Load-linker conjugates, antibody drug conjugates, and methods of making and using the same

Abstract

The present disclosure relates to the technical field of biological medicine, in particular to a payload-linker conjugate, an antibody drug conjugate and a preparation method and application thereof. The present disclosure has the following advantages: the present disclosure provides a payload-linker conjugate and an antibody drug conjugate, and the payload-linker conjugate or the antibody drug conjugate has good antitumor effect.

Description

Technical Field

[0001] This disclosure relates to the field of biomedical technology, specifically to load-linker conjugates, antibody-drug conjugates, their preparation methods and applications. Background Technology

[0002] Topoisomerase inhibitors (Top-1) are ribozymes that play a central role in key cellular processes such as DNA replication, transcription, repair, and chromosome segregation. They relieve DNA supercoiling tension and maintain genome stability by reversibly cleaving and rejoining single-stranded DNA. In rapidly proliferating tumor cells, Top-1 expression and activity are typically significantly upregulated to meet their abnormally high DNA metabolic demands. Therefore, Top-1 has become an important and clinically validated molecular target for anti-tumor drug development.

[0003] Camptothecin is a compound derived from the camptotheca tree (Campanula spp.), a plant in the Davidiaceae family of China. Camptotheca acuminata The natural alkaloid isolated from *Camptothecin* is the first discovered Top-1 specific inhibitor with potent antitumor activity. Its mechanism of action is unique: camptothecin and its derivatives can stably bind to the Top1-DNA cleavage complex (Top1cc), forming a so-called "toxic complex" that prevents the rejoining of cleaved DNA single strands. When the replication fork encounters this "frozen" complex, it causes a fatal DNA double-strand break, thereby inducing cell cycle arrest and apoptosis, selectively killing proliferating tumor cells.

[0004] Since its discovery in the 1960s, camptothecin has attracted widespread attention due to its remarkable in vitro and in vivo antitumor activity. However, its original natural form has some serious defects that limit its clinical application, including: 1) the lactone ring is easily converted into a carboxylate form with significantly reduced activity under physiological pH conditions; 2) poor water solubility; and 3) strong toxic side effects (such as bone marrow suppression and diarrhea). To overcome these limitations, researchers have developed a series of semi-synthetic camptothecin derivatives by systematically modifying its chemical structure (especially the A ring, B ring, and hydroxyl group at position 20).

[0005] Irinotecan (CPT-11) and topotecan are representative of the first-generation camptothecin drugs successfully applied in clinical practice, and have been approved for the treatment of various solid tumors such as colorectal cancer, ovarian cancer, and small cell lung cancer. However, existing clinically used camptothecin drugs still face many challenges, such as: the development of tumor resistance (involving mechanisms such as drug efflux pumps, Top1 mutations, and enhanced DNA repair), narrow therapeutic windows, complex management of adverse reactions, and the need to improve efficacy against certain refractory cancers. In addition, there is still room for improvement in the metabolic stability, tissue distribution characteristics, and ability to maintain blood drug concentrations.

[0006] Antibody-drug conjugates (ADCs) are conjugates formed by linking monoclonal antibodies targeting specific antigens with cytotoxic drugs via linkers. Through this conjugation, ADCs combine the targeting ability of monoclonal antibodies with the potent killing effect of cytotoxic drugs. Utilizing the specific binding of the antigen and antibody, cytotoxic drugs are accurately delivered to target cells. Through endocytosis and the bystander effect, they achieve specific killing of tumor cells and adjacent tumor stromal cells, tumor blood vessels, and other cells supporting tumor growth. This significantly reduces drug toxicity, expands the therapeutic window, and improves pharmacokinetic properties. It overcomes the disadvantage of monoclonal antibodies being ineffective when used alone and avoids the unfortunate situation where cytotoxic drugs are too toxic to be effective. Because ADCs have proven to be a promising treatment for hematologic malignancies and solid tumors, they have become a hot topic in antibody drug development both domestically and internationally.

[0007] Therefore, there is an urgent need in the field to develop next-generation camptothecin-based Top1 inhibitors, and it is essential to construct antibody-drug conjugates with good inhibitory effects on tumor cells. This disclosure provides a series of antibody-drug conjugates containing novel topoisomerase-1 (TOP-1) inhibitor loadings, which exhibit significant antitumor activity and have potential application value in tumor treatment. Summary of the Invention

[0008] This disclosure aims to provide load-linker conjugates, antibody-drug conjugates, their preparation methods, and applications.

[0009] To achieve the above-mentioned technical objectives, the technical solution adopted in this disclosure is as follows: On the one hand, this disclosure provides a load-linker conjugate or a pharmaceutically acceptable salt, stereoisomer, optical isomer, deuterated derivative, solvate, or prodrug thereof, said load-linker conjugate having a structure as shown in formula (I): (I) In the formula, L1 is a divalent structural unit that connects the cysteine ​​side chain group of the antibody to the L2 group; L2 is a bivalent structural unit connecting L1 and L3; L3 is a unit of n1 amino acids that can be digested by enzymes; A is selected from H and C. 1-6 Alkyl, C 3-6 cycloalkyl, C 1-6 Halogenated alkyl or C 1-6 Alkoxy; X is selected from H and C. 1-6 Alkyl, C 3-6 cycloalkyl, C 1-6 Halogenated alkyl or C 1-6 Alkoxy; R4 is selected from H and C. 1-6 Alkyl, C 3-6 cycloalkyl, C 1-6 Halogenated alkyl or C 1-6 Alkoxy; D is a TOP-1 inhibitor loading drug containing a hydroxylamine group, which forms -NX-CH2-NO- through the amide of L3 and the loaded hydroxylamine; n1 is an integer selected from 1 to 15; n2 is an integer selected from 0 to 10.

[0010] In some embodiments, the load-connector coupling has the structure shown in formula (II): (II); In formula (II), L1, L2, L3, A, X, R4, D, n1, and n2 are each defined in the same way as in formula (I); R1, R2, and R3 are each independently selected from H, halogens, and C. 1-6 Alkyl, C 3-6 cycloalkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl; or any two substituents from R1, R2, and R3 together form a methylene dioxy substituent group (-O-CH2-O-).

[0011] In some implementations, L1 is a divalent structural unit that connects the antibody thiol group and the L2 group.

[0012] In some embodiments, L1 is selected from C1-C8 alkylene groups, polyethylene glycol (PEG) fragments, polypeptide fragments, or divalent structural units containing functional groups that can be linked to antibody cysteine ​​side chain groups.

[0013] In some embodiments, L1 is selected from divalent structural units of maleimide, bromoacetyl, 2-bromomethylpyridine-4-carboxyl, or 2-bromomethyl-3-oxo-dihydroquinoxaline-6-carboxyl.

[0014] In some implementations, L1 is selected from , , and ; The position shown indicates that it is linked to the cysteine ​​side chain group of the antibody. The position shown indicates that it is connected to the L2 group.

[0015] n3 is selected from integers from 0 to 10.

[0016] In some implementations, n3 is selected from integers from 0 to 8.

[0017] In some implementations, n3 is selected from integers from 0 to 6.

[0018] In some implementations, n3 is selected from integers from 0 to 3.

[0019] In some implementations, n3 is selected from integers from 0 to 2.

[0020] In some implementations, n3 is selected from 0 or 1.

[0021] In some implementations, L1 is selected from , , and ; The position shown indicates that it is linked to the cysteine ​​side chain group of the antibody. The position shown indicates that it is connected to the L2 group.

[0022] In some embodiments, L2 is selected from C1-C8 alkylene groups, polyethylene glycol (PEG) fragments, polypeptide fragments, or divalent structural units containing functional groups that can be linked to the L1 group.

[0023] In some embodiments, L2 is selected from -C(=O)-C1-C8 alkylene groups, -C(=O)-CH2O-(CH2CH2O). 2-5 -CH2CH2NH-、-C(=O)-(CH2CH2O) 2-6 -CH2CH2-、-C(=O)-(CH2CH2O) 2-6 -CH2CH2NH- and -C(=O)-(CH2) 0-6 -NR5(CH2) 0-6 -, R5 is selected from -C1-C6 alkyl-carboxyl or -C1-C6 alkyl-amino.

[0024] In some embodiments, L2 is selected from -C(=O)-(CH2)5-, -C(=O)-CH2O-(CH2CH2O)3-CH2CH2NH-, -C(=O)-(CH2CH2O)2-CH2CH2-, -C(=O)-(CH2CH2O)2-CH2CH2NH- and -C(=O)-(CH2CH2O)4-CH2CH2NH-, .

[0025] In some embodiments, L2 is selected from -C(=O)-C1-C8 alkylene groups and -C(=O)-(CH2CH2O). 2-6 -CH2CH2NH-、 .

[0026] In some implementations, L1-L2 are selected from... , , , , , and “ The indicated position represents the link to the antibody's thiol group; The position indicated is linked to an amino acid group.

[0027] In some embodiments, L3 is a peptide residue consisting of 1-8 amino acids; said amino acids are selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit) and aspartic acid (Asn).

[0028] In some embodiments, L3 is selected from 2-5 peptide residues composed of amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit), and aspartic acid (Asn).

[0029] In some embodiments, L3 is selected from Val-Cit, Val-Ala, Gly-Val-Ala, Gly-Val-Ala-Gly, Gly-Lys, Gly-Gly-Lys, Gly-Gly-Lys-Gly , Val-Ala, Ala-Ala-Asn, Gly-Leu, Gly-Gly-Leu, Gly-Gly-Leu-Gly, Gly-Phe, Gly-Gly-Phe and Gly-Gly-Phe-Gly.

[0030] In some embodiments, L3 is selected from two or three peptide residues composed of amino acids selected from glycine, valine, alanine, lysine, and citrulline.

[0031] In some implementations, L3 is selected from , and Its carbonyl end is connected to -NH-, and the other end is connected to L2.

[0032] In some implementation schemes, A is selected from H and C. 1-5 Alkyl, C 3-5 cycloalkyl, C 1-5 Halogenated alkyl or C 1-5 Alkyl group.

[0033] In some implementation schemes, A is selected from H and C. 1-5 Alkyl, C 1-5 Halogenated alkyl or C 1-5 Alkyl group.

[0034] In some implementation schemes, A is selected from H and C. 1-4 Alkyl, C 1-4 Halogenated alkyl or C 1-4 Alkyl group.

[0035] In some implementation schemes, A is selected from H and C. 1-3 Alkyl, C 1-3 Halogenated alkyl or C 1-3 Alkyl group.

[0036] In some implementation schemes, A is selected from H and C. 1-3 alkyl.

[0037] In some implementations, A is selected from H, methyl, propyl, and isopropyl.

[0038] In some implementations, A is selected from H, methyl, and isopropyl.

[0039] In some implementation schemes, X is selected from H, C 1-5 Alkyl, C 3-5 cycloalkyl, C 1-5 Halogenated alkyl or C 1-5 Alkyl group.

[0040] In some implementation schemes, X is selected from H, C 1-5 Alkyl, C 1-5 Halogenated alkyl or C 1-5 Alkyl group.

[0041] In some implementation schemes, X is selected from H, C 1-4 Alkyl, C1-4 Halogenated alkyl or C 1-4 Alkyl group.

[0042] In some implementation schemes, X is selected from H, C 1-3 Alkyl, C 1-3 Halogenated alkyl or C 1-3 Alkyl group.

[0043] In some implementation schemes, X is selected from H, C 1-3 alkyl.

[0044] In some implementations, X is selected from H or methyl.

[0045] In some implementations, X is H.

[0046] In some implementations, R4 is selected from H, C 1-5 Alkyl, C 3-5 cycloalkyl, C 1-5 Halogenated alkyl or C 1-5 Alkyl group.

[0047] In some implementations, R4 is selected from H, C 1-5 Alkyl, C 1-5 Halogenated alkyl or C 1-5 Alkyl group.

[0048] In some implementations, R4 is selected from H, C 1-4 Alkyl, C 1-4 Halogenated alkyl or C 1-4 Alkyl group.

[0049] In some implementations, R4 is selected from H, C 1-3 Alkyl, C 1-3 Halogenated alkyl or C 1-3 Alkyl group.

[0050] In some implementations, R4 is selected from H, C 1-3 alkyl.

[0051] In some implementations, R4 is selected from methyl or ethyl.

[0052] In some implementations, R4 is methyl.

[0053] In some implementation schemes, R1, R2, and R3 are each independently selected from H, halogens, and C. 1-5 Alkyl, C 3-5 cycloalkyl, C 1-5 Haloalkyl, C 1-5 Alkoxy, C 1-5 Hydroxyalkyl; or any two substituents from R1, R2, and R3 together form a methylene dioxy substituent group (-O-CH2-O-).

[0054] In some implementation schemes, R1, R2, and R3 are each independently selected from H, halogens, and C. 1-3 Alkyl, C 3-4 cycloalkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy, C 1-3 Hydroxyalkyl; or any two substituents from R1, R2, and R3 together form a methylene dioxy substituent group (-O-CH2-O-).

[0055] In some implementation schemes, R1, R2, and R3 are each independently selected from H, fluorine, chlorine, bromine, and C. 1-3 Alkyl, C 3-4 cycloalkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy, C 1-3 Hydroxyalkyl; or any two substituents from R1, R2, and R3 together form a methylene dioxy substituent group (-O-CH2-O-).

[0056] In some implementation schemes, R1, R2, and R3 are each independently selected from H, fluorine, chlorine, bromine, and C. 1-3 Alkyl group; or any two substituents from R1, R2, and R3 together form a methylene dioxy substituent group (-O-CH2-O-).

[0057] In some embodiments, R1, R2, and R3 are each independently selected from H, fluorine, chlorine, methyl, and ethyl; or any two substituents from R1, R2, and R3 together form a methylene dioxy substituent group (-O-CH2-O-).

[0058] In some embodiments, R1, R2, and R3 are each independently selected from H, fluorine, methyl, and ethyl; or any two substituents from R1, R2, and R3 together form a methylene dioxy substituent group (-O-CH2-O-).

[0059] In some embodiments, R1, R2, and R3 are each independently selected from H, fluorine, and methyl; or any two substituents in R1, R2, and R3 together form a methylene dioxy substituent group (-O-CH2-O-).

[0060] In some implementations, n1 is selected from integers from 1 to 10.

[0061] In some implementations, n1 is selected from integers from 1 to 8.

[0062] In some implementations, n1 is selected from integers from 1 to 5.

[0063] In some implementations, n1 is selected from integers from 2 to 5.

[0064] In some implementations, n1 is 2 or 3.

[0065] In some implementations, n2 is selected from integers from 0 to 8.

