Substituted fused heteroaromatic bicyclic compounds, and their linker-drug and antibody drug conjugates

Abstract

The present application provides a kind of substituted fused heteroaromatic bicyclic compound, its linker-drug, antibody drug conjugate and purposes thereof. Specifically, the present application provides a kind of linker-drug and the antibody drug conjugate containing the linker-drug, the linker-drug is expressed as V-L-D, wherein V-L is linker, D is at least including the drug unit derived from the compound or its deuterium derivative shown in the following formula i, wherein the compound of formula i is ATR inhibitor.

Description

Technical Field

[0001] This invention relates to substituted fused heterocyclic aromatic compounds, their linkers-drugs, and antibody-drug conjugates. Background Technology

[0002] Antibody-drug conjugates (ADCs) are drugs that link antibodies or antibody fragments to small-molecule cytotoxic drugs via specific linkers. They leverage the specificity of antibodies binding to tumor surface antigens and the high efficiency of small-molecule cytotoxic drugs in killing tumor cells, overcoming the limitations of low efficacy of antibodies alone and the significant toxic side effects of small-molecule cytotoxic drugs. Several ADCs have been approved for marketing, such as brentuximab velituximab, trastuzumab, goxatuzumab, and lucanstatuzumab. In the past decade, ADCs have achieved significant success in cancer treatment. However, due to the heterogeneity and drug resistance of tumor cells, the efficacy of single-loaded drug ADCs has certain limitations. Dual toxins can reduce drug resistance because they can overcome intracellular or extracellular obstacles that single toxins may encounter, such as target expression downregulation, drug pump expression upregulation, and activation of DNA repair mechanisms. ADCs can utilize the high specificity and high affinity of antibodies to precisely deliver two synergistic drugs into tumor cells, thereby achieving a highly efficient killing effect. Currently, several dual-toxin ADCs have been publicly disclosed. These dual-toxin drug combinations mainly consist of different toxins, including combinations with the same or different mechanisms of action. Examples include: MMAE / MMAF (Nat Commun 2021, 12: 3528; Angew Chem Int Ed Engl 2017, 56:733-7; Bioconjugate Chem. 2023, 34, 4, 748-755); DM1 / MMAE (Nature Protocols 2017, 12: 1702-21; Pharmaceutics 2023, 15: 2020); PNU-159682 / MMAF (Antib Ther 2019, 2: 71-8); MMAE / a-Amanitin (Int J Mol Sci 2018, 19: 2098); and MMAE / PBD (Bioorg Med Chem Lett.). 2018, 28: 3617-21). Early studies have demonstrated that bitoxic ADC molecules can significantly improve drug efficacy and enhance the killing activity of ADC drugs against drug-resistant tumor cells.

[0003] TOPO I inhibitors (TOPOIi) are a class of anticancer drugs that inhibit the activity of topoisomerase I. Topoisomerase I plays a crucial role in DNA replication and transcription; its inhibitors stabilize the DNA-topoisomerase I complex, leading to single-strand breaks in DNA, thereby inducing replication stress and DNA damage. In recent years, TOPO I has demonstrated significant efficacy in the treatment of various tumors. For example, Trastuzumab deruxtecan (T-DXd), an ADC drug targeting HER2, has shown excellent antitumor effects in HER2-positive breast cancer and gastric cancer. However, drug resistance remains a major challenge in TOPO I inhibitor therapy, and its mechanism may be related to the activation of DNA damage repair pathways such as ATR / CHK1.

[0004] Rad3-associated kinase (ATR), a mutant gene associated with ataxia-telangiectasia, is a protein kinase that responds to DNA damage in the cell. Once activated, ATR regulates various cellular life processes through multiple signaling pathways, including cell cycle arrest, inhibition of replication origins, initiation of replication forks, and repair of DNA double strands (Enriquez-Rios V et al., 2017). ATR is a key member of the DDR pathway, the mechanism of DNA replication response to damage, and is crucial for maintaining genomic stability and integrity, and promoting cell survival. When intracellular DNA damage occurs, ATR is recruited to the site of DNA damage, and various proteins subsequently participate in regulating ATR activation. ATR activation then regulates several important cellular processes. Many cancer cells lack key tumor suppressor genes, which allows cancer cells to rely more heavily on the ATR pathway to regulate DNA damage repair and promote cell survival than normal cells. Because ATR inhibitors disrupt the DNA replication mechanism, they can be used alone or in combination with DNA-damaging agents for cancer treatment. To date, several ATR inhibitors are in clinical trials, such as Ceralaseritb, Alnodesertib, Tuvusertib, Camonsertib, and Berzosertib. These ATR inhibitors have been shown to be effective as single active agents in cancer cells and can also serve as effective sensitizers for radiotherapy and chemotherapy.

[0005] Developing dual-load drugs combining TOPO I inhibitors and ATR inhibitors has significant clinical implications. First, this combination strategy can simultaneously target DNA damage and repair pathways, overcoming the resistance issues associated with single-drug therapy. Second, dual-load technology enables precise delivery, reducing systemic toxicity and improving drug tolerability. Finally, this innovative treatment modality holds promise for providing more effective treatment options for various cancer patients, especially in tumor types with a high prevalence of drug resistance. Summary of the Invention

[0006] The present invention provides substituted fused heteroaromatic bicyclic compounds with structures as shown in Formula i (including Formula ii), which are ATR inhibitors and can be used as ADC loads.

[0007] The present invention also provides compounds with structures as shown in Formula I (including Formulas Ia, Ib, Ic, Id and Ie), which can be coupled to antibody molecules by coupling technology to form single, dual, triple or multiple ADCs.

[0008] The present invention also provides compounds of formula II (including formulas IIa, IIb, IIc, IId and IIe) and formula III, which are single, double, triple or multiple ADCs capable of releasing toxin molecules including ATR inhibitors.

[0009] Specifically, the present invention also provides a pharmaceutical composition comprising an effective amount of a compound of formula II (including formula IIa, IIb, IIc, IId and IIe) or formula III, which may be used to treat cancer.

[0010] In one specific embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or diluents.

[0011] In one specific embodiment, the pharmaceutical composition further contains at least one known anticancer drug or a pharmaceutically acceptable salt of the anticancer drug.

[0012] The present invention also relates to methods for preparing compounds of formula i (including formula ii), formula I (including formulas Ia, Ib, Ic, Id and Ie), formula II (including formulas IIa, IIb, IIc, IId and IIe) and formula III.

[0013] Instruction manual illustrations

[0014] Figure 1 Changes in tumor volume in HPAF-II xenograft tumor model mice of human pancreatic cancer after intravenous administration of solvent, C-ADC0-1 and C-ADC56-1-1.

[0015] Figure 2 Changes in tumor volume in HPAF-II xenograft tumor model mice of human pancreatic cancer after intravenous administration of solvent, C-ADC0-1 and C-ADC56-2-1.

[0016] Figure 3 Changes in tumor volume in tumor-bearing mice of the human colon cancer LS-1034 xenograft tumor model after intravenous administration of solvent, C-ADC0-1 and C-ADC56-2-1.

[0017] Figure 4Changes in tumor volume in tumor-bearing mice of the human colon cancer LS-1034 xenograft tumor model after intravenous administration of solvent, C-ADC0-2 and C-ADC56-2-2.

[0018] Figure 5 Changes in tumor volume in xT-84 xenograft tumor-bearing mice with human colon cancer after intravenous administration of solvent, C-ADC56-2-2 and C-ADC59-2-2.

[0019] Figure 6 Changes in tumor volume in tumor-bearing mice of the human breast cancer JIMT-1 xenograft tumor model after intravenous administration of solvent and H-ADC-L04-1. Detailed Implementation

[0020] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form preferred technical solutions.

[0021] definition

[0022] Unless otherwise stated, the terms used herein have their common meanings in the art.

[0023] In this article, the term "hydrogen (H)" includes its isotopes deuterium (D) and tritium (T).

[0024] In this article, "halogen" or halogen group includes fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0025] In this document, the term "alkyl" refers to a saturated straight-chain or branched aliphatic hydrocarbon group, which can be a straight-chain or branched group containing 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more carbon atoms. Useful alkyl groups include straight-chain or branched C4 groups. 1-12 Alkyl groups, preferably straight-chain or branched C4 groups. 1-6 Alkyl group. In some embodiments, the alkyl group is a straight-chain or branched C-chain. 1-4 Alkyl group. Typical C 1-12 Alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, 3-pentyl, hexyl, and octyl, which may be optionally substituted. In some embodiments, the alkyl group is substituted with deuterium, such as deuterated methyl.

[0026] In this document, the term "alkylene" refers to a divalent group derived by removing two hydrogen atoms from the same or different carbon atoms of a saturated straight-chain or branched aliphatic hydrocarbon (such as the alkyl groups described herein). An exemplary alkylene group is C10. 1-12 Alkylene, such as C 1-6 Alkylene or C1-4 Alkylenes. Specific alkylenes include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, and hexylene. The alkylenes may optionally be substituted.

[0027] In this document, the term "alkoxy" refers to an alkyl group substituted with an alkyl group as described herein, and can be represented as RO-, where R represents an alkyl group. Preferred alkoxy groups are C-. 1-6 Alkoxy or C 1-4 Alkoxy groups. Exemplary alkoxy groups include methoxy, ethoxy, etc. The alkyl group in an alkoxy group may optionally be substituted. Substituents of an alkoxy group include, but are not limited to, halogens, amino groups, and carboxyl groups (including their ester groups), wherein the amino group includes alkylamino and dialkylamino groups. The term "alkeneoxy" refers to an alkylene-substituted alkoxy group as described herein, and may be represented as -RO-, where R represents alkylene.

[0028] In this document, the term "alkenyl" refers to a group containing 2-10 carbon atoms in a straight or branched chain, unless otherwise limited by the chain length, wherein at least two carbon atoms in the chain are connected by a double bond. The number of carbon atoms in an alkenyl group can be 2, 3, 4, 5, 6, 7, 8, 9, or 10, or within any two of the above ranges. The alkenyl group is preferably C10. 2-6 alkenyl or C 2-4 Alkenyl groups. Typical alkenyl groups include vinyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, and 2-butenyl.

[0029] In this document, the term "alkenyl" refers to a divalent group derived from the same or different carbon atoms of an alkene described herein by removing two hydrogen atoms. It should be understood that an alkenyl still contains at least one carbon-carbon double bond. Exemplary alkenyl groups include C1... 2-6 imide or C 2-4 Alkenyl groups, such as vinylene, propenylene, butenylene, pentenylene, and hexenylene, etc. The alkenyl groups may optionally be substituted.

[0030] In this document, the term "alkynyl" refers to a group containing 2 to 10 carbon atoms in a straight or branched chain, wherein at least two carbon atoms in the chain are connected by a triple bond. The number of carbon atoms in an alkynyl group can be 2, 3, 4, 5, 6, 7, 8, 9, or 10, or within any two of the above ranges. The alkynyl group is preferably C10. 2-6 alkynyl or C 2-4 Alkynyl. Typical alkynyl groups include ethynyl, 1-propynyl, 1-methyl-2-propynyl, 2-propynyl, 1-butynyl, and 2-butynyl.

[0031] In this document, the term "ynynyl" refers to a divalent group derived from the removal of two hydrogen atoms from the same or different carbon atoms of an alkyne. It should be understood that an ynynyl group contains at least one carbon-carbon triple bond. Exemplary ynynyl groups include C1... 2-6 Ethyne or C 2-4 The ynyl group, such as ethynylene, propynylene, butynylene, pentyynylene, and hexynylene, etc., may be optionally substituted.

[0032] In this article, the term "amino" can be –NR ′ R ′′ It means that R ′ and R ′′ Each is independently hydrogen, and the C atoms can be optionally substituted. 1-10 Alkyl, optionally substituted C 3-8 Cycloalkyl, optionally substituted heterocyclic, optionally substituted aryl, or optionally substituted heteroaryl; preferably, R ′ and R ′′ Each is independently H, and the C can be arbitrarily substituted. 1-4 Alkyl, optionally substituted C 3-6 Cycloalkyl or optionally substituted 5-membered heteroaryl; or R ′ and R ′′ Together with the N atoms attached to them, they form optionally substituted nitrogen-containing heterocyclic groups or nitrogen-containing heteroaryl groups, such as 4- to 7-membered nitrogen-containing heterocyclic groups or nitrogen-containing heteroaryl groups, which optionally also contain one or more (e.g., 2 or 3) heteroatoms selected from O, N, and S. Preferred amino groups include –NH2, –NHR. ′ and –NHR ′′ , where R ′ and R ′′ Each can be independently replaced by C 1-4 Alkyl, optionally substituted C 3-6 Cycloalkyl groups and optionally substituted 3-6 membered heterocyclic groups.

[0033] In this document, the term "acyl" may be represented as RC(O)-, where R can be any of the alkyl, cycloalkyl, heterocycloalkyl, alkoxy, heterocycloalkoxy, alkenyl, heterocycloalkenyl, alkynyl, amino, carboxyl, hydroxyl, mercapto, alkylthio, silyl, phosphonic, phosphonyl, carbocyclic, heterocyclic, aryl, heteroaryl, alkylene, alkenyl, arylene, heteroarylene, carbocyclic, or heterocyclic groups described in any of the embodiments herein. For example, an alkyl acyl group may be C 1-6 Alkyl acyl (C 1-6 Alkyl-C(O)-). For example, the alkylene acyl group can be C. 1-6 alkylene acyl (C 1-6 alkylene-C(O)-).

[0034] In this document, the term "aryl" refers to a monocyclic, bicyclic, or tricyclic aromatic group containing 6 to 14 carbon atoms, either as a component of another group. An aryl group can be substituted by one or more substituents as described herein. Useful aryl groups include C... 6-14 Aryl, preferably C 6-10 Aryl. Typical C 6-14 Aryl groups include phenyl, naphthyl, phenanthryl, anthraceneyl, indyl, azulel, biphenyl, biphenylene, and fumonisinyl.

[0035] In this document, the term "arylene" refers to a divalent group derived by removing two hydrogen atoms from the same or different carbon atoms of an aromatic ring. Exemplary arylene groups include C 6-14 Aryl groups, preferably C 6-10 Arylenes. Specific arylene groups include phenylene or naphthylene. The arylene groups may optionally be substituted.

[0036] In this document, the term "heteroaryl" refers to a group containing 5–14, preferably 5–10, ring atoms, and having 6, 10, or 14 π electrons shared in the ring system. The ring atoms in a heteroaryl group are carbon atoms and 1–3 heteroatoms selected from oxygen, nitrogen, and sulfur. A heteroaryl group may be substituted with one or more of the substituents described herein. Exemplary heteroaryl groups, as described herein, include, but are not limited to, furanyl, thiophene, pyridyl, pyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, pyrazolyl, and tetrazolyl. The heteroaryl group may optionally be substituted.

[0037] In this document, the term "heteroaryl" refers to a divalent group derived by removing two hydrogen atoms from the same or different ring atoms (such as carbon and / or nitrogen atoms) of the heteroaryl group described herein. Exemplary heteroaryl groups include, but are not limited to, furanyl, thiopheneyl, pyridinyl, pyrrolidinyl, pyrimidinyl, pyrazinyl, imidazolyl, pyrazolyl, and tetrazolyl. The heteroaryl group may optionally be substituted.

[0038] In this document, the term "carbocyclic group" includes cycloalkyl groups and partially saturated carbocyclic groups. The number of carbon atoms in a carbocyclic group can range from 3 to 10. Carbocyclic groups include spirocyclic groups and bridging groups. Useful cycloalkyl groups are C16-3 ... 3-8 cycloalkyl groups, such as C 3-6 cycloalkyl or C 3-4 Cycloalkyl groups. Typical cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Useful partially saturated carbocyclic groups include cycloalkenyl groups, such as C... 3-8 Cycloalkenyl, C 3-6 Cycloalkenyl or C 3-4 Cycloalkenyl groups, such as cyclopentenyl, cycloheptenyl, and cyclooctenyl. The carbocyclic group may be substituted by one or more substituents as described herein.

[0039] In this document, the term "carbocyclic group" refers to a divalent group, such as a cycloalkyl group and a cycloolefin group, derived by removing two hydrogen atoms from the same or different carbon atoms of the carbocyclic ring described herein. Exemplary cycloalkyl groups include C10, C20, C30, C40, C50, C60, C7 ... 3-8 Cycloalkylene and C 3-6 Cycloalkylene groups, exemplary cycloalkylene groups include C 3-8 Cycloalkylene and C 3-6 Cycloalkylene groups. Exemplary carbocycloalkyl groups include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclohexane, cyclohexenyl, cyclohexadienyl, cycloheptane, cycloheptanetrienyl, and cyclooctylene. The carbocycloalkyl group may optionally be substituted.

[0040] In this document, "heterocyclic group" refers to a saturated or partially saturated 3-7 membered monocyclic group, 7-10 membered bicyclic group, 11-14 membered tricyclic group, helical ring group, fused ring group, or bridged ring group, which consists of a carbon atom and 1-4 heteroatoms selected from O, N, and S. In some embodiments, the nitrogen and sulfur heteroatoms in the heterocyclic group can be arbitrarily oxidized, and the nitrogen can also be arbitrarily quaternized. Heterocyclic groups also include fused heterocycles formed by the fusion of any heterocycle in the monocyclic or bicyclic system defined above with an aromatic ring (such as a benzene ring) or a heteroaromatic ring (such as a pyridine ring, pyrazole ring, imidazole ring, etc.). If the resulting compound is stable, the carbon or nitrogen atom of the heterocycle can be substituted. The heterocyclic group can be substituted by one or more substituents described herein.

[0041] In this document, the term "subheterocyclic group" refers to a divalent group derived by removing two hydrogen atoms from the same or different reductants (such as carbon and / or nitrogen atoms) of the heterocycle described herein. The subheterocyclic group may optionally be substituted.

[0042] In this document, the term "coupled compound" generally refers to a compound prepared by one or more chemical reactions, or a compound connected to each other by one or more connecting structures such as a bridge, spacer, or connecting portion.

[0043] Unless otherwise stated herein, when substituted, the alkyl, cycloalkyl, heterocycloalkyl, alkoxy, heterocycloalkoxy, alkenyl, heterocycloalkenyl, alkynyl, amino, amide, acyloxy, carboxyl, hydroxyl, mercapto, alkylthio, sulfonyl, sulfinyl, silyl, phosphonocarboxyl, phosphonyl, carbocyclic, heterocyclic, aryl, or heteroaryl groups described in any embodiment herein may be substituted by one or more (e.g., 1, 2, 3, or 4) substituents selected from the following groups: halogen, hydroxyl, carboxyl, amino, nitro, cyano, C 1-6 Acylamino, C 1-6 Acyloxy group, C 1-6 Alkoxy, aryloxy, alkylthio, C 1-6 Alkyl, C1-6 Acyl group, C 6-10 Aryl, C 3-8 cycloalkyl, C 2-6 alkenyl, C 2-6 Alkyne, heterocyclic or heteroaryl, methylenedioxy, ureo, mercapto, azide, carbonyl, alkylsulfonyl, aminosulfonyl, dialkylaminosulfonyl, and alkylsulfinyl, etc. The substituent itself may also be optionally substituted. More preferably, the substituents include, but are not limited to, halogen, hydroxyl, carboxyl, amino, C... 1-6 Acylamino, C 1-6 Acyloxy group, C 1-6 Alkoxy, C 1-6 Alkyl, C 1-6 Alkyl acyl and alkyl sulfonyl groups.

[0044] In this document, the term "group capable of coupling with a thiol group" generally refers to a group capable of reacting with a thiol group to covalently link two chemical molecules. Exemplary groups capable of coupling with thiol groups include, but are not limited to, maleimide groups.

[0045] In this document, the term "functional group coupled to antibody molecule" generally refers to a reactive chemical functional group located at the end of the linker-payload structure that can undergo a specific covalent reaction with a reactive group (including but not limited to the amino group of lysine, the thiol group of cysteine, and the aldehyde group generated by the oxidation of glycosyl groups) naturally present in the antibody molecule or introduced through modification, thereby achieving stable coupling between the linker-payload and the antibody. Exemplary functional groups may be the groups shown in Q1 of this document, particularly including one or more of the following: maleimide group, N-hydroxysuccinimide ester group, acylhydrazine group, haloacetyl group, and pyridyl disulfide group.

[0046] In this document, the term "linking group" generally refers to a group capable of being linked to another group. Linking groups can be monovalent, divalent, trivalent, or more. A divalent linking group typically refers to a chemical structure, fragment, or bond whose one end is linked to a group and the other end to another group. The linking group can be directly or indirectly linked to the pharmaceutical unit described herein. Direct or indirect linking to the pharmaceutical unit means that the linking group directly links the pharmaceutical unit via a covalent bond, or links the pharmaceutical unit via another linking group. For example, a compound having a linking group can achieve the linking of the compound to another group through a coupling reaction between the linking group and another group. For example, a maleimide group can serve as a linking group. In this document, exemplary linker groups may be functional groups coupled to antibody molecules (such as the structure shown in Q1), optionally substituted amino acid residues, optionally substituted polypeptide residues, branching groups, spacer groups, capping groups, or chemical structural fragments selected from acid-labile linker structures (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linker structures, photostable linker structures, dimethyl linker structures, and disulfide-containing linker structures.

[0047] In this article, the term "linker" refers to a linear or branched chemical structural unit in an antibody-drug conjugate (ADC) that covalently links an antibody molecule to a drug molecule (payload). One end of the linker is covalently coupled to the antibody via the aforementioned "functional group coupled to the antibody molecule," while the other end forms a stable link to the drug molecule (payload), constituting a complete linker-payload structure. Linkers include cleavable and non-cleavable linkers: cleavable linkers include, but are not limited to, peptidase-sensitive linkers (such as valine-citrulline (Val-Cit) and phenylalanine-lysine (Phe-Lys) peptide linkers), acid-sensitive linkers (such as hydrazone and acetal linkers), and reduction-sensitive linkers (such as disulfide linkers); non-cleavable linkers are mainly degraded after the antibody is internalized by target cells, releasing the drug molecule along with the antibody fragment (such as thioether linkers).

[0048] In this document, the term "drug unit" generally refers to a chemical entity that directly or indirectly conjugates an antibody or antigen-binding fragment to form an immunoconjugate. This drug unit is typically covalently linked to the remainder of the ADC via conventional chemical bonds, including but not limited to amide bonds, ester bonds, carbamate bonds, urea bonds, or hydrazone bonds. The drug unit is usually derived from a biologically active compound (active compound), including but not limited to compounds with antitumor activity as described herein (such as ATR inhibitors and topoisomerase inhibitors). Specifically, the drug unit is a chemical entity formed by the loss of one group (such as a hydrogen atom, hydroxyl group, etc.) from the aforementioned active compound, or a chemical entity covalently linked to the remainder of the ADC (linker) through functional groups inherent in its molecular structure (such as hydroxyl, amino, carboxyl groups, etc.). The drug units described herein retain the core biological activity (such as antitumor activity) of the corresponding active compound (i.e., the active compound that produces the drug unit).

[0049] In this article, the term "compound with antitumor activity" generally refers to a compound that has the ability to reduce the proliferation rate, viability, or metastatic activity of tumor cells. For example, antitumor activity may manifest as a reduced growth rate of abnormal cells that appear during treatment, or as stabilization or reduction of tumor size, or as longer survival due to treatment compared to a control. Antitumor activity can be assessed using recognized in vitro or in vivo tumor models, such as xenograft models.

[0050] In some embodiments of the present invention, the bioactive molecule in the conjugate is a compound entity with antitumor activity, such as a radioactive isotope, for example, At. 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 Pb 212Radioactive isotopes of Lu; metal complexes, such as platinum complexes (e.g., oxaliplatin) or gold complexes; glycopeptide antibiotics, such as bleomycin or bleomycin; topoisomerase inhibitors; drugs that interfere with DNA synthesis, such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, fludarabine, cladribine, or nerabine; drugs that act on structural proteins, such as microtubule inhibitors (e.g., vinblastine alkaloids, vincristine, paclitaxel, maytansine, auristatin, tubulysin B, or eribulin); tumor signaling pathway inhibitors, such as serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors, or histidine kinase inhibitors; proteasome inhibitors; epigenetic target inhibitors; tumor angiogenesis inhibitors; cyclin inhibitors, etc.

[0051] Topoisomerases are enzymes that correct the ring number of DNA by cleaving phosphodiester bonds in one or both strands and then rewinding and sealing them. In this document, the terms "topoisomerase inhibitor" or "drugs targeting topoisomerases" refer to compounds or derivatives thereof, including topoisomerase I inhibitors and topoisomerase II inhibitors. Examples of topoisomerase I inhibitors include, but are not limited to, camptothecin and its analogues; topoisomerase II inhibitors include, but are not limited to, voreloxin or its derivatives, actinomycin D, doxorubicin, doxorubicin, docarmicin, daunorubicin, mitoxantrone, podophyllotoxin, and etoposide.

[0052] In this article, the term "camptothecin analogue" generally refers to compounds that are structurally similar to or derived from camptothecin. Exemplary camptothecin analogues include, but are not limited to, Exatecan, DXd, 7-ethyl-10-hydroxycamptothecin (SN38), Belotecan, (4-NH2)-Exatecan (AZD'0132), 7-MAD-MDCPT, and 7-aminomethyl-10-methyl-11-fluorocamptothecin.

[0053] In this article, the term "ATR inhibitor" stands for Rad3-associated kinase inhibitor, a class of molecularly targeted therapeutic drugs that target and inhibit ATR.

[0054] In this article, the term "antibody" is used in its broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity. Antibodies can be mouse, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system capable of recognizing and binding to specific antigens. Target antigens generally have numerous binding sites, also called epitopes, recognized by the CDRs of various antibodies. Antibodies that specifically bind to different epitopes have different structures. Therefore, an antigen can have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or the immunoactive portion of full-length immunoglobulin molecules, i.e., molecules containing antigens or portions thereof that specifically bind to a target of interest, including but not limited to cancer cells or cells that produce autoantibodies associated with autoimmune diseases. The immunoglobulins described in this invention can have any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Immunoglobulins can be derived from any species. However, in one aspect, immunoglobulins are derived from humans, mice, or rabbits. "Antibody fragment" generally refers to a fragment that retains all or part of the function of an antibody, such as an antigen-binding fragment. Examples of antibody fragments include: Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; minibody; fragments prepared from Fab expression libraries; anti-idiotypic (anti-Id) antibodies; CDR (complementarity-determining region); and any of the above epitope-binding fragments that bind to cancer cell antigens, viral antigens, or microbial antigens in an immune-specific manner; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The aforementioned antibodies can also be modified using THIOMAB technology to specifically modify unpaired cysteine ​​residues, achieving more coupling sites and site-specific coupling. The antibodies constituting the antibody-drug conjugates of this invention can maintain their original wild-state antigen-binding ability. Therefore, the antibodies of this invention can, for example, specifically bind to antigens. The antigens involved include, but are not limited to: tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules related to tissue growth and differentiation (such as those known or anticipated to be functional), lymphokines, cytokines, molecules involved in cell circulation regulation, molecules involved in angiogenesis, and molecules related to angiogenesis (such as those known to bind to antigens). The antigen bound by the antibody can be one or a subset of the above categories, while other subsets include other molecules / antigens with specific properties (compared to the target antigen). Antibodies used in antibody-drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens.Such tumor-associated antigens are well-known in the industry and can be prepared using well-known antibody preparation methods and information. These targets can be specifically expressed on the surface of one or more cancer cells, while being expressed sparingly or not at all on the surface of one or more non-cancer cells. Typically, such tumor-associated peptides can be overexpressed on the surface of cancer cells compared to non-cancer cells.

[0055] In this document, the term "amino acid residue" generally refers to a chemical structural segment formed by the loss of a hydrogen atom from the amino group and / or the loss of a hydroxyl group from the carboxyl group of an amino acid. An amino acid residue can be a monovalent group (e.g., the amino group loses a hydrogen atom or the carboxyl group loses a hydroxyl group) or a divalent group (e.g., the amino group loses a hydrogen atom and the carboxyl group loses a hydroxyl group). In this document, amino acids can be natural or non-natural, and exemplary amino acids may be selected from phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, and glycine. The amino acid residue may be optionally substituted.

[0056] In this document, the term "peptide residue" generally refers to a group consisting of two or more amino acid residues linked together. One or more amino acid residues in a polypeptide residue may be optionally substituted. The number of amino acid residues in a polypeptide residue may be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, generally in the range of 2 to 10, such as 2 to 8, 2 to 6, or 2 to 5. Similarly, a polypeptide residue may be a monovalent or divalent group. It should be understood that, in this document, a monovalent group refers to a group that can only covalently bond with other groups at one site, and a divalent group refers to a group that can covalently bond with other groups at two sites.

[0057] In this article, "alanine" refers to either α-alanine or β-alanine.

[0058] In this article, the terms "polyethylene glycol group" and "glycol group" are used interchangeably, generally referring to a group consisting of two or more ethylene glycol residues linked together by -(CH2CH2O)-. For example, a polyethylene glycol group can contain -(CH2CH2O). r - where r is any integer between 1 and 500. The polyethylene glycol group may be optionally substituted.

[0059] In this article, the term "branched group" refers to a multifunctional linker structural fragment, including bifunctional, trifunctional, and multifunctional linker structural fragments. Bifunctional linker structural fragments are derived from small molecule chemical reagents with two different reactive ends. One end of the bifunctional linker structural fragment is used to directly or indirectly connect to a specific functional group on the antibody (such as the thiol group of cysteine ​​or the amino group of lysine), while the other end is directly or indirectly connected to the drug unit described in this article. Trifunctional linker structural fragments are derived from small molecule chemical reagents with three different reactive ends. One end of the trifunctional linker structural fragment is used to directly or indirectly connect to the antibody, while the other two ends can each be directly or indirectly connected to the same or different drug units; or one of the other two ends can be directly or indirectly connected to the drug unit, and the other can be directly or indirectly connected to a capping group. Using multifunctional linker structural fragments in ADC structures can increase the drug loading capacity of the antibody.

[0060] In the linker-drug described in this article, the branching group can be chemically bonded to Q1, A1, L1, L2, L3, the capping group, or another branching group.

[0061] In this paper, the bifunctional connector structural segment can be selected from -NH-(CH2). b1 -C(O)-, -NH-(CH2) b1 -、-(CH2CH2O) b2 -、-(CH2CH2O) b2 -、-(CH2CH2O) b2 -(CH2) b1 -C(O)-、-(CH2CH2O) b2 -(CH2) b1 -C(O)-NH-(CH2CH2O) b2 -(CH2) b1 -C(O)-, -NH-(CH2) b1 -(CH2OCH2) b2 -(CH2) b1 -C(O)-、-C(O)-(CH2CH2O) b2 -(CH2) b1 -NH-、-C(O)-(CH2CH2O) b2 -(CH2) b1 -NH-、-C(O)-(CH2CH2O) b2 -(CH2) b1 -C(O)-、-NH-(CH2CH2O) b2 -(CH2) b1 -C(O)-、-(OCH2CH2) b2-C(O)-、-C(O)-(CH2CH2O) b2 -(CH2) b1 -C(O)-、-NH-CO-NH-(CH2) b1 -CO-, -NH-(CH2) b1 -triazolyl-(CH2) b1 -O-(CH2) b1 -(OCH2CH2) b2 -NH-CO-(CH2) b1 -O-(CH2) b1 -、 , , and , where b1 and b2 are independent integers from 1 to 10.

[0062] In this article, the three-function connector structure segment can be selected from... , , , , , , , , , , and L4 and L5 are defined as described in any embodiment of this document.

[0063] In this document, the term "spacer group" refers to a specific divalent linker group that connects a drug unit to a linker backbone (such as a branched group, amino acid-based polypeptide residues, etc.) to regulate linker-drug stability, water solubility, and drug release efficiency. Its core function is to control the release of the drug unit at the target site. The spacer group described herein can be a self-eliminating spacer group conventional in the art, such as an optionally substituted PAB group (p-aminobenzyloxycarbonyl) or an optionally substituted m-aminobenzyloxycarbonyl. The spacer group can be modified with one, two, or three optionally substituted sugar rings and / or hydrophilic groups to improve ADC aggregation or solubility issues and enhance its pharmacokinetic properties. Exemplary spacer groups include... , , , , R t As defined in any of the embodiments described herein.

[0064] In this document, the term "terminating group" refers to a chemical group used to temporarily and selectively protect or block one of the three reactive functional groups of a trifunctional linker structural fragment during its synthesis and coupling. The terminating group described herein may be a conventional terminating group in the art. Preferred terminating groups may be terminating groups containing a polyethylene glycol structural fragment, such as -NH-(CH2CH2O). b2 -(CH2) b1 -C(O)-NH2、-NH-(CH2CH2O) b2 -CH3, -(OCH2CH2) b2 -CO-NH2, etc., can improve the hydrophilicity and stability of ADCs and improve their pharmacokinetic performance. b1 and b2 are defined as described in any embodiment of this document.

[0065] In this article, the term "multi-drug" (e.g., "two-drug" or "three-drug") refers to an ADC where two or more drug molecules with the same or different chemical structures are covalently linked to the same site or the same linker. When the chemical structures are different, the two or more drug molecules may have the same mechanism of action (e.g., both belonging to ATR inhibitors) or different mechanisms of action (e.g., belonging to ATR inhibitors and TOPO I inhibitors, respectively).

[0066] In this paper, the various parts of the ADC (antibody moiety, linker, drug unit) or the various groups can be covalently linked by means well known in the art, including but not limited to carbon-carbon bonds, amide bonds, ester bonds, carbamate bonds, urea bonds, and hydrazone bonds. In this paper, carbon-carbon bonds can be -(CH2). n - indicates that n is an integer from 1 to 20. An amide bond can be represented by "-CO-NH-", an ester bond by "-C(O)O-", a carbamate bond by "-NH-C(O)-O-", a urea bond by "-NH-CO-NH-", and a hydrazone bond by "-C(R')=N-NH-", where R' is H or C. 1-4 alkyl.

