNTMYLQMNSLRAEDTAVYYCARAYPDSGDGLDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0953] SEQ ID No:6 (Heavy chain constant region)

[0954] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0955] SEQ ID No:7 (Light chain variable region)

[0956] DVVMTQSPSSVSASVGDRVTITCQASEDISGWLAWYQQKPGQAPKLLIYWASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSTFYGTSDVAAFGGGTKVEIK

[0957] SEQ ID No:8 (Light chain variable region CDR1)

[0958] QASEDISGWLA

[0959] SEQ ID No:9 (Light chain variable region CDR2)

[0960] WASNLAS

[0961] SEQ ID No:10 (Light chain variable region CDR3)

[0962] QSTFYGTSDVAA

[0963] SEQ ID No:11 (Light Chain)

[0964] DVVMTQSPSSVSASVGDRVTITCQASEDISGWLAWYQQKPGQAPKLLIYWASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSTFYGTSDVAAFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0965] SEQ ID No:12 (Light chain constant region)

[0966] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0967] SEQ ID No:13 (Heavy chain variable region -- nucleic acid coding sequence)

[0968] CAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCTTCGACTTCAACACCTACTACATGACCTGGGTGAGGCAGGCCCCGGCAAGGGCCTGGAGTGGGTGGGCGTGATCTACGCCTCCGGCGGCACCTACT ACGCCACCTGGGCCAAGGGCAGGTTCACCATCTCCAAGTCCAAGAACACCATGTACCTGCGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGCCTACCCCGACAGCGGCGACGGCCTGGACATCTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCC

[0969] SEQ ID No:14 (Heavy chain CDR1 -- Nucleic acid coding sequence)

[0970] ACCTACTACATGACC

[0971] SEQ ID No:15 (Heavy chain CDR2 -- Nucleic acid coding sequence)

[0972] GTGATCTACGCCTCCGGCGGCACCTACTACGCCACCTGGGCCAAGGGC

[0973] SEQ ID No:16 (Heavy chain CDR3 -- Nucleic acid coding sequence)

[0974] GCCTACCCCGACAGCGGCCGACGGCCTGGACATC

[0975] SEQ ID No:17 (Heavy chain -- Nucleic acid coding sequence)

[0976]

[0977] SEQ ID No:18 (Heavy chain constant region -- nucleic acid coding sequence)

[0978] GCCTCTACCAAGGGCCCCAGCGTGTTTCCTCTGGCTCCCTCTTCTAAGTCCACCTCGGGCGGCACAGCCGCGCTGGGTTGCCTGGTGAAGGACTACTTCCCTGAACCCGTCACCGTGTCCTGGAACTCCGGCGCCTTAACATCTGGCGTGCACACCTTTCCTGCCGTCCTGCAGAGCTCCGGACTGTACTCTCTTAGTAGCGTGGTGACAGTGCCTAGCTCATCTCTGGGCACCCAGACCTACATCTGCAACGTCAACCACAAGCCCTCTAACACCAAGGTGGACAAGCGGGTTGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCACCTTGCCCCGCCCCTGAACTGCTGGGCGGCCCTTCCGTTTTTCTGTTCCCCCCTAAGCCTAAGGACACCCTGATGATCTCTCGGACACCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCATGAAGATCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCAAGAGAAGAACAGTACAACTCCACTTACAGAGTTGTGTCCGTGCTGACCGTGCTGCATCAAGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTGAGTAACAAGGCTCTGCCTGCCCCTATCGAGAAAACCATCTCTAAGGCTAAGGGACAACCTAGAGAGCCTCAGGTGTACACCCTGCCTCCTTCCCGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAAGGCTTCTACCCCTCTGACATCGCCGTGGAATGGGAGTCGAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGATCTTTCTTCCTGTACTCCAAGCTGACCGTCGACAAGTCTAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCTCTGCACAATCACTACACCCAGAAGTCCCTGTCCCTATCGCCAGGCTGA

[0979] SEQ ID No:19 (Light chain variable region -- nucleic acid coding sequence)

[0980] GACGTGGTGATGACCCAGTCCCCCTCCTCCGTGTCCGCCTCCGTGGGCGACAGGGTGACCATCACCTGCCAGGCCTCCGAGGACATCTCCGGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAAGCTGCTGATCTACTGGGCCTCCAACCTGGCC TCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGTCCACCTTTCTACGGCACCTCCGACGTGGCCGCCTTCGGCGGCGGCACCAAGGTGGAGATCAAG

[0981] SEQ ID No:20 (Light chain variable region CDR1 -- Nucleic acid coding sequence)

[0982] CAGGCCTCCGAGGACATCTCCGGCTGGCTGGCC

[0983] SEQ ID No:21 (Light chain variable region CDR2 -- Nucleic acid coding sequence)

[0984] TGGGCCTCCAACCTGGCCTCC

[0985] SEQ ID No:22 (Light chain variable region CDR3 -- Nucleic acid coding sequence)

[0986] CAGTCCACCTTTCTACGGCACCTCCGACGTGGCCGCC

[0987] SEQ ID No:23 (Light chain -- Nucleic acid coding sequence)

[0988] GACGTGGTGATGACCCAGTCCCCCTCCTCCGTGTCCGCCTCCGTGGGCGACAGGGTGACCATCACCTGCCAGGCCTCCGAGGACATCTCCGGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAAGCTGCTGATCTACTGGGCCTCCAACCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGTCCACCTTCTACGGCACCTCCGACGTGGCCGCCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCTCCTAGCGTGTTCATCTTCCCCCCTTCCGACGAACAGCTGAAGTCCGGAACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTAGAGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAATCGGGCAATTCCCAAGAGTCAGTCACTGAGCAGGACTCCAAGGATTCCACCTACTCCCTGTCTTCTACCCTGACCTTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCATCAGGGCCTGTCCTCTCCTGTGACAAAGAGCTTCAACCGGGGCGAATGTTGA

[0989] SEQ ID No:24 (Light chain constant region - nucleic acid coding sequence)

[0990] CGGACCGTGGCCGCTCCTAGCGTGTTCATCTTCCCCCCTCCGACGAACAGCTGAAGTCCGGAACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTAGAGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAATCGGGCAATTCCCAAGAG TCAGTCACTGAGCAGGACTCCAAGGATTCCACCTACTCCCTGTCTTCTACCCTGACCTTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCATCAGGGCCTGTCCTCTCCTGTGACAAAGAGCTTCAACCGGGGCGAATGTTGA

[0991] Example 91 Preparation of antibody-drug conjugates

[0992] Using an ultrafiltration centrifuge tube, transfer the antibody to acetate buffer and adjust the concentration to 5 mg / mL. Add 25 times the molar amount of TCEP (Total Chloride-Effective Fluid) and react at room temperature for 2 hours to open the disulfide bonds between the antibody chains. Add 20 times the molar amount of linker-drug compound (linker-payload) and react at room temperature for 2 hours. After the reaction, transfer the antibody to histidine buffer again using an ultrafiltration centrifuge tube, simultaneously removing any unconjugated linker-payload, thus obtaining the corresponding antibody-drug conjugate (ADC) sample.

[0993] In Examples 92 to 197 below, the compounds prepared in the examples of the present invention were conjugated to antibody 18M1 according to the preparation method to prepare ADC-1 to ADC-106, wherein the sum of n1, n2, and n3 represents the drug-antibody conjugation ratio (DAR) of the ADC.

