Bispecific antibody-camptothecin conjugate and medical use thereof

Abstract

The bispecific antibody targeting two different epitopes or targets is coupled with camptothecin drugs to form a bispecific antibody-toxin conjugate with stable mass and good uniformity, and the toxin-to-antibody ratio (DAR) is 6.0-8.0. The antibody-toxin conjugate has the structure shown in general formula I, wherein Ab refers to the bispecific antibody targeting two different epitopes or targets and coupled with the linker-camptothecin drugs. Meanwhile, the application also discloses a preparation and purification method of the ADC drug, and application in tumor treatment. The application also relates to the linker-drug compound which can be coupled with Ab to form the antibody-toxin conjugate.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, specifically to an antibody-drug conjugate formed by a bispecific antibody and a camptothecin-based drug, as well as a method for preparing and using the antibody-drug conjugate. This invention also relates to linker-drug compounds that can be conjugated with an antibody (Ab) to form antibody-toxin conjugates. Technical Background

[0002] Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 3 (also known as HER3 and ErbB3) are both receptor protein tyrosine kinases and belong to the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases. This family includes EGFR (ErbB-1), HER2 / c-neu (ErbB-2), Her 3 (ErbB-3), and Her 4 (ErbB-4).

[0003] Epidermal growth factor receptor (EGFR) is a glycoprotein with a molecular weight of 170 kDa that spans the cell membrane and is activated by binding to a ligand. Upon activation, EGFR transforms from a monomer into a self-dimer or forms a heterodimer with other members of the EGFR family. Dimer formation activates intracellular kinase pathways, guiding phosphorylation of downstream pathways, including the MAPK, Akt, and JNK pathways, and inducing cell proliferation. Studies have shown that high EGFR expression is associated with tumor cell proliferation, angiogenesis, and tumor invasion. EGFR-related signaling pathways play a crucial role in the maintenance and growth of epidermal tissues. Particularly in breast cancer, malignant glioma, and lung cancer, EGFR promotes tumorigenesis. In lung cancer tissues, the EGFR signaling pathway is in an activated state, and EGFR expression levels are positively correlated with cancer development stages. Furthermore, due to the discovery of secondary mutations in EGFR under drug stress, increasing research is using EGFR as a biomarker for tumor drug resistance.

[0004] Epidermal growth factor receptor 3 (Her3 or ErbB3) also has the typical structure of epidermal growth factor receptors, but Her3 lacks an intracellular protein tyrosine kinase domain and therefore cannot autophosphorylate. Her3 can bind to ligand proteins, promoting heterodimerization with other members of the epidermal growth factor receptor family, activating receptor-mediated signaling pathways. It not only acts as a means of signal diversification but also amplifies signals, accelerating tumor progression. Heregulins (glial growth factor, neudifferentiation factor) can activate intracellular kinase-dependent multi-step signaling pathways after binding to transmembrane receptors HER3 and HER4. Downregulation of this signaling pathway often leads to Alzheimer's disease, heart failure, atherosclerosis, and cancer. Upregulation of HER3 expression can promote tumor formation and growth through interaction with receptor tyrosine kinases (RTKs). Furthermore, because HER3 is a heterodimer chaperone for other EGFR family members, it has the potential to regulate EGFR / HER2 signaling pathway-mediated cancer cell drug resistance. The study also showed that HER3 causes cancer treatment failure by activating the PI3K / AKT, MAPK / ERK, and JAK / STAT signaling pathways.

[0005] Overexpression / dysregulation of EGFR and Her3 is closely associated with the development of various tumors. EGFR and Her3 have demonstrated their role in driving tumor development in solid tumors such as breast cancer, lung cancer, gastric cancer, and pancreatic cancer. Multiple studies have shown that high Her3 expression is associated with the clinical ineffectiveness of EGFR antibodies and inhibitors. Clinically, several combination therapies targeting EGFR and Her3 are currently underway.

[0006] Monoclonal antibodies have been widely used in clinical practice for anti-tumor treatment in recent years, but their efficacy still has many shortcomings. A significant number of cancer patients have poor clinical responses, and some patients with clinical responses develop drug resistance after a period of monoclonal antibody treatment, leading to tumor recurrence. Bispecific monoclonal antibodies refer to monoclonal antibody molecules with two different antigen recognition sequences, which can bind to protein molecules with two different antigenic epitopes. This allows them to achieve various new and unique anti-tumor mechanisms, such as mediating the killing of tumor cells by immune cells, mediating the killing of tumor cells by toxic small molecules, or blocking signaling pathways that promote tumor growth. The development of bispecific monoclonal antibodies is mainly due to the fact that multiple mediators participate in the pathogenesis of tumors through specific or overlapping mechanisms. Blocking multiple targets simultaneously will produce better therapeutic effects than inhibiting a single target. At the same time, the action of multiple targets greatly reduces the probability of drug resistance. Currently, two bispecific antibodies, Catumaxomab and Blinatumomab, have been approved for marketing in the United States, and more than fifty bispecific antibody molecules are undergoing clinical trials.

[0007] Antibody-drug conjugates (ADCs) are molecules with specific targeted killing effects obtained by attaching small-molecule toxins with cytotoxic effects to antibodies. They are mainly used for the treatment of tumors and other diseases. The antibodies used in ADCs can specifically bind to proteins on the surface of tumor cells, thus possessing tumor specificity and potential that traditional drugs cannot achieve. Currently, 12 ADC drugs have been approved for marketing worldwide, and hundreds of projects are undergoing clinical trials. However, the vast majority of ADC projects currently on the market or in clinical trials target only one target, failing to achieve the synergistic advantages of dual-target therapy. Summary of the Invention

[0008] Based on a comprehensive understanding of antibody-drug conjugates (ADCs), the inventors disclose a bispecific antibody-drug conjugate and its preparation method, a pharmaceutical composition comprising the conjugate, and the use of the conjugate or pharmaceutical composition. This invention also relates to linker-drug compounds that can be conjugated with bispecific antibodies to form antibody-toxin conjugates.

[0009] The first aspect of the present invention discloses a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharmaceutically acceptable salt or solvation thereof;

[0010]

[0011] in:

[0012] Ab is a bispecific antibody or its antigen-binding fragment that simultaneously targets two different epitopes or targets;

[0013] L1 is selected without restriction from:

[0014]

[0015] L1 is preferred

[0016] L1 is preferred

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

[0018]

[0019]

[0020] Y is a scaffold selected from C1-C6 alkyl, substituted C1-C6 alkyl, or C3-C8 cycloalkyl; preferably Y is C1-C6 alkyl; Ac is a hydrophilic structural unit; the second carbon atom connected to Y has absolute chirality in either the R or S configuration;

[0021] L3 may or may not exist; if it exists, L3 is selected from the PEG hydrophilic unit. o is an integer selected from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), preferably an integer from 2 to 8;

[0022] L4 is the enzyme digestion unit;

[0023] L5 is a connection unit;

[0024] In Formula I, the chiral carbon atom at position 1 bonded to N has absolute chirality in either the R or S configuration.

[0025] 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;

[0026] Preferably, R is selected from hydrogen atoms or C1-C6 alkyl groups;

[0027] 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;

[0028] R1 is preferably selected from hydrogen atoms or C1-C6 alkyl groups;

[0029] More preferably, R1 is selected from C1-C6 alkyl groups;

[0030] 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;

[0031] Preferably, R2 is selected from hydrogen atoms, halogens, or C1-C6 alkyl groups;

[0032] More preferably, R2 is selected from halogens;

[0033] X is selected from -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-;

[0034] X is preferably selected from -C(O)-CR a R b -(CR3R4) m -O-;

[0035] R a and R b Each is independently selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halo-C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C6-C10 aryl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, 3-7 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;

[0036] Preferred R a and R b Each is independently selected from hydrogen atoms, C1-C6 alkyl, halo-C1-C6 alkyl, C3-C8 cycloalkyl-C1-C6 alkyl, or C6-C10 aryl-C1-C6 alkyl;

[0037] Or, Ra R b The carbon atoms attached to it constitute C3-C8 cycloalkyl, C3-C8 cycloalkyl-C1-C6 alkyl, 3-7 membered heterocyclic group, or substituted 3-7 membered heterocyclic group; preferably R a R b The carbon atoms to which they are attached form C3-C8 cycloalkyl groups;

[0038] R3 and R4 may be the same or different, and are independently hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, halo-C1-C6 alkyl, deuterated C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, amino, cyano, nitro, hydroxy-C1-C6 alkyl, C3-C8 cycloalkyl, 3-7 membered heterocyclic group, substituted 3-7 membered heterocyclic group;

[0039] Preferably, R3 and R4 are each independently a hydrogen atom or a C1-C6 alkyl group;

[0040] Alternatively, R3, R4 and the carbon atoms attached to them can form C3-C8 cycloalkyl, C3-C8 cycloalkyl-C1-C6 alkyl, 3-7 membered heterocyclic group, or substituted 3-7 membered heterocyclic group;

[0041] m is selected from an integer from 0 to 4 (e.g., 0, 1, 2, 3 or 4), preferably 0 or 1; n is selected from an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0042] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof as shown in general formula I is disclosed, characterized in that the Ab is a bispecific antibody or antigen-binding fragment thereof that simultaneously targets two different epitopes or targets, preferably a bispecific antibody or antigen-binding fragment thereof that simultaneously targets EGFR and HER3.

[0043] In some embodiments of the first aspect of the invention, a ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, as shown in general formula I, is disclosed, characterized in that the antibody of the Ab comprises: an IgG1 heavy chain, a κ light chain, and a single-chain Fv (scFv) domain; wherein the single-chain Fv (scFv) domain forms a construct with the IgG1 heavy chain or the κ light chain; wherein the IgG1 heavy chain and the κ light chain form an IgG moiety having binding specificity to EGFR; the scFv domain has binding specificity to HER3, and the scFv domain is connected by a linker (e.g., having (gl... The amino acid sequence (y-gly-gly-gly-ser)n, where n is an integer of at least 1, preferably an integer from 1 to 10, is linked to the C-terminus or N-terminus of the IgG1 heavy chain or the C-terminus or N-terminus of the κ light chain; and wherein the single-chain Fv domain has a structural sequence of N-terminus-heavy chain variable region-linker-light chain variable region-C-terminus or N-terminus-light chain variable region-linker-heavy chain variable region-C-terminus (e.g., the linker consists of the amino acid sequence (gly-gly-gly-gly-ser)m, where m is an integer of at least 3, preferably m is 3, 4, 5, or 6).

[0044] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, as shown in general formula I, is disclosed, characterized in that the κ light chain of the antibody of Ab contains a CDR as shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, the IgG1 heavy chain contains a CDR as shown in SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and the single-chain Fv (scFv) domain contains a heavy chain variable region CDR as shown in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and a light chain variable region CDR as shown in SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37.

[0045] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the light chain of the antibody Ab contains a variable region as shown in SEQ ID NO: 28, the heavy chain of IgG1 contains a variable region as shown in SEQ ID NO: 38, and the single-chain Fv (scFv) domain contains a heavy chain variable region as shown in SEQ ID NO: 39 and a light chain variable region as shown in SEQ ID NO: 40.

[0046] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the antibody of Ab has a light chain amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of the construct of the antibody heavy chain and the single-chain Fv (scFv) domain is SEQ ID NO: 4.

[0047] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the antibody of Ab has a light chain nucleic acid coding sequence of SEQ ID NO: 1, and the nucleic acid coding sequence of the construct of the antibody heavy chain and the single-chain Fv (scFv) domain is SEQ ID NO: 3.

[0048] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the κ light chain of the antibody of Ab contains a CDR as shown in SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, the IgG1 heavy chain contains a CDR as shown in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, and the single-chain Fv (scFv) domain contains a heavy chain variable region CDR as shown in SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, and a light chain variable region CDR as shown in SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37.

[0049] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the light chain of the antibody Ab contains a variable region as shown in SEQ ID NO: 44, the heavy chain of IgG1 contains a variable region as shown in SEQ ID NO: 48, and the single-chain Fv (scFv) domain contains a heavy chain variable region as shown in SEQ ID NO: 39 and a light chain variable region as shown in SEQ ID NO: 40.

[0050] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the antibody of Ab has a light chain amino acid sequence of SEQ ID NO: 6, and the amino acid sequence of the construct of the antibody heavy chain and the single-chain Fv (scFv) domain is SEQ ID NO: 8.

[0051] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the light chain nucleic acid coding sequence of the antibody Ab is SEQ ID NO: 5, and the nucleic acid coding sequence of the construct of the antibody heavy chain and the single-chain Fv (scFv) domain is SEQ ID NO: 7.

[0052] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the light chain of the antibody Ab contains a variable region as shown in SEQ ID NO: 49, the heavy chain of IgG1 contains a variable region as shown in SEQ ID NO: 52, and the single-chain Fv (scFv) domain contains a heavy chain variable region as shown in SEQ ID NO: 50 and a light chain variable region as shown in SEQ ID NO: 51.

[0053] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the heavy chain amino acid sequence of the antibody Ab is SEQ ID NO: 12, and the amino acid sequence of the construct of the antibody light chain and the single-chain Fv (scFv) domain is SEQ ID NO: 10.

[0054] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the antibody of Ab has a heavy chain nucleic acid coding sequence of SEQ ID NO: 11, and the nucleic acid coding sequence of the construct of the antibody light chain and the single-chain Fv (scFv) domain is SEQ ID NO: 9.

[0055] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the antibody of Ab has a light chain amino acid sequence of SEQ ID NO: 14, and the amino acid sequence of the construct of the antibody heavy chain and the single-chain Fv (scFv) domain is SEQ ID NO: 16.

[0056] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the antibody of Ab has a light chain nucleic acid coding sequence of SEQ ID NO: 13, and the nucleic acid coding sequence of the construct of the antibody heavy chain and the single-chain Fv (scFv) domain is SEQ ID NO: 15.

[0057] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the heavy chain amino acid sequence of the antibody Ab is SEQ ID NO: 20, and the amino acid sequence of the construct of the antibody light chain and the single-chain Fv (scFv) domain is SEQ ID NO: 18.

[0058] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the antibody of Ab has a heavy chain nucleic acid coding sequence of SEQ ID NO: 19, and the nucleic acid coding sequence of the construct of the antibody light chain and the single-chain Fv (scFv) domain is SEQ ID NO: 17.

[0059] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the light chain of the antibody Ab contains a variable region as shown in SEQ ID NO: 53, the heavy chain of IgG1 contains a variable region as shown in SEQ ID NO: 54, and the single-chain Fv (scFv) domain contains a heavy chain variable region as shown in SEQ ID NO: 50 and a light chain variable region as shown in SEQ ID NO: 51.

[0060] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the heavy chain amino acid sequence of the antibody Ab is SEQ ID NO: 24, and the amino acid sequence of the construct of the antibody light chain and the single-chain Fv (scFv) domain is SEQ ID NO: 22.

[0061] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the antibody of Ab has a heavy chain nucleic acid coding sequence of SEQ ID NO: 23, and the nucleic acid coding sequence of the construct of the antibody light chain and the single-chain Fv (scFv) domain is SEQ ID NO: 21.

[0062] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the antibody of Ab comprises: two IgG1 heavy chains; two κ light chains; and two single-chain Fv (scFv) domains.

[0063] In some embodiments of the first aspect of the invention, ligand-camptothecin derivative conjugates or pharmaceutically acceptable salts or solvates thereof as shown in general formula I are disclosed, characterized in that X is not limited to being selected from the following structures or isomers thereof:

[0064]

[0065]

[0066] The wavy line on the left is connected to the camptothecin derivative, and the wavy line on the right is connected to L5.

[0067] In some embodiments of the first aspect of the invention, ligand-camptothecin derivative conjugates or pharmaceutically acceptable salts or solvates thereof as shown in general formula I are disclosed, characterized in that: L4 is not limited to peptide residues composed of amino acids.

[0068] Optionally, the amino acid is further substituted with one or more substituents selected from deuterium, halogen, hydroxyl, cyano, amino, nitro, carboxyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy and C3-C8 cycloalkyl or substituted C3-C8 cycloalkyl.

[0069] 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) or aspartic acid (D).

[0070] More preferably, the peptide residue is a tetrapeptide residue composed of glycine (G)-glycine (G)-phenylalanine (F)-glycine (G).

[0071] Particularly preferred is that the peptide residue is -GGFG-.

[0072] In some embodiments of the first aspect of the present invention, ligand-camptothecin derivative conjugates or pharmaceutically acceptable salts or solvates thereof as shown in general formula I are disclosed, characterized in that:

[0073] L5 is not limited to -NR5 (CR6R7). q - Or a chemical bond, where q is an integer from 0 to 6 (e.g., 0, 1, 2, 3, 4, 5, or 6);

[0074] R5, R6, and R7 may be the same or different, and each is independently selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, 3-7 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;

[0075] Preferably, R5, R6 and R7 are each independently selected from hydrogen atoms or C1-C6 alkyl groups;

[0076] More preferably, R5, R6 and R7 are each independently selected from hydrogen atoms.

[0077] In some implementations, L1 is not limited to being selected from:

[0078]

[0079] In some embodiments of the first aspect of the invention, ligand-camptothecin derivative conjugates or pharmaceutically acceptable salts or solvates thereof as shown in general formula I are disclosed, characterized in that: the connecting units -L1-L2-L3-L4-L5 - are not limited to being selected from the following structures;

[0080]

[0081]

[0082] Preferred

[0083]

[0084] in:

[0085] Ac is a hydrophilic structural unit;

[0086] R5, R6, and R7 may be the same or different, and each is independently selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, 3-7 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;

[0087] Preferably, R5, R6 and R7 are each independently selected from hydrogen atoms or C1-C6 alkyl groups;

[0088] More preferably, R5, R6 and R7 are each independently selected from hydrogen atoms;

[0089] The carbon atom bonded to N has absolute chirality in either the R or S configuration;

[0090] The wavy line on the left is connected to the antibody or its antigen-binding fragment, and the wavy line on the right is connected to the X.

[0091] o is an integer selected from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0092] A second aspect of the present invention discloses a ligand-camptothecin derivative conjugate as shown in general formula II, or a pharmaceutically acceptable salt or solvate thereof;

[0093]

[0094] in:

[0095] Ab is a bispecific antibody or its antigen-binding fragment that simultaneously targets EGFR and HER3;

[0096] L1 is a connecting unit connected to Ab, and can be selected without limitation from:

[0097]

[0098] L1 is preferred

[0099] L1 is preferred

[0100] L3 exists or does not exist; when L3 exists, L3 is selected from... o is an integer selected from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), preferably an integer from 2 to 8;

[0101] Ac is a hydrophilic structural unit;

[0102] Chiral carbon atoms at positions 1, 2, and 3 have either an R-absolute configuration or an S-absolute configuration;

[0103] 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;

[0104] Preferably, R is selected from hydrogen atoms or C1-C6 alkyl groups;

[0105] 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;

[0106] R1 is preferably selected from hydrogen atoms or C1-C6 alkyl groups;

[0107] More preferably, R1 is selected from C1-C6 alkyl groups;

[0108] 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;

[0109] Preferably, R2 is selected from hydrogen atoms, halogens, or C1-C6 alkyl groups;

[0110] More preferably, R2 is selected from halogens;

[0111] X is selected from -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-;

[0112] X is preferably selected from -C(O)-CR a R b -(CR3R4) m -O-;

[0113] R a and R b Each is independently selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halo-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, substituted 5-10 membered heteroaryl;

[0114] Preferred Ra and R b Each is independently selected from hydrogen atoms, C1-C6 alkyl, halo-C1-C6 alkyl, C3-C8 cycloalkyl-C1-C6 alkyl, or C6-C10 aryl-C1-C6 alkyl;

[0115] Or, R a R b The carbon atoms attached to it constitute C3-C8 cycloalkyl, C3-C8 cycloalkyl-C1-C6 alkyl, 3-7 membered heterocyclic group, or substituted 3-7 membered heterocyclic group; preferably R a R b The carbon atoms to which they are attached form C3-C8 cycloalkyl groups;

[0116] R3 and R4 may be the same or different, and are independently hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, halo-C1-C6 alkyl, deuterated C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, amino, cyano, nitro, hydroxy-C1-C6 alkyl, C3-C8 cycloalkyl, 3-7 membered heterocyclic group, substituted 3-7 membered heterocyclic group;

[0117] Preferably, R3 and R4 are each independently a hydrogen atom or a C1-C6 alkyl group;

[0118] Alternatively, R3, R4 and the carbon atoms attached to them can form C3-C8 cycloalkyl, C3-C8 cycloalkyl-C1-C6 alkyl, 3-7 membered heterocyclic group, or substituted 3-7 membered heterocyclic group;

[0119] m is selected from an integer between 0 and 4 (e.g., 0, 1, 2, 3 or 4), preferably 0 or 1;

[0120] n is an integer selected from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0121] In some embodiments of the first and second aspects of the present invention, the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate is disclosed, characterized in that: the Ac has the structure shown in formula B.

[0122]

[0123] in:

[0124] Z is not limited to being selected from one or more of the following groups: hydrophilic carboxyl groups, phosphoric acid, polyphosphoric acid, phosphorous acid, sulfonic acid, sulfinic acid, or polyethylene glycol (PEG).

[0125] Z is preferably selected from hydrophilic carboxyl groups, phosphoric acid, or polyethylene glycol (PEG);

[0126] Y' is an optional scaffold connecting the amino group and Z; preferably Y' is a C1-C6 alkylene group (e.g., methylene);

[0127] Ac is connected to the carbon at position 2, which is already indicated in structural formula I, via the support Y.

[0128] In some embodiments of the first and second aspects of the present invention, the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate is disclosed, characterized in that: the Ac is not limited to being selected from 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) cysteine, (D / L) arginine, (D / L) histidine, (D / L) methionine, (D / L) asparagine, (D / L) glutamine, (D / L) threonine, (D / L) aspartic acid, (D / L) glutamic acid, natural or non-natural amino acid derivatives, or the following structures or isomers thereof.

[0129]

[0130] Preferred

[0131] In some embodiments of the first and second aspects of the present invention, the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate is disclosed, characterized in that: the Ac is not limited to being selected from glycine, phosphoric acid, (D / L) glutamic acid or polyethylene glycol hydrophilic structures.

