Ligand-drug conjugate, preparation method therefor and use thereof

Abstract

The present disclosure relates to a ligand-drug conjugate, a preparation method therefor and a use thereof, relating to a ligand-drug conjugate having a structure represented by formula (I) or a pharmaceutically acceptable salt thereof, or a compound having a structure represented by formula (IA), formula (IIA), or formula (IIIA), or a tautomer, mesomer, racemate, enantiomer, or diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, as well as a preparation method thereof, a composition, and the use thereof in the treatment and / or prevention of tumors.

Description

A ligand-drug conjugate, its preparation method and application Technical Field

[0001] This disclosure pertains to the pharmaceutical field and relates to a ligand-drug conjugate using camptothecin compounds as cytotoxic drugs, its preparation method, and its application. Background Technology

[0002] Antibody-drug conjugates (ADCs), as an emerging drug form, have developed rapidly in recent years, providing new treatment options for patients with advanced cancer. ADCs consist of three parts: an antibody, a toxin molecule (payload), and a linker. The antibody specifically recognizes specific targets on the surface of tumor cells, guiding the ADC to the tumor cell surface and entering the lysosomes of cancer cells via endocytosis. Subsequently, the toxin molecule is cleaved and released within the lysosomes, selectively killing cancer cells with minimal damage to normal tissue cells. Furthermore, the toxin molecule, with its good membrane permeability, can also affect neighboring tumor cells through side-effect killing.

[0003] Camptothecin compounds, as inhibitors of topoisomerase I (TOP1), bind to the DNA-topoisomerase I complex, inhibiting the rejoining of supercoiled double-stranded DNA after a TOP1 cleavage, leading to DNA breakage and subsequently DNA damage and apoptosis. Research on camptothecin began in the 1960s with the isolation of this compound from the natural plant *Camptotheca acuminata*. Subsequently, scientists conducted extensive structural modifications and synthetic studies, developing a series of more potent derivatives, such as topotecan, irinotecan, and beloteccan. These derivatives have been approved as chemotherapy drugs and are used to treat various solid tumors, including metastatic colorectal cancer, ovarian cancer, small cell lung cancer, cervical cancer, and gastric cancer.

[0004] In recent years, camptothecin compounds have attracted widespread attention as toxins in ADC drugs. Among them, Enhertu (DS-8201a) and Trodelvy (IMMU-132) are the most well-known. Both drugs use camptothecin derivatives as payloads and have shown significant efficacy in clinical trials, and have been approved for marketing. These two drugs represent the development direction of third-generation ADC drugs, possessing the following characteristics: ① They use moderately potent drugs, such as DXd or SN38, whose killing activity against tumor cells is at the low nanomolar to sub-nanomolar level. Compared to other traditional ADC payloads such as microtubule inhibitors or DNA alkylating agents, camptothecin inhibitors are 10-100 times less potent. Their relatively low in vitro killing activity makes high-dose administration possible, thus fully leveraging the role of monoclonal antibodies in blocking signaling pathways. ② They employ moderately stable linkers to ensure the stability of the ADC in plasma, releasing toxins only within tumor tissues. ③ They have a high drug-to-antibody ratio (DAR4-8). ④ The toxin molecules have a short half-life and low systemic exposure, thereby reducing damage to peripheral normal cells. ⑤ High-dose administration can be repeated, sometimes even for more than 24 months, with manageable gastrointestinal and hematological toxicity.

[0005] Despite the significant efficacy demonstrated by camptothecin-based ADCs in clinical trials, several challenges remain: ① Camptothecin inhibitors typically exhibit moderate activity, requiring high DAR values ​​to ensure efficacy. However, improper control of payload hydrophobicity in high-DAR ADCs can lead to ADC aggregation, accelerated plasma clearance, and enhanced off-target toxicity, ultimately narrowing the therapeutic window; ② Some camptothecin toxins, especially those with high biological activity, have shown significant hematologic and gastrointestinal toxicity in clinical trials; ③ TOP1 gene mutations or upregulation of efflux transporters (such as ABCG2) may lead to tumor cell resistance to camptothecin toxins. Therefore, further balancing the cytotoxic activity of camptothecin ADCs with hydrophilicity / hydrophobicity and endocytic cleavage efficiency, as well as developing non-efflux substrate camptothecin toxins, is crucial for improving the physicochemical properties, pharmacodynamics, and pharmacokinetic characteristics of the final ADC product. Summary of the Invention

[0006] This disclosure provides a compound (including a ligand-drug conjugate) or its tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, or a pharmaceutically acceptable salt thereof, which may have one or more effects selected from the group consisting of: (1) better inhibitory activity against the in vitro proliferation of tumor cells or a better non-specific killing window; (2) better targeted inhibition; (3) better plasma stability; (4) better in vivo tumor suppression; (5) better bystander effect; (6) better antitransporter transport capability; (7) better in vivo tumor targeting capability; (8) better pharmacokinetic characteristics; (9) more effective cell affinity; (10) better endocytosis-promoting activity; and (11) good in vivo safety. The aforementioned compound provided in this disclosure can be used as a drug for treating cancer. This disclosure also provides methods and uses of the aforementioned compound for cancer treatment.

[0007] The first aspect of this disclosure relates to a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate has the structure shown in formula (I):

[0008] B#—(L—D)n Formula (I);

[0009] B# is the ligand that binds to the target.

[0010] n is the average coupling ratio of drug ligands, and n is an integer or decimal from 1 to 16;

[0011] L is a connecting unit with the following structure: —L3—L2—L1—; where,

[0012] The L3 is selected from Where m is an integer from 0 to 8, with 1 bit connected to the ligand and 2 bits connected to L2;

[0013] The L2 does not exist or is selected from C=O. Where r is 0 or 1, s is an integer from 1 to 10, t is an integer from 0 to 30, p is 0 or 1, and each q is an independent integer from 0 to 6, with 1 bit connected to L3 and 2 bits connected to L1; each R 2a and R 2b Each is independently hydrogen, -(CH2) s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X ; Each R X Independently hydrogen or C 1-6 Alkyl group; s1 is an integer from 1 to 30, s2 is an integer from 0 to 6; R 2c Selected from hydrogen, C 1-6 Alkyl or C 3-6 cycloalkyl;

[0014] L1 is Of these, bit 1 is connected to L2, and bit 2 is connected to D;

[0015] D is a drug having the structure shown in formula (II) or formula (III):

[0016] Wherein, R1 is a halogen or C 1-6 alkyl;

[0017] R2 is F;

[0018] W is In this configuration, position 1 is connected to the carbonyl group, and position 2 is connected to the methylene group.

[0019] The first aspect of this disclosure also relates to a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate has the structure shown in formula (I):

[0020] B#—(L—D)n Formula (I);

[0021] B# is the ligand that binds to the target.

[0022] n is the average coupling ratio of drug ligands, and n is an integer or decimal from 1 to 16;

[0023] L is a connecting unit with the following structure: —L3—L2—L1—; where,

[0024] The L3 is selected from Where m is an integer from 0 to 8, with 1 bit connected to the ligand and 2 bits connected to L2;

[0025] The L2 does not exist or is selected from C=O. Where s is an integer from 1 to 10, t is an integer from 1 to 30, 1 bit is connected to L3, and 2 bits are connected to L1; each R 2a and R 2b Each is independently hydrogen, -(CH2) s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X ; Each R X Independently hydrogen or C 1-6 Alkyl group; s1 is an integer from 1 to 30, s2 is an integer from 0 to 6; R 2c Selected from hydrogen, C 1-6 Alkyl or C 3-6 cycloalkyl;

[0026] L1 is Of these, bit 1 is connected to L2, and bit 2 is connected to D;

[0027] D is a drug having the structure shown in formula (II) or formula (III):

[0028] Wherein, R1 is a halogen or C 1-6 alkyl;

[0029] R2 is F;

[0030] W is In this configuration, position 1 is connected to the carbonyl group, and position 2 is connected to the methylene group.

[0031] Another aspect of this disclosure relates to a compound or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein said compound has the structure shown in formula (IA):

[0032] L-D formula (IA)

[0033] Wherein, L is a connecting unit, having the following structure: L3'—L2—L1—;

[0034] The L3' is selected from: Where m is an integer from 0 to 6, and 2 bits are connected to L2;

[0035] L2, L1, and D are as described in this article.

[0036] Another aspect of this disclosure relates to a compound or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein said compound has a structure represented by formula (IIA) or (IIIA):

[0037] Among them, R 1 R 2 And W as described in this article.

[0038] Another aspect of this disclosure relates to a method for preparing the ligand-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof, the compound having the structure shown in formula (I), the method comprising contacting ligand B# with the structure shown in formula (IA) of this disclosure.

[0039] Another aspect of this disclosure relates to a pharmaceutical composition comprising a ligand-drug conjugate of the present disclosure (e.g., formula (I)) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0040] Another aspect of this disclosure relates to a pharmaceutical composition comprising a compound of the present disclosure (e.g., formula (IIA), formula (IIIA)) or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0041] Another aspect of this disclosure relates to the use of a ligand-drug conjugate containing the ligand-drug conjugate of this disclosure (e.g., formula (I)) or a pharmaceutically acceptable salt thereof, or a compound of this disclosure (e.g., formula (IA), formula (IIA), formula (IIIA)) or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a medicament for treating and / or preventing tumors.

[0042] This disclosure also provides methods for treating and / or preventing tumors, including administering to a subject in need a ligand-drug conjugate of this disclosure (e.g., formula (I)) or a pharmaceutically acceptable salt thereof, or a compound of this disclosure (e.g., formula (IIA), formula (IIIA)) or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0043] This disclosure also provides a ligand-drug conjugate of the present disclosure (e.g., formula (I)) or a pharmaceutically acceptable salt thereof, or a compound of the present disclosure (e.g., formula (IIA), formula (IIIA)) or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the treatment and / or prevention of tumors.

[0044] This disclosure also provides a kit comprising a ligand-drug conjugate of the present disclosure (e.g., formula (I)) or a pharmaceutically acceptable salt thereof, or a compound of the present disclosure (e.g., formula (IIA), formula (IIIA)) or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0045] The details of this application are set forth in the accompanying description below. Although similar or equivalent methods and materials may be used in the practice or testing of this application, illustrative methods and materials are described hereafter. Other features, objects, and advantages of this application will be apparent from the description and claims. In the description and appended claims, the singular form also includes the plural form, unless the context clearly requires otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. All patents and publications referenced in this specification are incorporated herein by reference in their entirety.

[0046] All references cited throughout this application (including bibliographic references, granted patents, published patent applications, and co-pending patent applications) are expressly incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly known to one of ordinary skill in the art. Attached Figure Description

[0047] Figure 1 shows the serum stability assay results of the antibody-drug conjugate.

[0048] Figure 2 shows the pharmacokinetic analysis results of the antibody-drug conjugate in CD-1 mice.

[0049] Figure 3 shows the pharmacokinetic analysis results of the antibody-drug conjugate in SD rats.

[0050] Figure 4 shows the cell affinity activity assay results of the antibody-drug conjugate in SK-BR-3 cells. The anti-HER2 monoclonal antibody control was HLXB.

[0051] Figure 5 shows the results of the antibody-drug conjugate's endocytic activity assay in NCI-N87 cells. The anti-HER2 monoclonal antibody control was HLXB.

[0052] Figure 6 shows the results of the antibody-drug conjugate's endocytic activity assay in SK-BR-3 cells. The anti-HER2 monoclonal antibody control was HLXB.

[0053] Figure 7 shows the tumor volume change curve of the antibody-drug conjugate in a subcutaneous xenograft model (N87 cells). The monoclonal antibody control is HLXB.

[0054] Figure 8 shows the body weight change curves of the antibody-drug conjugate in a subcutaneous xenograft model (N87 cells). The monoclonal antibody control was HLXB.

[0055] Figure 9 shows the second curve of tumor volume change in a subcutaneous xenograft model (N87 cells) using the antibody-drug conjugate. The monoclonal antibody control was HLXB.

[0056] Figure 10 shows the second curve of body weight change in an animal using the antibody-drug conjugate in a subcutaneous xenograft model (N87 cells). The monoclonal antibody control was HLXB.

[0057] Figure 11 shows the tumor volume change curves of antibody-drug conjugates in subcutaneous co-xenograft tumor models (NCI-N87 and U87-MG-luc).

[0058] Figure 12 shows the in vivo imaging photon count results of antibody-drug conjugates in subcutaneous co-xenograft tumor models (NCI-N87 and U87-MG-luc).

[0059] Figure 13 shows the weight change curves of the antibody-drug conjugates in subcutaneous co-xenograft tumor models (NCI-N87 and U87-MG-luc). Detailed Implementation

[0060] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0061] In this disclosure, the term "ligand" generally refers to a macromolecular compound that recognizes and binds to antigens or receptors associated with target cells. The role of a ligand can be to present a drug to a target cell population that has bound the ligand. These ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules that can bind to cells, receptors, and / or antigens. In this disclosure, a ligand may be represented as B#, and the ligand antigen forms a linker bond with a linker unit via a heteroatom on the ligand. The ligand may be an antibody or its antigen-binding fragment, and the antibody may be selected from chimeric antibodies, humanized antibodies, fully human antibodies, or murine antibodies; the antibody may be a monoclonal antibody. For example, the antibody may be an antibody targeting the following targets: HER2, HER3, B7H3, TROP2, Claudin 18.2, CD30, CD33, CD70, or EGFR. For example, the antibody may be an antibody targeting the following targets: 5T4, ADAM9, AGS-16, ANGPTL4, ALPP, ALPP2, ApoE, CTGF, CXCR5, FGF2, MCPT8, MFI2, MS4A7, NCA, Sema5b, SLITRK6, STC2, TGF, 0772P, ACTA2, ADGRE1, AG-7, AIF1, AKR1C1, AKR1C2, ASLG659, Axl, B7H3, BAFF-R, BCMA, BMPR1B, BNIP3, C1QA, C1QB, CA6, CADM1, CCD79b, CCL5, CCR 5. CCR7, CD11c, CD123, CD138, CD142, CD147, CD166, CD19, CD21, CD20, CD205, CD22, CD223, CD228, CD25, CD30, CD33, CD37, CD38, CD40, CD45, CD45 ( PTPRC), CD46, CD47, CD49D(ITGA4), CD56, CD66e, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CDCP1, CDH11, CDH17, CD11b, CEA, CEACAM5, Claudin 18.2. c-Met, COL6A3, COL7A1, CRIPTO, CSF1R, CTSD, CTSS, CXCL11, CXCL10, DDIT4, DLL3, DLL4, DR5, E16, EFNA4, EGFR, EGF RvIII, EGLN, EGLN3, EMR2, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FcRH2, FcRHl, FGFR2, FGFR3, FLT3, FOLR-α, FRα, GD2, G EDA, GPC-1, GPNMB, GPR20, GZMB, HER2, HER3, HLA-DOB, HMOX1, IFI6, IFNG, IGF-1R, IGFBP3, IL10RA1, IL-13R, IL-2, IL 20Ra, IL-3, IL-4, IL-6, IRTA2, KISS1R, KRT33A, LIV1, LOX, LRP-1, LRRC15, LUM, LY64, LY6E, Ly86, LYPD3, MDP, MMP10, M MP14, MMP16, MPF, MSG783, MSLN, MUC-1, NaPi2b, Napi3b, Nectin-4, NOG, P2X5, pCAD, P-Cadherin, PDGFRA, PDK1, PD-L1, PFKFB3, PGF, PGK1, PIK3AP1, PIK3CD, PLOD2, PSCA, PSCAhlg, PSMA, PTK7, P-cadherin, RNF43, ROR1, ROR2, SERPINE1, SL C39A6, SLTRK6, STAT1, STEAP1, STEAP2, TCF4, TENB2, TGFB1, TGFB2, TGFBR1, TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, WNT5A, epidermal growth factor, short proteoglycans, mesothelin, endothelial peptide receptors, mucins (such as mucin 1 and mucin 16), guanylate cyclase C, integrin α4β7, integrin α5β6, trophoblast cell glycoproteins, or tissue factor.

[0062] In this disclosure, the term "drug" generally refers to a cytotoxic drug, which is a chemical molecule that can strongly disrupt the normal growth of tumor cells. Cytotoxic drugs can kill tumor cells at sufficiently high concentrations. The "cytotoxic drug" may include toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, and radioactive isotopes (e.g., At). 211 I 131 I 125 Y 90 Re186 Re 188 、Sm 153 Bi 212 P 32 (or radioactive isotopes of Lu), toxic drugs, chemotherapy drugs, antibiotics, and nucleolysins.

[0063] In this disclosure, the term "linking unit" generally refers to a chemical structural segment or bond that is connected to a ligand at one end and to a cytotoxic drug at the other end. The linking unit can be directly or indirectly connected to the cytotoxic drug; for example, it can be directly connected to the cytotoxic drug via a covalent bond, or it can be indirectly connected to the cytotoxic drug via other chemical structural segments. The linking unit can also be directly or indirectly connected to the ligand; for example, it can be directly connected to the ligand via a covalent bond, or it can be indirectly connected to the ligand via other chemical structural segments (linker units). The linking unit can include chemical structural segments such as self-cleaving units, conditionally cleaving units, hydrophilic units, and linker units. For example, the linking unit can be the structure shown in the -L3-L2-L1- diagram described in this disclosure. For example, the linking unit can include chemical structural segments containing acid-labile linker structures (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linker structures, light-labile linker structures, dimethyl linker structures, and / or disulfide-containing linker structures.

[0064] In this disclosure, the term "self-cleaving unit" refers to a linker structural unit embedded between a conditional cleavage unit and a cytotoxic drug. The mechanism of action of the self-cleavage unit is that when the conditional cleavage unit breaks under suitable conditions, the self-cleavage unit can spontaneously rearrange its structure, thereby releasing the linked cytotoxic drug. Common self-cleavage units include p-aminobenzyl alcohols (PABs) and substituted or unsubstituted aminomethylene groups. wait.

[0065] In this disclosure, the term "conditional cleavage unit" refers to a connecton structural unit that breaks under certain conditions within a target cell. Conditional cleavage units can be divided into two main categories: chemically unstable connecton structures and enzyme-instantaneous connecton structures. Chemically unstable connecton structures can selectively cleave due to differences in plasma and cytoplasmic properties. Such properties include pH value, glutathione concentration, etc. pH-sensitive connecton structures are also called acid-cleaving connecton structures. Such connecton structures will be hydrolyzed in weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0), for example, hydrazones, carbonates, acetals, and ketals. Glutathione-sensitive connecton structures are also called disulfide-bonded connecton structures. In tumor cells, the low oxygen content leads to enhanced reductase activity, resulting in higher glutathione concentrations. The difference between the high concentration (millimolar range) of glutathione in tumor cells and the relatively low concentration (micromolar range) of glutathione in the blood will cause the cleavage of connecton structures. Unstable enzyme linker structures, such as peptide linkers, can be effectively cleaved by lysosomal proteases (such as cathepsin B) or plasmin (which is present in increased amounts in some tumor tissues). Typical unstable enzyme linker structures include Val-Cit (VC) and Phe-Lys.

[0066] In this disclosure, the term "hydrophilic unit" refers to a linker structural unit with a strongly hydrophilic group. Hydrophilic units can typically modulate the overall hydrophilicity of the molecule. Common hydrophilic groups include polyethylene glycol groups, etc.

[0067] In this disclosure, the term "connector unit" refers to a linker structural unit used for connection with ligands. Typical connector units include maleimide groups, etc.

[0068] In this disclosure, the term "ligand-drug conjugate" generally refers to a ligand linked to a biologically active cytotoxic drug via a linker unit. In this disclosure, "ligand-drug conjugate" can also refer to an antibody-drug conjugate (ADC), where an ADC can refer to a monoclonal antibody or antibody fragment linked to a biologically active cytotoxic drug via a linker unit.

[0069] In this disclosure, the term "antibody or antigen-binding fragment thereof" generally refers to an immunologically binding agent extending to all antibodies from all species, including dimer, trimer, and multimer antibodies; bispecific antibodies; chimeric antibodies; fully human antibodies; humanized antibodies; recombinant and modified antibodies, and fragments thereof. The term "antibody or fragment thereof" can refer to any antibody-like molecule having an antigen-binding region, including small molecule fragments such as Fab′, Fab, F(ab′)2, single-domain antibodies (DABs), Fv, scFv (single-chain Fv), linear antibodies, biantibodies, etc. The term "antigen-binding fragment" can refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. For example, fragments of full-length antibodies can be used to perform the antigen-binding function of an antibody. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art. The antibodies may include: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-TROP2 antibody, and anti-Claudin antibody. 18.2 Antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody or anti-Mesothelin antibody, for example, trastuzumab or pertuzumab.

[0070] In this disclosure, the term "chimeric antibody" generally refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by murine antibodies. To establish a chimeric antibody, a hybridoma that secretes murine-specific monoclonal antibodies can be created. The variable region gene can then be cloned from the murine hybridoma cells, and the constant region gene of a human antibody can be cloned as needed. The murine variable region gene and the human constant region gene can be linked to form a chimeric gene, which is then inserted into an expression vector. The chimeric antibody molecule can then be expressed in eukaryotic or prokaryotic systems.

[0071] In this disclosure, the term "humanized antibody," also known as a CDR-grafted antibody, generally refers to an antibody generated by grafting a mouse CDR sequence into a human antibody variable region framework, i.e., a human germline antibody framework sequence of different types. This can overcome the heterologous response induced by chimeric antibodies carrying a large number of mouse protein components. Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available references. Germline DNA sequences of human heavy chain and light chain variable region genes, for example, can be found in the VBase human germline sequence database.

[0072] In this disclosure, the terms "fully human antibody," "fully human antibody," or "completely human antibody," also known as "fully human monoclonal antibody," refer to antibodies whose variable and constant regions can both be human-derived, thus eliminating immunogenicity and toxic side effects. The development of monoclonal antibodies has gone through four stages: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies. The antibodies or ligands described in this disclosure can be fully human monoclonal antibodies. Related technologies for the preparation of fully human antibodies include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology, etc.

[0073] In this disclosure, the term "CDR" generally refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. One of the most common definitions of the six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242; Chothia et al., "Canonical Structures For the Hypervariable Regions of Immunoglobulins", J. Mol. Biol. 196:901 (1987); and MacCallum et al., "Antibody-Antigen Interactions: Contact Analysis and Binding Site Topography", J. Mol. Biol. 262:732 (1996). As used in this disclosure, the Kabat definition of CDR can be applied to CDR1, CDR2, and CDR3 (CDRL1, CDR L2, CDR L3 or L1, L2, L3) of light chain variable structural domains, and CDR1, CDR2, and CDR3 (CDR H1, CDR H2, CDR H3 or H1, H2, H3) of heavy chain variable structural domains.

[0074] In this disclosure, the term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) carbon atoms. 1-20 Alkyl groups, preferably alkyl groups containing 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms (i.e., C14, C24, C34, C44, C44, C54, C64, C74, C84, C9 ... 1-20 Alkyl groups, more preferably alkyl groups containing 1 to 6 carbon atoms (i.e., C1646-C ... 1-6Alkyl groups). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers, etc. Alkyl groups can be substituted or unsubstituted. When substituted, they can be substituted at any usable connection point. The substituents are preferably selected from one or more of deuterium, halogen, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0075] In this disclosure, the term "methylene" refers to a residue derived from a group containing one carbon atom by removing two hydrogen atoms. The methylene group can be substituted or unsubstituted, substituted or non-substituted. The term "alkylene" refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane. It can be a straight-chain or branched group containing 1 to 20 carbon atoms, for example, containing 1 to 12 carbon atoms, such as an alkylene group containing 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH2CH2CH2CH2CH2-). The alkylene group can be substituted or unsubstituted, alternative or non-alternative. For example, when substituted, the substituent can be replaced at any usable connection point. The substituent is preferably independently selected independently from one or more substituents chosen from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo, for example, it can be hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, or C 1-6 Aliphatic groups.

[0076] In this disclosure, the term "cycloalkyl" refers to a cyclic hydrocarbon group containing 3 to 8 carbon atoms, which may be saturated or unsaturated, but cannot be aromatic. In some embodiments, the cycloalkyl group is fully saturated. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0077] In this disclosure, the term "halogen" generally refers to fluorine, chlorine, bromine, or iodine, for example, fluorine or chlorine.

[0078] In this disclosure, the term "hydroxyl" refers to -OH.

[0079] In this disclosure, the term "thiol" refers to -SH.

[0080] In this disclosure, the term "amino" refers to -NH2.

[0081] In this disclosure, the term "cyano" refers to -CN.

[0082] In this disclosure, the term "nitro" refers to -NO2.

[0083] In this disclosure, the term "oxo" or "oxo" means "=O".

[0084] In this disclosure, the term "carbonyl" refers to C=O.

[0085] In this disclosure, the term "aldehyde group" refers to -C(O)H

[0086] In this disclosure, the term "carboxyl group" refers to -C(O)OH.

[0087] In this disclosure, the terms “optional” or “optionally” generally mean that the event or environment described below may but does not have to occur, and the description includes situations in which the event or environment occurs or does not occur. For example, “optionally alkyl-substituted heterocyclic group” means that an alkyl group may but does not have to be present, and the description can include cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0088] In this disclosure, the term "substituted" generally refers to one or more hydrogen atoms in a group, for example, up to five, or for example, one to three hydrogen atoms, independently substituted by the corresponding number of substituents. Substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when bonded to a carbon atom with an unsaturated (e.g., alkene) bond.

[0089] In this disclosure, the term "replaced" for 0 or more (e.g., 0 or at least 1, 0 or 1, 0) methylene units generally means that when the structure contains 1 or more methylene units, the one or more methylene units may not be replaced, or may be replaced by one or more groups that are not methylene (e.g., -NHC(O)-, -C(O)NH-, -C(O)-, -OC(O)-, -C(O)O-, -NH-, -O-, -S-, -SO-, -SO2-, -PH-, -P(=O)H-, -NHSO2-, -SO2NH-, -C(=S)-, -C(=NH)-, -N=N-, -C=N-, -N=C- or -C(=N2)-).

[0090] One or more hydrogen atoms in a group, for example, up to five, or for example, one to three hydrogen atoms, are independently replaced by the corresponding number of substituents. The substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when bonded to a carbon atom with an unsaturated bond (such as an alkene).

[0091] In the chemical structure of the compounds disclosed herein, the bonds... This indicates that the configuration is not specified; that is, if chiral isomers exist in the chemical structure, the bond... It can be or Or it may contain both of these configurations.

[0092] In this disclosure, the term "absent" generally indicates that there is no substituent at the position of the group, or that the two groups connected to the group are directly linked. For example, in the structure —CH2—R, when R is absent, it means that CH2 has no R substituent, i.e., the structure is —CH3; as another example, in the structure —L3—L2—L1—, when L2 is absent, it means that L3 and L1 are directly linked, i.e., the structure is —L3—L1—.

[0093] In this disclosure, the term "compound" generally refers to a substance having two or more different elements. For example, the compounds of this disclosure can be organic compounds, compounds with a molecular weight of less than 500, less than 1000, more than 1000, or more than 10,000 or 100,000. In this disclosure, a compound can also refer to a compound linked by chemical bonds, such as a compound in which one or more molecules with a molecular weight of less than 1000 are linked by chemical bonds to a biological macromolecule, which can be a polysaccharide, protein, nucleic acid, polypeptide, etc. For example, the compounds of this disclosure can include compounds in which a protein is linked to one or more molecules with a molecular weight of less than 1000, compounds in which a protein is linked to one or more molecules with a molecular weight of less than 10,000, or compounds in which a protein is linked to one or more molecules with a molecular weight of less than 100,000. For example, the compounds of this disclosure can be ligand-drug conjugates.

[0094] This disclosure also includes compounds identical to those described herein, but in which one or more atoms are replaced by isotopes of the present disclosure with atomic weights or mass numbers different from those commonly found in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as... 2 H,3 H, 11 C 13 C 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0095] The compounds disclosed herein may contain atomic isotopes in non-natural proportions on one or more atoms constituting the compound. For example, the compounds may be labeled with radioactive isotopes, such as tritium. 3 Deuterated drugs can be formed by replacing hydrogen with deuterium (H). The bond between deuterium and carbon is stronger than that between ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have advantages such as reduced toxicity, increased drug stability, enhanced efficacy, and prolonged drug biological half-life. All isotopic variations of the compounds disclosed herein, regardless of radioactivity, are included within the scope of this disclosure.

[0096] In addition, heavier isotopes (such as deuterium) are used. 2 H)) substitution can provide certain therapeutic advantages resulting from higher metabolic stability (e.g., increased in vivo half-life or reduced dose requirement), and may therefore be preferred in certain situations, where deuterium substitution can be partial or complete, with partial deuterium substitution referring to at least one hydrogen being replaced by at least one deuterium.

[0097] Unless otherwise specified, when a position is specifically designated as deuterium (D), that position should be understood as having a deuterium abundance of at least 1000 times greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 10% deuterium doping). The natural abundance of deuterium in the example compounds can be at least 1000 times, at least 2000 times, at least 3000 times, at least 4000 times, at least 5000 times, at least 6000 times, or higher. This disclosure also includes various deuterated forms of compounds of formula (I) (including formulas (I), (II), (III-1), and (III-2)). Each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of compounds of formula (I) (including formulas (I), (II), (III-1), and (III-2)) by referring to relevant literature. Commercially available deuterated starting materials can be used in the preparation of deuterated forms of compounds of formula (I) (including formulas (I), (II), (III-1), and (III-2)), or they can be synthesized using conventional techniques with deuterating reagents, including but not limited to deuterated boranes, trideuterated borane tetrahydrofuran solutions, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane.

[0098] In this disclosure, the term "pharmaceutical composition" generally refers to a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. Pharmaceutical compositions can facilitate administration to organisms, promote the absorption of the active ingredient, and thereby exert its biological activity. Conventional methods for preparing pharmaceutical compositions can be found in the Chinese Pharmacopoeia.

[0099] In this disclosure, the terms "pharmaceutically acceptable salt" or "medicinal salt" generally refer to a salt of the compounds or ligand-drug conjugates of this disclosure, or a salt of the compounds described in this disclosure, which may be safe and / or effective when used in mammals and may have the desired biological activity. The antibody-antibody-drug conjugates of this disclosure may form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0100] In this disclosure, the terms "solvent" or "solvent compound" generally refer to a pharmaceutically usable solvate formed by the ligand-drug conjugate of this disclosure with one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0101] The term "drug loading" typically refers to the average amount of cytotoxic drug loaded onto each ligand, and can also be expressed as the ratio of cytotoxic drug to antibody. The range of cytotoxic drug loading can be 0-12 cytotoxic drugs per ligand (Ab), for example, 1-10 cytotoxic drugs. In embodiments of this disclosure, drug loading is expressed as N. a Examples can be the average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The drug loading of each ADC molecule after the coupling reaction can be identified using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, and HPLC characterization.

[0102] The pharmaceutical composition may be in the form of a sterile injectable aqueous or oil suspension for intramuscular and subcutaneous administration. This suspension may be formulated using suitable dispersants or wetting agents and suspending agents as described above, according to known techniques. The sterile injectable formulation may also be a sterile injectable solution or suspension prepared in a non-toxic, parenteral-acceptable diluent or solvent, such as a solution prepared in 1,3-butanediol. Furthermore, a sterile fixative oil may be conveniently used as a solvent or suspension medium. For example, any blended fixative oil, including synthetic mono- or diglycerides of glycerol, may be used. Additionally, fatty acids such as oleic acid may also be used to prepare the injectable formulation.

[0103] In this disclosure, the term "comprising" generally means including the expressly specified features, but does not exclude other elements. The terms "above" and "below" generally mean including the stated number.

[0104] In this disclosure, the singular forms “a”, “an”, and “the” include plural objects, and vice versa, unless the context clearly indicates otherwise.

[0105] In this disclosure, the term "about" generally refers to a variation within 10% (i.e., ±10%) above or below a specified value, for example, a variation within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value. For parameters such as pH, concentration, and temperature, it indicates that the parameter can vary within ±10%, and sometimes more preferably within ±5%. As those skilled in the art will understand, when a parameter is not critical, figures are generally given for illustrative purposes only and not as limitations.

[0106] The compounds disclosed herein

[0107] The first aspect of this disclosure relates to a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate has the structure shown in formula (I):

[0108] B#—(L—D)n formula (I);

[0109] B# is the ligand that binds to the target.

[0110] n is the average coupling ratio of drug ligands, and n is an integer or decimal from 1 to 16;

[0111] L is a connecting unit with the following structure: —L3—L2—L1—; where,

[0112] The L3 is selected from Where m is an integer from 0 to 8, with 1 bit connected to the ligand and 2 bits connected to L2;

[0113] The L2 does not exist or is selected from C=O. Where r is 0 or 1, s is an integer from 1 to 10, t is an integer from 0 to 30, p is 0 or 1, and each q is an independent integer from 0 to 6, with 1 bit connected to L3 and 2 bits connected to L1; each R 2a and R 2b Each is independently hydrogen, -(CH2) s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X ; Each R X Independently hydrogen or C 1-6 Alkyl group; s1 is an integer from 1 to 30, s2 is an integer from 0 to 6; R 2c Selected from hydrogen, C 1-6 Alkyl or C3-6 cycloalkyl;

[0114] L1 is Of these, bit 1 is connected to L2, and bit 2 is connected to D;

[0115] D is a drug having the structure shown in formula (II) or formula (III):

[0116] Wherein, R1 is a halogen or C 1-6 alkyl;

[0117] R2 is F;

[0118] W is In this configuration, position 1 is connected to the carbonyl group, and position 2 is connected to the methylene group.