[0994] Additionally, it should be noted that during the preparation of the ADC of the present invention, the compound (linker-drug) therein, when coupled with the antibody, readily reacts with water molecules under easily hydrolyzed conditions, undergoing a hydrolytic ring-opening reaction, with the hydrolysis site being the maleimide portion. When the antibody is coupled with multiple linker drugs, depending on the degree of hydrolysis, the following situations may occur: (1) Maleimide does not hydrolyze, that is, all maleimides are in closed-ring form. (2) Maleimide is not completely hydrolyzed, that is, some maleimide is in the closed ring form. The other portion of maleimide is in an open-ring form. (3) Maleimide is completely hydrolyzed, that is, all maleimide is in the open ring form. Therefore, in the ADCs prepared in Examples 92 to 197 below, n1, n2, and n3 are not simultaneously 0, and the sum of n1, n2, and n3 represents DAR, the specific value of which was measured in Example 199. Meanwhile, those skilled in the art will understand that, as mentioned above, the number of linker drugs conjugated to each antibody can be the same or different. Therefore, the average number of linker drugs conjugated to each antibody can be an integer or a decimal. Correspondingly, when the linker drug is conjugated with the antibody, the maleimide portion can undergo hydrolysis under easily hydrolyzed conditions. The degree of hydrolysis can be the same or different; therefore, n1, n2, and n3 can be integers or decimals.

[0995] Example 92

[0996]

[0997] Following the general conjugation method of Example 91, compound 2 was conjugated to antibody 18M1 to prepare ADC-1.

[0998] Example 93

[0999]

[1000] Following the general conjugation method of Example 91, compound 1 was conjugated to antibody 18M1 to prepare ADC-2.

[1001] Example 94

[1002]

[1003] Following the general conjugation method of Example 91, compound 4 was conjugated to antibody 18M1 to prepare ADC-3.

[1004] Example 95

[1005]

[1006] Following the general conjugation method of Example 91, compound 3 was conjugated to antibody 18M1 to prepare ADC-4.

[1007] Example 96

[1008]

[1009] Following the general conjugation method of Example 91, compound 5B was conjugated to antibody 18M1 to prepare ADC-5.

[1010] Example 97

[1011]

[1012] Following the general conjugation method of Example 91, compound 5A was conjugated to antibody 18M1 to prepare ADC-6.

[1013] Example 98

[1014]

[1015] Following the general conjugation method of Example 91, compound 6B was conjugated to antibody 18M1 to prepare ADC-7.

[1016] Example 99

[1017]

[1018] Following the general conjugation method of Example 91, compound 6A was conjugated to antibody 18M1 to prepare ADC-8.

[1019] Example 100

[1020]

[1021] Following the general conjugation method of Example 91, compound 8B was conjugated to antibody 18M1 to prepare ADC-9.

[1022] Example 101

[1023]

[1024] Following the general conjugation method of Example 91, compound 7B was conjugated to antibody 18M1 to prepare ADC-10.

[1025] Example 102

[1026]

[1027] Following the general conjugation method of Example 91, compound 8A was conjugated to antibody 18M1 to prepare ADC-11.

[1028] Example 103

[1029]

[1030] Following the general conjugation method of Example 91, compound 7A was conjugated to antibody 18M1 to prepare ADC-12.

[1031] Example 104

[1032]

[1033] Following the general conjugation method of Example 91, compound 9B was conjugated to antibody 18M1 to prepare ADC-13.

[1034] Example 105

[1035]

[1036] Following the general conjugation method of Example 91, compound 9A was conjugated to antibody 18M1 to prepare ADC-14.

[1037] Example 106

[1038]

[1039] Following the general conjugation method of Example 91, compound 10B was conjugated to antibody 18M1 to prepare ADC-15.

[1040] Example 107

[1041]

[1042] Following the general conjugation method of Example 91, compound 10A was conjugated to antibody 18M1 to prepare ADC-16.

[1043] Example 108

[1044]

[1045] Following the general conjugation method of Example 91, compound 11B was conjugated to antibody 18M1 to prepare ADC-17.

[1046] Example 109

[1047]

[1048] Following the general conjugation method of Example 91, compound 11A was conjugated to antibody 18M1 to prepare ADC-18.

[1049] Example 110

[1050]

[1051] Following the general conjugation method of Example 91, compound 12B was conjugated to antibody 18M1 to prepare ADC-19.

[1052] Example 111

[1053]

[1054] Following the general conjugation method of Example 91, compound 12A was conjugated to antibody 18M1 to prepare ADC-20.

[1055] Example 112

[1056]

[1057] Following the general conjugation method of Example 91, compound 13B was conjugated to antibody 18M1 to prepare ADC-21.

[1058] Example 113

[1059]

[1060] Following the general conjugation method of Example 91, compound 13A was conjugated to antibody 18M1 to prepare ADC-22.

[1061] Example 114

[1062]

[1063] Following the general conjugation method of Example 91, compound 14B was conjugated to antibody 18M1 to prepare ADC-23.

[1064] Example 115

[1065]

[1066] Following the general conjugation method of Example 91, compound 14A was conjugated to antibody 18M1 to prepare ADC-24.

[1067] Example 116

[1068]

[1069] Following the general conjugation method of Example 91, compound 16A was conjugated to antibody 18M1 to prepare ADC-25.

[1070] Example 117

[1071]

[1072] Following the general conjugation method of Example 91, compound 15A was conjugated to antibody 18M1 to prepare ADC-26.

[1073] Example 118

[1074]

[1075] Following the general conjugation method of Example 91, compound 16B was conjugated to antibody 18M1 to prepare ADC-27.

[1076] Example 119

[1077]

[1078] Following the general conjugation method of Example 91, compound 15B was conjugated to antibody 18M1 to prepare ADC-28.

[1079] Example 120

[1080]

[1081] Following the general conjugation method of Example 91, compound 18A was conjugated to antibody 18M1 to prepare ADC-29.

[1082] Example 121

[1083]

[1084] Following the general conjugation method of Example 91, compound 17A was conjugated to antibody 18M1 to prepare ADC-30.

[1085] Example 122

[1086]

[1087] Following the general conjugation method of Example 91, compound 18B was conjugated to antibody 18M1 to prepare ADC-31.

[1088] Example 123

[1089]

[1090] Following the general conjugation method of Example 91, compound 17B was conjugated to antibody 18M1 to prepare ADC-32.

[1091] Example 124

[1092]

[1093] Following the general conjugation method of Example 91, compound 20A was conjugated to antibody 18M1 to prepare ADC-33.

[1094] Example 125

[1095]

[1096] Following the general conjugation method of Example 91, compound 19B was conjugated to antibody 18M1 to prepare ADC-34.

[1097] Example 126

[1098]

[1099] Following the general conjugation method of Example 91, compound 20B was conjugated to antibody 18M1 to prepare ADC-35.

[1100] Example 127

[1101]

[1102] Following the general conjugation method of Example 91, compound 19A was conjugated to antibody 18M1 to prepare ADC-36.

[1103] Example 128

[1104]

[1105] Following the general conjugation method of Example 91, compound 21 was conjugated to antibody 18M1 to prepare ADC-37.

[1106] Example 129

[1107]

[1108] Following the general conjugation method of Example 91, compound 22 was conjugated to antibody 18M1 to prepare ADC-38.

[1109] Example 130

[1110]

[1111] Following the general conjugation method of Example 91, compound 23 was conjugated to antibody 18M1 to prepare ADC-39.

[1112] Example 131

[1113]

[1114] Following the general conjugation method of Example 91, compound 24 was conjugated to antibody 18M1 to prepare ADC-40.

