TW202532076A - Anti-claudin18.2 antibody-camptothecin drug conjugate and pharmaceutical use thereof - Google Patents

Abstract

An antibody targeting human Claudin18.2 was coupled with a camptothecin analog to form a stable and homogeneous antibody-drug conjugate with a drug-to-antibody ratio (DAR) of 6.0-8.0. This antibody-drug conjugate has the structure shown in general formula I, where Ab refers to an antibody targeting Claudin18.2 coupled with a linker-camptothecin analog. The present application also discloses methods of preparation and purification of conjugates, their applications in cancer therapy, and linker-drug compounds that can be coupled with Ab to form antibody-drug conjugates.

Description

Anti-CLAUDIN18.2 antibody-camptothecin drug conjugate and its medical use

This application claims the benefit of the filing date of CN 202311712634.5, filed on November 15, 2023, the entire disclosure of which is incorporated herein by reference.

The present invention relates to the field of biopharmaceuticals and, more particularly, to antibody-drug conjugates formed from an anti-Claudin 18.2 antibody and a camptothecin, as well as methods for preparing and using the antibody-drug conjugates. The present application also relates to linker-drug compounds that can be conjugated to the antibody to form the antibody-drug conjugate.

Claudin18.2 (or CLDN18.2 for short) is a protein encoded by the Claudin18 gene that has tight junction function and is a member of the Claudin protein family.

Claudin family proteins are tetraspanins containing two extracellular loops, N- and C-termini localized in the cytoplasm, and four transmembrane regions. Claudin family proteins are important members of tight junctions, playing a role in intercellular connectivity. Abnormal expression of these proteins can induce structural disruptions, such as those in epithelial cells, leading to functional alterations and a wide range of diseases. Current research indicates that the mammalian claudin protein family has 27 members, and humans contain all of the claudin proteins except claudin 13. The functions of each member are highly conserved, and each member is differentially expressed in different tissues and mediates different tumor types.

Claudin18.2 is normally expressed only in gastric mucosal cells and not in other normal tissues. Overexpression of claudin18.2 leads to gastric, pancreatic, esophageal, and non-small cell lung cancers. Claudin18.1, a splice variant of claudin18.2, is selectively expressed in lung cells. However, claudin18.1 and claudin18.1 differ in their first extracellular loop by only eight amino acids in normal tissues. Claudin18.1 is selectively expressed in lung cells in both normal and normal tissues. Therefore, the highly restricted expression of claudin18.2 in normal tissues and its specific expression in tumor tissues make it an important potential target for tumor immunotherapy.

Currently, the leading monoclonal antibody targeting claudin18.2 is zolbetuximab (IMAB362), developed by Ganymed in Germany. Its research for gastric and gastroesophageal cancers has advanced to Phase III clinical trials, and its research for pancreatic cancer has advanced to Phase II clinical trials. Ganymed's dual antibodies against claudin18.2 and CD3 are in preclinical stages, with over 60% of its dual claudin18.2 and CD3 targets in development. Dual antibodies targeting claudin18.2 and CD47/PD-L1/4-1BB are also under development. The most advanced anti-Claudin18.2 ADCs are in Phase I/II clinical trials, including LM-302 from Lixin Pharmaceuticals, RC118 from Rongchang Biologics, and GMG901 from Konoia/Leep Bio. The majority of Claudin18.2-targeting ADCs are in Phase I or preclinical stages.

Antibody-drug conjugates (ADCs) are a class of biopharmaceuticals that link antibodies or antibody fragments to biologically active small molecule toxins via stable chemical linkers. These drugs not only fully utilize the high specificity of antibody-antigen binding and the high lethality of cytotoxic agents, but also effectively circumvent the shortcomings of antibodies' weak therapeutic efficacy and small molecule toxins' indiscriminate attack.

There are currently 14 approved ADC drugs on the global market. ADC company target Approval Date Adcetris Seagen/Takeda CD30 2011.08 Kadcyla Roche HER2 2013.02 Besponsa Pfizer CD22 2017.08 Mylotarg Pfizer CD33 2017.09 Lumoxiti AstraZeneca CD22 2018.09 Polivy Roche CD79β 2019.06 Padcev Seagen NECTIN-4 2019.12 Enhertu Daiichi Sankyo/AstraZeneca HER2 2019.12 Trodelvy Gilead Sciences TROP2 2020.04 Blenrep GSK BCMA 2020.08 Akalux Rakuten Aspyrian EGFR 2020.09 Zynlonta ADC Therapeutics CD19 2021.04 Vidicilimumab Rongchang Bio HER2 2021.06 Tivdak Seagen/Genmab TF 2021.09

Mylotarg, one of the first ADCs marketed globally, was granted accelerated approval by the FDA in May 2000 for the treatment of patients with CD33-positive acute myeloid leukemia (AML) in first relapse who are over 60 years of age and are not candidates for cytotoxic chemotherapy. Following subsequent studies linking Mylotarg to severe and fatal liver damage, Pfizer voluntarily withdrew the drug from the market in June 2010. In 2017, Mylotarg was re-approved by the FDA at a reduced dose for the treatment of newly diagnosed CD33-positive AML (children and adults aged ≥1 month) and relapsed or refractory CD33-positive AML (children and adults aged ≥2 years). The recently approved Tivdak is the first FDA-approved ADC targeting tissue factor (TF) for the treatment of patients with recurrent or metastatic cervical cancer.

This application discloses a safer and more effective ADC drug targeting Claudin18.2 with camptothecin as the toxin molecule, which has great research value and therapeutic potential.

The following invention is illustrative only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the accompanying drawings and the following embodiments.

The present disclosure provides, inter alia, drugs of the ADC class, including anti-Claudin18.2 antibody-drug conjugates, compounds such as drug moieties and linker derivatives that can be used to form a portion of the antibody-drug conjugates, methods for preparing the conjugates, pharmaceutical compositions comprising such conjugates or compounds, and methods of using such conjugates or pharmaceutical compositions to treat diseases, such as cancer. The present disclosure further provides linker-drug compounds that can be conjugated to anti-Claudin18.2 antibodies to form the disclosed antibody-drug conjugates. In some embodiments, the anti-Claudin18.2 antibody-drug conjugates exhibit excellent clinical therapeutic efficacy, good molecular stability, and significantly improved preclinical efficacy compared to existing drugs of similar classes.

In one aspect, the present disclosure provides a ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvent thereof as shown in Formula I : wherein Ab is an antibody or an antigen-binding fragment thereof having binding affinity for human Claudin18.2; L ₁₁ , L ₁₂ , and L ₁₃ are different from each other and independently are: 、 、 、 、 、 、 、 、 or ; L ₂ has the structure shown in the following formula A. Formula A wherein Y is a scaffold selected from C1-C6 alkyl, substituted C1-C6 alkyl, or C3-C8 cycloalkyl; in one embodiment, Y is C1-C6 alkyl; Ac is a hydrophilic building block; and the carbon 2 attached to Y is absolutely chiral in the R or S configuration.

In one embodiment, _L3 is present or absent, and when present, _L3 is selected from PEG hydrophilic units: , o is an integer selected from 1-10; in one embodiment, o is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; in one embodiment, o is an integer from 2-8, such as 2, 3, 4, 5, 6, 7, or 8; in one embodiment, _L4 is an enzymatically cleavable unit.

In one embodiment, _L5 is a connection unit.

In one embodiment, the chiral carbon atom No. 1 attached to N in Formula I has an absolute chirality of R or S; R is selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 membered heteroaryl group, and a substituted 5-10 membered heteroaryl group.

In one embodiment, R is selected from a hydrogen atom or a C1-C6 alkyl group.

_R1 is selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a carboxyl group, a 3-7 heterocyclic group, a substituted 3-7 heterocyclic group, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 heteroaryl group, and a substituted 5-10 heteroaryl group.

In one embodiment, R ₁ is selected from a hydrogen atom or a C1-C6 alkyl group.

In one embodiment, _R is selected from a C1-C6 alkyl group. _R is selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a carboxyl group, a 3-7 heterocyclic ring, a substituted 3-7 heterocyclic ring, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 heteroaryl group, and a substituted 5-10 heteroaryl group.

In one embodiment, R ₂ is selected from a hydrogen atom, a halogen or a C1-C6 alkyl group. In one embodiment, R ₂ is selected from a halogen.

X is selected from -C(O) _{-CRaRb- ( CR3R4 ) m} -O-, -C(O) _{-CRaRb- ( CR3R4 ) m} -NH- or -C(O) _{-CRaRb- ( CR3R4} ) _m - _S- . In one embodiment, X is selected _from -C(O) _{-CRaRb- ( CR3R4} ) _m -O-.

R _a and R _b are each independently selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a halogenated C1-C6 alkyl group, a C3-C8 cycloalkyl group, a C3-C8 cycloalkylC1-C6 alkyl group, a C6-C10 arylC1-C6 alkyl group, a C1-C6 alkoxyC1-C6 alkyl group, a 3-7 heterocyclic group, a substituted 3-7 heterocyclic group, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 heteroaryl group, or a substituted 5-10 heteroaryl group.

In one embodiment, _Ra and _Rb are each independently selected from a hydrogen atom, a C1-C6 alkyl group, a halogen-C1-C6 alkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, or a C6-C10 aryl C1-C6 alkyl group.

Alternatively, _Ra , _Rb , and the carbon atoms to which they are attached constitute C3-C8 cycloalkyl, C3-C8 cycloalkyl, C1-C6 alkyl, 3-7 membered heterocycloalkyl, or substituted 3-7 membered heterocycloalkyl. In one embodiment, _Ra , _Rb , and the carbon atoms to which they are attached constitute C3-C8 cycloalkyl.

R ₃ and R ₄ are the same or different and independently represent a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a halogenated C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C1-C6 alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyl C1-C6 alkyl group, a C3-C8 cycloalkyl group, a 3-7 membered heterocyclic group, or a substituted 3-7 membered heterocyclic group.

In one embodiment, R ₃ and R ₄ are independently hydrogen or a C1-C6 alkyl group. Alternatively, R ₃ , R ₄ , and the carbon atom to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl-C1-C6 alkyl group, a 3-7 membered heterocyclic group, or a substituted 3-7 membered heterocyclic group.

m is selected from an integer between 0 and 4. In one embodiment, m can be 0, 1, 2, 3, or 4. In one embodiment, m can be 0 or 1; n1, n2, and n3 are each independently selected from any integer or decimal number between 0 and 10, n1, n2, and n3 are not all 0, and 1 ≤ n1+n2+n3 ≤ 10.

In some embodiments, the ligand-camptothecin derivative conjugate represented by Formula I or a pharmaceutically acceptable salt or solvent thereof is characterized in that the Ab is an antibody targeting Claudin18.2 or an antigen-binding fragment thereof.

In one embodiment, the ligand-camptotherine derivative conjugate, or a pharmaceutically acceptable salt or solvent thereof, is characterized in that the Ab is an antibody and comprises an IgG1 heavy chain and a κ light chain. In one embodiment, the antibody comprising the IgG1 heavy chain and a κ light chain specifically recognizes human Claudin 18.2 protein.

In one embodiment, the ligand-dendrimer derivative conjugate, or a pharmaceutically acceptable salt or solvent thereof, is characterized in that the CDRs comprised by the κ light chain of the antibody have an amino acid sequence that is at least 80%, 90%, 98%, 99% or 100% identical to SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, and the CDRs comprised by the IgG1 heavy chain have an amino acid sequence that is at least 80%, 90%, 98%, 99% or 100% identical to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

In one embodiment, the ligand-camptothecin derivative conjugate as shown in Formula I , or a pharmaceutically acceptable salt or solvent thereof, is characterized in that the antibody comprises a heavy chain and a light chain. In one embodiment, the light chain comprises CDRL1, CDRL2, and CDRL3, each having an amino acid sequence encoded by a nucleic acid encoding sequence having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively, and the heavy chain comprises CDRH1, CDRH2, and CDRH3, each having an amino acid sequence encoded by a nucleic acid sequence having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

In one embodiment, the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvent thereof, as shown in Formula I is characterized in that the antibody comprises a heavy chain and a light chain. In one embodiment, the heavy chain comprises a heavy chain variable region having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, and the light chain comprises a light chain variable region having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7.

In one embodiment, the ligand-dendrimer derivative conjugate, or a pharmaceutically acceptable salt or solvent thereof, as shown in Formula I is characterized in that the antibody comprises a heavy chain and a light chain. In one embodiment, the heavy chain variable region comprises a heavy chain variable region encoded by a nucleic acid sequence having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13, and the light chain variable region is encoded by a nucleic acid sequence having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 19.

In one embodiment, the ligand-camptothecin derivative conjugate as shown in Formula I , or a pharmaceutically acceptable salt or solvent thereof, is characterized in that Ab comprises a heavy chain having an amino acid sequence that is at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 5, and a light chain having an amino acid sequence that is at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 11.

In one embodiment, the ligand-camptothecin derivative conjugate as shown in Formula I , or a pharmaceutically acceptable salt or solvent thereof, is characterized in that the nucleic acid encoding the antibody heavy chain has at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17, and the nucleic acid encoding the antibody light chain has at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23.

In one embodiment, the ligand-dendrimer derivative conjugate represented by Formula I or a pharmaceutically acceptable salt or solvent thereof is characterized in that X can be the following structure or an isomer thereof: 、 、 、 、 、 、 、 、 、 、 、 、 or .

The position shown by the left wavy line is connected to the derivative portion, and the position shown by the right wavy line is connected to L ₅ .

In one embodiment, the ligand-dendrogenin derivative conjugate or a pharmaceutically acceptable salt or solvent thereof as shown in Formula I is characterized in that _L4 can be a peptide residue comprising an amino acid. In one embodiment, the amino acid is further substituted with one or more substituents selected from one or more of a deuterium atom, a halogen, a hydroxyl group, a cyano group, an amino group, a nitro group, a carboxyl group, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a C1-C6 alkoxy group, and a C3-C8 cycloalkyl group or a substituted C3-C8 cycloalkyl group.

In one embodiment, the peptide residue is a peptide residue formed from one, two, or more amino acids selected from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline (C), serine (S), glutamine (E), or aspartic acid (D). In one embodiment, the peptide residue is a tetrapeptide residue comprising glycine (G)-glycine (G)-phenylalanine (F)-glycine (G). In one embodiment, the peptide residue is -GGFG-.

In one embodiment, the ligand-dendrogenin derivative conjugate of Formula I or a pharmaceutically acceptable salt or solvent thereof is characterized as follows: L ₅ is selected from, but not limited to, -NR ₅ (CR ₆ R ₇ ) _q - or a chemical bond, q is selected from an integer of 0-6 (e.g., 0, 1, 2, 3, 4, 5, or 6); R ₅ , R ₆ and R R 5 , R 6 , and R ₇ are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C3-C8 cycloalkyl group, a C3-C8 cycloalkylC1-C6 alkyl group, a C1-C6 alkoxyC1-C6 alkyl group, a 3-7 heterocyclic group, a substituted 3-7 heterocyclic group, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 heteroaryl group, or a substituted 5-10 heteroaryl group. In one embodiment, R ₅ , R ₆ , and R ₇ are each independently selected from a hydrogen atom or a C1-C6 alkyl group. In one embodiment, R ₅ , R ₆ , and R ₇ are each independently selected from a hydrogen atom.

In some embodiments, _L1 may be: 、 、 、 、 or .

In one embodiment, the ligand-dendrimer derivative conjugate or a pharmaceutically acceptable salt or solvent thereof is characterized in that the linking units -L ₁₁ -L ₂ -L ₃ -L ₄ -L ₅ -, -L ₁₂ -L ₂ -L ₃ -L ₄ -L ₅ -, or -L ₁₃ -L ₂ -L ₃ -L ₄ -L ₅ - are mutually inconsistent and independently have the following structures: 、 、 、 、 、 、 、 、 、 、 or .

In one embodiment, -L ₁₁ -L ₂ -L ₃ -L ₄ -L ₅ -, -L ₁₂ -L ₂ -L ₃ -L ₄ -L ₅ -, or -L ₁₃ -L ₂ -L ₃ -L ₄ -L ₅ - are different from each other and are each independently and non-limitingly selected from: 、 、 、 、 ,or wherein: Ac is a hydrophilic structural unit; R ₅ , R ₆ , and R ₇ are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a 3-7 membered heterocyclic ring, a substituted 3-7 membered heterocyclic ring, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 membered heteroaryl group, and a substituted 5-10 membered heteroaryl group.

In one embodiment, R ₅ , R ₆ and R ₇ are each independently selected from a hydrogen atom or a C1-C6 alkyl group; In one embodiment, R ₅ , R ₆ and R ₇ are each independently selected from a hydrogen atom; Carbon atom #2 connected to N has an absolute chirality of R or S configuration; The position indicated by the left wavy line is connected to the antibody or antigen-binding fragment thereof, and the position indicated by the right wavy line is connected to X; o is an integer selected from 1-10; for example, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In another aspect, the present application discloses a ligand-dendrimer derivative conjugate as shown in Formula II , or a pharmaceutically acceptable salt or solvent thereof; II wherein: Ab is an antibody or antigen-binding fragment thereof having binding affinity (or specificity) for human Claudin18.2; L ₁₁ , L ₁₂ , and L ₁₃ are linker units, and L ₁₁ , L ₁₂ , and L ₁₃ are mutually identical and are independently and non-limitingly selected from: 、 、 、 、 、 、 、 、 、 ; L ₃ is present or absent, and when L ₃ is present, L ₃ is selected from , o is selected from an integer of 1-10; for example, o can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, it is an integer from 2-8; Ac is a hydrophilic structural unit; the chiral carbon atoms at positions 1, 2, and 3 independently have a chiral configuration of R or S; R is selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 membered heteroaryl group, a substituted 5-10 membered heteroaryl group; in one embodiment, R is selected from a hydrogen atom or a C1-C6 alkyl group; R _R is selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a carboxyl group, a 3-7 heterocyclic ring, a substituted 3-7 heterocyclic ring, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 heteroaryl group, or a substituted 5-10 heteroaryl group. In one embodiment, _R is selected from a hydrogen atom or a C1-C6 alkyl group. In one embodiment, _R is selected from a C1-C6 alkyl group; _R is selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a carboxyl group, a 3-7 heterocyclic ring, a substituted 3-7 heterocyclic ring, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 heteroaryl group, or a substituted 5-10 heteroaryl group. In one embodiment, _R is selected from a hydrogen atom, a halogen, or a C1-C6 alkyl group. In one embodiment, _R2 is selected from halogen; _X is -C(O) _-CRaRb- ( _CR3R4 ) _m -O- _{, -C} (O) _-CRaRb- ( _{CR3R4 ) m} - _NH- , or -C(O) _-CRaRb- ( _CR3R4 ) _m -S- _. In one embodiment, X is selected _from -C(O) _{-CRaRb- ( CR3R4} ) _m -O-; _Ra and _Rb are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a halogenated C1-C6 alkyl group, a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a 3-7 heterocyclic group, a substituted 3-7 heterocyclic group, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 membered heteroaryl group, or a substituted 5-10 membered heteroaryl group; In one embodiment, _Ra and R R _a , R _b , and R _b are each independently selected from a hydrogen atom, a C1-C6 alkyl group, a halogen-C1-C6 alkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, or a C6-C10 aryl C1-C6 alkyl group; or, Ra, R b , and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3-7 heterocycloalkyl group, or a substituted 3-7 heterocycloalkyl group; in one embodiment, _Ra , R _b , and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group; R ₃ , R _{R 4} are the same or different and independently represent a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a halogenated C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C1-C6 alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyl C1-C6 alkyl group, a C3-C8 cycloalkyl group, a 3-7 heterocyclic group, or a substituted 3-7 heterocyclic group; in one embodiment, R ₃ and R ₄ are independently a hydrogen atom or a C1-C6 alkyl group; or, R ₃ and R ₄ and the carbon atom to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3-7 heterocyclic group, or a substituted 3-7 heterocyclic group; m is selected from an integer 0-4; for example, m can be 0, 1, 2, 3, or 4. In one embodiment, m is 0 or 1; each of n1, n2, and n3 is independently selected from any integer or any decimal number from 0 to 10, n1, n2, and n3 are not all 0, and 1 ≤ n1+n2+n3 ≤ 10.

In one embodiment, the ligand-camptothenate derivative conjugate represented by Formula II or a pharmaceutically acceptable salt or solvent thereof is characterized in that Ac has a structure represented by Formula B: B wherein: Z may be a group consisting of one or more of a hydrophilic structure carboxyl, phosphate, polyphosphate, phosphite, sulfonic acid, sulfinic acid, or polyethylene glycol (PEG); in one embodiment, Z is selected from a hydrophilic structure carboxylate, phosphate, or polyethylene glycol (PEG); Y' is optionally a scaffold that connects the amine group to Z; in one embodiment, Y' is a C1-C6 alkylene group (e.g., a methylene group); and Ac is connected to the carbon 2 position marked by brackets Y in structural formula I.

In some embodiments, the ligand-camptotherine derivative conjugate, or a pharmaceutically acceptable salt or solvent thereof, is characterized in that the Ac is glycine, ( D/L ) alanine, ( D/L ) leucine, ( D/L ) isoleucine, ( D/L ) valine, (D/L) phenylalanine, ( D/L ) proline, ( D/L ) tryptophan, ( D/L ) serine, ( D/L ) tyrosine, ( D/L ) cysteine, (D/L) cystine, ( D/L ) arginine, ( D/L ) histidine, ( D/L ) methionine, ( D/L ) asparagine, (D/L) glutamine, ( D/L ) threonine, ( D/L) )aspartic acid, (D/L)glutamine, natural or unnatural amino acid derivatives, or the following structures or their isomers. 、 、 、 、 、 、 、 、 、 or ;

In one embodiment, Ac may be 、 、 or .

In some embodiments, the ligand-dendrimer derivative conjugate or a pharmaceutically acceptable salt or solvent thereof is characterized in that Ac is a hydrophilic structure of glycine, phosphate, ( D/L ) glutamine, or polyethylene glycol.

In some embodiments, the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvent thereof is characterized in that the camptothecin derivative has a structure shown in Formula d below; d wherein: R is selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 heteroaryl group, and a substituted 5-10 heteroaryl group; In one embodiment, R is selected from a hydrogen atom or a C1-C6 alkyl group; R _R is selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a carboxyl group, a 3-7 heterocyclic ring, a substituted 3-7 heterocyclic ring, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 heteroaryl group, or a substituted 5-10 heteroaryl group; In one embodiment, _R is selected from a hydrogen atom or a C1-C6 alkyl group; In one embodiment, _R is selected from a C1-C6 alkyl group; R _R is selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a carboxyl group, a 3-7 heterocyclic ring, a substituted 3-7 heterocyclic ring, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 heteroaryl group, or a substituted 5-10 heteroaryl group; In one embodiment, _R is selected from a hydrogen atom, a halogen, or a C1-C6 alkyl group; In one embodiment, _R is selected from a halogen; _R and R _b are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a halogenated C1-C6 alkyl group, a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a 3-7 heterocyclic group, a substituted 3-7 heterocyclic group, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 heteroaryl group, a substituted 5-10 heteroaryl group; in one embodiment, R _a and R R a and R _b are each independently selected from a hydrogen atom, a C1-C6 alkyl group, a halogen-C1-C6 alkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, or a C6-C10 aryl C1-C6 alkyl group; in one embodiment, R _a and R _b are each independently selected from a hydrogen atom, a C1-C6 alkyl group, a halogen-C1-C6 alkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, or a C6-C10 aryl C1-C6 alkyl group; in one embodiment, R _a and R _b are each independently selected from a hydrogen atom, a C1-C6 alkyl group, a halogen-C1-C6 alkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, or a C6-C10 aryl group; in one embodiment, R _a and R _{b are each independently selected from a hydrogen atom, a methyl group, an ethyl group, a trifluoromethyl group, a cyclopropylmethyl group, or a phenyl group; or, R a , R b} and the carbon atom to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3-7 heterocycloalkyl group, or a substituted 3-7 heterocycloalkyl group; in one embodiment, R _a , R _b and the carbon atom to which it is attached constitute a C3-C8 cycloalkyl group (e.g., a C3-C5 cycloalkyl group); the chiral carbon atom at position 1 may be in R or S configuration; m is selected from 0 or 1.

In some embodiments, the ligand-dendrimer derivative conjugate or a pharmaceutically acceptable salt or solvent thereof is characterized in that the structural formula d can be the following compound: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 .

In another aspect, the present application discloses a linker-drug compound having a structure represented by the following Formula III or a pharmaceutically acceptable salt or solvent thereof: III where: L is selected from 、 、 、 、 、 ,or , wherein the position on the right side is connected to the position marked 2 by the wavy line; R is selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 heteroaryl group, and a substituted 5-10 heteroaryl group; R _a is selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a halogenated C1-C6 alkyl group, a C3-C8 cycloalkyl group, a C3-C8 cycloalkylC1-C6 alkyl group, a C1-C6 alkoxyC1-C6 alkyl group, a 3-7 heterocyclic group, a substituted 3-7 heterocyclic group, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 heteroaryl group, and a substituted 5-10 heteroaryl group; R _{R a} is selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a halogenated C1-C6 alkyl group, a C3-C8 cycloalkyl group, a C3-C8 cycloalkylC1-C6 alkyl group, a C1-C6 alkoxyC1-C6 alkyl group, a 3-7 heterocyclic group, a substituted 3-7 heterocyclic group, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 heteroaryl group, a substituted 5-10 heteroaryl group; or, R _a , R _b and the carbon atom to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkylC1-C6 alkyl group, a 3-7 heterocyclic group, a substituted 3-7 heterocyclic group; in one embodiment, R _a and R _R and R are each independently selected from a hydrogen atom, a C1-C6 alkyl group, a halogen-C1-C6 alkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, and a C6-C10 aryl group; in one embodiment, _R and _R are each independently selected from a hydrogen atom, a methyl group, an ethyl group, a trifluoromethyl group, a cyclopropylmethyl group, and a phenyl group; or, _R , _R, and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3-7 heterocycloalkyl group, or a substituted 3-7 heterocycloalkyl group; in one embodiment, _R , _R , and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group (e.g., a C3-C5 cycloalkyl group); _L is absent or present, and when L is present, it is selected from , o is an integer selected from 1 to 10; in one embodiment, o is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; the chiral carbon atom at position 1 or position 2 independently has an R or S configuration; Ac is a hydrophilic structural unit; m is selected from 0 or 1; in one embodiment, the linker-drug compound or a pharmaceutically acceptable salt or solvent thereof is used to couple with the ligand Ab to form the ligand-dendrimer derivative conjugate of Formula I or Formula II.