[0119] In some embodiments of the ligand-drug conjugate shown in formula (I), the L2 is absent or selected from C=O, Where s is an integer from 1 to 10, t is an integer from 1 to 30, and each q is an independent integer from 0 to 6, with 1 bit connected to L3 and 2 bits connected to L1; each R 2a and R 2b Each is independently hydrogen, -(CH2) s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X ; Each R X Independently hydrogen or C 1-6 Alkyl group; s1 is an integer from 1 to 30, s2 is an integer from 0 to 6; R 2c Selected from hydrogen, C 1-6 Alkyl or C 3-6 Cycloalkyl.

[0120] The first aspect of this disclosure also relates to a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate has the structure shown in formula (I):

[0121] B#—(L—D)n formula (I);

[0122] B# is the ligand that binds to the target.

[0123] n is the average coupling ratio of drug ligands, and n is an integer or decimal from 1 to 16;

[0124] L is a connecting unit with the following structure: —L3—L2—L1—; where,

[0125] The L3 is selected from Where m is an integer from 0 to 8, with 1 bit connected to the ligand and 2 bits connected to L2;

[0126] The L2 does not exist or is selected from C=O. Where s is an integer from 1 to 10, t is an integer from 1 to 30, 1 bit is connected to L3, and 2 bits are connected to L1; each R 2a and R 2b Each is independently hydrogen, -(CH2) s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X ; Each R X Independently hydrogen or C 1-6 Alkyl group; s1 is an integer from 1 to 30, and s2 is an integer from 0 to 6;

[0127] L1 is Of these, bit 1 is connected to L2, and bit 2 is connected to D;

[0128] D is a drug having the structure shown in formula (II) or formula (III):

[0129] Among them, R 1 Halogen or C 1-6 alkyl;

[0130] R 2 For F;

[0131] W is In this configuration, position 1 is connected to the carbonyl group, and position 2 is connected to the methylene group.

[0132] In some embodiments of the ligand-drug conjugate shown in formula (I), the R 1 For F, Cl, Br or C 1-3 Alkyl group. Preferably, the R 1 It can be F, Cl, -CH3, -CH2CH3, -CH2CH2CH3, or -CH(CH3)2. In some embodiments, the R... 1 For Cl. In some implementations, the R 1 It is -CH3.

[0133] In some embodiments of the ligand-drug conjugate shown in formula (I), D is selected from the following structures:

[0134] In some embodiments of the ligand-drug conjugate shown in formula (I), the L3 is selected from... Where m is an integer from 0 to 6, with 1 bit connected to the ligand and 2 bits connected to L2. For example, m can be 0, 1, 2, 3, 4, 5, or 6. More preferably, m is an integer from 0 to 5. In some embodiments, m is 0. In some embodiments, m is 2. In some embodiments, m is 5.

[0135] In some embodiments of the ligand-drug conjugate shown in formula (I), the L2 is absent.

[0136] In some embodiments of the ligand-drug conjugate shown in formula (I), L2 is C=O.

[0137] In some embodiments of the ligand-drug conjugate shown in formula (I), L2 is Where s is an integer from 1 to 10, with 1 bit connected to L3 and 2 bits connected to L1; each R 2a and R 2b Each is independently hydrogen, -(CH2) s2 C(=O)NH(CH2CH2O) s1 -RX -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X ; Each R X Independently hydrogen or C 1-6 Alkyl group; s1 is an integer from 1 to 30, and s2 is an integer from 0 to 6.

[0138] In some embodiments of the ligand-drug conjugate shown in formula (I), the R of all methylene units of L2 2a and R 2b At least one of them is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X The rest of R 2a and R 2b All are hydrogen. Preferably, the R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L2 have R 2a and R 2b All are hydrogen. More preferably, the R of one methylene unit of L2... 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L2 have R 2a and R 2b All are hydrogen. More preferably, the R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L2 have R 2a and R 2b Both are hydrogen.

[0139] Preferably, s is an integer from 1 to 8. For example, s is 1, 2, 3, 4, 5, 6, 7, or 8. More preferably, s is an integer from 1 to 6. More preferably, s is an integer from 3 to 6. In some embodiments, s is 3.

[0140] Preferably, s1 is an integer from 1 to 28. For example, s1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. More preferably, s1 is an integer from 4 to 26. More preferably, s1 is an integer from 8 to 24. In some embodiments, s1 is 14. In some embodiments, s1 is 16. In some embodiments, s1 is 18.

[0141] Preferably, s2 is an integer from 0 to 5. For example, s2 is 0, 1, 2, 3, 4, or 5. More preferably, s2 is an integer from 0 to 4. More preferably, s2 is an integer from 0 to 3. In some embodiments, s2 is 0. In some embodiments, s2 is 1. In some embodiments, s2 is 2.

[0142] Preferably, each R X Independently hydrogen or C 1-3 Alkyl group. More preferably, R X C 1-3 Alkyl group. More preferably, R X It is -CH3 or -C2H5. In some implementations, R X It is -CH3.

[0143] In some implementations, the R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L2 have R 2a and R 2b All are hydrogen; where s2 is 0, s1 is an integer from 4 to 26, and R X The value is -CH3. Preferably, s1 is an integer from 8 to 24. More preferably, s1 is an integer from 12 to 20. In some embodiments, s1 is 14. In some embodiments, s1 is 16. In some embodiments, s1 is 18.

[0144] In some implementations, L2 is s1 is an integer from 4 to 26. Preferably, L2 is... Preferably, s1 is an integer from 8 to 24. More preferably, s1 is an integer from 12 to 20. In some embodiments, s1 is 14. In some embodiments, s1 is 16. In some embodiments, s1 is 18.

[0145] In some implementations, L2 is

[0146] In some embodiments of the ligand-drug conjugate shown in formula (I), L2 is Where r is 0 or 1, t is an integer from 0 to 30, p is 0 or 1, each q is an independent integer from 0 to 6, 1 bit is connected to L3, and 2 bits are connected to L1; R 2c Selected from hydrogen, C 1-6 Alkyl or C 3-6 Cycloalkyl.

[0147] In some implementations, r is 0. In some implementations, r is 1.

[0148] In some implementations, p is 0. In some implementations, p is 1.

[0149] In some implementations, L2 is selected from... Where t is an integer from 1 to 30, each q is an independent integer from 0 to 6, with 1 bit connected to L3 and 2 bits connected to L1; R 2c Selected from hydrogen, C 1-6 Alkyl or C 3-6 Cycloalkyl.

[0150] In some embodiments, t is an integer from 1 to 24. Preferably, t is an integer from 1 to 20. For example, t is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. More preferably, t is an integer from 1 to 12. More preferably, t is an integer from 1 to 6. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, t is 4.

[0151] In some implementations, each q is independently 0, 1, 2, 3, 4, or 5. Preferably, each q is independently 0, 1, 2, or 3.

[0152] In some implementation schemes, R 2c Selected from C 1-6 Alkyl or C 3-6 Cycloalkyl. In some embodiments, R 2c C 1-6 Alkyl group. Preferably, R 2cC 1-3 Alkyl group. More preferably, R 2c It is methyl or ethyl. In some embodiments, R 2c It is methyl. In some embodiments, R 2c It is an ethyl group.

[0153] In some embodiments of the ligand-drug conjugate shown in formula (I), L2 is Where t is an integer from 1 to 24. Preferably, t is an integer from 1 to 20. For example, t is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. More preferably, t is an integer from 1 to 12. More preferably, t is an integer from 1 to 6. In some embodiments, t is 2.

[0154] In some embodiments of the ligand-drug conjugate shown in formula (I), L2 is Where t is an integer from 1 to 24. Preferably, t is an integer from 1 to 20. For example, t is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. More preferably, t is an integer from 1 to 12. More preferably, t is an integer from 1 to 6. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, t is 4.

[0155] In some embodiments of the ligand-drug conjugate shown in formula (I), L is selected from the structures in Table 1 below:

[0156] Table 1

[0157] In Table 1, bit 1 is connected to B#, and bit 2 is connected to D.

[0158] In some embodiments of the ligand-drug conjugate shown in formula (I), B# can be an antibody or its antigen-binding fragment.

[0159] Preferably, the B# is selected from the group consisting of chimeric antibodies, humanized antibodies, and fully human antibodies.

[0160] In some embodiments of the ligand-drug conjugate shown in formula (I), B# may be an antibody targeting the following targets: 5T4, ADAM9, AGS-16, ANGPTL4, ALPP, ALPP2, ApoE, CTGF, CXCR5, FGF2, MCPT8, MFI2, MS4A7, NCA, Sema5b, SLITRK6, STC2, TGF, O772P, ACTA2, ADGRE1, AG-7, AIF1, AKR1C1, AKR1C2, ASLG659, Axl, B7H3, BAFF-R, BCMA, BMPR1B, BNIP3, C1QA, C1QB, CA6, CADM1, CCD7 9b, CCL5, CCR5, CCR7, CD11c, CD123, CD138, CD142, CD147, CD166, CD19, CD21, CD20, CD205, CD22, CD223, CD228, CD25, CD30, CD33, CD37, CD38, CD40, CD45 , CD45(PTPRC), CD46, CD47, CD49D(ITGA4), CD56, CD66e, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CDCP1, CDH11, CDH17, CD11b, CEA, CEACAM5, Claudin 18.2. c-Met, COL6A3, COL7A1, CRIPTO, CSF1R, CTSD, CTSS, CXCL11, CXCL10, DDIT4, DLL3, DLL4, DR5, E16, EFNA4, EGFR, EGF RvIII, EGLN, EGLN3, EMR2, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FcRH2, FcRHl, FGFR2, FGFR3, FLT3, FOLR-α, FRα, GD2, G EDA, GPC-1, GPNMB, GPR20, GZMB, HER2, HER3, HLA-DOB, HMOX1, IFI6, IFNG, IGF-1R, IGFBP3, IL10RA1, IL-13R, IL-2, IL 20Ra, IL-3, IL-4, IL-6, IRTA2, KISS1R, KRT33A, LIV1, LOX, LRP-1, LRRC15, LUM, LY64, LY6E, Ly86, LYPD3, MDP, MMP10, M MP14, MMP16, MPF, MSG783, MSLN, MUC-1, NaPi2b, Napi3b, Nectin-4, NOG, P2X5, pCAD, P-Cadherin, PDGFRA, PDK1, PD-L1, PFKFB3, PGF, PGK1, PIK3AP1, PIK3CD, PLOD2, PSCA, PSCAhlg, PSMA, PTK7, P-cadherin, RNF43, ROR1, ROR2, SERPINE1, SL C39A6, SLTRK6, STAT1, STEAP1, STEAP2, TCF4, TENB2, TGFB1, TGFB2, TGFBR1, TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, WNT5A, epidermal growth factor, short proteoglycans, mesothelin, endothelial peptide receptors, mucins (such as mucin 1 and mucin 16), guanylate cyclase C, integrin α4β7, integrin α5β6, trophoblast cell glycoproteins, or tissue factor.

[0161] In some embodiments of the ligand-drug conjugate shown in Formula (I), B# is an antibody targeting the following targets: ALPP, ALPP2, HER2, HER3, B7H3, TROP2, Claudin 18.2, CD30, CD33, CD70, EGFR, or FRα.

[0162] In some embodiments of the ligand-drug conjugate shown in formula (I), B# is an anti-Trop-2 antibody or an antigen-binding fragment thereof; preferably, B# is datopotamab, sacituzumab or an antigen-binding fragment thereof.

[0163] In some embodiments of the ligand-drug conjugate shown in formula (I), B# is an anti-Her 2 antibody or its antigen-binding fragment; preferably, B# is anbenitamab, coprelotamab, disitamab, gancotamab, margetuximab, pertuzumab, timigutuzumab, zanidatamab, trastuzumab, pertuzumab or its antigen-binding fragment; more preferably, B# is an anti-Her 2 antibody or its antigen-binding fragment; it comprises two heavy chains and two light chains, the heavy chains comprising heavy chain variable regions, the heavy chain variable regions comprising heavy chain complementarity-determining regions 1 (HCDR1) as shown in SEQ ID NO:7, 2 (HCDR2) as shown in SEQ ID NO:8, and 3 (HCDR3) as shown in SEQ ID NO:9, the light chains comprising light chain variable regions, the light chain variable regions comprising light chain complementarity-determining regions 1 (LCDR1) as shown in SEQ ID NO:10, and 3 (HCDR3) as shown in SEQ ID NO:9. The light chain complementarity-determining region 2 (LCDR2) shown in NO:11 and the light chain complementarity-determining region 3 (LCDR3) shown in SEQ ID NO:12; more preferably, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:13, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:14; more preferably, the heavy chain contains the amino acid sequence shown in SEQ ID NO:6, and the light chain contains the amino acid sequence shown in SEQ ID NO:5.

[0164] In some embodiments of the ligand-drug conjugate shown in formula (I), B# is an anti-Her3 antibody or an antigen-binding fragment thereof; preferably, B# is a barecetamab, duligotuzumab, elgemtumab, istiramumab, lumretuzumab, patritumab, seribantumab, zenocutuzumab, 202-2-1 antibody or an antigen-binding fragment thereof.

[0165] In some embodiments of the ligand-drug conjugate shown in formula (I), B# is an anti-EGFR antibody or an antigen-binding fragment thereof; preferably, B# is demupitamab, depatuxizumab, futuximab, imgatuzumab, lapatuximab, losatuxizumab, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, pimurutamab, serclutamab, tomuzotuximab, zalutumumab, Cetuximab or an antigen-binding fragment thereof.

[0166] In some embodiments of the ligand-drug conjugate shown in formula (I), B# is an anti-B7H3 antibody or its antigen-binding fragment; preferably, B# is an antibody such as 1D1, 1D1-01, 2E3, 2E3-02, enoblituzumab, mirzotamab, omburtamab or its antigen-binding fragment.

[0167] In some embodiments of the ligand-drug conjugate shown in formula (I), B# is an anti-LIV1 antibody or its antigen-binding fragment; preferably, B# is Ladiratuzumab or its antigen-binding fragment.

[0168] In some embodiments of the ligand-drug conjugate shown in formula (I), the average drug-ligand conjugation ratio n is an integer or fraction from about 1 to about 10. Preferably, the average drug-ligand conjugation ratio n is an integer or fraction from about 3 to about 8. Preferably, the average drug-ligand conjugation ratio n is an integer or fraction from about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, about 9 to about 10. Preferably, the average drug-ligand conjugation ratio n is about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8.

[0169] In some implementations, the ligand-drug conjugate represented by formula (I) is selected from the structures in Table 2 below:

[0170] Table 2

[0171] In some implementations, the ligand-drug conjugate represented by formula (I) is selected from the structures in Table 3 below:

[0172] Table 3

[0173] In Table 3, TZB or tzb represents trastuzumab, HLXB represents anti-Her2 antibody, hRS7 represents anti-Trop2 antibody, LIV1 represents anti-LIV1 antibody, FRα represents anti-FRα antibody, and ALPP represents anti-ALPP antibody.

[0174] In some embodiments, the ligand-drug conjugate represented by formula (I) is substituted with an isotope. Preferably, in some embodiments, the isotope substitution is deuterium substitution.

[0175] The second aspect of this disclosure relates to a compound or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein said compound has the structure shown in formula (IA):

[0176] L-D type (IA)

[0177] Wherein, L is a connecting unit, having the following structure: L3'—L2—L1—;

[0178] The L3' is selected from: Where m is an integer from 0 to 6, and 2 bits are connected to L2;

[0179] L2, L1 and D are defined as in equation (I).

[0180] In some embodiments of the compound of formula (IA), the R... 1 For F, Cl, Br or C 1-3 Alkyl group. Preferably, the R 1It can be F, Cl, -CH3, -CH2CH3, -CH2CH2CH3, or -CH(CH3)2. In some embodiments, the R... 1 It is Cl.

[0181] In some embodiments of the compound of formula (IA), D is selected from the following structures:

[0182] In some embodiments of the compound of formula (IA), the L3' is selected from... Where m is an integer from 0 to 6, with 1 bit connected to the ligand and 2 bits connected to L2. For example, m can be 0, 1, 2, 3, 4, 5, or 6. More preferably, m is an integer from 0 to 5. In some embodiments, m is 0. In some embodiments, m is 2. In some embodiments, m is 5.

[0183] In some embodiments of the compound of formula (IA), the L2 is absent.

[0184] In some embodiments of the compound of formula (IA), the L2 is C=O.

[0185] In some embodiments of the compound of formula (IA), the L2 is Where s is an integer from 1 to 10, with 1 bit connected to L3 and 2 bits connected to L1; each R 2a and R 2b Each is independently hydrogen, -(CH2) s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X ; Each R X Independently hydrogen or C 1-6 Alkyl group; s1 is an integer from 1 to 30, and s2 is an integer from 0 to 6.

[0186] In some embodiments of the ligand-drug conjugate shown in formula (I), the R of all methylene units of L2 2a and R 2b At least one of them is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X The rest of R 2a and R 2b All are hydrogen. Preferably, the R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L3 have R 2a and R 2bAll are hydrogen. More preferably, the R of one methylene unit of L2... 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L2 have R 2a and R 2b All are hydrogen. More preferably, the R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L2 have R 2a and R 2b Both are hydrogen.

[0187] Preferably, s is an integer from 1 to 8. For example, s is 1, 2, 3, 4, 5, 6, 7, or 8. More preferably, s is an integer from 1 to 6. More preferably, s is an integer from 3 to 6. In some embodiments, s is 3.

[0188] Preferably, s1 is an integer from 1 to 28. For example, s1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. More preferably, s1 is an integer from 4 to 26. More preferably, s1 is an integer from 8 to 24. In some embodiments, s1 is 14. In some embodiments, s1 is 16. In some embodiments, s1 is 18.

[0189] Preferably, s2 is an integer from 0 to 5. For example, s2 is 0, 1, 2, 3, 4, or 5. More preferably, s2 is an integer from 0 to 4. More preferably, s2 is an integer from 0 to 3. In some embodiments, s2 is 0. In some embodiments, s2 is 1. In some embodiments, s2 is 2.

[0190] Preferably, each RX Independently hydrogen or C 1-3 Alkyl group. More preferably, R X Ground C 1-3 Alkyl group. More preferably, R X It is -CH3 or -C2H5. In some implementations, R X It is -CH3.

[0191] In some implementations, the R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L2 have R 2a and R 2b All are hydrogen; where s2 is 0, s1 is an integer from 4 to 26, and R X The value is -CH3. Preferably, s1 is an integer from 8 to 24. More preferably, s1 is an integer from 12 to 20. In some embodiments, s1 is 14. In some embodiments, s1 is 16. In some embodiments, s1 is 18.

[0192] In some implementations, L2 is s1 is an integer from 4 to 26. Preferably, L2 is... Preferably, s1 is an integer from 8 to 24. More preferably, s1 is an integer from 12 to 20. In some embodiments, s1 is 14. In some embodiments, s1 is 16. In some embodiments, s1 is 18.

[0193] In some implementations, L2 is

[0194] In some embodiments of the compound of formula (IA), the L2 is Where r is 0 or 1, t is an integer from 0 to 30, p is 0 or 1, each q is an independent integer from 0 to 6, 1 bit is connected to L3, and 2 bits are connected to L1; R 2c Selected from hydrogen, C 1-6 Alkyl or C 3-6 Cycloalkyl.

[0195] In some implementations, r is 0. In some implementations, r is 1.

[0196] In some implementations, p is 0. In some implementations, p is 1.

[0197] In some implementations, L2 is selected from... Where t is an integer from 1 to 30, each q is an independent integer from 0 to 6, with 1 bit connected to L3 and 2 bits connected to L1; R 2c Selected from hydrogen, C 1-6 Alkyl or C 3-6 Cycloalkyl.

[0198] In some embodiments, t is an integer from 1 to 24. Preferably, t is an integer from 1 to 20. For example, t is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. More preferably, t is an integer from 1 to 12. More preferably, t is an integer from 1 to 6. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, t is 4.

[0199] In some implementations, each q is independently 0, 1, 2, 3, 4, or 5. Preferably, each q is independently 0, 1, 2, or 3.

[0200] In some implementation schemes, R 2c Selected from C 1-6 Alkyl or C 3-6 Cycloalkyl. In some embodiments, R 2c C 1-6 Alkyl group. Preferably, R 2c C 1-3 Alkyl group. More preferably, R 2c It is methyl or ethyl. In some embodiments, R 2c It is methyl. In some embodiments, R 2c It is an ethyl group.

[0201] In some embodiments of the compound of formula (IA), the L2 is Where t is an integer from 1 to 24. Preferably, t is an integer from 1 to 20. For example, t is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. More preferably, t is an integer from 1 to 12. More preferably, t is an integer from 1 to 6. In some embodiments, t is 2.

[0202] In some embodiments of the ligand-drug conjugate shown in formula (I), L2 is Where t is an integer from 1 to 24. Preferably, t is an integer from 1 to 20. For example, t is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. More preferably, t is an integer from 1 to 12. More preferably, t is an integer from 1 to 6. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, t is 4.

[0203] In some embodiments of the compound of formula (IA), the compound of formula (IA) is selected from the structures in Table 4 below:

[0204] Table 4

[0205] In some embodiments, the above-mentioned compound is substituted with an isotope. Preferably, in some embodiments, the isotope substitution is deuterium substitution.

[0206] A third aspect of this disclosure relates to a compound or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure represented by formula (IIA) or (IIIA):

[0207] Among them, R 1 Halogen or C 1-6 alkyl;

[0208] R 2 For F;

[0209] W is In this configuration, position 1 is connected to the carbonyl group, and position 2 is connected to the methylene group.

[0210] In some implementations, the R 1 For F, Cl, Br or C 1-3 Alkyl group. Preferably, the R 1 It can be F, Cl, -CH3, -CH2CH3, -CH2CH2CH3, or -CH(CH3)2. In some embodiments, the R... 1 For Cl. In some implementations, the R 1 It is -CH3.

[0211] In some embodiments, the compound is selected from the following structures:

[0212] In some embodiments, the above-mentioned compound is substituted with an isotope. Preferably, in some embodiments, the isotope substitution is deuterium substitution.

[0213] Preparation method

[0214] The fourth aspect of this disclosure relates to a method for preparing a compound of formula (I) of this disclosure or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, comprising contacting ligand B# with the structure shown in formula (IA) of this disclosure.

[0215] Composition

[0216] A fifth aspect of this disclosure relates to a pharmaceutical composition comprising, for example, a ligand-drug conjugate of formula (I) of this disclosure or a pharmaceutically acceptable salt thereof, or a compound of formula (IIA), (IIIA) or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The carrier may be selected from the group consisting of fillers (diluents), binders, wetting agents, disintegrants, and excipients. Depending on the method of administration, the composition may contain 0.1 to 99% by weight of the active compound.

[0217] Pharmaceutical compositions containing active ingredients may be in oral forms, such as tablets, sugar lozenges, tablets, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups. Oral compositions may be prepared according to any method known in the art for preparing pharmaceutical compositions, and may contain binders, fillers, lubricants, disintegrants, or pharmaceutically acceptable wetting agents, and may also contain one or more ingredients selected from the group consisting of sweeteners, flavoring agents, coloring agents, and preservatives.

[0218] Aqueous suspensions may contain active substances and excipients suitable for mixing. Aqueous suspensions may also contain one or more preservatives, such as one or more colorants, one or more flavoring agents, and one or more sweeteners. Oil suspensions can be prepared by suspending the active ingredient in vegetable oil. Oil suspensions may contain thickeners. The aforementioned sweeteners and flavoring agents may also be added.

[0219] The pharmaceutical composition may also provide the active ingredient as a dispersible powder or granules for preparing an aqueous suspension, by adding one or more of a water-mixing dispersant, wetting agent, suspending agent, or preservative. Other excipients such as sweeteners, flavoring agents, and coloring agents may also be added. These compositions are preserved by adding antioxidants such as ascorbic acid. The pharmaceutical compositions of this application may also be in the form of an oil-in-water emulsion.

[0220] The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Acceptable solvents or media that can be used include water, Ringer's solution, and isotonic sodium chloride solution. The sterile injectable formulation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase. For example, the active ingredient may be dissolved in a mixture of soybean oil and lecithin. The oil solution may then be treated with a mixture of water and glycerol to form a microemulsion. The injection solution or microemulsion may be injected into the patient's bloodstream via local large-volume injection. Alternatively, the solution and microemulsion may be administered in a manner that maintains a constant circulating concentration of the compound of this application. To maintain such a constant concentration, a continuous intravenous delivery device may be used. For example, the device may be a Deltec CADD-PLUS™ 5400 intravenous infusion pump.

[0221] The pharmaceutical composition may be in the form of a sterile injectable aqueous or oil suspension for intramuscular and subcutaneous administration. This suspension may be formulated using suitable dispersants or wetting agents and suspending agents described above, according to known techniques. The sterile injectable formulation may also be a sterile injectable solution or suspension prepared in a parenteral-acceptable, non-toxic diluent or solvent. Alternatively, a sterile fixative oil may be conveniently used as a solvent or suspension medium.

[0222] The disclosed compounds can be administered in suppository form for rectal administration. These pharmaceutical compositions can be prepared by mixing the drug with a suitable, non-irritating excipient that is solid at normal temperatures but liquid in the rectum, and thus dissolves in the rectum to release the drug. Such substances include cocoa butter, glycerin gelatin, hydrogenated vegetable oils, polyethylene glycol of various molecular weights, and mixtures of fatty acid esters of polyethylene glycol.

[0223] As is well known to those skilled in the art, the dosage of a drug depends on a variety of factors, including but not limited to: the activity of the specific compound used, the patient's age, the patient's weight, the patient's health status, the patient's behavior, the patient's diet, the timing of administration, the route of administration, the rate of excretion, and the combination of drugs; in addition, the optimal mode of treatment, such as the treatment regimen, the compound described in this disclosure or its tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, and / or the daily dosage or type of pharmaceutically acceptable salt of the compound or its tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, can be verified based on conventional treatment protocols.

[0224] Treatment and / or prevention

[0225] The sixth aspect of this disclosure relates to the use of a ligand-drug conjugate containing, for example, a ligand-drug conjugate of formula (I) of this disclosure or a pharmaceutically acceptable salt thereof, or a compound of formula (IA), (IIA), (IIIA) of this disclosure or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and / or a pharmaceutical composition thereof, in the preparation of a medicament for treating and / or preventing tumors.

[0226] This disclosure also provides the use of compounds of formula (IA), formula (IIA), formula (IIIA) or tautomers thereof, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, in the preparation of ADC medicaments for the treatment and / or prevention of tumors.

[0227] This disclosure also provides a method for treating and / or preventing tumors, comprising administering to a subject in need the ligand-drug conjugate described herein or a pharmaceutically acceptable salt thereof, or the compound described herein or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and / or a pharmaceutical composition thereof.

[0228] This disclosure also provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, or the compound thereof or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and / or a pharmaceutical composition thereof, for the treatment and / or prevention of tumors.

[0229] In some embodiments, the tumor may be selected from tumors associated with the expression of the following groups: 5T4, ADAM9, AGS-16, ANGPTL4, ALPP, ALPP2, ApoE, CTGF, CXCR5, FGF2, MCPT8, MFI2, MS4A7, NCA, Sema5b, SLITRK6, STC2, TGF, O772P, ACTA2, ADGRE1, AG-7, AIF1, AKR1C1, AKR1C2, ASLG659, Axl, B7H3, BAFF-R, BCMA, BMPR1B, BNIP3, C1QA, C1QB, CA6, CADM1, CCD79b, CCL 5. CCR5, CCR7, CD11c, CD123, CD138, CD142, CD147, CD166, CD19, CD21, CD20, CD205, CD22, CD223, CD228, CD25, CD30, CD33, CD37, CD38, CD40, CD45, CD 45(PTPRC), CD46, CD47, CD49D(ITGA4), CD56, CD66e, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CDCP1, CDH11, CDH17, CD11b, CEA, CEACAM5, Claudin 18.2. c-Met, COL6A3, COL7A1, CRIPTO, CSF1R, CTSD, CTSS, CXCL11, CXCL10, DDIT4, DLL3, DLL4, DR5, E16, EFNA4, EGFR, EGF RvIII, EGLN, EGLN3, EMR2, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FcRH2, FcRHl, FGFR2, FGFR3, FLT3, FOLR-α, FRα, GD2, G EDA, GPC-1, GPNMB, GPR20, GZMB, HER2, HER3, HLA-DOB, HMOX1, IFI6, IFNG, IGF-1R, IGFBP3, IL10RA1, IL-13R, IL-2, IL 20Ra, IL-3, IL-4, IL-6, IRTA2, KISS1R, KRT33A, LIV1, LOX, LRP-1, LRRC15, LUM, LY64, LY6E, Ly86, LYPD3, MDP, MMP10, MMP14, MMP16, MPF, MSG783, MSLN, MUC-1, NaPi2b, Napi3b, Nectin-4, NOG, P2X5, pCAD, P-Cadherin, PDGFRA, PDK1, PD-L1, PFKFB3, PGF, PGK1, PIK3AP1, PIK3CD, PLOD2, PSCA, PSCAhlg, PSMA, PTK7, P-cadherin, RNF43, ROR1, ROR2, SERPINE1, S LC39A6, SLTRK6, STAT1, STEAP1, STEAP2, TCF4, TENB2, TGFB1, TGFB2, TGFBR1, TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, WNT5A, epidermal growth factor, short proteoglycans, mesothelin, endothelial peptide receptors, mucins (such as mucin 1 and mucin 16), guanylate cyclase C, integrin α4β7, integrin α5β6, trophoblast cell glycoproteins, and tissue factor.

[0230] In some embodiments, the tumor may be selected from tumors associated with the expression of the following groups: ALPP, HER2, HER3, B7H3, TROP2, LIV1, Claudin 18.2, CD30, CD33, CD70, EGFR, or FRα.

[0231] In some implementations, the tumor is a tumor associated with ALPP expression.

[0232] In some implementations, the tumor is a tumor associated with HER2 expression.

[0233] In some implementations, the tumor is a tumor associated with HER3 expression.

[0234] In some implementations, the tumor is a tumor associated with B7H3 expression.

[0235] In some implementations, the tumor is a tumor associated with TROP2 expression.

[0236] In some implementations, the tumor is a tumor associated with LIV1 expression.

[0237] In some implementations, the tumor is a tumor associated with Claudin 18.2 expression.

[0238] In some implementations, the tumor is a tumor associated with CD30 expression.

[0239] In some implementations, the tumor is a tumor associated with CD33 expression.

[0240] In some implementations, the tumor is a tumor associated with CD70 expression.

[0241] In some implementations, the tumor is a tumor associated with EGFR expression.

[0242] In some implementations, the tumor is a tumor associated with FRα expression.

[0243] In some implementations, the tumor may be selected from the group consisting of: lung cancer, breast cancer, rectal cancer, colon cancer, esophageal cancer, gastric cancer, liver cancer, gallbladder cancer, bile duct cancer, kidney cancer, bladder cancer, urothelial carcinoma, head and neck cancer, nasopharyngeal carcinoma, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, pancreatic cancer, melanoma, bone cancer, mesothelioma, gastrointestinal stromal tumor, sarcoma, glioma, thyroid cancer, salivary gland tumor, glioblastoma, neuroblastoma, gastric mucinoma, lymphoma, leukemia, plasmacytoma, sinoatrial node cell tumor, giant cell tumor of the tendon sheath, brain cancer, squamous cell carcinoma, epidermal carcinoma, and non-Hodgkin's lymphoma.

[0244] Preferably, the tumor is selected from breast cancer, gastric cancer, prostate cancer, lung cancer, pancreatic cancer, ovarian cancer, cervical cancer, endometrial cancer, kidney cancer, bladder cancer, glioma, malignant lymphoma, liver cancer, and leukemia; or

[0245] Preferably, the tumor is HER2 positive, TROP2 positive, LIV1 positive, FRα positive and / or ALPP positive.

[0246] More preferably, the tumor is HER2-positive gastric cancer or HER2-positive breast cancer; or

[0247] More preferably, the tumor is TROP2-positive human pancreatic carcinoma in situ or TROP2-positive breast cancer; or

[0248] More preferably, the tumor is a LIV1-positive renal cell carcinoma; or

[0249] More preferably, the tumor is FRα-positive endometrial cancer or FRα-positive ovarian cancer; or

[0250] Preferably, the tumor is ALPP-positive non-small cell lung cancer.

