Linker-drug molecules, antibody drug conjugates, and uses thereof

Abstract

The present application belongs to the field of medicine, and relates to a linker-drug molecule, an antibody-drug conjugate and use thereof. Specifically, the present application relates to an antibody-drug conjugate represented by formula (I) or a pharmaceutically acceptable salt thereof. The antibody-drug conjugate involved in the present application has good cytotoxic activity and can inhibit the proliferation of tumor cells. (I)

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals. Specifically, this invention relates to a linker-drug molecule, an antibody-drug conjugate, and their uses, as well as their use in the prevention and / or treatment of diseases related to abnormal cellular activity, including but not limited to their use in the prevention and / or treatment of cancer. Background Technology

[0002] Antibody-drug conjugates (ADCs) are a promising type of targeted therapy. They consist of three main parts: an antibody (Ab) that selectively recognizes tumor cell surface antigens, a small molecule cytotoxic drug (Payload) that kills tumor cells, and a linker that links the Payload to the Ab. The antibody acts as a carrier to deliver the small molecule cytotoxic drug to the target cells.

[0003] Antibody-associated drug (ADC) drugs enter cells via endocytosis after binding to tumor cell surface antigens. Then, small-molecule cytotoxic drugs dissociate from the ADC to exert their biological functions. Therefore, ADCs combine the potent killing effect of traditional small-molecule cytotoxic drugs with the tumor-targeting properties of antibodies. Currently, several ADC drugs are used to treat liquid tumors and solid tumors. For example, POLIVY (Polatuzumab vedotin-piiq, CD79b-MMAE) is approved by the FDA for the treatment of relapsed or refractory diffuse large B-cell lymphoma (R / RDLBCL); PADCEV (Enfortumab vedotin-ejfv, Nectin4-MMAE) is approved by the FDA for the treatment of locally advanced or metastatic urothelial carcinoma (UC); Enhertu (Fam-trastuzumab deruxtecan-nxki, Her2-Dxd) is used to treat breast cancer, etc.; and Trodelvy (sacituzumab govitecan-hziy, TROP2-SN38) is used to treat patients with metastatic triple-negative breast cancer.

[0004] In typical antibody-drug conjugates (ADCs), the choice of antibody and drug depends on the specific disease, significantly impacting the safety and efficacy of the ADC. Factors determining the efficacy of ADCs include the stability and breakage sensitivity of the linker unit, and the activation of internalization, transport, and cytotoxicity at the cell surface. For example, T... DM1 has a much lower cytotoxicity than some anthracycline antibiotics, but it is prone to premature degradation and release toxins before being internalized by antigen-binding proteins, causing side effects. In addition, the drug / antibody ratio obtained by conjugating it with antibodies is low and the distribution varies greatly, making it difficult to precisely control its efficacy and safety (e.g., patent applications WO2012061590A1 and WO201139721A1).

[0005] Therefore, there is still a great clinical need and application value in finding linkers with high stability and good water solubility, and then developing new ADC drugs with excellent permeability, metabolic stability and good cytotoxicity. Summary of the Invention

[0006] This invention provides a novel compound containing a linker and an antitumor drug molecule. In this invention, the compound is also referred to as a "linker-drug molecule" or "linker-payload." Furthermore, this invention provides an antibody-drug conjugate formed by the compound and an antibody. The compound and the antibody-drug conjugate exhibit good antitumor activity, excellent permeability, low efflux rate, good plasma stability, and good metabolic stability, showing promise for the treatment of cancer. In addition, the antibody-drug conjugate of this invention has good tolerability, few toxic side effects, a wide therapeutic window, good tumor tissue targeting, and a strong bystander effect.

[0007] On the one hand, the present invention provides an antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof.

[0008] (I) in, Ab represents an antibody or antigen-binding fragment; L1 is a linker that connects an S atom to an antibody or antigen-binding fragment; L2 is the connecting segment between connectors L1 and L3; L3 is the linker segment connecting L2 and drug D; Drug D is selected from cytotoxic drugs, drugs for treating autoimmune diseases, or anti-inflammatory drugs; t can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0009] In some embodiments, the drug D of the present invention is selected from eribulin, monomethylolpropionate E (MMAE), monomethylolpropionate F (MMAF), oropontostatin E, oropontostatin F, maytansine DM1, maytansine DM4, maytansine alcohol, sandycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, anthracyclines, chalcogenide, dolalastatin 10, docamycin, doxorubicin, tylansstatin A, hookmycin, amatoxins, ricin, diphtheria toxin, interleukin, tumor necrosis factor, chemokine, SN38, or eczema and its derivatives.

[0010] In some embodiments, the drug D of the present invention is selected from... , , , , or .

[0011] In some embodiments, L1 of the present invention is selected from the following structures: , , , , or ;in The end is connected to Ab via an S atom. The terminal is connected to L2; X represents a bond, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH=CH-, -CH=CHCH2-, -CH2CH=CH-, -C≡C-, -CH2C≡C-, -C≡C-CH2-, -CH2CH2C≡C-, -CH2C≡CCH2-, -C≡CCH2CH2-, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, indole, naphthyl, pyrrolyl, pyrazolyl, imidazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, furanyl, thienyl, thiazolyl, oxazolyl, pyridyl, pyrimidinyl, pyrazinyl, or pyridazinyl; R a and R bIndependently, it is H, D, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, vinyl, propenyl, allyl, ethynyl, propargyl, 1-propynyl, 1-ynylbutyl, 2-ynylbutyl, 3-ynylbutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxetyl, aziryl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, piperazinyl, or morpholinyl; wherein the methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl... alkyl, tert-butyl, vinyl, propenyl, allyl, ethynyl, propargyl, 1-propynyl, 1-ynylbutyl, 2-ynylbutyl, 3-ynylbutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxacyclobutyl, aziroxybutyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, piperazinyl, and morpholinyl are each independently and optionally surrounded by 1, 2, 3, 4, or 5 groups selected from D, -F, -Cl, -Br, -I, -OH, -NH2, -CN, -SH, -COOH, oxo, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Alkoxy and C 1-6 Substituents of haloalkoxy groups; m can be 1, 2, 3, 4, 5, 6, 7 or 8.

[0012] In some embodiments, L1 is selected from the following structures: , , , , , , , or ;in The end is connected to Ab via an S atom. The terminal is connected to L2.

[0013] In some embodiments, L2 is selected from a structure consisting of one or more of the following segments: C 1-6 Alkylene, -NR x -、-C(=O)-、-O-、 , , , , , , , , , , , , or ; R x For H, D, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-8 Cycloalkyl or 3-8 membered heterocyclic groups.

[0014] In some embodiments, L2 is selected from a structure consisting of one or more of the following segments: C 1-6 Alkylene, -NR x -、-C(=O)-、-O-、 , , , , , , , , , , , , , , , , , or ; R x The derivatives are H, D, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, vinyl, propenyl, allyl, ethynyl, propynyl, 1-propynyl, 1-ynylbutyl, 2-ynylbutyl, 3-ynylbutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxetyl, aziryl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, piperazinyl, or morpholinyl. n can be 1, 2, 3, 4, 5, 6, 7 or 8.

[0015] In some embodiments, L3 is selected from key, -NR y CH2-, -C(=O)-, -O-, -NR y CH2CH2NR z C(=O)-、 or ; R y and R z H, D, C independently 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-8 Cycloalkyl or 3-8 membered heterocyclic groups.

[0016] In some embodiments, R y and R z H, D, C independently 1-4 Alkyl, C 2-4 alkenyl, C 2-4 alkynyl group, C 3-6 Cycloalkyl or 3-6 membered heterocyclic groups.

[0017] In some embodiments, R y and R z Independently, it is H, D, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, vinyl, propenyl, allyl, ethynyl, propynyl, 1-propynyl, 1-ynylbutyl, 2-ynylbutyl, 3-ynylbutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxacyclobutyl, aziroxybutyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, piperazinyl, or morpholinyl.

[0018] In some embodiments, the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt thereof, wherein Selected from the following structures: , , or .

[0019] On the one hand, the present invention provides a compound of formula (II), or a stereoisomer, tautomer, nitride, hydrate, solvate, or pharmaceutically acceptable salt thereof of a compound of formula (II). L-L2-L3-D (II), in, L is selected from the following structures: , , , , or ; The terminal is connected to L2; L2, L3, D, X, R a R b "and m" have the meanings described in this invention.

[0020] In some embodiments, L is selected from the following structures: , , , , , , , or ; The terminal is connected to L2.

[0021] In some embodiments, the compounds of the present invention have the following structures, or stereoisomers, tautomers, nitrides, hydrates, solvates, or pharmaceutically acceptable salts thereof, but are by no means limited to: (1) (2) (3) or (4).

[0022] In some embodiments, the Ab described in this invention is an anti-EGFR antibody, an anti-TROP2 antibody, or an anti-MUC1 antibody, wherein the anti-EGFR antibody is Cetuximab, demupitamab, depatuxizumab, futuximab, imgatuzumab, lapatuximab, losatuxizumab, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, pimurutamab, serclutamab, tomuzotuximab, or zalutumumab. The anti-TROP2 antibody is enoblituzumab, mirzotamab, or omburtamab; The anti-MUC1 antibody comprises heavy chain variable region CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, and light chain variable region CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

[0023] In some embodiments, the anti-EGFR antibody of the present invention comprises the heavy chain of SEQ ID NO: 1 and the light chain of SEQ ID NO: 2; The anti-MUC1 antibody comprises the heavy chain of SEQ ID NO: 3 and the light chain of SEQ ID NO: 4; The anti-TROP2 antibody comprises the heavy chain of SEQ ID NO: 5 and the light chain of SEQ ID NO: 6.

[0024] On one hand, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate of formula (I), a compound of formula (II), or a pharmaceutically acceptable salt thereof.

[0025] In some embodiments, the pharmaceutical composition of the present invention comprises one or more antibody-drug conjugates of formula (I) of the present invention, wherein the DAR of the composition is an integer or decimal of about 1 to 10, for example 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 39, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6 1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.58, 7.6, 7.64, 7.66, 7.7, 7.78, 7.8, 7.86, 7.9, 7.94, 7.96, 8.0, 8.02, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0.

[0026] In some embodiments, the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier.

[0027] On the other hand, the present invention relates to the use of antibody-drug conjugates of formula (I), compounds of formula (I), or pharmaceutical compositions thereof in the preparation of medicaments for the prevention or treatment of cancer.

[0028] In some embodiments, the cancers described in this invention are breast cancer, lung cancer, kidney cancer, liver cancer, ovarian cancer, urethral cancer, prostate cancer, glioblastoma multiforme, pancreatic cancer, colorectal cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, colon cancer, rectal cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, melanoma, bladder cancer, stomach cancer, or esophageal cancer.

[0029] In another aspect, the present invention relates to methods for the preparation, separation and purification of antibody-drug conjugates of formula (I) and compounds of formula (II).

[0030] The foregoing description only outlines certain aspects of the invention, but is not limited to these aspects. These and other aspects will be described in more detail below.

[0031] Detailed Description of the Invention Definitions and general terms Certain embodiments of the invention will now be described in detail, examples of which are illustrated by the accompanying structural and chemical formulas. The invention is intended to cover all alternatives, modifications, and equivalents, all of which are included within the scope of the invention as defined in the claims. Those skilled in the art will recognize that many similar or equivalent methods and materials can be used to practice the invention. The invention is by no means limited to the methods and materials described herein. In the event that one or more of the incorporated documents, patents, and similar materials differ from or contradict this application (including, but not limited to, defined terminology, application of terminology, described techniques, etc.), this application shall prevail.

[0032] It should be further appreciated that certain features of the invention, for clarity, have been described in multiple independent embodiments, but may also be provided in combination in a single embodiment. Conversely, various features of the invention, for brevity, have been described in a single embodiment, but may also be provided individually or in any suitable sub-combination.

[0033] Unless otherwise stated, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. All patents and publications related to this invention are incorporated herein by reference in their entirety.

[0034] The term "antibody" "Drug conjugate" refers to a ligand linked to a biologically active drug via a stable linker unit. In this disclosure, "antibody" is used. Antibody drug conjugates (ADCs) are monoclonal antibodies or antibody fragments linked to biologically active toxic drugs via stable linker units.

[0035] The term "antibody" refers to an immunoglobulin molecule that specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof, via at least one antigen recognition site within the variable region of the immunoglobulin molecule. It is denoted by Ab. This encompasses complete polyclonal antibodies, complete monoclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) mutants, multispecific antibodies (e.g., bispecific antibodies, antibodies with two complementary sites, etc.), multivalent antibodies (e.g., trivalent, tetravalent, etc. antibodies with three, four, or more antigen-binding sites), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing the antigen-determining portion of an antibody, and any other modified immunoglobulin molecule containing an antigen recognition site, provided that the antibody exhibits the desired biological activity. The antigenicity of immunoglobulins varies depending on the amino acid composition and sequence of the constant region of the heavy chain. Accordingly, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in its heavy chain, it can be further divided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ or λ chain. The antibodies described in this invention are preferably specific antibodies against cell surface antigens on target cells. Non-limiting examples include the following antibodies: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, and anti-B7 antibody. 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-MUGI antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin C antibody, anti-SLC44A4 antibody, anti-CD79 antibody, anti-TROP 2. Antibody, anti-CD79B antibody, anti-Mesothelin antibody, or their antigen-binding fragments. In some embodiments, the antibody is selected from trastuzumab, pertuzumab, nimotuzumab (trade name: Taixinsheng), enoblituzumab, emibetuzumab, Inotuzumab, and Vitin. Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, and Glematumamab.

[0036] The term "heavy chain variable region (VH)" refers to the amino-terminal variable region domain of the immunoglobulin heavy chain.

[0037] The term "light chain variable region (VL)" refers to the amino-terminal variable region domain of the immunoglobulin light chain.