[1115] Example 132

[1116]

[1117] Following the general conjugation method of Example 91, compound 25 was conjugated to antibody 18M1 to prepare ADC-41.

[1118] Example 133

[1119]

[1120] Following the general conjugation method of Example 91, compound 26 was conjugated to antibody 18M1 to prepare ADC-42.

[1121] Example 134

[1122]

[1123] Following the general conjugation method of Example 91, compound 27 was conjugated to antibody 18M1 to prepare ADC-43.

[1124] Example 135

[1125]

[1126] Following the general conjugation method of Example 91, compound 28 was conjugated to antibody 18M1 to prepare ADC-44.

[1127] Example 136

[1128]

[1129] Following the general conjugation method of Example 91, compound 29 was conjugated to antibody 18M1 to prepare ADC-45.

[1130] Example 137

[1131]

[1132] Following the general conjugation method of Example 91, compound 30 was conjugated to antibody 18M1 to prepare ADC-46.

[1133] Example 138

[1134]

[1135] Following the general conjugation method of Example 91, compound 31 was conjugated to antibody 18M1 to prepare ADC-47.

[1136] Example 139

[1137]

[1138] Following the general conjugation method of Example 91, compound 32 was conjugated to antibody 18M1 to prepare ADC-48.

[1139] Example 140

[1140]

[1141] Following the general conjugation method of Example 91, compound 34 was conjugated to antibody 18M1 to prepare ADC-49.

[1142] Example 141

[1143]

[1144] Following the general conjugation method of Example 91, compound 33 was conjugated to antibody 18M1 to prepare ADC-50.

[1145] Example 142

[1146]

[1147] Following the general conjugation method of Example 91, compound 35 was conjugated to antibody 18M1 to prepare ADC-51.

[1148] Example 143

[1149]

[1150] Following the general conjugation method of Example 91, compound 36 was conjugated to antibody 18M1 to prepare ADC-52.

[1151] Example 144

[1152]

[1153] Following the general conjugation method of Example 91, compound 37 was conjugated to antibody 18M1 to prepare ADC-53.

[1154] Example 145

[1155]

[1156] Following the general conjugation method of Example 91, compound 38 was conjugated to antibody 18M1 to prepare ADC-54.

[1157] Example 146

[1158]

[1159] Following the general conjugation method of Example 91, compound 39 was conjugated to antibody 18M1 to prepare ADC-55.

[1160] Example 147

[1161]

[1162] Following the general conjugation method of Example 91, compound 40 was conjugated to antibody 18M1 to prepare ADC-56.

[1163] Example 148

[1164]

[1165] Following the general conjugation method of Example 91, compound 42 was conjugated to antibody 18M1 to prepare ADC-57.

[1166] Example 149

[1167]

[1168] Following the general conjugation method of Example 91, compound 41 was conjugated to antibody 18M1 to prepare ADC-58.

[1169] Example 150

[1170]

[1171] Following the general conjugation method of Example 91, compound 43 was conjugated to antibody 18M1 to prepare ADC-59.

[1172] Example 151

[1173]

[1174] Following the general conjugation method of Example 91, compound 44 was conjugated to antibody 18M1 to prepare ADC-60.

[1175] Example 152

[1176]

[1177] Following the general conjugation method of Example 91, compound 47 was conjugated to antibody 18M1 to prepare ADC-61.

[1178] Example 153

[1179]

[1180] Following the general conjugation method of Example 91, compound 46 was conjugated to antibody 18M1 to prepare ADC-62.

[1181] Example 154

[1182]

[1183] Following the general conjugation method of Example 91, compound 48B was conjugated to antibody 18M1 to prepare ADC-63.

[1184] Example 155

[1185]

[1186] Following the general conjugation method of Example 91, compound 48A was conjugated to antibody 18M1 to prepare ADC-64.

[1187] Example 156

[1188]

[1189] Following the general conjugation method of Example 91, compound 49B was conjugated to antibody 18M1 to prepare ADC-65.

[1190] Example 157

[1191]

[1192] Following the general conjugation method of Example 91, compound 49A was conjugated to antibody 18M1 to prepare ADC-66.

[1193] Example 158

[1194]

[1195] Following the general conjugation method of Example 91, compound 51B was conjugated to antibody 18M1 to prepare ADC-67.

[1196] Example 159

[1197]

[1198] Following the general conjugation method of Example 91, compound 50B was conjugated to antibody 18M1 to prepare ADC-68.

[1199] Example 160

[1200]

[1201] Following the general conjugation method of Example 91, compound 51A was conjugated to antibody 18M1 to prepare ADC-69.

[1202] Example 161

[1203]

[1204] Following the general conjugation method of Example 91, compound 50A was conjugated to antibody 18M1 to prepare ADC-70.

[1205] Example 162

[1206]

[1207] Following the general conjugation method of Example 91, compound 53B was conjugated to antibody 18M1 to prepare ADC-71.

[1208] Example 163

[1209]

[1210] Following the general conjugation method of Example 91, compound 53A was conjugated to antibody 18M1 to prepare ADC-72.

[1211] Example 164

[1212]

[1213] Following the general conjugation method of Example 91, compound 54A was conjugated to antibody 18M1 to prepare ADC-73.

[1214] Example 165

[1215]

[1216] Following the general conjugation method of Example 91, compound 54B was conjugated to antibody 18M1 to prepare ADC-74.

[1217] Example 166

[1218]

[1219] Following the general conjugation method of Example 91, compound 55B was conjugated to antibody 18M1 to prepare ADC-75.

[1220] Example 167

[1221]

[1222] Following the general conjugation method of Example 91, compound 55A was conjugated to antibody 18M1 to prepare ADC-76.

[1223] Example 168

[1224]

[1225] Following the general conjugation method of Example 91, compound 57 was conjugated to antibody 18M1 to prepare ADC-77.

[1226] Example 169

[1227]

[1228] Following the general conjugation method of Example 91, the compound was conjugated to antibody 18M1 to prepare ADC-78.

[1229] Example 170

[1230]

[1231] Following the general conjugation method of Example 91, compound 60 was conjugated to antibody 18M1 to prepare ADC-79.

[1232] Example 171

[1233]

[1234] Following the general conjugation method of Example 91, compound 59 was conjugated to antibody 18M1 to prepare ADC-80.

[1235] Example 172

[1236]

[1237] Following the general conjugation method of Example 91, compound 62 was conjugated to antibody 18M1 to prepare ADC-81.

[1238] Example 173

[1239]

[1240] Following the general conjugation method of Example 91, compound 61 was conjugated to antibody 18M1 to prepare ADC-82.

[1241] Example 174

[1242]

[1243] Following the general conjugation method of Example 91, compound 64 was conjugated to antibody 18M1 to prepare ADC-83.

[1244] Example 175

[1245]

[1246] Following the general conjugation method of Example 91, compound 63 was conjugated to antibody 18M1 to prepare ADC-84.

[1247] Example 176

[1248]

[1249] Following the general conjugation method of Example 91, compound 66B was conjugated to antibody 18M1 to prepare ADC-85.

[1250] Example 177

[1251]

[1252] Following the general conjugation method of Example 91, compound 65B was conjugated to antibody 18M1 to prepare ADC-86.

[1253] Example 178

[1254]

[1255] Following the general conjugation method of Example 91, compound 66A was conjugated to antibody 18M1 to prepare ADC-87.