[0132] In some embodiments of the first and second aspects of the present invention, the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate is disclosed, characterized in that: the... It has the structure shown in equation d below;

[0133]

[0134] in:

[0135] 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;

[0136] Preferably, R is selected from hydrogen atoms or C1-C6 alkyl groups;

[0137] 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;

[0138] R1 is preferably selected from hydrogen atoms or C1-C6 alkyl groups;

[0139] More preferably, R1 is selected from C1-C6 alkyl groups;

[0140] 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;

[0141] Preferably, R2 is selected from hydrogen atoms, halogens, or C1-C6 alkyl groups;

[0142] More preferably, R2 is selected from halogens;

[0143] R a and R b Each is independently selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halo-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, substituted 5-10 membered heteroaryl;

[0144] Preferred R a and R b Each is independently selected from hydrogen atoms, C1-C6 alkyl, halo-C1-C6 alkyl, C3-C8 cycloalkyl-C1-C6 alkyl, or C6-C10 aryl-C1-C6 alkyl;

[0145] Preferred R a and R b Each is independently selected from hydrogen atoms, C1-C6 alkyl groups, halogenated C1-C6 alkyl groups, C3-C8 cycloalkyl groups, C1-C6 alkyl groups, and C6-C10 aryl groups; preferably R. a and R b Each is independently selected from hydrogen atom, methyl, ethyl, trifluoromethyl, cyclopropylmethyl, and phenyl;

[0146] Or, R aR b The carbon atoms attached to it constitute C3-C8 cycloalkyl, C3-C8 cycloalkyl-C1-C6 alkyl, 3-7 membered heterocyclic group, or substituted 3-7 membered heterocyclic group; preferably R a R b And the carbon atoms to which they are attached form C3-C8 cycloalkyl groups (e.g., C3-C5 cycloalkyl groups);

[0147] The 1st chiral carbon atom has two chiral configurations: R absolute configuration or S absolute configuration.

[0148] m is selected from 0 or 1.

[0149] In some embodiments of the first and second aspects of the present invention, the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate is disclosed, characterized in that: the structural formula d is not limited to a selection from the following compounds:

[0150]

[0151]

[0152] In some embodiments of the present invention, L1 may contain a succinimide group. In these embodiments, the ligand-drug conjugate can undergo hydrolysis under readily hydrolyzable conditions, with the hydrolysis site being the succinimide group of the linking unit. When the ligand contains multiple linker-drug groups, the following situations may occur depending on the degree of hydrolysis:

[0153] The succinimide group is completely non-hydrolyzed, meaning that all succinimide groups are in closed-ring form.

[0154] The succinimide group is not completely hydrolyzed, meaning that some of the succinimide groups are in a closed-ring form. The other part of the succinimide group is in an open-ring form.

[0155] The succinimide groups are completely hydrolyzed, meaning that all succinimide groups are in ring-open form.

[0156] Therefore, when multiple L1s containing succinimide groups are present in an ADC (i.e., Ab is linked to multiple drug-linkers containing succinimide groups), these succinimide groups can all be in closed-ring form, some in open-ring form, or all in open-ring form.

[0157] It is understood that although the succinimide group appearing in the chemical structure of the ADC in this application is in a closed-ring form, it actually covers three cases: completely closed ring, partially open ring, and completely open ring of succinimide. A third aspect of the invention discloses a linker-drug compound or a pharmaceutically acceptable salt or solvate thereof, characterized in that it has the structure shown in Formula III.

[0158]

[0159] in:

[0160] 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;

[0161] R a Selected from hydrogen atoms, deuterium atoms, halogens, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated 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, substituted 5-10 membered heteroaryl;

[0162] R b Selected from hydrogen atoms, deuterium atoms, halogens, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated 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, substituted 5-10 membered heteroaryl;

[0163] Or, R a R b The carbon atoms attached to them constitute C3-C8 cycloalkyl, C3-C8 cycloalkyl-C1-C6 alkyl, 3-7 membered heterocyclic groups, and substituted 3-7 membered heterocyclic groups;

[0164] Preferred R a and R b Each is independently selected from hydrogen atoms, C1-C6 alkyl groups, halogenated C1-C6 alkyl groups, C3-C8 cycloalkyl groups, C1-C6 alkyl groups, and C6-C10 aryl groups; preferably R. a and R b Each is independently selected from hydrogen atom, methyl, ethyl, trifluoromethyl, cyclopropylmethyl, and phenyl;

[0165] Or, R a R b The carbon atoms attached to it constitute C3-C8 cycloalkyl, C3-C8 cycloalkyl-C1-C6 alkyl, 3-7 membered heterocyclic group, or substituted 3-7 membered heterocyclic group; preferably R a R b And the carbon atoms to which they are attached form C3-C8 cycloalkyl groups (e.g., C3-C5 cycloalkyl groups);

[0166] Whether L3 exists or not, if L3 exists, it is selected from... o is an integer selected from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10);

[0167] A chiral carbon atom at position 1 or 2 can have either an absolute R configuration or an absolute S configuration;

[0168] Ac is a hydrophilic structural unit;

[0169] m is selected from 0 or 1.

[0170] In some embodiments of the third aspect of the invention, the linker-drug compound or a pharmaceutically acceptable salt or solvate thereof is disclosed, characterized in that: the Ac has the structure shown in formula B.

[0171]

[0172] in:

[0173] Z is not limited to being selected from one or more of the following groups: hydrophilic carboxyl groups, phosphoric acid, polyphosphoric acid, phosphorous acid, sulfonic acid, sulfinic acid, or polyethylene glycol (PEG).

[0174] Y' is an optional scaffold connecting the amino group and Z; preferably Y' is a C1-C6 alkylene group (e.g., methylene);

[0175] Ac is connected to the carbon at position 2, which is already indicated in structural formula I, via the support Y.

[0176] In some embodiments of the third aspect of the invention, the linker-drug compound or a pharmaceutically acceptable salt or solvate thereof is disclosed, characterized in that: the Ac is not limited to being selected from 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) cysteine, (D / L) arginine, (D / L) histidine, (D / L) methionine, (D / L) asparagine, (D / L) glutamine, (D / L) threonine, (D / L) aspartic acid, (D / L) glutamic acid, natural or non-natural amino acid derivatives, or the following structures.

[0177]

[0178] In some embodiments of the third aspect of the invention, the linker-drug compound or a pharmaceutically acceptable salt or solvate thereof is disclosed, characterized in that: Ac is not limited to being selected from glycine, phosphoric acid, (D / L) glutamic acid or a polyethylene glycol hydrophilic structure.

[0179] In some embodiments of the third aspect of the invention, the linker-drug compound or a pharmaceutically acceptable salt or solvate thereof is disclosed, characterized in that: the linker-drug compound is not limited to being selected from the following structures or isomers thereof,

[0180]

[0181]

[0182]

[0183]

[0184] Where: o is an integer selected from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0185] The linker-drug compound or its pharmaceutically acceptable salt or solvate disclosed in the third aspect of the present invention can be used as an intermediate for coupling with ligand Ab to form ligand-camptothecin derivative conjugates of Formula I or Formula II as described in the first and second aspects.

[0186] The fourth aspect of this invention discloses a method for preparing ligand-camptothecin derivative conjugates or pharmaceutically acceptable salts or solvates of general formula I or general formula II as described in the first and second aspects, characterized by comprising the following steps:

[0187]

[0188] 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.

[0189] The chiral carbon atoms at positions 1, 2, or 3 have absolute chirality in either the R or S configuration;

[0190] Ab, L1, L2, L3, L4, L5, X, R, R1, R2 and n are as described above.

[0191] This application also relates to the use of the linker-drug compound or its pharmaceutically acceptable salt or solvation as disclosed in the third aspect as an intermediate in the preparation of a ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvation. In some embodiments, the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvation is the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvation of the first, second, and fourth aspects of this invention. In some embodiments, the preparation is carried out according to the preparation method disclosed in the fourth aspect.

[0192] In some embodiments of the first, second, and fourth aspects of the present invention, the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate is disclosed, characterized in that: the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate is not limited to being selected from the following structures or their succinimide ring-opening structures or isomers thereof.

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203] in:

[0204] SI-1×6.4 is a bispecific antibody or its antigen-binding fragment that simultaneously targets EGFR and HER3;

[0205] n is an integer selected from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0206] In some embodiments of the first, second, and fourth aspects of the present invention, the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate is disclosed, characterized in that: the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate is not limited to being selected from the following structures or their succinimide ring-opening structures or isomers thereof.

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217] in:

[0218] SI-1×4 is a bispecific antibody or its antigen-binding fragment that simultaneously targets EGFR and HER3;

[0219] n is an integer selected from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0220] In some embodiments of the first, second, and fourth aspects of the present invention, the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate is disclosed, characterized in that: the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate is not limited to being selected from the following structures or their succinimide ring-opening structures or isomers thereof.

[0221]

[0222]

[0223] in:

[0224] SI-1×22 is a bispecific antibody or its antigen-binding fragment that simultaneously targets EGFR and HER3;

[0225] n is an integer selected from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0226] In some embodiments of the first, second, and fourth aspects of the present invention, the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate is disclosed, characterized in that: the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate is not limited to being selected from the following structures or their succinimide ring-opening structures or isomers thereof.

[0227]

[0228]

[0229] in:

[0230] SI-1×24 is a bispecific antibody or its antigen-binding fragment that simultaneously targets EGFR and HER3;

[0231] n is an integer selected from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0232] In some embodiments of the first, second, and fourth aspects of the present invention, the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate is disclosed, characterized in that: the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate is not limited to being selected from the following structures or their succinimide ring-opening structures or isomers thereof.

[0233]

[0234]

[0235] in:

[0236] SI-1×25 is a bispecific antibody or its antigen-binding fragment that simultaneously targets EGFR and HER3;

[0237] n is an integer selected from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0238] In some embodiments of the first, second, and fourth aspects of the present invention, the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate is disclosed, characterized in that: the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate is not limited to being selected from the following structures or their succinimide ring-opening structures or isomers thereof.

[0239]

[0240]

[0241] in:

[0242] SI-1×26 is a bispecific antibody or its antigen-binding fragment that simultaneously targets EGFR and HER3;

[0243] n is an integer selected from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0244] In some embodiments of the first, second, and third aspects of the present invention, the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate or linker-drug compound or its pharmaceutically acceptable salt or solvate is disclosed, characterized in that: the pharmaceutically acceptable salt includes sodium, potassium, calcium, or magnesium salts formed with acidic functional groups in the structure, and 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.

[0245] The fifth aspect of the present invention discloses a pharmaceutical composition comprising the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof as described in the first and second aspects, or the linker-pharmaceutical compound or a pharmaceutically acceptable salt or solvate thereof as described in the third aspect, and optionally a pharmaceutically acceptable carrier.

[0246] The sixth aspect of the present invention discloses a pharmaceutical formulation comprising the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof described in the first and second aspects, or the linker-pharmaceutical compound or a pharmaceutically acceptable salt or solvate thereof described in the third aspect.

[0247] The seventh aspect of the present invention discloses the use of the ligand-camptothecin derivative conjugates or pharmaceutically acceptable salts or solvates thereof described in the first and second aspects, or the linker-drug compound or pharmaceutically acceptable salts or solvates thereof described in the third aspect, or the pharmaceutical composition described in the fifth aspect and / or the pharmaceutical formulation described in the sixth aspect, in the preparation of a medicament for treating or preventing cancer or tumors.

[0248] Alternatively, the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate as described in the first and second aspects, or the linker-drug compound or its pharmaceutically acceptable salt or solvate as described in the third aspect, or the pharmaceutical composition as described in the fifth aspect and / or the pharmaceutical preparation as described in the sixth aspect, for the treatment or prevention of cancer or tumors.

[0249] Preferably, the cancer or tumor expresses EGFR and / or HER3;

[0250] 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, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioma multiforme, sarcoma, lymphoma, and leukemia.

[0251] The eighth aspect of the present invention discloses a method for treating or preventing cancer or tumors, comprising administering to a subject in need a preventive or therapeutically effective amount of the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof described in the first or second aspect, or the linker-drug compound or a pharmaceutically acceptable salt or solvate thereof described in the third aspect, or the pharmaceutical composition described in the fifth aspect and / or the pharmaceutical preparation described in the sixth aspect;

[0252] Preferably, the cancer or tumor expresses EGFR and / or HER3;

[0253] 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, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioma multiforme, sarcoma, lymphoma, and leukemia.

[0254] In the above aspects and embodiments of the present invention

[0255] 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”.

[0256] "C3-C8 cycloalkyl" and various complex groups involving "C3-C8 cycloalkyl" can be replaced with "C3-C20 cycloalkyl" or "C3-C10 cycloalkyl";

[0257] "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".

[0258] "C6-C10 aryl" and various complex groups involving it can be replaced with "C6-C12 aryl";

[0259] 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”.

[0260] Beneficial effects

[0261] The EGFR / Her3 bispecific antibody-drug conjugate provided by this invention is a bispecific antibody-drug conjugate that targets both EGFR and Her3 simultaneously. It has good molecular stability and good preclinical efficacy, and is expected to have excellent clinical therapeutic effects. Attached Figure Description

[0262] Figure 1A This demonstrates the detection of ADC-5 aggregation by SEC-HPLC.

[0263] Figure 1B This demonstrates the detection of ADC-6 aggregation by SEC-HPLC.

[0264] Figure 1C This describes the SEC-HPLC detection of ADC-64 aggregation.

[0265] Figure 1D This describes the SEC-HPLC detection of ADC-DS aggregation.

[0266] Figure 1E This demonstrates the SEC-HPLC detection of ADC-108 aggregation.

[0267] Figure 1FThis demonstrates the SEC-HPLC detection of ADC-112 aggregation.

[0268] Figure 1G This demonstrates the SEC-HPLC detection of ADC-215 aggregation.

[0269] Figure 1H This demonstrates the SEC-HPLC detection of ADC-219 aggregation.

[0270] Figure 1I This demonstrates the SEC-HPLC detection of ADC-227 aggregation.

[0271] Figure 1J This demonstrates the SEC-HPLC detection of ADC-235 aggregation.

[0272] Figure 2A This describes the RP-HPLC method for detecting the drug-antibody conjugation ratio (DAR) of ADC-5.

[0273] Figure 2B This describes the RP-HPLC method for detecting the drug-antibody conjugation ratio (DAR) of ADC-6.

[0274] Figure 2C This describes the RP-HPLC method for detecting the drug-antibody conjugation ratio (DAR) of ADC-10.

[0275] Figure 2D This describes the RP-HPLC method for detecting the drug-antibody conjugation ratio (DAR) of ADC-12.

[0276] Figure 2E This describes the RP-HPLC method for detecting the drug-antibody conjugation ratio (DAR) of ADC-64.

[0277] Figure 2F This describes the RP-HPLC method for detecting the drug-antibody conjugation ratio (DAR) of ADC-108.

[0278] Figure 2G This describes the RP-HPLC method for detecting the drug-antibody conjugation ratio (DAR) of ADC-112.

[0279] Figure 2H This describes the RP-HPLC method for detecting the drug-antibody conjugation ratio (DAR) of ADC-215.

[0280] Figure 2I This describes the RP-HPLC assay for the drug-antibody conjugate ratio (DAR) of ADC-219.

[0281] Figure 2J This describes the RP-HPLC method for detecting the drug-antibody conjugate ratio (DAR) of ADC-227.

[0282] Figure 2K This describes the RP-HPLC method for detecting the drug-antibody conjugation ratio (DAR) of ADC-235.

[0283] Figure 2L This describes the RP-HPLC method for detecting the drug-antibody conjugate ratio (DAR) of ADC-243.

[0284] Figure 3A This indicates that ADC-112 and SI-1×4 antibody maintain the same affinity for both antigens EGFR and HER3-Fc.

[0285] Figure 3B This indicates that ADC-6 and SI-1×6.4 antibodies maintain simultaneous affinity for both antigens EGFR and HER3-Fc.

[0286] Figure 3C This indicates that ADC-219 and SI-1×22 antibody maintain the same affinity for both antigens EGFR and HER3-Fc.

[0287] Figure 3D This indicates that ADC-227 and SI-1×24 antibody maintain the same affinity for both antigens EGFR and HER3-Fc.

[0288] Figure 3E This indicates that the ADC-235 antibody maintains the same affinity for both the EGFR and HER3-Fc antigens as the SI-1×25 antibody.

[0289] Figure 3F This indicates that ADC-243 and SI-1×26 antibody maintain the same affinity for both antigens EGFR and HER3-Fc.

[0290] Figure 4A This study describes the in vitro efficacy of six naked antibodies and six ADCs in A431.

[0291] Figure 4B This study describes the in vitro efficacy of six naked antibodies and six ADCs in BXPC-3.

[0292] Figure 4C This study describes the in vitro efficacy of six naked antibodies and six ADCs in FaDu.

[0293] Figure 4D This study describes the in vitro efficacy of six naked antibodies and six ADCs in HARA-B.

[0294] Figure 4E This study describes the in vitro efficacy of six naked antibodies and six ADCs in HCC827.

[0295] Figure 4FThis study describes the in vitro efficacy of six naked antibodies and six ADCs in SW620.

[0296] Figure 5A This study describes the in vivo efficacy results of ADC-6 and SI-1×6.4 naked antibodies in the A431 monotumor model.

[0297] Figure 5B This study describes the in vivo efficacy results of ADC-6, SI-1×6.4 naked antibody, and Cetuximab (Cet) and ADC-214 in the SW620 monotumor model.

[0298] Figure 5C This study describes the in vivo efficacy results of ADC-6 and SI-1×6.4 naked antibodies in A431+SW620 heterogeneous tumors.

[0299] Figure 6A This study describes the in vivo efficacy results of ADC-6, ADC-219, ADC-235, ADC-227, and ADC-112 in the A431 single-tumor model.

[0300] Figure 6B This study describes the in vivo efficacy results of ADC-6, ADC-219, ADC-235, ADC-227, and ADC-112 in the BXPC-3 monotumor model.

[0301] Figure 7A This study describes the in vitro efficacy of SI-1×6.4, Cetuximab, ADC-6, ADC-214, and d3 against the human poorly differentiated lung cancer squamous cell carcinoma line Oka-c-1.

[0302] Figure 7B This study describes the in vitro efficacy of SI-1×6.4, Cetuximab, ADC-6, ADC-214, and d3 against human lung squamous cell carcinoma cells SK-MES-1. Detailed implementation method.

[0303] Abbreviations and Definitions

[0304] 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.

[0305] Unless otherwise stated, the terms used in the claims and specification herein shall have the following meanings.

[0306] The term "ligand" is a macromolecular compound that recognizes and binds to antigens or receptors associated with target cells. The role of ligands is to deliver drugs to the target cell population that has bound to them. 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 designated 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.

[0307] A ligand unit is a targeting agent that specifically binds to a target moiety. The ligand is capable of specifically binding to cellular components or other target molecules of interest. The target moiety or target is typically located on the cell surface. In some aspects, the role of the ligand unit is 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}.

[0308] As used herein, "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 are capable of specifically binding 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).

[0309] 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.

[0310] The term "inhibition" or "suppression of" refers to reducing the amount that can be detected, or completely blocking it.

[0311] The term "cancer" refers to a physiological condition or disease characterized by disordered cell growth. "Tumor" includes cancer cells.

[0312] The term "autoimmune disease" refers to a disease or disorder that affects an individual's own tissues or proteins.

[0313] The term "drug" refers to cytotoxic drugs, denoted by 'd', which are chemical molecules that strongly disrupt the normal growth of tumor cells. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations; however, due to their lack of specificity, they can also induce apoptosis in 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.

[0314] The term "linker" or "linker fragment" or "linker unit" refers 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 linkers before being linked to a drug.

[0315] 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.

[0316] 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.

[0317] 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.

[0318] The amino acid three-letter codes and single-letter codes used in this disclosure are as described in J.boil.Chem.1968,243,3558.

[0319] 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.

[0320] The term "substituted alkyl" refers to an alkyl group in which hydrogen is replaced by a substituent group. Unless otherwise stated in the text, the substituent group of an alkyl 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.

[0321] 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 1 to 12 carbon atoms, and more preferably alkylene 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-butylene (-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.

[0322] 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.

[0323] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms (i.e., "C3-C20 cycloalkyl"), preferably 3 to 12 carbon atoms (i.e., "C3-C12 cycloalkyl"), more preferably 3 to 10 carbon atoms (i.e., "C3-C10 cycloalkyl"), and most preferably 3 to 8 carbon atoms (i.e., "C3-C8 cycloalkyl"). Non-limiting examples of 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.

[0324] 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.

[0325] The term "cycloalkyl" refers to an alkyl group that is substituted with one or more cycloalkyl groups, preferably 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.

[0326] The term "halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens, wherein the alkyl group is as defined above. For example, halogenated C1-C6 alkyl groups.

[0327] 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.

[0328] The term "C6-C12 aryl" refers to a group in a carbocyclic aromatic system having 6-12 carbon atoms.

[0329] The term "C6-C10 aryl" refers to a group in a carbocyclic aromatic system with 6-10 carbon atoms, such as phenyl, naphthyl, etc.

[0330] 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 attached 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.

[0331] 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 an aryl, heteroaryl, or heterocyclic group by a substituent group. Unless otherwise stated in the text, the substituents in 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 The following groups are listed: -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. The number of substituents ranges 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.

[0332] The term "hydroxyl group" refers to the -OH group.

[0333] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0334] The term "amino" refers to -NH2. The term "nitro" refers to -NO2.

[0335] The term "amide group" refers to -C(O)N(alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0336] The term "carboxylic acid ester group" refers to -C(O)O (alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0337] This invention also includes various deuterated forms of Formula I. 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 by referring 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.