[0251] The compounds described in this application can possess inhibitory activity against the in vitro proliferation of tumor cells. This inhibitory activity can be defined as a decrease in the cell proliferation capacity of tumor cells by more than 1%, 2%, 4%, 5%, 8%, 10%, 15%, 18%, 20%, 25%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% when the compound is added to the culture medium of tumor cells, compared to the addition of a negative control or control drug. For example, the inhibitory activity can be defined as an effect on the IC50 of tumor cells. 50The value (nM) can be below 10000, below 5000, below 4000, below 3000, below 2000, below 1000, below 500, below 400, below 300, below 200, below 150, below 120, below 110, below 100, below 99, below 98, below 97, below 95, below 90, below 80, below 75, below 70, below 65, below 62, below 60, below 50, below 40, below 30, below 25, below 23, below 22, below 20, below 19, below 18, below 18.5, below 17, below 15, below 12, below 10, below 9. Below 8.5, below 7, below 6.7, below 6, below 5.9, below 5.5, below 5.0, below 4.8, below 4.5, below 4.4, below 4, below 3.5, below 3, below 2.5, below 2, below 1.5, below 1.0, below 0.5, below 0.3, below 0.29, below 0.25, below 0.21, below 0.20, below 0.18, below 0.17, below 0.15, below 0.12, below 0.10, below 0.09, below 0.08, below 0.07, below 0.06, below 0.05, below 0.04, below 0.03, below 0.02, or below 0.01. For example, the tumor cells may include, but are not limited to, solid tumor cells, such as gastric cancer cells or breast cancer cells, such as MCF-7 cells, MCF7... Y537S Cells, PC-3 cells, DA-MB-361 cells, BT-20 cells, AGS cells, MDA-MB-468 cells, BT474 cells, T47D cells, MCF7-LIV1 cells, NCI-N87 cells, JIMT-1 cells, or MBA-MB-231 cells.

[0252] The compounds described in this application may possess targeted inhibitory properties. This targeted inhibitory property can be defined as follows: when the compound of this application is added to the culture medium of tumor cells highly expressing a specific target, compared to the addition of a negative control or control drug, the cell proliferation capacity of the tumor cells highly expressing the specific target decreases by more than 1%, 2%, 4%, 5%, 8%, 10%, 15%, 18%, 20%, 25%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. For example, the targeted inhibitory property can be defined as an IC50 effect on tumor cells highly expressing a specific target. 50The value (nM) can be below 10000, below 5000, below 4000, below 3000, below 2000, below 1000, below 500, below 400, below 300, below 200, below 185, below 150, below 120, below 110, below 100, below 99, below 98, below 97, below 95, below 91, below 80, below 74, below 70, below 65, below 62, below 60, below 50, below 40, below 30, below 25, below 23, below 22, below 20, below 19, below 18, below 18.5, below 17, below 15, below 12, below 10, 9 Below, below 8.5, below 7, below 6.7, below 6, below 5.9, below 5.5, below 5.0, below 4.8, below 4.5, below 4.4, below 4, below 3.5, below 3, below 2.5, below 2, below 1.5, below 1.0, below 0.5, below 0.3, below 0.29, below 0.25, below 0.21, below 0.20, below 0.18, below 0.17, below 0.15, below 0.12, below 0.10, below 0.09, below 0.08, below 0.07, below 0.06, below 0.05, below 0.04, below 0.03, below 0.02, or below 0.01. For example, tumor cells that highly express the specific target may include, but are not limited to, solid tumor cells. For instance, tumor cells that highly express the specific target may include, but are not limited to, gastric cancer cells or breast cancer cells. For example, tumor cells that highly express the specific target may include, but are not limited to, BT474 cells, T47D cells, or MCF7-LIV1 cells. The specific target may include, but is not limited to, HER2, TROP2, or LIV1.

[0253] The compounds described in this application may possess plasma stability. This plasma stability means that when the compound is added to plasma, the release rate of the cytotoxic drug released by the compound does not exceed 50%, 40%, 30%, 20%, 10%, 7%, 5%, 4%, 3%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%.

[0254] The compounds described in this application can have an in vivo tumor-suppressing effect. This tumor-suppressing effect can be demonstrated by the following: when the compounds of this application are administered to animals, compared to the addition of a negative control or control drug, the tumor volume of the animals is reduced by more than 1%, 2%, 4%, 5%, 8%, 10%, 15%, 18%, 20%, 25%, 40%, 50%, 55%, 60%, 70%, 73%, or 75% at 1, 3, 5, 7, 14, 20, 21, or 30 days, respectively. The tumor volume of the animal decreased by more than 1.1 times, 1.3 times, 1.5 times, 2 times, 3 times, 5 times, 10 times, 20 times, 22 times, 30 times, 50 times, 100 times, 500 times, 1000 times, or 1500 times or more at 1 day, 3 days, 5 days, 7 days, 14 days, 20 days, 21 days, or 30 days, or compared to the administration of a negative control or control drug. The animal may include, but is not limited to, mammals, such as cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, monkeys, or humans. The administration may include, but is not limited to, oral administration, intravenous injection, intravenous infusion, intraperitoneal injection, or local administration.

[0255] The compounds described in this application may exhibit a bystander effect. This bystander effect can mean that the compounds of this application have no significant inhibitory effect on the cell proliferation of tumor cells with low expression of a specific target, but in co-culture of tumor cells with low expression of a specific target and tumor cells with high expression of a specific target, the compounds of this application can simultaneously inhibit the cell proliferation of both types of tumor cells. For example, in co-culture of tumor cells with low expression of a specific target and tumor cells with high expression of a specific target, the inhibitory activity can be defined as an IC50 value against tumor cells with low expression of a specific target. 50The value (nM) can be below 10000, below 5000, below 4000, below 3000, below 2000, below 1000, below 500, below 400, below 300, below 200, below 185, below 150, below 120, below 110, below 100, below 99, below 98, below 97, below 95, below 91, below 80, below 74, below 70, below 65, below 62, below 60, below 50, below 40, below 30, below 25, below 23, below 22, below 20, below 19, below 18, below 18.5, below 17, below 15, below 12, below 10, 9 Below, below 8.5, below 7, below 6.7, below 6, below 5.9, below 5.5, below 5.0, below 4.8, below 4.5, below 4.4, below 4, below 3.5, below 3, below 2.5, below 2, below 1.5, below 1.0, below 0.5, below 0.3, below 0.29, below 0.25, below 0.21, below 0.20, below 0.18, below 0.17, below 0.15, below 0.12, below 0.10, below 0.09, below 0.08, below 0.07, below 0.06, below 0.05, below 0.04, below 0.03, below 0.02, or below 0.01. Compared to tumor cells that highly express a specific target, tumor cells with low expression of that target may exhibit a reduction in the expression of that specific target by more than 1%, 2%, 4%, 5%, 8%, 10%, 15%, 18%, 20%, 25%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. For example, tumor cells with high expression of the specific target may include, but are not limited to, solid tumor cells, such as gastric cancer cells or breast cancer cells, including but not limited to BT474 cells, T47D cells, or MCF7-LIV1 cells. Similarly, tumor cells with low expression of the specific target may include, but are not limited to, solid tumor cells, such as breast cancer cells, including but not limited to HCC1187 cells.

[0256] The compounds described in this application may possess antitransporter transport capabilities. This antitransporter capability can be defined as a reduction in the efflux ratio of the compound compared to a standard of the transport substrate, which is more than 1%, 2%, 4%, 5%, 8%, 10%, 15%, 18%, 20%, 25%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. For example, the efflux ratio test can be a method commonly used by those skilled in the art, or it can be described in the embodiments of this application.

[0257] The compounds described in this application may possess in vivo tumor-targeting capability. This in vivo targeting capability can refer to the distribution of the labeled compound in the tumor tissue of an animal, compared to other tissues and organs, being increased by more than 1%, 2%, 4%, 5%, 8%, 10%, 15%, 18%, 20%, 25%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or by more than 1.1 times, 1.3 times, 1.5 times, 2 times, 3 times, 5 times, 10 times, 20 times, 22 times, 30 times, 50 times, 100 times, 500 times, 1000 times, or 1500 times. The signaling substance may be a radioactive substance, and for example, the signaling substance includes, but is not limited to, radioactive substances. 125 I. The animal may include, but is not limited to, mammals, such as cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, monkeys, or humans. The administration may include, but is not limited to, oral administration, intravenous injection, intravenous infusion, intraperitoneal injection, or local administration. The tissue or organ may include, but is not limited to, the heart, liver, spleen, lungs, kidneys, brain, or bone marrow.

[0258] The compounds described in this application can exhibit good in vivo safety. This in vivo safety can be defined as the release rate of free toxins in animals after administration of the compounds to animals not exceeding 50%, 40%, 30%, 20%, 10%, 7%, 5%, 4%, 3%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%. For example, the in vivo safety can be defined as the absence of toxic effects in animals. The application concentration of the compound described in this application can be 0.5 mg / kg or higher, 1 mg / kg or higher, 2 mg / kg or higher, 3 mg / kg or higher, 4 mg / kg or higher, 5 mg / kg or higher, 10 mg / kg or higher, 20 mg / kg or higher, 30 mg / kg or higher, 50 mg / kg or higher, 70 mg / kg or higher, 100 mg / kg or higher, 200 mg / kg or higher, 500 mg / kg or higher, or 1000 mg / kg or higher. For example, the animals may include, but are not limited to, cats, dogs, horses, pigs, dairy cows, sheep, rabbits, mice, rats, monkeys, or humans. The application may include, but is not limited to, oral administration, intravenous injection, intravenous infusion, intraperitoneal injection, or local administration.

[0259] Reagent test kit

[0260] A seventh aspect of this disclosure relates to a kit containing, for example, a ligand-drug conjugate of formula (I) of this disclosure or a pharmaceutically acceptable salt thereof, or a compound of formula (IIA), formula (IIIA) or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and / or a pharmaceutical composition of this disclosure.

[0261] Other aspects and advantages of this disclosure will readily become apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this disclosure are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this disclosure enables them to make modifications to the specific embodiments disclosed without departing from the spirit and scope of the invention to which this disclosure pertains. Accordingly, the descriptions in the accompanying drawings and specification of this disclosure are merely exemplary and not restrictive.

[0262] In addition, this disclosure also provides the following implementation schemes:

[0263] 1. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate has the structure shown in formula (I):

[0264] B#—(L—D)n formula (I);

[0265] B# is the ligand that binds to the target.

[0266] n is the average conjugation ratio of the drug ligands, and n is an integer or decimal from about 1 to about 16;

[0267] L is a connecting unit with the following structure: —L3—L2—L1—; where,

[0268] The L3 is selected from Where m is an integer from 0 to 8, with 1 bit connected to the ligand and 2 bits connected to L2;

[0269] The L2 does not exist or is selected from C=O. Where r is 0 or 1, s is an integer from 1 to 10, t is an integer from 0 to 30, p is 0 or 1, and each q is an independent integer from 0 to 6, with 1 bit connected to L3 and 2 bits connected to L1; each R 2a and R 2b Each is independently hydrogen, -(CH2) s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X ; Each R X Independently hydrogen or C 1-6 Alkyl group; s1 is an integer from 1 to 30, s2 is an integer from 0 to 6; R 2c Selected from hydrogen, C 1-6 Alkyl or C 3-6 cycloalkyl;

[0270] Preferably, L2 is absent or selected from C=O. Where s is an integer from 1 to 10, t is an integer from 1 to 30, and each q is an independent integer from 0 to 6, with 1 bit connected to L3 and 2 bits connected to L1; each R 2a and R 2b Each is independently hydrogen, -(CH2) s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X ; Each R X Independently hydrogen or C 1-6 Alkyl group; s1 is an integer from 1 to 30, s2 is an integer from 0 to 6; R 2c Selected from hydrogen, C 1-6 Alkyl or C 3-6 cycloalkyl;

[0271] More preferably, L2 is absent or selected from C=O, Where s is an integer from 1 to 10, t is an integer from 1 to 30, 1 bit is connected to L3, and 2 bits are connected to L1; each R 2a and R 2b Each is independently hydrogen, -(CH2) s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X ; Each R X Independently hydrogen or C 1-6 Alkyl; R 2c Selected from hydrogen, C1-6 alkyl or C3-6 cycloalkyl; s1 is an integer from 1 to 30, and s2 is an integer from 0 to 6;

[0272] L1 is Of these, bit 1 is connected to L2, and bit 2 is connected to D;

[0273] D is a drug having the structure shown in formula (II) or formula (III):

[0274] Among them, R 1 Halogen or C 1-6 alkyl;

[0275] R 2 For F;

[0276] W is In this configuration, position 1 is connected to the carbonyl group, and position 2 is connected to the methylene group.

[0277] 2. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to embodiment 1, wherein the R 1 For F, Cl, Br or C 1-3 alkyl;

[0278] Preferably, the R 1 For F, Cl, -CH3, -CH2CH3, -CH2CH2CH3, or -CH(CH3)2;

[0279] More preferably, the R 1 For Cl; or

[0280] More preferably, the R 1 It is -CH3.

[0281] 3. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to embodiment 1 or 2, wherein D is selected from the following structures:

[0282] 4. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-3, wherein the L3 is selected from... Where m is an integer from 0 to 6, with 1 bit connected to the ligand and 2 bits connected to L2;

[0283] Preferably, m is an integer between 0 and 5;

[0284] More preferably, m is 0; or

[0285] More preferably, m is 2; or

[0286] Preferably, m is 5.

[0287] 5. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4, wherein the L2 is absent.

[0288] 6. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4, wherein the L2 is C=O.

[0289] 7. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4, wherein L2 is Where s is an integer from 1 to 10, with 1 bit connected to L3 and 2 bits connected to L1; each R 2a and R 2b Each is independently hydrogen, -(CH2) s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X ; Each R X Independently hydrogen or C 1-6 Alkyl group; s1 is an integer from 1 to 30, and s2 is an integer from 0 to 6.

[0290] 8. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4 or 7, wherein all methylene units of said L2 contain R 2a and R 2b At least one of them is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X The rest of R 2a and R 2b All are hydrogen;

[0291] Preferably, the R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L3 have R2a and R 2b All are hydrogen;

[0292] More preferably, the R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L2 have R 2a and R 2b All are hydrogen;

[0293] More preferably, the R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L2 have R 2a and R 2b Both are hydrogen.

[0294] 9. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4 or 7-8, wherein s is an integer from 1 to 8;

[0295] Preferably, s is an integer from 1 to 6;

[0296] Preferably, s is an integer between 3 and 6;

[0297] More preferably, s is 3.

[0298] 10. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4 or 7-9, wherein s1 is an integer from 1 to 28;

[0299] Preferably, s1 is an integer from 4 to 26;

[0300] More preferably, s1 is an integer between 8 and 24;

[0301] More preferably, s1 is 14; or

[0302] More preferably, s1 is 16; or

[0303] More preferably, s1 is 18.

[0304] 11. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4 or 7-10, wherein s2 is an integer from 0 to 5;

[0305] Preferably, s2 is an integer between 0 and 4;

[0306] Preferably, s2 is an integer between 0 and 3;

[0307] More preferably, s2 is 0; or

[0308] More preferably, s2 is 1; or

[0309] More preferably, s2 is 2.

[0310] 12. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4 or 7-11, wherein each R X Independently hydrogen or C 1-3 alkyl;

[0311] Preferably, R X C 1-3 alkyl;

[0312] More preferably, R X It is -CH3 or -C2H5;

[0313] More preferably, R X It is -CH3.

[0314] 13. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4 or 7-12, wherein the R of one methylene unit of said L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L2 have R 2a and R 2b All are hydrogen; where s2 is 0, s1 is an integer from 4 to 26, and R X -CH3;

[0315] Preferably, s1 is an integer between 8 and 24;

[0316] Preferably, s1 is an integer between 12 and 20;

[0317] More preferably, s1 is 14; or

[0318] More preferably, s1 is 16; or

[0319] More preferably, s1 is 18.

[0320] 14. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4 or 7-13, wherein L2 is s1 is an integer between 4 and 26;

[0321] Preferably, L2 is

[0322] Preferably, s1 is an integer between 8 and 24;

[0323] Preferably, s1 is an integer between 12 and 20;

[0324] More preferably, s1 is 14; or

[0325] More preferably, s1 is 16; or

[0326] More preferably, s1 is 18.

[0327] 15. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4, wherein L2 is Where r is 0 or 1, t is an integer from 0 to 30, p is 0 or 1, each q is an independent integer from 0 to 6, 1 bit is connected to L3, and 2 bits are connected to L1; R 2c Selected from hydrogen, C 1-6 Alkyl or C 3-6 Cycloalkyl.

[0328] 16. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4 or 15, wherein the L2 is selected from... Where t is an integer from 1 to 30, each q is an independent integer from 0 to 6, with 1 bit connected to L3 and 2 bits connected to L1; R 2c Selected from hydrogen, C 1-6 Alkyl or C 3-6 Cycloalkyl.

[0329] 17. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4 or 15-16, wherein t is an integer from 1 to 24;

[0330] Preferably, t is an integer from 1 to 20;

[0331] Preferably, t is an integer from 1 to 12;

[0332] More preferably, t is an integer from 1 to 6;

[0333] More preferably, t is 1; or

[0334] More preferably, t is 2; or

[0335] More preferably, t is 3; or

[0336] More preferably, t is 4.

[0337] 18. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4 or 15-17, wherein each q is independently 0, 1, 2, 3, 4 or 5;

[0338] Preferably, each q is 0, 1, 2 or 3 independently.

[0339] 19. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4 or 15-18, wherein R 2c Selected from C 1-6 Alkyl or C 3-6 cycloalkyl;

[0340] Preferably, R 2c C 1-6 alkyl;

[0341] More preferably, R 2c C 1-3 alkyl;

[0342] More preferably, R 2c It is methyl or ethyl;

[0343] More preferably, R 2c Methyl; or

[0344] More preferably, R 2c It is an ethyl group.

[0345] 20. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4 or 15-19, wherein L2 is Where t is an integer from 1 to 24;

[0346] Preferably, t is an integer from 1 to 20;

[0347] Preferably, t is an integer from 1 to 12;

[0348] More preferably, t is an integer from 1 to 6;

[0349] More preferably, t is 2.

[0350] 21. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4 or 15-19, wherein L2 is Where t is an integer from 1 to 24;

[0351] Preferably, t is an integer from 1 to 20;

[0352] Preferably, t is an integer from 1 to 12;

[0353] More preferably, t is an integer from 1 to 6;

[0354] More preferably, t is 1; or

[0355] More preferably, t is 2; or

[0356] More preferably, t is 3; or

[0357] More preferably, t is 4.

[0358] 22. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-21, wherein L is selected from the structures in Table 1.

[0359] 23. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-22, wherein B# may be an antibody or an antigen-binding fragment thereof.

[0360] 24. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-23, wherein the B# is selected from the group consisting of chimeric antibodies, humanized antibodies and fully human antibodies.

[0361] 25. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-24, wherein B# is an antibody targeting the following targets: 5T4, ADAM9, AGS-16, ANGPTL4, ALPP, ALPP2, ApoE, CTGF, CXCR5, FGF2, MCPT8, MFI2, MS4A7, NCA, Sema5b, SLITRK6, STC2, TGF, O772P, ACTA2, ADGRE1, AG-7, AIF1, AKR1C1, AKR1C2, ASLG659, Axl, B7H3, BAFF-R, BCMA, BMPR1B, BNIP3, C1QA, C1QB, CA6, CA DM1, CCD79b, CCL5, CCR5, CCR7, CD11c, CD123, CD138, CD142, CD147, CD166, CD19, CD21, CD20, CD205, CD22, CD223, CD228, CD25, CD30, CD33, CD37, CD38, CD40, CD45, CD45(PTPRC), CD46, CD47, CD49D(ITGA4), CD56, CD66e, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CDCP1, CDH11, CDH17, CD11b, CEA, CEACAM5, Claudin 18.2. c-Met, COL6A3, COL7A1, CRIPTO, CSF1R, CTSD, CTSS, CXCL11, CXCL10, DDIT4, DLL3, DLL4, DR5, E16, EFNA4, EGFR, EGF RvIII, EGLN, EGLN3, EMR2, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FcRH2, FcRHl, FGFR2, FGFR3, FLT3, FOLR-α, FRα, GD2, G EDA, GPC-1, GPNMB, GPR20, GZMB, HER2, HER3, HLA-DOB, HMOX1, IFI6, IFNG, IGF-1R, IGFBP3, IL10RA1, IL-13R, IL-2, IL 20Ra, IL-3, IL-4, IL-6, IRTA2, KISS1R, KRT33A, LIV1, LOX, LRP-1, LRRC15, LUM, LY64, LY6E, Ly86, LYPD3, MDP, MMP10, M MP14, MMP16, MPF, MSG783, MSLN, MUC-1, NaPi2b, Napi3b, Nectin-4, NOG, P2X5, pCAD, P-Cadherin, PDGFRA, PDK1, PD-L1, PFKFB3, PGF, PGK1, PIK3AP1, PIK3CD, PLOD2, PSCA, PSCAhlg, PSMA, PTK7, P-cadherin, RNF43, ROR1, ROR2, SERPINE1, SL C39A6, SLTRK6, STAT1, STEAP1, STEAP2, TCF4, TENB2, TGFB1, TGFB2, TGFBR1, TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, WNT5A, epidermal growth factor, short proteoglycans, mesothelin, endothelial peptide receptors, mucins (such as mucin 1 and mucin 16), guanylate cyclase C, integrin α4β7, integrin α5β6, trophoblast cell glycoproteins, or tissue factor.

[0362] 26. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-25, wherein the B# is an antibody targeting the following targets: ALPP, HER2, HER3, B7H3, TROP2, Claudin 18.2, CD30, CD33, CD70, EGFR, or FRα.

[0363] 27. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-26, wherein B# is an anti-Trop-2 antibody or an antigen-binding fragment thereof;

[0364] Preferably, B# is datopotamab, sacituzumab, or an antigen-binding fragment thereof.

[0365] 28. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-26, wherein B# is an anti-Her 2 antibody or an antigen-binding fragment thereof;

[0366] Preferably, B# is anbenitamab, coprelotamab, disitamab, gancotamab, margetuximab, timigutuzumab, zanidatamab, trastuzumab, pertuzumab, or an antigen-binding fragment thereof.

[0367] More preferably, B# is an anti-Her 2 antibody or its antigen-binding fragment; it comprises two heavy chains and two light chains, the heavy chains comprising heavy chain variable regions, the heavy chain variable regions comprising heavy chain complementarity-determining region 1 (HCDR1) as shown in SEQ ID NO:7, heavy chain complementarity-determining region 2 (HCDR2) as shown in SEQ ID NO:8, and heavy chain complementarity-determining region 3 (HCDR3) as shown in SEQ ID NO:9, the light chains comprising light chain variable regions, the light chain variable regions comprising light chain complementarity-determining region 1 (LCDR1) as shown in SEQ ID NO:10, light chain complementarity-determining region 2 (LCDR2) as shown in SEQ ID NO:11, and light chain complementarity-determining region 3 (LCDR3) as shown in SEQ ID NO:12;

[0368] More preferably, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:13, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:14;

[0369] More preferably, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:6, and the light chain comprises the amino acid sequence shown in SEQ ID NO:5.

[0370] 29. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-26, wherein B# is an anti-Her3 antibody or an antigen-binding fragment thereof;

[0371] Preferably, B# is a barecetamab, duligotuzumab, elgemtumab, istiramumab, lumretuzumab, patritumab, seribantumab, zenocutuzumab, 202-2-1 antibody or its antigen-binding fragment.

[0372] 30. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-26, wherein B# is an anti-EGFR antibody or an antigen-binding fragment thereof;

[0373] Preferably, B# is demupitamab, depatuxizumab, futuximab, imgatuzumab, lapatuximab, losatuxizumab, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, pimurutamab, serclutamab, tomuzotuximab, zalutumumab, Cetuximab, or an antigen-binding fragment thereof.

[0374] 31. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-26, wherein B# is an anti-B7H3 antibody or an antigen-binding fragment thereof;

[0375] Preferably, B# is 1D1, 1D1-01, 2E3, 2E3-02 antibody, enoblituzumab, mirzotamab, omburtamab, or an antigen-binding fragment thereof.

[0376] 32. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-26, wherein B# is an anti-LIV1 antibody or an antigen-binding fragment thereof;

[0377] Preferably, B# is Ladiratuzumab or its antigen-binding fragment.

[0378] 33. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-26, wherein the average conjugation ratio n of the drug ligand is an integer or decimal of about 1 to about 10;

[0379] Preferably, the average conjugation ratio n of the drug ligand is an integer or decimal of about 3 to about 8; or

[0380] Preferably, the average conjugation ratio n of the drug ligand is an integer or decimal of about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, or about 9 to about 10; or

[0381] Preferably, the average conjugation ratio n of the drug ligand is about 1, about 2, about 3, about 4, about 5, about 6, about 7 or about 8.

[0382] 34. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-33, wherein the ligand-drug conjugate is selected from the structures in Table 2.

[0383] 35. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-34, wherein the ligand-drug conjugate is selected from the structures in Table 3.

[0384] 36. A compound or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the compound has the structure shown in formula (IA):

[0385] L-D type (IA)

[0386] Wherein, L is a connecting unit, having the following structure: L3'—L2—L1—;

[0387] The L3' is selected from: Where m is an integer from 0 to 6, and 2 bits are connected to L2;

[0388] L2, L1, and D are as defined in any of the implementation schemes 1-35.

[0389] 37. The compound according to embodiment 36, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the R 1 For F, Cl, Br or C 1-3 alkyl;

[0390] Preferably, the R 1 For F, Cl, -CH3, -CH2CH3, -CH2CH2CH3, or -CH(CH3)2;

[0391] More preferably, the R 1 For Cl; or

[0392] More preferably, the R 1 It is -CH3.

[0393] 38. The compound according to embodiment 36 or 37, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein D is selected from the following structures:

[0394] 39. The compound according to any one of embodiments 36-38, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the L3' is selected from... Where m is an integer from 0 to 6, with 1 bit connected to the ligand and 2 bits connected to L2;

[0395] Preferably, m is an integer between 0 and 5;

[0396] More preferably, m is 0; or

[0397] More preferably, m is 2; or

[0398] Preferably, m is 5.

[0399] 40. The compound according to any one of embodiments 36-39, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the L2 is absent.

[0400] 41. The compound according to any one of embodiments 36-39, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the L2 is C=O.

[0401] 42. The compound according to any one of embodiments 36-39, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein L2 is Where s is an integer from 1 to 10, with 1 bit connected to L3 and 2 bits connected to L1; each R 2a and R 2b Each is independently hydrogen, -(CH2) s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -RX -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X ; Each R X Independently hydrogen or C 1-6 Alkyl group; s1 is an integer from 1 to 30, and s2 is an integer from 0 to 6.

[0402] 43. The compound according to any one of embodiments 36-39 or 42, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein all methylene units of said L2 contain R 2a and R 2b At least one of them is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X The rest of R 2a and R 2b All are hydrogen;

[0403] Preferably, the R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L3 have R 2a and R 2b All are hydrogen;

[0404] More preferably, the R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L2 have R 2a and R 2b All are hydrogen;

[0405] More preferably, the R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L2 have R 2a and R 2b Both are hydrogen.

[0406] 44. The compound or its tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, according to any one of embodiments 36-39 or 42-43, wherein s is an integer from 1 to 8;

[0407] Preferably, s is an integer from 1 to 6;

[0408] Preferably, s is an integer from 3 to 6.

[0409] More preferably, s is 3.

[0410] 45. The compound or its tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, according to any one of embodiments 36-39 or 42-44, wherein s1 is an integer from 1 to 28;

[0411] Preferably, s1 is an integer from 4 to 26;

[0412] Preferably, s1 is an integer between 8 and 24.

[0413] More preferably, s1 is 14; or

[0414] More preferably, s1 is 16; or

[0415] More preferably, s1 is 18.

[0416] 46. ​​The compound or its tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, according to any one of embodiments 36-39 or 42-45, wherein s2 is an integer from 0 to 6;

[0417] Preferably, s2 is an integer between 0 and 5;

[0418] Preferably, s2 is an integer between 0 and 3;

[0419] More preferably, s2 is 0; or

[0420] More preferably, s2 is 1; or

[0421] More preferably, s2 is 2.

[0422] 47. The compound or its tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, according to any one of embodiments 36-39 or 42-46, wherein each R X Independently hydrogen or C 1-3 alkyl;

[0423] Preferably, R X C 1-3 alkyl;

[0424] More preferably, R X It is -CH3 or -C2H5;

[0425] More preferably, R X It is -CH3.

[0426] 48. The compound according to any one of embodiments 36-39 or 42-47, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the R of one methylene unit of said L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L2 have R 2a and R 2b All are hydrogen; where s2 is 0, s1 is an integer from 4 to 26, and R X -CH3;

[0427] Preferably, s1 is an integer between 8 and 24;

[0428] Preferably, s1 is an integer between 12 and 20;

[0429] More preferably, s1 is 14; or

[0430] More preferably, s1 is 16; or

[0431] More preferably, s1 is 18.

[0432] 49. The compound or its tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, according to any one of embodiments 36-39 or 42-48, wherein L2 is s1 is an integer between 4 and 26;

[0433] Preferably, L2 is

[0434] Preferably, s1 is an integer between 8 and 24;

[0435] Preferably, s1 is an integer between 12 and 20;

[0436] More preferably, s1 is 14; or

[0437] More preferably, s1 is 16; or

[0438] More preferably, s1 is 18.

[0439] 50. The compound according to any one of embodiments 36-39, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein L2 is Where r is 0 or 1, t is an integer from 0 to 30, p is 0 or 1, each q is an independent integer from 0 to 6, 1 bit is connected to L3, and 2 bits are connected to L1; R 2c Selected from hydrogen, C 1-6 Alkyl or C 3-6 Cycloalkyl.

[0440] 51. The compound according to any one of embodiments 36-39 or 50, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the L2 is selected from... Where t is an integer from 1 to 30, each q is an independent integer from 0 to 6, with 1 bit connected to L3 and 2 bits connected to L1; R 2c Selected from hydrogen, C 1-6 Alkyl or C 3-6 Cycloalkyl.

[0441] 52. The compound or its tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, according to any one of embodiments 36-39 or 50-51, wherein t is an integer from 1 to 24;

[0442] Preferably, t is an integer from 1 to 20;

[0443] Preferably, t is an integer from 1 to 12;

[0444] More preferably, t is an integer from 1 to 6;

[0445] More preferably, t is 1; or

[0446] More preferably, t is 2; or

[0447] More preferably, t is 3; or

[0448] More preferably, t is 4.

[0449] 53. The compound or its tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, according to any one of embodiments 36-39 or 50-52, wherein each q is independently 0, 1, 2, 3, 4 or 5;

[0450] Preferably, each q is 0, 1, 2 or 3 independently.

[0451] 54. The compound or its tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, according to any one of embodiments 36-39 or 50-53, wherein R 2c Selected from C 1-6 Alkyl or C 3-6 cycloalkyl;

[0452] Preferably, R 2c C 1-6 alkyl;

[0453] More preferably, R 2c C 1-3 alkyl;

[0454] More preferably, R 2c It is methyl or ethyl;

[0455] More preferably, R 2c Methyl; or

[0456] More preferably, R 2c It is an ethyl group.

[0457] 55. The compound or its tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, according to any one of embodiments 36-39 or 50-54, wherein L2 is Where t is an integer from 1 to 24;

[0458] Preferably, t is an integer from 1 to 20;

[0459] Preferably, t is an integer from 1 to 12;

[0460] More preferably, t is an integer from 1 to 6;

[0461] More preferably, t is 2.

[0462] 56. The compound or its tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, according to any one of embodiments 36-39 or 50-54, wherein L2 is Where t is an integer from 1 to 24;

[0463] Preferably, t is an integer from 1 to 20;

[0464] Preferably, t is an integer from 1 to 12;

[0465] More preferably, t is an integer from 1 to 6;

[0466] More preferably, t is 1; or

[0467] More preferably, t is 2; or

[0468] More preferably, t is 3; or

[0469] More preferably, t is 4.

[0470] 57. The compound according to any one of embodiments 36-56, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from Table 4.

[0471] 58. A compound or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure represented by formula (IIA) or (IIIA):

[0472] Among them, R 1 Halogen or C 1-6 alkyl;

[0473] R 2 For F;

[0474] W is In this configuration, position 1 is connected to the carbonyl group, and position 2 is connected to the methylene group.

[0475] 59. The compound according to embodiment 58, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the R 1 For F, Cl, Br or C 1-3 alkyl;

[0476] Preferably, the R 1For F, Cl, -CH3, -CH2CH3, -CH2CH2CH3, or -CH(CH3)2;

[0477] More preferably, the R 1 For Cl;

[0478] More preferably, the R 1 It is -CH3.

[0479] 60. The compound according to embodiment 58 or 59, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the following structures:

[0480] 61. A method for preparing a ligand-drug conjugate or a pharmaceutically acceptable salt thereof as shown in formula (I) of any one of embodiments 1-35, characterized in that the method comprises contacting ligand B# with the structure shown in formula (IA) of any one of embodiments 36-57.

[0481] 62. A pharmaceutical composition, characterized in that the pharmaceutical composition contains a ligand-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1-35, and a pharmaceutically acceptable carrier.

[0482] 63. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the compound of any one of embodiments 58-60, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0483] 64. Use of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-35, or a pharmaceutical composition according to embodiment 62, in the preparation of a medicament for treating and / or preventing tumors.

[0484] 65. Use of a compound according to any one of embodiments 36-57, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating and / or preventing tumors;

[0485] Preferably, the drug is a ligand-drug conjugate.

[0486] 66. Use of a compound according to embodiments 58-60, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to embodiment 63, in the preparation of a medicament for treating and / or preventing tumors.

[0487] 67. A method of treating and / or preventing tumors, comprising administering to a subject in need the ligand-drug conjugate or a pharmaceutically acceptable salt thereof, as described in any one of embodiments 1-35, or the pharmaceutical composition described in embodiment 62.

[0488] 68. A method of treating and / or preventing tumors, comprising administering to a subject in need a compound or tautomer of any one of embodiments 58-60, or a racemic, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of embodiment 63.