In some embodiments, L may comprise a succinimide group. In such embodiments, the ligand-drug conjugate can undergo hydrolysis under readily hydrolyzable conditions, and the hydrolysis site is the succinimide portion of the linker unit. When the ligand contains multiple linker-drugs, the following situations may occur with varying degrees of hydrolysis: (1) The succinimide group is not hydrolyzed at all, i.e., the succinimide groups are all in a closed ring form. or (2) Incomplete hydrolysis of the succinimide groups, i.e., some succinimide groups are in a closed ring form or And some of the succinimidyl groups are in the ring-opened form 、 ,or 、 (3) Complete hydrolysis of the succinimide groups, i.e., all the succinimide groups are in the open-ring form 、 ,or 、 .

Thus, when multiple Ls containing a succinimide moiety are simultaneously present in an ADC (i.e., Ab is linked to multiple drug-linkers containing a succinimide moiety), all or some of these succinimide moieties may be in a closed ring form, partially in an open ring form, or all or some of them may be in an open ring form.

In some embodiments, the linker-drug compounds disclosed herein, or pharmaceutically acceptable salts or solvents thereof, are characterized in that: Ac has a structure shown in Formula B below, B wherein: Z may be a group consisting of one or more of the hydrophilic structures carboxyl, phosphate, polyphosphate, phosphite, sulfonic acid, sulfinic acid, or polyethylene glycol (PEG); Y' is optionally a scaffold that connects the amino group to Z; in one embodiment, Y' is a C1-C6 alkylene group (e.g., a methylene group); Ac is connected to the carbon 2 position indicated by brackets Y in structural formula I.

In some embodiments, the linker-drug compound or pharmaceutically acceptable salt or solvent thereof is characterized in that Ac is glycine, ( D/L ) alanine, ( D/L ) leucine, ( D/L ) isoleucine, ( D/L ) valine, (D/L) phenylalanine, ( D/L ) proline, ( D/L ) tryptophan, ( D /L) serine, ( D/L ) tyrosine, ( D/L ) cysteine, ( D/L ) cystine, ( D/L ) arginine, ( D/L ) histidine, ( D/L ) methionine, (D/L) asparagine, (D/L) glutamine, ( D/L ) threonine, (D/L) aspartic acid, ( D/L ) D/L ) glutamine, natural or unnatural amino acid derivatives, or the following structures. 、 、 、 、 、 、 、 or .

In some embodiments, the linker-drug compound or a pharmaceutically acceptable salt or solvent thereof is characterized in that Ac can be a hydrophilic structure of glycine, phosphate, ( D/L ) glutamine or polyethylene glycol.

In some embodiments, the linker-drug compound or a pharmaceutically acceptable salt or solvent thereof is characterized in that the linker-drug compound is selected from the following structures or isomers thereof, without limitation. 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or .

In one embodiment, o is selected from an integer between 1 and 10; for example, o can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

The linker-drug compounds disclosed herein, or pharmaceutically acceptable salts or solvents thereof, can be used as intermediates for coupling with ligand Ab to form ligand-camptothecin derivative conjugates of Formula I or Formula II.

In another aspect, the present application discloses a method for preparing a ligand-camptothecin derivative conjugate represented by Formula I or Formula II , or a pharmaceutically acceptable salt or solvent thereof. In one embodiment, the method comprises the step of coupling a reduced antibody or antigen-binding fragment thereof with a linker-drug compound to provide a ligand-camptothecin derivative conjugate represented by Formula I or Formula II : I The chiral carbon atom at position 1, position 2, or position 3 has absolute chirality of R or S configuration; Ab, L, L ₁₁ , L ₁₂ , L ₁₃ , L ₂ , L ₃ , L ₄ , L ₅ , X, R, R ₁ , R ₂ , n1, n2, or n3 are as described above.

The present disclosure also relates to the use of the linker-drug compounds described herein, or pharmaceutically acceptable salts or solvents thereof, as intermediates in the preparation of ligand-dendrimer derivative conjugates or pharmaceutically acceptable salts or solvents thereof.

In certain embodiments, the preparation is performed according to the preparation methods disclosed herein.

In some embodiments, the ligand-dendrogenin derivative conjugate, or a pharmaceutically acceptable salt thereof or a solvent thereof, is characterized in that the ligand-dendrogenin derivative conjugate, or a pharmaceutically acceptable salt thereof or a solvent thereof can be the following structure, or a succinimide ring-opened structure thereof or an isomer thereof. 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 ,or wherein: 5103F3-BSM is an antibody targeting human Claudin18.2 (i.e., having affinity or specificity for it) or an antigen-binding fragment thereof; each of n1, n2, and n3 is independently selected from any integer or any decimal number from 0 to 10, n1, n2, and n3 are not all 0, and 1 ≤ n1+n2+n3 ≤ 10.

In some embodiments, the ligand-dendrimer derivative conjugate, linker-drug compound, or pharmaceutically acceptable salt or solvent thereof is characterized in that the pharmaceutically acceptable salt comprises a sodium salt, a potassium salt, a calcium salt, or a magnesium salt formed with an acidic functional group in the structural formula, and an ethyl salt formed with a basic functional group in the structural formula. acid salt, trifluoroacetate, citrate, oxalate, tartrate, malate, nitrate, chloride, bromide, iodide, sulfate, hydrosulfate, phosphate, lactate, oleate, ascorbate, salicylate, formate, glutamine, methanesulfonate, ethanesulfonate, benzenesulfonate, or p-toluenesulfonate.

In another aspect, the present application provides methods for producing conjugates, compounds, salts thereof, solvents thereof, or derivatives thereof. In one embodiment, the anti-Claudin18.2 antibody-drug conjugate is obtained by conjugating a humanized anti-Claudin18.2 antibody to a camptothecin derivative.

In another aspect, the present application discloses a pharmaceutical composition comprising a ligand-dendrimer derivative conjugate as described herein and a linker-drug compound and a pharmaceutically acceptable salt thereof or a solvate thereof, and optionally a pharmaceutically acceptable carrier.

In another aspect, the present application discloses a pharmaceutical formulation comprising a ligand-camptotheine derivative conjugate as described herein and a linker-drug compound and a pharmaceutically acceptable salt thereof or a solvate thereof.

In another aspect, the present application discloses ligand-camptothenate derivative conjugates and linker-drug compounds and pharmaceutically acceptable salts or solvents thereof as described herein, pharmaceutical compositions and pharmaceutical formulations for the preparation of medicaments for treating or preventing cancer or tumors.

In another aspect, the present application discloses ligand-camptothenate derivative conjugates, linker-drug compounds, and pharmaceutically acceptable salts or solvents thereof, as described herein, pharmaceutical compositions and pharmaceutical formulations for treating or preventing cancer or tumors.

In another aspect, the present application discloses a method for treating or preventing cancer or tumors. In one embodiment, the method comprises administering to a subject in need thereof a preventatively or therapeutically effective amount of a ligand-dendrimer derivative conjugate, linker-drug compound, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition or formulation thereof, as described herein.

In one embodiment, the cancer or tumor expresses Claudin 18.2.

In one embodiment, the cancer or tumor can be adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urothelial carcinoma, bladder cancer, hepatocellular carcinoma, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, breast cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, neuroglioma, neuroblastoma, glioblastoma multiforme, or sarcoma.

In one embodiment, the cancer or tumor is a solid tumor. In one embodiment, the cancer or tumor is a hematological tumor such as lymphoma and leukemia.

In the above aspects, the "C1-C6 alkyl" in "C1-C6 alkyl" and various structural moieties containing "C1-C6 alkyl" (such as "substituted C1-C6 alkyl" and "deuterated C1-C6 alkyl") can be replaced with a "C1-C20 alkyl", "C1-C12 alkyl", or "C1-C10 alkyl"; The "C3-C8 cycloalkyl" in "C3-C8 cycloalkyl" and various structural moieties containing "C3-C8 cycloalkyl" can be replaced with a "C3-C20 cycloalkyl" or "C3-C10 cycloalkyl"; "C1-C6 alkoxy" and various structural moieties containing "C1-C6 alkoxy" may be replaced with "C1-C20 alkoxy", "C1-C12 alkoxy", or "C1-C10 alkoxy". "C6-C10 aryl" and various structural moieties containing "C6-C10 aryl" may be replaced with "C6-C12 aryl". "3-7 membered heterocyclic group" and various structural moieties containing "3-7 membered heterocyclic group" may be replaced with "3-20 membered heterocyclic group", "3-12 membered heterocyclic group", or "3-10 membered heterocyclic group".

Abbreviations and Definitions

Unless otherwise indicated, the following terms and phrases as used herein are intended to have the following stated meanings: When a trade name is used herein, unless the context indicates otherwise, the trade name includes product formulations, generic drugs and active ingredients of the trade name product.

Unless otherwise stated, capitalized terms used in the claims and description herein shall have the meanings specified below.

The term "ligand" refers to a macromolecular compound that recognizes and binds to an antigen or receptor associated with a target cell. Ligands are used to present drugs to the target cell population bound to the ligand, including but not limited to protein-like hormones, lectins, growth factors, antibodies, or other molecules that bind to cells. In this embodiment of the invention, the ligand is referred to as an Ab, and the ligand can form a bond with the linker unit via a heteroatom on the ligand, preferably an antibody or antigen-binding fragment thereof, wherein the antibody is selected from chimeric, humanized, fully human, or murine antibodies; preferably a monoclonal antibody.

The ligand unit is a targeting agent that specifically binds to a target component. The ligand is capable of specifically binding to a cell part or cell component or other target molecule of interest. The target part or target is typically on the surface of a cell. In some embodiments, the ligand unit is used to deliver the 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, and non-proteins such as sugars. Suitable ligand units include, for example, antibodies such as full-length (intact) antibodies and antigen-binding fragments thereof. In embodiments where the ligand unit is a 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 and colony stimulating factors, vitamins, nutrient transport molecules, or any other cell-binding molecule or substance. In some embodiments, the linker is covalently attached 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 of the antibody. In another aspect, the sulfur atom is a sulfur atom of a cysteine residue in an imported ligand unit that forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is a sulfur atom of a cysteine residue that is introduced into the ligand unit (e.g., by targeted induction or chemical reaction). In other aspects, the linker-binding sulfur atom is selected from cysteine residues that form interchain disulfide bonds in the antibody or cysteine residues introduced into the ligand unit (e.g., by targeted mutagenesis or chemical reaction). In some embodiments, the EU index numbering system is according to Kabat {[Kabat E.A et al. (1991)] Sequences of proteins of Immunological Interest, 5th ed., NIH Publication 91-3242}.

As used herein, the terms "antibody" or "antibody unit" include within their scope any portion of the antibody structure. This unit can bind, reactively associate, or complex with a receptor, antigen, or other receptor unit present in a target cell population. An antibody can be any protein or protein-like molecule that binds, complexes, or reacts with a portion of a cell population to be treated or biologically modified. The antibodies comprising the antibody-drug conjugate compounds of the present invention retain their native antigen-binding ability. Thus, the antibodies of the present invention are capable of binding exclusively to an antigen. Antigens of interest 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 (if known or predicted to be functional), lymphokines, cytokines, molecules involved in regulating cell cycle changes, and molecules involved in angiogenesis (if known or predicted to be functional). Tumor-associated factors may be cluster differentiation factors (e.g., CD proteins).

Antibodies used in antibody-drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens. These tumor-associated antigens are well known in the industry and can be prepared using methods and information for antibody preparation that are well known in the industry. In order to develop effective cell-level targets that can be used for cancer diagnosis and treatment, researchers have sought to discover transmembrane or other tumor-associated peptides. These targets may be specifically expressed on the surface of one or more cancer cells and have little or no expression on the surface of one or more non-cancerous cells. Typically, these tumor-associated polypeptides are overexpressed on the surface of cancer cells relative to the surface of non-cancerous cells. Identifying these tumor-associated factors can greatly enhance the specific targeting properties of antibody-based cancer therapies. For convenience, information related to antigens known in the industry is identified below, including the name, other names, and GenBank accession number. Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in publicly available databases such as Genbank, and antibodies targeting tumor-associated antigens include all amino acid sequence variants and isoforms that have at least 70%, 80%, 85%, 90%, or 95% homology to the sequences identified in the references, or have the same biological properties and characteristics as the sequences of the tumor-associated antigens identified in the cited documents.

The term "inhibition" or "inhibiting" means that a detectable effect is reduced or completely prevented.

The term "cancer" refers to a physiological condition or disease characterized by unregulated cell growth. "Tumor" includes cancer cells.

The term "autoimmune disease" refers to a disease or condition that originates by targeting an individual's own body tissues or proteins.

The term "drug" refers to chemical molecules called cytotoxic drugs that have a strong ability to disrupt normal growth in tumor cells. Cytotoxic drugs can, in principle, kill tumor cells at sufficiently high concentrations, but due to their lack of specificity, they can simultaneously kill tumor cells while inducing apoptosis in normal cells, leading to severe side effects. The term includes toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (e.g., the radioisotopes At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and Lu ¹⁷⁶ ), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes, preferably toxic drugs.

The term "linker" or "linker fragment" or "linker unit" refers to a fragment or bond of a chemical structure that is attached to a ligand at one end and to a drug at the other end, or that can be attached to another linker and then to a drug, or to another linker and then to a drug.

The linkage comprising the extension, spacer, and amino acid unit can be synthesized by methods known in the art, such as those described in US 2005-0238649 A1. The linkage can be a "cleavable linkage" that facilitates release of the drug in cells. For example, an acid-labile linkage (e.g., a hydrazone), a protease-sensitive (e.g., a peptidase-sensitive) linkage, a photolabile linkage, a dimethyl linkage, or a disulfide-containing linkage can be used (Chari et al. Cancer Research 52:127-131, 1992); U.S. Patent No. 5,208,020.

Based on the mechanism of intracellular drug release, "linkers" or "linker antibody-drug conjugates," as used herein, can be categorized into two types: non-destructible linkers and destructible linkers. For antibody-drug conjugates containing non-destructible linkers, the drug release mechanism is as follows: After the conjugate binds to the antigen and is internalized by the cell, the antibody is enzymatically cleaved in lysosomes, releasing the active molecule composed of the small drug molecule, the linker, and the amino acid residues of the antibody. The resulting change in the drug molecule's structure does not reduce its cytotoxicity, but because the active molecule is charged (amino acid residues), it is unable to penetrate into neighboring cells. As a result, these active drugs are unable to kill neighboring tumor cells that do not express the target antigen (antigen-negative cells) (a bystander effect) (Ducry et al., 2010, Bioconjugate Chem. 21: 5-13). For antibody-drug conjugates containing cleavable linkers, the drug release mechanism is that the conjugate binds to the antigen and is internalized by the cell, which then cleaves the active ingredient (the small molecule drug itself) and releases it into the target cell. Cleavable linkers are primarily categorized as chemosensitive linkers and enzyme-sensitive linkers. Chemosensitive linkers can be selectively cleaved due to differences in the properties of the plasma and cytoplasm or tumor microenvironment. These properties include pH, glutathione concentration, etc. pH-sensitive linkers, such as stilbene, acetyl carbonate, and ketones, which are relatively stable in the neutral or slightly alkaline environment of blood (pH 7.3-7.5), hydrolyze in the weakly acidic tumor microenvironment (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). Antibody-drug conjugates based on these linkers typically have a short half-life (2-3 days) due to the limited plasma stability of the acid-damaged linkers. This short half-life slightly limits the use of pH-sensitive linkers in new generation antibody-drug conjugates. Glutathione-sensitive linkers are also known as disulfide-bonded linkers. Drug release is based on the difference between high intracellular glutathione concentrations (millimolar range) and relatively low glutathione concentrations in the blood (micromolar range). This is particularly true for tumor cells, where low oxygen levels lead to increased reductase activity and, consequently, higher glutathione concentrations. Disulfide bonds are thermodynamically stable and therefore have better stability in plasma. Enzymatically destabilizing linkers, such as peptide linkers, provide better control of drug release. Peptide linkers can be efficiently cleaved by lysosomal proteases such as cathepsin (cathepsin B). This peptide bond is believed to be very stable in plasma circulation due to the unfavorable extracellular pH and the presence of serum protease inhibitors, which generate proteases that are normally inactive outside of cells. Due to their high plasma stability and good selectivity and efficiency for intracellular destruction, enzyme-labile linkers are widely used as cleavable linkers in antibody-drug conjugates.

The term "antibody-drug conjugate" refers to an antibody attached to a biologically active drug via a stabilizing binding unit. In this application, a "ligand-drug conjugate," preferably an antibody-drug conjugate (ADC), refers to a monoclonal antibody or antibody fragment attached to a biologically active toxic drug via a stabilizing binding unit.

The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. boil. chem . 1968, 243 , 3558.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms (i.e., a "C1-C20 alkyl group"), preferably an alkyl group containing 1 to 12 carbon atoms (i.e., a "C1-C12 alkyl group"), more preferably an alkyl group containing 1 to 10 carbon atoms (i.e., a "C1-C10 alkyl group"), and most preferably an alkyl group containing 1 to 6 carbon atoms (i.e., a "C1-C6 alkyl group"). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-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-trimethyl, 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-dimethyl, 2,4-dimethyl, 2,2-dimethyl, 2,3-dimethyl, 3,3-dimethyl pentyl, 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, n-ethylpentyl, 2,2-dimethyl, n-nonyl, 2,2-dimethyl, n-nonyl, 2,2-dimethyl, 2,2-methylpentyl, 2,2-methylpentylethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers thereof, and the like. More preferred are lower alkyl groups having 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-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-trimethyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, hydroxyl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

The term "substituted alkyl" means that the hydrogen of the alkyl group is replaced by a substituent which, unless otherwise indicated herein, may be a group 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) ₂ R'', -CN, and -NO ₂ , the number of substituents is 0 to (2m'+1), where m' is the total number of carbon atoms in the group. R', R'', and R''' each independently represent hydrogen, unsubstituted C _1-8 alkyl, unsubstituted C 6-C 12 aryl (or C 6-C 10 aryl), C 6-C 12 aryl (or C 6-C 10 aryl) substituted with 1-3 halogens, unsubstituted C _1-8 alkyl, C _1-8 alkoxy, or C _1-8 thioalkoxy, or unsubstituted C 6-C 12 aryl (or C 6-C 10 aryl)-C _1-4 alkyl. When R' and R'' are attached to the same nitrogen atom, they may form 3, 4, 5, 6, or 7 mesocyclic rings together with that nitrogen atom. For example, -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.

The term "substituted alkyl" refers to a saturated straight or branched aliphatic hydrocarbon group having two residual groups derived from the same carbon atom or two different carbon atoms of a parent alkyl group by removing two hydrogen atoms, wherein the hydrocarbon group is a straight or branched chain group containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. Non-limiting examples of alkyl groups include, but are not limited to, methylene ( _-CH2- , 1,1-ethylene (-CH( _CH3 )-), 1,2-ethylene ( _-CH2CH2 )-, 1,1-propylene (-CH _{( CH2CH3 )} -), 1,2-propylene ( _-CH2CH ( _CH3 ) _- ), 1,3-propylene ( _-CH2CH2CH2- ), 1,4 _- butylene ( _{-CH2CH2CH2CH2- ) ,} and 1,5 _{- butylene} ( _{-CH2CH2CH2CH2CH2CH2 ) - .} -). The alkylene group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, the substituent being preferably independently selected from the group consisting of alkyl, alkynyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxyl, nitro, cyano, cycloalkyl, heterocycle, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylsulfide, heterocycloalkylsulfide, and one or more substituents are independently selected from the group consisting of oxygen-containing substituents.

The term "alkoxy" refers to -O-(alkyl) and -O-(cycloalkyl), wherein alkyl or cycloalkyl is as defined above. Non-limiting examples of C1-C6 alkoxy groups include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexyloxy. The alkoxy group may be substituted or unsubstituted as appropriate. When substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkynyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic 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 groups (e.g., "C3-C8 cycloalkyl") include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, cycloheptatrienyl, cyclooctyl, and the like, and polycyclic cycloalkyl groups include spiro, fused, and bridged ring cycloalkyl groups.

The term "heterocyclic group" means a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms (i.e., a "3-20 membered heterocyclic group"), wherein one or more of the ring atoms is a heteroatom selected from nitrogen, oxygen, or S(O) _m (wherein m is an integer from 0 to 2), excluding the ring moiety -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. Preferably, the heterocyclic group contains 3 to 12 ring atoms (i.e., a "3-12 membered heterocyclic group"), of which 1 to 4 are heteroatoms. More preferably, the heterocyclic group contains 3 to 10 ring atoms (i.e., a "3-10 membered heterocyclic group"). Non-limiting examples of monocyclic heterocyclic groups (e.g., 3-7 membered heterocyclic groups) include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, and homopiperazinyl. Polycyclic heterocyclic groups include those having spiro, fused, or bridged rings.

The term "cycloalkylalkyl" means an alkyl group substituted with one or more cycloalkyl groups, preferably one cycloalkyl group, wherein alkyl is as defined above and cycloalkyl is as defined above, for example, C3-C8cycloalkylC1-C6alkyl.

The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above, for example, a halo C1-C6 alkyl group.

The term "deuterated alkyl" means an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above, for example, a deuterated C1-C6 alkyl group.

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

The term "C6-C10 aryl" refers to a group of a carbocyclic aromatic system having 6 to 10 carbon atoms, such as phenyl and naphthyl.

The term "5-10 membered heteroaryl" refers to an aromatic heterocyclic ring, typically a 5-, 6-, 7-, 8-, 9-, or 10-membered heterocyclic ring having 1 to 3 heteroatoms selected from N, O, or S; the heteroaryl ring may further incorporate or be attached to aromatic and non-aromatic carbon atoms and heterocyclic rings as appropriate. Non-limiting examples of the 5- to 10-membered heteroaryl ring are, for example, pyridyl, pyrazinyl, pyrimidinyl, oxazinyl, indolyl, imidazolyl, thiazolyl, isothiazolyl, thioxazolyl, pyrroloxy, phenyl-pyrroloxy, furanyl, phenyl-furanyl, phenyl-furanyl, oxazolyl, isoxazolyl, pyrazolyl, thienyl, benzo-furanyl, benzothienyl, benzo-1,3-dioxolane, (benzodioxolanyl), isodihydroindolyl, benzimidazolyl, indazolyl, quinolinyl, isoquinolinyl, 1,2,3-triazolyl, 1-phenyl-1,2,3-triazolyl, 2,3-dihydroindolyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothiophene, benzylpyranyl, 2,3-dihydrobenzoxazinyl, 2,3-dihydroquinoxalinyl, and other groups.

The term "substituted C6-C10 aryl", "substituted 5-10 membered heteroaryl", or "substituted 3-7 membered heterocyclic" means that, unless otherwise specified herein, a hydrogen atom in the aryl, heteroaryl, or heterocyclic group is replaced by a substituent. The substituents of the aryl or heteroaryl or heterocyclic group may be a group selected from the group consisting of: -halogen, -OR', -NRR''', -SR', -SiR'R''R''', -OC(O)R', -C(O)R', -CO ₂ R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O) ₂ R', -NH-C(NH ₂ )=NH, -NR'C(NH ₂ )=NH, -NH-C(NH ₂ )=NR', -S(O)R', -S(O) ₂ R', -S(O) ₂ NR'R'', -NR'S(O) ₂ R'', -CN and -NO ₂ , having 0 to (2m'+1) substituents, where m' is the total number of carbon atoms in the group. R', R'', and R''' each independently represent hydrogen, unsubstituted _C1-8 alkyl, unsubstituted C6-C12 aryl (or C6-C10 aryl), C6-C12 aryl (or C6-C10 aryl) substituted with 1-3 halogens, unsubstituted _C1-8 alkyl, _C1-8 alkoxy, or C1-8 thioalkoxy, or unsubstituted C6- _C12 aryl (or C6-C10 aryl) _-C1-4 alkyl. When R' and R'' are attached to the same nitrogen atom, they can form a 3-, 4-, 5-, 6-, or 7-membered ring with that nitrogen atom. For example, -NR'R'' includes 1-pyrrolidinyl and 4-morpholinyl.

The term "hydroxy" refers to an -OH group.

The term "halogen" means fluorine, chlorine, bromine, or iodine.

The term "amino" means -NH ₂ . The term "nitro" means -NO ₂ .

The term "amido" means -C(O)N(alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

The term "carboxylate group" means -C(O)O(alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

This application also encompasses various deuterated forms of Formula I. Each of the available hydrogen atoms attached to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can refer to the relevant literature for synthesizing deuterated forms of Formula I. Commercially available deuterated starting materials can be used to prepare deuterated forms of Formula I, or they can be synthesized by known techniques using deuterated reagents, non-limiting examples of which include deuterated borane, trideuterated borane in tetrahydrofuran, lithium-aluminum hydrogen deuteride, ethyl iodide deuteride, diiodomethane deuteride, and the like.

The term "antibody" refers to an immunoglobulin that is a tetrapeptide chain composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. Immunoglobulins differ in the composition and arrangement of amino acids in the constant regions of the heavy chains and, therefore, in their antigenicity. Thus, immunoglobulins can be classified into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, with corresponding heavy chains of μ, δ, γ, α, and ε chains, respectively. Ig proteins of the same class can be further divided into subclasses based on differences in the amino acid composition of their hinge regions and the number and location of disulfide bonds in their heavy chains. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into either kappa or lambda chains based on differences in the homeostatic regions. Each of the five Ig classes can possess either a kappa or lambda chain. The antibodies described in the present invention are preferably specific antibodies against cell surface antigens on target cells, and non-limiting examples are 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, anti-Claudin18.2 antibody, anti-mesothelin antibody, anti-HER2 Anti-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-binding protein 4 antibody, 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-β-calcineurin antibody, anti-GD3 antibody, anti-calcineurin 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.