[0338] The term "antibody" refers to immunoglobulin, 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. Based on this, 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, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further divide them 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 the constant region. Each of the five classes of Ig can have either a κ chain or a λ chain. The antibodies described in this invention are preferably specific antibodies against cell surface antigens on target cells. Non-limiting examples include the following antibodies: anti-EGFRvIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-1 antibody, anti-CanAg / AFP antibody, and anti-cladin antibody. 18.2 Antibodies, anti-Mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT / CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7 / CS1 antibody, anti-NaPi2B / SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD276) antibody, anti-PTK7 / CCK4 antibody, anti-PRLR antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-Nectin 4. One or more of the following antibodies: anti-TROP-2 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-Integrin Av antibody, anti-CAIX antibody, anti-P-cadherin antibody, anti-GD3 antibody, anti-Cadherin 6 antibody, anti-LAMP1 antibody, anti-FLT3 antibody, anti-BCMA antibody, anti-CD79b antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody, or anti-CD123 antibody.

[0339] The term "solvent" or "solvent compound" refers to the ligand-drug conjugate of the present 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.

[0340] 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 the present 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 assay, and HPLC characterization.

[0341] 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.

[0342] The loading of ligand-cytotoxic drug conjugates can be controlled using the following non-limiting methods, including:

[0343] (1) Control the molar ratio of the ligation reagent and the monoclonal antibody.

[0344] (2) Control the reaction time and temperature.

[0345] (3) Choose different reaction reagents.

[0346] For the preparation of conventional pharmaceutical compositions, please refer to the Chinese Pharmacopoeia.

[0347] The terms "pharmaceutically acceptable salt" or "medicinal salt" refer to the salt of the ligand-drug conjugate of the present invention, or the salt of the compound described in the present invention, which is safe and effective when used in mammals and has the intended biological activity. The ligand-drug conjugate of the present 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.

[0348] The term "pharmaceutically acceptable salt" or "medicinal salt" refers to a salt of the antibody-drug conjugate of the present invention, or a salt of the compound described in the present invention, which is safe and effective in mammalian use and has the intended biological activity. The ligand-drug conjugate of the present 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.

[0349] "Acidic amino acids" refer to amino acids with an isoelectric point of 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.

[0350] "Natural amino acids" refer to amino acids synthesized by organisms. 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.

[0351] "Non-natural amino acids" refer to amino acids obtained through synthetic means.

[0352] 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.

[0353] Example 1

[0354] Synthesis of compound M1:

[0355]

[0356] In a 5000 mL single-necked flask, N-fluorenemethoxycarbonyl-glycine-glycine (100 g, 282 mmol, 1.0 eq), lead tetraacetate (175 g, 395 mmol, 1.4 eq), 2000 mL of dry tetrahydrofuran, and 670 mL of toluene were added. 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.

[0357] Example 2

[0358] Synthesis of compound M3:

[0359]

[0360] Compound SM-2 (synthesized according to the method disclosed in patent 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 temperature was lowered to 50 °C, and the solvent was removed by rotary evaporation under reduced pressure. 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.

[0361] In a 1000 mL single-necked flask, 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. The mixture was reacted at room temperature for 30 min (monitored by TLC). Insoluble matter was filtered off. The reaction solution was directly purified by preparative purification. The preparative solution was concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile. The solution was then lyophilized to obtain compound M3 (31.5 g), yield 64%. LC-MS: [M+H] + =565.1.

[0362] Example 3

[0363] Synthesis of compound ent-M3:

[0364]

[0365] Following the synthetic route in Example 2, compound ent-M3 (27.8 g) was obtained; LC-MS: [M+H] + =565.2.

[0366] Example 4

[0367] Synthesis of Compound 1:

[0368]

[0369] Step 1: Compound 1a

[0370] 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 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, 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 = 10:1-5:1-1:1) to give 1a (4 g), yield 52%; LC-MS: [M+H] + =475.18.

[0371] Step 2: Compound 1b

[0372] Add 1a (2g, 4.2mmol) and 10mL DMF to a 25mL single-necked flask, stir at 0℃, add DBU (766mg, 5.04mmol), react for 1h, monitor by TLC until Fmoc deprotection is complete, and then set aside for use;

[0373] In a separate 25 mL single-necked flask, add M4 (prepared according to the method disclosed in patent 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. Add DIPEA (830 μL, 5.04 mmol) under ice-water bath conditions and continue stirring for 30 min. Add the above reaction solution to the reaction flask and allow the mixture to react at room temperature. After the reaction is complete as monitored by HPLC, the reaction solution is purified by preparative HPLC to obtain the product preparation solution. The preparation solution is extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure to obtain solid 1b (1.7 g), yield 63%; LCMS: [M+H] + =648.26.

[0374] Step 3: Compound 1c

[0375] Add 1b (900 mg, 1.39 mmol) to a 25 mL single-necked flask, dissolve it in 15 mL of DMF, then add 900 mg of 5% Pd / C, and hydrogenate for 2 hours. After the reaction is complete, filter to obtain the filtrate, which is used directly in the next step of the reaction without purification.

[0376] Step 4: Compound 1d

[0377] The crude product 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 brought to room temperature and reacted for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by HPLC to obtain the preparative solution, which was then lyophilized to obtain 1d (504 mg). LC-MS: [M+H] + =804.4.

[0378] Step 5: Compound 1e

[0379] Add 1d (500 mg, 0.62 mmol), M5 (310 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF to a 50 mL single-necked flask. Add DIPEA (378 μL, 2.29 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 HPLC to obtain the preparative solution of compound 1e. The preparative solution is lyophilized to obtain 1e (210 mg). LC-MS: [M+H] + =1221.6.

[0380] Step 6: Compound 1

[0381] Ie (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 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.

[0382] Example 5

[0383] Synthesis of compound 2:

[0384]

[0385] Following the synthetic route in Example 4, compound 2 (51 mg) was obtained; LC-MS: [M+H] + =1065.3.

[0386] Example 6

[0387] Synthesis of compound 3:

[0388]

[0389] Step 1: Compound 3a

[0390] 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, 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 3a (4.2 g), yield 52%; LC-MS: [M+H] + =503.3.

[0391] Step 2: Compound 3b

[0392] Add 3a (2g, 4.0mmol) and 10mL DMF to a 25mL single-necked flask, stir at 0℃, add DBU (760mg, 5.0mmol), react for 1h, monitor by TLC until Fmoc deprotection is complete, and set aside for use;

[0393] In a separate 25 mL single-necked flask, add 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. Add DIPEA (823 μL, 5.04 mmol) under ice-water bath conditions and continue stirring for 30 min. Add the above reaction solution to the reaction flask and allow the mixture to react at room temperature. After the reaction is complete as monitored by HPLC, the reaction solution is purified by preparative HPLC to obtain the product preparation solution. The preparation solution is extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure to obtain solid 3b (1.4 g), yield 53%; LC-MS: [M+H] + =676.2.

[0394] Step 3: Compound 3c

[0395] Add 3b (700 mg, 1.04 mmol) to a 25 mL single-necked flask, dissolve it in 10 mL of DMF, then add 700 mg of 5% Pd / C, and hydrogenate for 1.5 h. After the reaction is complete, filter to obtain the filtrate, which is used directly in the next step of the reaction without purification.

[0396] Step 4: Compound 3d

[0397] The crude product 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 brought to room temperature and reacted for 1 hour. The reaction was monitored by HPLC until completion. The reaction solution was purified by HPLC to obtain the preparative solution, which was then lyophilized to obtain 3d (486 mg). LC-MS: [MH] - =830.5.

[0398] Step 5: Compound 3e

[0399] Add 3d (300 mg, 0.36 mmol), M5 (180 mg, 0.36 mmol), PyBOP (260 mg, 0.5 mmol), HOBt (67 mg, 0.5 mmol), and 10 mL of DMF to a 50 mL single-necked flask. Add DIPEA (219.5 μL, 1.33 mmol) under ice-water bath conditions, and react at room temperature for 3 h. After the reaction is complete as monitored by HPLC, the reaction solution is purified by HPLC to obtain the preparative solution of compound 3e. The preparative solution is lyophilized to obtain 3e (157 mg). LC-MS: [M+H] + =1249.6.

[0400] Step 6: Compound 3

[0401] 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 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.

[0402] Example 7

[0403] Synthesis of compound 4:

[0404]

[0405] Following the synthetic route of Example 6, compound 4 (60 mg) was obtained; LC-MS: [M+H] + =1093.2.

[0406] Example 8

[0407] Synthesis of compound 5A:

[0408]

[0409] Step 1: Compound 5a

[0410] In a 25 mL single-necked flask, M1 (500 mg, 1.4 mmol, 1.0 eq), p-toluenesulfonic acid monohydrate (26 mg, 0.1 mmol, 0.1 eq), and 10 mL of THF were added. After stirring thoroughly, the mixture was cooled to 0 °C, and then L-benzyl lactate (1.2 g, 7.0 mmol, 5 eq) was slowly added. After the addition was complete, the mixture was brought 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 5a (400 mg).

[0411] LC-MS: [M+NH4] + =506.2.

[0412] 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).

[0413] Step 2: Compound 5b

[0414] 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.

[0415] In a separate 25 mL single-necked flask, add M4 (372 mg, 0.9 mmol, 1.1 eq), PyBOP (852 mg, 1.6 mmol, 2.0 eq), and 3 mL DMF. Stir at room temperature for 5 minutes, then add reaction solution ①. Incubate at room temperature and monitor the reaction by HPLC. After the reaction is complete, purify the reaction solution by HPLC to obtain compound 5b (326 mg); LC-MS: [M+NH4] + =679.2.

[0416] Step 3: Compound 5c

[0417] Add 5b (4.0 g, 6.05 mmol, 1.0 eq) to a 100 mL single-necked flask, dissolve in DMF (60 mL), then add 5% Pd / C (4 g), and hydrogenate at room temperature for 4 h (the reaction progress was monitored by HPLC). Filter the Pd / C, and place the filtrate directly in an ice-water bath (approximately 0 °C) without concentration until use.

[0418] Step 4: Compound 5d

[0419] The crude product 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 HPLC to obtain the preparative solution, which was then lyophilized to obtain 5d (3.15 g). LC-MS: [MH] - =816.3.

[0420] Step 5: Compound 5e

[0421] Add 5d (2.07 g, 2.53 mmol, 1.0 eq), 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) to a 100 mL single-necked flask. Add DIPEA (1.05 mL, 1.5 eq) under ice-water bath conditions, and 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 give compound 5e (1.92 g), yield 61%. LC-MS: [M+H] + =1235.4.

[0422] Step 6: Compound 5A

[0423] 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. 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). Compound 5A was prepared by acid method. The prepared solution was concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile, and then lyophilized to give compound 5A (786 mg) in 90% yield.

[0424] LC-MS: [M+H] + =1079.4;

[0425] 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).

[0426] Example 9

[0427] Synthesis of compound 5B:

[0428]

[0429] Step 1: Compound 5d-1

[0430] 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. 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 temperature was raised to 20 ± 5 °C and the reaction was carried out for 1 h. The reaction was monitored by HPLC until completion. After the reaction, the product was purified by HPLC, collected, and lyophilized to obtain compound 5d-1 (200 mg), yield 54%. LC-MS: [MH] - =816.3.

[0431] Step 2: Compound 5e-1

[0432] Compound 5d-1 (200 mg, 0.24 mmol, 1.0 eq), 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 HPLC, and the product solution was collected and lyophilized to obtain compound 5e-1 (162.8 mg). LC-MS: [M+H] + =1235.4.

[0433] Step 3: Compound 5B

[0434] 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 was monitored by HPLC. The evaporated solid was directly purified by HPLC, and 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.

[0435] Example 10

[0436] Preparation of compound 6A:

[0437]

[0438] Following the synthetic route in Example 8, compound 6A (71 mg) was obtained; LC-MS: [M+H] + =1079.4.

[0439] Example 11

[0440] Preparation of compound 6B:

[0441]

[0442] Following the synthetic route of Example 9, compound 6B (59 mg) was obtained; LC-MS: [M+H] + =1079.4.

[0443] Example 12

[0444] Preparation of compounds 7A and 7B:

[0445]

[0446] Step 1: Compound 7a

[0447] M1 (10 g, 27.1 mmol), benzyl 3,3,3-trifluorolactate (prepared according to the method disclosed in patent 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 obtain 5.15 g of the target analyte, with a yield of 35.1%; LC-MS: [M+H] + =543.17.

[0448] Step 2: Compound 7b

[0449] Add 7a (5g, 9.2mmol) and 15mL DMF to a 50mL single-necked flask. After dissolving, add DBU (1.68g, 11mmol) under an ice-water bath and react for 1h. This reaction solution is recorded as ①.

[0450] In a separate 50 mL single-necked flask, add M4 (3.8 g, 9.2 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol), and 10 mL of DMF. After dissolving completely, add DIPEA (1.82 mL, 11 mmol) under ice-water bath conditions and continue the reaction for 30 min. Then add reaction solution ① and allow the mixture to react at room temperature for 2 h. Monitor the reaction progress by HPLC. After the reaction is complete, purify the reaction solution by HPLC 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 4.1 g of solid, with a yield of 62.3%. LC-MS: [M+H] + =716.25.

[0451] Step 3: Compound 7d

[0452] 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 reaction was carried out for 2 h. After the reaction was complete, the mixture was filtered, and the filtrate was placed in an ice-water bath. DIPEA (228 μL, 1.38 mmol) was added, followed by M3 (712 mg, 1.26 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 HPLC to obtain the preparative solution. The preparative solution was lyophilized to obtain 525 mg of product, with a yield of 47.9%. LC-MS: [MH] - =870.33.

[0453] Step 4: Compound 7e

[0454] Add 7d (500 mg, 0.57 mmol), M5 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF to a 50 mL single-necked flask. Add DIPEA (378 μL, 2.29 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 HPLC to obtain preparative solutions of compounds 7e-1 and 7e-2. The preparative solutions are 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.

[0455] Step 5: Compound 7A

[0456]

[0457] 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 reaction was carried out 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 high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 52 mg of solid; TOF result: 1133.3613.

[0458] Step 6: Compound 7B

[0459]

[0460] 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 high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 63 mg of solid; TOF result: 1133.3668.

[0461] Example 13

[0462] Synthesis of compounds 8A and 8B:

[0463]

[0464] Step 1: Compound 8d

[0465] Add 7c (900 mg, 1.83 mmol) to a 25 mL single-necked flask, dissolve in 20 mL of LMF, then add DIPEA (303 μL, 1.83 mmol), followed by ent-M3 (1034 mg, 1.83 mmol). After the addition is complete, bring to room temperature and react for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by HPLC to obtain the preparative solution. Lyophilization of the preparative solution yielded 613 mg of product, with a yield of 38.5%. LC-MS: [MH] - =870.32.

[0466] Step 2: Compounds 8e-1 and 8e-2

[0467] Add 8d (500 mg, 0.57 mmol), M5 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF to a 50 mL single-necked flask. Add DIPEA (378 μL, 2.29 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 HPLC to obtain preparative solutions of compounds 8e-1 and 8e-2. The preparative solutions are 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.

[0468] Step 3: Compound 8A

[0469]

[0470] 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 reaction was carried out 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 high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 50 mg of solid; TOF result: 1133.3623.

[0471] Step 4: Compound 8B

[0472]

[0473] 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 reaction was carried out 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 high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 58 mg of solid; TOF result: 1133.3653.

[0474] Example 14

[0475] Synthesis of compound 9A:

[0476]

[0477] Step 1: Compound 9a

[0478] 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. 2-hydroxy-2-cyclopropylbenzyl acetate (prepared according to the method disclosed in patent US20050020645A1) (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 9a (3.7 g), yield 45%; LC-MS: [M+H] + =501.5.

[0479] Step 2: Compound 9b

[0480] Add 9a (2g, 4.0mmol) and 10mL DMF to a 25mL single-necked flask, stir at 0℃, add DBU (760mg, 5.0mmol), react for 1h, monitor by TLC until Fmoc deprotection is complete, and then set aside for use;

[0481] In a separate 25 mL single-necked flask, add 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. Add DIPEA (823 μL, 5.04 mmol) under ice-water bath conditions and continue stirring for 30 min. Add the above reaction solution to the reaction flask and allow the mixture to react at room temperature. After the reaction is complete as monitored by HPLC, the reaction solution is purified by preparative HPLC to obtain the product preparation solution. The preparation solution is extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure to obtain 1.5 g of solid, yield 56%. LC-MS: [M+H] + =674.7.

[0482] Step 3: Compound 9c

[0483] Add 9b (900 mg, 1.3 mmol) to a 25 mL single-necked flask, dissolve it in 10 mL of DMF, then add 900 mg of 5% Pd / C, and hydrogenate for 1.5 h. After the reaction is complete, filter to obtain the filtrate, which is used directly in the next step of the reaction without purification.

[0484] Step 4: Compound 9d

[0485] The crude product 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 brought to room temperature and reacted for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by HPLC to obtain the preparative solution, which was then lyophilized to obtain 9d (529 mg). LC-MS: [MH] - =828.4.

[0486] Step 5: Compound 9e

[0487] Add 9d (500 mg, 0.6 mmol), M5 (300 mg, 0.6 mmol), PyBOP (416 mg, 0.8 mmol), HOBt (108 mg, 0.5 mmol), and 15 mL of DMF to a 50 mL single-necked flask. Add DIPEA (351 μL, 2.13 mmol) under ice-water bath conditions, and react at room temperature for 3 h. After the reaction is complete as monitored by HPLC, the reaction solution is purified by HPLC to obtain the preparative solution of compound 9e. The preparative solution is lyophilized to obtain 9e (257 mg). LC-MS: [M+H] + =1247.5.

[0488] Step 6: Compound 9A

[0489] 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 reaction was carried out 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 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.

[0490] Example 15

[0491] Synthesis of compound 9B:

[0492]

[0493] Following the synthetic route of Example 14, compound 9B (44 mg) was obtained; LC-MS: [M+H] + =1091.3.

[0494] Example 16

[0495] Synthesis of compound 10A:

[0496]

[0497] Step 1: Compound 10a

[0498] 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 3-hydroxy-2-cyclopropylpropionate (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, 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 10a (4.9 g), yield 58%; LC-MS: [M+H] + =515.4.

[0499] Step 2: Compound 10b

[0500] Add 10a (4g, 7.8mmol) and 10mL DMF to a 25mL single-necked flask, stir at 0℃, add DBU (1.2g, 8.0mmol), react for 1h, monitor by TLC until Fmoc deprotection is complete, and set aside for use;

[0501] In a separate 25 mL single-necked flask, add 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. Add DIPEA (1.65 mL, 10.1 mmol) under ice-water bath conditions and continue stirring for 50 min. Add the above reaction solution to the reaction flask and allow the mixture to react at room temperature. After the reaction is complete as monitored by HPLC, the reaction solution is purified by preparative HPLC to obtain the product preparation solution. The preparation solution is extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure to obtain 2.3 g of solid, yield 42%. LC-MS: [M+H] + =688.8.

[0502] Step 3: Compound 10c

[0503] Add 10b (1.0 g, 1.45 mmol) to a 25 mL single-necked flask, dissolve it in 15 mL of DMF, then add 1.0 g of 5% Pd / C, and hydrogenate for 1.5 h. After the reaction is complete, filter to obtain the filtrate, which is used directly in the next step of the reaction without purification.

[0504] Step 4: Compound 10d

[0505] The crude product 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 HPLC to obtain the preparative solution, which was then lyophilized to obtain 10d (499 mg). LC-MS: [MH] - =842.4.

[0506] Step 5: Compound 10e

[0507] Add 10d (400 mg, 0.48 mmol), M5 (240 mg, 0.48 mmol), PyBOP (250 mg, 0.48 mmol), HOBt (104 mg, 0.48 mmol), and 15 mL of DMF to a 50 mL single-necked flask. Add DIPEA (330 μL, 2.0 mmol) under ice-water bath conditions, and react at room temperature for 3 h. After the reaction is complete as monitored by HPLC, the reaction solution is purified by HPLC to obtain the preparative solution of compound 10e. The preparative solution is lyophilized to obtain 10e (188 mg). LC-MS: [M+H] + =1261.5.

[0508] Step 6: Compound 10A

[0509] 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 HPLC to obtain the product preparation solution, which was then lyophilized to obtain solid compound 10A (61 mg); LC-MS: [M+H] + =1105.4.

[0510] Example 17

[0511] Synthesis of compound 10B:

[0512]

[0513] Following the synthetic route of Example 16, compound 10B (75 mg) was obtained; LC-MS: [M+H] + =1105.4.

[0514] Example 18

[0515] Synthesis of compound 11A:

[0516]

[0517] Step 1: Compound 11a

[0518] 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. 2-hydroxy-2-cyclobutylacetic 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 obtain 11a (5.1 g), yield 62%; LC-MS: [M+H] + =515.7.

[0519] Step 2: Compound 11b

[0520] Add 11a (4g, 7.8mmol) and 10mL DMF to a 25mL single-necked flask, stir at 0℃, add DBU (1.2g, 8.0mmol), react for 1h, monitor by TLC until Fmoc deprotection is complete, and then set aside for use;

[0521] In a separate 25 mL single-necked flask, add 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. Add DIPEA (1.63 mL, 10.0 mmol) under ice-water bath conditions and continue stirring for 40 min. Add the above reaction solution to the reaction flask and allow the mixture to react at room temperature. After the reaction is complete as monitored by HPLC, the reaction solution is purified by preparative HPLC to obtain the product preparation solution. The preparative solution is extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure to obtain 2.3 g of solid, yield 42%. LC-MS: [M+H] + =688.3.

[0522] Step 3: Compound 11c

[0523] 11b (2.0 g, 2.9 mmol) was added to a 25 mL single-necked flask and dissolved in 25 mL of DMF. Then, 2.0 g of 5% Pd / C was added, and the hydrogenation reaction was carried out for 3 h. After the reaction was completed, the mixture was filtered to obtain the filtrate, which was used directly in the next step of the reaction without purification.

[0524] Step 4: Compound 11d

[0525] The crude product 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 HPLC to obtain the preparative solution, which was then lyophilized to obtain 11d (934 mg). LC-MS: [MH] - =842.4.