[0038] The term "CDR (Complementarity Determinant Region)" refers to the amino acid sequence that defines the binding affinity and specificity of the Fv region of an antibody. This is because antibody specificity depends on the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site consists primarily of residues from the so-called hypervariable region or complementarity determinant (CDR), with relatively conserved framework regions (FRs) scattered throughout other regions. The light chain (L) and heavy chain (H) of an antibody each have three CDRs, named Light Chain Complementarity Determinant Region 1 (LCDR1), Light Chain Complementarity Determinant Region 2 (LCDR2), Light Chain Complementarity Determinant Region 3 (LCDR3), and Heavy Chain Complementarity Determinant Region 1 (HCDR1), Heavy Chain Complementarity Determinant Region 2 (HCDR2), Heavy Chain Complementarity Determinant Region 3 (HCDR3), respectively. Therefore, the antigen-binding site of a conventional antibody comprises six CDRs, containing a set of CDRs from each of the heavy chain and light chain variable regions. Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0039] The term "antigen-binding fragment" refers to an antibody fragment such as Fv, Fab, F(ab')2, or Fab', or any fragment that should be able to increase its half-life through chemical modification or incorporation into liposomes. This chemical modification includes, for example, the addition of a poly(alkylene) glycol such as polyethylene glycol ("PEGylated," or "PEGylated") (the PEGylated fragment referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" stands for polyethylene glycol), which has CD73 binding activity. Preferably, the functional fragment will consist of or contain a portion of the variable region of the heavy or light chain of its source antibody, the portion of which is sufficient to retain the same binding specificity and sufficient affinity as its source antibody. Such a functional fragment will contain at least 5 amino acids, preferably 10, 15, 25, 50, and 100 consecutive amino acids from the sequence of its source antibody. Examples of antigen-binding fragments include, but are not limited to: (1) Fab fragments, which may be monovalent fragments having VL-CL chains and VH-CH1 chains; (2) F(ab')2 fragments, which may be divalent fragments having two Fab' fragments connected by disulfide bridges (i.e., Fab' dimers) in the hinge region; and (3) Fv fragments with VL and VH domains on a single arm of antibody.

[0040] The term "drug" refers to a cytotoxic drug or immunomodulator. Cytotoxic drugs are chemical molecules that strongly disrupt the normal growth of tumor cells. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations; however, due to their lack of specificity, they can also cause apoptosis of normal cells while killing tumor cells, leading to serious side effects. The term includes toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals; radioactive isotopes (e.g., radioactive isotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and Lu); toxic drugs; chemotherapeutic drugs; antibiotics; and ribolysins. Immunomodulators are inhibitors of immune checkpoint molecules. In some embodiments of this disclosure, the drug is designated as D and belongs to the category of immunomodulators, particularly TLR8 agonists.

[0041] The terms “linker,” “linker unit,” “connector unit,” “connector,” or “linker fragment” refer to a chemical structural segment or bond that is connected to a ligand at one end and to a drug at the other end. It can also be connected to other linkers before being connected to a drug.

[0042] The term "DAR" or "drug loading" typically refers to the average number of cytotoxic drugs loaded onto each ligand, or it can be expressed as the ratio of cytotoxic drug to antibody. The range of cytotoxic drug loading can be 0-12 cytotoxic drugs linked to each ligand (Ab), for example, 1-10 cytotoxic drugs. In embodiments of this application, the drug loading is expressed as t, which can be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as exemplarily. The drug loading of each ADC molecule after the coupling reaction can be identified using conventional methods such as UV / Vis spectroscopy, mass spectrometry, ELISA assays, and HPLC characterization.

[0043] A joint may include one or more joint components. An exemplary joint component includes 6... Maleiminohexanoyl (MC), Maleiminopropionyl (MP), Valine Citrulline (Val Cit or vitamin C), alanine Phenylalanine (ala) phe), p-aminobenzyloxycarbonyl (PAB), and those derived from coupling with linker reagents: N Succinimide 4 (2 Pyridylthiovalerate (SPP), N Succinimide 4 (N (maleiminomethyl)cyclohexane 1. Carboxylic acid ester (SMCC, also referred to as MCC in this invention) and N Succinimide (4) iodine Acetylaminobenzoate (SIAB). The linker may include stretching units, spacer units, amino acid units, and extension units. It can be synthesized by methods known in the art, such as US2005. As described in 0238649A1, the adapter can be a "cleavable adapter" that facilitates drug release into the cell. For example, acid-labile adapters (e.g., hydrazones), protease-sensitive adapters (e.g., peptidase-sensitive adapters), light-labile adapters, dimethyl adapters, or disulfide-containing adapters can be used (Chari et al., Cancer Research 52:127). 131 (1992); U.S. Patent No. 5,208,020).

[0044] The term "antigen-binding fragment" refers to one or more fragments of an antibody that maintain its ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in "antigen-binding fragments" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab′)2 fragments, bivalent fragments comprising two Fab fragments connected by disulfide bridges on the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VH and VL domains of a single arm of the antibody; and (v) single-domain or dAb fragments (Ward et al., (1989) Nature 341:544). 546), which consists of the VH domain; and (vi) a separate complementarity-determining region (CDR) or (vii) a combination of two or more separate CDRs connected by a synthetic linker. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by a synthetic linker using recombination methods, thereby enabling the production of a single protein chain (called a single-stranded Fv (scFv)) in which the VL and VH regions pair to form a monovalent molecule; see, for example, Bird et al. (1988) Science 242: 423. 426; and Huston et al. (1988) Proc Natl Acad Sci USA 85: 5879 5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and are screened for functionality in the same manner as for intact antibodies. The antigen-binding portion can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0045] As used in this invention, the term "test subject" refers to an animal. Typically, the animal is a mammal. Test subjects also include, for example, primates (e.g., humans, males or females), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc. In some embodiments, the test subject is a primate. In other embodiments, the test subject is a human.

[0046] As used in this invention, the term "patient" refers to a person (including adults and children) or other animal. In some embodiments, "patient" refers to a person.

[0047] The term "comprising" is an open-ended expression, meaning it includes the contents specified in this invention, but does not exclude other aspects.

[0048] "Stereoisomers" refer to compounds that have the same chemical structure but differ in the spatial arrangement of their atoms or groups. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometrical isomers (cis / trans isomers), trans-blocking isomers, and so on. Unless otherwise stated, all stereoisomers or mixtures of stereoisomers of the structural formulas described in this invention are within the scope of this invention. Furthermore, unless otherwise stated, the structural formulas of the compounds described in this invention include enriched isotopes of one or more different atoms.

[0049] The stereochemical definitions and rules used in this invention generally follow those of S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., “Stereochemistry of Organic Compounds,” John Wiley & Sons, Inc., New York, 1994.

[0050] Any mixture of stereoisomers obtained can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on differences in the physicochemical properties of the components, for example, by chromatography and / or fractional crystallization.

[0051] The terms "tautomer" or "tautomer form" refer to structural isomers with different energies that can interconvert through a low energy barrier. If tautomerism is possible (e.g., in solution), chemical equilibrium can be achieved in the tautomer. For example, proton tautomers (also called prototropic tautomers) involve interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers involve interconversions via the rearrangement of some bonding electrons. A specific example of keto-enol tautomers is the interconversion between pentane-2,4-dione and 4-hydroxypent-3-en-2-one. Another example of tautomerism is phenol-keto tautomerism. A specific example of phenol-keto tautomerism is pyridine-4-ol and pyridine-4(1... HTautomerism of keto tautomers. Unless otherwise indicated, all tautomer forms of the compounds of this invention are within the scope of this invention.

[0052] As described in this invention, the compounds of this invention can be independently and optionally substituted by one or more substituents, such as the general formula compounds above, or as the specific examples, subclasses, and classes of compounds included in this invention, as described in the embodiments. It should be understood that the terms "independently and optionally substituted" or "optionally substituted" are used interchangeably with the term "substituted or unsubstituted." Generally, the term "substituted" means that one or more hydrogen atoms in the given structure are substituted by a specific substituent. Unless otherwise indicated, an optional substituent group may be substituted at each substituted position of the group. When more than one position in the given structural formula can be substituted by one or more substituents selected from a specific group, the substituents may be substituted at the same or different positions.

[0053] Additionally, it should be noted that, unless otherwise explicitly stated, the descriptive terms “each…independently is”, “…each independently is”, and “…independently is” used in this invention are interchangeable and should be interpreted broadly. They can mean that the specific options expressed by the same symbols in different groups do not affect each other, or that the specific options expressed by the same symbols in the same group do not affect each other.

[0054] In various parts of this specification, the substituents of the compounds disclosed herein are disclosed according to the type or scope of the groups. In particular, the invention includes every independent secondary combination of the various members of these group types and scopes. For example, the term "C..." 1-6 "Alkyl" specifically refers to independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0055] Linking substituents are described in various parts of this invention. When the structure clearly requires a linking group, the Markush variable listed for that group should be understood as the linking group. For example, if the structure requires a linking group and the Markush group definition for that variable lists "alkyl" or "aryl," it should be understood that "alkyl" or "aryl" represents a linked alkylene group or an arylene group, respectively.

[0056] The term "alkyl" refers to a saturated straight-chain or branched monovalent hydrocarbon group containing 1 to 20 carbon atoms, wherein the alkyl group may optionally be substituted by one or more substituents described in this invention. In one embodiment, the alkyl group contains 1 to 6 carbon atoms, denoted as C1. 1-6 Alkyl group; in yet another embodiment, the alkyl group contains 1-4 carbon atoms, denoted as C1 1-4Alkyl group; in another embodiment, the alkyl group contains 1-3 carbon atoms, denoted as C1 1-3 Alkyl groups. Examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), n-propyl (... n -Pr, -CH2CH2CH3), isopropyl ( i -Pr, -CH(CH3)2), n-butyl ( n -Bu、-CH2CH2CH2CH3), isobutyl ( i -Bu、-CH2CH(CH3)2), sec-butyl( s -Bu、-CH(CH3)CH2CH3), tert-butyl( t -Bu、-C(CH3)3), n-pentyl(-CH2CH2CH2CH2CH3), 2-pentyl(-CH(CH3)CH2CH2CH3), 3-pentyl(-CH(CH2CH3)2), 2-methyl-2-butyl(-C(CH3)2CH2CH3), 3-methyl-2-butyl(-CH(CH3)CH (CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), n-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl ( -CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3), n-heptyl, n-octyl, etc.

[0057] The term "alkenyl" refers to a straight-chain or branched monovalent hydrocarbon group containing 2-12 carbon atoms, with at least one unsaturated site, i.e., one carbon-carbon sp. 2 A double bond, wherein the alkenyl group may optionally be replaced by one or more substituents described in this invention, including " cis "and" trans The positioning of "or " E "and" ZThe positioning of "". In one embodiment, the alkenyl group contains 2-6 carbon atoms, denoted as C 2-6 Alkenyl group; in yet another embodiment, the alkenyl group comprises 2-4 carbon atoms, denoted as C1 2-4 Alkenyl groups. Examples of alkenyl groups include, but are not limited to, vinyl (-CH=CH2), allyl (-CH2CH=CH2), and 1-propenyl (i.e., propenyl, -CH=CH). - CH3), etc.

[0058] The term "alkynyl" refers to a straight-chain or branched monovalent hydrocarbon group containing 2-12 carbon atoms, wherein there is at least one unsaturated site, i.e., one carbon-carbon sp triple bond, wherein the alkynyl group may optionally be substituted by one or more substituents described in this invention. In one embodiment, the alkynyl group comprises 2-6 carbon atoms, denoted as C1. 2-6 Alkynyl group; in yet another embodiment, the alkynyl group comprises 2-4 carbon atoms, denoted as C0. 2-4 Alkynyl. Examples of alkynyl groups include, but are not limited to, ethynyl (-C≡CH), propynyl (-CH2C≡CH), 1-propynyl (-C≡C-CH3), etc.

[0059] The term "alkylene" refers to a saturated divalent hydrocarbon group obtained by removing two hydrogen atoms from a saturated straight-chain or branched hydrocarbon group. Unless otherwise specified, the alkylene group contains 1-12 carbon atoms. The alkylene group may optionally be substituted by one or more substituents described in this invention. In one embodiment, the alkylene group contains 1-6 carbon atoms; in another embodiment, the alkylene group contains 1-4 carbon atoms; in yet another embodiment, the alkylene group contains 1-3 carbon atoms; and in still another embodiment, the alkylene group contains 1-2 carbon atoms. Examples of such groups include methylene (-CH2-), ethylene (-CH2CH2-), and isopropylene (-CH(CH3)CH2-), etc.

[0060] The term "alkenyl" refers to an unsaturated divalent hydrocarbon group obtained by removing two hydrogen atoms from an alkylene group. Unless otherwise specified, the alkenyl group contains 1-12 carbon atoms. The alkenyl group may optionally be substituted by one or more substituents described in this invention. In one embodiment, the alkenyl group contains 1-6 carbon atoms; in another embodiment, the alkenyl group contains 1-4 carbon atoms; in yet another embodiment, the alkenyl group contains 1-3 carbon atoms; and in still another embodiment, the alkylene group contains 1-2 carbon atoms. Such examples include, but are not limited to, vinylene (-CH=CH-) and propenylene (-CH2CH=CH-), etc.

[0061] The term "ynynyl" refers to a straight-chain or branched divalent hydrocarbon group containing 2-12 carbon atoms, wherein there is at least one unsaturated site, i.e., one carbon-carbon sp triple bond, wherein the ynynyl group may optionally be substituted by one or more substituents described in this invention. In one embodiment, the ynynyl group contains 2-8 carbon atoms; in another embodiment, the ynynyl group contains 2-6 carbon atoms; and in yet another embodiment, the ynynyl group contains 2-4 carbon atoms. Examples of ynynyl groups include, but are not limited to, ethynylene (-C≡C-), etc.

[0062] The term "cycloalkyl" refers to a monocyclic or bicyclic carbocyclic system with 3-12 carbon atoms, wherein the -CH2- group in the carbocyclic ring may optionally be replaced by -C(=O)- (or -(CO)-). In one embodiment, the cycloalkyl group comprises 3-10 carbon atoms, i.e., C2... 3-10 Cycloalkyl; in another embodiment, the cycloalkyl group comprises 3-7 carbon atoms, i.e., C64-C7 ... 3-7 Cycloalkyl; in another embodiment, the cycloalkyl group comprises 3-6 carbon atoms, i.e., C64-C ... 3-6 Cycloalkyl; in another embodiment, the cycloalkyl group comprises 3-5 carbon atoms, i.e., C64-C74-C ... 3-5 Cycloalkyl. In another embodiment, the cycloalkyl group is a monocyclic cycloalkyl group containing 3-7 carbon atoms, i.e., C10-C2 ... 3-7 Monocyclic cycloalkyl groups. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and octahydro-1-ylyl groups. H -Indenyl, octahydrocyclopentadienyl, bicyclo[1.1.0]butyl, bicyclo[3.1.0]hexyl, etc. Examples of -CH2- groups in the carbide ring that can be replaced by -C(=O)- include, but are not limited to: cyclopentanone, cyclobutanone, etc.