[0066] In some implementations, n2 is selected from integers from 0 to 6.

[0067] In some implementations, n2 is selected from integers from 0 to 5.

[0068] In some implementations, n2 is selected from integers from 1 to 5.

[0069] In some implementations, n2 is selected from integers from 1 to 4.

[0070] In some implementations, n2 is selected from integers from 1 to 3.

[0071] In some embodiments, the load-linker conjugate is selected from compounds in a form that can be linked to antibody cysteine ​​side chain groups: .

[0072] On the other hand, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt, stereoisomer, optical isomer, deuterated derivative, solvate, or prodrug thereof, the structure of which is shown in formula (III): (III) In equation (Ⅲ), Ab is a target connector, which is a structure that can target and bind to the lesion site; S is a cysteine ​​side chain group of the antibody or its binding fragment; n is any number between 1 and 20; L1, L2, L3, A, X, R4, D, n1, and n2 are each defined in the same formula (I).

[0073] In some embodiments, the structure of the antibody-drug conjugate is shown in formula (Ⅳ): (IV) In equation (Ⅳ), Ab is a target connector, which is a structure that can target and bind to the lesion site; S is a cysteine ​​side chain group of the antibody or its binding fragment; n is any number between 1 and 20; L1, L2, L3, A, X, R4, D, n1, and n2 are each defined in the same way as in formula (I); R1, R2, and R3 are each defined in the same way as in formula (II).

[0074] In some embodiments, the structure of the antibody-drug conjugate is shown in formula (V): (V) Ab is a target connector, which is a structure that can target and bind to the lesion site; S is a cysteine ​​side chain group of the antibody or its binding fragment; n is any number between 1 and 20; L1, L2, L3, A, X, R4, and D are each defined in the same way as in formula (I); R1, R2, and R3 are each defined in the same way as in formula (II).

[0075] In some embodiments, the antibody-drug conjugate is designated as ADC, and the antibody-drug conjugate is selected from the following compounds: in, Ab is a target connector, which is a structure that can target and bind to the lesion site; S is a cysteine ​​side chain group of the antibody or its binding fragment; n is any number between 1 and 20.

[0076] In some embodiments, the antibody-drug conjugate is selected from the following compounds: in, S is a cysteine ​​side chain group of the antibody or its binding fragment; n is any number between 1 and 20.

[0077] In some embodiments, the antibody-drug conjugate is selected from the following compounds: In some embodiments, the antibody-drug conjugate exhibits good inhibitory activity against colorectal cancer cells GP2D, HT55, NCI-H716, SW403, LoVo, gastric cancer cells KATO-III, SNU16, or lung cancer cells NCI-H292.

[0078] In some implementations, the Ab is selected from antibodies or their binding fragments, proteins, small polypeptides, glycopeptides, mimic peptides, or nucleic acid oligonucleotide aptamers.

[0079] In some implementations, the Ab is an antibody or a binding fragment thereof that targets a tumor antigen.

[0080] In some embodiments, the antibody is selected from anti-HS627 antibody, anti-Trop2 antibody, anti-CDH17 antibody, anti-HER3 antibody, anti-B7H3 antibody, anti-HER2 antibody, anti-Nectin-4 antibody, anti-CD20 antibody, anti-ROR1 antibody, or anti-CEACAM5 antibody.

[0081] In some embodiments, the antibody is selected from anti-HS627 antibody, anti-Trop2 antibody, and anti-CDH17 antibody.

[0082] In some implementations, n is any number between 1 and 15.

[0083] In some implementations, n is any number between 1 and 12.

[0084] In some implementations, n is any number between 1 and 10.

[0085] In some implementations, n is any number between 1 and 8.

[0086] In some implementations, n is any number between 2 and 8.

[0087] In some implementations, the antibody-drug conjugates of this disclosure can maintain or enhance their targeted antitumor efficacy while improving their pharmaceutical properties (such as chemical stability, solubility, and metabolic stability), reducing toxic side effects, and overcoming or delaying the development of drug resistance.

[0088] On the other hand, this disclosure provides pharmaceutical compositions comprising, as described above, a load-linker conjugate or a pharmaceutically acceptable salt, stereoisomer, optical isomer, deuterated derivative, solvate or prodrug thereof, or an antibody-drug conjugate or a pharmaceutically acceptable salt, stereoisomer, optical isomer, deuterated derivative, solvate or prodrug thereof, and optionally a pharmaceutically acceptable carrier.

[0089] On the other hand, this disclosure provides the use of the aforementioned load-linker conjugate or its pharmaceutically acceptable salt, stereoisomer, optical isomer, deuterated derivative, solvate or prodrug, the aforementioned antibody-drug conjugate or its pharmaceutically acceptable salt, stereoisomer, optical isomer, deuterated derivative, solvate or prodrug, or the aforementioned pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of tumors.

[0090] In some implementations, the tumor is selected from breast cancer, lung cancer, colorectal cancer, esophageal cancer, gastric cancer, kidney cancer, ovarian cancer, cervical cancer, bladder cancer, head and neck cancer, pancreatic cancer, liver cancer, urethral cancer, prostate cancer, squamous cell carcinoma, peritoneal cancer, salivary gland cancer, thyroid cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.

[0091] In some implementations, the tumor is selected from breast cancer, lung cancer, colorectal cancer, esophageal cancer, gastric cancer, kidney cancer, ovarian cancer, cervical cancer, bladder cancer, head and neck cancer, pancreatic cancer, or liver cancer.

[0092] In some implementations, the tumor is colorectal cancer or gastric cancer.

[0093] The antibody-drug conjugate disclosed herein targets the corresponding antigen on tumor cells, enters the tumor cells via endocytosis, and releases the drug 7-camptothecin (containing a hydroxylamine substituent) or its hydroxyacetyl derivative to kill the tumor cells, leading to tumor cell apoptosis.

[0094] The antibody-drug conjugate (ADC) disclosed herein, containing a novel topoisomerase-1 (TOP-1) inhibitor load, can be applied in the field of anti-tumor therapy.

[0095] This disclosure has the following advantages: (1) This disclosure provides a load-linker conjugate suitable for constructing antibody-drug conjugates.

[0096] (2) This disclosure provides a load-linker conjugate and an antibody-drug conjugate, wherein the load-linker conjugate or the antibody-drug conjugate has good anti-tumor activity.

[0097] (3) This disclosure provides payload-linker conjugates and antibody-drug conjugates, which are expected to provide new strategies for cancer treatment. Attached Figure Description

[0098] To more clearly illustrate the technical solutions in the specific embodiments of this disclosure or the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. The accompanying drawings are incorporated in and constitute a part of this specification, illustrating embodiments consistent with this specification, and are used together with the specification to explain the principles of this specification.

[0099] Figure 1 The RP-HPLC-MS test results of antibody-drug conjugate ACD234141 are shown.

[0100] Figure 2 The RP-HPLC-MS test results of the antibody-drug conjugate ACD234144 are shown.

[0101] Figure 3 The RP-HPLC-MS test results of antibody-drug conjugate ACD251541 are shown.

[0102] Figure 4 The RP-HPLC-MS test results of antibody-drug conjugate ACP227541 are shown.

[0103] Figure 5 The RP-HPLC-MS test results of antibody-drug conjugate ACP251541 are shown.

[0104] Figure 6 The tumor suppression curve of the GP2D colorectal cancer model is shown.

[0105] Figure 7 The tumor inhibition curve of the HT55 colorectal cancer model is shown.

[0106] Figure 8 The tumor inhibition curve of the NCI-H716 colorectal cancer model is shown.

[0107] Figure 9 The tumor inhibition curve of the SW403 colorectal cancer model is shown.

[0108] Figure 10 The tumor inhibition curve of the LoVo colorectal cancer model is shown.

[0109] Figure 11 The tumor inhibition curve of the KATO III gastric cancer model is shown.

[0110] Figure 12 The tumor inhibition curve of the SNU16 gastric cancer model is shown. Detailed Implementation

[0111] Definitions and Explanations To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. In this disclosure, unless otherwise stated, the scientific and technical terms used herein have meanings commonly understood by those skilled in the art. Furthermore, the cell and tissue culture, microbiology-related terms, and laboratory procedures used herein are all widely used terms and routine procedures in their respective fields. Meanwhile, to better understand this disclosure, definitions and explanations of relevant terms are provided below. It should be understood that this disclosure is not limited to specific methods, reagents, compounds, compositions, or biological systems, and variations thereof are certainly possible. It should also be understood that the terminology used in this application is for describing specific embodiments only and is not intended to be limiting.

[0112] Unless otherwise expressly stated, the terms “a,” “an,” and “the” as used in this specification and the appended claims cover one or more types.

[0113] As used herein, the terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0114] In the description herein, references to “some embodiments,” “some implementations,” or “some implementation schemes” describe a subset of all possible embodiments. However, it is understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0115] As used herein, the term “substitution” means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, provided that the valence state of the particular atom is normal and the substituted compound is stable. The terms “optional substitution” or “optionally substituted” mean that a substance may or may not be substituted, and unless otherwise specified, the type and number of substituents may be arbitrary on a chemically feasible basis.

[0116] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition is independent in each case. Thus, for example, if a group is substituted by 0-2 Rs, the group can optionally be substituted by at most two Rs, and the Rs in each case have independent options. Furthermore, combinations of substituents and / or their variants are only permitted if such combinations produce a stable compound.

[0117] In any embodiment, any or all hydrogen atoms present in the compound, or hydrogen atoms in a specific group or portion of the compound, may be replaced by deuterium or tritium. One to a maximum number of hydrogen atoms present in the compound may be replaced by deuterium. One to a maximum number of hydrogen atoms present in any group of the general formula compound or a specific compound may be replaced by deuterium. For example, when a group is described as ethyl, the ethyl group may be C2H5 or a C2H5 in which x (1 to 5) hydrogen atoms are replaced by deuterium, such as C2D. x H 5-x When a group is described as a deuterated ethyl group, the deuterated ethyl group can be a C2H5 with x (1 to 5) hydrogen atoms replaced by deuterium, such as C2D. x H 5-xThe stable deuterated derivatives described in this disclosure are preferably stable deuterated isotope derivatives obtained by replacing any deuterated hydrogen atom in each formula with 1 to a maximum number (e.g., 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, etc.) of deuterium atoms.

[0118] The terms "tautomer" or "tautomer form" refer to isomers with different functional groups that are in dynamic equilibrium at room temperature and can rapidly interconvert to each other. A "tautomer" or "tautomer form" is one of two or more structural isomers that exist in equilibrium and readily transform from one isomer form to another. This transformation results in the formal migration of hydrogen atoms, accompanied by the conversion of adjacent conjugated double bonds. Tautomers exist as a mixture of tautomer groups in solution. In solutions where tautomerization is possible, chemical equilibrium of the tautomers will be reached. The exact proportions of tautomers depend on several factors, including temperature, solvent, and pH conditions. The concept of tautomers that can interconvert through tautomerization is called tautomerism.

[0119] When this specification describes a compound that is readily tautomerizable, but only one of its tautomers is described, it should be understood that all tautomers are included as part of the chemical meaning described. It should be understood that when a compound has tautomeric forms, it is intended to include all tautomeric forms, and the naming of the compound does not exclude any tautomeric form.

[0120] Of the various possible types of tautomerism, two are typically observed. In keto-enol tautomerism, both electrons and hydrogen atoms move simultaneously.

[0121] Common tautomer pairs are: keto-enol, amide-nitrile, lactam-lactam, amide-imine tautomer in heterocycles, imine-enamine, and enamine-enamine.

[0122] Based on their structure, the compounds disclosed herein can exist in various stable isotopic forms. These forms include those in which one or more hydrogen atoms have been replaced by deuterium atoms, and those in which one or more nitrogen atoms have been replaced by deuterium atoms. 15 Those that are replaced by N atoms, or one or more of the carbon, fluorine, chlorine, bromine, sulfur, or oxygen atoms, have been replaced by stable isotopes of their respective original atoms.

[0123] According to this disclosure, some compounds and salts can exist in different crystalline forms (polymorphs) within the scope of this disclosure.

[0124] The term "solvent" as used in this disclosure refers to a complex formed by the compound of this disclosure with a solvent. These complexes either react in the solvent or precipitate or crystallize from the solvent. For example, a complex formed with water is called a "hydrate". Solvents of the compounds represented by formula (I) of this disclosure are within the scope of this disclosure.

[0125] This disclosure includes prodrugs of the compounds described herein. A "prodrug" refers to a precursor or derivative of a pharmaceutically active substance that exhibits lower cytotoxicity to tumor cells compared to the parent drug and can be activated by enzymatic action or converted into a more active parent form. Prodrugs of this disclosure include, but are not limited to, phosphate (ester)-containing prodrugs, thiophosphate (ester)-containing prodrugs, sulfate (ester)-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, prodrugs containing optionally substituted phenoxyacetamide or phenylacetamide, 5-fluorocytosine, and other 5-fluorouridine prodrugs capable of being converted into more active cytotoxic free drugs. Examples of cytotoxic drugs that can be derived into prodrug forms for use in this disclosure include, but are not limited to, the chemotherapeutic agents described above.

[0126] In this disclosure, “ "and" "Represents the absolute configuration of the center of a solid." “ in "" refers to the point where chemical bonds are joined.

[0127] When the ring appears " ", and if the connection location is uncertain, it means the connection point is located at " "Any atom on the single ring, as long as the valence allows."

[0128] The term "alkyl" refers to a chain-like (straight-chain or branched) saturated aliphatic hydrocarbon group. The term "alkyl" can refer to a straight-chain or branched alkyl group containing 1 to 10 carbon atoms. 1-10 Alkyl groups, preferably alkyl groups containing 1 to 6 carbon atoms (C 1-6Alkyl groups. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and various branched isomers thereof. More preferably are lower alkyl groups containing 1 to 3 carbon atoms (C... 1-3 Alkyl groups, including methyl, ethyl, n-propyl, isopropyl, etc., are used in non-limiting embodiments. Alkyl groups may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups described in this application.

[0129] The amino acid sequences disclosed herein contain conventional single-letter or three-letter codes for naturally occurring amino acids, as well as generally accepted three-letter codes for other amino acids, such as Gly (α-aminoacetic acid) or G (glycine).

[0130] An amino acid is a molecule that contains both amino and carboxyl functional groups. In α-amino acids, the amino and carboxyl groups are attached to the same carbon atom (α-carbon). The α-carbon may also have one or two additional organic substituents. Amino acids include L and D isomers and racemic mixtures.