[1256] Example 179

[1257]

[1258] Following the general conjugation method of Example 91, compound 65A was conjugated to antibody 18M1 to prepare ADC-88.

[1259] Example 180

[1260]

[1261] Following the general conjugation method of Example 91, compound 68B was conjugated to antibody 18M1 to prepare ADC-89.

[1262] Example 181

[1263]

[1264] Following the general conjugation method of Example 91, compound 68A was conjugated to antibody 18M1 to prepare ADC-90.

[1265] Example 182

[1266]

[1267] Following the general conjugation method of Example 91, compound 69A was conjugated to antibody 18M1 to prepare ADC-91.

[1268] Example 183

[1269]

[1270] Following the general conjugation method of Example 91, compound 69B was conjugated to antibody 18M1 to prepare ADC-92.

[1271] Example 184

[1272]

[1273] Following the general conjugation method of Example 91, compound 70B was conjugated to antibody 18M1 to prepare ADC-93.

[1274] Example 185

[1275]

[1276] Following the general conjugation method of Example 91, compound 70A was conjugated to antibody 18M1 to prepare ADC-94.

[1277] Example 186

[1278]

[1279] Following the general conjugation method of Example 91, compound 72 was conjugated to antibody 18M1 to prepare ADC-95.

[1280] Example 187

[1281]

[1282] Following the general conjugation method of Example 91, compound 71 was conjugated to antibody 18M1 to prepare ADC-96.

[1283] Example 188

[1284]

[1285] Following the general conjugation method of Example 91, compound 74 was conjugated to antibody 18M1 to prepare ADC-97.

[1286] Example 189

[1287]

[1288] Following the general conjugation method of Example 91, compound 73 was conjugated to antibody 18M1 to prepare ADC-98.

[1289] Example 190

[1290]

[1291] Following the general conjugation method of Example 91, compound 76 was conjugated to antibody 18M1 to prepare ADC-99.

[1292] Example 191

[1293]

[1294] Following the general conjugation method of Example 91, compound 77 was conjugated to antibody 18M1 to prepare ADC-100.

[1295] Example 192

[1296]

[1297] Following the general conjugation method of Example 91, compound 75 was conjugated to antibody 18M1 to prepare ADC-101.

[1298] Example 193

[1299]

[1300] Following the general conjugation method of Example 91, compound 78 was conjugated to antibody 18M1 to prepare ADC-102.

[1301] Example 194

[1302]

[1303] Following the general conjugation method of Example 91, compound 82 was conjugated to antibody 18M1 to prepare ADC-103.

[1304] Example 195

[1305]

[1306] Following the general conjugation method of Example 91, compound 81 was conjugated to antibody 18M1 to prepare ADC-104.

[1307] Example 196

[1308]

[1309] Following the general conjugation method of Example 91, compound 79 was conjugated to antibody 18M1 to prepare ADC-105.

[1310] Example 197

[1311]

[1312] Following the general conjugation method of Example 91, compound 80 was conjugated to antibody 18M1 to prepare ADC-106.

[1313] Example 198

[1314] The gene encoding the following light and heavy chain variable regions (sequence from WO2024012523A1) was constructed into an expression vector containing the 18M1 constant region, and an antibody was prepared according to the general antibody preparation method of Example 90. Compound 5A was conjugated to the prepared antibody according to the general conjugation method of Example 91 to prepare ADC-107, which has the same quality and stability as ADC-6.

[1315] SEQ ID No:25(ADC-107-VH)

[1316] EVQLVQSGAEVKKPGASVKVSCKASGFTFTDYYMHWVRQAPGQSLEWMGLVYPYNGDISYAQKFQGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARQGNYVNNAIDYWGQGTLVTVSS

[1317] SEQ ID No:26(ADC-107-VL)

[1318] DIQMTQSPSSSLSASVGDRVTITCKASQNVGNIIAWYQQKPGKSPKALIYLASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYFCQQYSDSPYTFGQGTKVEIK

[1319] Example 199

[1320] The gene encoding the following light and heavy chain variable regions (sequence from WO2024012523A1) was constructed into an expression vector containing the constant region 18M1, and an antibody was prepared according to the general antibody preparation method of Example 90. Compound 5A was conjugated to the prepared antibody according to the general conjugation method of Example 91 to prepare ADC-108 with equivalent quality and stability to ADC-6.

[1321] SEQ ID No:27(ADC-108-VH)

[1322] EVQLVQSGAEVKKPGASVKVSCKASGFTFTDYYMHWVRQAPGQSLEWMGLVYPYSGDISYAQKFQGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARQGNYVNNAIDYWGQGTLVTVSS

[1323] SEQ ID No:28(ADC-108-VL)

[1324] DIQMTQSPSSSLSASVGDRVTITCKASQNVGNIIAWYQQKPGKSPKALIYLASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYFCQQYSDSPYTFGQGTKVEIK

[1325] Example 200: Determination of monomer yield of ADC

[1326] Monomer content was determined using the SEC-HPLC method.

[1327] Column: Biocore SEC-300 5μm, 4.6×300mm

[1328] Manufacturer: NanoChrom, Part Number: B213-050030-04630S

[1329] Mobile phase: 50 mM PB + 300 mM NaCl + 200 mM Arg + 5% IPA, pH = 6.5

[1330] See Table 1 for specific parameters.

[1331] Table 1: Method Parameters

[1332] parameter set up Flow rate 0.3 mL / min wavelength 280nm Column temperature 30℃ Sample tray temperature room temperature Injection volume 20ug Maximum pressure 150 bar / 15 MPa / 2175 PSI gradient isometry Runtime 20 minutes

[1333] Table 2. Monomer content data of ligand-drug conjugates (ADCs) disclosed in this invention.

[1334] Molecular name batch Aggregate % Monomer percentage degradation% ADC-6 C230714D101A 1.38 97.5 1.12

[1335] The test results are shown in Table 2 and Figure 1 The results show that the ADCs disclosed in the embodiments of the present invention, such as ADC-1 to ADC-106, especially ADC-6, have the characteristics of low degradation rate, low aggregation rate, and excellent properties of high monomer content.

[1336] Example 201: Determination of the drug-antibody conjugate ratio (DAR) of ADCs

[1337] The drug antibody conjugation ratio (DAR) was determined using the RP-HPLC method.

[1338] Column name: Proteomix RP-1000 4.6×100mm 5μm 1000A

[1339] Manufacturer: Sepax Part Number: 465950-4610

[1340] See Table 3 for specific parameters.

[1341] Table 3: Method Parameters

[1342]

[1343] Table 4: Detailed Data on Drug-Antibody Conjugate Ratio (DAR) for ADCs

[1344] Sample Name batch number RP-DAR ADC-6 C230714D101A 7.47

[1345] The test results are shown in Table 4 and Figure 2 The results show that the ADCs disclosed in the embodiments of the present invention, such as ADC-1 to ADC-106, especially ADC-6, have the excellent property of high DAR value, and can significantly increase the drug concentration at the target site at the same dose of ADC drug.

[1346] Example 202

[1347] ADC plasma stability data:

[1348] A mixture of ADC and IgG-free plasma was prepared to achieve a final ADC concentration of 0.6 mg / mL. This mixture was incubated in a water bath at 37°C for 0, 3, and 7 days. A control group without plasma incubation was also included. After incubation, the drug-antibody ratio (DAR) was measured after purification and extraction to reflect the stability of the ADC in plasma. The results are shown in the table below:

[1349]

[1350] The results show that the ADCs disclosed in the embodiments of the present invention, such as ADC-1 to ADC-106, especially ADC-6, exhibit excellent plasma stability.