[0063] The term "heterocycle" or "heterocyclic group" refers to a saturated or partially unsaturated monocyclic, bicyclic, or tricyclic system comprising 3 to 12 ring atoms, wherein at least one ring atom is selected from nitrogen, sulfur, and oxygen atoms; wherein the heterocycle or heterocyclic group is non-aromatic and does not contain any aromatic ring. When a heterocycle is attached to other parts of a molecule via a linking site, the heterocycle is represented as a monovalent heterocyclic group. Unless otherwise stated, the heterocyclic group can be carbonyl or nitrogenyl, and the -CH2- group may optionally be replaced by -C(=O)-. The sulfur atom of the ring may optionally be oxidized to... S -Oxides. The nitrogen atom in the ring can optionally be oxidized to... N-Oxides. In some embodiments, the heterocycle or heterocyclic group consists of 3-10 atoms, represented as a 3-10 membered heterocycle or a 3-10 membered heterocyclic group; in other embodiments, the heterocycle or heterocyclic group consists of 3-9 atoms, represented as a 3-9 membered heterocycle or a 3-9 membered heterocyclic group; in other embodiments, the heterocycle or heterocyclic group consists of 5-9 atoms, represented as a 5-9 membered heterocycle or a 5-9 membered heterocyclic group; in other embodiments, the heterocycle or heterocyclic group consists of 3-6 atoms, represented as a 3-6 membered heterocycle or a 3-6 membered heterocyclic group; in other embodiments, the heterocycle or heterocyclic group consists of 5-6 atoms, represented as a 5-6 membered heterocycle or a 5-6 membered heterocyclic group. In still other embodiments, the heterocycle or heterocyclic group is a monocyclic ring consisting of 3-7 atoms, represented as a 3-7 membered monocyclic heterocycle or a 3-7 membered monocyclic heterocyclic group. Examples of the heterocycles include, but are not limited to, ethylene oxide, aziridine, aziridine, oxacyclobutane, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, thiazoline, pyrazolidine, pyrazolidine, pyrazoline, oxazolidine, imidazoline, piperidine, piperazine, morpholine, 3,8-diazabicyclo[3.2.1]octane, 3,6-diazabicyclo[3.1.1]heptane, and 2,5-diazabicyclo[2.2.2]octane. The heterocyclic groups include, but are not limited to, ethylene oxide, aziridine, aziridine, oxazolidine, pyrrolidine, tetrahydrofuranyl, tetrahydrothiopheneyl, thiazoline, pyrazolidine, pyrazolidine, pyrazolidine, oxazolidine, imidazoline, piperidinyl, piperazinyl, or morpholinyl.

[0064] The term "aryl" refers to a monocyclic, bicyclic, or tricyclic carbocyclic system containing 6-14, 6-12, or 6-10 ring atoms, wherein at least one ring system is aromatic, and each ring system comprises a ring of 3-7 atoms. In some embodiments, the aryl group contains 6-12 ring atoms, denoted as C 6-12 Aryl or 6-12-membered aryl. In some embodiments, the aryl group contains 6-10 ring atoms, denoted as C. 6-10 Aryl or 6-10 aryl groups. Examples of aryl groups may include phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, and anthracene.

[0065] The term "heteroaryl" or "heteroaromatic ring" refers to a monovalent monocyclic, bicyclic, or tricyclic system containing 5-14, 5-12, 5-10, or 5-6 ring atoms, wherein at least one ring is aromatic and at least one ring contains one or more heteroatoms selected from nitrogen, oxygen, and sulfur. The heteroaryl group is typically, but not necessarily, linked to the parent molecule via its aromatic ring. When a -CH2- group is present in the heteroaryl group, the -CH2- group may optionally be replaced by -C(=O)-. Unless otherwise stated, the heteroaryl group may be linked to the remainder of the molecule (e.g., the main structure in the general formula) through any reasonable site (which may be C or N). The term "heteroaryl" may be used interchangeably with the terms "heteroaromatic ring" or "heteroaromatic compound". In some embodiments, the heteroaryl group is a heteroaryl group containing 5-12 ring atoms, denoted as a 5-12-membered heteroaryl group; in other embodiments, the heteroaryl group is a heteroaryl group containing 5-10 ring atoms, denoted as a 5-10-membered heteroaryl group; in still other embodiments, the heteroaryl group is a heteroaryl group containing 5-6 ring atoms, denoted as a 5-6-membered heteroaryl group. Examples of heteroaryl groups include, but are not limited to, furanyl, imidazolyl, isoxazolyl, oxazolyl, pyrrolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyridazinyl, thiophene, thiazolyl, triazolyl, tetrazolyl, benzofuran, benzothiazole, benzopyridinyl, benzoimidazolyl, benzopyrrolyl, benzopyrazolyl, benzopyrrolylalkyl, 2,3-dihydrobenzofuran, benzo[2,3-b][1,4]dioxin, pyrido[2,3-b][1,4]dioxin, benzo[2,3-b][1,4]oxazine, pyrido[2,3-b][1,4]oxazine, etc.

[0066] The term "halogen" refers to F (fluorine), Cl (chlorine), Br (bromine), or I (iodine).

[0067] The term "oxo" means =O.

[0068] The term "cyano" means -CN or -C≡N.

[0069] The term "thiol" represents -SH.

[0070] The term "hydroxyl group" represents -OH.

[0071] The term "carboxyl group" represents -C(=O)OH.

[0072] The term "composed of jk atoms" or "of jk elements" means that the cyclic group is composed of jk ring atoms, including carbon atoms and / or heteroatoms such as O, N, S, P, etc.; j and k are each independently any non-zero natural number, and k > j; "jk" includes j, k, and any natural number between them. For example, "composed of 3-8 atoms" or "3-8-element", "composed of 3-6 atoms" or "3-6-element", "composed of 5-10 atoms" or "5-10-element", or "composed of 5-6 atoms" or "5-6-element" indicates that the cyclic group is composed of 3-8 (i.e., 3, 4, 5, 6, 7 or 8), 3-6 (i.e., 3, 4, 5 or 6), 5-10 (i.e., 5, 6, 7, 8, 9 or 10), or 5-6 (i.e., 5 or 6) ring atoms, wherein the ring atoms include carbon atoms and / or heteroatoms such as O, N, S, P, etc.

[0073] As described in this invention, the substructure of group L1 has two linkage sites that can be linked to the rest of the antibody-drug conjugate. For example, when L1 is... When, it means on L1 The end is connected to Ab via an S atom. The terminal is connected to L2, as shown in equation a. Where X and m have the meanings described in this invention.

[0074] As described in this invention, group L2 is selected from any structure composed of one or more segments. For example, when L2 is selected from... and When the composition is obtained, L2 can be , , or Group L2 has two attachment sites that can connect to the rest of the molecule, and the connection methods of the two attachment sites can be interchanged. For example, when L2 is... In this case, the mm connection terminal of L2 is connected to L1, and the other nn connection terminal is connected to L3, as shown in equation b. ; or it could mean that the mm connection end of L2 is connected to L3, and the other nn connection end is connected to L1, as shown in equation c. .

[0075] The term "prodrug" as used in this invention refers to the conversion of a compound into the compound represented by formula (I) in vivo. Such conversion is influenced by the hydrolysis of the prodrug in the blood or its enzymatic conversion into the parent structure in the blood or tissues. The prodrug compounds of this invention can be esters; among existing inventions, esters that can serve as prodrugs include phenyl esters and aliphatic (C) esters. 1-24Esters, acyloxymethyl esters, carbonates, carbamates, and amino acid esters. For example, one compound in this invention contains a hydroxyl group, meaning it can be acylated to yield a prodrug form. Other prodrug forms include phosphate esters, such as those obtained by phosphorylation of a parent compound with a hydroxyl group.

[0076] "Metabolic products" refer to the products obtained from the metabolism of a specific compound or its salt in the body. The metabolites of a compound can be identified using techniques known in the art, and their activity can be characterized by experimental methods as described in this invention. Such products can be obtained by subjecting the compound to oxidation, reduction, hydrolysis, acylation, deacylation, esterification, defatting, enzymatic cleavage, etc. Accordingly, this invention includes the metabolites of compounds, including metabolites produced by sufficiently exposing the compounds of this invention to mammals for a period of time.

[0077] Unless otherwise specified, the "compounds" and "antibody-drug conjugates" of this invention may exist independently in the form of salts, mixed salts, or non-salts (e.g., free acids or free bases). When present in the form of salts or mixed salts, they may be pharmaceutically acceptable salts.

[0078] As used in this invention, "pharmaceutically acceptable salt" refers to the compounds or antibodies of this invention. Organic and inorganic salts of drug conjugates. Pharmaceutically acceptable salts are well-known in their respective fields, as seen in literature such as SMBerge. et al. The invention describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19. Pharmaceutically acceptable salts formed from non-toxic acids include, but are not limited to, inorganic acid salts formed by reaction with amino groups. The invention also contemplates quaternary ammonium salts formed from any compound containing an N group. Water-soluble or oil-soluble or dispersed products can be obtained by quaternization. Pharmaceutically acceptable salts further include suitable, non-toxic ammonium, quaternary ammonium salts, and amine cations that resist the formation of equilibrium ions.

[0079] In this invention, "solvent" refers to an association formed by one or more solvent molecules and a compound of this invention. Solvents forming solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, ethanolamine, or mixtures thereof. The term "hydrate" refers to an association formed when the solvent molecules are water.

[0080] When the solvent is water, the term "hydrate" may be used. In one embodiment, a molecule of the compound of the present invention may bind to one water molecule, such as a monohydrate; in another embodiment, a molecule of the compound of the present invention may bind to more than one water molecule, such as a dihydrate; in yet another embodiment, a molecule of the compound of the present invention may bind to fewer than one water molecule, such as a hemihydrate. It should be noted that the hydrates of the present invention retain the bioavailability of the non-hydrated form of the compound. As used herein, the term "treatment" refers to any disease or condition, in some embodiments meaning to improve the disease or condition (i.e., slow down or stop or alleviate the development of the disease or at least one of its clinical symptoms). In other embodiments, "treatment" means to alleviate or improve at least one bodily parameter, including bodily parameters that may not be perceptible to the patient. In other embodiments, "treatment" means to regulate the disease or condition from a physical (e.g., stabilizing perceptible symptoms) or physiological (e.g., stabilizing bodily parameters) or both. In other embodiments, "treatment" means to prevent or delay the onset, occurrence, or worsening of the disease or condition.

[0081] The term “prevention” or “protection” refers to the reduction of the risk of acquiring a disease or disorder (i.e., stopping the development of at least one clinical symptom of the disease in a subject who may be facing or predisposed to facing the disease, but has not yet experienced or exhibited symptoms of the disease).

[0082] The term "therapeutic effective dose" refers to the amount of a compound that is sufficient to treat a disease when administered to a subject. The "therapeutic effective dose" can vary depending on the compound, the disease and its severity, and the condition, age, weight, and sex of the subject being treated.

[0083] Unless otherwise stated, all suitable isotopic variations, stereoisomers, tautomers, hydrates, solvates, metabolites, pharmaceutically acceptable salts, and prodrugs of the compounds of this invention are included within the scope of this invention.

[0084] In the structures disclosed in this invention, when the stereochemistry of any particular chiral atom is not specified, all stereoisomers of that structure are considered within the scope of this invention and are included in this invention as disclosed compounds. When the stereochemistry is indicated by a solid wedge or dashed line representing a particular configuration, the stereoisomers of that structure are thus clearly defined.

[0085] The nitrogen oxides of the compounds of this invention are also included within the scope of this invention. The nitrogen oxides of the compounds of this invention can be prepared by oxidizing the corresponding nitrogen-containing basic substances in the presence of an acid, such as acetic acid, using a common oxidizing agent (e.g., hydrogen peroxide) at elevated temperature, or by reacting with a peracid in a suitable solvent, such as with peracetic acid in dichloromethane, ethyl acetate, or methyl acetate, or with 3-chloroperoxybenzoic acid in chloroform or dichloromethane.

[0086] The antibody-drug conjugate of formula (I) or the compound of formula (II) described in this invention may exist in the form of a salt.

[0087] Any structural formulas provided in this invention are intended to represent both the unenriched and isotopically enriched forms of these compounds. Isotopically enriched compounds have the structures described by the general formulas provided in this invention, except that one or more atoms are replaced by atoms having a chosen atomic weight or mass number. Exemplary isotopes that may be introduced into the compounds of this invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, such as... 2 H, 3 H, 11 C 13 C 14 C 15 N、 17 O、 18 O、 18 F, 31 P, 32 P, 35 S, 36 Cl and 125 I.

[0088] Detailed description of the compounds of the present invention This invention provides a novel linker-drug molecule and antibody-drug conjugate. This antibody-drug conjugate exhibits good antitumor activity, excellent permeability, low efflux rate, and good metabolic stability, making it a promising candidate for cancer treatment. Furthermore, the antibody-drug conjugate of this invention demonstrates good tolerability, low toxicity, a wide therapeutic window, good tumor tissue targeting, and a strong bystander effect.

[0089] On the one hand, the present invention provides an antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof. (I) in, Ab, L1, L2, L3, D, and t have the meanings described in this invention.

[0090] In some embodiments, the Ab described in this invention is an antibody or an antigen-binding fragment.

[0091] In some embodiments, the Ab described in this invention is an antibody or an antigen-binding fragment, wherein the antibody or its antigen-binding fragment targets HER2, HER3, EGFR, ROR1, CLDN6, CLDN9, CLDN18.2, and B7. H3, B7 H4, TROP 2. CD20, CD22, CD30, CD33, CD47, CD56, CD70, CD79b, VEGF, VEGFR, MUC1, c MET, RET, LIV 1. PD 1 and PD Antibodies or antigen-binding fragments targeting one or two L1 targets.

[0092] In some embodiments, the Ab mentioned in this invention is selected from trastuzumab, pertuzumab, vidictetumab, maggotuximab, or inetetumab.

[0093] In some embodiments, L1 in this invention is a connector that is linked to an antibody or antigen-binding fragment via an S atom.

[0094] In some embodiments, L1 of the present invention is selected from the following structures: , , , , or ;in The end is connected to Ab via an S atom. The terminal is connected to L2; X represents a bond, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH=CH-, -CH=CHCH2-, -CH2CH=CH-, -C≡C-, -CH2C≡C-, -C≡C-CH2-, -CH2CH2C≡C-, -CH2C≡CCH2-, -C≡CCH2CH2-, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, indole, naphthyl, pyrrolyl, pyrazolyl, imidazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, furanyl, thienyl, thiazolyl, oxazolyl, pyridyl, pyrimidinyl, pyrazinyl, or pyridazinyl; R a and R bIndependently, it is H, D, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, vinyl, propenyl, allyl, ethynyl, propargyl, 1-propynyl, 1-ynylbutyl, 2-ynylbutyl, 3-ynylbutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxetyl, aziryl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, piperazinyl, or morpholinyl; wherein the methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl... alkyl, tert-butyl, vinyl, propenyl, allyl, ethynyl, propargyl, 1-propynyl, 1-ynylbutyl, 2-ynylbutyl, 3-ynylbutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxacyclobutyl, aziroxybutyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, piperazinyl, and morpholinyl are each independently and optionally surrounded by 1, 2, 3, 4, or 5 groups selected from D, -F, -Cl, -Br, -I, -OH, -NH2, -CN, -SH, -COOH, oxo, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Alkoxy and C 1-6 Substituents of haloalkoxy groups; m can be 1, 2, 3, 4, 5, 6, 7 or 8.