[0131] The term "antibody-drug conjugate (ADC)" refers to a small molecule drug with biological activity linked to an antibody via a chemical link. The antibody then acts as a carrier to target and deliver the small molecule drug to the target cell.

[0132] The term "antibody" as used in this disclosure refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain differ, thus their antigenicity also differs.

[0133] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. The antibody fragment retains at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, the antibody fragment retains at least 10% of the parent antibody's binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity to the target. Examples of antigen-binding fragments include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies.

[0134] Typically, the linker drug conjugates of this disclosure, or their tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, or their pharmaceutically acceptable salts, solvates, prodrugs, or antibody drug conjugates, can be formulated with one or more pharmaceutical carriers into suitable dosage forms for administration. These dosage forms are suitable for oral, rectal, topical, intraoral, and other non-gastrointestinal administration (e.g., subcutaneous, intramuscular, intravenous, etc.). For example, suitable dosage forms for oral administration include capsules, tablets, granules, and syrups. The compounds of this disclosure contained in these formulations can be solid powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; water-in-oil or oil-in-water emulsions, etc. The above dosage forms can be prepared from the active compound and one or more carriers or excipients using conventional pharmaceutical methods. The carriers described above need to be compatible with the active compound or other excipients. For solid dosage forms, commonly used non-toxic carriers include, but are not limited to, mannitol, lactose, starch, magnesium stearate, cellulose, glucose, and sucrose. Carriers used in liquid formulations include water, physiological saline, glucose solution, ethylene glycol, and polyethylene glycol. The active compound can form a solution or suspension with these carriers.

[0135] The pharmaceutical compositions disclosed herein are formulated, quantified, and administered in accordance with medical practice guidelines.

[0136] The "therapeutic effective dose" is determined by factors such as the specific disease to be treated, the individual being treated, the cause of the disease, the drug's target, and the method of administration.

[0137] As used herein, “pharmaceuticalally acceptable carrier” means a non-toxic, inert, solid, semi-solid substance or liquid, diluent, encapsulating material or excipient or any type of excipient that is compatible with patients, preferably mammalian, more preferably human, and suitable for delivering an active agent to a target site without terminating the agent’s activity.

[0138] As used in this article, "patient" or "subject" refers to an animal, preferably a mammal, and more preferably a human. The term "mammal" refers to warm-blooded vertebrate mammals, including animals such as cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, rats, pigs, and humans.

[0139] As used in this article, the term "tumor" refers to any of the various types of malignant tumors, most of which invade surrounding tissues, can metastasize to several sites, and may recur after attempted resection, leading to the patient's death unless adequately treated. As used in this article, tumor formation includes cancer. Representative tumors include, for example, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, hepatocellular carcinoma and renal cell carcinoma, bladder cancer, colorectal cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, head cancer, kidney cancer, liver cancer, lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, and stomach cancer; leukemia, including non-acute and acute leukemia, such as acute myeloid leukemia, acute lymphoblastic leukemia, acute promyelocytic leukemia (APL), acute T-cell lymphoblastic leukemia, T-cell acute lymphoblastic leukemia (T-ALL), adult T-cell leukemia, basophilic leukemia, eosinophilic leukemia, granulocytic leukemia, hairy cell leukemia, leukopenic leukemia, lymphocytic leukemia, lymphocytic leukemia, megakaryocytic leukemia, microcytic leukemia, monocytic leukemia, neutrophilic leukemia, and stem cell leukemia; Benign and malignant lymphomas, particularly Burkitt lymphoma and non-Hodgkin's lymphoma; benign and malignant melanomas; myeloproliferative disorders; sarcomas, including Ewing sarcoma, angiosarcoma, Kaposi's sarcoma, liposarcoma, sarcoma, myoma, peripheral neuroepithelial tumor, synovial sarcoma, glioma, astrocytoma, oligodendroglioma, ependymoma, glioblastoma, neuroblastoma, ganglioneuroma, ganglioglioma, medulloblastoma, pineal cell carcinoma, hemangioma, meningeal sarcoma, neurofibroma, and schwannoma; colorectal cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, gastric cancer, liver cancer, colon cancer, melanoma; carcinosarcoma, Hodgkin's disease, nephroblastoma, and teratoma, etc., can be treated with one or more of the compounds disclosed herein.

[0140] The term "pharmaceutical composition" refers to a mixture of at least one compound that can be used in this disclosure with a pharmaceutically acceptable carrier. This pharmaceutical composition facilitates the administration of the compound to a patient or subject. Various techniques for administering compounds exist in the art, including but not limited to intravenous, oral, aerosol, parenteral, ocular, pulmonary, and topical administration. The term "pharmaceutical composition" may refer to, but is by no means limited to, compositions or formulations that allow for the effective distribution of the pharmaceutical agents provided in this disclosure, in a form suitable for administration to the physical site most optimal for their desired activity, such as systemic administration.

[0141] Example To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this disclosure, not all embodiments. The following is merely a further description of this disclosure, and the scope of protection of this disclosure is not limited thereto. In the embodiments and experimental examples of this disclosure, the technical means or methods, etc., not specifically described, are conventional technical means or methods in the art. Unless otherwise specified, the materials, reagents, experimental equipment, etc., used in the embodiments and experimental examples of this disclosure can all be obtained commercially.

[0142] Example 1: Synthesis of AK34141 Manufacturing process of AK34141: 1. AK34141-01: At room temperature (25℃), D2341 (1.0 g, 2.2 mmol), N,N-dimethylformamide (DMF, 20 mL), N,N-diisopropylethylamine (DIPEA, 852.98 mg, 6.6 mmol), and AK10246-06 (586.69 mg, 2.2 mmol) were added sequentially to a 50 mL single-necked flask, and the mixture was stirred at room temperature. After 2 h of reaction, HPLC monitoring showed that D2341 had reacted completely. The reaction solution was poured into a 5% citric acid aqueous solution (40 mL), stirred for 10 min, and filtered. The filtrate was extracted with dichloromethane (DCM, 20 mL × 3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation under reduced pressure to obtain a light yellow solid AK34141-01 (1.3 g, 1.9 mmol, yield 86%, purity 80%). LCMS: (M+1) + 684.24 (Calculated value: 683.69).

[0143] 2. AK34141-02: At 25°C, AK34141-01 (1.3 g, 1.9 mmol), dichloromethane (DCM, 13 mL), and hydrazine hydrate (1.32 g) were added sequentially to a 50 mL single-necked flask, and the mixture was stirred overnight at room temperature. Samples were taken and analyzed by HPLC and LC-MS to confirm the complete reaction of AK34141-01, at which point the reaction was terminated. Dichloromethane (20 mL × 3) and distilled water (20 mL) were added to the reaction solution for extraction. After the target product was no longer present in the organic phase, the aqueous phase was purified by reverse-phase column chromatography, eluting with a 0.1% trifluoroacetic acid / acetonitrile system. The product was lyophilized to obtain a pale yellow solid AK34141-02 (650 mg, yield 44.3%, purity 98%). LCMS: (M+1)+ 554.21 (Calculated value: 553.59). 1 H NMR (600 MHz, DMSO-d6) δ 8.84 (t, J=6.1 Hz, 1H), 8.16 (d, J=8.1 Hz, 1H), 8.07–8.02 (m, 3H), 7.88 (d, J=10.7 Hz, 1H), 7.31 (s,1H), 6.54 (s, 1H), 5.46–5.41 (m, 2H), 5.31–5.27 (m, 2H), 4.38 (dd, J=13.4,6.3 Hz, 1H), 4.28 (dd, J=13.3, 5.8 Hz, 1H), 3.85 (dd, J=11.4, 5.8 Hz, 1H),3.57 (s, 3H), 3.47–3.38 (m, 1H), 2.97 (t, J=7.9 Hz, 2H), 2.51 (s, 2H), 1.87(d, J=21.4, 7.3 Hz, 2H), 1.31 (d, J=7.0 Hz, 3H), 0.88 (t, J=7.3 Hz, 3H).

[0144] 3. AK34141-03: At room temperature (25℃), AK34141-02 (650 mg, 1.17 mmol), N,N-dimethylformamide (DMF, 7 mL), Fmoc-PEG-GV-OSu (633.1 mg, 1.17 mmol), and N,N-diisopropylethylamine (DIPEA, 250.7 mg, 1.9 mmol) were added sequentially to a 50 mL single-necked flask, and the mixture was stirred at room temperature for 2 h. Samples were taken and analyzed by HPLC and LC-MS to confirm the complete reaction of AK34141-02, at which point the reaction was terminated. Post-treatment: The reaction solution was diluted with 25 mL of dichloromethane, washed three times with 5% sodium chloride aqueous solution (15 mL × 3), and separated. The organic phase was dried over anhydrous sodium sulfate (5.0 g) for 0.5 h and concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal-phase preparative chromatography using a gradient elution system of dichloromethane / methanol. The target product was collected when the methanol content reached approximately 5.8%, and evaporated to dryness under reduced pressure to obtain AK34141-03 (800 mg, yield 73%, purity 95%). LCMS: (M+1) + : 1092.19 (Calculated value: 1091.52). 1HNMR (600 MHz, DMSO-d6) δ 8.28 (t, J=6.2 Hz, 1H), 8.17–8.05 (m, 3H), 7.91–7.82 (m, 3H), 7.66 (dd, J=17.7, 8.1 Hz, 3H), 7.39 (t, J=7.5 Hz, 2H), 7.36–7.27 (m,4H), 6.53 (s, 1H), 5.44 (s, 2H), 5.28 (s, 2H), 4.34–4.05 (m, 8H), 3.79–3.67(m, 2H), 3.59 (t, J=6.5 Hz, 2H), 3.54 (s, 3H), 3.47 (s, 3H), 3.49–3.42 (m,0H), 3.38 (dt, J=12.5, 6.9 Hz, 4H), 3.15 (dd, J=28.9, 5.4 Hz, 5H), 2.98–2.82(m, 2H), 2.37 (t, J=6.5 Hz, 2H), 1.87 (dp, J=21.4, 7.1 Hz, 2H), 1.75 (dp, J=13.6, 6.8 Hz, 1H), 1.16 (d, J=7.1 Hz, 3H), 0.88 (t, J=7.3 Hz, 3H), 0.66 (d, J=6.8 Hz, 6H).

[0145] 4. AK34141-04: At room temperature (25℃), AK34141-03 (600 mg, 0.54 mmol), N,N-dimethylformamide (DMF, 6 mL), and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 41.85 mg, 0.27 mmol) were added sequentially to a 50 mL single-necked flask, and the mixture was stirred at room temperature for 2 h. Samples were taken and analyzed by HPLC and LC-MS to confirm that the reaction of AK34141-03 was complete, and the reaction was terminated. Post-processing: The reaction solution was added dropwise to 60 mL of methyl tert-butyl ether, stirred for 30 min, and filtered to obtain a white solid AK34141-04 (350 mg, 0.40 mmol, yield 74%, purity 92%). LCMS: (M+1) + : 870.33, calculated value: 869.42. 1H NMR (600 MHz, DMSO-d6) δ 8.14 (dd, J=22.5, 8.6 Hz, 1H), 7.95 (s,2H), 7.87 (dd, J=17.3, 10.9 Hz, 1H), 7.33–7.26 (m, 1H), 5.44 (d, J=3.2 Hz,1H), 5.32 (s, 1H), 5.23 (s, 1H), 4.31–4.15 (m, 1H), 4.15–4.02 (m, 1H), 3.78–3.69 (m, 1H), 3.68–3.45 (m, 5H), 3.44 (t, J=5.9 Hz, 3H), 3.23 (t, J=5.8 Hz,1H), 3.08 (s, 2H), 3.02 (s, 1H), 2.92 (d, J=8.2 Hz, 1H), 2.89 (s, 5H), 2.73(s, 5H), 2.63 (s, 1H), 2.67–2.59 (m, 0H), 2.53–2.43 (m, 6H), 2.41–2.28 (m,1H), 1.89 (s, 1H), 1.90–1.80 (m, 1H), 1.70–1.55 (m, 4H), 1.17 (dd, J=10.4,6.9 Hz, 3H), 1.11 (s, 9H), 0.87 (q, J=7.3 Hz, 2H), 0.76–0.62 (m, 4H).

[0146] 5. AK34141: At room temperature (25℃), crude AK34141-04 (300 mg, 0.34 mmol) and N,N-dimethylformamide (DMF, 6 mL) were added to a 50 mL single-necked flask and stirred at room temperature for 10 min until dissolved. Linker41 (422.64 mg, 1.64 mmol) and N,N-diisopropylcarbodiimide (DIC, 69.39 mg, 0.54 mmol) were added sequentially, and the mixture was stirred at room temperature for 2 h. Samples were taken and analyzed by HPLC and LC-MS to confirm the complete reaction of AK34141-04, at which point the reaction was terminated. Post-processing: The reaction solution was added dropwise to 60 mL of methyl tert-butyl ether, stirred for 30 min, and filtered to obtain crude AK34141. The crude product was purified by reversed-phase preparative chromatography, with acetonitrile increasing from 0 to 35% over 60 min while maintaining the gradient. The target fraction was collected and lyophilized to obtain AK34141 (200 mg, 0.18 mmol / L, yield 52%, purity 95.0%). LCMS: (M+1) + : 1108.13, calculated value (M+1)+ : 1107.48. 1 H NMR (600 MHz, DMSO-d6)δ 8.29 (t, J=6.3 Hz, 1H), 8.16 (d, J=8.2 Hz, 1H), 8.14–8.05 (m, 2H), 7.96 (s,1H), 7.88 (d, J=10.7 Hz, 1H), 7.65 (d, J=8.9 Hz, 1H), 7.31 (s, 1H), 7.02 (s,2H), 6.52 (s, 1H), 5.44 (s, 2H), 5.37–5.27 (m, 2H), 4.31 (dd, J=13.2, 6.4 Hz,1H), 4.22 (dp, J=19.1, 6.5, 6.0 Hz, 2H), 4.12 (dd, J=8.9, 6.7 Hz, 1H), 3.78–3.68 (m, 2H), 3.63–3.51 (m, 8H), 3.40 (dd, J=7.3, 4.1 Hz, 4H), 3.22 (q, J=5.8Hz, 2H), 2.99–2.83 (m, 3H), 2.51 (s, 3H), 2.36 (t, J=6.5 Hz, 2H), 1.86 (dhept, J=21.4, 7.2 Hz, 2H), 1.74 (dq, J=13.6, 6.9 Hz, 1H), 1.15 (d, J=7.1Hz, 3H), 0.87 (t, J=7.3 Hz, 3H), 0.66 (d, J=6.8 Hz, 6H).