The term "solvate" or "solvated compound" refers to a pharmaceutically acceptable solvent compound that is used with one or more solvent molecules to form the ligand-drug conjugate of the present application. Non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

The term "drug loading" refers to the average amount of cytotoxic drug loaded on each antibody in Formula I. It can also be expressed as a ratio of drug to antibody, and drug loading can range from 0-12, preferably 1-10 cytotoxic drug (d) per antibody (Ab). In the examples of this application, drug loading is referred to as n, and illustratively can be an average of, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The average amount of drug per ADC molecule after the coupling reaction can be identified by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA, and HPLC.

In one embodiment of the present application, a cytotoxic drug is coupled to an open cysteine hydroxyl-SH group and/or a hydroxyl-SH group of a site-specifically mutated cysteine residue between antibody chains via a linker unit. In general, the number of drug molecules that can be coupled to the antibody in the coupling reaction is less than or equal to the theoretical maximum.

The loading of the ligand-cytotoxic drug conjugate can be controlled by using the following non-limiting methods, including: (1) controlling the molar ratio of the linker reagent to the monoclonal antibody, (2) controlling the reaction time and temperature, and (3) selecting different reaction reagents.

For preparation of pharmaceutical compositions, refer to pharmacopoeias such as the Chinese Pharmacopoeia or USP.

The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a salt of the ligand-drug conjugate of the present application, or a salt of the compound described herein, that is safe and effective for use in mammals and, if desired, biologically active. The ligand-drug conjugate compounds of the present application contain at least one carboxyl group and are therefore capable of forming salts with bases. Non-limiting examples of pharmaceutically acceptable salts include sodium salts, potassium salts, calcium salts, or magnesium salts.

The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a salt of the ligand-drug conjugate of the present application, or a salt of the compound described in the present application, that is safe and effective for use in mammals and, if desired, biologically active. The ligand-drug conjugate of the present invention contains at least one amine group and is therefore capable of forming a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include hydrochlorides, hydrobromides, hydroiodates, sulfates, hydrosulfates, citrates, acetates, succinates, ascorbates, oxalates, nitrates, pyrites, hydrophosphates, dihydrophosphates, salicylates, citrates, tartrates, maleates, fumarates, formates, benzoates, methanesulfonates, ethanesulfonates, benzenesulfonates, and p-toluenesulfonic acid.

The term "acidic amino acid" refers to an amino acid with an isoelectric point less than 7. Acidic amino acid molecules typically contain one or more acidic groups, such as carboxyl groups, that efficiently ionize to anionic forms within the structure, increasing hydrophilicity. Acidic amino acids can be natural or non-natural.

The term "natural amino acid" refers to amino acids that are biologically synthesized. Natural amino acids are usually L-form, but there are a few exceptions that occur naturally and are biosynthesized by living organisms, such as glycine.

The term "unnatural amino acids" refers to amino acids obtained by synthetic means.

This application may be more readily understood by reference to the following detailed description of the specific embodiments and examples contained herein. Although this application is described with reference to specific details of certain embodiments thereof, such details are not intended to limit the scope of this disclosure. The experimental procedures indicated in the following examples without specific conditions were generally performed under normal conditions or under conditions recommended by the manufacturer. Unless otherwise indicated, all percentages, ratios, proportions, or parts are by weight. Example 1

Synthesis of compound M1 : ; In a 5000 mL flask, N-fluorenylmethoxycarbonyl-glycine-glycine (100 g, 282 mmol, 1.0 equivalent), lead tetraacetate (175 g, 395 mmol, 1.4 equivalent), 2000 mL of dry tetrahydrofuran and 670 mL of toluene were added, stirred evenly, protected by nitrogen, and heated to 85°C for 2.5 h. The reaction was monitored by TLC, and the raw material completed the reaction. After the reaction, it was cooled to room temperature, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain compound M1 (87 g); LC-MS: [M+NH] ₄ ⁺ =386.0. Example 2

Synthesis of compound M3 : In a 1000 mL flask, compound SM-2 (synthesized according to the method disclosed in CN108452321A) (40 g, 96 mmol, 1.0 equivalent), triethylamine (26.7 mL, 2.0 equivalent), and toluene (400 mL) were added, and the compound was refluxed at 120°C for 2 h to perform the reaction. The reaction was monitored by TLC until almost complete, and then the solvent was separated by reducing the temperature to 50°C and reducing the pressure. The solvent was removed by rotating at 50°C under reduced pressure. Dissolved with ethyl acetate (150 mL) and water (40 mL), adjusted to pH 2-3 with 1M HCl while stirring in an ice bath, and partitioned. The aqueous layer was extracted again with ethyl acetate, and the organic layers were combined and dried over anhydrous sodium sulfate. After filtration, the residue was concentrated to afford a pale yellow oily crude product, which was purified by column chromatography (DCM:MeOH = 40:1) to obtain compound M2 (26.6 g); LC-MS: [M+H] ⁺ = 399.3.

In a 1000 mL flask, compound M2 (26.5 g, 60.5 mmol, 1.0 equivalent), pentafluorophenol (12.2 g, 66.5 mmol, 1.1 equivalent), DCC (13.7 g, 66.5 mmol, 1.1 equivalent), and THF (300 mL) were added, and the reaction was carried out at room temperature for 30 min (monitored by TLC), and the insoluble material was filtered off. The reaction solution was directly purified by the preparation, and the preparation solution was concentrated at 35° C. in a reduced pressure water bath to remove acetonitrile and freeze-dried to obtain compound M3 (31.5 g) in 64% yield; LC-MS: [M+H] ⁺ = 565.1. Example 3

Synthesis of compound ent-M3 : ; Referring to the synthetic route of Example 2, compound ent-M3 (27.8 g) was obtained; LC-MS: [M+H] ⁺ = 565.2. Example 4

Synthesis of compound 1 : Step 1: Compound 1a was placed in a 250 mL flask, and 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. Benzylhydroxyacetic acid (5.4 g, 32.6 mmol) was added dropwise. The mixture was naturally warmed to room temperature (reaction time: about 2-4 h) and monitored by TLC. At the end of the reaction, saturated NaHCO ₃ solution was added, 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 (PE:EA=10:1-5:1-1:1) to obtain 1a (4 g) in 52% yield; LC-MS: [M+H] ⁺ =475.18. Step 2: Compound 1b

In a 25 mL flask, 1a (2 g, 4.2 mmol) and 10 mL of DMF were added, and the mixture was stirred at 0°C. DBU (766 mg, 5.04 mmol) was added, and the mixture was reacted for 1 h. After the completion of Fmoc deprotection as monitored by TLC, the reaction was set aside; another 25 mL flask was charged with M4 (prepared by the method disclosed in 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, and DIPEA (830 uL, 5.04 mmol) was added to the reaction vial under an ice-water bath, and stirring was continued for 30 min. The above reaction solution was added to the reaction flask, and the reaction was raised to room temperature. The reaction was completed by HPLC monitoring, and the reaction solution was purified by preparative liquid phase to obtain a product preparation. The preparation was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain solid 1b (1.7 g) in a 63% yield; LCMS: [M+H] ⁺ = 648.26. Step 3: Compound 1c

1b (900 mg, 1.39 mmol) was added to a 25 mL flask, and after dissolving in 15 mL of DMF, 900 mg of 5% Pd/C was added, and the reaction was hydrogenated for 2 h. The reaction was completed, and the filtrate was filtered to obtain a filtrate, which was directly used in the next step without purification. Step 4: Compound 1d

The crude product 1c was placed in an ice-water bath, DIPEA (235 μL, 1.39 mmol) and compound M3 (784 mg, 1.39 mmol) were added, and the reaction was allowed to reach room temperature for 1 h. The reaction was monitored for completion by HPLC, and the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution, which was lyophilized to obtain 1d (504 mg); LC-MS: [M+H] ⁺ = 804.4. Step 5: Compound 1e

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 were added to a 50 mL single-necked vial. DIPEA (378 μL, 2.29 mmol) was added to the vial under an ice-water bath, and the reaction was carried out at room temperature for 2 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to give a preparative solution of compound 1e, and the preparative solution was lyophilized to give 1e (210 mg). The reaction was monitored by LCLC, and the preparative solution was lyophilized to give 1e (210 mg). The reaction was carried out at room temperature for 2 h. After the reaction was completed and monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a solution of compound 1e . The solution was lyophilized to obtain 1e (210 mg); LC-MS: [M+H] ⁺ = 1221.6. Step 6: Compound 1

1e (100 mg, 0.081 mmol), zinc bromide (368 mg, 1.63 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was freeze-dried to obtain solid compound 1 (60 mg); LC-MS: [M+H] ⁺ = 1065.3. Example 5

Synthesis of compound 2 : ; Referring to the synthetic route of Example 4, compound 2 (51 mg) was obtained; LC-MS: [M+H] ⁺ = 1065.3. Example 6

Synthesis of compound 3 : Step 1: Compound 3a was added to a 250 mL flask with M1 (6 g, 16.3 mmol), 100 mL THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). 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 reaction was allowed to cool, the mixture was naturally warmed to room temperature for reaction (about 2-4 h) and monitored by TLC. At the end of the reaction, saturated NaHCO ₃ solution was added, 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 (PE:EA=10:1-5:1-2:1) to give 3a (4.2 g) in 52% yield; LC-MS: [M+H] ⁺ = 503.3. Step 2: Compound 3b

To a 25 mL flask, 3a (2 g, 4.0 mmol) and 10 mL of DMF were added, stirred at 0°C, and DBU (760 mg, 5.0 mmol) was added, followed by reaction for 1 h. After completion of Fmoc deprotection, monitored by TLC, the reaction was allowed to stand. M4 (1.65 g, 4.0 mmol), PyBOP (2.59 g, 5.0 mmol), HOBt (675 mg, 5.0 mmol), and 10 mL of DMF were added to another 25 mL flask and stirred continuously. DIPEA (823 μL, 5.04 mmol) was added in an ice-water bath and stirred for 30 min. The above reaction solution was then added to the reaction flask and the reaction was allowed to warm to room temperature. After the reaction was monitored by HPLC, the reaction solution was purified by preparative liquid phase to obtain a product preparation solution, which was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain solid 3b (1.4 g) in 53% yield; LC-MS: [M+H] ⁺ = 676.2. Step 3: Compound 3c

3b (700 mg, 1.04 mmol) was added to a 25 mL flask and dissolved in 10 mL of DMF. 700 mg of 5% Pd/C was then added and the reaction was hydrogenated for 1.5 h. The reaction was completed and the filtrate was filtered to obtain a filtrate, which was used directly in the next step without purification. Step 4: Compound 3d

The crude product 3c was placed in an ice-water bath, DIPEA (210 μL, 1.25 mmol) was added, and compound M3 (704 mg, 1.25 mmol) was added, and the reaction was allowed to reach room temperature for 1 h. The reaction completion was monitored by HPLC, and the reaction solution was purified by HPLC to give a product, which was lyophilized to give 3d (486 mg); LC-MS: [MH] ⁻ = 830.5. Step 5: Compound 3e

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 were added to a 50 mL flask. DIPEA (219.5 μL, 1.33 mmol) was added to the vial under an ice-water bath, and the reaction was carried out at room temperature for 3 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to give a prepared solution of compound 3e . The reaction was carried out at room temperature for 3 h. After the reaction was completed and monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a solution of compound 3e , which was then lyophilized to obtain 3e (157 mg); LC-MS: [M+H] ⁺ = 1249.6. Step 6: Compound 3

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 vial and the reaction was carried out at 40° C. for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain solid compound 3 (64 mg); LC-MS: [M+H] ⁺ = 1093.1. Example 7

Synthesis of compound 4 : Compound 4 (60 mg) was obtained by following the synthetic route of Example 6; LC-MS: [M+H] ⁺ = 1093.2. Example 8

Synthesis of compound 5A : Step 1: Compound 5a : M1 (500 mg, 1.4 mmol, 1.0 equivalent), p-toluenesulfonic acid monohydrate (26 mg, 0.1 mmol, 0.1 equivalent), and 10 mL of THF were added to a 25 mL flask and stirred thoroughly. The temperature was then lowered to 0°C, followed by the slow addition of benzyl L-lactate (1.2 g, 7.0 mmol, 5 equivalents). After the addition of the reactants, the temperature was raised to room temperature and monitored by TLC. At the end of the reaction, a saturated _NaHCO₃ solution was added, 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 afford 5a (400 mg); LC-MS: [M+NH] _₄⁺ = ^506.2 .

¹ H NMR (400 Mz, CDCl ₃ /CD ₃ OD): 1.39 (3H, d, J = 6.8 Hz), 3.78 (2H, t, J = 4.0 Hz), 4.17-4.27 (2H, m), 4.42 (2H, d, J = 4.0 Hz), 4.72-4.85 (2H, m), 5.11-5.58 (2H, m), 5.43 (1H, s), 7.06 (1H, t, J = 8.0 Hz), 7.25-7.33 (6H, m), 7.38 (2H, t,, 7.38 (2H, t, 7.06 (1H, t, 7.06), 7.25-7.33 (6H, m), 7.25-7.33 (7.25-7.33) 5.43 (1H, s), 7.06 (1H, t, J = 8.0 Hz), 7.25-7.33 (6H, m), 7.38 (2H, t, J = 8.0 Hz), 7.57 (2H, d, J = 8.0 Hz), 7.75 (2H, d, J = 8.0 Hz). Step 2: Compound 5b

Compound 5a (400 mg, 0.8 mmol, 1.0 equivalent) and 4 mL of DMF were added to a 25 mL flask, stirred thoroughly, and then cooled to 0°C. DBU (137 mg, 0.9 mmol, 1.1 equivalent) was then slowly added, and the reaction was allowed to reach room temperature. The reaction was monitored by TLC, and the completion of the reaction was recorded as reaction solution ①. Another 25 mL single-necked vial was obtained, and M4 (372 mg, 0.9 mmol, 1.1 equivalent), PyBOP (852 mg, 1.6 mmol, 2.0 equivalent), and 3 mL of DMF were added. The mixture was stirred at room temperature for 5 min, and reaction solution ① was added. The reaction was carried out at room temperature and monitored by HPLC. The reaction was completed, and the reaction solution was purified by HPLC to produce compound 5b (326 mg); LC-MS: [M+NH] ₄ ⁺ = 679.2. Step 3: Compound 5c

5b (4.0 g, 6.05 mmol, 1.0 equivalent) was added to a 100 mL flask, dissolved in DMF (60 mL), and then 5% Pd/C (4 g) was added and hydrogenated at room temperature for 4 h (HPLC was used to monitor the progress of the reaction). The Pd/C was filtered and the filtrate was directly placed in an ice-water bath (about 0°C) without concentration and retained for use. Step 4: Compound 5d

The crude product 5c was placed in an ice-water bath, DIPEA (1.1 mL, 1.1 equivalents) was added, and compound M3 (3.4 g, 6.05 mmol) was added, and the reaction was allowed to reach room temperature for 2 h. The completion of the reaction was detected by HPLC, and the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution, which was lyophilized to obtain 5d (3.15 g); LC-MS: [MH] ⁻ = 816.3. Step 5: Compound 5e

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) were added to a 100 mL single-necked vial and the reaction was allowed to reach room temperature for 2 h. The reaction was monitored by HPLC. DIPEA (1.05 mL, 1.5 eq) was added and the reaction was allowed to reach room temperature for 2 h (monitored by HPLC). The reaction solution was directly purified by preparative method, and the preparative solution was concentrated by pumping water at 35° C. under reduced pressure to remove acetonitrile and lyophilized to obtain compound 5e (1.92 g) in 61% yield; LC-MS: [M+H] ⁺ = 1235.4. Step 6: Compound 5A

Compound 5e (1.0 g, 0.8 mmol, 1.0 equivalent) and 35 mL of nitromethane were added to a 100 mL flask and dissolved. Zinc bromide (3.64 g, 16 mmol, 20.0 equivalents) was then added and reacted in an oil bath at 40°C for 30 min (pre-stabilized by preheating). The mixture was then concentrated under reduced pressure in a water bath at 45°C to remove the nitromethane, yielding a solid yellow residue (monitored by HPLC). After the acid was prepared, a preparative solution of compound 5A was obtained. The preparative solution was concentrated and the acetonitrile was separated by pumping under reduced pressure in a water bath at 35°C. The solution was then lyophilized to obtain compound 5A (786 mg) in a 90% yield.

LC-MS:[M+H] ⁺ =1079.4; ¹ H NMR (400 MHz, DMSO-d6) δ 9.39 - 9.02 (m, 1H), 8.70 (t, J = 6.5 Hz, 1H), 8.64 (t, J = 5.7 Hz, 1H), 8.56 (d, J = 8.8 Hz, 1H), 8.34 (t, J = 5.7 Hz, 1H), 8.16 (d, J = 8.2 Hz, 1H), 8.01 (t, J = 5.5 Hz, 1H), 7.71 (d, J = 10.9 Hz, 1H), 7.30 (s, 1H), 7.28 – 7.15 (m, 4H), 7.14 (s, 2H), 5.53 (dd, J = 14.5, 6.4 Hz, 1H), 5.49 - 5.34 (m, 2H), 5.22 (d, J = 18.8 Hz, 1H), 5.09 (d, J = 18.7 Hz, 1H), 5.03 (dd, J = 9.6, 3.9 Hz, 1H), 4.73 (dd, J = 9.9, 6.9 Hz, 1H), 4.59 (dd, J = 10.1, 6.5 Hz, 1H), 4.49 (dd, J = 13.2, 8.6, 4.4 Hz, 1H), 4.14 (dd, J = 13.3, 6.6 Hz, 2H), 3.93 (s, 2H), 3.84 (dd, J = 16.5, 6.3 Hz, 1H), 3.76 (dd, J = 16.9, 5.7 Hz, 2H), 3.70 (d, J = 9.9, 6.9 Hz, 2H) 3.70 (dd, J = 5.2 Hz, 2H), 3.60 (dd, J = 16.7, 5.4 Hz, 1H), 3.52 (dd, J = 16.4, 5.1 Hz, 1H), 3.45 (dd, J = 12.8, 10.1 Hz, 1H), 3.25 - 3.15 (m, 1H), 3.14 - 3.05 (m, 1H), 3.01 (dd, J = 13.7, 4.1 Hz, 1H), 2.73 (dd, J = 13.5, 9.8 Hz, 1H), 2.54 - 2.47 (m, 1H), 2.33 (s, 2H), 2.17 (d, J = 5.5 Hz, 2H ), 1.91 - 1.79 (m, 2H), 1.33 (d, J = 6.6 Hz, 2H), 0.87 (t, J = 7.3 Hz, 2H). Example 9

Synthesis of compound 5B : Step 1: Compound 5d-1 was stirred in a 25 mL flask. Compound 5b (300 mg, 0.45 mmol, 1.0 eq) and DMF (3 mL) were added to clarify the solution. 5% Pd/C (300 mg) was added, and hydrogen was purged three times. The hydrogenation reaction was continued for 2 h. The reaction was monitored by HPLC. At the end of the reaction, the Pd/C was removed by filtration, and the filtrate was cooled to 0-5°C. DIPEA (65 mg, 0.5 mmol, 1.1 eq) was added, followed by ent-M3 (255 mg, 0.45 mmol). The reaction was then heated to 20±5°C for 1 h. The reaction was monitored by HPLC. At the end of the reaction, it was purified by HPLC preparation, and the product preparation was collected and lyophilized to obtain 54% yield of compound 5d-1 (200 mg); LC-MS: [MH] ^- = 816.3. Step 2: Compound 5e-1

Compound 5d-1 (200 mg, 0.24 mmol, 1.0 equiv), M5 (127 mg, 0.24 mmol, 1.0 equiv), PyBOP (187 mg, 0.36 mmol, 1.2 equiv), HOBt (48 mg, 0.36 mmol, 1.2 equiv), and DMF (6 mL) were added to a 25 mL flask. The water bath was lowered to 0-5°C, DIPEA (62 mg, 0.48 mmol, 2.0 equiv) was added, and the reaction temperature was raised to 20±5°C for 2 h. Completion of the reaction was monitored by HPLC. The reaction solution was directly purified by HPLC preparative, and the product preparation was collected and lyophilized to obtain compound 5e-1 (162.8 mg); LC-MS: [M+H] ⁺ = 1235.4. Step 3: Compound 5B

Compound 5e-1 (110 mg, 0.089 mmol, 1.0 eq), ZnBr ₂ (400 mg, 1.78 mmol, 20.0 eq) and CH ₃ NO ₂ (10 mL) were added sequentially to a 25 mL flask. After addition, the reaction was heated to 40° C. for 0.5 h, after which the reaction was stopped and the reaction solution was directly dried at 45° C. by rotary drying under reduced pressure to obtain a yellow solid. The reaction was monitored by HPLC. The rotary dried solid was directly purified by HPLC preparation, and the product preparation was collected and freeze-dried to obtain compound 5B (73.4 mg) in 76.5% yield; LC-MS: [M+H] ⁺ =1079.4. Example 10

Preparation of compound 6A : Referring to the synthetic route of Example 8, compound 6A (71 mg) was obtained; LC-MS: [M+H] ⁺ = 1079.4. Example 11

Preparation of compound 6B : Compound 6B (59 mg) was obtained by following the synthetic route of Example 9; LC-MS: [M+H] ⁺ = 1079.4. Example 12

Preparation of compounds 7A and 7B : Step 1: Compound 7a was added to a 250 mL flask with M1 (10 g, 27.1 mmol), benzyl 3,3,3-trifluorolactate (prepared according to the method disclosed in WO2020063673A1) (12.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene. The mixture was reacted at 100°C for 4 h. The reaction was allowed to complete. After the reaction was complete, the mixture was cooled to room temperature, filtered to remove insoluble material, and the filtrate was concentrated to obtain a 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 compound, with a yield of 35.1%; LC-MS: [M+H] ⁺ = 543.17. Step 2: Compound 7b

7a (5 g, 9.2 mmol) and 15 mL of DMF were added to a 50 mL flask, dissolved, and clarified. DBU (1.68 g, 11 mmol) was then added in an ice-water bath and reacted for 1 h. This was recorded as reaction solution ①. Another 50 mL flask was prepared, and M4 (3.8 g, 9.2 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol), and 10 mL of DMF were added and dissolved. DIPEA (1.82 mL, 11 mmol) was added in an ice-water bath and the reaction continued for 30 min. Reaction solution ① was then added, and the reaction was allowed to reach room temperature for 2 h. The reaction was monitored by HPLC. The reaction was monitored by HPLC for 2 h. After the reaction was complete, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a preparative solution. The preparative solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.1 g of a solid with a yield of 62.3%; LC-MS: [M+H] ⁺ = 716.25. Step 3: Compound 7d

7b (900 mg, 1.26 mmol) was added to a 25 mL flask and dissolved in 15 mL of DMF. 900 mg of 5% Pd/C was added and hydrogenated for 2 h. After the reaction was completed, the filtrate was filtered and placed in an ice-water bath. DIPEA (228 uL, 1.38 mmol) was added, followed by M3 (712 mg, 1.26 mmol), and the solution was allowed to warm to room temperature. The reaction was monitored by HPLC for 1 h. The reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a preparative solution, which was then freeze-dried to obtain 525 mg of the product in a 47.9% yield; LC-MS: [MH] ^- = 870.33. Step 4: Compound 7e

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 were added to a 50 mL flask. DIPEA (378 μL, 2.29 mmol) was added to the flask under an ice-water bath, and the reaction was carried out at room temperature for 2 h. After monitoring the reaction by HPLC, the reaction solution was purified by HPLC to obtain a preparative solution of compound 7e-1 and compound 7e-2, and the preparative solution was lyophilized to obtain 150 mg of compound 7e-2. The reaction was carried out at room temperature for 2 h. After HPLC monitoring, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain solutions of compound 7e-1 and compound 7e-2 . These solutions were lyophilized to obtain 150 mg of compound 7e-1 (LC-MS: [M+H] ⁺ = 1289.46) and 220 mg of compound 7e-2 (LC-MS: [M+H] ⁺ = 1289.46), respectively. Step 5: Compound 7A

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 flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was freeze-dried to obtain 52 mg of a solid; TOF result: 1133.3613. Step 6: Compound 7B

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 flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was freeze-dried to obtain 63 mg of a solid; TOF result: 1133.3668. Example 13

Synthesis of compounds 8A and 8B : Step 1: Compound 8d was added to a 25 mL flask, 7c (900 mg, 1.83 mmol) was dissolved in 20 mL of DMF, and then DIPEA (303 uL, 1.83 mmol) was added, and ent-M3 (1034 mg, 1.83 mmol) was added, and the reaction was allowed to reach room temperature for 1 h. The completion of the reaction was detected by HPLC, and the reaction solution was purified by high-performance liquid chromatography (HPLC) to obtain a preparative solution, which was lyophilized to give 613 mg of the product in a 38.5% yield. The preparative solution was lyophilized to obtain 613 mg of the product in a 38.5% yield; LC-MS: [MH] ^- = 870.32. Step 2: Compound 8e-1 and Compound 8e-2

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 were added to a 50 mL flask. DIPEA (378 μL, 2.29 mmol) was added to the ice-water bath in the vial, and the reaction was carried out at room temperature for 2 h. The reaction was monitored by HPLC, and the reaction solution was purified by HPLC to obtain preparations of compounds 8e-1 and 8e-2 . After the reaction was completed by HPLC, the reaction solution was purified by high-performance liquid chromatography (HPLC) to obtain solutions of Compound 8e-1 and Compound 8e-2 . These solutions were lyophilized to obtain 140 mg of Compound 8e-1 and 210 mg of Compound 8e-2 , respectively. LC-MS for Compound 8e-1 : [M+H] ⁺ = 1289.47; LC-MS for Compound 8e-2 : [M+H] ⁺ = 1289.47. Step 3: Compound 8A

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 flask and reacted at 40°C for 1 h. After the reaction was completed by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was freeze-dried to obtain 50 mg of a solid; TOF result: 1133.3623. Step 4: Compound 8B