[0095] In other embodiments, L1 of the present invention is selected from the following structures: , , , , , , , or ;in The end is connected to Ab via an S atom. The terminal is connected to L2.

[0096] In some embodiments, L2 in this invention refers to the connection segment between connectors L1 and L3.

[0097] In some embodiments, the L2 selection of the present invention is a structure composed of one or more of the following segments: C 1-6 Alkylene, -NR x -、-C(=O)-、-O-、 , , , , , , , , , , , , or ; R x For H, D, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-8 Cycloalkyl or 3-8 membered heterocyclic groups.

[0098] In other embodiments, L2 is selected from a structure consisting of one or more of the following segments: C 1-6 Alkylene, -NR x -、-C(=O)-、-O-、 , , , , , , , , , , , , , , , , , or ; R x The derivatives are H, D, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, vinyl, propenyl, allyl, ethynyl, propynyl, 1-propynyl, 1-ynylbutyl, 2-ynylbutyl, 3-ynylbutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxetyl, aziryl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, piperazinyl, or morpholinyl. n can be 1, 2, 3, 4, 5, 6, 7 or 8.

[0099] In some embodiments, L3 in this invention is a connecting segment that connects L2 and drug D.

[0100] In some embodiments, L3 in this invention is selected from key, -NR y CH2-, -C(=O)-, -O-, -NR y CH2CH2NR z C(=O)-、 or Among them, R y and Rz Each has the meaning described in this invention.

[0101] In some embodiments, R y and R z H, D, C independently 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-8 Cycloalkyl or 3-8 membered heterocyclic groups.

[0102] In some embodiments, R y and R z H, D, C independently 1-4 Alkyl, C 2-4 alkenyl, C 2-4 alkynyl group, C 3-6 Cycloalkyl or 3-6 membered heterocyclic groups.

[0103] In other embodiments, R y and R z Independently, it is H, D, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, vinyl, propenyl, allyl, ethynyl, propynyl, 1-propynyl, 1-ynylbutyl, 2-ynylbutyl, 3-ynylbutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxacyclobutyl, aziroxybutyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, piperazinyl, or morpholinyl.

[0104] In some embodiments, the drug D of the present invention is selected from cytotoxic drugs, drugs for treating autoimmune diseases, or anti-inflammatory drugs. In some embodiments, the drug D of the present invention is selected from eribulin, monomethylolpropionate E (MMAE), monomethylolpropionate F (MMAF), oropontostatin E, oropontostatin F, maytansine DM1, maytansine DM4, maytansine alcohol, sandycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, anthracyclines, chalcogenide, dolalastatin 10, docamycin, doxorubicin, tylansstatin A, hookmycin, amatoxins, ricin, diphtheria toxin, interleukin, tumor necrosis factor, chemokine, SN38, or eczema and its derivatives.

[0105] In some embodiments, the drug D of the present invention is selected from... , , , , or .

[0106] In some embodiments, t is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0107] In some embodiments, the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt thereof, wherein Selected from the following structures: , , or .

[0108] In some embodiments, the Ab described in this invention is an anti-EGFR antibody, an anti-TROP2 antibody, or an anti-MUC1 antibody or an antigen-binding fragment thereof.

[0109] In some embodiments, the anti-EGFR antibody of the present invention is Cetuximab, demupitamab, depatuxizumab, futuximab, imgatuzumab, lapatuximab, losatuxizumab, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, pimurutamab, serclutamab, tomuzotuximab, or zalutumumab.

[0110] In some embodiments, the anti-EGFR antibody of the present invention is cetuximab, which comprises the heavy chain of SEQ ID NO:1 and the light chain of SEQ ID NO:2, the sequence information of SEQ ID NO:1 and SEQ ID NO:2 are shown below: SEQ ID NO: 1 (heavy chain sequence) QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQG TLVTVSAASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0111] SEQ ID NO: 2 (light chain sequence) DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0112] In some embodiments, the anti-TROP2 antibody described in this invention is enoblituzumab, mirzotamab, or omburtamab.

[0113] In some embodiments, the anti-TROP2 antibody of the present invention comprises the heavy chain of SEQ ID NO: 5 and the light chain of SEQ ID NO: 6, and in the present invention, the antibody is numbered 83. The sequence information for SEQ ID NO: 5 and SEQ ID NO: 6 is shown below: SEQ ID NO: 5 (heavy chain sequence) MEWSWVFLFFLSVTTGVHSQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0114] SEQ ID NO: 6 (light chain sequence) MRFFFVFLAIVLFQGIHGDIQLTQSPSSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTF GAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0115] According to some embodiments of the present invention, the MUC1 antibody or its antigen-binding fragment contains a CDR sequence selected from at least one of the following: heavy chain variable region CDR sequence: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9; light chain variable region CDR sequence: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12.

[0116] Table 1

[0117] According to embodiments of the present invention, the CDR sequence of the antibody can be defined according to the Kabat, IMGT, Chothia, AbM, Contact, or North numbering systems. In some embodiments, the CDR sequences of the antibodies listed in Table 1 are defined according to the Kabat numbering system. Regardless of the numbering method used to define the CDR sequence, it is within the scope of protection of the present invention.

[0118] In some embodiments, the anti-MUC1 antibody of the present invention comprises heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12.

[0119] In some embodiments, the anti-MUC1 antibody of the present invention has a heavy chain as in SEQ ID NO: 3 and a light chain as in SEQ ID NO: 4, and in the present invention, this antibody is numbered 82. The sequence information of SEQ ID NO: 3 and SEQ ID NO: 4 is as follows: SEQ ID NO: 3 (heavy chain sequence) QVQLVQSGAEVKKPGASVKVSCKASGFYTTTYWMHWVRQAPGQGLEWMGMIHPSDGETRLNQNFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREDMGGYPWFAYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0120] SEQ ID NO: 4 (light chain sequence) DIVMTQSPDSLAVSLGERATISCKASKSVSSSAYSYVHWYQQKPGQPPKLLIYLASNLGSGVPARFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWALPYTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0121] In some embodiments of the present invention, the positive control antibody 1H7 has a heavy chain variable region and a light chain variable region as shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

[0122] EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWMHWVQQAPGKGLEWIGYINPGTGYIEYNQKFKDRVTITADKSTTDTAYMELSSLRSEDTAVYYCASSTAPFDYWGQGTLVTVSS (SEQ ID NO: 13) EIVLTQSPGTLSLSPGERATLSCKASQDIKSYLSWYQQKPGQAPRLLIYYATRLADGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCLQYDESPYTFGQGTKLEIKR (SEQ ID NO: 14) On the one hand, the present invention provides a compound of formula (II), or a stereoisomer, tautomer, nitride, hydrate, solvate, or pharmaceutically acceptable salt thereof of a compound of formula (II). L-L2-L3-D (II), in, L is selected from the following structures: , , , , or ; The terminal is connected to L2; L2, L3, D, X, R a R b "and m" have the meanings described in this invention.

[0123] In some embodiments, L is selected from the following structures: , , , , , , , or ; The terminal is connected to L2.

[0124] In some embodiments, the compounds of the present invention have the following structures, or stereoisomers, tautomers, nitrides, hydrates, solvates, or pharmaceutically acceptable salts thereof, but are by no means limited to: (1) (2) (3) or (4).

[0125] On one hand, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate of formula (I), a compound of formula (II), or a pharmaceutically acceptable salt thereof.

[0126] In some embodiments, the pharmaceutical composition of the present invention comprises one or more antibody-drug conjugates of formula (I) of the present invention, wherein the DAR of the composition is an integer or decimal of about 1 to 10, for example 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 39, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6 1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.58, 7.6, 7.64, 7.66, 7.7, 7.78, 7.8, 7.86, 7.9, 7.94, 7.96, 8.0, 8.02, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0.

[0127] In some embodiments, the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier.

[0128] On the other hand, the present invention relates to the use of antibody-drug conjugates of formula (I), compounds of formula (I), or pharmaceutical compositions thereof in the preparation of medicaments for the prevention or treatment of cancer.

[0129] In some embodiments, the cancers described in this invention are breast cancer, lung cancer, kidney cancer, liver cancer, ovarian cancer, urethral cancer, prostate cancer, glioblastoma multiforme, pancreatic cancer, colorectal cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, colon cancer, rectal cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, melanoma, bladder cancer, stomach cancer, or esophageal cancer.

[0130] In another aspect, the present invention relates to methods for the preparation, separation and purification of antibody-drug conjugates of formula (I) and compounds of formula (II).

[0131] Pharmaceutical compositions, formulations, administration and uses of the compounds of this invention The pharmaceutical compositions of the present invention are characterized by comprising an antibody-drug conjugate of formula (I) or a compound of formula (II), an antibody-drug conjugate or compound listed in the present invention, or an antibody-drug conjugate or compound of the examples, and a pharmaceutically acceptable carrier. The amount of the compound in the pharmaceutical compositions of the present invention is sufficient to effectively treat or alleviate diseases such as cancer in patients.

[0132] The antibody-drug conjugates or compounds of the present invention exist in free form or as suitable, pharmaceutically acceptable derivatives. According to the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable prodrugs, salts, esters, salts of esters, or any other adducts or derivatives that can be administered directly or indirectly as needed by a patient, compounds described in other aspects of the present invention, their metabolites, or their residues.

[0133] As described in this invention, pharmaceutically acceptable compositions of the present invention further comprise pharmaceutically acceptable excipients, such as those used in this invention, including any solvent, diluent, or other liquid excipient, dispersant or suspending agent, surfactant, isotonic agent, thickener, emulsifier, preservative, solid binder or lubricant, etc., suitable for a particular target dosage form. As described in the following literature: In Remington: The Science and Practice of Pharmacy, 21st edition, 2005, ed. DB Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and JCBoylan, 1988-1999, Marcel Dekker, New York, the contents of which, taken together, show that various excipients can be used in the formulation of pharmaceutically acceptable compositions and their known methods of preparation. The use of any conventional excipients, except those incompatible with the compounds of the present invention, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0134] The terms “pharmaceutical-grade carrier” or “pharmaceutical-acceptable carrier” include, but are not limited to, any adjuvant, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that has been approved by the U.S. Food and Drug Administration for use in humans or livestock.

[0135] The pharmaceutical compositions of the present invention can be in the form of sterile injectable aqueous solutions. Water, Ringer's solution, and isotonic sodium chloride solution may be used as acceptable solvents and media. The sterile injectable formulation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then treated with a mixture of water and glycerol to form a microemulsion. The injectable solution or microemulsion can be injected into the bloodstream of the subject by local large-volume injection. Alternatively, it is preferable to administer the solution and microemulsion in a manner that maintains a constant circulating concentration of the compounds disclosed herein. To maintain such a constant concentration, a continuous intravenous delivery device can be used. An example of such a device is the Deltec CADD. PLUS.TM.5400 intravenous infusion pump.

[0136] The pharmaceutical compositions of the present invention may be in the form of sterile injectable aqueous or oil suspensions for intramuscular and subcutaneous administration. These suspensions may be formulated using suitable dispersants or wetting agents and suspending agents as described above, according to known techniques. The sterile injectable formulations may also be sterile injectable solutions or suspensions prepared in non-toxic, parenteral-acceptable diluents or solvents, such as 1, 3 Solutions prepared in butanediol. Furthermore, sterile fixative oils can be conveniently used as solvents or suspension media. For this purpose, any blended fixative oil, including synthetic mono- or diglycerides of glycerol, can be used. Additionally, fatty acids such as oleic acid can also be used to prepare injectable formulations.

[0137] The compounds or pharmaceutical compositions of the present invention can also be administered in the form of liposome delivery systems, such as small monolayer vesicles, large monolayer vesicles, and multilayer vesicles. Liposomes can be formed from different phospholipids, such as cholesterol, stearamine, or phosphatidylcholine.

[0138] The compounds or pharmaceutical compositions of the present invention are also coupled with soluble polymers that act as targeted drug carriers. Such polymers include polyvinylpyrrolidone, pyran copolymers, polyhydroxypropyl methacrylate-phenol, polyhydroxyethyl asparagine, or polyvinyl oxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds of the present invention can be coupled with a class of biodegradable polymers for controlled drug release, such as polylactic acid, polyglycolic acid, copolymers of polylactic acid and polyglycolic acid, polycaprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyran, polycyanoacrylates, and crosslinked or amphiphilic blocking copolymers of hydrogels.

[0139] The administration regimen of the compounds or pharmaceutical compositions of this invention will vary depending on a variety of known factors, such as the pharmacokinetic characteristics and patterns of the specific agent and the route of administration; the recipient's race, age, sex, health status, medical condition, and weight; the nature and severity of symptoms; the types of concurrent treatments; the frequency of treatment; the route of administration; the patient's renal and hepatic function; and the desired effect. A physician or veterinarian can make a decision and prescribe an effective amount of the drug to prevent, counteract, or halt the progression of a disease.

[0140] According to general guidelines, in order to achieve the specified effect, the dosage of each active ingredient used ranges from about 0.001 to 1000 mg / kg body weight per day, preferably from about 0.01 to 100 mg / kg body weight. The compounds of the present invention can be administered once daily, or divided into two, three, or four daily doses.

[0141] Each unit dose of a suitable dosage form (pharmaceutical composition) may contain from about 0.1 mg to about 100 mg of the active ingredient. In these pharmaceutical compositions, the weight of the active ingredient will generally account for about 0.5-95% of the total weight of the composition.

[0142] The compounds and compositions described in this invention can be administered alone or in combination with other compounds or other therapeutic agents. The compounds or compositions of this invention can be administered simultaneously or sequentially with other therapeutic agents via the same or different routes of administration. The compounds of this invention can be included in a single formulation along with other therapeutic agents or in a separate formulation.

[0143] When the compounds of the present invention are administered together with other therapeutic agents, the amount of each component in a typical daily dose and typical dosage form may generally be reduced relative to the usual dose when administered alone, taking into account the additional or synergistic effects of the therapeutic agents when administered in combination.