[0489] 69. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-35, or a pharmaceutical composition according to embodiment 62, for the treatment and / or prevention of tumors.

[0490] 70. A compound according to any one of embodiments 58-60, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to embodiment 63, for the treatment and / or prevention of tumors.

[0491] 71. The use according to any one of embodiments 64-66, the method of embodiment 67 or 68, the ligand-drug conjugate of embodiment 69 or a pharmaceutically acceptable salt or pharmaceutical composition thereof, the compound of embodiment 70 or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, characterized in that the tumor is selected from tumors associated with expression of the following group: 5T4, ADAM9, AGS-16, ANGPTL4, ALPP, ALPP2, ApoE, CTGF, CXCR5, FGF2, MCPT8, MFI2, MS4A7, NCA, Sema5b, SLITRK6, STC2, TGF, O772P, ACTA2, ADGRE1, AG-7, AIF1, AKR1C1, AKR1C2, ASLG6 59, Axl, B7H3, BAFF-R, BCMA, BMPR1B, BNIP3, C1QA, C1QB, CA6, CADM1, CCD79b, CCL5, CCR5, CCR7 , CD11c, CD123, CD138, CD142, CD147, CD166, CD19, CD21, CD20, CD205, CD22, CD223, CD228, CD25 , CD30, CD33, CD37, CD38, CD40, CD45, CD45(PTPRC), CD46, CD47, CD49D(ITGA4), CD56, CD66e, C D70, CD71, CD72, CD74, CD79a, CD79b, CD80, CDCP1, CDH11, CDH17, CD11b, CEA, CEACAM5, Claudin 18.2. c-Met, COL6A3, COL7A1, CRIPTO, CSF1R, CTSD, CTSS, CXCL11, CXCL10, DDIT4, DLL3, DLL4, DR5, E16, EFNA4, EGFR, EGF RvIII, EGLN, EGLN3, EMR2, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FcRH2, FcRHl, FGFR2, FGFR3, FLT3, FOLR-α, FRα, GD2, G EDA, GPC-1, GPNMB, GPR20, GZMB, HER2, HER3, HLA-DOB, HMOX1, IFI6, IFNG, IGF-1R, IGFBP3, IL10RA1, IL-13R, IL-2, IL 20Ra, IL-3, IL-4, IL-6, IRTA2, KISS1R, KRT33A, LIV1, LOX, LRP-1, LRRC15, LUM, LY64, LY6E, Ly86, LYPD3, MDP, MMP10, MMP14, MMP16, MPF, MSG783, MSLN, MUC-1, NaPi2b, Napi3b, Nectin-4, NOG, P2X5, pCAD, P-Cadherin, PDGFRA, PDK1, PD-L1, PFKFB3, PGF, PGK1, PIK3AP1, PIK3CD, PLOD2, PSCA, PSCAhlg, PSMA, PTK7, P-cadherin, RNF43, ROR1, ROR2, SERPINE1, S LC39A6, SLTRK6, STAT1, STEAP1, STEAP2, TCF4, TENB2, TGFB1, TGFB2, TGFBR1, TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, WNT5A, epidermal growth factor, short proteoglycans, mesothelin, endothelial peptide receptors, mucins (such as mucin 1 and mucin 16), guanylate cyclase C, integrin α4β7, integrin α5β6, trophoblast cell glycoproteins, and tissue factor.

[0492] 72. The use according to any one of embodiments 64-66, the method of embodiment 67 or 68, the ligand-drug conjugate of embodiment 69 or a pharmaceutically acceptable salt or pharmaceutical composition thereof, the compound of embodiment 70 or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, characterized in that the tumor is selected from tumors associated with expression of the following group: ALPP, HER2, HER3, B7H3, TROP2, LIV1, Claudin 18.2, CD30, CD33, CD70, EGFR, or FRα.

[0493] 73. The use according to any one of embodiments 64-66, the method of embodiment 67 or 68, the ligand-drug conjugate of embodiment 69 or a pharmaceutically acceptable salt or pharmaceutical composition thereof, the compound of embodiment 70 or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, characterized in that the tumor is a tumor associated with HER2 expression.

[0494] 74. The use according to any one of embodiments 64-66, the method of embodiment 67 or 68, the ligand-drug conjugate of embodiment 69 or a pharmaceutically acceptable salt or pharmaceutical composition thereof, the compound of embodiment 70 or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, characterized in that the tumor is a tumor associated with HER3 expression.

[0495] 75. The use according to any one of embodiments 64-66, the method of embodiment 67 or 68, the ligand-drug conjugate of embodiment 69 or a pharmaceutically acceptable salt or pharmaceutical composition thereof, the compound of embodiment 70 or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, characterized in that the tumor is a tumor associated with B7H3 expression.

[0496] 76. The use according to any one of embodiments 64-66, the method of embodiment 67 or 68, the ligand-drug conjugate of embodiment 69 or a pharmaceutically acceptable salt or pharmaceutical composition thereof, the compound of embodiment 70 or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, characterized in that the tumor is a tumor associated with TROP2 expression.

[0497] 77. The use according to any one of embodiments 64-66, the method of embodiment 67 or 68, the ligand-drug conjugate of embodiment 69 or a pharmaceutically acceptable salt or pharmaceutical composition thereof, the compound of embodiment 70 or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, characterized in that the tumor is a tumor associated with LIV1 expression.

[0498] 78. The use according to any one of embodiments 64-66, the method of embodiment 67 or 68, the ligand-drug conjugate of embodiment 69 or a pharmaceutically acceptable salt or pharmaceutical composition thereof, the compound of embodiment 70 or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, characterized in that the tumor is a tumor associated with Claudin 18.2 expression.

[0499] 79. The use according to any one of embodiments 64-66, the method of embodiment 67 or 68, the ligand-drug conjugate of embodiment 69 or a pharmaceutically acceptable salt or pharmaceutical composition thereof, the compound of embodiment 70 or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, characterized in that the tumor is a tumor associated with CD30 expression.

[0500] 80. The use according to any one of embodiments 64-66, the method of embodiment 67 or 68, the ligand-drug conjugate of embodiment 69 or a pharmaceutically acceptable salt or pharmaceutical composition thereof, the compound of embodiment 70 or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, characterized in that the tumor is a tumor associated with CD33 expression.

[0501] 81. The use according to any one of embodiments 64-66, the method according to embodiment 67 or 68, the ligand-drug conjugate according to embodiment 69 or a pharmaceutically acceptable salt or pharmaceutical composition thereof, the compound according to embodiment 70 or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, characterized in that the tumor is a tumor associated with CD70 expression.

[0502] 82. The use according to any one of embodiments 64-66, the method of embodiment 67 or 68, the ligand-drug conjugate of embodiment 69 or a pharmaceutically acceptable salt or pharmaceutical composition thereof, the compound of embodiment 70 or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, characterized in that the tumor is a tumor associated with EGFR expression.

[0503] 83. The use according to any one of embodiments 64-66, the method of embodiment 67 or 68, the ligand-drug conjugate of embodiment 69 or a pharmaceutically acceptable salt or pharmaceutical composition thereof, the compound of embodiment 70 or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, characterized in that the tumor is a tumor associated with ALPP expression.

[0504] 84. The use according to any one of embodiments 64-66, the method of embodiment 67 or 68, the ligand-drug conjugate of embodiment 69 or a pharmaceutically acceptable salt or pharmaceutical composition thereof, the compound of embodiment 70 or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, characterized in that the tumor is a tumor associated with FRα expression.

[0505] 85. The use according to any one of embodiments 64-66, the method of embodiment 67 or 68, the ligand-drug conjugate of embodiment 69 or a pharmaceutically acceptable salt or pharmaceutical composition thereof, the compound of embodiment 70 or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, characterized in that the tumor is selected from the group consisting of: lung cancer, breast cancer, rectal cancer, colon cancer, Esophageal cancer, stomach cancer, liver cancer, gallbladder cancer, bile duct cancer, kidney cancer, bladder cancer, urothelial carcinoma, head and neck cancer, nasopharyngeal carcinoma, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, pancreatic cancer, melanoma, bone cancer, mesothelioma, gastrointestinal stromal tumor, sarcoma, glioma, thyroid cancer, salivary gland tumor, glioblastoma, neuroblastoma, gastric mucinous tumor, lymphoma, leukemia, plasmacytoma, sinoatrial node cell tumor, giant cell tumor of the tendon sheath, brain cancer, squamous cell carcinoma, epidermal carcinoma, non-Hodgkin's lymphoma;

[0506] Preferably, the tumor meets one or more of the following conditions:

[0507] (1) The tumors are selected from breast cancer, gastric cancer, prostate cancer, lung cancer, pancreatic cancer, ovarian cancer, cervical cancer, endometrial cancer, kidney cancer, bladder cancer, glioma, malignant lymphoma, liver cancer and leukemia;

[0508] (2) The tumor is HER2 positive, TROP2 positive, LIV1 positive, FRα positive and / or ALPP positive;

[0509] (3) The tumor is HER2-positive gastric cancer or HER2-positive breast cancer;

[0510] (4) The tumor is TROP2-positive human pancreatic carcinoma in situ or TROP2-positive breast cancer;

[0511] (5) The tumor is a LIV1-positive renal cell carcinoma;

[0512] (6) The tumor is FRα-positive endometrial cancer or FRα-positive ovarian cancer;

[0513] (7) The tumor is ALPP-positive non-small cell lung cancer.

[0514] 86. A kit comprising a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-35, a compound or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and / or a pharmaceutical composition according to embodiment 62 or 63.

[0515] Example

[0516] The following embodiments are used to further describe this disclosure, but these embodiments are not intended to limit the scope of this disclosure.

[0517] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰. -6 The unit (ppm) is given. NMR measurements were performed using a Bruker AVANCE NEO 500M / 400M / 300M NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), with tetramethylsilane (TMS) as the internal standard.

[0518] MS measurements were performed using an Agilent 1200 / 1290DAD-6110 / 6120 Quadrupole MS LC-MS system (manufacturer: Agilent, MS model: 6110 / 6120 Quadrupole MS), a Waters ACQuity UPLC-QD / SQD system (manufacturer: Waters, MS model: Waters ACQuity Qda Detector / Waters SQ Detector), or a THERMO Ultimate 3000-Q Exactive system (manufacturer: THERMO, MS model: THERMO Q Exactive).

[0519] High-performance liquid chromatography (HPLC) analysis was performed using an Agilent HPLC 1200DAD, an Agilent HPLC 1200VWD, and a Waters HPLC e2695-2489 HPLC system.

[0520] Chiral HPLC analysis was performed using an Agilent 1260DAD high-performance liquid chromatograph.

[0521] High-performance liquid chromatography (HPLC) preparative chromatography was performed using Waters 2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP, and Gilson-281 preparative chromatographs.

[0522] Chiral preparative chromatography was performed using a Shimadzu LC-20AP preparative chromatograph.

[0523] The CombiFlash rapid preparation system uses a CombiFlash Rf200 (TELEDYNE ISCO).

[0524] The silica gel plates used for thin-layer chromatography are Yantai Huanghai HSGF254 silica gel plates. The silica gel plates used in thin-layer chromatography (TLC) have a diameter of 0.15 mm to 0.2 mm, and the diameter of the silica gel plates used for thin-layer chromatography separation and purification products is 0.4 mm to 0.5 mm.

[0525] Silica gel column chromatography typically uses 200–300 mesh or 300–400 mesh silica gel as the carrier.

[0526] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as Shanghai Titan Technology, ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, and Bid Pharmaceuticals.

[0527] Unless otherwise specified in the examples, all reactions can be carried out under an argon or nitrogen atmosphere.

[0528] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0529] A hydrogen atmosphere refers to a reaction vessel connected to a hydrogen balloon with a volume of approximately 1L.

[0530] The pressurized hydrogenation reaction was performed using a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

[0531] The hydrogenation reaction is usually carried out under vacuum, filled with hydrogen gas, and repeated 3 times.

[0532] The microwave reaction was performed using a CEM Discover-S 908860 microwave reactor.

[0533] Unless otherwise specified in the examples, "solution" refers to an aqueous solution.

[0534] Unless otherwise specified in the examples, the reaction temperature is room temperature, which is 20℃~30℃.

[0535] The reaction process in the examples was monitored using thin-layer chromatography (TLC). The developing solvent used in the reaction, the eluent system used for column chromatography to purify the compounds, and the developing solvent system for TLC included, but were not limited to: A: n-hexane / ethyl acetate system, B: dichloromethane / methanol system. The volume ratio of the solvent was adjusted according to the polarity of the compounds. Small amounts of basic or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0536] The specific enantiomers in the examples can be obtained by separation using chiral HPLC. When a specific geometrical isomer is desired, chromatography, recrystallization, and other conventional separation operations can also be used. Alternatively, undesirable enantiomers can be racemiced to desired enantiomers in the presence of an acid or base, according to methods known to those skilled in the art or as described in the accompanying examples.

[0537] Example 1: Preparation of drug D

[0538] Example 1-1: Synthesis of Compound 1

[0539] Step a:

[0540] 2-Chloro-3,4-Dihydroxybenzaldehyde

[0541] 2-chloro-3,4-dihydroxybenzaldehyde

[0542] Under nitrogen protection, boron tribromide (0.18 L, 0.36 mol, 2 M in CH2Cl2) was added dropwise to a solution of 2-chloro-3,4-dimethoxybenzaldehyde (24.00 g, 0.12 mol) in dichloromethane (240 mL) at -78 °C, and then stirred at 25 °C for 4 hours. After the reaction was completed, the reaction solution was quenched with ice water (500 mL), and then stirred at 25 °C for 1 hour. The mixture was then filtered to obtain compound Int 1-a (18.20 g, yield 88.2%).

[0543] 1 H NMR (400MHz, DMSO-d6) δ10.96(brs,1H),10.13(s,1H),9.59(brs,1H),7.30(d,1H),6.89(d,1H).

[0544] Step b:

[0545] 4-Chlorobenzo[d][1,3]dioxacyclopentene-5-carboxaldehyde

[0546] 4-chlorobenzo[d][1,3]dioxole-5-carbaldehyde

[0547] Under nitrogen protection, cesium carbonate (45.31 g, 139.07 mmol) and dibromomethane (17.73 g, 101.99 mmol) were added to a solution of Int 1-a (16.00 g, 93.02 mmol) in N,N-dimethylformamide (420 mL) at 25 °C, and the mixture was stirred at 100 °C for 6 hours. After the reaction was complete, the reaction solution was concentrated and poured into an aqueous solution (200 mL), and then extracted with ethyl acetate (200 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (petroleum ether:tetrahydrofuran (v / v) = 5:1) to give compound Int 1-b (8.00 g, yield 46.6%).

[0548] MS m / z (ESI): 185.0 [M+H] + .

[0549] Step c:

[0550] 1-(4-Chlorobenzo[d][1,3]dioxacyclopenten-5-yl)ethane-1-ol

[0551] 1-(4-chlorobenzo[d][1,3]dioxol-5-yl)ethan-1-ol

[0552] Under nitrogen protection, methyl magnesium bromide (46.3 mL, 46.32 mmol, 1 M in THF) was added dropwise to a tetrahydrofuran (60 mL) solution of Int 1-b (5.70 g, 31.00 mmol) at -78 °C, and then stirred at 25 °C for 4 hours. After the reaction was complete, the reaction solution was quenched with ice water (60 mL) and then extracted with ethyl acetate (100 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (petroleum ether:tetrahydrofuran (v / v) = 6:1) to give compound Int 1-c (6.10 g, yield 98.4%).

[0553] 1 H NMR (400MHz, CDCl3) δ7.05(d,1H),6.74(d,1H),6.03(s,2H),5.20-5.16(m,1H),2.04-1.84(m,1H),1.46(d,3H).

[0554] Step d:

[0555] 1-(4-Chlorobenzo[d][1,3]dioxacyclopenten-5-yl)ethane-1-one

[0556] 1-(4-chlorobenzo[d][1,3]dioxol-5-yl)ethan-1-one

[0557] In a solution of Int 1-c (6.10 g, 30.50 mmol) in dichloromethane (120 mL), Dys-Martin oxidant (14.19 g, 33.46 mmol) was slowly added at -5 °C, and the mixture was stirred at -5 °C for 45 minutes. After the reaction was complete, the reaction mixture was poured into an aqueous solution (80 mL) and extracted with dichloromethane (80 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (petroleum ether:ethyl acetate (v / v) = 4:1) to give compound Int 1-d (4.70 g, yield 77.8%).

[0558] 1 H NMR (400MHz, DMSO-d6) δ7.46(d,1H),7.00(d,1H),6.23(s,2H),2.54(s,3H).

[0559] Step e:

[0560] 1-(4-chloro-6-nitrobenzo[d][1,3]dioxacyclopenten-5-yl)ethane-1-one

[0561] 1-(4-chloro-6-nitrobenzo[d][1,3]dioxol-5-yl)ethan-1-one

[0562] A solution of Int 1-d (6.40 g, 32.32 mmol) in dichloromethane (60 mL) was added dropwise to a solution of Int 1-d at -5 °C with concentrated sulfuric acid (6.90 mL, 129.45 mmol), followed by fuming nitric acid (5.40 mL, 118.44 mmol). The mixture was stirred at 25 °C for 1 hour. After the reaction was complete, the reaction solution was poured into an ice-water solution (50 mL) and extracted with dichloromethane (50 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by rapid silica gel column chromatography (petroleum ether:ethyl acetate (v / v) = 5:1) to give compound Int 1-e (4.10 g, yield 52.1%).

[0563] 1 H NMR (300MHz, DMSO-d6) δ7.83 (s, 1H), 6.41 (s, 2H), 2.54 (s, 3H).

[0564] Step f:

[0565] 1-(6-amino-4-chlorobenzo[d][1,3]dioxacyclopenten-5-yl)ethane-1-one

[0566] 1-(6-amino-4-chlorobenzo[d][1,3]dioxol-5-yl)ethan-1-one

[0567] Under nitrogen protection, iron filings (9.40 g, 168.31 mmol) and ammonium chloride (9.00 g, 168.26 mmol) were added to a mixed solution of Int 1-e (4.10 g, 16.83 mmol) in ethanol / water (120 / 20 mL) at 25 °C, and the mixture was stirred at 90 °C for 5 hours. After the reaction was completed, the mixture was cooled to room temperature, filtered, and the filter cake was washed with aqueous solution to give compound Int 1-f (3.20 g, yield 89.0%).

[0568] MS m / z (ESI): 214.0 [M+H] + .

[0569] Step g:

[0570] N-(6-acetyl-7-chlorobenzo[d][1,3]dioxacyclopenten-5-yl)acetamide

[0571] N-(6-acetyl-7-chlorobenzo[d][1,3]dioxol-5-yl)acetamide

[0572] In a solution of Int 1-f (1.60 g, 7.51 mmol) in dichloromethane (32 mL), N,N-diisopropylethylamine (1.45 g, 11.22 mmol) and acetyl chloride (676.2 mg, 8.61 mmol) were added. After stirring at 0 °C for 0.5 hours, acetyl chloride (205.8 mg, 2.62 mmol) was added, and stirring was continued at 0 °C for another 0.5 hours. After the reaction was complete, the reaction solution was poured into an aqueous solution (30 mL) and extracted with dichloromethane (30 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (dichloromethane) to give compound Int 1-g (1.56 g, yield 81.3%).

[0573] 1 ¹H NMR (300MHz, DMSO-d⁶) δ 9.68 (s, ¹H), 6.87 (s, ¹H), 6.18 (s, 2H), 2.42 (s, 3H), 1.95 (s, 3H). Step h:

[0574] N-(6-(2-bromoacetyl)-7-chlorobenzo[d][1,3]dioxacyclopenten-5-yl)acetamide

[0575] N-(6-(2-bromoacetyl)-7-chlorobenzo[d][1,3]dioxol-5-yl)acetamide

[0576] In a solution of Int 1-g (200.0 mg, 0.78 mmol) in acetic acid (8 mL) at 10 °C, hydrobromic acid solution (211.0 mg, 0.86 mmol, 33% in AcOH) was added, followed by dropwise addition of elemental bromine (125.0 mg, 0.78 mmol). The mixture was stirred at 25 °C for 48 hours. After the reaction was complete, the reaction mixture was poured into an aqueous solution (20 mL) and extracted with ethyl acetate (25 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The crude product was recrystallized from isopropanol and filtered to give compound Int 1-h (100.1 mg, yield 38.3%).

[0577] 1¹H NMR (300MHz, DMSO-d⁶) δ 9.82 (s, ¹H), 6.89 (s, ¹H), 6.21 (s, 2H), 4.57 (s, 2H), 1.97 (s, 3H). Step i:

[0578] 1-(6-amino-4-chlorobenzo[d][1,3]dioxacyclopenten-5-yl)-2-chloroethane-1-one

[0579] 1-(6-amino-4-chlorobenzo[d][1,3]dioxol-5-yl)-2-chloroethan-1-one

[0580] A solution of Int 1-h (200.0 mg, 0.60 mmol) in ethanol (5 mL) was added to a concentrated hydrochloric acid aqueous solution (2 mL, 12N in H2O) at 25 °C, and then stirred at 60 °C for 16 hours. After the reaction was completed, the reaction solution was poured into an aqueous solution (10 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (petroleum ether:tetrahydrofuran (v / v) = 3:1) to give compound Int 1-i (80.3 mg, yield 54.1%).

[0581] MS m / z (ESI): 248.0 [M+H] + .

[0582] Step j:

[0583] (S)-15-chloro-14-(chloromethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione

[0584] In a solution of Int 1-i (180.2 mg, 0.73 mmol) in dichloroethane (8 mL), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyran[3,4-f]indoleazine-3,6,10(4H)-trione (191.0 mg, 0.73 mmol) and p-toluenesulfonic acid (125.2 mg, 0.73 mmol) were added, and the mixture was stirred at 90 °C for 16 hours. After the reaction was completed, the reaction solution was cooled to room temperature and filtered to obtain the crude solid. The solid was then slurried with a dichloromethane:methanol (v / v) mixture of 10:1, filtered, washed, and dried to obtain compound Int 1-j (160.4 mg, yield 46.5%).

[0585] 1 H NMR(300MHz,DMSO-d6)δ7.58(s,1H),7.23(s,1H),6.53(s,1H),6.41(s,2H), 5.54(s,2H),5.43(s,2H),5.33(s,2H),1.91-1.82(m,2H),0.90-0.85(m,3H).

[0586] Step k:

[0587] (S)-14-(aminomethyl)-15-chloro-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxacyclopentene[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-8,11(7H)-dione hydrochloride

[0588] (S)-14-(aminomethyl)-15-chloro-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione HCl salt

[0589] Under nitrogen protection, hexamethylenetetramine (177.1 mg, 1.26 mmol) was added to a 20 mL ethanol solution of Int 1-j (150.0 mg, 0.32 mmol) at 25 °C, and then stirred at 90 °C for 2 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by reversed silica gel column chromatography (water:acetonitrile (v / v) = 70:30, containing 0.1% hydrochloric acid) to obtain compound Int 1-k (33.3 mg, yield 21.4%).

[0590] 1H NMR(400MHz,DMSO-d6)δ8.69(s,3H),7.67(s,1H),7.27(s,1H),6.54(brs,1H),6.4 5(s,2H),5.60(s,2H),5.44(s,2H),4.87(brs,2H),1.92-1.81(m,2H),0.87(t,3H);

[0591] MS m / z (ESI): 455.9 [M+H] + .

[0592] Step 1:

[0593] (S)-N-((15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopentene[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-14-yl)methyl)-1-(hydroxymethyl)cyclopropane-1-carboxamide

[0594] (S)-N-((15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)-1-(hydroxymethyl)cyclopropane-1-carboxamide

[0595] At 25°C, a solution of Int 1-k (35.1 mg, 0.071 mmol) in N,N-dimethylformamide (2 mL) was mixed with 1-(hydroxymethyl)cyclopropane-1-carboxylic acid (16.5 mg, 0.14 mmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride hydrate (41.8 mg, 0.14 mmol), and N-methylmorpholine (14.4 mg, 0.14 mmol). The mixture was then stirred at 30°C for 1 hour. After the reaction was complete, the reaction mixture was poured into a solution of water (10 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the mother liquor was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (Welchrom C18, 20×150mm, 5μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound 1 (10.2 mg, yield 25.8%).

[0596] 1 H NMR(400MHz,DMSO-d6)δ8.34(t,1H),7.59(s,1H),7.24(s,1H),6.51(s,1H),6.41(s,2H),5.49(s,2H), 5.42(s,2H),5.36(t,1H),5.08(d,2H),3.45(d,2H),1.89-1.82(m,2H),0.90-0.85(m,5H),0.57(d,2H)

[0597] MS m / z (ESI): 554.0 [M+H] + .

[0598] Examples 1-2: Synthesis of Compound 2

[0599] Step a:

[0600] 2-((tert-butyldiphenylsilyl)oxy)ethane-1-ol

[0601] 2-((tert-butyldiphenylsilyl)oxy)ethan-1-ol

[0602] At 25 °C, a mixture of ethylene glycol (2.23 g, 35.86 mmol) and pyridine (2 mL) was mixed with tert-butyldiphenylchlorosilane (10 mL, 38.46 mmol), and the mixture was stirred at 30 °C for 1 hour. After the reaction was complete, the reaction mixture was poured into water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with saturated sodium bicarbonate solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (petroleum ether:ethyl acetate (v / v) = 10:1) to give compound Int 2-a (7.5 g, yield 69.6%).

[0603] 1 H NMR (400MHz, DMSO-d6) δ7.66-7.64(m,4H),7.46-7.38(m,6H),4.65(t,1H),3.66(t,2H),3.55-3.51(m,2H),1.00(s,9H).

[0604] Step b:

[0605] 2-((tert-butyldiphenylsilyl)oxy)ethyl(4-nitrophenyl)carbonate

[0606] 2-((tert-butyldiphenylsilyl)oxy)ethyl(4-nitrophenyl)carbonate

[0607] In a solution of compound Int 2-a (1.0 g, 3.33 mmol) in dichloromethane (30 mL), p-nitrophenyl chloroformate (1.33 g, 6.60 mmol) and triethylamine (1.34 g, 13.24 mmol) were added, and the mixture was stirred at 25 °C for 16 hours. After the reaction was complete, the reaction mixture was poured into an aqueous solution (30 mL) and extracted with dichloromethane (30 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (petroleum ether:ethyl acetate (v / v) = 10:1) to give compound Int 2-b (1.2 g, yield 77.3%).

[0608] 1 H NMR (400MHz, DMSO-d6) δ8.33(d,2H),7.70-7.64(m,4H),7.54-7.37(m,8H),4.43-4.40(m,2H),3.93-3.91(m,2H),1.00(s,9H).

[0609] Step c:

[0610] 2-((tert-butyldiphenylsilyl)oxy)ethyl(S)-((15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)carbamate

[0611] Compound Int 2-b (60.0 mg, 0.13 mmol) and N,N-diisopropylethylamine (25.0 mg, 0.19 mmol) were added to a solution of compound Int 1-k (63.5 mg, 0.13 mmol) in N,N-dimethylformamide (3 mL) at 25 °C, and the mixture was stirred at 25 °C for 2 hours. After the reaction was complete, the reaction mixture was poured into an aqueous solution (10 mL) and extracted with dichloromethane (15 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (petroleum ether:ethyl acetate (v / v) = 3:1) to give compound Int 2-c (37.1 mg, yield 36.8%).

[0612] 1 H NMR(400MHz,DMSO-d6)δ7.87-7.86(m,1H),7.58-7.54(m,5H),7.41-7.32(m,7H),6.51(s,1H),6.39(s, 2H),5.42(s,4H),5.02(t,2H),4.11-4.10(m,2H),3.73(t,2H),1.87-1.83(m,2H),0.87-0.85(m,12H).

[0613] Step d:

[0614] (S)-2-((((15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopentene[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline-14-yl)methyl)carbamoyl)oxy)ethyl 2,2,2-trifluoroacetate

[0615] (S)-2-((((15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]py rano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)carbamoyl)oxy)ethyl2,2,2-trifluoroacetate

[0616] In a 1 mL solution of dichloromethane containing 37.1 mg (0.047 mmol) of compound Int 2-c, trifluoroacetic acid (1 mL) was added, and the mixture was stirred at 25 °C for 1 hour. After the reaction was complete, the reaction solution was concentrated under reduced pressure to obtain a crude product containing compound Int 2-d, which could be directly proceeded to the next step without purification.

[0617] MS m / z (ESI): 640.0 [M+H] + .

[0618] Step e:

[0619] 2-Hydroxyethyl (S)-((15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopentene[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline-14-yl)methyl)carbamate

[0620] 2-hydroxyethyl(S)-((15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)carbamate

[0621] At 25°C, sodium hydroxide aqueous solution (1 mL, 2N) was added to a methanol (1 mL) solution of compound Int 2-d (crude product), and the mixture was stirred at 25°C for 1 hour. After the reaction was completed, the reaction solution was poured into an aqueous solution (10 mL) and extracted with dichloromethane (15 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the mother liquor was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (Welchrom C18, 20 × 150 mm, 5 μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound 2 (10.1 mg, two-step yield 39.2%).

[0622] 1H NMR(400MHz,DMSO-d6)δ7.63(t,1H),7.56(s,1H),7.24(s,1H),6.50(s,1H),6.38(s,2H),5.42(d ,4H),5.01(d,2H),4.69(d,1H),3.95(t,2H),3.52-3.50(m,2H),1.88-1.83(m,2H),0.87(t,3H);

[0623] MS m / z (ESI): 544.0 [M+H] + .

[0624] Examples 1-3: Synthesis of Compound 3

[0625] Step a:

[0626] 4-Fluoro-6-iodobenzo[d][1,3]dioxacyclopenten-5-amine

[0627] 4-fluoro-6-iodobenzo[d][1,3]dioxol-5-amine

[0628] In a 2 L solution of acetic acid containing 100.0 g (0.64 mol) of 4-fluorobenzo[d][1,3]dioxane-5-amine, N-iodosuccinimide (159.5 g, 0.71 mol) was added, and the mixture was stirred at 25 °C for 16 hours. After the reaction was complete, the reaction solution was concentrated and poured into water (5 L), and then extracted with ethyl acetate (5 L × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (petroleum ether:ethyl acetate (v / v) = 50:1) to give compound Int 3-a (70.1 g, yield 38.7%).

[0629] 1 H NMR(400MHz,DMSO-d6)δ7.02(d,1H),6.00(s,2H),4.81(brs,2H);

[0630] MS m / z (ESI): 281.9 [M+H] + .

[0631] Step b:

[0632] N-(4-fluoro-6-iodobenzo[d][1,3]dioxacyclopenten-5-yl)acetamide

[0633] N-(4-fluoro-6-iodobenzo[d][1,3]dioxol-5-yl)acetamide

[0634] Under nitrogen protection, potassium carbonate (56.53 g, 409.01 mmol) and acetyl chloride (48.16 g, 613.50 mmol) were added to a solution of Int 3-a (11.50 g, 40.92 mmol) in dichloromethane (250 mL) at 0 °C, and the mixture was stirred at 0 °C for 2 hours. After the reaction was complete, the reaction solution was poured into water (300 mL) and extracted with ethyl acetate (300 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (petroleum ether:ethyl acetate (v / v) = 3:1) to give compound Int 3-b (11.00 g, yield 83.2%).

[0635] MS m / z (ESI): 324.1 [M+H] + .

[0636] Step c:

[0637] N-(4-fluoro-6-(1-hydroxycyclobutyl)benzo[d][1,3]dioxacyclopenten-5-yl)acetamide

[0638] N-(4-fluoro-6-(1-hydroxycyclobutyl)benzo[d][1,3]dioxol-5-yl)acetamide

[0639] Under nitrogen protection, isopropyl magnesium chloride (4.7 mL, 9.40 mmol, 2 M in THF) was added dropwise to a tetrahydrofuran (30 mL) solution of Int 3-b (1.00 g, 3.10 mmol) at 0 °C and stirred for 1 hour at 0 °C. Cyclobutanone (0.43 g, 6.13 mmol) was added to the reaction mixture, and the mixture was stirred for another hour at 0 °C. After the reaction was complete, the reaction mixture was diluted with saturated ammonium chloride solution (50 mL) and extracted with ethyl acetate (70 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (petroleum ether:ethyl acetate (v / v) = 1:1) to give compound Int 3-c (220.5 mg, yield 26.6%).

[0640] MS m / z (ESI): 268.0 [M+H] + .

[0641] Step d:

[0642] N-(4-fluoro-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxacyclopenten-5-yl)acetamide

[0643] N-(4-fluoro-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-5-yl)acetamide

[0644] Silver nitrate (63.6 mg, 0.37 mmol) and potassium persulfate (505.8 mg, 1.87 mmol) were added to a dichloromethane / water (6 / 6 mL) mixture of Int 3-c (200.0 mg, 0.75 mmol) at 25 °C, and the mixture was stirred at 25 °C for 16 hours. After the reaction was complete, the reaction mixture was poured into water (15 mL) and extracted with dichloromethane (40 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (petroleum ether:ethyl acetate (v / v) = 1:1) to give compound Int 3-d (60.0 mg, yield 30.2%).

[0645] 1 H NMR (400MHz, DMSO-d6) δ9.74(s,1H),6.25(s,2H),2.77(t,2H),2.56(t,2H),2.04(s,3H),2.01-1.94(m,2H);

[0646] MS m / z (ESI): 266.2 [M+H] + .