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 vial and reacted at 40°C for 1 h. After the reaction was completed by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain 58 mg of a solid; TOF result: 1133.3653. Example 14

Synthesis of compound 9A : Step 1: Compound 9a was added to a 250 mL flask with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). The mixture was stirred and cooled to 0°C. Benzyl 2-hydroxy-2-cyclopropylacetate (prepared according to the method disclosed in US20050020645 A1) (6.3 g, 32.6 mmol) was added dropwise. After the reaction mixture was allowed to cool to room temperature (reaction time: approximately 2-4 h) and monitored by TLC. At the end of the reaction, saturated NaHCO ₃ solution was added, 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 (PE:EA=10:1-5:1-2:1) to give 9a (3.7 g) in 45% yield; LC-MS: [M+H] ⁺ =501.5. Step 2: Compound 9b

To a 25 mL flask, 9a (2 g, 4.0 mmol) and 10 mL of DMF were added, stirred at 0°C, and DBU (760 mg, 5.0 mmol) was added, followed by reaction for 1 h. After completion of Fmoc deprotection, monitored by TLC, the reaction was allowed to stand. M4 (1.65 g, 4.0 mmol), PyBOP (2.59 g, 5.0 mmol), HOBt (675 mg, 5.0 mmol), and 10 mL of DMF were added to another 25 mL flask and stirred continuously. DIPEA (823 μL, 5.04 mmol) was added in an ice-water bath and stirred for 30 min. The above reaction solution was then added to the reaction flask and the reaction was allowed to warm to room temperature. After the reaction was monitored by HPLC, the reaction solution was purified by preparative liquid phase to obtain a product preparation solution, which was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 1.5 g of a solid in a 56% yield; LC-MS: [M+H] ⁺ = 674.7. Step 3: Compound 9c

Compound 9b (900 mg, 1.3 mmol) was added to a 25 mL flask and dissolved in 10 mL of DMF and clarified. 900 mg of 5% Pd/C was added and hydrogenation was performed for 1.5 h. After the reaction was completed, the filtrate was filtered to obtain a filtrate, which was directly used in the next step without purification. Step 4: Compound 9d

The crude product 9c was placed in an ice-water bath, DIPEA (223 μL, 1.3 mmol) was added, and compound M3 (750 mg, 1.3 mmol) was added, and the reaction was allowed to reach room temperature for 1 h. The reaction was monitored for completion by HPLC, and the reaction solution was purified by HPLC to give a product, which was lyophilized to give 9d (529 mg); LC-MS: [MH] ⁻ = 828.4. Step 5: Compound 9e

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 were added to a 50 mL vial. DIPEA (351 μL, 2.13 mmol) was added to the flask under an ice-water bath, and the reaction was allowed to proceed for 3 h. After the reaction was completed by HPLC, the reaction solution was purified by HPLC to obtain a preparative solution of compound 9e . After monitoring the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography (HPLC) to obtain a preparative solution of compound 9e . The solution was lyophilized to obtain 9e (257 mg); LC-MS: [M+H]+ = 1247.5. Step 6: Compound 9A

9e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain solid compound 9A (55 mg); LC-MS: [M+H] + = 1091.3. Example 15

Synthesis of compound 9B : Following the synthetic route of Example 14, compound 9B (44 mg) was obtained; LC-MS: [M+H]+ = 1091.3. Example 16

Synthesis of compound 10A : Step 1: Compound 10a was added to a 250 mL flask with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). The mixture was stirred and cooled to 0°C. Benzyl 3-hydroxy-2-cyclopropylpropionate (prepared according to the method disclosed in WO2013187496A1) (6.7 g, 32.6 mmol) was then added dropwise. After the reaction was allowed to cool to room temperature (approximately 2-4 h), the reaction was monitored by TLC. At the end of the reaction, saturated NaHCO ₃ solution was added, 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 (PE:EA=10:1-5:1-2:1) to give 10a (4.9 g) in 58% yield; LC-MS: [M+H] ⁺ =515.4. Step 2: Compound 10b

To a 25 mL flask, 10a (4 g, 7.8 mmol) and 10 mL of DMF were added, stirred at 0°C, and DBU (1.2 g, 8.0 mmol) was added, and the reaction was allowed to proceed for 1 h. After completion of Fmoc deprotection, monitored by TLC, the reaction was allowed to proceed. Another 25 mL flask was charged with 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. With continuous stirring, DIPEA (1.65 mL, 10.1 mmol) was added in an ice-water bath and the mixture was stirred for 50 min. The above reaction solution was then added to the flask and the temperature was raised to room temperature. After the reaction was monitored by HPLC, the reaction solution was purified by preparative liquid phase to obtain a product preparation solution, which was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.3 g of a solid in a 42% yield; LC-MS: [M+H] + = 688.8. Step 3: Compound 10c

Compound 10b (1.0 g, 1.45 mmol) was added to a 25 mL flask and dissolved in 15 mL of DMF for clarification. Then, 1.0 g of 5% Pd/C was added and hydrogenation was carried out for 1.5 h. The reaction was completed and the filtrate was filtered to obtain a filtrate, which was directly used in the next step without purification. Step 4: Compound 10d

The crude product 10c was placed in an ice-water bath, DIPEA (258 μL, 1.5 mmol) was added, and compound M3 (837 mg, 1.45 mmol) was added, and the reaction was allowed to reach room temperature for 1 h. The reaction completion was monitored by HPLC, and the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution, which was lyophilized to obtain 10d (499 mg); LC-MS: [MH] ⁻ = 842.4. Step 5: Compound 10e

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 were added to a 50 mL flask in an ice-water bath. DIPEA (330 μL, 2.0 mmol) was added, and the reaction was allowed to proceed for 3 h. After monitoring the reaction by HPLC, the reaction solution was purified by HPLC to give a prepared solution of compound 10e . The reaction was carried out at room temperature for 3 h. After the reaction was completed and monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a solution of compound 10e . The solution was lyophilized to obtain 10e (188 mg); LC-MS: [M+H]+ = 1261.5. Step 6: Compound 10A

10e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain solid compound 10A (61 mg); LC-MS: [M+H] + = 1105.4. Example 17

Synthesis of compound 10B : Referring to the synthetic route of Example 16, compound 10B (75 mg) was obtained; LC-MS: [M+H]+ = 1105.4. Example 18

Synthesis of compound 11A : Step 1: Compound 11a was added to a 250 mL flask with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). The mixture was stirred and cooled to 0°C. Benzyl 2-hydroxy-2-cyclobutylacetate (see Journal of Medicinal Chemistry , 2013, 56 (13), 5541-5552. The method of synthesis) (6.7 g, 32.6 mmol) was added dropwise. After the reaction was allowed to cool naturally to room temperature (the reaction was carried out for about 2-4 h) and monitored by TLC. At the end of the reaction, saturated NaHCO ₃ solution was added, 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 (PE:EA=10:1-5:1-2:1) to give 11a (5.1 g) in 62% yield; LC-MS: [M+H] ⁺ =515.7. Step 2: Compound 11b

To a 25 mL flask, 11a (4 g, 7.8 mmol) and 10 mL of DMF were added, stirred at 0°C, and DBU (1.2 g, 8.0 mmol) was added, and the reaction was allowed to proceed for 1 h. After the completion of Fmoc deprotection, monitored by TLC, the reaction was allowed to proceed. M4 (3.3 g, 8.0 mmol), PyBOP (5.2 g, 10.0 mmol), HOBt (1.35 g, 10.0 mmol), and 10 mL of DMF were added to another 25 mL single-necked vial and stirred continuously. DIPEA (1.63 mL, 10.0 mmol) was added and the mixture was stirred in an ice-water bath for 40 min. The reaction solution was then added to the flask. After the reaction was monitored by HPLC, the reaction solution was purified by preparative liquid phase to obtain a product preparation solution, which was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.3 g of a solid in a 42% yield; LC-MS: [M+H] + = 688.3. Step 3: Compound 11c

Compound 11b (2.0 g, 2.9 mmol) was added to a 25 mL flask and dissolved in 25 mL of DMF and clarified. Then, 2.0 g of 5% Pd/C was added and hydrogenation was performed for 3 hours. After the reaction was completed, the filtrate was filtered to obtain a filtrate, which was directly used in the next step without purification. Step 4: Compound 11d

The crude product 11c was placed in an ice-water bath, DIPEA (516 μL, 3.0 mmol) was added, and compound M3 (1.7 g, 2.9 mmol) was added, and the reaction was allowed to reach room temperature for 2 h. The reaction completion was monitored by HPLC, and the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution, which was lyophilized to give 11d (934 mg); LC-MS: [MH] ⁻ = 842.4. Step 5: Compound 11e

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 to a 50 mL flask in an ice-water bath. DIPEA (660 μL, 4.0 mmol) was added, and the reaction was allowed to proceed for 4 h. After monitoring the reaction by HPLC, the reaction solution was purified by HPLC to give a prepared solution of compound 11e . The reaction was carried out at room temperature for 4 h. After the reaction was completed and monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a solution of compound 11e . The solution was lyophilized to obtain 11e (401 mg); LC-MS: [M+H]+ = 1261.4. Step 6: Compound 11A

11e (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol) and 10 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain solid compound 11A (86 mg); LC-MS: [M+H] + = 1105.4. Example 19 Synthesis of compound 11B

Compound 11B (50 mg) was obtained by following the synthetic route of Example 18. LC-MS: [M+H] ⁺ 1105.4. Example 20

Synthesis of compound 12A : Step 1: Compound 12a was added to a 250 mL flask with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). The mixture was stirred and cooled to 0°C. Benzyl 3-hydroxy-2-cyclobutylpropionate (prepared according to the method disclosed in WO2009011285A1) (7.2 g, 32.6 mmol) was added dropwise. The reaction was allowed to warm to room temperature (approximately 2-4 h) and monitored by TLC. At the end of the reaction, saturated NaHCO ₃ solution was added, 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 (PE:EA=10:1-5:1-2:1) to give 12a (4.5 g) in 52% yield; LC-MS: [M+H] ⁺ = 529.4. Step 2: Compound 12b

To a 25 mL flask, 12a (4 g, 7.6 mmol) and 10 mL of DMF were added, stirred at 0°C, and DBU (1.2 g, 8.0 mmol) was added, and the reaction was allowed to proceed for 1 h. After the completion of Fmoc deprotection, monitored by TLC, the reaction was allowed to proceed. M4 (3.2 g, 7.6 mmol), PyBOP (4.7 g, 9.0 mmol), HOBt (1.22 g, 9.0 mmol), and 10 mL of DMF were added to another 25 mL flask. DIPEA (1.49 mL, 0.9 mmol) was added under an ice-water bath, and stirring was continued for 30 min. The reaction solution was then added to the reaction flask and the temperature was raised to room temperature. After the reaction was monitored by HPLC, the reaction solution was purified by preparative liquid phase to obtain a product preparation solution, which was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.0 g of a solid in a 37% yield; LC-MS: [M+H] + = 702.8. Step 3: Compound 12c

Compound 12b (1.0 g, 1.43 mmol) was added to a 25 mL flask and dissolved in 15 mL of DMF and clarified. Then, 1.0 g of 5% Pd/C was added and hydrogenation was performed for 1.5 h. After the reaction was completed, the filtrate was filtered to obtain a filtrate, which was directly used in the next step without purification. Step 4: Compound 12d

The crude product 12c was placed in an ice-water bath, DIPEA (258 μL, 1.5 mmol) was added, and compound M3 (825 mg, 1.43 mmol) was added, and the reaction was allowed to reach room temperature for 1 h. The reaction completion was monitored by HPLC, and the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution, which was lyophilized to obtain 12d (522 mg); LC-MS: [MH] ⁻ = 856.4. Step 5: Compound 12e

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 were added to a 50 mL flask in an ice-water bath. DIPEA (330 μL, 2.0 mmol) was added, and the reaction was allowed to proceed for 3 h. After monitoring the reaction by HPLC, the reaction solution was purified by HPLC to give a prepared solution of compound 12e . The reaction was carried out at room temperature for 3 h. After the reaction was completed and monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a solution of compound 12e . The solution was lyophilized to obtain 12e (198 mg); LC-MS: [M+H]+ = 1275.4. Step 6: Compound 12A

12e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain solid compound 12A (55 mg); LC-MS: [M+H] + = 1119.4. Example 21

Synthesis of compound 12B : Referring to the synthetic route of Example 20, compound 12B (50 mg) was obtained; LC-MS: [M+H]+ = 1119.4. Example 22

Synthesis of compound 13A : Step 1: Compound 13a was added to a 250 mL flask with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). The mixture was stirred and cooled to 0°C. Benzyl 2-hydroxy-2-cyclopentylacetate (see Journal of Medicinal Chemistry , 2013, 56 (13), 5541-5552. For the method of synthesis) (7.2 g, 32.6 mmol) was added dropwise. After the addition, the reaction was naturally warmed to room temperature (the reaction was carried out for about 2-4 h) and monitored by TLC. At the end of the reaction, saturated NaHCO ₃ solution was added, 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 (PE:EA=10:1-5:1-2:1) to give 13a (4.6 g) in 53% yield; LC-MS: [M+H] ⁺ = 529.5. Step 2: Compound 13b

To a 25 mL flask, 13a (4 g, 7.6 mmol) and 10 mL of DMF were added, stirred at 0°C, and DBU (1.17 g, 7.8 mmol) was added, and the reaction was allowed to proceed for 1 h. After the completion of Fmoc deprotection, monitored by TLC, the reaction was allowed to proceed. M4 (3.14 g, 7.6 mmol), PyBOP (4.42 g, 8.5 mmol), HOBt (1.15 g, 8.5 mmol), and 10 mL of DMF were added to another 25 mL flask and stirred continuously. DIPEA (1.39 mL, 0.85 mmol) was added and the mixture was stirred in an ice-water bath for 30 min. The reaction solution was then added to the flask. After the reaction was monitored by HPLC, the reaction solution was purified by preparative liquid phase to obtain a product preparation solution, which was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.1 g of a solid in a 39% yield; LC-MS: [M+H] + = 702.8. Step 3: Compound 13c

Compound 13b (1.5 g, 1.87 mmol) was added to a 25 mL flask and dissolved in 25 mL of DMF for clarification. Then, 1.5 g of 5% Pd/C was added and hydrogenation was carried out for 3 h. After the reaction was completed, the filtrate was filtered and used directly in the next step without purification. Step 4: Compound 13d

The crude product 13c was placed in an ice-water bath, DIPEA (333 μL, 1.93 mmol) was added, and compound M3 (1.1 g, 1.87 mmol) was added, and the reaction was allowed to reach room temperature for 1 h. The reaction completion was monitored by HPLC, and the reaction solution was subjected to high-performance liquid chromatography to obtain a preparative solution, which was lyophilized to give 13d (519 mg); LC-MS: [MH] ⁻ = 856.6. Step 5: Compound 13e

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 flask in an ice-water bath. DIPEA (330 μL, 2.0 mmol) was added, and the reaction was allowed to proceed for 4 h. After HPLC monitoring, the reaction solution was purified by HPLC to obtain a preparative solution of compound 13e , which was then freeze-dried to obtain 13e (187 mg). LC-LLC was performed to monitor the completion of the reaction. The reaction was carried out at room temperature for 4 h. After the reaction was completed and monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a solution of compound 13e . The solution was lyophilized to obtain 13e (187 mg); LC-MS: [M+H]+ = 1275.5. Step 6: Compound 13A

13e (100 mg, 0.08 mmol), zinc bromide (355 mg, 0.16 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain solid compound 13A (60 mg); LC-MS: [M+H] + = 1119.6. Example 23

Synthesis of compound 13B : Compound 13B (51 mg) was obtained by following the synthetic route of Example 22; LC-MS: [M+H]+ = 1119.6. Example 24

Synthesis of compound 14A : Step 1: Compound 14a was added to a 250 mL flask with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). The mixture was stirred and cooled to 0°C. Benzyl 3-hydroxy-2-cyclopentylpropionate (synthesized according to the method disclosed in WO2009011285A1) (7.6 g, 32.6 mmol) was then added dropwise. The reaction was allowed to proceed at room temperature (approximately 2-4 h). After cooling, the mixture was allowed to warm naturally and monitored by TLC. At the end of the reaction, saturated NaHCO ₃ solution was added, 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 (PE:EA=10:1-5:1-2:1) to give 14a (4.4 g) in 49% yield; LC-MS: [M+H] ⁺ = 543.6. Step 2: Compound 14b

To a 25 mL flask, 14a (4 g, 7.4 mmol) and 10 mL of DMF were added, stirred at 0°C, and DBU (1.2 g, 8.0 mmol) was added, and the reaction was allowed to proceed for 1 h. After the completion of Fmoc deprotection, monitored by TLC, the reaction was allowed to proceed. M4 (3.1 g, 7.4 mmol), PyBOP (4.6 g, 8.8 mmol), HOBt (1.19 g, 8.8 mmol), and 10 mL of DMF were added to another 25 mL flask, and DIPEA (1.49 mL, 9.0 mmol) was added in an ice-water bath. Stirring was continued for 30 min, and then the above reaction solution was added to the reaction flask and the temperature was raised to room temperature. After the reaction was monitored by HPLC, the reaction solution was purified by preparative liquid phase to obtain a product preparation solution, which was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.6 g of a solid in a 49% yield; LC-MS: [M+H] + = 716.4. Step 3: Compound 14c

In a 25 mL flask, compound 14b (1.0 g, 1.4 mmol) was added and dissolved in 15 mL of DMF and clarified. Then, 1.0 g of 5% Pd/C was added and hydrogenation was performed for 1.5 h. After the reaction was completed, it was filtered and the filtrate was directly used in the next step without purification. Step 4: Compound 14d

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), and the reaction was allowed to reach room temperature for 1 h. The reaction was monitored for completion by HPLC, and the reaction solution was purified by HPLC to obtain a preparative solution, which was lyophilized to obtain 14d (500 mg); LC-MS: [MH] ⁻ = 870.5. Step 5: Compound 14e

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 were added to a 50 mL flask in an ice-water bath. DIPEA (331 μL, 2.0 mmol) was added, and the reaction was allowed to proceed for 3 h. After monitoring the reaction by HPLC, the reaction solution was purified by HPLC to give a prepared solution of compound 14e . The reaction was carried out at room temperature for 3 h. After the reaction was completed and monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a solution of compound 14e . The solution was lyophilized to obtain 14e (146 mg); LC-MS: [M+H]+ = 1289.5. Step 6: Compound 14A

14e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain solid compound 14A (52 mg); LC-MS: [M+H] + = 1133.4. Example 25

Synthesis of compound 14B : Following the synthetic route of Example 24, compound 14B (48 mg) was obtained; LC-MS: [M+H]+ = 1133.4. Example 26

Synthesis of compounds 15A and 15B : Step 1: Compound 15a was added to a 250 mL flask with M1 (10 g, 27.1 mmol), 2-hydroxy-butyric acid benzyl ester (prepared by the method disclosed 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. The mixture was heated to 100°C for 4 h. After the reaction was completed, the temperature was lowered to room temperature, filtered to remove insoluble materials, and the filtrate was concentrated to obtain a 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 in 42% yield; LC-MS: [M+H] ⁺ = 503.5. Step 2: Compound 15b

15a (5 g, 9.95 mmol) and 15 mL of DMF were added to a 50 mL flask to dissolve the solution, and then DBU (1.68 g, 11 mmol) was added in an ice-water bath and reacted for 1 h. The reaction was recorded as reaction solution ①; Another 50 mL flask was obtained, M4 (4.1 g, 10.0 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol) and 10 mL of DMF were added, and after dissolution, DIPEA (1.82 mL, 11 mmol) was added in an ice-water bath and the reaction was continued for 40 min, and then reaction solution (1) was added and the reaction was allowed to reach room temperature for 2 h. The reaction was monitored by HPLC. The reaction was monitored by HPLC for 2 h. After the reaction was complete, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a preparative solution. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.6 g of a solid with a yield of 68%; LC-MS: [M+H]+ = 676.7. Step 3: Compound 15d

15b (2.0 g, 2.96 mmol) was added to a 25 mL flask, dissolved in 15 mL of DMF, and then 2.0 g of 5% Pd/C was added and hydrogenated for 2 h. After the reaction was complete, the filtrate was filtered and placed in an ice-water bath. DIPEA (496 μL, 3.0 mmol) was added, followed by M3 (1.7 g, 2.96 mmol). After the reaction was complete, the reaction was allowed to reach room temperature. The reaction was monitored by HPLC for 1 h. The reaction solution was purified by high-performance liquid chromatography (HPLC) to obtain a preparative solution, which was then lyophilized to afford 1120.0 mg of the product in a 45% yield; LC-MS: [MH] ⁻ = 830.3. Step 4: Compound 15e

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 were added to a 50 mL flask. DIPEA (446 μL, 2.7 mmol) was added to the vial in an ice-water bath, and the reaction was carried out at room temperature for 2 h. After monitoring the reaction by HPLC, the reaction solution was purified by HPLC to obtain preparative solutions of compound 15e-1 and compound 15e-2 , and the preparative solutions were lyophilized to obtain 138 mg of compound 15e-2 , respectively. The reaction was carried out at room temperature for 2 h. After HPLC monitoring, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain solutions of compound 15e-1 and compound 15e-2 . These solutions were lyophilized to obtain 138 mg of compound 15e-1 (LC-MS: [M+H]+ = 1249.5) and 140 mg of compound 15e-2 (LC-MS: [M+H]+ = 1249.5), respectively. Step 5: Compound 15A

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 flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was freeze-dried to obtain 59 mg of a solid; LC-MS: [M+H]+ = 1093.4. Step 6: Compound 15B

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 flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was freeze-dried to obtain 60 mg of a solid; LC-MS: [M+H]+=1093.4. Example 27

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

Referring to the synthetic route of Example 26, compound 16B (54 mg) was obtained; LC-MS: [M+H]+ = 1093.4. Example 28

Synthesis of compounds 17A and 17B : Step 1: Compound 17a M1 (10 g, 27.1 mmol), 2-hydroxy-phenylpropionic acid benzyl ester (synthesized by the method disclosed 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 flask. Toluene was heated to 100 ° C for 4 h. After the reaction was completed, it was cooled to room temperature, filtered to remove insoluble materials, and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (PE: EA = 10: 1-5: 1-2: 1) to obtain 6.13 in 40% yield. g target; LC-MS: [M+H]+=565.6. Step 2: Compound 17b

In a 50 mL flask, 17a (5 g, 8.86 mmol) and 15 mL of DMF were added, dissolved, and clarified. DBU (1.53 g, 10 mmol) was then added in an ice-water bath and the reaction was carried out for 1 h, recorded as reaction solution ①. Another 50 mL flask was prepared, and M4 (3.6 g, 8.86 mmol), PyBOP (5.23 g, 10 mmol), HOBt (1.36 g, 10 mmol), and 10 mL of DMF were added and dissolved. DIPEA (1.65 mL, 10 mmol) was added in an ice-water bath and the reaction was continued for 30 min. Then, reaction solution ① was added, and the reaction was allowed to reach room temperature for 2 h. The reaction was monitored by HPLC. The reaction was monitored by HPLC for 2 h. After the reaction was complete, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a preparative solution. The preparative solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 5.0 g of a solid in a 77% yield; LC-MS: [M+H]+ = 738.3. Step 3: Compound 17d

17b (3.0 g, 4.07 mmol) was added to a 25 mL flask, and after dissolving 15 mL of DMF, 3.0 g of 5% Pd/C was added, and the reaction was completed for 2 h. The reaction was completed by filtration, and the filtrate was placed in an ice-water bath, and DIPEA (744 uL, 4.5 mmol) was added, followed by M3 (2.34 g, 4.07 mmol), and after the addition, the reaction was allowed to reach room temperature. The reaction was monitored by HPLC for 1 h. The reaction solution was purified by HPLC to obtain a preparative solution, and the solution was freeze-dried to give 1.2 g of the product in 33% yield; LC-MS: [MH] ^- = 892.4. Step 4: Compound 17e

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 were added to a 50 mL flask. DIPEA (330 μL, 2.0 mmol) was added to the flask under an ice-water bath, and the reaction was carried out at room temperature for 2 h. After monitoring the reaction by HPLC, the reaction solution was purified by HPLC to obtain preparative solutions of compound 17e-1 and compound 17e-2 , and the preparative solutions were lyophilized to obtain 156 mg of compound 17e-2 , respectively. The reaction was carried out at room temperature for 2 h. After HPLC monitoring, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain solutions of compound 17e-1 and compound 17e-2 . These solutions were lyophilized to obtain 156 mg of compound 17e-1 (LC-MS: [M+H]+ = 1311.4) and 150 mg of compound 17e-2 (LC-MS: [M+H]+ = 1311.7), respectively. Step 5: Compound 17A

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 flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was freeze-dried to obtain 43 mg of a solid; LC-MS: [M+H]+=1155.4. Step 6: Compound 17B

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 flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was freeze-dried to obtain 40 mg of a solid; LC-MS: [M+H]+=1155.4. Example 29

Synthesis of compounds 18A and 18B : Compound 18A (54 mg) was obtained according to the synthetic route of Example 28; LC-MS: [M+H]+ = 1155.4.