[0526] Step 5: Compound 11e

[0527] In a 50 mL single-necked flask, 11d (800 mg, 0.96 mmol), 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. 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 complete as monitored by HPLC, the reaction solution was purified by HPLC to obtain the preparative solution of compound 11e. The preparative solution was lyophilized to obtain 11e (401 mg). LC-MS: [M+H] + =1261.4.

[0528] Step 6: Compound 11A

[0529] 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 HPLC to obtain the product preparation solution, which was then lyophilized to obtain solid compound 11A (86 mg); LC-MS: [M+H] + =1105.4.

[0530] Example 19

[0531] Synthesis of Compound 11B

[0532]

[0533] Following the synthetic route of Example 18, compound 11B (50 mg) was obtained. LC-MS: [M+H] + 1105.4.

[0534] Example 20

[0535] Synthesis of compound 12A:

[0536]

[0537] Step 1: Compound 12a

[0538] 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 3-hydroxy-2-cyclobutylpropionate (prepared according to the method disclosed in patent WO2009011285A1) (7.2 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 12a (4.5 g), yield 52%; LC-MS: [M+H] + =529.4.

[0539] Step 2: Compound 12b

[0540] Add 12a (4g, 7.6mmol) and 10mL DMF to a 25mL single-necked flask, stir at 0℃, add DBU (1.2g, 8.0mmol), react for 1h, monitor by TLC until Fmoc deprotection is complete, and then set aside for use;

[0541] In a separate 25 mL single-necked flask, add M4 (3.2 g, 7.6 mmol), PyBOP (4.7 g, 9.0 mmol), HOBt (1.22 g, 9.0 mmol), and 10 mL LDM. Add DIPEA (1.49 mL, 0.9 mmol) under ice-water bath conditions and continue stirring for 30 min. Add the above reaction solution to the reaction flask and allow the mixture to react at room temperature. After the reaction is complete as monitored by HPLC, the reaction solution is purified by preparative HPLC to obtain the product preparation solution. The preparative solution is extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure to obtain 2.0 g of solid, yield 37%. LC-MS: [M+H] + =702.8.

[0542] Step 3: Compound 12c

[0543] Add 12b (1.0 g, 1.43 mmol) to a 25 mL single-necked flask, dissolve it in 15 mL of DMF, then add 1.0 g of 5% Pd / C, and hydrogenate for 1.5 h. After the reaction is complete, filter to obtain the filtrate, which is used directly in the next step of the reaction without purification.

[0544] Step 4: Compound 12d

[0545] The crude product 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 brought to room temperature and reacted for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by HPLC to obtain the preparative solution, which was then lyophilized to obtain 12d (522 mg). LC-MS: [MH] - =856.4.

[0546] Step 5: Compound 12e

[0547] Add 12d (400 mg, 0.47 mmol), M5 (240 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt (101 mg, 0.47 mmol), and 15 mL of DMF to a 50 mL single-necked flask. Add DIPEA (330 μL, 2.0 mmol) under ice-water bath conditions, and react at room temperature for 3 h. After the reaction is complete as monitored by HPLC, the reaction solution is purified by HPLC to obtain the preparative solution of compound 12e. The preparative solution is lyophilized to obtain 12e (198 mg). LC-MS: [M+H] + =1275.4.

[0548] Step 6: Compound 12A

[0549] 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 HPLC to obtain the product preparation solution, which was then lyophilized to obtain solid compound 12A (55 mg); LC-MS: [M+H] + =1119.4.

[0550] Example 21

[0551] Synthesis of compound 12B:

[0552]

[0553] Following the synthetic route of Example 20, compound 12B (50 mg) was obtained; LC-MS: [M+H] + =1119.4.

[0554] Example 22

[0555] Synthesis of compound 13A:

[0556]

[0557] Step 1: Compound 13a

[0558] 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. 2-hydroxy-2-cyclopentylacetic 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 obtain 13a (4.6 g), yield 53%; LC-MS: [M+H] + =529.5.

[0559] Step 2: Compound 13b

[0560] Add 13a (4g, 7.6mmol) and 10mL DMF to a 25mL single-necked flask, stir at 0℃, add DBU (1.17g, 7.8mmol), react for 1h, monitor by TLC until Fmoc deprotection is complete, and set aside for use;

[0561] In a separate 25 mL single-necked flask, add 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. Add DIPEA (1.39 mL, 0.85 mmol) under ice-water bath conditions and continue stirring for 30 min. Add the above reaction solution to the reaction flask and allow the mixture to react at room temperature. After the reaction is complete as monitored by HPLC, the reaction solution is purified by preparative HPLC to obtain the product preparation solution. The preparative solution is extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure to obtain 2.1 g of solid, yield 39%. LC-MS: [M+H] + =702.8.

[0562] Step 3: Compound 13c

[0563] Add 13b (1.5 g, 1.87 mmol) to a 25 mL single-necked flask, dissolve it in 25 mL of DMF, then add 1.5 g of 5% Pd / C, and hydrogenate for 3 h. After the reaction is complete, filter to obtain the filtrate, which is used directly in the next step of the reaction without purification.

[0564] Step 4: Compound 13d

[0565] The crude product 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 brought to room temperature and reacted for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by HPLC to obtain the preparative solution, which was then lyophilized to obtain 13d (519 mg). LC-MS: [MH] - =856.6.

[0566] Step 5: Compound 13e

[0567] 13d (400 mg, 0.47 mmol), 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 HPLC to obtain the preparative solution of compound 13e. The preparative solution was lyophilized to obtain 13e (187 mg). LC-MS: [M+H] + =1275.5.

[0568] Step 6: Compound 13A

[0569] 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 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.

[0570] Example 23

[0571] Synthesis of compound 13B:

[0572]

[0573] Following the synthetic route of Example 22, compound 13B (51 mg) was obtained; LC-MS: [M+H] + =1119.6.

[0574] Example 24

[0575] Synthesis of compound 14A:

[0576]

[0577] Step 1: Compound 14a

[0578] 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 3-hydroxy-2-cyclopentylpropionate (synthesized according to the method disclosed in patent WO2009011285A1) (7.6 g, 32.6 mmol) 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 14a (4.4 g), yield 49%; LC-MS: [M+H] + =543.6.

[0579] Step 2: Compound 14b

[0580] Add 14a (4g, 7.4mmol) and 10mL DMF to a 25mL single-necked flask, stir at 0℃, add DBU (1.2g, 8.0mmol), react for 1h, monitor by TLC until Fmoc deprotection is complete, and then set aside for use;

[0581] In a separate 25 mL single-necked flask, add M4 (3.1 g, 7.4 mmol), PyBOP (4.6 g, 8.8 mmol), HOBt (1.19 g, 8.8 mmol), and 10 mL LDM. Add DIPEA (1.49 mL, 9.0 mmol) under ice-water bath conditions and continue stirring for 30 min. Add the above reaction solution to the reaction flask and allow the mixture to react at room temperature. After the reaction is complete as monitored by HPLC, the reaction solution is purified by preparative HPLC to obtain the product preparation solution. The preparative solution is extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure to obtain 2.6 g of solid, yield 49%. LC-MS: [M+H] + =716.4.

[0582] Step 3: Compound 14c

[0583] Add 14b (1.0 g, 1.4 mmol) to a 25 mL single-necked flask, dissolve it in 15 mL of DMF, then add 1.0 g of 5% Pd / C, and hydrogenate for 1.5 h. After the reaction is complete, filter to obtain the filtrate, which is used directly in the next step of the reaction without purification.

[0584] Step 4: Compound 14d

[0585] The crude product 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 reach room temperature and reacted for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by HPLC to obtain the preparative solution, which was then lyophilized to obtain 14d (500 mg). LC-MS: [MH] - =870.5.

[0586] Step 5: Compound 14e

[0587] Add 14d (400 mg, 0.46 mmol), M5 (235 mg, 0.46 mmol), PyBOP (245 mg, 0.46 mmol), HOBt (99 mg, 0.46 mmol), and 15 mL of DMF to a 50 mL single-necked flask. Add DIPEA (331 μL, 2.0 mmol) under ice-water bath conditions, and react at room temperature for 3 h. After the reaction is complete as monitored by HPLC, the reaction solution is purified by HPLC to obtain the preparative solution of compound 14e. The preparative solution is lyophilized to obtain 14e (146 mg). LC-MS: [M+H] + =1289.5.

[0588] Step 6: Compound 14A

[0589] 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 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.

[0590] Example 25

[0591] Synthesis of compound 14B:

[0592]

[0593] Following the synthetic route of Example 24, compound 14B (48 mg) was obtained; LC-MS: [M+H] + =1133.4.

[0594] Example 26

[0595] Synthesis of compounds 15A and 15B:

[0596]

[0597] Step 1: Compound 15a

[0598] 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 obtain 5.67 g of the target compound, with a yield of 42%; LC-MS: [M+H] + =503.5.

[0599] Step 2: Compound 15b

[0600] Add 15a (5g, 9.95mmol) and 15mL DMF to a 50mL single-necked flask, dissolve them, and then add DBU (1.68g, 11mmol) under an ice-water bath. React for 1h and record the reaction solution as ①.

[0601] In a separate 50 mL single-necked flask, add M4 (4.1 g, 10.0 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol), and 10 mL of DMF. After dissolving completely, add DIPEA (1.82 mL, 11 mmol) under an ice-water bath and continue the reaction for 40 min. Then add reaction solution ① and raise the temperature to room temperature for 2 h. Monitor the reaction progress by HPLC. After the reaction is complete, purify the reaction solution by HPLC 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 4.6 g of solid, yield 68%; LC-MS: [M+H] + =676.7.

[0602] Step 3: Compound 15d

[0603] Add 2.0 g (2.96 mmol) of 15b to a 25 mL single-necked flask, dissolve in 15 mL of DMF, then add 2.0 g of 5% Pd / C. Hydrogenate for 2 h. After the reaction is complete, filter. Place the filtrate in an ice-water bath, add 496 μL (3.0 mmol) of DIPEA, then add 1.7 g (2.96 mmol) of M3. After the addition is complete, bring to room temperature and react for 1 h. Monitor the reaction completion by HPLC. Purify the reaction solution by HPLC to obtain the preparative solution. Lyophilize the preparative solution to obtain 1120.0 mg of product, yield 45%. LC-MS: [MH] - =830.3.

[0604] Step 4: Compound 15e

[0605] Add 15d (500 mg, 0.60 mmol), 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 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.

[0606] Step 5: Compound 15A

[0607]

[0608] 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 HPLC to obtain the product preparation solution, which was then lyophilized to obtain 59 mg of solid. LC-MS: [M+H] + =1093.4.

[0609] Step 6: Compound 15B

[0610]

[0611] 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 HPLC to obtain the product preparation solution, which was then lyophilized to obtain 60 mg of solid. LC-MS: [M+H] + =1093.4.

[0612] Example 27

[0613] Synthesis of compounds 16A and 16B:

[0614]

[0615] Following the synthetic route of Example 26, compound 16A (55 mg) was obtained; LC-MS: [M+H] + =1093.4.

[0616]

[0617] Following the synthetic route of Example 26, compound 16B (54 mg) was obtained; LC-MS: [M+H] + =1093.4.

[0618] Example 28

[0619] Synthesis of compounds 17A and 17B:

[0620]

[0621] Step 1: Compound 17a

[0622] M1 (10 g, 27.1 mmol), benzyl 2-hydroxyphenylpropionate (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 obtain 6.13 g of the target compound, with a yield of 40%; LC-MS: [M+H] + =565.6.

[0623] Step 2: Compound 17b

[0624] Add 17a (5g, 8.86mmol) and 15mL DMF to a 50mL single-necked flask. After dissolving, add DBU (1.53g, 10mmol) under an ice-water bath and react for 1h. This reaction solution is recorded as ①.

[0625] In a separate 50 mL single-necked flask, add M4 (3.6 g, 8.86 mmol), PyBOP (5.23 g, 10 mmol), HOBt (1.36 g, 10 mmol), and 10 mL of DMF. After dissolving completely, add DIPEA (1.65 mL, 10 mmol) under ice-water bath conditions and continue the reaction for 30 min. Then add reaction solution ① and allow the mixture to react at room temperature for 2 h. Monitor the reaction progress by HPLC. After the reaction is complete, purify the reaction solution by HPLC 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 5.0 g of solid, yield 77%; LC-MS: [M+H]+ =738.3.

[0626] Step 3: Compound 17d

[0627] 17b (3.0 g, 4.07 mmol) was added to a 25 mL single-necked flask and dissolved in 15 mL of DMF. Then, 3.0 g of 5% Pd / C was added, and the reaction was carried out for 2 h. After the reaction was complete, the mixture was filtered, and the filtrate was placed in an ice-water bath. DIPEA (744 μL, 4.5 mmol) was added, followed by M3 (2.34 g, 4.07 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 HPLC to obtain the preparative solution. The preparative solution was lyophilized to obtain 1.2 g of product, with a yield of 33%. LC-MS: [MH] - =892.4.

[0628] Step 4: Compound 17e

[0629] Add 17d (500 mg, 0.56 mmol), M5 (300 mg, 0.56 mmol), PyBOP (438 mg, 0.84 mmol), HOBt (113 mg, 0.84 mmol), and 15 mL of DMF to a 50 mL single-necked flask. Add DIPEA (330 μL, 2.0 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 HPLC to obtain preparative solutions of compounds 17e-1 and 17e-2. The preparative solutions are 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.

[0630] Step 5: Compound 17A

[0631]

[0632] 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 high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 43 mg of solid. LC-MS: [M+H] + =1155.4.

[0633] Step 6: Compound 17B

[0634]

[0635] 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 high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 40 mg of solid. LC-MS: [M+H] + =1155.4.

[0636] Example 29

[0637] Synthesis of compounds 18A and 18B:

[0638]

[0639] Following the synthetic route of Example 28, compound 18A (54 mg) was obtained; LC-MS: [M+H]+=1155.4.

[0640]

[0641] Following the synthetic route of Example 28, compound 18B (55 mg) was obtained; LC-MS: [M+H] + =1155.4.

[0642] Example 30

[0643] Synthesis of compounds 19A and 19B:

[0644]

[0645] Step 1: Compound 19a

[0646] 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 obtain 4.97 g of the target analyte, with a yield of 36%; LC-MS: [M+H] + =515.2.

[0647] Step 2: Compound 19b

[0648] Add 19a (4g, 7.8mmol) and 10mL DMF to a 50mL single-necked flask, dissolve it, and then add DBU (1.42g, 9.3mmol) under an ice-water bath. React for 1h and record the reaction solution as ①.

[0649] In a separate 50 mL single-necked flask, add 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. After dissolving completely, add DIPEA (1.65 mL, 10 mmol) under ice-water bath conditions and continue the reaction for 30 min. Add reaction solution ① and allow the mixture to react at room temperature for 2 h. Monitor the reaction progress using HPLC. After the reaction is complete, purify the reaction solution using HPLC 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, yield 78%. LC-MS: [M+H] + =688.3.

[0650] Step 3: Compound 19d

[0651] 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 reaction was carried out for 2 h. After the reaction was complete, the mixture was filtered, and the filtrate was placed in an ice-water bath. DIPEA (248 μL, 1.5 mmol) was added, followed by M3 (720 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 HPLC to obtain the preparative solution. The preparative solution was lyophilized to obtain 503 mg of product, with a yield of 41%. LC-MS: [MH] - =842.3.

[0652] Step 4: Compounds 19e-1 and 19e-2

[0653] Add 19d (500 mg, 0.59 mmol), M5 (317 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol), and 10 mL of DMF to a 50 mL single-necked flask. Add DIPEA (292 μL, 1.77 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 HPLC to obtain preparative solutions of compounds 19e-1 and 19e-2. The preparative solutions are 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.

[0654] Step 5: Compound 19A

[0655]

[0656] 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 HPLC to obtain the product preparation solution, which was then lyophilized to obtain 55 mg of solid. LC-MS: [M+H] + =1105.4.

[0657] Step 6: Compound 19B

[0658]

[0659] 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 high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 58 mg of solid. LC-MS: [M+H] + =1105.4.

[0660] Example 31

[0661] Synthesis of compounds 20A and 20B:

[0662]

[0663] Step 1: Compound 20a

[0664] M1 (10 g, 27.1 mmol), benzyl 2-hydroxycyclopropylpropionate (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 obtain 5.09 g of the target analyte; LC-MS: [M+H] + =529.2.

[0665] Step 2: Compound 20b

[0666] Add 20a (4g, 7.6mmol) and 10mL DMF to a 50mL single-necked flask. After dissolving, add DBU (1.39g, 9.1mmol) under an ice-water bath and react for 1h. This reaction solution is recorded as ①.

[0667] In a separate 50 mL single-necked flask, add 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. After dissolving completely, add DIPEA (1.65 mL, 10 mmol) under ice-water bath conditions and continue the reaction for 30 min. Add reaction solution ① and allow the mixture to react at room temperature for 2 h. Monitor the reaction progress with HPLC. After the reaction is complete, purify the reaction solution using HPLC 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 4.5 g of solid, yield 84%; LC-MS: [M+H] + =702.3.

[0668] Step 3: Compound 20d

[0669] 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 reaction was carried out for 2 h. After the reaction was complete, the mixture was filtered, and the filtrate was placed in an ice-water bath. DIPEA (248 μL, 1.5 mmol) was added, followed by M5 (708 mg, 1.42 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 HPLC to obtain the preparative solution. The preparative solution was lyophilized to obtain 443 mg of product, with a yield of 36%. LC-MS: [MH] - =856.4.

[0670] Step 4: Compounds 20e-1 and 20e-2

[0671] Add 20d (400 mg, 0.47 mmol), eczema mesylate (250 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol), and 10 mL of DMF to a 50 mL single-necked flask. Add DIPEA (248 μL, 1.5 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 HPLC to obtain preparative solutions of compounds 20e-1 and 20e-2. The preparative solutions are 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.

[0672] Step 5: Compound 20A

[0673]

[0674] 8A (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 HPLC to obtain the product preparation solution, which was then lyophilized to obtain 51 mg of solid. LC-MS: [M+H] + =1119.4.

[0675] Step 6: Compound 20B

[0676]

[0677] 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 HPLC to obtain the product preparation solution, which was then lyophilized to give 47 mg of solid. LC-MS: [M+H] + =1119.4.

[0678] Example 32

[0679] Synthesis of compound 21:

[0680]

[0681] Step 1: Compound SM3-1

[0682] Add 77087-60-6 (100 g, 458 mmol), maleic acid (53.4 g, 460 mmol), TEA (64 mL, 460 mmol), and 1000 mL of toluene to a 2000 mL single-necked flask, and heat to 100 °C for 5 h. After the reaction is complete, cool to room temperature, filter to remove insoluble matter, and concentrate the filtrate to obtain the crude product. The crude product is purified by silica gel column chromatography (PE:EA = 100:1-50:1-20:1) to obtain 75.6 g of the target analyte; LC-MS: [M+H] + =299.1.

[0683] Step 2: Compound (R)-2-hydroxy-1,5-pentanoic acid tert-butyl ester

[0684] 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 obtain 91.2 g of the target analyte; LC-MS: [M+H] + =261.4.

[0685] Step 3: Compound SM3

[0686] (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 SM3-1 (57.3 g, 192 mmol) were added sequentially. The mixture was then 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.

[0687] 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 obtain 50.4 g of SM3; LC-MS: [MH]. - =525.5.

[0688] Step 4: Compound M6

[0689] 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). Insoluble matter was filtered off. The reaction solution was directly purified by preparative purification. The preparative solution was concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile. The solution was then lyophilized to give compound M6 (51.9 g), yield 79%. LC-MS: [M+H] + =693.3.

[0690] Step 5: Compound 21a

[0691] Add 1 g (2.36 mmol) of DMF to a 25 mL single-necked flask, dissolve the DMF in 25 mL of solution, then add 430 μL (2.6 mmol) of DIPEA, followed by 1177 mg (2.36 mmol) of M6. After the addition is complete, bring the mixture to room temperature and react for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by HPLC to obtain the preparative solution, which was then lyophilized to yield 555 mg of the product. LC-MS: [MH] - =931.0.

[0692] Step 6: Compound 21b

[0693] Add 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 to a 100 mL single-necked flask. Add DIPEA (248 μL, 1.5 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 HPLC to obtain the preparative solution of compound 21b. The preparative solution is lyophilized to obtain compound 231 mg. LC-MS: [M+H] + =1349.5.

[0694] Step 7: Compound 21

[0695] 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 HPLC to obtain the product preparation solution, which was then lyophilized to give 103 mg of solid. LC-MS: [M+H] + =1137.5.

[0696] Example 33

[0697] Synthesis of compound 22:

[0698]

[0699] 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.

[0700] Example 34

[0701] Synthesis of compounds 23 and 24:

[0702]

[0703] Starting with compounds M6 and 5c, and following the synthetic route of Example 32, 102 mg of compound 23 was obtained. LC-MS: [M+H] + =1151.4; 99 mg of compound 24 was obtained, LC-MS: [M+H] + =1151.4.

[0704] Example 35

[0705] Synthesis of compounds 25 and 26:

[0706]

[0707] 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.

[0708] Example 36

[0709] Synthesis of compounds 27 and 28:

[0710]

[0711] 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.

[0712] Example 37

[0713] Synthesis of compound 29:

[0714]

[0715] Step 1: Compound SM4-1

[0716] 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 SM4-1 (64.7 g), yield 50%; LC-MS: [M+H] + =299.2.

[0717] Step 2: Compound SM4-2

[0718] SM4-1 (64 g, 215 mmol) was added to a 2000 mL single-necked flask, dissolved in 1000 mL of DMF, followed by DIPEA (71 mL, 430 mmol), and then 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 product; LC-MS: [M+H] + =709.4.

[0719] Step 3: Compound SM4

[0720] 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 purification. 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.

[0721] Step 4: Compound M7

[0722] 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). Insoluble matter was filtered off. The reaction solution was directly purified by preparative purification. The preparative solution was concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile. The solution was then lyophilized to give compound M7 (23.3 g), yield 93%. LC-MS: [M+H] + =695.8.