[0147] Example 2: Synthesis of AK34144 Manufacturing process of AK34144: 1. AK34141-03: At room temperature, AK34141-02 (200 mg, 0.361 mmol), Fmoc-PEG-GV-OSu (195 mg), N,N-dimethylformamide (DMF, 4 mL), and N,N-diisopropylethylamine (DIEA, 95 μL, 0.542 mmol) were added sequentially to a 25 mL single-necked flask, and the mixture was stirred at room temperature. After 140 min of reaction, a sample was taken and analyzed by HPLC to confirm that the AK34141-02 reaction was complete, and the reaction was terminated. Dichloromethane (DCM, 20 mL) and water (15 mL) were added to the reaction solution, and the mixture was stirred for 10 min. The solution was then separated, and the organic phase was washed with water (15 mL × 2). Silica gel was added to the organic phase and stirred. The mixture was then separated by normal-phase column chromatography, eluted with a dichloromethane / methanol system. The target product fraction was collected and concentrated under reduced pressure to obtain solid AK34141-03 (225 mg, 0.206 mmol / L, yield 57%, purity 95%). LCMS: (M+1) + 1091.45 (calculated value: 1090.48).

[0148] 2. AK34141-04: At room temperature, AK34141-03 (225 mg, 0.206 mmol), N,N-dimethylformamide (DMF, 4 mL), and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 15.68 mg, 15 μL) were added sequentially to a single-necked flask, and the mixture was stirred at room temperature. After 30 min of reaction, a sample was taken and analyzed by HPLC to confirm that AK34141-03 had reacted completely, and the reaction was terminated. Post-processing: The reaction solution was added dropwise to methyl tert-butyl ether (40 mL), stirred for 10 min, centrifuged, and filtered to obtain a pale yellow solid AK34141-04 (120 mg, 0.148 mmol, yield 72%, purity 90%). LCMS: (M+1) + 869.92 (Calculated value: 868.96).

[0149] 3. Preparation of AK34144 At room temperature, AK51541-04 (120 mg, 0.138 mmol) and acetonitrile (10 mL) were added sequentially to a 100 mL single-necked flask and sonicated until dissolved. The reaction solution was stirred in an ice bath, and N-(methoxycarbonyl)maleimide (107 mg, 0.69 mmol) was added. After stirring for 8 min, saturated sodium bicarbonate solution (2 mL) was added dropwise, and the reaction was continued with stirring in an ice bath. After 1 h of reaction, a sample was taken for HPLC analysis. AK51541-04 was found to be incompletely reacted. Acetonitrile (2 mL) and saturated sodium bicarbonate solution (0.5 mL) were added, and the reaction was carried out overnight at 8 °C with stirring. HPLC and LC-MS analysis confirmed that AK51541-04 had reacted completely, and the reaction was terminated. Dichloromethane (20 mL) and 10% citric acid solution (35 mL) were added to the reaction solution, and the mixture was separated. The aqueous phase was extracted again with dichloromethane (20 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The concentrate was separated by normal-phase column chromatography, wet loading, elution with a dichloromethane / methanol system, and the target product fraction was collected and concentrated under reduced pressure to obtain solid AK34144 (33 mg, yield 24%, 0.034 mmol / L, purity 95%). LCMS: (M+1) + 949 (Calculated value: 949); 1H NMR (600MHz, DMSO-d6) δ 8.29 (t, J=6.3 Hz, 1H), 8.16 (d, J=8.2 Hz, 1H), 8.14–8.05 (m,2H), 7.96 (s, 1H), 7.88 (d, J=10.7 Hz, 1H), 7.65 (d, J=8.9 Hz, 1H), 7.31 (s,1H), 7.02 (s, 2H), 6.52 (s, 1H), 5.44 (s, 2H), 5.37–5.27 (m, 2H), 4.31 (dd, J=13.2, 6.4 Hz, 1H), 4.22 (dp, J=19.1, 6.5, 6.0 Hz, 2H), 4.12 (dd, J=8.9, 6.7Hz, 1H), 3.78–3.68 (m, 2H), 3.63–3.51 (m, 8H), 3.40 (dd, J=7.3, 4.1 Hz, 4H), 3.22 (q, J=5.8 Hz, 2H), 2.99–2.83 (m, 3H), 2.51 (s, 3H), 2.36 (t, J=6.5 Hz,2H), 1.86 (dhept, J=21.4, 7.2 Hz, 2H), 1.74 (dq, J=13.6, 6.9 Hz, 1H), 1.15(d, J=7.1 Hz, 3H), 0.87 (t, J=7.3 Hz, 3H), 0.66 (d, J=6.8 Hz, 6H).

[0150] Example 3: Synthesis of AK34110 Manufacturing process of AK34110: 1. Preparation of AK34141pfp-01 At room temperature (25℃), AK51541-04 (145.46 mg, 165 μmol), dichloromethane (DCM, 5 mL), 4-(4-methoxycarbonyl-1-piperidinyl)-4-oxobutyric acid (80.33 mg, 330 μmol), and N,N-diisopropylcarbodiimide (DIC, 14.58 mg, 115.5 μmol, 18 μL) were added sequentially to a 100 mL single-necked flask, and the mixture was stirred at room temperature. After 40 min of reaction, a sample was taken for HPLC and LC-MS analysis. The reaction showed that AK51541-04 had not reacted completely, so DIC (8 μL, 0.3 eq.) was added. After another 30 min of reaction, the starting material was still not completely converted, and the mixture was stirred overnight at room temperature. The next day, a sample was taken for HPLC and LC-MS analysis, confirming that AK51541-04 had reacted completely. The reaction solution was subjected to normal-phase column chromatography via wet loading, using dichloromethane / methanol as the elution system. The target product fraction was collected under reduced pressure at a dichloromethane:methanol ratio of 92.5:7.5, and concentrated to obtain a pale yellow solid AK51546pfp-01 (115 mg, 105 μmol / L, yield 63%, purity 95%). LCMS: (M+1) + 1095.13 (Calculated value: 1094.21). 1 H NMR (600MHz, DMSO) δ 8.29 (t, J =6.1 Hz, 1H), 8.16–8.07 (m, 3H), 7.87 (dd, J =12.6, 8.1Hz, 2H), 7.65 (d, J =8.8 Hz, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 5.44 (s, 2H), 5.30 (s, 2H), 4.35–4.10 (m, 6H), 3.83–3.71 (m, 4H), 3.63–3.58 (m, 7H), 3.55(s, 3H), 3.50–3.46 (m, 5H), 3.17 (q, J =5.8 Hz, 2H), 3.06 (dd, J =18.1, 6.9 Hz, 1H), 2.91 (tdd, J =15.6, 13.2, 7.5 Hz, 2H), 2.68 (dd, J =17.7, 6.8 Hz, 1H), 2.60(tt, J =11.0, 3.9 Hz, 2H), 2.37 (t, J =6.5 Hz, 2H), 2.31 (t,J =7.0 Hz, 2H), 1.92–1.72 (m, 6H), 1.49 (td, J =15.0, 3.8 Hz, 2H), 1.32–1.22 (m, 11H), 1.16 (d, J =7.1Hz, 3H), 0.88 (t, J =7.3 Hz, 4H), 0.67 (d, J =6.7 Hz, 7H).

[0151] 2. Preparation of AK34141pfp-02 At room temperature, AK34141pfp-01 (100 mg, 0.09 mmol) and methanol (20 mL) were added to a 50 mL single-necked flask and stirred until dissolved. Lithium hydroxide solution (500 μL, 2 mol / L) was added, and the reaction was stirred at room temperature. After 40 min of reaction, HPLC monitoring showed that AK34141pfp-01 had not reacted completely, so lithium hydroxide solution (500 μL, 2 mol / L) was added to continue the reaction. After 1 h of reaction, HPLC monitoring showed that the starting material was completely consumed. The pH of the reaction solution was adjusted to 3–4 with 15% citric acid aqueous solution, and the mixture was extracted with dichloromethane (DCM, 30 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain AK34141pfp-02 (78 mg, 72 μmol / L, yield 63%, purity 95%), LCMS: (M+1). + 1080.15 (Calculated value: 1080.18).

[0152] 3. AK34110: At room temperature, AK34141pfp-02 (78 mg, 0.072 mmol), N,N-dimethylformamide (DMF, 1 mL), dichloromethane (DCM, 1 mL), pentafluorophenol (66.3 mg, 0.36 mmol), and N,N-dicyclohexylcarbodiimide (DCC, 14.86 mg, 0.072 mmol) were added sequentially to a 50 mL single-necked flask, and the mixture was stirred at room temperature. After 40 min of reaction, HPLC monitoring showed that AK34141pfp-02 had reacted completely. The reaction solution was added dropwise to methyl tert-butyl ether (20 mL), stirred for 10 min, and centrifuged to obtain solid AK34110 (50 mg, 72 μmol / L, yield 56%, purity 95%). LCMS: (M+1) + 1245.77 (Calculated value: 1246.23). 1H NMR (600 MHz, DMSO) δ 8.29 (s, 1H), 8.17 (d, J =7.6 Hz, 1H), 8.12–8.05 (m, 2H), 7.88 (d, J =9.4 Hz, 2H), 7.65 (d, J =8.4 Hz, 1H), 7.32 (s, 1H), 6.54 (s, 1H), 5.45 (s, 2H), 5.34 (s, 2H), 4.37–4.10 (m, 5H), 3.87 (d, J =12.9Hz, 1H), 3.73 (s, 2H), 3.64–3.51 (m, 6H), 3.46–3.35 (m, 55H), 3.17 (d, J =4.8Hz, 4H), 3.01–2.88 (m, 9H), 2.85–2.77 (m, 1H), 2.41–2.25 (m, 4H), 2.10–1.80(m, 4H), 1.51 (d, J =10.2 Hz, 2H), 1.35–1.12 (m, 7H), 1.04 (dd, J =21.2, 10.6 Hz, 4H), 0.88 (t, J =6.6 Hz, 3H), 0.67 (d, J =5.9 Hz, 6H).

[0153] Example 4: Synthesis of AK51541 AK51541 Manufacturing Process: 1. AK51541-01: At room temperature (25℃), D2515 (260 mg, 515.31 μmol), N,N-dimethylformamide (DMF, 6 mL), N,N-diisopropylethylamine (DIPEA, 199.80 mg, 1.55 mmol, 269.27 μL), and AK10246-06 (137.42 mg, 449.42 μmol) were added sequentially to a 10 mL single-necked flask, and the mixture was stirred at room temperature. After 1 h of reaction, HPLC monitoring showed that D2515 had completely reacted. 5g of silica gel was added to the reaction solution and stirred. The mixture was then separated by normal-phase column chromatography, eluting with a dichloromethane / methanol system. The target product fraction was collected under reduced pressure at a dichloromethane:methanol ratio of 96:4. The fraction was concentrated to obtain a brownish-yellow solid AK51541-01 (310mg, 409.96μmol, yield 79.56%, purity 92%). LCMS: (M+1) + 696.42 (Calculated value: 695.69). 1 H NMR (600 MHz, DMSO-) d 6) δ 8.62 (t, J =6.2 Hz, 1H), 7.87 (dt, J =14.9, 5.3 Hz, 1H),7.81 (s, 4H), 7.56 (s, 1H), 7.52 (s, 1H), 7.26 (s, 1H), 6.50 (s, 1H), 6.28(s, 2H), 5.43 (s, 2H), 5.29 (d, J =3.1 Hz, 2H), 4.72 (q, J =7.0 Hz, 1H), 4.29–4.20 (m, 2H), 3.53 (s, 3H), 2.97–2.84 (m, 2H), 1.87 (dhept, J =21.6, 7.3 Hz, 2H), 1.56 (d, J =7.4 Hz, 0H), 1.47 (d, J =7.3 Hz, 3H), 1.23 (s, 1H), 0.88 (t, J =7.4 Hz, 3H).

[0154] 2. AK51541-02: At room temperature (25℃), AK51541-01 (310 mg, 421.90 μmol), methanol (6 mL), and hydrazine hydrate (253.44 mg, 5.06 mmol) were added sequentially to a 10 mL single-necked flask, and the mixture was stirred at room temperature. After 2 h of reaction, a sample was taken and analyzed by HPLC and LC-MS to confirm that AK51541-01 had reacted completely, and the reaction was terminated. Dichloromethane (100 mL) and distilled water (20 mL) were added to the reaction solution for extraction. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain a pale yellow solid AK51541-02 (200 mg, 281.14 μmol, yield 66.64%, purity 85%). LC-MS: (M+1) + 566.32 (Calculated value: 565.58).

[0155] 3. AK51541-03: At room temperature (25℃), AK51541-02 (200 mg, 330.76 μmol), N,N-dimethylformamide (DMF, 3 mL), and Fmoc-PEG-GV-OSu (215.88 mg, 312.06 μmol) were added sequentially to a 10 mL single-necked flask, and the mixture was stirred at room temperature. After 1 h of reaction, a sample was taken and analyzed by HPLC and LC-MS to confirm that AK51541-02 had reacted completely, and the reaction was terminated. 5 g of silica gel was added to the reaction solution and stirred. The mixture was then separated by normal-phase column chromatography, eluted with a dichloromethane / methanol system. The target product fraction was collected under reduced pressure at a dichloromethane:methanol ratio of 91:9, concentrated, and yielded a pale yellow solid AK51541-03 (250 mg, 203.54 μmol, yield 61.54%, purity 93%). LC-MS: (M+1) + 1104.05 (Calculated value: 1103.20).

[0156] 4. AK51541-04: At room temperature (25℃), AK51541-03 (150 mg, 131.32 μmol), N,N-dimethylformamide (DMF, 3.75 mL), and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 10.00 mg, 65.66 μmol, 9.82 μL) were added sequentially to a 10 mL single-necked flask, and the mixture was stirred at room temperature. After 1 h of reaction, a sample was taken and analyzed by HPLC and LC-MS to confirm that AK51541-03 had reacted completely, and the reaction was terminated. 10 mL of methyl tert-butyl ether was added to the reaction solution, and a white solid precipitated. After centrifugation at 10000 r / min for 5 min, a pale yellow solid AK51541-04 (105 mg, 95.87 μmol, yield 73.00%, purity 84%) was obtained; LCMS: (M+1) + 881.67 (Calculated value: 880.95).