[0067] In this article, "DAR" stands for Drug-to-Antibody Ratio, which refers to the average number of drug molecules conjugated to a monoclonal antibody molecule. Its value is usually 1 to 8 or 2 to 8, preferably 2 to 6 or 3 to 6.

[0068] In this text, “treatment” includes the following meanings: (i) curbing the development of a disease or symptom; (ii) alleviating a disease or symptom, that is, causing the state of the disease or symptom to subside; or (iii) reducing the symptoms caused by the disease or symptom. “Prevention” has the following meaning: preventing the occurrence of a disease or symptom in mammals, especially when such mammals are susceptible to the disease or symptom but have not yet been diagnosed with it.

[0069] In this document, the terms "individual," "subject," "patient," and "object" are used interchangeably to refer to vertebrates. In some embodiments, vertebrates refer to mammals. Mammals include, but are not limited to, livestock (such as cattle), sporting animals, pets (such as cats, dogs, and horses), primates (such as humans), and rodents (such as mice and rats). Preferably, mammals refer to humans.

[0070] In this document, "optional" or "optionally" means that the event described below may or may not occur, and the description includes instances where said event or situation occurs and instances where it does not occur. For example, "optionally substituted aryl" means that the aryl group may or may not be substituted, and the description includes both substituted and unsubstituted aryl groups.

[0071] Linker-drug

[0072] The linker-payload described in this article is a compound that uses a linker to modify a drug molecule (payload), facilitating the covalent attachment of the drug to an antibody molecule to form an ADC.

[0073] The linker-drug unit in this paper can be represented by VLD, where VL is the linker and D is the drug unit.

[0074] In this document, the linker VL can be any linker molecule commonly used in various ADCs and is well known in the art. Exemplary linker molecules may be as described in US 6214345, US 6436912, US 5688931, US 7591994, US 2011 / 0256157, WO 2012 / 015912, WO 2014 / 100443, US 2015 / 0337040, WO 2024 / 238872, WO 2025 / 051241, WO 2025 / 031307 or WO 2025 / 077814, the entire contents of which are incorporated herein by reference.

[0075] In this document, D includes at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) pharmaceutical unit. The pharmaceutical unit may be derived from a small molecule compound having biological activities of interest in the art (e.g., anticancer, anti-inflammatory, etc.). In this document, "derived," "derived from," or its synonyms such as "from" indicate that the pharmaceutical unit retains the main chemical structure and biological activity of the small molecule compound, but is directly covalently connected to VL by losing an atom, such as a hydrogen atom, or is covalently connected to VL through a linker.

[0076] Exemplary small molecule compounds include, but are not limited to, ATR inhibitors, such as those disclosed in PCT / EP2015 / 067804, PCT / EP2019 / 073520, PCT / CN2020 / 130326, PCT / CA2020 / 051014, PCT / CN2020 / 098159, PCT / CN2021 / 141153, PCT / CN2022 / 111893, and PCT / CN2022 / 143770. The entire contents of these documents are included in this paper. ; Targeted DNA damage response (DDR) inhibitors; Targeted microtubule drugs, such as vinca alkaloids, vincristine, vinblastine, paclitaxel, maytansine, auristatin, tubulolysin, sea haretoxin, nostoc cyclic peptide, spindle kinesin inhibitors or eribulin; DNA damaging agents, such as enediynes, topoisomerase inhibitors, pyrrolo[2,1-c][1,4]benzodiazepines (PBDs), ducamycin or trabectedin; RNA damaging agents, such as tyrannostatin or amatoxins; and immune agonists, such as Toll-type receptor agonists or STING agonists.

[0077] In a preferred embodiment, D includes at least an ATR inhibitor-derived pharmaceutical unit, which may include, for example, the ATR inhibitors disclosed in PCT / EP2015 / 067804, PCT / EP2019 / 073520, PCT / CN2020 / 130326, PCT / CA2020 / 051014, PCT / CN2020 / 098159, PCT / CN2021 / 141153, PCT / CN2022 / 111893, and PCT / CN2022 / 143770, compounds of formula i or ii herein, or their deuterated derivatives. In some embodiments, D also includes a pharmaceutical unit as described in any embodiment herein, such as the pharmaceutical unit shown in D2 and / or D3.

[0078] In this article, VL and D can be covalently linked by various chemical bonds well known in the art, including but not limited to carbon-carbon single bonds (-CC-), ester bonds (-CO-O-), amide bonds (-NH2-CO-), disulfide bonds (-SS-), hydrazone bonds (-C=N-NH-), glucuronide bonds, pyrophosphate diester bonds, p-aminobenzyl (PAB), p-aminobenzyloxycarbonyl (PABC), and carbamate bonds (-NH-CO-O-).

[0079] In some embodiments, the linker-drug described herein is a compound of Formula I, its stereoisomers, tautomers, N-oxides, hydrates, solvates, isotopically labeled compounds, or pharmaceutically acceptable salts, or mixtures thereof: (I) In the formula: Q1 is a functional group that couples with antibody molecules, especially functional groups that can couple with cysteine, lysine, non-natural amino acids or glycosyl groups of antibody molecules. A1 is selected from: bonded, optionally substituted alkylene, optionally substituted alkylene acyl, optionally substituted alkylene-polyethylene glycol, optionally substituted polyethylene glycol, optionally substituted alkylene-polyethylene glycol-alkylene, optionally substituted alkylene-C(O)NH-alkylene, optionally substituted alkylene-NHC(O)-alkylene, optionally substituted alkylene-C(O)NH-alkylene-acyl, optionally substituted alkylene-cycloalkylene acyl, optionally substituted polyethylene glycol-alkylene-acyl, optionally substituted alkenyl, optionally substituted alkyneyl, optionally substituted alkyneyl- Alkylene, optionally substituted carbocyclic, optionally substituted heterocyclic, optionally substituted aryl, optionally substituted heterocyclic, optionally substituted heterocyclic-alkylene-acyl, optionally substituted aryl-alkylene-acyl, and optionally substituted heterocyclic-alkylene-acyl; preferably, when A1 is substituted, the number of substituents may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, and the substituents are preferably selected from hydrophilic groups, halogens, hydroxyl groups, amino groups, carboxyl groups, sulfonic acid groups, sulfone groups, phosphate groups, alkoxy groups and alkylene-NH-alkylene-carboxyl groups; B1, B2, B3, B4, B5, B6, B7, and B8 are each independently a bond, an optional substituted amino acid residue, an optional substituted polypeptide residue, or a branched group, and when A1 is a bond, B4 is not a bond; L1, L2, and L3 are each independently a bond, an optional substituted amino acid residue, or an optional substituted polypeptide residue. T1, T2, and T3 are each independently bonded or optionally substituted spacer groups, wherein at most two of T1, T2, and T3 are bonds; At least one of D1, D2 and D3 is a first pharmaceutical unit, and the remaining one or two are each independently selected from a second pharmaceutical unit and a third pharmaceutical unit, wherein the first pharmaceutical unit is a pharmaceutical unit derived from an ATR inhibitor, preferably a pharmaceutical unit derived from a compound of formula i or formula ii or its deuterated derivative. E is a capping group; t, m, and n are each independently 0, 1, 2, or 3.

[0080] In some implementations, B1, B2, B3, B4, B5, B6, B7, and B8 are each independently a bond or branching group, and when A1 is a bond, B4 is not a bond.

[0081] In some implementations, L1, L2, and L3 are each independently a bonded or optionally substituted polypeptide residue.

[0082] In some embodiments, at least one of A1, B1, B2, B3, B4, B5, B6, B7, B8, T1, T2, and T3 may optionally be substituted with a hydrophilic group.

[0083] In some embodiments, the hydrophilic group is selected from: optionally substituted polysarcosine residues, polyglycerol groups, polyol groups, glycosyl groups, cyclodextrin groups, optionally substituted polyethylene glycol groups, optionally substituted glycosylated polyethylene glycol groups, optionally substituted glycosylated polyglycerol groups, optionally substituted cyclodextrin polyethylene glycol groups, or combinations thereof.

[0084] Preferably, the optionally substituted polysarcosine residues are: or , Where n2 is an integer between 4 and 20, for example, an integer between 4 and 16; R is selected from: H, C 1-6 Alkyl, C 1-6 cycloalkyl and C 1-6 Alkyl group.

[0085] Preferably, the optionally substituted glycosylated polyethylene glycol group is: , Where n3 is an integer between 4 and 18, for example, an integer between 4 and 12.

[0086] Preferably, the optionally substituted glycosylated polyglycerol group is: , Where n4 is an integer between 4 and 12, for example, an integer between 4 and 10.

[0087] Preferably, the optionally substituted polyethylene glycol group is: , , or , where n5 is an integer between 1 and 24, for example, an integer between 1 and 8.

[0088] Preferably, the substituted polyethylene glycol group is , where n5 is an integer between 4 and 24, for example, an integer between 8 and 18.

[0089] In some embodiments, when B4 is a bond, m is 0, B5 is a bond, n is 0, and t is 0 in Formula I of the present invention, the compound of Formula I is a compound represented by Formula Ia, its stereoisomers, tautomers, N-oxides, hydrates, solvates, isotopically labeled compounds, or pharmaceutically acceptable salts, or mixtures thereof: (Ia) Q1, A1, B6, L1, T1 and D1 are each as described in any of the embodiments herein.

[0090] In some embodiments, when m is 1, n is 0, B6 is a bond, and t is 1 in Formula I of the present invention, the compound of Formula I is a compound represented by Formula Ib, its stereoisomers, tautomers, N-oxides, hydrates, solvates, isotopically labeled compounds, or pharmaceutically acceptable salts, or mixtures thereof: (Ib) Wherein, Q1, A1, B1, B2, B4, B5, L1, L2, T1, T2, D1, D2 and E are each as described in any of the embodiments herein.

[0091] In some embodiments, when m, n, and t are 1 in Formula I of the present invention, the compound of Formula I is a compound represented by Formula Ic, its stereoisomers, tautomers, N-oxides, hydrates, solvates, isotopically labeled compounds, or pharmaceutically acceptable salts, or mixtures thereof:

[0092] (Ic)

[0093] Wherein, Q1, A1, B1, B2, B3, B4, B5, B6, L1, L2, L3, T1, T2, T3, D1, D2, D3 and E are each as described in any of the embodiments herein.

[0094] In some embodiments, when m is 1, n is 1, and t is 0 in Formula I of the present invention, the compound of Formula I is a compound represented by Formula Id, its stereoisomers, tautomers, N-oxides, hydrates, solvates, isotopically labeled compounds, or pharmaceutically acceptable salts, or mixtures thereof:

[0095] (Id)

[0096] Wherein, Q1, A1, B2, B3, B4, B5, B6, B7, B8, L1, L2, L3, T1, T2, T3, D1, D2 and D3 are each as described in any of the embodiments herein.

[0097] In some embodiments, when m is 1, n is 0, t is 0, and B6 is a bond in Formula I of the present invention, the compound of Formula I is a compound represented by Formula Ie, its stereoisomers, tautomers, N-oxides, hydrates, solvates, isotopically labeled compounds, or pharmaceutically acceptable salts, or mixtures thereof:

[0098] (Ie)

[0099] Q1, A1, B2, B4, B5, B7, L1, L2, T1, T2, D1, and D2 are each as described in any of the embodiments herein.

[0100] In some embodiments of Formulas I, Ia, Ib, Ic, Id, and Ie, the ATR inhibitor is any ATR inhibitor disclosed in PCT / EP2015 / 067804, PCT / EP2019 / 073520, PCT / CN2020 / 130326, PCT / CA2020 / 051014, PCT / CN2020 / 098159, PCT / CN2021 / 141153, PCT / CN2022 / 111893, or PCT / CN2022 / 143770, the entire contents of which are incorporated herein by reference.

[0101] In some embodiments, in Formula I: Q1 is a functional group capable of coupling with cysteine, lysine, non-natural amino acid, or glycosyl group of an antibody molecule; A1 is selected from alkylene, optionally substituted alkylene, optionally substituted alkylene acyl, optionally substituted alkylene-polyethylene glycol, optionally substituted polyethylene glycol, optionally substituted alkylene-polyethylene glycol-alkylene, optionally substituted alkylene-C(O)NH-alkylene, optionally substituted alkylene-NHC(O)-alkylene, optionally substituted alkylene-C(O)NH-alkylene-acyl, optionally substituted alkylene-cyclic Alkyl acyl, optionally substituted alkenyl, optionally substituted alynyl, optionally substituted alynyl-alkylene, optionally substituted carbocyclic, optionally substituted heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl, wherein, when A1 is substituted, the substituent may be selected from hydrophilic groups, halogens, hydroxyl groups, amino groups, carboxyl groups, sulfonic acid groups, sulfone groups, phosphoric acid groups, alkoxy groups, and alkylene-NH-alkylene-carboxyl groups, such as those selected from halogens, hydroxyl groups, amino groups, carboxyl groups, sulfonic acid groups, sulfone groups, phosphoric acid groups, and alkoxy groups; L1, B4, B6, B7, and B8 are all bonds; B2 and B3 Each of the following groups is an independent bond or branched group, and when A1 is a bond, B2 is not a bond; B5, L2, and L3 are each an independent bond or optionally substituted polypeptide residue; T1, T2, and T3 are each an independent bond or optionally substituted spacer group, wherein at most two of T1, T2, and T3 are bonds; D1 is the first pharmaceutical unit, which is a pharmaceutical unit derived from an ATR inhibitor, preferably PCT / EP2015 / 067804, PCT / EP2019 / 073520, PCT / CN2020 / 130326, or PCT / CA2020 / 0510. 14. A pharmaceutical unit derived from an ATR inhibitor disclosed in any of PCT / CN2020 / 098159, PCT / CN2021 / 141153, PCT / CN2022 / 111893 or PCT / CN2022 / 143770; D2 and D3 are the second and third pharmaceutical units, respectively; D1, D2 and D3 may be the same or different; t is 0; m and n are each independently 0, 1, 2 or 3; preferably, at least one of B3, T1, T2 and T3 is substituted with a hydrophilic group; preferably, m and n are both 1.

[0102] In some embodiments, in Formula I: Q1 is a functional group capable of coupling with cysteine, lysine, non-natural amino acids, or glycosyl groups of the antibody molecule; A1 is selected from the group consisting of a bond, optionally substituted alkylene, optionally substituted alkylene acyl, optionally substituted alkylene-polyethylene glycol, optionally substituted polyethylene glycol, optionally substituted alkylene-polyethylene glycol-alkylene, optionally substituted alkylene-C(O)NH-alkylene, optionally substituted alkylene-NHC(O)-alkylene, optionally substituted alkylene-C(O)NH-alkylene-acyl, and optionally substituted alkylene-C(O)NH-alkylene-acyl. The substituents are selected from alkylene-cycloalkylene acyl, alkenylene, alkyneene, alkyneene-alkylene, carbocyclic, heterocyclic, aryl, and heteroaryl groups, wherein when A1 is substituted, the substituents may be selected from hydrophilic groups, halogens, hydroxyl groups, amino groups, carboxyl groups, sulfonic acid groups, sulfone groups, phosphoric acid groups, alkoxy groups, and alkylene-NH-alkylene-carboxyl groups, such as those selected from halogens, hydroxyl groups, amino groups, carboxyl groups, sulfonic acid groups, sulfone groups, phosphoric acid groups, and alkoxy groups; L1, B4 and B7 are bonds; B2 and B6 are each independently bonds or branched groups, and when A1 is a bond, B2 is not a bond; B5 and L2 are each independently bonds or optionally substituted polypeptide residues; T1 and T2 are each independently bonds or optionally substituted spacer groups, wherein at most two of T1 and T2 are bonds; D1 is the first pharmaceutical unit, which is a pharmaceutical unit derived from an ATR inhibitor, preferably PCT / EP2015 / 067804, PCT / EP2019 / 073520, PCT / CN2020 / 130326, P The pharmaceutical unit derived from any of the ATR inhibitors disclosed in CT / CA2020 / 051014, PCT / CN2020 / 098159, PCT / CN2021 / 141153, PCT / CN2022 / 111893 or PCT / CN2022 / 143770; D2 is the second pharmaceutical unit; D1 and D2 may be the same or different; n and t are both 0; m is 0, 1, 2 or 3; preferably, at least one of B6, T1 and T2 is substituted with a hydrophilic group; preferably, m is 1.

[0103] In some embodiments, in Formula I: Q1 is a functional group capable of coupling with cysteine, lysine, non-natural amino acids, or glycosyl groups of the antibody molecule; A1 is selected from the group consisting of a bond, optionally substituted alkylene, optionally substituted alkylene acyl, optionally substituted alkylene-polyethylene glycol, optionally substituted polyethylene glycol, optionally substituted alkylene-polyethylene glycol-alkylene, optionally substituted alkylene-C(O)NH-alkylene, optionally substituted alkylene-NHC(O)-alkylene, optionally substituted alkylene-C(O)NH-alkylene-acyl, and optionally substituted alkylene-C(O)NH-alkylene-acyl. The substituents are selected from alkylene-cycloalkylene acyl, alkenylene, alkyneene, alkyneene-alkylene, carbocyclic, heterocyclic, aryl, and heteroaryl groups, wherein when A1 is substituted, the substituents may be selected from hydrophilic groups, halogens, hydroxyl groups, amino groups, carboxyl groups, sulfonic acid groups, sulfone groups, phosphoric acid groups, alkoxy groups, and alkylene-NH-alkylene-carboxyl groups, such as those selected from halogens, hydroxyl groups, amino groups, carboxyl groups, sulfonic acid groups, sulfone groups, phosphoric acid groups, and alkoxy groups; L1, B6 and B8 are both bonds; B3 and B4 are each independently a bond or a branched group, and when A1 is a bond, B4 is not a bond; B5 and L3 are each independently a bond or an optionally substituted polypeptide residue; T1 and T3 are each independently a bond or an optionally substituted spacer group, wherein at most two of T1 and T3 are bonds; D1 is the first pharmaceutical unit, which is a pharmaceutical unit derived from an ATR inhibitor, preferably PCT / EP2015 / 067804, PCT / EP2019 / 073520, or PCT / CN2020 / 130326. The pharmaceutical unit derived from any of the ATR inhibitors disclosed in PCT / CA2020 / 051014, PCT / CN2020 / 098159, PCT / CN2021 / 141153, PCT / CN2022 / 111893 or PCT / CN2022 / 143770; D3 is a second pharmaceutical unit; D1 and D3 may be the same or different; m and t are 0; n is 0, 1, 2 or 3; preferably, at least one of B3, T1 and T3 is substituted with a hydrophilic group; preferably, n is 1.

[0104] In some embodiments, in Formula I: Q1 is a functional group capable of coupling with cysteine, lysine, non-natural amino acids, or glycosyl groups of an antibody molecule; A1 is selected from alkylene groups, optionally substituted alkylene groups, optionally substituted alkylene acyl groups, optionally substituted alkylene-polyethylene glycol groups, optionally substituted polyethylene glycol groups, optionally substituted alkylene-polyethylene glycol-alkylene groups, optionally substituted alkylene-C(O)NH-alkylene groups, optionally substituted alkylene-NHC(O)-alkylene groups, optionally substituted... The group comprises alkylene-C(O)NH-alkylene-acyl, optionally substituted alkylene-cycloalkylene-acyl, optionally substituted alkenyl, optionally substituted ynylene, optionally substituted ynylene-alkylene, optionally substituted carbocyclic, optionally substituted heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl, wherein when A1 is substituted, the substituent may be selected from hydrophilic groups, halogens, hydroxyl groups, amino groups, carboxyl groups, sulfonic acid groups, sulfone groups, phosphate groups, alkoxy groups, and alkylene-NH-alkylene. A1 is a carboxyl group, selected from halogen, hydroxyl, amino, carboxyl, sulfonic acid, sulfone, phosphate, and alkoxy groups; L1 is a bond; B4 and B6 are each independently a bond or branched group, and when A1 is a bond, B4 is not a bond; B5 is a bond or an optionally substituted polypeptide residue; T1 is a bond or an optionally substituted spacer group; D1 is a first pharmaceutical unit, which is a pharmaceutical unit derived from an ATR inhibitor, preferably PCT / EP2015 / 067804 or PCT / EP2019 / 0735. 20. A pharmaceutical unit derived from any of the ATR inhibitors disclosed in PCT / CN2020 / 130326, PCT / CA2020 / 051014, PCT / CN2020 / 098159, PCT / CN2021 / 141153, PCT / CN2022 / 111893 or PCT / CN2022 / 143770; m, n and t are all 0; preferably, at least one of B6 and T1 is substituted with a hydrophilic group.

[0105] In some embodiments, the ATR inhibitor described herein is a compound represented by formula i or a deuterated derivative thereof:

[0106] (i)

[0107] in: R0 is an optional substituted 5-membered heteroaryl group; R1 is H, halogen, or C with optional substitution. 1-6 Alkyl, optionally substituted C 3-6 cycloalkyl, optionally substituted C 2-6 alkenyl or optionally substituted C 2-6 alkynyl group; R2 is an optionally substituted carbocyclic group, an optionally substituted heterocyclic group, an optionally substituted aryl group, or an optionally substituted heteroaryl group; In this configuration, any H atom can be optionally replaced by deuterium.

[0108] In some embodiments of formulas I, Ia, Ib, Ic, Id, and Ie, the ATR inhibitor described herein is a compound represented by formula ii or a deuterated derivative thereof: (ii) Wherein, R1 and R2 are as described in equation i; R3 is hydrogen, halogen, or optionally substituted C. 1-6 alkyl; In this configuration, any H atom can be optionally replaced by deuterium.

[0109] Therefore, in any embodiment of this document, the ATR inhibitor-derived pharmaceutical unit or the first pharmaceutical unit in D1, D2 and D3 may be a pharmaceutical unit derived from the compound shown in formula i or ii above or its deuterated derivative.

[0110] In some embodiments of formulas I, Ia, Ib, Ic, Id, and Ie, the ATR inhibitor described herein is a compound or a deuterated derivative thereof:

[0111] In some embodiments of Formulas I, Ia, Ib, Ic, Id, and Ie, the second and third pharmaceutical units are each independently derived from an ATR inhibitor (such as the ATR inhibitor described in any embodiment herein), a DNA damage response (DDR) inhibitor, a microtubule-targeting drug (such as vinca alkaloids, vincristine, vinblastine, paclitaxel, maytansine, auristatin, tubulolysin, sea haretoxin, nostoc cyclic peptide, spindle kinesin inhibitor, or eribulin), a DNA damaging agent (such as enediyne, topoisomerase inhibitor, pyrrolo[2,1-c][1,4]benzodiazepine PBDs, ducamycin, or trabectedin), an RNA damaging agent (such as tyrannostatin or amatoxins), or an immune agonist (such as Toll-type receptor agonists or STING agonists). In some implementations, D2 and D3 are each independently derived from drug units targeting DNA damage response (DDR) inhibitors, microtubule-targeting drugs (such as vinca alkaloids, vincristine, vinblastine, paclitaxel, maytansine, auristatin, tubulolysin, sea haretoxin, nostoc cyclic peptide, spindle kinesin inhibitors, or eribulin), DNA damaging agents (such as enediynes, topoisomerase inhibitors, pyrrolo[2,1-c][1,4]benzodiazepines PBDs, ducamycin, or trabectedin), RNA damaging agents (such as tyrannostatin or amatoxins), or immune agonists (such as Toll-type receptor agonists or STING agonists).

[0112] In some embodiments of Formulas I, Ia, Ib, Ic, Id, and Ie, the second and third pharmaceutical units may be independently derived from ATR inhibitors or topoisomerase inhibitors as described in any of the embodiments herein. In some preferred embodiments, one of the second and third pharmaceutical units may be an ATR inhibitor-derived pharmaceutical unit and the other a topoisomerase inhibitor-derived pharmaceutical unit; or both the second and third pharmaceutical units may be ATR inhibitor-derived pharmaceutical units; or both the second and third pharmaceutical units may be independently derived from topoisomerase I (TOPO I) inhibitors.

[0113] In some implementations, D2 and D3 are each independently derived from ATR inhibitors (such as the ATR inhibitors described in any of the embodiments herein), DNA damage response (DDR) inhibitors, microtubule-targeting drugs (such as vinca alkaloids, vincristine, vinblastine, paclitaxel, maytansine, auristatin, tubulolysin, sea haretoxin, nostoc cyclic peptide, spindle kinin inhibitors, or eribulin), DNA damaging agents (such as enediynes, topoisomerase inhibitors, pyrrolo[2,1-c][1,4]benzodiazepines PBDs, ducamycin, or trabectedin), RNA damaging agents (such as tyrannostatin or amatoxins), or immune agonists (such as Toll-type receptor agonists or STING agonists).

[0114] In some embodiments, D2 and D3 may be independently pharmaceutical units derived from the ATR inhibitor or topoisomerase inhibitor described in any of the embodiments herein. In some preferred embodiments, D2 and D3 may be independently pharmaceutical units derived from topoisomerase I inhibitors.

[0115] In this article, exemplary topoisomerase I inhibitors include the compounds described below:

[0116] In some implementations of formulas I, Ia, Ib, Ic, Id, and Ie, D1, D2, and D3 are selected from combinations in the following table:

[0117] In some implementations, D1, D2, and D3 are selected from combinations 1 to 4 in the table above.

[0118] In some implementations, the combination of D1, D2, and D3 can be selected from the combinations in the table below: .

[0119] In some implementations, D1, D2 and D3 are selected from combinations 1-1 to 1-22, 2-1 to 2-28, 3-1 to 3-28 and 4-1 to 4-4 in the table above.

[0120] In some implementations of formulas I, Ia, Ib, Ic, Id, and Ie, E is selected from: H, -(CH2). a1 CH3, -(CH2) a1 -C(O)-(CH2) a1 CH3, -(CH2) a1 -(O-CH2-CH2) a2 -(CH2) a1 CH3, CH3(CH2) a1 -(O-CH2-CH2) a2 -(CH2) a3 -、-(CH2) a1 -C(O)NH-(CH2) a2 CH3, -(CH2) a1 -NH-(CH2) a1 CH3, -(CH2) a1 -NHC(O)-(CH2) a1 CH3, -(CH2) a1 -C(O)NH-(CH2) a2 -C(O)-(CH2)a1 CH3, -(CH2) a1 -(cycloalkylene)-C(O)-(CH2) a1 CH3、-C≡C-(CH2) a1 CH3、-C≡C-(CH2) a1 -CO-(CH2) a1 CH3, -NH-(CH2) b1 -C(O)-(CH2) a1 CH3, -NH-(CH2) b1 -C(O)-NH2、-NH-(CH2CH2O) b2 -(CH2) b1 -C(O)-(CH2) a1 CH3、-NH-(CH2CH2O) b2 -(CH2) b1 -C(O)-NH2、-NH-(CH2CH2O) b2 -CH3, -(OCH2CH2) b2 -CO-NH2 and -NH-CO-NH-(CH2) b1 -CO-NH2, wherein a1, a2, a3, b1, and b2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, E is selected from the above-mentioned -NH-(CH2CH2O). b2 -(CH2) b1 -C(O)-NH2 and -NH-(CH2) b1 -(CH2OCH2) b2 -(CH2) b1 -C(O)-NH.

[0121] In some embodiments of formulas I, Ia, Ib, Ic, Id, and Ie, Q1 is a group with the following characteristics: , , , , , , , , , , or , in, 1 indicates the connection position between Q1 and A1; X is Cl, Br, or I; Y is independently Br or phenyl-S-, which is optionally substituted by 1-3 substituents selected from halogens, nitro groups, alkyl groups, alkoxy groups, ester groups, and -C(O)NR'R''-, wherein R' and R'' are independently selected from H and alkyl groups, or R' and R'' together with the commonly connected N atom form a 5-7 membered heterocycle; Z is -(CH2CH2O). z -CH2CH2-OH, where z is any integer from 0 to 100; Q1 may be optionally substituted, and the substituent may be halogen or alkyl, preferably unsubstituted.

[0122] In some implementations, Q1 is a group that includes: , , , , , , , , or ,in, 1 indicates the connection position of Q1 and A1; X is Cl, Br or I; Y is Br or phenyl-S-, which is optionally substituted by 1-3 substituents selected from halogen, nitro, alkyl, alkoxy, ester and -C(O)NR'R''-, wherein R' and R'' are each independently selected from H and alkyl, or R' and R'' together with the N atom to which they are connected form a 5-7 membered heterocycle; Q1 may be optionally substituted, and the substituents may be halogen, alkyl, etc., preferably unsubstituted.

[0123] In some preferred embodiments, Q1 is: , or ; In some more preferred embodiments, Q1 is: .

[0124] In some implementations of equations I, Ia, Ib, Ic, Id, and Ie, A l For bond, -(CH2) a1 -、-(CH2) a1 -C(O)-, -(CH2) a1 -C(O)NH-(O)-、-(CH2) a1 -(O-CH2-CH2) a2 -、-(CH2) a1 -(O-CH2-CH2) a2 -(CH2) a3 -、-(CH2)a1 -C(O)NH-(CH2) a2 -、-(CH2) a1 -C(O)NH-(CH2) a2 -NH-, -(CH2) a1 -NH-, -(CH2) a1 -NHC(O)-, -(CH2) a1 -NHC(O) -(CH2) a2 -、-(CH2) a1 -C(O)NH-(CH2) a2 -C(O)-, -(CH2) a1 -(cycloalkylene)-C(O)-, -C≡C-(CH2) a1 -、-C≡C-(CH2) a1 -CO-, -(heterocyclic)-(CH2) a1 -C(O)-、-(aryl)-(CH2) a1 -C(O)- and -(heteroaryl)-(CH2) a1 -C(O)-, where a1, a2 and a3 are independent integers from 1 to 6, and the substituents shown are connected to Q1 on the left and to B4 or B6 on the right.

[0125] In some implementation schemes, A l For bond, -(CH2) a1 -、-(CH2) a1 -C(O)-, -(CH2) a1 -(O-CH2-CH2) a2 -、-(CH2) a1 -(O-CH2-CH2) a2 -(CH2) a3 -、-(CH2) a1 -C(O)NH-(CH2) a2 -、-(CH2) a1 -NHC(O)-(CH2) a2 -、-(CH2) a1 -C(O)NH-(CH2) a2 -C(O)-, -(CH2) a1 -(cycloalkylene)-C(O)-, -C≡C-(CH2) a1 - and -C≡C-(CH2) a1 -CO-, where a1, a2, and a3 are independent integers from 1 to 6, and the substituents shown are connected to Q1 on the left and to B2 or B4 on the right.

[0126] In some implementations, A1 may optionally be replaced by a hydrophilic group.

[0127] In some embodiments of formulas I, Ia, Ib, Ic, Id, and Ie, B4, B5, B6, B7, and B8 are each independently a bond or are each independently selected from the following groups: -NH-(CH2) b1 -C(O)- , -NH-(CH2) b1 - , -(CH2CH2O) b2 - , -(CH2CH2O) b2 - , -(CH2CH2O) b2 -(CH2) b1 -C(O)- , -(CH2CH2O) b2 -(CH2) b1 -C(O)-NH-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -NH-(CH2) b1 -(CH2OCH2) b2 -(CH2) b1 -C(O)- , -C(O)-(CH2CH2O) b2 -(CH2) b1 -NH- , -C(O)-(CH2CH2O) b2 -(CH2) b1 -NH- , -C(O)-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -NH-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -(OCH2CH2) b2 -C(O)- , -C(O)-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -NH-CO-NH-(CH2) b1 -CO- , -NH-(CH2) b1 -triazolyl-(CH2) b1 -O-(CH2) b1 -(OCH2CH2) b2 -NH-CO-(CH2) b1 -O-(CH2) b1 - , -NH-CH[(CH2) b1 -C(O)-NH-(CH2CH2O) b2 -(CH2) b1 CH3]-C(O)- , -NH-(CH2) b1 -triazolyl-(CH2) b1 -O-(CH2) b1 -(OCH2CH2) b2 -NH-CO-(CH2) b1 -O-(CH2) b1 -CO- , -NH-(CH2) b1 -triazolyl-(CH2) b1 -(OCH2CH2) b2 -NH-CO-(CH2) b1 -O-(CH2) b1 -CO- , -triazolyl-(CH2) b1 -CO- , , , , and In the formula, This indicates the position where B4 connects to A1, the position where B5 connects to B2 or A2, the position where B6 connects to B3 or A1, the position where B7 connects to B2, or the position where B8 connects to B3. This indicates the position where B4 connects to B2, the position where B5 connects to B3 or L1 or B1 or L2, the position where B6 connects to B1 or L1, the position where B7 connects to L2, or the position where B8 connects to L3; b1 and b2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; m1, m2 and m3 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; X is selected from H, halogen, C. 1-6 Alkyl, C 1-6 Alkyl groups and -(CH2CH2O) x1 -CH3, x1 is an integer selected from 1 to 15.

[0128] In some embodiments of formulas I, Ia, Ib, Ic, Id, and Ie, B2, B3, and B1 are each independently bonds or are each independently selected from the following groups: , , , , , , , , , , , and , In the formula, 1 indicates the position where B1 connects to B6 or B5, the position where B3 connects to B5, or the position where B2 connects to B4; 2 indicates the position where B1 connects to E, the position where B3 connects to B6, or the position where B2 connects to B5; 3 indicates the position where B1 connects to L1, the position where B3 connects to L3 or B8, or the position where B2 connects to L2 or B7; L4 is selected from -(CH2). m4 -、-(CH2) m4 -C(O)NH-(CH2) m5 -、-(CH2) m4 -NHC(O)-(CH2) m5 -; L5 is -(CH2) m4 -or-(CH2-CH2-O) m6 m4, m5, m6 and u are each an integer from 1 to 10.

[0129] In some embodiments, B2, B3, and B1 are each independently a bond or are each independently selected from the following groups: and ,in, 1. 2. 3 and L4 are as described in any of the preceding embodiments; preferably, L4 is -(CH2). m1 -, m1 is 1, 2, 3, 4, 5 or 6.

[0130] In some implementation schemes, B2, B3, and B1 are each independently: , , , or , In the formula, 1. 2. 3. L4 and L5 are as described in any of the above implementation schemes.