[0647] Step e:

[0648] N-(4-fluoro-7-(hydroxyimino)-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxacyclopenten-5-yl)acetamide

[0649] Under nitrogen protection, N-(4-fluoro-7-(hydroxyimino)-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-5-yl)acetamide was added to a tetrahydrofuran (5 mL) solution of Int 3-d (100.2 mg, 0.38 mmol) at 0 °C. Potassium tert-butoxide (212.1 mg, 1.89 mmol) dissolved in a tetrahydrofuran / isopropanol mixture (5 / 1, 6 mL) was then added, followed by amyl nitrite (220.8 mg, 1.88 mmol). The mixture was stirred at 0 °C for 2 hours. After the reaction was complete, the reaction solution was poured into water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by rapid silica gel column chromatography (petroleum ether: ethyl acetate (v / v) = 1:1) to obtain compound Int 3-e (45.0 mg, yield 40.5%).

[0650] MS m / z (ESI): 295.0 [M+H] + .

[0651] Step f:

[0652] (9H-fluorene-9-yl)methyl(5-acetamido-4-fluoro-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxacyclopenten-7-yl)carbamate

[0653] (9H-fluoren-9-yl)methyl(5-acetamido-4-fluoro-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-7-yl)carbamate

[0654] In a methanol / tetrahydrofuran (20 / 20 mL) mixture of Int 3-e (1.00 g, 3.40 mmol), palladium on carbon (300.0 mg, w / w, 10%) and hydrochloric acid aqueous solution (6.8 mL, 1 M) were added at 25 °C. The reaction mixture was then stirred at 25 °C for 2 hours under hydrogen atmosphere. The reaction mixture was filtered and the filtrate was concentrated. Under nitrogen protection, the concentrate was dissolved in a 5 / 1 mixture of 1,4-dioxane / water (40 mL), and sodium bicarbonate (1.14 g, 13.60 mmol) and 9-fluorenemethyl-N-succinimide carbonate (1.38 g, 4.09 mmol) were added. The reaction mixture was then stirred at 25 °C for 2 hours. After the reaction was complete, the reaction mixture was poured into water (100 mL) and extracted with ethyl acetate (150 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by rapid silica gel column chromatography (petroleum ether: ethyl acetate (v / v) = 1:2) to obtain compound Int 3-f (670.0 mg, yield 39.2%).

[0655] MS m / z (ESI): 503.5 [M+H] + .

[0656] Step g:

[0657] (9H-fluorene-9-yl)methyl(5-amino-4-fluoro-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxacyclopenten-7-yl)carbamate

[0658] (9H-fluoren-9-yl)methyl(5-amino-4-fluoro-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-7-yl)carbamate

[0659] Under nitrogen protection, concentrated hydrochloric acid aqueous solution (3 mL, 12N) was added to a 1,4-dioxane (15 mL) solution of Int 3-f (630.6 mg, 1.25 mmol) at 0 °C, and then stirred at 70 °C for 2 hours. After the reaction was completed, the reaction solution was cooled to room temperature and adjusted to pH > 9 with saturated sodium bicarbonate aqueous solution, and then extracted with ethyl acetate (50 mL × 3). The combined organic layers were washed with saturated saline aqueous solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by high performance liquid chromatography (Welchrom C18, 20 × 150 mm, 5 μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound Int 3-g (302.1 g, yield 52.3%).

[0660] 1 H NMR(400MHz,DMSO-d6)δ7.91(d,2H),7.76(d,2H),7.52(d,1H),7.42(t,2H),7.39-7.33( m,4H),6.08(s,1H),6.06(s,1H),4.33-4.21(m,4H),2.85-2.73(m,2H),2.08-1.99(m,2H)

[0661] MS m / z (ESI): 461.5 [M+H] + .

[0662] Step h:

[0663] (9H-fluorene-9-yl)methyl((10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)carbamate

[0664] (9H-fluoren-9-yl)methyl((10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahyd ro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0665] Under nitrogen protection, (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indoleazine-3,6,10(4H)-trione (171.5 mg, 0.65 mmol) and p-toluenesulfonic acid (112.1 mg, 0.65 mmol) were added to a 15 mL toluene solution of Int 3-g (301.2 mg, 0.65 mmol) at 25 °C, and then the mixture was refluxed and stirred at 110 °C for 4 hours. After the reaction was completed, the reaction solution was cooled to room temperature and concentrated under reduced pressure. The crude product was purified by rapid silica gel column chromatography (dichloromethane:methanol (v / v) = 4:1) to give compound Int 3-h (368.1 mg, yield 81.8%).

[0666] MS m / z (ESI): 688.0 [M+H] + .

[0667] Step i:

[0668] (1S,10S)-1-amino-10-ethyl-7-fluoro-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxacyclopentene[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline-11,14-dione trifluoroacetate

[0669] (1S,10S)-1-amino-10-ethyl-7-fluoro-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione 2,2,2-trifluoroacetic acid salt

[0670] Under nitrogen protection, diethylamine (114.9 mg, 1.58 mmol) was added to a solution of Int 3-h (540.0 mg, 0.79 mmol) in N,N-dimethylformamide (15 mL) at 25 °C, and the mixture was stirred at 25 °C for 0.5 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by high performance liquid chromatography (Welchrom C18, 20 × 150 mm, 5 μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound Int 3-i (50.4 mg, yield 11.1%).

[0671] 1H NMR(400MHz,DMSO-d6)δ8.45(br,2H),7.30(s,1H),6.56(s,1H),6.45(s,1H),6.41(s,1H),5.70(d,1H),5.45(s,2H),5.39( d,1H),5.10(s,1H),3.15-3.10(m,1H),3.02-2.94(m,1H),2.45(br,1H),2.16-2.07(m,1H),1.94-1.83(m,2H),0.89(t,3H);

[0672] MS m / z (ESI): 466.0 [M+H] + .

[0673] Step j:

[0674] 2-((tert-butyldiphenylsilyl)oxy)ethyl((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopentene[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)carbamate

[0675] 2-((tert-butyldiphenylsilyl)oxy)ethyl((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16 -hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3′,4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0676] In a solution of Int 3-i (30.1 mg, 0.053 mmol) in N,N-dimethylformamide (2 mL), 2-((tert-butyldiphenylsilyl)oxy)ethyl(4-nitrophenyl) carbonate (30.0 mg, 0.064 mmol) and N,N-diisopropylethylamine (16.7 mg, 0.13 mmol) were added, and the mixture was stirred at 25 °C for 16 hours. After the reaction was complete, the reaction solution was poured into water (10 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the mother liquor was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (petroleum ether: ethyl acetate = 1:4) to give compound Int 3-j (25.2 mg, yield 59.6%).

[0677] MS m / z (ESI): 792.0 [M+H] + .

[0678] Step k:

[0679] 2-((((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopentene[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)carbamoyl)oxy)ethyl 2,2,2-trifluoroacetate

[0680] 2-((((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3] dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)ethyl2,2,2-trifluoroacetate

[0681] A solution of Int 3-j (25.2 mg, 0.032 mmol) in dichloromethane (3 mL) was added to trifluoroacetic acid (0.5 mL) at 25 °C, and the mixture was stirred at 25 °C for 16 hours. After the reaction was complete, the pH was adjusted to 8–9 with saturated sodium bicarbonate aqueous solution, and then extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the mother liquor was concentrated under reduced pressure to obtain the crude compound Int 3-k (21.0 mg, proceed directly to the next step).

[0682] MS m / z (ESI): 650.4 [M+H] + .

[0683] Step 1:

[0684] 2-Hydroxyethyl ((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopentene[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)carbamate

[0685] 2-hydroxyethyl((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro- 1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0686] In a methanol (3 mL) solution of Int 3-k (21.0 mg, 0.032 mmol), sodium hydroxide aqueous solution (0.12 mL, 0.06 mmol, 0.5 M) was added, and the mixture was stirred at 25 °C for 1 hour. After the reaction was complete, the pH was adjusted to 4–5 with formic acid solution, and the mother liquor was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (Welchrom C18, 20 × 150 mm, 5 μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%–95%) to obtain compound 3 (4.2 mg, two-step yield 23.8%).

[0687] 1 H NMR(400MHz,DMSO-d6)δ7.98(d,1H),7.27(s,1H),6.51(s,1H),6.38(t,2H),5.42(s,2H),5.26-5.24(m,1H),5.22(s,2H), 4.78(t,1H),4.15-4.03(m,2H),3.63-3.59(m,2H),3.11-2.92(m,2H),2.14-2.06(m,2H),1.92-1.81(m,2H),0.88(t,3H);

[0688] MS m / z (ESI): 554.0 [M+H] + .

[0689] Examples 1-4: Synthesis of Compound 4

[0690] Step a:

[0691] N-((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-1-yl)-1-hydroxymethylcyclopropane-1-carboxamide

[0692] N-((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dio xolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-(hydroxymethyl)cyclopropane-1-carboxamide

[0693] At 25°C, a solution of Int 3-i (20.0 mg, 0.035 mmol) in N,N-dimethylformamide (3 mL) was mixed with 1-(hydroxymethyl)cyclopropane-1-carboxylic acid (9.9 mg, 0.085 mmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride hydrate (25.3 mg, 0.086 mmol), and N-methylmorpholine (8.7 mg, 0.086 mmol). The mixture was then stirred at 25°C for 1 hour. After the reaction was complete, the reaction solution was poured into a solution of water (10 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (Welchrom C18, 20×150mm, 5μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound 4 (4.3 mg, yield 21.5%).

[0694] 1 H NMR(400MHz,DMSO-d6)δ8.11(d,1H),7.25(s,1H),6.51(s,1H),6.38(d,2H),5.62-5.55(m,1H),5.42(s,2H),5.26-5.08(m,3H),3 .61(dd,1H),3.47(dd,1H),3.00(t,2H),2.13-2.08(m,2H),1.91-1.82(m,2H),1.13-1.02(m,2H),0.88(t,3H),0.73-0.65(m,2H);

[0695] MS m / z (ESI): 564.1 [M+H] + .

[0696] Examples 1-5: Synthesis of Compound 5

[0697] Step a:

[0698] 2-(6-amino-4-chlorobenzo[d][1,3]dioxacyclopenten-5-yl)-2-oxoethyl acetate

[0699] 2-(6-amino-4-chlorobenzo[d][1,3]dioxol-5-yl)-2-oxoethyl acetate

[0700] Sodium acetate (992.1 mg, 12.09 mmol) was added to a solution of compound Int 1-i (1.00 g, 4.03 mmol) in N,N-dimethylformamide (30 mL) at 30 °C, and the mixture was stirred at 30 °C for 16 hours. After the reaction was complete, the reaction mixture was poured into an aqueous solution (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (petroleum ether:ethyl acetate (v / v) = 1:1) to give compound Int 5-a (301.2 mg, yield 27.5%).

[0701] MS m / z (ESI): 272.0 [M+H] + .

[0702] Step b:

[0703] 2-(6-amino-4-methylbenzo[d][1,3]dioxacyclopenten-5-yl)-2-oxoethyl acetate

[0704] 2-(6-amino-4-methylbenzo[d][1,3]dioxol-5-yl)-2-oxoethyl acetate

[0705] Under nitrogen protection, at 25 °C, a solution of compound Int 5-a (270.0 mg, 0.99 mmol) in 1,4-dioxane (25 mL) was mixed with potassium carbonate (274.7 mg, 1.99 mmol), chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (78.1 mg, 0.099 mmol), and trimethylcycloboroxane (1.1 mL, 3.85 mmol, 3.5 M in THF). The mixture was then stirred at 80 °C for 1.5 hours. After the reaction was complete, the reaction solution was poured into an aqueous solution (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by rapid silica gel column chromatography (petroleum ether: ethyl acetate (v / v) = 5:1) to obtain compound Int 5-b (180.1 mg, yield 72.1%).

[0706] MS m / z (ESI): 252.2 [M+H] + .

[0707] Step c:

[0708] (S)-(7-ethyl-7-hydroxy-15-methyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopentene[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinolino-14-yl)methyl acetate

[0709] (S)-(7-ethyl-7-hydroxy-15-methyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl acetate

[0710] In a 16 mL solution of dichloroethane containing 160.0 mg (0.64 mmol) of compound Int 5-b, (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indoleazine-3,6,10(4H)-trione (167.7 mg, 0.64 mmol) and pyridine 4-methylbenzenesulfonic acid (160.1 mg, 0.64 mmol) were added, and the mixture was stirred at 90 °C for 2 hours. After the reaction was complete, the reaction solution was cooled to room temperature and concentrated under reduced pressure. The crude product was purified by rapid silica gel column chromatography (dichloromethane:methanol (v / v) = 20:1) to give compound Int 5-c (60.1 mg, yield 19.7%).

[0711] MS m / z (ESI): 479.2 [M+H] + .

[0712] Step d:

[0713] (S)-7-ethyl-7-hydroxy-14-(hydroxymethyl)-15-methyl-10,13-dihydro-11H-[1,3]dioxacyclopentene[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-8,11(7H)-dione

[0714] (S)-7-ethyl-7-hydroxy-14-(hydroxymethyl)-15-methyl-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione

[0715] A methanol (5 mL) solution of compound Int 5-c (50.4 mg, 0.11 mmol) was added to a sodium hydroxide aqueous solution (0.2 mL, 0.2 mmol, 1 M) at 30 °C, and the mixture was stirred at 30 °C for 0.5 hours. After the reaction was complete, the pH of the reaction solution was adjusted to 6 with acetic acid solution, and the mixture was filtered to obtain compound Int 5-d (30.1 mg, yield 65.5%).

[0716] 1 H NMR(400MHz,DMSO-d6)δ7.39(s,1H),7.22(s,1H),6.48(s,1H),6.25(s,2H),5.73(t,1H ),5.42(s,2H),5.37(s,2H),5.11(d,2H),2.79(s,3H),1.90-1.83(m,2H),0.88(t,3H);

[0717] MS m / z (ESI): 437.0 [M+H] + .

[0718] Step e:

[0719] (S)-14-(chloromethyl)-7-ethyl-7-hydroxy-15-methyl-10,13-dihydro-11H-[1,3]dioxacyclopentene[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-8,11(7H)-dione

[0720] (S)-14-(chloromethyl)-7-ethyl-7-hydroxy-15-methyl-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione

[0721] A solution of compound Int 5-d (15.0 mg, 0.03 mmol) in dichloromethane (3 mL) was added with thionyl chloride (61.4 mg, 0.52 mmol) and N,N-dimethylformamide (2.5 mg, 0.03 mmol), and the mixture was stirred at 30 °C for 0.5 hours. After the reaction was complete, the reaction solution was quenched with water, concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (petroleum ether:ethyl acetate (v / v) = 2:1) to give compound Int 5-e (8.1 mg, yield 51.8%).

[0722] MS m / z (ESI): 455.2 [M+H] + .

[0723] Step f:

[0724] (S)-14-(aminomethyl)-7-ethyl-7-hydroxy-15-methyl-10,13-dihydro-11H-[1,3]dioxacyclopentene[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-8,11(7H)-dione trifluoroacetate

[0725] (S)-14-(aminomethyl)-7-ethyl-7-hydroxy-15-methyl-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione TFA salt

[0726] Hexamethylenetetramine (9.9 mg, 0.07 mmol) was added to a 2 mL ethanol solution of compound Int 5-e (8.1 mg, 0.02 mmol) at 30 °C, and the mixture was stirred at 90 °C for 3 hours. After the reaction was complete, the reaction solution was cooled to room temperature and concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (Welchrom C18, 20 × 150 mm, 5 μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound Int 5-f (5.5 mg, yield 56.2%).

[0727] 1 H NMR(400MHz,DMSO-d6)δ8.33(s,2H),7.51(s,1H),7.26(s,1H),6.53(s,1H),6.31(s,2H ),5.49(s,2H),5.44(s,2H),4.73(s,2H),2.72(s,3H),1.89-1.85(m,2H),0.88(t,3H);

[0728] MS m / z (ESI): 436.0 [M+H] + .

[0729] Step g:

[0730] 2-((tert-butyldimethylsilyl)oxy)ethyl(S)-((7-ethyl-7-hydroxy-15-methyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-14-yl)methyl)carbamate

[0731] 2-((tert-butyldimethylsilyl)oxy)ethyl(S)-((7-ethyl-7-hydroxy-15-methyl-8,11-dioxo-7,8,11,13-tet rahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)carbamate

[0732] In a solution of compound Int 5-f (50.0 mg, 0.09 mmol) in N,N-dimethylformamide (2 mL), 2-((tert-butyldimethylsilyl)oxy)ethyl(4-nitrophenyl) carbonate (40.4 mg, 0.12 mmol), pyridine (36.0 mg, 0.46 mmol), 1-hydroxybenzotriazole (12.3 mg, 0.09 mmol), and N,N-diisopropylethylamine (23.5 mg, 0.18 mmol) were added, and the mixture was stirred at 25 °C for 3 hours. After the reaction was complete, the reaction solution was poured into an aqueous solution (10 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by rapid silica gel column chromatography (petroleum ether:tetrahydrofuran (v / v) = 1:1) to obtain compound Int 5-g (25.0 mg, yield 43.1%).

[0733] MS m / z (ESI): 638.3 [M+H] + .

[0734] Step h:

[0735] 2-Hydroxyethyl (S)-((7-ethyl-7-hydroxy-15-methyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-14-yl)methyl)carbamate

[0736] 2-hydroxyethyl(S)-((7-ethyl-7-hydroxy-15-methyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)carbamate

[0737] At 25°C, a solution of dioxane (1 mL, 4 M) containing 5-g (25.0 mg, 0.04 mmol) of compound Int was added to a solution of dichloromethane (2 mL), and the mixture was stirred at 25°C for 0.5 hours. After the reaction was complete, the reaction solution was poured into an aqueous solution (10 mL) and extracted with dichloromethane (15 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (Welchrom C18, 20 × 150 mm, 5 μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound 5 (8.0 mg, 39.0%).

[0738] 1 H NMR(400MHz,DMSO-d6)δ7.80(brs,1H),7.41(s,1H),7.23(s,1H),6.48(s,1H),6.26(s,2H),5.42(s,2H),5.33( s,2H),4.85(d,2H),4.70(t,1H),4.00(t,2H),3.55-3.52(m,2H),2.72(s,3H),1.92-1.81(m,2H),0.87(t,3H);

[0739] MS m / z (ESI): 524.1 [M+H] + .

[0740] Examples 1-6: Synthesis of Compound 6

[0741] Step a:

[0742] (S)-N-((7-ethyl-7-hydroxy-15-methyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-14-yl)methyl)-1-(hydroxymethyl)cyclopropane-1-carboxamide

[0743] (S)-N-((7-ethyl-7-hydroxy-15-methyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)-1-(hydroxymethyl)cyclopropane-1-carboxamide

[0744] At 25°C, a solution of Int 5-f (40.0 mg, 0.073 mmol) in N,N-dimethylformamide (2 mL) was mixed with 1-(hydroxymethyl)cyclopropane-1-carboxylic acid (16.9 mg, 0.15 mmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride hydrate (42.8 mg, 0.15 mmol), and N-methylmorpholine (14.7 mg, 0.15 mmol). The mixture was then stirred at 25°C for 0.5 hours. After the reaction was complete, the reaction solution was poured into a solution of water (10 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (Welchrom C18, 20×150mm, 5μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound 6 (8.5mg, yield 21.9%).

[0745] 1 H NMR(400MHz,DMSO-d6)δ8.04(t,1H),7.43(s,1H),7.23(s,1H),6.48(s,1H),6.27(s,2H),5.42(s,2H),5.33(s,2H),5.1 1(t,1H),4.93(d,2H),3.49(d,2H),2.72(s,3H),1.91-1.80(m,2H),0.97-0.95(m,2H),0.87(t,3H),0.63-0.61(m,2H);

[0746] MS m / z (ESI): 534.1 [M+H] + .

[0747] The compounds of the typical drug D prepared in this embodiment are shown in Table 5 below:

[0748] Table 5

[0749] Example 2 Preparation of LD compounds

[0750] Example 2-1 Synthesis of LD-Int1

[0751] Solid-phase synthesis steps:

[0752] Step a:

[0753] N,N-diisopropylethylamine (7.10 g, 54.94 mmol) was added to a solution of 2-chlorotriphenylmethyl chloride resin (2-CTC Resin, 5.50 g, 1.0 mmol / g, 5.5 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)glycylglycine (9.74 g, 27.5 mmol) in dichloromethane (80 mL). The reaction mixture was stirred at room temperature under nitrogen protection for 12 hours. After the reaction was complete, dichloromethane was removed by vacuum filtration. Then, methanol (80 mL) was added to the resin, and nitrogen was bubbled into the mixture and stirred for 30 min. The methanol was then removed by vacuum filtration. The resin was then washed with dichloromethane and methanol alternately, and this process was repeated 5 times.

[0754] A solution of N,N-dimethylformamide (80 mL) containing 20% ​​piperidine was added to the above system, and nitrogen gas was bubbled into the mixture and stirred for 2 hours. After the reaction was completed, the reaction system was filtered under reduced pressure, and the filter cake was washed with dichloromethane and methanol in turn, and this process was repeated 5 times alternately to finally obtain compound LD-Int1-a.

[0755] Step b:

[0756] Under a nitrogen atmosphere, 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP, 8.58 g, 16.46 mmol), Fmoc-L-phenylalanine (6.39 g, 16.49 mmol), and N,N-diisopropylethylamine (4.26 g, 32.96 mmol) were added to an N,N-dimethylformamide (80 mL) solution of compound LD-Int1-a, and the mixture was stirred for 3 hours at room temperature under nitrogen atmosphere. After the reaction was completed, the reaction system was filtered under reduced pressure, and the filter cake was washed sequentially with dichloromethane and methanol, and this process was repeated 5 times alternately to finally obtain compound LD-Int1-b.

[0757] Step c:

[0758] A solution of N,N-dimethylformamide (80 mL) containing 20% ​​piperidine was added to the above system, and nitrogen gas was bubbled into the mixture and stirred for 2 hours. After the reaction was completed, the reaction system was filtered under reduced pressure, and the filter cake was washed with dichloromethane and methanol in turn, and this process was repeated 5 times alternately to finally obtain compound LD-Int1-c.

[0759] Step d:

[0760] Under a nitrogen atmosphere, 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP, 8.58 g, 16.46 mmol), N-fluorenylmethoxycarbonyl-glycine (5.85 g, 16.51 mmol), and N,N-diisopropylethylamine (4.26 g, 32.96 mmol) were added to an N,N-dimethylformamide (80 mL) solution of compound LD-Int1-c, and the mixture was stirred for 2 hours at room temperature under nitrogen atmosphere. After the reaction was completed, the reaction system was filtered under reduced pressure, and the filter cake was washed sequentially with dichloromethane and methanol, and this process was repeated 5 times alternately to finally obtain compound LD-Int1-d.

[0761] Step e:

[0762] Add 50 mL of lysis buffer (30% hexafluoroisopropanol / 70% dichloromethane) to the crude LD-Int1-d, and stir the mixture for 10 minutes at room temperature under nitrogen puffing. Then filter the reaction mixture under reduced pressure, dissolve the filter cake in lysis buffer (30% hexafluoroisopropanol / 70% dichloromethane) (50 mL), and continue stirring the mixture for 10 minutes at room temperature under nitrogen puffing. Filter the reaction mixture again under reduced pressure, combine the filtrates, and concentrate to obtain the crude product. The obtained crude LD-Int1 (2.50 g, 73.9% yield in 6 steps) is directly used for the next step.

[0763] 1 H NMR(400MHz,DMSO-d6)δ11.96(s,1H),8.34(t,1H),8.14(d,1H),8.04(t,1H),8.00(t,1H),7.89(d,2H),7.71(d,2H),7.62(t,1H), 7.42(t,2H),7.33(t,2H),7.25-7.15(m,5H),4.55-4.49(m,1H),4.30-4.20(m,3H),3.80-3.57(m,8H),3.05(dd,1H),2.79(dd,1H);

[0764] MS m / z (ESI): 616.3 [M+H] + .

[0765] Example 2-2 Synthesis of LD1

[0766] Step a:

[0767] (9H-fluorene-9-yl)methyl(S)-(2-((((1-(((15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopentene[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-14-yl)methyl)carbamoyl)cyclopropyl)methoxy)methyl)amino)-2-oxoethyl)carbamate

[0768] (9H-fluoren-9-yl)methyl(S)-((((1-(((15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g ]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)carbamoyl)cyclopropyl)methoxy)methyl)amino)-2-oxoethyl)carbamate

[0769] At 25 °C, (2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (53.2 mg, 0.14 mmol) and trifluoroacetic acid (41.2 mg, 0.36 mmol) were added to a solution of compound 1 (20.0 mg, 0.036 mmol) in dichloroethane (4 mL). The reaction system was stirred at 60 °C for 2 hours. After the reaction was completed, the reaction solution was cooled to room temperature and concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (Welchrom C18, 20 × 150 mm, 5 μm; mobile phase A: 0.1% aqueous trifluoroacetic acid, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD-1a (15.1 mg, yield 48.6%).

[0770] MS m / z (ESI): 862.2 [M+H] + .

[0771] Step b:

[0772] (S)-1-(((2-aminoacetamido)methoxy)methyl)-N-((15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopentene[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-14-yl)methyl)cyclopropane-1-carboxamide

[0773] (S)-1-(((2-aminoacetamido)methoxy)methyl)-N-((15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro -10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)cyclopropane-1-carboxamide

[0774] Piperidine (0.2 mL) was added to a 2 mL solution of N,N-dimethylformamide containing compound LD-1a (45.3 mg, 0.053 mmol), and the reaction mixture was stirred at 30 °C for 0.5 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the crude product was purified by high-performance liquid chromatography (Welchrom C18, 20 × 150 mm, 5 μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD-1b (23.0 mg, 68.4%). MS m / z (ESI): 640.2 [M+H] + .

[0775] Step c:

[0776] 1-((S)-9-benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14,17-pentoxo-2-oxa-4,7,10,13,16-pentazadocosyl)-N-(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-14-yl)methyl)cyclopropane-1-carboxamide

[0777] 1-((S)-9-benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17-pentaoxo-2-oxa-4,7,10,13,16-pentaazadocosyl)-N-(((S)-15-chloro-7-ethyl-7- hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)cyclopropane-1-carboxamide

[0778] At 0°C, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (19.8 mg, 0.10 mmol) and 2-hydroxypyridine-N-oxide (11.5 mg, 0.10 mmol) were added to a solution of (6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl)glycylglycyl-L-phenylalanine (48.8 mg, 0.10 mmol) in N,N-dimethylformamide (1 mL), and the reaction mixture was stirred at 0°C for 0.5 h. Then, a solution of compound LD-1b (23.0 mg, 0.036 mmol) and N,N-diisopropylethylamine (20.0 mg, 0.15 mmol) in N,N-dimethylformamide (1 mL), was added, and the reaction mixture was stirred at 0°C for 0.5 h. After the reaction was completed, acetic acid was added to the reaction solution to quench the reaction, and the solution was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (Welchrom C18, 20×150mm, 5μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD1 (3.2mg, 8.1%).

[0779] 1H NMR (400MHz, DMSO-d6) δ8.50(t,1H),8.28(t,1H),8.12(d,1H),8.06(t,1H),7.99(t,2H),7.57(s,1H),7.26-7. 15(m,6H),6.99(s,2H),6.50(s,1H),6.38(d,2H),5.48(s,2H),5.42(s,2H),5.10-5.08(m,2H),4.60(d,2H),4. 48-4.43(m,1H),3.76-3.59(m,6H),3.46(s,2H),3.37(s,2H),3.04-2.99(m,1H),2.80-2.74(m,1H),2.09(t,2H ),1.90-1.79(m,2H),1.51-1.42(m,4H),1.23-1.14(m,2H),0.95-0.94(m,2H),0.86(t,3H),0.64-0.63(m,2H);

[0780] MS m / z (ESI): 1094.1 [M+H] + .

[0781] Example 2-3 Synthesis of LD2

[0782] Step a:

[0783] (9H-fluorene-9-yl)methyl((S)-9-benzyl-1-(1-((((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopentene[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-14-yl)methyl)carbamoyl)cyclopropyl)-5,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-yl)carbamate

[0784] (9H-fluoren-9-yl)methyl((S)-9-benzyl-1-(1-((((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3 ′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)carbamoyl)cyclopropyl)-5,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-yl)carbamate

[0785] At 25°C, (E)-4-oxo-4-phenylbut-2-en-2-yl(((9H-fluorene-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine ester (36.7 mg, 0.057 mmol) was added to a solution of compound LD-1b (33.0 mg, 0.052 mmol) in N,N-dimethylformamide (2 mL). The reaction mixture was stirred at 25°C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (dichloromethane:methanol = 10:1) to give compound LD-2a (33.2 mg, yield 57.3%).

[0786] 1 H NMR(400MHz,DMSO-d6)δ8.52(t,1H),8.31(t,1H),8.15(d,1H),8.02-7.97(m,2H),7.87(d,2H),7.68(d,2 H),7.58(t,1H),7.56(s,1H),7.39(t,2H),7.30(t,2H),7.25-7.14(m,6H),6.51(s,1H),6.37(d,2H),5.4 7(s,2H),5.41(s,2H),5.10-5.04(m,2H),4.60(d,2H),4.50-4.44(m,1H),4.30-4.16(m,3H),3.78-3.43( m,8H),3.02(dd,1H),2.76(dd,1H),1.88-1.79(m,2H),0.95-0.92(m,2H),0.86(t,3H),0.64-0.59(m,2H);

[0787] MS m / z (ESI): 1123.3 [M+H] + .

[0788] Step b:

[0789] 1-((S)-15-amino-9-benzyl-5,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecanyl)-N-(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopentene[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-14-yl)methyl)cyclopropane-1-carboxamide

[0790] 1-((S)-15-amino-9-benzyl-5,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecyl)-N-(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo -7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)cyclopropane-1-carboxamide

[0791] At 25°C, piperidine (0.3 mL) was added to a 2 mL solution of compound LD-2a (30.0 mg, 0.027 mmol) in N,N-dimethylformamide, and the reaction mixture was stirred at 25°C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the resulting concentrate (24.0 mg, crude product) was used directly in the next step of the reaction.

[0792] MS m / z (ESI): 901.3 [M+H] + .

[0793] Step c:

[0794] (S)-N 5 -((S)-9-benzyl-1-(1-((((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-14-yl)methyl)carbamoyl)cyclopropyl)-5,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoamide)-N 1-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47-hexadecanopentadecan-49-yl)pentadiamide

[0795] (S)-N 5 -((S)-9-benzyl-1-(1-((((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4′:6,7]indolizino[1,2-b]quinolin- 14-yl)methyl)carbamoyl)cyclopropyl)-5,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N 1 -

[0796] (2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47-hexadecaoxanonatetracontan-49-yl)pentanediamide at 25°C, was added to a solution of compound LD-2b (11.5 mg, 0.027 mmol) in N,N-dimethylformamide (2 mL) with 2,5-dimethylformamide. Oxypyrrolidine-1-yl(S)-52-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-51-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47-hexadecoxa-50-azapentaane-55-ester (33.8 mg, 0.029 mmol) was reacted with acetic acid at 25 °C for 1 hour. After the reaction was completed, the reaction solution was quenched with acetic acid, concentrated under reduced pressure, and the crude product was purified by high performance liquid chromatography (Welchrom C18, 20 × 150 mm, 5 μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD2 (10.4 mg, 20.1% yield in two steps).

[0797] 1H NMR(400MHz,DMSO-d6)δ8.50(t,1H),8.29(br,1H),8.11-7.98(m,4H),7.90-7.87(m,2H),7.57(s,1H),7.24-7.15( m,6H),6.99(s,2H),6.49(s,1H),6.39(d,2H),5.48(s,2H),5.42(s,2H),5.10-5.05(m,2H),4.61(d,2H),4.48-4.42 (m,1H),4.22-4.16(m,1H),3.76-3.34(m,72H),3.23(s,3H),3.21-3.14(m,2H),3.01(dd,1H),2.80-2.67(m,1H),2. 13-2.05(m,4H),1.89-1.64(m,4H),1.49-1.41(m,4H),1.21-1.13(m,2H),0.94(br,2H),0.86(t,3H),0.63(br,2H);

[0798] MS m / z (ESI): 992.9 [1 / 2M+Na] + .

[0799] Example 2-4 Synthesis of LD3

[0800] Step a:

[0801] (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentoxo-2-oxa-4,7,10,13,16-pentazaheptadecane-17-ylacetate

[0802] (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazaheptadecan-17-yl acetate

[0803] At 25 °C, anhydrous copper acetate (24.8 mg, 0.14 mmol), acetic acid (4.7 mg, 0.078 mmol), and lead tetraacetate (181.5 mg, 0.41 mmol) were added to a solution of compound LD-Int1 (210.0 mg, 0.34 mmol) in N,N-dimethylformamide (3 mL). The reaction mixture was stirred at 60 °C for 20 minutes. After the reaction was complete, the reaction solution was cooled to room temperature, poured into a solution of water (10 mL), and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the solution was concentrated under reduced pressure to give compound LD-3a (190.4 mg, yield 88.6%).