[0157] 5. AK51541: At room temperature (25℃), AK51541-04 (105 mg, 114.13 μmol), N,N-dimethylformamide (DMF, 7.5 mL), 2-[carboxymethyl-[2-(2,5-dioxopyrrole-1-yl)ethyl]amino]acetic acid (29.24 mg, 99.02 μmol), and N,N-diisopropylcarbodiimide (DIC, 14.40 mg, 114.13 μmol, 17.67 μL) were added sequentially to a 10 mL single-necked flask, and the mixture was stirred at room temperature. After 1 h of reaction, a sample was taken and analyzed by HPLC and LC-MS to confirm that the reaction of AK51541-04 was complete, and the reaction was terminated. The reaction solution was purified by reversed-phase preparative chromatography using a C18 Spherical 20–35 μm, 100 Å, 120 g reversed-phase column with 0.1% acetic acid aqueous solution as mobile phase A2 and acetonitrile as mobile phase B2, monitored at wavelengths of 254 nm and 214 nm. 5 mL of the sample DMF solution was injected into the reversed-phase column, and eluted for 5 min with A2:B2 = 95:5 to remove DMF. Then, the acetonitrile ratio was increased from 5% to 35% over 90 min and maintained at this ratio for another 30 min. The target product fraction was collected. After purity detection by HPLC (220 nm) and LC-MS, the qualified fractions were combined, the product was extracted with dichloromethane, separated, and the organic phase was concentrated under reduced pressure. The concentrate was added to a water / acetonitrile (volume ratio 4:1) mixture and lyophilized to obtain a white solid powder AK51541 (54 mg, 45.22 μmol, yield 39.63%, purity 97%); LCMS: (M+1) + 1120.08 (Calculated value: 1119.15). 1 H NMR (500 MHz, DMSO- d6) δ 8.12–8.05(m, 3H), 7.72–7.63 (m, 2H), 7.53 (d, J =7.5 Hz, 2H), 7.00 (s, 2H), 6.30 (s,3H), 5.44 (s, 2H), 5.31 (s, 2H), 4.24 (dtd, J =27.3, 13.3, 6.5 Hz, 4H), 3.74(dd, J =5.8, 2.4 Hz, 3H), 3.57 (d, J =16.0 Hz, 7H), 3.48 (d, J =7.6 Hz, 7H), 3.39(t, J =5.9 Hz, 2H), 3.21 (s, 4H), 2.96–2.83 (m, 2H), 2.73 (t, J =6.4 Hz, 3H),1.93–1.80 (m, 2H), 1.17 (d, J =7.0 Hz, 4H), 0.88 (t, J =7.3 Hz, 5H), 0.68 (dd, J =6.7, 1.6 Hz, 8H).

[0158] Example 5: Synthesis of AK51546 AK51546 Manufacturing Process: 1. AK51546-01: At 25°C, D2515 (220 mg, 471.64 μmol), AK10246-06 (126.25 mg, 471.64 μmol), dichloromethane (DCM, 5 mL), and triethylamine (TEA, 47.72 mg, 471.64 μmol, 65.74 μL) were added sequentially to a 100 mL round-bottom flask, and the mixture was stirred at 25°C for 4 h. The reaction solution was concentrated under reduced pressure and purified by reverse-phase chromatography to obtain a pale yellow solid AK51546-01 (35.4 mg, 22.87 μmol, yield 33.54%); MS: (M+1) + 969.58 (Calculated value: 695.69).

[0159] 2. AK51546-02: AK51546-01 (0.2 g, 287.08 μmol) was added to a 100 mL round-bottom flask at 25 °C, followed by N,N-dimethylformamide (DMF, 3 mL) and hydrazine hydrate (57.48 mg, 1.15 mmol). The mixture was stirred at 25 °C for 4 h. Extraction yielded a pale yellow solid AK51546-02 (0.175 g, 247.10 μmol, yield 86.07%). MS: (M+1) + 566.59 (Calculated value: 565.58).

[0160] 3. AK51546-03: AK51546-02 (218.75 mg, 308.87 μmol) was added to a 100 mL round-bottom flask at 25 °C and dissolved in N,N-dimethylformamide (DMF, 3 mL). Then, Fmoc-PEG-GV-OSu (201.91 mg, 308.87 μmol) was added, and the mixture was stirred at 25 °C for 4 h. The reaction solution was purified by silica gel stirring and normal-phase column chromatography to obtain a pale yellow solid AK51546-03 (0.15 g, 126.34 μmol, yield 40.90%). MS: (M+1) + 1104.18 (Calculated value: 1103.20).

[0161] 4. AK51546-04: AK51546-03 (0.15 g, 135.85 μmol) was added to a 25 mL round-bottom flask at 25 °C, followed by N,N-dimethylformamide (DMF, 3 mL) and piperidine (0.3 mL). The mixture was stirred at 25 °C for 0.5 h. After reverse-phase purification, a pale yellow powder solid AK51546-04 (101 mg, 108.79 μmol, yield 80.09%) was obtained; the pale yellow powder solid MS: (M+1) + 881.87 (Calculated value: 880.95).

[0162] 5. AK51546: At 25°C, bromoacetic acid (19.28 mg, 137.73 μmol) was added to a 25 mL round-bottom flask and dissolved in dichloromethane (DCM, 2.98 mL). N,N-diisopropylcarbodiimide (DIC, 14.48 mg, 114.78 μmol, 17.77 μL) was then added, and the mixture was stirred for 5 min. This mixture was then added dropwise to a mixture containing AK51546-04 (0.101 g, 114.78 μmol) and N,N-dimethylformamide (DMF, 2.98 mL), and the mixture was stirred at 25°C for 0.5 h. HPLC monitoring showed that the reaction was complete. The reaction solution was purified by reverse-phase chromatography to obtain a pale yellow powdery solid AK51546 (0.035 g, 33.29 μmol, yield 29.00%). MS: (M+1) + 1001.87 (Calculated value: 1001.89). 1 H NMR (500 MHz, DMSO-d6) δ 9.59 (s, 1H), 8.92 (t, J=6.7 Hz, 1H), 8.14 (dq, J=22.6, 9.4, 7.6 Hz, 1H), 7.87–7.80 (m, 1H), 7.20(dddd, 3.75 (t, J=5.5 Hz,2H), 3.51–3.45 (m, 4H), 3.41–3.31 (m, 4H), 3.28–3.10 (m, 5H), 3.01 (d, J=3.8Hz, 1H), 2.78 (t, J=9.7 Hz, 3H), 2.63–2.53 (m, 2H), 2.46–2.35 (m, 2H), 2.29 (tt, J=16.2, 7.5 Hz, 1H), 2.14–1.91 (m, 3H), 1.47 (p, J=7.5 Hz, 3H), 1.26–1.12 (m, 4H), 1.11–1.01 (m, 2H), 0.88 (dddd, J=55.8, 28.8, 13.3, 6.6 Hz, 13H).

[0163] Example 6: Synthesis of AK51546pfp AK51546pfp manufacturing process: 1. AK51546pfp-01: At 25°C, AK51541-04 (100 mg, 113.52 μmol), dichloromethane (5 mL), 4-(4-methoxycarbonyl-1-piperidinyl)-4-oxobutyric acid (55.23 mg, 227.03 μmol), and N,N'-diisopropylcarbodiimide (14.33 mg, 113.52 μmol, 17.58 μL) were added sequentially to a 10 mL single-necked flask, and the mixture was stirred at room temperature. After 1 hour of reaction, a sample was taken for HPLC and LCMS analysis, which showed that AK51541-04 was completely consumed, indicating that the reaction was complete. 5 g of silica gel was added to the reaction solution and stirred. Column chromatography was performed using a dichloromethane / methanol system, with the target product eluting at a dichloromethane:methanol ratio of 92:8. The product fraction was collected and concentrated under reduced pressure to give a pale yellow solid AK51546pfp-01 (110 mg, 92.48 μmol, yield 81.47%, purity 93%). LCMS: (M+1) + 1107.07 (Calculated value: 1106.20).

[0164] 2. AK51546pfp-02: At 25°C, AK51546pfp-01 (110 mg, 99.44 μmol), water (2 mL), methanol (2 mL), and lithium hydroxide (23.81 mg, 994.41 μmol) were added sequentially to a 10 mL single-necked flask. The mixture was stirred at room temperature for 16 hours. HPLC and LCMS analysis showed complete consumption of AK51546pfp-01, indicating the reaction was complete. The pH of the reaction solution was adjusted to approximately 4 with 15% citric acid aqueous solution, and 10 mL of distilled water was added. The mixture was extracted with dichloromethane (30 mL × 3). The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain a pale yellow solid AK51546pfp-02 (85 mg, 72.38 μmol, yield 72.79%, purity 93%). LCMS: (M+1) + 1093.08 (Calculated value: 1092.17).

[0165] 3. AK51546pfp: At 25°C, AK51546pfp-02 (85 mg, 77.83 μmol), N,N-dimethylformamide (0.5 mL), dichloromethane (0.5 mL), pentafluorophenol (71.63 mg, 389.15 μmol, 40.77 μL), and N,N'-dicyclohexylcarbodiimide (16.06 mg, 77.83 μmol) were added sequentially to a 10 mL single-necked flask. The mixture was stirred at room temperature for 1 hour. HPLC and LCMS analysis showed that AK51546pfp-02 was completely consumed, indicating the reaction was complete. The reaction mixture was added dropwise to 10 mL of anhydrous diethyl ether, centrifuged for 5 minutes (10000 r / min), the supernatant was discarded, and the mixture was dried to obtain a white solid AK51546pfp (60 mg, 45.06 μmol, yield 57.90%, purity 94.5%). LCMS: (M+1) + 1259.13 (Calculated value: 1258.22). 1 H NMR (500 MHz, DMSO- d 6) δ 8.28 (t, J =6.3 Hz, 1H), 8.13–8.05 (m, 2H), 7.87 (t, J =5.6 Hz, 1H), 7.65 (d, J =8.8 Hz, 1H), 7.53 (d, J =8.4 Hz, 2H), 7.25 (s, 1H), 6.49 (s, 1H), 6.32–6.28 (m, 2H), 5.57 (d, J =7.9 Hz, 1H), 5.43 (s, 2H), 5.30 (s, 2H), 4.33–4.10(m, 3H), 3.88 (d, J =13.6 Hz, 1H), 3.74 (dd, J =5.8, 2.9 Hz, 2H), 3.60 (t, J =6.5Hz, 2H), 3.56 (s, 3H), 3.48 (s, 3H), 3.38 (t, J =5.9 Hz, 2H), 3.31 (s, 1H), 3.18 (dd, J =6.9, 4.5 Hz, 2H), 2.97–2.78 (m, 2H), 2.55 (dd, J =6.8, 4.4 Hz, 1H), 2.35 (dt, J=25.9, 6.9 Hz, 3H), 2.02 (dd, J =19.9, 12.7 Hz, 1H), 1.93–1.81 (m,2H), 1.74 (td, J =13.6, 5.4 Hz, 2H), 1.66–1.59 (m, 1H), 1.54–1.48 (m, 1H), 1.24(s, 3H), 1.17 (d, J =7.1 Hz, 3H), 1.13–0.99 (m, 2H), 0.88 (t, J =7.3 Hz, 3H), 0.67 (dd, J =6.9, 1.8 Hz, 5H).

[0166] Example 7: Synthesis of AK27541 AK27541 Manufacturing Process: 1. AK27541-1 D2275 (450 mg, 1.05 mmol) was added to a 100 mL single-necked reaction flask and dissolved in N,N-dimethylformamide (10 mL). Then, AK10246-06 (304.15 mg, 1.05 mmol) and N,N-diisopropylethylamine (203.56 mg, 1.58 mmol, 274.34 μL) were added, and the mixture was stirred at 25°C for 2 hours. HPLC showed complete conversion of the starting material and the formation of a new product peak; LCMS showed the target product signal. 10 g of silica gel was added to the reaction mixture, and the mixture was separated by column chromatography to obtain AK27541-1 (500 mg, 756.86 μmol, yield 72.08%). LCMS: (M+1) + =638.22. 1H NMR (500 MHz, DMSO-d6) δ 8.92(s, 1H), 8.41 (d, J=8.5 Hz, 1H), 8.20 (d, J= 8.5 Hz, 1H), 8.03 (d, J=2.8 Hz,2H), 7.96 (s, 2H), 7.89 (d, J=2.3 Hz, 1H), 7.87 (s, 1H), 7.75 (t, J=7.5 Hz,1H), 7.37 (s, 1H), 6.54 (s, 1H), 5.47 (s, 2H), 5.35 (d, J=19.2 Hz, 1H), 4.87(q, J=7.0 Hz, 1H), 4.59–4.52 (m, 1H), 4.46–4.36 (m, 2H), 4.31 (d, J=13.9 Hz,1H), 2.89 (s, 3H), 1.90 (tt, J=14.4, 7.4 Hz, 2H), 1.60 (d, J=7.1 Hz, 3H), 0.91 (t, J=7.0 Hz, 3H).

[0167] 2. AK27541-2 AK27541-1 (480 mg, 726.58 μmol) was added to a 100 mL single-necked reaction flask and dissolved in methanol (10 mL). Hydrazine hydrate (545.59 mg, 8.72 mmol, 80% purity) was added, and the mixture was stirred at 25°C for 16 hours. HPLC showed complete conversion of the starting material and the formation of a new product peak; LCMS showed the target product signal. The mixture was extracted with 30 mL of water and 30 mL of dichloromethane, repeated five times. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and the organic solvent was removed under reduced pressure to obtain AK27541-2 (700 mg, 689.60 μmol, yield 94.91%, purity 50%). LCMS: (M+1) + =508.37.

[0168] 3. AK27541-3 AK27541-2 (700 mg, 659.72 μmol) was added to a 100 mL single-necked reaction flask and dissolved in a mixture of dichloromethane (5 mL) and N,N-dimethylformamide (5 mL). Fmoc-PEG-GV-OSu (713.22 mg, 1.06 mmol) was then added, and the mixture was stirred at 25°C for 2 hours. HPLC showed complete conversion of the starting material and the formation of a new product peak; LCMS showed the target product signal. 5 g of silica gel was added to the reaction mixture, and the mixture was separated by column chromatography to obtain AK27541-3 (450 mg, 421.30 μmol, yield 63.86%). LCMS: (M+1) + =1045.23. 1 H NMR (500 MHz, DMSO-d6) δ 8.54 (s, 1H), 8.46 (d, J=8.4Hz, 1H), 8.23 ​​(t, J=8.1 Hz, 2H), 8.15 (t, J=5.7 Hz, 1H), 7.91 (dd, J=7.2, 4.2Hz, 4H), 7.84 (d, J=8.9 Hz, 1H), 7.79–7.75 (m, 1H), 7.71 (d, J=7.3 Hz, 2H), 7.42 (dd, J=15.5, 8.1 Hz, 4H), 7.38–7.29 (m, 4H), 6.57 (s, 1H), 5.48 (s, 2H),5.41 (s, 2H), 4.52 (d, J=6.2 Hz, 1H), 4.36–4.29 (m, 4H), 4.24 (d, J=6.8 Hz, 1H), 3.83 (d, J=5.6 Hz, 2H), 3.62 (t, J=6.4 Hz, 2H), 3.51 (d, J=6.7 Hz, 4H), 3.42 (t, J=5.8 Hz, 2H), 3.16 (q, J=5.6 Hz, 2H), 2.99 (s, 3H), 2.41 (t, J=6.5Hz, 2H), 2.03 (d, J=6.7 Hz, 1H), 1.95–1.87 (m, 2H), 1.35 (d, J=7.1 Hz, 3H), 0.93 (t, J=7.3 Hz, 3H), 0.89 (d, J=6.8 Hz, 3H), 0.84 (d, J=6.8 Hz, 3H).