[0144] The antibody-drug conjugates, pharmaceutical salts thereof, or hydrates thereof, or pharmaceutical compositions thereof involved in this invention can be effectively used to prevent, treat, or alleviate diseases in patients, especially cancer.

[0145] In some embodiments, the compounds of the present invention or pharmaceutical compositions thereof are effective in preventing, treating or alleviating symptoms in patients, including, but not limited to: breast cancer, lung cancer, kidney cancer, liver cancer, ovarian cancer, urethral cancer, prostate cancer, glioblastoma multiforme, pancreatic cancer, colorectal cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, colon cancer, rectal cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, melanoma, bladder cancer, stomach cancer, or esophageal cancer. Attached Figure Description

[0146] Figure 1 This is a graph showing the changes in tumor volume in different groups of Balb / c-nu mice after inoculation with NUGC4 human gastric cancer cell xenografts. Figure 2 This is a graph showing the changes in tumor volume in different groups of Balb / c-nu mice after drug administration, inoculated with NCI-H1975 human lung cancer cell xenograft tumor model. General Synthesis Process To describe this invention, the following embodiments will be used to further illustrate the technical solution of this invention. These embodiments are only used to illustrate specific implementation methods of this invention so that those skilled in the art can understand it, but are not intended to limit the scope of protection of this invention. In the specific implementation methods of this invention, technical means or methods not specifically described are conventional technical means or methods in the art.

[0147] Unless otherwise specified, the substituents are defined as described in this invention. The following reaction schemes and examples are provided to further illustrate the content of this invention.

[0148] Those skilled in the art will recognize that the chemical reactions described in this invention can be suitably used to prepare other compounds of this invention, and that other methods for preparing the compounds of this invention are considered to be within the scope of this invention. For example, the synthesis of those non-illustrative compounds according to this invention can be successfully accomplished by those skilled in the art through modification methods, such as by appropriately protecting interfering groups, by utilizing other known reagents besides those described in this invention, or by making some conventional modifications to the reaction conditions. Furthermore, the reactions disclosed in this invention or the known reaction conditions are also generally accepted to be applicable to the preparation of other compounds of this invention.

[0149] In the examples described below, unless otherwise specified, all temperatures are in degrees Celsius (°C). Room temperature in the examples represents 15 °C–30 °C; in some examples, room temperature is 20 °C–30 °C. Reagents were purchased from commodity suppliers such as Aldrich Chemical Company, Arco Chemical Company, and Alfa Chemical Company and were used without further purification. Unless otherwise specified, general reagents were purchased from Shantou Xilong Chemical Plant, Guangdong Guanghua Chemical Reagent Plant, Guangzhou Chemical Reagent Plant, Tianjin Haoyuyu Chemical Co., Ltd., Tianjin Fuchen Chemical Reagent Plant, Wuhan Xinhuayuan Technology Development Co., Ltd., Qingdao Tenglong Chemical Reagent Co., Ltd., and Qingdao Haiyang Chemical Plant.

[0150] When this invention relates to one or more of the following reagents and solvents, the solvents can be used in experiments after being treated as follows: anhydrous tetrahydrofuran, dioxane, toluene, and diethyl ether are obtained by reflux drying with metallic sodium; anhydrous dichloromethane and chloroform are obtained by reflux drying with calcium hydride; ethyl acetate, petroleum ether, and n-hexane... N , N -dimethylacetamide and N , N - Dimethylformamide is used after being dried with anhydrous sodium sulfate.

[0151] The reaction can be carried out under positive pressure of nitrogen or argon or over an anhydrous solvent with a drying tube attached (unless otherwise indicated). All reaction flasks are sealed with suitable rubber stoppers, and the substrate is injected via syringe. All glassware is dried.

[0152] The chromatographic column used was a silica gel column. The silica gel (300-400 mesh) was purchased from Qingdao Ocean Chemical Plant.

[0153] 1 H NMR spectra were recorded using a Bruker 400 MHz, 600 MHz, or 599 MHz nuclear magnetic resonance spectrometer. 1 ¹H NMR spectra with CDCl₃ and DMSO- d 6. CD3OD or acetone - d 6 represents the solvent (in ppm), with TMS (0 ppm) or chloroform (7.26 ppm) as the reference standard. When multiplets are observed, the following abbreviations will be used: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broadened), br s (broadened singlet), dd (doublet of doublets), dt (doublet of triplets), qt (quartet of triplets). Coupling constant. J It is represented by Hertz (Hz).

[0154] The determination conditions for low-resolution mass spectrometry (MS) data were as follows: Agilent 6120 quadrupole HPLC-MS (column model: Zorbax SB-C18, 2.1 × 30 mm, 3.5 μm, 6 min, flow rate: 0.6 mL / min; mobile phase: 5%–95% (CH3CN containing 0.1% formic acid) in (H2O containing 0.1% formic acid), electrospray ionization (ESI) at 210 nm / 254 nm, detection by UV.

[0155] Pure compounds were detected using UV at 210 nm / 254 nm using an Agilent 1260 pre-HPLC or a Calesep pump 250 pre-HPLC (column model: NOVASEP 50 / 80 mm DAC).

[0156] The following abbreviations or English words are used throughout this invention:

[0157] The following examples further illustrate the compounds, pharmaceutical compositions, and their applications provided by the present invention.

[0158] Example Example 1 Compound I-15

[0159]

[0160] Step 1: Synthesis of compound I-2 Compound I-1 (5.0 g, 26.56 mmol), sodium carbonate (7.04 g, 66.40 mmol), water (200 mL), and solvent 1,4-dioxane (200 mL) were added to a reaction flask. While stirring at 0 °C, a solution of 1,4-dioxane (200 mL) containing FmocCl (7.21 g, 27.89 mmol) was added. The reaction was maintained at this temperature for 2 h, then transferred to room temperature and reacted overnight (approximately 12 h). The mixture was concentrated under reduced pressure, and water (700 mL) was added to adjust the pH to 2. Extraction was performed using EA (200 mL × 2), followed by washing with saturated brine, drying over anhydrous sodium sulfate, filtration, and concentration of the filtrate under reduced pressure until evaporated to dryness, yielding a white solid (10.24 g, 93.91% yield).

[0161] MS (ESI, pos. ion) m / z: 411.05 [M+H] + ; 1 H NMR (400 MHz, CD3OD) δ 7.82 (d, J = 7.5 Hz, 2H), 7.69 (t, J = 7.1 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 4.40 (tt, J = 17.9, 8.9Hz, 3H), 4.25 (t, J = 6.7 Hz, 1H), 3.97 (d, J = 7.1 Hz, 1H), 2.08 (dd, J = 13.6, 6.8 Hz, 1H), 1.42 (d, J = 7.3 Hz, 3H), 1.00 (d, J = 6.7 Hz, 3H), 0.96 (d, J = 6.7Hz, 3H). Step 2: Synthesis of compound I-14 Add 10.0 g (37.27 mmol) of 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl acetic acid to the reaction flask. N6.43 g (55.91 mmol) of hydroxysuccinimide, 10.72 g (55.91 mmol) of EDCI, and 100 mL of dichloromethane were reacted at room temperature for 12 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane) to give a white solid (13.23 g, 97.15% yield).

[0162] MS (ESI, pos. ion) m / z: 366.10 [M+H] + . Step 3: Synthesis of Compound I-5 Compound I-3 (10.0 g, 25.18 mmol), acetonitrile (200 mL), compound I-4 (6.31 g, 37.77 mmol), and silver oxide (11.67 g, 50.36 mmol) were added to a reaction flask. The mixture was stirred at room temperature for approximately 25 h under nitrogen protection. The reaction solution was directly filtered, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure to give a crude solid. The crude solid was washed with ethyl acetate (100 mL) and 10% sodium bicarbonate aqueous solution (100 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a light brown solid (9.2 g, yield 75.59%).

[0163] Step 4: Synthesis of compound I-6 Compound I-5 (9.2 g, 19.03 mmol), isopropanol (30 mL), chloroform (150 mL), silica gel (6.86 g), and sodium borohydride (1.8 g, 47.58 mmol) were added to a reaction flask. After the addition was complete, the mixture was stirred at room temperature for about 5 h. The reaction mixture was quenched with ice water and filtered through diatomaceous earth. The filtrate was extracted with dichloromethane (200 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 50 / 1) to give an off-white solid product (7.0 g, yield 75.77%).

[0164] MS (ESI, pos. ion) m / z: 503.0 [M+H3O] + ; Step 5: Synthesis of Compound I-7 Compound I-6 (5.0 g, 10.3 mmol) was added to a reaction flask, followed by dichloromethane (100 mL), TBDPSCl (4.25 g, 15.45 mmol), and imidazole (2.10 g, 30.90 mmol). After the addition was complete, the mixture was stirred overnight at room temperature (approximately 12 h). The reaction solution was washed with water (300 mL × 3). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE / EA (v / v) = 1 / 1) to give a pale yellow, transparent liquid (7.0 g, 93.89% yield).

[0165] 1 H NMR (599 MHz, CDCl3) δ 7.72 (d, J = 1.8 Hz, 1H), 7.67 – 7.63 (m, 4H), 7.49 (dd, J = 8.6, 1.9 Hz, 1H), 7.46 – 7.42 (m, 2H), 7.38 (dd, J = 11.1, 4.0 Hz, 4H), 7.33 (d, J = 8.6 Hz, 1H), 5.37 – 5.29 (m, 3H), 5.18 (d, J = 6.8 Hz, 1H), 4.72 (s, 2H), 4.20 (d, J = 8.9 Hz, 1H), 3.75 (s, 3H), 2.13 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 1.10 (s, 9H). Step 6: Synthesis of Compound I-8 Compound I-7 (7.0 g, 9.67 mmol) was added to a reaction flask, along with activated carbon (2.0 g) in ethyl acetate (100 mL) and tetrahydrofuran (100 mL). The mixture was stirred at room temperature for approximately 30 h under hydrogen protection. The mixture was then placed on a diatomaceous earth liner and filtered under reduced pressure. The filtrate was rotary evaporated under reduced pressure to give a colorless oily liquid (6.70 g, 99.85% yield).

[0166] MS (ESI, pos.ion) m / z: 694.0 [M+H] + ; Step 7: Synthesis of Compound I-9 Compound I-8 (4.0 g, 5.77 mmol), compound I-2 (2.61 g, 6.35 mmol), and EEDQ (2.14 g, 8.65 mmol) were added to dichloromethane (70 ml) in a reaction flask and stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (PE / EA (v / v) = 10 / 1-5 / 2) to give an off-white solid (2.67 g, yield 42.63%).

[0167] 1 H NMR (400 MHz, CDCl3) δ 8.25 (s, 1H), 8.17 (s, 1H), 7.76 (d, J = 7.4Hz, 2H), 7.68 (d, J = 7.3 Hz, 4H), 7.61 (d, J = 3.5 Hz, 2H), 7.40 (dq, J = 14.0, 7.0 Hz, 8H), 7.31 (t, J = 7.4 Hz, 2H), 7.15 (d, J = 7.9 Hz, 1H), 6.93 (d, J = 8.4Hz, 1H), 6.73 (d, J = 7.4 Hz, 1H), 5.54 – 5.45 (m, 1H), 5.43 (d, J = 9.4 Hz, 1H), 5.34 (dd, J = 20.2, 10.2 Hz, 2H), 5.10 (d, J = 7.5 Hz, 1H), 4.76 (dd, J = 14.4, 7.2Hz, 1H), 4.70 (s, 2H), 4.48 – 4.40 (m, 1H), 4.39 – 4.31 (m, 1H), 4.23 (t, J =9.8 Hz, 2H), 4.14 (dd, J = 14.4, 7.3 Hz, 1H), 3.76 (s, 3H), 2.21 (d, J = 7.0 Hz,1H), 2.12 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 1.50 (d, J= 6.9 Hz, 3H), 1.09(s, 9H), 1.02 (d, J = 6.6 Hz, 3H), 0.95 (d, J = 6.6 Hz, 3H). Step 8: Synthesis of Compound I-10 Compound I-9 (2.67 g, 2.46 mmol) was added to a reaction flask and then to tetrahydrofuran (20 mL). The mixture was transferred to 0 °C and stirred. Pyridine hydrogen fluoride (3.66 g, 36.9 mmol) was slowly added. After the addition was complete, the mixture was transferred to room temperature and stirred for approximately 7 h. TLC was monitored until the reaction was complete. The reaction solution was then slowly added dropwise to a 10% sodium bicarbonate aqueous solution for neutralization. The aqueous phase remained alkaline. Extraction was performed using EA (50 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was evaporated to dryness under reduced pressure. The residue was purified by column chromatography (petroleum ether / ethyl acetate (v / v) = 1 / 1 to 1 / 2) to give a white solid product (1.7 g, 81.57%).

[0168] MS (ESI, pos. ion) m / z: 847.95 [M+H] + ; Step 9: Synthesis of Compound I-11 Compound I-10 (1.7 g, 2.01 mmol) was added to a reaction flask and then to DMF (6 mL). The mixture was transferred to 0 °C and stirred. DIPEA (0.52 g, 4.02 mmol) was added, followed by the slow addition of di(p-nitrobenzene) carbonate (1.22 g, 4.02 mmol). After the addition was complete, the mixture was transferred to room temperature and stirred for approximately 1.5 h. Water (50 mL) was added to the reaction mixture, and the mixture was extracted with EA (50 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was evaporated to dryness under reduced pressure. The residue was purified by column chromatography (petroleum ether / ethyl acetate (v / v) = 5 / 1 to 1 / 1) to give a white solid (1.836 g, yield 90.40%).

[0169] Step 10: Synthesis of Compound I-12 Compound I-11 (1.83 g, 1.81 mmol) was added to a reaction flask and then to DMF (18 mL). DIPEA (0.47 g, 3.62 mmol), HOBT (0.42 g, 3.12 mmol), and Exatecan (0.90 g, 2.07 mmol) were added. After the addition was complete, the mixture was stirred at room temperature for approximately 5 hours. The reaction mixture was then added directly to water (100 mL) and stirred at room temperature. The mixture was filtered, and the filter cake was washed with water. The filter cake was dissolved in DCM and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was evaporated to dryness under reduced pressure. The residue was purified by column chromatography (dichloromethane / methanol (v / v) = 100 / 1-50 / 1) to give a pale yellow solid (1.92 g, yield 81.35%).

[0170] HRMS-ESI excess mass calcd for C 68 H 69 FN6O 20 [M + H] + requires m / z:1309.4623, 1310.4657; found 1309.4687, 1310.4706.