[0723] Step 5: Compound 29a

[0724] Add 1 g (2.36 mmol) of DMF to a 25 mL single-necked flask, dissolve the DMF in 25 mL of solution, then add 430 μL (2.6 mmol) of DIPEA, followed by 1640 mg (2.36 mmol) of M7. After the addition is complete, bring the mixture to room temperature and react for 1 h. The reaction was monitored by HPLC until completion. The reaction solution was purified by HPLC to obtain the preparative solution, which was then lyophilized to yield 609 mg of the product. LC-MS: [MH] - =1098.5.

[0725] Step 6: Compound 29b

[0726] Add 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 to a 100 mL single-necked flask. Add DIPEA (248 μL, 1.5 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 HPLC to obtain the preparative solution of compound 29b. The preparative solution is lyophilized to obtain 187 mg of compound. LC-MS: [M+H] + =1517.6.

[0727] Step 7: Compound 29

[0728] 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 HPLC to obtain the product preparation solution, which was then lyophilized to give 114 mg of solid. LC-MS: [M+H] + =1517.9.

[0729] Example 38

[0730] Synthesis of compound 30:

[0731]

[0732] Starting with compounds M7 and 3c, and following the synthetic route of Example 37, compound 30 (125 mg) was obtained; LC-MS: [M+H] + =1445.6.

[0733] Example 39

[0734] Synthesis of compounds 31 and 32:

[0735]

[0736] 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; 63 mg of compound 32 was obtained, LC-MS: [M+H] + =1431.7.

[0737] Example 40

[0738] Synthesis of compounds 33 and 34:

[0739]

[0740] 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.

[0741] Example 41

[0742] Synthesis of compounds 35 and 36:

[0743]

[0744] 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.

[0745] Example 42

[0746] Synthesis of compound 37:

[0747]

[0748] Step 1: Compound SM5-1

[0749] 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 SM5-1 (110.1 g), yield 87%; LC-MS: [M+H] + =429.2.

[0750] Step 2: Compound SM5-2

[0751] 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 SM5-2 (37.1 g); LC-MS: [M+H] + =443.3.

[0752] Step 3: Compound SM5 (refer to Org. Lett., 2006, 8, 3387-3390.)

[0753] 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 SM5 (28 g); LC-MS: [M+H] + =501.3.

[0754] Step 4: Compound SM6

[0755] 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 purification. 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.

[0756] Step 5: Compound SM7

[0757] Add 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. Set aside for later use.

[0758] In a separate 250 mL single-necked flask, add the above crude product, 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 HPLC 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, yield 36%; LC-MS: [M+H] + =507.3.

[0759] Step 6: Compound M8

[0760] 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). Insoluble matter was filtered off. The reaction solution was directly purified by preparative purification. The preparative solution was concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile. The solution was then lyophilized to give compound M8 (3.7 g), yield 70%. LC-MS: [M+H] + =673.2.

[0761] Step 7: Compound 37a

[0762] Add 1 g (2.36 mmol) of methyl methacrylate (DMF) to a 25 mL single-necked flask, dissolve the DMF in 25 mL of DMF, then add 430 μL (2.6 mmol) of DIPEA, followed by 1.2 g (2.36 mmol) of M8. After the addition is complete, bring the mixture to room temperature and react for 1 h. Monitor the reaction completion by HPLC. Purify the reaction solution by HPLC to obtain the preparative solution. Lyophilize the preparative solution to obtain 488 mg of product. LC-MS: [MH] - =911.0.

[0763] Step 8: Compound 37b

[0764] Add 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 to a 100 mL single-necked flask. Add DIPEA (218 μL, 1.32 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 HPLC to obtain the preparative solution of compound 37b. The preparative solution is lyophilized to obtain 201 mg of compound. LC-MS: [M+H] + =1329.6.

[0765] Step 9: Compound 37

[0766] 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 HPLC to obtain the product preparation solution, which was then lyophilized to give 96 mg of solid. LC-MS: [M+H] + =1117.4.

[0767] Example 43

[0768] Synthesis of compound 38:

[0769]

[0770] 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.

[0771] Example 44

[0772] Synthesis of compounds 39 and 40:

[0773]

[0774] 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; 60 mg of compound 40 was obtained, LC-MS: [M+H] + =1131.4.

[0775] Example 45

[0776] Synthesis of compounds 41 and 42:

[0777]

[0778] Starting with compounds M7 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.

[0779] Example 46

[0780] Synthesis of compounds 43 and 44:

[0781]

[0782] 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.

[0783] Example 47 (Control Example)

[0784] Synthesis of compound 45:

[0785]

[0786] Compound 45 was synthesized according to the method provided in Example 58 of patent "CN104755494A".

[0787] Example 48

[0788] Synthesis of compound 46:

[0789]

[0790] Step 1: Compound 46a

[0791] Add 1d (500 mg, 0.62 mmol), M9 (310 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF to a 50 mL single-necked flask. Add DIPEA (378 μL, 2.29 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 HPLC to obtain the preparative solution. The preparative solution is lyophilized to obtain 46a (210 mg). LC-MS: [M+H] + =1221.6.

[0792] Step 2: Compound 46

[0793] 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 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.

[0794] Example 49

[0795] Synthesis of compound 47:

[0796]

[0797] Following the synthetic route of Example 48, compound 47 (81 mg) was obtained; LC-MS: [M+H] + =1065.3.

[0798] Example 50

[0799] Synthesis of compound 48A:

[0800]

[0801] Step 1: Compound 48a

[0802] 5d (1.66 g, 2.02 mmol, 1.0 eq), 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 reacted 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 give compound 48a (1.54 g), yield 61%. LC-MS: [M+H] + =1235.4.

[0803] Step 6: Compound 48A

[0804] 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. The mixture was reacted in an oil bath at 40 °C (preheated for stability) for 30 min. The mixture was then concentrated under reduced pressure in a water bath at 45 °C to remove the nitromethane, yielding a yellow solid residue (monitored by HPLC). The solution was prepared by acidification. The solution was then concentrated under reduced pressure in a water bath at 35 °C to remove acetonitrile, and lyophilized to give compound 48A (786 mg) in 90% yield.

[0805] Example 51

[0806] Synthesis of compound 48B:

[0807]

[0808] Step 1: Compound 48b

[0809] Compound 5d-1 (200 mg, 0.24 mmol, 1.0 eq), 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 HPLC, and the product solution was collected and lyophilized to obtain compound 48b (150.2 mg). LC-MS: [M+H] + =1235.4.

[0810] Step 2: Compound 48B

[0811] 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 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 was monitored by HPLC. The evaporated solid was directly purified by HPLC, and the product solution was collected and lyophilized to obtain compound 48B (70.0 mg). LC-MS: [M+H] + =1079.4.

[0812] Example 52

[0813] Synthesis of compound 49A:

[0814]

[0815] Following the route described in Example 50, compound 49A (71 mg) was obtained; LC-MS: [M+H] + =1079.4.

[0816] Example 53

[0817] Preparation of compound 49B:

[0818]

[0819] Following the synthetic route of Example 51, compound 49B (65 mg) was obtained; LC-MS: [M+H] + =1079.4.

[0820] Example 54

[0821] Synthesis of compounds 50A and 50B:

[0822]

[0823] Step 1: Compounds 50a and 50b

[0824] Add 7d (500 mg, 0.57 mmol), M9 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF to a 50 mL single-necked flask. Add DIPEA (378 μL, 2.29 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 HPLC to obtain the preparative solutions of compounds 50a and 50b. The preparative solutions are lyophilized to obtain 170 mg of compound 50a. LC-MS: [M+H]+ =1289.46; 202 mg compound 50b, LC-MS: [M+H] + =1289.46.

[0825] Step 2: Compound 50A

[0826]

[0827] 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 high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 44 mg of solid.

[0828] Step 3: Compound 50B

[0829]

[0830] 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 high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 45 mg of solid.

[0831] Example 55

[0832] Synthesis of compounds 51A and 51B:

[0833]

[0834] Step 1: Compound 51a and Compound 51b

[0835] Add 8d (500 mg, 0.57 mmol), M9 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF to a 50 mL single-necked flask. Add DIPEA (378 μL, 2.29 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 HPLC to obtain the preparative solutions of compounds 51a and 51b. The preparative solutions are lyophilized to obtain 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.

[0836] Step 2: Compound 51A

[0837]

[0838] 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 high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 39 mg of solid.

[0839] Step 3: Compound 51B

[0840]

[0841] 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 high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 60 mg of solid.

[0842] Example 56

[0843] Synthesis of compound 52A:

[0844]

[0845] Step 1: Compound 52a

[0846] Add 11d (800 mg, 0.96 mmol), M9 (480 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol), and 30 mL of DMF to a 50 mL single-necked flask. Add DIPEA (660 μL, 4.0 mmol) under ice-water bath conditions, and react at room temperature for 4 h. After the reaction is complete as monitored by HPLC, the reaction solution is purified by HPLC to obtain the preparative solution of compound 52a. The preparative solution is lyophilized to obtain 52a (388 mg). LC-MS: [M+H] + =1261.4.

[0847] Step 2: Compound 52A

[0848] 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 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.

[0849] Example 57

[0850] Synthesis of Compound 52B

[0851]

[0852] Following the synthetic route of Example 56, compound 52B (50 mg) was obtained. LC-MS: [M+H] + 1105.4.

[0853] Example 58

[0854] Synthesis of compound 53A:

[0855]

[0856] Step 1: Compound 53a

[0857] Add 12d (400 mg, 0.47 mmol), M9 (240 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt (101 mg, 0.47 mmol), and 15 mL of DMF to a 50 mL single-necked flask. Add DIPEA (330 μL, 2.0 mmol) under ice-water bath conditions, and react at room temperature for 3 h. After the reaction is complete as monitored by HPLC, the reaction solution is purified by HPLC to obtain the preparative solution of compound 53a. The preparative solution is lyophilized to obtain 53a (200 mg). LC-MS: [M+H] + =1275.4.

[0858] Step 2: Compound 53A

[0859] 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 HPLC to obtain the product preparation solution, which was then lyophilized to obtain solid compound 53A (51 mg); LC-MS: [M+H] + =1119.4.

[0860] Example 59

[0861] Synthesis of compound 53B:

[0862]

[0863] Following the synthetic route of Example 58, compound 53B (50 mg) was obtained; LC-MS: [M+H] + =1119.4.

[0864] Example 60

[0865] Synthesis of compounds 54A and 54B:

[0866]

[0867] Step 1: Compounds 54a and 54b

[0868] Add 19d (500 mg, 0.59 mmol), M9 (317 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol), and 10 mL of DMF to a 50 mL single-necked flask. Add DIPEA (292 μL, 1.77 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 HPLC to obtain the preparative solutions of compounds 54a and 54b. The preparative solutions are lyophilized to obtain 103 mg of compound 54a. LC-MS: [M+H] + =1261.5; 111 mg compound 54b, LC-MS: [M+H] + =1261.5.

[0869] Step 2: Compound 54A

[0870]

[0871] 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 high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to give 61 mg of solid. LC-MS: [M+H] + =1105.4.

[0872] Step 3: Compound 54B

[0873]

[0874] 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 high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 57 mg of solid. LC-MS: [M+H] + =1105.4.

[0875] Example 61

[0876] Synthesis of compounds 55A and 55B:

[0877]

[0878] Step 1: Compounds 55a and 55b

[0879] Add 20d (400 mg, 0.47 mmol), M9 (250 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol), and 10 mL of DMF to a 50 mL single-necked flask. Add DIPEA (248 μL, 1.5 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 HPLC to obtain the preparative solutions of compounds 55a and 55b. The preparative solutions are lyophilized to obtain 100 mg of compound 55a. LC-MS: [M+H] + =1275.5; 101 mg compound 55b, LC-MS: [M+H] + =1275.5.

[0880] Step 2: Compound 55A

[0881]

[0882] 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 high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 42 mg of solid. LC-MS: [M+H] + =1119.4.

[0883] Step 3: Compound 55B

[0884]

[0885] 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 high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 45 mg of solid. LC-MS: [M+H] + =1119.4.

[0886] Example 62

[0887] Synthesis of compound 56:

[0888]

[0889] Following the synthetic route of Example 60, compound 56 (50 mg) was obtained; LC-MS: [M+H] + =1119.3.

[0890] Example 63

[0891] Synthesis of compound 57:

[0892]

[0893] Following the synthetic route of Example 61, compound 57 (50 mg) was obtained; LC-MS: [M+H] + =1119.4.

[0894] Example 64

[0895] Synthesis of compound 58:

[0896]

[0897] Step 1: Synthesis of compound 58a

[0898] 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.

[0899] Step 2: Synthesis of compound 58b

[0900] 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.

[0901] Step 3: Synthesis of Compound 58

[0902] 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 a hydrogen balloon, 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.

[0903] Example 65

[0904] Synthesis of compound 59:

[0905]

[0906] Step 1: Compound 59a

[0907] Add 1d (500 mg, 0.62 mmol), 58 (279 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF to a 50 mL single-necked flask. Add DIPEA (378 μL, 2.29 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 HPLC to obtain the preparative solution. The preparative solution is lyophilized to obtain 59a (166 mg). LC-MS: [M+H] + =1235.6.

[0908] Step 2: Compound 59

[0909] 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 reaction was carried out 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 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.

[0910] Example 66

[0911] Synthesis of compound 60:

[0912]

[0913] Following the synthetic route of Example 65, compound 60 (40 mg) was obtained; LC-MS: [M+H] + =1079.3.

[0914] Example 67

[0915] Synthesis of compound 61:

[0916]

[0917] Step 1: Compound 61a

[0918] 5d (1.66 g, 2.02 mmol, 1.0 eq), 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 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 61a (1.21 g). LC-MS: [M+H] + =1249.4.

[0919] Step 2: Compound 61

[0920] 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 under reduced pressure in a water bath at 45 °C to remove the nitromethane, yielding a yellow solid residue (monitored by HPLC). The solution was prepared by acidification and concentrated under reduced pressure in a water bath at 35 °C to remove acetonitrile. The solution was then lyophilized to obtain compound 61 (786 mg). LC-MS: [M+H] + =1093.6.

[0921] Example 68

[0922] Synthesis of compound 62:

[0923]

[0924] Step 1: Compound 62a

[0925] Compounds 5d-1 (200 mg, 0.24 mmol, 1.0 eq), 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 HPLC, and the product solution was collected and lyophilized to obtain compound 62a (120.9 mg). LC-MS: [M+H] + =1249.4.

[0926] Step 2: Compound 62

[0927] 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 HPLC, and the product solution was collected and lyophilized to obtain compound 62 (61 mg). LC-MS: [M+H] + =1093.4.

[0928] Example 69

[0929] Preparation of compound 63:

[0930]

[0931] Following the route described in Example 67, compound 63 (60 mg) was obtained; LC-MS: [M+H] + =1093.4.

[0932] Example 70

[0933] Preparation of compound 64:

[0934]

[0935] Following the synthetic route of Example 68, compound 64 (65 mg) was obtained; LC-MS: [M+H] + =1093.4.

[0936] Example 71

[0937] Preparation of compounds 65A and 65B:

[0938]

[0939] Step 1: Compounds 65a and 65b

[0940] Add 7d (500 mg, 0.57 mmol), 58 (256.8 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF to a 50 mL single-necked flask. Add DIPEA (378 μL, 2.29 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 HPLC to obtain preparative solutions of compounds 65a and 65b. The preparative solutions are lyophilized to obtain 155 mg of compound 65a. LC-MS: [M+H] + =1303.4; 158 mg compound 65b, LC-MS: [M+H] + =1303.6.

[0941] Step 2: Compound 65A

[0942]

[0943] 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 high-performance liquid chromatography to obtain the preparative solution, which was then lyophilized to obtain 49 mg of solid.

[0944] Step 3: Compound 65B

[0945]

[0946] 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 high-performance liquid chromatography to obtain the preparative solution, which was then lyophilized to obtain 47 mg of solid.

[0947] Example 72

[0948] Synthesis of compounds 66A and 66B:

[0949]

[0950] Step 1: Compound 66a and Compound 66b

[0951] Add 8d (500 mg, 0.57 mmol), 58 (256.8 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF to a 50 mL single-necked flask. Add DIPEA (378 μL, 2.29 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 HPLC to obtain the preparative solutions of compounds 66a and 66b. The preparative solutions are 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.

[0952] Step 2: Compound 66A

[0953]

[0954] 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 HPLC to obtain the product preparation solution, which was then lyophilized to give 57 mg of solid. LC-MS: [M+H] + =1147.5.

[0955] Step 3: Compound 66B

[0956]

[0957] 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 HPLC to obtain the product preparation solution, which was then lyophilized to give 57 mg of solid. LC-MS: [M+H] + =1147.5.

[0958] Example 73

[0959] Synthesis of compound 67A:

[0960]

[0961] Step 1: Compound 67a

[0962] In a 50 mL single-necked flask, 11d (800 mg, 0.96 mmol), 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. 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 complete as monitored by HPLC, the reaction solution was purified by HPLC to obtain the preparative solution of compound 67a. The preparative solution was lyophilized to obtain 67a (402 mg). LC-MS: [M+H] + =1275.4.

[0963] Step 2: Compound 67A

[0964] 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 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.

[0965] Example 74

[0966] Synthesis of compound 67B:

[0967]

[0968] Following the synthetic route of Example 73, compound 67B (50 mg) was obtained. LC-MS: [M+H] +1119.4.

[0969] Example 75

[0970] Synthesis of compound 68A:

[0971]

[0972] Step 1: Compound 68a

[0973] Add 12d (400 mg, 0.47 mmol), 58 (211.7 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt (101 mg, 0.47 mmol), and 15 mL of DMF to a 50 mL single-necked flask. Add DIPEA (330 μL, 2.0 mmol) under ice-water bath conditions, and react at room temperature for 3 h. After the reaction is complete as monitored by HPLC, the reaction solution is purified by HPLC to obtain the preparative solution of compound 68a. The preparative solution is lyophilized to obtain 68a (177 mg). LC-MS: [M+H] + =1289.4.

[0974] Step 2: Compound 68A

[0975] 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 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.

[0976] Example 76

[0977] Synthesis of compound 68B:

[0978]

[0979] Following the synthetic route of Example 75, compound 68B (50 mg) was obtained; LC-MS: [M+H] + =1133.4.

[0980] Example 77

[0981] Synthesis of compounds 69A and 68B:

[0982]

[0983] Step 1: Compounds 69a and 69b

[0984] Add 19d (500 mg, 0.59 mmol), 58 (266 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol), and 10 mL of DMF to a 50 mL single-necked flask. Add DIPEA (292 μL, 1.77 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 HPLC to obtain the preparative solutions of compounds 69a and 69b. The preparative solutions are lyophilized to obtain 109 mg of compound 69a. LC-MS: [M+H] + =1275.5; 111 mg compound 69b, LC-MS: [M+H] + =1275.7.

[0985] Step 2: Compound 69A

[0986]

[0987] 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 high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 53 mg of solid. LC-MS: [M+H] + =1119.4.

[0988] Step 3: Compound 69B

[0989]

[0990] 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 high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 54 mg of solid. LC-MS: [M+H] + =1119.4.

[0991] Example 78

[0992] Synthesis of compounds 70A and 70B:

[0993]

[0994] Step 1: Compounds 70a and 70b

[0995] Add 20d (400 mg, 0.47 mmol), 58 (211.7 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol), and 10 mL of DMF to a 50 mL single-necked flask. Add DIPEA (248 μL, 1.5 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 HPLC to obtain the preparative solutions of compounds 70a and 70b. The preparative solutions are lyophilized to obtain 106 mg of compound 70a. LC-MS: [M+H] + =1289.5; 101 mg compound 70b, LC-MS: [M+H] + =1289.4.

[0996] Step 2: Compound 70A

[0997]

[0998] 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 high-performance liquid chromatography to obtain the product preparation solution, which was then lyophilized to obtain 39 mg of solid. LC-MS: [M+H] + =1133.4.

[0999] Step 3: Compound 70B

[1000]

[1001] 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 HPLC to obtain the product preparation solution, which was then lyophilized to obtain 35 mg of solid. LC-MS: [M+H] + =1133.4.

[1002] Example 79

[1003] Synthesis of compound 71:

[1004]

[1005] Following the synthetic route of Example 78, compound 71 (30 mg) was obtained; LC-MS: [M+H] + =1133.3.

[1006] Example 80

[1007] Synthesis of compound 72:

[1008]

[1009] Following the synthetic route of Example 78, compound 72 (33 mg) was obtained; LC-MS: [M+H] + =1133.4.

[1010]

[1011] Synthesis of compound M11:

[1012] 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 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 M10 (13.2 g), yield 78%; LC-MS: [M+H] + =866.5.

[1013] 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). Insoluble matter was filtered off. The reaction solution was directly purified by preparative purification. The preparative solution was concentrated in a water bath at 35 °C under reduced pressure to remove acetonitrile. The solution was then lyophilized to obtain compound M11 (14.2 g), yield 92%. LC-MS: [M+H] + =1032.5.

[1014] Example 82

[1015] Synthesis of compound 73:

[1016]

[1017] Step 1: Synthesis of compound 73a

[1018] 10 mL of DMF was added to 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.

[1019] Step 2: Synthesis of compound 73b

[1020] Add 73a (1.2 g, 0.94 mmol), M5 (500 mg, 0.94 mmol), PyBOP (625 mg, 1.2 mmol), HOBt (162 mg, 1.2 mmol), and 15 mL of DMF to a 25 mL single-necked flask. Add DIPEA (310 mg, 2.4 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 HPLC to obtain the preparative solution. The preparative solution is lyophilized to obtain 73b (709 mg). LC-MS: [M+H] + =1720.8.

[1021] Step 3: Synthesis of Compound 73

[1022] 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 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.

[1023] Example 83

[1024] Synthesis of compound 74:

[1025]

[1026] Following the synthetic route of Example 82, compound 74 (90 mg) was obtained; LC-MS: [M+H] + =1532.6.

[1027] Example 84

[1028] Synthesis of compound 75:

[1029]

[1030] Step 1: Synthesis of compound 75a

[1031] 10 mL of DMF was added to 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.