[0131] In some implementation schemes, B2, B3, and B1 are each independently: , , , , , and In the formula, 1. 2. 3. L4, L5 and u are as described in any of the above implementation schemes.

[0132] In some embodiments, B2, B3, B4, and B6 are each independently bonds or are each independently selected from the following groups: -NH-(CH2) b1 -C(O)- , -NH-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -(OCH2CH2) b2 -CO- , -NH-CO-NH-(CH2) b1 -CO- , -NH-(CH2) b1 -triazolyl-(CH2) b1 -O-(CH2) b1 -(OCH2CH2) b2 -NH-CO-(CH2) b1 -O-(CH2) b1 - , , , , , , , and , In the formula, This indicates the position where B6 connects to B5 or where B4 connects to A1; Indicates the position where B6 connects to T1 or the position where B4 connects to B5; 1 indicates the position where B2 connects to A1, the position where B3 connects to B5, the position where B6 connects to B5, or the position where B4 connects to A1; 2 indicates the position where B2 connects to B5, the position where B3 connects to T1, the position where B6 connects to T1, or the position where B4 connects to B5; 3 indicates the position where B2 connects to L2 or B3 connects to L3; L4 is selected from -(CH2). m1 -、-(CH2) m1 -C(O)NH-(CH2) m2 -、-(CH2) m1 -NHC(O)-(CH2) m2 -; L5 is -(CH2) m1 -or-(CH2-CH2-O) m2 X is H or a halogen, such as F. Preferably, when X is a halogen, X is located adjacent to the position where B4 connects to A1; b1, b2, m l m1, m2, m3 and v are each an integer from 1 to 10.

[0133] In some implementations, B2 or B3 are each independent of: , , , or , In the formula, 1 indicates the position where B2 connects to A1, or the position where B3 connects to B5; 2 indicates the position where B2 connects to B5, or the position where B3 connects to T1; 3 indicates the position where B2 connects to L2, or the position where B3 connects to L3; L4 and L5 are as described in any of the preceding embodiments.

[0134] In some embodiments of Formulas I, Ia, Ib, Ic, Id, and Ie, B5, L1, L2, and L3 are each independently an optionally substituted amino acid residue or an optionally substituted polypeptide residue. Preferably, the amino acid residues are selected from the group consisting of phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, and glycine. The amino acid residues in the polypeptide residues are selected from the group consisting of phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, and glycine. Preferably, the amino acid residues in the polypeptide residues are selected from the group consisting of glycine, phenylalanine, valine, alanine, glutamic acid, arginine, citrulline, aspartic acid, asparagine, and lysine. More preferably, the polypeptide residues are selected from the group consisting of: phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala), valine-citrulline (Val-Cit), glutamic acid-valine-alanine (Glu-Val-Ala), glutamic acid-valine-citrulline (Glu-Val-Cit), glutamic acid-glycine-citrulline (Glu-Gly-Cit), valine-lysine (Val-Lys), alanine-alanine (Ala-Ala), and alanine amino acid. Alanine-Alanine (Ala-Ala-Ala), Alanine-Alanine-Asparagine (Ala-Ala-Asn), Alanine-Leucine (Ala-Leu), Leucine-Leucine (Leu-Leu), Phenylalanine-Arginine (Phe-Arg), Phenylalanine-Lysine (Phe-Lys), Glycine-Phenylalanine-Glycine (Gly-Phe-Gly), Alanine-Alanine-Phenylalanine-Alanine (Ala-Ala-Phe-Ala) (cBu-Cit) , , Glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) and glycine-proline (Gly-Pro).

[0135] In some embodiments, B5, L2, and L3 are each independently an amino acid residue selected from the group consisting of glycine, phenylalanine, valine, alanine, arginine, citrulline, aspartic acid, asparagine, and lysine, wherein said amino acid residues are optionally substituted. In some embodiments, B5, L2, and L3 are each independently a polypeptide residue composed of amino acids selected from the group consisting of glycine, phenylalanine, valine, alanine, arginine, citrulline, aspartic acid, asparagine, and lysine, wherein said polypeptide residues are optionally substituted. In some embodiments, B5, L2, and L3 are each independently selected from the group consisting of polypeptide residues consisting of Val-Ala, Ala-Ala, Gly-Phe-Gly, Ala-Ala-Phe-Ala, and Gly-Gly-Phe-Gly.

[0136] In some implementation schemes, B5, L l L2 and L3 can each be independently a bond or a group with the following characteristics: , , , , or , In the formula, 3 indicates the position where B5 connects to B2 or B4, the position where L1 connects to B1, B5, or B6, the position where L2 connects to B2 or B7, or the position where L3 connects to B8 or B3. 4 indicates the position where B5 connects to B6 or B3 or L1 or B1, the position where L1 connects to T1, the position where L2 connects to T2, and the position where L3 connects to T3.

[0137] In some implementation schemes, B5, L2, and L3 are each independently a bond or a group with the following characteristics: , , , or , In the formula, 3 indicates the position where B5 connects to B2 or B4, the position where L2 connects to B2, or the position where L3 connects to B3. 4 represents the position where B5 connects to B3 or B6, the position where L2 connects to T2, or the position where L3 connects to T3; b is an integer from 1 to 10.

[0138] In some implementations of equations I, Ia, Ib, Ic, Id, and Ie, T l T2 and T3 are each independently selected from the following groups: 4 -NH-(CH2) L6 - 5. , , , , , , , , , , , , , and , In the formula, 4 indicates the position where T1 connects to L1, the position where T2 connects to L2, and the position where T3 connects to L3. 5 represents the position where T1 connects to D1, the position where T2 connects to D2, and the position where T3 connects to D3; where R t H, optionally substituted alkyl groups, or -(CH2CH2O) t1 -CH3, t1 is an integer selected from 0-15, and L6 is an integer selected from 0-15. The T... l T2 and T3 may optionally be replaced by hydrophilic groups selected from -O(CH2CH2O). L6 -(CH2) L7 CH3 or -(CH2) L6 -N(R N1 )-[C(O)-(CH2) L7 -N(R N2 )] L8 -C(O)-R N3 L6, L7, or L8 are each independently assigned the numbers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, R N1 R N2 and R N3 Each is independent as C 1-6 alkyl.

[0139] In some implementations, T l T2 and T3 are each independently selected from the following groups: , , , , or , In the formula, 4 indicates the position where T1 connects to B3 or B6, the position where T2 connects to L2, and the position where T3 connects to L3. 5 indicates the position where T1 connects to D1, the position where T2 connects to D2, and the position where T3 connects to D3.

[0140] In some implementations, at least one of T1, T2, and T3 is: , in, 1 indicates the position where T1, T2, and T3 are connected to B3 or B6, L2, and L3 respectively. 2 indicates the position where T1, T2, T3 are connected to D1, D2, D3; where X is an optional substituted group: , The hydrophilic group described in any embodiment of this document is connected to T1, T2, or T3 via X, wherein the connection point 1. Connected to a hydrophilic group, the connection point 2 is connected to one of T1, T2, and T3.

[0141] In some implementations, T l T2 and T3 can each be arbitrarily selected by hydrophilic groups (such as...) ) is replaced, and n5 is preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10; when T l When the structures of T2 and T3 include a benzene ring, the hydrophilic group is preferably substituted on the benzene ring, and more preferably on the 4th ring. -NH adjacent.

[0142] In some implementations, T l T2 and T3 (e.g.) Each of the components may be optionally fitted with a hydrophilic group, preferably -(CH2). L6 -N(R N1 )-[C(O)-(CH2) L7 -N(R N2 )] L8 -C(O)-R N3 L6 and L7 are preferably 1, L8 is preferably 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, R N1 R N2 and R N3 Each is independent as C 1-4 Alkyl group. Preferably, T l T2 and T3 are independent of each other. In this case, the hydrophilic group is preferably substituted on the benzene ring, more preferably on the 5' ring. The adjacent position of the substituent.

[0143] In some implementations, in formula Ia: Q1 is: , or Preferred ; A l For bond, -(CH2) a1 -、-(CH2) a1 -C(O)-, -(CH2) a1 -(O-CH2-CH2) a2 -、-(CH2) a1 -(O-CH2-CH2) a2 -(CH2) a3 -、-(CH2) a1 -C(O)NH-(CH2) a2 -、-(CH2) a1 -NHC(O)-(CH2) a2 -、-(CH2) a1 -C(O)NH-(CH2) a2 -C(O)-, -(CH2) a1 -(cycloalkylene)-C(O)-, -C≡C-(CH2) a1 - and -C≡C-(CH2) a1 -CO-, where a1, a2, and a3 are independently integers from 1 to 6, and the indicated substituent is connected to Q1 on the left and to B6 on the right; preferably -(CH2). a1 -C(O)-, a1 is preferably 1, 2, 3 or 4; B6 is a bond or is selected from the following groups: -NH-(CH2) b1 -C(O)- , -NH-(CH2) b1 - , -(CH2CH2O) b2 - , -(CH2CH2O) b2 - , -(CH2CH2O) b2 -(CH2) b1 -C(O)- , -(CH2CH2O) b2 -(CH2) b1 -C(O)-NH-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -NH-(CH2) b1 -(CH2OCH2) b2 -(CH2) b1 -C(O)- , -C(O)-(CH2CH2O) b2 -(CH2) b1 -NH- , -C(O)-(CH2CH2O) b2 -(CH2) b1 -NH- , -C(O)-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -NH-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -(OCH2CH2) b2 -C(O)- , -C(O)-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -NH-CO-NH-(CH2) b1 -CO- In the formula, Indicates the position where B6 connects to A1. The position where B6 connects to L1 is indicated, with b1 and b2 each independently being 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably... -NH-(CH2CH2O) b2 -(CH2) b1 -C(O)- b2 is preferably 6, 7, 8 or 9, and b1 is preferably 1, 2, 3 or 4; L l For bonds or the following groups: , , , , or In the formula, 3 indicates the position where L1 connects to B6. 4 indicates the position where L1 and T1 are connected; preferably... ; T l Selected from the following groups: 4 -NH-(CH2) L6 - 5. , , , , , , , , , , , , , and In the formula, 4 indicates the position where T1 and L1 are connected. 5 indicates the position where T1 and D1 are connected; where R t H, optionally substituted alkyl groups, or -(CH2CH2O) t1 -CH3, t1 is an integer selected from 0-15, and L6 is an integer selected from 0-15; preferably R t Preferably -(CH2CH2O) t1 -CH3, t1 is preferably 1, 2, 3, 4, 5 or 6; D1 is defined as described in any embodiment of this document, and is preferably a pharmaceutical unit derived from an ATR inhibitor as described in any embodiment of this document.

[0144] In some implementations, in formula Ib: Q1 is: , or Preferred ; A l For bond, -(CH2) a1 -、-(CH2) a1 -C(O)-, -(CH2) a1 -(O-CH2-CH2) a2 -、-(CH2) a1 -(O-CH2-CH2) a2 -(CH2) a3 -、-(CH2) a1 -C(O)NH-(CH2) a2 -、-(CH2) a1 -NHC(O)-(CH2) a2 -、-(CH2) a1-C(O)NH-(CH2) a2 -C(O)-, -(CH2) a1 -(cycloalkylene)-C(O)-, -C≡C-(CH2) a1 - and -C≡C-(CH2) a1 -CO-, where a1, a2, and a3 are independently integers from 1 to 6, and the indicated substituent is connected to Q1 on the left and to B6 on the right; preferably -(CH2). a1 -C(O)-, a1 is preferably 1, 2, 3 or 4; B4 and B5 can each form a bond independently or be selected from the following groups: -NH-(CH2) b1 -C(O)- , -NH-(CH2) b1 - , -(CH2CH2O) b2 - , -(CH2CH2O) b2 - , -(CH2CH2O) b2 -(CH2) b1 -C(O)- , -(CH2CH2O) b2 -(CH2) b1 -C(O)-NH-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -NH-(CH2) b1 -(CH2OCH2) b2 -(CH2) b1 -C(O)- , -C(O)-(CH2CH2O) b2 -(CH2) b1 -NH- , -C(O)-(CH2CH2O) b2 -(CH2) b1 -NH- , -C(O)-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -NH-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -(OCH2CH2) b2 -C(O)- , -C(O)-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -NH-CO-NH-(CH2) b1 -CO- In the formula, This indicates the position where B4 connects to A1 or where B5 connects to B2. This indicates the position where B4 connects to B2 or where B5 connects to B1, where b1 and b2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably... -NH-(CH2CH2O) b2 -(CH2) b1 -C(O)- b2 is preferably 1, 2, 3, 4, 5, 6, 7, 8 or 9, and b1 is preferably 1, 2, 3 or 4; B2 and B1 are either independently bonds or independently selected from the following groups: and L4 is -(CH2) m1 -, m1 is 1, 2, 3, 4, 5 or 6, where, 1 indicates the position where B1 connects to B5, or the position where B2 connects to B4; 2 indicates the position where B1 connects to E, or the position where B2 connects to B5; 3 indicates the position where B1 connects to L1, or the position where B2 connects to L2; preferably... ; L l Each of L2 and L2 can be independently bonded or have the following groups: , , , , or In the formula, 3 indicates the position where L1 connects to B1 or L2 connects to B2. 4 indicates the position where L1 connects to T1 or the position where L2 connects to T2; preferably... ; T l T2 and T2 are each independently selected from the following groups: 4 -NH-(CH2) L6 - 5. , , , , , , , , , , , , , and In the formula, 4 indicates the position where T1 connects to L1 or the position where T2 connects to L2. 5 indicates the position where T1 connects to D1 or the position where T2 connects to D2; where R t H, optionally substituted alkyl groups, or -(CH2CH2O) t1 -CH3, t1 is an integer selected from 0-15, L6 is an integer from 0-15; T1 is preferably... R t Preferably -(CH2CH2O) t1 -CH3, t1 is preferably 1, 2, 3, 4, 5 or 6; T2 is preferably ; E is selected from: H, -(CH2) a1 CH3, -(CH2) a1 -C(O)-(CH2) a1 CH3, -(CH2) a1 -(O-CH2-CH2) a2 -(CH2) a1 CH3, CH3(CH2) a1 -(O-CH2-CH2) a2 -(CH2) a3 -、-(CH2) a1 -C(O)NH-(CH2) a2 CH3, -(CH2) a1 -NH-(CH2) a1 CH3, -(CH2) a1 -NHC(O)-(CH2) a1 CH3, -(CH2) a1 -C(O)NH-(CH2) a2 -C(O)-(CH2) a1 CH3, -(CH2) a1 -(cycloalkylene)-C(O)-(CH2)a1 CH3、-C≡C-(CH2) a1 CH3、-C≡C-(CH2) a1 -CO-(CH2) a1 CH3, -NH-(CH2) b1 -C(O)-(CH2) a1 CH3, -NH-(CH2) b1 -C(O)-NH2、-NH-(CH2CH2O) b2 -(CH2) b1 -C(O)-(CH2) a1 CH3、-NH-(CH2CH2O) b2 -(CH2) b1 -C(O)-NH2、-NH-(CH2CH2O) b2 -CH3, -(OCH2CH2) b2 -CO-NH2 and -NH-CO-NH-(CH2) b1 -CO-NH2, wherein a1, a2, a3, b1, and b2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably -NH-(CH2CH2O). b2 -(CH2) b1 -C(O)-NH2; D1 and D2 are defined as described in any embodiment of this document, wherein D1 is preferably a drug unit derived from an ATR inhibitor as described in any embodiment of this document, and D2 is preferably a drug unit derived from a topoisomerase I inhibitor as described in any embodiment of this document.

[0145] In some implementations, the compound of formula I herein may be selected from: .

[0146] Antibody-drug conjugates

[0147] Antibodies can be directly or via linkers to drugs. As is well known in the art, linkers can be classified into two categories: unbreakable linkers and breakable linkers. Breakable linkers can be further divided into two main categories: chemically unstable linkers and enzyme-unstable linkers. Chemically unstable linkers can be selectively broken due to differences in plasma and cytoplasmic properties. Such properties include pH value and glutathione concentration. pH-sensitive linkers, often called acid-breaking linkers, are relatively stable in the neutral environment of blood (pH 7.3-7.5), but will be hydrolyzed in weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). Glutathione-sensitive linkers, also known as disulfide linkers, release drugs based on the difference between the high intracellular glutathione concentration (millimolar range) and the relatively low blood glutathione concentration (micromolar range). Enzyme-unstable linkers, such as peptide linkers, offer better control over drug release. Peptide linkers can be effectively cleaved by lysosomal proteases, such as cathepsin B or plasmin (these enzymes are present in increased amounts in some tumor tissues). Typical enzyme-unstable linkers include Val-Cit (VC) and Phe-Lys. Self-cleaving linkers are generally embedded between the cleavable linker and the active drug, or are themselves part of the cleavable linker. Common self-cleaving linkers include p-aminobenzyl alcohol (PAB) and β-glucuronide.

[0148] Linkers may comprise one or more linker components. For example, in some embodiments, the linker may have a VL structure, where the V component may or may not be present, or may be a tridentate linker component or a tetramaleimide-type linker component as described below, and the L component may be an unbreakable linker or a breakable linker as described above, such as an acid-labile linker (e.g., hydrazone), a protease-sensitive linker (e.g., peptidase-sensitive), a light-labile linker, a dimethyl linker, or a disulfide-containing linker. Exemplary linker components include 6-maleiminohexanoyl, maleiminopropionylvaline-citrulline, alanine-phenylalanine, p-aminobenzyloxycarbonyl, N-succinimino-4-(2-pyridylthio)valerate, N-succinimino-4-(N-maleiminomethyl)cyclohexane-1-carboxylate, and N-succinimino-(4-iodo-acetyl)aminobenzoate. Other exemplary linker components may also be linkers containing amino acid units to allow protease cleavage, thereby facilitating the release of the drug from the immunoconjugate upon exposure to intracellular proteases, such as lysosomal enzymes. Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include: valine-citrulline; alanine-phenylalanine; phenylalanine-lysine; or N-methyl-valine-citrulline. Exemplary tripeptides include: glycine-valine-citrulline and glycine-glycine-glycine.

[0149] An exemplary three-tooth (or bismaleimide) connector may have the following structure: .

[0150] An exemplary tetramaleimide-type connector may have the following structure: , , , , , , , , , , , , .

[0151] Other suitable linkers, drugs, or linker-drug examples can be found in CN 103933575 A and CN107652219 A. The entire contents of these two applications are incorporated herein by reference.

[0152] In some implementations, the antibody-drug conjugates described herein may have the following structures: Ab-(VLD)p Wherein, Ab is an antibody; VL is a linker, V may or may not be present, and may be any tridentate linker component or tetramaleimide linker component as described above, L is a cleavable linker or an uncleavable linker, and at least one of V and L is present; D is a pharmaceutical unit as described in any embodiment of the present invention, and it includes at least the pharmaceutical unit of formula (i) as described in any embodiment of the present invention; p is the drug-antibody ratio (DAR), the value of which is in the range of 1-8 (e.g., an integer or decimal of 1-8).

[0153] In some embodiments, the VLD may be the “linker-drug” portion described herein, wherein the VLD is covalently linked to the antibody Ab via a functional group in the V portion corresponding to the Q1-derived antibody molecule.

[0154] In some embodiments, the antibody-drug conjugates described herein are compounds of Formula II, their stereoisomers, tautomers, N-oxides, hydrates, solvates, isotopically labeled compounds, or pharmaceutically acceptable salts, or mixtures thereof: (II) Wherein, n, m, t, A1, B1, B2, B3, B4, B5, B6, B7, B8, L1, L2, L3, T1, T2, T3, D1, D2, and D3 are each as described in any embodiment of this document; Ab is an antibody molecule; Q'1 is a functional group derived from Q1 and coupled to the antibody molecule, such as a functional group coupled to cysteine, lysine, non-natural amino acid, or glycosyl group of the antibody molecule; p is an integer or decimal from 1 to 8.

[0155] In some embodiments, one group of preferred compounds of Formula II of the present invention is represented as compounds of Formula IIa, their stereoisomers, tautomers, N-oxides, hydrates, solvates, isotopically labeled compounds, or pharmaceutically acceptable salts, or mixtures thereof: (IIa) Wherein, Ab, Q'1, A1, B6, L1, T1, D1 and p are each as described in any of the embodiments herein.

[0156] In some embodiments, one group of preferred compounds of Formula II of the present invention is represented as compounds of Formula IIb, their stereoisomers, tautomers, N-oxides, hydrates, solvates, isotopically labeled compounds, or pharmaceutically acceptable salts, or mixtures thereof: (IIb) Wherein, Ab, Q'1, A1, B1, B2, B4, B5, L1, L2, T1, T2, D1, D2, E and p are each as described in any of the embodiments herein.

[0157] In some embodiments, one group of preferred compounds of Formula II of the present invention is represented as compounds of Formula IIc, their stereoisomers, tautomers, N-oxides, hydrates, solvates, isotopically labeled compounds, or pharmaceutically acceptable salts, or mixtures thereof:

[0158] (IIc)

[0159] Wherein, Ab, Q'1, A1, B1, B2, B3, B4, B5, B6, L1, L2, L3, T1, T2, T3, D1, D2, D3, E and p are each as described in any of the embodiments herein.

[0160] In some embodiments, one group of preferred compounds of Formula II of the present invention is represented as compounds of Formula IId, their stereoisomers, tautomers, N-oxides, hydrates, solvates, isotopically labeled compounds, or pharmaceutically acceptable salts, or mixtures thereof:

[0161] (IId)

[0162] Wherein, Ab, Q'1, A1, B2, B3, B4, B5, B6, B7, B8, L1, L2, L3, T1, T2, T3, D1, D2, D3 and p are each as described in any of the embodiments herein.

[0163] In some embodiments, one group of preferred compounds of Formula II of the present invention is represented as compounds of Formula IIe, their stereoisomers, tautomers, N-oxides, hydrates, solvates, isotopically labeled compounds, or pharmaceutically acceptable salts, or mixtures thereof:

[0164] (IIe)

[0165] Wherein, Ab, Q'1, A1, B2, B4, B5, B7, L1, L2, T1, T2, D1, D2 and p are each as described in any of the embodiments herein.

[0166] In some implementations, the compound of formula II herein may be selected from:

[0167] In each formula, p is an integer or decimal from 1 to 8.

[0168] In some embodiments of Formula II, p is an integer or decimal of 2 to 6, preferably p is 4 or 6.

[0169] In some embodiments, the present invention provides compounds of Formula III, stereoisomers thereof, tautomers, N-oxides, hydrates, solvates, isotopically labeled compounds, or pharmaceutically acceptable salts thereof, or mixtures thereof:

[0170] (III)

[0171] in: Ab, Q'1, A1, B6, B5, L1, L2, T1, T2, D1, D2 and p are each as described in any embodiment herein, and at least one of D1 and D2 is a pharmaceutical unit derived from a compound of formula (i) or formula (ii) described herein or a deuterated derivative thereof; Q'2 is a functional group derived from Q1 that can couple with antibody molecules, especially functional groups that can couple with cysteine, lysine, non-natural amino acids or glycosyl groups of antibody molecules. A2 is selected from: bonded, optionally substituted alkylene, optionally substituted alkylene acyl, optionally substituted alkylene-polyethylene glycol, optionally substituted polyethylene glycol, optionally substituted alkylene-polyethylene glycol-alkylene, optionally substituted alkylene-C(O)NH-alkylene, optionally substituted alkylene-NHC(O)-alkylene, optionally substituted alkylene-C(O)NH-alkylene-acyl, optionally substituted alkylene-cycloalkylene acyl, optionally substituted polyethylene glycol-alkylene-acyl, optionally substituted alkenyl, optionally substituted alkyneyl, optionally substituted alkyneyl- Alkylene, optionally substituted carbocyclic, optionally substituted heterocyclic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclic-alkylene-acyl, optionally substituted aryl-alkylene-acyl, and optionally substituted heteroaryl-alkylene-acyl; preferably, when A2 is substituted, the number of substituents may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, and the substituents are preferably selected from hydrophilic groups, halogens, hydroxyl groups, amino groups, carboxyl groups, sulfonic acid groups, sulfone groups, phosphate groups, alkoxy groups and alkylene-NH-alkylene-carboxyl groups; q is the drug-antibody ratio (DAR), which is an integer or decimal from 1 to 8.

[0172] In some implementations, A2 has the same definition as A1.

[0173] In some embodiments of Formula III, D1 and D2 are preferably as described in any of the preferred examples above.

[0174] In some implementations, in Equation III: Q1 is: , or Preferred ; A l Each of A2 and -(CH2) is an independent bond. a1 -、-(CH2) a1 -C(O)-, -(CH2) a1 -(O-CH2-CH2) a2 -、-(CH2) a1 -(O-CH2-CH2) a2 -(CH2) a3 -、-(CH2) a1 -C(O)NH-(CH2) a2 -、-(CH2) a1 -NHC(O)-(CH2)a2 -、-(CH2) a1 -C(O)NH-(CH2) a2 -C(O)-, -(CH2) a1 -(cycloalkylene)-C(O)-, -C≡C-(CH2) a1 - and -C≡C-(CH2) a1 -CO-, wherein a1, a2, and a3 are independently integers from 1 to 6, and the indicated substituent is connected to Q'1 or Q'2 on the left and to B5 or B6 on the right; preferably -(CH2). a1 -C(O)-, a1 is preferably 1, 2, 3 or 4; B5 and B6 can each form a bond independently or be selected from the following groups: -NH-(CH2) b1 -C(O)- , -NH-(CH2) b1 - , -(CH2CH2O) b2 - , -(CH2CH2O) b2 - , -(CH2CH2O) b2 -(CH2) b1 -C(O)- , -(CH2CH2O) b2 -(CH2) b1 -C(O)-NH-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -NH-(CH2) b1 -(CH2OCH2) b2 -(CH2) b1 -C(O)- , -C(O)-(CH2CH2O) b2 -(CH2) b1 -NH- , -C(O)-(CH2CH2O) b2 -(CH2) b1 -NH- , -C(O)-(CH2CH2O) b2 -(CH2) b1-C(O)- , -NH-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -(OCH2CH2) b2 -C(O)- , -C(O)-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -NH-CO-NH-(CH2) b1 -CO- In the formula, This indicates the position where B6 connects to A1 or where B5 connects to A2. This indicates the position where B6 connects to L1 or B5 connects to L2, where b1 and b2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably... -NH-(CH2CH2O) b2 -(CH2) b1 -C(O)- b2 is preferably 6, 7, 8 or 9, and b1 is preferably 1, 2, 3 or 4; L l Each of L2 and L2 can be independently bonded or have the following groups: , , , , or In the formula, 3 indicates the position where L1 connects to B6 or the position where L2 connects to B5. 4 indicates the position where L1 connects to T1 or the position where L2 connects to T2; preferably... ; T l T2 and T2 are each independently selected from the following groups: 4 -NH-(CH2) L6 - 5. , , , , , , , , , , , , , and In the formula, 4 indicates the position where T1 connects to L1 or the position where T2 connects to L2. 5 indicates the position where T1 connects to D1 or the position where T2 connects to D2; where R t H, optionally substituted alkyl groups, or -(CH2CH2O) t1 -CH3, t1 is an integer selected from 0-15, L6 is an integer from 0-15; T1 is preferably... R t Preferably -(CH2CH2O) t1 -CH3, t1 is preferably 1, 2, 3, 4, 5 or 6; T2 is preferably ; The definitions of Ab, D1, D2, p, and q are as described in any embodiment of this document; D1 is preferably a drug unit derived from an ATR inhibitor as described in any embodiment of this document, and D2 is preferably a drug unit derived from a topoisomerase I inhibitor as described in any embodiment of this document.

[0175] In some implementations, the compound of formula III herein may be selected from:

[0176] In each expression, p and q are each an independent integer or decimal from 1 to 8.

[0177] In some implementations of Equation III, p and q are both integers or decimals of 3-5.

[0178] In this article, antibody (Ab) includes the antibody itself and its antigen-binding fragment. Antibodies can be monoclonal antibodies.

[0179] In a preferred embodiment, the antibody (especially a monoclonal antibody) is an antibody against the following antigens: HER2, HER3, TROP2, B7-H3, B7-H4, GPC20, GPRC5D, CDH6, EGFR, EGFRvIIL, AXL, Nectin-4, Tissuefactor, TIM-1, PSMA, PTK7, EpCAM, MUCI, STEAP1, GPNMB, FGF2, FOLRI, FolRα, C-MET, GFR, AGS-I6, and Guanylyl cyclase. C, Mesothelin, SLC44A4, EPhA2, AGS-5, GPC-3, c-KIT, RORl, ROR2, PD1, PD-Ll, CD27L, 5T4, MerTK, Mucin16, NaPi2b, STEAP, SLlTRK6, ETBR, BCMA, CEACAM5, ICAM1, SC-16, SLC39A6, Delta-like protein3, Claudin 18.2, Claudin 3, Claudin 6, Claudin 9. CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD73, CD74, CD79b, CDl23, CDI38, CDI47, CDl66, CD223, MUCl6, MSLN, ENPP3, SLTRK6, FGFR, LIV-1, Lewis Y, av-integrin, ITGB6 (integrin beta-6), ASCT2, C4.4a, CA-IX, CD324, CD352, CD44v6, CD48a, CLL-1, Cripto, CS1, DPEP3, Ephrin-A2. Ephrin-A4, ETBR, FGFR2, FGFR3, FLT3, GD3, Globo H, GPC3, LAMP-1, LRRC15, Ly6E, TM4SF1, MFI2, NOTCH3, p-cadherin, PRLR, CLDNl, CD228, CUB domain-containing protein 1(CDCPl), CXCR4, LGR4, FZD7, CTR, GPR56, CCR7, DDRl, RNF43, CEACAM1, CEACAM6, GCC, GPA33, TEM8, AMHR2, NTSE, FLT1, DKK1, sema4D, Her2 / neu , HER1, VWF, IGF-1, GRP78, MART-1 / Melan-A, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2, MAGE-Xp3, MAGE-Xp4, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE- 5. GAGE-6, GAGE-7, GAGE-8, GAGE-9, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, SSX-1, SSX-2, SSX-4, SSX-5, SCP-1, CT-7, c-erbB-2, HPV-16 E6, HPV-16E7, HPV-18 E6, HPV-18 E7, EBNA-1, βhCG, WT1, TRP-2, NY-BR-1, NY-CO-58, MN, Lea, Leb, Lex, H-2, B-1, B-2, CD25, CD40, CD44, CD45R, CD49, CD93, CD163, CD205, CD206, CD303, CD304, BMPR1B, E16, 0772P, Napi3b, Sema 5b. PSCA The antibodies used include hlg, MSG783, STEAP2, TrpM4, CD21, FcRH2, NCA, MDP, IL20Rα, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, TMEFF1, GDNF-Ra1, Ly6E, TMEM46, Ly6G6D, LGR5, RET, LY6K, GPR19, GPR54, ASPHD1, TMEM118, GPR172A, CD151, folate receptor β, transferrin receptor, IL4 receptor, and IL2 receptor. In some embodiments, the antibodies used are antibodies against HER2, CEACAM5, or their antigen-binding fragments. In some embodiments, the antibodies used are anti-HER2 antibodies or anti-CEACAM5 antibodies or their antigen-binding fragments.

[0180] In this document, the antibody is an anti-CEACAM5 antibody or its antigen-binding fragment. In some embodiments, the anti-CEACAM5 antibody used herein is any anti-CEACAM5 antibody described in WO2023198019A1.

[0181] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment described herein comprises the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3: (a) HCDR1, selected from any amino acid sequence of SEQ ID NO: 1-14 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with any amino acid sequence of SEQ ID NO: 1-14, or an amino acid sequence having one or more (e.g., 1-5, preferably 2 or 3) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to any amino acid sequence of SEQ ID NO: 1-14; (b) HCDR2, selected from any amino acid sequence of SEQ ID NO: 15-28, 121, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of identity with any amino acid sequence of SEQ ID NO: 15-28, 121, or an amino acid sequence having one or more (e.g., 1-5, preferably 2 or 3) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to any amino acid sequence of SEQ ID NO: 15-28, 121; (c) HCDR3, selected from any amino acid sequence of SEQ ID NO: 29-41, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of identity with any amino acid sequence of SEQ ID NO: 29-41, or an amino acid sequence having one or more (e.g., 1-5, preferably 2 or 3) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to any amino acid sequence of SEQ ID NO: 29-41; (d) LCDR1, selected from any amino acid sequence of SEQ ID NO: 42-54, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of identity with any amino acid sequence of SEQ ID NO: 42-54, or an amino acid sequence having one or more (e.g., 1-5, preferably 2 or 3) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to any amino acid sequence of SEQ ID NO: 42-54; (e) LCDR2, selected from any amino acid sequence of SEQ ID NO: 55-65, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with any amino acid sequence of SEQ ID NO: 55-65, or an amino acid sequence having one or more (e.g., 1-5, preferably 2 or 3) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to any amino acid sequence of SEQ ID NO: 55-65; and / or (f) LCDR3, selected from any amino acid sequence of SEQ ID NO: 66-79, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with any amino acid sequence of SEQ ID NO: 66-79, or an amino acid sequence having one or more (e.g., 1-5, preferably 2 or 3) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to any amino acid sequence of SEQ ID NO: 66-79.