[0171] 1 H NMR (400 MHz, CDCl3) δ 8.44 (s, 1H), 8.24 (s, 1H), 7.71 (d, J = 7.3Hz, 2H), 7.63 – 7.53 (m, 3H), 7.49 (d, J = 10.4 Hz, 1H), 7.36 (t, J = 7.1 Hz,2H), 7.28 (m, 2H), 7.12 (d, J = 7.6 Hz, 1H), 6.99 (d, J = 6.9 Hz, 1H), 6.91 (d, J =8.0 Hz, 1H), 6.12 (s, 1H), 5.64 (d, J = 16.1 Hz, 1H), 5.58 (d, J = 9.1 Hz, 1H),5.49 – 5.39 (m, 1H), 5.31 (m, 4H), 5.19 (m, 3H), 5.09 (d, J= 7.3 Hz, 1H), 5.02(s, 2H), 4.79 – 4.64 (m, 1H), 4.44 – 4.36 (m, 1H), 4.36 – 4.27 (m, 1H), 4.20(m, 3H), 3.74 (s, 3H), 3.07 (s, 2H), 2.40 (m, 1H), 2.34 (s, 3H), 2.25 – 2.16(m, 1H), 2.12 (s, 3H), 2.06 (s, 6H), 1.96 (m, 2H), 1.46 (d, J = 6.7 Hz, 3H), 1.09 (t, J = 7.2 Hz, 3H), 0.99 (d, J = 6.4 Hz, 3H), 0.91 (d, J = 6.6 Hz, 3H). Step 11: Synthesis of Compound I-13 Compound I-12 (0.5 g, 0.38 mmol) was added to a reaction flask, followed by 2 mL of DMF and diethylamine (0.06 g, 0.76 mmol). The mixture was stirred at room temperature for approximately 40 min. Water (10 mL) was added to the reaction mixture, and the mixture was extracted with EA (30 mL × 2), washed once with saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a pale yellow crude liquid. The crude product was added to methanol (5 mL), followed by 2 mL of an aqueous solution of potassium carbonate (0.32 g, 2.28 mmol). The mixture was stirred at room temperature for approximately 40 min. Methanol (50 mL) and dichloromethane (50 mL) were added to the reaction mixture, which was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a pale yellow solid (0.36 g, 99.55% yield).

[0172] Step 12: Synthesis of Compound I-15 Compound I-13 (0.18 g, 0.19 mmol) was added to a reaction flask, followed by DMF (6 mL). DIPEA (0.074 g, 0.57 mmol) was then added, and the mixture was stirred at room temperature. Compound I-14 (0.062 g, 0.17 mmol) dissolved in DMF (1 mL) was slowly added dropwise. After the addition was complete, the mixture was stirred overnight at room temperature for approximately 13 h. Methyl tert-butyl ether (50 mL) was added to the reaction mixture, and the mixture was stirred at room temperature. A solid precipitated, and the mixture was stirred for 10 min. The mixture was then filtered under reduced pressure. The filter cake was collected. The solution was purified by pre-HPLC (under preparative high-performance liquid chromatography conditions as follows), extracted with ethyl acetate (100 mL × 3), and the organic phases were combined and concentrated under reduced pressure to obtain a pale yellow oily liquid (0.043 g, yield 18.90%).

[0173] Prep-HPLC conditions: Feromon C18 column, 50 mm 250mm 10 μm, mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile, flow rate: 100.0 ml / min, detection wavelength: 260 nm, isocratic elution with phase A: phase B - 55: 45 for 20 min.

[0174] HRMS-ESI excess mass calcd for C 57 H 61 FN8O 18 S [M+H] + requires 1197.3881,1198.3915; found 1197.3925, 1198.3940.

[0175] Example 2: Compound II-4

[0176]

[0177] Step 1: Synthesis of Compound II-3 Compound II-1 (7.0 g, 33.14 mmol), compound II-2 (5.72 g, 49.71 mmol), EDCI (9.53 g, 49.71 mmol), and dichloromethane (70 mL) were added to a reaction flask. The reaction was carried out under nitrogen protection at room temperature for approximately 15 h. The reaction solution was washed with water (100 mL × 3), dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated and evaporated to dryness. The residue was purified by column chromatography (DCM) to give a white solid (5.94 g, yield 58.14%).

[0178] MS (ESI, pos. ion) m / z: 309.10 [M+H] + ; 1 H NMR (400 MHz, DMSO- d 6) δ 7.04 (s, 2H), 3.43 (t, J = 7.0 Hz, 2H), 2.84(s, 4H), 2.69 (t, J = 7.3 Hz, 2H), 1.70 – 1.62 (m, 2H), 1.55 (dt, J = 14.7, 7.2Hz, 2H), 1.38 – 1.30 (m, 2H). Step 2: Synthesis of Compound II-4 Compound I-13 (0.11 g, 0.12 mmol) was added to a reaction flask, followed by DMF (2 mL). DIPEA (0.078 g, 0.60 mmol) was added, and the mixture was stirred at room temperature. Compound II-3 (0.041 g, 0.13 mmol) was then added, and the mixture was stirred at room temperature for approximately 3.5 h. Methyl tert-butyl ether (50 mL) was added to the reaction mixture, and the mixture was stirred at room temperature. A solid precipitated. The mixture was stirred for 10 min. The mixture was then filtered. The filter cake was collected. The mixture was purified by prep-HPLC (preparative high-performance liquid chromatography, under the following conditions), extracted with ethyl acetate (100 mL × 3), and the organic phases were combined and concentrated under reduced pressure to obtain a pale yellow oily liquid (0.020 g, yield 15.10%).

[0179] Prep-HPLC conditions: Feromon C18 column, 50 mm 250mm 10 μm, mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile, flow rate: 100.0 ml / min, detection wavelength: 260 nm, isocratic elution with phase A: phase B - 55: 45 for 20 min.

[0180] HRMS-ESI excess mass calcd for C 56 H 62 FN7O 18 [M+H] + requires 1140.4208, 1141.4242; found 1140.4228, 1141.4248.

[0181] Example 3: Compound III-10

[0182]

[0183] Step 1: Synthesis of Compound III-6 Acetyl bromide-Alpha-D-glucuronide methyl ester (1.00 g, 2.52 mmol), dichloromethane (10 mL), silver trifluoromethanesulfonate (0.97 g, 3.78 mmol), and benzyl glycolate (0.46 g, 2.77 mmol) were added to a single-necked flask. The mixture was stirred at room temperature for approximately 0.5 h. TLC was used to confirm the complete reaction of the starting material. The mixture was filtered, washed once with 30 mL of water, and the organic phase was collected, dried over anhydrous sodium sulfate, and purified by column chromatography (PE:EA(v / v) = 2:1) to give a white solid (0.31 g, yield 25.52%). MS (ESI, pos. ion) m / z: 500.0 [M+H2O] + ; Step 2: Synthesis of Compound III-7 Compound III-6 (0.21 g, 0.44 mmol), palladium on carbon (0.094 g, 0.088 mmol), and THF (5 mL) were added to a single-necked flask. The mixture was placed under hydrogen protection and reacted at room temperature after displacement. The reaction proceeded completely. The mixture was filtered, washed with tetrahydrofuran (10 mL), and evaporated to dryness to give a white solid (172 mg, 100% yield).

[0184] MS (ESI, pos.ion) m / z: 410.0 [M+H2O] + .

[0185] Step 3: Synthesis of Compound III-1 In a single-necked flask, N-(fluorenylmethoxycarbonyl)-L-valine-L-alanine (2.50 g, 6.09 mmol), tert-butyl(5-amino-2-((tert-butyldiphenylsiloxy)methyl)benzyl)(methyl)carbamate (3.07 g, 6.09 mmol), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (2.26 g, 9.13 mmol), and DCM (100 mL) were added, and the mixture was stirred overnight at room temperature. The mixture was washed once with water (50 mL), dried over anhydrous sodium sulfate, and purified by column chromatography (PE:EA(v / v) = 2:1) to give a pale yellow solid (5.40 g, yield 98.82%).

[0186] 1H NMR (400 MHz, DMSO-d6) δ 10.04 (s, 1H), 8.20 (s, 1H), 7.89 (d, J =7.5 Hz, 2H), 7.75 (t, J = 7.1 Hz, 2H), 7.64 (d, J = 6.9 Hz, 4H), 7.56 (s,1H), 7.45 (dt, J = 13.7, 6.8 Hz, 10H), 7.36 – 7.30 (m, 3H), 4.71 (s, 2H), 4.42 (d, J = 13.3 Hz, 3H), 4.23 (d, J = 3.7 Hz, 2H), 3.92 (t, J = 8.0 Hz,1H), 3.81 (d, J = 6.5 Hz, 1H), 2.68 (s, 3H), 1.90 – 1.83 (m, 1H), 1.35 – 1.25(m, 9H), 1.10 (d, J=4.0, 3H), 1.01 (s, 9H), 0.89 (d, J = 6.7 Hz, 6H). Step 4: Synthesis of Compound III-2 Compound III-1 (1.90 g, 2.12 mmol) and THF (20 mL) were added to a single-necked flask. Pyridine hydrogen fluoride (3.15 g, 31.8 mmol) was added at 0 °C, and the reaction was maintained at this temperature for approximately 27 h. The mixture was neutralized with saturated sodium bicarbonate solution, extracted with EA (30 mL × 3), washed once with saturated brine (50 mL), and purified by column chromatography (PE:EA (v / v) = 1:2) to give a white solid (0.86 g, yield 61.64%).

[0187] MS (ESI, pos.ion) m / z: 586.1 [M-OC4H9] + .

[0188] 1H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.21 (d, J = 6.9 Hz, 1H), 7.93 (d, J = 7.4 Hz, 2H), 7.78 (t, J = 7.3 Hz, 2H), 7.55 (s, 1H), 7.46 (dd, J= 15.5, 8.2 Hz, 3H), 7.35 (dd, J = 14.6, 7.3 Hz, 4H), 5.08 (t, J = 4.8 Hz,1H), 4.52 – 4.40 (m, 5H), 4.32 (d, J = 11.0 Hz, 1H), 4.26 (t, J = 5.1 Hz,2H), 3.95 (t, J = 8.0 Hz, 1H), 2.78 (s, 3H), 2.03 – 1.94 (m, 1H), 1.43 (d, J= 27.3 Hz, 9H), 1.33 (d, J = 7.0 Hz, 3H), 0.91 (dd, J = 12.4, 6.7 Hz, 6H). Step 5: Synthesis of Compound III-3 Compound III-2 (0.50 g, 0.76 mmol), DMF (3 mL), DIPEA (0.20 g, 1.52 mmol), and di(p-nitro)carbonate (0.28 g, 0.91 mmol) were added to a single-necked flask. The reaction was carried out under nitrogen protection at room temperature for approximately 4 h. TLC was monitored until the reactants had completely reacted. The reaction mixture was then extracted with water (10 mL), ethyl acetate (30 mL × 3), washed once with saturated brine, and purified by column chromatography (DCM:MeOH (v / v) = 99:1) to give a white solid (360 mg, yield 57.58%).

[0189] 1H NMR (400 MHz, CDCl3) δ 8.68 (s, 1H), 8.26 (d, J = 9.1 Hz, 2H), 7.75 (d, J = 7.5 Hz, 2H), 7.55 (dd, J = 11.9, 7.6 Hz, 2H), 7.38 (dd, J = 13.5, 8.2Hz, 5H), 7.33 – 7.27 (m, 2H), 6.61 (d, J = 6.0 Hz, 1H), 5.35 (d, J = 6.7 Hz, 1H), 5.29 (s, 2H), 4.70 – 4.60 (m, 1H), 4.54 (s, 1H), 4.48 (d, J = 6.4 Hz, 2H), 4.19 (t, J = 6.3 Hz, 1H), 4.04 - 3.96 (m, 1H), 2.78 (s, 3H), 2.22 - 2.10(m, 1H), 1.47 (s, 9H), 1.44 (d, 3H), 0.94 (dd, J = 9.8, 7.1 Hz, 6H).

[0190] Step 6: Synthesis of Compound III-4 Compound III-3 (0.12 g, 0.15 mmol), Exatecan (0.078 g, 0.18 mmol), and DMF (1 mL) were added to a single-necked flask and dissolved. HOBT (0.030 g, 0.22 mmol) and DIPEA (0.039 g, 0.30 mmol) were added, and the reaction was carried out at room temperature for approximately 8 hours under nitrogen protection. TLC monitoring showed that the starting material had completely reacted. The reaction mixture was extracted with tap water (10 mL), EA (30 mL × 3), washed once with saturated brine, and dried over anhydrous sodium sulfate. Column chromatography purification (DCM:MeOH (v / v) = 98:2) yielded a brownish-yellow solid (127 mg, yield 77.84%).

[0191] 1H NMR (400 MHz, DMSO-d6) δ 10.04 (s, 1H), 8.19 (d, J = 7.0 Hz, 1H), 8.04 (d, J = 8.3 Hz, 1H), 7.89 (d, J = 7.4 Hz, 2H), 7.80 – 7.71 (m, 3H), 7.56(s, 1H), 7.44 – 7.36 (m, 5H), 7.33 – 7.30 (m, 3H), 6.53 (s, 1H), 5.44 (s,2H), 5.29 (s, 3H), 5.12 (s, 2H), 4.46 (s, 2H), 4.44 – 4.38 (m, 1H), 4.33 –4.27 (m, 1H), 4.24 – 4.19 (m, 2H), 3.95 (t, J = 7.8 Hz, 1H), 3.26 – 3.20 (m, 1H), 2.76 (s, 3H), 2.38 (s, 3H), 2.23 – 2.13 (m, 2H), 2.03 – 1.95 (m, 2H), 1.91 – 1.81 (m, 2H), 1.36 – 1.27 (m, 9H), 1.25 – 1.24 (m, 3H), 0.90 – 0.85(m, 9H). HRMS-ESI excess mass calcd for C 62 H 66 FN7O 12 [M + H] + requires 1120.4826; found 1120.4847 Step 7: Synthesis of Compound III-5 Compound III-4 (0.758 g, 0.68 mmol) and DCM (8 mL) were added to a single-necked flask, and TFA (7.75 g, 68 mmol) was added at 0 °C. The reaction mixture was then reacted for approximately 1 h. The reaction solution was concentrated to give a yellow solid (0.81 g, 100% yield). MS (ESI, pos.ion) m / z: 1019.9 [M+H] + .

[0192] Step 8: Synthesis of Compound III-8 Compound III-7 (0.33 g, 0.85 mmol), HATU (0.81 g, 2.13 mmol), DIPEA (0.46 g, 3.55 mmol), and DMF (9 mL) were added to a single-necked flask and stirred at room temperature. Then, III-5 (0.81 g, 0.71 mmol) was added, and the reaction proceeded for approximately 22 h. The starting material was completely reacted by TLC. The mixture was extracted with water (100 mL) and ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine (50 mL × 2), and purified by column chromatography (DCM:MeOH (v / v) = 97:3) to give a brownish-gray solid (0.264 g, yield 26.5%).