[0169] 4. AK27541-4 AK27541-3 (300 mg, 280.86 μmol) was added to a 50 mL round-bottom flask and dissolved in N,N-dimethylformamide (15 mL). Then, 1,8-diazabicyclo[5.4.0]undec-7-ene (21.38 mg, 140.43 μmol, 21.00 μL) was added, and the mixture was stirred at 25°C for 1 hour. HPLC and LCMS analysis showed complete conversion of the starting material to the target product. The reaction solution was used directly in the next step of the reaction.

[0170] 5. AK27541 1-Hydroxybenzotriazole (48.66 mg, 317.77 μmol) was added to the above reaction solution; N,N'-diisopropylcarbodiimide (40.10 mg, 317.77 μmol, 49.21 μL) and 2-[carboxymethyl-[2-(2,5-dioxo-3H-pyrrolo-3-onthiol-1-yl)ethyl]amino]acetic acid (88.40 mg, 317.77 μmol) were dissolved in 0.5 mL of a mixture of N,N-dimethylformamide and dichloromethane, and then added to the above reaction solution. The mixture was stirred at 25°C for 1 hour. HPLC and LCMS analysis showed complete conversion of the starting material, yielding the target product. The organic solvent was removed by vacuum distillation. The residue was dissolved in 2 mL of N,N-dimethylformamide and purified using a C18 spherical reversed-phase silica column (20–35 μm, 100 Å, 120 g). Acetonitrile (HPLC grade) was used as mobile phase B2, and 0.1% trifluoroacetic acid aqueous solution was used as mobile phase A2. The detection wavelengths were 254 nm and 214 nm. After loading the sample solution onto the column, N,N-dimethylformamide was removed by elution with A2:B2 = 95:5 for 5 minutes. Then, the acetonitrile ratio was linearly increased to 36% (A2:B2 changed from 95:5 to 64:36) over 90 minutes, and elution was continued at 36% acetonitrile for 30 minutes until the product was completely eluted. The product fraction was collected, and its purity was confirmed by HPLC (254 nm) and LCMS. The preparation solution was extracted with dichloromethane (100 mL) and concentrated under reduced pressure to obtain AK27541 (100 mg, 94.24 μmol, yield 35.59%). LCMS: (M+1) + =1062.68. 1H NMR (500 MHz, DMSO-d6) δ 8.52 (d, J=5.9 Hz, 1H), 8.43 (d, J=8.6 Hz, 1H), 8.21 (dd, J=10.4, 7.7 Hz, 2H), 8.12 (t, J=5.4Hz, 1H), 7.88 (t, J=7.3 Hz, 1H), 7.82 (d, J=8.7 Hz, 1H), 7.77–7.68 (m, 2H),7.37 (s, 1H), 7.00 (s, 1H), 6.54 (s, 1H), 5.42 (d, J=33.3 Hz, 4H), 4.57–4.47(m, 1H), 4.45–4.24 (m, 5H), 3.79 (d, J=5.6 Hz, 2H), 3.59 (t, J=6.5 Hz, 2H), 3.47 (d, J=3.1 Hz, 6H), 3.42–3.37 (m, 5H), 3.21 (d, J=3.7 Hz, 4H), 2.96 (s,3H), 2.73 (t, J=6.3 Hz, 2H), 2.38 (t, J=6.5 Hz, 2H), 2.00 (d, J=6.7 Hz, 1H),1.92–1.84 (m, 2H), 1.32 (d, J=7.1 Hz, 3H), 0.90 (t, J=7.3 Hz, 3H), 0.86 (d, J=6.7 Hz, 3H), 0.81 (d, J=6.8 Hz, 3H).

[0171] Example 8: Synthesis of AK26541 AK26541 Manufacturing Process: 1. AK26541-01: At 25°C, D2265 (200 mg, 473.43 μmol) was added to a 50 mL reaction flask and dissolved in N,N-dimethylformamide (3 mL). N,N-diisopropylethylamine (183.56 mg, 1.42 mmol, 247.39 μL) was added and stirred until homogeneous. Then AK10246-06 (126.25 mg, 471.64 μmol) was added, and the mixture was stirred at 25°C for 2 hours. Samples were taken for HPLC and LCMS analysis to confirm the completion of the reaction. Silica gel was added to the reaction solution for stirring, and normal-phase column chromatography was performed using dichloromethane / methanol as the eluent. The product eluted at a dichloromethane:methanol ratio of 93:7. The product fraction was collected, concentrated under reduced pressure, and yielded a white solid AK26541-01 (210 mg, 312.10 μmol, yield 65.92%, purity 97%). LCMS: (M+1) + 652.14 (Calculated value: 651.23).

[0172] 2. AK26541-02: AK26541-01 (210 mg, 321.75 μmol) was added to the reaction flask and dissolved in methanol (4 mL). Hydrazine hydrate (193.29 mg, 3.86 mmol) was added, and the mixture was reacted in a closed system at 50°C in an oil bath for 2 hours. HPLC analysis showed the reaction was complete, and the reaction was stopped. 5 mL of water was added to the reaction solution and the mixture was shaken well. The solution was adjusted to neutral with 15% citric acid aqueous solution. The crude product was purified by reverse-phase chromatography using a C18 spherical reverse-phase silica column (20–35 μm, 100 Å, 40 g), with acetonitrile as mobile phase B2 and 0.1% acetic acid aqueous solution as mobile phase A2. Detection wavelengths were 254 nm and 214 nm. After loading the sample onto the column, N,N-dimethylformamide was removed by elution with A2:B2 = 95:5 for 5 minutes. Then, the acetonitrile ratio was increased to 25% over 30 minutes and maintained at 25% acetonitrile for 20 minutes until the product was completely eluted. The product was collected to obtain AK26541-02 (100 mg, 179.88 μmol, yield 55.91%, purity 94%); LCMS: (M+1) + 522.16 (Calculated value: 521.23).

[0173] 3. AK26541-03: AK26541-02 (100 mg, 191.36 μmol) was dissolved in N,N-dimethylformamide (3 mL), and Fmoc-PEG-GV-OSu (125.09 mg, 191.36 μmol) was added. The mixture was stirred at 25°C for 2 hours. The reaction was stopped after HPLC monitoring showed completion. Silica gel was added to the reaction solution, and normal-phase column chromatography was performed using dichloromethane / methanol as eluent. The product fraction was collected and dried under reduced pressure to obtain AK26541-03 (186 mg, 164.92 μmol, yield 86.18%, purity 94%). LCMS: (M+1) + 1059.45 (Calculated value: 1058.47). 1 H NMR (500 MHz, DMSOd6) δ 8.32–8.22 (m, 1H), 8.13–8.06 (m, 1H), 7.92–7.85 (m, 1H), 7.67 (dd, J=20.3, 8.1 Hz, 1H), 7.42 (dt, J=14.6, 7.4 Hz, 1H), 7.37–7.29 (m, 2H), 5.45 (s, 1H), 5.38 (s, 1H), 4.34–4.17(m, 2H), 3.74 (dd, J=5.7, 3.6 Hz, 1H), 3.55 (s, 1H), 3.51–3.42 (m, 1H), 3.40(q, , 2H), 1.19 (s, 4H), 1.24–1.14 (m, 3H), 0.92–0.83 (m, 1H), 0.76 (s, 2H), 0.70–0.64 (m, 2H).

[0174] 4. AK26541-04: AK26541-03 (150 mg, 141.49 μmol) was dissolved in N,N-dimethylformamide (3 mL), and 1,8-diazabicyclo[5.4.0]undec-7-ene (10.77 mg, 70.74 μmol, 10.58 μL) was added with stirring. The mixture was stirred at 25°C for 1 hour. HPLC showed that the reaction was complete. 20 mL of methyl tert-butyl ether was added to the reaction solution, and the product precipitated. After filtration and drying, AK26541-04 (110 mg, 123.40 μmol, yield 87.22%, purity 94%) was obtained. LCMS: (M+1) + 837.33 (Calculated value: 836.41).

[0175] 5. AK26541: AK26541-04 (110 mg, 131.27 μmol), 2-[carboxymethyl-[2-(2,5-dioxopyrrole-1-yl)ethyl]amino]acetic acid (33.63 mg, 130.76 μmol), and N,N'-diisopropylcarbodiimide (16.57 mg, 131.27 μmol, 20.33 μL) were added sequentially to a 20 mL single-necked flask, and the mixture was stirred at 25°C for 30 minutes. Samples were taken for HPLC and LCMS analysis to confirm the completeness of the reaction. The reaction solution was purified by a C18 spherical reversed-phase silica column (20–35 μm, 100 Å, 40 g) using acetonitrile as mobile phase B2 and 0.1% trifluoroacetic acid aqueous solution as mobile phase A2, with a detection wavelength of 254 nm. After loading the sample onto the column, N,N-dimethylformamide was removed by elution with A2:B2 = 95:5 for 5 minutes. Then, the acetonitrile ratio was linearly increased to 35% over 90 minutes and maintained at this ratio for elution for 30 minutes until the product was completely eluted. The product was collected, and its purity was confirmed by HPLC (220 nm) and LCMS. Lyophilization yielded a white solid powder AK26541 (40 mg, 34.94 μmol, yield 26.62%, purity 94%). LCMS: (M+1) + 1075.34 (Calculated value: 1074.47). 1H NMR (500 MHz, DMSO– d6) δ 8.32–8.23 (m, 2H), 8.19 (d, J=8.4 Hz,1H), 8.09 (t, J=5.7 Hz, 2H), 7.88 (t, J=7.7 Hz, 1H), 7.77 (s, 1H), 7.72–7.63(m, 2H), 7.36 (s, 1H), 7.00 (s, 2H), 6.54 (s, 1H), 5.46 (s, 2H), 5.40 (s,2H), 4.26 (dtd, J=27.8, 13.2, 6.1 Hz, 2H), 4.13 (dd, J=8.9, 6.6 Hz, 1H), 3.73(dd, J=5.6, 2.9 Hz, 2H), 3.59 (t, J=6.5 Hz, 2H), 3.56 (s, 3H), 3.47 (s, 6H),3.51–3.43 (m, 2H), 3.40 (d, J=5.9 Hz, 1H), 3.21 (d, J=4.6 Hz, 3H), 3.00–2.89 (m, 1H), 2.73 (t, J=6.3 Hz, 2H), 2.37 (t, J=6.5 Hz, 2H), 2.05–1.96 (m, 1H), 1.88 (dp, J=21.3, 7.0 Hz, 2H), 1.76 (dt, J=13.4, 6.8 Hz, 1H), 1.24 (s, 3H), 1.16 (d, J=7.1 Hz, 3H), 0.87 (dt, J=14.3, 7.2 Hz, 3H), 0.67 (d, J=6.7 Hz, 5H).

[0176] Example 9: Synthesis of AK53941 Manufacturing process of AK53941: 1. AK53941-1 At 25°C, D2539 (300 mg, 665.18 μmol) was added to a 50 mL reaction flask and dissolved in N,N-dimethylformamide (5 mL). N,N-diisopropylethylamine (171.61 mg, 1.33 mmol) was added and stirred until homogeneous. Then, AK10246-06 (177 mg, 665.18 μmol) was added, and the mixture was stirred at 25°C for 2 hours. HPLC and LCMS showed the reaction was complete. Silica gel was added to the reaction solution for stirring, and normal-phase column chromatography was performed using dichloromethane / methanol as eluent. The product eluted at a dichloromethane:methanol ratio of 95:5. The product fraction was collected, concentrated under reduced pressure, and yielded a white solid AK53941-1 (365 mg, 535.97 μmol, yield 80.5%, purity 95%). LCMS: (M+1) + 682.11 (Calculated value: 681.21).

[0177] 2. AK53941-2 AK53941-1 (365 mg, 535.97 μmol) was added to the reaction flask and dissolved in methanol (8 mL). Hydrazine hydrate (170.24 mg, 5.35 mmol) was added, and the mixture was reacted in a closed system at 50°C in an oil bath for 2 hours. HPLC and LCMS showed the reaction was complete, and the reaction was stopped. 5 mL of water was added to the reaction solution and the mixture was shaken well. The solution was adjusted to neutral with 15% citric acid aqueous solution. The crude product was purified by reverse-phase chromatography using a C18 spherical reverse-phase silica column (20–35 μm, 100 Å, 40 g), with acetonitrile as mobile phase B2 and 0.1% trifluoroacetic acid aqueous solution as mobile phase A2. After loading the sample onto the column, N,N-dimethylformamide was removed by elution with A2:B2 at a ratio of 95:5 for 5 minutes. Then, the acetonitrile ratio was increased to 28% (A2:B2 changed from 95:5 to 72:28) over 30 minutes, and elution was maintained at 28% acetonitrile for 10 minutes until the product was completely eluted. The product was collected to obtain AK53941-2 (235 mg, 426.49 μmol, yield 79.67%, purity 96%). LCMS: (M+1) + 552.13 (Calculated value: 551.20).

[0178] 3. AK53941-3 AK53941-2 (200 mg, 348.11 μmol) was added to a 100 mL single-necked reaction flask and dissolved in a mixture of dichloromethane (5 mL) and N,N-dimethylformamide (5 mL). Fmoc-PEG-GV-OSu (376.33 mg, 556.97 μmol) was then added, and the mixture was stirred at 25°C for 2 hours. HPLC showed complete conversion of the starting material and the formation of a new product peak; LCMS showed the target product signal. 5 g of silica gel was added to the reaction mixture, and the mixture was separated by column chromatography to obtain AK53941-3 (260 mg, 233.78 μmol, yield 67.16%). LCMS: (M+1) + 1189.13 (Calculated value: 1188.45).