[1351] Example 203 In vitro efficacy detection 1

[1352] In this invention, various human tumor cell lines, including NCI-H446 DLL3#B5C, A375 DLL3#B2G, NCI-H1339 DLL3#C3G, NCI-H2286 DLL3#B6G, SHP-77#Wild Type, and NCI-H460, are used as in vitro cell models to evaluate the cytotoxic effects of bioactive molecules such as antibodies or antibody-drug conjugates (ADCs) on in vitro cells.

[1353] A certain number of tumor cells were seeded into 96-well culture plates. After the cells adhered, serially diluted test samples were added to the wells. The initial concentration of the antibody or ADC was 500 nM, and the dilution factor was 7-fold. Each detection concentration was tested in triplicate, for a total of 8 detection concentrations. The plates were incubated at 37°C and 5% CO2 for 5 days. Cell viability was assessed using the Luminescent Cell Viability Assay (CTG). Cell viability was calculated as follows: Cell viability = (Experimental group - Blank) / (Negative control group - Blank) × 100%. Using Graph Pad Prism 9.0 statistical software, with the X-axis representing the molar concentration of the test sample (nM) and the Y-axis representing cell viability (%), a four-parameter model was used to fit a curve and calculate the half-maximal inhibitory concentration (IC50) of the test sample. 50 To evaluate the in vitro tumor cell killing activity of the test samples.

[1354] Table 5. IC50 of the test samples in in vitro cell models 50 (nM) Statistical Table

[1355]

[1356] The test results are shown in Table 5 and Figures 3 to 8 .

[1357] Test results show that in the above-mentioned in vitro cell model, the bioactive molecule 18M1 of the present invention has no tumor cell killing activity; the ADCs disclosed in the embodiments of the present invention, such as ADC-1 to ADC-106, especially ADC-6, have tumor cell killing activity, particularly against NCI-H446 DLL3#B5C, A375DLL3#B2G, NCI-H1339 DLL3#C3G, and NCI-H2286DLL3#B6G. 50 It exhibits excellent lethality.

[1358] Example 204 In vitro efficacy detection 2

[1359] In this invention, various human tumor cell lines, including A375 DLL3#B2G, NCI-H1339 DLL3#C3G, NCI-H2286 DLL3#B6G, SHP-77, and NCI-H460, are used as in vitro cell models to evaluate the cytotoxic effects of bioactive molecules such as antibodies or antibody-drug conjugates (ADCs) on in vitro cells.

[1360] A certain number of tumor cells were seeded into 96-well culture plates. After cell attachment, serially diluted test samples were added to the wells. The initial concentration of ADC was 500 nM, with a dilution factor of 7-fold. Each detection concentration was tested in triplicate, for a total of 8 detection concentrations. The plates were incubated at 37°C and 5% CO2 for 5 days. Cell viability was detected using an MTS (Promega, G3581) assay. Cell viability was calculated as (experimental group - blank) / (negative control group - blank) × 100%. The half-maximal inhibitory concentration (IC50) of the test sample was calculated using Graph PadPrism 10.0 statistical software, with the X-axis representing the molar concentration of the test sample (nM) and the Y-axis representing cell viability (%). A four-parameter model was used to fit the curve, and the IC50 of the test sample was calculated. 50 To evaluate the in vitro tumor cell killing activity of the test samples.

[1361] Table 6. IC50 of the test samples in in vitro cell models 50 (nM) Statistical Table

[1362]

[1363] The test results are shown in Table 6 and Figures 9 to 13 .

[1364] The test results show that, in the above-mentioned in vitro cell model, the ADC-6 disclosed in this embodiment of the invention has a specific killing effect on DLL3-positive tumor cells, and the killing effect is superior to that of ADC-107 and ADC-108. No significant killing effect was observed in the tested ADCs on the DLL3-negative cell line (H460).

[1365] Example 205 In vivo efficacy detection 1

[1366] This invention established a mouse subcutaneous xenograft model of human small cell lung cancer cells SHP-77-DLL3#B7GNOD Scid with positive target expression to evaluate the in vivo efficacy of the ADCs disclosed in this invention, ADC-1 to ADC-106, especially ADC-6. (The text then repeats the process of using 5 × 10⁻⁶ cells.) 6One SHP-77-DLL3#B7G cell (0.1 mL / mouse) was subcutaneously injected into the right scapular region of 6-7 week old NOD Scid mice. The tumors were allowed to grow to an average size of 200 mm. 3 Six animals were randomly assigned to three groups: a vehicle control group, an 18M1 antibody control group (2 mg / kg), and an ADC-6 treatment group (1 mg / kg, 1.5 mg / kg, and 2 mg / kg), with six animals in each group starting treatment (D0). All animals received the drug via tail vein injection at a dose of 10 mL / kg body weight, once weekly for four weeks (QW×4).

[1367] On day 28 (D28) after administration to all groups, two-factor statistical analysis showed that the ADC-61 mg / kg, 1.5 mg / kg, and 2 mg / kg dose groups had significant tumor-suppressing effects compared to the solvent control group (P < 0.05) (see [link to relevant documentation]). Figure 14 Univariate statistical analysis of the mean tumor volume on day 21 (D21) after group administration showed that the tumor-suppressing effects of ADC-6 at doses of 1.5 mg / kg and 2 mg / kg were significantly better than those of the solvent control group (P<0.05) (see Table 7).

[1368] Table 7. Efficacy analysis of each group in the mouse subcutaneous xenograft model of human small cell lung cancer cells SHP-77-DLL3#B7G NOD Scid.

[1369]

[1370] Note: 1. Data is expressed as mean ± standard error;

[1371] 2. T / C%=T RTV / C RTV ×100%; TGI%=(1-T / C)×100%;

[1372] 3. The P value was obtained by comparing the tumor volume of the treatment group and the solvent control group on D21 (day 21 after drug administration).

[1373] Example 206 In vivo efficacy detection 2

[1374] This invention established a mouse subcutaneous xenograft model of human malignant melanoma cells A375-DLL3#B2GNOD Scid with positive target expression to evaluate the in vivo efficacy of the ADCs disclosed in this invention, ADC-1 to ADC-106, especially ADC-6. At 5 × 10⁻⁶... 6 One A375-DLL3#B2G cell (0.1 mL / mouse) was subcutaneously injected into the right scapular region of 6-7 week old NOD Scid mice. The tumors were allowed to grow to an average size of 170 mm.3 Six animals were randomly assigned to three groups: a vehicle control group, an 18M1 antibody control group (2 mg / kg), and an ADC-6 treatment group (1 mg / kg, 1.5 mg / kg, and 2 mg / kg), with six animals in each group starting treatment (D0). All animals received the drug via tail vein injection at a dose of 10 mL / kg body weight, once weekly for four weeks (QW×4).

[1375] On day 28 (D28) after administration to all groups, two-factor statistical analysis showed that the ADC-61 mg / kg, 1.5 mg / kg, and 2 mg / kg dose groups had significant tumor-suppressing effects compared to the solvent control group (P < 0.05) (see [link to relevant documentation]). Figure 15 Univariate statistical analysis of the mean tumor volume on day 28 (D28) after group administration showed that the tumor-suppressing effects of ADC-6 at doses of 1.5 mg / kg and 2 mg / kg were significantly better than those of the solvent control group (P<0.05) (see Table 8).