[0193] HRMS-ESIexcess mass calcd for C 72 H 76 FN7O 21 [M + H] + requires 1394.5151, found 1394.5171. Step 9: Synthesis of Compound III-9 Compound III-8 (0.27 g, 0.19 mmol) was added to THF (6 mL), and the mixture was stirred at 0°C. Methanol (6 mL) was slowly added dropwise. Potassium carbonate (0.26 g, 1.90 mmol) and water (4 mL) solution were added dropwise, and the mixture was stirred at room temperature. The reaction proceeded for approximately 12 hours, and liquid chromatography confirmed that the reactants had reacted completely. The pH of the reaction mixture was adjusted to approximately 7 using sodium bisulfate aqueous solution. The pH was then further adjusted to approximately 6 using a small amount of trifluoroacetic acid. A portion of the solvent was concentrated by rotary evaporation at 45°C. Methyl tert-butyl ether (20 mL) was added, and the mixture was stirred at room temperature for 20 minutes. The lower turbid layer was collected. The mixture was directly concentrated to dryness at 50°C. The concentrate was then mixed with dichloromethane (50 mL) and methanol (25 mL), and stirred at room temperature for 1 hour. The mixture was filtered, and the filtrate was concentrated at 50°C to obtain a pale yellow solid (0.38 g), which was used directly in the next reaction step.

[0194] Step 10: Synthesis of Compound III-10 Compound III-9 (0.19 g, 0.18 mmol) was added to DMF (2 mL), followed by DIPEA (0.047 g, 0.36 mmol). Then, intermediate I-14 (0.053 g, 0.14 mmol) solution dissolved in DMF (0.5 mL) was added dropwise. The reaction was allowed to proceed at room temperature for approximately 2.5 h, under HPLC control. The reaction solution was then directly prepared using Pre-HPLC. The prepared solution was concentrated to remove some acetonitrile and lyophilized to give a pale yellow solid (0.057 g, yield 24.14%).

[0195] HRMS-ESI excess mass calcd for C 61 H 68 FN9O 19 S [M+H] + requires 1282.4409, found 1282.4453. Prep-HPLC conditions: Feromon C18 column, 50 mm 250mm 10 μm, mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile, flow rate: 100.0 ml / min, detection wavelength: 260 nm, eluted isocratically with phase A: phase B - 55: 45 for 20 min.

[0196] Example 4: Compound IV-1

[0197] Step 1: Synthesis of compound IV-1 Compound III-9 (0.088 g, 0.085 mmol), DMF (1 mL), and DIPEA (0.022 g, 0.17 mmol) were added dropwise to a single-necked flask. A solution of II-3 (0.026 g, 0.085 mmol) and DMF (0.1 mL) was then added dropwise. The reaction was carried out under nitrogen atmosphere at room temperature for approximately 2 hours, controlled by HPLC. Methyl tert-butyl ether (10 mL) was added to the reaction mixture, precipitating an oily liquid. The oily liquid was sent for pre-HPLC preparation. Most of the organic solvent was removed by vortexing, and the remaining solvent was lyophilized to give a yellow solid (0.036 g, yield 34.46%).

[0198] HRMS-ESI excess mass calcd for C 60 H 69 FN8O 19 [M + H] +requires 1225.4736, found 1225.4786.

[0199] Prep-HPLC conditions: Feromon C18 column, 50 mm 250mm 10 μm, mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile, flow rate: 100.0 ml / min, detection wavelength: 260 nm, eluted isocratically with phase A: phase B - 55: 45 for 20 min.

[0200] Example 5: Phage screening technology and flow cytometry screening of MUC1 monoclonal antibodies A gene encoding the C-terminus and SEA domain of the MUC1 protein (120 amino acids) was designed using the NCBI database and constructed into a mammalian eukaryotic expression system. The resulting protein, MUC1-P120, containing the C-terminus and SEA domain, was expressed and used for mouse immunization and screening of monoclonal antibodies from a mouse immune library. After four intramuscular injections into Balb / C mice, serum antibody titers were measured by ELISA, reaching 10... 5 Level. Lymphocyte mRNA was extracted from mouse spleens and then reverse transcribed to obtain corresponding cDNA. Using the corresponding cDNA as a template, the VH and VK sequences of the antibody were amplified. Gene fragments were linked using a linker to establish an immune library. Phages in the immune library were panned using the MUC1-P120 protein. After three rounds of panning, monoclonal antibody phages that could bind to the MUC1-P120 protein were screened using ELISA. The binding results of candidate phage 82 with MUC1-P120 are shown in Table 2 (1H7 is the positive control scfv antibody). The ELISA results showed that both the positive antibody 1H7 and the screened phage 82 could specifically bind to the MUC1 protein.

[0201] Table 2. ELISA results of candidate substance binding to MUC1-P120

[0202] Example 6: Antibody Expression and Purification A series of antibody expression vectors were constructed using molecular cloning methods and recombinantly expressed in the 293F expression system. The nucleotide sequences encoding the light and heavy chains of a series of monoclonal antibodies were obtained by extracting plasmids from panning phage cultures. Heavy and light chain expression plasmids were constructed separately, and finally co-transformed to obtain Fab-form monoclonal antibodies.

[0203] Translational-level analysis was performed on a series of antibodies. Cell culture media were purified using a Protein A chromatography column, and absorption peaks were collected for mass spectrometry analysis. Mass spectrometry analysis revealed that the molecular weights of the Fab antibodies were approximately 150 kDa, consistent with the theoretical molecular weight, indicating a dimeric form. Simultaneously, the collected samples were analyzed by 10% SDS-PAGE electrophoresis after reduction and non-reduction. Reduced SDS-PAGE electrophoresis showed bands of approximately 50 kDa and 25 kDa, while non-reduced SDS-PAGE electrophoresis showed a single band at approximately 150 kDa, consistent with theoretical band sizes. The purified samples were dialyzed overnight at 4°C using 0.02 M PBS buffer (pH 7.4).

[0204] Example 7: The binding specificity of the antibody to the antigen described in this invention The binding of HEK23T cells stably overexpressing human MUC1 to the candidate antibody was detected by flow cytometry. The positive control antibody 1H7 is derived from patent CN111201240A. 1.5 10 5 Cells were resuspended in 96-well plates and incubated with different concentrations of antibody at 4°C for 2 h. The cells were then washed twice in PBS. PE-Goat anti-human IgG Fc (Jackson Immuno Research) was diluted 1:500 and incubated at 4°C for 30 min to detect the binding of candidate antibodies. The cells were then washed three times in PBS and analyzed on FACS (Beckman). The results are shown in Table 3. The experimental results indicate that the candidate antibody has a higher affinity (top value) for HEK23T cells overexpressing human MUC1 than the positive antibody 1H7.

[0205] Table 3. Binding of candidate antibodies to HEK293T human MUC1 overexpressing cells (EC) 50

[0206] The binding of candidate antibodies to NCI-H1650 tumor cells endogenously expressing human MUC1 was detected using flow cytometry. The positive control antibody 1H7 is derived from patent CN111201240A. 1.5 105 cells were resuspended in 96-well plates and incubated with different concentrations of antibody at 4°C for 2 h. The cells were then washed twice in PBS. PE-Goat anti-human IgG Fc (Jackson Immuno Research) was diluted 1:500 and incubated at 4°C for 30 min to detect the binding of candidate antibodies. The cells were then washed three times in PBS and analyzed on FACS (Beckman). The results are shown in Table 4. The experimental results indicate that the candidate antibody has a better affinity and TOP value than the positive antibody 1H7 for NCI-H1650 tumor cells overexpressing human MUC1.

[0207] Table 4. Binding of candidate antibodies to NCI-H1650 human MUC1 overexpressing cells.

[0208] The binding of antibodies to the cross-species recombinant cynomolgus monkey and human MUC1 was detected using ELISA. Recombinant cynomolgus monkey and human MUC1 proteins were purchased from Acro. Antigen at 1 μg / ml was coated onto ELISA plates and incubated overnight at 4°C. Non-specific binding sites were then blocked with 3% BSA for 1 h at RT, followed by incubation with 3 μg / ml antibody for 1 h. The bound mAbs were detected using HRP-goat anti-human antibody, followed by washing, addition of TMB substrate (Yunqiao Biotechnology), and readings at 450 nm. The experimental results are shown in Table 5. The results indicate that the candidate antibodies showed better affinity for human MUC1 protein than the positive antibody 1H7; all MUC1 candidate antibodies bound to the monkey antigen, while the 1H7 antibody did not show any binding to the monkey antigen.

[0209] Table 5. Binding of candidate antibodies to human and monkey-derived MUC1 proteins.

[0210] The binding of antibodies to human and monkey MUC1 antigens was confirmed using biomembrane interference technology. An AHC2 (octet) sensor chip was activated in SD buffer (PBS solution, 0.1% BSA, 0.02% Tween), and antibodies (5 μg / ml) were immobilized on the sensor chip. Binding to different concentrations of human and monkey MUC1 was detected, and the association and dissociation constants were calculated using ForteBio Data Analysis software (version 9.0). The results are shown in Table 6. As can be seen from Table 6, compared with 1H7, the screened MUC1 candidate antibodies exhibited stronger affinity for both human and monkey MUC1 proteins.

[0211] Table 6

[0212] Preparation of antibody-drug conjugates Example 8 Preparation of conjugate MUC1-ADC-01 Add 900 μL of LMUC1 monoclonal antibody (antibody 82 of this invention) in PBS solution (1.8 mg, 6.0 nmol, 1.0 eq, concentration 2.00 mg / mL) to a 2.5 mL PE tube, then add 10 mM TCEP solution (36 μL, 30.0 eq) and mix well. Adjust the pH of the reaction solution to 7.2 ± 0.1 using 0.5 M Na2HPO4 aqueous solution, and then incubate the reaction at 37 °C for 2 h. After that, slowly add 18.0 μL of compound I-15 in DMSO solution (15 eq, concentration 10 mM), and then add 80 μL of the solution. DMSO solvent was added to the reaction mixture, and after mixing, it was incubated at 25°C for 2 hours. After the reaction was completed, DMSO and small molecule compound I-15 were removed by ultrafiltration using a 50 kDa filter membrane (10 times of 2x ultrafiltration, replacement with 20 mM acetate-sodium acetate buffer, pH=4.5). The sample obtained was 800 μl, the sample concentration was 1.85 mg / ml, the DAR value calculated by mass spectrometry was 7.94, and the purity detected by SEC was 96.5%.

[0213] The conjugates MUC1-ADC-02, MUC1-ADC-03, and MUC1-ADC-04 were prepared using the same method as MUC1-ADC-01, and the results are shown in Table 7 below. Table 7: Linker-Payload Usage and Characterization Results of MUC1 Couplings

[0214] Example 9 Preparation of conjugate EGFR-ADC-05 720 μL of EGFR monoclonal antibody (cetuximab) in PBS solution (1.8 mg, 6.0 nmol, 1.0 eq, concentration 2.5 mg / mL) was added to a 2.5 mL PE tube, followed by the addition of 10 mM TCEP solution (30 μL, 25.0 eq.). The mixture was then incubated at 37 °C for 2 h. Next, 18.0 μL of compound I-15 DMSO solution (14 eq, concentration 10 mM) was slowly added, followed by the addition of 65 μL of DMSO solution. The mixture was then incubated at 25 °C for 2 h. After the reaction was complete, the DMSO and small molecule compound I-15 were removed by ultrafiltration using a 50 kDa membrane (15 cycles of 2x ultrafiltration, 20...). The sample was replaced with mM acetate-sodium acetate buffer (pH=4.5) to obtain 400 μl of sample. The sample concentration was 2.12 mg / ml. The DAR value calculated by mass spectrometry was 7.98, and the purity detected by SEC was 97.4%.

[0215] The conjugates EGFR-ADC-06, EGFR-ADC-06, and EGFR-ADC-06 were prepared using the same method as EGFR-ADC-05, and the results are shown in Table 8 below. Table 8: Characterization results of Linker-Payload usage and EGFR conjugates

[0216] Example 10 Preparation of conjugate TROP2-ADC-09 Add 500 μL of TROP2 monoclonal antibody (antibody 83 of this invention) in PBS solution (0.9 mg, 6.0 nmol, 1.0 eq, concentration 1.8 mg / mL) to a 2.5 mL PE tube, then add 10 mM TCEP solution (19.2 μL, 32 eq.) and mix well. Adjust the pH of the reaction solution to 7.2 ± 0.1 using 0.5 M Na2HPO4 aqueous solution, and then incubate the reaction at 37 °C for 2 h. After that, slowly add 9.0 μL of compound I-15DMSO solution (15 eq, concentration 10 mM), and then add 50 μL of the remaining solution. Add DMSO solution to the reaction mixture, mix well, and incubate at 25°C for 4 hours. After the reaction is complete, use a 50 kDa ultrafiltration membrane to concentrate and remove DMSO and small molecule compound I-15 (15 times of 2x ultrafiltration, replacement with 20 mM PBS buffer, pH=6.5), to obtain a sample of 400 μl. The sample concentration was tested at 1.52 mg / ml. The DAR value calculated by mass spectrometry was 7.88, and the purity detected by SEC was 93.6%.

[0217] The conjugates TROP2-ADC-10, TROP2-ADC-11, and TROP2-ADC-12 were prepared using the same method as TROP2-ADC-09, and the results are shown in Table 9 below. Table 9. Linker-Payload Usage and Characterization Results of TROP2 Conjugate

[0218] Biological tests Example A: Detection of the cytotoxic activity of the antibody-drug conjugate (ADC molecule) of the present invention against cells in vitro. Tumor cells (human skin squamous cell carcinoma cell line A431, human non-small cell lung cancer cell line HCC827, human non-small cell lung cancer cell line NCI-H1650, human lung adenocarcinoma cell line NCI-H1975, human gastric cancer cell line NUGC4, and human pancreatic cancer cell line HPAC) in the logarithmic growth phase were digested with trypsin, and cell counts and viability were recorded. Cells were resuspended in complete culture medium and added to 96-well plates at 3000 cells / well / 90 μL, and incubated overnight at 37°C in a 5% CO2 incubator. The next day, after cell attachment, the ADC of this embodiment was diluted with complete culture medium, and 10 μL was added to each well. The final ADC concentration started at 500 nM and was 3-fold diluted (9 concentration gradients). The cells were incubated at 37°C in a 5% CO2 incubator for 5 days. The inhibitory effect of ADCs on tumor cell growth was evaluated using the Promega CellTiter-Glo chemiluminescence immunoassay (CELISA) on cell viability in the CellTiter-Glo 2.0 Assay (Promega, G9243). Relative light units (RLU) were read from cell culture plates using a microplate reader. The IC50 was calculated using Graphpad Prism 8.0 software to analyze the experimental data. 50 The results are shown in Table A.