[1032] Step 2: Synthesis of compound 75b

[1033] Add 75a (700 mg, 0.54 mmol), M5 (289 mg, 0.54 mmol), PyBOP (313 mg, 0.6 mmol), HOBt (81 mg, 0.6 mmol), and 10 mL of DMF to a 25 mL single-necked flask. Add DIPEA (155 mg, 1.2 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 HPLC to obtain the preparative solution. The preparative solution is lyophilized to obtain 75b (304 mg). LC-MS: [M+H] + =1734.8.

[1034] Step 3: Synthesis of Compound 75

[1035] 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 HPLC to obtain the product preparation solution, which was then lyophilized to obtain solid compound 75 (96 mg); LC-MS: [M+H] + =1546.6.

[1036] Example 85

[1037] Synthesis of compound 76:

[1038]

[1039] Following the synthetic route of Example 84, compound 76 (92 mg) was obtained; LC-MS: [M+H] + =1546.5.

[1040] Example 86

[1041] Synthesis of compound 77:

[1042]

[1043] Following the synthetic route of Example 84, compound 77 (87 mg) was obtained; LC-MS: [M+H] + =1546.5.

[1044] Example 87

[1045] Synthesis of compound 78:

[1046]

[1047] Following the synthetic route of Example 84, compound 78 (94 mg) was obtained; LC-MS: [M+H] + =1546.7.

[1048] Example 88

[1049] Synthesis of compounds 79 and 80:

[1050]

[1051] Step 1: Synthesis of compound 79a

[1052] 10 mL of DMF was added to 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.

[1053] Step 2: Synthesis of compounds 79b-1 and 79b-2

[1054] 79a (600 mg, 0.45 mmol), M5 (240 mg, 0.45 mmol), PyBOP (261 mg, 0.5 mmol), HOBt (68 mg, 0.5 mmol), and 10 mL of DMF were added to a 25 mL single-necked flask. DIPEA (130 mg, 1 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 complete as monitored by HPLC, the reaction solution was purified by HPLC to obtain preparative solutions of compounds 79b-1 and 79b-2. The preparative solutions were lyophilized to obtain 79b-1 (124 mg). LC-MS: [M+H] +=1743.0; yielded 79b-1 (122 mg); LC-MS: [M+H] + =1743.0.

[1055] Step 3: Synthesis of Compound 79

[1056]

[1057] 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 HPLC to obtain the product preparation solution, which was then lyophilized to obtain solid compound 79 (30 mg); LC-MS: [M+H] + =1586.9.

[1058] Step 4: Synthesis of Compound 80

[1059]

[1060] 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 HPLC to obtain the product preparation solution, which was then lyophilized to give solid compound 80 (33 mg); LC-MS: [M+H] + =1587.0.

[1061] Example 89

[1062] Synthesis of compound 81:

[1063]

[1064] Following the synthetic route of Example 88, compound 81 (24 mg) was obtained; LC-MS: [M+H] + =1586.9.

[1065] Example 90

[1066] Synthesis of compound 82:

[1067]

[1068] Following the synthetic route of Example 88, compound 82 (29 mg) was obtained; LC-MS: [M+H] + =1586.9.

[1069] Example 91

[1070] 1) Expression and purification of SI-1×6.4 antibody:

[1071] SI-1 × 6.4 antibody was expressed using Expi293 (Shanghai Aopuma Biotechnology Co., Ltd.) suspension cells. The day before transfection, 0.9 × 10⁴ cells were used. 6 Cells were seeded at a density of cells / mL in 1L shake flasks containing 300mL of OPM-293CD05 Medium (81075-001, Shanghai Aopumai Biotechnology Co., Ltd.) and cultured overnight at 37℃, 5% CO2, and 120rpm on a cell culture shaker. The next day, the antibody expression plasmid was transfected using PEI-MAX, with a plasmid:PEI-MAX mass ratio of 1:3. On the first day after transfection, OPM-293ProFeed was added at 5% (v / v), and on the third day after transfection, OPM-293ProFeed was added again at 5% (v / v). On the sixth day after transfection, the supernatant was collected by centrifugation.

[1072] The collected cell expression supernatant was eluted with 0.05M sodium acetate (pH 3.6) using a Protein A affinity chromatography column (UniMab 50, Suzhou Nanomicro Technology Co., Ltd.). The captured antibody was adjusted to pH 7.0 with 1M Tris-HCl (pH 8.8) at a ratio of 0.7 / 10 (v / v). Then, it was passed through a gel filtration chromatography column (SEC, Superdex 200, GE) to remove impurities such as polymers. At the same time, the antibody buffer was replaced with 20mM PB (pH 6.5).

[1073] Antibody SI-1 × 6.4:

[1074] Light chain nucleic acid coding sequence

[1075] SEQ ID NO 1

[1076] GACATCTTGCTGACTCAGTCTCCAGTCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGTATTGGCACAAACATACACTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGATTCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAATAATAACTGGCCAACCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

[1077] Light chain amino acid sequence

[1078] SEQ ID NO 2

[1079] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGT DFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[1080] Among them, the variable region is:

[1081] SEQ ID NO 28

[1082] DILLTQSPVILSVSPGERVSFSC RASQSIGTNIH WYQQRTNGSPRLLIK YASESIS GIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNNNWPTT FGAGTKLELK

[1083] The underlined parts, from left to right, are CDR1 (SEQ ID NO 25), CDR2 (SEQ ID NO 26), and CDR3 (SEQ ID NO 27).

[1084] Nucleic acid coding sequences of constructs of heavy-chain and single-chain Fv (scFv) domains

[1085] SEQ ID NO 3

[1086]

[1087] Amino acid sequences of the heavy chain and single-chain Fv (scFv) domain constructs

[1088] SEQ ID NO 4

[1089] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGS QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRD DAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS GGGGSGGGGSGGGGS QSALTQPASVSGSPGQS ITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYG SSSTHVIFGGGTKVTVL

[1090] The variable region of the heavy chain is:

[1091] SEQ ID NO 38

[1092] QVQLKQSGPGLVQPSQSLSITCTVSGFSLT NYGVH WVRQSPGKGLEWLG VIWSGGNTDYNTPFTS RLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR ALTYYDYEFAY WGQGTLVTVSS

[1093] The underlined parts, from left to right, are CDR1 (SEQ ID NO 29), CDR2 (SEQ ID NO 30), and CDR3 (SEQ ID NO 31).

[1094] The variable region of the heavy chain in the single-chain Fv(scFv) structural domain is:

[1095] SEQ ID NO 39

[1096] QVQLQESGGGLVKPGGSLRLSCAASGFTFS SYWMS WVRQAPGKGLEWVA NINRDGSASYYVDSVKG RFTISRDDAKNSLYLQMNSLRAEDTAVYYCAR DRGVGYFDL WGRGTLVTVSS

[1097] The underlined parts, from left to right, are CDR1 (SEQ ID NO 32), CDR2 (SEQ ID NO 33), and CDR3 (SEQ ID NO 34).

[1098] The variable region of the light chain in the single-chain Fv(scFv) structural domain is:

[1099] SEQ ID NO 40

[1100] QSALTQPASVSGSPGQSITISC TGTSSDVGGYNFVS WYQQHPGKAPKLMIY DVSDRPS GVSDRFSGSSKSGNTASLIISGLQADDEADYYC SSYGSSSTHVI FGGGTKVTVL

[1101] The underlined parts, from left to right, are CDR1 (SEQ ID NO 35), CDR2 (SEQ ID NO 36), and CDR3 (SEQ ID NO 37).

[1102] 2) Expression and purification of SI-1×22 antibody: SI-1×22 antibody was expressed and purified using a similar method.

[1103] Antibody SI-1×22:

[1104] Nucleic acid coding sequences of constructs of light chain and single-stranded Fv (scFv) domains

[1105] SEQ ID NO 9

[1106]

[1107] Amino acid sequences of the light chain and single-chain Fv (scFv) domain constructs

[1108] SEQ ID NO 10

[1109] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGT DFTLSINSVESEDIADYYCQQNNNWPTTFGCGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGS QVQLQESGGGLVKPGGSLSLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTIS RDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS GGGGSGGGGSGGGGS QSALTQPASVSGSPG QSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSS YGSSSTHVIFGGGTKVTVL

[1110] The variable region of the light chain is:

[1111] SEQ ID NO 49

[1112] DILLTQSPVILSVSPGERVSFSC RASQSIGTNIH WYQQRTNGSPRLLIK YASESIS GIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNNNWPTT FGCGTKLELK

[1113] The underlined parts, from left to right, are CDR1 (SEQ ID NO 25), CDR2 (SEQ ID NO 26), and CDR3 (SEQ ID NO 27).

[1114] The variable region of the heavy chain in the single-chain Fv(scFv) structural domain is:

[1115] SEQ ID NO 50

[1116] QVQLQESGGGLVKPGGSLSLSCAASGFTFS SYWMS WVRQAPGKGLEWVA NINRDGSASYYVDSVKG RFTISRDDAKNSLYLQMNSLRAEDTAVYYCAR DRGVGYFDL WGRGTLVTVSS

[1117] The underlined parts, from left to right, are CDR1 (SEQ ID NO 32), CDR2 (SEQ ID NO 33), and CDR3 (SEQ ID NO 34).

[1118] The variable region of the light chain in the single-chain Fv(scFv) structural domain is:

[1119] SEQ ID NO 51

[1120] QSALTQPASVSGSPGQSITISC TGTSSDVGGYNFVS WYQQHPGKAPKLMIY DVSDRPS GVSDRFSGSSKSGNTASLIISGLQADDEADYYC SSYGSSSTHVI FGGGTKVTVL

[1121] The underlined parts, from left to right, are CDR1 (SEQ ID NO 35), CDR2 (SEQ ID NO 36), and CDR3 (SEQ ID NO 37).

[1122] Nucleic acid coding sequence of heavy chain

[1123] SEQ ID NO 11

[1124]

[1125] amino acid sequence of heavy chain

[1126] SEQ ID NO 12

[1127] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKCLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[1128] The variable region of the heavy chain is:

[1129] SEQ ID NO 52

[1130] QVQLKQSGPGLVQPSQSLSITCTVSGFSLT NYGVH WVRQSPGKCLEWLG VIWSGGNTDYNTPFTS RLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR ALTYYDYEFAY WGQGTLVTVSA

[1131] The underlined parts, from left to right, are CDR1 (SEQ ID NO 29), CDR2 (SEQ ID NO 30), and CDR3 (SEQ ID NO 31).

[1132] 3) Expression and purification of SI-1×24 antibody: SI-1×24 antibody was expressed and purified using a similar method.

[1133] Antibody SI-1×24:

[1134] Light chain nucleic acid coding sequence

[1135] SEQ ID NO 13

[1136] GACATCTTGCTGACTCAGTCTCCAGTCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGTATTGGCACAAACATACACTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGATTCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAATAATAACTGGCCAACCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

[1137] Light chain amino acid sequence

[1138] SEQ ID NO 14

[1139] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGT DFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[1140] Among them, the variable region is:[[]]

[1141] SEQ ID NO 28

[1142] DILLTQSPVILSVSPGERVSFSC RASQSIGTNIH WYQQRTNGSPRLLIK YASESIS GIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNNNWPTT FGAGTKLELK

[1143] The underlined parts, from left to right, are CDR1 (SEQ ID NO 25), CDR2 (SEQ ID NO 26), and CDR3 (SEQ ID NO 27).

[1144] Nucleic acid coding sequences of constructs of heavy-chain and single-chain Fv (scFv) domains

[1145] SEQ ID NO 15

[1146]

[1147] Amino acid sequences of the heavy chain and single-chain Fv (scFv) domain constructs

[1148] SEQ ID NO 16

[1149] QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFT ISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS GGGGSGGGGSGGGGS QSALTQPASVSGS PGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYC SSYGSSSTHVIFGGGTKVTVL GGGGSGGGGS QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLE WLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[1150] The variable region of the heavy chain is:

[1151] SEQ ID NO 38

[1152] QVQLKQSGPGLVQPSQSLSITCTVSGFSLT NYGVH WVRQSPGKGLEWLG VIWSGGNTDYNTPFTS RLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR ALTYYDYEFAY WGQGTLVTVSS

[1153] The underlined parts, from left to right, are CDR1 (SEQ ID NO 29), CDR2 (SEQ ID NO 30), and CDR3 (SEQ ID NO 31).

[1154] The variable region of the heavy chain in the single-chain Fv(scFv) structural domain is:

[1155] SEQ ID NO 39QVQLQESGGGLVKPGGSLRLSCAASGFTFS SYWMS WVRQAPGKGLEWVA NINRDGS ASYYVDSVKG RFTISRDDAKNSLYLQMNSLRAEDTAVYYCAR DRGVGYFDL WGRGTLVTVSS

[1156] The underlined parts, from left to right, are CDR1 (SEQ ID NO 32), CDR2 (SEQ ID NO 33), and CDR3 (SEQ ID NO 34).

[1157] The variable region of the light chain in the single-chain Fv(scFv) structural domain is:

[1158] SEQ ID NO 40QSALTQPASVSGSPGQSITISC TGTSSDVGGYNFVS WYQQHPGKAPKLMIY DVSDR PS GVSDRFSGSSKSGNTASLIISGLQADDEADYYC SSYGSSSTHVI FGGGTKVTVL

[1159] The underlined parts, from left to right, are CDR1 (SEQ ID NO 35), CDR2 (SEQ ID NO 36), and CDR3 (SEQ ID NO 37).

[1160] Example 92

[1161] 1) Expression and purification of SI-1×4 antibody:

[1162] SI-1×4 antibody was expressed using Expi293 (Shanghai Aopuma Biotechnology Co., Ltd.) suspension cells. The day before transfection, 0.9×10⁴ cells were used. 6 Cells were seeded at a density of cells / mL in 1L shake flasks containing 300mL of OPM-293CD05 Medium (81075-001, Shanghai Aopumai Biotechnology Co., Ltd.) and cultured overnight at 37℃, 5% CO2, and 120rpm on a cell culture shaker. The next day, the antibody expression plasmid was transfected using PEI-MAX, with a plasmid:PEI-MAX mass ratio of 1:3. On the first day after transfection, OPM-293ProFeed was added at 5% (v / v), and on the third day after transfection, OPM-293ProFeed was added again at 5% (v / v). On the sixth day after transfection, the supernatant was collected by centrifugation.

[1163] The collected cell expression supernatant was eluted with 0.05M sodium acetate (pH 3.6) using a Protein A affinity chromatography column (UniMab 50, Suzhou Nanomicro Technology Co., Ltd.). The captured antibody was adjusted to pH 7.0 with 1M Tris-HCl (pH 8.8) at a ratio of 0.7 / 10 (v / v). Then, it was passed through a gel filtration chromatography column (SEC, Superdex 200, GE) to remove impurities such as polymers. At the same time, the antibody buffer was replaced with 20mM PB (pH 6.5).

[1164] Antibody SI-1 × 4:

[1165] Light chain nucleic acid coding sequence

[1166] SEQ ID NO 5

[1167] GATATTCAAATGACTCAATCTCCTTCTCTCTTTCTGCTTCTGTTGGTGATCGTGTTACTATTACTTGTCGTTCTTCTCAAAATATTGTTCATTCTAATGGTAATACTTATCTTGATTGGTATCAACAAACTCCTGGTAAAGCTCCTAAACTTCTTATTTATAAA GTTTCTAATCGTTTTTCTGGTGTTCCTTCTCGTTTTTCTGGTTCTGGTTCTGGTACTGATTTTACTTTTACTATTTCTTTCTCTTCAACCTGAAGATATTGCTACTTATTATTGTTTTCAATATTCTCATGTTCCTTGGACTTTTGGTCAAGGTACTAAACTTCAA ATTACTCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAG GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

[1168] Light chain amino acid sequence

[1169] SEQ ID NO 6

[1170] DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSG SGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQIT RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[1171] The variable region is:

[1172] SEQ ID NO 44

[1173] DIQMTQSPSSLSASVGDRVTITC RSSQNIVHSNGNTYLD WYQQTPGKAPKLLIY KVSNRFS GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC FQYSHVPWT FGQGTKLQIT

[1174] The underlined parts, from left to right, are CDR1 (SEQ ID NO 41), CDR2 (SEQ ID NO 42), and CDR3 (SEQ ID NO 43).

[1175] Nucleic acid coding sequences of constructs of heavy-chain and single-chain Fv (scFv) domains

[1176] SEQ ID NO 7

[1177]

[1178] Amino acid sequences of the heavy chain and single-chain Fv (scFv) domain constructs

[1179] SEQ ID NO 8

[1180] QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVT ITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGS QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFT ISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS GGGGSGGGGSGGGGS QSALTQPASVSGS PGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYC SSYGSSSTHVIFGGGTKVTVL

[1181] The variable region of the heavy chain is:

[1182] SEQ ID NO 48

[1183] QVQLQQSGAEVKKPGSSVKVSCKASGYTFT NYYIY WVRQAPGQGLEWIG GINPTSGGSNFNEKFKT RVTITADESSTTAYMELSSLRSEDTAFYFCTR QGLWFDSDGRGFDF WGQGTTVTVSS

[1184] The underlined parts, from left to right, are CDR1 (SEQ ID NO 45), CDR2 (SEQ ID NO 46), and CDR3 (SEQ ID NO 47).

[1185] The variable region of the heavy chain in the single-chain Fv(scFv) structural domain is:

[1186] SEQ ID NO 39

[1187] QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFT ISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS

[1188] The underlined parts, from left to right, are CDR1 (SEQ ID NO 32), CDR2 (SEQ ID NO 33), and CDR3 (SEQ ID NO 34).

[1189] The variable region of the light chain in the single-chain Fv(scFv) structural domain is:

[1190] SEQ ID NO 40

[1191] QSALTQPASVSGSPGQSITISC TGTSSDVGGYNFVS WYQQHPGKAPKLMIY DVSDRPS GVSDRFSGSSKSGNTASLIISGLQADDEADYYC SSYGSSSTHVI FGGGTKVTVL

[1192] The underlined parts, from left to right, are CDR1 (SEQ ID NO 35), CDR2 (SEQ ID NO 36), and CDR3 (SEQ ID NO 37).

[1193] 2) Expression and purification of SI-1×25 antibody: SI-1×25 antibody was expressed and purified using a similar method.

[1194] Antibody SI-1×25:

[1195] Nucleic acid coding sequences of constructs of light chain and single-stranded Fv (scFv) domains

[1196] SEQ ID NO 17

[1197]

[1198] Amino acid sequences of the light chain and single-chain Fv (scFv) domain constructs

[1199] SEQ ID NO 18

[1200] QVQLQESGGGLVKPGGSLSLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFT ISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS GGGGSGGGGSGGGGS QSALTQPASVSGS PGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYC SSYGSSSTHVIFGGGTKVTVL GGGGSGGGGS DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRL LIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGCGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[1201] The variable region of the light chain is:

[1202] SEQ ID NO 49

[1203] DILLTQSPVILSVSPGERVSFSC RASQSIGTNIH WYQQRTNGSPRLLIK YASESIS GIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNNNWPTT FGCGTKLELK

[1204] The underlined parts, from left to right, are CDR1 (SEQ ID NO 25), CDR2 (SEQ ID NO 26), and CDR3 (SEQ ID NO 27).

[1205] The variable region of the heavy chain in the single-chain Fv(scFv) structural domain is:

[1206] SEQ ID NO 50

[1207] QVQLQESGGGLVKPGGSLSLSCAASGFTFS SYWMS WVRQAPGKGLEWVA NINRDGSASYYVDSVKG RFTISRDDAKNSLYLQMNSLRAEDTAVYYCAR DRGVGYFDL WGRGTLVTVSS

[1208] The underlined parts, from left to right, are CDR1 (SEQ ID NO 32), CDR2 (SEQ ID NO 33), and CDR3 (SEQ ID NO 34).

[1209] The variable region of the light chain in the single-chain Fv(scFv) structural domain is:

[1210] SEQ ID NO 51

[1211] QSALTQPASVSGSPGQSITISC TGTSSDVGGYNFVS WYQQHPGKAPKLMIY DVSDRPS GVSDRFSGSSKSGNTASLIISGLQADDEADYYC SSYGSSSTHV IFGGGTKVTVL

[1212] The underlined parts, from left to right, are CDR1 (SEQ ID NO 35), CDR2 (SEQ ID NO 36), and CDR3 (SEQ ID NO 37).

[1213] Nucleic acid coding sequence of heavy chain

[1214] SEQ ID NO 19

[1215]

[1216] amino acid sequence of heavy chain

[1217] SEQ ID NO 20

[1218] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKCLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[1219] The variable region of the heavy chain is:

[1220] SEQ ID NO 52

[1221] QVQLKQSGPGLVQPSQSLSITCTVSGFSLT NYGVH WVRQSPGKCLEWLG VIWSGGNTDYNTPFTS RLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR ALTYYDYEFAY WGQGTLVTVSA

[1222] The underlined parts, from left to right, are CDR1 (SEQ ID NO 29), CDR2 (SEQ ID NO 30), and CDR3 (SEQ ID NO 31).

[1223] 3) Expression and purification of SI-1×26 antibody: SI-1×26 antibody was expressed and purified using a similar method.

[1224] Antibody SI-1×26:

[1225] Nucleic acid coding sequences of constructs of light chain and single-stranded Fv (scFv) domains

[1226] SEQ ID NO 21

[1227]

[1228] Amino acid sequences of the light chain and single-chain Fv (scFv) domain constructs

[1229] SEQ ID NO 22

[1230] DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSG SGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGCGTKLQIT RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGS QVQLQESGGGLVKPGGSLSLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKG RFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS GGGGSGGGGSGGGGS QSALTQPASV SGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEAD YYCSSYGSSSTHVIFGGGTKVTVL

[1231] The variable region of the light chain is:

[1232] SEQ ID NO 53

[1233] DIQMTQSPSSLSASVGDRVTITC RSSQNIVHSNGNTYLD WYQQTPGKAPKLLIY KVSNRFS GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC FQYSHVPWT FGCGTKLQIT

[1234] The underlined parts, from left to right, are CDR1 (SEQ ID NO 41), CDR2 (SEQ ID NO 42), and CDR3 (SEQ ID NO 43).