[0182] In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the antibody are selected from any combination shown in (1)-(17) below: (1) HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 15, HCDR3 shown in SEQ ID NO: 29, LCDR1 shown in SEQ ID NO: 42, LCDR2 shown in SEQ ID NO: 55, and LCDR3 shown in SEQ ID NO: 66; (2) HCDR1 shown in SEQ ID NO: 2, HCDR2 shown in SEQ ID NO: 16, HCDR3 shown in SEQ ID NO: 30, LCDR1 shown in SEQ ID NO: 43, LCDR2 shown in SEQ ID NO: 56, and LCDR3 shown in SEQ ID NO: 67; (3) HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 17, HCDR3 shown in SEQ ID NO: 31, LCDR1 shown in SEQ ID NO: 44, LCDR2 shown in SEQ ID NO: 57, and LCDR3 shown in SEQ ID NO: 68; (4) HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 18, HCDR3 shown in SEQ ID NO: 32, LCDR1 shown in SEQ ID NO: 45, LCDR2 shown in SEQ ID NO: 58, and LCDR3 shown in SEQ ID NO: 69; (5) HCDR1 shown in SEQ ID NO: 5, HCDR2 shown in SEQ ID NO: 19, HCDR3 shown in SEQ ID NO: 33, LCDR1 shown in SEQ ID NO: 46, LCDR2 shown in SEQ ID NO: 59, and LCDR3 shown in SEQ ID NO: 70; (6) HCDR1 shown in SEQ ID NO: 6, HCDR2 shown in SEQ ID NO: 20, HCDR3 shown in SEQ ID NO: 34, LCDR1 shown in SEQ ID NO: 47, LCDR2 shown in SEQ ID NO: 60, and LCDR3 shown in SEQ ID NO: 67; (7) HCDR1 shown in SEQ ID NO: 6, HCDR2 shown in SEQ ID NO: 20, HCDR3 shown in SEQ ID NO: 34, LCDR1 shown in SEQ ID NO: 48, LCDR2 shown in SEQ ID NO: 61, and LCDR3 shown in SEQ ID NO: 71; (8) HCDR1 shown in SEQ ID NO: 7, HCDR2 shown in SEQ ID NO: 21, HCDR3 shown in SEQ ID NO: 35, LCDR1 shown in SEQ ID NO: 49, LCDR2 shown in SEQ ID NO: 62, and LCDR3 shown in SEQ ID NO: 72; (9) HCDR1 shown in SEQ ID NO: 8, HCDR2 shown in SEQ ID NO: 22, HCDR3 shown in SEQ ID NO: 36, LCDR1 shown in SEQ ID NO: 50, LCDR2 shown in SEQ ID NO: 63, and LCDR3 shown in SEQ ID NO: 73; (10) HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 23, HCDR3 shown in SEQ ID NO: 30, LCDR1 shown in SEQ ID NO: 51, LCDR2 shown in SEQ ID NO: 56, and LCDR3 shown in SEQ ID NO: 74; (11) HCDR1 shown in SEQ ID NO: 10, HCDR2 shown in SEQ ID NO: 24, HCDR3 shown in SEQ ID NO: 37, LCDR1 shown in SEQ ID NO: 50, LCDR2 shown in SEQ ID NO: 63, and LCDR3 shown in SEQ ID NO: 75; (12) HCDR1 shown in SEQ ID NO: 11, HCDR2 shown in SEQ ID NO: 24, HCDR3 shown in SEQ ID NO: 37, LCDR1 shown in SEQ ID NO: 50, LCDR2 shown in SEQ ID NO: 63, and LCDR3 shown in SEQ ID NO: 76; (13) HCDR1 shown in SEQ ID NO: 12, HCDR2 shown in SEQ ID NO: 25, HCDR3 shown in SEQ ID NO: 38, LCDR1 shown in SEQ ID NO: 52, LCDR2 shown in SEQ ID NO: 64, and LCDR3 shown in SEQ ID NO: 67; (14) HCDR1 shown in SEQ ID NO: 2, HCDR2 shown in SEQ ID NO: 26, HCDR3 shown in SEQ ID NO: 39, LCDR1 shown in SEQ ID NO: 51, LCDR2 shown in SEQ ID NO: 56, and LCDR3 shown in SEQ ID NO: 77; (15) HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 27, HCDR3 shown in SEQ ID NO: 40, LCDR1 shown in SEQ ID NO: 53, LCDR2 shown in SEQ ID NO: 57, and LCDR3 shown in SEQ ID NO: 78; (16) HCDR1 shown in SEQ ID NO: 14, HCDR2 shown in SEQ ID NO: 28, HCDR3 shown in SEQ ID NO: 41, LCDR1 shown in SEQ ID NO: 54, LCDR2 shown in SEQ ID NO: 65, and LCDR3 shown in SEQ ID NO: 79; (17) HCDR1 shown in SEQ ID NO: 2, HCDR2 shown in SEQ ID NO: 121, HCDR3 shown in SEQ ID NO: 30, LCDR1 shown in SEQ ID NO: 43, LCDR2 shown in SEQ ID NO: 56, and LCDR3 shown in SEQ ID NO: 67.

[0183] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein: (a) The heavy chain variable region has any of the amino acid sequences given in SEQ ID NO: 80-94, 122-125, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same as the amino acid sequences given in SEQ ID NO: 80-94, 122-125, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to any of the amino acid sequences in SEQ ID NO: 80-94, 122-125; (b) The light chain variable region has any of the amino acid sequences given in SEQ ID NO: 95-110, 126-128; or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same as any of the amino acid sequences given in SEQ ID NO: 95-110, 126-128; or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to any of the amino acid sequences in SEQ ID NO: 95-110, 126-128.

[0184] In some embodiments, the heavy chain variable region and light chain variable region of the antibody against CEACAM5 described herein or its antigen-binding fragment are selected from any combination of the following (1)-(28): (1) SEQ ID NO: 80 and SEQ ID NO: 95; (2) SEQ ID NO: 81 and SEQ ID NO: 96; (3) SEQ ID NO: 82 and SEQ ID NO: 97; (4) SEQ ID NO: 83 and SEQ ID NO: 98; (5) SEQ ID NO: 84 and SEQ ID NO: 99; (6) SEQ ID NO: 85 and SEQ ID NO: 100; (7) SEQ ID NO: 85 and SEQ ID NO: 101; (8) SEQ ID NO: 86 and SEQ ID NO: 102; (9) SEQ ID NO: 87 and SEQ ID NO: 103; (10) SEQ ID NO: 88 and SEQ ID NO: 104; (11) SEQ ID NO: 89 and SEQ ID NO: 105; (12) SEQ ID NO: 90 and SEQ ID NO: 106; (13) SEQ ID NO: 91 and SEQ ID NO: 107; (14) SEQ ID NO: 92 and SEQ ID NO: 108; (15) SEQ ID NO: 93 and SEQ ID NO: 109; (16) SEQ ID NO: 94 and SEQ ID NO: 110; (17) SEQ ID NO: 122 and SEQ ID NO: 126; (18) SEQ ID NO: 122 and SEQ ID NO: 127; (19) SEQ ID NO: 122 and SEQ ID NO: 128; (20) SEQ ID NO: 123 and SEQ ID NO: 126; (21) SEQ ID NO: 123 and SEQ ID NO: 127; (22) SEQ ID NO: 123 and SEQ ID NO: 128; (23) SEQ ID NO: 124 and SEQ ID NO: 126; (24) SEQ ID NO: 124 and SEQ ID NO: 127; (25) SEQ ID NO: 124 and SEQ ID NO: 128; (26) SEQ ID NO: 125 and SEQ ID NO: 126; (27) SEQ ID NO: 125 and SEQ ID NO: 127; (28) SEQ ID NO: 125 and SEQ ID NO: 128.

[0185] In some implementations, the heavy and light chains of the anti-CEACAM5 antibody described herein are selected from any one of the following groups (1)-(13): (1) The antibody contains a heavy chain full-length sequence containing an amino acid sequence SEQ ID NO: 136, and the antibody contains a light chain full-length sequence containing an amino acid sequence SEQ ID NO: 137; (2) The antibody contains a heavy chain full-length sequence containing an amino acid sequence SEQ ID NO: 129, and the antibody contains a light chain full-length sequence containing an amino acid sequence SEQ ID NO: 133; (3) The antibody contains a heavy chain full-length sequence containing an amino acid sequence SEQ ID NO: 129, and the antibody contains a light chain full-length sequence containing an amino acid sequence SEQ ID NO: 134; (4) The antibody contains a heavy chain full-length sequence containing an amino acid sequence SEQ ID NO: 129, and the antibody contains a light chain full-length sequence containing an amino acid sequence SEQ ID NO: 135; (5) The antibody contains a heavy chain full-length sequence containing an amino acid sequence SEQ ID NO: 130, and the antibody contains a light chain full-length sequence containing an amino acid sequence SEQ ID NO: 133; (6) The antibody contains a heavy chain full-length sequence containing an amino acid sequence SEQ ID NO: 130, and the antibody contains a light chain full-length sequence containing an amino acid sequence SEQ ID NO: 134; (7) The antibody contains a heavy chain full-length sequence containing an amino acid sequence SEQ ID NO: 130, and the antibody contains a light chain full-length sequence containing an amino acid sequence SEQ ID NO: 135; (8) The antibody contains a heavy chain full-length sequence containing an amino acid sequence SEQ ID NO: 131, and the antibody contains a light chain full-length sequence containing an amino acid sequence SEQ ID NO: 133; (9) The antibody contains a heavy chain full-length sequence containing an amino acid sequence SEQ ID NO: 131, and the antibody contains a light chain full-length sequence containing an amino acid sequence SEQ ID NO: 134; (10) The antibody contains a heavy chain full-length sequence containing an amino acid sequence SEQ ID NO: 131, and the antibody contains a light chain full-length sequence containing an amino acid sequence SEQ ID NO: 135; (11) The antibody contains a heavy chain full-length sequence containing an amino acid sequence SEQ ID NO: 132, and the antibody contains a light chain full-length sequence containing an amino acid sequence SEQ ID NO: 133; (12) The antibody contains a heavy chain full-length sequence containing an amino acid sequence SEQ ID NO: 132, and the antibody contains a light chain full-length sequence containing an amino acid sequence SEQ ID NO: 134; (13) The antibody contains a heavy chain full-length sequence containing an amino acid sequence (SEQ ID NO: 132) and a light chain full-length sequence containing an amino acid sequence (SEQ ID NO: 135).

[0186] It should be understood that the amino acid sequences shown by the above sequence numbers in this document are the same as the amino acid sequences of the corresponding sequence numbers recorded in WO2023198019A1. All content disclosed in WO2023198019A1 is incorporated herein by reference.

[0187] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment further comprises an Fc region selected from mouse IgG1, IgG2a, IgG2b and / or IgG3, or rat IgG1, IgG2a, IgG2b and / or IgG2c.

[0188] In some embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment further comprises an Fc region selected from human IgG1, IgG2, IgG3 and / or IgG4, or an Fc region amino acid sequence having one or more amino acid mutations (preferably substitutions, insertions or deletions) with human IgG1, IgG2, IgG3, IgG4.

[0189] In a preferred embodiment, the at least two compounds linked to the antibody molecule are linked to different coupling sites on the antibody molecule; preferably, the different coupling sites are selected from natural or modified thiol, amino, and non-natural amino acids of the antibody.

[0190] Pharmaceutical Composition

[0191] This invention provides a pharmaceutical composition comprising the linker-drug or antibody-drug conjugate described herein. The pharmaceutical composition may also contain a pharmaceutically acceptable carrier or excipient well known in the art.

[0192] In this article, pharmaceutically acceptable carriers or excipients can be any pharmaceutically acceptable carrier or excipient, which may vary depending on the dosage form and route of administration. Pharmaceutically acceptable carriers are generally safe and non-toxic and may contain any known substance used in the formulation of pharmaceutical compositions in the pharmaceutical industry, including one or more of fillers, diluents, coagulants, binders, lubricants, flow aids, stabilizers, colorants, wetting agents, and disintegrants. Suitable pharmaceutically acceptable carriers include sugars such as lactose or sucrose, mannitol or sorbitol; cellulose preparations and / or calcium phosphates such as tricalcium phosphate or calcium hydrogen phosphate; starches, including corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone; silica, talc, stearic acid or its salts, such as magnesium stearate or calcium stearate; and / or polyethylene glycol, etc. When selecting a pharmaceutically acceptable carrier, the route of administration of the drug dosage form is the primary consideration. This is well known in the field.

[0193] In some embodiments, the pharmaceutical composition comprises the antibody-drug conjugate of the present invention at a concentration of 0.1-100 mg / mL, such as antibody-drug conjugates at concentrations of 1 to 20 mg / mL, 5 to 15 mg / mL, 8 to 12 mg / mL, or 9 to 11 mg / mL.

[0194] In some embodiments, the pharmaceutical composition comprises at least one selected from L-histidine, TWEEN-20, trehalose dihydrate, and sucrose. In some embodiments, the pharmaceutical composition provided herein further comprises hydrochloric acid or succinic acid.

[0195] In some embodiments, the pharmaceutical composition has a pH in the range of 5.5 to 6.5, such as a pH in the range of 5.7 to 6.3. In some embodiments, the pH is measured at room temperature. In other embodiments, the pH is measured at 15°C to 27°C. In other embodiments, the pH is measured at 4°C. In other embodiments, the pH is measured at 25°C. In some embodiments, the pH is adjusted using HCl. In some embodiments, the pH is adjusted using succinic acid.

[0196] In one embodiment, the pharmaceutical compositions described herein may be formulated for single-dose or multiple-dose administration. Single-dose formulations are packaged in ampoules, vials, or syringes. Multiple-dose parenteral formulations may contain antimicrobial agents at concentrations that inhibit bacteria or fungi. As is known and practiced in the art, all parenteral formulations must be sterile.

[0197] In one embodiment, the pharmaceutical composition is provided as a ready-to-use sterile solution. In another embodiment, the pharmaceutical composition is provided as a sterile, dried, soluble product to be reconstituted with a carrier prior to use, including lyophilized powders and subcutaneous tablets. In another embodiment, the pharmaceutical composition is provided as a ready-to-use sterile suspension. In another embodiment, the pharmaceutical composition is provided as a sterile, dried, insoluble product to be reconstituted with a carrier prior to use. In yet another embodiment, the pharmaceutical composition is provided as a ready-to-use sterile emulsion.

[0198] In one embodiment, the pharmaceutical composition provided herein can be formulated as an immediate-release dosage form or an improved release dosage form, including delayed-release, sustained-release, pulsatile-release, controlled-release, targeted-release, and programmed-release forms.

[0199] Pharmaceutical compositions may also include excipients to protect the composition from rapid degradation or excretion from the body, such as controlled-release formulations, including implants, liposomes, hydrogels, prodrugs, and microencapsulation delivery systems. For example, time-delaying materials, such as glyceryl monostearate or glyceryl stearate, may be used alone or in combination with waxes. Prolonged absorption of injectable pharmaceutical compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate or gelatin). Microbial action can be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.

[0200] The pharmaceutical compositions provided herein can be stored at -80°C, 4°C, 25°C, or 37°C.

[0201] Lyophilized compositions can be prepared by freeze-drying the liquid pharmaceutical compositions provided herein. In one specific embodiment, the pharmaceutical compositions provided herein are lyophilized pharmaceutical compositions. In some embodiments, the pharmaceutical preparations are lyophilized powders that can be reconstituted for application in the form of solutions, emulsions, and other mixtures. They can also be reconstituted and formulated into solids or gels.

[0202] In some embodiments, the preparation of the lyophilized formulations provided herein involves batch filling of a bulk solution for lyophilization and aseptic filtration into vials, freezing the vials in a freeze dryer chamber, followed by lyophilization, stoppering, and capping.

[0203] Lyophilizers can be used to prepare lyophilized formulations. For example, the VirTis Genesis Model EL experimental unit can be used. This unit has a chamber with three working shelves (up to approximately 0.4 square meters of total usable shelf area), an external condenser, and a mechanical vacuum pump system. Cascaded mechanical refrigeration allows the shelves to be cooled to -70°C or lower, and the external condenser to -90°C or lower. The shelf temperature and chamber pressure are automatically controlled to + / -0.5°C and + / -2 micrometers (millitors), respectively. The unit is equipped with a capacitive pressure vacuum gauge, a Pirani vacuum gauge, a pressure transducer (measuring range: 0 to 1 atmosphere), and a relative humidity sensor.

[0204] The lyophilized powder can be prepared by dissolving the antibody-drug conjugate provided herein in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solution is then sterilely filtered and subsequently lyophilized under standard conditions known to those skilled in the art to obtain the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial will contain a single or multiple doses of the antibody-drug conjugate. The lyophilized powder can be stored under suitable conditions, such as from about 4°C to room temperature.

[0205] The lyophilized powder is reconstituted with water for injection to obtain a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable excipients. This amount can be determined empirically and adjusted as needed.

[0206] The exemplary recovery procedure is described below: (1) Fit an 18 or 20 needle into a 5 mL or 3 mL syringe and fill the syringe with water for injection (WFI) grade water; (2) Measure an appropriate amount of WFI using the syringe scale to ensure the syringe is free of air bubbles; (3) Insert the needle through the rubber stopper; (4) Dispense the entire contents of the syringe down the vial wall into the container, remove the syringe and needle and place them in the tip container; (5) Continuously vortex the vial to carefully dissolve the entire contents of the vial until fully recovered (e.g., about 20-40 seconds), and minimize excessive agitation of the protein solution that may cause foaming.

[0207] In some embodiments, the pharmaceutical compositions provided herein are supplied in the form of a dry, sterile lyophilized powder or anhydrous concentrate in a hermetically sealed container and can be reconstituted to an appropriate concentration with, for example, water or saline for administration to a subject. In some embodiments, the antibody-drug conjugate is supplied in the form of a dry, sterile lyophilized powder in a hermetically sealed container at unit doses of at least 0.1 mg, at least 0.5 mg, at least 1 mg, at least 2 mg, at least 3 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 60 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mg. The lyophilized antibody-drug conjugate can be stored in its original container between 2 and 8°C, and the antibody-drug conjugate can be administered within 12 hours, for example, within 6 hours, 5 hours, 3 hours, or 1 hour after reconstitution. In an alternative embodiment, the pharmaceutical composition comprising the antibody-drug conjugate provided herein is supplied in liquid form in a hermetically sealed container, the container indicating the quantity and concentration of the antibody-drug conjugate. In some embodiments, the liquid form of the antibody-drug conjugate is supplied in the hermetically sealed container at doses of at least 0.1 mg / mL, at least 0.5 mg / mL, at least 1 mg / mL, at least 5 mg / mL, at least 10 mg / mL, at least 15 mg / mL, at least 25 mg / mL, at least 30 mg / mL, at least 40 mg / mL, at least 50 mg / mL, at least 60 mg / mL, at least 70 mg / mL, at least 80 mg / mL, at least 90 mg / mL, or at least 100 mg / mL.

[0208] The pharmaceutical composition can be formulated into various suitable dosage forms, including but not limited to tablets, capsules, injections, etc., and can be administered via any suitable route to achieve the intended purpose. For example, it can be administered parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, transdermally, or orally, intrathecally, intracranially, intranasally, or externally. The dosage of the drug may depend on the patient's age, health condition, weight, concurrent treatments, and frequency of treatment. The pharmaceutical compositions disclosed herein can be administered to any subject in need, such as mammals, especially humans.

[0209] In some embodiments, the pharmaceutical compositions described herein further contain at least one known anticancer drug or a pharmaceutically acceptable salt of an anticancer drug. In some embodiments, a kit is provided herein containing an active ingredient (i) the linker-drug or antibody-drug conjugate described in any embodiment herein, and an active ingredient (ii) at least one known anticancer drug or a pharmaceutically acceptable salt of an anticancer drug; preferably, the active ingredient (i) and the active ingredient (ii) are in separate pharmaceutical compositions, or the active ingredient (i) and the active ingredient (ii) are formulated in the same pharmaceutical composition.

[0210] In this article, the anticancer drugs particularly suitable for use with the linker-drug or antibody-drug conjugates described herein include: (1) Anticancer drugs related to DNA damage and repair mechanisms, including PARP inhibitors such as olaparib, niraparib, rucaparib, talazoparib, senaparib and saruparib; HDAC inhibitors such as vorinostat, romidesin, pabistat and belistat; (2) Anticancer drugs related to cell division checkpoints, including Chk1 / 2 inhibitors, CDK4 / 6 inhibitors such as palbociclib, and ATM / ATR inhibitors; (3) Other anticancer drugs that can be used in combination anticancer therapy, including but not limited to: (i) Alkylating agents, such as busulfan, melphalan, chlorambucil, cyclophosphamide, ifosfamide, temozolomide, bendamustine, cisplatin, mitomycin C, bleomycin and carboplatin; (ii) Topoisomerase I inhibitors, such as camptothecin, irinotecan and topotecan; (iii) Topoisomerase II inhibitors, such as doxorubicin, epirubicin, aclarubicin, mitoxantrone, methyl hydroxyrosine, and methyltopopeptide; (iv) RNA / DNA antimetabolites, such as 5-azacytidine, gemcitabine, 5-fluorouracil, and methotrexate; (v) DNA antimetabolites, such as 5-fluoro-2′-deoxyuridine, fludarabine, nerabine, cytarabine, pralatrexate, pemetrexed, hydroxyurea and thioguanine; (vi) Antimitotic agents, such as colchicine, vincristine, vinorelbine, paclitaxel, ixaspirin, cabazitaxel and docetaxel; (vii) Antibodies, such as monoclonal antibodies, panitumumab, nezotuzumab, nivolumab, pembrolizumab, ramucirumab, bevacizumab, pertuzumab, trastuzumab, cetuximab, oxantuzumab, ofamumab, rituximab, alemtuzumab, tiimumab, tosimob, babutuximab, daratumumab, erlotuzumab, T-DM1, ofatumumab, dinutuximab, blinatumomab, iprimumab, avastin, Herceptin, and rituximab; (viii) Kinase inhibitors, such as imatinib, gefitinib, erlotinib, ostinib, afatinib, celitinib, alectinib, crizotinib, erlotinib, lapatinib, sorafenib, regorafenib, vemurafenib, dabrafenib, aflibercept, sunitinib, nilotinib, dasatinib, bosutinib, prambutinib, ibrutinib, cabozantinib, lenvatinib, vandetanib, trametinib, carbitinib, axitinib, tesiromoximib, idalalisib, pazopanib, tesiromoximib, and everolimus; (4) Other anticancer drugs that can be used in combination therapy include tamoxifen, letrozole, fulvestrant, mitoxantrone, octreotide, retinoid, arsenic, zoledronic acid, bortezomib, carfilzomib, Ixazomib, vemodega, sinedega, denosumab, thalidomide, lenalidomide, venetoclax, Aldesleukin (recombinant human interleukin-2), and Sipueucel-T (prostate cancer treatment vaccine).

[0211] The pharmaceutical compositions described herein may contain an effective amount of all the linker-drug or antibody-drug conjugates described in any of the embodiments herein. Herein, "effective amount" refers to an amount that effectively achieves its intended purpose (e.g., treatment or prevention). Although individual needs vary, those skilled in the art can determine the optimal dosage of each component of the pharmaceutical composition. Generally, when administered orally daily to mammals, an effective amount of the linker-drug or antibody-drug conjugates described herein may be given at a dose of about 0.0025 to 50 mg / kg body weight, preferably about 0.01 to 10 mg / kg body weight orally. If simultaneously administered, at least one known anticancer drug described herein should be effective in achieving its intended purpose. The optimal dosages of these known anticancer drugs are well known to those skilled in the art.

[0212] Treatment methods and medicinal uses

[0213] This article also includes methods for treating or preventing diseases such as tumors or cancer, which include administering to a subject in need a therapeutically or preventively effective amount of a linker-drug, antibody-drug conjugate, or pharmaceutical composition thereof as described herein. This method may be used in combination with any known radiotherapy or immunotherapy.

[0214] This document also includes the use of the linker-drug described in any embodiment of this document in the construction or preparation of antibody-drug conjugates, and the use of the linker-drug and antibody-drug conjugates described in any embodiment of this document in the preparation of drugs for the treatment or prevention of diseases such as tumors or cancer.

[0215] This document also includes linker-drugs as described in any embodiment of this document for the construction or preparation of antibody-drug conjugates.

[0216] This document also includes linker-drug or antibody-drug conjugates as described in any of the embodiments herein for the treatment or prevention of diseases such as tumors or cancer.

[0217] It should be understood that the term "construction" as used herein includes the preparation of antibody-drug conjugates using the linker-drug itself or its components (such as the linker itself, drug units, or groups within the linker). The linker-drug itself can be used directly during preparation, or its components can be sequentially linked together. The resulting antibody-drug conjugate will contain the linker-drug described herein.

[0218] Diseases suitable for treatment or prevention using the linker-drug, antibody-drug conjugate, or pharmaceutical composition thereof disclosed herein can be determined based on the pharmaceutical unit linked in the linker-drug and antibody-drug conjugate. In some embodiments, the diseases include various conditions caused by abnormal ATR activity and various conditions caused by abnormal topoisomerase I activity.

[0219] In this article, tumor refers to a physiological disorder in mammals, typically characterized by unregulated cell growth. Tumors can be classified as benign or malignant, with malignant tumors also known as cancer. Specifically, the diseases described in this article include tumors or cancers caused by abnormal ATR activity and / or abnormal topoisomerase I activity. In some embodiments, the tumors or cancers include, but are not limited to, liver cancer, melanoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer (such as small cell lung cancer), Wilms' tumor, cervical cancer, testicular cancer, soft tissue sarcoma, primary macroglobulinemia, bladder cancer, chronic myeloid leukemia, primary brain cancer, gastric cancer, colon cancer, malignant pancreatic islet cell tumor, choriocarcinoma, mycosis fungoides, head and neck cancer, osteosarcoma, pancreatic cancer, acute myeloid leukemia, piloblastic leukemia, rhabdomyosarcoma, Kaposi's sarcoma, thyroid cancer, esophageal cancer, cervical dysplasia, renal cell carcinoma, endometrial cancer, adrenocortical carcinoma, skin cancer, and prostate cancer. In some embodiments, the tumors or cancers include, but are not limited to, colorectal cancer, gastric cancer, pancreatic cancer, colon cancer, and breast cancer.

[0220] The linker-drug, antibody-drug conjugate, or pharmaceutical composition thereof of the present invention may be administered using methods well known in the art, including but not limited to parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, intrathecal, intracranial, intranasal, or external administration. The dosage may depend on the patient's age, health condition, weight, concurrent treatments, and frequency of treatment.

[0221] Preparation method of antibody drug conjugate

[0222] The present invention provides a general preparation method for antibody-drug conjugates, comprising the following steps: (a) The reducing agent and the antibody to be conjugated are incubated in a buffer system in the presence of an effective amount of transition metal ions to selectively reduce the disulfide bonds between the inner chains of the antibody; (b) Reacting an excess of the effective load carrying the reactive group with the reduced thiol group obtained in step (a); and (c) Add an effective amount of oxidant to reoxidize the unreacted thiol groups, and then recover the obtained antibody-drug conjugate.

[0223] The transition metal ions in step (a) are selected from Zn 2+ Cd 2+ and Hg 2+ One or more of the following, preferably Zn 2+ .

[0224] The buffer system used in step (a) is selected from Hepes, histidine buffer, PBS and MES, and the pH is 5.5 to 8.

[0225] The final concentration of the antibody to be conjugated added in step (a) is 0.01-0.1 mM.

[0226] Step (a) is performed at a temperature of approximately -10°C to 37°C, for example, at approximately 0°C to 20°C.

[0227] The reducing agent in step (a) is preferably TCEP.

[0228] The oxidant in step (c) is preferably DHAA.

[0229] It should be understood that the method for preparing antibody-drug conjugates described in WO2020164561 can be used to prepare the ADC described in this paper, and all the contents disclosed in WO2020164561 are incorporated herein by reference.

[0230] amino acid sequence

[0231] The amino acid sequences of SEQ ID NO: 1-137 described herein are identical to those of SEQ ID NO: 1-137 recorded in WO2023198019A1. The amino acid sequences shown in SEQ ID NO: 138 and SEQ ID NO: 139 are the light chain amino acid sequence and heavy chain amino acid sequence of the HER2 antibody used herein, respectively.

[0232] The present invention will be described below by way of specific embodiments. It should be understood that the embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the materials and methods used in the embodiments are conventional materials and methods in the art.

[0233] A. Synthesis of ATR inhibitors

[0234] Examples P-1 to P-5 below can be prepared using a synthetic method similar to WO 2020 / 259601A1, and Examples P-6 to P-22 can be prepared using a synthetic method similar to WO 2022 / 135560A1. The compounds are as follows:

[0235] B. Linker-Drug Synthesis

[0236] Example B-1: Compound LP01

[0237] a) Preparation of intermediate 1-1: Compound P-1 (2.00 g, 5.49 mmol) was dissolved in DMF (30 mL), and cesium carbonate (1.79 g, 5.49 mmol) and tert-butyl 2-bromoacetate (1.07 g, 5.49 mmol) were added sequentially. The mixture was stirred at 25 °C for 3 hours. The reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with brine (30 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to obtain the crude product, and purified by column chromatography to give intermediate 1-1 (2.10 g, 77.0% yield) as a yellow solid. LCMS: m / z = 479.1 [M+H] + .

[0238] b) Preparation of intermediate 1-2: Intermediate 1-1 (2.10 g, 4.39 mmol) was dissolved in dichloromethane (20 mL), and trifluoroacetic acid (7.68 g, 67.3 mmol, 5 mL) was added. The mixture was stirred at 25 °C for 8 hours. After the reaction was complete, the solution was concentrated under reduced pressure to obtain intermediate 1-2 (1.84 g, 99.3% yield) as a yellow solid. LCMS: m / z = 423.2 [M+1] + .

[0239] c) Preparation of intermediates 1-3: Intermediate 1-2 (640 mg, 1.42 mmol) was dissolved in toluene (30 mL), and compound 1-B (928 mg, 1.52 mmol) and TEA (229 mg, 2.27 mmol, 315 μL) were added sequentially. DPPA (416 mg, 1.52 mmol, 327 μL) was added dropwise at 100 °C, and the mixture was stirred for 3 hours. The reaction mixture was concentrated under reduced pressure to obtain a residue, which was purified by high performance liquid chromatography to give intermediate 1-3 (310 mg, yield 19.3%) as a yellow solid. LCMS: m / z = 1032.8 [M+H] + .

[0240] d) Preparation of intermediates 1-4: Intermediate 1-3 (250 mg, 242 μmol) was dissolved in water (5 mL) and tetrahydrofuran (5 mL), and then lithium hydroxide monohydrate (50.8 mg, 1.21 mmol) was added. The mixture was stirred at 0 °C for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude product, which was purified by high performance liquid chromatography to give intermediate 1-4 (150 mg, yield 67.9%) as a white solid. LCMS: m / z = 892.0 [M+H] + .

[0241] e) Preparation of intermediates 1-5: Intermediate 1-4 (150 mg, 168 μmol) was dissolved in 10 mL of 2% TFA HFIP solution at 0 °C, and reacted in an ice bath for 2 h. The reaction mixture was concentrated under reduced pressure to give intermediate 1-5 (130 mg, yield 97.6%) as a yellow solid. LCMS: m / z = 792.2 [M+H] + .

[0242] f) Preparation of compound LP01: Intermediate 1-5 (130 mg, 164 μmol) and compound 1-C (65.5 mg, 246 μmol) were dissolved in DMF (8 mL), and DIEA (42.4 mg, 328 μmol, 57.2 μL) was added. The mixture was stirred at 25 °C for 1 hour. After the reaction was completed, the solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain a light yellow compound LP01 (61.1 mg, yield 39.4%, purity 99.84%). LCMS: m / z = 943.4 [M+1] + . 1 H NMR(400 MHz, DMSO-d6) δ 9.11 (s, 1H), 8.66 (t, J = 6.0 Hz, 1H), 8.37 (t, J = 5.6Hz, 1H), 8.16 (s, 1H), 8.00 (s, 1H), 7.82 (s, 1H), 7.67 (d, J = 1.6 Hz, 1H),7.20-7.30 (m, 2H), 7.07 (q, J = 8.4 Hz, 2H), 6.99 (s, 2H), 6.84 (d, J = 1.6Hz, 1H), 6.40 (d, J = 12.4 Hz, 1H), 5.47 (d, J = 6.4 Hz, 2H), 5.00 (s, 2H), 4.88 (d, J = 7.6 Hz, 1H), 4.44 (d, J = 6.4 Hz, 1H), 3.98-4.03 (m, 5H), 3.87-3.91 (m, 3H), 3.76-3.81 (m, 2H), 3.72 (d, J = 9.6 Hz, 2H), 3.63 (s, 4H), 3.40(s, 2H), 3.32 (s, 2H), 2.46 (t, J = 7.2 Hz, 2H), 1.29 (d, J = 6.4 Hz, 3H).

[0243] Example B-2: Compound LP02

[0244] a) Preparation of intermediate 2-1: Compound 1-2 (600 mg, 1.42 mmol) was dissolved in toluene (24 mL), followed by the addition of compound 2-A (614 mg, 1.56 mmol), DPPA (586 mg, 2.13 mmol, 459 μL), and TEA (215 mg, 2.13 mmol, 296 μL). After stirring at 100 °C for 2 hours, the reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to give intermediate 2-1 (80.0 mg, yield 6.58%) as a yellow solid. LCMS: m / z = 813.7 [M+H] + .

[0245] d) Preparation of intermediate 2-2: Intermediate 2-1 (80.0 mg, 98.4 μmol) was dissolved in 10 mL of 2% TFA HFIP solution at 0 °C. The reaction was carried out in an ice bath for 1 hour, then stirred for another 4 hours at room temperature. After drying and concentration, a white solid intermediate 2-2 (65.0 mg, yield 92.7%) was obtained and used directly in the next step without purification. LCMS: m / z = 713.5 [M+H] + .