Compound 18B (55 mg) was obtained by following the synthetic route of Example 28; LC-MS: [M+H]+ = 1155.4. Example 30

Synthesis of compounds 19A and 19B : Step 1: Compound 19a : M1 (10 g, 27.1 mmol), benzyl 2-cyclopropyl-2-hydroxyacetate (prepared according to the method disclosed in 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 flask and heated to 100°C for 4 h. The reaction was allowed to complete. After the reaction was completed, the mixture was cooled to room temperature, filtered to remove insoluble material, 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 compound in 36% yield; LC-MS: [M+H] + = 515.2. Step 2: Compound 19b

19a (4 g, 7.8 mmol) and 10 mL of DMF were added to a 50 mL flask to dissolve the solution. DBU (1.42 g, 9.3 mmol) was then added in an ice-water bath and reacted for 1 h. The reaction was recorded as reaction solution ①. Another 50 mL single-necked vial was prepared and M4 (3.2 g, 7.8 mmol), PyBOP (4.5 g, 8.6 mmol), HOBt (1.16 g, 8.6 mmol), and 10 mL of DMF were added and dissolved. DIPEA (1.65 mL, 10 mmol) was then added in an ice-water bath and the reaction continued for 30 min. Reaction solution ① was then added and the reaction was allowed to reach room temperature for 2 h. The reaction progress was monitored by HPLC, and the preparation was purified by HPLC. The reaction was monitored by HPLC for 2 h. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a preparative solution. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.2 g of a solid with a yield of 78%; LC-MS: [M+H]+ = 688.3. Step 3: Compound 19d

In a 25 mL flask, 19b (1000 mg, 1.45 mmol) was added, dissolved in 15 mL of DMF, and then 1000 mg of 5% Pd/C was added. The reaction was hydrogenated for 2 h. The reaction was completed, 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), and the reaction was allowed to reach room temperature for 1 h. The reaction was monitored by HPLC, and the reaction solution was purified by HPLC to obtain a preparative solution, which was lyophilized to obtain 503 mg of the product in a 41% yield. The reaction was monitored by HPLC, and the reaction solution was purified by HPLC to obtain a preparative solution, which was lyophilized to obtain 503 mg of the product in a 41% yield; LC-MS: [MH] ⁻ = 842.3. Step 4: Compounds 19e-1 and 19e-2

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 were added to a 50 mL flask. DIPEA (292 μL, 1.77 mmol) was added to the flask under an ice-water bath, and the reaction was carried out at room temperature for 2 h. The reaction was monitored by HPLC, and the reaction solution was purified by HPLC to obtain preparative solutions of compound 19e-1 and compound 19e-2 . The preparative solutions were lyophilized to obtain 112 mM of compound 19e-2 , respectively. The reaction was carried out at room temperature for 2 h. After HPLC monitoring, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain solutions of compound 19e-1 and compound 19e-2 . These solutions were lyophilized to obtain 112 mg of compound 19e-1 (LC-MS: [M+H]+ = 1261.5) and 131 mg of compound 19e-2 (LC-MS: [M+H]+ = 1261.5), respectively. Step 5: Compound 19A

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 flask and the reaction was carried out at 40°C for 1 h. After the reaction was completed and monitored by HPLC, the solvent was concentrated under reduced pressure and removed to obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was freeze-dried to obtain 55 mg of a solid; LC-MS: [M+H]+=1105.4. Step 6: Compound 19B

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 flask and the reaction was carried out at 40°C for 1 h. After the reaction was completed by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was freeze-dried to obtain 58 mg of a solid; LC-MS: [M+H]+=1105.4. Example 31

Synthesis of compounds 20A and 20B : Step 1: Compound 20a : M1 (10 g, 27.1 mmol), benzyl 2-hydroxycyclopropylpropionate (synthesized by the method disclosed in 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 flask and reacted at 100°C for 4 h. Upon completion of the reaction, the temperature was lowered to room temperature. After completion of the reaction, the temperature was lowered to room temperature, filtered to remove insoluble material, and the filtrate was concentrated to obtain a 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 compound; LC-MS: [M+H] + = 529.2. Step 2: Compound 20b

20a (4 g, 7.6 mmol) and 10 mL of DMF were added to a 50 mL flask, and after dissolution and clarification, DBU (1.39 g, 9.1 mmol) was added in an ice-water bath and the reaction was carried out for 1 h, which was recorded as reaction solution ①; another 50 mL flask was obtained, and M4 (3.12 g, 7.6 mmol), PyBOP (4.5 g, 8.6 mmol), HOBt (1.16 g, 8.6 mmol) and 10 mL of DMF were added and dissolved, and DIPEA (1.65 mL, 10 mmol) was added in an ice-water bath and the reaction was continued for 30 min, and then reaction solution (1) was added to the reaction solution and the reaction was allowed to reach room temperature for 2 h. HPLC was performed to monitor the progress of the reaction, and the reaction solution was purified by high performance liquid chromatography to obtain a preparative solution. The reaction was monitored by HPLC, and the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a preparative solution. The preparative solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.5 g of a solid with a yield of 84%; LC-MS: [M+H]+=702.3. Step 3: Compound 20d

Compound 20b (1000 mg, 1.42 mmol) was added to a 25 mL flask, dissolved in 15 mL of DMF, and then 1000 mg of 5% Pd/C was added. The reaction was hydrogenated for 2 h. After the reaction was complete, filtration was performed, 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), and the reaction was allowed to reach room temperature for 1 h. The reaction was monitored by HPLC, and the reaction solution was purified by HPLC to obtain a preparative solution, which was then lyophilized to obtain 443 mg of the product in a 36% yield. The reaction was monitored by HPLC and the reaction solution was purified by HPLC to obtain a preparative solution, which was lyophilized to obtain 443 mg of the product in a 36% yield; LC-MS: [MH] ⁻ = 856.4. Step 4: Compounds 20e-1 and 20e-2

20d (400 mg, 0.47 mmol), ezetimibe methanesulfonic acid (250 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol), and 10 mL of DMF were added to a 50 mL vial, and DIPEA (248 uL, 1.5 mmol) was added to the vial under an ice-water bath. DIPEA (248 uL, 1.5 mmol) was added to the ice-water bath, and the reaction was allowed to reach room temperature for 2 h. After the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography to obtain solutions of compound 20e-1 and compound 20e-2 . These solutions were lyophilized to obtain 103 mg of compound 20e-1 (LC-MS: [M+H] ⁺ = 1275.5) and 103 mg of compound 20e-2 (LC-MS: [M+H] ⁺ = 1275.5). Step 5: Compound 20A

In a 25 mL flask, 8A (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol) and 5 mL of nitromethane were added, and the reaction was carried out at 40°C for 1 h. After the completion of the reaction was detected by HPLC, the solvent was concentrated under reduced pressure to obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was freeze-dried to obtain 51 mg of a solid; LC-MS: [M+H]+=1119.4. Step 6: Compound 20B

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 vial and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was freeze-dried to obtain 47 mg of a solid; LC-MS: [M+H]+=1119.4. Example 32

Synthesis of compound 21 : Step 1: Compound SM3-1 : 77087-60-6 (100 g, 458 mmol), maleic acid (53.4 g, 460 mmol), TEA (64 mL, 460 mmol), and 1000 mL of toluene were added to a 2000 mL flask and heated to 100°C for 5 hours. After the reaction was complete, the reaction mixture was cooled to room temperature, insoluble material was removed by filtration, and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA = 100:1-50:1-20:1) to obtain 75.6 g of the target compound; LC-MS: [M+H] ⁺ = 299.1. Step 2: Compound ( R )-tert-butyl 2-hydroxy-1,5-pentanedioate

172793-31-6 (100 g, 338 mmol) and 1000 mL of water were added to a 2000 mL flask, followed by the addition of sodium nitrite (35 g, 507 mmol) and concentrated sulfuric acid (32 mL, 35 mmol). The temperature was slowly raised to room temperature for 24 h. After the reaction was complete, the product was extracted three times with 500 mL of ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA = 50:1-30:1-2:1) to obtain 91.2 g of the target compound; LC-MS: [M+H] ⁺ = 261.4. Step 3: Compound SM3

( R )-tert-Butyl 2-hydroxy-1,5-pentanedioate (50 g, 192 mmol) and 1000 mL of anhydrous tetrahydrofuran were added to a 2000 mL flask. The temperature was cooled to 0°C, and then _PPh3 (87.7 g, 288 mmol), DEAD (50.2 g, 288 mmol), and SM3-1 (57.3, 192 mmol) were added sequentially. The reaction was slowly warmed to room temperature for 13 h. After the reaction was complete, the insoluble material 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 product. 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. After the reaction, the pH was adjusted to 3 by adding HCl (6 M/L), the mixture was extracted five times with 500 mL of dichloromethane, and the mixture was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the crude product was purified by column chromatography (DCM/MeOH = 50/1-20/1-2/1) to obtain SM3 50.4 g; LC-MS: [MH] ^- = 525.5. Step 4: Compound M6

In a 2000 mL flask, compound SM3 (50 g, 95 mmol, 1.0 equivalent), pentafluorophenol (19.2 g, 104.5 mmol, 1.1 equivalent), DCC (21.5 g, 104.5 mmol, 1.1 equivalent) and THF (600 mL) were added, and the reaction was carried out at room temperature for 1 h (monitored by TLC), and the insoluble material was filtered off. The reaction solution was directly purified by the preparative method, and the preparative solution was concentrated by pumping water at 35° C. under reduced pressure to remove acetonitrile, and lyophilized to obtain compound M6 (51.9 g) in 79% yield; LC-MS: [M+H] + = 693.3. Step 5: Compound 21a

In a 25 mL flask, 1c (1 g, 2.36 mmol) was added, dissolved in 25 mL of DMF, and then DIPEA (430 uL, 2.6 mmol) was added, followed by M6 (1177 mg, 2.36 mmol), and the reaction was allowed to reach room temperature for 1 h. The completion of the reaction was monitored by HPLC, and the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a preparative solution, which was lyophilized to produce the following product 555 mg; LC-MS: [MH] ^- = 931.0. Step 6: Compound 21b

In a 100 mL flask, 21a (500 mg, 0.54 mmol), ezetimibe methanesulfonic acid M5 (285 mg, 0.54 mmol), PyBOP (239 mg, 0.6 mmol), HOBt (239 mg, 0.6 mmol), and 10 mL of DMF were added, and DIPEA (248 uL, 1.5 mmol) was added to the reaction in an ice-water bath. DIPEA (248 uL, 1.5 mmol) was added to the ice-water bath, and the reaction was allowed to reach room temperature for 2 h. After the completion of the reaction monitored by HPLC, the reaction solution was subjected to HPLC purification to obtain a preparative solution of compound 21b , which was lyophilized to give 231 mg of the compound; LC-MS: [M+H] ⁺ = 1349.5. Step 7: Compound 21

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 flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain 103 mg of a solid; LC-MS: [M+H]+=1137.5. Example 33

Synthesis of compound 22 : Compound M6 and 3c were used as starting materials and the synthetic route of Example 32 was referred to obtain compound 22 (91 mg); LC-MS: [M+H]+ = 1165.5. Example 34

Synthesis of compounds 23 and 24 : , Using compounds M6 and 5c as starting materials and referring to 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. Example 35

Synthesis of compounds 25 and 26 : , Using compounds M6 and 7c as starting materials and referring to 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. Example 36

Synthesis of compounds 27 and 28 : , Using compounds M6 and 19c as starting materials and referring to 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. Example 37

Synthesis of compound 29 : Step 1: Compound SM4-1 was added to a 5000 mL flask with maleic acid (50 g, 431 mmol, 1.0 equivalent), 114559-25-0 (110 g, 431 mmol, 1 equivalent), TEA (263 g, 2.16 mol, 5 equivalents), and toluene (2000 mL). The reaction was heated to reflux for 5 h (monitored by TLC), and the insoluble material was filtered off. The reaction solution was directly removed from the solvent by rotary distillation under reduced pressure and the residue was subjected to silica gel column chromatography (PE/EA = 50/1-20/1-1-1/1) to obtain SM4-1 (64.7 g) in 50% yield; LC-MS: [M+H] ⁺ = 299.2. Step 2: Compound SM4-2

SM4-1 (64 g, 215 mmol) was added to a 2000 mL flask and dissolved in 1000 mL of DMF. DIPEA (71 mL, 430 mmol) was then added, followed by nonaethylene glycol monomethyl ether methanesulfonic acid (111.5 g, 220 mmol). The reaction was then allowed to reach room temperature for 2 hours. Reaction completion was monitored by HPLC, and the reaction solution was purified by silica gel column chromatography (PE/EA = 50/1-20/1-1/1) to obtain 59.9 g; LC-MS: [M+H] = 709.4. The reaction solution was purified by silica gel column chromatography (PE/EA = 50/1-20/1-1/1) to obtain 59.9 g of the product; LC-MS: [M+H] ⁺ = 709.4. Step 3: Compound SM4

SM4-2 (59 g, 83 mmol) was added to a 2000 mL flask and dissolved in 1000 mL of MeOH. K ₂ CO ₃ (11.75 g, 85 mmol) was then added and the reaction was allowed to proceed at room temperature for 4 h. The reaction was monitored by HPLC, and the insoluble material was filtered. The reaction solution was directly purified by preparation and the prepared solution was pumped to concentrate in a water bath under reduced pressure at 35° C. to remove acetonitrile, followed by freeze drying to obtain compound SM4 (27 g); LC-MS: [MH] ⁼ 693.5. Step 4: Compound M7

In a 500 mL flask, compound SM4 (25 g, 36 mmol, 1.0 equivalent), pentafluorophenol (7.3 g, 40 mmol, 1.1 equivalent), DCC (8.2 g, 40 mmol, 1.1 equivalent) and THF (200 mL) were added, and the reaction was carried out at room temperature for 1 h (monitored by TLC), and the insoluble material was filtered off. The reaction solution was directly purified by the preparative method, and the preparative solution was concentrated by pumping water at 35° C. under reduced pressure to remove acetonitrile, and lyophilized to obtain compound M7 (23.3 g) in 93% yield; LC-MS: [M+H] + = 695.8. Step 5: Compound 29a

In a 25 mL flask, 1c (1 g, 2.36 mmol) was added, dissolved in 25 mL of DMF, and then DIPEA (430 uL, 2.6 mmol) was added, followed by M7 (1640 mg, 2.36 mmol), and the reaction was allowed to reach room temperature for 1 h. The reaction completion was monitored by HPLC, and the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a preparative solution, which was lyophilized to obtain 609 mg of the product; by LC-MS, [MH] = 1098.5. The product was 609 mg; LC-MS: [MH] ^- = 1098.5. Step 6: Compound 29b

29a (500 mg, 0.45 mmol), ezetimibe methanesulfonic acid M5 (240 mg, 0.45 mmol), PyBOP (215 mg, 0.54 mmol), HOBt (215 mg, 0.54 mmol), and 10 mL of DMF were added to a 100 mL vial, and DIPEA (248 μL, 1.5 mmol) was added to the flask under an ice-water bath. The reaction was allowed to reach room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by high-performance liquid chromatography (HPLC) to obtain a preparative solution of compound 29b , and lyophilization of the preparative solution yielded 187 mg of the compound; LC-MS: [M+H]+ = 1517.6. Step 7: Compound 29

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 flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain 114 mg of a solid; LC-MS: [M+H]+ = 1517.9. Example 38

Synthesis of compound 30 : Compounds M7 and 3c were used as starting materials and the synthetic route of Example 37 was followed to obtain compound 30 (125 mg); LC-MS: [M+H]+ = 1445.6. Example 39

Synthesis of compounds 31 and 32 : , Using compounds M7 and 5c as starting materials and referring to 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. Example 40

Synthesis of compounds 33 and 34 : Using compounds M7 and 7c as starting materials, and referring to 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. Example 41

Synthesis of compounds 35 and 36 : , Using compounds M7 and 19c as starting materials and referring to 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. Example 42

Synthesis of compound 37 : Step 1: Compound SM5-1 was added to a 2000 mL flask with compound 16947-84-5 (100 g, 295 mmol, 1.0 equivalent), DIPEA (50 mL, 300 mmol), benzyl bromide (51.3 g, 300 mmol), and THF (1000 mL). The mixture was reacted at room temperature for 12 h (monitored by TLC), and the insoluble material was filtered off. The reaction solution was directly removed from the solvent by rotary distillation under reduced pressure, and the residue was subjected to silica gel column chromatography (PE/EA = 50/1-20/1-1-1/1) to obtain SM5-1 (110.1 g) in 87% yield; LC-MS: [M+H] ⁺ = 429.2. Step 2: Compound SM5-2

In a 2000 mL flask, compound SM5-1 (100 g, 233.4 mmol, 1.0 equivalent) 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 portionwise, and the reaction was maintained at 0°C for 24 h (monitored by TLC). The reaction was quenched by adding 500 mL of saturated aqueous NH ₄ Cl solution, and 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 from the solvent by rotary distillation under reduced pressure, and the residue was analyzed by silica gel column chromatography (PE/EA = 100/1-50/1-10/1) to obtain SM5-2 (37.1 g), LC-MS: [M+H] ⁺ = 443.3. Step 3: Compound SM5 (See Org. Lett. , 2006, 8 , 3387-3390.)

Compound SM5-2 (35 g, 79 mmol, 1.0 equivalent) and DCE (500 mL) were added to a 1000 mL flask. Palladium diacetate (180 mg, 0.8 mmol), _I2 (20 g, 79 mmol), and iodobenzene diacetate (40.8 g, 126.4 mmol) were also added, and the reaction was carried out at 60°C for 40 h (monitored by TLC). The reaction was quenched by adding 500 mL of saturated aqueous sodium thiosulfate solution, extracted three times with 500 mL of dichloromethane, and the organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was directly removed from the solvent by rotary distillation under reduced pressure, and the residue was analyzed by silica gel column chromatography (PE/EA = 100/1-50/1-10/1) to obtain SM5 (28 g), LC-MS: [M+H] ⁺ = 501.3. Step 4: Compound SM6

In a 500 mL flask, compound SM5 (25 g, 50 mmol, 1.0 equivalent), di-tert-butyl potassium phosphate (13.66 g, 55 mmol, 1.1 equivalent), p-toluenesulfonic acid monohydrate (951 mg, 5 mmol, 0.1 equivalent) and THF (200 mL) were added, and the reaction was carried out at room temperature for 1 h (monitored by TLC), and the insoluble material was filtered off. The reaction solution was directly purified by the preparation, and the preparation solution was concentrated by pumping water at 35° C. under reduced pressure to remove acetonitrile, and lyophilized to obtain compound SM6 (15.1 g) in 46% yield; LC-MS: [M+H] + = 651.4. Step 5: Compound SM7

SM6 (15 g, 23 mmol) and 100 mL of DMF were added to a 250 mL flask and dissolved. Then, 15 g of 5% Pd/C was added to an ice-water bath. The atmosphere in the system was replaced with hydrogen three times, and the reaction was carried out at room temperature for 12 h. The Pd/C was removed by filtration, and the solvent was evaporated under reduced pressure using an oil pump and set aside for future use.

In another 250 mL flask, add the above crude product and 100 mL of toluene, triethylamine (6.4 mL, 46 mmol), maleic anhydride (2.4 g, 24 mmol), dissolve and clarify, raise the temperature to 100 ° C and continue the reaction for 2 h. The progress of the reaction is monitored by HPLC. After the reaction is completed, the reaction solution is purified by high performance liquid chromatography to obtain a preparation solution. The preparation solution is extracted with dichloromethane, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain 4.2 g of solid with a yield of 36%; LC-MS: [M+H] + = 507.3. Step 6: Compound M8

In a 100 mL flask, compound SM7 (4 g, 7.9 mmol, 1.0 equivalent), pentafluorophenol (1.6 g, 8.7 mmol, 1.1 equivalent), DCC (1.8 g, 8.7 mmol, 1.1 equivalent) and THF (60 mL) were added, and the reaction was carried out at room temperature for 1 h (monitored by TLC), and the insoluble material was filtered off. The reaction solution was directly purified by the preparative method, and the preparative solution was concentrated by pumping water at 35° C. under reduced pressure to remove acetonitrile, and lyophilized to obtain compound M8 (3.7 g) in 70% yield; LC-MS: [M+H] + = 673.2. Step 7: Compound 37a

In a 25 mL flask, 1c (1 g, 2.36 mmol) was added, dissolved in 25 mL of DMF, and then DIPEA (430 μL, 2.6 mmol) was added, followed by M8 (1.2 g, 2.36 mmol), and the reaction was allowed to reach room temperature for 1 h. The completion of the reaction was detected by HPLC, and the reaction solution was purified by high-performance liquid chromatography (HPLC) to obtain a preparative solution. The solution was lyophilized to obtain the following product (488 mg); LC-MS: [MH] ⁻ = 911.0. Step 8: Compound 37b

37a (400 mg, 0.44 mmol), ezetimibe methanesulfonic acid M5 (235 mg, 0.44 mmol), PyBOP (199 mg, 0.5 mmol), HOBt (69 mg, 0.5 mmol), and 10 mL of DMF were added to a 100 mL flask, and DIPEA (218 uL, 1.32 mmol) was added to the reaction in an ice-water bath. DIPEA (218 uL, 1.32 mmol) was added to the ice-water bath, and the reaction was allowed to reach room temperature for 2 h. After monitoring the reaction by HPLC, the reaction solution was subjected to high-performance liquid chromatography to obtain a preparative solution of compound 37b , which was lyophilized to give 201 mg of the compound, LC-MS: [M+H] ⁺ = 1329.6. Step 9: Compound 37

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 flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain 96 mg of a solid; LC-MS: [M+H]+ = 1117.4. Example 43

Synthesis of compound 38 : Compound M8 and 3c were used as starting materials and the synthetic route of Example 42 was referred to to obtain compound 38 (51 mg); LC-MS: [M+H]+ = 1145.6. Example 44

Synthesis of compounds 39 and 40 : , Using compounds M8 and 5c as starting materials and referring to 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. Example 45

Synthesis of compounds 41 and 42 : Using compounds M7 and 7c as starting materials, and referring to 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. Example 46

Synthesis of compounds 43 and 44 : , Using compounds M8 and 19c as starting materials and referring to 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. Example 47 (Comparative Example)

Synthesis of compound 45 : Compound 45 was synthesized according to the method provided in Example 58 of CN104755494A. Example 48

Synthesis of compound 46 : Step 1: Compound 46a1d ( 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 were added to a 50 mL flask. DIPEA (378 μL, 2.29 mmol) was added to the vial under an ice-water bath and the reaction was allowed to proceed for 2 h. The reaction was monitored by HPLC, and the reaction solution was purified by HPLC to obtain a preparative solution. This solution was lyophilized to obtain 46a (210 mg). LC-MS: The preparative solution was lyophilized to obtain 46a (210 mg). LC-MS: The preparative solution was purified by HPLC. The reaction was carried out at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a preparative solution, which was lyophilized to give 46a (210 mg); LC-MS: [M+H]+ = 1221.6. Step 2: Compound 46

46a (200 mg, 0.162 mmol), zinc bromide (736 mg, 3.26 mmol) and 10 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain solid compound 46 (120 mg); LC-MS: [M+H] + = 1065.3. Example 49

Synthesis of compound 47 : ; Referring to the synthetic route of Example 48, compound 47 (81 mg) was obtained; LC-MS: [M+H]+=1065.3. Example 50

Synthesis of compound 48A : Step 1: Compound 48a -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 flask and allowed to reach room temperature for 2 h. The reaction was monitored by HPLC. DIPEA (0.84 mL, 1.5 eq) was added and the reaction was allowed to reach room temperature for 2 h (monitored by HPLC). The reaction solution was directly purified by preparative method, and the preparative solution was concentrated by pumping water at 35° C. under reduced pressure to remove acetonitrile and lyophilized to obtain compound 48a (1.54 g) in 61% yield; LC-MS: [M+H]+=1235.4. Step 2: Compound 48A

In a 100 mL flask, compound 48a (1.0 g, 0.8 mmol, 1.0 equivalent) and 35 mL of nitromethane were added, and zinc bromide (3.64 g, 16 mmol, 20.0 equivalent) was dissolved and then added. The reaction was carried out in an oil bath at 40°C (stabilized in advance by preheating) for 30 min, and then concentrated in a water bath at 45°C under reduced pressure to remove nitromethane, resulting in a yellow residual solid (monitored by HPLC). After the acid was prepared, the prepared solution was obtained, and the prepared solution was concentrated in a water bath at 35°C under reduced pressure to remove acetonitrile and lyophilized to obtain compound 48A (786 mg) in 90% yield. Example 51

Synthesis of compound 48B : Step 1: Compound 48b : Compound 5d-1 (200 mg, 0.24 mmol, 1.0 equiv), M9 (127 mg, 0.24 mmol, 1.0 equiv), PyBOP (187 mg, 0.36 mmol, 1.2 equiv), HOBt (48 mg, 0.36 mmol, 1.2 equiv), and DMF (6 mL) were added to a 25 mL flask. The water bath was lowered to 0-5°C, DIPEA (62 mg, 0.48 mmol, 2.0 equiv) was added, and the reaction temperature was raised to 20±5°C for 2 h. Completion of the reaction was monitored by HPLC. The reaction solution was directly purified by HPLC preparation, and the product preparation was collected and lyophilized to obtain compound 48b (150.2 mg); LC-MS: [M+H] ⁺ = 1235.4. Step 2: Compound 48B

Compound 48b (100 mg, 0.081 mmol, 1.0 eq), ZnBr ₂ (364 mg, 1.62 mmol, 20.0 eq) and CH ₃ NO ₂ (10 mL) were added sequentially to a 25 mL flask, and the reaction was then heated to 40° C. for 0.5 h. The reaction was stopped, and the reaction solution was directly rotary dried at 45° C. under reduced pressure to obtain a yellow solid, which was monitored by sampling HPLC. The reaction was monitored by HPLC. The rotary dried solid was directly purified by HPLC preparation, and the product preparation was collected and lyophilized to obtain compound 48B (70.0 mg); LC-MS: [M+H] ⁺ =1079.4. Example 52

Synthesis of compound 49A : Compound 49A (71 mg) was obtained by following the procedure of Example 50; LC-MS: [M+H] ⁺ = 1079.4. Example 53

Preparation of compound 49B : Compound 49B (65 mg) was obtained by following the synthetic route of Example 51; LC-MS: [M+H]+ = 1079.4. Example 54

Synthesis of compounds 50A and 50B : Step 1: Compounds 50a and 50b (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 were added to a 50 mL flask. DIPEA (378 μL, 2.29 mmol) was added to the flask under an ice-water bath, and the reaction was carried out at room temperature for 2 h. The reaction was monitored by HPLC, and the reaction solution was purified by HPLC to obtain compounds 50a and 50b . The product was lyophilized to obtain 170 mg of compound 50a . The reaction was carried out at room temperature for 2 h. After HPLC monitoring, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain solutions of Compound 50a and Compound 50b . These solutions were lyophilized to obtain 170 mg of Compound 50a (LC-MS: [M+H]+ = 1289.46) and 202 mg of Compound 50b (LC-MS: [M+H]+ = 1289.46), respectively. Step 2: Compound 50A