[0804] 1 H NMR(400MHz,DMSO-d6)δ8.84(t,1H),8.33(t,1H),8.15(d,1H),8.01(t,1H),7.89(t,2H),7.71(d,2H),7.59(t,1H),7.42(t,2H),7.33(t ,2H),7.25-7.16(m,5H),5.10(d,2H),4.54-4.50(m,1H),4.30-4.20(m,3H),3.81-3.57(m,6H),3.05(dd,1H),2.79(dd,1H),1.99(s,3H);

[0805] MS m / z (ESI): 652.3 [M+Na] + .

[0806] Step b:

[0807] (9H-fluorene-9-yl)methyl(S)-(10-benzyl-1-hydroxy-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)carbamate

[0808] (9H-fluoren-9-yl)methyl(S)-(10-benzyl-1-hydroxy-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)carbamate

[0809] At 25°C, ethylene glycol (1.1 mL, 19.72 mmol) and trifluoroacetic acid (4 mL) were added to a 20 mL solution of compound LD-3a (600.0 mg, 0.95 mmol) in dichloromethane. The reaction mixture was stirred at 25°C for 30 minutes. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by rapid silica gel column chromatography (dichloromethane:methanol = 10:1) to obtain compound LD-3b (570.3 mg, yield 94.7%).

[0810] 1 H NMR(400MHz,DMSO_d6)δ8.48(t,1H),8.33(t,1H),8.16(d,1H),8.03(t,1H),7.89(d,2H),7.71(d,2H),7.60(t,1H),7.42(d,2H),7.33(d ,2H),7.26-7.16(m,5H),4.56-4.49(m,3H),4.30-4.21(m,3H),3.81-3.58(m,7H),3.47(t,2H),3.40(t,2H),3.06(dd,1H),2.80(dd,1H);

[0811] MS m / z (ESI): 654.3 [M+Na] + .

[0812] Step c:

[0813] (9H-fluorene-9-yl)methyl(S)-(12-benzyl-1-(4-nitrophenoxy)-1,8,11,14,17-pentoxo-2,5-dioxa-7,10,13,16-tetraazaoctadecane-18-yl)carbamate

[0814] (9H-fluoren-9-yl)methyl(S)-(12-benzyl-1-(4-nitrophenoxy)-1,8,11,14,17-pentaoxo-2,5-dioxa-7,10,13,16-tetraazaoctadecan-18-yl)carbamate

[0815] At 25°C, pyridine (15 mL) and p-nitrophenyl chloroformate (507.3 mg, 2.52 mmol) were added to a tetrahydrofuran (10 mL) solution of compound LD-3b (530.5 mg, 0.84 mmol). The reaction mixture was stirred at 45°C for 4 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by rapid silica gel column chromatography (dichloromethane:methanol = 10:1) to give compound LD-3c (390.0 mg, yield 58.3%).

[0816] 1 H NMR(400MHz,DMSO_d6)δ8.58(t,1H),8.35(t,1H),8.35-8.28(m,2H),8.16(d,1H),8.02(t,1H),7.89(d,2H),7.70(d,2H),7.60-7.53(m,3H),7 .41(t,2H),7.32(t,2H),7.26-7.16(m,5H),4.61(d,2H),4.53-4.50(m, 1H),4.36-4.21(m,5H),3.80-3.58(m,8H),3.06(dd,1H),2.79(dd,1H).

[0817] Step d:

[0818] (9H-fluorene-9-yl)methyl((S)-14-benzyl-1-((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopentene[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-14-yl)-3,10,13,16,19-pentoxo-4,7-dioxa-2,9,12,15,18-pentazaeicosano-20-yl)carbamate

[0819] (9H-fluoren-9-yl)methyl((S)-14-benzyl-1-((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g ]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)-3,10,13,16,19-pentaoxo-4,7-dioxa-2,9,12,15,18-pentaazaicosan-20-yl)carbamate

[0820] At 25°C, compounds Int 1-k (123.1 mg, 0.25 mmol), 1-hydroxybenzotriazole (27.7 mg, 0.21 mmol), and pyridine (97.1 mg, 1.23 mmol) were added to a solution of compound LD-3c (159.4 mg, 0.20 mmol) in N,N-dimethylformamide (5 mL). The reaction mixture was stirred at 45°C for 3 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by rapid silica gel column chromatography (dichloromethane:methanol = 10:1) to obtain compound LD-3d (170.0 mg, yield 76.3%).

[0821] 1 H NMR(400MHz,DMSO_d6)δ8.49(t,1H),8.31(t,1H),8.14(d,1H),8.01(t,1H),7.86(d,2H),7.72(t ,1H),7.69(d,2H),7.55(s,2H),7.39(t,2H),7.30(t,2H),7.24-7.15(m,6H),6.51(s,1H),6.37( s,2H),5.42(s,2H),5.40(s,2H),5.00(d,2H),4.53-4.46(m,3H),4.28-4.18(m,3H),4.04(t,2H) ,3.79-3.57(m,6H),3.52(t,2H),3.03(dd,1H),2.78(dd,1H),1.88-1.81(m,2H),0.87(t,3H);MS m / z(ESI):1113.3[M+H] + .

[0822] Step e:

[0823] (S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecyl(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopentene[4,5-g]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-14-yl)methyl)carbamate

[0824] (S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecyl(((S)-15-chloro-7-ethyl-7-hydroxy-8,11- dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)carbamate

[0825] At 25°C, piperidine (0.3 mL) was added to a 2 mL solution of compound LD-3d (56.0 mg, 0.05 mmol) in N,N-dimethylformamide. The reaction mixture was stirred at 25°C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain compound LD-3e, which was then directly used for the next reaction.

[0826] MS m / z (ESI): 891.3 [M+H] + .

[0827] Step f:

[0828] (S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-6,9,12,15,18-pentoxo-3-oxa-5,8,11,14,17-pentazatrialkyl(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopentene[4,5-g]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-14-yl)methyl)carbamate

[0829] (S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazatricosyl(((S)-15-chloro-7-et hyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)carbamate

[0830] At 25°C, 2,5-dioxopyrrolidone-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate (41.5 mg, 0.13 mmol) was added to a solution of compound LD-3e (80.0 mg, 0.090 mmol) in N,N-dimethylformamide (2 mL). The reaction mixture was stirred at 25°C for 2 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by high performance liquid chromatography (Welchrom C18, 20 × 150 mm, 5 μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD3 (20.5 mg, 2-step yield 37.6%).

[0831] 1 H NMR(400MHz,DMSO-d6)δ8.48(t,1H),8.28(t,1H),8.11(d,1H),8.06(t,1H),8.00(t,1H),7. 73(t,1H),7.57(s,1H),7.26-7.14(m,6H),6.99(s,2H),6.38(s,2H),5.42(s,4H),5.01(d,2H ),4.53-4.44(m,3H),4.05-4.02(m,2H),3.98-3.50(m,9H),3.36(t,2H),3.04(dd,1H),2.78 (dd,1H),2.10(t,2H),1.91-1.80(m,2H),1.51-1.41(m,4H),1.23-1.14(m,2H),0.87(t,3H);

[0832] MS m / z (ESI): 1084.0 [M+H] + .

[0833] Examples 2-5 Synthesis of LD4

[0834] Step a:

[0835] (52S,63S)-63-benzyl-52-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl)-51,55,58,61,64,67-hexaoxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,70-heptadecoxa-50 ,56,59,62,65,68-Hexaazaheptadecane-72-yl(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopentene[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-14-yl)methyl)carbamate

[0836] (52S,63S)-63-benzyl-52-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-51,55,58,61,64 ,67-hexaoxa-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,70-heptadecaoxa-50,56,59,62,65,68 -hexaazadoheptacontan-72-yl(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)carbamate

[0837] At 30 °C, 2,5-dioxopyrrolidone-1-yl(S)-52-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-51-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47-hexadecoxa-50-azapentadecane-55-ester (57.1 mg, 0.049 mmol) was added to a solution of compound LD-3e (40.0 mg, 0.044 mmol) in N,N-dimethylformamide (1 mL). The reaction system was stirred at 30 °C for 0.5 h. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by high performance liquid chromatography (Welchrom C18, 20×150mm, 5μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD4 (25.3mg, yield 29.2%).

[0838] 1 H NMR(400MHz,DMSO-d6)δ8.48(t,1H),8.30(t,1H),8.11-8.02(m,3H),7.88-7.92(m,2H),7.73-7.71(m,1H),7.56(s,1H), 7.24-7.16(m,6H),6.99(s,2H),6.38(s,3H),5.42(s,4H),5.01(d,2H),4.53-4.45(m,3H),4.22-4.17(m,1H),4.04-4.03 (m,2H),3.75-3.66(m,5H),3.50(s,62H),3.43-3.35(m,6H),3.24(s,3H),3.21-3.18(m,1H),3.05-3.01(m,1H),2.81-2. 75(m,1H),2.14-2.07(m,4H),1.89-1.80(m,3H),1.73-1.66(m,1H),1.50-1.43(m,4H),1.21-1.13(m,2H),0.87(t,3H); MS m / z(ESI):966.1[1 / 2M+H] + .

[0839] Examples 2-6 Synthesis of LD5

[0840] Step a:

[0841] (9H-fluorene-9-yl)methyl((S)-12-benzyl-1-(((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopentene[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-1-yl)amino)-1,8,11,14,17-pentoxo-2,5-dioxa-7,10,13,16-tetraazaoctadecane-18-yl)carbamate

[0842] (9H-fluoren-9-yl)methyl((S)-12-benzyl-1-(((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dio xolo[4,5-g]pyrano[3′,4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,8,11,14,17-pentaoxo-2,5-dioxa-7,10,13,16-tetraazaoctadecan-18-yl)carbamate

[0843] At 25 °C, compounds Int 3-i (50.1 mg, 0.089 mmol), 1-hydroxybenzotriazole (11.7 mg, 0.087 mmol), and pyridine (41.0 mg, 0.52 mmol) were added to a solution of compound LD-3c (137.5 mg, 0.17 mmol) in N,N-dimethylformamide (2 mL). The reaction mixture was stirred at 45 °C for 3 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by rapid silica gel column chromatography (dichloromethane:methanol = 12:1) to give compound LD-5a (83.0 mg, yield 83.2%). MS m / z (ESI): 1123.4 [M+H] + .

[0844] Step b:

[0845] (S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecyl((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxa[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-1-yl)carbamate

[0846] (S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecyl((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-di oxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0847] At 25°C, piperidine (0.5 mL) was added to a solution of compound LD-5a (80.0 mg, 0.071 mmol) in N,N-dimethylformamide (3 mL), and the reaction mixture was stirred at 25°C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the concentrate was recrystallized from ethyl acetate (15 mL). The solution was filtered to obtain compound LD-5b (60.0 mg, crude product), which was then directly used for the next reaction.

[0848] MS m / z (ESI): 901.3 [M+H] + .

[0849] Step c:

[0850] (S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-6,9,12,15,18-pentoxo-3-oxa-5,8,11,14,17-pentazatrialkyl((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxa[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate

[0851] (S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazatricosyl((1S,10S)-10-ethyl-7-fluoro- 10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0852] At 25 °C, 2,5-dioxopyrrolidone-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate (24.6 mg, 0.08 mmol) was added to a solution of compound LD-5b (24.0 mg, 0.027 mmol) in N,N-dimethylformamide (2 mL). The reaction mixture was stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by high performance liquid chromatography (Welchrom C18, 20 × 150 mm, 5 μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD5 (8.3 mg, 26.6% yield in 2 steps).

[0853] 1 H NMR(400MHz,DMSO-d6)δ8.52(t,1H),8.29(t,1H),8.12(d,1H),8.08-8.04(m,2H),8.00(t,1H),7 .27-7.12(m,6H),6.99(s,2H),6.52(brs,1H),6.37(d,2H),5.42(s,2H),5.21(brs,3H),4.62-4.5 3(m,2H),4.52-4.44(m,1H),4.22-4.14(m,2H),3.76-3.51(m,10H),3.11-2.93(m,3H),2.81-2.73 (m,1H),2.14-2.04(m,4H),1.92-1.80(m,2H),1.52-1.42(m,4H),1.25-1.14(m,2H),0.88(t,3H);

[0854] MS m / z (ESI): 1094.2 [M+H] + .

[0855] Example 2-7 Synthesis of LD6

[0856] Step a:

[0857] (52S,63S)-63-benzyl-52-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-51,55,58,61,64,67-hexaoxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,70-heptaaza-50,56,59,6 2,65,68-Hexaazaheptadecane-72-yl((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopentene[4,5-g]pyran[3',4':6,7]indolo[1,2-b]quinoline-1-yl)carbamate

[0858] (52S,63S)-63-benzyl-52-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-51,55,58,61,64,67- hexaoxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,70-heptadecaoxa-50,56,59,62,65,68-hexaaza doheptacontan-72-yl((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydr o-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0859] At 25°C, 2,5-dioxopyrrolidone-1-yl(S)-52-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-51-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47-hexadecoxa-50-azapentadecane-55-ester (76.9 mg, 0.067 mmol) and N,N-diisopropylethylamine (12.9 mg, 0.10 mmol) were added to a solution of compound LD-5b (60.0 mg, 0.067 mmol) in N,N-dimethylformamide (2 mL). The reaction system was stirred at 25°C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by high performance liquid chromatography (Welchrom C18, 20×150mm, 5μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD6 (27.7mg, yield 21.4%).

[0860] 1 H NMR(400MHz,DMSO_d6)δ8.53(t,1H),8.32(t,1H),8.12-8.03(m,4H),7.97-7.89(m,2H),7.27(s,1H),7.24-7.1 4(m,5H),7.00(s,2H),6.53(s,1H),6.38(d,2H),5.42(s,2H),5.37-5.20(m,3H),4.60-4.56(m,2H),4.50-4.45 (m,1H),4.22-4.14(m,3H),3.77-3.31(m,72H),3.23(s,3H),3.20-3.13(m,2H),3.07-2.93(m,3H),2.78(dd,1H ),2.14-2.03(m,6H),1.92-1.79(m,3H),1.73-1.64(m,1H),1.50-1.42(m,4H),1.21-1.13(m,2H),0.88(t,3H);

[0861] MS m / z (ESI): 993.0 [1 / 2M+Na] + .

[0862] Example 2-8 Synthesis of LD7

[0863] Step a:

[0864] Dibenzylcyclopropane-1,1-dicarboxylate

[0865] dibenzyl cyclopropane-1,1-dicarboxylate

[0866] At 25 °C, 1,2-dibromoethane (16.15 g, 85.97 mmol), tetrabutylammonium bromide (90.0 mg, 0.28 mmol), and potassium carbonate (19.80 g, 143.26 mmol) were added to a solution of dibenzyl malonate (16.30 g, 57.33 mmol) in N,N-dimethylformamide (150 mL). The reaction mixture was stirred at 25 °C for 7 hours. After the reaction was complete, water (200 mL) was added to the solution, and the mixture was extracted with ethyl acetate (250 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by rapid silica gel column chromatography (petroleum ether:ethyl acetate = 15:1) to give compound LD-7a (15.70 g, yield 88.2%).

[0867] 1 H NMR (400MHz, CDCl3) δ7.33-7.28(m,10H),5.16(s,4H),1.49(s,4H);

[0868] MS m / z (ESI): 311.4 [M+H] + .

[0869] Step b:

[0870] Benzyl 1-(hydroxymethyl)cyclopropane-1-carboxylate

[0871] benzyl 1-(hydroxymethyl)cyclopropane-1-carboxylate

[0872] Under nitrogen protection, diisobutylaluminum hydride (66.1 mL, 99.20 mmol, 1.5 M in toluene) was slowly added dropwise to a dichloromethane (200 mL) solution of compound LD-7a (14.00 g, 45.11 mmol) at -65 °C. The reaction mixture was stirred at -15 °C for 30 min. After the reaction was complete, the reaction mixture was quenched with hydrochloric acid (150 mL) and stirred at room temperature for 15 min, followed by extraction with dichloromethane (300 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by rapid silica gel column chromatography (petroleum ether: ethyl acetate = 15:1) to give compound LD-7b (4.45 g, yield 47.8%).

[0873] 1H NMR (400MHz, CDCl3) δ7.39-7.30(m,5H),5.14(s,2H),3.65(s,2H),2.60(brs,1H),1.34-1.31(m,2H),0.92-0.87(m,2H);

[0874] MS m / z (ESI): 207.2 [M+H] + .

[0875] Step c:

[0876] Benzyl(S)-1-(9-benzyl-19-(9H-fluorene-9-yl)-5,8,11,14,17-pentoxo-2,18-dioxa-4,7,10,13,16-pentazanonadecanyl)cyclopropane-1-carboxylate

[0877] benzyl(S)-1-(9-benzyl-19-(9H-fluoren-9-yl)-5,8,11,14,17-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazanonadecyl)cyclopropane-1-carboxylate

[0878] Compound LD-3a (780.0 mg, 1.24 mmol) and p-toluenesulfonic acid monohydrate (258.9 mg, 1.36 mmol) were added to a tetrahydrofuran (24 mL) solution of compound LD-7b (511.0 mg, 2.48 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 2 hours. After the reaction was complete, the mixture was poured into a water (20 mL) solution and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by rapid silica gel column chromatography (petroleum ether:ethyl acetate = 15:1) to give compound LD-7c (241.0 mg, yield 25.1%).

[0879] 1H NMR(400MHz,DMSO_d6)δ8.48(t,1H),8.30(t,1H),8.14(d,1H),8.01(t,1H),7.89( d,2H),7.71(d,2H),7.59(t,1H),7.43-7.31(m,9H),7.27-7.16(m,5H),5.09(s,2H) ,4.57-4.48(m,3H),4.30-4.18(m,3H),3.79-3.68(m,3H),3.68-3.58(m,3H),3.55( s,2H),3.08-3.03(m,1H),2.82-2.76(m,1H),1.34-1.10(m,2H),0.92-0.89(m,2H);

[0880] MS m / z (ESI): 776.6 [M+H] + .

[0881] Step d:

[0882] (S)-1-(9-benzyl-19-(9H-fluoren-9-yl)-5,8,11,14,17-pentoxo-2,18-dioxa-4,7,10,13,16-pentazanonadecanyl)cyclopropane-1-carboxylic acid

[0883] (S)-1-(9-benzyl-19-(9H-fluoren-9-yl)-5,8,11,14,17-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazanonadecyl)cyclopropane-1-carboxylic acid

[0884] Palladium on carbon (133.7 mg, w / w, 10%) was added to a dichloromethane / methanol (10 / 20 mL) mixed solution of compound LD-7c (650.5 mg, 0.84 mmol) at 25 °C. The reaction system was stirred at 25 °C for 2 hours in a hydrogen atmosphere. After the reaction was complete, the reaction solution was filtered and concentrated under reduced pressure to give compound LD-7d (510.0 mg, yield 88.7%).

[0885] MS m / z (ESI): 708.2 [M+Na] + .

[0886] Step e:

[0887] (9H-fluorene-9-yl)methyl((S)-9-benzyl-1-(1-(((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopentene[4,5-g]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-1-yl)carbamoyl)cyclopropyl)-5,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-yl)carbamate

[0888] (9H-fluoren-9-yl)methyl((S)-9-benzyl-1-(1-(((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxol o[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)cyclopropyl)-5,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-yl)carbamate

[0889] At 25°C, compounds Int 3-i (202.8 mg, 0.35 mmol), 1-hydroxybenzotriazole (52.0 mg, 0.38 mmol), 1H-benzotriazole-1-yloxytripyrrolyl hexafluorophosphate (200.3 mg, 0.38 mmol), and N,N-diisopropylethylamine (124.4 mg, 0.96 mmol) were added to a 2 mL solution of N,N-dimethylformamide (2 mL) containing compound LD-7d (220.1 mg, 0.32 mmol). The reaction mixture was stirred at 25°C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by rapid silica gel column chromatography (dichloromethane:methanol = 20:1) to obtain compound LD-7e (320.0 mg, yield 88.0%).

[0890] 1H NMR(400MHz,DMSO-d6)δ8.45(t,1H),8.29(t,1H),8.15(d,1H),8.03-7.96(m,2H),7.87(d,2H),7.68(d,2H),7.59(t,1H),7.4 2-7.38(m,2H),7.33-7.28(m,2H),7.25-7.21(m,3H),7.18-7.14(m,3H),6.51(s,1H),6.34(d,2H),5.54-5.53(m,1H),5.45-5. 35(m,2H),5.27-5.02(m,2H),4.56-4.52(m,2H),4.40-4.38(m,1H),4.28-4.19(m,3H),3.78-3.72(m,1H),3.61-3.49(m,6H),3 .17-2.90(m,4H),2.72-2.67(m,1H),2.14-2.07(m,2H),1.85-1.79(m,2H),1.12-1.09(m,2H),0.87(t,3H),0.79-0.72(m,2H);

[0891] MS m / z (ESI): 1133.2 [M+H] + .

[0892] Step f:

[0893] 1-((S)-15-amino-9-benzyl-5,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecanyl)-N-((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxa[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide

[0894] 1-((S)-15-amino-9-benzyl-5,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecyl)-N-((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2 ,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide

[0895] Piperidine (0.5 mL) was added to a solution of compound LD-7e (100.0 mg, 0.088 mmol) in N,N-dimethylformamide (3 mL) at 25 °C, and the reaction mixture was stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the concentrate was recrystallized from ethyl acetate (15 mL). The solution was filtered to obtain compound LD-7f (70.0 mg, yield 87.0%).

[0896] MS m / z (ESI): 911.3 [M+H] + .

[0897] Step g:

[0898] 1-((S)-9-benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14,17-pentoxo-2-oxa-4,7,10,13,16-pentazadocosyl)-N-((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxa[4,5-g]pyrano[3',4′:6,7]indolo[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide

[0899] 1-((S)-9-benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17-penta oxo-2-oxa-4,7,10,13,16-pentaazadocosyl)-N-((1S,10S)-10-ethyl-7-fluoro-10-hyd roxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide

[0900] At 25 °C, 2,5-dioxopyrrolidone-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate (12.2 mg, 0.04 mmol) was added to a solution of compound LD-7f (30.0 mg, 0.033 mmol) in N,N-dimethylformamide (1 mL). The reaction mixture was stirred at 25 °C for 20 minutes. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by high performance liquid chromatography (Welchrom C18, 20 × 150 mm, 5 μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD7 (11.1 mg, yield 30.5%).

[0901] 1 H NMR(400MHz,DMSO-d6)δ8.43(t,1H),8.26(t,1H),8.12-8.06(m,2H),7.99-7.97(m,2H),7.26-7.16 (m,6H),6.99(s,2H),6.52(s,1H),6.35(d,2H),5.55-5.54(m,1H),5.45-5.36(m,2H),5.28-5.04(m ,2H),4.59-4.50(m,2H),4.40-4.37(m,1H),3.75-3.45(m,10H),3.07-2.89(m,3H),2.73-2.67(m,1 H),2.10-2.06(m,4H),1.86-1.81(m,2H),1.46-1.45(m,4H),1.21-1.11(m,4H),0.88-0.72(m,5H);

[0902] MS m / z (ESI): 1104.2 [M+H]+ .

[0903] Example 2-9 Synthesis of LD8

[0904] Step a:

[0905] (S)-N 5 -((S)-9-benzyl-1-(1-(((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)carbamoyl)cyclopropyl)-5,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoamide)-N 1 -(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47-hexadecanopentadecan-49-yl)pentadiamide

[0906] (S)-N 5 -((S)-9-benzyl-1-(1-(((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,1 1,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino [1,2-b]quinolin-1-yl)carbamoyl)cyclopropyl)-5,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N 1 -(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47-hexadecaoxanonatetracontan-49-yl)pentanediamide

[0907] At 25 °C, 2,5-dioxopyrrolidone-1-yl(S)-52-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-51-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47-hexadecoxa-50-azapentapentadecane-55-ester (53.0 mg, 0.046 mmol) and N,N-diisopropylethylamine (5.4 mg, 0.042 mmol) were added to a solution of compound LD-7f (38.0 mg, 0.042 mmol) in N,N-dimethylformamide (1 mL). The reaction system was stirred at 25 °C for 30 minutes. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by high performance liquid chromatography (Welchrom C18, 20×150mm, 5μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD8 (14.1mg, yield 17.3%).

[0908] 1 H NMR(400MHz,DMSO-d6)δ8.43(t,1H),8.27(t,1H),8.11-7.89(m,6H),7.26-7.16(m,6H),7.00(s,2H),6.50(b rs,1H),6.35(d,2H),5.58-5.52(m,1H),5.45-5.36(m,2H),5.26(d,1H),5.07(d,1H),4.60-4.51(m,2H),4.41 -4.35(m,1H),4.21-4.16(m,1H),3.75-3.32(m,64H),3.20-3.13(m,5H),3.08-2.89(m,4H),2.73-2.67(m,1H) ,2.10-1.99(m,8H),1.86-1.65(m,5H),1.50-1.40(m,5H),1.23-1.09(m,7H),0.86(t,3H),0.82-0.71(m,2H);

[0909] MS m / z (ESI): 975.9 [1 / 2M+H] + .

[0910] Example 2-10 Synthesis of LD9

[0911] Step a:

[0912] 1-((S)-9-benzyl-29-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14,17,27-hexaoxo-2,20,23-trioxa-4,7,10,13,16,26-hexaazaeicosaprenyl)-N-(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indo[1,2-b]quinoline-14-yl)methyl)cyclopropane-1-carboxamide

[0913] 1-((S)-9-benzyl-29-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17,27-he xaoxo-2,20,23-trioxa-4,7,10,13,16,26-hexaazanonacosyl)-N-(((S)-15-chloro-7-e thyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)cyclopropane-1-carboxamide

[0914] At 25°C, 3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propamido)ethoxy)ethoxy)propionic acid (32.8 mg, 0.010 mmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride hydrate (39.2 mg, 0.13 mmol) and N-methylmorpholine (13.5 mg, 0.13 mmol) were added to a solution of compound LD-2b (60.0 mg, 0.067 mmol) in N,N-dimethylformamide (1 mL), and the mixture was stirred at 25°C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by high performance liquid chromatography (Welchrom C18, 20×150mm, 5μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD9 (21.5mg, yield 26.7%).

[0915] 1H NMR(400MHz,DMSO-d6)δ8.50(t,1H),8.27(t,1H),8.15-8.09(m,2H),7.98(t,3H),7.57-7.54(m,1H ),7.26-7.17(m,6H),6.99(s,2H),6.49(s,1H),6.38(s,2H),5.47(s,2H),5.42(s,2H),5.09(brs,2H ),4.60(d,2H),4.50-4.43(m,1H),3.76-3.45(m,18H),3.15-3.11(m,2H),3.04-2.99(m,1H),2.80- 2.74(m,1H),2.38(t,2H),2.32(t,2H),1.90-1.80(m,2H),0.94(brs,2H),0.86(t,3H),0.63(s,2H);

[0916] MS m / z (ESI): 1211.1 [M+H] + .

[0917] Example 2-11 Synthesis of LD10

[0918] Step a:

[0919] (S)-10-benzyl-30-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-6,9,12,15,18,28-hexaoxo-3,21,24-trioxa-5,8,11,14,17,27-hexaazatridodecyl(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxa[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)methyl)carbamate

[0920] (S)-10-benzyl-30-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15,18,28-hexaoxo-3,21,24-trioxa-5,8,11,14,17,27-hexaazatriacontyl(((S)-15-chlo ro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)carbamate

[0921] At 25°C, 3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propamido)ethoxy)ethoxy)propionic acid (27.6 mg, 0.084 mmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride hydrate (33.1 mg, 0.11 mmol) and N-methylmorpholine (11.4 mg, 0.11 mmol) were added to a solution of compound LD-3e (50.0 mg, 0.056 mmol) in N,N-dimethylformamide (2 mL), and the mixture was stirred at 25°C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by high performance liquid chromatography (Welchrom C18, 20×150mm, 5μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD10 (17.1mg, yield 25.4%).

[0922] 1H NMR(400MHz,DMSO-d6)δ8.51-8.48(m,1H),8.31-8.28(m,1H),8.18-8.11(m,2H),8.01(m,2H),7.75 -7.73(m,1H),7.57(s,1H),7.24-7.17(m,6H),7.00(s,2H),6.51(s,1H),6.38(s,2H),5.42(s,4H), 5.01(d,2H),4.53-4.45(m,3H),4.05-4.04(m,2H),3.77-3.52(m,16H),3.35-3.32(m,2H),3.15-3. 11(m,2H),3.05-3.01(m,1H),2.81-2.75(m,1H),2.40-2.30(m,4H),1.91-1.80(m,2H),0.88(t,3H);

[0923] MS m / z (ESI): 1201.0 [M+H] + .

[0924] Example 2-12 Synthesis of LD11

[0925] Step a:

[0926] 1-((S)-9-benzyl-28-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14,17-pentoxo-2,20,23,26-tetraoxa-4,7,10,13,16-pentazaoctadecyl)-N-(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-14-yl)methyl)cyclopropane-1-carboxamide

[0927] 1-((S)-9-benzyl-28-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17-penta oxo-2,20,23,26-tetraoxa-4,7,10,13,16-pentaazaoctacosyl)-N-(((S)-15-chloro-7- ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)cyclopropane-1-carboxamide

[0928] At 25°C, 3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethoxy)ethoxy)ethoxy)propionic acid (17.5 mg, 0.058 mmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride hydrate (22.9 mg, 0.08 mmol) and N-methylmorpholine (7.9 mg, 0.08 mmol) were added to a solution of compound LD-2b (35.0 mg, 0.039 mmol) in N,N-dimethylformamide (4 mL), and the mixture was stirred at 25°C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by high performance liquid chromatography (Welchrom C18, 20×150mm, 5μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD11 (14.0mg, yield 30.4%).

[0929] 1H NMR(400MHz,DMSO-d6)δ8.52(t,1H),8.30(t,1H),8.16-8.12(m,2H),8.00(t,2H),7.57(s,1 H),7.24-7.16(m,6H),7.01(s,2H),6.41(br,1H),6.38(s,2H),5.47(s,2H),5.42(s,2H),5.0 9(br,2H),4.61(d,2H),4.46-4.45(m,1H),3.75-3.43(m,22H),3.15-3.11(m,1H),2.79-2.73 (m,1H),2.33-2.30(m,2H),1.88-1.81(m,2H),0.94-0.87(m,2H),0.86(t,3H),0.61(br,2H);

[0930] MS m / z (ESI): 1184.1 [M+H] + .

[0931] Example 2-13 Synthesis of LD12

[0932] Step a:

[0933] 1-((S)-9-benzyl-31-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14,17-pentoxo-2,20,23,26,29-pentoxa-4,7,10,13,16-pentazatridodecyl)-N-(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-14-yl)methyl)cyclopropane-1-carboxamide

[0934] 1-((S)-9-benzyl-31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17-pentaox o-2,20,23,26,29-pentaoxa-4,7,10,13,16-pentaazahentriacontyl)-N-(((S)-15-chloro -7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)cyclopropane-1-carboxamide

[0935] At 25 °C, 1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12-tetraoxapentadecan-15-acid (28.8 mg, 0.083 mmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride hydrate (32.7 mg, 0.11 mmol) and N-methylmorpholine (11.2 mg, 0.11 mmol) were added to a solution of compound LD-2b (50.0 mg, 0.055 mmol) in N,N-dimethylformamide (2 mL), and the mixture was stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by high performance liquid chromatography (Welchrom C18, 20×150mm, 5μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD12 (15.0 mg, yield 22.0%).

[0936] 1H NMR(400MHz,DMSO-d6)δ8.49(t,1H),8.26(t,1H),8.14(t,1H),8.10(d,1H),7.98(br,2H),7 .56(s,1H),7.25-7.14(m,6H),7.01(s,2H),6.50(br,1H),6.38(s,2H),5.46(s,2H),5.41(s ,2H),5.09(br,2H),4.61(d,2H),4.46-4.45(m,1H),3.81-3.41(m,26H),3.04-2.99(m,1H), 2.80-2.74(m,1H),2.37(t,2H),1.88-1.81(m,2H),0.94(br,2H),0.86(t,3H),0.63(br,2H);

[0937] MS m / z (ESI): 1228.1 [M+H] + .

[0938] Example 2-14 Synthesis of LD13

[0939] Step a:

[0940] 1-((S)-9-benzyl-32-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14,17,30-hexaoxo-2,20,23,26-tetraoxa-4,7,10,13,16,29-hexaazatridodecyl)-N-(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-14-yl)methyl)cyclopropane-1-carboxamide

[0941] 1-((S)-9-benzyl-32-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17,30-hexa oxo-2,20,23,26-tetraoxa-4,7,10,13,16,29-hexaazadotriacontyl)-N-(((S)-15-chloro -7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)cyclopropane-1-carboxamide

[0942] At 25 °C, 1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3-oxo-7,10,13-trioxa-4-azahexadecane-16-acid (36.0 mg, 0.097 mmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride hydrate (38.0 mg, 0.13 mmol), and N-methylmorpholine (13.0 mg, 0.13 mmol) were added to a solution of compound LD-2b (58.0 mg, 0.064 mmol) in N,N-dimethylformamide (2 mL), and the mixture was stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by high performance liquid chromatography (Welchrom C18, 20×150mm, 5μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD13 (13.2mg, yield 16.3%).

[0943] 1H NMR(400MHz,DMSO-d6)δ8.50(br s,1H),8.28(br,1H),8.15(d,2H),7.99(br,3H),7.56(s,1H),7.25-7.14( m,6H),6.99(s,2H),6.50-6.38(m,3H),5.46(s,2H),5.41(s,2H),5.09(br s,2H),4.60(s,2H),4.46-4.45(m,1H),3.89-3.41(m,22H),3.14(s,2H),3.03(d,1H),2.68-2.67(m ,1H),2.41-2.32(m,4H),1.86-1.85(m,2H),0.95-0.94(m,2H),0.87-0.85(m,3H),0.63(br,2H); MS m / z(ESI):1255.1[M+H] + .