[0179] 4. AK53941-4 AK53941-3 (100 mg, 89.92 μmol) was added to a 50 mL round-bottom flask and dissolved in dichloromethane (5 mL). Then, 1,8-diazabicyclo[5.4.0]undec-7-ene (6.84 mg, 44.96 μmol, 6.72 μL) was added, and the mixture was stirred at 25°C for 0.5 hours. HPLC analysis showed complete conversion of the starting material to the new product. 10 mL of methyl tert-butyl ether was added to the reaction mixture, resulting in the precipitation of a white solid. The solid was filtered to obtain AK53941-4 (72 mg, 67.28 μmol, yield 80%). LCMS: (M+1) + 1075.34 (Calculated value: 1074.47).

[0180] 5. AK53941 N,N-dimethylformamide (2 mL), AK53941-4 (72 mg, 67.28 μmol), N,N'-diisopropylcarbodiimide (13.62 mg, 107.90 μmol, 16.71 μL), and 2-[carboxymethyl-[2-(2,5-dioxopyrrole-1-yl)ethyl]amino]acetic acid (30.13 mg, 107.90 μmol) were added sequentially to a 50 mL round-bottom flask, and the mixture was stirred at 25°C for 1 hour. HPLC and LCMS analysis showed complete conversion of the starting material to the target product. The reaction solution was purified by C18 spherical reversed-phase silica column (20–35 μm, 100 Å, 120 g), and the product fraction was collected. The purity was confirmed by HPLC (254 nm) and LCMS, and the product was lyophilized to obtain AK53941 (47.72 mg, 42.30 μmol, yield 47.04%). LCMS: (M+1) + 1105.86 (Calculated value: 1104.96); 1 H NMR (500 MHz, DMSO- d6) δ 12.49 (s, 1H), 8.43 (t, J =6.2 Hz, 1H), 8.20 (d, J =6.6 Hz, 1H), 8.14(t, J =5.8 Hz, 1H), 7.85 (d, J =8.6 Hz, 1H), 7.72 (d, J =10.2 Hz, 2H), 7.51 (s,1H), 7.26 (s, 1H), 7.00 (s, 2H), 6.30 (d, J =4.1 Hz, 2H), 5.43 (d, J =2.4 Hz,2H), 5.28 (s, 2H), 4.30 (s, 2H), 4.25 (q, J =7.4, 6.9 Hz, 2H), 4.19 (t, J =5.4Hz, 2H), 3.79 (d, J =5.7 Hz, 2H), 3.59 (t, J =6.5 Hz, 2H), 3.47 (s, 6H), 3.21 (d, J =5.0 Hz, 4H), 2.73 (t, J =6.3 Hz, 2H), 2.38 (t, J =6.5 Hz, 2H), 1.99 (h, J =6.8Hz, 1H), 1.88 (tt, J =14.2, 7.1 Hz, 2H), 1.26 (d, J =7.1 Hz, 4H), 1.21 (s, 3H), 1.18 (s, 1H), 0.92–0.86 (m, 5H), 0.88–0.75 (m, 6H).

[0181] Example 10: Preparation of CDH17 antibody-drug conjugate 1. Preparation of ACD234141 Take CDH17 antibody (10.0 mg / mL, 50 mg, 0.33 μL), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate solution (125 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) solution (10 mM, 0.2 mL), and react at room temperature (25 °C) by rotating the disc for 2 h.

[0182] AK34141 (3.67 mg, 3.3 μmol) was dissolved in 0.4 mL of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature using a rotating disc for 2 h. After the reaction was complete, small molecules were removed using a NAP-5 gel column (Cytiva), and the buffer solution was replaced with a 20 mM histidine-histidine hydrochloride solution at pH 6.2 to obtain the antibody-drug conjugate ACD234141 (5.2 mg / mL, 6 mL). The RP-HPLC-MS results are shown below. Figure 1 The average value calculated by RP-MS was n=7.9. ​​The MS results showed that the antibody light chain (L) was linked to one linker-payload (retention time was 13.71 min), and the heavy chain (H) was linked to three linker-payloads (retention time was 13.98 min).

[0183] 2. Preparation of ACD234144 Take CDH17 antibody (10.0 mg / mL, 50 mg, 0.33 μL), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate solution (125 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) solution (10 mM, 0.2 mL), and react with a rotating disc at room temperature (25 °C) for 4 h.

[0184] AK34144 (3.4 mg, 3.3 μmol / L) was dissolved in 0.3 mL of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature using a rotating disc for 16 h. After the reaction was complete, small molecules were removed using a NAP-5 gel column (Cytiva), and the buffer solution was replaced with a 20 mM histidine-histidine hydrochloride solution at pH 6.2 to obtain the antibody-drug conjugate ACD234144 (6.1 mg / mL, 6 mL). The RP-HPLC-MS results are shown below. Figure 2 The average value calculated by RP-MS was n=7.8. The MS results showed that the antibody light chain (L) was linked to one linker-payload (retention time was 14.02 min), and the heavy chain (H) was linked to three linker-payloads (retention time was 14.52 min).

[0185] 3. Preparation of ACD251541 Take CDH17 antibody (10.0 mg / mL, 50 mg, 0.33 μL), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate solution (125 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) solution (10 mM, 0.2 mL), and react with a rotating disc at room temperature (25 °C) for 4 h.

[0186] AK51541 (3.5 mg, 3.3 μmol) was dissolved in 0.3 mL of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature using a rotating disc for 16 h. After the reaction was complete, small molecules were removed using a NAP-5 gel column (Cytiva), and the buffer solution was replaced with a 20 mM histidine-histidine hydrochloride solution at pH 6.2 to obtain the antibody-drug conjugate ACD251541 (5.2 mg / mL, 6 mL). The RP-HPLC-MS results are shown below. Figure 3 The average value calculated by RP-MS was n=8.0. The MS results showed that the antibody light chain (L) was linked to one linker-payload (retention time was 13.07 min), and the heavy chain (H) was linked to three linker-payloads (retention time was 13.07 min).

[0187] Example 11: Preparation of Trop2 antibody-drug conjugate 1. Preparation of ACP227541 Take Trop2 antibody (7.0 mg / mL, 70 mg, 0.46 μL), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 250 μL of 0.1 M disodium ethylenediaminetetraacetate solution, and add 0.12 mL of prepared TCEP·HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) solution. Rotate the plate at room temperature (25 °C) for 2 h.

[0188] AK27541 (5.1 mg, 4.6 μmol / L) was dissolved in 0.5 mL of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature using a rotating disc for 2 h. After the reaction was complete, small molecules were removed using a NAP-5 gel column (Cytiva), and the buffer solution was replaced with a 20 mM histidine-histidine hydrochloride solution at pH 6.2 to obtain the antibody-drug conjugate ACP227541 (4.7 mg / mL, 10 mL). The RP-HPLC-MS results are shown below. Figure 4The average value calculated by RP-MS was n=7.2. The MS results showed that the antibody light chain (L) was linked to one linker-payload (retention time was 24.14 min), and the heavy chain (H) was linked to three linker-payloads (retention time was 23.37 min).

[0189] 2. Preparation of ACP251541 Take Trop2 antibody (7.0 mg / mL, 70 mg, 0.46 μL), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 250 μL of 0.1 M disodium ethylenediaminetetraacetate solution, and add 0.12 mL of prepared TCEP·HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) solution. Rotate the plate at room temperature (25 °C) for 4 h.

[0190] AK51541 (5.2 mg, 4.6 μmol / L) was dissolved in 0.1 mL of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature using a rotating disc for 16 h. After the reaction was complete, small molecules were removed using a NAP-5 gel column (Cytiva), and the buffer solution was replaced with a 20 mM histidine-histidine hydrochloride solution at pH 6.2 to obtain the antibody-drug conjugate ACP251541 (4.9 mg / mL, 10 mL). The RP-HPLC-MS results are shown below. Figure 5 The average value calculated by RP-MS was n=7.8. The MS results showed that the antibody light chain (L) was linked to one linker-payload (retention time was 14.44 min), and the heavy chain (H) was linked to three linker-payloads (retention time was 13.94 min).

[0191] Experimental Example 1: In vivo inhibition of tumor growth activity by ADCs Methods for testing the tumor-inhibiting activity of ADC in nude mouse cell-derived xenograft (CDX) models: Colorectal cancer cells GP2D, HT55, NCI-H716, SW403, and LoVo, and gastric cancer cells KATO-III and SNU16 were cultured in vitro in a monolayer. When the cell saturation reached 80%-90%, the cells were digested with trypsin-EDTA, centrifuged, and the supernatant was discarded. The cells were resuspended in PBS, and the cell suspension was adjusted to an appropriate concentration. Cells (2-10 × 10⁻⁶ cells / mL) were then... 6Subcutaneous inoculation of 0.1 mL of tumor cells into BALB / c nude mice was performed. Animals and tumor growth were observed regularly. Once the tumor volume reached approximately 100-200 mm³, mice were randomly assigned to groups of 6 animals each based on tumor volume and body weight. The medication was administered intravenously regularly. The major diameter (a) and minor diameter (b) of the tumor, along with the mouse's body weight, were measured using calipers 2-3 times per week. Tumor volume (V) was calculated using the following formula: V = 1 / 2 × a × b 2 (mm) 3 (a) and (b) represent the tumor length and width, respectively, and growth curves are plotted. Statistical analysis was performed using GraphPad Prism software based on tumor volume data at the end of the experiment to obtain tumor suppression results.

[0192] Key evaluation indicators: Tumor growth inhibition rate (TGI): TGI (%) = [1 - (avTi-0 / avCi-0)] × 100%; where avTi-0 is the average tumor volume of the treatment group on a specific day, minus the average tumor volume of the treatment group on the first day of administration; where avCi-0 is the average tumor volume of the solvent control group on a specific day, minus the average tumor volume of the solvent control group on the first day of administration.

[0193] 1. GP2D colorectal cancer model The drug was administered twice, on days 0 and 4, and the observation period ended on day 29. The tumor inhibition curve of the GP2D colorectal cancer model is shown in [Figure number missing]. Figure 6 .

[0194] Among them, IgG1 is a monoclonal antibody tool sequence without target selectivity, and it has no affinity for human cells.

[0195] The structural formula of ACD210002 is: .

[0196] Table 2. Tumor suppression in the GP2D colorectal cancer model Experimental results: ACD234144 at a dose of 5 mg / kg showed stronger tumor growth inhibitory activity than non-targeting ADC (IgG1-234144) and targeted ADC (ACD210002).

[0197] 2. HT55 colorectal cancer model The drug was administered three times, on days 0, 3, and 16, and observation was completed on day 25. The tumor inhibition curve of the HT55 colorectal cancer model is shown in [Figure number missing]. Figure 7.

[0198] Table 3. Tumor suppression in the HT55 colorectal cancer model Experimental results: ACD234144 at a dose of 5 mg / kg showed stronger tumor growth inhibitory activity than non-targeting ADC (IgG1-234144) and targeted ADC (ACD210002).

[0199] 3. NCI-H716 colorectal cancer model The drug was administered twice, on days 0 and 4, and observation continued until day 25. Tumor inhibition curves for the NCI-H716 colorectal cancer model are shown below. Figure 8 .

[0200] Table 4. Tumor suppression in the NCI-H716 colorectal cancer model Experimental results: ACD234144 at a dose of 5 mg / kg showed stronger tumor growth inhibitory activity than non-targeting ADC (IgG1-234144) and targeted ADC (ACD210002).

[0201] 4. SW403 colorectal cancer model The drug was administered twice, on days 0 and 4, and the observation period ended on day 25. The tumor inhibition curve of the SW403 colorectal cancer model is shown in [Figure number missing]. Figure 9 .

[0202] Table 5. Tumor suppression in the NCI-H716 colorectal cancer model Experimental results: ACD234144 at a dose of 5 mg / kg showed stronger tumor growth inhibitory activity than non-targeting ADC (IgG1-234144) and targeted ADC (ACD210002).

[0203] 5. LoVo colorectal cancer model The drug was administered three times, on days 0, 4, and 19, and observation was completed on day 25. Tumor inhibition curves for the LoVo colorectal cancer model are shown below. Figure 10 .

[0204] Table 6. Tumor suppression in the LoVo colorectal cancer model Experimental results: ACD234144 at a dose of 5 mg / kg showed stronger tumor growth inhibition activity than the targeted ADC (ACD210002).

[0205] 6. KATO III gastric cancer model The drug was administered three times, on days 0, 3, and 10, and the observation period ended on day 35. The tumor inhibition curve for the KATO III gastric cancer model is shown in [Figure number missing]. Figure 11 .

[0206] Table 7. Tumor suppression in the KATO III gastric cancer model Experimental results: ACD234144 at a dose of 5 mg / kg showed stronger tumor growth inhibition activity than the targeted ADC (ACD210002).

[0207] 7. SNU16 gastric cancer model The drug was administered twice, on days 0 and 4, and the observation period ended on day 31. The tumor inhibition curve of the SNU16 gastric cancer model is shown in [Figure number missing]. Figure 12 .

[0208] Table 8. Tumor suppression in the SNU16 gastric cancer model Experimental results: ACD234144 at a dose of 5 mg / kg showed stronger tumor growth inhibition activity than the targeted ADC (ACD210002).

[0209] The specific sequence of the HS627 antibody is as follows: HS627 antibody heavy chain amino acid sequence SEQ ID NO.1: EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the light chain of HS627 antibody SEQ ID NO.2: DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC The specific sequence of the CDH17 antibody is as follows: Amino acid sequence of the heavy chain of CDH17 antibody SEQ ID NO.3: QVQLVQSGAEVKKPGASVKVSCKASGYTFSSRYMHWVRQAPGQGLEWMGIINPKTGSRNHAQKFQGRVTLTRDTATDTVYMELSSLRSEDTAVYYCASQGHSSLFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Amino acid sequence of the light chain of the CDH17 antibody, SEQ ID NO.4: DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC The specific sequence of the Trop2 antibody is as follows: Amino acid sequence of the heavy chain of the Trop2 antibody, SEQ ID NO.5: EVQLVESGGGLVQPGGSLRLSCAASGFTFNNYAMSWVRQAPGKGLEWVSTISSDGTYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHPPSYYYAFDYWGQ GTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Trop2 antibody light chain amino acid sequence SEQ ID NO.6: EIVLTQSPGTLSLSPGERATLSCRASESVDSYGNSFIHWYQQKPGQAPRLLIYLASNLESGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQNNEDLWTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC The foregoing description of specific exemplary embodiments of this disclosure is for illustrative and explanatory purposes. These descriptions are not intended to limit this disclosure to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of this disclosure and their practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of this disclosure, as well as various different choices and variations. The scope of this disclosure is intended to be defined by the claims and their equivalents.