[1235] The variable region of the heavy chain in the single-chain Fv(scFv) structural domain is:

[1236] SEQ ID NO 50

[1237] QVQLQESGGGLVKPGGSLSLSCAASGFTFS SYWMS WVRQAPGKGLEWVA NINRDGSASYYVDSVKG RFTISRDDAKNSLYLQMNSLRAEDTAVYYCAR DRGVGYFDL WGRGTLVTVSS

[1238] The underlined parts, from left to right, are CDR1 (SEQ ID NO 32), CDR2 (SEQ ID NO 33), and CDR3 (SEQ ID NO 34).

[1239] The variable region of the light chain in the single-chain Fv(scFv) structural domain is:

[1240] SEQ ID NO 51

[1241] QSALTQPASVSGSPGQSITISC TGTSSDVGGYNFVS WYQQHPGKAPKLMIY DVSDRPS GVSDRFSGSSKSGNTASLIISGLQADDEADYYC SSYGSSSTHV IFGGGTKVTVL

[1242] The underlined parts, from left to right, are CDR1 (SEQ ID NO 35), CDR2 (SEQ ID NO 36), and CDR3 (SEQ ID NO 37).

[1243] Nucleic acid coding sequence of heavy chain

[1244] SEQ ID NO 23

[1245]

[1246] amino acid sequence of heavy chain

[1247] SEQ ID NO 24

[1248] QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQCLEWIGGINPTSGGSNFNEKFKTRVT ITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[1249] The variable region of the heavy chain is:

[1250] SEQ ID NO 54

[1251] QVQLQQSGAEVKKPGSSVKVSCKASGYTFT NYYIY WVRQAPGQCLEWIG GINPTSGGSNFNEKFKT RVTITADESSTTAYMELSSLRSEDTAFYFCTR QGLWFDSDGRGFDF WGQGTTVTVSS

[1252] The underlined parts, from left to right, are CDR1 (SEQ ID NO 45), CDR2 (SEQ ID NO 46), and CDR3 (SEQ ID NO 47).

[1253] Example 93

[1254] 1) Preparation of SI-1×6.4 antibody-drug conjugate samples by conjugating SI-1×6.4 antibody with a payload:

[1255] After cell expression and purification via Protein A affinity chromatography and molecular sieve chromatography, the SI-1×6.4 antibody was replaced with 20 mM PB, pH 6.5 buffer to concentrate or dilute the SI-1×6.4 antibody to a protein concentration of 3 mg / mL. The payload, a white powder, was dissolved in DMA to a concentration of 20 mg / mL for later use. To break the interchain disulfide bonds of the SI-1×6.4 antibody, 20 times the molecular weight of TECP was added, and the reaction was carried out at room temperature for 3 hours. Then, 20 times the molecular weight of the payload solution was added, and the reaction was carried out at room temperature for 1 hour. After the reaction, ultrafiltration was performed using 30 kDa ultrafiltration centrifuge tubes to remove the payload that was not conjugated with SI-1×6.4, thus obtaining the SI-1×6.4 antibody-drug conjugate sample.

[1256] 2) Following a similar method, SI-1×22 antibody-drug conjugate samples were prepared by conjugating SI-1×22 antibody with a payload.

[1257] 3) Following a similar method, SI-1×24 antibody-drug conjugate samples were prepared by conjugating SI-1×24 antibody with a payload.

[1258] Example 94

[1259] 1) Preparation of SI-1×4 antibody-drug conjugate samples by conjugating SI-1×4 antibody with payload:

[1260] After cell expression and purification via Protein A affinity chromatography and molecular sieve chromatography, the SI-1×4 antibody was replaced with 20 mM PB, pH 6.5 buffer to concentrate or dilute the SI-1×4 antibody to a protein concentration of 3 mg / mL. The payload, a white powder, was dissolved in DMA to a concentration of 20 mg / mL for later use. To break the interchain disulfide bonds of the SI-1×4 antibody, 20 times the molecular weight of TECP was added, and the reaction was carried out at room temperature for 3 hours. Then, 20 times the molecular weight of the payload solution was added, and the reaction was carried out at room temperature for 1 hour. After the reaction, ultrafiltration was performed using 30 kDa ultrafiltration centrifuge tubes to remove the payload that was not conjugated with SI-1×4, thus obtaining the SI-1×4 antibody-drug conjugate sample.

[1261] 2) Following a similar method, SI-1×25 antibody-drug conjugate samples were prepared by conjugating SI-1×25 antibody with a payload.

[1262] 3) Following a similar method, SI-1×26 antibody-drug conjugate samples were prepared by conjugating SI-1×26 antibody with a payload.

[1263] Example 95

[1264] ADC-1 was prepared according to the general coupling method of Example 93.

[1265]

[1266] ADC-2 was prepared according to the general coupling method of Example 93.

[1267]

[1268] ADC-3 was prepared according to the general coupling method of Example 93.

[1269]

[1270] ADC-4 was prepared according to the general coupling method of Example 93.

[1271]

[1272] ADC-5 was prepared according to the general coupling method of Example 93.

[1273]

[1274] ADC-6 was prepared according to the general coupling method of Example 93.

[1275]

[1276] The ADC-7 was prepared according to the general coupling method of Example 93.

[1277]

[1278] The ADC-8 was prepared according to the general coupling method of Example 93.

[1279]

[1280] The ADC-9 was prepared according to the general coupling method of Example 93.

[1281]

[1282] The ADC-10 was prepared according to the general coupling method of Example 93.

[1283]

[1284] ADC-11 was prepared according to the general coupling method of Example 93.

[1285]

[1286] ADC-12 was prepared according to the general coupling method of Example 93.

[1287]

[1288] ADC-13 was prepared according to the general coupling method of Example 93.

[1289]

[1290] ADC-14 was prepared according to the general coupling method of Example 93.

[1291]

[1292] The ADC-15 was prepared according to the general coupling method of Example 93.

[1293]

[1294] The ADC-16 was prepared according to the general coupling method of Example 93.

[1295]

[1296] The ADC-17 was prepared according to the general coupling method of Example 93.

[1297]

[1298] The ADC-18 was prepared according to the general coupling method of Example 93.

[1299]

[1300] The ADC-19 was prepared according to the general coupling method of Example 93.

[1301]

[1302] The ADC-20 was prepared according to the general coupling method of Example 93.

[1303]

[1304] ADC-21 was prepared according to the general coupling method of Example 93.

[1305]

[1306] ADC-22 was prepared according to the general coupling method of Example 93.

[1307]

[1308] ADC-23 was prepared according to the general coupling method of Example 93.

[1309]

[1310] ADC-24 was prepared according to the general coupling method of Example 93.

[1311]

[1312] ADC-25 was prepared according to the general coupling method of Example 93.

[1313]

[1314]

[1315] ADC-26 was prepared according to the general coupling method of Example 93.

[1316]

[1317] ADC-27 was prepared according to the general coupling method of Example 93.

[1318]

[1319] The ADC-28 was prepared according to the general coupling method of Example 93.

[1320]

[1321] ADC-29 was prepared according to the general coupling method of Example 93.

[1322]

[1323] The ADC-30 was prepared according to the general coupling method of Example 93.

[1324]

[1325] ADC-31 was prepared according to the general coupling method of Example 93.

[1326]

[1327] The ADC-32 was prepared according to the general coupling method of Example 93.

[1328]

[1329] ADC-33 was prepared according to the general coupling method of Example 93.

[1330]

[1331] The ADC-34 was prepared according to the general coupling method of Example 93.

[1332]

[1333] The ADC-35 was prepared according to the general coupling method of Example 93.

[1334]

[1335] The ADC-36 was prepared according to the general coupling method of Example 93.

[1336]

[1337] The ADC-37 was prepared according to the general coupling method of Example 93.

[1338]

[1339] The ADC-38 was prepared according to the general coupling method of Example 93.

[1340]

[1341] ADC-39 was prepared according to the general coupling method of Example 93.

[1342]

[1343] The ADC-40 was prepared according to the general coupling method of Example 93.

[1344]

[1345] ADC-41 was prepared according to the general coupling method of Example 93.

[1346]

[1347] The ADC-42 was prepared according to the general coupling method of Example 93.

[1348]

[1349] ADC-43 was prepared according to the general coupling method of Example 93.

[1350]

[1351] The ADC-44 was prepared according to the general coupling method of Example 93.

[1352]

[1353] The ADC-45 was prepared according to the general coupling method of Example 93.

[1354]

[1355] The ADC-46 was prepared according to the general coupling method of Example 93.

[1356]

[1357] ADC-47 was prepared according to the general coupling method of Example 93.

[1358]

[1359] The ADC-48 was prepared according to the general coupling method of Example 93.

[1360]

[1361] The ADC-49 was prepared according to the general coupling method of Example 93.

[1362]

[1363] The ADC-50 was prepared according to the general coupling method of Example 93.

[1364]

[1365] ADC-51 was prepared according to the general coupling method of Example 93.

[1366]

[1367]

[1368] The ADC-52 was prepared according to the general coupling method of Example 93.

[1369]

[1370] ADC-53 was prepared according to the general coupling method of Example 93.

[1371]

[1372] The ADC-54 was prepared according to the general coupling method of Example 93.

[1373]

[1374] The ADC-55 was prepared according to the general coupling method of Example 93.

[1375]

[1376] The ADC-56 was prepared according to the general coupling method of Example 93.

[1377]

[1378] The ADC-57 was prepared according to the general coupling method of Example 93.

[1379]

[1380] The ADC-58 was prepared according to the general coupling method of Example 93.

[1381]

[1382] The ADC-59 was prepared according to the general coupling method of Example 93.

[1383]

[1384] The ADC-60 was prepared according to the general coupling method of Example 93.

[1385]

[1386] ADC-61 was prepared according to the general coupling method of Example 93.

[1387]

[1388] The ADC-62 was prepared according to the general coupling method of Example 93.

[1389]

[1390] ADC-63 was prepared according to the general coupling method of Example 93.

[1391]

[1392] The ADC-64 was prepared according to the general coupling method of Example 93.

[1393]

[1394] The ADC-65 was prepared according to the general coupling method of Example 93.

[1395]

[1396] The ADC-66 was prepared according to the general coupling method of Example 93.

[1397]

[1398] The ADC-67 was prepared according to the general coupling method of Example 93.

[1399]

[1400] The ADC-68 was prepared according to the general coupling method of Example 93.

[1401]

[1402] The ADC-69 was prepared according to the general coupling method of Example 93.

[1403]

[1404] The ADC-70 was prepared according to the general coupling method of Example 93.

[1405]

[1406] The ADC-71 was prepared according to the general coupling method of Example 93.

[1407]

[1408] The ADC-72 was prepared according to the general coupling method of Example 93.

[1409]

[1410]

[1411] ADC-73 was prepared according to the general coupling method of Example 93.

[1412]

[1413] The ADC-74 was prepared according to the general coupling method of Example 93.

[1414]

[1415] The ADC-75 was prepared according to the general coupling method of Example 93.

[1416]

[1417] The ADC-76 was prepared according to the general coupling method of Example 93.

[1418]

[1419] The ADC-77 was prepared according to the general coupling method of Example 93.

[1420]

[1421] The ADC-78 was prepared according to the general coupling method of Example 93.

[1422]

[1423] The ADC-79 was prepared according to the general coupling method of Example 93.

[1424]

[1425] The ADC-80 was prepared according to the general coupling method of Example 93.

[1426]

[1427] The ADC-81 was prepared according to the general coupling method of Example 93.

[1428]

[1429] The ADC-82 was prepared according to the general coupling method of Example 93.

[1430]

[1431] ADC-83 was prepared according to the general coupling method of Example 93.

[1432]

[1433] The ADC-84 was prepared according to the general coupling method of Example 93.

[1434]

[1435] The ADC-85 was prepared according to the general coupling method of Example 93.

[1436]

[1437] The ADC-86 was prepared according to the general coupling method of Example 93.

[1438]

[1439] The ADC-87 was prepared according to the general coupling method of Example 93.

[1440]

[1441] The ADC-88 was prepared according to the general coupling method of Example 93.

[1442]

[1443] The ADC-89 was prepared according to the general coupling method of Example 93.

[1444]

[1445] The ADC-90 was prepared according to the general coupling method of Example 93.

[1446]

[1447] The ADC-91 was prepared according to the general coupling method of Example 93.

[1448]

[1449] The ADC-92 was prepared according to the general coupling method of Example 93.

[1450]

[1451] ADC-93 was prepared according to the general coupling method of Example 93.

[1452]

[1453] The ADC-94 was prepared according to the general coupling method of Example 93.

[1454]

[1455] The ADC-95 was prepared according to the general coupling method of Example 93.

[1456]

[1457] The ADC-96 was prepared according to the general coupling method of Example 93.

[1458]

[1459] The ADC-97 was prepared according to the general coupling method of Example 93.

[1460]

[1461] The ADC-98 was prepared according to the general coupling method of Example 93.

[1462]

[1463] The ADC-99 was prepared according to the general coupling method of Example 93.

[1464]

[1465] The ADC-100 was prepared according to the general coupling method of Example 93.

[1466]

[1467] ADC-101 was prepared according to the general coupling method of Example 93.

[1468]

[1469] ADC-102 was prepared according to the general coupling method of Example 93.

[1470]

[1471] ADC-103 was prepared according to the general coupling method of Example 93.

[1472]

[1473] ADC-104 was prepared according to the general coupling method of Example 93.

[1474]

[1475] ADC-105 was prepared according to the general coupling method of Example 93.

[1476]

[1477] ADC-106 was prepared according to the general coupling method of Example 93.

[1478]

[1479] Compound 45 was prepared into ADC-DS according to the general coupling method of Example 93.

[1480]

[1481] ADC-107 was prepared according to the general coupling method of Example 94.

[1482]

[1483] The ADC-108 was prepared according to the general coupling method of Example 94.

[1484]

[1485] ADC-109 was prepared according to the general coupling method of Example 94.

[1486]

[1487] The ADC-110 was prepared according to the general coupling method of Example 94.

[1488]

[1489] ADC-111 was prepared according to the general coupling method of Example 94.

[1490]

[1491] ADC-112 was prepared according to the general coupling method of Example 94.

[1492]

[1493] ADC-113 was prepared according to the general coupling method of Example 94.

[1494]

[1495] ADC-114 was prepared according to the general coupling method of Example 94.

[1496]

[1497] ADC-115 was prepared according to the general coupling method of Example 94.

[1498]

[1499] ADC-116 was prepared according to the general coupling method of Example 94.

[1500]

[1501] ADC-117 was prepared according to the general coupling method of Example 94.

[1502]

[1503] ADC-118 was prepared according to the general coupling method of Example 94.

[1504]

[1505] ADC-119 was prepared according to the general coupling method of Example 94.

[1506]

[1507] The ADC-120 was prepared according to the general coupling method of Example 94.

[1508]

[1509] ADC-121 was prepared according to the general coupling method of Example 94.

[1510]

[1511] ADC-122 was prepared according to the general coupling method of Example 94.

[1512]

[1513] ADC-123 was prepared according to the general coupling method of Example 94.

[1514]

[1515] ADC-124 was prepared according to the general coupling method of Example 94.

[1516]

[1517] ADC-125 was prepared according to the general coupling method of Example 94.

[1518]

[1519] ADC-126 was prepared according to the general coupling method of Example 94.

[1520]

[1521] ADC-127 was prepared according to the general coupling method of Example 94.

[1522]

[1523] ADC-128 was prepared according to the general coupling method of Example 94.

[1524]

[1525]

[1526] ADC-129 was prepared according to the general coupling method of Example 94.

[1527]

[1528] The ADC-130 was prepared according to the general coupling method of Example 94.

[1529]

[1530] ADC-131 was prepared according to the general coupling method of Example 94.

[1531]

[1532] ADC-132 was prepared according to the general coupling method of Example 94.

[1533]

[1534] ADC-133 was prepared according to the general coupling method of Example 94.

[1535]

[1536] ADC-134 was prepared according to the general coupling method of Example 94.

[1537]

[1538] ADC-135 was prepared according to the general coupling method of Example 94.

[1539]

[1540] ADC-136 was prepared according to the general coupling method of Example 94.

[1541]

[1542] ADC-137 was prepared according to the general coupling method of Example 94.

[1543]

[1544] The ADC-138 was prepared according to the general coupling method of Example 94.

[1545]

[1546] ADC-139 was prepared according to the general coupling method of Example 94.

[1547]

[1548] The ADC-140 was prepared according to the general coupling method of Example 94.

[1549]

[1550] ADC-141 was prepared according to the general coupling method of Example 94.

[1551]

[1552] ADC-142 was prepared according to the general coupling method of Example 94.

[1553]

[1554] ADC-143 was prepared according to the general coupling method of Example 94.

[1555]

[1556] The ADC-144 was prepared according to the general coupling method of Example 94.

[1557]

[1558] The ADC-145 was prepared according to the general coupling method of Example 94.

[1559]

[1560] The ADC-146 was prepared according to the general coupling method of Example 94.

[1561]

[1562] ADC-147 was prepared according to the general coupling method of Example 94.

[1563]

[1564] The ADC-148 was prepared according to the general coupling method of Example 94.

[1565]

[1566] ADC-149 was prepared according to the general coupling method of Example 94.

[1567]

[1568] The ADC-150 was prepared according to the general coupling method of Example 94.

[1569]

[1570] ADC-151 was prepared according to the general coupling method of Example 94.

[1571]

[1572] ADC-152 was prepared according to the general coupling method of Example 94.

[1573]

[1574] ADC-153 was prepared according to the general coupling method of Example 94.

[1575]

[1576] The ADC-154 was prepared according to the general coupling method of Example 94.

[1577]

[1578] The ADC-155 was prepared according to the general coupling method of Example 94.

[1579]

[1580] ADC-156 was prepared according to the general coupling method of Example 94.

[1581]

[1582] ADC-157 was prepared according to the general coupling method of Example 94.

[1583]

[1584] The ADC-158 was prepared according to the general coupling method of Example 94.

[1585]

[1586] ADC-159 was prepared according to the general coupling method of Example 94.

[1587]

[1588] The ADC-160 was prepared according to the general coupling method of Example 94.

[1589]

[1590] ADC-161 was prepared according to the general coupling method of Example 94.

[1591]

[1592] ADC-162 was prepared according to the general coupling method of Example 94.

[1593]

[1594] ADC-163 was prepared according to the general coupling method of Example 94.

[1595]

[1596] The ADC-164 was prepared according to the general coupling method of Example 94.

[1597]

[1598] The ADC-165 was prepared according to the general coupling method of Example 94.

[1599]

[1600] The ADC-166 was prepared according to the general coupling method of Example 94.

[1601]

[1602] ADC-167 was prepared according to the general coupling method of Example 94.

[1603]

[1604] The ADC-168 was prepared according to the general coupling method of Example 94.

[1605]

[1606] The ADC-169 was prepared according to the general coupling method of Example 94.

[1607]

[1608] The ADC-170 was prepared according to the general coupling method of Example 94.

[1609]

[1610] ADC-171 was prepared according to the general coupling method of Example 94.

[1611]

[1612] The ADC-172 was prepared according to the general coupling method of Example 94.

[1613]

[1614] ADC-173 was prepared according to the general coupling method of Example 94.

[1615]

[1616] The ADC-174 was prepared according to the general coupling method of Example 94.

[1617]

[1618] The ADC-175 was prepared according to the general coupling method of Example 94.

[1619]

[1620] The ADC-176 was prepared according to the general coupling method of Example 94.

[1621]

[1622] ADC-177 was prepared according to the general coupling method of Example 94.

[1623]

[1624] The ADC-178 was prepared according to the general coupling method of Example 94.

[1625]

[1626] ADC-179 was prepared according to the general coupling method of Example 94.

[1627]

[1628] The ADC-180 was prepared according to the general coupling method of Example 94.

[1629]

[1630] ADC-181 was prepared according to the general coupling method of Example 94.

[1631]

[1632] ADC-182 was prepared according to the general coupling method of Example 94.

[1633]

[1634] ADC-183 was prepared according to the general coupling method of Example 94.

[1635]

[1636] The ADC-184 was prepared according to the general coupling method of Example 94.

[1637]

[1638] The ADC-185 was prepared according to the general coupling method of Example 94.

[1639]

[1640] The ADC-186 was prepared according to the general coupling method of Example 94.

[1641]

[1642] The ADC-187 was prepared according to the general coupling method of Example 94.

[1643]

[1644] The ADC-188 was prepared according to the general coupling method of Example 94.

[1645]

[1646] The ADC-189 was prepared according to the general coupling method of Example 94.

[1647]

[1648] The ADC-190 was prepared according to the general coupling method of Example 94.

[1649]

[1650] ADC-191 was prepared according to the general coupling method of Example 94.

[1651]

[1652] ADC-192 was prepared according to the general coupling method of Example 94.

[1653]

[1654] ADC-193 was prepared according to the general coupling method of Example 94.

[1655]

[1656] ADC-194 was prepared according to the general coupling method of Example 94.

[1657]

[1658] ADC-195 was prepared according to the general coupling method of Example 94.

[1659]

[1660] ADC-196 was prepared according to the general coupling method of Example 94.

[1661]

[1662] ADC-197 was prepared according to the general coupling method of Example 94.

[1663]

[1664]

[1665] ADC-198 was prepared according to the general coupling method of Example 94.

[1666]

[1667] ADC-199 was prepared according to the general coupling method of Example 94.

[1668]

[1669] The ADC-200 was prepared according to the general coupling method of Example 94.

[1670]

[1671] The ADC-201 was prepared according to the general coupling method of Example 94.

[1672]

[1673] ADC-202 was prepared according to the general coupling method of Example 94.

[1674]

[1675] ADC-203 was prepared according to the general coupling method of Example 94.

[1676]

[1677] ADC-204 was prepared according to the general coupling method of Example 94.

[1678]

[1679] ADC-205 was prepared according to the general coupling method of Example 94.

[1680]

[1681] ADC-206 was prepared according to the general coupling method of Example 94.

[1682]

[1683] ADC-207 was prepared according to the general coupling method of Example 94.

[1684]

[1685] The ADC-208 was prepared according to the general coupling method of Example 94.

[1686]

[1687] ADC-209 was prepared according to the general coupling method of Example 94.