[0944] Example 2-15 Synthesis of LD14

[0945] Step a:

[0946] (S)-10-benzyl-29-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-6,9,12,15,18-pentoxo-3,21,24,27-tetraoxa-5,8,11,14,17-pentazanonadecanyl(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolo[1,2-b]quinoline-14-yl)methyl)carbamate

[0947] (S)-10-benzyl-29-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15,18-pentaoxo-3,21,24,27-tetraoxa-5,8,11,14,17-pentaazanonacosyl(((S)-15-chlo ro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)carbamate

[0948] At 25°C, 3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethoxy)ethoxy)ethoxy)propionic acid (43.6 mg, 0.14 mmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride hydrate (56.9 mg, 0.19 mmol) and N-methylmorpholine (19.5 mg, 0.19 mmol) were added to a solution of compound LD-3e (86.0 mg, 0.096 mmol) in N,N-dimethylformamide (2 mL), and the mixture was stirred at 25°C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by high performance liquid chromatography (Welchrom C18, 20×150mm, 5μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD14 (20.9mg, yield 18.4%).

[0949] 1 H NMR(400MHz,DMSO-d6)δ8.49(t,1H),8.27(t,1H),8.14(t,1H),8.09(d,1H),7. 99(t,1H),7.72(t,1H),7.56(s,1H),7.26-7.15(m,6H),7.01(s,2H),6.38(s,2H ),5.42(s,4H),5.01(d,2H),4.53-4.45(m,3H),4.05-4.02m,2H),3.77-3.41(m, 23H),3.03(dd,1H),2.78(dd,1H),2.38(t,2H),1.89-1.82(m,2H),0.87(t,3H);

[0950] MS m / z (ESI): 1174.1 [M+H] + .

[0951] Example 2-16 Synthesis of LD15

[0952] Step a:

[0953] (S)-10-benzyl-33-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-6,9,12,15,18,31-hexaoxo-3,21,24,27-tetraoxa-5,8,11,14,17,30-hexaazatrianealkyl(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-14-yl)methyl)carbamate

[0954] (S)-10-benzyl-33-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15,18,31-hexaoxo-3,21,24,27-tetraoxa-5,8,11,14,17,30-hexaazatritriacontyl((S)-15- chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)carbamate

[0955] At 25 °C, 1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3-oxo-7,10,13-trioxa-4-azahexadecane-16-acid (31.3 mg, 0.084 mmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride hydrate (33.1 mg, 0.11 mmol) and N-methylmorpholine (11.4 mg, 0.11 mmol) were added to a solution of compound LD-3e (50.0 mg, 0.056 mmol) in N,N-dimethylformamide (2 mL), and then stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by high performance liquid chromatography (Welchrom C18, 20×150mm, 5μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD15 (13.0 mg, yield 18.6%).

[0956] 1H NMR(400MHz,DMSO-d6)δ8.48(t,1H),8.29(t,1H),8.16(t,1H),8.12(d,1H),8.01-7.99(m,2H),7.73-7.7 2(m,1H),7.57(s,1H),7.26-7.16(m,6H),7.00(s,2H),6.47(br,1H),6.38(s,2H),5.42(s,4H),5.01(d,2 H),4.53-4.47(m,3H),4.03(t,2H),3.76-3.71(m,5H),3.69-3.63(m,5H),3.60-3.46(m,12H),3.15-3.11 (m,2H),3.04-3.01(m,1H),2.81-2.77(m,1H),2.38(t,2H),2.32(t,2H),1.87-1.83(m,2H),0.86(t,3H);

[0957] MS m / z (ESI): 1245.2 [M+H] + .

[0958] Example 2-17 Synthesis of LD16

[0959] Step a:

[0960] (S)-10-benzyl-32-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-6,9,12,15,18-pentoxo-3,21,24,27,30-pentoxa-5,8,11,14,17-pentazatridodecyl(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-14-yl)methyl)carbamate

[0961] (S)-10-benzyl-32-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15,18-pentaoxo-3,21,24,27,30-pentaoxa-5,8,11,14,17-pentaazadotriacontyl(((S)-15-c hloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)carbamate

[0962] At 25°C, 1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12-tetraoxapentadecan-15-acid (31.3 mg, 0.09 mmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride hydrate (31.7 mg, 0.11 mmol) and N-methylmorpholine (10.9 mg, 0.11 mmol) were added to a solution of compound LD-3e (48.0 mg, 0.054 mmol) in N,N-dimethylformamide (2 mL), and the mixture was stirred at 25°C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by high performance liquid chromatography (Welchrom C18, 20×150mm, 5μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD16 (17.3mg, yield 26.4%).

[0963] 1 H NMR(400MHz,DMSO-d6)δ8.49(t,1H),8.29(t,1H),8.16(t,1H),8.11(d,1H)8.00(t ,1H),7.74(t,1H),7.57(s,1H),7.26-7.15(m,6H),7.02(s,2H),6.51(s,1H),6.38( s,2H),5.43(s,4H),5.00(d,2H),4.53-4.46(m,3H),4.05-4.02(m,2H),3.77-3.39 (m,26H),3.03(dd,1H),2.78(dd,1H),2.38(t,2H),1.89-1.82(m,2H),0.87(t,3H);

[0964] MS m / z (ESI): 1218.1 [M+H] + .

[0965] Example 2-18 Synthesis of LD17

[0966] Step a:

[0967] 1-((S)-9-benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14,17-pentoxo-2,20-dioxa-4,7,10,13,16-pentazadocosyl)-N-(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indo[1,2-b]quinoline-14-yl)methyl)cyclopropane-1-carboxamide

[0968] 1-((S)-9-benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17-pentaoxo-2,20-dioxa-4,7,10,13,16-pentaazadocosyl)-N-(((S)-15-chloro-7-ethyl- 7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)cyclopropane-1-carboxamide

[0969] At 25°C, 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethoxy)propionic acid (15.6 mg, 0.073 mmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride hydrate (39.3 mg, 0.13 mmol) and N-methylmorpholine (13.5 mg, 0.13 mmol) were added to a solution of compound LD-2b (60.0 mg, 0.067 mmol) in N,N-dimethylformamide (1 mL), and the mixture was stirred at 25°C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by high performance liquid chromatography (Welchrom C18, 20×150mm, 5μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD17 (15.9mg, yield 21.8%).

[0970] 1 H NMR(400MHz,DMSO-d6)δ8.51-8.48(m,1H),8.28-8.25(m,1H),8.11-8.09(m,2H),7.99-7.96(m,2H),7 .57(s,1H),7.26-7.15(m,6H),6.99(s,2H),6.49(s,1H),6.38(s,2H),5.44(d,4H),5.09-5.06(m,2H), 4.60(d,2H),4.49-4.43(m,1H),3.76-3.65(m,4H),3.63-3.51(m,6H),3.47-3.44(m,4H),3.04-2.99(m ,1H),2.80-2.74(m,1H),2.34-2.30(m,2H),1.89-1.81(m,2H),0.94(s,2H),0.87(t,3H),0.63(s,2H);

[0971] MS m / z (ESI): 1096.1 [M+H] + .

[0972] Example 2-19 Synthesis of LD18

[0973] Step a:

[0974] (S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-6,9,12,15,18-pentoxo-3,21-dioxa-5,8,11,14,17-pentazatrialkyl(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-14-yl)methyl)carbamate

[0975] (S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15,18-pentaoxo-3,21-dioxa-5,8,11,14,17-pentaazatricosyl(((S)-15-chloro-7- ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)carbamate

[0976] At 25°C, 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethoxy)propionic acid (17.9 mg, 0.084 mmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride hydrate (33.1 mg, 0.11 mmol) and N-methylmorpholine (11.4 mg, 0.11 mmol) were added to a solution of compound LD-3e (50.0 mg, 0.056 mmol) in N,N-dimethylformamide (2 mL), and the mixture was stirred at 25°C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by high performance liquid chromatography (Welchrom C18, 20×150mm, 5μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD18 (13.9mg, yield 22.8%).

[0977] 1H NMR(400MHz,DMSO-d6)δ8.47(t,1H),8.27(t,1H),8.10-8.08(m,2H),7.98(t,1H) ,7.72(t,1H),7.56(s,1H),7.25-7.15(m,6H),6.99(s,2H),6.49(s,1H),6.38(s, 2H),5.42(s,4H),5.01(d,2H),4.53-4.45(m,3H),4.05-4.03(m,2H),3.76-3.33( m,14H),3.03(dd,1H),2.78(dd,1H),2.32(t,2H),1.89-1.82(m,2H),0.87(t,3H);

[0978] MS m / z (ESI): 1086.1 [M+H] + .

[0979] Example 2-20 Synthesis of LD20

[0980] Step a:

[0981] tert-butyl 3-(methylamino)propionate

[0982] tert-butyl 3-(methylamino)propanoate

[0983] At -45°C, methylamine hydrochloride (1.58 g, 23.40 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (7.13 g, 46.83 mmol) were added to a solution of tert-butyl acrylate (1.00 g, 7.80 mmol) in N,N-dimethylformamide (15 mL), and the mixture was stirred at 0°C for 2.5 hours. After the reaction was complete, the mixture was poured into a solution of water (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with a saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure to give compound LD-20a (0.84 g, yield 67.6%).

[0984] 1 H NMR(400MHz, CDCl3)δ2.81(t,2H),2.45-2.41(m,2H),2.43(s,3H),1.45(s,9H)

[0985] Step b:

[0986] 3-((2-(((benzyloxy)carbonyl)amino)ethyl)(methyl)amino)tert-butyl propionate

[0987] tert-butyl 3-((2-(((benzyloxy)carbonyl)amino)ethyl)(methyl)amino)propanoate

[0988] At 25 °C, benzyl (2-bromoethyl)carbamate (4.37 g, 16.93 mmol), potassium carbonate (2.60 g, 18.84 mmol), and sodium iodide (0.28 g, 1.87 mmol) were added to a solution of compound LD-20a (3.00 g, 18.84 mmol) in N,N-dimethylformamide (90 mL), and the mixture was stirred at 25 °C for 16 hours. After the reaction was complete, the mixture was poured into a solution of water (100 mL) and extracted with ethyl acetate (150 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (dichloromethane:methanol = 20:1) to give compound LD-20b (1.90 g, yield 30.0%).

[0989] 1 H NMR(400MHz,DMSO-d6)δ7.38-7.29(m,5H),7.02-6.99(m,1H),5.00(s,2H),3.09-3.04(m ,2H),2.58-2.54(m,2H),2.38-2.35(m,2H),2.31-2.28(m,2H),2.15(s,3H),1.39(s,9H).

[0990] Step c:

[0991] 3-((2-aminoethyl)(methyl)amino)propionate tert-butyl ester

[0992] tert-butyl 3-((2-aminoethyl)(methyl)amino)propanoate

[0993] At 25°C, 5% palladium on carbon (0.56 g) was added to a methanol (25 mL) solution of compound LD-20b (1.89 g, 5.62 mmol), and the reaction mixture was stirred at 25°C under hydrogen atmosphere for 1 hour. After the reaction was completed, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain compound LD-20c (1.10 g, crude product was used directly in the next step).

[0994] MS m / z (ESI): 203.1 [M+H] + .

[0995] Step d:

[0996] 4-((2-((3-(tert-butoxy)-3-oxopropyl)(methyl)amino)ethyl)amino)-4-oxobut-2-enoic acid

[0997] Maleic anhydride (460.5 mg, 4.70 mmol) was added to a solution of compound LD-20c (950.0 mg, 4.70 mmol) in dichloromethane (30 mL) at 0 °C, and the mixture was stirred at 15 °C for 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure, recrystallized from ethyl acetate, filtered, and dried to give compound LD-20d (1070.0 mg, yield 75.9%).

[0998] 1 H NMR(400MHz,DMSO-d6)δ9.07(s,1H),6.36(d,1H),6.21(d,1H),3.30-3.25(m,2H), 2.68-2.65(m,2H),2.54-2.51(m,2H),2.38-2.35(m,2H),2.24(s,3H),1.39(s,9H);

[0999] MS m / z (ESI): 301.2 [M+H] + .

[1000] Step e:

[1001] 3-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethyl)(methyl)amino)tert-butyl propionate

[1002] tert-butyl 3-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)propanoate

[1003] At 15 °C, acetic anhydride (659.4 mg, 6.46 mmol) and sodium acetate (39.7 mg, 0.48 mmol) were added to a 20 mL acetonitrile solution of compound LD-20d (970.0 mg, 3.23 mmol), and the mixture was stirred at 85 °C for 2 hours. After the reaction was completed, the mixture was cooled to room temperature, and the reaction solution was concentrated under reduced pressure. The crude product was purified by rapid silica gel column chromatography (dichloromethane:methanol = 20:1) to give compound LD-20e (896.0 mg, yield 98.3%).

[1004] 1 H NMR (400MHz, DMSO-d6) δ7.00(s,2H),3.48(t,2H),2.55(t,2H),2.45(t,2H),2.23(t,2H),2.16(s,3H),1.37(s,9H);

[1005] MS m / z (ESI): 283.2 [M+H] + .

[1006] Step f:

[1007] 3-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethyl)(methyl)amino)propionic acid

[1008] 3-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)propanoic acid

[1009] At 25°C, trifluoroacetic acid (2 mL) was added to a dichloromethane (2 mL) solution of compound LD-20e (100.0 mg, 0.35 mmol), and the mixture was stirred at 25°C for 4 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain compound LD-20f (80.0 mg, the crude product was used directly in the next step).

[1010] MS m / z (ESI): 227.1 [M+H] + .

[1011] Step g:

[1012] 3-(2-(3-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)propanamido)ethoxy)propanoate tert-butyl 3-(2-(3-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)propanamido)ethoxy)propanoate

[1013] At 25°C, tert-butyl 3-(2-aminoethoxy)propionate (234.2 mg, 1.24 mmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride hydrate (363.9 mg, 1.23 mmol), and N-methylmorpholine (0.41 mL, 3.71 mmol) were added to a solution of compound LD-20f (280.0 mg, 1.24 mmol) in N,N-dimethylformamide (10 mL), and the mixture was stirred at 25°C for 0.5 hours. After the reaction was complete, the solution was poured into water (15 mL), and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated saline solution and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure. The crude product was purified by rapid silica gel column chromatography (dichloromethane:methanol = 20:1) to obtain compound LD-20g (250.0 mg, yield 50.8%).

[1014] MS m / z (ESI): 398.3 [M+H] + .

[1015] Step h:

[1016] 3-(2-(3-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethyl)(methyl)amino)propamido)ethoxy)propionic acid

[1017] 3-(2-(3-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)propanamido)ethoxy)propanoic acid

[1018] At 20°C, trifluoroacetic acid (2 mL) was added to a solution of compound LD-20 g (250.0 mg, 0.63 mmol) in dichloromethane (2 mL), and the mixture was stirred at 20°C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain compound LD-20 h (250.0 mg, the crude product was used directly in the next step).

[1019] MS m / z (ESI): 342.2 [M+H] + .

[1020] Step i:

[1021] 1-((S)-9-benzyl-29-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-27-methyl-5,8,11,14,17,24-hexaoxo-2,20-dioxa-4,7,10,13,16,23,27-heptaazaeicosaprenyl)-N-(((S)-15-chloro-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indo[1,2-b]quinoline-14-yl)methyl)cyclopropane-1-carboxamide

[1022] 1-((S)-9-benzyl-29-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-27-methyl-5,8,11,14, 17,24-hexaoxo-2,20-dioxa-4,7,10,13,16,23,27-heptaazanonacosyl)-N-(((S)-15-chlor o-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)cyclopropane-1-carboxamide

[1023] At 25 °C, compound LD-20h (45.5 mg, 0.13 mmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride hydrate (39.2 mg, 0.13 mmol), and N-methylmorpholine (33.7 mg, 0.33 mmol) were added to a solution of compound LD-2b (60.0 mg, 0.067 mmol) in N,N-dimethylformamide (1 mL), and the mixture was stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by high performance liquid chromatography (Welchrom C18, 20 × 150 mm, 5 μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD20 (32.5 mg, yield 39.9%).

[1024] 1H NMR(400MHz, DMSO-d6)δ9.24(brs,1H),8.52(t,1H),8.29(t,1H),8.17-8.11(m,3H),8.03(t,1H),7.99(t,1H),7.57(s,1H),7. 26-7.15(m,6H),7.10(s,2H),6.49(s,1H),6.38(d,2H),5.47(s,2H),5.42(s,2H),5.10-5.08(m,2H),4.60(d,2H),4.50-4.45(m ,1H),3.80-3.76(m,2H),3.73-3.66(m,4H),3.62-3.54(m,4H),3.47-3.37(m,8H),3.25-3.20(m,2H),3.04-3.00(m,1H),2.80(s ,3H),2.80-2.74(m,1H),2.57-2.54(m,1H),2.39(t,2H),1.91-1.80(m,2H),0.95-0.94(m,2H),0.86(t,3H),0.64-0.63(m,2H);

[1025] MS m / z (ESI): 1224.2 [M+H] + .

[1026] Example 2-21 Synthesis of LD25

[1027] Step a:

[1028] (9H-fluorene-9-yl)methyl((S)-9-benzyl-1-(1-((((S)-7-ethyl-7-hydroxy-15-methyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4':6,7]indolazino[1,2-b]quinoline-14-yl)methyl)carbamoyl)cyclopropyl)-5,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-yl)carbamate

[1029] (9H-fluoren-9-yl)methyl((S)-9-benzyl-1-(1-((((S)-7-ethyl-7-hydroxy-15-methyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3 ',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)carbamoyl)cyclopropyl)-5,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-yl)carbamate

[1030] At 25°C, compounds Int 5-f (178.0 mg, 0.32 mmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride hydrate (190.5 mg, 0.65 mmol), and N-methylmorpholine (98.3 mg, 0.97 mmol) were added to a solution of compound LD-7d (226.6 mg, 0.33 mmol) in N,N-dimethylformamide (2 mL). The reaction mixture was stirred at 25°C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by rapid silica gel column chromatography (dichloromethane:methanol = 12:1) to obtain compound LD-25a (240.0 mg, yield 67.2%).

[1031] 1H NMR(400MHz,DMSO-d6)δ8.45(t,1H),8.29(t,1H),8.15(d,1H),8.03-7.96(m,2H),7.87(d,2H),7.68(d,2H),7.59(t,1H),7.4 2-7.38(m,2H),7.33-7.28(m,2H),7.25-7.21(m,3H),7.18-7.14(m,3H),6.51(s,1H),6.34(d,2H),5.54-5.53(m,1H),5.45-5. 35(m,2H),5.27-5.02(m,2H),4.56-4.52(m,2H),4.40-4.38(m,1H),4.28-4.19(m,3H),3.78-3.72(m,1H),3.61-3.49(m,6H),3 .17-2.90(m,4H),2.72-2.67(m,1H),2.14-2.07(m,2H),1.85-1.79(m,2H),1.12-1.09(m,2H),0.87(t,3H),0.79-0.72(m,2H);

[1032] MS m / z (ESI): 1103.4 [M+H] + .

[1033] Step b:

[1034] 1-((S)-15-amino-9-benzyl-5,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecanyl)-N-(((S)-7-ethyl-7-hydroxy-15-methyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline-14-yl)methyl)cyclopropane-1-carboxamide

[1035] 1-((S)-15-amino-9-benzyl-5,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecyl)-N-(((S)-7-ethyl-7-hydroxy-15-methyl-8,11-dioxo -7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)cyclopropane-1-carboxamide

[1036] Piperidine (0.6 mL) was added to a solution of compound LD-25a (240.0 mg, 0.22 mmol) in N,N-dimethylformamide (3 mL) at 25 °C, and the reaction mixture was stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the concentrate was recrystallized from ethyl acetate (15 mL). The solution was filtered to obtain compound LD-25b (140.0 mg, yield 73.1%).

[1037] MS m / z (ESI): 881.4 [M+H] + .

[1038] Step c:

[1039] 1-((S)-9-benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14,17-pentoxo-2-oxa-4,7,10,13,16-pentazadocosyl)-N-(((S)-7-ethyl-7-hydroxy-15-methyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)methyl)cyclopropane-1-carboxamide

[1040] 1-((S)-9-benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17-pentaoxo-2-oxa-4,7,10,13,16-pentaazadocosyl)-N-(((S)-7-ethyl-7-hydroxy-15 -methyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)cyclopropane-1-carboxamide

[1041] At 25 °C, 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid (35.0 mg, 0.17 mmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride hydrate (48.8 mg, 0.17 mmol) were added to a solution of compound LD-25b (73.0 mg, 0.083 mmol) in N,N-dimethylformamide (2 mL). The reaction mixture was stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by high performance liquid chromatography (Welchrom C18, 20 × 150 mm, 5 μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD25 (10.0 mg, yield 11.2%).

[1042] 1 H NMR(400MHz,DMSO-d6)δ8.46(t,1H),8.28(t,1H),8.14-8.12(m,1H),8.08-8.05(m,2H),7.99-7.94(m,1H) ,7.41(s,1H),7.25-7.13(m,6H),6.99(s,2H),6.50(s,1H),6.25(s,2H),5.39(s,2H),5.30(s,2H),4.93(s ,2H),4.57-4.51(m,2H),4.45-4.42(m,1H),3.75-3.44(m,10H),3.00-2.96(m,1H),2.78-2.56(m,4H),2.0 8(t,2H),1.87-1.78(m,2H),1.49-1.41(m,4H),1.23-1.15(m,2H),0.99(m,2H),0.87(t,3H),0.72(s,2H);

[1043] MS m / z (ESI): 1074.1 [M+H] + .

[1044] Example 2-22 Synthesis of LD26

[1045] Step a:

[1046] 1-((S)-9-benzyl-29-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14,17,27-hexaoxo-2,20,23-trioxa-4,7,10,13,16,26-hexaazanonane)-N-(((S)-7-ethyl-7-hydroxy-15-methyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline-14-yl)methyl)cyclopropane-1-carboxamide

[1047] 1-((S)-9-benzyl-29-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17,27-he xaoxo-2,20,23-trioxa-4,7,10,13,16,26-hexaazanonacosyl)-N-(((S)-7-ethyl-7-hyd roxy-15-methyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)cyclopropane-1-carboxamide

[1048] At 25°C, 3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propamido)ethoxy)ethoxy)propionic acid (50.7 mg, 0.15 mmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride hydrate (45.4 mg, 0.15 mmol) and N-methylmorpholine (23.4 mg, 0.23 mmol) were added to a 2 mL solution of N,N-dimethylformamide containing compound LD-25b (68.0 mg, 0.077 mmol), and N-methylmorpholine (23.4 mg, 0.23 mmol) were stirred at 25°C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by high performance liquid chromatography (Welchrom C18, 20×150mm, 5μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD26 (8.5mg, yield 9.2%).

[1049] 1H NMR (400MHz, DMSO-d6) δ8.48(t,1H),8.31(t,1H),8.18-8.13(m,2H),8.03-7.96(m,3H),7.41(s,1H),7. 25-7.18(m,6H),7.00(s,2H),6.48(s,1H),6.25(s,2H),5.42(s,2H),5.32(s,2H),4.94(s,2H),4.59-4. 52(m,2H),4.44-4.42(m,1H),3.77-3.44(m,16H),3.33(t,2H),3.14-3.12(m,2H),3.01-2.96(m,1H),2. 78-2.67(m,4H),2.37(t,2H),2.32(t,2H),1.88-1.82(m,2H),1.00(br,2H),0.86(t,3H),0.68(br,2H);

[1050] MS m / z (ESI): 1191.1 [M+H] + .

[1051] Example 2-23 Synthesis of LD31

[1052] Step a:

[1053] (S)-10-benzyl-31-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-6,9,12,15,18,29-hexaoxo-3,19,22,25-tetraoxa-5,8,11,14,17,28-hexaazatridecyl((1S,10S)-10-ethyl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate

[1054] (S)-10-benzyl-31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15,18,29-hexa oxo-3,19,22,25-tetraoxa-5,8,11,14,17,28-hexaazahentriacontyl((1S,10S)-10-eth yl-7-fluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[1055] At 25°C, 2-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propamido)ethoxy)ethoxy)ethyl(4-nitrophenyl)carbonate (31.0 mg, 0.067 mmol), 1-hydroxybenzotriazole (11.3 mg, 0.08 mmol), and N,N-diisopropylethylamine (10.8 mg, 0.08 mmol) were added to a 1 mL solution of N,N-dimethylformamide containing compound LD-5b (50.0 mg, 0.056 mmol). The reaction mixture was stirred at 25°C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by high performance liquid chromatography (Welchrom C18, 20×150mm, 5μm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound LD31 (18.3mg, yield 26.9%).

[1056] 1H NMR(400MHz,DMSO-d6)δ8.51(t,1H),8.30(t,1H),8.11(d,2H),8.05-7.96(m,2H),7.41(t,1H),7.27 -7.15(m,6H),6.99(s,2H),6.49(brs,1H),6.37(d,2H),5.42(s,2H),5.25-5.22(m,3H),4.59-4.48( m,3H),4.18-4.17(m,2H),4.06-4.04(m,2H),3.77-3.71(m,3H),3.62-3.55(m,12H),3.36(t,2H),3. 16-2.97(m,5H),2.79-2.74(m,1H),2.32(t,3H),2.11-2.08(m,2H),1.90-1.83(m,2H),0.88(t,3H);

[1057] MS m / z (ESI): 1227.2 [M+H] + .

[1058] The typical LD ​​compounds prepared in this embodiment are shown in Table 6 below:

[1059] Table 6

[1060] Example 3: Preparation of Antibody-Drug Conjugates (ADCs)

[1061] (1) The antibody sequences used in this disclosure are shown in Table 7 below:

[1062] Table 7

[1063] In Table 7, the anti-LIV1 antibody (SEQ ID No. 15, SEQ ID No. 16) is disclosed in CN103533957B, the anti-FRα antibody (SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19) is disclosed in CN119137154A, and the anti-ALPP antibody (SEQ ID No. 20, SEQ ID No. 21) is disclosed in WO2022197890A1.

[1064] In addition, hIgG1 was purchased from Shanghai Biointron Biotechnology Co., Ltd. (product number B117901).

[1065] (2) The ADC in this disclosure can be prepared using the following method:

[1066] Random coupling (DAR8)

[1067] The antibody was incubated with 4-15 equivalents of TCEP·HCl (Sigma Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 6.5-8.5 PB, 1 mM DTPA) for 120 minutes to open the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) of organic solvent (DMSO, DMF, DMAc, etc.) was added to the reduced antibody to help the toxin linker dissolve in the buffer. Finally, 4-15 equivalents of the toxin linker solution was added to the reaction system and reacted at 25°C for 40-180 minutes. The obtained antibody-drug conjugate (ADC) was then subjected to high-rate buffer exchange to remove the free linker-toxin molecules and organic solvent.

[1068] (3) Determination of the purity of antibody-drug conjugates (SECs)

[1069] To determine the aggregation of the obtained antibody-drug conjugates, the prepared ADCs were subsequently characterized by size exclusion chromatography. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) Size exclusion chromatography was performed on an Agilent 1260 HPLC system with a G3000SWxL column (Tosoh Bioscience LLC, 7.8 mm × 30 cm, 5 μm pore size). Antibody or conjugate samples were eluted from the column for 30 minutes at a flow rate of 0.5–1.0 mL / min with 0.1 M sodium phosphate containing 0.15 M sodium chloride and 10–15% isopropanol (pH 7.4). All data were analyzed using Agilent ChemStation software. The aggregation percentage of the antibody or antibody-drug conjugate was calculated as follows:

[1070] Where: PA = peak area of ​​the integral

[1071] (4) Determination of DAR value

[1072] To determine the drug-antibody ratio (i.e., the average drug-ligand conjugation ratio n) of the obtained antibody-drug conjugates, the prepared ADCs were subsequently analyzed by liquid chromatography-mass spectrometry (LC-MS). LC-MS analysis of the drug loading value of the conjugates was initiated after the antibody-drug conjugate sample was reduced with DTT at 37°C for 10 minutes. The reduced sample was then injected into an Acquity UPLCBEH C4 reversed-phase column (Waters Corporation, 2.1 mm × 50 mm, 1.7 μm). The LC-MS system (Agilent 1290 Infinity II LC system and Agilent 6545XT AdvanceBio Q-TOF) provides deconvolutioned LC-MS results for DAR computation.

[1073] In the antibody-drug conjugate (ADC) structures described in the following examples, TZB or tzb represents trastuzumab, HLXB represents anti-Her2 antibody, hRS7 represents anti-Trop2 antibody, LIV1 represents anti-LIV1 antibody, FRα represents anti-FRα antibody, and ALPP represents anti-ALPP antibody.

[1074] Example 3-1: Preparation of ADC-01a

[1075] The antibody trastuzumab (SEQ ID No. 1, SEQ ID No. 2) was incubated with 7 equivalents of TCEP·HCl (Sigma Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of Deruxtecan DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove the free linker-toxin molecules and organic solvent. The DAR value of ADC-01a was determined to be n = 8.2 based on the LC-MS deconvolution pattern.

[1076] Example 3-2: Preparation of ADC-01b

[1077] Antibody hRS7 (SEQ ID No. 3, SEQ ID No. 4) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of Deruxtecan DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-01b was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1078] Example 3-3: Preparation of ADC-01c

[1079] Anti-Her2 antibody HLXB (SEQ ID No. 5, SEQ ID No. 6) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of Deruxtecan DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-01c was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1080] Examples 3-4: Preparation of ADC-01d

[1081] The isotype antibody hIgG1 (purchased from Baiying Biotechnology, catalog number B117901) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of Deruxtecan DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-01d was determined to be n = 8.0 based on the LC-MS deconvolution pattern.

[1082] Examples 3-5: Preparation of ADC-01e

[1083] Anti-LIV1 antibody (LIV1) (SEQ ID No. 15, SEQ ID No. 16) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of Deruxtecan DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-01e was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1084] Examples 3-6: Preparation of ADC-01f

[1085] Anti-FRα antibodies (SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19) were incubated with 7 equivalents of TCEP·HCl (Sigma Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibodies. Subsequently, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of Deruxtecan DMAc solution were added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-01f was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1086] Examples 3-7: Preparation of ADC-01g

[1087] Anti-ALPP antibodies (SEQ ID No. 20, SEQ ID No. 21) were incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibodies. Subsequently, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of Deruxtecan DMAc solution were added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-01g was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1088] Examples 3-8: Preparation of ADC-02a

[1089] The antibody trastuzumab (SEQ ID No. 1, SEQ ID No. 2) was incubated with 7 equivalents of TCEP·HCl (Sigma Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD1 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove the free linker-toxin molecules and organic solvent. The DAR value of ADC-02a was determined to be n = 7.9 based on the LC-MS deconvolution pattern.

[1090] Examples 3-9: Preparation of ADC-02b

[1091] Antibody hRS7 (SEQ ID No. 3, SEQ ID No. 4) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD1 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-02b was determined to be n = 7.9 based on LC-MS deconvolution plot.

[1092] Examples 3-10: Preparation of ADC-02c

[1093] Anti-Her2 antibody HLXB (SEQ ID No. 5, SEQ ID No. 6) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Subsequently, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD1 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-02c was determined to be n = 7.4 based on LC-MS deconvolution plot.

[1094] Example 3-11: Preparation of ADC-02d

[1095] The isotype antibody hIgG1 (purchased from Baiying Biotechnology, catalog number B117901) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD1 of DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-02d was determined to be n = 8.0 based on the LC-MS deconvolution pattern.

[1096] Example 3-12: Preparation of ADC-02e

[1097] Anti-LIV1 antibody (LIV1) (SEQ ID No. 15, SEQ ID No. 16) was incubated with 7 equivalents of TCEP·HCl (SigmaAldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD1 of DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-02e was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1098] Example 3-13: Preparation of ADC-02f

[1099] Anti-FRα antibodies (SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19) were incubated with 7 equivalents of TCEP·HCl (Sigma Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibodies. Subsequently, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD1 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-02f was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1100] Example 3-14: Preparation of ADC-02g

[1101] Anti-ALPP antibodies (SEQ ID No. 20, SEQ ID No. 21) were incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibodies. Subsequently, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD1 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-02g was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1102] Example 3-15: Preparation of ADC-03a

[1103] The antibody trastuzumab (SEQ ID No. 1, SEQ ID No. 2) was incubated with 7 equivalents of TCEP·HCl (Sigma Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD2 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove the free linker-toxin molecules and organic solvent. The DAR value of ADC-03a was determined to be n = 7.9 based on the LC-MS deconvolution pattern.

[1104] Example 3-16: Preparation of ADC-03b

[1105] Antibody hRS7 (SEQ ID No. 3, SEQ ID No. 4) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD2 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-03b was determined to be n = 8.1 based on LC-MS deconvolution plot.

[1106] Example 3-17: Preparation of ADC-03c

[1107] Anti-Her2 antibody HLXB (SEQ ID No. 5, SEQ ID No. 6) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD2 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-03c was determined to be n = 8.1 based on LC-MS deconvolution plot.