In a 25 mL flask, 50a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added, and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain 44 mg of a solid. Step 3: Compound 50B

In a 25 mL test, 50b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added, and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain 45 mg of a solid. Example 55

Synthesis of compounds 51A and 51B : Step 1: Compound 51a and Compound 51b 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 were added to a 50 mL flask. DIPEA (378 μL, 2.29 mmol) was added to the flask in an ice-water bath, and the reaction was carried out at room temperature for 2 h. The reaction was monitored by HPLC, and the reaction solution was purified by HPLC to obtain the preparations of Compound 51a and Compound 51b . After the reaction was completed by HPLC, the reaction solution was purified by high-performance liquid chromatography (HPLC) to obtain solutions of Compound 51a and Compound 51b . These solutions were lyophilized to obtain 190 mg of Compound 51a and 186 mg of Compound 51b , respectively. LC-MS for Compound 51a : [M+H] ⁺ = 1289.47; LC-MS for Compound 51b : [M+H] ⁺ = 1289.47. Step 2: Compound 51A

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 flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain 39 mg of a solid. Step 3: Compound 51B

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 flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain 60 mg of a solid. Example 56

Synthesis of compound 52A : Step 1: Compound 52a 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 were added to a 50 mL flask in an ice-water bath. DIPEA (660 μL, 4.0 mmol) was added and the reaction was allowed to proceed for 4 h. After monitoring the reaction by HPLC, the reaction solution was purified by HPLC to give a prepared solution of compound 52a . The reaction was carried out at room temperature for 4 h. After the reaction was completed and monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a solution of compound 52e . The solution was lyophilized to obtain 52e (388 mg); LC-MS: [M+H]+ = 1261.4. Step 2: Compound 52A

52A (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol) and 10 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain solid compound 52A (79 mg); LC-MS: [M+H]+ = 1105.4. Example 57 Synthesis of Compound 52B

Compound 52B (50 mg) was obtained by following the synthetic route of Example 56. LC-MS: [M+H] ⁺ 1105.4. Example 58

Synthesis of compound 53A : Step 1: Compound 53a 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 were added to a 50 mL flask with an ice-water bath and DIPEA (330 μL, 2.0 mmol). The reaction was carried out at room temperature for 3 h. The reaction was monitored by HPLC, and the reaction solution was purified by HPLC. The reaction was carried out at room temperature for 3 h. After the reaction was completed and monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a solution of compound 53e . The solution was lyophilized to obtain 53e (200 mg); LC-MS: [M+H]+ = 1275.4. Step 2: Compound 53A

53A (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain solid compound 53A (51 mg); LC-MS: [M+H] + = 1119.4. Example 59

Synthesis of compound 53B : Referring to the synthetic route of Example 58, compound 53B (50 mg) was obtained; LC-MS: [M+H]+ = 1119.4. Example 60

Synthesis of compounds 54A and 54B : Step 1: Compounds 54a and 54b 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 were added to a 50 mL flask with an ice-water bath, and DIPEA (292 uL, 1.77 mmol) was added. The reaction was allowed to warm to room temperature and run for 2 h. After monitoring the reaction by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain solutions of Compounds 54a and 54b . These solutions were lyophilized to obtain 103 mg of Compound 54a (LC-MS: [M+H]+ = 1261.5) and 111 mg of Compound 54b (LC-MS: [M+H]+ = 1261.5), respectively. Step 2: Compound 54A

In a 25 mL flask, 54a (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added, and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was freeze-dried to obtain 61 mg of a solid; LC-MS: [M+H]+ = 1105.4. Step 3: Compound 54B

In a 25 mL flask, 54b (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added, and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure and removed to obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was freeze-dried to obtain 57 mg of a solid; LC-MS: [M+H]+=1105.4. Example 61

Synthesis of compounds 55A and 55B : Step 1: Compounds 55a and 55b 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 were added to a 50 mL flask in an ice-water bath, and DIPEA (248 uL, 1.5 mmol) was added, and the reaction was allowed to reach room temperature and run for 2 h. After the reaction was completed and monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain solutions of Compounds 55a and 55b . These solutions were lyophilized to obtain 100 mg of Compound 55a (LC-MS: [M+H]+ = 1275.5) and 101 mg of Compound 55b (LC-MS: [M+H]+ = 1275.5), respectively. Step 2: Compound 55A

55a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain 42 mg of a solid; LC-MS: [M+H]+ = 1119.4. Step 3: Compound 55B

55b (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain 45 mg of a solid; LC-MS: [M+H]+=1119.4. Example 62

Synthesis of compound 56 : Referring to the synthetic route of Example 60, compound 56 (50 mg) was obtained; LC-MS: [M+H]+ = 1119.3. Example 63

Synthesis of compound 57 : Referring to the synthetic route of Example 61, compound 57 (50 mg) was obtained; LC-MS: [M+H]+ = 1119.4. Example 64

Synthesis of compound 58 : Step 1: Synthesis of Compound 58a : 400 mL of DMF was added to exitecan mesylate M5 (15 g, 28 mol, prepared by the method disclosed in EP0737683A1). The reaction 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 reaction mixture was warmed to room temperature (25°C) for 1 hour. The reaction was completed as monitored by TLC. The reaction solution was concentrated under reduced pressure, and the crude product was purified by preparative high-performance liquid chromatography (acetonitrile/pure water system). The target peak was collected, and after removing the acetonitrile under reduced pressure, the mixture was lyophilized to obtain approximately 11 g of compound 58a as a yellow solid. , yield about 74%, MS m/z: [M+H] ⁺ 526.3. Step 2: Synthesis of compound 58b

At room temperature, compound 58a (11 g, 21 mol) was added to a 250 mL flask, 120 mL of formic acid was dissolved, 30 mL of formaldehyde (40% aqueous solution) was added to the resulting bright yellow solution, and the reaction was heated to 50°C for 1 h. The reaction was monitored by TLC to observe whether the reaction was complete, and then the reaction solution was cooled to room temperature. The reaction solution was then purified by preparative high-performance liquid chromatography (acetonitrile/water), and the target peak was collected. After removing the acetonitrile under reduced pressure, the target peak was freeze-dried to give approximately 4.5 g of compound 58b as a yellow powder solid, with a yield of approximately 40%, MS m/z: [M+H] ⁺ 540.6. Step 3: Synthesis of compound 58 :

Compound 58b (2.3 g, 4.3 mol) was placed in a 250 mL flask at room temperature, and 100 mL of DMF was added until dissolved. 2.3 g of 5% Pd/C was added to the resulting bright yellow solution. The atmosphere in the system was replaced with a hydrogen balloon, and the reaction was maintained at room temperature for 1.5 hours. The reaction was complete as detected by HPLC. The reactant was then filtered to remove Pd/C, and the resulting reaction solution was concentrated and purified by preparative HPLC (acetonitrile/pure water system). The obtained reaction solution was concentrated and purified by preparative high performance liquid chromatography (acetonitrile/water system). The target peak was collected and acetonitrile was removed under reduced pressure, followed by freeze-drying to obtain about 1.0 g of compound 58 as a yellow powdery solid in about 52% yield. MS m/z: [M+H] ⁺ 450.5. Example 65

Synthesis of compound 59 : Step 1: Compound 59a1d ( 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 were added to a 50 mL flask. DIPEA (378 μL, 2.29 mmol) was added to the flask under an ice-water bath and the reaction was allowed to proceed for 2 h. The reaction was monitored by HPLC, and the reaction solution was purified by HPLC to obtain a preparative solution, which was then lyophilized to obtain 59a (166 mg). LC-MS was used to obtain the preparative solution. The reaction was carried out at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a preparative solution, which was lyophilized to give 59a (166 mg); LC-MS: [M+H]+ = 1235.6. Step 2: Compound 59

59a (100 mg, 0.081 mmol), zinc bromide (368 mg, 1.63 mmol) and 10 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain solid compound 59 (43 mg); LC-MS: [M+H] + = 1079.3. Example 66

Synthesis of compound 60 : ; Referring to the synthetic route of Example 65, compound 60 (40 mg) was obtained; LC-MS: [M+H]+=1079.3. Example 67

Synthesis of compound 61 : Step 1: Compound 61a -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 flask and allowed to reach room temperature for 2 h. The reaction was monitored by HPLC. DIPEA (0.84 mL, 1.5 eq) was added and the reaction was allowed to reach room temperature for 2 h (monitored by HPLC). The reaction solution was directly purified by preparative method, and the preparative solution was concentrated by pumping water at 35° C. under reduced pressure to remove acetonitrile, and lyophilized to obtain compound 61a (1.21 g); LC-MS: [M+H]+=1249.4. Step 2: Compound 61

In a 100 mL flask, compound 61a (1.0 g, 0.8 mmol, 1.0 equivalent) and 35 mL of nitromethane were added, and zinc bromide (3.64 g, 16 mmol, 20.0 equivalents) was dissolved. The reaction was carried out in an oil bath at 40°C (stabilized in advance by preheating) for 30 min, and then concentrated in a water bath at 45°C under reduced pressure to remove nitromethane, resulting in a yellow solid residue (monitored by HPLC). After the acid was prepared, the prepared liquid was obtained. The prepared liquid was concentrated by pumping down the pressure in a water bath at 35°C to separate acetonitrile and then lyophilized to obtain compound 61 (786 mg). LC-MS: [M+H] ⁺ = 1093.6. Example 68

Synthesis of compound 62 : Step 1: Compound 62a Compound 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 flask. The reaction was carried out at 20±5°C for 2 h. The reaction was monitored by HPLC. The reaction solution was purified by direct HPLC preparation, and the product preparation was collected and freeze-dried to obtain compound 62a (120.9 mg); LC-MS: [M+H] ⁺ =1249.4. Step 2: Compound 62

Compound 62a (100 mg, 0.081 mmol, 1.0 eq), ZnBr ₂ (364 mg, 1.62 mmol, 20.0 eq) and CH ₃ NO ₂ (10 mL) were added to a 25 mL flask. After the addition, the reaction was heated to 40° C. for 0.5 h, after which the reaction was stopped and the reaction solution was dried directly at 45° C. by rotary drying under reduced pressure to obtain a yellow solid, which was sampled for HPLC monitoring. The reaction was monitored by HPLC. The rotary dried solid was directly purified by HPLC preparation, and the product preparation was collected and lyophilized to obtain compound 62 (61 mg); LC-MS: [M+H] + = 1093.4. Example 69

Preparation of compound 63 : Compound 63 (60 mg) was obtained by following the method of Example 67; LC-MS: [M+H] ⁺ = 1093.4. Example 70

Preparation of compound 64 : Referring to the synthetic route of Example 68, compound 64 (65 mg) was obtained; LC-MS: [M+H]+ = 1093.4. Example 71

Preparation of compounds 65A and 65B : Step 1: Compounds 65a and 65b (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 were added to a 50 mL flask. DIPEA (378 μL, 2.29 mmol) was added to the vial in an ice bath. The reaction was carried out at room temperature for 2 h. After monitoring the reaction by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain solutions of compounds 65a and 65b . These solutions were lyophilized to obtain 155 mg of compound 65a (LC-MS: [M+H]+ = 1303.4) and 158 mg of compound 65b (LC-MS: [M+H]+ = 1303.6), respectively. Step 2: Compound 65a

In a 25 mL flask, 50a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added, and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a preparative solution, and the solution was lyophilized to obtain 49 mg as a solid. Step 3: Compound 65B

In a 25 mL flask, 65b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added, and the reaction was carried out at 40°C for 1 h. After the reaction was completed by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a preparation solution, and the solution was lyophilized to obtain 47 mg of a solid. Example 72

Synthesis of compounds 66A and 66B : Step 1: Compound 66a and Compound 66b were added to a 50 mL flask with 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. DIPEA (378 μL, 2.29 mmol) was also added to the flask in an ice bath. After the reaction was complete by HPLC, the reaction solution was purified by high-performance liquid chromatography (HPLC) to obtain preparative solutions of Compound 66a and Compound 66b . These solutions were lyophilized to obtain 160 mg of Compound 66a and 160 mg of Compound 66b , respectively. LC-MS of compound 66a : [M+H] ⁺ = 1303.7, and LC-MS of compound 66b : [M+H] ⁺ = 1303.6. Step 2: Compound 66A

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 flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain 57 mg of a solid, LC-MS: [M+H]+=1147.5. Step 3: Compound 66B

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 flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to obtain the product as a crude substance. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain 57 mg of a solid, LC-MS: [M+H]+=1147.5. Example 73

Synthesis of compound 67A : Step 1: Compound 67a 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 to a 50 mL flask, and DIPEA (660 μL, 4.0 mmol) was added to the vial under an ice-water bath. The reaction was allowed to reach room temperature for 4 h. After HPLC monitoring, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a prepared solution of compound 67a , which was lyophilized to obtain 67a (402 mg); LC-MS: [M+H]+ = 1275.4. Step 2: Compound 67A

67A (100 mg, 0.78 mmol), zinc bromide (356 mg, 1.57 mmol) and 10 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain solid compound 67A (47 mg); LC-MS: [M+H] + = 1119.5. Example 74

Synthesis of compound 67B : Compound 67B (50 mg) was obtained by following the synthetic route of Example 73. LC-MS: [M+H] ⁺ 1119.4. Example 75

Synthesis of compound 68A : Step 1: Compounds 68a and 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 were added to a 50 mL flask. DIPEA (330 μL, 2.0 mmol) was added to the flask under an ice-water bath. The reaction was allowed to reach room temperature for 3 h. After HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography (HPLC) to obtain a preparative solution of compound 68a . The solution was lyophilized to afford 68a (177 mg); LC-MS: [M+H]+ = 1289.4. Step 2: Compound 68A 68A (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain solid compound 68A (45 mg); LC-MS: [M+H] + = 1133.4. Example 76

Synthesis of compound 68B : Referring to the synthetic route of Example 75, compound 68B (50 mg) was obtained; LC-MS: [M+H]+ = 1133.4. Example 77

Synthesis of compounds 69A and 68B : Step 1: Compounds 69a and 69b

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 were added to a 50 mL flask with an ice-water bath, and DIPEA (292 uL, 1.77 mmol) was added, and the reaction was warmed to room temperature and allowed to react for 2 h. After the reaction was completed and monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain solutions of compounds 69a and 69b . These solutions were lyophilized to obtain 109 mg of compound 69a (LC-MS: [M+H]+ = 1275.5) and 111 mg of compound 69b (LC-MS: [M+H]+ = 1275.7), respectively. Step 2: Compound 69a

69a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain 53 mg of a solid; LC-MS: [M+H]+ = 1119.4. Step 3: Compound 69B

69b (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain 54 mg of a solid; LC-MS: [M+H]+ = 1119.4. Example 78

Synthesis of compounds 70A and 70B : Step 1: Compounds 70a and 70b , 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 were added to a 50 mL flask. DIPEA (248 μL, 1.5 mmol) was also added to the flask under an ice-water bath. The reaction was carried out at room temperature for 2 h. After HPLC monitoring, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain solutions of compounds 70a and 70b . These solutions were lyophilized to obtain 106 mg of compound 70a (LC-MS: [M+H]+ = 1289.5) and 101 mg of compound 70b (LC-MS: [M+H]+ = 1289.4), respectively. Step 2: Compound 70A

In a 25 mL flask, 70a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol) and 5 mL of nitromethane were added, and the reaction was carried out at 40°C for 1 h. After the completion of the reaction was detected by HPLC, the solvent was concentrated under reduced pressure to obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was freeze-dried to obtain 39 mg of a solid; LC-MS: [M+H]+=1133.4. Step 3: Compound 70B

In a 25 mL flask, 70b (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol) and 5 mL of nitromethane were added and the reaction was carried out at 40°C for 1 h. After the completion of the reaction was detected by HPLC, the solvent was concentrated under reduced pressure and removed to obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was freeze-dried to obtain 35 mg of a solid; LC-MS: [M+H]+=1133.4. Example 79

Synthesis of compound 71 : Referring to the synthetic route of Example 78, compound 71 (30 mg) was obtained; LC-MS: [M+H]+ = 1133.3. Example 80

Synthesis of compound 72 : Referring to the synthetic route of Example 78, compound 72 (33 mg) was obtained; LC-MS: [M+H]+ = 1133.4. Example 81

Synthesis of Compound M11 : In a 100 mL flask, Compound M3 (11.0 g, 19.5 mmol, 1.0 equiv), DIPEA (2.8 g, 21.4 mmol, 1.1 equiv), 27-amino-4,7,10,13,16,19,22,25-octaoxoheptadecanoic acid (9.7 g, 20.5 mmol, 1.05 equiv), and DMF (60 mL) were reacted at room temperature for 20 min (monitored by TLC). The reaction solution was directly purified by preparative purification, and the preparative solution was concentrated by pumping water under reduced pressure at 35°C to remove acetonitrile and lyophilized to obtain Compound M10 (13.2 g) in 78% yield; LC-MS: [M+H]+ = 866.5.

In a 100 mL flask, compound M10 (13.0 g, 15 mmol, 1.0 equivalent), pentafluorophenol (3 g, 16.5 mmol, 1.1 equivalent), DCC (3.4 g, 16.5 mmol, 1.1 equivalent) and THF (30 mL) were added, and the reaction was carried out at room temperature for 30 min (monitored by TLC), and the insoluble material was filtered off. The reaction solution was directly purified by the preparation, and the preparation solution was concentrated by pumping water at 35° C. under reduced pressure to remove acetonitrile, and lyophilized to obtain compound M11 (14.2 g) in 92% yield; LC-MS: [M+H] + = 1032.5. Example 82

Synthesis of compound 73 : Step 1: Synthesis of Compound 73a To M11 (1 g, 0.79 mol), 10 mL of DMF was added, 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 added, and the reaction was maintained under these conditions for 1 h. The reaction was monitored by TLC to determine completion, and the reaction solution was purified by preparative HPLC (acetonitrile/water system), and the target peak was collected. The target peak was collected, and after removing acetonitrile under reduced pressure, the mixture was freeze-dried to give approximately 1.2 g of Compound 73a . MS m/z: [M+H] ⁺ = 1271.9. Step 2: Synthesis of Compound 73b

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 were added to a 25 mL flask. DIPEA (310 mg, 2.4 mmol) was added to the vial under an ice-water bath, and the reaction was carried out for 2 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by high-performance liquid chromatography (HPLC) to obtain a preparative solution, which was then lyophilized to give 73b (709 mg); LC-MS: [M+H]+ = 1720.8.

Step 3: Synthesis of compound 73 : 73b (200 mg, 0.116 mmol), zinc bromide (523 mg, 2.32 mmol) and 10 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain solid compound 73 (88 mg); LC-MS: [M+H] + = 1532.6. Example 83

Synthesis of compound 74 : ; Referring to the synthetic route of Example 82, compound 74 (90 mg) was obtained; LC-MS: [M+H]+=1532.6. Example 84

Synthesis of compound 75 : Step 1: Synthesis of Compound 75a : To M11 (1 g, 0.79 mol), 10 mL of DMF was added, 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 added, and the reaction was maintained under these conditions for 1 h. The reaction was monitored by TLC to determine completion, and the reaction solution was purified by preparative HPLC (acetonitrile/water system), and the target peak was collected. The target peak was collected, and after removing the acetonitrile under reduced pressure, the mixture was freeze-dried to give 0.9 g of Compound 75a . MS m/z: [M+H] ⁺ = 1285.6. Step 2: Synthesis of Compound 75b

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 were added to a 25 mL flask. DIPEA (155 mg, 1.2 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain a preparative solution, which was then lyophilized to obtain 75b (304 mg). LC-MS: [M+H]+ = 1734.8.

Step 3: Synthesis of compound 75 : 75b (200 mg, 0.116 mmol), zinc bromide (523 mg, 2.32 mmol) and 10 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain solid compound 75 (96 mg); LC-MS: [M+H] + = 1546.6. Example 85

Synthesis of compound 76 : Referring to the synthetic route of Example 84, compound 76 (92 mg) was obtained; LC-MS: [M+H]+ = 1546.5. Example 86

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

Synthesis of compound 78 : Referring to the synthetic route of Example 84, compound 78 (94 mg) was obtained; LC-MS: [M+H]+ = 1546.7. Example 88

Synthesis of compounds 79 and 80 : Step 1: Synthesis of Compound 79a : To M11 (1 g, 0.79 mol), 10 mL of DMF was added, 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 added, and the reaction was maintained under these conditions for 1 hour. The reaction was monitored for completion by TLC, and the reaction solution was purified by preparative HPLC (acetonitrile/water system) to collect the desired peak. After collecting the desired peak and removing the acetonitrile under reduced pressure, the product was lyophilized to give 783 mg of Compound 79a . MS m/z: [M+H] ⁺ = 1325.8. Step 2: Synthesis of Compounds 79b-1 and 79b-2 : 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 flask. DIPEA (130 mg, 1 mmol) was also added to the flask under an ice-water bath, and the reaction was allowed to proceed for 2 h. The reaction was monitored by HPLC. After the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain a preparative solution of compound 79b-1 and 79b-2 . The preparative solution was lyophilized to obtain 79b-1 (124 mg); LC-MS: [M+H]+ = 1743.0; 79b-1 (122 mg); LC-MS: [M+H]+ = 1743.0. Step 3: Synthesis of Compound 79

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 flask and the reaction was carried out at 40°C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to obtain the product as a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain solid compound 79 (30 mg); LC-MS: [M+H]+ = 1586.9. Step 4: Synthesis of compound 80

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 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 concentrated under reduced pressure and removed to obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a product preparation solution, and the preparation solution was lyophilized to obtain solid compound 80 (33 mg); LC-MS: [M+H] + = 1587.0. Example 89

Synthesis of compound 81 : Following the synthetic route of Example 88, compound 81 (24 mg) was obtained; LC-MS: [M+H]+ = 1586.9. Example 90

Synthesis of compound 82 : Referring to the synthetic route of Example 88, compound 82 (29 mg) was obtained; LC-MS: [M+H]+ = 1586.9. Example 91

1) Expression and Purification of the Humanized Anti-Claudin18.2 Antibody 5103F3-BSM: Expi293 (Shanghai OPM Biotechnology Co., Ltd.) suspension cells were used to express the 5103F3-BSM antibody. One day prior to transfection, cells were seeded at a density of 0.9 × ^10⁶ cells/mL in 1 L shaker flasks containing 300 mL of OPM-293 CD05 medium (81075-001, Shanghai Oppermax Bio-technology Co., Ltd.) and incubated overnight at 37°C, 5% _CO₂ , and 120 rpm on a shaker. The next day, PEI-MAX was used to transfect the antibody-expressing plasmid at a plasmid:PEI-MAX mass ratio of 1:3. OPM-293 ProFeed supplement was added at 5% (v/v) on the first day after transfection, and then at 5% (v/v) on the third day after transfection, and then centrifuged on the sixth day after transfection to collect the supernatant.

The resulting cell expression supernatant was collected and eluted using a protein A affinity chromatography column (UniMab 50, Suzhou Nano Technology Co., Ltd.) with 0.05 M sodium acetate buffer (pH 3.6). The captured antibody was adjusted to pH 7.0 with 1 M Tris-HCl (pH 8.8) at 0.7/10 (v/v) and then passed through a gel filtration chromatography column (SEC) (Superdex 200 Increase, Cytiva) to remove impurities such as aggregates. The antibody buffer was replaced with 20 mM sodium acetate buffer (pH 6.0). Example 92

5103F3-BSM antibody-drug conjugate samples were prepared by conjugating the 5103F3-BSM antibody to a payload. After cell expression and purification by protein A affinity chromatography and molecular sieving, the anti-Claudin 18.2 antibody was replaced in 20 mM NaAc-HAc, pH 6.0 buffer and concentrated or diluted to a protein concentration of 5 mg/mL. The white powder of the linker-drug combination was dissolved in DMA to 10 mg/mL and set aside. To open the interchain disulfide bonds of the anti-Claudin 18.2 antibody, 15x TCEP (based on the molecular weight ratio) was added and the reaction was incubated at room temperature for 2 hours. A 16-fold ligand-drug solution was then added based on the molecular ratio and allowed to react at room temperature for 2 hours. At the end of the reaction, the ligand-drug residue not bound to the anti-Claudin18.2 antibody was removed by ultrafiltration using a 30 KDa ultrafiltration centrifuge tube.

The resulting sample of the anti-Claudin18.2 antibody-drug conjugate was used to determine the monomer yield by SEC-HPLC and the drug loading by RP-HPLC or HIC-HPLC. Example 93

After cell expression and purification by protein A affinity chromatography and molecular weight screening, the anti-Claudin18.2 antibody was replaced in 20 mM NaAc-HAc, pH 6.0 buffer and concentrated or diluted to a protein concentration of 5 mg/mL. Samples of the 64C9-drug conjugate were prepared by conjugating the 64C9 antibody to the payload. The linker-drug white powder was dissolved in DMA to 10 mg/mL for use. To open the interchain disulfide bonds of the anti-Claudin18.2 antibody, 8-12 times TCEP was added, depending on the molecular ratio, and the reaction was carried out at room temperature for 1-2 hours. A 5-8x linker-drug solution was then added, depending on the molecular ratio, and the reaction was allowed to proceed at room temperature for 1-2 hours. Unbound linker-drug residues were removed by ultrafiltration using a 30 kDa ultrafiltration centrifuge tube at the end of the reaction. At the end of the reaction, a 30 kDa ultrafiltration centrifuge tube was used to remove unbound linker-drug residues, yielding a sample of the anti-Claudin18.2 antibody-drug conjugate.