[1376] Table 8. Efficacy analysis of each group in the human malignant melanoma cell A375-DLL3#B2G NOD Scid mouse subcutaneous xenograft tumor model.

[1377]

[1378] Note: 1. Data is expressed as mean ± standard error;

[1379] 2. T / C%=T RTV / C RTV ×100%; TGI%=(1-T / C)×100%;

[1380] 3. The P value was obtained by comparing the tumor volume of the treatment group and the solvent control group on D28 (day 28 after drug administration).

[1381] Example 207 In vivo efficacy detection 3

[1382] This invention established a mouse subcutaneous xenograft model of human small cell lung cancer cells H446-DLL3#B5C NOD Scid with positive target expression to evaluate the in vivo efficacy of the ADCs disclosed in this invention, ADC-1 to ADC-106, especially ADC-6. At 5 × 10⁻⁶... 6 One H446-DLL3#B5C cell (0.1 mL / mouse) was subcutaneously injected into the right scapular region of 6-7 week old NOD Scid mice. The tumors were allowed to grow to an average size of 170 mm. 3Five animals were randomly assigned to either a solvent control group (Vehicle) or an ADC-6 treatment group (1 mg / kg, 1.5 mg / kg, and 2 mg / kg) and started medication (D0). All animals were administered the drug via tail vein injection at a dose of 10 mL / kg body weight once a week for 4 weeks (QW×4).

[1383] On day 28 (D28) after administration to all groups, two-factor statistical analysis showed that the ADC-61 mg / kg, 1.5 mg / kg, and 2 mg / kg dose groups had significant tumor-suppressing effects compared to the solvent control group (P < 0.05) (see [link to relevant documentation]). Figure 16 Univariate statistical analysis of the mean tumor volume on day 28 (D28) after group administration showed that the tumor inhibition effect of ADC-6 at doses of 1.5 mg / kg and 2 mg / kg was significantly better than that of the solvent control group, but there was no statistical significance (P>0.05) (see Table 9).

[1384] Table 9. Efficacy analysis of each group in the mouse subcutaneous xenograft model of human small cell lung cancer cells H446-DLL3#B5C NOD Scid.

[1385]

[1386] Note: 1. Data is expressed as mean ± standard error;

[1387] 2. T / C%=T RTV / C RTV ×100%; TGI%=(1-T / C)×100%;

[1388] 3. The P value was obtained by comparing the tumor volume of the treatment group and the solvent control group on D28 (day 28 after drug administration).

[1389] Example 208 In vivo efficacy detection 4

[1390] This invention established a mouse subcutaneous xenograft model of human malignant melanoma cells A375-DLL3#B2GNOD Scid with positive target expression to evaluate the in vivo efficacy of the ADC-6, ADC-107, and ADC-108 disclosed in this invention. (5 × 10⁻⁶ cells / mL) 6 One A375-DLL3#B2G cell (0.1 mL / mouse) was subcutaneously injected into the right scapular region of 6-7 week old NOD Scid mice. The tumors were allowed to grow to an average size of 200 mm. 3Five animals were randomly assigned to four treatment groups: a solvent control group (Vehicle), an ADC-6 treatment group (1 mg / kg, 2 mg / kg), an ADC-107 treatment group (1 mg / kg), and an ADC-108 treatment group (1 mg / kg), with five animals in each group. Drug administration began on day 0 (D0). All animals received the drug via tail vein injection at a dose of 10 mL / kg body weight, once weekly for three consecutive weeks (QW×3).

[1391] All groups were observed on day 21 (D21) after drug administration. Statistical analysis of the mean tumor volume on day 21 (D21) showed that the ADC-6 treatment group (1 mg / kg, 2 mg / kg) had a significantly better tumor-suppressing effect than the solvent control group (P<0.05) (see Table 11); the ADC-6 treatment group (1 mg / kg) had a significantly better tumor-suppressing effect than the ADC-107 treatment group (1 mg / kg) and the ADC-108 treatment group (1 mg / kg) (see Table 11). Figure 17 ).

[1392] Table 10. Efficacy analysis of each group in the human malignant melanoma cell A375-DLL3#B2G NOD Scid mouse subcutaneous xenograft model.

[1393]

[1394] Note: 1. Data is expressed as mean ± standard error;

[1395] 2. T / C%=T RTV / C RTV ×100%; TGI%=(1-T / C)×100%;

[1396] 3. The P value was obtained by comparing the tumor volume of the treatment group and the solvent control group on D21 (day 21 after drug administration).

Claims

1. A ligand-camptothecin derivative conjugate as shown in general formula I, its stereoisomer or a pharmaceutically acceptable salt or solvate thereof; in: Ab is an antibody or its antigen-binding fragment that targets human DLL3; L 11 L 12 L 13 Each is selected independently from: L2 has the structure shown in equation A below. Y is a support selected from C1-C6 alkylene, substituted C1-C6 alkylene, or C3-C8 cycloalkylene; Ac is a hydrophilic structural unit; the second carbon atom attached to Y has absolute chirality in either the R or S configuration; L3 may or may not exist; if present, L3 is selected from the PEG hydrophilic unit. Where o is an integer selected from 1 to 10; L4 is the enzyme digestion unit; L5 is a connection unit; X is -C(O)-CR a R b -(CR3R4) m -O-、-C(O)-CR a R b -(CR3R4) m -NH- or -C(O)-CR a R b -(CR3R4) m -S-, preferably -C(O)-CR a R b -(CR3R4) m -O-, where R a and R b Each is independently selected from hydrogen atoms, deuterium atoms, halogens, C1-C6 alkyl groups, deuterated C1-C6 alkyl groups, halogenated C1-C6 alkyl groups, C3-C8 cycloalkyl groups, C3-C8 cycloalkyl C1-C6 alkyl groups, C6-C10 aryl C1-C6 alkyl groups, C1-C6 alkoxy C1-C6 alkyl groups, 3-7 membered heterocyclic groups, substituted 3-7 membered heterocyclic groups, C6-C10 aryl groups, substituted C6-C10 aryl groups, 5-10 membered heteroaryl groups, substituted 5-10 membered heteroaryl groups; or, R a R b The carbon atoms attached to it constitute C3-C8 cycloalkyl, substituted C3-C8 cycloalkyl, 3-7 membered heterocyclic group or substituted 3-7 membered heterocyclic group; R3 and R4 are each independently a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a halo-C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C1-C6 alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxy C1-C6 alkyl group, a C3-C8 cycloalkyl group, a 3-7 membered heterocyclic group, or a substituted 3-7 membered heterocyclic group; or, R3, R4 and the carbon atoms attached thereto constitute C3-C8 cycloalkyl, substituted C3-C8 cycloalkyl, 3-7 membered heterocyclic group or substituted 3-7 membered heterocyclic group; m is selected from 0, 1, 2, 3 or 4; The first chiral carbon atom bonded to N has absolute chirality, either in the R or S configuration. R is selected from 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, 5-10 heteroaryl, substituted 5-10 heteroaryl; R1 is selected from 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, 3-7 membered heterocyclic group, substituted 3-7 membered heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, substituted 5-10 membered heteroaryl; R2 is selected from 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, 3-7 membered heterocyclic group, substituted 3-7 membered heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, substituted 5-10 membered heteroaryl; n1, n2, and n3 are each an independent integer or decimal from 0 to 10, and n1, n2, and n3 are not all 0 at the same time, and 1 ≤ n1 + n2 + n3 ≤ 10.