[1108] Example 3-18: Preparation of ADC-03d

[1109] The isotype antibody hIgG1 (purchased from Baiying Biotechnology, catalog number B117901) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD2 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-03d was determined to be n = 7.9 based on LC-MS deconvolution plot.

[1110] Example 3-19: Preparation of ADC-03e

[1111] Anti-LIV1 antibody (SEQ ID No. 15, SEQ ID No. 16) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD2 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-03e was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1112] Example 3-20: Preparation of ADC-03f

[1113] Anti-FRα antibodies (SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19) were incubated with 7 equivalents of TCEP·HCl (Sigma Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibodies. Subsequently, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD2 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-03f was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1114] Example 3-21: Preparation of ADC-03g

[1115] Anti-ALPP antibody (ALPP) (SEQ ID No. 20, SEQ ID No. 21) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD2 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-03g was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1116] Example 3-22: Preparation of ADC-04a

[1117] The anti-HER2 antibody trastuzumab (TZB) (SEQ ID No. 1, SEQ ID No. 2) was incubated with 7 equivalents of TCEP·HCl (Sigma Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD3 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove the free linker-toxin molecules and organic solvent. The DAR value of ADC-04a was determined to be n = 8.0 based on the LC-MS deconvolution pattern.

[1118] Example 3-23: Preparation of ADC-04b

[1119] Anti-TROP2 antibody hRS7 (SEQ ID No. 3, SEQ ID No. 4) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD3 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-04b was determined to be n = 7.9 based on LC-MS deconvolution plot.

[1120] Example 3-24: Preparation of ADC-04c

[1121] Anti-Her2 antibody HLXB (SEQ ID No. 5, SEQ ID No. 6) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD3 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-04c was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1122] Example 3-25: Preparation of ADC-04d

[1123] The isotype antibody hIgG1 (purchased from Baiying Biotechnology, catalog number B117901) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD3 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-04d was determined to be n = 8.0 based on the LC-MS deconvolution pattern.

[1124] Example 3-26: Preparation of ADC-04e

[1125] Anti-LIV1 antibody (LIV1) (SEQ ID No. 15, SEQ ID No. 16) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD3 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-04e was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1126] Example 3-27: Preparation of ADC-04f

[1127] Anti-FRα antibodies (SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19) were incubated with 7 equivalents of TCEP·HCl (Sigma Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibodies. Subsequently, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD3 DMAc solution were added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-04f was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1128] Example 3-28: Preparation of ADC-04g

[1129] Anti-ALPP antibodies (SEQ ID No. 20, SEQ ID No. 21) were incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibodies. Subsequently, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD3 DMAc solution were added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-04g was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1130] Example 3-29: Preparation of ADC-05a

[1131] The antibody trastuzumab (SEQ ID No. 1, SEQ ID No. 2) was incubated with 7 equivalents of TCEP·HCl (Sigma Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD4 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove the free linker-toxin molecules and organic solvent. The DAR value of ADC-05a was determined to be n = 8.1 based on the LC-MS deconvolution pattern.

[1132] Examples 3-30: Preparation of ADC-05b

[1133] Antibody hRS7 (SEQ ID No. 3, SEQ ID No. 4) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD4 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-05b was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1134] Examples 3-31: Preparation of ADC-05c

[1135] Anti-Her2 antibody HLXB (SEQ ID No. 5, SEQ ID No. 6) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD4 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-05c was determined to be n = 8.1 based on LC-MS deconvolution plot.

[1136] Examples 3-32: Preparation of ADC-05d

[1137] The homotype antibody hIgG1 (purchased from Baiying Biotechnology, catalog number B117901) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD4 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-05d was determined to be n = 8.1 based on the LC-MS deconvolution pattern.

[1138] Examples 3-33: Preparation of ADC-05e

[1139] Anti-LIV1 antibody (SEQ ID No. 15, SEQ ID No. 16) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD4 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-05e was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1140] Examples 3-34: Preparation of ADC-05f

[1141] Anti-FRα antibodies (SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19) were incubated with 7 equivalents of TCEP·HCl (Sigma Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibodies. Subsequently, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD4 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-05f was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1142] Examples 3-35: Preparation of ADC-05g

[1143] Anti-ALPP antibodies (SEQ ID No. 20, SEQ ID No. 21) were incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibodies. Subsequently, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD4 DMAc solution were added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-05g was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1144] Examples 3-36: Preparation of ADC-06a

[1145] The antibody trastuzumab (SEQ ID No. 1, SEQ ID No. 2) was incubated with 7 equivalents of TCEP·HCl (Sigma Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD5 DMAc solution were added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove the free linker-toxin molecules and organic solvent. The DAR value of ADC-06a was determined to be n = 8.1 based on the LC-MS deconvolution pattern.

[1146] Examples 3-37: Preparation of ADC-06b

[1147] Antibody hRS7 (SEQ ID No. 3, SEQ ID No. 4) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD5 DMAc solution were added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-06b was determined to be n = 8.1 based on LC-MS deconvolution plot.

[1148] Examples 3-38: Preparation of ADC-06c

[1149] Anti-Her2 antibody HLXB (SEQ ID No. 5, SEQ ID No. 6) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD5 DMAc solution were added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-06c was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1150] Examples 3-39: Preparation of ADC-06d

[1151] The homotype antibody hIgG1 (purchased from Baiying Biotechnology, catalog number B117901) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD5 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-06d was determined to be n = 8.0 based on the LC-MS deconvolution pattern.

[1152] Examples 3-40: Preparation of ADC-06e

[1153] Anti-LIV1 antibody (SEQ ID No. 15, SEQ ID No. 16) was incubated with 7 equivalents of TCEP·HCl (Sigma-Aldrich, catalog number: 75259-1G) in buffer A (10-50 mM pH 7.4 PB, 1 mM DTPA) for 120 minutes to break the interchain disulfide bonds of the antibody. Then, 5-15% (v / v) organic solvent (DMAc) was added to the reduced antibody to help dissolve the toxin linker in the buffer. Finally, 10 equivalents of LD5 DMAc solution was added to the reaction system, and the reaction was carried out at 25°C for 120 minutes. The obtained antibody conjugate was then subjected to ultrafiltration to remove free linker-toxin molecules and organic solvent. The DAR value of ADC-06e was determined to be n = 8.0 based on LC-MS deconvolution plot.

[1154] Examples 3-41: Preparation of ADC-06f

[1155] Anti-FRα antibodies (SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19) were incubated with 7 equivalents of TCEP·HCl (Sigma Aldrich, catalog numbe...

Claims

1. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof, characterized in that, The ligand-drug conjugate has the structure shown in formula (I): B#—(L—D)n formula (I); B# is the ligand that binds to the target. n is the average conjugation ratio of the drug ligands, and n is an integer or decimal from about 1 to about 16; L is a connection unit with the following structure: —L3—L2—L1—; where, The L3 is selected from Where m is an integer from 0 to 8, with 1 bit connected to the ligand and 2 bits connected to L2; The L2 does not exist or is selected from C=O. Where r is 0 or 1, s is an integer from 1 to 10, t is an integer from 0 to 30, p is 0 or 1, and each q is an independent integer from 0 to 6, with 1 bit connected to L3 and 2 bits connected to L1; each R 2a and R 2b Each is independently hydrogen, -(CH2) s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X ; Each R X Independently hydrogen or C 1-6 Alkyl group; s1 is an integer from 1 to 30, s2 is an integer from 0 to 6; R 2c Selected from hydrogen, C 1-6 Alkyl or C 3-6 cycloalkyl; Preferably, L2 is absent or selected from C=O. Where s is an integer from 1 to 10, t is an integer from 1 to 30, and each q is an independent integer from 0 to 6, with 1 bit connected to L3 and 2 bits connected to L1; each R 2a and R 2b Each is independently hydrogen, -(CH2) s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X ; Each R X Independently hydrogen or C 1-6 Alkyl group; s1 is an integer from 1 to 30, s2 is an integer from 0 to 6; R 2c Selected from hydrogen, C 1-6 Alkyl or C 3-6 cycloalkyl; More preferably, L2 is absent or selected from C=O, Where s is an integer from 1 to 10, t is an integer from 1 to 30, 1 bit is connected to L3, and 2 bits are connected to L1; each R 2a and R 2b Each is independently hydrogen, -(CH2) s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X ; Each R X Independently hydrogen or C 1-6 Alkyl; R 2c Selected from hydrogen, C1-6 alkyl or C3-6 cycloalkyl; s1 is an integer from 1 to 30, and s2 is an integer from 0 to 6; L1 is Of these, bit 1 is connected to L2, and bit 2 is connected to D; D is a drug having the structure shown in formula (II) or formula (III): Among them, R 1 Halogen or C 1-6 alkyl; R 2 For F; W is In this configuration, position 1 is connected to the carbonyl group, and position 2 is connected to the methylene group.

2. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The R 1 For F, Cl, Br or C 1-3 alkyl; Preferably, the R 1 For F, Cl, -CH3, -CH2CH3, -CH2CH2CH3, or -CH(CH3)2; More preferably, the R 1 For Cl; or More preferably, the R 1 It is -CH3.

3. The ligand-drug conjugate or its pharmaceutically acceptable salt according to claim 1 or 2, characterized in that, D is selected from the following structures:

4. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, characterized in that, The L3 is selected from Where m is an integer from 0 to 6, with 1 bit connected to the ligand and 2 bits connected to L2; Preferably, m is an integer between 0 and 5; More preferably, m is 0; or More preferably, m is 2; or Preferably, m is 5.

5. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, characterized in that, The L2 does not exist.

6. The ligand-drug conjugate or its pharmaceutically acceptable salt according to any one of embodiments 1-4, characterized in that, L2 is C=O.

7. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, characterized in that, The L2 is Where s is an integer from 1 to 10, with 1 bit connected to L3 and 2 bits connected to L1; each R 2a and R 2b Each is independently hydrogen, -(CH2) s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X ; Each R X Independently hydrogen or C 1-6 Alkyl group; s1 is an integer from 1 to 30, and s2 is an integer from 0 to 6.

8. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-4 or 7, characterized in that, R of all methylene units of L2 2a and R 2b At least one of them is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X The rest of R 2a and R 2b All are hydrogen; Preferably, the R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L3 have R 2a and R 2b All are hydrogen; More preferably, the R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L2 have R 2a and R 2b All are hydrogen; More preferably, the R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L2 have R 2a and R 2b Both are hydrogen.

9. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-4 or 7-8, characterized in that, s is an integer from 1 to 8; Preferably, s is an integer from 1 to 6; Preferably, s is an integer between 3 and 6; More preferably, s is 3.

10. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-4 or 7-9, characterized in that, s1 is an integer from 1 to 28; Preferably, s1 is an integer from 4 to 26; Preferably, s1 is an integer between 8 and 24; More preferably, s1 is 14; or More preferably, s1 is 16; or More preferably, s1 is 18.

11. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-4 or 7-10, characterized in that, s2 is an integer between 0 and 5; Preferably, s2 is an integer between 0 and 4; Preferably, s2 is an integer between 0 and 3; More preferably, s2 is 0; or More preferably, s2 is 1; or More preferably, s2 is 2.

12. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-4 or 7-11, characterized in that, Each R X Independently hydrogen or C 1-3 alkyl; Preferably, R X C 1-3 alkyl; More preferably, R X It is -CH3 or -C2H5; More preferably, R X It is -CH3.

13. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-4 or 7-12, characterized in that, The R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L2 have R 2a and R 2b All are hydrogen; where s2 is 0, s1 is an integer from 4 to 26, and R X -CH3; Preferably, s1 is an integer between 8 and 24; Preferably, s1 is an integer between 12 and 20; More preferably, s1 is 14; or More preferably, s1 is 16; or More preferably, s1 is 18.

14. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-4 or 7-13, characterized in that, The L2 is s1 is an integer between 4 and 26; Preferably, s1 is an integer between 8 and 24; Preferably, s1 is an integer between 12 and 20; More preferably, s1 is 14; or More preferably, s1 is 16; or More preferably, s1 is 18.

15. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, characterized in that, The L2 is Where r is 0 or 1, t is an integer from 0 to 30, p is 0 or 1, each q is an independent integer from 0 to 6, 1 bit is connected to L3, and 2 bits are connected to L1; R 2c Selected from hydrogen, C 1-6 Alkyl or C 3-6 Cycloalkyl.

16. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-4 or 15, characterized in that, L2 is selected from Where t is an integer from 1 to 30, each q is an independent integer from 0 to 6, with 1 bit connected to L3 and 2 bits connected to L1; R 2c Selected from hydrogen, C 1-6 Alkyl or C 3-6 Cycloalkyl.

17. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-4 or 15-16, characterized in that, t is an integer from 1 to 24; Preferably, t is an integer from 1 to 20; Preferably, t is an integer from 1 to 12; More preferably, t is an integer from 1 to 6; More preferably, t is 1; or More preferably, t is 2; or More preferably, t is 3; or More preferably, t is 4.

18. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-4 or 15-17, characterized in that, Each q is independently 0, 1, 2, 3, 4 or 5; Preferably, each q is 0, 1, 2 or 3 independently.

19. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-4 or 15-18, characterized in that, R 2c Selected from C 1-6 Alkyl or C 3-6 cycloalkyl; Preferably, R 2c C 1-6 alkyl; More preferably, R 2c C 1-3 alkyl; More preferably, R 2c It is methyl or ethyl; More preferably, R 2c Methyl; or More preferably, R 2c It is an ethyl group.

20. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-4 or 15-19, characterized in that, The L2 is Where t is an integer from 1 to 24; Preferably, t is an integer from 1 to 20; Preferably, t is an integer from 1 to 12; More preferably, t is an integer from 1 to 6; More preferably, t is 2.

21. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-4 or 15-19, characterized in that, The L2 is Where t is an integer from 1 to 24; Preferably, t is an integer from 1 to 20; Preferably, t is an integer from 1 to 12; More preferably, t is an integer from 1 to 6; More preferably, t is 1; or More preferably, t is 2; or More preferably, t is 3; or More preferably, t is 4.

22. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-21, characterized in that, L is selected from the structures in Table 1.

23. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-22, characterized in that, B# is an antibody or its antigen-binding fragment; Preferably, the B# is selected from the group consisting of chimeric antibodies, humanized antibodies, and fully human antibodies; More preferably, B# is an antibody targeting the following targets: 5T4, ADAM9, AGS-16, ANGPTL4, ALPP, ALPP2, ApoE, CTGF, CXCR5, FGF2, MCPT8, MFI2, MS4A7, NCA, Sema5b, SLITRK6, STC2, TGF, O772P, ACTA2, ADGRE1, AG-7, AIF1, AKR1C1, AKR1C2, ASLG659, Axl, B7H3, BAFF-R, BCMA, BMPR1B, BNIP3, C1QA, C1QB, CA6, CADM1, CCD79b, CCL5, CCR 5. CCR7, CD11c, CD123, CD138, CD142, CD147, CD166, CD19, CD21, CD20, CD205, CD22, CD223, CD228, CD25, CD30, CD33, CD37, CD38, CD40, CD45, CD45 ( PTPRC), CD46, CD47, CD49D(ITGA4), CD56, CD66e, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CDCP1, CDH11, CDH17, CD11b, CEA, CEACAM5, Claudin 18.

2. c-Met, COL6A3, COL7A1, CRIPTO, CSF1R, CTSD, CTSS, CXCL11, CXCL10, DDIT4, DLL3, DLL4, DR5, E16, EFNA4, EGFR, EGF RvIII, EGLN, EGLN3, EMR2, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FcRH2, FcRHl, FGFR2, FGFR3, FLT3, FOLR-α, FRα, GD2, G EDA, GPC-1, GPNMB, GPR20, GZMB, HER2, HER3, HLA-DOB, HMOX1, IFI6, IFNG, IGF-1R, IGFBP3, IL10RA1, IL-13R, IL-2, IL 20Ra, IL-3, IL-4, IL-6, IRTA2, KISS1R, KRT33A, LIV1, LOX, LRP-1, LRRC15, LUM, LY64, LY6E, Ly86, LYPD3, MDP, MMP10, M MP14, MMP16, MPF, MSG783, MSLN, MUC-1, NaPi2b, Napi3b, Nectin-4, NOG, P2X5, pCAD, P-Cadherin, PDGFRA, PDK1, PD-L1, PFKFB3, PGF, PGK1, PIK3AP1, PIK3CD, PLOD2, PSCA, PSCAhlg, PSMA, PTK7, P-cadherin, RNF43, ROR1, ROR2, SERPINE1, SL C39A6, SLTRK6, STAT1, STEAP1, STEAP2, TCF4, TENB2, TGFB1, TGFB2, TGFBR1, TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, WNT5A, epidermal growth factor, short proteoglycans, mesothelin, endothelial peptide receptors, mucins (such as mucin 1 and mucin 16), guanylate cyclase C, integrin α4β7, integrin α5β6, trophoblast cell glycoproteins, and tissue factor. More preferably, B# is an antibody targeting the following targets: ALPP, HER2, HER3, B7H3, TROP2, Claudin 18.2, CD30, CD33, CD70, EGFR, or FRα; For example, B# is an anti-Trop-2 antibody or its antigen-binding fragment; or, for example, B# is datopotamab, sacituzumab or its antigen-binding fragment; or For example, B# is an anti-Her 2 antibody or its antigen-binding fragment; or, for example, B# is anbenitamab, coprelotamab, disitamab, gancotamab, margetuximab, timigutuzumab, zanidatamab, trastuzumab, pertuzumab or its antigen-binding fragment; or For example, B# is an anti-Her 2 antibody or its antigen-binding fragment; it comprises two heavy chains and two light chains, the heavy chains comprising a heavy chain variable region, the heavy chain variable region comprising heavy chain complementarity-determining region 1 (HCDR1) as shown in SEQ ID NO:7, heavy chain complementarity-determining region 2 (HCDR2) as shown in SEQ ID NO:8, and heavy chain complementarity-determining region 3 (HCDR3) as shown in SEQ ID NO:9, the light chains comprising a light chain variable region, the light chain variable region comprising light chain complementarity-determining region 1 (LCDR1) as shown in SEQ ID NO:10, light chain complementarity-determining region 2 (LCDR2) as shown in SEQ ID NO:11, and light chain complementarity-determining region 3 (LCDR3) as shown in SEQ ID NO:12; for another example, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:13, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:14; for yet another example, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:6, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:

5. For example, B# is an anti-Her3 antibody or its antigen-binding fragment; or, for example, B# is a barecetamab, duligotuzumab, elgemtumab, istiramumab, lumretuzumab, patritumab, seribantumab, zenocutuzumab, 202-2-1 antibody or its antigen-binding fragment. For example, B# is an anti-EGFR antibody or its antigen-binding fragment; or, for example, B# is demupitamab, depatuxizumab, futuximab, imgatuzumab, lapatuximab, losatuxizumab, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, pimurutamab, serclutamab, tomuzotuximab, zalutumumab, Cetuximab or its antigen-binding fragment. For example, B# is an anti-B7H3 antibody or its antigen-binding fragment; or, for example, B# is 1D1, 1D1-01, 2E3, 2E3-02 antibody, enoblituzumab, mirzotamab, omburtamab or its antigen-binding fragment. For example, B# is an anti-LIV1 antibody or its antigen-binding fragment; or, for another example, B# is Ladiratuzumab or its antigen-binding fragment.

24. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-23, characterized in that, The average conjugation ratio n of the drug ligand is an integer or decimal of about 1 to about 10; Preferably, the average conjugation ratio n of the drug ligand is an integer or decimal of about 3 to about 8; or Preferably, the average conjugation ratio n of the drug ligand is an integer or decimal of about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, or about 9 to about 10; or Preferably, the average conjugation ratio n of the drug ligand is about 1, about 2, about 3, about 4, about 5, about 6, about 7 or about 8.

25. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-24, characterized in that, The ligand-drug conjugates are selected from the structures in Table 2 or Table 3.

26. A compound or its tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The compound has the structure shown in formula (IA): L-D formula (IA); Wherein, L is a connecting unit, having the following structure: L3'—L2—L1—; The L3' is selected from: Where m is an integer from 0 to 6, and 2 bits are connected to L2; L2, L1 and D are as defined in claim 1.

27. The compound according to claim 26, or its tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, characterized in that, The R 1 For F, Cl, Br or C 1-3 alkyl; Preferably, the R 1 For F, Cl, -CH3, -CH2CH3, -CH2CH2CH3, or -CH(CH3)2; More preferably, the R 1 For Cl; or More preferably, the R 1 It is -CH3.

28. The compound according to claim 26 or 27, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, D is selected from the following structures:

29. The compound according to any one of claims 26-28, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, L3' is selected from Where m is an integer from 0 to 6, with 1 bit connected to the ligand and 2 bits connected to L2; Preferably, m is an integer between 0 and 5; More preferably, m is 0; or More preferably, m is 2; or Preferably, m is 5.

30. The compound according to any one of claims 26-29, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The L2 does not exist.

31. The compound according to any one of claims 26-29, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, L2 is C=O.

32. The compound according to any one of claims 26-29, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The L2 is Where s is an integer from 1 to 10, with 1 bit connected to L3 and 2 bits connected to L1; each R 2a and R 2b Each is independently hydrogen, -(CH2) s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X ; Each R X Independently hydrogen or C 1-6 Alkyl group; s1 is an integer from 1 to 30, and s2 is an integer from 0 to 6.

33. The compound according to any one of claims 26-29 or 32, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, R of all methylene units of L2 2a and R 2b At least one of them is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X The rest of R 2a and R 2b All are hydrogen; Preferably, the R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 C(=O)N((CH2CH2O) s1 -R X 2、-(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X -(CH2) s2 OC(=O)N((CH2CH2O) s1 R X 2. or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L3 have R 2a and R 2b All are hydrogen; More preferably, the R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X -(CH2) s2 NHC(=O)(CH2CH2O) s1 -R X -(CH2) s2 O(CH2CH2O) s1 -R X or -(CH2) s2 OC(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L2 have R 2a and R 2b All are hydrogen; More preferably, the R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L2 have R 2a and R 2b Both are hydrogen.

34. The compound according to any one of claims 26-29 or 32-33, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, s is an integer from 1 to 8; Preferably, s is an integer from 1 to 6; Preferably, s is an integer from 3 to 6. More preferably, s is 3.

35. The compound according to any one of claims 26-29 or 32-34, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, s1 is an integer from 1 to 28; Preferably, s1 is an integer from 4 to 26; Preferably, s1 is an integer between 8 and 24. More preferably, s1 is 14; or More preferably, s1 is 16; or More preferably, s1 is 18.

36. The compound according to any one of claims 26-29 or 32-35, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, s2 is an integer between 0 and 6; Preferably, s2 is an integer between 0 and 5; Preferably, s2 is an integer between 0 and 3; More preferably, s2 is 0; or More preferably, s2 is 1; or More preferably, s2 is 2.

37. The compound according to any one of claims 26-29 or 32-36, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, Each R X Independently hydrogen or C 1-3 alkyl; Preferably, R X C 1-3 alkyl; More preferably, R X It is -CH3 or -C2H5; More preferably, R X It is -CH3.

38. The compound according to any one of claims 26-29 or 32-37, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The R of one methylene unit of L2 2a and R 2b One is hydrogen, and the other is -(CH2). s2 C(=O)NH(CH2CH2O) s1 -R X The remaining methylene units of L2 have R 2a and R 2b All are hydrogen; where s2 is 0, s1 is an integer from 4 to 26, and R X -CH3; Preferably, s1 is an integer between 8 and 24; Preferably, s1 is an integer between 12 and 20; More preferably, s1 is 14; or More preferably, s1 is 16; or More preferably, s1 is 18.

39. The compound according to any one of claims 26-29 or 32-38, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The L2 is s1 is an integer between 4 and 26; Preferably, s1 is an integer between 8 and 24; Preferably, s1 is an integer between 12 and 20; More preferably, s1 is 14; or More preferably, s1 is 16; or More preferably, s1 is 18.

40. The compound according to any one of claims 26-29, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The L2 is Where r is 0 or 1, t is an integer from 0 to 30, p is 0 or 1, each q is an independent integer from 0 to 6, 1 bit is connected to L3, and 2 bits are connected to L1; R 2c Selected from hydrogen, C 1-6 Alkyl or C 3-6 Cycloalkyl.

41. The compound according to any one of claims 26-29 or 40, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, L2 is selected from Where t is an integer from 1 to 30, each q is an independent integer from 0 to 6, with 1 bit connected to L3 and 2 bits connected to L1; R 2c Selected from hydrogen, C 1-6 Alkyl or C 3-6 Cycloalkyl.

42. The compound according to any one of claims 26-29 or 40-41, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, t is an integer from 1 to 24; Preferably, t is an integer from 1 to 20; Preferably, t is an integer from 1 to 12; More preferably, t is an integer from 1 to 6; More preferably, t is 1; or More preferably, t is 2; or More preferably, t is 3; or More preferably, t is 4.

43. The compound according to any one of claims 26-29 or 40-42, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, Each q is independently 0, 1, 2, 3, 4 or 5; Preferably, each q is 0, 1, 2 or 3 independently.

44. The compound according to any one of claims 26-29 or 40-43, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, R 2c Selected from C 1-6 Alkyl or C 3-6 cycloalkyl; Preferably, R 2c C 1-6 alkyl; More preferably, R 2c C 1-3 alkyl; More preferably, R 2c It is methyl or ethyl; More preferably, R 2c Methyl; or More preferably, R 2c It is an ethyl group.

45. The compound according to any one of claims 26-29 or 40-44, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The L2 is Where t is an integer from 1 to 24; Preferably, t is an integer from 1 to 20; Preferably, t is an integer from 1 to 12; More preferably, t is an integer from 1 to 6; More preferably, t is 2.

46. ​​The compound according to any one of claims 26-29 or 40-44, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The L2 is Where t is an integer from 1 to 24; Preferably, t is an integer from 1 to 20; Preferably, t is an integer from 1 to 12; More preferably, t is an integer from 1 to 6; More preferably, t is 1; or More preferably, t is 2; or More preferably, t is 3; or More preferably, t is 4.

47. The compound according to any one of claims 26-46, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The compounds mentioned are selected from those in Table 4.

48. A compound or its tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The compound has the structure shown in formula (IIA) or formula (IIIA): Among them, R 1 Halogen or C 1-6 alkyl; R 2 For F; W is In this configuration, position 1 is connected to the carbonyl group, and position 2 is connected to the methylene group.

49. The compound according to claim 48, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The R 1 For F, Cl, Br or C 1-3 alkyl; Preferably, the R 1 For F, Cl, -CH3, -CH2CH3, -CH2CH2CH3, or -CH(CH3)2; More preferably, the R 1 For Cl; More preferably, the R 1 It is -CH3.

50. The compound according to claim 48 or 49, or its tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, characterized in that, The compound is selected from:

51. A method for preparing a ligand-drug conjugate of formula (I) as described in any one of claims 1-25, or a pharmaceutically acceptable salt thereof, characterized in that, The method includes contacting ligand B# with the structure of formula (IA) as described in any one of claims 26-47.

52. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains a ligand-drug conjugate of formula (I) as described in any one of claims 1-25 or a pharmaceutically acceptable salt thereof, or a compound as described in any one of claims 48-50 or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

53. The use of a ligand-drug conjugate of formula (I) according to any one of claims 1-25, or a pharmaceutically acceptable salt thereof, a compound according to any one of claims 26-47, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, a compound according to any one of claims 48-50, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and / or the use of the pharmaceutical composition of claim 52 in the preparation of a medicament for treating and / or preventing tumors; Preferably, the drug is a ligand-drug conjugate.

54. A method of treating and / or preventing tumors, comprising administering to a subject in need a ligand-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1-25, a compound or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 52.

55. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-25, a compound or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 52, for the treatment and / or prevention of tumors.

56. The use according to claim 53, the method according to claim 54, the ligand-drug conjugate or its pharmaceutically acceptable salt or pharmaceutical composition according to claim 55, wherein the compound or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, or pharmaceutical composition, is characterized in that, The tumors were selected from those associated with the expression of the following groups: 5T4, ADAM9, AGS-16, ANGPTL4, ALPP, ALPP2, ApoE, CTGF, CXCR5, FGF2, MCPT8, MFI2, MS4A7, NCA, Sema5b, SLITRK6, STC2, TGF, 0772P, ACTA2, ADGRE1, AG-7, AIF1, AKR1C1, AKR1C2, ASLG659, Axl, B7H3, BAFF-R, BCMA, BMPR1B, BNIP3, C1QA, C1QB, CA6, CADM1, CCD79b, CCL5, CCR5. , CCR7, CD11c, CD123, CD138, CD142, CD147, CD166, CD19, CD21, CD20, CD205, CD22, CD223, CD228, CD25, CD30, CD33, CD37, CD38, CD40, CD45, CD45 ( PTPRC), CD46, CD47, CD49D(ITGA4), CD56, CD66e, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CDCP1, CDH11, CDH17, CD11b, CEA, CEACAM5, Claudin 18.

2. c-Met, COL6A3, COL7A1, CRIPTO, CSF1R, CTSD, CTSS, CXCL11, CXCL10, DDIT4, DLL3, DLL4, DR5, E16, EFNA4, EGFR, EGF RvIII, EGLN, EGLN3, EMR2, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FcRH2, FcRHl, FGFR2, FGFR3, FLT3, FOLR-α, FRα, GD2, G EDA, GPC-1, GPNMB, GPR20, GZMB, HER2, HER3, HLA-DOB, HMOX1, IFI6, IFNG, IGF-1R, IGFBP3, IL10RA1, IL-13R, IL-2, IL 20Ra, IL-3, IL-4, IL-6, IRTA2, KISS1R, KRT33A, LIV1, LOX, LRP-1, LRRC15, LUM, LY64, LY6E, Ly86, LYPD3, MDP, MMP10, M MP14, MMP16, MPF, MSG783, MSLN, MUC-1, NaPi2b, Napi3b, Nectin-4, NOG, P2X5, pCAD, P-Cadherin, PDGFRA, PDK1, PD-L1, PFKFB3, PGF, PGK1, PIK3AP1, PIK3CD, PLOD2, PSCA, PSCAhlg, PSMA, PTK7, P-cadherin, RNF43, ROR1, ROR2, SERPINE1, SL C39A6, SLTRK6, STAT1, STEAP1, STEAP2, TCF4, TENB2, TGFB1, TGFB2, TGFBR1, TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, WNT5A, epidermal growth factor, short proteoglycans, mesothelin, endothelial peptide receptors, mucins (such as mucin 1 and mucin 16), guanylate cyclase C, integrin α4β7, integrin α5β6, trophoblast cell glycoproteins, and tissue factor. Preferably, the tumor is selected from tumors associated with the expression of the following groups: ALPP, HER2, HER3, B7H3, TROP2, LIV1, Claudin 18.2, CD30, CD33, CD70, EGFR, or FRα; For example, the tumor is a tumor associated with HER2 expression; For example, the tumor is a tumor associated with HER3 expression; For example, the tumor is a tumor associated with B7H3 expression; For example, the tumor described is a tumor associated with TROP2 expression; For example, the tumor is a tumor associated with LIV1 expression; For example, the tumor is one associated with Claudin 18.2 expression; For example, the tumor is a tumor associated with CD30 expression; For example, the tumor is a tumor associated with CD33 expression; For example, the tumor is a tumor associated with CD70 expression; For example, the tumor is a tumor associated with EGFR expression; For example, the tumor is a tumor associated with ALPP expression; For example, the tumor is a tumor associated with FRα expression.

57. The use according to claim 53, the method according to claim 54, the ligand-drug conjugate or its pharmaceutically acceptable salt or pharmaceutical composition according to claim 55, wherein the compound or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, or pharmaceutical composition, is characterized in that, The tumors are selected from the following group: lung cancer, breast cancer, rectal cancer, colon cancer, esophageal cancer, gastric cancer, liver cancer, gallbladder cancer, bile duct cancer, kidney cancer, bladder cancer, urothelial carcinoma, head and neck cancer, nasopharyngeal carcinoma, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, pancreatic cancer, melanoma, bone cancer, mesothelioma, gastrointestinal stromal tumor, sarcoma, glioma, thyroid cancer, salivary gland tumor, glioblastoma, neuroblastoma, gastric mucinous tumor, lymphoma, leukemia, plasmacytoma, sinoatrial node cell tumor, giant cell tumor of the tendon sheath, brain cancer, squamous cell carcinoma, epidermal carcinoma, and non-Hodgkin's lymphoma. Preferably, the tumor meets one or more of the following conditions: (1) The tumors are selected from breast cancer, gastric cancer, prostate cancer, lung cancer, pancreatic cancer, ovarian cancer, cervical cancer, endometrial cancer, kidney cancer, bladder cancer, glioma, malignant lymphoma, liver cancer and leukemia; (2) The tumor expressed HER2, TROP2, LIV1, FRα and / or ALPP positive; (3) The tumor is HER2-positive gastric cancer or HER2-positive breast cancer; (4) The tumor is TROP2-positive human pancreatic carcinoma in situ or TROP2-positive breast cancer; (5) The tumor is a LIV1-positive renal cell carcinoma; (6) The tumor is FRα-positive endometrial cancer or FRα-positive ovarian cancer; (7) The tumor is ALPP-positive non-small cell lung cancer.

58. A kit comprising a ligand-drug conjugate of formula (I) as described in any one of claims 1-25 or a pharmaceutically acceptable salt thereof, a compound as described in claim 48 or 50 or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and / or a pharmaceutical composition as described in claim 52.

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