Claims

1. A load-linker conjugate or a pharmaceutically acceptable salt, stereoisomer, optical isomer, deuterated derivative, solvate, or prodrug thereof, said load-linker conjugate having the structure shown in formula (I): （I） In the formula, L1 is a divalent structural unit that connects the cysteine ​​side chain group of the antibody to the L2 group; L2 is a bivalent structural unit connecting L1 and L3; L3 is a unit of n1 amino acids that can be digested by enzymes; A is selected from H and C. 1-6 Alkyl, C 3-6 cycloalkyl, C 1-6 Halogenated alkyl or C 1-6 Alkoxy; X is selected from H and C. 1-6 Alkyl, C 3-6 cycloalkyl, C 1-6 Halogenated alkyl or C 1-6 Alkyl groups; R4 is selected from H and C. 1-6 Alkyl, C 3-6 cycloalkyl, C 1-6 Halogenated alkyl or C 1-6 Alkyl groups; D is a TOP-1 inhibitor loading drug containing a hydroxylamine group, which forms -NX-CH2-NO- through the amide of L3 and the loaded hydroxylamine; n1 is an integer selected from 1 to 15; n2 is an integer selected from 0 to 10.

2. The load-linker conjugate according to claim 1, or its pharmaceutically acceptable salt, stereoisomer, optical isomer, deuterated derivative, solvate, or prodrug, characterized in that, The load-connector coupling has the structure shown in formula (II): （Ⅱ）； In formula (II), L1, L2, L3, A, X, R4, D, n1, and n2 are each defined in the same way as in formula (I); R1, R2, and R3 are each independently selected from H, halogens, and C. 1-6 Alkyl, C 3-6 cycloalkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl; or any two substituents from R1, R2, and R3 together form a methylene dioxy substituent group (-O-CH2-O-).

3. The load-linker conjugate according to claim 1 or 2, or its pharmaceutically acceptable salt, stereoisomer, optical isomer, deuterated derivative, solvate, or prodrug, characterized in that, L1 is a divalent structural unit that connects the antibody thiol group and the L2 group; Preferably, L1 is selected from divalent structural units of maleimide, bromoacetyl, 2-bromomethylpyridine-4-carboxyl, or 2-bromomethyl-3-oxo-dihydroquinoxaline-6-carboxyl. Preferably, L1 is selected from , , and ;" The indicated position represents the attachment to the cysteine ​​side chain group of the antibody. The position shown indicates that it is connected to the L2 group; n3 is selected from integers from 0 to 10; Preferably, n3 is selected from integers from 0 to 8; Preferably, n3 is selected from integers from 0 to 6; Preferably, n3 is selected from integers from 0 to 3; Preferably, n3 is selected from integers from 0 to 2; Preferably, n3 is selected from 0 or 1; Preferably, L1 is selected from , , and ;" The indicated position represents the attachment to the cysteine ​​side chain group of the antibody. The position shown indicates that it is connected to the L2 group; Preferably, L2 is selected from -C(=O)-C1-C8 alkylene groups and -C(=O)-CH2O-(CH2CH2O). 2-5 -CH2CH2NH-、-C(=O)-(CH2CH2O) 2-6 -CH2CH2-、-C(=O)-(CH2CH2O) 2-6 -CH2CH2NH- and -C(=O)-(CH2) 0-6 -NR5(CH2) 0-6 -, R5 is selected from -C1-C6 alkyl-carboxyl or -C1-C6 alkyl-amino; Preferably, L2 is selected from -C(=O)-(CH2)5-, -C(=O)-CH2O-(CH2CH2O)3-CH2CH2NH-, -C(=O)-(CH2CH2O)2-CH2CH2-, -C(=O)-(CH2CH2O)2-CH2CH2NH- and -C(=O)-(CH2CH2O)4-CH2CH2NH-, ; Preferably, L2 is selected from -C(=O)-C1-C8 alkylene groups and -C(=O)-(CH2CH2O). 2-6 -CH2CH2NH-、 ; Preferably, L1-L2 are selected from , , , , , and " The indicated position represents the link to the antibody's thiol group; The position indicated is linked to an amino acid group; Preferably, L3 is a peptide residue composed of 1-8 amino acids; the amino acids are selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit) and aspartic acid (Asn); Preferably, L3 is selected from 2-5 peptide residues composed of amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit) and aspartic acid (Asn). Preferably, L3 is selected from Val-Cit, Val-Ala, Gly-Val-Ala, Gly-Val-Ala-Gly, Gly-Lys, Gly-Gly-Lys, Gly-Gly-Lys-Gly, Va l-Ala, Ala-Ala-Asn, Gly-Leu, Gly-Gly-Leu, Gly-Gly-Leu-Gly, Gly-Phe, Gly-Gly-Phe and Gly-Gly-Phe-Gly; Preferably, L3 is selected from two or three peptide residues composed of amino acids selected from glycine (Gly), valine (Val), alanine (Ala), lysine (Lys), and citrulline (Cit). Preferably, L3 is selected from , and Its carbonyl end is connected to -NH-, and the other end is connected to L2; Preferably, A is selected from H and C. 1-5 Alkyl, C 3-5 cycloalkyl, C 1-5 Halogenated alkyl or C 1-5 Alkyl groups; Preferably, A is selected from H and C. 1-5 Alkyl, C 1-5 Halogenated alkyl or C 1-5 Alkyl groups; Preferably, A is selected from H and C. 1-4 Alkyl, C 1-4 Halogenated alkyl or C 1-4 Alkyl groups; Preferably, A is selected from H and C. 1-3 Alkyl, C 1-3 Halogenated alkyl or C 1-3 Alkyl groups; Preferably, A is selected from H and C. 1-3 alkyl; Preferably, A is selected from H, methyl, propyl, and isopropyl; Preferably, A is selected from H, methyl, or isopropyl; Preferably, X is selected from H and C. 1-5 Alkyl, C 3-5 cycloalkyl, C 1-5 Halogenated alkyl or C 1-5 Alkyl groups; Preferably, X is selected from H and C. 1-5 Alkyl, C 1-5 Halogenated alkyl or C 1-5 Alkyl groups; Preferably, X is selected from H and C. 1-4 Alkyl, C 1-4 Halogenated alkyl or C 1-4 Alkyl groups; Preferably, X is selected from H and C. 1-3 Alkyl, C 1-3 Halogenated alkyl or C 1-3 Alkyl groups; Preferably, X is selected from H and C. 1-3 alkyl; Preferably, X is selected from H or methyl; Preferably, X is H; Preferably, R4 is selected from H and C. 1-5 Alkyl, C 3-5 cycloalkyl, C 1-5 Halogenated alkyl or C 1-5 Alkyl groups; Preferably, R4 is selected from H and C. 1-5 Alkyl, C 1-5 Halogenated alkyl or C 1-5 Alkyl groups; Preferably, R4 is selected from H and C. 1-4 Alkyl, C 1-4 Halogenated alkyl or C 1-4 Alkyl groups; Preferably, R4 is selected from H and C. 1-3 Alkyl, C 1-3 Halogenated alkyl or C 1-3 Alkyl groups; Preferably, R4 is selected from H and C. 1-3 alkyl; Preferably, R4 is selected from methyl or ethyl; Preferably, R4 is a methyl group; Preferably, R1, R2, and R3 are each independently selected from H, halogens, and C. 1-5 Alkyl, C 3-5 cycloalkyl, C 1-5 Haloalkyl, C 1-5 Alkoxy, C 1-5 Hydroxyalkyl; or any two substituents in R1, R2, and R3 together form a methylene dioxy substituent group (-O-CH2-O-). Preferably, R1, R2, and R3 are each independently selected from H, halogens, and C. 1-3 Alkyl, C 3-4 cycloalkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy, C 1-3 Hydroxyalkyl; or any two substituents in R1, R2, and R3 together form a methylene dioxy substituent group (-O-CH2-O-). Preferably, R1, R2, and R3 are each independently selected from H, fluorine, chlorine, bromine, and C. 1-3 Alkyl, C 3-4 cycloalkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy, C 1-3 Hydroxyalkyl; or any two substituents in R1, R2, and R3 together form a methylene dioxy substituent group (-O-CH2-O-). Preferably, R1, R2, and R3 are each independently selected from H, fluorine, chlorine, bromine, and C. 1-3 Alkyl group; or any two substituents in R1, R2, and R3 together form a methylene dioxy substituent group (-O-CH2-O-). Preferably, R1, R2, and R3 are each independently selected from H, fluorine, chlorine, methyl, and ethyl; or any two substituents from R1, R2, and R3 together form a methylene dioxy substituent group (-O-CH2-O-). Preferably, R1, R2, and R3 are each independently selected from H, fluorine, methyl, and ethyl; or any two substituents in R1, R2, and R3 together form a methylene dioxy substituent group (-O-CH2-O-). Preferably, R1, R2, and R3 are each independently selected from H, fluorine, and methyl; or any two substituents in R1, R2, and R3 together form a methylene dioxy substituent group (-O-CH2-O-). Preferably, n1 is selected from integers from 1 to 10; Preferably, n1 is selected from integers from 1 to 8; Preferably, n1 is selected from integers from 1 to 5; Preferably, n1 is selected from integers from 2 to 5; Preferably, n1 is 2 or 3; Preferably, n2 is selected from integers from 0 to 8; Preferably, n2 is selected from integers from 0 to 6; Preferably, n2 is selected from integers from 0 to 5; Preferably, n2 is selected from integers from 1 to 5; Preferably, n2 is selected from integers from 1 to 4; Preferably, n2 is selected from integers from 1 to 3.

4. The load-linker conjugate according to claim 1 or 2, or its pharmaceutically acceptable salt, stereoisomer, optical isomer, deuterated derivative, solvate, or prodrug, characterized in that, The load-linker conjugate is selected from the following compounds in a form that can be linked to the antibody cysteine ​​side chain group: 。 5. An antibody-drug conjugate or its pharmaceutically acceptable salt, stereoisomer, optical isomer, deuterated derivative, solvate, or prodrug, characterized in that, The structure of the antibody-drug conjugate is shown in formula (III): （Ⅲ） In equation (Ⅲ), Ab is a target connector, which is a structure that can target and bind to the lesion site; S is a cysteine ​​side chain group of the antibody or its binding fragment; n is any number between 1 and 20; L1, L2, L3, A, X, R4, D, n1, and n2 are each defined in the same formula (I).

6. The antibody-drug conjugate according to claim 5, or a pharmaceutically acceptable salt, stereoisomer, optical isomer, deuterated derivative, solvate, or prodrug thereof, characterized in that, The structure of the antibody-drug conjugate is shown in formula (Ⅳ): （Ⅳ） In equation (Ⅳ), Ab is a target connector, which is a structure that can target and bind to the lesion site; S is a cysteine ​​side chain group of the antibody or its binding fragment; n is any number between 1 and 20; L1, L2, L3, A, X, R4, D, n1, and n2 are each defined in the same way as in formula (I); R1, R2, and R3 are each defined in the same way as in formula (II).

7. The antibody-drug conjugate according to claim 6, or its pharmaceutically acceptable salt, stereoisomer, optical isomer, deuterated derivative, solvate, or prodrug, characterized in that, The antibody-drug conjugate is denoted as ADC, and the antibody-drug conjugate is selected from the following compounds: in, Ab is a target connector, which is a structure that can target and bind to the lesion site; S is a cysteine ​​side chain group of the antibody or its binding fragment; n is any number between 1 and 20; Preferably, the antibody-drug conjugate is selected from the following compounds: in, S is a cysteine ​​side chain group of the antibody or its binding fragment; n is any number between 1 and 20.

8. The antibody-drug conjugate according to any one of claims 5 to 7, or a pharmaceutically acceptable salt, stereoisomer, optical isomer, deuterated derivative, solvate, or prodrug thereof, characterized in that, The Ab is selected from antibodies or their binding fragments, proteins, small molecule polypeptides, glycopeptides, mimic peptides, or nucleic acid oligonucleotide aptamers; Preferably, the Ab is an antibody targeting a tumor antigen or a binding fragment thereof; Preferably, the antibody is selected from anti-HS627 antibody, anti-Trop2 antibody, anti-CDH17 antibody, anti-HER3 antibody, anti-B7H3 antibody, anti-HER2 antibody, anti-Nectin-4 antibody, anti-CD20 antibody, anti-ROR1 antibody, or anti-CEACAM5 antibody. Preferably, the antibody is selected from anti-HS627 antibody, anti-Trop2 antibody, and anti-CDH17 antibody; Preferably, n is any number between 1 and 15; Preferably, n is any number between 1 and 12; Preferably, n is any number between 1 and 10; Preferably, n is any number between 1 and 8; Preferably, n is any number between 2 and 8.

9. A pharmaceutical composition comprising the load-linker conjugate of any one of claims 1 to 4 or a pharmaceutically acceptable salt, stereoisomer, optical isomer, deuterated derivative, solvate, or prodrug thereof, or the antibody-drug conjugate of any one of claims 5 to 8 or a pharmaceutically acceptable salt, stereoisomer, optical isomer, deuterated derivative, solvate, or prodrug thereof, and optionally a pharmaceutically acceptable carrier.

10. Use of the load-linker conjugate of any one of claims 1 to 4 or a pharmaceutically acceptable salt, stereoisomer, optical isomer, deuterated derivative, solvate or prodrug thereof, the antibody-drug conjugate of any one of claims 5 to 8 or a pharmaceutically acceptable salt, stereoisomer, optical isomer, deuterated derivative, solvate or prodrug thereof, or the pharmaceutical composition of claim 9 in the preparation of a medicament for the prevention and / or treatment of tumors; Preferably, the tumor is selected from breast cancer, lung cancer, colorectal cancer, esophageal cancer, gastric cancer, kidney cancer, ovarian cancer, cervical cancer, bladder cancer, head and neck cancer, pancreatic cancer, liver cancer, urethral cancer, prostate cancer, squamous cell carcinoma, peritoneal cancer, salivary gland cancer, thyroid cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma. Preferably, the tumor is selected from breast cancer, lung cancer, colorectal cancer, esophageal cancer, gastric cancer, kidney cancer, ovarian cancer, cervical cancer, bladder cancer, head and neck cancer, pancreatic cancer, or liver cancer; Preferably, the tumor is colorectal cancer or gastric cancer.

Images