[0219] Table A: Inhibitory IC50 of the ADC of the present invention on a series of tumor cells 50 (nM)

[0220] Test results: The ADC molecule of this invention has a strong tumor cell killing effect, and the tumor killing ability is positively correlated with the expression abundance of the target.

[0221] Example B: Pharmacodynamic experiment of the ADC molecule of the present invention in a human gastric cancer NUGC4 xenograft model. Human gastric cancer cells NUGC4 in logarithmic growth phase were harvested, centrifuged, and counted. The cell density was then adjusted to 5.0 g / cm³ using serum-free RPMI-1640 medium and Matrigel mixture (1:1 ratio). 10 7 / mL. Inject 0.1 mL / mouse subcutaneously into the back of Babl / c-nude mice. Wait until the average tumor volume reaches 200 mm. 3and randomly grouped for administration. The corresponding ADC was intravenously administered once a week for 2 weeks. Starting from day 0 of administration, tumor diameter was measured and the body weight of mice was weighed twice a week, for calculating the tumor volume and the trend of body weight change. The relative tumor growth inhibition rate (TGI) or relative tumor proliferation rate T / C (%) was calculated based on the results on the 19th day after administration for efficacy evaluation, and safety evaluation was performed based on animal body weight changes and mortality. SPSS software was used for statistical analysis. When there are three or more groups, if the data of each group are in normal distribution, one-way ANOVA test is used for inter-group comparison, LSD is used for inter-group comparison with homogeneous variance, and Dunnett's T3 is used for inter-group comparison with heterogeneous variance. P < 0.5 indicates statistically significant difference.

[0222] T / C % is the relative tumor proliferation rate, that is, the percentage value of the relative tumor volume of the treatment group and the control group at a certain time point. T and C are the relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively.

[0223] The calculation formula is as follows: T / C % = T RTV / C RTV ×100% (T RTV : mean RTV of the treatment group; C RTV : mean RTV of the control group; RTV=V t / / V0, where V0 is the tumor volume of the animal at the time of grouping, and V t is the mean tumor volume at a certain measurement, and T RTV and C RTV use data from the same day).

[0224] The calculation method of TGI (%) is as follows: (1) If V t >V0, relative tumor growth inhibition rate (TGI)% = [1-T / C] ×100%; (2) If V t <V0, relative tumor growth inhibition rate (TGI)% = [1-(V t - V0) / V0] ×100%.

[0225] The experimental results are shown in the attached Figure 1 , on the 19th day after grouped administration, the mean tumor volume of the control group mice was 1789.82mm 3 ; the mean tumor volume of the MUC1-ADC-01 4.53mpk group was 24.18mm 3The T / C value was 1.35%, and the TGI value was 188.78%; the mean tumor volume in the EGFR-ADC-05 4.53mpk group was 73.91 mm. 3 The T / C value was 4.12%, and the TGI value was 165.67%.

[0226] The ADC molecules of the present invention can effectively kill human gastric cancer NUGC4 cells in vivo, producing an anti-tumor effect, and the ADC molecules of the present invention are well tolerated in mice.

[0227] Example C: Pharmacodynamic experiment of the ADC molecule of the present invention in a human non-small cell lung cancer NCI-H1975 xenograft model. Logarithmic growth phase human non-small cell lung cancer cells (NCI-H1975) were harvested, centrifuged, and counted. The cell density was then adjusted to 5.0 g / cm³ using serum-free RPMI-1640 medium and Matrigel mixture (1:1 ratio). 10 7 / mL. Inject 0.1 mL / mouse subcutaneously into the back of Babl / c-nude mice. Wait until the average tumor volume reaches 200 mm. 3 Mice were randomly assigned to groups and administered the corresponding ADC intravenously for 2 weeks, once a week. Starting from day 0 of administration, tumor diameter and body weight were measured twice a week to calculate the trend of tumor volume and body weight changes. The relative tumor inhibition rate (TGI) or relative tumor proliferation rate (T / C) (%) was calculated based on the results on day 18 after administration to evaluate efficacy. Safety was evaluated based on changes in body weight and mortality. Statistical analysis was performed using SPSS software. When there were three or more groups and the data were normally distributed, one-way ANOVA was used for comparisons between groups. LSD was used for comparisons between groups with homogeneous variances, and Dunnett's T3 was used for comparisons between groups with unequal variances. P < 0.05 was considered statistically significant.

[0228] T / C % represents the relative tumor proliferation rate, which is the percentage of tumor volume in the treatment group and the control group at a specific time point. T and C are the relative tumor volumes (RTV) in the treatment group and the control group at a specific time point, respectively.

[0229] The calculation formula is as follows: T / C % = T RTV / C RTV 100% (T RTV : Mean RTV in the treatment group; C RTV : Average RTV of the control group; RTV=V t / V0, where V0 is the tumor volume of the animal at the time of grouping, V tis the average tumor volume at a certain measurement, T RTV and C RTV take data from the same day).

[0230] The calculation method of TGI (%) is as follows: (1) If V t >V0, relative tumor growth inhibition rate (TGI)% = [1-T / C] 100%; (2) If V t <V0, relative tumor growth inhibition rate (TGI)% = [1-(V t - V0) / V0] 100%.

[0231] The experimental results are shown in the attached Figure 2 as follows: on day 18 after grouped administration, the average tumor volume of mice in the control group was 1036.43 mm 3 ; the average tumor volume of the MUC1-ADC-01 2.5mpk group was 308.61 mm 3 , the T / C value was 29.53%, and the TGI value was 70.50%; the average tumor volume of the EGFR-ADC-05 2.5mpk group was 403.49 mm 3 , the T / C value was 38.93%, and the TGI value was 61.60%.

[0232] The ADC molecules of the present invention can effectively kill human lung cancer NCI-H1975 cells in vivo to produce an anti-tumor effect, and the ADC molecules of the present invention have good tolerance in mice.

[0233] Example D: Detection of plasma stability of the ADC molecules of the present invention in human, monkey and mouse plasma The ADC molecule to be tested was diluted to 100 μg / mL with human, monkey and mouse plasma. The prepared drug-containing plasma was placed in an incubator at 37°C for incubation. On day 0, day 3, day 5, day 9, day 15, day 22 and day 25, 100 μL of plasma from each species was taken out and added to a 1.5 mL EP tube, then 300 μL of internal standard (propranolol) working solution was added for precipitation. After centrifugation at 4000 rpm for 5 min, 120 μL of the supernatant was taken, 120 μL of ultrapure water was added and mixed uniformly for LC-MS / MS analysis to detect the concentration of free payload and calculate the payload release rate. The experimental results are shown in Table B. According to the experimental results in Table B, after the ADC molecules of the present invention were incubated in plasma of different species for 25 days, the payload release rate did not exceed 1.2%. In particular, no payload was detected in the plasma of humans and monkeys for MUC1-ADC-01, indicating that the ADC molecules of the present invention maintain good plasma stability in the plasma of different species. The results are shown in Table B.

[0234] Table B: Payload release rate (%) of the ADC molecule of the present invention in plasma of different species

[0235] / indicates that the payload was not detected in the blood plasma.

[0236] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "some implementations," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments, implementations, or examples described in this specification, as well as the features of different embodiments, implementations, or examples.

[0237] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. An antibody-drug conjugate as shown in formula (I) or a pharmaceutically acceptable salt thereof, (I) in, Ab represents an antibody or antigen-binding fragment; L1 is a linker that connects an S atom to an antibody or antigen-binding fragment; L2 is the connecting segment between connectors L1 and L3; L3 is the linker segment connecting L2 and drug D; Drug D is selected from cytotoxic drugs, drugs for treating autoimmune diseases, or anti-inflammatory drugs; t can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

2. The antibody-drug conjugate according to claim 1, wherein, Drug D is selected from eribulin, monomethyl aurestatin E (MMAE), monomethyl aurestatin F (MMAF), aurestatin E, aurestatin F, maytansine DM1, maytansine DM4, maytansine alcohol, sandycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, anthracyclines, chachiin, dollasstatin 10, docamycin, doxorubicin, tylansstatin A, hookmycin, amatoxins, ricin, diphtheria toxin, interleukins, tumor necrosis factor, chemokines, SN38, or eczema and its derivatives.

3. The antibody-drug conjugate according to claim 1 or 2, wherein, Drug D is selected from , , , , or .

4. The antibody-drug conjugate according to any one of claims 1-3, wherein, L1 is selected from the following structures: , , , , or ;in The end is connected to Ab via an S atom. The terminal is connected to L2; Each X is independently a bond, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH=CH-, -CH=CHCH2-, -CH2CH=CH-, -C≡C-, -CH2C≡C-, -C≡C-CH2-, -CH2CH2C≡C-, -CH2C≡CCH2-, -C≡CCH2CH2-, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, indole, naphthyl, pyrrolyl, pyrazolyl, imidazoleyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, furanyl, thienyl, thiazolyl, oxazolyl, pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl; R a and R b Independently, it is H, D, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, vinyl, propenyl, allyl, ethynyl, propargyl, 1-propynyl, 1-ynylbutyl, 2-ynylbutyl, 3-ynylbutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxetyl, aziryl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, piperazinyl, or morpholinyl; wherein the methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl... alkyl, tert-butyl, vinyl, propenyl, allyl, ethynyl, propargyl, 1-propynyl, 1-ynylbutyl, 2-ynylbutyl, 3-ynylbutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxacyclobutyl, aziroxybutyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, piperazinyl, and morpholinyl are each independently and optionally surrounded by 1, 2, 3, 4, or 5 groups selected from D, -F, -Cl, -Br, -I, -OH, -NH2, -CN, -SH, -COOH, oxo, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 1-6 Alkoxy and C 1-6 Substituents of haloalkoxy groups; m can be 1, 2, 3, 4, 5, 6, 7 or 8.

5. The antibody-drug conjugate according to any one of claims 1-4, wherein, L1 is selected from the following structures: , , , , , , , or ;in The end is connected to Ab via an S atom. The terminal is connected to L2.

6. The antibody-drug conjugate according to any one of claims 1-5, wherein, L2 selects a structure composed of one or more of the following segments: C 1-6 Alkylene, -NR x -、-C(=O)-、-O-、 , , , , , , , , , , , , or ; R x For H, D, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-8 Cycloalkyl or 3-8 membered heterocyclic groups.

7. The antibody-drug conjugate according to any one of claims 1-6, wherein, L2 selects a structure composed of one or more of the following segments: C 1-6 Alkylene, -NR x -、-C(=O)-、-O-、 , , , , , , , , , , , , , , , , , or ; R x The derivatives are H, D, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, vinyl, propenyl, allyl, ethynyl, propynyl, 1-propynyl, 1-ynylbutyl, 2-ynylbutyl, 3-ynylbutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxetyl, aziryl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, piperazinyl, or morpholinyl. n can be 1, 2, 3, 4, 5, 6, 7 or 8.

8. The antibody-drug conjugate according to any one of claims 1-7, wherein, L3 is selected from the bond, -NR y CH2-, -C(=O)-, -O-, -NR y CH2CH2NR z C(=O)-、 or ; R y and R z H, D, C independently 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-8 Cycloalkyl or 3-8 membered heterocyclic groups.

9. The antibody-drug conjugate according to any one of claims 1-8, wherein, R y and R z H, D, C independently 1-4 Alkyl, C 2-4 alkenyl, C 2-4 alkynyl group, C 3-6 Cycloalkyl or 3-6 membered heterocyclic groups.

10. The antibody-drug conjugate according to any one of claims 1-9, wherein, R y and R z Independently, it is H, D, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, vinyl, propenyl, allyl, ethynyl, propynyl, 1-propynyl, 1-ynylbutyl, 2-ynylbutyl, 3-ynylbutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxacyclobutyl, aziroxybutyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, piperazinyl, or morpholinyl.

11. The antibody-drug conjugate according to any one of claims 1-10, wherein... Selected from the following structures: 、 、 or 。 12. The antibody-drug conjugate according to any one of claims 1-11, wherein... Ab is an antibody against EGFR, an antibody against TROP2, or an antibody against MUC1, wherein the antibody against EGFR is Cetuximab, demupitamab, depatuxizumab, futuximab, imgatuzumab, lapatuximab, losatuxizumab, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, pimurutamab, serclutamab, tomuzotuximab, or zalutumumab. The anti-TROP2 antibody is enoblituzumab, mirzotamab, or omburtamab; The anti-MUC1 antibody comprises heavy chain variable region CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, and light chain variable region CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO:

12.

13. The antibody-drug conjugate according to any one of claims 1-12, wherein the anti-EGFR antibody comprises the heavy chain of SEQ ID NO: 1 and the light chain of SEQ ID NO: 2; The anti-MUC1 antibody comprises the heavy chain of SEQ ID NO: 3 and the light chain of SEQ ID NO: 4; The anti-TROP2 antibody comprises the heavy chain of SEQ ID NO: 5 and the light chain of SEQ ID NO:

6.

14. A compound that is a compound of formula (II), or a stereoisomer, tautomer, nitride, hydrate, solvate, or pharmaceutically acceptable salt of a compound of formula (II). L-L2-L3-D (II), in, L is selected from the following structures: , , , , or ; The terminal is connected to L2; L2, L3, D, X, R a R b "and m" have the meaning as described in any one of claims 1-10.

15. The compound according to claim 14, wherein, L is selected from the following structures: , , , , , , , or ; The terminal is connected to L2.

16. The compound according to claim 14 or 15, having the following structure, or a stereoisomer, tautomer, nitride, hydrate, solvate, or pharmaceutically acceptable salt thereof, (1) (2) (3) or (4).

17. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of claims 1-13, a compound according to any one of claims 14-16, or a pharmaceutically acceptable salt thereof, further comprising a pharmaceutically acceptable carrier.

18. Use of the antibody-drug conjugate of any one of claims 1-13, the compound of any one of claims 14-16, or the pharmaceutical composition of claim 17 in the preparation of a medicament for the prevention or treatment of cancer.

19. The use according to claim 18, wherein, The cancers mentioned are breast cancer, lung cancer, kidney cancer, liver cancer, ovarian cancer, urethral cancer, prostate cancer, glioblastoma multiforme, pancreatic cancer, colorectal cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, colon cancer, rectal cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, melanoma, bladder cancer, stomach cancer, or esophageal cancer.

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