2. The conjugate of claim 1, its stereoisomer, or a pharmaceutically acceptable salt or solvate thereof, wherein L 11 L 12 L 13 Each is selected independently from: or L 11 L 12 L 13 Each is selected independently from:

3. The conjugate, its stereoisomer, or a pharmaceutically acceptable salt or solvate thereof as described in claim 1 or 2, wherein Ac has a structure as shown in formula B. in: Z represents a carboxyl group, a phosphoryloxy group, or -(OCH2CH2). i OCH3, where i is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably i is 1, 2, 3, 4, 5, 6, 7 or 8; Y' is a support connecting the amino group and Z, and is a C1-C6 alkylene group or a C1-C6 alkylene group substituted with a carboxyl group, preferably methylene, ethylene, carboxyl-substituted methylene, or carboxyl-substituted ethylene, more preferably methylene, ethylene, or carboxyl-substituted methylene; or Ac is a residue formed by removing a hydrogen atom from the amino terminus of glycine, alanine, leucine, isoleucine, valine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, cystine, arginine, histidine, methionine, asparagine, glutamine, threonine, aspartic acid, or glutamic acid. Preferably, Ac is: More preferably, Ac is:

4. The conjugate, its stereoisomer, or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-3, wherein L4 is a peptide residue consisting of an amino acid, wherein the amino acid is optionally substituted by one or more substituents selected from the group consisting of deuterium, 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 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 from glycine (G)-glycine (G)-phenylalanine (F)-glycine (G).

5. The conjugate, its stereoisomer, or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-4, wherein L5 is -NR5 (CR6R7). q - or chemical bond, wherein q is 0, 1, 2, 3, 4, 5 or 6, preferably 0, 1, 2 or 3, more preferably 0, 1 or 2, and more preferably 0 or 1; R5, R6, and R7 are each independently selected from hydrogen atoms, deuterium atoms, halogens, 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, 3-7 membered heterocyclic group, substituted 3-7 membered heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, and substituted 5-10 membered heteroaryl. Preferably, R5, R6 and R7 are each independently selected from hydrogen atoms or C1-C6 alkyl groups; More preferably, R5, R6 and R7 are each independently selected from hydrogen atoms or C1-C4 alkyl groups; More preferably, R5, R6 and R7 are each independently selected from hydrogen atoms, methyl, ethyl, n-propyl or n-butyl; More preferably, R5, R6 and R7 are each independently selected from hydrogen atoms.

6. The conjugate, its stereoisomer, or its pharmaceutically acceptable salt or solvate according to any one of claims 1-5, characterized in that the following features One or more: i)R a and R b Each is independently selected from hydrogen atom, C1-C6 alkyl, halo-C1-C6 alkyl, C3-C8 cycloalkyl-C1-C6 alkyl, or C6-C10 aryl-C1-C6 alkyl; or R a R b The carbon atoms to which they are attached form C3-C8 cycloalkyl groups; Preferably, R a R is a hydrogen atom or a C1-C4 alkyl group. b It is a hydrogen atom, a C1-C4 alkyl group, a halo-C1-C4 alkyl group, a C3-C6 cycloalkyl group, a C1-C4 alkyl group, or a phenyl C1-C4 alkyl group, or R a R b The carbon atoms to which they are attached form C3-C6 cycloalkyl groups; Preferably, R a For hydrogen atoms, methyl, ethyl, n-propyl or n-butyl, R b The following are possible meanings: hydrogen atom, methyl, ethyl, n-propyl, n-butyl, halomethyl, haloethyl, halopropyl or halobutyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclopropylethyl, cyclopentylethyl, cyclohexylethyl, benzyl, phenethyl or phenylpropyl, or R a R b The carbon atoms attached to it form cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl groups; Preferably, preferably, R a R is a hydrogen atom or a methyl group. b It can be hydrogen atom, methyl, ethyl, trifluoromethyl, cyclopropylmethyl, benzyl, or R a R b The carbon atoms attached to it form cyclopropyl, cyclobutyl, or cyclopentyl groups; Preferably, X is Preferably, the position indicated by the wavy line on the right is connected to L5. ii) R3 and R4 are each independently a hydrogen atom or a C1-C6 alkyl group; iii) m is 0, 1 or 2, more preferably 0 or 1; iv) R is a hydrogen atom or a C1-C6 alkyl group; preferably, R is a hydrogen atom or a C1-C4 alkyl group; preferably, R is a hydrogen atom, methyl, ethyl, n-propyl, or n-butyl; preferably, R is a hydrogen atom or a methyl group. v) R1 is a hydrogen atom or a C1-C6 alkyl group; preferably, R1 is a C1-C6 alkyl group; preferably, R1 is a C1-C4 alkyl group; preferably, R1 is methyl, ethyl, n-propyl or n-butyl; preferably, R1 is methyl. vi) R2 is a hydrogen atom, a halogen, or a C1-C6 alkyl group; preferably, R2 is a hydrogen atom, a halogen, or a C1-C4 alkyl group; preferably, R2 is a 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 a C1-C6 alkylene; preferably, Y is a C1-C4 alkylene; preferably, Y is methylene, ethylene, propylene, or butylene; preferably, Y is methylene; ix) n1, n2, and n3 are each independently any integer or decimal from 0 to 8, and n1, n2, and n3 are not all 0 at the same time, and 1 ≤ n1 + n2 + n3 ≤ 8; preferably, 5 ≤ n1 + n2 + n3 ≤ 10; preferably, 6 ≤ n1 + n2 + n3 ≤ 8; preferably, 7 ≤ n1 + n2 + n3 ≤ 8.

7. The conjugate, its stereoisomer, or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-6, wherein the antibody targeting human DLL3 or its antigen-binding fragment comprises: (a) The following three heavy chain variable region (VH) complementarity-determining regions (CDRs): (i) VH CDR1, having the sequence of CDR1 contained in VH as shown in SEQ ID NO:1, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of CDR1 contained in VH. (ii) VH CDR2, having the sequence of CDR2 contained in VH as shown in SEQ ID NO:1, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of one or two amino acids) compared to the sequence of CDR2 contained in VH; and (iii) VH CDR3, having the sequence of CDR3 contained in VH as shown in SEQ ID NO:1, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1 amino acid) compared to the sequence of CDR3 contained in VH. and / or (b) The following three light chain variable region (VL) CDRs: (iv) VL CDR1, having the sequence of CDR1 contained in VL as shown in SEQ ID NO:7, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of CDR1 contained in VL. (v) VL CDR2, having the sequence of CDR2 contained in VL as shown in SEQ ID NO:7, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of one or two amino acids) compared to the sequence of CDR2 contained in VL; and (vi) VL CDR3, having the sequence of CDR3 contained in VL as shown in SEQ ID NO:7, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of CDR3 contained in VL. Preferably, the substitution described in any one of (i)-(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 the Kabat, Chothia, or IMGT numbering system.