The resulting sample of the anti-Claudin18.2 antibody-drug conjugate was used to determine the monomer yield by SEC-HPLC and the drug loading by RP-HPLC or HIC-HPLC. Example 94

ADC-1 was prepared according to the general coupling method of Example 92: Example 95

ADC-2 was prepared according to the general coupling method of Example 92: Example 96

ADC-3 was prepared according to the general coupling method of Example 92: Example 97

ADC-4 was prepared according to the general coupling method of Example 92: Example 98

ADC-5 was prepared according to the general coupling method of Example 92: Example 99

ADC-6 was prepared according to the general coupling method of Example 92: Example 100

ADC-7 was prepared according to the general coupling method of Example 92: Example 101

ADC-8 was prepared according to the general coupling method of Example 92: Example 102

ADC-9 was prepared according to the general coupling method 92 of Example 9. Example 103

ADC-10 was prepared according to the general coupling method of Example 92: Example 104

ADC-11 was prepared according to the general coupling method of Example 92: Example 105

ADC-12 was prepared according to the general coupling method of Example 92: Example 106

ADC-13 was prepared according to the general coupling method of Example 92: Example 107

ADC-14 was prepared according to the general coupling method of Example 92: Example 108

ADC-15 was prepared according to the general coupling method of Example 92: Example 109

ADC-16 was prepared according to the general coupling method of Example 92: Example 110

ADC-17 was prepared according to the general coupling method of Example 92: Example 111

ADC-18 was prepared according to the general coupling method of Example 92: Example 112

ADC-19 was prepared according to the general coupling method of Example 92: Example 113

ADC-20 was prepared according to the general coupling method of Example 92: Example 114

ADC-21 was prepared according to the general coupling method of Example 92: Example 115

ADC-22 was prepared according to the general coupling method of Example 92: Example 116

ADC-23 was prepared according to the general coupling method of Example 92: Example 117

ADC-24 was prepared according to the general coupling method of Example 92: Example 118

ADC-25 was prepared according to the general coupling method of Example 92: Example 119

ADC-26 was prepared according to the general coupling method of Example 92: ; Example 120

ADC-27 was prepared according to the general coupling method of Example 92: Example 121

ADC-28 was prepared according to the general coupling method of Example 92: Example 122

ADC-29 was prepared according to the general coupling method of Example 92. Example 123

ADC-30 was prepared according to the general coupling method of Example 92: Example 124

ADC-31 was prepared according to the general coupling method of Example 92: Example 125

ADC-32 was prepared according to the general coupling method of Example 92: Example 126

ADC-33 was prepared according to the general coupling method of Example 92: ; Example 127

ADC-34 was prepared according to the general coupling method of Example 92: Example 128

ADC-35 was prepared according to the general coupling method of Example 92: Example 129

ADC-36 was prepared according to the general coupling method of Example 92: Example 130

ADC-37 was prepared according to the general coupling method of Example 92: Example 131

ADC-38 was prepared according to the general coupling method of Example 92: Example 132

ADC-39 was prepared according to the general coupling method of Example 92: Example 133

ADC-40 was prepared according to the general coupling method of Example 92: Example 134

ADC-41 was prepared according to the general coupling method of Example 92: Example 135

ADC-42 was prepared according to the general coupling method of Example 92: Example 136

ADC-43 was prepared according to the general coupling method of Example 92: Example 137

ADC-44 was prepared according to the general coupling method of Example 92: Example 138

ADC-45 was prepared according to the general coupling method of Example 92: Example 139

ADC-46 was prepared according to the general coupling method of Example 92: Example 140

ADC-47 was prepared according to the general coupling method of Example 92: ; Example 141

ADC-48 was prepared according to the general coupling method of Example 92: ; Example 142

ADC-49 was prepared according to the general coupling method of Example 92: Example 143

ADC-50 was prepared according to the general coupling method of Example 92: Example 144

ADC-51 was prepared according to the general coupling method of Example 92: Example 145

ADC-52 was prepared according to the general coupling method of Example 92: Example 146

ADC-53 was prepared according to the general coupling method of Example 92: Example 147

ADC-54 was prepared according to the general coupling method of Example 92: Example 148

ADC-55 was prepared according to the general coupling method of Example 92: Example 149

ADC-56 was prepared according to the general coupling method of Example 92: Example 150

ADC-57 was prepared according to the general coupling method of Example 92: Example 151

ADC-58 was prepared according to the general coupling method of Example 92: Example 152

ADC-59 was prepared according to the general coupling method of Example 92: Example 153

ADC-60 was prepared according to the general coupling method of Example 92: Example 154

ADC-61 was prepared according to the general coupling method of Example 92: Example 155

ADC-62 was prepared according to the general coupling method of Example 92: Example 156

ADC-63 was prepared according to the general coupling method of Example 92: Example 157

ADC-64 was prepared according to the general coupling method of Example 92: Example 158

ADC-65 was prepared according to the general coupling method of Example 92: Example 159

ADC-66 was prepared according to the general coupling method 92 of Example Example 160

ADC-67 was prepared according to the general coupling method of Example 92: Example 161

ADC-68 was prepared according to the general coupling method of Example 92: Example 162

ADC-69 was prepared according to the general coupling method of Example 92: Example 163

ADC-70 was prepared according to the general coupling method of Example 92: Example 164

ADC-71 was prepared according to the general coupling method of Example 92: Example 165

ADC-72 was prepared according to the general coupling method of Example 92: Example 166

ADC-73 was prepared according to the general coupling method of Example 92: Example 167

ADC-74 was prepared according to the general coupling method of Example 92: Example 168

ADC-75 was prepared according to the general coupling method of Example 92: Example 169

ADC-76 was prepared according to the general coupling method of Example 92: Example 170

ADC-77 was prepared according to the general coupling method of Example 92: Example 171

ADC-78 was prepared according to the general coupling method of Example 92: Example 172

ADC-79 was prepared according to the general coupling method of Example 92: Example 173

ADC-80 was prepared according to the general coupling method of Example 92: Example 174

ADC-81 was prepared according to the general coupling method of Example 92: Example 175

ADC-82 was prepared according to the general coupling method of Example 92: Example 176

ADC-83 was prepared according to the general coupling method of Example 92: Example 177

ADC-84 was prepared according to the general coupling method of Example 92: Example 178

ADC-85 was prepared according to the general coupling method of Example 92: Example 179

ADC-86 was prepared according to the general coupling method of Example 92: . Example 180

ADC-87 was prepared according to the general coupling method of Example 92: Example 181

ADC-88 was prepared according to the general coupling method of Example 92: Example 182

ADC-89 was prepared according to the general coupling method of Example 92: Example 183

ADC-90 was prepared according to the general coupling method of Example 92: Example 184

ADC-91 was prepared according to the general coupling method of Example 92: Example 185

ADC-92 was prepared according to the general coupling method of Example 92: Example 186

ADC-93 was prepared according to the general coupling method of Example 92: Example 187

ADC-94 was prepared according to the general coupling method of Example 92: Example 188

ADC-95 was prepared according to the general coupling method of Example 92: Example 189

ADC-96 was prepared according to the general coupling method of Example 92: . Example 190

ADC-97 was prepared according to the general coupling method of Example 92: . Example 191

ADC-98 was prepared according to the general coupling method of Example 92: . Example 192

ADC-99 was prepared according to the general coupling method of Example 92: . Example 193

ADC-100 was prepared according to the general coupling method of Example 92: Example 194

ADC-101 was prepared according to the general coupling method of Example 92: Example 195

ADC-102 was prepared according to the general coupling method of Example 92: Example 196

ADC-103 was prepared according to the general coupling method of Example 92: Example 197

ADC-104 was prepared according to the general coupling method of Example 92: Example 198

ADC-105 was prepared according to the general coupling method of Example 92: . Example 199

ADC-106 was prepared according to the general coupling method of Example 92: . Example 200

ADC-107 was prepared according to the general coupling method of Example 93: , where 64C9 is an anti-Claudin18.2 antibody. Example 309

To determine the monomer rate, the following SEC-HPLC method was used: Column: Biocore SEC-300 5 μm, 4.6×300 mm Manufacturer: NanoChrom, Catalog Number: B213-050030-04630S Mobile phase: 50 mM PB + 300 mM NaCl + 200 mM Arg + 5% IPA, pH = 6.5 Table 1. Method and parameters Parameters settings Flow rate 0.3 mL/min Wavelength 214 nm and 280 nm Column temperature 30℃ Sample plate temperature Indoor temperature Injection volume 20 ug Maximum pressure 150 bar/15 MPa/2175 PSI gradient Equality running time 20 minutes Table 2. ADC Monomer Rate ADC Demotion % Aggregate % Single rate % ADC-6 0 3.66 96.34 ADC-107 0.46 2.00 97.54 Conclusion: The antibody-drug conjugate (ADC) disclosed in this application has the excellent properties of low degradation rate and low aggregation rate, as well as high monomer rate. Example 310

To determine the drug antibody ratio (DAR), the following RP-HPLC method was used: Column name: Proteomix RP-1000 4.6×100mm 5μm 1000A Manufacturer: Sepax Catalog number: 465950-4610 Table 3. Method and parameters Parameters settings Phase shift A: 0.1% TFA in water; B: 0.1% TFA in acetonitrile Flow rate 0.5 mL/min Wavelength 214 nm and 280 nm Column temperature 65 ℃ Sample plate temperature Indoor temperature Injection volume 25 ug Maximum pressure 100 bar/10 MPa/1450 PSI gradient Time(min) Flow rate (mL/min) Shift phase A (%) Shift phase B (%) 0.0 0.5 75 25 3 0.5 75 25 28 0.5 50 50 30 0.5 5 95 32 0.5 5 95 33 0.5 75 25 40 0.5 75 25 Table 4. ADC Drug-Antibody Conjugation Ratio (DAR) ADC RP-DAR ADC-6 7.33 ADC-107 7.42 Conclusion: The ADC in this application has the excellent property of high DAR value. When the same dose of ADC is administered, the ADC drug concentration at the target site can be significantly increased. Example 311

Plasma Stability of ADCs: ADCs were mixed with IgG-free plasma to produce a final ADC concentration of 0.6 mg/mL and incubated in a 37°C water bath for 0, 3, and 7 days. Unincubated plasma was used as a control. After incubation, samples were purified and extracted, and the drug-antibody ratio (DAR) was measured to reflect the plasma stability of the ADCs. Table 5. Plasma Stability of ADCs ADC DAR Unincubated plasma Plasma incubation day 0 Plasma incubation for 3 days Plasma incubation for 7 days ADC-6 7.51 7.56 7.61 7.67 ADC-107 7.31 7.23 7.49 7.43 Conclusion: The ADC disclosed in this disclosure has good plasma stability, and there is no significant change in the DAR value during the plasma incubation period. Example 312

ADC maintains the same affinity for claudin18.2 as the original anti-Claudin18.2 antibody, 5103F3-BSM. The relative affinities of 5103F3-BSM compared to ADC-6, and 64C9 compared to ADC-107, for claudin18.2 were compared using a double-antigen sandwich ELISA.

The specific steps of the Claudin18.2 relative binding activity assay are as follows: The antigen is diluted with PBS to an appropriate concentration, added to the plate and incubated overnight at 4°C. After coating, the plate is washed and sealed at 37°C for 1 hour. After sealing, the plate is washed, the test material is diluted in a gradient according to the appropriate concentration, the sample is added to the sealed enzyme plate and incubated. The rabbit anti-human antibody is diluted with buffer to an appropriate concentration, the sample is added and incubated. A homemade color development solution is added to develop color for 7 minutes, and then a stop solution is added to terminate the reaction. The plate is read at 450 nm, four-parameter simulation is performed to read the results, the EC50 value is calculated, and then the relative binding activity results are obtained.

Conclusion: As shown in Figures 3A and 3B , the conjugated ADC maintained similar affinity to the corresponding antibody, and there was no significant difference in the EC50 value, indicating that conjugation of the antibody to the toxin did not affect its affinity for the antigen. Example 313

Analysis of Antibody Binding Specificity: In this application, HEK293T-18.1 and HEK293T-18.2, two human embryonic kidney cell lines that overexpress Claudin18.1 and Claudin18.2, respectively, were used as experimental models. Specific binding of each naked ADC antibody to Claudin18.2 was analyzed by flow cytometry. HEK293T-18.1/18.2 cells in logarithmic growth phase were harvested, trypsinized, washed twice with pre-chilled 1× PBS, and resuspended to 1× ¹⁰⁷ cells/mL in 1× PBS containing 3% BSA. 2 × 10 ⁵ cells were plated in a 96-well plate, and serial dilutions of the test antibody were added to the cells for incubation on ice for 1 hour. At the end of the incubation, cells were washed with pre-chilled PBS and then incubated with FITC-mouse anti-human fluorescent secondary antibody for 30 minutes on ice, washed, resuspended, and analyzed using a Beckman Coulter CytoFlex flow cytometer. The gating strategy was set as follows: cells to be analyzed were circled using the SSC/FSC scatter plot; single cells were circled using the FSC-H/FSC-A scatter plot to exclude adherent and debris; single-cell gating was applied to PB450-A to identify viable cells. Finally, the cell populations to be analyzed were identified in FITC-A, and fluorescence values corresponding to different antibody concentrations were statistically calculated. Graph Pad Prism 9.0 data analysis software using a four-parameter model was used for data statistics and analysis.

Table 6. Specific binding of Claudin18.2 by subjects ADC-107 naked antibody and ADC-6 naked antibody (see also FIG . 4A and FIG. 4B ). Group antibody EC50 (μg/mL) HEK293T-18.1 64C9 ND 5103F3-BSM ND HEK293T-18.2 64C9 0.24 5103F3-BSM 0.12

Note: ND means not detected.

Conclusion: Although naked antibodies 64C9 and 5103F3-BSM do not recognize claudin18.1, they demonstrate highly specific recognition of claudin18.2. The binding affinity of 5103F3-BSM is characteristically superior to that of 64C9. Example 314

In vitro pharmacodynamics: In this application, various human gastric adenocarcinoma tumor cell lines, including HEK293T-18.2, BxPC-3#A9F, SNU-5, and SNU-16, that overexpress Claudin18.2, were used as experimental models to evaluate the efficacy of ADC drugs in vitro . Multiple tumor cell lines were seeded in 96-well plates, and serial dilutions of the test antibody and the corresponding ADC drug were added to the cells. The cells were treated for 5 days, and cell viability was monitored using MTS assay. The inhibitory effect of the test antibody and ADC on the tumor cell lines was evaluated by calculating the IC50. The antibody was initially administered at a concentration of 500 nM and diluted sevenfold for a total of eight concentration points, with treatment lasting five days. The final calculation was based on the formula: survival rate = (experimental group - blank) / ( control group - blank) × 100%. Graph Pad Prism was then used to fit the curve ( Figure 5 ) and calculate the half-inhibitory concentration (IC50).

Table 7. In vitro efficacy results of naked antibody 64C9 compared to ADC-107, and naked antibody 5103F3-BSM compared to ADC-6 at the cellular level (see also Figures 5A , 5B , 5C , and 5D ). Group Antibodies/ ADCs IC50 (nM) HEK293T-18.2 64C9 >500 ADC-107 0.538 5103F3-BSM >500 ADC-6 0.149 BxPC-3 #A9F 64C9 >500 ADC-107 0.127 5103F3-BSM >500 ADC-6 0.100 SNU-5 64C9 >500 ADC-107 119.66 5103F3-BSM >500 ADC-6 10.07 SNU-16 64C9 >500 ADC-107 66.56 5103F3-BSM >500 ADC-6 7.061

Conclusion: No significant difference in tumor cell cytotoxicity was found between 64C9 and 5103F3-BSM in HE293T-18.2, BxPC-3#A9F, SNU-5, and SNU-16 cell models. However, ADC-6 (DAR=8) exhibited significantly higher in vitro tumor cell cytotoxicity, i.e., efficacy and potency, than ADC-107 (DAR=8). Example 315

This application established a subcutaneous xenograft tumor model of target-positive human gastric cancer cells in SNU-5 NOD Scid mice to evaluate the in vivo efficacy of ADC-6. 5×10 ⁶ SNU-5 cells (0.1 mL/cell) were subcutaneously injected into the right scapula of 6-7 week-old NOD Scid mice. When the average tumor size reached approximately 180 mm ³ , mice were randomly divided into a lysate control group (vehicle) and an ADC-6 treatment group (5 mg/kg), each containing 5 mice. ADC-6 administration began on D0. All groups received 10 mL/kg of body weight via tail vein injection once weekly for 4 weeks (QW x 4). All groups were observed until day 28 (D28) after subgroup administration ( Table 8 , Figure 6 ). Statistical analysis of the mean tumor volume on day 28 (D28) after subgroup administration showed that ADC - 6 administered intravenously at a dose of 5 mg/kg (QW x 4) had a significant tumor inhibitory effect (P < 0.05).

Table 8. Efficacy analysis of each group in the subcutaneous tumor transplantation model of human gastric cancer cells (SNU-5) in NOD Scid mice . Experimental group Day 0 after administration 28 days after administration Mean tumor volume (±S) mm ³ Mean tumor volume (±S) mm ³ Relative tumor volume (±S) TGI (%) T/C (%) P -value Group vehicle 177.98±8.91 1493.75±190.25 8.34±0.87 - - - ADC-6 (5 mg/kg) 177.71±10.43 53.60±3.17 0.30±0.01 96.38 3.62 <0.001 Note: 1. Data are expressed as "mean ± standard error"; 2. T/C % = _TRTV / C _RTV × 100%; TGI% = (1-T/C) × 100%; 3. p values were obtained by comparing the tumor volume of the treatment group with the lysate control group on D 28 (28 days after administration). Example 316

This application establishes a subcutaneous tumor xenograft model in BALB/c nude mice using human gastric cancer cells (e.g., MKN-45) that overexpress Claudin18.2 to evaluate the in vivo efficacy of ADC-6. MKN-45 cells (2×10 ⁶ ) were injected subcutaneously (0.1 mL/mouse) into the right scapula of 6- to 7-week-old BALB/c nude mice. When the average tumor size reached approximately 160 mm ³ , mice were randomly divided into a vehicle control group (Vehicle) and an ADC-6 treatment group (2 mg/kg, 5 mg/kg), with 5 mice per group, and drug administration began (D0). Each group received 10 mL/kg of body weight once weekly for 4 consecutive weeks (QW×4). All groups were observed on day 28 (D28) after administration ( Table 9 , Figure 7 ), and statistical analysis of the mean tumor volume of each subgroup on day 28 (D28) after administration showed that ADC-6 administered via the tail vein at doses of 2 mg/kg and 5 mg/kg (QW×4) had a significant tumor inhibitory effect (P<0.05). Table 9. Efficacy analysis of each group in the subcutaneous tumor transplantation model of human gastric cancer cells MKN-45 in BALB/c nude mice Experimental group Day 0 after administration 28 days after administration Mean tumor volume (±S) mm ³ Mean tumor volume (±S) mm ³ Relative tumor volume (±S) TGI (%) T/C (%) P -value Group vehicle 167.08±12.44 1240.62±274.11 7.98±2.40 - - - ADC-6 (2 mg/kg) 166.79±16.44 72.00±27.44 0.43±0.17 94.55 5.45 <0.001 ADC-6 (5 mg/kg) 167.76±14.39 6.12±0.95 0.04±0.00 99.55 0.45 <0.001 Note: 1. Data are expressed as mean ± standard error; 2. T/C % = _TRTV / _CRTV × 100%; TGI % = (1-T/C) × 100%; 3. p -values were calculated by comparing tumor volume in the treatment group with that in the lysate control group on D28 (day 28 after administration). Sequence Listing > Seq ID No.1: 5103F3 BSM VH amino acid sequence QVQLQESGGRLIKPGEPLRLSCKTSGIDLS GFAMG WVRQAPGKGLEYIG FIDSGGGAFYATWAR GRFTISRTTSTNTVYLQMNSLTAEDTAVYYCAR HGGNTYYYAMDP WGPGTLVTVSS > Seq ID No.2: 5103F3 BSM VH-CDRH1 GFAMG ＞ Seq ID No.3: 5103F3 BSM VH-CDRH2 FIDSGGGAFYATWARG ＞ Seq ID No.4: 5103F3 BSM VH-CDRH3 HGGNTYYYAMDP ＞ Seq ID No.5: 5103F3 BSM heavy chain amino acid sequence QVQLQESGGRLIKPGEPLRLSCKTSGIDLSGFAMGWVRQAPGKGLEYIGFIDSGGGAFYATWARGRFTISRTSTNTVYLQMNSLTAEDTAVYYCARHGGNTYYY AMDPWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG ＞Seq ID No.6: 5103F3 BSM heavy chain constant region amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG ＞Seq ID No.7: 5103F3 BSM VL amino acid sequence ALRMTQSPSSLAATTGQRVTITC QASQSISNYLA WYQQKPGQPPKLLIY SASTLAS GVPSRFKGSGSGTQFTLTISCVQCEDFATYYC QQVYSVTNIDNA FGGGTRVEIK ＞Seq ID No.8: 5103F3 BSM VL-CDRL1 QASQSISNYLA ＞ Seq ID No.9: 5103F3 BSM VL-CDRL2 SASTLAS ＞ Seq ID No.10: 5103F3 BSM VL-CDRL3 QQVYSVTNIDNA ＞ Seq ID No.11: 5103F3 BSM light chain amino acid sequence ALRMTQSPSSLAATTGQRVTITCQASQSISNYLAWYQQKPGQPPKLLIYSASTLASGVPSRFKGSGSGTQFTLTISSVQPEDFATYYCQQVYSVTNIDNAFGG GTRVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ＞ Seq ID No.12: 5103F3 BSM light chain constant region amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ＞ Seq ID No.13: 5103F3 BSM VH nucleic acid sequence CAGGTGCAGCTGCAGGAGTCCGGCGGCAGGCTGATCAAGCCCGGCGAGCCCCTGAGGCTGTCCTGCAAGACCTCCGGCATCGACCTGTCCGGCTTCGCCATGGGCTGGGGTGAGGCAGGCCCCGGCAAGGGCCTGGAGTACATCGGCTTCATCGACTCCGGCGGCGGCGCCTTCT ACGCCACCTGGGCCAGGGGCAGGTTCACCATCTCCAGGACCTCCACCAACACCGTGTACCTGCGATGAACTCCCTGACCGCCGAGGACACCGCCGTGTACTACTGCGCCAGGCACGGCGGCAACACCTACTACGCCATGGACCCCTGGGGCCCCGGCACCCTGGTGACCGTGTCCTCC ＞ Seq ID No.14: 5103F3 BSM VH-CDRH1 nucleic acid sequence GGCTTCGCCATGGGC ＞ Seq ID No.15: 5103F3 BSM VH-CDRH2 nucleic acid sequence TTCATCGACTCCGGCGGCGGCGCCTTCTACGCCACCTGGGCCAGGGGC > Seq ID No.16: 5103F3 BSM VH-CDRH3 nucleic acid sequence CACGGCGGCAACACCTACTACTACGCCATGGACCCC > Seq ID No.17: 5103F3 BSM heavy chain nucleic acid sequence > Seq ID No.18: 5103F3 BSM heavy chain constant region nucleic acid sequence > Seq ID No.19: 5103F3 BSM light chain constant region nucleic acid sequence GCCCTGAGGATGACCCAGTCCCCTCCTCCCTGGCCGCCACCACCGGCCAGAGGGTGACCATCACCTGCCAGGCCTCCCAGTCCATCTCCAACTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACTCCGCCTCCACC CTGGCCTCCGGCTGCCCTCCAGGTTCAAGGGCTCCGGCTCCGGCACCCAGTTCACCCTGACCATCTCCAGCGTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGTACTCCGTGACCAACATCGACAACGCCTTCGGCGGCGGCACCAGGGTGGAGATCAAG ＞ Seq ID No.20: 5103F3 BSM VL-CDRL1 nucleic acid sequence CAGGCCTCCCAGTCCATCTCCAACTACCTGGCC ＞ Seq ID No.21: 5103F3 BSM VL-CDRL2 nucleic acid sequence TCCGCCTCCACCCTGGCCTCC > Seq ID No.22: 5103F3 BSM VL-CDRL3 nucleic acid sequence CAGCAGGTGTACTCCGTGACCAACATCGACAACGCC > Seq ID No.23: 5103F3 BSM light chain nucleic acid sequence > Seq ID No.24: 5103F3 BSM light chain constant region nucleic acid sequence CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCC CAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG ＞ Seq ID No.25: 64C9 VH amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFT TYPIE WVRQAPGQRLEWMG NFHPYNDDTKYNEKFK GRVTITRDTSASTAYMELSSLRSEDTAVYYCAR RAYGYPYAMDY WGQGTLVTVSS ＞ Seq ID No.26: 64C9 VH-CDRH1 TYPIE ＞ Seq ID No.27: 64C9 VH-CDRH2 NFHPYNDDTKYNEKFKG ＞ Seq ID No.28: 64C9 VH-CDRH3 RAYGYPYAMDY ＞Seq ID No.29: 64C9 heavy chain amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYPIEWVRQAPGQRLEWMGNFHPYNDDTKYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARRAYGYPYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG > Seq ID No.30: 64C9 heavy chain constant region amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG ＞Seq ID No.31: 64C9 Light Chain Variable Region amino acid sequence DIVMTQSPDSLAVSLGERATINC KSSQSLLNSGNQKNYLT WYQQKPGQPPKLLIY RASSRES GVPDRFSGSGSGTTDFTLTISSLQAEDVAVYYC QNDYIYPYT FGGGTKLEIK ＞ Seq ID No.32: 64C9 VL-CDRL1 KSSQSLLNSGNQKNYLT ＞ Seq ID No.33: 64C9 VL-CDRL2 RASSRES ＞ Seq ID No.34: 64C9 VL-CDRL3 QNDYIYPYT ＞ Seq ID No.35: 64C9 light chain amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYRASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYIYPYTF GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ＞ Seq ID No.36: 64C9 light chain constant region amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ＞ Seq ID No.37: 64C9 heavy chain variable region nucleic acid sequence CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCACCTACCCCATCGAGTGGGTGAGGCAGGCCCCCGGCCAGAGGCTGGAGTGGATGGGCAACTTCCACCCCTACAACGACGACAC CAAGTACAACGAGAAGTTCAAGGGCAGGGTGACCATCACCAGGGACACCAGCGCCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGAGGGCCTACGGCTACCCCTACGCCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC ＞ Seq ID No.38: 64C9 VH-CDRH1 nucleic acid sequence ACCTACCCCATCGAG ＞ Seq ID No.39: 64C9 VH-CDRH2 nucleic acid sequence AACTTCCACCCCTACAACGACGACACCAAGTACAACGAGAAGTTCAAGGGC > Seq ID No.40: 64C9 VH-CDRH3 nucleic acid sequence AGGGCCTACGGCTACCCCTACGCCATGGACTAC > Seq ID No.41: 64C9 heavy chain nucleic acid sequence > Seq ID No.42: 64C9 heavy chain constant region nucleic acid sequence > Seq ID No.43: 64C9 light chain constant region nucleic acid sequence GACATCGTGATGACCCAGAGCGACAGCCTGGCCGTGAGCCTGGGCGAGAGGGCCACCATCAACTGCAAGAGCAGCCAGAGCCTGCTGAACAGCGGCAACCAGAAGAACTACCTGACCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACAGGGCCAGCAGCAGGGAGAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGC ACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGAACGACTACATCTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG ＞Seq ID No.44: 64C9 VL-CDRL1 nucleic acid sequence AAGAGCAGCCAGAGCCTGCTGAACAGCGGCAACCAGAAGAACTACCTGACC ＞Seq ID No.45: 64C9 VL-CDRL2 nucleic acid sequence AGGGCCAGCAGCAGGGAGAGC > Seq ID No.46: 64C9 VL-CDRL3 nucleic acid sequence CAGAACGACTACATCTACCCCTACACC > Seq ID No.47: 64C9 light chain nucleic acid sequence > Seq ID No.48: 64C9 light chain constant region nucleic acid sequence CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTC CCAGGAGGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

without

The foregoing and other features of the present disclosure will become more fully apparent from the following description and the appended claims when read in conjunction with the accompanying drawings. Understanding that these drawings merely depict various embodiments arranged in accordance with the present disclosure and, therefore, are not to be considered limiting of its scope, the present disclosure may be described with additional specificity and detail through the use of the accompanying drawings, in which: FIG1 shows SEC-HPLC detection of ADC-6 aggregation (A) and ADC-107 aggregation (B); FIG2 shows SEC-HPLC detection of ADC-6 (A) and ADC-107 aggregation ( B ) ; RP-HPLC detection of drug-antibody coupling ratio (DAR) (B); Figure 3 shows that ADC-6 maintains equal affinity of the 5103F3-BSM antibody for the antigen Claudin18.2 (A) and ADC-107 also maintains equal affinity of the 64C9 antibody for the antigen Claudin18.2 (B); Figure 4 shows the binding curves of the antibodies to Claudin18.1-expressing cells (A) and Claudin18.2-expressing cells (B); Figure 5 shows the human embryonic kidney cell line HEK293T-18.2 expressing Claudin18.2 (A); the human pancreatic cancer cell line BxPC-3#A9F expressing Claudin18.2 (B); in vitro tumor inhibitory activity of ADC-6 and ADC-107 in experimental models of SNU-5, a human gastric cancer cell line expressing Claudin18.2 (C); and SNU-16 , another human gastric cancer cell line expressing Claudin18.2 (D); Figure 6 shows the in vivo efficacy of ADC-6 in the SNU-5 isolated tumor model; and Figure 7 shows the in vivo efficacy of ADC-6 in the MKN-45 single tumor model.