[1688]

[1689] The ADC-210 was prepared according to the general coupling method of Example 94.

[1690]

[1691] ADC-211 was prepared according to the general coupling method of Example 94.

[1692]

[1693] ADC-212 was prepared according to the general coupling method of Example 94.

[1694]

[1695] Compound 45 was prepared according to the general coupling method of Example 94 to obtain ADC-213.

[1696]

[1697] Compound 5A was prepared according to the general coupling method of Example 93 to obtain ADC-214.

[1698]

[1699]

[1700] Compound 5A was prepared as HER3 ADC-215 according to the general coupling method of Example 93.

[1701]

[1702] The H3 antibody is the portion of SI-1×6.4 antibody that knocks out the EGFR-targeting component.

[1703] Example 311

[1704] ADC-216 to ADC-223 were prepared according to the general coupling method of Example 93.

[1705]

[1706] ADC-224 to ADC-231 were prepared according to the general coupling method of Example 93.

[1707]

[1708]

[1709] ADC-232 to ADC-239 were prepared according to the general coupling method of Example 94.

[1710]

[1711]

[1712] ADC-240 to ADC-247 were prepared according to the general coupling method of Example 94.

[1713]

[1714] Monomer content was determined using the SEC-HPLC method.

[1715] Column: Biocore SEC-300 5μm, 4.6×300mm

[1716] Manufacturer: NanoChrom, Part Number: B213-050030-04630S

[1717] Mobile phase: 50 mM PB + 300 mM NaCl + 200 mM Arg + 5% IPA, pH = 6.5

[1718] Table 1: Method Parameters

[1719]

[1720]

[1721] Table 2. Monomer content data of ligand-drug conjugates (ADCs) disclosed in this invention.

[1722]

[1723]

[1724] Conclusion: The ADC disclosed in this invention has the characteristics of low degradation rate and low aggregation rate, and has the excellent property of high monomer content.

[1725] Example 316

[1726] The drug antibody ratio (DAR) was determined using the RP-HPLC method.

[1727] Column Name: Proteomix RP-1000 4.6×100mm 5μm 1000A Manufacturer: Sepax

[1728] Table 3: Method Parameters

[1729]

[1730] Table 4: Detailed Data on Drug-Antibody Conjugate Ratio (DAR) for ADCs

[1731]

[1732]

[1733] Conclusion: The ADC disclosed in this invention has the excellent property of high DAR value, and can significantly increase the drug concentration at the target site under the same dosage of ADC drug.

[1734] Example 317

[1735] The ADCs maintained the affinity for EGFR and HER3 of the corresponding original bispecific antibodies SI-1×6.4, SI-1×4, SI-1×22, SI-1×24, SI-1×25, and SI-1×26.

[1736] The relative affinities of SI-1×6.4 with ADC-6, and SI-1×4 with ADC-112 for EGFR and HER3 were compared using a double-antigen sandwich ELISA method. The specific steps are as follows:

[1737] Recombinant EGFR-His*6 antigen was coated onto ELISA plates and blocked with 1% bovine serum albumin. SI-1×6.4, ADC-6, SI-1×4, and ADC-112 were then serially diluted 3-fold at an initial concentration of 5000 ng / mL, resulting in 11 concentrations. After incubation on the coated ELISA plates for a period of time, the plates were incubated with biotin-labeled HER3-Fc antigen, followed by incubation with streptavidin-HRP-labeled antigen. Finally, TMB color development was performed, and the reaction was terminated with sulfuric acid. The absorbance at 450 nm was measured using an ELISA reader. The results were plotted against concentration at 0D450 nm.

[1738] Conclusion: As attached Figure 3A , 3B The results showed that the conjugated ADC-6 and ADC-112 maintained similar affinity to SI-1×6.4 and SI-1×4, respectively, with no significant difference in EC50 values; indicating that the conjugation of SI-1×6.4 and SI-1×4 with the toxin did not affect their affinity for the antigen.

[1739] Similarly, using a test method similar to the one described above, the results are as follows: Figure 3C , Figure 3D , Figure 3E and Figure 3F The results showed that the conjugated ADC-219, ADC-227, ADC-235, and ADC-243 maintained similar affinity to SI-1×22, SI-1×24, SI-1×25, and SI-1×26, respectively, with no significant difference in EC50 values. This indicates that conjugation of SI-1×22, SI-1×24, SI-1×25, and SI-1×26 toxins did not affect their affinity for the antigen.

[1740] Example 318

[1741] In vitro efficacy testing:

[1742] This invention utilizes various human tumor cell lines (human epidermal carcinoma A431, human orthotopic pancreatic adenocarcinoma BXPC-3, human pharyngeal squamous cell carcinoma FaDu, human lung cancer squamous cell carcinoma line HARA-B, human non-small cell lung cancer cell line HCC827, and human colon cancer cell line SW620) as experimental models to evaluate the in vitro efficacy of ADC-conjugated drugs. A certain number of tumor cells were seeded in 96-well plates, and serially diluted test antibodies and corresponding ADC drugs were added to the cells. After 5 days of treatment, cell viability was detected using AlamarBlue or MTS, and the inhibitory effect of the test antibodies and ADCs on the tumor cell lines was evaluated by calculating the IC50. The initial antibody drug concentration was 500 nM, with a 7-fold dilution, for a total of 8 concentration points, and the treatment lasted for 5 days. The final algorithm calculated the survival rate as (experimental group - blank) / (control group - blank) × 100%, followed by curve fitting using Graph Pad Prism to calculate the half-maximal inhibitory concentration (IC50) and efficacy (%).

[1743] Table 5: In vitro efficacy of six naked antibodies (SI-1X4, SI-1X6.4, SI-1X22, SI-1X24, SI-1X25, SI-1X26) and six ADCs (ADC-112, ADC-6, ADC-219, ADC-227, ADC-235, ADC-243) in A431.

[1744]

[1745] Table 6: In vitro efficacy of six naked antibodies (SI-1X4, SI-1X6.4, SI-1X22, SI-1X24, SI-1X25, SI-1X26) and six ADCs (ADC-112, ADC-6, ADC-219, ADC-227, ADC-235, ADC-243) in BXPC-3

[1746]

[1747]

[1748] Table 7: In vitro efficacy of 6 naked antibodies (SI-1X4, SI-1X6.4, SI-1X22, SI-1X24, SI-1X25, SI-1X26) and 6 ADCs (ADC-112, ADC-6, ADC-219, ADC-227, ADC-235, ADC-243) in FaDu.

[1749]

[1750] Table 8: In vitro efficacy of 6 naked antibodies (SI-1X4, SI-1X6.4, SI-1X22, SI-1X24, SI-1X25, SI-1X26) and 6 ADCs (ADC-112, ADC-6, ADC-219, ADC-227, ADC-235, ADC-243) in HARA-B

[1751]

[1752]

[1753] Table 9: In vitro efficacy of 6 naked antibodies (SI-1X4, SI-1X6.4, SI-1X22, SI-1X24, SI-1X25, SI-1X26) and 6 ADCs (ADC-112, ADC-6, ADC-219, ADC-227, ADC-235, ADC-243) in HCC827.

[1754]

[1755] Table 10: In vitro efficacy of 6 naked antibodies (SI-1X4, SI-1X6.4, SI-1X22, SI-1X24, SI-1X25, SI-1X26) and 6 ADCs (ADC-112, ADC-6, ADC-219, ADC-227, ADC-235, ADC-243) in SW620

[1756]

[1757]

[1758] Conclusion: In human epidermal carcinoma A431, human orthotopic pancreatic adenocarcinoma BXPC-3, human pharyngeal squamous cell carcinoma FaDu, human lung cancer squamous cell carcinoma line HARA-B, human non-small cell lung cancer cells HCC827, and human colon cancer cells SW620, six ADCs (ADC-112, ADC-6, ADC-219, ADC-227, ADC-235, and ADC-243) showed more sensitive efficacy and stronger tumor cell growth inhibition compared to their corresponding naked antibodies (SI-1X4, SI-1X6.4, SI-1X22, SI-1X24, SI-1X25, and SI-1X26). (See attached image) Figures 4A - 4F (and Tables 5-10).

[1759] Example 319

[1760] In vivo efficacy testing:

[1761] In this invention, BALB / c nude mice subcutaneously inoculated with various human tumor cell lines (A431, SW620, A431+SW620) serve as an experimental model to evaluate the in vivo efficacy of ADC-conjugated drugs. A certain number of tumor cell lines are inoculated subcutaneously into BALB / c nude mice, and the tumor volume is increased to 150-300 mm². 3 During the treatment, the antibody and the corresponding ADC drug were injected via the tail vein once a week for four weeks. The patients were continuously observed, and tumors were tested twice a week to evaluate the inhibitory effect of the test antibody and ADC on tumor cell lines.

[1762] in conclusion:

[1763] In an A431 monocytoma model with high EGFR expression, 10 mg / kg ADC-6 (DAR=8) showed stronger tumor-suppressive activity compared to SI-1 × 6.4 naked antibody (see appendix). Figure 5A ).

[1764] In the SW620 monotumor model with low EGFR expression, ADC-6 at 10 and 15 mg / kg showed stronger tumor-suppressive activity compared to 15 mg / kg of SI-1 × 6.4 naked antibody; and its tumor-suppressive activity was stronger than that of Cet-ADC (ADC-214, DAR = 8) at the corresponding molecular molar concentration (see appendix). Figure 5B ).

[1765] In A431+SW620 heterogeneous tumors, ADC-6 at concentrations of 5, 15, and 30 mg / kg also showed stronger tumor-suppressive activity compared to 30 mg / kg of SI-1 × 6.4 naked antibody (see appendix). Figure 5C ).

[1766] Example 320

[1767] In vivo efficacy testing:

[1768] In this invention, BALB / c Nude mice were subcutaneously inoculated with human epidermal cancer cells A431 and human pancreatic cancer cells BxPC3 as experimental models to evaluate the in vivo efficacy of ADC-conjugated drugs. A certain number of tumor cell lines were inoculated subcutaneously on the right shoulder of female BALB / c Nude mice. When the average tumor volume reached 180-250 mm², the in vivo efficacy was evaluated. 3 During the treatment, the corresponding ADC drug was injected via the tail vein once a week for four consecutive weeks. The tumor volume was measured twice a week to evaluate the inhibitory effect of the test ADC drug on tumor growth.

[1769] %change to D0=(Dn-D0) / D0*100;

[1770] %TGI=1-[changes of tumor volume in treatment group / changes of tumor volume in control group]×100.

[1771] In an A431 monosomy model with high EGFR expression, 42 days after the first administration, all tested ADC drugs significantly inhibited tumor growth in a human epidermal cancer cell line A431 BALB / c-Nude mouse subcutaneous xenograft model (P < 0.05). Among them, ADC-6, ADC-219, ADC-235, ADC-227, and ADC-112 showed comparable tumor-suppressive effects, all demonstrating strong tumor-suppressive efficacy. (See appendix) Figure 6A );

[1772] In a BxPC3 monosomy model expressed in EGFR, 35 days after the first administration, all tested ADC drugs significantly inhibited tumor growth in a human epidermal cancer cell line BxPC3 BALB / c-Nude mouse subcutaneous xenograft model (P < 0.05). Among them, ADC-6, ADC-219, ADC-235, ADC-227, and ADC-112 showed comparable tumor-suppressive effects, all demonstrating strong tumor-suppressive efficacy. (See appendix) Figure 6B ).

[1773] Example 321

[1774] In vitro efficacy of payload compound 5A:

[1775] 1) Experimental materials:

[1776] Cells: The cells used in the tests were sourced from the Cell Bank of the Chinese Academy of Sciences;

[1777] Cell culture medium DMEM: Gibco;

[1778] FBS: BIOWEST.

[1779] 2) Preparation of culture medium:

[1780] Growth medium (with 10% FBS, Penicillin / streptomycin (100 U / mL);

[1781] Test medium (with 1% FBS, Penicillin / streptomycin (100 U / mL)).

[1782] 3) Operation:

[1783] Irradiate the biosafety cabinet with UV light for 30 minutes beforehand, then ventilate for 3 minutes. Preheat the growth medium, assay medium, D-PBS, and trypsin in a 37°C water bath, then disinfect the surfaces with alcohol before placing them in the biosafety cabinet. Place cells with approximately 80% confluence in the biosafety cabinet, aspirate the old medium, rinse with D-PBS, discard the rinse, digest with trypsin for 2-3 minutes, then add growth medium to neutralize, and centrifuge at 1200 rpm for 3 minutes. Aspirate the supernatant, mix with 4 mL of assay medium, and take 100 μL for counting (take 50 μL of cell suspension, add 50 μL of Trypan Blue Stain and mix well, then count). Plate cells according to the pre-optimized cell deposition density, 80 μL / well in a 96-well plate. Add only 80 μL of assay medium to wells E11 and F11, and add 150 μL of DPBS to the edge wells. After 24 hours of plating, add diluted antibody (20 μL per well) and set up a control. Add only 20 μL of detection medium to column 11. Set up two replicates for each concentration. After adding, mix on a cell vortex mixer at 550 rpm for 3 min.

[1784] Solution dilution: Prepare an initial concentration of 5 μM, 300 μL of test solution in the first column of a V-type 96-well plate using the test medium. Add 240 μL of test medium to each of the 2nd to 10th columns. Take 60 μL from the mixed first column and add it to the second column. Mix 10 times up and down with a pipette and discard the pipette tip. Repeat the same process for the next 7 concentrations.

[1785] 4) Testing:

[1786] Four days later, the MTS reagent was removed, thawed at room temperature in the dark, and thoroughly vortexed. Then, in a biosafety cabinet, 20 μL of CellTiter One Solution Reagen MTS reagent was added per 100 μL of cell culture volume along the sidewall of each well. The plate was gently tapped to ensure thorough mixing of the MTS solution, and then incubated in a cell culture incubator in the dark for 2 hours. After the reaction, the 96-well plate was removed, and the OD490 nm absorbance was measured using a microplate reader. The data was recorded, organized, analyzed, and stored.

[1787] 5) Results:

[1788] Table 11 shows that compound 5A (Payload) has a good inhibitory effect on the following solid tumor cells and hematologic malignancies.

[1789] Table 11. In vitro inhibitory effects of compound 5A on human non-small cell lung adenocarcinoma cells H1975, human non-small cell lung cancer cells HCC827, human epidermal carcinoma cells A431, human gastric carcinoma cells NCI-N87, human in situ pancreatic adenocarcinoma cells BXPC-3, human epidermal carcinoma cells A431 + human colon cancer cells SW620, human breast cancer cells ZR-75-1, human plasma cell leukemia cells H929, human multiple myeloma cells RPMI8226, human leukemia cells JJN-3, human breast cancer cells MDA-MB-361, and human breast cancer cells SK-BR-3.

[1790]

[1791]

[1792] Example 322: In vitro efficacy data of ADC-6 and ADC-214:

[1793] The in vitro efficacy of ADC-conjugated drugs was evaluated using two human tumor cell lines (human poorly differentiated lung cancer squamous cell carcinoma cell line Oka-c-1 and human lung squamous cell carcinoma cell line SK-MES-1) as experimental models.

[1794] A certain number of tumor cells were seeded in 96-well plates. Serially diluted test antibodies and corresponding ADC drugs were added to the cells, and the treatment lasted for 5 days. Cell viability was detected using Alamar Blue or MTS assays. The inhibitory effects of the test antibody, ADC, and small molecule drug d3 on the tumor cell lines were evaluated by calculating the IC50. The initial antibody drug concentration was 500 nM, with 7-fold dilutions, for a total of 7 concentration points, and the treatment lasted for 5 days. The final algorithm was calculated as follows: survival rate = (experimental group - blank) / (control group - blank) × 100%, followed by curve fitting using Graph Pad Prism to calculate the half-maximal inhibitory concentration (IC50). Results are attached. Figure 7A ,7B And Table 12.

[1795] Table 12. In vitro efficacy of SI-1×6.4, Cetuximab, ADC-6, ADC-214, and d3 against human poorly differentiated lung cancer squamous cell carcinoma lines Oka-c-1 and SK-MES-1.

[1796]

Claims

1. A ligand-camptothecin derivative conjugate as shown in general formula I, or a pharmaceutically acceptable salt or solvate thereof; I in: In Formula I, the chiral carbon atom 1 bonded to N has an S configuration; R is a hydrogen atom; R1 is a methyl group; R2 is a fluorine atom; X is wherein the left-hand wavy line is attached to the camptothecin derivative moiety and the right-hand wavy line is attached to L5; Connection units - L1-L2-L3-L4-L5 - are selected from the following structures: 、 、 ； Ac is ; R5, R6and R7are each independently selected from a hydrogen atom; The 2-chiral carbon atom has an S absolute configuration; n is an integer selected from 1 to 10; Ab is a bispecific antibody or its antigen-binding fragment that simultaneously targets EGFR and HER3; The antibody of Ab comprises: an IgG1 heavy chain, a κ light chain, and a single-chain Fv (scFv) domain; wherein the single-chain Fv (scFv) domain forms a construct with the IgG1 heavy chain or the κ light chain; wherein the IgG1 heavy chain and the κ light chain form an IgG moiety that is specific for binding to EGFR; the scFv domain is specific for binding to HER3, and the scFv domain is linked to the C-terminus or N-terminus of the IgG1 heavy chain or the C-terminus or N-terminus of the κ light chain via a linker; and wherein the single-chain Fv domain has a structural sequence of N-terminus-heavy chain variable region-linker-light chain variable region-C-terminus or N-terminus-light chain variable region-linker-heavy chain variable region-C-terminus; The κ light chain of the antibody Ab contains CDR1 as shown in SEQ ID NO: 25, CDR2 as shown in SEQ ID NO: 26, and CDR3 as shown in SEQ ID NO: 27; the IgG1 heavy chain contains CDR1 as shown in SEQ ID NO: 29, CDR2 as shown in SEQ ID NO: 30, and CDR3 as shown in SEQ ID NO: 31; and the single-chain Fv (scFv) domain contains heavy chain variable region CDR1 as shown in SEQ ID NO: 32, heavy chain variable region CDR2 as shown in SEQ ID NO: 33, heavy chain variable region CDR3 as shown in SEQ ID NO: 34, light chain variable region CDR1 as shown in SEQ ID NO: 35, light chain variable region CDR2 as shown in SEQ ID NO: 36, and light chain variable region CDR3 as shown in SEQ ID NO:

37.

2. The ligand-camptothecin derivative conjugate according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, characterized in that, The light chain of the antibody Ab contains the variable region shown in SEQ ID NO: 28, the heavy chain of IgG1 contains the variable region shown in SEQ ID NO: 38, and the single-chain Fv (scFv) domain contains the heavy chain variable region shown in SEQ ID NO: 39 and the light chain variable region shown in SEQ ID NO:

40.

3. The ligand-camptothecin derivative conjugate as shown in general formula I according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, characterized in that, The amino acid sequence of the light chain of the antibody Ab is SEQ ID NO: 2, and the amino acid sequence of the construct of the antibody heavy chain and the single-chain Fv (scFv) domain is SEQ ID NO:

4.

4. The ligand-camptothecin derivative conjugate as shown in general formula I according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, characterized in that, The light chain nucleic acid coding sequence of the antibody Ab is SEQ ID NO: 1, and the nucleic acid coding sequence of the construct of the antibody heavy chain and the single-chain Fv (scFv) domain is SEQ ID NO:

3.

5. The ligand-camptothecin derivative conjugate as shown in general formula I according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, characterized in that, The antibody Ab contains: two IgG1 heavy chains; two κ light chains; and two single-chain Fv (scFv) domains.

6. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 5, wherein: The ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate is selected from the following structures or their succinimide ring-opening structures. in: Ab is a bispecific antibody or its antigen-binding fragment that simultaneously targets EGFR and HER3, as described in any one of claims 1-5; n is an integer selected from 1 to 10.

7. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 5, wherein: The ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate is selected from the following structures. 、 ， in: Ab is a bispecific antibody or its antigen-binding fragment that simultaneously targets EGFR and HER3, as described in any one of claims 1-5; n is an integer selected from 1 to 10.

8. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 5, wherein: The toxin-to-antibody ratio (DAR) of the ligand-camptothecin derivative conjugate is 6.0-8.

0.

9. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 5, wherein: The pharmaceutically acceptable salts include sodium, potassium, calcium, or magnesium salts formed with acidic functional groups in the structure, and 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 basic functional groups in the structure.

10. A method for preparing ligand-camptothecin derivative conjugates as shown in general formula I, or pharmaceutically acceptable salts or solvates thereof, characterized in that: Includes the following steps, ， By coupling reduced antibodies or their antigen-binding fragments with linkers-drug compounds, ligand-camptothecin derivative conjugates as shown in general formula I are obtained. Ab, connecting units -L1-L2-L3-L4-L5-, X, R, R1, R2 and n as described in any one of claims 1-5.

11. A pharmaceutical composition comprising a ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof as described in any one of claims 1-9, and optionally a pharmaceutically acceptable carrier.

12. A pharmaceutical formulation comprising the ligand-camptothecin derivative conjugate of any one of claims 1-9 or a pharmaceutically acceptable salt or solvate thereof.

13. Use of the ligand-camptothecin derivative conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 1-9, or the pharmaceutical composition of claim 11 and / or the pharmaceutical formulation of claim 12, in the preparation of a medicament for treating or preventing cancer or tumors; wherein the cancer or tumor expresses EGFR and / or HER3; and wherein the cancer or tumor is selected from adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, esophageal cancer, lung cancer, colorectal cancer, bone cancer, skin cancer, melanoma, glioma, neuroblastoma, glioma multiforme, sarcoma, lymphoma, head and neck cancer, and leukemia.

14. Use of the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof as described in any one of claims 1-9, or the pharmaceutical composition of claim 11 and / or the pharmaceutical formulation of claim 12, in the preparation of a medicament for treating or preventing cancer or tumors; wherein the cancer or tumor expresses EGFR and / or HER3; and wherein the cancer or tumor is selected from prostate cancer, salivary gland cancer, colon cancer, rectal cancer, thyroid cancer, pancreatic cancer, and breast cancer.

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