8. The conjugate, its stereoisomer, or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-7, wherein the antibody targeting human DLL3 or its antigen-binding fragment comprises: The sequences of CDR1, CDR2, and CDR3 contained in VH as shown in SEQ ID NO:1; and / or The CDR1, CDR2 and CDR3 sequences contained in VL as shown in SEQ ID NO:7; 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 the Kabat, Chothia or IMGT numbering system; Preferably, the antibody targeting human DLL3 or its antigen-binding fragment comprises: (a) The following three heavy chain variable region (VH) CDRs: (i) VH CDR1, which consists of the sequence shown in SEQ ID NO:

2. (ii) VH CDR2, which consists of the sequence shown in SEQ ID NO:3, and (iii) VH CDR3, which consists of the sequence shown in SEQ ID NO:4; and / or (b) The following three light chain variable region (VL) CDRs: (iv) VL CDR1, which consists of the sequence shown in SEQ ID NO:

8. (v)VL CDR2, which consists of the sequence shown in SEQ ID NO:9, and (vi)VL CDR3, which consists of the sequence shown in SEQ ID NO:10; Preferably, the VH of the antibody targeting human DLL3 or its antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO:2; VH CDR2 as shown in SEQ ID NO:3; and VH CDR3 as shown in SEQ ID NO:4; and / or, the VL of the antibody targeting human DLL3 or its antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO:8; VL CDR2 as shown in SEQ ID NO:9; and VL CDR3 as shown in SEQ ID NO:10; Preferably, the VH of the antibody targeting human DLL3 or its antigen-binding fragment comprises: VHCDR1 as shown in SEQ ID NO:2; VH CDR2 as shown in SEQ ID NO:3; and VH CDR3 as shown in SEQ ID NO:4; and the VL of the antibody targeting human DLL3 or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:8; VL CDR2 as shown in SEQ ID NO:9; and VL CDR3 as shown in SEQ ID NO:10; Preferably, the antibody targeting human DLL3 or its antigen-binding fragment comprises: (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following: (i) The sequence shown in SEQ ID NO: 1; (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 1; or (iii) A sequence having 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% sequence identity with the sequence shown in SEQ ID NO: 1; and (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following: (iv) The sequence shown in SEQ ID NO: 7; (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 7; or (vi) A sequence having 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% sequence identity with the sequence shown in SEQ ID NO: 7; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: The heavy chain variable region (VH) comprises a sequence having the sequence shown in SEQ ID NO:1 or having 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% sequence identity with the sequence shown in SEQ ID NO:1; and the light chain variable region (VL) comprises a sequence having the sequence shown in SEQ ID NO:7 or having 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% sequence identity with the sequence shown in SEQ ID NO:7; Preferably, the antibody targeting human DLL3 or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:1 and VL having the sequence shown in SEQ ID NO:

7.

9. The conjugate, its stereoisomer, or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-8, wherein the antibody targeting human DLL3 or its antigen-binding fragment is humanized; Preferably, the antibody targeting human DLL3 or its antigen-binding fragment further comprises the framework region of a human immunoglobulin; Preferably, the antibody targeting human DLL3 or its antigen-binding fragment further comprises a heavy chain framework region of human immunoglobulin (e.g., a heavy chain framework region contained in the amino acid sequence encoded by the human heavy chain germline antibody gene), and / or a light chain framework region of human immunoglobulin (e.g., a light chain framework region contained in the amino acid sequence encoded by the human light chain germline antibody gene). Preferably, the heavy chain framework region and / or the light chain framework region optionally include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) reversion mutations from human residues to mouse residues; Preferably, the antibody targeting human DLL3 or its antigen-binding fragment comprises: A heavy chain having a sequence as shown in SEQ ID NO:5 and a light chain having a sequence as shown in SEQ ID NO:11; Preferably, the antibody or its antigen-binding fragment further comprises a constant region derived from human immunoglobulin; Preferably, the heavy chain of the antibody or its antigen-binding fragment includes a heavy chain constant region derived from human immunoglobulins (e.g., IgG1, IgG2, IgG3, or IgG4). Preferably, the light chain of the antibody or its antigen-binding fragment includes a light chain constant region derived from human immunoglobulins (e.g., κ or λ); Preferably, the antibody targeting human DLL3 or its antigen-binding fragment comprises: Having a heavy chain constant region with a sequence as shown in SEQ ID NO:6 and a light chain constant region with a sequence as shown in SEQ ID NO:12; Preferably, the antibody targeting human DLL3 or its antigen-binding fragment is selected from monoclonal antibodies, mouse antibodies, rabbit antibodies, humanized antibodies, fully human antibodies, chimeric antibodies (e.g., human-mouse chimeric antibodies), bispecific antibodies, multispecific antibodies, single-chain antibodies, dAbs, complementarity-determining region fragments, 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 a species other than rodents, such as from human antibodies.

10. The conjugate, its stereoisomer, or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-9, wherein... -L 11 -L2-L3-L4-L5-、-L 12 -L2-L3-L4-L5-or-L 13 -L2-L3-L4-L5- are each independently selected from the following structures; Preferably, -L 11 -L2-L3-L4-L5-、-L 12 -L2-L3-L4-L5-or-L 13 -L2-L3-L4-L5- are each selected independently and without restriction from: in: The definitions of Ac, o, R5, R6 and R7 are as described in any one of claims 1-9; The carbon atom bonded to N has absolute chirality in either the R or S configuration; The position indicated by the wavy line on the left is connected to the antibody or its antigen-binding fragment, and the position indicated by the wavy line on the right is connected to X.

11. The conjugate, its stereoisomer, or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-10, wherein the conjugate has a structure as shown in general formula II. in: Ab、L 11 L 12 L 13 The definitions of Ac, L3, X, R, R1, R2, n1, n2, and n3 are as described in any one of claims 1-10; Chiral carbon atoms at positions 1, 2, or 3 have either an R-absolute configuration or an S-absolute configuration.

12. The conjugate, its stereoisomer, or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-11, wherein the conjugate is selected from: in: 18M1 represents Ab; The definitions of Ab, n1, n2, and n3 are as described in any one of claims 1-11.

13. A linker-drug conjugate of Formula III-A or Formula III-B, its stereoisomer, or a pharmaceutically acceptable salt or solvate thereof, wherein: L is selected from L2, L4, L5, X, R, R1, R2, Ac, L3, m, R a R b The definition is as described in any one of claims 1-11; Chiral carbon atoms at positions 1, 2, or 3 have either an R-absolute configuration or an S-absolute configuration.

14. The linker-drug conjugate, its stereoisomer, or a pharmaceutically acceptable salt or solvate thereof according to claim 13, wherein the linker-drug conjugate is selected from: in: 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.

15. The use of the linker-drug conjugate, its stereoisomer, or a pharmaceutically acceptable salt or solvate thereof as described in any one of claims 13-14 in the preparation of antibody-drug conjugates.

16. The conjugate, its stereoisomer, or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-12, or the linker-drug conjugate, its stereoisomer, or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 13-14, wherein the pharmaceutically acceptable salt is a sodium, potassium, calcium, or magnesium salt formed with an acidic functional group in the structural formula, or an acetate, trifluoroacetate, citrate, oxalate, tartrate, malate, nitrate, chloride, bromide, iodide, sulfate, bisulfate, phosphate, lactate, oleate, ascorbate, salicylate, formate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, or p-toluenesulfonate formed with a basic functional group in the structural formula.

17. A pharmaceutical composition comprising a conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-12, or a linker-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 13-14, and optionally a pharmaceutically acceptable carrier.

18. A pharmaceutical formulation comprising a conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-12, or a linker-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 13-14.

19. Use of the conjugate, stereoisomer or pharmaceutically acceptable salt or solvate of any one of claims 1-12, or the linker-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate of any one of claims 13-14, or the pharmaceutical composition or pharmaceutical formulation of claim 17, in the preparation of a medicament for the treatment or prevention of cancer or tumors; Preferably, the cancer or tumor expresses DLL3; More preferably, the cancer or tumor is selected from solid tumors or hematologic malignancies such as adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach 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, glioma multiforme, sarcoma, lymphoma, and leukemia.

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