[0246] c) Preparation of compound LP02: Intermediate 2-2 (60.0 mg, 84.2 μmol) and compound 2-B (69.7 mg, 101 μmol) were dissolved in DMF (3 mL) at 20 °C. DIEA (21.8 mg, 168 μmol, 29.3 μL) was added, and the mixture was stirred at 20 °C for 3 hours. After the reaction was complete, the solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by high performance liquid chromatography to obtain white compound LP02 (52.5 mg, yield 47.3%, purity 97.7%). LCMS: m / z = 1288.0 [M+H] + . 1 HNMR (400 MHz, DMSO-d6) δ 9.97 (s, 1H), 8.67 (s, 1H), 8.19 (d, J = 6.8 Hz,1H), 7.98-8.08 (m, 2H), 7.86-7.96 (m, 2H), 7.68 (d, J= 2.0 Hz, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.25-7.34 (m, 4H), 6.99 (s, 2H), 6.86 (d, J = 2.0 Hz, 1H), 5.49 (d, J = 6.4 Hz, 2H), 5.01 (s, 2H), 4.46 (d, J = 6.0 Hz, 1H), 4.35-4.40(m, 1H), 4.19 (d, J = 1.2 Hz, 1H), 4.03 (s, 1H), 4.00 (s, 3H), 3.78-3.81 (m,1H), 3.73 (d, J = 2.0 Hz, 1H), 3.59 (s, 3H), 3.47-3.50 (m, 25H), 3.35 (t, J =5.6 Hz, 2H), 3.14 (q, J = 5.6 Hz, 2H), 2.46 (d, J = 6.8 Hz, 1H), 2.35-2.41(m, 1H), 2.32 (t, J = 7.2 Hz, 2H), 1.95 (d, J = 6.8 Hz, 1H), 1.30 (d, J = 6.8Hz, 6H), 0.85 (dd, J = 15.6, 6.8 Hz, 6H).

[0247] Example B-3: Compound LP03 MW: 1358.52. Compound LP03 can be prepared using a synthetic method similar to that in Example B-2.

[0248] Example B-4: Compound LP04

[0249] a) Preparation of intermediate 4-1: DIEA (3.2 g, 24.7 mmol) and 4-nitrophenylcarbonyl chloride (2.0 g, 9.9 mmol) were added to a DCM (40 mL) solution of compound 4-A (4.0 g, 8.2 mmol) at 0 °C. The mixture was stirred at 0 °C under a N2 atmosphere for 2 hours. After the reaction was complete, water (50 mL) was added to the reaction mixture, and the mixture was extracted with DCM (50 mL × 3). The organic layer was washed with brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel chromatography to give intermediate 4-1 (3.8 g, yield: 71%) as a white solid.

[0250] b) Preparation of intermediate 4-2: 2-Methoxyethylamine (526.0 mg, 7.0 mmol) and DIEA (2.3 g, 17.5 mmol) were added to a DMF (40 mL) solution of intermediate 4-1 (3.8 g, 5.8 mmol). The mixture was stirred at room temperature under a N2 atmosphere for 16 hours. After the reaction was complete, water (50 mL) was added to the reaction mixture, and the mixture was extracted with EA (50 mL × 3). The organic layer was washed with brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel chromatography to give intermediate 4-2 (2.6 g, yield: 76%) as a white solid.

[0251] c) Preparation of intermediate 4-3: TMSCl (962 mg, 8.86 mmol) and (HCHO) were added to a DCM (30 mL) solution of intermediate 4-2 (2.6 g, 4.4 mmol). n (266.0 mg, 8.8 mmol). The mixture was stirred at room temperature for 2 hours under a nitrogen atmosphere. After the reaction was complete, the reaction mixture was filtered and the filtrate was concentrated to give crude intermediate 4-3.

[0252] d) Preparation of intermediate 4-4: Compound P-1 (1.6 g, 4.4 mmol) and PMP (1.4 g, 8.8 mmol) were added to a DCM (30 mL) solution containing intermediate 4-3 (crude product). The mixture was stirred at 40 °C under a N2 atmosphere for 16 hours. After the reaction was complete, the reaction mixture was concentrated. The residue was filtered through C... 18 Purification was performed using an (HCOOH / H2O / ACN) column to give a yellow solid intermediate 4-4 (0.7 g, two-step yield: 49%). LC-MS: m / z = 963.25 [M+H] + .

[0253] e) Preparation of intermediate 4-5: Iron powder (465.0 mg, 8.3 mmol) and NH4Cl (446.0 mg, 8.3 mmol) were added to a solution of intermediate 4-4 (0.8 g, 0.8 mmol) in ethanol / water (10 mL / 4 mL). The mixture was stirred at 85 °C for 2 hours under a nitrogen atmosphere. After the reaction was complete, water (20 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic layer was washed with brine, dried over sodium sulfate, and concentrated. The residue was purified by silica gel chromatography to give intermediate 4-5 (0.4 g, yield: 52%) as a yellow solid. LC-MS: m / z = 933.30 [M+H] + .

[0254] f) Preparation of intermediate 4-6: Glycine tert-butyl ester (113.0 mg, 0.6 mmol), HATU (245.0 mg, 0.6 mmol), and DIEA (166.0 mg, 1.3 mmol) were added to intermediate 4-5 (430.0 mg, 0.43 mmol) dissolved in DMF (8 mL). The mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction mixture was concentrated. The residue was purified by silica gel chromatography to give intermediate 4-6 (0.4 g, yield: 86%) as a white solid. LC-MS: m / z = 1089.90 [M+H] + .

[0255] j) Preparation of intermediate 4-7: LiOH (12.0 mg, 0.28 mmol) was added to a THF / H₂O (3 mL / 1 mL) solution of intermediate 4-6 (30.0 mg, 0.03 mmol). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was concentrated. The residue was purified by preparative high-performance liquid chromatography (Prep-HPLC) to give intermediate 4-7 (10.8 mg, yield: 41%) as a white solid. LC-MS: m / z = 949.70 [M+H] + 947.65 [MH] - . 1H NMR (400 MHz, DMSO-d6) δ 9.66 (d, J = 16.2 Hz, 1H), 8.52 (d, J = 17.9 Hz, 1H), 8.35 (s,1H), 7.86 (d, J = 8.9 Hz, 1H), 7.62 (d, J = 1.9 Hz, 1H), 7.36 (d, J = 3.1 Hz, 1H), 7.24 – 7.02 (m, 3H), 6.92 (s, 1H), 6.77 (d, J = 1.9 Hz, 1H), 5.66 (d, J= 10.5 Hz, 2H), 5.55 (s, 1H), 5.09 (d, J = 18.7 Hz, 3H), 4.53 (d, J = 7.3 Hz,1H), 4.41-4.35 (m, 1H), 3.99-3.93 (m, 4H), 3.91 – 3.68 (m, 5H), 3.59 – 3.51(m, 5H), 3.46-3.41 (m, 5H), 3.23 – 3.19 (m, 5H), 1.39 (s, 9H), 1.23 (d, J =6.6 Hz, 3H).

[0256] h) Preparation of intermediate 4-8: TFA (3 mL) was added to a DCM (5 mL) solution of intermediate 4-7 (0.2 g, 0.2 mmol). The mixture was stirred at room temperature under a N2 atmosphere for 1 hour. After the reaction was complete, the reaction mixture was concentrated to give intermediate 4-8 (0.2 g, crude product, colorless oil). LC-MS: m / z = 850.20 [M+H] + .

[0257] i) Preparation of compound LP04: DIEA (82.0 mg, 0.63 mmol) and compound 1-C (56.0 mg, 0.21 mmol) were added to a DMF (5 mL) solution of intermediate 4-8 (0.2 g, crude). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction mixture was purified by preparative high-performance liquid chromatography (Prep-HPLC) to give the target compound LP04 as a white solid (102.7 mg, two-step yield: 49%). LC-MS: m / z = 1000.65 [M+H] + 998.60 [MH] - . 1H NMR (400 MHz, DMSO-d6) δ 9.10 (d, J = 7.5 Hz, 1H), 8.42-8.31 (m, 1H), 8.17 (d, J = 14.1 Hz, 1H), 8.02-7.96 (m, 1H), 7.68 (s, 1H), 7.65 (d, J = 2.0Hz, 1H), 7.25-7.16 (m, 1H), 7.15-7.01 (m, 3H), 6.96 (s, 2H), 6.80 (d, J = 2.0Hz, 1H), 5.69 (s, 2H), 5.14-5.00 (m, 2H), 4.87 (d, J = 7.6 Hz, 1H), 4.45 –4.35 (m, 1H), 4.00-3.92 (m, 5H), 3.90-3.85 (m, 3H), 3.77 – 3.68 (m, 3H), 3.63-3.58 (m, 2H), 3.56 – 3.53 (m, 4H), 3.52-3.50 (m, 3H), 3.30-3.27 (m, 4H), 3.22-3.17 (m, 3H), 2.46 – 2.41 (m, 2H), 1.25 (d, J = 6.6 Hz, 3H).

[0258] Example B-5: Compound LP05

[0259] a) Preparation of intermediate 5-1: 2-Methoxyethylamine (2.6 g, 35.26 mmol) and DIEA (7.6 g, 58.76 mmol) were added to a THF (200 mL) solution of intermediate 5-A (20.0 g, 29.38 mmol). The mixture was stirred at room temperature under a N2 atmosphere for 16 hours. After the reaction was complete, the reaction mixture was used directly for the next step.

[0260] b) Preparation of intermediate 5-2: Piperidine (50.0 g, 587.6 mmol) was added to the reaction solution from the previous step, and the mixture was stirred at room temperature under a nitrogen atmosphere for 2 hours. After the reaction was complete, the reaction mixture was concentrated. The residue was filtered through C... 18 Purification was performed using an (HCOOH / H2O / ACN) column to give a yellow solid intermediate 5-2 (9.0 g, two-step yield: 77%). LC-MS: m / z = 395.10 [M+H] + .

[0261] c) Preparation of intermediate 5-3: K₂CO₃ (5.6 g, 40.56 mmol) and Cu₂SO₄·5H₂O (506.0 mg, 2.03 mmol) were added to a MeOH / H₂O / DCM (100 mL / 20 mL / 40 mL) solution of intermediate 5-2, and the mixture was stirred for 10 min. 1H-imidazolium-1-sulfonyl azide (4.2 g, 24.34 mmol) was added to the mixture. The mixture was stirred at room temperature under a N₂ atmosphere for 16 h. After completion, the reaction mixture was concentrated. Water (50 mL) was added to the residue, and the mixture was extracted with DCM (50 mL × 3). The organic layer was washed with brine, dried over Na₂SO₄, and concentrated. The residue was purified by silica gel chromatography (DCM: MeOH = 25: 1) to give a white solid compound 5-3 (5.8 g, yield: 68%). LC-MS: m / z = 419.05 [MH] - .

[0262] d) Preparation of intermediate 5-4: TMSCl (517.0 mg, 4.76 mmol) and (HCHO) were added to a DCM (15 mL) solution of intermediate 5-3 (1.0 g, 2.38 mmol). n (143.0 mg, 4.8 mmol). The mixture was stirred in a nitrogen atmosphere for 1 hour at room temperature. After the reaction was complete, the reaction mixture was filtered and the filtrate was concentrated to give crude intermediate 5-4.

[0263] e) Preparation of Intermediate 5-5: Compound P-1 (867.0 mg, 2.38 mmol) and PMP (739.0 mg, 4.76 mmol) were added to a solution of intermediate 5-4 (crude product) dissolved in DCM (10 mL). The mixture was stirred at 40 °C for 16 hours under a N2 atmosphere. After completion, the reaction mixture was concentrated. The residue was subjected to C... 18 Purification with (HCOOH / H2O / ACN) yielded a yellow solid intermediate 5-5 (0.5 g, two-step yield: 26%). LC-MS: m / z = 797.35 [M+H] + .

[0264] f) Preparation of intermediate 5-6: PMe3 (1.0 M, 1.0 mL, 1.00 mmol) was added to a solution of intermediate 5-5 (400.0 mg, 0.50 mmol) dissolved in THF / H2O (10 mL / 1 mL). The mixture was stirred at room temperature under a N2 atmosphere for 1 hour. After the reaction was complete, water (20 mL) was added to the reaction mixture, and the mixture was extracted with EA (30 mL × 3). The organic layer was washed with brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel chromatography (DCM: MeOH = 20: 1) to give intermediate 5-6 (250.0 mg, yield: 65%) as a yellow solid. LC-MS: m / z = 771.25 [M+H] + .

[0265] g) Preparation of compound LP05: Compound 2-B (85.0 mg, 0.14 mmol), HATU (54.0 mg, 0.14 mmol), and DIEA (65.0 mg, 0.5 mmol) were added to a DMF (3 mL) solution of intermediate 5-6 (110.0 mg, 0.14 mmol). The mixture was stirred at room temperature for 30 minutes. After the reaction was complete, the reaction mixture was concentrated. The residue was purified by preparative high-performance liquid chromatography (Prep-HPLC) to give the target compound LP05 (100.0 mg, yield: 65%) as a white solid. LC-MS: m / z = 1345.55 [M+H] + 1343.50 [MH] - . 1H NMR (400 MHz, DMSO-d6) δ 9.92(s, 1H), 8.16 (d, J = 6.9 Hz, 1H), 7.98 (t, J = 5.6 Hz, 1H), 7.92 – 7.75 (m,2H), 7.63 (d, J = 1.9 Hz, 1H), 7.58 (s, 2H), 7.45 – 7.35 (m, 2H), 7.30 (d, J= 8.1 Hz, 1H), 7.10 (d, J = 8.6 Hz, 1H), 6.97 (s, 2H), 6.92 (s, 1H), 6.77 (d,J = 1.9 Hz, 1H), 5.66 (s, 2H), 5.08 (d, J = 18.4 Hz, 2H), 4.43 – 4.29 (m,2H), 4.18 (dd, J = 8.6, 6.7 Hz, 1H), 4.01-3.92 (m, 4H), 3.87 (d, J = 13.3 Hz,1H), 3.78-3.66 (m, 2H), 3.60 – 3.51 (m, 7H), 3.49 – 3.43 (m, 30H), 3.36-3.32(m, 2H), 3.27 – 3.24 (m, 2H), 3.23-3.17 (m, 2H), 3.12 (q, J = 5.8 Hz, 2H),2.46-2.41 (m, 1H), 2.40-2.34 (m, 1H), 2.30 (t, J = 7.3 Hz, 2H), 1.99 – 1.90(m, 1H), 1.28 (d, J = 7.1 Hz, 3H), 1.22 (d, J = 6.5 Hz, 3H), 0.85 (d, J = 6.7Hz, 3H), 0.81 (d, J = 6.7Hz, 3H).

[0266] Compounds LP06 to LP46 can be prepared using methods known to those skilled in the art or synthetic methods similar to those in Examples B-1, B-2, or B-5. The compounds are shown below: Example B-6: Compound LP06 , MW: 1416.60.

[0267] Example B-7: Compound LP07 , MW: 913.86.

[0268] Example B-8: Compound LP08 , MW: 950.03.

[0269] Example B-9: Compound LP09 , MW: 1329.48.

[0270] Example B-10: Compound LP10 , MW: 992.11.

[0271] Example B-11: Compound LP11 , MW: 963.07.

[0272] Example B-12: Compound LP12 , MW: 890.96.

[0273] Example B-13: Compound LP13 , MW: 1467.65.

[0274] Example B-14: Compound LP14 , MW: 1512.71.

[0275] Example B-15: Compound LP15 , MW: 956.93.

[0276] Example B-16: Compound LP16 , MW: 1301.47.

[0277] Example B-17: Compound LP17 , MW: 1372.55.

[0278] Example B-18: Compound LP18 , MW: 1015.01.

[0279] Example B-19: Compound LP19 , MW: 1359.55.

[0280] Example B-20: Compound LP20 , MW: 1430.63.

[0281] Example B-21: Compound LP21 , MW: 927.89.

[0282] Example B-22: Compound LP22 , MW: 1272.43.

[0283] Example B-23: Compound LP23 , MW: 1343.50.

[0284] Example B-24: Compound LP24 , MW: 1006.14.

[0285] Example B-25: Compound LP25 , MW: 977.10.

[0286] Example B-26: Compound LP26 , MW: 904.99.

[0287] Example B-27: Compound LP27 , MW: 1481.68.

[0288] Example B-28: Compound LP28 , MW: 1526.74.

[0289] Example B-29: Compound LP29 , MW: 951.02.

[0290] Example B-30: Compound LP30 , MW: 1593.70.

[0291] Example B-31: Compound LP31 , MW: 1827.94.

[0292] Example B-32: Compound LP32 , MW: 1841.96.

[0293] Example B-33: Compound LP33 , MW: 1769.88.

[0294] Example B-34: Compound LP34 , MW: 1551.66.

[0295] Example B-35: Compound LP35 , MW: 1593.70.

[0296] Example B-36: Compound LP36 , MW: 1846.96.

[0297] Example B-37: Compound LP37 , MW: 1744.81.

[0298] Example B-38: Compound LP38 , MW: 1660.82.

[0299] Example B-39: Compound LP39 , MW: 1768.93.

[0300] Example B-40: Compound LP40 , MW: 965.04.

[0301] Example B-41: Compound LP41 , MW: 939.90.

[0302] Example B-42: Compound LP42 , MW: 1284.44.

[0303] Example B-43: Compound LP43 , MW: 1355.52.

[0304] Example B-44: Compound LP44 , MW: 960.96.

[0305] Example B-45: Compound LP45 , MW: 1305.50.

[0306] Example B-46: Compound LP46 , MW: 1376.57.

[0307] Compounds LP47 to LP52 can be prepared using a synthetic method similar to that of Example B-62; compounds LP53 and LP54 can be prepared using synthetic methods similar to those of Examples B-5 and B-2, respectively. The compounds are shown below: Example B-47: Compound LP47 , MW: 2576.86.

[0308] Example B-48: Compound LP48 , MW: 2590.89.

[0309] Example B-49: Compound LP49 , MW: 2573.86.

[0310] Example B-50: Compound LP50 , MW: 2594.91.

[0311] Example B-51: Compound LP51 , MW: 3403.73.

[0312] Example B-52: Compound LP52 , MW: 3417.76.

[0313] Example B-53: Compound LP53 , MW: 1333.42. LCMS: m / z =1333.7 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 9.03 (s, 1H), 8.32 (d, J= 6.5 Hz, 1H), 8.12 (t, J = 6.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.84–7.49(m, 3H), 7.12 (d, J = 18.2 Hz, 3H), 6.97 (d, J = 5.4 Hz, 3H), 6.82 (s, 1H), 5.73 (s, 2H), 5.10 (d, J = 15.9 Hz, 2H), 4.58–4.50 (m, 1H), 4.36 (d, J = 7.9Hz, 2H), 4.33–4.21 (m, 2H), 4.16–4.06 (m, 3H), 4.03–3.92 (m, 5H), 3.86–3.67(m, 5H), 3.58–3.43 (m, 17H), 3.41–3.36 (m, 3H), 3.33–3.13 (m, 13H), 3.07 (d,J = 8.5 Hz, 1H), 2.07 (d,J = 6.6 Hz, 1H), 1.34–1.24 (m, 6H), 0.89–0.78 (m,6H).

[0314] Example B-54: Compound LP54 , MW: 1373.53. LC-MS: m / z = 898.75 [M+H] + 896.40 [MH] - . 1H NMR (400 MHz, DMSO-d6) δ9.99 (s, 1H), 8.56 (d, J = 6.6 Hz, 1H), 8.10 (d, J = 7.5 Hz, 1H), 8.01-7.94(m, 1H), 7.90–7.79 (m, 2H), 7.62 (d, J = 1.9 Hz, 1H), 7.57 (d, J = 8.4 Hz,2H), 7.36 (s, 1H), 7.27 (d, J = 8.2 Hz, 2H), 7.13 (d, J = 2.3 Hz, 1H), 6.97(s, 2H), 6.92 (s, 1H), 6.77 (d, J = 2.0 Hz, 1H), 5.99 (t, J = 5.6 Hz, 1H), 5.38 (d, J = 5.5 Hz, 4H), 4.98 (s, 2H), 4.41-4.30 (m, 2H), 4.26–4.17 (m, 1H), 4.01-3.92 (m, 4H), 3.87 (d, J = 13.2 Hz, 1H), 3.77-3.67 (m, 2H), 3.59–3.54(m, 4H), 3.50–3.43 (m, 30H), 3.36-3.33 (m, 1H), 3.25-3.21 (m, 1H), 3.14–3.10(m, 2H), 3.01-2.89 (m, 2H), 2.45–2.34 (m, 2H), 2.30 (t, J = 7.3 Hz, 2H), 2.00-1.89 (m, 1H), 1.76–1.52 (m, 2H), 1.47-1.29 (m, 2H), 1.23 (d, J = 6.6 Hz, 3H), 0.82 (dd, J = 12.8, 6.7 Hz, 6H).

[0315] Example B-55: Compound LP55

[0316] a) Preparation of intermediate 55-1: Compound 55-A (3.0 g, 9.5 mmol), 2-methoxyethylamine (1.4 g, 19 mmol), and DIPEA (3.7 g, 28.6 mmol) were dissolved in DCM (50 mL) and stirred at room temperature for 3 hours. After the reaction was complete, the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give intermediate 55-1 (1.6 g, 66% yield) as a white solid.

[0317] b) Preparation of compound 55-2: Intermediate 55-1 (2 g, 8.0 mmol) and (CHO) were prepared. n TMSBr (2.45 g, 16.0 mmol) was added to a mixture of dichloromethane (DCM, 50 mL). The reaction mixture was stirred in air at room temperature for 2 hours. The solid was filtered off and concentrated to give 2.8 g of a red, oily crude product, which was used directly in the next step.

[0318] c) Preparation of compound 55-3: Intermediate 55-2 (2.7 g, 7.87 mmol) was added to a DCM (50 mL) mixture containing compound P-1 (2.4 g, 6.56 mmol) and Cs₂CO₃ (4.27 g, 13.12 mmol). The reaction was carried out in a sealed tube under a nitrogen atmosphere at 40°C for 16 hours. After the reaction was completed, the residue was concentrated and purified by silica gel column chromatography (MeOH / DCM: 5%) to give intermediate 55-3 (2.4 g, yield 58%) as a yellow solid. LCMS: m / z = 627 [M+H] + .

[0319] d) Preparation of intermediate 55-4: Me3P (9.57 mL, 1 M THF solution) was added to a mixture containing intermediate 55-3 (2 g, 3.19 mmol), 4A MS (20 g), and 100 mL of anhydrous THF. The reaction mixture was stirred at room temperature under nitrogen protection for 10 min. Compound 55-B (1.98 g, 3.51 mmol) dissolved in anhydrous THF (100 mL) was added. The mixture was stirred at room temperature for 1 h. The solid was filtered off and concentrated. The residue was purified by silica gel column chromatography to give intermediate 55-4 (2.45 g, 78% yield) as a yellow solid. LCMS: m / z = 980 [M+H] + .

[0320] e) Preparation of intermediate 55-5: Et2NH (1.76 g, 25 mmol) was added to a mixture of intermediate 55-4 (2.45 g, 2.5 mmol) dissolved in DMF (25 mL), and the mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was purified by reversed-phase chromatography to obtain intermediate 55-5 (1.9 g, 100% yield) as a yellow solid.

[0321] f) Preparation of intermediate 55-6: HATU (1.15 g, 3.01 mmol) was added to a mixture of intermediate 55-5 (1.9 g, 2.51 mmol), (9H-fluorene-9-ylmethoxy)carbonyl-L-valine (850 mg, 2.51 mmol), and DIPEA (973 mg, 7.53 mmol) dissolved in DMF (50 mL). The reaction mixture was stirred at room temperature for 2 hours. The mixture was then poured into ice water (250 mL) and filtered to give intermediate 55-6 (2.8 g, 100% yield) as a white solid. LCMS: m / z = 1080 [M+H] + .

[0322] g) Preparation of intermediate 55-7: Diethylamine (1.9 g, 25.9 mmol) was added to a mixture of intermediate 55-6 (2.8 g, 2.59 mmol) dissolved in DMF (30 mL), and the mixture was stirred at room temperature for 3 hours. The mixture was purified by reversed-phase chromatography to give intermediate 55-7 (2.15 g, 100% yield) as a yellow solid. LCMS: m / z = 429 [M / 2+H] + .

[0323] h) Preparation of intermediate 55-8: EDCI (704 mg, 3.67 mmol) and HOBT (496 mg, 3.67 mmol) were added to a mixture of intermediate 55-7 (2.1 g, 2.45 mmol), compound 55-C (1.04 g, 2.45 mmol), and DIPEA (950 mg, 7.35 mmol) dissolved in DMF (25 mL). The mixture was then stirred at room temperature for 2 hours. The reaction mixture was diluted with EA (50 mL), washed with water (20 mL × 4), concentrated, and the residue was purified by silica gel column chromatography to give intermediate 55-8 (1.4 g, 45% yield) as a yellow solid. LCMS: m / z = 1264 [M+H] + .

[0324] i) Preparation of intermediate 55-9: A mixture of intermediate 55-8 (140 mg, 0.11 mmol) and TFA / DCM (4 mL, 1 / 4 volume ratio) was stirred at room temperature for 2 hours. The reaction mixture was concentrated. The residue was dissolved in DMF (5 mL), and DEA (81 mg, 1.11 mmol) was added. The mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction solution was concentrated, and the residue was purified by preparative high-performance liquid chromatography (HPLC) to give intermediate 55-9 (60 mg, 55% yield) as a white solid. LCMS: m / z = 494[M / 2+H] + .

[0325] j) Preparation of compound LP55: HATU (57 mg, 0.15 mmol) was added to a mixture of compound 2-B (90 mg, 0.15 mmol) and DIPEA (52 mg, 0.4 mmol) dissolved in DMF (2 mL). The reaction mixture was stirred at room temperature for 30 min. Intermediate 55-9 (100 mg, 0.1 mmol) dissolved in DMF (2 mL) was added to the above mixture. The mixture was stirred at room temperature for 2 h. The reaction mixture was purified by preparative high-performance liquid chromatography (Prep-HPLC) to give the target compound LP55 (67.9 mg, yield 43%) as a white solid. LCMS: m / z = 781 [M / 2+H] + . 1 H NMR (400 MHz, DMSO- d6) δ 10.05 (s, 1H), 8.19 (d, J = 7.1 Hz, 1H), 8.14 – 7.88 (m, 4H), 7.71 (dd, J = 13.4, 5.2 Hz, 2H), 7.60 (d, J = 8.5 Hz, 2H), 7.41 – 7.20 (m, 4H), 7.01 (s, 2H), 6.85 (d, J = 1.9 Hz, 1H), 6.00 (s, 1H), 5.74 (s, 2H), 5.44 (s, 2H), 5.09 (d, J = 13.9 Hz, 3H), 4.46 (s, 1H), 4.34 (dd, J = 13.7, 8.1 Hz, 2H), 4.23 – 4.15 (m, 1H), 4.01 (d, J = 11.8 Hz, 5H), 3.75 (dd, J = 32.5, 10.4 Hz, 1H), 3.63 – 3.53 (m, 6H), 3.51 – 3.46 (m, 28H), 3.36 (t, J = 5.8 Hz, 4H), 3.23 (d, J = 16.8 Hz, 4H), 3.14 (dd, J = 11.5, 5.7 Hz, 2H), 2.98 (d, J = 24.8 Hz, 2H), 2.46 – 2.28 (m, 8H), 2.26 – 2.21 (m, 2H), 2.02 – 1.82 (m, 3H), 1.77 – 1.50 (m, 4H), 1.45 – 1.26 (m, 5H), 0.83 (dd, J = 14.3, 6.7 Hz, 6H).

[0326] Example B-56: Compound LP56

[0327] a) Preparation of intermediate 56-1: NH4Cl (30.0 g, 0.565 mol), HATU (64.4 g, 0.169 mol), and DIEA (29.1 g, 0.226 mol) were added to a DMF (300 mL) solution of compound 56-A (75.0 g, 0.113 mol). The resulting solution was stirred at room temperature for 12 hours. The reaction mixture was purified by reversed-phase chromatography to give intermediate 56-1 (56.7 g, 75.8% yield) as a brown oil. LCMS: m / z = 663.5 [M+H] + .

[0328] b) Preparation of intermediate 56-2: Diethylamine (DEA, 61.2 g, 0.85 mol) was added to a solution of intermediate 56-1 (56.7 g, 0.085 mol) in dichloromethane (DCM, 400 mL) at room temperature, and the reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was concentrated and purified by silica gel rapid column chromatography to give a brown oily intermediate 56-2 (35.0 g, yield: 93%). LCMS: m / z = 441.5 [M+H] + .

[0329] c) Preparation of intermediate 56-3: Compound 56-B (84.0 g, 0.197 mol), DIEA (30.57 g, 0.237 mol), HOBt (21.3 g, 0.158 mol), and EDCI (30.3 g, 0.158 mol) were added to a DMF (500 mL) solution of intermediate 56-2 (35.0 g, 0.079 mol). The resulting solution was stirred at room temperature for 12 hours. The mixture was quenched with water (2 L) and extracted with ethyl acetate (600 mL × 3). The organic layer was washed with water (1 L) and brine (500 mL), dried over anhydrous sodium sulfate, concentrated, and purified by silica gel rapid column chromatography to give intermediate 56-3 (43.0 g, yield 63.7%) as a brown oil. LCMS: m / z = 848.5 [M+H] + .

[0330] d) Preparation of intermediate 56-4: Et₂NH (40 mL) was added to a mixture of intermediate 56-3 (43.0 g, 0.05 mol) dissolved in DCM (300 mL), and the mixture was stirred at room temperature for 12 hours. The reaction mixture was concentrated and purified by silica gel rapid column chromatography to give intermediate 56-4 (20 g, yield 63.1%) as a brown oil. LCMS: m / z = 626.5 [M+H] +.

[0331] e) Preparation of intermediate 56-5: HATU (18.2 g, 0.048 mol) was added to a mixture of intermediate 56-4 (20.0 g, 0.032 mol), compound 56-A (31.8 g, 0.048 mol), and DIPEA (12.3 g, 0.096 mol) dissolved in DMF (300 mL). The reaction was stirred at room temperature for 3 hours. The mixture was quenched with water (2.5 L) and extracted with ethyl acetate (600 mL × 3). The organic layer was washed with water (1 L) and brine (500 mL), dried over anhydrous sodium sulfate, concentrated, and purified by silica gel rapid column chromatography to give intermediate 56-5 (35.0 g, yield 86.2%) as a colorless oil. LCMS: m / z = 1271.8 [M+H] + .

[0332] f) Preparation of intermediate 56-6: Et₂NH (40 mL) was added to a mixture of intermediate 56-5 (35.0 g, 0.027 mol) dissolved in DCM (300 mL), and the mixture was stirred at room temperature for 12 hours. The reaction mixture was concentrated and purified by silica gel rapid column chromatography to give intermediate 56-6 (28 g, yield 96.8%) as a colorless oil. LCMS: m / z = 1049.7 [M+H] + .

[0333] g) Preparation of intermediate 56-7: Compounds 56-D (5.48 g, 12.9 mmol), DIEA (4.99 g, 38.7 mmol), and HATU (5.89 g, 15.5 mmol) were added to a DMF (100 mL) solution of compound 56-C (9.7 g, 12.9 mmol). The mixture was stirred at room temperature for 1 hour. The mixture was filtered through a diatomaceous earth mat, and the filtrate was dried under vacuum to give intermediate 56-7 (12.3 g, 88% yield) as a gray solid. LCMS: m / z = 1162 [M+H] + .

[0334] h) Preparation of intermediate 56-8: 9.7 g (8.34 mmol) of intermediate 56-7 was dissolved in 50 mL of formic acid. The mixture was stirred at 50 °C for 2 hours. The mixture was cooled to room temperature and poured into ice water (500 mL), and stirred at 0 °C for 30 minutes. After filtration, intermediate 56-8 (7.97 g, yield 86%) was obtained as a gray solid. LCMS: m / z = 1106.4 [M+H] + .

[0335] i) Preparation of intermediate 56-9: HATU (1.61 g, 4.24 mmol) was added to a mixture of intermediate 56-8 (3.9 g, 3.53 mmol), intermediate 56-6 (3.70 g, 3.53 mmol), and DIPEA (1.37 g, 10.59 mmol) in DMF (60 mL). The reaction was stirred at room temperature for 1 hour. The mixture was purified by reversed-phase chromatography to give intermediate 56-9 (2.96 g, 39% yield) as a yellow solid. LCMS: m / z = 1069.3 [M / 2+H] + .

[0336] j) Preparation of intermediate 56-10: A mixture of intermediate 56-9 (3.73 g, 1.75 mmol) dissolved in formic acid (40 mL) was stirred at room temperature for 16 hours. The mixture was purified by preparative high-performance liquid chromatography (Prep-HPLC) to give intermediate 56-10 (1.06 g, yield 29%) as a yellow solid. MS (ESI) m / z: 1041.4 [M / 2+H] + .

[0337] k) Preparation of intermediate 56-11: Intermediate 5-6 (850 mg, 1.103 mmol), DIEA (0.9 mL), and HATU (500 mg, 1.315 mmol) were added to a DMF (18 mL) solution of intermediate 56-10 (2.1 g, 1.009 mmol). The mixture was stirred at room temperature for 1 hour. The mixture was then purified directly by preparative high-performance liquid chromatography (Prep-HPLC) to give intermediate 56-11 (2.7 g, 94% yield) as a yellow solid. LCMS: m / z = 1417.7 [M / 2+H] + .

[0338] l) Preparation of intermediate 56-12: Diethylamine (0.3 mL) was added dropwise to intermediate 56-11 (2.29 g, 0.808 mmol) dissolved in DMF (10 mL). After stirring at room temperature for 2 hours, the solution was dried over N2 to remove DMF. The residue was prepared with MTBE (200 mL) and then dissolved in water (100 mL). The solution was lyophilized to give intermediate 56-12 (2.1 g, 99% yield) as a yellow solid. LCMS: m / z = 1307.2 [M / 2+H] + .