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TW202532076A_113148725_SEQL.xml

Claims (31)

A ligand-camptothecin derivative conjugate represented by general formula I , or a pharmaceutically acceptable salt or solvent thereof; I wherein Ab is an antibody targeting human Claudin18.2 or an antigen-binding fragment thereof; L ₁₁ , L ₁₂ , and L ₁₃ are different and independently selected from the group consisting of: 、 、 、 、 、 、 、 、 or ; L ₂ has the structure shown in the following formula A: Formula A wherein 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; and the carbon 2 attached to Y has absolute chirality in the R or S configuration; _L3 is present or absent, and when present, _L3 comprises a PEG hydrophilic unit: , o is an integer of 1-10, preferably an integer of 2-8; L ₄ is an enzymatically cleavable unit; L ₅ is a linking unit; the chiral carbon atom No. 1 attached to N in Formula I has an absolute chirality of R or S configuration; R is hydrogen, deuterium, 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, or substituted 5-10 heteroaryl; preferably R is hydrogen or C1-C6 alkyl; R _{R 1} is hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxylic acid, 3-7 heterocyclic group, substituted 3-7 heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 heteroaryl, or substituted 5-10 heteroaryl; preferably, R ₁ is hydrogen or C1-C6 alkyl; more preferably, R ₁ is C1-C6 alkyl; R _{R 2} is hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxylic acid, a 3-7 heterocyclic group, a substituted 3-7 heterocyclic group, a C6-C10 aryl, a substituted C6-C10 aryl, a 5-10-membered heteroaryl, or a substituted 5-10 heteroaryl; preferably, R ₂ is hydrogen, halogen, or C1-C6 alkyl; more preferably, R ₂ is halogen; X is -C(O)-CR _a R _b -(CR ₃ R ₄ ) _m -O-, -C(O)-CR _a R _b -(CR ₃ R ₄ ) _m -NH-, or -C(O)-CR _aRb- ( _CR3R4 ) _m - _S- ; Preferably, X is -C(O) _-CRaRb- ( _{CR3R4 )m -} O-; Ra and Rb are each independently hydrogen, deuterium, halogen, C1- _C6 alkyl, deuterium-substituted C1-C6 _alkyl , halogen-substituted C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C6-C10 arylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, 3-7 heterocyclic group, substituted 3-7 heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 heteroaryl, or substituted 5-10 heteroaryl; Preferably, _Ra and R R _a and R _b are each independently hydrogen, C1-C6 alkyl, halogen-C1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, or C6-C10 arylC1-C6 alkyl; alternatively, Ra, R _b and the carbon atoms to which they are attached constitute C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, 3-7 heterocycloalkyl, or substituted 3-7 heterocycloalkyl; preferably, _Ra , R _b and the carbon atoms to which they are attached constitute C3-C8 cycloalkyl; R ₃ , R _{R 4} are the same or different and independently represent hydrogen, deuterium, a halogen, a C1-C6 alkyl group, a halogenated C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C1-C6 alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyl C1-C6 alkyl group, a C3-C8 cycloalkyl group, a 3-7 heterocyclic group, or a substituted 3-7 heterocyclic group; preferably, R ₃ and R ₄ are independently hydrogen or a C1-C6 alkyl group; alternatively, R ₃ , R ₄ and the carbon atom to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3-7 heterocyclic group, or a substituted 3-7 heterocyclic group; m is an integer between 0 and 4, preferably 0 or 1. n1, n2, and n3 are each independently selected from any integer or decimal number between 0 and 10. n1, n2, and n3 are not all 0 at the same time, and 1 ≤ n1 + n2 + n3 ≤ 10. The ligand-camptothenate derivative conjugate or a pharmaceutically acceptable salt or solvent thereof as described in claim 1, wherein Ab is an antibody having binding affinity to human Claudin18.2. The ligand-camptotherine derivative conjugate or a pharmaceutically acceptable salt or solvent thereof as described in claim 2, wherein the antibody comprises an IgG heavy chain and a light chain. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvent thereof as described in claim 3, wherein the light chain comprises the CDRs of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and the IgG heavy chain comprises the CDRs of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. The ligand-dendrogenin derivative conjugate or a pharmaceutically acceptable salt or solvent thereof as described in claim 1, wherein Ab comprises a light chain comprising CDRL1, CDRL2, and CDRL3, each having an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively, and a heavy chain comprising CDRH1, CDRH2, and CDRH3, each having an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively. The ligand-dendrimer derivative conjugate of claim 1, as represented by general formula I , or a pharmaceutically acceptable salt or solvent thereof, wherein Ab comprises a heavy chain comprising a heavy chain variable region of SEQ ID NO: 1 and a light chain comprising a light chain variable region of SEQ ID NO: 7. The ligand-arbenzidine derivative conjugate or a pharmaceutically acceptable salt or solvent thereof as described in claim 1, wherein Ab comprises a heavy chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 13, and a light chain variable region comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 19. The ligand-camptothenate derivative conjugate or a pharmaceutically acceptable salt or solvent thereof as described in claim 1, comprising: a heavy chain having the amino acid sequence of SEQ ID NO: 5, and a light chain having the amino acid sequence of SEQ ID NO: 11. The ligand-dendrogenin derivative conjugate or a pharmaceutically acceptable salt or solvent thereof as described in claim 1, comprising: a heavy chain having an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 17, and a light chain having an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 23. The ligand-dendritic alkaloid derivative conjugate or a pharmaceutically acceptable salt or solvent thereof as described in any one of claims 1 to 9, wherein X comprises the following structure or an isomer thereof: 、 、 、 、 、 、 、 、 、 、 、 、 or , wherein the position indicated by the wavy line on the left side is connected to the dendrobium derivative, and the position indicated by the wavy line on the right side is connected to L5. The ligand-dendrimer derivative conjugate or a pharmaceutically acceptable salt or solvent thereof as described in claim 10, wherein _L4 comprises a peptide residue composed of an amino acid; wherein, optionally, the amino acid is further substituted with one or more substituents selected from one or more of deuterium, a halogen, a hydroxyl group, a cyano group, an amino group, a nitro group, a carboxyl group, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a C1-C6 alkoxy group, a C3-C8 cycloalkyl group, or a substituted C3-C8 cycloalkyl group; 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), glutamine (E) or aspartic acid (D); More preferably, the peptide residue is a tetrapeptide residue comprising glycine (G)-glycine (G)-phenylalanine (F)-glycine (G). The ligand-arbenzylamine derivative conjugate or a pharmaceutically acceptable salt or solvent thereof as claimed in claim 11, wherein L ₅ is -NR ₅ (CR ₆ R ₇ ) _q - or a chemical bond, and q is an integer from 0 to 6; R ₅ , R ₆ and R _{R 7} is the same or different and is each independently hydrogen, deuterium, a halogen, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C3-C8 cycloalkyl group, a C3-C8 cycloalkylC1-C6 alkyl group, a C1-C6 alkoxyC1-C6 alkyl group, a 3-7 heterocyclic heteroaryl group, a substituted 3-7 heterocyclic heteroaryl group, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 heteroaryl group, or a substituted 5-10 heteroaryl group; preferably, R ₅ , R ₆ , and R ₇ are each independently hydrogen or C1-C6 alkyl group; more preferably, R ₅ , R ₆ , and R ₇ are each independently hydrogen. The ligand-dendrimer derivative conjugate or a pharmaceutically acceptable salt or solvent thereof as described in any one of claim 1, wherein the linking units -L ₁₁ -L ₂ -L ₃ -L ₄ -L ₅ -, -L ₁₂ -L ₂ -L ₃ -L ₄ -L ₅ -, or -L ₁₃ -L ₂ -L ₃ -L ₄ -L ₅ - are different from each other and are each independently: 、 、 、 、 、 、 、 、 、 、 or Preferably, -L ₁₁ -L ₂ -L ₃ -L ₄ -L ₅ -, -LL ₁₂₂ -L ₃ -L ₄ -L ₅ - or -L ₁₃ -LL ₂₃ -L ₄ -L ₅ - are different from each other and are each independently: 、 、 、 、 or wherein Ac is a hydrophilic structural unit; R ₅ , R ₆ , and R ₇ are the same or different and are each independently hydrogen, deuterium, a halogen, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a 3-7 heterocyclic group, a substituted 3-7 heterocyclic group, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 heteroaryl group, or a substituted 5-10 heteroaryl group; preferably, R ₅ , R ₆ , and R ₇ are each independently hydrogen or C1-C6 alkyl group; more preferably, R ₅ , R ₆ , and R ₇ are each independently hydrogen; The carbon atom #2 attached to N has an absolute chirality of R or S configuration; it is connected to Ab at the position shown by the left wavy line, and to X at the position shown by the right wavy line; o is an integer from 1 to 10. A ligand-camptothenate derivative conjugate represented by formula II , or a pharmaceutically acceptable salt or solvent thereof; II wherein Ab is an antibody or an antigen-binding fragment thereof having binding affinity for human Claudin18.2; L ₁₁ , L ₁₂ , and L ₁₃ are linking units, and L ₁₁ , L ₁₂ , and L ₁₃ are different from each other and independently are: 、 、 、 、 、 、 、 、 or ; L ₃ is present or absent, and when L ₃ is present, L ₃ is , o is an integer of 1-10, preferably an integer of 2-8; Ac is a hydrophilic structural unit; the chiral carbon atoms at positions 1, 2, and 3 are each independently in R or S configuration; R is hydrogen, deuterium, a halogen, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 heteroaryl group, or a substituted 5-10 heteroaryl group; preferably, R is hydrogen or a C1-C6 alkyl group; R _R1 is hydrogen, deuterium, 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 heterocyclic group, substituted 3-7 meta heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 meta heteroaryl, or substituted 5-10 heteroaryl; preferably, _R1 is hydrogen or C1-C6 alkyl; more preferably, _R1 is C1-C6 alkyl; R _R2 is hydrogen, deuterium, 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 heterocyclic ring, substituted 3-7 heterocyclic ring, C6-C10 aryl, substituted C6-C10 aryl, 5-10 heteroaryl, or substituted 5-10 heteroaryl; preferably, _R2 is hydrogen, halogen, or C1-C6 alkyl; more preferably, _R2 is halogen; _X is -C(O)-CRaRb-( _CR3R4 ) _{m - O-} , _{-C (} O) _-CRaRb- ( _CR3R4 ) _m -NH-, or -C(O) _{-CRaR b} -(CR ₃ R ₄ ) _m -S-; Preferably, X is -C(O)-CR _a R _b -(CR ₃ R ₄ ) _m -O-; _Ra and R _b are each independently hydrogen, deuterium, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, 3-7 heterocyclic group, substituted 3-7 heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 heteroaryl, or substituted 5-10 heteroaryl; Preferably, _Ra and R R _a and R _b are each independently hydrogen, C1-C6 alkyl, halogen-C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, or C6-C10 aryl C1-C6 alkyl; alternatively, Ra, R _b and the carbon atoms to which they are attached constitute C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, 3-7 heterocycloalkyl, or substituted 3-7 heterocycloalkyl; preferably, _Ra , R _b and the carbon atoms to which they are attached constitute C3-C8 cycloalkyl; R ₃ , R _{R 4} is the same or different and independently represents hydrogen, deuterium, a halogen, a C1-C6 alkyl group, a halogenated C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C1-C6 alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxy C1-C6 alkyl group, a C3-C8 cycloalkyl group, a 3-7 heterocyclic group, or a substituted 3-7 heterocyclic group; preferably, R ₃ and R ₄ are independently hydrogen or a C1-C6 alkyl group; alternatively, R ₃ , R ₄ and the carbon atom to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3-7 heterocyclic group, or a substituted 3-7 heterocyclic group; m is an integer between 0 and 4, preferably 0 or 1; n1, n2, and n3 are each independently an integer or decimal between 0 and 10, n1, n2, and n3 are not all 0 at the same time, and 1 ≤ n1 + n2 + n3 ≤ 10. The ligand-dendrimer derivative conjugate or a pharmaceutically acceptable salt or solvent thereof as described in any one of claims 1 or 14, wherein Ac has a structure represented by formula B: B wherein Z comprises a hydrophilic structure of carboxyl, phosphate, polyphosphate, phosphite, sulfonic acid, sulfinic acid, or polyethylene glycol (PEG); preferably, Z comprises a hydrophilic structure of carboxylate, phosphate, or polyethylene glycol (PEG); Y' is optionally a scaffold that connects the amino group to Z; preferably, Y' is a C1-C6 alkyl group; the position indicated by the wavy line is the attachment site. The ligand-camptotherine derivative conjugate or a pharmaceutically acceptable salt or solvent thereof as described in any one of claims 1 or 14, wherein the Ac comprises glycine, ( D/L ) alanine, (D/L) leucine, ( D/L ) isoleucine, ( D/L ) valine, ( D/L ) phenylalanine, ( D/L ) proline, ( D/L ) tryptophan, ( D/L ) serine, ( D/L ) tyrosine, ( D/L ) cysteine, ( D/L ) cystine, ( D/L ) arginine, ( D/L ) histidine, ( D/L ) methionine, (D/L) asparagine, (D/L) glutamine, ( D/L ) threonine, ( D/L) ) aspartic acid, ( D/L ) glutamine, natural or unnatural amino acid derivatives, or the following structures or their isomers: 、 、 、 、 、 、 、 、 、 or ; Better 、 、 or . The ligand-dendrimer derivative conjugate or a pharmaceutically acceptable salt or solvent thereof as described in any one of claims 1 to 14, wherein the Ac comprises a hydrophilic structure of glycine, phosphate, ( D/L ) glutamine, or polyethylene glycol. The ligand-dendrogenin derivative conjugate or a pharmaceutically acceptable salt or solvent thereof as described in any one of claims 1 to 14, wherein the dendrogenin derivative has a structure represented by the following formula d; d wherein R comprises hydrogen, deuterium, 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, or substituted 5-10 heteroaryl; preferably, R is hydrogen or C1-C6 alkyl; R _{R 1} comprises hydrogen, deuterium, 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 metacyclic, substituted 3-7 metacyclic, C6-C10 aryl, substituted C6-C10 aryl, 5-10 metacyclic, or substituted 5-10 heteroaryl; preferably, R ₁ is hydrogen or C1-C6 alkyl; more preferably, R ₁ comprises C1-C6 alkyl; R _{R 2} includes hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxylic acid, 3-7 heterocyclic group, substituted 3-7 heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 heteroaryl, or substituted 5-10 heteroaryl; preferably, R ₂ is hydrogen, halogen or C1-C6 alkyl; more preferably, R ₂ is halogen; R _a and R _{R and R} are each independently hydrogen, deuterium, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, 3-7 heterocyclic groups, substituted 3-7 heterocyclic groups, C6-C10 aryl, substituted C6-C10 aryl, 5-10 heteroaryl, or substituted 5-10 heteroaryl; preferably, _R and _R are each independently hydrogen, C1-C6 alkyl, halogen-C1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, or C6-C10 aryl; or, _R , R _{R a} and the carbon atom to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3-7 heterocycloalkyl group, or a substituted 3-7 heterocycloalkyl group; preferably, R _a , R _b and the carbon atom to which they are attached constitute a C3-C8 cycloalkyl group; the chiral carbon atom at position 1 is in the R or S configuration; and m is 0 or 1. The ligand-dendrimer derivative conjugate or a pharmaceutically acceptable salt or solvent thereof as described in any one of claims 1 or 14, wherein the structural formula d comprises the following: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 , 、 、 、 、 , , or A linker-drug compound or a pharmaceutically acceptable salt or solvent thereof has a structure shown in Formula III below: III where L contains 、 、 、 、 、 or , wherein the position on the right side indicated by the wavy line is connected to the position marked 2; R comprises hydrogen, deuterium, 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, or substituted 5-10 heteroaryl; R _a comprises hydrogen, deuterium, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, 3-7 membered heterocyclic group, substituted 3-7 membered heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, or substituted 5-10 membered heteroaryl; R _b comprises hydrogen, deuterium, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, 3-7 membered heterocyclic group, substituted 3-7 membered heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, or substituted 5-10 membered heteroaryl; or, R _a , R _b and the carbon atom to which they are attached constitute a C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, 3-7 membered heterocyclic group, or substituted 3-7 membered heterocyclic group; L ₃ is present or absent; when L ₃ is present, L ₃ is , o is an integer from 1 to 10; the chiral carbon atom at position 1, position 2 or position 3 is in R or S configuration; Ac is a hydrophilic structural unit; m comprises 0 or 1; preferably, the linker-drug compound or a pharmaceutically acceptable salt or solvent thereof is combined with the ligand Ab to form a ligand-dendrimer derivative conjugate of formula I or formula II as described in any one of claims 1-19. The linker-drug compound or a pharmaceutically acceptable salt or solvent thereof as described in claim 20, wherein Ac has a structure represented by the following formula B: B wherein: Z comprises a hydrophilic structure of carboxyl, phosphate, polyphosphate, phosphite, sulfonic acid, sulfinic acid, or polyethylene glycol (PEG); Y' is an optional unit linking the amino group to Z; the position indicated by the wavy line is the attachment site. The linker-drug compound or pharmaceutically acceptable salt or solvent thereof as described in claim 20, wherein the Ac comprises glycine, ( D/L ) alanine, ( D/L ) leucine, ( D/L ) isoleucine, ( D/L ) valine, ( D/L ) phenylalanine, ( D/L ) proline, ( D/L ) tryptophan, ( D/L ) serine, ( D/L ) tyrosine, ( D/L ) cysteine, ( D/L ) cystine, ( D/L ) arginine, ( D/L ) histidine, ( D/L ) methionine, (D/L) asparagine, (D/L) glutamine, ( D/L ) threonine, ( D/L ) aspartic acid, ( D/L) )glutamine, a natural or unnatural amino acid derivative, or the following structure: 、 、 、 、 、 、 、 or . The linker-drug compound or pharmaceutically acceptable salt or solvent thereof as described in claim 20, wherein Ac comprises a hydrophilic structure of glycine, phosphate, ( D/L ) glutamine, or polyethylene glycol. The linker-drug compound or a pharmaceutically acceptable salt or solvent thereof as described in claim 20, wherein the linker-drug compound comprises the following structure or an isomer thereof: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or ; where o is an integer from 1 to 10. A method for preparing a ligand-camptothecin derivative conjugate represented by Formula I or Formula II , or a pharmaceutically acceptable salt or solvent thereof, wherein the method comprises the following steps: coupling the reduced antibody or antigen-binding fragment thereof with the linker-drug compound to obtain a ligand-camptothecin derivative conjugate represented by Formula I or Formula II ; , , the chiral carbon atoms at position 1, position 2, or position 3 have absolute chirality of R- or S-configuration; Ab, L, L ₁₁ , L ₁₂ , L ₁₃ , L ₂ , L 3 , L ₄ , L ₅ , X, R, R ₁ , R ₂ , _n1 , n2, or n3 are as described in any one of claims 1 to 24. The ligand-dendrogenin derivative conjugate or a pharmaceutically acceptable salt or solvent thereof as described in any one of claim 1 or 14, or the method as described in claim 25, wherein the ligand-dendrogenin derivative conjugate or a pharmaceutically acceptable salt or solvent thereof comprises the following structure or its succinimide ring-opened structure or an isomer thereof. 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 ; 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、. 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or wherein 5103F3-BSM is an antibody or an antigen-binding fragment thereof that has binding affinity for human Claudin18.2; n1, n2, and n3 are each independently selected from any integer or decimal number between 0 and 10, n1, n2, and n3 are not simultaneously 0, and 1 ≤ n1 + n2 + n3 ≤ 10. The ligand-dendrimer derivative conjugate or pharmaceutically acceptable salt or solvent complex thereof as described in any one of claims 1, 14, or 20, or the linker-drug compound or pharmaceutically acceptable salt or solvent complex thereof as described in any one of claims 20-24, wherein the pharmaceutically acceptable salts include sodium salts, potassium salts, calcium salts, calcium salts or magnesium salts formed with the acidic functional groups in the structural formula and acetate salts, trifluoroacetic acid salts formed with the basic functional groups in the structure. acid salts, citrates, oxalates, tartrates, bromides, iodides, malates, nitrates, chlorides, iodates, iodine salts, or magnesium salts, and acetates, trifluoroacetates, citrates, oxalates, tartrates, malates, nitrates, chlorides, bromides, iodides, sulfates, hydrosulfates, phosphates, lactates, oleates, ascorbic acid, salicylate, formates, glutamine salts, methanesulfonates, ethanesulfonates, benzenesulfonates, or p-toluenesulfonates. A pharmaceutical composition comprising the ligand-dendrimer derivative conjugate of any one of claims 1-19 or 26-27, or a pharmaceutically acceptable salt or solvent thereof, or the linker-drug compound of any one of claims 20-24, or a pharmaceutically acceptable salt or solvent thereof, and optionally a pharmaceutically acceptable carrier. A pharmaceutical preparation comprising a ligand-dendrimer derivative conjugate compound as described in any one of claims 1-19 or 26-28, or a pharmaceutically acceptable salt or solvent thereof, or a linker-drug compound as described in any one of claims 20-24, or a pharmaceutically acceptable salt or solvent thereof. A ligand-dendritic alkaloid derivative conjugate as described in any one of claims 1-19 or 26-27, or a pharmaceutically acceptable salt or solvent thereof, a linker-drug compound as described in any one of claims 20-24, or a pharmaceutically acceptable salt or solvent thereof, a pharmaceutical composition as described in claim 28, or a pharmaceutical formulation as described in claim 29, in the preparation of a drug for treating or preventing cancer or tumors; Alternatively, a ligand-dendritic alkaloid derivative conjugate as described in any one of claims 1-19 or 26-27, or a pharmaceutically acceptable salt or solvent thereof, a linker-drug compound as described in any one of claims 20-24, or a pharmaceutically acceptable salt or solvent thereof, a pharmaceutical composition as described in claim 27, or a pharmaceutical formulation as described in claim 29 for use in treating or preventing cancer or tumors; preferably, the cancer or tumor expresses Claudin 18.2; More preferably, the cancer or tumor comprises adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urothelial carcinoma, bladder cancer, hepatocellular carcinoma, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, triple-negative breast cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, neuroglioma, neuroblastoma, neuroglioblastoma multiforme, solid or blood tumors such as sarcoma, lymphoma and leukemia. Use of a preventively or therapeutically effective amount of a ligand-dendrimer derivative conjugate compound as described in any one of claims 1 or 14, or a pharmaceutically acceptable salt or solvent thereof, a linker-drug compound as described in claim 20, or a pharmaceutically acceptable salt or solvent thereof, a pharmaceutical composition as described in claim 28, or a pharmaceutical formulation as described in claim 29 in the manufacture of a medicament for treating or preventing cancer or tumors, comprising administering the compound to a subject in need thereof; Preferably, the cancer or tumor expresses Claudin18.2; More preferably, the cancer or tumor comprises adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urothelial carcinoma, bladder cancer, hepatocellular carcinoma, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, triple-negative breast cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, neuroglioma, neuroblastoma, neuroglioblastoma multiforme, solid or blood tumors such as sarcoma, lymphoma and leukemia.