[0339] m) Preparation of compound LP56: Compound 56-E (700 mg, 1.681 mmol), DIEA (1.1 mL), and HATU (800 mg, 2.104 mmol) were added to intermediate 56-12 (4.23 g, 1.619 mmol) dissolved in DMF (31 mL). The mixture was stirred at room temperature for 1 hour. The mixture was then purified directly by preparative high-performance liquid chromatography (Prep-HPLC) to give the target compound LP56 (2.4 g, 49% yield) as a yellow solid. LCMS: m / z = 1004.3 [M / 3+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 9.95 (s, 2H), 8.22 (d, J = 6.4 Hz, 2H), 8.07 –7.98 (m, 6H), 7.92 (t, J = 5.2 Hz, 2H), 7.86 – 7.75 (m, 3H), 7.68 (d, J = 2.0Hz, 1H), 7.60 – 7.57 (m, 4H), 7.35 – 7.30 (m, 8H), 6.99 (s, 2H), 6.85 (d, J =2.0 Hz, 1H), 6.79 (s, 1H), 5.74 (s, 2H), 5.44 (s, 2H), 5.28 – 5.27 (m, 3H),5.11 – 5.07 (m, 4H), 4.47 (brs, 1H), 4.40 – 4.35 (m, 2H), 4.21– 4.15 (m, 4H),4.02– 3.99 (m, 5H), 3.81 – 3.70 (m, 2H), 3.60 – 3.55 (m, 12H), 3.49 – 3.47(m, 68H), 3.43 – 3.36 (m, 4H), 3.35 – 3.31 (m, 3H), 3.25 – 3.17 (m, 8H), 3.16– 3.11 (m, 3H), 2.39 – 2.25 (m, 12H), 2.19 – 2.15 (m, 6H), 1.97 – 1.81 (m,6H), 1.72 – 1.67 (m, 2H), 1.30 – 1.28 (m, 9H), 0.89 – 0.81 (m, 15H).

[0340] Compounds LP57 to LP60 can be prepared using a synthetic method similar to that in Examples B-56, and compound LP61 can be prepared using a synthetic method similar to that in Examples B-62. The examples are shown below: Example B-57: Compound LP57 MW: 3225.57, LCMS: m / z = 806 [M / 4+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 9.99 (d, J =38.8 Hz, 2H), 8.28 – 8.13 (m, 2H), 8.09 – 7.89 (m, 8H), 7.85 – 7.71 (m, 4H), 7.68 (d, J = 1.9 Hz, 1H), 7.59 (d, J = 8.4 Hz, 4H), 7.41 – 7.19 (m, 8H), 6.99(s, 2H), 6.82 (t, J = 9.3 Hz, 2H), 5.98 (s, 1H), 5.73 (s, 2H), 5.45 (s, 3H), 5.26 (d, J = 17.8 Hz, 3H), 5.09 (d, J = 15.0 Hz, 4H), 4.35 (ddd, J = 22.1,20.3, 14.0 Hz, 5H), 4.18 (dd, J = 13.1, 5.1 Hz, 4H), 4.06 – 3.92 (m, 6H), 3.75 (dd, J = 30.8, 11.6 Hz, 6H), 3.68 – 3.51 (m, 39H), 3.48 (d, J = 1.5 Hz, 29H), 3.37 (ddd, J = 24.6, 12.0, 6.2 Hz, 15H), 3.27 – 3.11 (m, 12H), 3.07 – 2.89 (m, 3H), 2.42 – 2.08 (m, 20H), 2.01 – 1.78 (m, 8H), 1.74 – 1.56 (m, 5H),1.35 (dt, J= 13.4, 7.4 Hz, 8H), 0.84 (ddd, J = 11.0, 10.5, 5.7 Hz, 15H).

[0341] Example B-58: Compound LP58 , MW: 3089.32. LC-MS: m / z = 1030.5 [M / 3+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 13.36–11.88 (m, 1H), 9.94 (s, 1H), 9.13 (s, 1H), 8.21 (d, J = 6.7 Hz, 3H), 8.00(ddd, J = 35.5, 22.2, 8.7 Hz, 8H), 7.79 (dd, J = 13.7, 9.7 Hz, 3H), 7.68 (d,J = 1.9 Hz, 1H), 7.59 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 8.5 Hz, 2H), 7.31 (s,1H), 7.29–7.17 (m, 3H), 7.10 (d, J = 8.4 Hz, 2H), 6.99 (s, 2H), 6.83 (d, J =1.9 Hz, 1H), 6.79 (s, 1H), 5.72 (s, 2H), 5.45 (s, 2H), 5.28 (s, 3H), 5.09 (d,J = 12.1 Hz, 4H), 4.90 (d, J = 7.5 Hz, 1H), 4.49–3.55 (m, 41H), 3.49 (s, 21H), 3.48–3.05 (m, 50H), 2.42–2.24 (m, 14H), 2.18 (d, J = 7.2 Hz, 7H), 2.01–1.62 (m, 9H), 1.29 (t, J = 6.7 Hz, 7H), 0.94–0.76 (m, 10H).

[0342] Example B-59: Compound LP59 ,MW:3010.36. LC-MS: m / z = 1004.3 [M / 3+H] + . 1H NMR (400 MHz, DMSO-d6) δ 9.94 (s, 2H), 8.20 (d, J= 6.9 Hz, 2H), 8.10–7.95 (m, 5H), 7.90 (t, J = 5.5 Hz, 2H), 7.79 (t, J = 10.2Hz, 3H), 7.67 (d, J = 1.9 Hz, 2H), 7.59 (dd, J = 8.4, 3.3 Hz, 4H), 7.27 (ddd,J = 50.3, 24.1, 14.0 Hz, 8H), 6.99 (s, 2H), 6.81 (t, J = 9.2 Hz, 2H), 5.71(s, 2H), 5.45(s, 2H), 5.31 (d, J = 22.4 Hz, 3H), 5.09 (d, J = 15.1 Hz, 4H), 4.38 (dd, J = 8.2, 5.9 Hz, 3H), 4.23–4.16 (m, 4H), 4.02–3.94 (m, 5H), 3.74(dd, J = 27.5, 11.8 Hz, 4H), 3.60–3.55 (m, 14H), 3.48–3.28 (m, 72H), 3.17(ddd, J = 22.1, 11.3, 5.8 Hz, 11H), 2.39–2.24 (m, 12H), 2.17 (t, J = 7.8 Hz,6H), 1.98–1.80 (m, 6H), 1.69 (dd, J = 13.6, 8.2 Hz, 2H), 1.32–1.23 (m, 9H), 0.91–0.77 (m, 16H).

[0343] Example B-60: Compound LP60 , MW: 3039.32. LCMS: m / z = 1520.7 [M / 2+H] + . 1H NMR (400 MHz, DMSO-d6) δ 9.95 (s, 2H), 8. 23–8.20 (m, 2H), 8.07–7.75 (m, 12H), 7.68 (d, J = 1.6 Hz, 1H), 7.60–7.57(m, 4H), 7.37–7.24 (m, 8H), 6.99 (s, 2H), 6.84 (d, J = 2.0 Hz, 1H), 6.78(brs, 1H), 5.73 (s, 2H), 5.45 (s, 2H), 5.29–5.28 (m, 3H), 5.11–5.07 (m, 4H),4.44 (brs, 1H), 4.39–4.32 (m, 3H), 4.26–4.15 (m, 5H), 4.06–3.99 (m, 8H), 3.80–3.70 (m, 3H), 3.62–3.55 (m, 9H), 3.49–3.47 (m, 58H), 3.42–3.32 (m, 8H), 3.26–3.18 (m, 9H), 3.14–3.06 (m, 6H), 2.39–2.37 (m, 5H), 2.33 – 2.25 (m, 4H), 2.19 – 2.15 (m, 6H), 1.96 – 1.70 (m, 8H), 1.31 – 1.27 (m, 9H), 0.89 – 0.81(m, 15H).

[0344] Example B-61: Compound LP61 , MW: 2604.91. LC-MS: m / z = 1303.05 [M / 2+H] + 869.05 [M / 3+H] + 652.00 [M / 4+H] + . 1HNMR (400 MHz, DMSO-d6) δ 9.91 (s, 2H), 8.55 (s, 1H), 8.16 (d, J = 6.1 Hz,2H), 8.03 (d, J = 8.6 Hz, 1H), 7.91–7.81 (m, 6H), 7.74 (d, J = 10.8 Hz, 1H),7.62 (d, J = 2.0 Hz, 1H), 7.56 (t, J = 8.9 Hz, 4H), 7.34 (d, J = 7.8 Hz, 3H),7.30–7.24 (m, 3H), 7.12 (d, J = 2.3 Hz, 1H), 6.95 (s, 2H), 6.90 (s, 1H), 6.76(d, J = 2.0 Hz, 1H), 6.51 (s, 1H), 5.42 (s, 2H), 5.38 (d, J = 6.6 Hz, 2H),5.32-5.20 (m, 3H), 5.05 (s, 2H), 4.97 (s, 2H), 4.39–4.30 (m, 3H), 4.17 (t, J= 7.5 Hz, 3H), 3.99-3.91 (m, 4H), 3.86 (d, J = 13.3 Hz, 1H), 3.77-3.67 (m,2H), 3.61–3.53 (m, 10H), 3.49–3.45 (m, 54H), 3.29-3.22 (m, 2H), 3.20-3.14 (m,2H), 2.97-2.87 (m, 2H), 2.42 (t, J = 6.6 Hz, 2H), 2.39-2.32 (m, 7H), 2.27 (t,J = 7.3 Hz, 2H), 2.21-2.07 (m, 1H), 2.04–1.76 (m, 6H), 1.59-1.35 (m, 2H),1.31-1.25 (m, 7H), 1.24-1.19 (m, 8H), 0.87–0.78 (m, 18H).

[0345] Example B-62: Compound LP62

[0346] a) Preparation of intermediate 62-1: A mixture of compound 62-A (6.00 g, 22.56 mmol), compound 62-B (9.25 g, 22.56 mmol), and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (11.16 g, 45.11 mmol) in dichloromethane (150 mL) and methanol (75 mL) was stirred at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give intermediate 62-1 (5.00 g, yield 33.69%) as a pale yellow solid. LCMS: m / z = 681.5 [M+Na] + .

[0347] b) Preparation of intermediate 62-2: Diethylamine (3.03 g, 41.49 mmol) was added to a solution of intermediate 62-1 (9.10 g, 13.83 mmol) dissolved in N,N-dimethylformamide (100 mL). The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was purified by preparative high-performance liquid chromatography (HPLC) to give intermediate 62-2 (5.75 g, 95.36% yield) as a white solid. LCMS: m / z = 437.5 [M+H] + .

[0348] c) Preparation of intermediate 62-3: Under nitrogen atmosphere at 0°C, 1H-imidazolium-1-sulfonyl azide (3.65 g, 21.10 mmol) was added to a methanol (60 mL) mixture containing intermediate 62-2 (5.75 g, 13.19 mmol), potassium carbonate (3.64 g, 26.38 mmol), and copper sulfate (II) pentahydrate (0.33 g, 1.32 mmol). The reaction mixture was heated to room temperature and stirred for 16 hours. The residue was filtered off. The filtrate was concentrated, and the residue was purified by silica gel column chromatography to give intermediate 62-3 (5.13 g, 84.20% yield) as a white solid. LCMS: m / z = 485.4 [M+Na] + .

[0349] d) Preparation of intermediate 62-4: A mixture of intermediate 62-3 (4.00 g, 8.66 mmol), 4-nitrophenylcarbonyl chloride (3.48 g, 17.32 mmol), and pyridine (2.05 g, 25.97 mmol) in dichloromethane (40 mL) was stirred at room temperature for 5 hours. The reaction mixture was purified by silica gel column chromatography to give intermediate 62-4 (4.20 g, 77.37% yield) as a white solid. LCMS: m / z = 650.3 [M+Na] + .

[0350] e) Preparation of compound 62-5: A mixture of intermediate 62-4 (4.20 g, 6.70 mmol), 2-methoxyethylamine (0.60 g, 8.04 mmol), and N,N-diisopropylethylamine (1.73 g, 13.40 mmol) in N,N-dimethylformamide (30 mL) was stirred at room temperature for 2 hours. The reaction mixture was diluted with water (500 mL). The resulting solution was diluted with ethyl acetate (200 mL). 3) Extraction. The organic layers were combined and concentrated. The residue was purified by silica gel column chromatography to give intermediate 62-5 (3.00 g, yield 79.53%) as a white solid. LCMS: m / z = 586.3 [M+Na] + .

[0351] f) Preparation of intermediate 62-6: Trimethylchlorosilane (0.74 g, 6.82 mmol) was added to a solution of dichloromethane (40 mL) containing compound 62-5 (3.2 g, 5.68 mmol) and paraformaldehyde (0.85 g, 28.42 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was filtered. The filtrate was concentrated under vacuum to give intermediate 62-6. LCMS: m / z = 616.4 [M-Cl+OMe] + .

[0352] g) Preparation of intermediate 62-7: A mixture of intermediate 62-6 (3.47 g, 5.68 mmol), compound P-1 (1.65 g, 4.54 mmol), and 1,2,2,6,6-pentamethylpiperidine (1.76 g, 11.36 mmol) dissolved in dichloromethane (30 mL) was stirred in a sealed tube at 40 °C for 24 h. The reaction mixture was purified by preparative high-performance liquid chromatography (HPLC) to give intermediate 62-7 (1.2 g, two-step yield 22.50%) as a yellow solid. LCMS: m / z = 940.5 [M+H] + .

[0353] h) Preparation of intermediate 62-8: Under nitrogen protection, trimethylphosphine (3.58 mL, 1N THF solution) was added dropwise to a solution of tetrahydrofuran (10 mL) and water (1 mL) containing intermediate 62-7 (1.12 g, 1.19 mmol) at room temperature. The reaction mixture was stirred for 4 hours. The resulting solution was concentrated, and the residue was purified by preparative high-performance liquid chromatography (HPLC) to give intermediate 62-8 (500 mg, yield 45.91%) as a yellow solid. LCMS: m / z = 914.5 [M+H] + .

[0354] h) Preparation of intermediate 62-9: A mixture of compound 62-C (2.00 g, 4.26 mmol), compound 62-D (1.96 g, 4.26 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (1.22 g, 6.39 mmol), 1-hydroxybenzotriazole (1.15 g, 8.52 mmol), and N,N-diisopropylethylamine (1.65 g, 12.77 mmol) in N,N-dimethylformamide (20 mL) was stirred at room temperature for 16 hours. The reaction mixture was purified by preparative high-performance liquid chromatography (HPLC) to give intermediate 62-9 (2.15 g, 57.64% yield) as a pale yellow oil. LCMS: m / z = 877.5 [M+H] + .

[0355] i) Preparation of compound 62-10: A mixture of intermediate 62-9 (2.00 g, 2.28 mmol), compound 56-C (1.72 g, 2.28 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.66 g, 3.42 mmol), 1-hydroxybenzotriazole (0.62 g, 4.57 mmol), and N,N-diisopropylethylamine (0.88 g, 6.85 mmol) in N,N-dimethylformamide (20 mL) was stirred at room temperature for 16 hours. The reaction mixture was purified by preparative high-performance liquid chromatography (HPLC) to give intermediate 62-10 (1.7 g, 46.19% yield) as a grayish-white solid. LCMS: m / z = 807.5 [M / 2+H] + .

[0356] j) Preparation of intermediate 62-11: Intermediate 62-10 (1.70 g, 1.05 mmol) was dissolved in a solution of trifluoroacetic acid (4 mL) and dichloromethane (12 mL) and stirred at room temperature for 5 hours. The reaction mixture was dried by purging with nitrogen. The residue was diluted with N,N-dimethylformamide (5 mL). The residue was purified by preparative high-performance liquid chromatography (HPLC) to give intermediate 62-11 (460 mg, yield 28.03%) as a pale yellow solid. LCMS: m / z = 779.5 [M / 2+H] + .

[0357] h) Preparation of intermediate 62-12: Compound 56-A (28 mg, 0.042 mmol), DIEA (0.1 mL), and HATU (18 mg, 0.047 mmol) were added to intermediate 62-8 (35 mg, 0.038 mmol) dissolved in DMF (1.5 mL). The mixture was stirred at room temperature for 1 hour. The mixture was then purified directly by Prep-HPLC to give intermediate 62-12 (45 mg, 75% yield) as a yellow solid. LCMS: m / z = 780.6 [M / 2+H] + .

[0358] i) Preparation of intermediate 62-13: Diethylamine (0.1 mL) was added dropwise to a solution of intermediate 62-12 (45 mg, 0.029 mmol) dissolved in DMF (1.5 mL). After stirring at room temperature for 1 hour, the mixture was purified by preparative HPLC to give intermediate 62-13 (25 mg, 65% yield) as a yellow solid. MS (ESI) m / z: 669.5 [M / 2+H] + .

[0359] j) Preparation of intermediate 62-14: Intermediate 62-13 (25 mg, 0.019 mmol), DIEA (0.1 mL), and HATU (9 mg, 0.024 mmol) were added to a solution of intermediate 62-11 (29 mg, 0.019 mmol) dissolved in DMF (1.5 mL). The mixture was stirred at room temperature for 1 hour. The mixture was then purified directly by preparative high-performance liquid chromatography (Prep-HPLC) to give intermediate 62-14 (20 mg, 37% yield) as a yellow solid. LCMS: m / z = 960.0 [M / 3+H] + .

[0360] k) Preparation of intermediate 62-15: TFA (0.1 mL) was added to intermediate 62-14 (20 mg, 0.007 mmol) dissolved in DCM (0.5 mL). The mixture was stirred at room temperature for 2 hours. The mixture was concentrated to dryness to give intermediate 62-15 as a yellow solid (crude product, directly used in the next step). LCMS: m / z = 926.6 [M / 3+H] + .

[0361] l) Preparation of intermediate 62-16: Compound 62-E (7 mg, 0.009 mmol), DIEA (0.1 mL), and HATU (4 mg, 0.011 mmol) were added to intermediate 62-15 (crude product) dissolved in DMF (0.5 mL). The mixture was stirred at room temperature for 1 hour. The mixture was then purified directly by preparative high-performance liquid chromatography (Prep-HPLC) to give intermediate 62-16 (12 mg, 49% yield) as a yellow solid. LCMS: m / z = 1765.8 [M / 2+H] + .

[0362] m) Preparation of intermediate 62-17: Diethylamine (0.05 mL) was added dropwise to intermediate 62-16 (12 mg, 0.003 mmol) dissolved in DMF (0.5 mL). After stirring at room temperature for 1 hour, the mixture was purified by preparative high-performance liquid chromatography (Prep-HPLC) to give intermediate 62-17 (9 mg, 80% yield) as a yellow solid. LCMS: m / z = 1103.6 [M / 3+H] + .

[0363] n) Preparation of the target compound LP62: Compound 62-F (1.2 mg, 0.004 mmol) and DIEA (0.01 mL) were added to intermediate 62-17 (9 mg, 0.003 mmol) dissolved in DMF (0.4 mL). The mixture was stirred at room temperature for 1 hour. The mixture was then purified directly by preparative high-performance liquid chromatography (Prep-HPLC) to give the target compound LP62 (5.1 mg, 54% yield) as a yellow solid. LCMS: m / z = 1729.9 [M / 2+H] + . 1H NMR (400 MHz, DMSO-d6) δ9.93 (s, 2H), 8.18 (d, J = 6.8 Hz, 2H), 8.07 – 8.02 (m, 2H), 7.94 – 7.87 (m,6H), 7.92 (d, J = 11.2 Hz, 2H), 7.68 – 7.59 (m, 4H), 7.36 (d, J = 8.4 Hz,3H), 7.31 (s, 1H), 7.24 – 7.19 (m, 2H), 6.99 (s, 2H), 6.83 (s, 1H), 5.72 (s,2H), 5.45 (s, 2H), 5.28 (s, 3H), 5.15 – 5.07 (m, 4H), 4.64 (brs, 2H), 4.42–4.33 (m, 9H), 4.27– 4.18 (m, 10H), 4.08– 3.87 (m, 17H), 3.79 – 3.72 (m, 3H), 3.59 – 3.56 (m, 10H), 3.49 – 3.47 (m, 48H), 3.39 – 3.36 (m, 4H), 3.23 – 3.18(m, 6H), 2.94 – 2.67 (m, 39H), 2.47 – 2.28 (m, 12H), 2.22 – 2.14 (m, 2H),1.99 – 1.75 (m, 7H), 1.58 – 1.56 (m, 1H), 1.46 – 1.43 (m, 1H), 1.31 – 1.16 (m, 14H), 0.89 – 0.82 (m, 15H).

[0364] Compounds LP63 and LP64 can be prepared using a synthetic method similar to that in Examples B-62, as shown in the examples below: Example B-63: Compound LP63 , MW: 2737.07. LCMS: m / z = 913.4 [M / 3+H] + . 1H NMR (400 MHz, CDCl3) δ 9.17 (s, 1H),8.60-8.55 (m, 1H), 8.27-8.21 (m, 1H), 7.80-7.30 (m, 9H), 7.23-6.44 (m, 8H),5.74-5.68 (m, 2H), 5.39-5.08 (m,7H), 4.65-3.85(m, 12H), 3.73-3.14 (m, 66H), 2.61-1.64 (m, 28H), 1.18-1.06 (m, 33H), 1.06 -0.83 (m, 18H).

[0365] Example B-64: Compound LP64 , MW: 2413.6. LCMS: m / z =1207.7 [M / 2+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 9.96 (s,3H), 8.23-8.21 (m, 3H), 8.08-7.98 (m, 5H), 7.94-7.90 (m, 4H), 7.83-7.76 (m,5H), 7.65-7.59 (m, 8H), 7.43-7.29 (m, 10H), 7.14-7.12 (m, 2H), 7.00 (s, 2H), 6.95 (s, 2H), 6.79 (s, 3H), 6.53 (s, 1H), 5.69-5.68 (m, 4H), 5.45 (s, 2H),5.29-5.26 (m, 3H), 5.12-5.08 (m, 6H), 4.40-4.37 (m, 5H), 4.23-4.17 (m, 6H), 4.00-3.98 (m, 8H), 3.91-3.88 (m, 2H), 3.78-3.48 (m, 121H), 3.42-3.39 (m, 9H),3.25-3.14 (m, 16H), 2.37-2.28 (m, 12H), 2.19-2.16 (m, 7H), 1.98-1.82 (m, 9H),1.73-1.67 (m, 3H), 1.31-1.24 (m, 18H), 0.90-0.82 (m, 21H).

[0366] Example B-65: Compound LP65

[0367] a) Preparation of intermediate 65-1: Compound 65-A (48 mg, 0.0642 mmol), HATU (36.5 mg, 0.0963 mmol), and DIPEA (41.4 mg, 0.321 mmol) were added to a DMF solution (62 mg, 0.0278 mmol) containing intermediate 62-11 (100 mg, 0.0642 mmol) at room temperature. The mixture was stirred at room temperature for 1 hour. The crude product was purified by preparative high-performance liquid chromatography (HPLC) to obtain intermediate 65-1 (81 mg, yield 55.1%) as a white solid. LCMS: m / z = 1143.0 [M / 2+H] +

[0368] b) Preparation of intermediate 65-2: Intermediate 65-1 (81 mg, 0.0355 mmol) was dissolved in a mixed solution of dichloromethane (DCM, 50 mL) and trifluoroacetic acid (TFA, 25 mL) and stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography to give a white solid intermediate 65-2 (40 mg, yield 50.6%). LCMS: m / z = 1115.0 [M / 2+H] +

[0369] c) Preparation of intermediate 65-3: Compound 56-A (87 mg, 0.131 mmol), DIEA (0.1 mL), and HATU (60 mg, 0.158 mmol) were added to a solution of intermediate 5-6 (100 mg, 0.130 mmol) dissolved in DMF (2 mL). The mixture was stirred at room temperature for 1 hour. The mixture was then purified directly by Prep-HPLC (preparative high-performance liquid chromatography) to give intermediate 65-3 (135 mg, 73% yield) as a yellow solid. LCMS: m / z = 709.0 [M / 2+H] + .

[0370] d) Preparation of intermediate 65-4: Diethylamine (0.1 mL) was added dropwise to intermediate 65-3 (135 mg, 0.095 mmol) dissolved in DMF (2 mL). After stirring at room temperature for 1 hour, the mixture was purified by preparative high-performance liquid chromatography (Prep-HPLC) to give intermediate 65-4 (105 mg, 93% yield) as a yellow solid. LCMS: m / z = 598.0 [M / 2+H] + .

[0371] e) Preparation of intermediate 65-5: DIPEA (18 mg, 0.1392 mmol) was added to a mixture of DMF (2.5 mL) containing intermediate 65-2 (62 mg, 0.0278 mmol), intermediate 65-4 (33.2 mg, 0.0278 mmol), and HATU (15.9 mg, 0.0417 mmol), and the mixture was stirred at room temperature for 1 hour. The crude product was purified by preparative high-performance liquid chromatography (HPLC) to obtain intermediate 65-5 (75 mg, yield 79.2%) as a yellow solid. LCMS: m / z = 1135.7 [M / 2+H] + .

[0372] f) Preparation of intermediate 65-6: Intermediate 65-5 (4.5 mg, 0.0617 mmol) dissolved in DMF (1.5 mL) was stirred at room temperature for 30 minutes. The crude product was purified by preparative high-performance liquid chromatography (HPLC) to obtain intermediate 65-6 (63 mg, 96.9% yield) as a yellow solid. LCMS: m / z = 1061.6 [M / 3+H] + .

[0373] g) Preparation of target compound LP65: DIPEA (6.1 mg, 0.0471 mmol) was added to a 1.5 mL DMF solution containing intermediate 65-6 (50 mg, 0.0157 mmol) and intermediate 62-F (6.3 mg, 0.0188 mmol) at room temperature, and the mixture was stirred for 30 minutes at room temperature. The reaction mixture was purified by preparative high-performance liquid chromatography to obtain the target compound LP65. LCMS: m / z = 1112.0 [M / 3+H] + . 1HNMR (400 MHz, DMSO-d6) δ 9.94 (s,2H), 8.19 (d,J = 6.8 Hz, 2H), 8.07 (d,J = 8.8 Hz, 2H), 8.00 (d,J = 5.3 Hz,1H), 7.94 (d,J = 4.7 Hz, 2H), 7.91 – 7.80 (m, 7H), 7.78 (d,J = 10.6 Hz, 1H),7.69 (d,J = 1.9 Hz, 1H), 7.59 (d,J = 8.4 Hz, 4H), 7.40 – 7.30 (m, 6H), 7.26(d,J = 9.3 Hz, 2H), 7.00 (s, 2H), 6.85 (s, 1H), 5.74 (s, 2H), 5.45 (s, 2H),5.29 (s, 3H), 5.09 (d,J = 12.7 Hz, 5H), 4.45 (d,J = 8.7 Hz, 2H), 4.38 (d,J =7.3 Hz, 4H), 4.23 – 4.16 (m, 8H), 4.04 – 3.97 (m, 8H), 3.79 (d,J = 11.8 Hz,2H), 3.71 (d,J = 8.8 Hz, 2H), 3.67 (d,J = 1.4 Hz, 1H), 3.59 (t,J = 7.1 Hz,13H), 3.48 (d,J = 8.4 Hz, 109H), 3.44 – 3.37 (m, 9H), 3.25 – 3.16 (m, 12H),2.98 – 2.93 (m, 2H), 2.44 (d,J = 4.4 Hz, 2H), 2.38 (d,J = 5.9 Hz, 6H), 2.36(s, 1H), 2.30 (d,J = 6.5 Hz, 2H), 2.22 – 2.14 (m, 2H), 2.07 (t,J = 6.1 Hz,2H), 1.98 – 1.91 (m, 3H), 1.89 – 1.81 (m, 3H), 1.76 – 1.69 (m, 1H), 1.59 (d,J= 14.6 Hz, 1H), 1.47 (d,J = 11.0 Hz, 1H), 1.30 (d,J = 6.0 Hz, 11H), 0.85 (dd,J = 19.4, 9.5 Hz, 18H)。

[0374] Compounds LP66 and LP67 can be prepared using a synthetic method similar to that in Examples B-62, as shown in the examples below: Example B-66: Compound LP66 , MW: 2665.99.

[0375] Example B-67: Compound LP67 , MW: 4818.50.

[0376] Example B-68: Compound LP68

[0377] a) Preparation of intermediate 68-1: HATU (2.18 g, 5.73 mmol) and DIEA (1.48 g, 11.5 mmol) were added to a 20.0 mL solution of DMF containing intermediate 56-6 (4.00 g, 3.82 mmol) and compound 56-B (1.62 g, 3.82 mmol). The mixture was stirred at room temperature for 2 hours. After the reaction, the mixture was purified by reverse-phase column chromatography (C-18) to give intermediate 68-1 (2.74 g, 49% yield) as a white solid. LCMS: m / z = 729.0 [M / 2+H] + .

[0378] b) Preparation of intermediate 68-2: DEA (686 mg, 9.40 mmol) was added to a 10.0 mL solution of DMF containing intermediate 68-1 (2.74 g, 1.88 mmol). The mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated, and the residue was passed through a reverse-phase column (C10). 18 Purification yielded intermediate 68-2 (1.85 g, 80% yield), a yellow oily substance. LCMS: m / z = 618.0 [M / 2+H] + .

[0379] c) Preparation of intermediate 68-3: HATU (855 mg, 2.25 mmol) and DIEA (581 mg, 4.50 mmol) were added to a 10.0 mL solution of DMF containing intermediate 68-2 (1.85 g, 1.50 mmol) and compound 56-A (995 mg, 1.50 mmol). The mixture was stirred at room temperature for 2 hours. After the reaction, the product was passed through a reverse-phase column (C10). 18The mixture was purified to give a yellow oily intermediate 68-3 (2.10 g, 75% yield). LCMS: m / z = 941.0 [M / 2+H] + .

[0380] d) Preparation of intermediate 68-4: A solution of intermediate 68-3 (1.0 g, 0.53 mmol) dissolved in DCM / TFA (6.0 mL, v / v ratio 3:1) was stirred at 25 °C for 2 hours. After the reaction, the mixture was concentrated by purging with nitrogen and passed through a reverse-phase column (C10). 18 Purification yielded a yellow, oily intermediate 68-4 (800 mg, 85% yield). LCMS: m / z = 1249.6 [M+H] + .

[0381] e) Preparation of intermediate 68-5: HATU (258 mg, 0.68 mmol) and DIEA (174 mg, 1.35 mmol) were added to a 6.0 mL solution of DMF containing intermediate 68-4 (800 mg, 0.45 mmol) and intermediate 68-6 (349 mg, 0.45 mmol). The mixture was stirred at room temperature for 2 hours. After the reaction, the mixture was passed through a reverse-phase column (C10). 18 Purification yielded intermediate 68-5 (1.02 g, 69% yield) as a yellow solid. LCMS: m / z = 1091.9 [M / 3+H] + .

[0382] f) Preparation of intermediate 68-6: DEA (113 mg, 1.55 mmol) was added to intermediate 68-5 (1.02 g, 0.31 mmol) dissolved in DMF (5.0 mL). The mixture was stirred at room temperature for 2 hours. After the reaction, the mixture was concentrated and passed directly through a reversed-phase column (C10). 18 The solution was purified to give intermediate 68-6 (500 mg, 53% yield) as a yellow solid. LCMS: m / z = 1018.0 [M / 3+H] + .

[0383] g) Preparation of intermediate 68-7: Intermediate 56-8 (27 mg, 0.04 mmol), HATU (6.80 mg, 0.05 mmol), and DIEA (14 mg, 0.11 mmol) were added to a dry DMF (3.0 mL) solution containing intermediate 68-6 (106 mg, 0.04 mmol). The mixture was stirred at room temperature for 1 hour. After the reaction, the mixture was concentrated and purified directly by preparative high-performance liquid chromatography (Prep-HPLC) to give intermediate 68-7 (33 mg, 23% yield) as a grayish-white solid. LCMS: m / z = 1035.7 [M / 4+H] + .

[0384] h) Preparation of intermediate 68-8: DEA (3.00 mg, 0.008 mmol) was added to intermediate 68-7 (33.0 mg, 0.008 mmol) dissolved in DMF (2.0 mL). The mixture was stirred at room temperature for 2 hours. After the reaction, the mixture was concentrated and purified directly by preparative high-performance liquid chromatography (Prep-HPLC) to obtain intermediate 68-8 (16 mg, yield 64%) as a yellow oil. LCMS: m / z = 980.5 [M / 4+H] + .

[0385] i) Preparation of the target compound LP68: Compound 2-B (1.70 mg, 0.004 mmol), HATU (2.30 mg, 0.006 mmol), and DIEA (1.60 mg, 0.012 mmol) were added to a dry DCM (3.0 mL) containing intermediate 68-8 (16.0 mg, 0.004 mmol). The mixture was stirred at room temperature for 2 hours. After the reaction, the mixture was concentrated and purified directly by preparative high-performance liquid chromatography (Prep-HPLC) to obtain the target compound LP68 (10 mg, 56% yield) as a grayish-white solid. LCMS: m / z = 913.4 [M / 3+H] + . 1H NMR (400 MHz, DMSO-d6) δ 9.96(s, 3H), 8.23-8.21 (m, 3H), 8.08-7.98 (m, 5H), 7.94-7.90 (m, 4H), 7.83-7.76(m, 5H), 7.65-7.59 (m, 8H), 7.43-7.29 (m, 10H), 7.14-7.12 (m, 2H), 7.00 (s,2H), 6.95 (s, 2H), 6.79 (s, 3H), 6.53 (s, 1H), 5.69-5.68 (m, 4H), 5.45 (s,2H), 5.29-5.26 (m, 3H), 5.12-5.08 (m, 6H), 4.40-4.37 (m, 5H), 4.23-4.17 (m,6H), 4.00-3.98 (m, 8H), 3.91-3.88 (m, 2H), 3.78-3.48 (m, 121H), 3.42-3.39 (m,9H), 3.25-3.14 (m, 16H), 2.37-2.28 (m, 12H), 2.19-2.16 (m, 7H), 1.98-1.82 (m,9H), 1.73-1.67 (m, 3H), 1.31-1.24 (m, 18H), 0.90-0.82 (m, 21H).

[0386] Compounds LP69 and LP70 can be prepared using a synthetic method similar to that of Example B-68, and compounds LP71 and LP72 can be prepared using a synthetic method similar to that of Example B-62. The examples are shown below: Example B-69: Compound LP69 , MW: 4394.82. LCMS: m / z = 1099.6 [M / 4+H] + . 1H NMR (400 MHz, DMSO-d6) δ 9.97 (s,2H), 9.13 (s, 1H), 8.20 (dd, J = 18.4, 9.4 Hz, 5H), 8.09–7.89 (m, 11H), 7.79 (dd, J = 19.6, 9.9 Hz, 5H), 7.68 (d, J = 1.8 Hz, 2H), 7.59 (d, J = 8.3 Hz, 4H), 7.40–7.29 (m, 6H), 7.18 (dd, J = 29.0, 16.8 Hz, 6H), 6.99 (s, 2H), 6.82(t, J = 6.1 Hz, 3H), 5.72 (s, 5H), 5.44 (s, 2H), 5.07 (t, J = 13.6 Hz, 7H), 4.89 (d, J = 7.3 Hz, 1H), 4.38 (dd, J = 17.2, 10.2 Hz, 5H), 4.19 (dt, J =15.2, 7.8 Hz, 5H), 4.02–3.88 (m, 15H), 3.74 (dd, J = 29.1, 11.7 Hz, 8H), 3.60–3.55 (m, 25H), 3.47–3.33 (m, 86H), 3.26–3.11 (m, 22H), 2.42–2.11 (m,28H), 1.99–1.62 (m, 14H), 1.31–1.25 (m, 12H), 0.84 (dd, J = 17.0, 6.6 Hz, 15H).

[0387] Example B-70: Compound LP70 , MW: 4825.24.

[0388] Example B-71: Compound LP71 , MW: 3403.86.

[0389] Example B-72: Compound LP72 , MW: 2662.99.

[0390] Compounds LP73, LP74, and LP77 can be prepared using a synthetic method similar to that in Example B-56, and compounds LP75, LP76, and LP78 can be prepared using a synthetic method similar to that in Example B-68. The examples are shown below: Example B-73: Compound LP73 , MW: 3039.32.

[0391] Example B-74: Compound LP74 , MW: 3118.28.

[0392] Example B-75: Compound LP75 , MW: 4315.86. LCMS: m / z =1079.7 [M / 4+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 9.95 (s,3H), 8.22 (d, J = 6.8 Hz, 3H), 8.13 – 7.96 (m, 7H), 7.92 (d, J = 5.9 Hz, 4H), 7.79 (dd, J = 16.8, 9.6 Hz, 6H), 7.68 (d, J = 1.9 Hz, 2H), 7.64 – 7.54 (m,6H), 7.41 – 7.27 (m, 8H), 7.27 – 7.14 (m, 4H), 6.99 (s, 2H), 6.83 (d, J = 1.8Hz, 3H), 5.72 (s, 4H), 5.45 (s, 2H), 5.28 (s, 3H), 5.09 (d, J = 15.5 Hz, 6H), 4.38 (dd, J = 17.9, 11.0 Hz, 6H), 4.24 – 4.14 (m, 7H), 4.03 – 3.94 (m, 11H), 3.74 (dd, J = 29.7, 11.4 Hz, 12H), 3.60 – 3.55 (m, 20H), 3.43 – 3.32 (m,84H), 3.25 – 3.11 (m, 20H), 2.40 – 2.25 (m, 15H), 2.17 (t, J = 7.8 Hz, 9H),2.00 – 1.79 (m, 9H), 1.69 (dd, J = 13.5, 8.0 Hz, 3H), 1.49 (d, J = 6.9 Hz, 1H), 1.28 (t, J = 7.5 Hz, 16H), 0.85 (dt, J = 17.0, 7.0 Hz, 22H).

[0393] Example B-76: Compound LP76 , MW: 4315.86.

[0394] Example B-77: Compound LP77 , MW: 3026.44.

[0395] Example B-78: Compound LP78 , MW: 4331.94.

[0396] C. Synthesis of antibody-drug conjugates

[0397] Each antibody-drug conjugate (ADC) can be prepared using the methods described in the following examples. The drug-antibody ratio (DAR) of the ADC was determined by liquid chromatography-mass spectrometry (LC-MS) using an Agilent 1290 liquid chromatography system in series with an Agilent 6224 time-of-flight mass spectrometer. The purity of the ADC was analyzed by size exclusion chromatography (SEC-HPLC) using an Agilent 1260 series high performance liquid chromatography system.

[0398] Example C-1

[0399] The preparation of antibody-drug conjugates (ADCs) followed the general method described in WO2020164561, namely: selectively reducing the antibody under appropriate conditions in the presence of transition metal ions, and then conjugating it with a linker-drug to obtain ADCs with an average drug-antibody ratio (DAR) of approximately 4.

[0400] Using this conjugation method, anti-CEACAM5 antibodies (whose heavy chain amino acid sequences and light chain amino acid sequences are shown in SEQ ID NO: 129 and 134, respectively, as described in WO2023198019A1) were conjugated to each linker-drug to prepare the following ADCs:

[0401] Note: The average DAR value is the drug-antibody ratio determined by mass spectrometry.

[0402] LP0-1 is a linker-drug synthesized using synthetic methods known to those skilled in the art, and its structure is as follows: (LP0-1).

[0403] Example C-2

[0404] The preparation of antibody-drug conjugates (ADCs) followed the general method described in WO2025011561, namely: selectively reducing the antibody under appropriate conditions in the presence of transition metal ions, and then conjugating it with a linker-drug to obtain ADCs with an average drug-antibody ratio (DAR) of approximately 6.

[0405] Using this conjugation method, an anti-CEACAM5 antibody (whose heavy chain amino acid sequence and light chain amino acid sequence are shown in SEQ ID NO: 129 and 134 respectively, as recorded in WO2023198019A1, and whose sequence numbers in this paper are SEQ ID NO: 129 and 134 respectively) was conjugated with the linker-drug LP56 to prepare the following ADC:

[0406] Note: The average DAR value is the drug-antibody ratio determined by mass spectrometry.

[0407] Example C-3

[0408] Add conjugation buffer (e.g., PBS, pH 7.4, or 20 mM histidine, pH 6.0), 200 mM EDTA (final concentration 2 mM), and 5 mM TCEP solution (3.75 eq) to a 1.5 mL tube containing 1 mg of antibody. The antibody concentration in the reaction solution is 7 mg / mL. Mix the reaction solution thoroughly and incubate at 22°C for 18 hours. First, add a certain amount of DMSO to the reaction solution, and then add 10 mM DMSO solution of the linker-drug LP (10 eq) to the reaction, with the total volume of DMSO added being 10% of the total volume of the reaction solution. Mix thoroughly and incubate at 22°C for another hour. The antibody concentration during the conjugation process is 6.3 mg / mL. Purify the crude conjugate using Zeba (40 kDa, 0.5 mL) and a buffer of 20 mM histidine / histidine-HCl, 8% sucrose, pH 5.5. The purified samples were characterized by drug-antibody ratio (DAR) analysis using LC-MS and product purity detection using SEC-HPLC.

[0409] Using a conjugation method similar to that described above, the anti-CEACAM5 antibody (whose heavy chain amino acid sequence and light chain amino acid sequence are the amino acid sequences shown in SEQ ID NO: 129 and 134 as described in WO2023198019A1, respectively, and whose sequence numbers in this paper are SEQ ID NO: 129 and 134, respectively) was conjugated with each linker-drug to prepare the following ADCs:

[0410] Note: The average DAR value is the drug-antibody ratio determined by mass spectrometry.

[0411] LP0-2 is a linker-drug synthesized using synthetic methods known to those skilled in the art, and its structure is as follows: (LP0-2).

[0412] Example C-4

[0413] First, ADC intermediates were prepared according to the general method described in WO2020164561, namely: selectively reducing the antibody in the presence of transition metal ions under appropriate conditions and coupling it with a linker-drug, such that each antibody is coupled with an average of 4 linker-drugs. Subsequently, an additional reduction step was performed under suitable conditions to reduce the remaining interchain disulfide bonds, followed by re-coupling, thereby obtaining ADCs with an average of 4+4 linker-drugs per antibody.

[0414] Using this conjugation method, an anti-CEACAM5 antibody (whose heavy chain amino acid sequence and light chain amino acid sequence are shown in SEQ ID NO: 129 and 134 as described in WO2023198019A1, respectively, and whose sequence numbers in this paper are SEQ ID NO: 129 and 134, respectively) was conjugated with the linker-drugs LP0 and LP05 to prepare the following ADC:

[0415] Note: The average DAR value is the drug-antibody ratio determined by mass spectrometry.

[0416] Using this conjugation method, an anti-HER2 antibody (Trastuzumab, purchased from WuXi Biologics) was conjugated with linkers-drugs LP0 and LP05 to prepare the following ADC:

[0417] Note: The average DAR value is the drug-antibody ratio determined by mass spectrometry.

[0418] The sequence of the HER2 antibody used in this article is shown in "Physicochemical and Biological Characterization of a Biosimilar Trastuzumab" (http: / / dx.doi.org / 10.1155 / 2015 / 427235). Figure 3As shown, the amino acid sequence of its light chain is shown in SEQ ID NO: 138 of this paper, and the amino acid sequence of its heavy chain is shown in SEQ ID NO: 139 of this paper.

[0419] D. Bioactivity / Affinity Detection

[0420] Example D-1: The inhibitory effect of the ATR inhibitor loaded with the present invention on ATR enzyme activity

[0421] The compound was serially diluted with DMSO in a 384-well plate (Labcyte, #PP-0200), and then 0.05 μL of the compound (P-1 to P-22) was transferred to a 384-well microplate (Greiner, #784075) using an Echo apparatus, ensuring a final DMSO concentration of 1%. 2.5 μL of ATR enzyme solution was added to each well of the 384-well microplate and incubated at 25°C for 10 min. Then, 2.5 μL of a mixed substrate solution of ATP and His6-P53 was added to each well and incubated at 25°C for 180 min. Add 5 μL of a mixed assay solution of Mab-anti-phospho-P53-Euclidean acid (Revvity, #61P08KAZ) and MAb Anti 6HIS-d2 (Revvity, #61HISDLB) to each well and incubate at 25°C for 180 min (final concentration: 2.5 nM ATR, 25 nM His6-P53, 1 μM ATP, 1X Mab-anti-phospho-P53-Euclidean acid and MAb Anti 6HIS-d2). Wells containing DMSO and the enzyme serve as high controls, and wells containing the same amounts of DMSO and buffer serve as low controls. Read the HTRF signal on a BMG (PHERAstar FSX) microplate reader and calculate the HTRF ratio for each well using the following formula: Ratio = 665nm / 620nm × 10 4 Inhibition rate % = (ratio) 高对照 - Ratio 化合物处理 ) / (ratio 高对照 - Ratio 低对照 ()×100). The data was analyzed using Xlfit 5.5.0 software, with the curve equation Y = Bottom + (Top-Bottom) / (1+10^((LogIC)). 50 -X) HillSlope) fitted IC 50 value.

[0422] The ATR inhibitor compounds P-1 to P-22 of this invention have a strong inhibitory effect on ATR, IC50. 50 All are less than 10 nM.

[0423] Example D-2: Inhibitory effect of the ATR inhibitor-loaded drug of the present invention on the growth of human colorectal cancer cells LoVo

[0424] Resuscitated human colorectal cancer cells (LoVo) were passaged until they reached a good growth state and approximately 90% confluence, at which point they were used for experiments. Cells were digested with trypsin, centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in fresh medium (1640 medium + 10% FBS). Cells were counted and seeded at a density of 2000 / 4000 cells per well in 96-well cell culture plates and incubated overnight at 37°C with 5% CO2. Stock solutions of the test substances (including test compounds P-1 to P-22) were serially diluted with DMSO at 1:3 and 1:10 ratios to eight concentrations. 5 μL of each concentration was added to 120 μL of medium (25-fold dilution) and vortexed. Cells cultured overnight were removed from the medium, and 195 μL of fresh medium was added to each well, followed by 5 μL of the diluted medium containing the corresponding compound (DMSO final concentration 1‰). The culture plates were then incubated at 37°C with 5% CO2 for 4 days. After removing the stock solution, add 100 μL of fresh serum-free DMEM medium containing MTT (0.5 mg / mL) to each well and continue culturing. After 4 hours, remove the stock solution, add 100 μL of DMSO to each well, shake in the dark for 10 minutes, and read the absorbance at 552 / 690 nm wavelengths using a multi-functional absorber. Cell viability (%) = (OD) / (D) 化合物 -OD 背景 ) / (OD DMSO -OD 背景 () × 100. Data were analyzed using Graph Pad Prism 6.0 software. The inhibitory activity of the compound on cell proliferation was plotted as cell viability versus the logarithm of the compound concentration. IC 50 The value was fitted with an S-shaped dose-response curve equation, which is: Y = 100 / (1 + 10^(LogC - LogIC)) 50 C is the concentration of the compound.

[0425] The ATR inhibitor compounds P-1 to P-22 of this invention have a strong inhibitory effect on the proliferation of human colorectal adenocarcinoma cells LoVo, IC50. 50 All are less than 50 nM.

[0426] Example D-3: Synergistic effect of loading drug eczema and ATR inhibitors in colorectal cancer cells The synergistic killing effect of the loading drug eicenthecan and the ATR inhibitor on colorectal cancer cells was determined by cell viability assay. The experimental method is as follows: One day prior to treatment, colorectal cancer cells were seeded at a specific density of 90 μL in 96-well plates. Ecinotecan, an ATR inhibitor, and a 1:1 mixture of eccinotecan and an ATR inhibitor were prepared in the cell culture medium at a starting concentration of 10-fold. The test compounds were serially diluted 4-fold, with 10 μL of the diluted compound added to each well. The 96-well plates were incubated at 37°C in a CO2 incubator for 3 days. Cell Counting-Lite 2.0 Luminescent Cell Viability Assay reagent (Vazyme, DD1101-03) was added to each well and treated according to the manufacturer's instructions. The luminescence signal was measured using a microplate reader (BMG, PHERAstar FSX), and the cell viability (%) corresponding to each concentration of the test compound was calculated from the measured values. The half-inhibitory concentration (IC50) of the compound was determined by fitting a dose-response curve. 50 )value.

[0427] The test results are summarized in the table below. Max Inh% represents the highest inhibition rate of the test compound against cancer cell proliferation at the highest concentration. Compound P0 is eczema, and ++++ indicates IC50. 50 <10nM, +++ means 10 nM≤IC 50 <100nM, ++ means 100nM≤IC 50 <1μM, + indicates IC 50 >1μM.

[0428] The results showed that ixotecan and ATR inhibitors had a good synergistic effect in killing cancer cells.

[0429]

[0430] Example D-4: Naked antibody and affinity detection of ADC molecules with CEACAM5

[0431] Biomembrane interference (BLI) was used to monitor the intermolecular interaction between CEACAM5 and both naked antibody and anti-CEACAM5 ADC with CEACAM5 in real time. The CEACAM5 protein information is as follows:

[0432] The detection method is as follows: 1) Sensor pre-wetting: Prepare two 96-well plates, one as the sample plate and the other as the pre-wetting plate. Add 200 μl of 1XPBST buffer to the pre-wetting plate per well. Place the pre-wetting plate in the designated area of ​​the instrument (ForteBIO, Octet RED 96) and place the sensor in the corresponding position on the pre-wetting plate. Before starting the experiment, the sensor should be pre-wetted by at least 20 molecules.

[0433] 2) Dilution and Sample Loading: Dilute the naked antibody and ADC to 20 μg / mL with 1XPBST buffer as the assay; serially dilute CEACAM5 two-fold with 1XPBST buffer as the analyte, with a dilution range of 100-3.125 nM; add 200 μL of 1XPBST buffer to each well in column 1 of a 96-well plate; add 200 μL of 1XPBST buffer to well 1 in column 2 of a 96-well plate as a blank control; add 200 μL of diluted CAECAM5 protein to wells 2-8, with concentrations of 100, 50, 25, 12.5, 6.23, 3.125, and 100 nM respectively; add 200 μL of 1XPBST buffer to each well in column 3 of a 96-well plate; add 200 μL of 10 mM regeneration buffer (10 mM Glycine-HCl, pH 10) to each well in column 11 of a 96-well plate. 1.5) Add 200 μL of 1XPBST buffer to each well in column 12 of the 96-well plate as a neutralizing agent; add 200 μL of sample to wells 1-7 in columns 5-10 of the 96-well plate, and add 200 μL of 1XPBST buffer to each well in column 8 as a blank control.

[0434] 3) Set up the program and perform the test: After adding the sample, place the sensor disk containing the pre-wetted plate and the sample plate into the instrument, and set the program sequentially for testing. Program: Binding time 120 seconds; Dissociation time 60 seconds.

[0435] The experimental results are shown in the table below. The results show that the affinity of the ADC molecule of the present invention for CEACAM5 is consistent with that of the naked antibody for CEACAM5.

[0436]

[0437] Example D-5: Killing effect of ADC molecules on TOPO I inhibitor-sensitive cancer cells

[0438] The killing effect of anti-CEACAM5 ADC molecules on cancer cell lines was determined by cell viability assay. Cancer cell information is shown in the table below:

[0439] The experimental method is as follows: One day prior to treatment, SNU-16, LS174T, and HPAF-II cancer cells were seeded in 96-well plates at 2500 cells / 90 μL per well. The test molecule was prepared in the cell culture medium at a 10-fold starting concentration (750 nM). The test compound was serially diluted 4-fold, with 10 μL of the diluted compound added to each well. The 96-well plates were incubated at 37 °C in a CO2 incubator for four days (SNU-16 and LS174T cells) or six days (HPAF-II cells). To determine cell viability, Cell Counting-Lite 2.0 Luminescent Cell Viability Assay reagent (Vazyme, DD1101-03) was added to each well and treated according to the manufacturer's instructions. The luminescence signal was measured using a microplate reader (BMG, PHERAstar FSX), and the cell viability (%) at each concentration of the test compound was calculated from the measured values. The half-inhibitory concentration (IC50) of the compound was determined by fitting a dose-response curve. 50 )value.

[0440] The results are summarized in the table below. Max Inh% represents the highest inhibition rate of ADC molecules against cancer cell proliferation at the highest concentration (750 nM for SNU-16 and LS174T, and 100 nM for HPAF-II). The results show that the dual-load ADC has a better cancer cell killing effect than the single-load ADC of eczema.

[0441]

[0442] Example D-6: Killing effect of ADC molecules on TOPO I inhibitor-insensitive cancer cells

[0443] The killing effect of anti-CEACAM5 ADC molecules on cancer cell lines was determined by cell viability assay. Cancer cell information is shown in the table below:

[0444] The experimental method is as follows: One day prior to treatment, LS1034, xT-84, and SW403 cancer cells were seeded in 96-well plates at 2500 cells / 90 μL per well. The test molecule was prepared in the cell culture medium at a 10-fold starting concentration (750 nM). The test compound was serially diluted 4-fold, with 10 μL of the diluted solution added to each well. The 96-well plates were incubated at 37°C in a CO2 incubator for six days. To determine cell viability, Cell Counting-Lite 2.0 Luminescent Cell Viability Assay reagent (Vazyme, DD1101-03) was added to each well and treated according to the manufacturer's instructions. The luminescence signal was measured using a microplate reader (BMG, PHERAstar FSX), and the cell viability (%) at each concentration of the test compound was calculated from the measured values. The half-inhibitory concentration (IC50) of the compound was determined by fitting a dose-response curve. 50 )value.

[0445] The results are summarized in the table below. Max Inh% represents the highest inhibition rate of ADC molecules against cancer cell proliferation at the highest concentration (750 nM). The results show that the dual-load ADC has a better killing effect on cancer cells than the single-load ADC of ethionecan.

[0446]

[0447] E. In vivo efficacy studies

[0448] Example E-1: Antitumor effect of ADC in a human pancreatic cancer HPAF-II cell tumor model

[0449] Human pancreatic cancer HPAF-II cells were cultured in EMEM medium supplemented with 10% fetal bovine serum and 1% antibiotic-antifungal agent, and incubated at 37 °C in a 5% CO2 incubator. The cells were passaged twice a week using trypsin digestion. When cell saturation reached 80%-90% and the desired number was achieved, the cells were harvested, counted, and seeded. 0.2 mL (3 × 10⁻⁶ cells) was used as the seeding medium. 6 HPAF-II cells (1:1 ratio of matrix gel to PBS) were subcutaneously seeded into the right posterior dorsal region of each mouse, resulting in an average tumor volume of approximately 120-150 mm². 3 Dosing will begin in groups at that time.

[0450] The tumor diameter was measured twice a week using calipers. The tumor volume was calculated using the formula: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0451] The antitumor efficacy of compounds was evaluated using TGI (%) or relative tumor proliferation rate (T / C) (%). TGI (%) reflects the tumor growth inhibition rate. Tumor regression efficacy was evaluated using Reg (%).

[0452] Calculation of TGI(%): TGI(%) = [(1 - (mean tumor volume at the end of treatment - mean tumor volume at the beginning of treatment)) / (mean tumor volume at the end of treatment in solvent control group - mean tumor volume at the beginning of treatment in solvent control group)] × 100%.

[0453] Relative tumor proliferation rate T / C (%): The calculation formula is as follows: T / C % = T RTV / C RTV × 100% (T) RTV Treatment group RTV; C RTV : Negative control group (RTV).

[0454] Calculation of Reg(%): Reg(%) = [(average tumor volume at the start of administration in a treatment group - average tumor volume at the end of administration in the treatment group) / average tumor volume at the start of administration in the treatment group) × 100%.

[0455] The relative tumor volume (RTV) is calculated based on the tumor measurement results, using the formula R. TV = V t / V0, where V0 is the average tumor volume measured at the time of grouped drug administration (i.e., d0), V t T represents the average tumor volume at a given measurement. RTV With C RTV Data from the same day will be collected. Tumor weight will be measured and T / C calculated after the experiment. 重量 Percentage, T 重理 and C 重量 The values ​​represent the tumor weight in the drug administration group and the solvent control group, respectively.

[0456] E-1-1)

[0457] The antitumor effects of eczemab single-loaded C-ADC0-1 and dual-loaded C-ADC56-1-1 were evaluated in a human pancreatic cancer HPAF-II cell tumor model. Grouping and dosing regimens are shown in the table below.

[0458]

[0459] Note: a. N is the number of animals per group, 6 tumor-bearing nude mice per group; b. The solvent is PBS; c. Adjust the administration volume according to the mouse's body weight at 10 μL / g; if the body weight decreases by more than 15%, adjust the administration regimen; div refers to intravenous injection; e. q2w means administration once every two weeks.

[0460] The results are as follows Figure 1 As shown, compared with the solvent control group, all treatment groups exhibited significant tumor growth inhibition. C-ADC56-1-1 showed a good dose-dependent relationship at doses of 1 mg / kg, 3 mg / kg, and 6 mg / kg. At doses of 3 mg / kg and 6 mg / kg, tumor regression was observed from day 14, with an 82% regression rate observed at the 6 mg / kg dose until day 49. In contrast, the tumor regression rate of eiceletane-only C-ADC0-1 was only 43% at day 49 at the 6 mg / kg dose. The dual-load C-ADC56-1-1 demonstrated better sustained efficacy than the eiceletane-only C-ADC0-1.

[0461] During the study, no significant weight loss was observed in any group, animal behavior was normal, and there were no signs of death or morbidity. C-ADC56-1-1 was well tolerated in mice at all dose levels.

[0462] E-1-2)

[0463] The antitumor effects of eczemab single-loaded C-ADC0-1 and dual-loaded C-ADC56-2-1 were evaluated in a human pancreatic cancer HPAF-II cell tumor model. Grouping and dosing regimens are shown in the table below.

[0464]

[0465] Note: a. N is the number of animals per group, 6 tumor-bearing nude mice per group; b. The solvent is PBS; c. Adjust the administration volume according to the mouse's body weight at 10 μL / g; if the body weight decreases by more than 15%, adjust the administration regimen; div refers to intravenous injection.

[0466] The results are as follows Figure 2 As shown, all treatment groups exhibited tumor regression compared to the solvent control group. A single dose of C-ADC56-2-1 at 3 mg / kg resulted in 75% tumor regression by day 25, and a further 67% regression by day 34. Ecinotecan-loaded C-ADC0-1 at 3 mg / kg showed 51% tumor regression by day 25, but tumor regeneration exceeded the initial tumor size by day 34. The dual-loaded C-ADC56-2-1 demonstrated better sustained efficacy than the eczema-loaded C-ADC0-1.

[0467] During the study, no significant weight loss was observed in any group, animal behavior was normal, and there were no signs of death or morbidity. C-ADC56-2-1 was well tolerated in mice at all dose levels.

[0468] Example E-2: Antitumor effect of ADC in a human colon cancer LS-1034 cell tumor model

[0469] Human colon cancer LS-1034 cells were cultured in vitro in adherent medium supplemented with 10% fetal bovine serum and 1% antibiotic-antifungal agent, and incubated at 37 °C in a 5% CO2 incubator. The cells were passaged twice a week using trypsin digestion. When cell saturation reached 80%-90% and the desired number was achieved, the cells were harvested, counted, and seeded. 0.2 mL (5 × 10⁻⁶ cells) was used as the seeding medium. 6 LS-1034 cells (1:1 ratio of matrix gel to PBS) were subcutaneously seeded into the right posterior dorsal region of each mouse. For pharmacodynamic experiments, the average tumor volume reached approximately 120-150 mm². 3 Dosing will begin in groups at that time.

[0470] The experimental endpoint is to examine whether tumor growth is inhibited, slowed, or cured. Tumor diameter is measured twice weekly using calipers. The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0471] The antitumor efficacy of the compounds was evaluated using TGI (%) or relative tumor proliferation rate (T / C) (%). TGI (%) reflects the tumor growth inhibition rate.

[0472] Calculation of TGI(%): TGI(%) = [(1 - (mean tumor volume at the end of treatment - mean tumor volume at the beginning of treatment)) / (mean tumor volume at the end of treatment in solvent control group - mean tumor volume at the beginning of treatment in solvent control group)] × 100%.

[0473] Relative tumor proliferation rate T / C (%): The calculation formula is as follows: T / C % = T RTV / C RTV × 100% (T) RTV Treatment group RTV; C RTV The negative control group's RTV was calculated based on tumor measurements, using the formula: RTV = V t / V0, where V0 is the average tumor volume measured at the time of grouped drug administration (i.e., d0), V t T represents the average tumor volume at a given measurement. RTV With CRTV Data from the same day will be collected. Tumor weight will be measured and T / C calculated after the experiment. 重量 Percentage, T 重量 and C 重量 The values ​​represent the tumor weight in the drug administration group and the solvent control group, respectively.

[0474] E-2-1)

[0475] The antitumor effects of eczemab single-loaded C-ADC0-1 and dual-loaded C-ADC56-2-1 in a human colon cancer LS-1034 cell tumor model were evaluated. Grouping and dosing regimens are shown in the table below.

[0476]

[0477] Note: a. N is the number of animals per group, with 6 tumor-bearing nude mice per group; b. The solvent is PBS; c. Adjust the dosage volume according to the mouse's body weight at 10 μL / g; if the body weight decreases by more than 15%, adjust the dosage regimen; d. iv e. Administer via intravenous injection; Administer on days 0, 14, 21, and 26 in each group.

[0478] The results are as follows Figure 3 As shown, C-ADC56-2-1 exhibited a favorable dose-dependent relationship at 3 mg / kg and 6 mg / kg. Compared to the solvent control group, C-ADC56-2-1 had a TGI of 63% on day 28 at a dose of 6 mg / kg, while the TGI of eiceletane-only C-ADC0-1 was 41% on day 28 at a dose of 6 mg / kg. The dual-load C-ADC56-2-1 demonstrated better efficacy than the eiceletane-only C-ADC0-1.

[0479] During the study, no significant weight loss was observed in any group, animal behavior was normal, and there were no signs of death or morbidity. C-ADC56-2-1 was well tolerated in mice at all dose levels.

[0480] E-2-2)

[0481] The antitumor effects of eczemab single-loaded C-ADC0-2 and dual-loaded C-ADC56-2-2 were evaluated in a human colon cancer LS-1034 cell tumor model. Grouping and dosing regimens are shown in the table below.

[0482]

[0483] Note: a. N is the number of animals per group, with 6 tumor-bearing nude mice per group; b. The solvent is PBS; c. Adjust the dosage volume according to the mouse's body weight at 10 μL / g; if the body weight decreases by more than 15%, adjust the dosage regimen; d. ivIntravenous injection refers to administration; e. biw refers to administration twice a week.

[0484] The results are as follows Figure 4 As shown, compared to the solvent control group, C-ADC56-2-2 had a TGI of 91% on day 28 at a dose of 6 mg / kg, while the TGI of eiceletane-only C-ADC0-2 was 87% on day 28 at a dose of 6 mg / kg. The dual-load C-ADC56-2-2 demonstrated better efficacy than the eiceletane-only C-ADC0-2.

[0485] During the study, no significant weight loss was observed in any group, animal behavior was normal, and there were no signs of death or morbidity. C-ADC56-2-2 was well tolerated in mice at all dose levels.

[0486] Example E-3: Antitumor effect of ADC in a human colorectal cancer xT-84 cell tumor model

[0487] Human colon cancer xT84 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% Antibiotic-Antimycotic in a 37 °C, 5% CO2 incubator. Cells were passaged twice a week using trypsin digestion. When cell saturation reached 80%-90% and the desired number was achieved, cells were harvested, counted, and seeded. 0.2 mL (10 × 10⁻⁶) of the medium was added to each cell. 6 xT-84 cells (1:1 ratio of matrix gel to PBS) were subcutaneously seeded into the right posterior dorsal region of each mouse. For pharmacodynamic experiments, the average tumor volume reached approximately 120-180 mm². 3 Dosing will begin in groups at that time.

[0488] The experimental endpoint is to examine whether tumor growth is inhibited, slowed, or cured. Tumor diameter is measured twice weekly using calipers. The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0489] The antitumor efficacy of compounds was evaluated using TGI (%) or relative tumor proliferation rate (T / C) (%). TGI (%) reflects the tumor growth inhibition rate. Tumor regression efficacy was evaluated using Reg (%).

[0490] Calculation of TGI(%): TGI(%) = [(1 - (mean tumor volume at the end of treatment - mean tumor volume at the beginning of treatment)) / (mean tumor volume at the end of treatment in solvent control group - mean tumor volume at the beginning of treatment in solvent control group)] × 100%.

[0491] Relative tumor proliferation rate T / C (%): The calculation formula is as follows: T / C % = T RTV / C RTV × 100% (T) RTV Treatment group RTV; C RTV : Negative control group (RTV).

[0492] Calculation of Reg(%): Reg(%) = [(average tumor volume at the start of administration in a treatment group - average tumor volume at the end of administration in the treatment group) / average tumor volume at the start of administration in the treatment group) × 100%.

[0493] The relative tumor volume (RTV) is calculated based on the tumor measurement results, using the formula R. TV = V t / V0, where V0 is the average tumor volume measured at the time of grouped drug administration (i.e., d0), V t T represents the average tumor volume at a given measurement. RTV With C RTV Data from the same day will be collected. Tumor weight will be measured and T / C calculated after the experiment. 重量 Percentage, T 重理 and C 重量 The values ​​represent the tumor weight in the drug administration group and the solvent control group, respectively.

[0494] The antitumor effects of dual-load C-ADC56-2-2 and C-ADC59-2-2 in a human colon cancer xT84 cell tumor model were evaluated. Grouping and administration regimens are shown in the table below.

[0495]

[0496] Note: a. N is the number of animals per group, with 6 tumor-bearing nude mice per group; b. The solvent is PBS; c. Adjust the dosage volume according to the mouse's body weight at 10 μL / g; if the body weight decreases by more than 15%, adjust the dosage regimen; d. iv Intravenous injection.

[0497] The results are as follows Figure 5 As shown, all treatment groups exhibited significant tumor growth inhibition. At a dose of 6 mg / kg, C-ADC56-2-2 achieved 69% tumor regression on day 25. C-ADC59-2-2 demonstrated good efficacy at doses of 3 mg / kg and 6 mg / kg, achieving tumor regression rates of 70% and 76%, respectively, on day 25.

[0498] During the study, no significant weight loss was observed in any group, animal behavior was normal, and there were no signs of death or morbidity. C-ADC56-2-2 and C-ADC59-2-2 were well tolerated in mice at all dose levels.

[0499] Example E-4: Antitumor effect of ADC in human breast cancer JIMT-1 cell tumor model

[0500] Human breast cancer JIMT-1 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% Antibiotic-Antimycotic, and then placed in a 37 °C, 5% CO2 incubator for in vitro adherence. Cells were passaged twice a week using trypsin digestion. When cell saturation reached 80%-90% and the desired number was achieved, cells were harvested, counted, and seeded. 0.2 mL (5 × 10⁻⁶ cells) of the medium was added to each cell. 6 JIMT-1 cells (1:1 matrix gel and PBS ratio) were subcutaneously seeded into the right posterior dorsal region of each mouse. For pharmacodynamic experiments, the average tumor volume reached approximately 120-150 mm². 3 Dosing will begin in groups at that time.

[0501] The experimental endpoint is to examine whether tumor growth is inhibited, slowed, or cured. Tumor diameter is measured twice weekly using calipers. The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0502] The antitumor efficacy of compounds was evaluated using TGI (%) or relative tumor proliferation rate (T / C) (%). TGI (%) reflects the tumor growth inhibition rate. Tumor regression efficacy was evaluated using Reg (%).

[0503] Calculation of TGI(%): TGI(%) = [(1 - (mean tumor volume at the end of treatment - mean tumor volume at the beginning of treatment)) / (mean tumor volume at the end of treatment in solvent control group - mean tumor volume at the beginning of treatment in solvent control group)] × 100%.

[0504] Relative tumor proliferation rate T / C (%): The calculation formula is as follows: T / C % = T RTV / C RTV × 100% (T) RTV Treatment group RTV; C RTV : Negative control group (RTV).

[0505] Calculation of Reg(%): Reg(%) = [(average tumor volume at the start of administration in a treatment group - average tumor volume at the end of administration in the treatment group) / average tumor volume at the start of administration in the treatment group) × 100%.

[0506] The relative tumor volume (RTV) is calculated based on the tumor measurement results, using the formula R. TV = V t / V0, where V0 is the average tumor volume measured at the time of grouped drug administration (i.e., d0), V t T represents the average tumor volume at a given measurement. RTV With C RTV Data from the same day will be collected. Tumor weight will be measured and T / C calculated after the experiment. 重量 Percentage, T 重理 and C 重量 The values ​​represent the tumor weight in the drug administration group and the solvent control group, respectively.

[0507] The antitumor effect of dual-load H-ADC-L04-1 in a human breast cancer JIMT-1 cell tumor model was evaluated. Grouping and administration regimens are shown in the table below.

[0508]

[0509] Note: a. N is the number of animals per group, with 6 tumor-bearing nude mice per group; b. The solvent is PBS; c. Adjust the dosage volume according to the mouse's body weight at 10 μL / g; if the body weight decreases by more than 15%, adjust the dosage regimen; d. iv e. Administer via intravenous injection; Administer on days 0, 4, and 7 in each group.

[0510] The results are as follows Figure 6 As shown, compared with the solvent control group, all treatment groups exhibited significant tumor growth inhibition. H-ADC-L04-1 showed a good dose-dependent relationship at 3 mg / kg and 10 mg / kg. In the low-dose group (3 mg / kg), tumor regression was observed from day 14 to day 42, with a TGI of 76%. In the high-dose group (10 mg / kg), tumor regression was observed from day 11 to day 42, with 7% of the tumors still showing regression.

[0511] During the study, no significant weight loss was observed in any group, animal behavior was normal, and there were no signs of death or morbidity. H-ADC-L04-1 was well tolerated in mice at all dose levels.

[0512] While the invention has been fully described, those skilled in the art will understand that the same practices can be carried out under broad and equivalent conditions, formulations, and other parameters without affecting the scope of the invention or any embodiments thereof. All patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety.

Claims

1. A linker-drug, denoted as VLD, wherein, VL is a linker, and D is a pharmaceutical unit, characterized in that the pharmaceutical unit D comprises at least a pharmaceutical unit derived from a compound of formula i or a deuterated derivative thereof, wherein the compound of formula i is an ATR inhibitor: （i） in: R0 is an optional substituted 5-membered heteroaryl group; R1 is H, halogen, or C with optional substitution. 1-6 Alkyl, optionally substituted C 3-6 cycloalkyl, optionally substituted C 2-6 alkenyl or optionally substituted C 2-6 alkynyl group; R2 is an optionally substituted carbocyclic group, an optionally substituted heterocyclic group, an optionally substituted aryl group, or an optionally substituted heteroaryl group; In this configuration, any H atom can be optionally replaced by deuterium.

2. The linker-drug as described in claim 1, characterized in that, The linker-drug is a compound represented by formula I, Ia, Ib, Ic, Id, or Ie, its stereoisomers, tautomers, N-oxides, hydrates, solvates, isotopically labeled compounds, or pharmaceutically acceptable salts, or mixtures thereof: （I） (It) (One) (Ic) (Id) (Ie) In the formula: Q1 is a functional group that couples with antibody molecules, especially functional groups that can couple with cysteine, lysine, non-natural amino acids or glycosyl groups of antibody molecules. A1 is selected from: bonded, optionally substituted alkylene, optionally substituted alkylene acyl, optionally substituted alkylene-polyethylene glycol, optionally substituted polyethylene glycol, optionally substituted alkylene-polyethylene glycol-alkylene, optionally substituted alkylene-C(O)NH-alkylene, optionally substituted alkylene-NHC(O)-alkylene, optionally substituted alkylene-C(O)NH-alkylene-acyl, optionally substituted alkylene-cycloalkylene acyl, optionally substituted polyethylene glycol-alkylene-acyl, optionally substituted alkenyl, optionally substituted alkyneyl, optionally substituted alkyneyl- Alkylene, optionally substituted carbocyclic, optionally substituted heterocyclic, optionally substituted aryl, optionally substituted heterocyclic, optionally substituted heterocyclic-alkylene-acyl, optionally substituted aryl-alkylene-acyl, and optionally substituted heterocyclic-alkylene-acyl; preferably, when A1 is substituted, the number of substituents may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, and the substituents are preferably selected from hydrophilic groups, halogens, hydroxyl groups, amino groups, carboxyl groups, sulfonic acid groups, sulfone groups, phosphate groups, alkoxy groups and alkylene-NH-alkylene-carboxyl groups; B1, B2, B3, B4, B5, B6, B7, and B8 are each independently a bond, an optional substituted amino acid residue, an optional substituted polypeptide residue, or a branched group, and when A1 is a bond, B4 is not a bond; L1, L2, and L3 are each independently a bond, an optional substituted amino acid residue, or an optional substituted polypeptide residue. T1, T2, and T3 are each independently bonded or optionally substituted spacer groups, wherein at most two of T1, T2, and T3 are bonds; At least one of D1, D2 and D3 is a first pharmaceutical unit, and the remaining one or two are each independently selected from a second pharmaceutical unit and a third pharmaceutical unit, wherein the first pharmaceutical unit is a pharmaceutical unit derived from an ATR inhibitor, preferably a pharmaceutical unit derived from the compound shown in Formula i or its deuterated derivative. E is a capping group; t, m, and n are each independently 0, 1, 2, or 3; Preferably, in Formula I: Q1 is a functional group capable of coupling with cysteine, lysine, non-natural amino acids, or glycosyl groups of the antibody molecule; A1 is selected from bonds, optionally substituted alkylene groups, optionally substituted alkylene acyl groups, optionally substituted alkylene-polyethylene glycol groups, optionally substituted polyethylene glycol groups, optionally substituted alkylene-polyethylene glycol-alkylene groups, optionally substituted alkylene-C(O)NH-alkylene groups, optionally substituted alkylene-NHC(O)-alkylene groups, optionally substituted alkylene-C(O)NH-alkylene-acyl groups, optionally substituted alkylene-cycloalkylene-acyl groups, optionally substituted alkenyl groups, optionally substituted alkyne groups, optionally substituted alkyne-alkylene groups, optionally substituted carbocyclic groups, optionally substituted heterocyclic groups, optionally substituted aryl groups, and optionally substituted heteroaryl groups. When A1 is substituted, the substituent is selected from hydrophilic groups. Groups, halogens, hydroxyl groups, amino groups, carboxyl groups, sulfonic acid groups, sulfone groups, phosphate groups, alkoxy groups, and alkylene-NH-alkylene-carboxyl groups; L1, B4, B6, B7, and B8 are all bonds; B2 and B3 are each independently bonds or branched groups, and when A1 is a bond, B2 is not a bond; B5, L2, and L3 are each independently bonds or optionally substituted polypeptide residues; T1, T2, and T3 are each independently bonds or optionally substituted spacer groups, wherein at most two of T1, T2, and T3 are bonds; D1 is a first pharmaceutical unit, which is a pharmaceutical unit derived from an ATR inhibitor; D2 and D3 are the second and third pharmaceutical units, respectively, and D1, D2, and D3 may be the same or different; t is 0; m and n are each independently 0, 1, 2, or 3, preferably, m and n are both 1; preferably, at least one of B2, T1, and T2 is substituted with a hydrophilic group; or Preferably, in Formula I: Q1 is a functional group capable of coupling with cysteine, lysine, non-natural amino acids, or glycosyl groups of the antibody molecule; A1 is selected from the following groups: alkylene group, optionally substituted alkylene group, optionally substituted alkylene acyl group, optionally substituted alkylene-polyethylene glycol group, optionally substituted polyethylene glycol group, optionally substituted alkylene-polyethylene glycol-alkylene group, optionally substituted alkylene-C(O)NH-alkylene group, optionally substituted alkylene-NHC(O)-alkylene group, optionally substituted alkylene-C(O)NH-alkylene-acyl group, optionally substituted alkylene-cycloalkylacyl group, optionally substituted alkenyl group, optionally substituted alkyneyl group, optionally substituted alkyneyl-alkylene group, optionally substituted carbocyclic group, optionally substituted heterocyclic group. Optionally substituted arylene and optionally substituted heteroarylene, wherein when A1 is substituted, the substituent is selected from hydrophilic groups, halogens, hydroxyl groups, amino groups, carboxyl groups, sulfonic acid groups, sulfone groups, phosphate groups, alkoxy groups, and alkylene-NH-alkylene-carboxyl groups; L1, B4, and B7 are bonds; B2 and B6 are each independently bonds or branched groups, and when A1 is a bond, B2 is not a bond; B5 and L2 are each independently bonds or optionally substituted polypeptide residues; T1 and T2 are each independently bonds or optionally substituted spacer groups, wherein at most two of T1 and T2 are bonds; D1 is a first pharmaceutical unit, which is a pharmaceutical unit derived from an ATR inhibitor; D2 is a second pharmaceutical unit; n and t are both 0; m is 1, 2, or 3, preferably m is 1; or Preferably, in Formula I: Q1 is a functional group capable of coupling with cysteine, lysine, non-natural amino acids, or glycosyl groups of the antibody molecule; A1 is selected from the following groups: alkylene group, optionally substituted alkylene group, optionally substituted alkylene acyl group, optionally substituted alkylene-polyethylene glycol group, optionally substituted polyethylene glycol group, optionally substituted alkylene-polyethylene glycol-alkylene group, optionally substituted alkylene-C(O)NH-alkylene group, optionally substituted alkylene-NHC(O)-alkylene group, optionally substituted alkylene-C(O)NH-alkylene-acyl group, optionally substituted alkylene-cycloalkyl acyl group, optionally substituted alkenyl group, optionally substituted alkyneyl group, optionally substituted alkyneyl-alkylene group, optionally substituted carbocyclic group, optionally substituted heterocyclic group, optionally substituted aryl group, and optionally substituted... A heteroaryl group, wherein when A1 is substituted, the substituent is selected from hydrophilic groups, halogens, hydroxyl groups, amino groups, carboxyl groups, sulfonic acid groups, sulfone groups, phosphate groups, alkoxy groups, and alkylene-NH-alkylene-carboxyl groups; L1, B6, and B8 are all bonds; B3 and B4 are each independently bonds or branched groups, and when A1 is a bond, B4 is not a bond; B5 and L3 are each independently bonds or optionally substituted polypeptide residues; T1 and T3 are each independently bonds or optionally substituted spacer groups, wherein at most two of T1 and T3 are bonds; D1 is a first pharmaceutical unit, which is a pharmaceutical unit derived from an ATR inhibitor; D3 is a second pharmaceutical unit; m and t are 0; n is 1, 2, or 3, preferably n is 1; preferably, at least one of B3, T1, and T3 is substituted with a hydrophilic group; or Preferably, in Formula I: Q1 is a functional group capable of coupling with cysteine, lysine, non-natural amino acids, or glycosyl groups of the antibody molecule; A1 is selected from: alkylene groups, optionally substituted alkylene groups, optionally substituted alkylene acyl groups, optionally substituted alkylene-polyethylene glycol groups, optionally substituted polyethylene glycol groups, optionally substituted alkylene-polyethylene glycol-alkylene groups, optionally substituted alkylene-C(O)NH-alkylene groups, optionally substituted alkylene-NHC(O)-alkylene groups, optionally substituted alkylene-C(O)NH-alkylene-acyl groups, optionally substituted alkylene-cycloalkylene acyl groups, optionally substituted alkenyl groups, optionally substituted alkyne groups, optionally substituted alkylene groups, etc. The substituents are alkylene-alkylene, optionally substituted carbocyclic, optionally substituted heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl, wherein when A1 is substituted, the substituent is selected from hydrophilic groups, halogens, hydroxyl groups, amino groups, carboxyl groups, sulfonic acid groups, sulfone groups, phosphate groups, alkoxy groups, and alkylene-NH-alkylene-carboxyl groups; L1 is a bond; B4 and B6 are each independently a bond or a branched group, and when A1 is a bond, B4 is not a bond; B5 is a bond or an optionally substituted polypeptide residue; T1 is a bond or an optionally substituted spacer group; D1 is the first pharmaceutical unit, which is a pharmaceutical unit derived from an ATR inhibitor; m, n, and t are all 0.

3. The linker-drug as described in claim 2, characterized in that, The compound represented by formula i or its deuterated derivative is the compound represented by formula ii or its deuterated derivative: (ii) Wherein, R1 and R2 are as described in claim 1; R3 is hydrogen, halogen, or optionally substituted C. 1-6 Alkyl group; any H atom may be optionally substituted with deuterium; Preferably, the compound represented by formula i or its deuterated derivative is one of the following compounds or its deuterated derivative: 。 4. The linker-drug as described in claim 2 or 3, characterized in that: The first pharmaceutical unit is a pharmaceutical unit derived from the ATR inhibitor described in claim 1 or 3. The second and third pharmaceutical units are each independently derived from the ATR inhibitor described in claim 1 or 3, the DNA damage response (DDR) inhibitor, the microtubule-targeting drug (such as vinca alkaloids, vincristine, vinblastine, paclitaxel, maytansine, auristatin, tubulolysin, sea haretoxin, nostoc cyclic peptide, spindle kinesin inhibitor or eribulin), the DNA damaging agent (such as enediyne, topoisomerase inhibitor, pyrrolo[2,1-c][1,4]benzodiazepine PBDs, ducamycin or trabectedin), the RNA damaging agent (such as tyrannostatin or amatoxins), or the immune agonist (such as Toll-type receptor agonists or STING agonists). Preferably, the second and third pharmaceutical units are each independently a pharmaceutical unit derived from the ATR inhibitor or topoisomerase inhibitor as described in claim 1 or 3. Preferably, one of the second and third drug units is an ATR inhibitor-derived drug unit, and the other is a topoisomerase inhibitor-derived drug unit; or both the second and third drug units are ATR inhibitor-derived drug units; or both the second and third drug units are topoisomerase inhibitor-derived drug units. Preferably, the topoisomerase I inhibitor is selected from: More preferably, the topoisomerase I inhibitor is: 。 5. The linker-drug as described in any one of claims 2 to 4, characterized in that, The combination of D1, D2, and D3 is selected from the combination of drug units derived from the compounds in the table below: 。 6. The linker-drug as described in any one of claims 2 to 5, characterized in that, Q1 is a group with the following characteristics: , , , , , , , , , , or , in, 1 indicates the connection position between Q1 and A1; X is Cl, Br, or I; Y is independently Br or phenyl-S-, which is optionally substituted by 1-3 substituents selected from halogens, nitro groups, alkyl groups, alkoxy groups, ester groups, and -C(O)NR'R''-, wherein R' and R'' are independently selected from H and alkyl groups, or R' and R'' together with the commonly connected N atom form a 5-7 membered heterocycle; Z is -(CH2CH2O). z -CH2CH2-OH, where z is any integer from 0 to 100; Q1 may be optionally substituted, preferably with a halogen or alkyl substituent, and preferably without substitution; Preferably, Q1 is: , or ; More preferably, Q1 is: 。 7. The linker-drug according to any one of claims 2 to 6, characterized in that, A l For bond, -(CH2) a1 -、-(CH2) a1 -C(O)-, -(CH2) a1 -C(O)NH-(O)-、-(CH2) a1 -C(O)NH-(CO)-、-(CH2) a1 -(O-CH2-CH2) a2 -、-(CH2) a1 -(O-CH2-CH2) a2 -(CH2) a3 -、-(CH2) a1 -C(O)NH-(CH2) a2 -、-(CH2) a1 -C(O)NH-(CH2) a2 -NH-, -(CH2) a1 -NH-, -(CH2) a1 -NHC(O)-, -(CH2) a1 -NHC(O)-(CH2) a2 -、-(CH2) a1 -C(O)NH-(CH2) a2 -C(O)-, -(CH2) a1 -(cycloalkylene)-C(O)-, -(CH2CH2O) a1 -(CH2) a2 -C(O)-, -C≡C-(CH2) a1 -、-C≡C-(CH2) a1 -CO-, -(heterocyclic)-(CH2) a1 -C(O)-、-(aryl)-(CH2) a1 -C(O)- and -(heteroaryl)-(CH2) a1 -C(O)-, where a1, a2 and a3 are each independent integers from 1 to 6, and the substituents shown are connected to Q1 on the left and to B4 or B6 on the right.

8. The linker-drug according to any one of claims 2 to 7, characterized in that: B4, B5, B6, B7, and B8 are each independently bonded or independently selected from the following groups: -NH-(CH2) b1 -C(O)- , -NH-(CH2) b1 - , -(CH2CH2O) b2 - , -(CH2CH2O) b2 - , -(CH2CH2O) b2 -(CH2) b1 -C(O)- , -(CH2CH2O) b2 -(CH2) b1 -C(O)-NH-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -NH-(CH2) b1 -(CH2OCH2) b2 -(CH2) b1 -C(O)- , -C(O)-(CH2CH2O) b2 -(CH2) b1 -NH- , -C(O)-(CH2CH2O) b2 -(CH2) b1 -NH- , -C(O)-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -NH-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -(OCH2CH2) b2 -C(O)- , -C(O)-(CH2CH2O) b2 -(CH2) b1 -C(O)- , -NH-CO-NH-(CH2) b1 -CO- , -NH-(CH2) b1 -triazolyl-(CH2) b1 -O-(CH2) b1 -(OCH2CH2) b2 -NH-CO-(CH2) b1 -O-(CH2) b1 - , -NH-CH[(CH2) b1 -C(O)-NH-(CH2CH2O) b2 -(CH2) b1 CH3]-C(O)- , -NH-(CH2) b1 -triazolyl-(CH2) b1 -O-(CH2) b1 -(OCH2CH2) b2 -NH-CO-(CH2) b1 -O-(CH2) b1 -CO- , -NH-(CH2) b1 -triazolyl-(CH2) b1 -(OCH2CH2) b2 -NH-CO-(CH2) b1 -O-(CH2) b1 -CO- , -triazolyl-(CH2) b1 -CO- , , , , and In the formula, This indicates the position where B4 connects to A1, the position where B5 connects to B2 or A2, the position where B6 connects to B3 or A1, the position where B7 connects to B2, or the position where B8 connects to B3. This indicates the position where B4 connects to B2, the position where B5 connects to B3 or L1 or B1 or L2, the position where B6 connects to B1 or L1, the position where B7 connects to L2, or the position where B8 connects to L3; b1 and b2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; m1, m2 and m3 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; X is selected from H, halogen, C. 1-6 Alkyl, C 1-6 Alkyl groups and -(CH2CH2O) x1 -CH3, x1 is an integer selected from 1-15; and / or, B2, B3, and B1 are each independently bonded or each independently selected from the following groups: , , , , , , , , , , , and , In the formula, 1 indicates the position where B1 connects to B6 or B5, the position where B3 connects to B5, or the position where B2 connects to B4; 2 indicates the position where B1 connects to E, the position where B3 connects to B6, or the position where B2 connects to B5; 3 indicates the position where B1 connects to L1, the position where B3 connects to L3 or B8, or the position where B2 connects to L2 or B7; L4 is selected from -(CH2). m4 -、-(CH2) m4 -C(O)NH-(CH2) m5 -、-(CH2) m4 -NHC(O)-(CH2) m5 -; L5 is -(CH2) m4 -or-(CH2-CH2-O) m6 -; m4, m5, m6 and u are each an integer from 1 to 10 independently.

9. The linker-drug as described in any one of claims 2 to 8, characterized in that, B5, L1, L2, and L3 are each independently an optionally substituted amino acid residue or an optionally substituted polypeptide residue, wherein the amino acid residue is selected from the group consisting of phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, and glycine; and the amino acid residue in the polypeptide residue is selected from the group consisting of phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, and glycine. Preferably, the amino acid residues in the polypeptide residues are selected from the group consisting of glycine, phenylalanine, valine, alanine, glutamic acid, arginine, citrulline, aspartic acid, asparagine, and lysine. More preferably, the polypeptide residues are selected from the group consisting of: phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala), valine-citrulline (Val-Cit), glutamic acid-valine-alanine (Glu-Val-Ala), glutamic acid-valine-citrulline (Glu-Val-Cit), glutamic acid-glycine-citrulline (Glu-Gly-Cit), valine-lysine (Val-Lys), alanine-alanine (Ala-Ala), alanine-alanine-alanine (Ala-Ala-Ala), alanine-alanine-asparagine (Ala-Ala-Asn), alanine-leucine (Ala-Leu), leucine-leucine (Leu-Leu), phenylalanine-arginine (Phe-Arg), phenylalanine-lysine (Phe-Lys), and glycine-phenylalanine-glycine (Gly-Phe-Gly). (cBu-Cit), 、 、 Glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) and glycine-proline (Gly-Pro); Preferably, B5, L l L2 and L3 can each be independently a bond or a group with the following characteristics: , , , , or , In the formula, 3 indicates the position where B5 connects to B2 or B4, the position where L1 connects to B1, B5, or B6, the position where L2 connects to B2 or B7, or the position where L3 connects to B8 or B3. 4 indicates the position where B5 connects to B6, B3, L1, or B1; the position where L1 connects to T1; the position where L2 connects to T2; or the position where L3 connects to T3.

10. The linker-drug according to any one of claims 2 to 9, characterized in that, T l T2 and T3 are each independently selected from the following groups: 4 -NH-(CH2) L6 - 5. , , , , , , , , , , , , , and , In the formula, 4 indicates the position where T1 connects to L1, the position where T2 connects to L2, and the position where T3 connects to L3. 5 represents the position where T1 connects to D1, the position where T2 connects to D2, and the position where T3 connects to D3; where R t H, optionally substituted alkyl groups, or -(CH2CH2O) t1 -CH3, t1 is an integer from 0 to 15, L6 is an integer from 0 to 15; the T l T2 and T3 may optionally be replaced by hydrophilic groups selected from -O(CH2CH2O). L6 -(CH2) L7 CH3 or -(CH2) L6 -N(R N1 )-[C(O)-(CH2) L7 -N(R N2 )] L8 -C(O)-R N3 Among them, L6, L7, or L8 are each independently represented as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, R N1 R N2 and R N3 Each is independent as C 1-6 Alkyl groups; and / or E is selected from: H, -(CH2) a1 CH3, -(CH2) a1 -C(O)-(CH2) a1 CH3, -(CH2) a1 -(O-CH2-CH2) a2 -(CH2) a1 CH3, CH3(CH2) a1 -(O-CH2-CH2) a2 -(CH2) a3 -、-(CH2) a1 -C(O)NH-(CH2) a2 CH3, -(CH2) a1 -NH-(CH2) a1 CH3, -(CH2) a1 -NHC(O)-(CH2) a1 CH3, -(CH2) a1 -C(O)NH-(CH2) a2 -C(O)-(CH2) a1 CH3, -(CH2) a1 -(cycloalkylene)-C(O)-(CH2) a1 CH3、-C≡C-(CH2) a1 CH3、-C≡C-(CH2) a1 -CO-(CH2) a1 CH3, -NH-(CH2) b1 -C(O)-(CH2) a1 CH3, -NH-(CH2) b1 -C(O)-NH2、-NH-(CH2CH2O) b2 -(CH2) b1 -C(O)-(CH2) a1 CH3、-NH-(CH2CH2O) b2 -(CH2) b1 -C(O)-NH2、-NH-(CH2CH2O) b2 -CH3, -(OCH2CH2) b2 -CO-NH2、-NH-(CH2) b1 -(CH2OCH2) b2 -(CH2) b1 -C(O)-NH and -NH-CO-NH-(CH2) b1 -CO-NH2, where a1, a2, a3, b1 and b2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

11. The linker-drug according to any one of claims 1 to 10, characterized in that, The linker-drug is a compound numbered LP01 to LP78.

12. An antibody-drug conjugate having the following structure: Ab-(V-L-D) p in, Ab is an antibody; VL is a linker, V may or may not be present, L is a cleavable linker or a non-cleavable linker, and at least one of V and L is present; D is a pharmaceutical unit or combination thereof as described in claim 1 or 3, and includes at least a pharmaceutical unit derived from the compound of formula (i) or its deuterated derivative; p is an integer or decimal of 1-8; Preferably, the VLD is a linker-drug according to any one of claims 1 to 11, wherein V is a functional group derived from Q1 according to any one of claims 2 to 11 that is coupled to a cysteine, lysine, non-natural amino acid, or glycosyl group of the antibody molecule.

13. The antibody-drug conjugate of claim 12, having a structure represented by formula II, IIa, IIb, IIc, IId, IIe, or III: (II) (IIa) (IIb) (IIc) (IId) (IIe) (III) in, n, m, t, A1, A2, B1, B2, B3, B4, B5, B6, B7, B8, L1, L2, L3, T1, T2 and T3 as described in any one of claims 2 to 9; D1, D2 and D3 are pharmaceutical units derived from the compound of claim 1 or 3 or its deuterated derivative, and at least one of D1, D2 and D3 is a pharmaceutical unit derived from the compound of formula (i) or its deuterated derivative. Ab is an antibody molecule; Q'1 and Q'2 are each independently functional groups derived from Q1 that are coupled to cysteine, lysine, non-natural amino acids, or glycosyl groups of the antibody molecule. p and q are each independent integers or decimals from 1 to 8; A2 is selected from: bonded, optionally substituted alkylene, optionally substituted alkylene acyl, optionally substituted alkylene-polyethylene glycol, optionally substituted polyethylene glycol, optionally substituted alkylene-polyethylene glycol-alkylene, optionally substituted alkylene-C(O)NH-alkylene, optionally substituted alkylene-NHC(O)-alkylene, optionally substituted alkylene-C(O)NH-alkylene-acyl, optionally substituted alkylene-cycloalkylene acyl, optionally substituted polyethylene glycol-alkylene-acyl, optionally substituted alkenyl, optionally substituted alkyneyl, optionally substituted alkyneyl- Alkylene, optionally substituted carbocyclic, optionally substituted heterocyclic, optionally substituted aryl, optionally substituted heterocyclic, optionally substituted heterocyclic-alkylene-acyl, optionally substituted aryl-alkylene-acyl, and optionally substituted heterocyclic-alkylene-acyl; preferably, when A2 is substituted, the number of substituents is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, said substituents being selected from hydrophilic groups, halogens, hydroxyl groups, amino groups, carboxyl groups, sulfonic acid groups, sulfone groups, phosphate groups, alkoxy groups and alkylene-NH-alkylene-carboxyl groups.

14. The antibody-drug conjugate as described in claim 11, characterized in that, The antibody Ab is an antibody (such as a monoclonal antibody) or its antigen-binding fragment that specifically binds to an antigen. The antigens include, but are not limited to: HER2, HER3, TROP2, B7-H3, B7-H4, GPC20, GPRC5D, CDH6, EGFR, EGFRvIIL, AXL, Nectin-4, Tissue Factor, TIM-1, PSMA, PTK7, EpCAM, MUCI, STEAP1, GPNMB, FGF2, FOLRI, FolRα, C-MET, GFR, AGS-I6, and Guanylylcyclase. C, Mesothelin, SLC44A4, EPhA2, AGS-5, GPC-3, c-KIT, RORl, ROR2, PD1, PD-Ll, CD27L, 5T4, MerTK, Mucin16, NaPi2b, STEAP, SLlTRK6, ETBR, BCMA, CEACAM5, ICAM1, SC-16, SLC39A6, Delta-like protein3, Claudin 18.2, Claudin 3, Claudin 6, Claudin 9. CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD73, CD74, CD79b, CDl23, CDI38, CDI47, CDl66, CD223, MUCl6, MSLN, ENPP3, SLTRK6, FGFR, LIV-1, Lewis Y, av-integrin, ITGB6 (integrin beta-6), ASCT2, C4.4a, CA-IX, CD324, CD352, CD44v6, CD48a, CLL-1, Cripto, CS1, DPEP3, Ephrin-A2. Ephrin-A4, ETBR, FGFR2, FGFR3, FLT3, GD3, Globo H, GPC3, LAMP-1, LRRC15, Ly6E, TM4SF1, MFI2, NOTCH3, p-cadherin, PRLR, CLDNl, CD228, CUB domain-containing protein 1(CDCPl), CXCR4, LGR4, FZD7, CTR, GPR56, CCR7, DDRl, RNF43, CEACAM1. CEACAM6, GCC, GPA33, TEM8, AMHR2, NTSE, FLT1, DKK1, sema4D, Her2 / neu HER1, VWF, IGF-1, GRP78, MART-1 / Melan-A, MAGE-A1, MAGE-A2, MAGE-A 3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A1 MAGE-A11, MAGE-A12, MAGE-Xp2, MAGE-Xp3, MAGE-Xp4, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE- 5、GAGE-6、GAGE-7、GAGE-8、GAGE-9、BAGE、RAGE、LAGE-1、NAG、GnT-V、MU M-1, CDK4, SSX-1, SSX-2, SSX-4, SSX-5, SCP-1, CT-7, c-erbB-2, HPV-16 E6, HPV-16 E7, HPV-1 E6, HPV-18E7, EBNA-1, βhCG, WT1, TRP-2, NY-BR-1, NY-CO-58, MN, Lea, Leb, LeX, H-2, B-1, B-2, CD25. CD40, CD44, CD45R, CD49, CD93, CD163, CD205, CD206, CD303, CD304, BMPR1B, E16, 0772P, Napi3b, Sema 5b、PSCA hlg, MSG783, STEAP2, TrpM4, CD21, FcRH2, NCA, MDP, IL20Rα, EphB2R, ASLG 659. PSCA, GEDA, BAFF-R, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2 TENB2, TMEFF1, GDNF-Ra1, Ly6E, TMEM46, Ly6G6D, LGR5, RET, LY6K, GPR19 GPR54, ASPHD1, TMEM118, GPR172A, CD151, free radical β, IL4 complex, and IL2 complex.

15. The antibody-drug conjugate as described in claim 12, characterized in that, The antibody-drug conjugates are selected from the antibody-drug conjugates numbered ADC01~ADC78 and ADC-L01~ADC-L04.

16. A pharmaceutical composition comprising a linker-drug according to any one of claims 1 to 11 or an antibody-drug conjugate according to any one of claims 12 to 15 and a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition further contains at least one known anticancer drug or a pharmaceutically acceptable salt of an anticancer drug; Preferably, the known anticancer drugs or pharmaceutically acceptable salts of anticancer drugs are selected from: (1) Anticancer drugs related to DNA damage and repair mechanisms, including PARP inhibitors such as olaparib, niraparib, rucaparib, talazoparib, senaparib and saruparib; HDAC inhibitors such as vorinostat, romidesin, pabistat and belistat; (2) Anticancer drugs related to cell division checkpoints, including Chk1 / 2 inhibitors, CDK4 / 6 inhibitors such as palbociclib, and ATM / ATR inhibitors; (3) Other anticancer drugs that can be used in combination anticancer therapy, including but not limited to: (i) Alkylating agents, such as busulfan, melphalan, chlorambucil, cyclophosphamide, ifosfamide, temozolomide, bendamustine, cisplatin, mitomycin C, bleomycin and carboplatin; (ii) Topoisomerase I inhibitors, such as camptothecin, irinotecan and topotecan; (iii) Topoisomerase II inhibitors, such as doxorubicin, epirubicin, aclarubicin, mitoxantrone, methyl hydroxyrosine, and methyltopopeptide; (iv) RNA / DNA antimetabolites, such as 5-azacytidine, gemcitabine, 5-fluorouracil, and methotrexate; (v) DNA antimetabolites, such as 5-fluoro-2′-deoxyuridine, fludarabine, nerabine, cytarabine, pralatrexate, pemetrexed, hydroxyurea and thioguanine; (vi) Antimitotic agents, such as colchicine, vincristine, vinorelbine, paclitaxel, ixaspirin, cabazitaxel and docetaxel; (vii) Antibodies, such as monoclonal antibodies, panitumumab, nezotuzumab, nivolumab, pembrolizumab, ramucirumab, bevacizumab, pertuzumab, trastuzumab, cetuximab, oxantuzumab, ofamumab, rituximab, alemtuzumab, tiimumab, tosimob, babutuximab, daratumumab, erlotuzumab, T-DM1, ofatumumab, dinutuximab, blinatumomab, iprimumab, avastin, Herceptin, and rituximab; (viii) Kinase inhibitors, such as imatinib, gefitinib, erlotinib, ostinib, afatinib, celitinib, alectinib, crizotinib, erlotinib, lapatinib, sorafenib, regorafenib, vemurafenib, dabrafenib, aflibercept, sunitinib, nilotinib, dasatinib, bosutinib, prambutinib, ibrutinib, cabozantinib, lenvatinib, vandetanib, trametinib, carbitinib, axitinib, tesiromoximib, idalalisib, pazopanib, tesiromoximib, and everolimus; (4) Other anticancer drugs that can be used in combination therapy include tamoxifen, letrozole, fulvestrant, mitoxantrone, octreotide, retinoid, arsenic, zoledronic acid, bortezomib, carfilzomib, Ixazomib, vemodega, sinedega, denosumab, thalidomide, lenalidomide, venetoclax, Aldesleukin (recombinant human interleukin-2), and Sipueucel-T (prostate cancer treatment vaccine).

17. Selected from the following applications: (1) The use of the linker-drug of any one of claims 1 to 11 in the construction or preparation of antibody-drug conjugates; and (2) The use of the linker-drug conjugate of any one of claims 1 to 11 and the antibody-drug conjugate of any one of claims 12 to 15 in the preparation of a medicament for treating a disease; Preferably, the disease includes tumors or cancer; Preferably, the tumors or cancers include, but are not limited to, liver cancer, melanoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer (such as small cell lung cancer), Wilms' tumor, cervical cancer, testicular cancer, soft tissue sarcoma, primary macroglobulinemia, bladder cancer, chronic myeloid leukemia, primary brain cancer, gastric cancer, colon cancer, malignant pancreatic islet cell tumor, choriocarcinoma, mycosis fungoides, head and neck cancer, osteosarcoma, pancreatic cancer, acute myeloid leukemia, piloblastic leukemia, rhabdomyosarcoma, Kaposi's sarcoma, thyroid cancer, esophageal cancer, cervical dysplasia, renal cell carcinoma, endometrial cancer, adrenocortical carcinoma, skin cancer, and prostate cancer.

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